From the lecture

1. Understand the processes of preparing and viewing tissues by light and electron microscopy.
2. Understand the physical bases for the appearance of tissues in the light and electron microscopes (e.g. What is basophilia and what causes structures to be basophilic? What creates the contrasting light and dark regions in an electron micrograph?)

From the lab session

3. A brief listing of some common stains is present at the end of this section. You should have a general familiarity with H&E (Hematoxylin and Eosin), Masson, PAS, and elastic stains.
4. Become familiar with the various ways to access and view images in the Michigan virtual slide collection

 

General And Connective Tissue Stains

Hematoxylin and Eosin
Hematoxylin is the most commonly used nuclear stain in histology and pathology although, despite its long use and honorable history, the chemistry of the dye is still not fully understood. Essentially, hematoxylin is a basic dye and complexes with nucleic acids (DNA and RNA in the nucleus; RNA in the cytoplasm) or other negatively charged molecules (such as sulfate groups). Structures that bind hematoxylin are therefore termed "basophilic" (base loving). Cells actively secreting proteins exhibit basophilic cytoplasm. Why? (CT6)

Eosin is an acidic dye and the basic structures it stains are termed "eosinophilic" or less commonly "acidophilic" (acid loving). It stains membranes and most proteins. Cells that have large quantities of folded membranes stain intensely with eosin, because of basic amino acids in the membranes (e.g. macrophages contain lots of membrane in the form of phagocytic vesicles as well as basic lysosomal enzymes within those vesicles that stain with eosin). Collagen is generally stained some shade of red/orange whereas actin (such as in smooth muscle cells) is a bit more pink. Elastin, when present in relatively large amounts (such in the walls of blood vessels, in elastic cartilage, and in the esophagus and trachea), will appear glassy red.

A note about acids/bases and their charges: It always seems to a point of confusion as to how it is that an acid such as DNA can have a negative charge when we generally think of something that is acidic as being positively charged (i.e. a solution with lots of H+ ions is "acidic"). However, the better way to think of acids is as proton donors --in solution, an acid such as DNA donates H+ protons (which makes the solution acidic). Upon donating protons, the DNA therefore becomes negatively charged and it is in this state that it binds hematoxylin.

Masson Triple Stain (or "Trichrome")
This dye combination stains mucus as well as collagenous and reticular fibers blue (aniline blue) or green (fast green) depending on the mixes of dyes used; muscle red; nuclei red (they are black if preceded by an iron hematoxylin). This is a commonly used connective tissue stain in both histology and pathology. On your slides the stain is designated "Masson" or "Mass"; but the blue or green collagen is the tip-off.

Elastic stains

• Aldehyde fuchsin
• Aldehyde Fuchsin is a deep purple dye. It stains elastic fibers and granules of beta cells in the islets of Langerhans, cartilage matrix, and stored neurosecretory product in the hypophyseal pars nervosa, among other things. In some of your slides, it is the only stain and therefore only elastin is demonstrated. Other times it is combined with Masson's trichrome.
• Weigert's stain
• Uses a different kind of fuchsin (basic fuchsin), but the result is similar: elastic fibers stain a deep purple color.
• Verhoeff/van Gieson elastic tissue stain
• Verhoeff's hematoxylin contains ferric chloride and iodide which causes it to stain elastic fibers deep purple/black. Frequently counterstained van Gieson's solution with which stains collagen red/orange and cytoskeletal elements (such as actin) yellow-brown.

Silver Stain
In this case silver nitrate is reduced to metallic (black) silver. The process of development and fixation is similar to developing a photograph (stains reticular fibers).

Periodic Acid Schiff (PAS)
This is an extremely useful technique for demonstrating glycoproteins, mucins and some proteoglycans -anything that contains a relatively high amount of sugar groups. It involves the generation of dialdehydes from hexoses (present as the carbohydrate portion of the aforementioned compounds. One of its main uses is the demonstration of basement membranes, especially in the kidney, and/or in sections with epithelia atypia, where breech of the basement membrane is suspected in early carcinomas. An excellent example is slide 210 from the kidney WebScope ImageScope where PAS staining demonstrates the basement membranes (pink lines) of the simple cuboidal epithelium lining the tubules and squamous epithelium in the glomeruli (the round tangles of cells). Note that PAS staining also shows the glycocalyx associated with microvilli (appears as a fuzzy pink border) on epithelia lining some of the tubules.

Virtual Microscopy: Installing, Configuring, and Annotating with ImageScope!

Click the link above to find more information about virtual slides: Set up to access the virtual slides on campus; Installing Aperio ImageScope; Navigating digital slides using Aperio Imagescope; Using Aperio Imagescope Annotations; Using WebScope to Create "Bookmarks" to Regions of Interest

Load Testing

Each year we make improvements to the Virtual Slides collections and infrastructure. This section will help us collect metrics on whether we have enough resources available. This will only take a few minutes. We are evaluating the time it takes to completely open an image.

PLEASE DO NOT CONTINUE UNTIL ASKED TO DO SO.
Test Link 1: WebScope

PLEASE WAIT TO CONTINUE UNTIL ASKED TO DO SO.
Test Link 2: WebScope

Getting Started: Using Annotations in ImageScope

One of the main advantages of viewing the virtual slides in ImageScope is the ability to annotate the images in a "layer" that you can then save to your own computer (the original image on the server is not altered in any way).

1. To begin making annotations, go to a slide of interest, in this case we will use slide #29, which may be opened by clicking on this ImageScope.
2. Begin by opening the "Annotations" window (under the "View" menu or by pressing Crtl+N)
3. Go to an area of interest, then select an annotation tool from the toolbar (see example)
4. Once an annotation tool is selected, the pointer will change to a small "pen" icon. Click and drag to draw the the annotation on the image; finish by releasing the mouse button. If you do not like the placement of the annotation graphic, it may be moved by pressing the Ctrl key and then clicking on the graphic with the mouse. Or you can delete the annotation by clicking on the "X" in the annotation window (see example).
5. Corresponding text can be entered in the "Annotations" window to go along with the annotation graphic:
• in the "Text" column: text entered here will also be displayed on the image
• in the "Description" column: text here is NOT displayed on the screen
6. After generating additional annotations, you may quickly move from one annotated region to another by clicking on rows within the "Annotations" window. You can also "hide" the annotations by clicking on the "Show/Hide Layers" icon (it looks like an eye).
7. Save the annotation file by Exporting it to your computer's hard drive. Be sure to use the "Export Annotations To File" icon (see example) and name the file accordingly --BE SURE to include the slide number in the filename.
8. Now, CLOSE slide #29. You should get an alert box saying "One or more annotation layers have been modified... Do you want to save the changes?" Select "No". (click here for an explanation)

9. Now, we'll see how to use the annotation files once they've been saved to your computer. Re-open slide #29 using this ImageScope. Click on the "Import Annotations To File" button in the "Annotations" window (see example) and select the file that you just saved; you should see the annotations that you generated in steps 4 through 6 above.
10. One thing to note is that ANY annotation file can be imported and overlaid on ANY slide --this is why it is important to include the slide number the annotation filename, so you'll know which file to import for a given slide. To illustrate this point, try opening slide #176 using this ImageScope and then import the slide #29 annotation file that you generated in the steps above. Notice that the annotations are overlaid on the slide, but probably in a manner that doesn't make sense.

 

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Electron Microscopy

 

Normal Structures

Structure	Description
Neutrophil	Normal polymorphonuclear leukocyte
Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 Lymphocyte	Normal lymphocyte
Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 Plasma cell	Normal plasma cell with prominent cytoplasmic smooth endoplasmic reticulum
macrophage	Normal macrophage with oblong nucleus, nucleolus, and cytoplasm with a variety of inclusions
platelets	Normal platelets
mitochondria	Happy mitochondria within a cell
skeletal muscle	Normal skeletal muscle, normal (inset shows a single sarcomere with dark Z discs, and a central M line, with thin actin and thick myosin filaments)
collagen	Normal collagen fibrils
cillia	Normal cilia of respiratory tract epithelium

 

Lymphocyte, normal, electron micrograph

This is a plasma cell with prominent cytoplasmic rough endoplasmic reticulum. The nucleus has radially arranged clumps of chromatin. The perinuclear Golgi apparatus does not appear in this plane of sectioning

Plasma cell with prominent cytoplasmic smooth endoplasmic reticulum, normal, electron micrograph

Platelets, normal, electron micrograph

Skeletal muscle, normal (inset shows a single sarcomere with dark Z discs, and a central M line, with thin actin and thick myosin filaments), electron micrograph

Collagen firbils, normal, electron micrograph

This electron micrograph of the cilia of the pseudostratified epithelium of the tracheal mucosa reveals the arrangement of microtubules, with 9 peripheral doublets and two central singlets. The radial spokes projecting from doublets to a central sheath around the singlets, and the dynein arms projecting between doublets, are hardly visible in this preparation

Special Stains:

 

Mucin stains

There are a variety of mucin stains, all attempting to demonstrate one or more types of mucopolysaccharide substances in tissues. The types of mucopolysaccharides are as follows:

    * Neutral - These can be found in glands of the GI tract and in prostate. They stain with PAS but not with Alcian blue, colloidal iron, mucicarmine, or metachromatic dyes.

    * Acid (simple, or non-sulfated) - Are the typical mucins of epithelial cells containing sialic acid. They stain with PAS, Alcin blue at pH 2.5, colloidal iron, and metachromatic dyes. They resist hyaluronidase digestion.

    * Acid (simple, mesenchymal) - These contain hyaluronic acid and are found in tissue stroma. They do not stain with PAS, but do stain with Alcian blue at pH 2.5, colloidal iron, and metachromatic dyes. They digest with hyaluronic acid. They can be found in sarcomas.

    * Acid (complex, or sulfated, epithelial) - These are found in adenocarcinomas. PAS is usually positive. Alcian blue is positive at pH 1, and colloidal iron, mucicarmine, and metachromatic stains are also positive. They resist digestion with hyaluronidase.

    * Acid (complex, connective tissue) - Found in tissue stroma, cartilage, and bone and include substances such as chondroitin sulfate or keratan sulfate. They are PAS negative but do stain selectively with Alcian blue at pH 0.5.

There are a variety of stains for mucin:

    * Colloidal iron ("AMP") - Iron particles are stabilized in ammonia and glycerin and are attracted to acid mucopolysaccharides. It requires formalin fixation. Phospholipids and free nucleic acids may also stain. The actual blue color comes from a Prussian blue reaction. Tissue can be pre-digested with hyaluronidase to provide more specificity.

    * Alcian blue - The pH of this stain can be adjusted to give more specificity.

    * PAS (peroidic acid-Schiff) - Stains glycogen as well as mucins, but tissue can be pre-digested with diastase to remove glycogen.

    * Mucicarmine - Very specific for epithelial mucins.

The mucin stain with the most specificity is mucicarmine, but it is very insensitive, so it is not really very useful. The stain that is the most sensitive is PAS, but you must learn how to interpret it in order to gain specificity. Colloidal iron stains are unpredictable. Alcian blue stains are simple, but have a lot of background staining.

Gastrointestinal tract goblet cells stained with Alcian blue

The goblet cells of the gastrointestinal tract are filled with abundant acid mucin and stain pale blue with this Alcian blue stain.

Colonic adenocarcinoma stained with mucicarmine

The cytoplasm of the cells lining this neoplastic gland in a colonic adenocarcinoma are pink with the mucicarmine stain, indicative of mucin production typical for an adenocarcinoma at this site

Stains for biogenic amines

Cells that produce polypeptide hormones, active amines, or amine precursors (epinephrine, norepinephrine) can be found individually (Kulchitsky cell of GI tract) or as a group (adrenal medulla). The following is a traditional classification of the staining patterns based upon the ability of the cells to reduce ammoniacal silver nitrate to metallic silver (black deposit in tissue section):

    * Chromaffin

    * Argentaffin

    * Argyrophil (pre-reduction step necessary)

The distinction between chromaffin and argentaffin is artificial, since this depends upon the fixative used. "Chromaffin" cells have cytoplasmic granules that appear brown when fixed with a dichromate solution. "Argentaffin" cells reduce a silver solution to metallic silver after formalin fixation. Either reaction can be produced depending upon which fixative was used. Traditionally, chromaffin reaction is associated with adrenal medulla or extraadrenal paraganglion tissues (pheochromocytomas) whereas argentaffin reaction is associated with carcinoid tumors of the gut. Using a pre-reduction step may get more cells to stain, but they are called "argyrophil" then.

Types of stains for argentaffin include:

   1. Diazo (diazonium salts)

   2. Fontana-Masson

   3. Schmorl's

   4. Autofluorescence

Types of stains for chromaffin include:

   1. Modified Giemsa

   2. Schmorl's

   3. Wiesel's

Types of stains for argyrophil include:

   1. Grimelius (Bouin's fixative preferred)

   2. Pascual's

Gastrointestinal tract enteroendocrine cells stained with Fontana-Masson argentaffin stain

The areas of black, stippled staining identify the scattered enteroendocrine cells in the mucosal crypts of the small intestine (Kulchitsky cells, or if you don't really believe in their existence, replace the K with a B)

Melanin stains

Melanin is normally found in the skin, eye, and substantia nigra. It may also be found in melanomas.

The commonly used Fontana-Masson ("melanin stain") method relies upon the melanin granules to reduce ammoniacal silver nitrate (but argentaffin, chromaffin, and some lipochrome pigments also will stain black as well).

Schmorl's method uses the reducing properties of melanin to stain granules blue-green.

The most specific method of all is an enzyme histochemical method called DOPA-oxidase. It requires frozen sections for best results, but paraffin sections of well-fixed tissues may be used. The stain works because the DOPA substrate is acted upon by DOPA-oxidase in the melanin-producing cells to produce a brownish black deposit.

Bleaching techniques remove melanin in order to get a good look at cellular morphology. They make use of a strong oxidizing agent such as potassium permanganate or hydrogen peroxide. Ocular melanin takes hours to bleach, while that from skin takes minutes.

Formaldehyde-induced fluorescence can be used to highlight biogenic amines (chromaffin, argentaffin) and melanin in tissues. Formalin fixation imparts a strong yellow autofluorescence to unstained tissues with these substances.

The pseudomelanin of melanosis coli is PAS positive whereas true melanin is not. Moreover, pseudomelanin pigment is usually found in macrophages.

                Melanin pigment in cells of malignant melanoma, Fontana-Masson stain

Melanin pigment in cells of malignant melanoma, Fontana-Masson stain.

Lipochrome (lipofuschin) pigments

These are the breakdown products within cells from oxidation of lipids and lipoproteins. They are the wear-and-tear pigments found most commonly in heart, liver, CNS, and adrenal cortex (zona reticularis). The less highly oxidized "ceroid" pigment of testis interstitium and seminal vesicle is another form of lipochrome.

Lipochrome can be stained by Sudan black B, long Ziehl-Neelson acid fast, and Schmorl's methods. Lipochrome may also exihibit a strong orange autofluorescence in formalin-fixed, unstained paraffin sections.

Lipochrome in liver, H and E stain

Lipochrome pigment in the cytoplasm of hepatocytes, H&E stain.

Iron (hemosiderin)

Hemosiderin (storage iron granules) may be present in areas of old hemorrhage or be deposited in tissues with iron overload (hemosiderosis is the term used if the iron does not interfere with organ function; hemochromatosis refers to a condition of iron overload associated with organ failure).

Perl's iron stain is the classic method for demonstrating iron in tissues. The section is treated with dilute hydrochloric acid to release ferric ions from binding proteins. These ions then react with potassium ferrocyanide to produce an insoluble blue compound (the Prussian blue reaction). Mercurial fixatives seem to do a better job of preserving iron in bone marrow than formalin.

Hemosiderin, liver, iron stain.

Hemosiderin, liver, iron stain

Calcium

Only calcium that is bound to an anion (such as PO4 or CO3) can be demonstrated. Calcium forms a blue-black lake with hematoxylin to give a blue color on H&E stain, usually with sharp edges.

VonKossa stain is a silver reduction method that demonstrates phosphates and carbonates, but these are usually present along with calcium. This stain is most useful when large amounts are present, as in bone.

Alizarin red S forms an orange-red lake with calcium at a pH of 4.2. It works best with small amounts of calcium (such as in Michaelis-Gutman bodies). The alizarin method is also used on the Dupont ACA analyzer to measure serum calcium photometrically.

Azan stain can be used to differentiate osteoid from mineralized bone.

Urates

Uric acid crystals are seen in acid urine. In tissue, urates are present as sodium urate. They are soluble in aqueous solutions and slightly soluble in weak alcoholic solutions. Therefore, tissues must be fixed in 95% or absolute alcohol to prevent leaching of urates.

Methenamine silver stains urates black. Sodium urate crystals are also birefringent on polarization. Using a red plate, the crystals show negative birefringence (yellow color) when the crystal's long axis is aligned in the direction of the slow wave. At 90 degrees to this, the crystals will be blue.

Uric acid crystals, polarized, with red plate

Uric acid crystans, polarized, with red plate

Copper

The rare autosomal recessive disorder known as Wilson's disease results from decreased serum ceruloplasmin, the blood protein that transports serum copper. This leads to excessive copper accumulation in brain, eye, and liver. Hepatic copper accumulation results in fatty change, acute hepatitis, chronic hepatitis, and eventual cirrhosis. Urinary copper excretion is increased.

 

The rubeanic acid and rhodanine stains are utilized to detect the cytoplasmic accumulation of copper in the liver.

Copper accumulation in liver, medium power microscopic

The red-brown granular material seen here is excessive lysosomal copper in a patient with the rare autosomal recessive disorder Wilson's disease. Hepatic copper accumulation results in fatty change (seen here with cholestasis as well), acute hepatitis, chronic hepatitis, and eventual cirrhosis

Exogenous pigments and minerals

These come from industrial or environmental exposure by inhalation, ingestion, or contact. Sometimes exposure comes from work-related activities (miners). Sometimes they are planned (tattoo).

Carbon appears as anthracotic pigment in the lungs. It can be distinguished from melanin by doing a melanin bleach. Poorly fixed tissues may contain formalin-heme pigment, which is black and finely granular, but this is widely scattered in the tissues without regard to cellular detail. Formalin-heme pigment is also birefringent on polarization.

Asbestos is a special type of long-thin silica crystal, usually of the mineral group chrysotile. In tissue, these crystals are highly irritative and highly fibrogenic. The fibers become coated with a protein-iron-calcium matrix, giving them a shish-kebab appearance. These are called "ferruginous bodies" because they are highlighted with an iron stain.

Asbestos body, unstained

.

Asbestos bodies, iron stain

Silica is present in many minerals and building materials. Most forms are very inert and cannot be stained in tissue but can be demonstrated by white birefringence on polarization. It is most often present in lung, but can make its way into lymph node.

Silica crystals in silicosis of lung, polarized

Street drugs for injection often are diluted with compounds containing minerals such as silica or talc. These crystals can be found throughout the body, but especially in lymphoreticular tissues.

Polarizable crystals in lung with intravenous drug use

Tattoo pigment is usually black and is inert and non-polarizable. Red tattoo pigment often contains cinnabar (which has mercury in it).

Tattoo pigment in dermis of skin, H and E stain.

In general, minerals are best demonstrated by microincineration techniques or by scanning electron microscopy with energy dispersive analysis (SEM-EDA).

Another use for SEM-EDA in forensic pathology is for analysis of gunshot residue. The primer residue has a characteristic pattern because of the elemental composition which contains antimony, barium, and lead.

SEM-EDA pattern of gunshot residue

Fat stains

The oil red O (ORO) stain can identify neutral lipids and fatty acids in smears and tissues. Fresh smears or cryostat sections of tissue are necessary because fixatives containing alcohols, or routine tissue processing with clearing, will remove lipids. The ORO is a rapid and simple stain. It can be useful in identifying fat emboli in lung tissue or clot sections of peripheral blood.

Oil red O stain of fat emboli in lung

.

Connective tissue stains

The trichrome stain helps to highlight the supporting collagenous stroma in sections from a variety of organs. This helps to determine the pattern of tissue injury. Trichrome will also aid in identifying normal structures, such as connective tissue capsules of organs, the lamina propria of gastrointestinal tract, and the bronchovascular structures in lung.

Chronic active hepatitis with collapse in liver, trichrome stain.

Cerebral abscess in brain, trichrome stain.

Scleroderma with fibrosis of submucosa in stomach, trichrome stain.

Heart with acute myocardial infarction and contraction bands, trichrome stain.

The reticulin stain is useful in parenchymal organs such as liver and spleen to outline the architecture. Delicate reticular fibers, which are argyrophilic, can be seen. A reticulin stain occasionally helps to highlight the growth pattern of neoplasms.

Normal liver at medium magnification, reticulin stain.

Normal spleen at high magnification, reticulin stain.

An elastic tissue stain helps to outline arteries, because the elastic lamina of muscular arteries, and the media of the aorta, contain elastic fibers. The van Gieson method for elastic fibers provides good contrast.

Normal aorta, media with parallel elastic fibers, van Gieson stain.

Hematologic stains

There are a number of special stains employed to identify specific inflammatory cells seen in peripheral blood and tissues. These include the all-purpose Wright-Giemas and Giemsa stains, leukocyte alkaline phosphatase (LAP), tartrate-resistant acid phosphatase (TRAP), and myeloperoxidase (MPO).

Giemsa stain

There are a variety of "Romanowsky-type" stains with mixtures of methylene blue, azure, and eosin compounds. Among these are the giemsa stain and the Wright's stain (or Wright-Giemsa stain). The latter is utilized to stain peripheral blood smears. The giemsa stain can be helpful for identifying components in a variety of tissues.

One property of methylene blue and toluidine blue dyes is metachromasia. This means that a tissue component stains a different color than the dye itself. For example, mast cell graules, cartilage, mucin, and amyloid will stain purple and not blue, which is helpful in identifying these components.

Skin with mast cells in dermis, giemsa stain.

Esophagus with eosinophils, giemsa stain.

Peripheral blood smear, Wright's stain.

Leukocyte alkaline phosphatase

The leukocyte alkaline phosphatase (LAP) stain is helpful in determining whether a high peripheral blood leukocytosis is a reactive process or a leukemia (chronic myelogenous leukemia, or CML). The more differentiated cells in the reactive process will stain more readily with LAP, while leukemic cells will not. The cells on a smear can be assessed and an "LAP score" can be generated. A high score generally indicates a "leukemoid reaction" or reactive condition (with an infection or other inflammatory process) while a low score suggests CML.

Neutrophils in peripheral blood, leukocyte alkaline phosphatase stain.

Tartrate-resistant acid phosphatase

The tartrate-resistant acid phosphatase (TRAP) stain has one major usefulness--to help diagnose a rare leukemia known as hairy cell leukemia. This neoplastic B lymphocyte proliferation affects mainly bone marrow and spleen. There is typically pancytopenia, so the peripheral WBC count is not high. The circulating hairy cells get their name from the cytoplasmic projections. However, positive staining with TRAP helps make the diagnosis.

Hairy cell leukemia with lymphocytes in peripheral blood, tartrate-resistant acid phosphatase stain.

Myeloperoxidase

The myeloperoxidase (MPO) stain is helpful to identify cytoplasmic granules characteristic of myeloid cells. This is useful when there are large, immature white blood cells in the peripheral blood, and it is not clear whether they are of myeloid or of lymphoid origin. Staining with MPO in this setting suggests a myeloid leukemia.

               

 

Acute myelogenous leukemia in peripheral blood smear, myeloperoxidase stain.

Microorganisms

Bacteria appear on H and E as blue rods or cocci regardless of gram reaction. Colonies appear as fuzzy blue clusters. Tissue gram stains are all basically the same as that used in the microbiology lab except that neutral red is used instead of safranin. Gram positive organisms usually stain well, but gram negatives do not (because the lipid of the bacterial walls is removed in tissue processing). Brown and Brenn (or the Brown and Hopps modification) is the method most commonly used.

Fungi stain blue with H and E and red with PAS. The most sensitive method for demonstrating them is Methenamine silver.

A Giemsa stain may help demonstrate donovan bodies and leishmania organisms in tissue sections.

Spirochetes are very difficult to stain. When these organisms are sought in tissues, the patient is generally in the tertiary stage of syphilis and there are few organisms. The best method is the Warthin-Starry. However, the small, thin, spiral organisms are difficult to see and a careful search must be made. In primary syphilis, a lesion called a "chancre" may be present on genitalia, and a scraping made from this lesion can be placed on a slide, and the organisms observed under darkfield microscopy (but you need a darkfield condenser on a microscope).

Spirochetes with Warthin-Starry silver stain.

Spirochetes under darkfield microscopy.

AFB (acid fast bacilli) stain

This stain uses carbol-fuchsin to stain the lipid walls of acid fast organisms such as M. tuberculosis. The most commonly used method is the Ziehl-Neelsen method, though there is also a Kinyoun's method. A modification of this stain is known as the Fite stain and has a weaker acid for supposedly more delicate M. leprae bacilli. However, much of the lipid in mycobacteria is removed by tissue processing, so this stain can, at times, be very frustrating and cause you to search extensively for organsisms you are sure are in a big granuloma. The most sensitive stain for mycobacteria is the auramine stain which requires a fluorescence microscope for viewing.

There are things other than mycobacteria that are acid fast. Included are cryptosporidium, isospora, and the hooklets of cysticerci.

Mycobacterium tuberculosis in lung, Ziehl-Neelsen acid fast stain.

Mycobacterium avium-complex, Ziehl-Neelsen acid fast stain.

Cryptosporidium in stool specimen, Ziehl-Neelsen acid fast stain.

Mycobacterium tuberculosis in lung, Auramine stain, fluorescence.

Nocardia asteroides in lung, acid fast stain.

Gomori methenamine silver stain

This stain, often abbreviated as "GMS", is used to stain for fungi and for Pneumocystis jiroveci (carinii). The cell walls of these organisms are stained, so the organisms are outlined by the brown to black stain. There is a tendency for this stain to produce a lot of artefact from background staining, so it is essential to be sure of the morphology of the organism being sought.

GMS stain for Pneumocystis jiroveci (carinii).

GMS stain for Cryptococcus neoformans.

GMS stain for Coccidioides immitis.

PAS (periodic acid-Schiff)

This an all-around useful stain for many things. It stains glycogen, mucin, mucoprotein, glycoprotein, as well as fungi. A predigestion step with amylase will remove staining for glycogen. PAS is useful for outlining tissue structures--basement membranes, capsules, blood vessels, etc. It does stain a lot of things and, therefore, can have a high background. It is very sensitive, but specificity depends upon interpretation.

Candida in lung, PAS stain.

Glycogen in Ewing's sarcoma, PAS stain.

Nodular glomerulosclerosis in kidney, PAS stain.

Histotechniques:

Tissue Processing

Tissues from the body taken for diagnosis of disease processes must be processed in the histology laboratory to produce microscopic slides that are viewed under the microscope by pathologists. The techniques for processing the tissues, whether biopsies, larger specimens removed at surgery, or tissues from autopsy, are described below. The persons who do the tissue processing and make the glass microscopic slides are histotechnologists.

Specimen Accessioning

 

Tissue specimens received in the surgical pathology laboratory have a request form that lists the patient information and history along with a description of the site of origin. The specimens are accessioned by giving them a number that will identify each specimen for each patient.

    Specimen accessioning.

Gross Examination

Tissues removed from the body for diagnosis arrive in the Pathology Department and are examined by a pathologist, pathology assistant, or pathology resident. Gross examination consists of describing the specimen and placing all or parts of it into a small plastic cassette which holds the tissue while it is being processed to a paraffin block. Initially, the cassettes are placed into a fixative.

    Gross specimen examination.

    Gross specimen examination

.

When a malignancy is suspected, then the specimen is often covered with ink in order to mark the margins of the specimen. Different colored inks can be used to identify different areas if needed. When sections are made and processed, the ink will mark the actual margin on the slide.

    Inking a gross specimen for margins

Fixation - types of fixatives

The purpose of fixation is to preserve tissues permanently in as life-like a state as possible. Fixation should be carried out as soon as possible after removal of the tissues (in the case of surgical pathology) or soon after death (with autopsy) to prevent autolysis. There is no perfect fixative, though formaldehyde comes the closest. Therefore, a variety of fixatives are available for use, depending on the type of tissue present and features to be demonstrated.

There are five major groups of fixatives, classified according to mechanism of action:

    * Aldehydes

    * Mercurials

     Alcohols

    * Oxidizing agents

    * Picrates

Aldehydes include formaldehyde (formalin) and glutaraldehyde. Tissue is fixed by cross-linkages formed in the proteins, particularly between lysine residues. This cross-linkage does not harm the structure of proteins greatly, so that antigenicity is not lost. Therefore, formaldehyde is good for immunoperoxidase techniques. Formalin penetrates tissue well, but is relatively slow. The standard solution is 10% neutral buffered formalin. A buffer prevents acidity that would promote autolysis and cause precipitation of formol-heme pigment in the tissues.

Glutaraldehyde causes deformation of alpha-helix structure in proteins so is not good for immunoperoxidase staining. However, it fixes very quickly so is good for electron microscopy. It penetrates very poorly, but gives best overall cytoplasmic and nuclear detail. The standard solution is a 2% buffered glutaraldehyde

Mercurials fix tissue by an unknown mechanism. They contain mercuric chloride and include such well-known fixatives as B-5 and Zenker's. These fixatives penetrate relatively poorly and cause some tissue hardness, but are fast and give excellent nuclear detail. Their best application is for fixation of hematopoietic and reticuloendothelial tissues. Since they contain mercury, they must be disposed of carefully.

Alcohols, including methyl alcohol (methanol) and ethyl alcohol (ethanol), are protein denaturants and are not used routinely for tissues because they cause too much brittleness and hardness. However, they are very good for cytologic smears because they act quickly and give good nuclear detail. Spray cans of alcohol fixatives are marketed to physicians doing PAP smears, but cheap hairsprays do just as well.

Oxidizing agents include permanganate fixatives (potassium permanganate), dichromate fixatives (potassium dichromate), and osmium tetroxide. They cross-link proteins, but cause extensive denaturation. Some of them have specialized applications, but are used very infrequently.

Picrates include fixatives with picric acid. Foremost among these is Bouin's solution. It has an unknown mechanism of action. It does almost as well as mercurials with nuclear detail but does not cause as much hardness. Picric acid is an explosion hazard in dry form. As a solution, it stains everything it touches yellow, including skin.

Fixation - factors affecting fixation

There are a number of factors that will affect the fixation process:

    * Buffering

    * Penetration

    * Volume

    * Temperature

    * Concentration

    * Time interval

Fixation is best carried out close to neutral pH, in the range of 6-8. Hypoxia of tissues lowers the pH, so there must be buffering capacity in the fixative to prevent excessive acidity. Acidity favors formation of formalin-heme pigment that appears as black, polarizable deposits in tissue. Common buffers include phosphate, bicarbonate, cacodylate, and veronal. Commercial formalin is buffered with phosphate at a pH of 7.

Penetration of tissues depends upon the diffusability of each individual fixative, which is a constant. Formalin and alcohol penetrate the best, and glutaraldehyde the worst. Mercurials and others are somewhere in between. One way to get around this problem is sectioning the tissues thinly (2 to 3 mm). Penetration into a thin section will occur more rapidly than for a thick section.

The volume of fixative is important. There should be a 10:1 ratio of fixative to tissue. Obviously, we often get away with less than this, but may not get ideal fixation. One way to partially solve the problem is to change the fixative at intervals to avoid exhaustion of the fixative. Agitation of the specimen in the fixative will also enhance fixation.

Increasing the temperature, as with all chemical reactions, will increase the speed of fixation, as long as you don't cook the tissue. Hot formalin will fix tissues faster, and this is often the first step on an automated tissue processor.

Concentration of fixative should be adjusted down to the lowest level possible, because you will expend less money for the fixative. Formalin is best at 10%; glutaraldehyde is generally made up at 0.25% to 4%. Too high a concentration may adversely affect the tissues and produce artefact similar to excessive heat.

Also very important is time interval from of removal of tissues to fixation. The faster you can get the tissue and fix it, the better. Artefact will be introduced by drying, so if tissue is left out, please keep it moist with saline. The longer you wait, the more cellular organelles will be lost and the more nuclear shrinkage and artefactual clumping will occur.

Fixatives - general usage

There are common usages for fixatives in the pathology laboratory based upon the nature of the fixatives, the type of tissue, and the histologic details to be demonstrated.

Formalin is used for all routine surgical pathology and autopsy tissues when an H and E slide is to be produced. Formalin is the most forgiving of all fixatives when conditions are not ideal, and there is no tissue that it will harm significantly. Most clinicians and nurses can understand what formalin is and does and it smells bad enough that they are careful handling it.

 

Zenker's fixatives are recommended for reticuloendothelial tissues including lymph nodes, spleen, thymus, and bone marrow. Zenker's fixes nuclei very well and gives good detail. However, the mercury deposits must be removed (dezenkerized) before staining or black deposits will result in the sections.

Bouin's solution is sometimes recommended for fixation of testis, GI tract, and endocrine tissue. It does not do a bad job on hematopoietic tissues either, and doesn't require dezenkerizing before staining.

Glutaraldehyde is recommended for fixation of tissues for electron microscopy. The glutaraldehyde must be cold and buffered and not more than 3 months old. The tissue must be as fresh as possible and preferably sectioned within the glutaraldehyde at a thickness no more than 1 mm to enhance fixation.

Alcohols, specifically ethanol, are used primarily for cytologic smears. Ethanol (95%) is fast and cheap. Since smears are only a cell or so thick, there is no great problem from shrinkage, and since smears are not sectioned, there is no problem from induced brittleness.

For fixing frozen sections, you can use just about anything--though methanol and ethanol are the best.

Tissue Processing

Once the tissue has been fixed, it must be processed into a form in which it can be made into thin microscopic sections. The usual way this is done is with paraffin. Tissues embedded in paraffin, which is similar in density to tissue, can be sectioned at anywhere from 3 to 10 microns, usually 6-8 routinely. The technique of getting fixed tissue into paraffin is called tissue processing. The main steps in this process are dehydration and clearing.

Wet fixed tissues (in aqueous solutions) cannot be directly infiltrated with paraffin. First, the water from the tissues must be removed by dehydration. This is usually done with a series of alcohols, say 70% to 95% to 100%. Sometimes the first step is a mixture of formalin and alcohol. Other dehydrants can be used, but have major disadvantages. Acetone is very fast, but a fire hazard, so is safe only for small, hand-processed sets of tissues. Dioxane can be used without clearing, but has toxic fumes.

The next step is called "clearing" and consists of removal of the dehydrant with a substance that will be miscible with the embedding medium (paraffin). The commonest clearing agent is xylene. Toluene works well, and is more tolerant of small amounts of water left in the tissues, but is 3 times more expensive than xylene. Chloroform used to be used, but is a health hazard, and is slow. Methyl salicylate is rarely used because it is expensive, but it smells nice (it is oil of wintergreen).

There are newer clearing agents available for use. Many of them are based on limolene, a volatile oil found in citrus peels. Another uses long chain aliphatic hydrocarbons (Clearite). Although they represent less of a health hazard, they are less forgiving with poorly fixed, dehydrated, or sectioned tissues.

 

Finally, the tissue is infiltrated with the embedding agent, almost always paraffin. Paraffins can be purchased that differ in melting point, for various hardnesses, depending upon the way the histotechnologist likes them and upon the climate (warm vs. cold). A product called paraplast contains added plasticizers that make the paraffin blocks easier for some technicians to cut. A vacuum can be applied inside the tissue processor to assist penetration of the embedding agent

The above processes are almost always automated for the large volumes of routine tissues processed. Automation consists of an instrument that moves the tissues around through the various agents on a preset time scale. The "technicon" tissue processor is one of the commonest and most reliable (a mechanical processor with an electric motor that drives gears and cams), though no longer made. Newer processors have computers, not cam wheels, to control them and have sealed reagent wells to which a vacuum and/or heat can be applied.

    Tissue processor

    Automated tissue processor

Tissues that come off the tissue processor are still in the cassettes and must be manually put into the blocks by a technician who must pick the tissues out of the cassette and pour molten paraffin over them. This "embedding" process is very important, because the tissues must be aligned, or oriented, properly in the block of paraffin.

    Tissue embedding

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Tissue embedding

Alternatives to paraffin embedding include various plastics that allow thinner sections. Such plastics include methyl methacrylate, glycol methacrylate, araldite, and epon. Methyl methacrylate is very hard and therefore good for embedding undecalcified bone. Glycol methacrylate has the most widespread use since it is the easiest to work with. Araldite is about the same as methacrylate, but requires a more complex embedding process. Epon is routinely used for electron microscopy where very thin sections are required.

Plastics require special reagents for deydration and clearing that are expensive. For this reason, and because few tissues are plastic embedded, the processing is usually done by hand. A special microtome is required for sectioning these blocks. Small blocks must be made, so the technique lends itself to small biopsies, such as bone marrow or liver.

Sectioning

Once the tissues have been embedded, they must be cut into sections that can be placed on a slide. This is done with a microtome. The microtome is nothing more than a knife with a mechanism for advancing a paraffin block standard distances across it. There are three important necessities for proper sectioning: (1) a very sharp knife, (2) a very sharp knife, and (3) a very sharp knife.

 

    Sectioning with microtome

    MPEG movie [672k] demonstrating sectioning technique with microtome.

Knives are either of the standard thick metal variety or thin disposable variety (like a disposable razor blade). The former type allows custom sharpening to one's own satisfaction, but is expensive (more than $100 per blade). The latter cost about $1 per blade and are nearly as good. The advantage of the disposable blade becomes apparent when sectioning a block in which is hidden a metal wire or suture.

Plastic blocks (methacrylate, araldite, or epon) are sectioned with glass or diamond knives. A glass knife can section down to about 1 micron. Thin sections for electron microscopy (1/4 micron) are best done with a diamond knife which is very expensive ($2500).

Microtomes have a mechanism for advancing the block across the knife. Usually this distance can be set, for most paraffin embedded tissues at 6 to 8 microns. The more expensive the microtome ($15,000 to $30,000), the better and longer-lasting this mechanism will be.

Sectioning tissues is a real art and takes much skill and practice. Histotechnologists are the artists of the laboratory. It is important to have a properly fixed and embedded block or much artefact can be introduced in the sectioning. Common artefacts include tearing, ripping, "venetian blinds", holes, folding, etc. Once sections are cut, they are floated on a warm water bath that helps remove wrinkles. Then they are picked up on a glass microscopic slide.

    Picking sections up from water bath.

    Unstained section on glass slide

.

The glass slides are then placed in a warm oven for about 15 minutes to help the section adhere to the slide. If this heat might harm such things as antigens for immunostaining, then this step can be bypassed and glue-coated slides used instead to pick up the sections.

    Tray of unstained slides in drying oven.

Frozen Sections

At times during performance of surgical procedures, it is necessary to get a rapid diagnosis of a pathologic process. The surgeon may want to know if the margins of his resection for a malignant neoplasm are clear before closing, or an unexpected disease process may be found and require diagnosis to decide what to do next, or it may be necessary to determine if the appropriate tissue has been obtained for further workup of a disease process. This is accomplished through use of a frozen section. The piece(s) of tissue to be studied are snap frozen in a cold liquid or cold environment (-20 to -70 Celsius). Freezing makes the tissue solid enough to section with a microtome.

 

Frozen sections are performed with an instrument called a cryostat. The cryostat is just a refrigerated box containing a microtome. The temperature inside the cryostat is about -20 to -30 Celsius. The tissue sections are cut and picked up on a glass slide. The sections are then ready for staining.

    Cutting a frozen section

Stainin

The embedding process must be reversed in order to get the paraffin wax out of the tissue and allow water soluble dyes to penetrate the sections. Therefore, before any staining can be done, the slides are "deparaffinized" by running them through xylenes (or substitutes) to alcohols to water. There are no stains that can be done on tissues containing paraffin.

The staining process makes use of a variety of dyes that have been chosen for their ability to stain various cellular components of tissue. The routine stain is that of hematoxylin and eosion (H and E). Other stains are referred to as "special stains" because they are employed in specific situations according to the diagnostic need.

    Slides being stained on automated stainer.

    Slides being stained on automated stainer

Frozen sections are stained by hand, because this is faster for one or a few individual sections. The stain is a "progressive" stain in which the section is left in contact with the stain until the desired tint is achieved.

    Staining a frozen section

H and E staining

Hematoxylin is the oxidized product of the logwood tree known as hematein. Since this tree is very rare nowadays, most hematein is of the synthetic variety. In order to use it as a stain it must be "ripened" or oxidized. This can be done naturally by putting the hematein solution on the shelf and waiting several months, or by buying commercially ripened hematoxylin or by putting ripening agents in the hematein solution.

Hematoxylin will not directly stain tissues, but needs a "mordant" or link to the tissues. This is provided by a metal cation such as iron, aluminum, or tungsten. The variety of hematoxylins available for use is based partially on choice of metal ion used. They vary in intensity or hue. Hematoxylin, being a basic dye, has an affinity for the nucleic acids of the cell nucleus.

Hematoxylin stains are either "regressive" or "progressive". With a regressive stain, the slides are left in the solution for a set period of time and then taken back through a solution such as acid-alcohol that removes part of the stain. This method works best for large batches of slides to be stained and is more predictable on a day to day basis. With a progressive stain the slide is dipped in the hematoxylin until the desired intensity of staining is achieved, such as with a frozen section. This is simple for a single slide, but lends itself poorly to batch processing.

Eosin is an acidic dye with an affinity for cytoplasmic components of the cell. There are a variety of eosins that can be synthesized for use, varying in their hue, but they all work about the same. Eosin is much more forgiving than hematoxylin and is less of a problem in the lab. About the only problem you will see is overstaining, especially with decalcified tissues.

Coverslipping

The stained section on the slide must be covered with a thin piece plastic or glass to protect the tissue from being scratched, to provide better optical quality for viewing under the microscope, and to preserve the tissue section for years to come. The stained slide must go through the reverse process that it went through from paraffin section to water. The stained slide is taken through a series of alcohol solutions to remove the water, then through clearing agents to a point at which a permanent resinous substance beneath the glass coverslip, or a plastic film, can be placed over the section.

    Coverslipping

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    Coverslipping

Decalcification

Some tissues contain calcium deposits which are extremely firm and which will not section properly with paraffin embedding owing to the difference in densities between calcium and parffin. Bone specimens are the most likely type here, but other tissues may contain calcified areas as well. This calcium must be removed prior to embedding to allow sectioning. A variety of agents or techniques have been used to decalcify tissue and none of them work perfectly. Mineral acids, organic acids, EDTA, and electrolysis have all been used.

Strong mineral acids such as nitric and hydrochloric acids are used with dense cortical bone because they will remove large quantities of calcium at a rapid rate. Unfortunately, these strong acids also damage cellular morphology, so are not recommended for delicate tissues such as bone marrow.

Organic acids such as acetic and formic acid are better suited to bone marrow, since they are not as harsh. However, they act more slowly on dense cortical bone. Formic acid in a 10% concentration is the best all-around decalcifier. Some commercial solutions are available that combine formic acid with formalin to fix and decalcify tissues at the same time.

EDTA can remove calcium and is not harsh (it is not an acid) but it penetrates tissue poorly and works slowly and is expensive in large amounts.

Electrolysis has been tried in experimental situations where calcium had to be removed with the least tissue damage. It is slow and not suited for routine daily use.

 

Artefacts in Histologic Sections

A number of artefacts that appear in stained slides may result from improper fixation, from the type of fixative, from poor dehydration and paraffin infiltration, improper reagents, and poor microtome sectioning.

The presence of a fine black precipitate on the slides, often with no relationship to the tissue (i.e., the precipitate appears adjacent to tissues or within interstices or vessels) suggests formalin-heme pigment has formed. This can be confirmed by polarized light microscopy, because this pigment will polarize a bright white (and the slide will look like many stars in the sky). Formalin-heme pigment is most often seen in very cellular or bloody tissues, or in autopsy tissues, because this pigment forms when the formalin buffer is exhausted and the tissue becomes acidic, promoting the formation of a complex of heme (from red blood cells) and formalin. Tissues such as spleen and lymph node are particularly prone to this artefact. Making thin sections and using enough neutral-buffered formalin (10 to 1 ratio of fixative to tissue) will help. If the fixative solution in which the tissues are sitting is grossly murky brown to red, then place the tissues in new fixative.

The presence of large irregular clumps of black precipitate on slides of tissues fixed in a mercurial fixative such as B-5 suggests that the tissues were not "dezenkerized" prior to staining. These black precipitates will also appear white with polarized light microscopy.

Tissues that are insufficiently dehydrated prior to clearing and infiltration with paraffin wax will be hard to section on the microtome, with tearing artefacts and holes in the sections. Tissue processor cycles should allow sufficient time for dehydration, and final ethanol dehydrant solution should be at 100% concentration. In humid climates, this is difficult to achieve. Covering or sealing the solutions from ambient air will help. Air conditioning (with refrigerants, not with evaporative coolers) will also reduce humidity in the laboratory. Toluene as a clearing agent is more forgiving of poorly dehydrated tissues, but it is more expensive and presents more of a health hazard than other non-xylene clearing agents

Though alcohols such as ethanol make excellent fixatives for cytologic smears, they tend to make tissue sections brittle, resulting in microtome sectioning artefacts with chattering and a "venetian blind" appearance.

Bubbles under the coverslip may form when the mounting media is too thin, and as it dries air is sucked in under the coverslip. Contamination of clearing agents or coverslipping media may also produce a bubbled appearance under the microscope.

    Artefact with undezenkerized tissue.

On the left with H&E staining black mercuric chloride precipitate is seen in this lymphoma fixed in B-5 and not properly dezenkerized. This precipitate is seen on the right under polarized light microscopy

Problems in Tissue Processing

"Floaters" are small pieces of tissue that appear on a slide that do not belong there--they have floated in during processing. Floaters may arise from sloppy procedure on the cutting bench-- dirty towels, instruments, or gloves can have tissue that is carried over to the next case. Therefore, it is essential that you do only one specimen at a time and clean thoroughly before opening the container of the next case.

The best way to guard against unrecognized floaters is to always separate like specimens in the numbering sequence. For example, if you have three cases with prostate chips, separate them in accessioning with totally different specimens such as uterus or stomach. That way, if numbers are transposed or labels written wrong or tissue carried over, then you will have an obvious mismatch. Carrying over one prostate to another, or transposing the numbers of identical tissues may never be recognized.

If reusable cassettes are employed, you must be aware that tissue may potentially be carried over and appear as "floaters" even several days later, when the cassette is re-used. The problem arises when, during embedding, not all the tissue is removed from the cassette. Then, in the cleaning process, not all of the wax is removed. Then, the next person using the cassette does not pay attention to the fact that there is tissue already in the cassette and puts his specimen in it. The floater that appears on the slide will look well-preserved--it should, because it was processed to paraffin.

Always be sure that you properly identify the tissue! This means that you make sure that the patient label on the specimen container matches that of the request slip. An accession number is given to the specimen. This number must appear with the tissue at all times. You must never submit a cassette of tissue without a label. You must never submit a cassette of tissue with the wrong label. Mislabelling or unlabelling of tissues is courting disaster.

Safety in the Lab

The lab should be well-ventilated. There are regulations governing formalin and hydrocarbonds such as xylene and toluene. There are limits set by the Occupational Safety and Health Administration (OSHA) that should not be exceeded. These limits have recently been revised to reduced levels.

Every chemical compound used in the laboratory should have a materials safety data sheet on file that specifies the nature, toxicity, and safety precautions to be taken when handling the compound.

The laboratory must have a method for disposal of hazardous wastes. Health care facilities processing tissues often contract this to a waste management company. Tissues that are collected should be stored in formalin and may be disposed by incineration or by putting them through a "tissue grinder" attached to a large sink (similar to a large garbage disposal unit).

 

Every instrument used in the laboratory should meet electrical safety specifications (be U.L. approved) and have written instructions regarding its use.

Flammable materials may only be stored in approved rooms and only in storage cabinets that are designed for this purpose.

Fire safety procedures are to be posted. Safety equipment including fire extinguishers, fire blankets, and fire alarms should be within easy access. A shower and eyewash should be readily available.

Laboratory accidents must be documented and investigated with incident reports and industrial accident reports.

Specific hazards that you should know about include:

      Bouin's solution is made with picric acid. This acid is only sold in the aqueous state. When it dries out, it becomes explosive.

Many reagent kits have sodium azide as a preservative. You are supposed to flush solutions containing sodium azide down the drain with lots of water, or there is a tendency for the azide to form metal azides in the plumbing. These are also explosive.

 Benzidine, benzene, anthracene, and napthol containing compounds are carcinogens and should not be used.

  Mercury-containing solutions (Zenker's or B-5) should always be discarded into proper containers. Mercury, if poured down a drain, will form amalgams with the metal that build up and cannot be removed.

Smudge or basket cell

Smudge cells or basket cells are leukocytes that have been damaged during preparation of the peripheral blood smear. This usually occurs due to the fragility of the cell. They are usually seen in chronic lymphocytic leukemia (CLL).

Spherocytes

Spherocytes are red blood cells that are almost spherical in shape. They have no area of central pallor like a normal red blood cell. Large spherocytes (macrospherocytes) are seen in hemolytic anemia. Small spherocytes (microspherocytes) are sometimes seen in severe burn cases.

A variety of spherical forms are seen in hereditary spherocytosis. The cells depicted in this image are from a patient with hereditary spherocytosis.

Stomatocyte

Stomatocytes are red blood cells with an oval or rectangular area of central pallor, sometimes referred to as a "mouth". These cells have lost the indentation on one side and may be found in liver disease, electrolyte imbalance, and hereditary stomatocytosis.

Target cells

Target cells (codocytes) are erythrocytes with a central color spot in the area of pallor, resembling a target. They are seen in many hemolytic anemias, especially sickle cell, HbC disease, and thalassemia.

Teardrop cells (dacrocytes)

Teardrop shaped red blood cells are found in myelofibrosis and other myeloproliferative disorders, pernicious anemia, thalassemia, myeloid metaplasia, and some hemolytic anemias.

Toxic granulation

Toxic granulation appears as dark blue-black granules in the cytoplasm of neutrophils. These granules are thought to be primary granules.

Artifactual heavy granulation caused by poor staining is seen evenly spread throughout each cell and in all granulocytes, whereas toxic granulation is unevenly spread throughout the cytoplasm of certain cells. Large amounts of toxic granulation can give the neutrophil a bluish appearance.Toxic granulation is a stress response to acute infections, burns, and drug poisoning.

Yeast

The image below illustrates a neutrophil with intracellular yeast. The major function of the neutrophil is to stop or retard the action of foreign matter or infectious agents. The neutrophil accomplishes this by moving to the area of inflammation or infection, phagocytizing (ingesting) the foreign material (in this case, the yeast), and killing and digesting the material.

Metamyelocyte

Metamyelocytes have a cell diameter from 10 to 18 µm, and the nucleus to cytoplasm ratio is 1:1. Indentation of the nucleus begins at this stage, forming an outline that varies from slightly kidney shaped, to that of a broad V shape.

Monoblast

The monoblast is the first stage of monocyte-macrophage maturation. It is about 12 to 20 µm in diameter, has a nuclear to cytoplasm ration of 4:1 to 3:1, and, like most myeloid blasts, has a round to oval nucleus with fine, lightly dispersed chromatin.

From one to four nucleoli may be visible. The nucleus may be either central or eccentric and it may show evidence of indentation or folding. The cytoplasm is agranular, stains moderately to lightly basophilic, and often has an intensely stained periphery and a prominent perinuclear zone. Monoblasts never appear in the normal peripheral blood.

Myeloblast

The myeloblast is the first stage of the granulocytic series that is identifiable by light microscopy. It may be dificult to distinguish myeloblasts from other blasts in the peripheral blood unless one uses special stains or infers their identity from the presence of other immature cells of the same line.

A myeloblast can be distinguished from a promyelocyte by its lack of cytoplamic granulation. The nucleus is composed of very fine nonaggregated chromatin that stains light blue to reddish-purple with Wright's stain. Two to five distinct nucleoli are usually present. The nucleus is often bordered at one side by a distinct perinuclear zone.

Myelocyte

Myelocytes have a cell diameter from 10 to 18 µm. The nucleus to cytoplasm ratio is 1:1, and the oval or round nucleus is often eccentrically located. Chromatin is finely granulated in early myelocytes and more aggregated in later cells.

Nucleated red blood cell

Nucleated red blood cells, (nrbcs or normoblasts), represent the stages of a red blood cell before it matures. Cells of this stage are usually seen in newborn infants, and in patients with responses to hemolytic crises, such as in megaloblastic anemia and iron deficiency anemia.

The average size of the normoblast is 7-12 µm in diameter. The cytoplasm is pink. The nucleus is pyknotic (a homogeneous blue-black mass with no structure).

Pappenheimer bodies

appenheimer bodies are iron containing granules in red blood cells that are seen because the iron is aggregated with mitochondria and ribosomes. They appear as faint violet or magenta specks, often in small clusters, due to staining of the associated protein. They are associated with severe anemias and thalassemias.

Parasites

The most common parasites encountered in the peripheral blood include malaria and babesia. Malaria is discussed in detail on the malaria link.

Babesia are small ringlike protozoa within erythrocytes that resemble the ring stages of falciparum malaria. Most infections result from Babesia microti which is transmitted from wild feral deer mice to humans by the tick Ixodes dammini. It occurs in Nantucket Island, on coastal regions of the northeastern US, and in California. It has been found in France, Ireland, Scotland, and other European countries.

The diagnosis of babesiosis can be made by demonstration of the ring-shaped parasites on Wright-Giemsa stained smears. Babesia are tiny rings with a minute chromatin dot and a minimal amount of cytoplasm. They may be round, oval, elongated, or ameboid. One or two chromatin dots, which stain dark purple, may be observed. More than one ring can be seen in an RBC. Tetrad forms may be noted and aid in positively identifying babesia.

Plasma cell

A plasma cell is a mature B lymphocyte that is specialized for antibody (immunoglobulin) production. Plasma cells are rarely found in the peripheral blood. They comprise from 0.2% to 2.8% of the bone marrow white cell count.

Mature plasma cells are often oval or fan shaped, measuring 8-15 µm. The nucleus is eccentric and oval in shape. The nucleus to cytoplasm ratio is typically 2:1 to 1:1. The nucleus may be bilobed or multilobed, especially in patients with lymphoid blood dyscrasias. The perinuclear zone is very distinct, appearing white in the deeply basophilic cytoplasm. Nuclear chromatin is condensed and very patchy, appearing as dark blocks on a reddish-purple background. The cytoplasm stains deep blue to gray blue, depending on the stain and the ribosomal content of the individual cell. Plasma cells are seen in multiple myeloma, plasma cell leukemia, Waldenström's macroglobulinemia, and MGUS (monoclonal gammopathy of uncertain significance. The cells depicted in this image are from a patient with plasma cell leukemia.

Platelet satellitosis

Platelet satellitosis, (platelets encircling a neutrophil) occurs when a patient has a serum factor that reacts to the anticoagulant EDTA.

Promyelocyte

Promyelocytes are the second largest stage in the granulocytic series. The large, reddish-purple granules that characterize promyelocytes are nonspecific in that they are shared by the other granulocytes (eosinophils and basophils). These granules are peroxidase positive, and a lipid component reacts with Sudan black stain, providing a second cytochemical reaction for the identification of large mononuclear cells in blood and bone marrow smears. The cell size, 12 to 20 µm, is the same as that of a myeloblast, but the nucleus to cytoplasm ratio is less, usually from 3:1 to 2:1. The chromatin is still fine, but some aggregation is evident. One to three nucleoli are also visible, although these can be obscured by heavy granulation.

Rouleaux

Rouleaux formation occurs when red blood cells form stacks or rolls. This is due to either an artifact (such as a result of not preparing the blood smear soon enough after placing the blood on the slide), or it may be due to the presence of high concentrations of abnormal globulins or fibrinogen. This formation of the red blood cells is found in multiple myeloma and macroglobulinemia

Schistocyte

Schistocytes are red blood cell fragments that result from membrane damage encountered during passage through vessels. They occur in microangiopathic hemolytic anemia, severe burns, uremia, and hemolytic anemias cause by physical agents, as in disseminated intravascular coagulation (DIC). They are sometimes referred to as "bite cells".

Schüffner's granules

Schüffner's granules may be found in cases of Plasmodium vivax. These granules appear as orange to pink colored stippling throughout the red blood cell. They may not be visible when normal staining times are used. To detect these granules, the smears should be allowed to stain for three hours.

Sickle cells

Sickle cells are red blood cells that have become crescent shaped. When a person with sickle cell anemia is exposed to dehydration, infection, or low oxygen supply, their fragile red blood cells form liquid crystals and assume a crescent shape causing red cell destruction and thickening of the blood. Since the life span of the red blood cell is shortened, there is a temporary depression of red cell production in the bone marrow, and a subsequent fall in hemoglobin (and therefore the resultant anemia).

Through the Microscope: Blood Cells

Atypical Lymphocyte

Atypical, or reactive, lymphocytes are lymphocytes that, as a result of antigen stimulation, have become quite large, sometimes more than 30 µm in diameter. The cells vary greatly in size and shape. The nucleus is less clumped than that of the normal lymphocyte. The shape of the nucleus ranges from elliptic to cleft to folded. The chromatin patterns appear similar to those of a blast and faintly stained multiple nucleoli are visible. The cytoplasm may range from large, deeply basophilic, and abundant to unevenly stained and granular. Causes of reactive lymphocytosis may be: ß-Streptococcus, cytomegalovirus, drugs, Epstein-Barr virus (infectious mononucleosis), syphilis, toxoplasmosis, vaccination, and viral hepatitis.

 

Auer rod

Auer rods are elongated, bluish-red rods composed of fused lysosomal granules, seen in the cytoplasm of myeloblasts, promyelocytes and monoblasts and in patients with acute myelogenous leukemia.

Bacteria

The image below illustrates a neutrophil with bacteria (Gram positive cocci). The major function of the neutrophil is to stop or retard the action of foreign matter or infectious agents. The neutrophil accomplishes this by moving to the area of inflammation or infection, phagocytizing (ingesting) the foreign material, and killing and digesting the material. Bacterial sepsis can result in a leukemoid reaction, involving white cell counts of 100,000 X 10⁶/L (normal WBC=4,500 to 11,000 X 10⁶/L), the presence of myelocytes, and the appearance of toxic granulation.This image was taken from a patient with Streptococcus pnemoniae infection.

Basophilic stippling

Basophilic stippling appears as round, dark-blue granules in red blood cells on smears stained with supra vital stains such as brilliant cresyl blue. They may be observed in lead poisoning, exposure to some drugs, severe burns, anemias, or septicemia. The granules are precipitated ribosomes and mitochondria.

Döhle bodies

Döhle bodies appear as a small, light blue-gray staining area in the cytoplasm of the neutrophil. They are found in poisoning, burns, infections, and following chemotherapy.

Echinocytes (crenated red blood cells)

Echinocytes are red blood cells with many blunt spicules, resulting from faulty drying of the blood smear or from exposure to hyperosmotic solutions. Pathological forms are associated with uremia. Echinocytes contain adequate hemoglobin and the spiny knobs are regularly dispersed over the cell surface, unlike those of acanthocytes.

Elliptocytes

Elliptocytes are red blood cells that are oval or cigar shaped. They may be found in various anemias, but are found in large amounts in hereditary elliptocytosis

Erythrocyte - polychromatophilic

Polychromatophilia may be defined as increased numbers of immature peripheral red blood cells that have a blue-gray tint on Wright-stained smears, indicating the presence of cytoplasmic RNA. These cells are usually larger than normal. Many of these cells prove to be reticulocytes when stained with supravital stains such as brilliant cresyl blue. They appear under conditions of accelerated red cell production.

Giant platelet

Giant platelets are platelets that are larger than 6.5 µm, or 75 to 100% the size of a normal red blood cell. A normal red cell is 6-8 µm in diameter. Normal platelets are approximately 1-4 µm, large platelets are approximately 4-6.5 µm.

Hairy cell

Hairy cells are characterized by their fine, irregular pseudopods and immature nuclear features. They are seen only in hairy cell leukemia.

Howell-Jolly Bodies

Howell-Jolly bodies are spherical blue-black inclusions of red blood cells seen on Wright-stained smears. They are nuclear fragments of condensed DNA, 1 to 2m in diameter, normally removed by the spleen.

They are seen in severe hemolytic anemias, in patients with dysfunctional spleens or after splenectomy.

Hypersegmented neutrophil

Hypersegmented neutrophils are neutrophils with 5 or more nuclear lobes. They are seen in disorders of nuclear maturation, such as the megaloblastic anemias.

Lymphoblast

Lymphoblasts are 12-20 µm in diameter with a round to oval nucleus, sometimes eccentric in location. The nucleus to cytoplasm ratio is about 4:1 and the periphery of both the nucleus and the cell may be irregular in outline.

The fine, highly dispersed nuclear chromatin stains a light reddish-purple, and one or two pale blue or colorless large nucleoli are visible. The cytoplasm is usually agranular and deeply to moderately basophilic, with marginal (peripheral) intensity a common characteristic.

Through the Microscope: Blood Cells

These images were taken by Wadsworth scientists. Images similar to these are used as part of Wadsworth's laboratory certification proficiency testing program and in continuing education programs for laboratories processing New York State specimens.

Use these images to learn more about your Life's Blood, or to enliven science or health papers with accurate images.

Normal peripheral blood

The usual diagnostic approach to blood disorders is blood counting and blood film examination. Blood films on glass slides are stained with a Romanowsky stain (usually Wright's, Giemsa, or May-Grünwald). Red cells in normal peripheral blood are circular and fairly uniform in size. Mild variation in shape (poikilocytosis) and size (anisocytosis) is seen. Platelets appear as small bluish-purple discs. During blood film examination, the individual types of white blood cells are enumerated; this is referred to as the differential count.

Band neutrophil

Band neutrophils comprise approximately 1 to 3% of the peripheral leukocytes. They are usually 9 to 15 µm in diameter. The nucleus forms a "U" or curled rod prior to segmentation. The chromatin pattern is coarse and clumped. The cytoplasm is moderate to abundant with a few nonspecific granules and many specific granules.

 

Basophil

Basophils are granulocytes that contain purple-blue granules that contain heparin and vasoactive compounds. They comprise approximately 0.5% of the total leukocyte count. Basophils participate in immediate hypersensitivity reactions, such as allergic reactions to wasp stings, and are also involved in some delayed hypersensitivity reactions. Basophils are the smallest circulating granulocytes, averaging 10 to 15 µm in diameter. The nucleus to cytoplasm ratio is about 1:1, and the nucleus is often unsegmented or bilobed, rarely with three or four lobes. The chromatin pattern is coarse and patchy, staining a deep blue to reddish-purple. The cytoplasm is a homogenous pale blue, but this is often obscured by the large dark granules.

Eosinophil

Eosinophils are the mature granulocytes that respond to parasitic infections and allergic conditions. Eosinophils comprise about 1 to 4% of the peripheral leukocytes. They are usually 9 to 15 µm in diameter. Granules stain a bright reddish-orange with Wright's or Giemsa stains. The nucleus contains one to three lobes. The chromatin pattern is coarse and clumped. The cytoplasm is abundant with a full complement of bright reddish-orange specific granules.

Erythrocytes (red blood cells)

The mature red blood cell (rbc) consists primarily of hemoglobin (about 90%). The membrane is composed of lipids and proteins. In addition, there are numerous enzymes present which are necessary for oxygen transport and cell viability. The main function of the red cell is to carry oxygen to the tissues and return carbon dioxide from the tissues to the lungs. The protein hemoglobin is responsible for most of this exchange. Normal red blood cells are round, have a small area of central pallor, and show only a slight variation in size. A normal red cell is 6-8 µm in diameter. As the relative amount of hemoglobin in the red cell decreases or increases, the area of central pallor will decrease or increase accordingly.

Lymphocyte

Lymphocytes in the peripheral blood have been described on the basis of size and cytoplasmic granularity. Small lymphocytes are the most common, ranging in size from 6 to 10 µm. The nucleus is usually round or slightly oval, occasionally showing a small indentation due to the adjacent centrosome. Except in the smallest cells, the nucleus is about 7 µm in diameter, a size that has been convenient for estimating the size of the surrounding erythrocytes. Nuclear chromatin stains a dark reddish-purple to blue with large dark patches of condensed chromatin. The nuclear cytoplasm ratio is 5:1 to 3:1, and the cytoplasm is often seen only as a peripheral ring around part of the nucleus.

 

Monocyte

Monocytes are large mononuclear phagocytes of the peripheral blood. They are the immature stage of the macrophage. Monocytes vary considerably, ranging in size from10 to 30 µm in diameter. The nucleus to cytoplasm ratio ranges from 2:1 to 1:1. The nucleus is often band shaped (horseshoe), or reniform (kindey-shaped). It may fold over on top of itself, thus showing brainlike convolutions. No nucleoli are visible. The chromatin pattern is fine, and arranged in skeinlike strands. The cytoplasm is abundant and blue gray with many fine azurophilic granules, giving a ground glass appearance. Vacuoles may be present

Platelets

Platelets are the cytoplasmic fragments of megakaryocytes, circulating as small discs in the peripheral blood. They are responsible for hemostasis (the stoppage of bleeding) and maintaining the endothelial lining of the blood vessels. During hemostasis, platelets clump together and adhere to the injured vessel in this area to form a plug and further inhibit bleeding. Platelets average 1 to 4 µm in diameter. The cytoplasm stains light blue to purple, and is very granular. There is no nucleus present. Normal blood concentrations range from 130,000 to 450,000/µL.

 

Segmented neutrophil (seg)

Segmented neutrophils (polymorphonuclear leukocytes, or segs) are the mature phagocytes that migrate through tissues to destroy microbes and respond to inflammatory stimuli. Segmented neutrophils comprise 40-75 % of the peripheral leukocytes. They are usually 9 to 16 µm in diameter. The nuclear lobes, normally numbering from 2 to 5, may be spread out so that the connecting filaments are clearly visible, or the lobes may overlap or twist. The chromatin pattern is coarse and clumped. The cytoplasm is abundant with a few nonspecific granules and a full complement of rose-violet specific granules.

Cells and findings in disease states

Listed below are several types of white and red blood cells. Some may be observed only in specific diseases, while others may be seen occasionally in normal peripheral blood.

White and Red Blood Cells

Acanthocyte

Acanthocytes are red blood cells with irregularly spaced projections. These projections vary in width but usually contain a rounded end. They may be found in abetalipoproteinemia and certain liver disorders.

blood cells 2

INTRODUCTION

What are blood cells? What do they look like? What functions do they perform? How can I recognize the different categories? This is a short description of the blood cells and includes a simple experiment which allows you to become familiar with the cells of this precious liquid.

The blood consists of a suspension of special cells in a liquid called plasma. In an adult man, the blood is about 1/12th of the body weight and this corresponds to 5-6 litres. Blood consists of 55 % plasma, and 45 % by cells called formed elements.
The blood performs a lot of important functions. By means of the hemoglobin contained in the erythrocytes, it carries oxygen to the tissues and collects the carbon dioxide (CO₂). It also conveys nutritive substances (e.g. amino acids, sugars, mineral salts) and gathers the excreted material which will be eliminated through the renal filter. The blood also carries hormones, enzymes and vitamins. It performs the defense of the organism by mean of the phagocitic activity of the leukocytes, the bactericidal power of the serum and the immune response of which the lymphocytes are the protagonists.

THE PLASMA

Cells free serum or plasma, can be obtained by centrifugation. The plasma is a slightly alkaline fluid, with a typical yellowish color. It consists of 90 % water and 10% dry matter. Nine parts of it are made up by organic substances, whereas one part is made up by minerals. These organic substances are composed of glucides (glucose), lipids (cholesterol, triglycerides, phospholipids, lecithin, fats), proteins (globulins, albumins, fibrinogen), glycoproteins, hormones (gonadothropins, erythropoietin, thrombopoietin), amino acids and vitamins. The mineral substances are dissolved in ionic form, that is dissociated into positive and negative ions.

THE HEMATIC CELLS

In the blood are present special cells, classified in: erythrocytes and leukocytes. There are also platelets which are not considered real cells. In the following, we will deal the different categories of blood cells.

ERYTHROCYTES (red cells)

The erythrocytes are the most numerous blood cells i.e. about 4-6 millions/mm^3. They are also called red cells. In man and in all mammals, erythrocytes are devoid of a nucleus and have the shape of a biconcave lens. In the other vertebrates (e.g. fishes, amphibians, reptilians and birds), they have a nucleus. The red cells are rich in hemoglobin, a protein able to bind in a faint manner to oxygen. Hence, these cells are responsible for providing oxygen to tissues and partly for recovering carbon dioxide produced as waste. However, most CO2 is carried by plasma, in the form of soluble carbonates.

In the red cells of the mammalians, the lack of nucleus allows more room for hemoglobin and the biconcave shape of these cells raises the surface and cytoplasmic volume ratio. These characteristics make more efficient the diffusion of oxygen by these cells. In so-called "sickle-cell anaemia", erythrocytes become typically sickle-shaped. With the electron microscope, biologists saw that red cells can have different shapes: normal (discocyte), berry (crenated), burr (echinocyte), target (codocyte), oat, sickled, helmet, pinched, pointed, indented, poikilocyte, etc. The mean life of erythrocytes is about 120 days. When they come to the end of their life, they are retained by the spleen where they are phagocyted by macrophages.

PLATELETS

The main function of platelets, or thrombocytes, is to stop the loss of blood from wounds (hematostasis). To this purpose, they aggregate and release factors which promote the blood coagulation. Among them, there are the serotonin which reduces the diameter of lesioned vessels and slows down the hematic flux, the fibrin which trap cells and forms the clotting. Even if platelets appears roundish in shape, they are not real cells. In the smears stained by Giemsa, they have an intense purple color. Their diameter is 2-3 µm about, hence they are much smaller than erythrocytes. Their density in the blood is 200000-300000 /mm³.

LEUKOCYTES (white cells)

Leukocytes, or white cells, are responsible for the defense of the organism. In the blood, they are much less numerous than red cells. The density of the leukocytes in the blood is 5000-7000 /mm³. Leukocytes divide in two categories: granulocytes and lymphoid cells or agranulocytes. The term granulocyte is due to the presence of granules in the cytoplasm of these cells. In the different types of granulocytes, the granules are different and help us to distinguish them. In fact, these granules have a different affinity towards neutral, acid or basic stains and give the cytoplasm different colors. So, granulocytes distinguish themselves in neutrophil, eosinophil (or acidophil) and basophil. The lymphoid cells, instead, distinguish themselves in lymphocytes and monocytes. As we will see later, even the shape of the nucleus helps us in the recognition of the leukocytes.

Each type of leukocyte is present in the blood in different proportions:

neutrophil 50 - 70 %
eosinophil 2 - 4 %
basophil 0,5 - 1 %
lymphocyte 20 - 40 %
monocyte 3 - 8 %

Neutrophils are very active in phagocyting bacteria and are present in large amount in the pus of wounds. Unfortunately, these cells are not able to renew the lysosomes used in digesting microbes and dead after having phagocyted a few of them.

 

Eosinophils attack parasites and phagocyte antigen-antibody complexes.

 

Basophil secrete anti-coagulant and vasodilatory substances as histamines and serotonin. Even if they have a phagocytory capability, their main function is secreting substances which mediate the hypersensitivity reaction.

Lymphocytes are cells which, besides being present in the blood, populate the lymphoid tissues and organs too, as well as the lymph circulating in the lymphatic vessel. The lymphoid organs include thymus, bone marrow (in birds bursa), spleen, lymphoid nodules, palatine tonsils, Peyer's patches and lymphoid tissue of respiratory and gastrointestinal tracts.

Most lymphocytes circulating in the blood is in a resting state. They look like little cells with a compact round nucleus which occupies nearly all the cellular volume. As a consequence, the cytoplasm is very reduced. The lymphocytes of the lymphoid tissues and organs can be activated in a different amount following antigenic stimulation. In the blood, lymphocytes are 20-40 % of all leukocytes and are slight larger than red blood cells.

The lymphocytes are the main constituents of the immune system which is a defense against the attack of pathogenic micro-organisms such as viruses, bacteria, fungi and protista. Lymphocytes yield antibodies and arrange them on their membrane. An antibody is a molecule able to bind itself to molecules of a complementary shape called antigens, and recognize them. As for all proteins, even the antibodies are coded by genes. On the basis of a recombination mechanism of some of these genes, every lymphocyte produces antibodies of a specific shape.

Hence, lymphocytes perform an action which is called specific in that each of them recognize the complementary antigen only. Even if every lymphocyte is so selective to recognize only one molecule, the number of circulating lymphocytes is so large that they are able to recognize practically all substances which are in the organism, both its own and foreign. It is a question of recognizing hundreds of millions of different molecules.

The cells of the immune system, chiefly lymphocytes, cooperate amongst themselves to activate, boost or make more precise the immune response. To attain this scope, there exist different types of lymphocytes, with different functions: T and B lymphocytes. When the B cells are activated, they breed quickly (clonal selection) and they become plasmacells which secrete a great deal of antibodies in the blood stream (humoral response). When free antibodies meet micro-organisms with complementary shape (epitopes), they bind to them and form complexes which immobilize the micro-organisms. Later, other cells which are not specific, but which are able to recognize antibodies, phagocyte these complexes.
In their turn, the T cells are divided into three categories: Tc (cytotoxic), Th (helpers), Ts (suppressors). Even the Cytotoxic lymphocytes breed quickly when they are activated. They do not release antibodies in the bloodstream, but they keep the antibodies on their membrane and use them to recognize cells mainly of its own organism infected by virus or tumoral cells. The cytotoxic lymphocytes kill cells by means of the release of perforins, substances which produces lesions in the membrane of the target cell and cause its death by osmotic lysis (cell-mediated response). The helper lymphocytes are needed to activate both B and Tc lymphocytes which, even though they recognize extraneous agents, seldom enter into direct action. Suppressor lymphocytes reduce the intensity of the immune response.

However, the immune system must not attack the cells of it's body as the autoimmune reaction can damage the organism and lead to death. How does the immune system distinguish between self and not self? We have seen that B and Tc lymphocytes which have recognized an antigen, do not enter in action, but they need to be activated by a helper lymphocyte. A few times after the organism's birth, some of the new lymphocytes pass through the thymus where they become T lymphocytes. Here, these cells are compared with all antigens of the organism (autoantigens). It seems that lymphocytes which recognize an antigen, as they are still immature, will die. In this way, as the autoreactive Th lymphocytes are been killed, only the B and Tc lymphocytes which have recognized extraneous antigens can be activated. The system of cellular cytotoxicity mediated by Th cells is evolved as a defense against their own infected, modified or aberrant cells. In fact, B and Tc lymphocytes can activate themselves against bacteria even without the agreement of the helpers.

The B and Tc activated lymphocytes, besides to producing antibodies and killing foreign cells, multiply quickly. During the cellular division, rearrangements often occur in the sequence of the genes which code for the antibody. In this way, the antibody of the new cell takes a slightly different shape in comparison to that of its "mitotic parent". If the new shape matches the antigen better, this cell will be induced to divide more. The next generation of clones is therefore more efficient and, in its turn, can induce more selective varieties. This process and that of clonal selection make the immune response more effective. Finally, the immune system produces memory cells, i.e. deactivated lymphocytes ready to be reactivated on the occasion of further meeting with the same antigen.

Besides the Th and B cells, there is a third population of lymphocytes in the peripheral blood and lymphoid organs which do not have receptors for antigens. These lymphocytes have a non-specific defense function which is not activated by Th lymphocytes. These cells represent the more ancient component of the immune system and they are characterized by their cytotoxic activity. For these reasons, they are named NK, Natural Killer. Apart from killing viruses, bacteria, infected and neoplastic cells, these lymphocytes also regulate the production of other hematic cells such as erythrocytes and granulocytes.

Monocytes are the precursors of macrophages. They are larger blood cells, which after attaining maturity in the bone marrow, enter the blood circulation where they stay for 24-36 hours. Then they migrate into the connective tissue, where they become macrophages and move within the tissues. In the presence of an inflammation site, monocytes quickly migrate from the blood vessel and start an intense phagocytory activity. The role of these cells is not solely in phagocytosis because they have also have an intense secretory activity. They produce substances which have defensive functions such as lysozime, interferons and other substances which modulate the functionality of other cells. Macrophages cooperate in the immune defense. They expose molecules of digested bodies on the membrane and present them to more specialized cells, such as B and Th lymphocytes.

PREPARATION OF THE BLOOD SMEAR

This experiment is intended for adults examining their own blood. If you want observe or let observe the blood of others (in school or other organization), you have to obtain the appropriate authorization to do so. You need to protect yourself and the others against the biohazard posed by; taking, working with and disposing of blood samples and you have to work according the suitable protocols.

In order to take a blood sample, you have to use latex gloves and special lancets which allow you to safely pierce the skin and take the sample. Following use, the lancets and glass slides must be disposed of in an appropriately labeled Sharps Bin. All materials such as tissues, wipes, stains etc that have been in contact with blood must be disposed of safely according to the protocols of the competent organization. In any case, read our page of Warnings.

MATERIALS
- sterilized lancets or needles
- 20 clean microscope slides and coverslips
- Canada balsam or other medium for permanent preparations
- 95% ethyl or methyl alcohol
- distilled water
- Giemsa stain
- low containers (you can make them with aluminum sheet also) or Petri dishes
- microscope which magnifies 200 times at least

TAKING THE BLOOD

Cleanse a finger. With a sterile lancet, make a puncture on a fingertip. If you have difficulties in doing this, you can wait until you have a casual wound. In the meantime, keep all the materials needed ready and protected from dust, particularly the clean microscope slides.

MAKING THE SMEAR

Place a small drop of blood near an end of a slide. According to figure 7, bring the edge of another slide in contact with the drop and allow the drop to bank evenly behind the spreader. The angle between the two slides has to be 30-40 degrees. Now, push to the left in a smooth, quick motion. The smear should cover about half the slide. It is important that the quantity of blood is not excessive, otherwise the red cells could hide the leukocytes. So, if you succeed in making a gradual transition from thick to thin in your smear, you should get a zone with a satisfactory distribution of cells. With a single drop of blood, you can make several smears. In fact, to make a smear, it is enough to leave a spot of blood of 3 mm about in diameter on the slide. It is useful to perform many smears. In fact, not always they are successful, and with some attempts, it is easier to get one well prepared. To avoid producing clots, you must make each smear with fresh blood and straight after having deposited it. To this purpose, it is useful to be helped by another person where one deposits the blood, and the other makes the smears. With the microscope, you should observe the smears to check that some of them are properly made. The red cells must not overlap each other, nor be so scarce as to be too spread out.

FIXING

If you apply the stain to a smear without having fixed it beforehand, the cells will explode because of the so-called osmotic or hypotonic shock. This happens because the saline concentration inside the cells is much higher than that of staining fluid which is diluted in distilled water. In the attempt to equal the internal saline concentration to the values of the external one, the cells undergo swelling by osmosis. To attain the same saline concentration of the external liquid, the cells should swell more than their membrane allows, in fact they explode. The cell contents are released, and the preparation becomes unusable. To avoid this, before staining, you have to fix the smear. This operation hinders the inflation of the cells which keep sound when they are stained.
A simple and effective fixing technique consists of dipping the smear in a vessel containing 95% ethyl or methyl alcohol for 3-5 minutes. In order to put alcohol on the smear, you can also use a dropper or a bottle dispenser.

STAINING

If you observe the smear as it is after fixing, you will see very little because all cells are very transparent. The erythrocytes are slightly visible, but the leukocytes are too pale, almost invisible and you will not see anything inside them. To be able to observe and recognize the different kinds of leukocyte, you must stain them. For this purpose, normally Giemsa stain is used. It is a mixture of stains, based on methylene blue and eosin. It is cheaply available commercially in volumes of 100 cc. It consists of a concentrated solution which you have to dilute in the proportion1/10, that is one part of Giemsa in nine of distilled water, or buffer solution (pH = 6,8-7,2). You can buy the stain in a store of chemicals and laboratory equipment.
To stain a smear, take a slide with a fixed and dry smear. Put on the slide a drop of stain until it is fully covered. Stain for about 16 minutes, renewing the stain about four times. Then rinse the slide with distilled water at room temperature. Drain off the water and leave the slide to dry.

CHECKING

With the microscope, verify that the cells are well stained. If necessary, apply the stain for a few more minutes. If you were planning to mount the slide with Canada balsam, the staining has to be stronger.

COVER-SLIPPING

At this point, your smear is ready to be observed, but if you want to keep it for a long time, you should make the preparation permanent. To this purpose, after drying the slide, place a drop of Canada balsam or another medium mountant on the smear, then mount the coverslip. If the balsam is too viscous, you may heat a few of the slides (but not over 40 degrees C) to help the balsam flow between the slide and coverslip.

OBSERVATION

A magnification of 200 times is enough to allow you to observe and identify the different types of cells. If you use a higher power, you can also see the cells details better. You can examine either with dry objectives or with the oil immersion technique. In this last case, if you have put on a coverslip, you must wait a day to allow the balsam to set, otherwise, when you move the slide, oil will displace the coverslip.

-- ERYTHROCYTES

The red cells are very numerous in the blood. Usually, they measure 6,6-7,5 µm in diameter. However, cells with a diameter higher than 9 µm (macrocytes) or lower than 6 µm (microcytes) have been observed. In the observation field of the microscope, you will see a lot of erythrocytes and, sometimes, some isolated leukocytes. Erythrocytes are without nucleus (among vertebrates, only the red cells of mammalians are lacking a nucleus). Their typical shape is that of a cake depressed in the center (fig. 1). Under the microscope, they look like pink discs clearer in the middle (fig. 2-6: pink cells around the leukocytes). Sometimes, they are piled up like coins. As we saw, the red cells can also have different shapes from those we described. Sometimes, this is normal, other times, this is due to diseases or to defective process of preparation and staining of the smear.

-- PLATELETS

Platelets are not true cells. They gemmate from big leukocytes called megakaryocytes. They are small sized diskettes about 3µm in diameter. They appear a purple color and are more intense than red cells (you can see some platelets in figures 5 and 6).

LEUKOCYTES

Unlike red cells, leukocytes have a nucleus. It is easily visible under the microscope, but only after having stained the smear. The nucleus of these cells can show multiple lobes, or be indented or kidney-shaped (reniform). Usually, the shape of the nucleus of various kind of leukocytes is different. Together with the different colors of granules, the shape of nucleus helps us to recognize these cells. Leukocytes are divided into granulocytes and lymphoid cells. In the drawings which follow, besides nuclei and granules, you can see even mitochondria, Golgi apparatus, endoplasmic reticula and ribosomes.

- GRANULOCYTES

They come from the marrow bone. Their cytoplasm is rich in granules which take typical colors which help their recognition. The nucleus is condensed in a little masses or lobes. In the blood, there are immature cells as well. They distinguish themselves by having a less segmented nucleus. As we have said, there are three types of granulocyte: neutrophil, eosinophil, basophil.

-- NEUTROPHIL Granulocytes

The neutrophil are the more common leukocytes. They have a diameter of 12-15 µm. You can recognize them as their nucleus is divided into 2 - 5 lobes connected by a fine nuclear strand or filament (fig. 8). The cytoplasm is transparent because its granules are small and faintly pink colored. Immature neutrophils have a band-shaped or horseshoe-shaped nucleus and are known as band cells. In the nucleus of the neutrophil of cells from females, you may see an appendage like a little drumstick (Barr body). It is the second X chromosome, inactivated.

-- EOSINOPHIL Granulocytes

The eosinophils are quite rare in the blood. They have the same size as the neutrophils. Generally their nucleus is bi-lobed. But even nuclei with three or four lobes have been observed. The cytoplasm is full of granules which assume a characteristic pink-orange color (fig. 9). As for the neutrophil, the nucleus is still easily visible.

-- BASOPHIL Granulocytes

Basophils are the rarest leukocytes: less than 1 %. They are quite small: 9-10 µm in diameter. Cytoplasm is very rich in granules which take a dark purple color. The nucleus is bi- or tri-lobed, but it is hard to see because of the number of granules which hide it (fig. 10).

- LYMPHOID CELLS (or agranulocytes)

Because usually these cells appear lacking in granules, they are also named agranulocytes. They have a compact nucleus and a transparent cytoplasm. There are two types of lymphoid cells: lymphocytes and monocytes. Their look is similar, but their origin is different. In fact, whereas lymphocytes spring from lymphatic organs, monocytes have the same origin as the granulocytes.

-- LYMPHOCYTES

Lymphocytes are quite common in the blood: 20-40%, 8-10 µm in diameter and generally they are smaller than the other leukocytes but they are still a few larger than red cells (fig. 11). The cytoplasm is transparent. The nucleus is round and large in comparison to the cell and it occupies most of it. In any case, some of the cytoplasm remains visible, generally in a lateral position. According to the quantity of cytoplasm, lymphocytes are divided into small, medium and large. With Giemsa stain, we cannot distinguish the different types of lymphocyte (B, T, NK), either in the blood because they are not activated, or because it would be necessary to perform special immunochemical staining.

-- MONOCYTES

Monocytes are the biggest leukocytes: 16-20 µm. They have a great reniform or horseshoe-shaped nucleus, in some cases even bi-lobed. The cytoplasm is transparent, but with an appearance of "ground glass" (fig. 12).

CONCLUSION

Now that you have learned this technique, you can apply it to analyze the blood of other animals. For example, observe the blood of earthworms, which are easy to find. What you see even in the blood of simple animals is very interesting. As the blood of vertebrates is the fruit of a long evolution, as you descend the evolutionary ladder, you will see simpler types of blood, but you will also find a continuity which will help you to understand the blood of different animals.
In the more primitive beings, the liquid which flows among the cells has a composition very close to that of the water and performs modest functions. If you go up the evolutionary tree, this liquid assumes new and more complex functions. While in the invertebrates, the blood, called hemolymph, wet the organs and only in a part flows inside vessels, in the vertebrates blood flows in a vascular system which is entirely contained by walls and the cells are wetted by lymph instead. In the vertebrates, the blood also carries out complex functions of transport, homeostasis and defense.

As for the immune system, even in the protists there is a very rudimentary form of recognition of what is extraneous. Of course, in this case it is too far removed to be called an immune system! However, in the comparatively simple multicellular organisms, such as annelids and arthropods, cells do exist with defensive functions but they do not perform a specific action. Often, they are generically named phagocytes, other times with more specific terms. In the vertebrates, lymphocytes occur, defensive cells endowed with specific roles.

We hope this experiment has helped you to become familiar with the blood and the methods for the microscopic observation of its cells. This may be useful as a science experiment during school, or even as general knowledge. To an amateur microscopist it may be a stimulating experiment. However, what you will observe during these experiments will help you to follow the development of life in our planet, even if from an unusual point of view.

blood cells

NEUTROPHIL

This granulocyte has very tiny light staining granules (the granules are very difficult to see). The nucleus is frequently multi-lobed with lobes connected by thin strands of nuclear material. These cells are capable of phagocytizing foreign cells, toxins, and viruses.

When taking a Differential WBC Count of normal blood, this type of cell would be the most numerous. Normally, neutrophils account for 50-70% of all leukocytes. If the count exceeds this amount, the cause is usually due to an acute infection such as appendicitis, smallpox or rheumatic fever. If the count is considerably less, it may be due to a viral infection such as influenza, hepatitis, or rubella.

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EOSINOPHIL

This granulocyte has large granules (A) which are acidophilic and appear pink (or red) in a stained preparation. This micrograph was color enhanced to illustrate this feature. The nucleus often has two lobes connected by a band of nuclear material. (Does it looks like a telephone receiver?) The granules contain digestive enzymes that are particularly effective against parasitic worms in their larval form. These cells also phagocytize antigen - antibody complexes.

These cells account for less than 5% of the WBC's. Increases beyond this amount may be due to parasitic diseases, bronchial asthma or hay fever. Eosinopenia may occur when the body is severely stressed.

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BASOPHIL

The basophilic granules in this cell are large, stain deep blue to purple, and are often so numerous they mask the nucleus. These granules contain histamines (cause vasodilation) and heparin (anticoagulant).

In a Differential WBC Count we rarely see these as they represent less than 1% of all leukocytes. If the count showed an abnormally high number of these cells, hemolytic anemia or chicken pox may be the cause.

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LYMPHOCYTE

The lymphocyte is an agranular cell with very clear cytoplasm which stains pale blue. Its nucleus is very large for the size of the cell and stains dark purple. (Notice that the nucleus almost fills the cell leaving a very thin rim of cytoplasm.) This cell is much smaller than the three granulocytes (which are all about the same size). These cells play an important role in our immune response. The T-lymphocytes act against virus infected cells and tumor cells. The B-lymphocytes produce antibodies.

This is the second most numerous leukocyte, accounting for 25-35% of the cells counted in a Differential WBC Count. When the number of these cells exceeds the normal amount, one would suspect infectious mononucleosis or a chronic infection. Patients with AIDS keep a careful watch on their T-cell level, an indicator of the AIDS virus' activity.

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MONOCYTE

This cell is the largest of the leukocytes and is agranular. The nucleus is most often "U" or kidney bean shaped; the cytoplasm is abundant and light blue (more blue than this micrograph illustrates). These cells leave the blood stream (diapedesis) to become macrophages. As a monocyte or macrophage, these cells are phagocytic and defend the body against viruses and bacteria.

These cells account for 3-9% of all leukocytes. In people with malaria, endocarditis, typhoid fever, and Rocky Mountain spotted fever, monocytes increase in number.

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ERYTHROCYTE

The background cells in this micrograph are erythrocytes (red blood cells). These cells are non-nucleated, biconcave discs that are filled with hemoglobin. The primary function of these cells is to carry oxygen from the lungs to the body cells.

Woman usually have 4-5 million erythrocytes per cubic millimeter of blood, men have 5-6 million. If this number is considerably higher, polycythemia may be the cause. If the number is considerably less, the person has anemia.

Sickle cell anemia is an inherited condition which results in some erythrocytes being malformed. The gene for this condition causes the hemoglobin to be incorrectly formed, which in turn causes some erythrocytes to take on a crescent shape. These cells are not able to carry adequate amounts of oxygen to cells.

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THROMBOCYTES - PLATELETS

Platelets, which are cell fragments, are seen next to the "t's" above. (Many of the other micrographs on this page contain them as well.) Platelets are important for proper blood clotting.

Each cubic millimeter of blood should contain 250,000 to 500,000 of these. If the number is too high, spontaneous clotting may occur. If the number is too low, clotting may not occur when necessary.

Red blood cell 1

 

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Human red blood cells (6-8μm)

Red blood cells (also referred to as erythrocytes) are the most common type of blood cell and the vertebrate organism's principal means of delivering oxygen (O₂) to the body tissues via the blood flow through the circulatory system. They take up oxygen in the lungs or gills and release it while squeezing through the body's capillaries.

These cells' cytoplasm is rich in hemoglobin, an iron-containing biomolecule that can bind oxygen and is responsible for the blood's red color.

In humans, mature red blood cells are flexible biconcave disks that lack a cell nucleus and most organelles. 2.4 million new erythrocytes are produced per second.^[1] The cells develop in the bone marrow and circulate for about 100–120 days in the body before their components are recycled by macrophages. Each circulation takes about 20 seconds. Approximately a quarter of the cells in the human body are red blood cells.^[2][3]

Red blood cells are also known as RBCs, red blood corpuscles (an archaic term), haematids, erythroid cells or erythrocytes (from Greek erythros for "red" and kytos for "hollow", with cyte translated as "cell" in modern usage). Packed red blood cells, which are made from whole blood with the plasma removed, are used in transfusion medicine.^[4]

Contents

[hide]

• 1 History
• 2 Vertebrate erythrocytes
  • 2.1 Nucleus
  • 2.2 Secondary functions
• 3 Mammalian erythrocytes
• 4 Human erythrocytes
  • 4.1 Life cycle
    • 4.1.1 Erythropoiesis
    • 4.1.2 Functional lifetime
    • 4.1.3 Senescence
  • 4.2 Membrane composition
    • 4.2.1 Membrane lipids
    • 4.2.2 Membrane proteins
  • 4.3 Separation and blood doping
  • 4.4 Artificially grown red blood cells
• 5 Diseases and diagnostic tools
• 6 See also
• 7 References
• 8 External links

History

The first person to describe red blood cells was the young Dutch biologist Jan Swammerdam, who had used an early microscope in 1658 to study the blood of a frog.^[5] Unaware of this work, Anton van Leeuwenhoek provided another microscopic description in 1674, this time providing a more precise description of red blood cells, even approximating their size, "25,000 times smaller than a fine grain of sand".

In 1901 Karl Landsteiner published his discovery of the three main blood groups—A, B, and C (which he later renamed to O). Landsteiner described the regular patterns in which reactions occurred when serum was mixed with red blood cells, thus identifying compatible and conflicting combinations between these blood groups. A year later Alfred von Decastello and Adriano Sturli, two colleagues of Landsteiner, identified a fourth blood group—AB.

In 1959, by use of X-ray crystallography, Dr. Max Perutz was able to unravel the structure of hemoglobin, the red blood cell protein that carries oxygen.^[6]

Vertebrate erythrocytes

There is an immense size variation in vertebrate erythrocytes, as well as a correlation between cell and nucleus size. Mammalian erythrocytes, which do not contain nuclei, are considerably smaller than those of most other vertebrates.^[7]

Erythrocytes consist mainly of hemoglobin, a complex metalloprotein containing heme groups whose iron atoms temporarily bind to oxygen molecules (O₂) in the lungs or gills and release them throughout the body. Oxygen can easily diffuse through the red blood cell's cell membrane. Hemoglobin in the erythrocytes also carries some of the waste product carbon dioxide back from the tissues; most waste carbon dioxide, however, is transported back to the pulmonary capillaries of the lungs as bicarbonate (HCO₃^-) dissolved in the blood plasma. Myoglobin, a compound related to hemoglobin, acts to store oxygen in muscle cells.^[8]

The color of erythrocytes is due to the heme group of hemoglobin. The blood plasma alone is straw-colored, but the red blood cells change color depending on the state of the hemoglobin: when combined with oxygen the resulting oxyhemoglobin is scarlet, and when oxygen has been released the resulting deoxyhemoglobin is of a dark red burgundy color, appearing bluish through the vessel wall and skin. Pulse oximetry takes advantage of this color change to directly measure the arterial blood oxygen saturation using colorimetric techniques.

The sequestration of oxygen carrying proteins inside specialized cells (rather than having them dissolved in body fluid) was an important step in the evolution of vertebrates as it allows for less viscous blood, higher concentrations of oxygen, and better diffusion of oxygen from the blood to the tissues. The size of erythrocytes varies widely among vertebrate species; erythrocyte width is on average about 25% larger than capillary diameter and it has been hypothesized that this improves the oxygen transfer from erythrocytes to tissues.^[9]

The only known vertebrates without erythrocytes are the crocodile icefishes (family Channichthyidae); they live in very oxygen rich cold water and transport oxygen freely dissolved in their blood.^[10] While they do not use hemoglobin any more, remnants of hemoglobin genes can be found in their genome.^[11]

Nucleus

Erythrocytes in mammals are anucleate when mature, meaning that they lack a cell nucleus. In comparison, the erythrocytes of other vertebrates have nuclei; the only known exceptions are salamanders of the Batrachoseps genus and fish of the Maurolicus genus with closely related species.^[12][13]

Secondary functions

When erythrocytes undergo shear stress in constricted vessels, they release ATP which causes the vessel walls to relax and dilate so as to promote normal blood flow.^[14]

When their hemoglobin molecules are deoxygenated, erythrocytes release S-nitrosothiols which also acts to dilate vessels,^[15] thus directing more blood to areas of the body depleted of oxygen.

It has been recently demonstrated that erythrocytes can also synthesize nitric oxide enzymatically, using L-arginine as substrate, just like endothelial cells.^[16] Exposure of erythrocytes to physiological levels of shear stress activates nitric oxide synthase and export of nitric oxide,^[17] which may contribute to the regulation of vascular tonus.

Erythrocytes can also produce hydrogen sulfide, a signalling gas that acts to relax vessel walls. It is believed that the cardioprotective effects of garlic are due to erythrocytes converting its sulfur compounds into hydrogen sulfide.^[18]

Erythrocytes also play a part in the body's immune response: when lysed by pathogens such as bacteria, their hemoglobin releases free radicals which break down the pathogen's cell wall and membrane, killing it.^[19][20]

Mammalian erythrocytes

Typical mammalian erythrocytes: (a) seen from surface; (b) in profile, forming rouleaux; (c) rendered spherical by water; (d) rendered crenate by salt. (c) and (d) do not normally occur in the body.

Mammalian erythrocytes are unique among the vertebrates as they are non-nucleated cells in their mature form. These cells have nuclei during early phases of erythropoiesis, but extrude them during development as they mature in order to provide more space for hemoglobin. In mammals, erythrocytes also lose all other cellular organelles such as their mitochondria, golgi apparatus and endoplasmic reticulum. As a result of not containing mitochondria, these cells use none of the oxygen they transport; instead they produce the energy carrier ATP by lactic acid fermentation of glucose. Because of the lack of nuclei and organelles, mature red blood cells do not contain DNA and cannot synthesize any RNA, and consequently cannot divide and have limited repair capabilities.^[21]

Mammalian erythrocytes are typically shaped as biconcave disks: flattened and depressed in the center, with a dumbbell-shaped cross section, and a torus-shaped rim on the edge of the disk. This distinctive biconcave shape optimises the flow properties of blood in the large vessels, such as maximization of laminar flow and minimization of platelet scatter, which suppresses their atherogenic activity in those large vessels.^[22] However, there are some exceptions concerning shape in the artiodactyl order (even-toed ungulates including cattle, deer, and their relatives), which displays a wide variety of bizarre erythrocyte morphologies: small and highly ovaloid cells in llamas and camels (family Camelidae), tiny spherical cells in mouse deer (family Tragulidae), and cells which assume fusiform, lanceolate, crescentic, and irregularly polygonal and other angular forms in red deer and wapiti (family Cervidae). Members of this order have clearly evolved a mode of red blood cell development substantially different from the mammalian norm.^[7][23] Overall, mammalian erythrocytes are remarkably flexible and deformable so as to squeeze through tiny capillaries, as well as to maximize their apposing surface by assuming a cigar shape, where they efficiently release their oxygen load.^[24]

In large blood vessels, red blood cells sometimes occur as a stack, flat side next to flat side. This is known as rouleaux formation, and it occurs more often if the levels of certain serum proteins are elevated, as for instance during inflammation.

The spleen acts as a reservoir of red blood cells, but this effect is somewhat limited in humans. In some other mammals such as dogs and horses, the spleen sequesters large numbers of red blood cells which are dumped into the blood during times of exertion stress, yielding a higher oxygen transport capacity.

Scanning electron micrograph of blood cells. From left to right: human erythrocyte, thrombocyte (platelet), leukocyte.

Human erythrocytes

Two drops of blood are shown with a bright red oxygenated drop on the left and a deoxygenated drop on the right.

An animation of a typical human red blood cell cycle in the circulatory system. This animation occurs at real time (20 seconds of cycle) and shows the red blood cell deform as it enters capillaries, as well as changing color as it alternates in states of oxygenation along the circulatory system.

A typical human erythrocyte has a disk diameter of 6–8 µm and a thickness of 2 µm, being much smaller than most other human cells. These cells have a volume of about 90 fL with a surface of about 136 μm², and can swell up to a sphere shape containing 150 fL, without membrane distension.

Adult humans have roughly 2–3 × 10¹³ (20-30 trillion) red blood cells at any given time, comprising approximately one quarter of the total human body cell number (women have about 4 to 5 million erythrocytes per microliter (cubic millimeter) of blood and men about 5 to 6 million; people living at high altitudes with low oxygen tension will have more). Red blood cells are thus much more common than the other blood particles: there are about 4,000–11,000 white blood cells and about 150,000–400,000 platelets in each microliter of human blood.

Human red blood cells take on average 20 seconds to complete one cycle of circulation.^[2][3][25] As red blood cells contain no nucleus, protein biosynthesis is currently assumed to be absent in these cells, although a recent study indicates the presence of all the necessary biomachinery in human red blood cells for protein biosynthesis.^[21]

The blood's red color is due to the spectral properties of the hemic iron ions in hemoglobin. Each human red blood cell contains approximately 270 million of these hemoglobin biomolecules, each carrying four heme groups; hemoglobin comprises about a third of the total cell volume. This protein is responsible for the transport of more than 98% of the oxygen (the remaining oxygen is carried dissolved in the blood plasma). The red blood cells of an average adult human male store collectively about 2.5 grams of iron, representing about 65% of the total iron contained in the body.^[26][27] (See Human iron metabolism.)

Life cycle

Human erythrocytes are produced through a process named erythropoiesis, developing from committed stem cells to mature erythrocytes in about 7 days. When matured, these cells live in blood circulation for about 100 to 120 days. At the end of their lifespan, they become senescent, and are removed from circulation.

Erythropoiesis

Erythropoiesis is the development process in which new erythrocytes are produced, through which each cell matures in about 7 days. Through this process erythrocytes are continuously produced in the red bone marrow of large bones, at a rate of about 2 million per second in a healthy adult. (In the embryo, the liver is the main site of red blood cell production.) The production can be stimulated by the hormone erythropoietin (EPO), synthesised by the kidney. Just before and after leaving the bone marrow, the developing cells are known as reticulocytes; these comprise about 1% of circulating red blood cells.

Functional lifetime

This phase lasts about 100–120 days, during which the erythrocytes are continually moving by the blood flow push (in arteries), pull (in veins) and squeezing through microvessels such as capillaries as they compress against each other in order to move.

Senescence

The aging erythrocyte undergoes changes in its plasma membrane, making it susceptible to selective recognition by macrophages and subsequent phagocytosis in the reticuloendothelial system (spleen, liver and bone marrow), thus removing old and defective cells and continually purging the blood. This process is termed eryptosis, erythrocyte programmed cell death. This process normally occurs at the same rate of production by erythropoiesis, balancing the total circulating red blood cell count. Eryptosis is increased in a wide variety of diseases including sepsis, haemolytic uremic syndrome, malaria, sickle cell anemia, beta-thalassemia, glucose-6-phosphate dehydrogenase deficiency, phosphate depletion, iron deficiency and Wilson's disease. Eryptosis can be elicited by osmotic shock, oxidative stress, energy depletion as well as a wide variety of endogenous mediators and xenobiotics. Excessive eryptosis is observed in erythrocytes lacking the cGMP-dependent protein kinase type I or the AMP-activated protein kinase AMPK. Inhibitors of eryptosis include erythropoietin, nitric oxide, catecholamines and high concentrations of urea.

Much of the resulting important breakdown products are recirculated in the body. The heme constituent of hemoglobin are broken down into Fe³⁺ and biliverdin. The biliverdin is reduced to bilirubin, which is released into the plasma and recirculated to the liver bound to albumin. The iron is released into the plasma to be recirculated by a carrier protein called transferrin. Almost all erythrocytes are removed in this manner from the circulation before they are old enough to hemolyze. Hemolyzed hemoglobin is bound to a protein in plasma called haptoglobin which is not excreted by the kidney.^[28]

Membrane composition

The membrane of the red blood cell plays many roles that aid in regulating their surface deformability, flexibility, adhesion to other cells and immune recognition. These functions are highly dependent on its composition, which defines its properties. The red blood cell membrane is composed of 3 layers: the glycocalyx on the exterior, which is rich in carbohydrates; the lipid bilayer which contains many transmembrane proteins, besides its lipidic main constituents; and the membrane skeleton, a structural network of proteins located on the inner surface of the lipid bilayer. In human erythrocytes, like in most mammal erythrocytes, half of the membrane mass is represented by proteins and the other half are lipids, namely phospholipids and cholesterol.^[29]

Membrane lipids

The most common erythrocyte cell membrane lipids, schematically disposed as they are distributed on the bilayer. Relative abundances are not at scale.

The erythrocyte cell membrane comprises a typical lipid bilayer, similar to what can be found in virtually all human cells. Simply put, this lipid bilayer is composed of cholesterol and phospholipids in equal proportions by weight. The lipid composition is important as it defines many physical properties such as membrane permeability and fluidity. Additionally, the activity of many membrane proteins is regulated by interactions with lipids in the bilayer.

Unlike cholesterol which is evenly distributed between the inner and outer leaflets, the 5 major phospholipids are asymmetrically disposed, as shown below:

Outer monolayer

• Phosphatidylcholine (PC);
• Sphingomyelin (SM).

Inner monolayer

• Phosphatidylethanolamine (PE);
• Phosphoinositol (PI) (small amounts).
• Phosphatidylserine (PS);

This asymmetric phospholipid distribution among the bilayer is the result of the function of several energy-dependent and energy-independent phospholipid transport proteins. Proteins called “Flippases” move phospholipids from the outer to the inner monolayer while others called “floppases” do the opposite operation, against a concentration gradient in an energy dependent manner. Additionally, there are also “scramblase” proteins that move phospholipids in both directions at the same time, down their concentration gradients in an energy independent manner. There is still considerable debate ongoing regarding the identity of these membrane maintenance proteins in the red cell membrane.

The maintenance of an asymmetric phospholipid distribution in the bilayer (such as an exclusive localization of PS and PIs in the inner monolayer) is critical for the cell integrity and function due to several reasons:

• Macrophages recognize and phagocytose red cells that expose PS at their outer surface. Thus the confinement of PS in the inner monolayer is essential if the cell is to survive its frequent encounters with macrophages of the reticuloendothelial system, especially in the spleen.

• Premature destruction of thallassemic and sickle red cells has been linked to disruptions of lipid asymmetry leading to exposure of PS on the outer monolayer.

• An exposure of PS can potentiate adhesion of red cells to vascular endothelial cells, effectively preventing normal transit through the microvasculature. Thus it is important that PS is maintained only in the inner leaflet of the bilayer to ensure normal blood flow in microcirculation.

• Both PS and phosphatidylinositol-4,5-bisphosphate (PIP2) can regulate membrane mechanical function, due to their interactions with skeletal proteins such as spectrin and protein 4.1R. Recent studies have shown that binding of spectrin to PS promotes membrane mechanical stability. PIP2 enhances the binding of protein band 4.1R to glycophorin C but decreases its interaction with protein band 3, and thereby may modulate the linkage of the bilayer to the membrane skeleton.

The presence of specialized structures named "lipid rafts" in the erythrocyte membrane have been described by recent studies. These are structures enriched in cholesterol and sphingolipids associated with specific membrane proteins, namely flotillins, stomatins (band 7), G-proteins, and β-adrenergic receptors. Lipid rafts that have been implicated in cell signaling events in nonerythroid cells have been shown in erythroid cells to mediate β2-adregenic receptor signaling and increase cAMP levels, and thus regulating entry of malarial parasites into normal red cells.^[30][31]

Membrane proteins

Red blood cell membrane proteins separated by SDS-Page and silverstained ^[32]

The proteins of the membrane skeleton are responsible for the deformability, flexibility and durability of the red blood cell, enabling it to squeeze through capillaries less than half the diameter of the erythrocyte (7-8 μm) and recovering the discoid shape as soon as these cells stop receiving compressive forces, in a similar fashion to an object made of rubber.

There are currently more than 50 known membrane proteins, which can exist in a few hundred up to a million copies per erythrocyte. Approximately 25 of these membrane proteins carry the various blood group antigens, such as the A, B and Rh antigens, among many others. These membrane proteins can perform a wide diversity of functions, such as transporting ions and molecules across the red cell membrane, adhesion and interaction with other cells such as endothelial cells, as signaling receptors, as well as other currently unknown functions. The blood types of humans are due to variations in surface glycoproteins of erythrocytes. Disorders of the proteins in these membranes are associated with many disorders, such as hereditary spherocytosis, hereditary elliptocytosis, hereditary stomatocytosis, and paroxysmal nocturnal hemoglobinuria.^[29][30]

The red blood cell membrane proteins organized according to their function:

Red Blood Cell membrane major proteins

Transport

• Band 3 - Anion transporter, also an important structural component of the erythrocyte cell membrane, makes up to 25% of the cell membrane surface, each red cell contains approximately one million copies. Defines the Diego Blood Group;^[33]
• Aquaporin 1 - water transporter, defines the Colton Blood Group;
• Glut1 - glucose and L-dehydroascorbic acid transporter;
• Kidd antigen protein - urea transporter;
• RhAG - gas transporter, probably of carbon dioxide, defines Rh Blood Group and the associated unusual blood group phenotype Rh_null;
• Na⁺/K⁺ - ATPase;
• Ca²⁺ - ATPase;
• Na⁺ K⁺ 2Cl^- - cotransporter;
• Na⁺-Cl^- - cotransporter;
• Na-H exchanger;
• K-Cl - cotransporter;
• Gardos Channel.

Cell adhesion

• ICAM-4 - interacts with integrins;
• BCAM - a glycoprotein that defines the Lutheran blood group and also known as Lu or laminin-binding protein.

Structural role - The following membrane proteins establish linkages with skeletal proteins and may play an important role in regulating cohesion between the lipid bilayer and membrane skeleton, likely enabling the red cell to maintain its favorable membrane surface area by preventing the membrane from collapsing (vesiculating).

• Ankyrin-based macromolecular complex - proteins linking the bilayer to the membrane skeleton through the interaction of their cytoplasmic domains with Ankyrin.
  • Band 3 - also assembles various glycolytic enzymes, the presumptive CO₂ transporter, and carbonic anhydrase into a macromolecular complex termed a “metabolon,” which may play a key role in regulating red cell metabolism and ion and gas transport function);
  • RhAG - also involved in transport, defines associated unusual blood group phenotype Rh_mod.

• Protein 4.1R-based macromolecular complex - proteins interacting with Protein 4.1R.
  • Protein 4.1R - weak expression of Gerbich antigens;
  • Glycophorin C and D - glycoprotein, defines Gerbich Blood Group;
  • XK - defines the Kell Blood Group and the Mcleod unusual phenotype (lack of Kx antigen and greatly reduced expression of Kell antigens);
  • RhD/RhCE - defines Rh Blood Group and the associated unusual blood group phenotype Rh_null;
  • Duffy protein - has been proposed to be associated with chemokine clearance;^[34]
  • Adducin - interaction with band 3;
  • Dematin- interaction with the Glut1 glucose transporter.

^[29][30]

Separation and blood doping

Red blood cells can be obtained from whole blood by centrifugation, which separates the cells from the blood plasma. During plasma donation, the red blood cells are pumped back into the body right away and the plasma is collected. Some athletes have tried to improve their performance by blood doping: first about 1 litre of their blood is extracted, then the red blood cells are isolated, frozen and stored, to be reinjected shortly before the competition. (Red blood cells can be conserved for 5 weeks at −79 °C.) This practice is hard to detect but may endanger the human cardiovascular system which is not equipped to deal with blood of the resulting higher viscosity.

Artificially grown red blood cells

In 2008 it was reported that human embryonic stem cells had been successfully coaxed into becoming erythrocytes in the lab. The difficult step was to induce the cells to eject their nucleus; this was achieved by growing the cells on stromal cells from the bone marrow. It is hoped that these artificial erythrocytes can eventually be used for blood transfusions.^[35]

Diseases and diagnostic tools

Affected by Sickle-cell disease, red blood cells alter shape and threaten to damage internal organs.

Blood diseases involving the red blood cells include:

• Anemias (or anaemias) are diseases characterized by low oxygen transport capacity of the blood, because of low red cell count or some abnormality of the red blood cells or the hemoglobin.

• Iron deficiency anemia is the most common anemia; it occurs when the dietary intake or absorption of iron is insufficient, and hemoglobin, which contains iron, cannot be formed

• Sickle-cell disease is a genetic disease that results in abnormal hemoglobin molecules. When these release their oxygen load in the tissues, they become insoluble, leading to mis-shaped red blood cells. These sickle shaped red cells are less deformable and viscoelastic meaning that they have become rigid and can cause blood vessel blockage, pain, strokes, and other tissue damage.

• Thalassemia is a genetic disease that results in the production of an abnormal ratio of hemoglobin subunits.

• Spherocytosis is a genetic disease that causes a defect in the red blood cell's cytoskeleton, causing the red blood cells to be small, sphere-shaped, and fragile instead of donut-shaped and flexible.

• Pernicious anemia is an autoimmune disease wherein the body lacks intrinsic factor, required to absorb vitamin B₁₂ from food. Vitamin B₁₂ is needed for the production of hemoglobin.

• Aplastic anemia is caused by the inability of the bone marrow to produce blood cells.

• Pure red cell aplasia is caused by the inability of the bone marrow to produce only red blood cells.

Effect of osmotic pressure on blood cells

• Hemolysis is the general term for excessive breakdown of red blood cells. It can have several causes and can result in hemolytic anemia.

• The malaria parasite spends part of its life-cycle in red blood cells, feeds on their hemoglobin and then breaks them apart, causing fever. Both sickle-cell disease and thalassemia are more common in malaria areas, because these mutations convey some protection against the parasite.

• Polycythemias (or erythrocytoses) are diseases characterized by a surplus of red blood cells. The increased viscosity of the blood can cause a number of symptoms.

• In polycythemia vera the increased number of red blood cells results from an abnormality in the bone marrow.

• Several microangiopathic diseases, including disseminated intravascular coagulation and thrombotic microangiopathies, present with pathognomonic (diagnostic) red blood cell fragments called schistocytes. These pathologies generate fibrin strands that sever red blood cells as they try to move past a thrombus.

• Inherited hemolytic anemias caused by abnormalities of the erythrocyte membrane comprise an important group of inherited disorders. These disorders are characterized by clinical and biochemical heterogeneity and also genetic heterogeneity, as evidenced by recent molecular studies.

• The Hereditary spherocytosis (HS) syndromes are a group of inherited disorders characterized by the presence of spherical-shaped erythrocytes on the peripheral blood smear. HS is found worldwide. It is the most common inherited anemia in individuals of northern European descent, affecting approximately 1 in 1000-2500 individuals depending on the diagnostic criteria. The primary defect in hereditary spherocytosis is a deficiency of membrane surface area. Decreased surface area may produced by two different mechanisms: 1) Defects of spectrin, ankyrin, or protein 4.2 lead to reduced density of the membrane skeleton, destabilizing the overlying lipid bilayer and releasing band 3-containing microvesicles. 2) Defects of band 3 lead to band 3 deficiency and loss of its lipid-stabilizing effect. This results in the loss of band 3-free microvesicles. Both pathways result in membrane loss, decreased surface area, and formation of spherocytes with decreased deformability. These deformed erythrocytes become trapped in the hostile environment of the spleen where splenic conditioning inflicts further membrane damage, amplifying the cycle of membrane injury.
• Hereditary elliptocytosis
• Hereditary pyropoikilocytosis
• Hereditary stomatocytosis^[36]

• Hemolytic transfusion reaction is the destruction of donated red blood cells after a transfusion, mediated by host antibodies, often as a result of a blood type mismatch.

Several blood tests involve red blood cells, including the RBC count (the number of red blood cells per volume of blood), the hematocrit (percentage of blood volume occupied by red blood cells), and the erythrocyte sedimentation rate. The blood type needs to be determined to prepare for a blood transfusion or an organ transplantation.

Main Index

Peripheral Blood Cells

Tissues, Layers, and Organs

Epithelial Tissue, Surface Specializations, and Glands

Connective Tissue

Cartilage and Bone and Bone Histogenesis

Bone Marrow and Hemopoiesis

Nervous Tissue and Neuromuscular Junction

Muscle Tissue

Integument

Circulatory System

Lymphoid Tissues and Organs

Urinary System

Digestive System: Oral Cavity and Teeth

Digestive System: Alimentary Canal

Liver, Gall Bladder, and Pancreas

Endocrine System

Respiratory System

Male Reproductive System

Female Reproductive System (including Organs of Pregnancy)

Eye

Ultrastructure of the Cell (Electron Micrographs)

 

 

Section 17: Central Nervous System

DIENCEPHALON
Ventral lateral nucleus coronal

Human, 10% formalin, Pal-Weigert, 2.0 x.

 

Fornix: A paired C-shaped fiber tract connecting the hippocampus with several brain regions, including the mamillary body, anterior thalamic nucleus, septal nuclei, and cingulate gyrus. Seen in two locations in this figure, below the corpus callosurn and within the hypothalamus as it approaches its termination in the mamillary body.

Massa intermedia: Connects the two thalami across the third ventricle. Present in about 70 percent of human brains. Also referred to as the interthalamic adhesion.

Lateral dorsal (thalamic) nucleus: Belongs to the dorsal subgroup of the lateral thalamic nuclei. Has an association function.

Ventral lateral (thalamic) nucleus: Traversed by bundles of myelinated fibers. Located lateral to the internal medullary lamina. Receives fibers from the cerebellum via the brachium conjunctivum (dentatorubrothalamic fiber system). Has reciprocal connections with the primary motor cortex. Plays a role in motor control.

External medullary lamina: Defines the lateral boundary of the thalamus.

Thalamic fasciculus: A bundle of myelinated fibers destined for several thalamic nuclei from the basal ganglia and cerebellum.

Lenticular fasciculus: A bundle of myelinated fibers originating from the basal ganglia (globus pallidus) and destined for the thalamus via the thalamic fasciculus.

Mamillothalamic tract: Seen in cross section. Connects the mamillary body with the anterior thalamic nucleus.

Hypothalamus: Ventral to the thalamus. Forms part of the lateral wall of the third ventricle. The fornix separates medial and lateral zones of the hypothalamus.

Third ventricle: Between the two hypothalami and two thalami. Bisected by the massa intermedia.

Optic tract: Myelinated fiber bundle conveying visual impulses from the contralateral field of vision to the lateral geniculate nucleus.

Ansa lenticularis: Axons of neurons in globus pallidus coursing around the internal capsule. Projects to ventral anterior thalamic nucleus. Together with fibers from lenticular fasciculus forms the thalamic fasciculus.

Zona incerta: Sandwiched between the lenticular fasciculus and thalamic fasciculus. Rostral continuation of mesencephalic reticular formation. Continuous with the reticular nucleus of the thalamus.

Globus pallidus: Belongs to the basal ganglia along with the caudate nucleus and putamen. Located medial to the putamen and traversed by heavily myelinated fiber bundles. Comprises the principal efferent nucleus of the basal ganglia. Receives fibers from caudate, putamen, and subthalamic nuclei; and projects fibers to the thalamus (ventral anterior nucleus) and the subthalamic nucleus.

Internal capsule: The posterior limb of the internal capsule separates the basal ganglia and the thalamus. Massive cortical afferent and efferent bundle.

Internal medullary lamina: A band of myelinated fibers that separate the medial from the lateral group of thalamic nuclei. Contains fibers that interconnect different thalamic nuclei.

Reticular nucleus: A thalamic nucleus located between the internal capsule and external medullary lamina.

Dorsomedial (thalamic) nucleus: Belongs to the medial group of thalamic nuclei. Most highly developed in man. Located medial to the internal medullary lamina. Has reciprocal connections with the prefrontal cortex and hypothalamus. Receives input from other thalamic nuclei. Concerned with affective behavior and memory.

Stria medullaris thalami: Located clorsomedial to the thalamus. A component of the epithalamus. Connects the septal area and the habenular nuclei.  

 

DIENCEPHALON
Anterior commissure coronal

Human, 10% formalin, Weigert's hematoxylin (Loyez), 0.9 x.

 

Corpus callosum: A massive bundle of myelinated fibers connecting the two hemispheres. Important in interhemispheric transfer of information.

Fornix: C-shaped, paired fiber system connecting the hippocampus with several brain regions, including the mamillary body, anterior thalamic nucleus, septal nuclei, and cingulate gyrus. Seen in two locations in this figure, beneath the corpus callosurn and above the anterior commissure.

Ventral anterior (thalamic) nucleus: One of the lateral group of thalamic nuclei. Characteristically traversed by heavily myelinated fiber bundles. Receives fibers from the basal ganglia and is reciprocally connected with the cerebral cortex. Plays a role in motor control.

Internal capsule (posterior limb): Separates the thalamus from the basal ganglia (putamen and globus pallidus). Carries fibers from and to the cerebral cortex. Lesions result in contralateral motor and sensory deficits.

External capsule: An efferent cortical bundle sandwiched between the putamen and claustrum.

Insula: Also referred to as the island of Reil,* lies deep in the sylvian fissure. Concerned primarily with autonomic function.

Caudate nucleus (body): A component of the basal ganglia. The body of the caudate is continuous rostrally with the head of the caudate and caudally with its tail. Plays a role in motor control.

Anterior (thalamic) nucleus: Belongs to the anterior group of thalamic nuclei. Has reciprocal connections with the mamillary body via the mamillothalamic tract and with the cingulate gyrus via the internal capsule. Considered part of the limbic system and thus plays a role in emotional behavior and memory.

Putamen: One of the basal ganglia nuclei. Concerned with motor control.

Extreme capsule: An efferent cortical bundle. Situated between the claustrum and the insula.

Globus pallidus: One of the basal ganglia nuclei. Located medial to putamen and characteristically traversed by heavily myelinated fiber bundles. Receives fibers from the caudate and putamen and projects to the thalamus (ventral anterior nucleus). Has also reciprocal connections with the subthalamic nucleus.

Claustrum: A thin layer of gray substance located between the external and extreme capsules.

Sylvian fissure: A major fissure on the lateral surface of the hemisphere separating the temporal from the frontal and parietal lobes.

Anterior commissure: A compact fiber bundle in close proximity to the fornix. Interconnects the olfactory bulbs and the temporal cortices.

*Reil was an eighteenth -century German physician, neurologist, and histologist.

 

DIENCEPHALON
Ventral anterior nucleus coronal

Human, 10% formalin, Pal-Weigert, 2.0 x.

 

Corpus callosum: Massive fiber bundle connecting the two cerebral hemispheres. Important in interhemispheric transfer of information.

Caudate nucleus: Basal ganglia nucleus. Important in motor control.

Anterior (thalamic) nucleus: Located in rostral diencephalon. Receives the mamillothalarnic tract from the mamillary body and projects to the cingulate gyrus of the cerebral cortex. A component of the limbic system.

Ventral anterior (thalamic) nucleus: Medial to the internal capsule. Traversed by heavily myelinated fiber bundles. Receives fibers from the basal ganglia and is reciprocally connected with the motor cortex. Plays a role in motor control.

Putamen: One of the basal ganglia nuclei. Similar to the caudate nucleus in structure and connections. Along with the caudate it forms the neostriaturn. Lies ventral and lateral to the internal capsule.

Globus pallidus: Another of the basal ganglia nuclei. Receives fibers from caudate and putamen and projects to the thalamus (ventral anterior nucleus). Also reciprocally connected with the subthalamic nucleus.

Mamillothalamic tract: Seen entering the anterior nucleus of the thalamus. Axons of neurons in the mamillary body.

External medullary lamina: Between the internal capsule and ventral anterior nucleus. Defines the lateral boundary of the thalamus.

Internal capsule: The posterior limb, separates the basal ganglia (putamen and globus pallidus) from the thalamus.

Fornix: At this rostral level, the fornix bundle separates as the columns of the fornix arch ventrally and caudally en route to the mamillary body

 

BASAL GANGLIA

Human, 10% formalin, Weigert's hematoxylin (Loyez), 1.0 x.

 

Corpus callosum: A massive myelinated fiber bundle connecting the two hemispheres. Important in interhemispheric transfer of information.

Putamen: Located lateral to the anterior limb of the internal capsule. Part of the basal ganglia (along with the caudate nucleus and globus pallidus). Concerned with regulation of movement.

Globus pallidus: Belongs to the basal ganglia (along with caudate nucleus and putamen). Located medial to putamen, and traversed by heavily myelinated fiber bundles. Constitutes the principal efferent nucleus of the basal ganglia. Receives fibers from the caudate, putamen, and subthalamic nuclei and projects fibers to the thalamus (ventral anterior nucleus) and to the subthalamic nucleus.

Corona radiata: White matter core of the cerebral hemispheres. Contains afferent and efferent fibers to and from the cerebral cortex.

Caudate nucleus: C-shaped mass of gray matter closely related to the lateral ventricle. Component of the basal ganglia. Plays a role in motor control.

Internal capsule (anterior limb): Separates the caudate nucleus and putamen. Carries fibers from and to the cerebral cortex

 

BASAL GANGLIA
Neostriatum (caudate nucleus and putamen)
coronal

Human, 10% formalin, Weigert's hematoxylin (Loyez), 8 x.

 

Longitudinal (interhemispheric) cerebral fissure: Separates the two cerebral hemispheres. Filled with a dural fold, the falx cerebri, which has been stripped away in this preparation.

Cingulate gyrus: Located dorsal to the corpus callosum. A component of the limbic lobe, which, in addition to the cingulate gyrus, includes the subcallosal gyrus, isthmus, parahippocampal gyrus, and uncus.

Corpus callosum: A massive myelinated fiber bundle connecting the two hemispheres. important in interhemispheric transfer of information.

Septum pellucidum: A thin septum, ventral to the corpus callosum, separates the two lateral ventricles. May contain a cavity, the cavurn septi pellucidi.

External capsule: One of the efferent cortical bundles. The others include the internal and extreme capsules. Located lateral to the putamen.

Extreme capsule: One of the efferent cortical bundles. Lateral to the external capsule. Between the external and extreme capsules is the claustrum (not seen in this preparation).

Lateral ventricle: Anterior horn of the lateral ventricle. Note the characteristic bulging of the head of the caudate nucleus into the cavity of the ventricle.

Caudate nucleus: C-shaped mass of gray matter closely related to the lateral ventricle. The part seen in this figure is the head of the caudate, which has a characteristic bulge into the lateral ventricle. This characteristic bulge is lost in the disease Huntington's chorea. The caudate nucleus is part of the basal ganglia, and thus plays a role in the regulation of movement. Huntington was a nineteenth-century American physician.

Internal capsule: Heavily myelinated broad band of white substance that arises from wide areas of the cerebral cortex and descends to the brain stem and spinal cord. Carries fibers from and to the cerebral cortex. The anterior limb of the internal capsule separates the caudate nucleus and putamen.

Putamen: Located lateral to the anterior limb of the internal capsule and medial to the external capsule. The putamen, like the caudate, is a component of the basal ganglia and thus plays a role in motor control.  

 

CEREBELLUM
Deep cerebellar nuclei

Human, 10% formalin, Weigert-carmine, 2.5 x.

 

Cerebellar folia: Leaf-like folds of the cerebellar cortex separated from each other by sulci and supported by a core of white matter.

Dentate nucleus: One of the deep cerebellar nuclei embedded in the deep white matter. Resembles the inferior olive in its purse-like appearance. Receives axons of Purkinje cells of the cerebellum as well as collaterals from the two major afferent bundles to the cerebellum; and projects through the brachium conjunctivurn to the red nucleus and ventrolateral nucleus of the thalamus. Lesions of the dentate nucleus result in homolateral volitional tremor.

Brachium conjunctivum: Outflow tract of the cerebellum. Heavily myelinated. Projects to the red nucleus and ventrolateral nucleus of the thalamus. Lesions of this tract result in homolateral volitional tremor.

Deep white matter: A compact mass of white matter, which is continuous between cerebellar hemispheres. In it are embedded the deep cerebellar nuclei. Extends into the folia as a core of white matter.  

 

ANTERIOR CEREBRAL
ARTERY INFARCT
Coronal

Human, 10% formalin, Weigert's hematoxylin (Loyez), 1.0 x.

 

This is a coronal section of the brain showing bilateral anterior cerebral artery infarcts. The infarcts involve structures supplied by the anterior cerebral artery such as the superior frontal gyrus, the cingulate gyrus, and the corpus callosum. Patients with this type of infarct will show bilateral lower extremity weakness or paralysis associated with upper motor neuron signs in the affected extremities. The upper extremities and the face will be spared in this type of lesion because their cortical areas are supplied by the middle cerebral artery.  

 

MIDDLE CEREBRAL
ARTERY INFARCT
Coronal

Human, 10% formalin, Weigert's hematoxylin (Loyez), 9 x.

 

This is a coronal section of the brain at the level of the corpus striatum, showing a middle cerebral artery infarct. The structures involved in the infarct are those supplied by the middle cerebral artery and include, at this level, the caudate nucleus, internal capsule, putamen, insula, globus pallidus, and the lateral surface of the hemisphere. Patients with this type of infarct will show contralateral weakness or paralysis of the upper motor neuron variety involving mainly the face and upper extremities. The lower extremity will be largely spared because its area of cortical representation is supplied by the anterior cerebral artery. Middle cerebral artery infarcts in the dominant left hemisphere will also be associated with disturbance in language known as aphasia.

 

CENTRUM SEMIOVALE
Axial

 

Human, 10% formalin, Weigert's hematoxylin (Loyez), 1 x.

 

Centrum semiovale: White matter core of the cerebral hemispheres. Contains myelinated nerve fibers entering or leaving the cerebral hemispheres.

Gray matter: Contains neurons whose axons contribute to the centrum semiovale.

 

 

BASAL GANGLIA
Axial

Human, 10% formalin, Weigert's hematoxylin (Loyez), 1 x.

 

Cingulate gyrus: Located dorsal to the corpus callosum. A component of the limbic lobe, which also includes the subcallosal gyrus, isthmus, parahippocampal gyrus and uncus.

Caudate nucleus: C-shaped mass of gray matter closely related to the lateral ventricle. A component of the basal ganglia and thus plays a role in motor control.

Internal capsule (anterior limb): Separates the caudate nucleus and cerebral cortex.

Putamen: One of the basal ganglia nuclei. Located lateral to the anterior limb of the internal capsule. Concerned with motor control.

Insula: Also referred to as the island of Reil, lies deep in the sylvian fissure. Concerned with autonomic function.

Choroid plexus: Vascular pial fold in the body of the lateral ventricle. One of the sites of formation of cerebrospinal fluid.

Corpus callosum: C-shaped bundle of heavily myelinated fibers connecting the two cerebral hemispheres. Important in interhemispheric transfer of information. Section is through the genu (anterior) and splenium (posterior) parts of the corpus callosum.

Lateral ventricle: Note the characteristic bulge of the caudate into the cavity of the lateral ventricle.

Septum pellucidum: Separates the two lateral ventricles. May contain a cavity, cavurn septi pellucidi

 

STRIATUM AND THALAMUS
Axial

Human, 10% formalin, Weigert's hematoxylin (Loyez), 1.1 x.

 

Corpus callosum: C-shaped bundle of heavily myelinated fibers. Connects the two hemispheres. Important in interhemispheric transfer of information. Section is through the genu (anterior) and splenium (posterior) parts of the corpus callosum.

Cingulate gyrus: A component of the limbic lobe. Located dorsal to the corpus callosurn on the medial surface of the hemisphere.

Caudate nucleus: C-shaped mass of gray matter closely related to the lateral ventricle. A component of the basal ganglia and thus concerned with motor control. Section shows the head of the caudate (anterior) and the much smaller tail (posterior).

Internal capsule: A massive bundle of myelinated fibers conveying motor and sensory impulses from and to the cerebral cortex. The anterior limb separates the caudate nucleus and putamen, whereas the posterior limb separates the putamen and thalamus.

Putamen: A component of the basal ganglia. Concerned with motor control.

Insula: Also referred to as the island of Reil. Lies deep in the sylvian fissure. Concerned with autonomic function.

Anterior thalamic nucleus: Belongs to the anterior group of thalamic nuclei. Has reciprocal connections with the mamillary body via the mamillothalamic tract and with the cingulate gyrus via the internal capsule. Considered part of the limbic system and thus plays a role in emotional behavior and memory.

Ventral lateral thalamic nucleus: Traversed by bundles of myelinated fibers. Belongs to the lateral group of thalamic nuclei. Relay thalamic nucleus for cerebellar fibers to the motor cortex. Reciprocally connected with the primary motor cortex. Concerned with motor control.

Pulvinar: One of the lateral group of thalamic nuclei. Has reciprocal connections with the medial and lateral geniculate bodies caudally and the association parietal, temporal, and occipital cortices rostrally. involved in several neural functions, including vision, audition, speech, and pain.

Fornix: A paired C-shaped fiber tract connecting the hippocampus with several brain regions, including the mamillary body, anterior thalamic nucleus, septal nuclei, and cingulate gyrus.

Lateral ventricle (anterior horn): Note characteristic bulge of head of caudate into the anterior horn.

Septum pellucidum: Separates the two cavities of the lateral ventricle. May contain a cavity, cavurn septi pellucidi.

Ventral anterior thalamic nucleus: One of the lateral group of thalamic nuclei. Characteristically traversed by heavily myelinated fiber bundles. Receives fibers from the basal ganglia and projects to the motor cortex. The posterior limb separates the thalamus from the putamen

 

THALAMUS AND BASAL GANGLIA
Axial

Human, 10% formalin, Weigert's hematoxylin (Loyez), 1 x.

 

Corpus callosum: C-shaped bundle of heavily myelinated fibers. Connects the two hemispheres. Important in interhemispheric transfer of information. Section shows the genu (anterior) and splenium (posterior) parts of the corpus callosum.

Caudate nucleus: C-shaped mass of gray matter closely related to the lateral ventricle. A component of the basal ganglia and thus concerned with motor control. Section shows the head of the caudate (anterior) and the much smaller tail (posterior).

Putamen: A component of the basal ganglia. Plays a role in motor control. Medial to the external capsule and lateral to the caudate and globus pallidus.

Globus pallidus: One of the basal ganglia nuclei. Located medial to the putamen. The posterior limb of the internal capsule separates it from the thalamus. Traversed by heavily myelinated fiber bundles. Receives fibers from the caudate and putamen and projects to the thalamus (ventral anterior nucleus). Has reciprocal connections with the subthalamic nucleus. Plays a role in motor control.

External capsule: An efferent cortical fiber bundle. Lateral to the putamen.

Claustrum: A thin zone of gray substance located between the external and extreme capsules.

Extreme capsule: An efferent cortical fiber bundle. Situated lateral to the claustrum.

Stria medullaris thalami: A bundle of myelinated fibers connecting the septal nuclei with the habenular complex. Characteristically located dorsomedial to the thalamus.

Fimbria of fornix: Efferent fibers from the hippocampus. Continues as the body of the fornix.

Fornix: C-shaped, paired fiber system connecting the hippocampus with several brain regions, including the mamillary body, anterior thalamic nucleus, septal nuclei, and cingulate gyrus. The part of the fornix seen here represents the columns of the fornix.

Anterior thalamic nucleus: Belongs to the anterior group of thalamic nuclei. Has reciprocal connections with the mamillary body via the mamillothalamic tract and with the cingulate gyrus via the internal capsule. Considered part of the limbic system. Plays a role in emotional behavior and memory.

Internal capsule (posterior limb): Separates the putamen from the thalamus. Massive fiber bundle conveying impulses to and from the cerebral cortex. Lesions in internal capsule result in contralateral motor and sensory deficits.

Dorsomedial thalamic nucleus: Belongs to the medial group of thalamic nuclei. Most highly developed in man. Located medial to the internal medullary lamina. Has reciprocal connections with the prefrontal cortex and hypothalamus. Receives input from other thalamic nuclei. Concerned with affective behavior and memory.

Forceps major: Extension of the splenium of the corpus callosurn posteriorly into the occipital lobes.

Lateral ventricle: Extension of the cavity of the lateral ventricle into the occipital lobe (occipital horn).  

 

ANTERIOR COMMISSURE
Axial

Human, 10% formalin, Weigert's hematoxylin (Loyez), 1.1 x.

 

Columns of fornix: Body of fornix separates into two columns, each of which curves ventrally and courses through the hypothalamus on its way to the mamillary body.

Anterior commissure: A compact fiber bundle in close proximity to anterior extent of the fornix. Interconnects olfactory bulbs and the temporal cortices.

Centromedion thalamic nucleus: Belongs to the intralaminar group of thalamic nuclei. Receives fibers from several sources, motor and sensory, that project diffusely to cerebral cortex either directly or indirectly via other thalamic nuclei. Plays a role in cortical arousal response and in pain mechanism.

Pulvinar: Belongs to the lateral group of thalamic nuclei. Has reciprocal connections with the medial and lateral geniculate bodies caudally and the association parietal, temporal, and occipital cortices rostrally. Plays a role in several neural functions, including vision, audition, speech, and pain.

Fimbria of fornix: Efferent fibers from the hippocampus that merge with the fornix.

Putamen: Lateral to globus pallidus. One of the basal ganglia nuclei. Concerned with motor control.

Globus pallidus: Medial to the putamen. Separated from the thalamus by the posterior limb of the internal capsule. Has two segments: outer, close to the putamen, and inner, close to the internal capsule. Characterized by heavily myelinated fibers traversing it. A component of the basal ganglia. Concerned with motor control.

Internal capsule (posterior limb): Separates putamen and globus pallidus from thalamus. Carries motor and sensory fibers from and to the cerebral cortex.

Dorsomedial thalamic nucleus: Belongs to the medial group of thalamic nuclei. Most highly developed in man. Concerned with affective behavior and memory.

Habenula: A nuclear mass located dorsal to the thalamus at the junction of the diencephalon and midbrain. A component of the limbic system.

 

MESENCEPHALON-DIENCEPHALON JUNCTION
Axial

Human, 10% formalin, Weigert's hematoxylin (Loyez), 1 x.

 

Gyrus rectus: inferior surface of the frontal lobe. Medial to orbital gyrus.

Mamillary body: A pair of spherical nuclear masses caudal to the optic chiasma and protruding from the ventral surface of the posterior hypothalamus in the interpeduncular fossa (between the cerebral peduncles). Concerned with memory function.

Substantia nigra: A pigmented (melanin) nuclear mass located in the base of the midbrain above the cerebral peduncles. invariably the site of pathologic changes in Parkinson's disease.

Red nucleus: So-called because of a pinkish color in the fresh state owing to its high vascularity. Links the cerebellum, cerebral cortex, and spinal cord.

Periaqueductal gray: A nuclear mass surrounding the aqueduct of Sylvius. Related to pain mechanisms.

Cerebellar folia: Leaf-like folds of the cerebellar cortex separated from each other by sulci and supported by a core of white matter.

Aqueduct of Sylvius: Connects the fourth with the third ventricle.

Cerebral peduncle: Descending corticofugal fiber system. Lesion results in contralateral muscle weakness or paralysis.

Superior colliculus: Cellular mass concerned with visual reflexes.  

 

CAUDATE NUCLEUS
Axial

Human, 10% formalin, Weigert's hematoxylin (Loyez), 4 x.

 

Corpus callosum: C-shaped massive bundle of heavily myelinated fibers. Connects the two hemispheres. Important in interhemispheric transfer of information. Figure shows the body of the corpus callosurn and extensions into the frontal pole (forceps minor) and into the occipital pole (forceps major).

Caudate nucleus: Another C-shaped mass of gray matter closely related to the lateral ventricle. A component of the basal ganglia. Important for motor control.

Lateral ventricle: Contains cerebrospinal fluid. Note characteristic bulge of caudate nucleus into the cavity of the lateral ventricle.

Corona radiata: White matter core of the cerebral hemispheres. Contains afferent and efferent fibers to and from the cerebral cortex.

 

SECTION THROUGH PUTAMEN
Parasagittal

Human, 10% formalin, Weigert's hematoxylin (Loyez), 1 x.

 

Centrum semiovale: White matter core of the cerebral hemispheres. Contains myelinated nerve fibers entering or leaving the cerebral hemispheres.

Putamen: One of the basal ganglia nuclei. Concerned with motor control. Note that at this most lateral parasagittal section, only the putamen of the basal ganglia nuclei is seen. In subsequent more medial sections, both the caudate nucleus and globus pallidus will also be visible.

Anterior commissure: A compact fiber bundle ventral to putamen. Interconnects olfactory bulbs and the temporal cortices.

Amygdala: From the Greek word amygdala, almond. The nuclei resemble almonds in shape and are located in the tip of the temporal lobe beneath the cortex of the uncus. Part of the limbic system and intimately connected with different components of the limbic system. Concerned with a variety of functions related to the limbic system such as emotional behavior, food intake, arousal, sexual activity, and a variety of motor activities.

Cerebellum: Located underneath the posterior part of the cerebral hemisphere.

Dentate gyrus: So-named because of its toothed or beaded surface appearance. A component of the hippocampal formation. Occupies the interval between the hippocampus and the parahippocampal gyrus.

Internal capsule: A component of the internal capsule, known as the retrolenticular part and characteristically located behind the putamen, is seen here. Note the different components of the internal capsule (anterior limb, posterior limb) in other sections. The retrolenticular component of the internal capsule contains the visual radiation, corticotectal, corticonigral, and corticotegmental fibers.

 

SECTION THROUGH LENTICULAR NUCLEUS
Parasagittal

Human, 10% formalin, Weigert's hematoxylin (Loyez), 1 x.

 

Centrum semiovale: White matter core of the cerebral hemispheres. Contains myelinated nerve fibers entering or leaving the cerebral hemispheres.

Putamen: One of the basal ganglia nuclei. Concerned with motor control. Note that this section also shows the globus pallidus, another component of the basal ganglia.

Globus pallidus: Another of the basal ganglia nuclei. Notice the difference in appearance from putamen. Globus pallidus is traversed by heavily myelinated fiber bundles. Note relationship to the anterior commissure.

Anterior commissure: A compact fiber bundle in close proximity to globus pallidus. Interconnects the olfactory bulbs and the temporal cortices.

Hippocampus: From the Greek word hippocampos, seahorse. The hippocampus is a major component of the limbic system. In man, the hippocampus is the largest component of the hippocampal formation. It plays an important role in memory function.

Cerebellum: Located ventral to the posterior part of the cerebral hemisphere.

Dentate gyrus: Another component of the hippocampal formation. So-named because of its toothed or beaded surface appearance. Occupies the interval between the hippocampus and the parahippocampal gyrus.

Lateral geniculate nucleus: One of the thalamic nuclei. Concerned with visual function. Receives fibers from the optic tract and projects into the primary visual cortex via the geniculocalcarine fiber pathway.

Pulvinar: One of the lateral group of thalamic nuclei. Has reciprocal connections with the medial and lateral geniculate bodies caudally and the association parietal, temporal, and occipital cortices rostrally. Involved in several neural functions, including vision, audition, speech, and pain.

 

SECTION THROUGH LENTICULAR NUCLEUS
AND LATERAL GENICULATE NUCLEUS
Parasagittal

Human, 10% formalin, Weigert's hematoxylin (Loyez), 1 x.

 

Internal capsule: A major fiber pathway carrying fibers from and to the cerebral cortex.

Putamen: One of the basal ganglia nuclei, concerned with motor control. Note relationship to globus pallidus.

Globus pallidus: Another of the basal ganglia nuclei. The two components of globus pallidus are shown in this section, an outer and an inner component. Note the difference in appearance of globus pallidus and putamen. Globus pallidus is characteristically traversed by heavily myelinated fiber bundles.

Anterior commissure: Compact fiber bundle inclose proximity to globus pallidus. interconnects the olfactory bulbs and the temporal cortices.

Optic tract: A bundle of heavily myelinated fibers conveying impulses from the retinae to the lateral geniculate nucleus as well as to the pretectal area.

Lateral geniculate nucleus: One of the thalamic nuclei. Concerned with visual functions. Receives fibers from the optic tract and projects to the primary visual cortex via the geniculocalcarine fiber pathway.

Pulvinar: One of the lateral group of thalamic nuclei. Has reciprocal connections with the medial and lateral geniculate bodies caudally and the association parietal, temporal, and occipital cortices rostrally. Involved in several neural functions, including vision, audition, speech, and pain.

Fornix (fimbria): Outflow tract from the hippocampus. When traced posteriorly on the floor of the inferior horn of the lateral ventricle, the fimbria continues as the crus of the fornix, which begins beneath the splenium of the corpus callosurn (see Plates 362, 363 and 364). The fornix connects the hippocampus with several brain regions, including the mamillary body, anterior thalamic nucleus, septal nuclei, and cingulate gyrus.  

 

SECTION THROUGH CORPUS STRIATUM
AND MEDIAL GENICULATE NUCLEUS
Parasagittal

Human, 10% formalin, Weigert's hematoxylin (Loyez), 1 x.

 

Ventral lateral (thalamic) nucleus: One of the lateral group of thalamic nuclei. Note the heavy bundle of myelinated fibers traversing the nucleus. Receives fibers from the cerebellum via the brachium conjunctivum. Has reciprocal connections with the primary motor cortex. Plays a role in motor control.

Caudate nucleus: One of the basal ganglia nuclei. Note its characteristic bulge into the cavity of the lateral ventricle. Concerned with motor control. This section shows in addition the putamen and globus pallidus.

Internal capsule (genu): The part of the internal capsule between the anterior and posterior limbs. Contains corticobulbar fibers, which terminate upon motor nuclei of the brain stem.

Internal capsule (anterior limb): Separates the caudate nucleus and putamen.

Globus pollidus: One of the basal ganglia nuclei. Characterized by heavily myelinated bundles of nerve fibers coursing through it. Note the difference in appearance between globus pallidus and both the caudate and putamen. Both components of the globus pallidus are shown in this section, the lateral (outer) and the medial (inner) segments. Concerned with motor control.

Putamen: Another of the basal ganglia nuclei. Similar in structure to caudate nucleus. Together with caudate and globus pallidus, it constitutes the corpus striatum.

Internal capsule (posterior limb): This part of the internal capsule separates the thalamus from the basal ganglia. The posterior limb of the internal capsule contains corticospinal, corticorubral, corticothalamic, and thalamocortical fibers.

Substantia innominata (nucleus basalis of Meynert): Located ventral to internal capsule and anterior commissure. The substantia innominata contains the nucleus basalis of Meynert, the neurons of which are rich in acetylcholine. Neurons in this nucleus project diffusely to the cerebral cortex and are believed to be involved in Alzheimer's disease. Alzheimer was a nineteenth-century German neurologist.

Optic tract: Carrying fibers from the retinae to the lateral geniculate nucleus and the pretectal area.

Cerebellum: Located ventral to the posterior part of the cerebral hemisphere.

Dentate nucleus: The largest of the deep cerebellar nuclei. Receives fibers from the Purkinje neurons in the hemispheres of the cerebellum and projects to the thalamus via the dentatorubrothalamic fiber system. Purkinje was a nineteenth-century Bohemian anatomist and physiologist.

Medial geniculate nucleus: One of the thalamic nuclei. Concerned with audition. Receives auditory fibers from the brain stem and projects to the primary auditory cortex in the temporal lobe.

Ventral posterior lateral (thalamic) nucleus: One of the lateral group of thalamic nuclei. Receives fibers of the medial lemniscus and spinothalamic tract. Reciprocally connected with the primary somesthetic cortex.

Fornix (fimbria): Axons of hippocampal neurons, continuous with the crus of the fornix. The fornix connects the hippocampus with several brain regions, including the mamillary body, anterior thalamic nucleus, septal nuclei, and cingulate gyrus.

Pulvinar: One of the lateral group of thalamic nuclei. Reciprocally connected with the medial and lateral geniculate bodies caudally and the association parietal, temporal, and occipital cortices rostrally. Involved in several neural functions, including vision, audition, speech, and pain.

Lateral posterior (thalamic) nucleus: One of the lateral group of thalamic nuclei. The borderline between this nucleus and the pulvinar is vague; the term pulvinar-lateral posterior complex has been used to refer to this nuclear complex.

 

SECTION THROUGH CEREBRAL PEDUNCLE
Parasagittal

Human, 10% formalin, Weigert's hematoxylin (Loyez), 1 x.

 

Ventral lateral (thalamic) nucleus: One of the lateral group of thalmic nuclei. Characteristically traversed by heavily myelinated fiber bundles. Receives fibers from the cerebellum and is reciprocally connected with the primary motor cortex. Concerned with motor control.

Internal capsule (posterior limb): Contains fibers destined for and leaving the cerebral cortex. This part of the internal capsule separates the thalamus from the basal ganglia.

Caudate nucleus: One of the basal ganglia nuclei. Concerned with motor function. Note the similarity in appearance between caudate and putamen.

Globus pallidus: Another of the basal ganglia nuclei. Characterized by heavily myelinated bundles traversing it. The two components of globus pallidus, the outer and inner segments, are seen in this section. Note proximity to anterior commissure.

Putamen: One of the basal ganglia nuclei. Concerned with motor function.

Anterior commissure: Compact fiber bundle located in close proximity to globus pallidus. interconnects the olfactory bulbs and the temporal cortices.

Optic tract: Myelinated fiber bundle conveying visual impulses from the contralateral field of vision to the lateral geniculate nucleus and the pretectal area.

Pons: Note the characteristic ventral bulge known as the basis pontis.

Cerebral peduncle: Located ventral to the substantia nigra. Contains descending corticofugal fibers, including the corticospinal, corticobulbar, and corticopontine fiber systems. Lesions result in weakness (paresis) or paralysis of the contralateral half of the body, including the face.

Dentate nucleus: One of the deep cerebellar nuclei. Receives fibers from Purkinje neurons in the hemispheres of the cerebellum and projects to the thalamus (ventral lateral nucleus) and red nucleus via the dentatorubro thalamic system.

Substantia nigra: A large nuclear mass in the midbrain. Contains pigmented cells (melanin). Connected with the basal ganglia and thalamus. Important in motor control. This area is invariably the site of pathological changes associated with Parkinson's disease.

Medial geniculate nucleus: One of the thalamic nuclei. Concerned with audition. Receives auditory fibers from the brain stem and is reciprocally connected with the primary auditory cortex.

Pulvinar: One of the lateral group of thalamic nuclei. Reciprocally connected with the medial and lateral geniculate bodies caudally and the association parietal, temporal, and occipital cortices rostrally. Involved in several neural functions, including vision, audition, speech, and pain.

Corpus callosum (splenium): The caudal part of the corpus callosum. Important in transfer of visual information between the two hemispheres.

Fornix (crus): Continuation of the same system seen in other sections. The fimbria of the fornix arising from the hippocampus continues as the crus. Note relationship to splenium of corpus callosum.

Ventral posterior lateral (thalamic) nucleus: One of the lateral group of thalamic nuclei. Receives the medial lemniscus and the spinothalamic tract and projects to the primary somesthetic cortex in the postcentral gyrus.

External medullary lamina: A bundle of myelinated fibers separating the medial from the lateral group of thalamic nuclei.

 

SECTION THROUGH CENTROMEDIAN THALAMIC NUCLEUS
Parasagittal

Human, 10% formalin, Weigert's hematoxylin (Loyez), 1 x.

 

Internal capsule (posterior limb): The part of the internal capsule separating the thalamus from the basal ganglia. Contains fibers destined for and leaving the cerebral cortex.

Caudatenucleus: One of the basal ganglia nuclei. Concerned with motor control. Note the characteristic bulge into the cavity of the lateral ventricle.

Anterior commissure: A compact fiber bundle connecting the olfactory bulbs and the temporal cortices.

Ventral posterior medial thalamic nucleus: One of the lateral group of thalamic nuclei. Receives the trigeminothalamic fiber system, including taste fibers, and projects to the primary somesthetic cortex in the postcentral gyrus.

Optic tract: Conveys impulses from the contralateral visual field to the lateral geniculate nucleus and the pretectal area.

Cerebral peduncle: A bundle of corticofugal fibers located inferior to the substantia nigra in the midbrain. Contains corticospinal, corticobulbar, and corticopontine fiber bundles. Lesions produce weakness or paralysis in the contralateral half of the body, including the face.

Oculomotor nerve: Seen leaving the ventral surface of the midbrain.

Subthalamic nucleus: Also known as corpus Luysii. Shaped like a biconcave lens. Receives fibers from and projects to the globus pallidus. Discrete lesions here result in an abnormal type of flinging movement known as ballism.

Basis pontis: Contains descending corticofugal fibers, pontine nuclei, and pontocerebellar fibers.

Restiform body: Also known as inferior cerebellar peduncle. A compact bundle of nerve fibers connecting the medulla with the cerebellum. Tracts and fibers forming this bundle originate in the medulla and the spinal cord.

Dentate nucleus: One of the deep cerebellar nuclei. Receives fibers from the Purkinje neurons of the cerebellar hemispheres and projects via the dentatorubrothalarnic system to the contralateral red nucleus and ventrolateral nucleus of the thalamus. Concerned with motor control.

Substantia nigra: The largest nuclear mass in the midbrain. Contains pigmented neurons (melanin). Connected with basal ganglia and thalamus. important in motor control. This area is invariably the site of pathological changes associated with Parkinson's disease.

Centromedian (thalamic) nucleus: Belongs to the intralaminar group of thalamic nuclei. Concerned with a variety of sensory and motor functions and arousal.

Pulvinar: One of the lateral group of thalamic nuclei. Has reciprocal connections with the medial and lateral geniculate bodies caudally and association parietal, temporal, and occipital cortices rostrally. involved in several neural functions, including vision, audition, speech, and pain.

Corpus callosum (splenium): The posterior part of the corpus callosurn. Concerned with transfer of visual information between the hemispheres.

Fornix (crus): Note the relationship of the crus of the fornix to the splenium of the corpus callosurn. The crus is a continuation of the fimbria of the fornix (see Plates 354, 355, 360, and 361). Rostrally, the crus continues as the body of the fornix. The fornix connects the hippocampus with several brain regions, including the mamillary body, anterior thalamic nucleus, septal nuclei, and cingulate gyrus.

Corpus callosum (body): The largest part of the corpus callosum. Between the splenium and the genu. Important in interhemispheric transfer of information.

Lateral dorsal (thalamic) nucleus: One of the dorsal subgroup of the lateral group of thalamic nuclei. An association thalamic nucleus.

Ventral posterior lateral (thalamic) nucleus: One of the lateral group of thalamic nuclei. Receives the medial lemniscus and the spinothalamic tract and is reciprocally connected with the somesthetic cortex of the postcentral gyrus.

Ventral lateral (thalamic) nucleus: Another of the lateral group of thalamic nuclei. Concerned with motor function. Characteristically traversed by heavily myelinated fiber bundles. Receives fibers from deep cerebellar nuclei and is reciprocally connected with the primary motor cortex.  

 

SECTION THROUGH MEDIAL THALAMUS
Midsagittal

Human, 10% formalin, Weigert's hematoxylin (Loyez), 1 x.

 

Mamillothalamic tract: A myelinated fiber bundle connecting the mamillary body with the anterior thalamic nucleus. Seen here approaching the anterior thalamic nucleus.

Corpus callosum (genu): The part of the corpus callosurn between the rostrum and the body. important in interhemispheric transfer of information.

Ventral anterior thalamic nucleus: One of the lateral group of thalamic nuclei. Concerned with motor function. Characteristically traversed by heavily myelinated fiber bundles. Receives fibers from the basal ganglia and projects to the motor cortex.

Corpus callosum (rostrum): The most rostral part of the corpus callosum. Concerned with interhemispheric transfer of information.

Anterior commissure: A compact bundle of myelinated fibers. Interconnects olfactory bulbs and the temporal cortices.

Optic chiasma: Site of partial crossing of optic nerve fibers before formation of the optic tracts. Lesions in the optic chiasma result in a characteristic visual field loss known as bitemporal hemianopia.

Cerebral peduncle: A bundle of corticofugal fibers located ventral to the substantia nigra in the midbrain. Contains corticospinal, corticobulbar, and corticopontine fibers. Lesions result in contralateral weakness or paralysis, including the face.

Substantia nigra: A large nuclear mass in the ventral part of the-midbrain. Located dorsal to the cerebral peduncles. Melanin-containing neurons in this nucleus are connected with the basal ganglia and the thalamus. The substantia nigra is an important structure in the genesis of movement. Invariably the site of pathology in Parkinson's disease.

Inferior Olive: Located in ventral part of the medulla oblongata. Receives cortical and subcortical fibers and projects to the cerebellum via the restiform body.

Brachium conjunctivum: Also known as superior cerebellar peduncle. Contains axons of deep cerebellar nuclei destined for the red nucleus and thalamus.

Inferior colliculus: An elevation on the dorsal surface of the midbrain. Contains neurons related to the auditory system.

Superior colliculus: An elevation in the dorsal surface of the midbrain. Related to the visual system.

Centromedian (thalamic) nucleus: Belongs to the intralaminar group of thalamic nuclei. Related to a variety of sensory and motor functions as well as arousal.

Corpus callosum (splenium): The most caudal part of the corpus callosum. Concerned with transfer of visual information between the hemispheres.

Pulvinar: One of the lateral group of thalamic nuclei. Reciprocally connected with the medial and lateral geniculate nuclei caudally and the association parietal, temporal, and occipital cortices rostrally. Plays a role in several neural functions, including vision, audition, speech, and pain.

Fornix (crus): Continuation of the fimbria of the fornix. Continues rostrally as the body of the fornix. The fornix connects the hippocampus with several brain regions, including the mamillary body, anterior thalamic nucleus, septal nuclei, and cingulate gyrus.

Dorsomedial (thalamic) nucleus: One of the medial group of thalamic nuclei. Most highly developed in man. Has reciprocal connections with the prefrontal cortex and hypothalamus. Receives input from other thalamic nuclei. Concerned with affective behavior and memory.

Anterior (thalamic) nucleus: Belongs to the anterior group of thalamic nuclei. Has reciprocal connections with the mamillary body via the mamillothalarnic tract and with the cingulate gyrus via the internal capsule. Considered part of the limbic system and plays a role in emotional behavior and memory.

Corpus callosum (body): The largest part of the corpus callosum. Between the genu and the splenium. Concerned with interhemispheric transfer of information.

Section 17: Central Nervous System

Knowledge of the structural organization of the nervous system is essential to the proper understanding of its normal, as well as altered, function.

For didactic purposes, the nervous system is generally divided into central and peripheral components. The central nervous system includes the brain and the spinal cord. The peripheral nervous system includes cranial and peripheral nerves and associated ganglia. The autonomic nervous system includes parts of the central and peripheral nervous systems.

The brain includes the cerebral hemispheres, the cerebellum, and the brain stem. The latter includes the diencephalon; mesencephalon, or midbrain; pons; and medulla oblongata. Each of these components is made up of cell groups and fibers, arranged in a manner that characterizes the particular component. Bundles of nerve fibers serving a common function and sharing a common origin and destination are grouped together in tracts or fasciculi. A group of neurons serving a common function forms a nucleus.

Knowledge of the existence and location of tracts has been gained through years of clinical observation and experimentation in both animals and man. Some of the methods used in the tracing of neural pathways follow:

1. Study of normal preparations: Many aspects of fiber connectivity of the nervous system have been elucidated by early studies using normal material and methods that demonstrate myelin sheaths (Weigert and Weil methods) or that impregnate cell bodies and their processes (Golgi method). The disadvantage of these methods is the difficulty of determining the site of termination of these fibers.
   
2. Myelinogenesis: This method, introduced by Flechsig, makes use of the fact that different fiber tracts become myelinated at different times in their development. Thus, study of the nervous system in embryos and in early neonatal life often affords information about the existence and locality of the different fiber tracts. This method is infrequently used today.
   
3. Study of pathological conditions in man and experimental lesions in animals: This method accounts for most of our current knowledge of neural connectivity. Although human material has been of use, experimentally produced lesions in animals have the major advantage of selectivity of site and size. Caution should be exercised, however, in applying to humans results achieved in experimental animals.
   
   After a lesion has been produced in animals or man and sufficient time has elapsed for anterograde degeneration to set in, the brains and spinal cords can be studied, and degenerated tracts can be localized by one of the following three methods:
   1. Methods that stain normal myelin (Weigert, Weil): In such preparations, normal myelin appears dark blue or black, and the degenerated tracts will be conspicuous by their failure to pick up the stain.
   2. Methods that stain degenerating myelin (Marchi): In such preparations, only degenerating myelinated tracts pick up the stain and can be followed from origin to termination. Normal myelinated tracts remain unstained. A major advantage of the Marchi method is that positive results may be obtained years after degeneration has occurred, making it particularly useful in the study of human material postmortem. One disadvantage of this method is that thinly myelinated or unmyelinated tracts will not stain. Another disadvantage is that it does not stain degenerating terminals; hence, the exact site of termination of a tract cannot be determined with certainty.
   3. Methods that stain degenerating axons (Nauta-Gygax, Fink-Heimer, De Olmos): These are silver impregnation techniques that stain degenerating axons and pre-terminals (Nauta-Gygax) or terminals (Fink-Heimer, De Olmos). These methods have a distinct advantage over myelin methods, since they are capable of revealing poorly myelinated as well as unmyelinated nerve fibers, because the axon, and not the myelin, is stained by these methods.
      
      A neuroanatomist is interested not only in the location and course of fiber tracts but also in their site of termination. To determine the latter, methods that stain the terminal boutons (Glees, Bodian) are used. Electron microscopy can also be used for this purpose.
      
4. Retrograde cell changes: By this method, the position of neurons giving rise to the tract is determined. Such neurons undergo chromatolytic changes of their Nissl substance or disappear completely (retrograde degeneration) if their axon is severed. These changes can be demonstrated by any of the methods that stain ribonucleic acids (Nissl material), the Nissl stains.
   
5. Autoradiography: This is a relatively recent pathway tracing technique used in brain research. it utilizes the principle that radioactive amino acids injected in the vicinity of neuronal perikarya will be taken up by the neuron, incorporated into its macromolecules, and transported anterograde along the axon to its terminal. After a finite time following injection, the radioactive amino acid can be demonstrated by autoradiography. By this method, the path of a neural tract can be traced from its origin to its termination.
   
6. Enzymatic method: When the enzyme horseradish peroxidase (HRP) is injected at the site of termination of nerve fibers, it is taken up by the nerve terminals and transported retrograde to the perikaryon where it is visualized by an enzyme histochernical technique as brown granules in the soma and dendrites.
   
7. Fluorescence method: This method, introduced in the early 1960s, is used to trace the fiber pathways of adrenergic and monaminergic neural systems. It relies on the observation that primary amines form fluorescent condensation products when treated with formaldehyde in the presence of protein. Fluorescent condensation products are demonstrated in cells, axons, and terminals by fluorescence microscopy.
   
8. Physiological exploration: By this method, stimulation and recording techniques are used to establish the presence or absence of structural and/or functional relationships between two or more loci in the nervous system. The stimulation and recording of evoked potentials may be orthodromic (recording of activity in the terminal projection site of a fiber system) or antidromic (recording of activity in the cells of origin when their axon terminals or axons are stimulated). Gross stimulation and recording techniques reflect the relationship between groups of neurons; intracellular recordings reflect the relationship between pairs of neurons.

These methods used to study neural connectivity are based on the principle of the neuron as a trophic unit. If an axon is transected, its peripheral parts, including its termination, undergo degeneration. This is referred to as anterograde degeneration. The methods described in 3 above are used to show this type of degeneration. Simultaneously with anterograde degeneration, changes occur in the proximal components of the neuron, namely in the proximal axon, the cell body, and dendrites. These changes are known as retrograde changes.

When considered together, anterograde and retrograde methods allow a detailed mapping of neural connectivity.

 

SPINAL CORD
Cervical region

Human, Müller's fluid, carmine stain, 8 x.

 

Dorsal root: The dorsal root carries both myelinated and unmyelinated afferent fibers to the spinal cord. Each fiber is the central process of a dorsal root ganglion cell.

Posterior gray column: Long and narrow column of gray matter reaches almost to the surface of the spinal cord. Primarily concerned with sensory input.

Anterior gray column: Short and broad column of gray matter. Concerned with motor function. Both posterior and anterior gray columns are sites where sensory and motor cell bodies, respectively, are found.

Ventral root: Bundle of somatic motor fibers (axons of somatic motor neurons) and preganglionic fibers (axons of autonomic motor neurons). Constitute the efferent outflow of the spinal cord.

Anterior median fissure: About 3 mm deep. Contains blood vessels (anterior spinal artery) supplying the anterior two thirds of the cord.

Anterior lateral sulcus: Site of exit of ventral root. Hardly distinguishable in this preparation.

Anterior funiculus: Between the anterior median fissure and anterolateral sulcus (ventral root). Merges with the lateral funiculus. Contains ascending and descending tracts.

Lateral funiculus: Between the dorsal and ventral roots. Merges with the anterior funiculus. Contains ascending and descending tracts.

Posterior lateral sulcus: Site of entry of dorsal root.

Posterior funiculus: Between posterior median sulcus and dorsal root. Contains ascending tracts. Posterior intermediate septum: Found only in cervical and upper thoracic segments. Posterior median sullcus: About 5 mm deep, reaches the deep-lying gray matter.  

 

SPINAL CORD
Cervical level

Human, 10% formalin, Weigert-carmine, 11 x.

 

Posterior column: The white matter located between the posterior median septum and the medial border of the posterior horn. Contains heavily myelinated nerve fibers that form the ascending gracile and cuneate tracts. These tracts carry impulses from proprioceptive and tactile receptors (Pacinian corpuscles, Golgi tendon organs, neuromuscular spindles, and Meissner's corpuscles), which give rise to sensations of position, movement, and tactile localization.

Substantia gelatinosa: Appears as a cap-like structure at the head of the posterior horn. It is largest in the first cervical and in the lumbosacral segments but extends the whole length of the cord. This nucleus contains small cells about 6 to 20 µm in diameter and is the primary associative center of the posterior horn for incoming impulses carried by the dorsal root. This nucleus is an important part of the pathway for pain, temperature, and some tactile impulses.

Dorsal root: Sensory nerve fibers entering the spinal cord. Follow some of these fibers to the anterior horn where they terminate on motor neurons.

Spinocerebellar tracts: The spinocerebellar tracts convey impulses to the cerebellum from muscle, tendon, and joint proprioceptors, thus enabling the cerebellum to coordinate skeletal muscle activity (posture and movement).

Reticular process: Contains small and medium-sized cells, which send their axons to the adjacent as well as to the opposite anterolateral white column. Also known as the nucleus reticularis.

Anterior gray horn: Contains cells whose axons pass to the extrafusal fibers of striated skeletal muscle. The cells are the largest found in the spinal cord. These multipolar neurons have as many as 20 dendrites, and axons approximately 12 µm in diameter. Smaller neurons with thin axons (gamma efferent fibers) supply the intrafusal muscle fibers of the neuromuscular spindle  

SPINAL CORD
Thoracic region

Human, Müller's fluid, Weigert's method, 10 x.

 

Dorsal root fibers: Central processes of dorsal root ganglia.

Reticular process: Characteristic of cervical levels of the spinal cord but also located at other spinal levels. Located between the posterior and anterior horns and produced by an extension of gray matter into the adjacent white substance. Constitutes the lateral zone of lamina V of Rexed. 

Nucleus dorsallis: Distinct nuclear mass located in the medial part of the base of the posterior horn. In this nucleus, dorsal root fibers synapse with neurons destined to form the dorsal (posterior) spinocerebellar tract. The nucleus extends between C₈ and L₂ spinal segments. Also known as the column of Clarke*.

Ventral root fibers.

Posterior funiculus: The white matter of the cord located between the posterior central (median) septum and the medial border of the posterior horn. Contains heavily myelinated fibers that form the gracile and cuneate tracts. Note the large size of this funiculus at this level compared to lower levels of the spinal cord. .

Posterior gray horn: A mass of neurons in the posterolateral part of the spinal cord. Receive collaterals or terminals of dorsal root fibers. Sends axons to anterior horn cells, interneurons, or to ascending tracts.

Lateral gray horn: Characteristic of thoracic level, this projection is formed by the intermediolateral nucleus. Contains visceral efferent neurons of the sympathetic nervous system. Extends from C₈ to L_2-4. Axons of neurons here exit with the ventral horn fibers to terminate in the chain of ganglia (sympathetic), where they synapse with ganglion cells whose axons are widely distributed to the iris (dilator smooth muscle); lacrimal, salivary and sweat glands; bronchi; heart; smooth muscle of the gastrointestinal tract; sex organs; urinary bladder; adrenal medulla; and blood vessels.

Anterior gray horn: A mass of large neurons in the anterolateral part of the spinal cord. Contains somatic efferent neurons. Compare the size of the horn at this level with those seen at higher and lower levels.

 

SPINAL CORD LESION
Thoracic level dorsal funiculus

Human, 10% formalin, Weigert-carmine, 9.4 x.

 

Gracile tract: Note that the gracile tract stains lighter than the adjacent cuneate tract. See Plates 317 and 322. This is due to the degenerating myelinated fibers in the gracile tract, which are not stained by this method. This slide was taken from a human with a spinal cord lesion in the dorsal (posterior) funiculus below the sixth thoracic segment. Fibers entering the spinal cord below this level form the gracile tract. The fibers entering above T₆ form the cuneate tract and therefore escape degeneration.

Substantia gelatinosa: Contains 6 to 20 µm diameter neurons. Note small size compared with that of cervical cord. Concerned with sensory relay and integration.

Intermediolateral horn: Lateral gray horn. Characteristic of dorsal (thoracic) and upper lumbar segments. Contains sympathetic neurons.

Nerve roots: Incoming dorsal roots.

 

SPINAL CORD
Primary lateral sclerosis

Human, 10% formalin, Weil, 8 x.

 

This is a section of the spinal cord stained with Weil's method. This method stains normal myelinated fiber tracts black. The lightly stained lateral corticospinal tract stands out in contrast to the darkly stained normal myelin elsewhere in the spinal cord. This selective loss of myelinated fibers in the lateral corticospinal tract is characteristic of a spinal cord disorder known as primary lateral sclerosis. Patients afflicted with this disorder show all the signs of upper motor neuron lesion, including weakness, spasticity, hyperreflexia, Babinski* reflex, and clonus.

 

SPINAL CORD
Lumbar region

Human, 10% formalin, Well, 8 x.

 

Dorsal roots: Central processes of dorsal root ganglion cells. Convey afferent (sensory) impulses to the spinal cord from peripheral receptor organs..

Substantia gelatinosa: Cap-like structure at the head of the posterior horn. Extends the whole length of the cord. Contains small neurons about 6 to 20 µm in diameter and is the primary associative center of the posterior horn for incoming impulses carried by the dorsal root. This nuclear mass is an important part of the pathway for pain, temperature, and some tactile impulses.

Dorsal root fiber collaterals: Heavily myelinated dorsal root fiber collaterals that enter the spinal cord to modify pain transmission or establish segmental reflexes.

Anterior funiculus: Between the anterior median fissure and anterolateral sulcus (ventral roots). Merges with the lateral funiculus. Contains several ascending and descending tracts.

Ventral roots: Bundles of somatic motor fibers (axons of somatic motor neurons in the anterior horn). Constitute the efferent outflow of the spinal cord.

Anterior gray horn: A mass of large multipolar motor neurons and interneurons. Axons of motor neurons form the ventral roots. Compare size of anterior gray horn at this level with those seen at higher and lower levels.

Lateral funiculus: Between the dorsal and ventral roots. Merges with the anterior funiculus. Contains major ascending and descending fiber tracts, including the lateral corticospinal, spinothalamic, and spinocerebellar tracts.

Posterior funiculus (fasciculus gracilis): The posterior funiculus at this level contains the fasciculus gracilis only, whereas at higher levels (above the sixth thoracic spinal segment), it contains the fasciculi gracilis and cuneatus. Compare size of the posterior funiculus at this level with that at levels below and above

 

SPINAL CORD
Sacral region

Human, Müller's fluid, Weigert's method with carmalum stain, 14 x.

 

Dorsal root fibers: Bundles of heavily myelinated nerve fibers entering the spinal cord. Represent central processes of dorsal root ganglion neurons. Convey afferent impulses from peripheral organs to the spinal cord. Some of these fibers go directly to form tracts (fasciculus gracilis), others give collaterals or terminate on neurons in the spinal cord.

Zone of Lissauer*: Also known as fasciculus dorsolateralis. Composed of fine myelinated and non- myelinated fibers that carry pain, thermal, and light touch impulses or that interconnect different levels of the substantia gelatinosa.

Ventral root fibers: Axons of somatic, fusimotor, and visceral motor neurons in the anterior (ventral) and lateral gray columns. Heavily myelinated.

Fasciculus gracilis: Heavily myelinated ascending fiber system. Conveys kinesthetic sense and discriminative touch. Note the absence of the fasciculus cuneatus, which appears at spinal cord levels above T₆.

Substantia gelatinosa: An expanded cell mass that forms the cap of the posterior gray horn of the spinal cord. Its size is related to that of the dorsal root. This area functions as an association region for incoming impulses. This region corresponds to lamina II of Rexed.

Central canal: Runs throughout the length of the cord. Partially obliterated in the adult.

Ventral white commissure: Bundle of myelinated fibers crossing from one side of the spinal cord to the other.

 

SPINAL CORD
Dorsal root collaterals coronal

Dog, Müller's fluid, Weigert's method, 3.75 x.

 

This is a section of the spinal cord showing the distribution of dorsal root collaterals. Note that the coarser, heavily myelinated collaterals are medially located. They are seen passing to the posterior horn, intermediate gray, and anterior horn. Many fibers of this bundle enter the posterior funiculus. Finer, poorly myelinated collaterals are more laterally located and are seen entering the substantia gelatinosa. Some heavily myelinated fibers also enter the substantia gelatinosa. The medial bundle is the larger of the two. Collaterals that pass directly to the anterior horn constitute components of monosynaptic reflex arc. Their number is relatively small since most of the collaterals to the anterior horn terminate on at least one interneuron before reaching the final efferent neuron. Note the bundle of motor fibers (axons of motor neurons) leaving the anterior horn to form the ventral roots.

 

MEDULLA OBLONGATA
Motor decussation coronal

Human, 10% formalin, Pal-Weigert and carmine stains, 11 x.

 

Fasciculus gracilis: Rostral continuation of the same tract seen at several spinal cord levels The lightly stained islands within the fasciculus represent neurons of the nucleus gracilis. This nucleus is larger at more rostral levels.

Fasciculus cuneatus: Rostral continuation of the same tract seen at spinal cord levels .

Spinal nucleus of nerve V: Functionally analogous to and structurally a continuation of the substantia gelatinosa seen at several spinal cord levels. In it terminate fibers of the descending (spinal) tract of cranial nerve V (trigeminal), which enters the neuraxis at a rostral level. The nucleus is primarily concerned with the perception of pain and thermal sense from the homolateral face.

Spinal tract of nerve V: Thinly myelinated fibers, hence less densely stained than the heavily myelinated fibers of the fasciculi gracilis and cuneatus or the spinocerebellar tracts. This tract is composed of descending trigeminal fibers and extends from the site of entry of the trigeminal nerve in the pons down to at least the second cervical spinal segment. Primarily concerned with pain and thermal sense. Synapse in the spinal nucleus of nerve V.

Spinocerebellar tracts: Heavily myelinated. Continuation of the same tracts seen at several spinal cord levels on their way to the cerebellum.

Motor decussation: Constitutes one of the most conspicuous features of sections at this level. Site of crossing of the pyramids to form the lateral corticospinal tracts. Approximately 75 to 90 per cent of descending pyramidal fibers cross at this level. The motor decussation forms the basis for voluntary motor control of one half of the body by the contralateral cerebral hemisphere.

Lateral corticospinal tract: Formed by decussation of the pyramidal tracts. Descends throughout the extent of the spinal cord .

Pyramid: Heavily myelinated motor fiber system. Represents descending fibers from the cerebral cortex that pass through the internal capsule, cerebral peduncle, and pons before reaching the medullary pyramids. Fibers in the pyramid undergo partial crossing in the motor decussation to give rise to the lateral corticospinal tracts. It is estimated that, in man, about one million fibers are present in each pyramid.  

 

MEDULLA OBLONGATA

Cat, 10% formalin, Marchi stain, 11.5 x.

 

This is a section of the medulla oblongata, stained by the Marchi* method, which reveals degenerated myelin. Note the degenerated fibers (black) in the pyramid and the crossing of these fibers to form the lateral corticospinal tract. An artifact of tissue preparation is seen in the upper right hand corner of the section. The tissue became folded during handling.

 

MEDULLA OBLONGATA
Motor (pyramidal) decussation coronal

Human, 10% formalin, Pal-Weigert, 7.5 x.

 

Nucleus gracilis: One of the dorsal column nuclei. Receives ascending fibers in the dorsal column (fasciculus gracilis) of the spinal cord entering below the level of the sixth thoracic spinal segment. Axons of neurons in this nucleus form the internal arcuate fiber system, cross in the midline (at more rostral levels), and form the medial lemniscus.

Fasciculus gracilis: Dorsal (posterior) column fibers capping the nucleus gracilis. Size is inversely proportional to that of nucleus gracilis, becoming smaller as more fibers terminate in the nucleus.

Nucleus cuneatus: One of the dorsal column nuclei. Located lateral to nucleus gracilis. Receives ascending fibers in the dorsal column of the spinal cord entering above the sixth thoracic spinal segment (fasciculus cuneatus). Axons of neurons in this nucleus, along with axons of the gracile neurons form the internal arcuate fiber system, which crosses in the midline to form the medial lemniscus.

Fasciculus cuneatus: Dorsal (posterior) column fibers capping the nucleus cuneatus. Size is inversely proportional to that of nucleus, becoming smaller as more fibers terminate in nucleus.

Spinal tract of trigerninal (CN V) nerve: Thinly myelinated fibers, hence less densely stained than the heavily myelinated fibers of the spinocerebellar tract. This tract is composed of descending trigeminal fibers and extends from the site of entry of the trigerninal nerve in the pons down to at least the second cervical spinal segment. Synapse in the nucleus of spinal tract of trigerninal nerve. Primarily concerned with pain and thermal sense from the homolateral face.

Nucleus of spinal tract of trigerninal (CN V) nerve: Functionally analogous to and structurally a continuation of the substantia gelatinosa seen at several spinal cord levels. In it terminate fibers of the descending (spinal) tract of cranial nerve V (trigeminal), which enters the neuraxis at a rostral level. The nucleus is primarily concerned with pain and thermal sense from the homolateral face.

Motor (pyramidal) decussation: Constitutes one of the most conspicuous features of sections at this level. Site of crossing of the pyramids to form the lateral corticospinal tracts. Approximately 75 to 90 percent of descending pyramidal fibers cross at this level. The motor decussation forms the basis for voluntary motor control of one half of the body by the contralateral cerebral hemisphere.

Pyramid: Corticospinal fibers prior to decussation.

Medial longitudinal fasciculus: Descending portion of a fiber system with ascending and descending components. Neurons of origin are from various brain stem nuclei with a major vestibular system component. Concerned with movement of neck and head in response to vestibular stimulation.

Spinocerebellar tract: Heavily myelinated. Continuation of the same tracts seen at several spinal cord levels. Destination is the cerebellum

 

MEDULLA OBLONGATA
Spinocerebellar tract coronal

Dog, Müller's fluid, Marchi's method, 12 x.

 

This cross section of the lower medulla is stained with the Marchi method, which selectively stains degenerated myelinated tracts black. In this section, black staining outlines the dorsal and ventral spinocerebellar tracts. These are fiber tracts that convey proprioceptive impulses to the cerebellum. They are concerned with unconscious proprioception. Three types of end organs are associated with proprioception: Pacinian corpuscles, muscle spindles, and Golgi tendon organs These receptors detect movements of joints and changes in stretch and tension in muscles and tendons

 

MEDULLA OBLONGATA
Sensory decussation
coronal

Human, 10% formalin, Pal-Weigert and carmine stains, 7 x.

 

Fasciculus and nucleus gracilis: Note the reduction in size of the fasciculus gracilis as the nucleus gracilis develops. Fibers of the fasciculus synapse on neurons of the nucleus gracilis. The nucleus gracilis appears caudal to the appearance of and terminates caudal to the termination of nucleus cuneatus .

Fasciculus and nucleus cuneatus: Note that the nucleus cuneatus is not as well developed at this level as the nucleus gracilis. The fasciculus cuneatus is voluminous.

Internal arcuate fibers: Second-order fibers arise from gracile and cuneate nuclei, course in the tegmentum of the medulla, and cross in the sensory decussation to form the medial lemniscus. They convey the same modalities of sensation as the gracile and cuneate tracts (proprioception, touch, and vibratory sense).

Medial longitudinal fasciculus: Descending portion of a fiber system with ascending and descending components. Arises from various brain stem nuclei, but with a major vestibular component. This system is concerned with eye and neck movements. The fibers in it are destined to synapse on the motor neurons in the cervical region supplying neck musculature.

Spinal nucleus of V: Continuation of same nucleus seen at more caudal levels .

Spinocerebellar tract: Continuation of the sme tract seen at several caudal levels

Sensory decussation: Also known as decussation of the medial lemniscus. Internal arcuate fibers cross here to form the contralateral medial lemniscus. Provides an anatomical basis for sensory representation of one half of the body in the contralateral cerebral cortex.

Pyramid:  Descending corticospinal fibers. Lesion will result in contralateral weakness or paralysis of the upper motor neuron variety.

 

MEDULLA OBLONGATA
Sensory (lemniscal) decussation
coronal

Human, 10% formalin, Pal-Weigert, 5.6 x.

 

Nucleus gracilis: Fully developed at this level. Only a small remnant of the fasciculus gracilis caps the nucleus.

Nucleus and fasciculus cuneatus: Note that a definite portion of the fasciculus cuneatus caps the nucleus cuneatus compared with that seen in the adjacent nucleus gracilis. The lightly stained island in the fasciculus cuneatus represents neurons of the accessory (lateral) cuneate nucleus.

Spinal tract and nucleus of trigerninal nerve: Continuation of similar structures seen at more caudal levels.

Medial lemniscus: Formed by the decussating internal arcuate fibers. Constitutes the second-order neurons of the posterior column pathways (fasciculi gracilis and cuneatus and their nuclei), conveying kinesthetic sense and discriminative touch to higher levels of the neuraxis.

Pyramid: Corticospinal fibers descending to decussate at more caudal levels.

Sensory (lemniscal) decussation: Also known as decussation of the medial lemniscus. Internal arcuate fibers cross here to form the contralateral medial lemniscus. Provides an anatomical basis for sensory representation of one half of the body in the contralateral cerebral cortex.

Internal arcuate fibers: Second-order fibers arise from gracile and cuneate nuclei, course in the tegmenturn of the medulla, and cross in the sensory decussation to form the medial lemniscus. They convey the same modalities of sensation as the gracile and cuneate tracts (proprioception, touch, and vibratory sense).

Hypoglossal (CN XII) nucleus: A group of large neurons located in a paramedian position dorsal to the medial lemniscus

 

MEDULLA OBLONGATA
Inferior olive
coronal

Human, 10% formalin, Pal-Weigert and carmine stains, 7 x.

 

Nucleus gracilis: Fully developed at this level. Only a small remnant of the fasciculus gracilis caps the nucleus.

Cuneate nucleus and tract: Note that a definite portion of the fasciculus cuneatus caps the nucleus cuneatus as compared to that seen on the adjacent nucleus gracilis. The lightly stained island in the fasciculus cuneatus represents neurons of the accessory cuneate nucleus.

Spinal tract and nucleus of nerve V: Continuation of similar structures seen at more caudal levels .

Medial longitudinal fasciculus: Note the change of position of the fasciculus in this figure as compared to a more caudal level .This is a result of the formation of the medial lemniscus, which displaces the medial longitudinal fasciculus to a more dorsal location.

Principal and medial accessory inferior olive: This nuclear group distinguishes sections of the medulla at this level. The principal olive is the larger component with its hilum directed medially. The medial accessory olive is found along the border of the medial lemniscus. Inferior olive neurons give rise to olivocerebellar fibers that project into the cerebellum.

Pyramid: See the same structure at more caudal levels.

Internal arcuate fibers: Axons of gracile and cuneate neurons.

Spinocerebellar tract: Continuation of the same tract seen in the spinal cord.

Medial lemniscus: Formed by the decussating internal arcuate fibers. Constitutes the second-order neurons of the posterior column pathways (fasciculi gracilis and cuneatus and their nuclei), conveying kinesthetic sense and discriminative touch to higher levels of the neuraxis.

Nerve XII rootlets: Hypoglossal cranial nerve. Note their characteristic location medial to the inferior olive and lateral to the pyramid. This proximity to the pyramid is the anatomical basis for the inferior or hypoglossal alternating hemiplegia resulting from lesions in this area. This syndrome (also known as medial medullary syndrome) consists of lower motor neuron paralysis of the ipsilateral half of the tongue and contralateral (upper motor neuron) hemiplegia. The hypoglossal nerve supplies all the intrinsic and extrinsic muscles of the tongue except the palatoglossus muscle.  

 

MEDULLA OBLONGATA
Inferior olive
coronal

 

Choroid plexus: Located in the caudal part of the roof of the fourth ventricle.

Inferior vestibular nucleus: One of four vestibular nuclei. Characteristically located medial and dorsal to the restiform body, and traversed by myelinated bundles.

Tractus solitarius: Contains general visceral as well as special visceral (taste) fibers from the vagus, glossopharyngeal, and facial nerves. Fibers project onto neurons in the nucleus solitarius located in close proximity to the tract.

Nucleus solitarius: Located in close proximity to the tractus solitarius, from which it receives fibers.

Hypoglossal (CN XII) nucleus: A group of large neurons located dorsal to the medial longitudinal fasciculus in the floor of the fourth ventricle in a paramedian position. Rootlets of hypoglossal nerve course in tegmentum of medulla between the medial lemniscus and inferior olive.

Hypoglossal (CN XII) rootlets: Coursing in the tegmentum of the medulla oblongata between the medial lemniscus and the inferior olive. Exit from the ventral surface of the medulla between the pyramid and inferior olive.

Dorsal accessory olive: A component of the inferior olivary complex located dorsal to the principal olive.

Medial longitudinal fasciculus: Descending portion of a fiber system with ascending and descending components. Neurons of origin are from various brain stem nuclei, but with a major vestibular component. The fibers descending in this fasciculus are destined to synapse on motor neurons in the cervical region of the spinal cord, which supply neck musculature.

Accessory cuneate nucleus: Receives fibers of the dorsal spinocerebellar tract entering the spinal cord above the eighth cervical segment. Projects to the cerebellum via the restiform body.

Restiform body: Also known as the inferior cerebellar peduncle. A compact bundle of nerve fibers connecting the medulla with the cerebellum. Described first in 1695 and named by Humphrey Ridley, an English anatomist. Tracts and fibers forming this bundle originate in the medulla and the spinal cord.

Nucleus of spinal tract of trigeminal (CN V) nerve: Receives exteroceptive fibers from the ipsilateral side of the face via the spinal tract of the trigeminal nerve. Lesions result in loss of pain sensation in the ipsilateral face.

Medial lemniscus: Axons of gracile and cuneate nuclei. Forward continuation of the same structure seen in more caudal sections.

Principal inferior olive: Located dorsal and lateral to the pyramid. Note the characteristic convoluted appearance. The principal inferior olive is the largest component of the inferior olivary complex, which includes, in addition, the dorsal accessory inferior olive and the medial accessory inferior olive.

Pyramid: Heavily myelinated motor fiber system. Represents descending fibers from the cerebral cortex that pass through the internal capsule, cerebral peduncle, and pons before reaching the medullary pyramids. Fibers in the pyramid undergo partial crossing in the motor decussation to give rise to the lateral corticospinal tract. It is estimated that, in man, about one million fibers are present in each pyramid.  

 

MEDULLA OBLONGATA
Cochleovestibular and
Glossopharyngeal nerves
coronal

Human, 10% formalin, Pal-Weigert, 3.8 x.

 

Medial longitudinal fasciculus: Descending portion of a fiber system with ascending and descending components. Neurons of origin are from various brain stem nuclei, but with a major vestibular component. This system is concerned with eye and neck movements. The fibers in the descending component are destined to synapse on motor neurons in the cervical spinal cord that supply neck musculature.

Medial lemniscus: Continuation of the same system noted in caudal sections.

Glossopharyngeal (CN IX) nerve: A mixed nerve. Characteristically enters the medulla, inferior and medial to the restiform body.

Amiculurn olivae: A bundle of fibers surrounding the inferior olivary complex. Contains fibers that terminate on neurons of the olivary complex.

Inferior olive: Convoluted laminae of gray matter dorsal to the pyramid. Receives fibers from cortical and subcortical sites and projects fibers primarily to the contralateral but also to the ipsilateral cerebellum via the restiform body. Concerned with motor control.

Pyramid: Heavily myelinated motor fiber system. Contains descending fibers from the cerebral cortex that pass through the internal capsule, cerebral peduncle, and pons before reaching the pyramids. Fibers in the pyramid undergo partial crossing in the motor decussation caudal to this level.

Arcuate nucleus: Motor neurons ventral to the pyramid. Receives cortical input and projects to the cerebellum via the stria medullaris and restiform body. Homologous to pontine nuclei.

Olivocerebellar tract: Axons of neurons in the inferior olivary complex. Fibers arise from both olivary complexes but primarily from the contralateral complex. Destined for the cerebellum via inferior cerebellar peduncle (restiform body). Olivocerebellar fibers constitute the major component of the restiform body.

Cochlear (CN VIII) nerve: Central processes of bipolar neurons in the spiral ganglion. Enters the lateral surface of the pons lateral and dorsal to the restiform body. Projects upon the dorsal and ventral cochlear nuclei. Lesions in the cochlear nerve result in ipsilateral loss of hearing.

Ventral cochlear nucleus: Located ventral and lateral to the restiform body. Receives axons of the cochlear nerve originating in the upper turns of the cochlea.

Restiform body: Also known as inferior cerebellar peduncle. A compact bundle of nerve fibers connecting the medulla with the cerebellum. Tracts and fibers forming this bundle originate in the medulla and the spinal cord.

Dorsal cochlear nucleus: Characteristically located dorsal and lateral to the restiform body. Receives axons of the cochlear nerve originating in the lower turns of the cochlea.

Inferior vestibular nucleus: One of four vestibular nuclei. Characteristically located medial to the restiform body and traversed by myelinated bundles.

Stria medullaris: Axons of arcuate neurons. Courses in the floor of the fourth ventricle. Joins the restiform body to reach the cerebellum.

Medial vestibular nucleus: One of four vestibular nuclei. Characteristically located medial to the inferior vestibular nucleus in the floor of the fourth ventricle. Axons of neurons in this nucleus form the medial vestibulospinal tract.  

 

PONS
Trapezoid body coronal

Human, 10% formalin, Pal-Weigert, 2.5 x.

 

Vermis of the cerebellum: Overlying the fourth ventricle. Midline portion of cerebellum.

Medial longitudinal fasciculus: Continuation of the same structure seen at more caudal levels. Concerned with ocular movement in response to vestibular stimulation.

Central tegmental tract: Compact fiber bundle located dorsal to the medial lemniscus. Carries fibers from midbrain tegmenturn, red nucleus, and periaqueductal gray matter to the inferior olivary complex.

Superior olivary nucleus: One of the tegmental nuclei that belong to the cochlear system. Receives fibers from the trapezoid body and contributes to the formation of the lateral lemniscus.

Trapezoid body: Also known as the inferior acoustic stria. Axons of neurons in the inferior cochlear nucleus form the trapezoid body.

Pontocerebellar tract: Axons of pontine nuclei on their way to the cerebellum via the brachiurn pontis.

Pontine nuclei: Scattered between the descending corticospinal, corticopontine, and corticobulbar fibers and the horizontally oriented pontocerebellar fibers. Receive input from the cerebral cortex via the corticopontine tract and project to cerebellum via the pontocerebellar tract.

Brachium conjunctivum: Also known as the superior cerebellar peduncle. Most important efferent fiber system of the deep cerebellar nuclei. Located dorsolateral to the fourth ventricle. Later in its course, it dips into the tegmenturn of the pons and midbrain .Nerve fibers in this bundle are destined to reach the contralateral red nucleus and ventrolateral nucleus of the thalamus.

Restiform body: Also known as the inferior cerebellar peduncle. Continuation of the same fiber system seen at more caudal levels. The restiform body is shown here entering the cerebellum.

Brachium pontis: A massive bundle of fibers connecting the basal portion of the pons with the cerebellum. Also known as the middle cerebellar peduncle. Contains pontocerebellar tract.

Spinal tract and nucleus of trigerninal nerve: Continuation of the same structures described at caudal levels.

Medial lemniscus: Continuation of the same fiber system described at more caudal levels (see .Note change in orientation of fibers from (previously) vertical in medulla oblongata to horizontal here in the pons.

Corticospinal, corticopontine, corticobulbar tracts: Descending fiber system sectioned transversely. Destined for the pontine nuclei, cranial nerve nuclei, and the spinal cord motoneurons.  

 

PONS
Facial and abducens
nerves coronal

 

Human, 10% formalin, Pal-Weigert, 2.2 x.

 

Genu of facial (CN VII) nerve: A bundle of facial nerve fibers in the floor of the fourth ventricle.

Brachium conjunctivum: Also known as the superior cerebellar peduncle. Most important efferent fiber system of the deep cerebellar nuclei. Located clorsolateral to the fourth ventricle.

Superior vestibular nucleus: Located dorsal and medial to the restiform body. One of four vestibular nuclei. Receives fibers from the vestibular component of the vestibulocochlear (CN VIII) nerve and projects fibers to the cerebellum via the restiform body and to nuclei of extraocular movement via the medial longitudinal fasciculus.

Facial (CN VII) nerve: Coursing ventrolaterally to emerge at the lateral border of the pons.

Facial nucleus: Located medial to the facial nerve. Axons of neurons in the facial nucleus course medially and dorsally to reach the floor of the fourth ventricle (genu of facial nerve) before turning laterally and ventrally to exit from the lateral surface of the pons.

Abducens nerve: Rootlets of the abducens nerve are seen coursing in the tegmenturn of the pons. They arise from the medial aspect of the nucleus and exit from the ventral surface at the caudal border of the pons. Supply the lateral rectus muscle of the eye.

Medial lemniscus: Continuation of the same structure seen at more caudal and more rostral levels.

Pontocerebellar tract: Continuation of the same structure seen at more caudal levels. Axons of pontine nuclei destined for the cerebellum.

Pontine nuclei: Scattered between pontocerebellar fibers and the corticospinal, corticopontine, and corticobulbar fibers. Relay station between the cerebral cortex and cerebellum.

Corticospinal, corticopontine, corticobulbar tracts: Long descending fiber system originating in the cerebral cortex. Sectioned transversely as it passes through the basal portion of the pons.

Abducens nucleus: Located in a paramedian position in the floor of the fourth ventricle. Axons of neurons in this nucleus emerge from the medial aspect of the nucleus to form the abducens nerve. Lesions of the abducens nucleus result in ipsilateral paralysis of lateral gaze. The abducens nucleus and the adjacent genu of facial nerve together form the facial colliculus, a paramedian elevation in the floor of the fourth ventricle.

Brachium pontis: A massive bundle of fibers connecting the basal portion of the pons with the cerebellum. Also known as the middle cerebellar peduncle.

Restiform body: Continuation of the same structure seen at more caudal levels. Seen entering the cerebellum.

Dentate nucleus: The largest of the deep cerebellar nuclei. Axons of this nucleus are major components of the brachium conjunctivum.

Facial colliculus: A paramedian elevation in the floor of the fourth ventricle overlying the abducens nucleus and the genu of the facial nerve

 

PONS
Trigeminal nerve coronal

Human, 10% formalin, Pal-Weigert, 2.4 x.

 

Fourth ventricle: The anterior part of the fourth ventricle overlying the pons.

Brachium conjunctivum: Massive outflow tract from the cerebellum seen at this level prior to decussation. Lesions in this area will result in a disorder of coordinated movement. Note the change in position of this structure in more rostral sections .

Principal (main) sensory nucleus of trigerninal (CN V) nerve: Located lateral to the motor nucleus of the trigeminal. Receives touch sensations from the ipsilateral face via the trigeminal nerve.

Motor nucleus of trigerninal (CN V) nerve: Located in the dorsal part of the tegmentum. Axons form the motor root of the trigerninal nerve and supply muscles of mastication, and the tensor tympani, tensor palati, mylohyoid, and the anterior belly of the digastric muscles.

Brachium pontis: Also known as the middle cerebellar peduncle. A massive bundle of fibers connecting the basal portion of the pons with the cerebellum. Contains pontocerebellar fibers.

Pontocerebellar tract: Axons of pontine nuclei destined for the cerebellum via the brachium pontis.

Corticospinal, corticopontine, corticobulbar tracts: Long descending fiber system originating in the cerebral cortex. Sectioned transversely as it passes through the basal portion of the pons. Note the horizontally oriented pontocerebellar tract.

Medial lemniscus: Continuation of the same system seen in more caudal levels.

Trigeminal nerve: Sensory-motor cranial nerve. Seen coursing in the lateral part of the pons.

Central tegmental tract: Compact fiber bundle located in the tegmenturn of the pons. Carries fibers from midbrain tegmentum, red nucleus, and periaqueductal gray matter to the inferior olivary complex.

Medial longitudinal fasciculus: Ascending component of a fiber system originating in vestibular nuclei and destined to synapse with neurons in nuclei of extraocular movement (CN III, IV, and VI). Concerned with control of eye movement.

Superior medullary velum: Forms the anterior (superior) part of the roof of the fourth ventricle.  

 

PONS
Trigeminal nerve coronal

Human, 10% formalin, Pal-Weigert, 2.5 x.

 

Brachium conjunctivum: Also known as superior cerebellar peduncle. Contains axons of deep cerebellar nuclei destined for the red nucleus and thalamus. Forms part of the lateral wall of the fourth ventricle.

Central tegmental tract: A compact fiber bundle located dorsal to the lateral part of the medial lemniscus. Carries fibers from midbrain tegmentum, red nucleus, and periaqueductal gray matter to the inferior olivary complex.

Brachium pontis: Also known as the middle cerebellar peduncle. Massive bundle of fibers connecting the basal portion of the pons with the cerebellum. Contains pontocerebellar fibers from the contralateral half of the pons. Some pontocerebellar fibers from the ipsilateral half of the pons are also contained in the brachium pontis.

Trigeminal (CN V) nerve: A mixed nerve with a larger sensory component (portio major) and a smaller motor component (portio minor).

Pontine nuclei: Located in the basal part of the pons. Continuous caudally with arcuate nuclei in the medulla oblongata. Receive corticofugal fibers and project (pontocerebellar tract) mainly to the contralateral cerebellum.

Corticospinal, corticopontine, corticobulbar tracts: A long descending fiber system sectioned transversely as it courses through the basal part of the pons.

Pontocerebellar tract: Axons of pontine nuclei destined for the cerebellum. Constitutes the major component of the middle cerebellar peduncle (brachium pontis).

Medial lemniscus: Continuation of the same fiber system noted in several more caudal levels. Note the change of orientation of this fiber bundle from a vertical orientation in the medulla to a horizontal orientation at this level.

Lateral lemniscus: Continuation of trapezoid body. Conveys auditory impulses.

Medial longitudinal fasciculus: The ascending component of this fasciculus. Contains fibers from the vestibular nuclei destined for the nuclei of extraocular movement. Lesions of the medial longitudinal fasciculus at this level will result in a characteristic clinical picture known as internuclear ophthalmoplegia

 

PONS- MESENCEPHALIC JUNCTION
Trochlear nerve coronal

Human, 10% formalin, Pal-Weigert, 2.9 x.

 

Brachium conjunctivum: Massive outflow tract of the cerebellum. Fibers are seen just prior to and beginning decussation. Fibers project, after decussation, into the red nucleus and ventrolateral nucleus of the thalamus. Lesions in this tract result in a disorder of coordinated movement.

Pontocerebellar tract: The same structure seen at more caudal levels.

Corticospinal, corticopontine, corticobulbar tracts: The same structures seen at more caudal levels. Cut in cross section as they descend to lower caudal levels.

Pontine nuclei: Scattered between pontocerebellar fibers and the corticospinal, corticopontine, and corticobulbar tracts.

Brachium pontis: Axons of pontine nuclei on their way to the cerebellum.

Medial lemniscus: Continuation of the same structure seen at more caudal levels.

Spinal lemniscus: Continuation of the same structure seen at more caudal levels. Contains spinothalamic and spinotectal fibers.

Lateral lemniscus: Contains cochlear fibers. Located laterally and dorsally on its way to the inferior colliculus and medial geniculate body. Concerned with audition.

Trochlear (CN IV) nerve: Seen exiting from the dorsal aspect of the midbrain after decussating. The fourth cranial nerve supplies the superior oblique extraocular muscle. The only cranial nerve to decussate (cross) completely prior to leaving the neuraxis.

Central tegmental tract: Compact fiber bundle located medial to the brachium conjunctivum. Carries fibers from the midbrain tegmentum, red nucleus, and periaqueductal gray matter to the inferior olivary complex. Note change in position of this tract in more caudal levels.

Medial longitudinal fasciculus: Continuation of same structure seen at more rostral and more caudal levels.

Decussation of trochlear (CN IV) nerve: Axons of neurons in the trochlear nucleus seen decussating prior to exit from the dorsal surface of the neuraxis

 

MESENCEPHALON
Inferior colliculus coronal

Human, 10% formalin, Pal-Weigert, 2.7 x.

 

Aqueduct of Sylvius: Connecting the third and fourth ventricles. Sylvius (Jacques Dubois) was a sixteenth -century French anatomist.

Trochlear (CN IV) nucleus: Motor neurons located in a paramedian position dorsal to the medial longitudinal fasciculus. Axons of neurons in trochlear nucleus decussate prior to leaving the neuraxis).

Inferior colliculus: Ovoid cellular mass in the tecturn of the mesencephalon. Belongs to the auditory system.

Lateral lemniscus: Located laterally and dorsally as it enters the inferior colliculus. Concerned with audition.

Spinal and medial lemnisci: Continuation of the same structures seen at more caudal levels.

Corticospinal, corticopontine, corticobulbar tracts: Sectioned transversely on their way to pontine nuclei, cranial nerve nuclei, and motor neurons of the spinal cord.

Pontocerebellar tract: Axons of pontine nuclei on their way to the cerebellum.

Decussation of brachium conjunctivum: Massive outflow tract of the cerebellum seen decussating at this level. Fibers project, after decussation, into the red nucleus and ventral lateral nucleus of the thalamus.

Tract of the mesencephalic nucleus of trigerninal (CN V) nerve: Processes of pseudounipolar neurons in the mesencephalic nucleus of the trigeminal nerve. Neurons are sparsely scattered on each side of the tract.

Medial longitudinal fasciculus: Continuation of the same structure seen at more rostral and more caudal levels.

 

MESENCEPHALON
Inferior colliculus coronal

Human, 10% formalin, Pal-Weigert, 3.0 x.

 

Inferior colliculus: Ovoid cellular mass belonging to the auditory system. Receives fibers from the lateral lemniscus and is reciprocally connected to the medial geniculate body.

Central (periaqueductal) gray: An area of gray matter surrounding the aqueduct of Sylvius. Contains scattered neurons, several nuclei, and some finely myelinated and unmyelinated fibers. Recent interest in this area has focused on its role in pain. The neuropeptide enkephalin has been identified in the central gray.

Spinal lemniscus (spinothalamic and spinotectal tracts): Continuation of the same fiber system seen at more caudal levels.

Medial lemniscus: Continuation of the same fiber system seen at more caudal levels.

Central tegmental tract: A compact fiber bundle located in the dorsal part of the mesencephalon dorsal to the decussation of brachiurn conjunctivurn. Carries fibers from the midbrain tegmentum, red nucleus, and periaqueductal gray matter to the inferior olivary complex. Note how the position of this tract changes in more caudal levels ).

Decussation of brachium conjunctivum: Massive outflow tract of the cerebellum seen crossing in the tegmenturn of the midbrain. Fibers project, after decussation, into the red nucleus and the ventral lateral nucleus of the thalamus. Lesion results in a disorder of coordinated movement.

Basis pontis: Basal part of pons. Contains pontine nuclei as well as corticospinal, corticobulbar, corticopontine, and pontocerebellar fibers.

Cerebral peduncle: Descending corticofugal fiber system. Lesion results in weakness (paresis) or paralysis of the contralateral half of the body, including the face.

Medial longitudinal fasciculus: The ascending component of this bundle. Connects vestibular nuclei with nuclei of extraocular movement (CN III, IV, VI).

Trochlear (CN IV) nucleus: Lies in the V-shaped ventral part of the central gray. Axons arch around the central gray, cross in the anterior medullary velum, and emerge from the dorsal aspect of the mesencephalon. Axons supply the superior oblique extraocular muscle.

Brachium of inferior colliculus: Also known as inferior quadrigeminal brachium. A bundle of nerve fibers from the lateral lemniscus and the inferior colliculus on their way to the medial geniculate body. This fiber bundle conveys auditory impulses from the midbrain to the thalamus.

Aqueduct of Sylvius: Named after the French anatomist Jacobus Sylvius (1478-1555). Connects the third and fourth ventricles.

 

MESENCEPHALON
Superior colliculus coronal

Human, 10% formalin, Pal-Weigert, 2.6 x.

 

Aqueduct of Sylvius: Connecting the third and fourth ventricles.

Periaqueductal (central) gray: Surrounds the aqueduct of Sylvius. Contains neurons related to both pain inhibition and stimulation.

Superior colliculus: Laminated cellular mass in the tecturn of the mesencephalon. Related to the visual system.

Brachium of inferior colliculus: Also known as inferior quadrigeminal brachium. Bundle of nerve fibers from the lateral lemniscus and the inferior colliculus to the medial geniculate body. Conveys auditory impulses to the thalamus.

Medial longitudinal fasciculus: Continuation of the same structure seen at more rostral and more caudal levels.

Substantia nigra: Largest nuclear mass in mesencephalon. Sandwiched between the medial lemniscus and the cerebral peduncle. A mass of pigmented cells (melanin) connected with basal ganglia and thalamus. Important in motor control. This area is invariably the site of pathological changes associated with Parkinson's disease. Parkinson was an eighteenth-century English physician.

Cerebral peduncle: Descending corticofugal fiber system. Lesions here result in weakness or paralysis of the contralateral half of the body, including the face.

Red nucleus: So-called because of a pinkish color in the fresh state owing to its high vascularity. This nucleus links the cerebellum, motor cortex, and spinal cord. Major input is from the brachium conjunctivurn and the cerebral cortex. Projects into the motor area of the cortex and spinal cord.

Oculomotor (CN III) nerve: Rootlets of the third cranial nerve seen coursing through the tegmenturn of the midbrain. Note relationship to red nucleus. Supplies the levator palpebrae superioris, the superior rectus, inferior rectus, medial rectus, inferior oblique, and constrictor pupillae muscles. Lesions of the oculomotor nerve result in ipsilateral paralysis of the eye muscles supplied by the nerve as well as a dilated, nonresponsive pupil.

Medial lemniscus: Continuation of the same tract seen at more caudal levels.

Medial geniculate nucleus: A thalamic nucleus concerned with audition. Receives auditory fibers from the inferior quadrigeminal brachiurn (brachiurn of inferior colliculus). Projects to the primary auditory cortex (transverse gyri of Heschl). Heschl was a nineteenth-century Austrian pathologist.

Spinal lemniscus: Continuation of the same structure seen at more caudal levels.

Oculomotor (CN III) nucleus: V-shaped collection of motor neurons located in a paramedian position, dorsal and medial to the medial longitudinal fasciculus. Axons form the oculomotor nerve. The oculomotor nucleus has two components: a somatic motor component, which supplies the extraocular (extrinsic) muscles, and a visceral, parasympathetic component (Edinger-Westphal nucleus), which supplies the intrinsic, constrictor pupillae muscle. Edinger was a nineteenth-century German anatomist and Westphal was a nineteenth-century German neurologist

 

MESENCEPHALON-DIENCEPHALON JUNCTION
coronal

Human, 10% formalin, Pal-Welgert, 2.3 x.

 

Pulvinar: Belongs to the lateral group of thalamic nuclei. Has reciprocal connections with the medial and lateral geniculate bodies caudally and the association parietal, temporal, and occipital cortices rostrally. Plays a role in several neural functions, including vision, audition, speech, and pain.

Lateral geniculate body: A thalamic relay nucleus concerned with vision. Receives fibers from the optic tract and projects to the primary visual cortex.

Substantia nigra: A mass of pigmented cells containing melanin located dorsal to the cerebral peduncle. This area is invariably the site of pathologic changes associated with Parkinson's disease.

Cerebral peduncle: Descending corticofugal fiber system. Lesion results in contralateral muscle weakness or paralysis.

Oculomotor (CN III) nerve: Coursing in the tegmentum of the midbrain medial to the substantia nigra and cerebral peduncle.

Red nucleus: So-called because of a pinkish color in the fresh state owing to its high vascularity. Links the cerebellum, cerebral cortex, and spinal cord.

Medial geniculate body: A thalamic relay nucleus concerned with audition. Receives fibers from brachium of the inferior colliculus and projects to the primary auditory cortex.

Brachium of superior colliculus: Fiber bundle connecting the superior colliculus and the lateral geniculate nucleus.

Pretectal nucleus: Rostral extension of the superior colliculus. Receives optic tract fibers and projects bilaterally to oculornotor nuclei. Important relay in pupillary light reflex.

Pineal gland: Located dorsal to the mesencephalon. Part of the epithalamus. Has endocrine function and is an important landmark radiologically.  

 

DIENCEPHALON
Mamillary body coronal

Human, 10% formalin, Pal-Weigert, 2.4 x.

 

Pulvinar nucleus: A thalamic nucleus. Belongs to the lateral group of thalamic nuclei. Has reciprocal connections with the medial and lateral geniculate bodies caudally and with the association parietal, temporal, and occipital cortices rostrally. Plays a role in several neural functions, including vision, audition, speech, and pain.

Lateral geniculate nucleus: A thalamic relay nucleus concerned with vision. Receives fibers from the optic tract and projects to the primary visual cortex (calcarine gyrus, area 17).

Red nucleus: So-called because of a pinkish color in the fresh state owing to its high vascularity. Links the cerebellum, cerebral cortex, and spinal cord.

Cerebral peduncle: A massive

Section 16: Special Senses

The different sensations perceived by the human body are grouped into two major categories: those concerned with general sensations (touch, pressure, pain, and temperature) and those concerned with special sensations (olfaction, taste, vision, audition, and sense of position and movement). Illustrations of nerve endings concerned with general sensibility are found in Section 6, Nervous Tissue. This section is devoted to a consideration of the organs of special senses. Whereas nerve endings concerned with general sensibility are widely distributed, those concerned with special sensations are limited to specific areas of the body.

Olfaction

The olfactory organ is located in the mucous membrane lining the uppermost part of the roof of the nasal cavity. From the roof, the olfactory epithelium extends down both sides of the nasal cavity to cover most of the superior concha laterally and 1 cm of nasal septum medially. Man is a microsmatic animal in whom the surface area of olfactory mucous membrane in both nostrils is small (approximately 5 cm). The specialized nerve cells of the olfactory epithelium are highly sensitive to different odors. There are approximately 25 million nerve cells in each half of the nasal cavity. Olfactory neurons are continuously produced from basal cells of the olfactory epithelium and are continuously lost by the normal wear and tear process. The presence of these nerve cells at the surface exposes them unduly to damage; it is estimated that 1 per cent of the fibers of the olfactory nerves (axons of olfactory neurons) is lost each year of life because of injury to the perikarya. The sense of smell thus diminishes in the elderly as a result of the exposure of the olfactory epithelium to repeated infections and trauma in life. The presence of olfactory neurons at the surface represents the only exception to the evolutionary rule by which nerve cell bodies of afferent neurons migrate along their axons to take up more central and well-protected positions. The olfactory neurons (see Plate 298) are bipolar nerve cells with short peripheral processes (dendrites) reaching the surface of the epithelium and longer central processes (unmyelinated axons) that constitute the olfactory nerves. Olfactory nerve fibers enter the cranial cavity through foramina in the cribriform plate of the ethmoid bone and synapse on neurons in the olfactory bulb. The peripheral short processes end as bulbous enlargements (olfactory vesicles) bearing sensory receptor hairs. The surface of the epithelium is constantly moistened by secretions of Bowman's glands. The moistening of the epithelium helps dissolve the gaseous substances, facilitating stimulation of the olfactory epithelium. The continuous secretion prevents retention of dissolved odors.

It is believed that different basic odors stimulate different olfactory neurons that are not evenly distributed throughout the olfactory mucosa. Stimulation of different combinations of receptors for basic odors is believed to be the basis for man's ability to recognize all the varieties of odors to which he is exposed.

Taste

The gustatory (taste) sense organs in higher vertebrates are limited to the cavity of the mouth. The sensory organ of taste is the taste bud which is a pale, ovoid structure within the stratified squamous epithelium. It is estimated that one vallate papilla of the tongue contains 200 taste buds on its sides and about 50 buds in the wall of the trench opposite the papilla. This number decreases progressively with age. In addition to the vallate and fungiform papillae of the tongue, taste buds are found in the soft palate, oropharynx, and epiglottis. The taste bud contains neuroepithelial and supporting cells. There are approximately 4 to 20 receptor cells in the center of each taste bud. The apex of each receptor cell is modified into microvilli, which increase the receptor surface and project into an opening, the taste pore. Receptor cells decrease in number with age. The neuroepithelial cells are stimulated by substances in solution.

Although all taste buds look histologically alike, sensitivity to the four basic taste modalities is different in different regions of the tongue. Like olfaction, the sense of taste is a chemical sense. Although man can taste a large number of substances, only four primary taste sensations are identified: sour, salty, sweet, and bitter. Most taste receptors respond to all four primary taste modalities at varying thresholds but respond preferentially at a very low threshold to only one or two. Thus, taste buds at the tip of the tongue respond best to sweet and salty substances, and those at the lateral margins and posterior part of the tongue respond best to sour and bitter substances, respectively. The mechanism by which a substance is tasted is not well understood. Substances in solution enter the pore of the taste bud and come in contact with the surface of taste receptors. This will induce a change in the electrical potential of the membrane of the receptor cells (receptor or generator potential). The receptor potential will in turn generate an action potential in nerve terminals in opposition to the receptor cell surface.

Taste sensations from the anterior two thirds of the tongue are mediated to the central nervous system via the chorda tympani of the seventh (facial) cranial nerve, those from the posterior one third of the tongue via the ninth (glossopharyngeal) cranial nerve, and those from the epiglottis and lower pharynx via the tenth (vagus) nerve. These nerves contain the peripheral processes of pseudounipolar sensory nerve cells located in the geniculate ganglion (seventh nerve), petrous ganglion (ninth nerve), and nodose ganglion (tenth nerve). These peripheral processes enter the deep ends of the taste buds and establish intimate contact with the neuroepithelial cells of the buds. The central processes of these sensory neurons project to the nucleus of the tractus solitarius in the brain stem.

Vision

Vision is by far the most important of man's senses. Most of our perception of the environment around us comes through our eyes. Our visual system is capable of adapting to extreme changes in light intensity to allow us to see clearly; it is also capable of color discrimination and depth perception.

The organ of vision is the eye; accessory structures include the eyelids, lacrimal glands, and the extrinsic eye muscles. The eye has been compared to a camera. Whereas structurally the two are similar, the camera lacks the intricate automatic control mechanism involved in vision. As an optical instrument, the eye has four functional components: a protective coat, a nourishing lightproof coat, a dioptric system, and a receptive integrating layer. The protective coat is the tough, opaque sclera, which covers the posterior five sixths of the eyeball; it is continuous with the dura mater around the optic nerve. The anterior one sixth is covered by the transparent cornea, which belongs to the dioptric system. The nourishing coat is made up of the vascular choroid, which supplies nutrients to the retina and, because of its rich content of melanocytes, acts as a light-absorbing layer. it corresponds to the pia-arachnoid layer of the nervous system. Anteriorly, this coat becomes the ciliary body and iris. The iris ends at a circular opening, the pupil. The dioptric system includes the cornea, the lens, the aqueous humor within the anterior eye chamber, and the vitreous body. The dioptric system helps focus the image on the retina. The greatest refraction of incoming light takes place at the air-cornea interface. The lens is supported by the suspensory ligament from the ciliary body and changes in its shape permit change of focus. This is a function of the ciliary muscle, which is supplied by the parasympathetic nervous system. In late middle age, the lens loses its elastic properties and a condition known as presbyopia results, wherein accommodative power is diminished, especially to near vision. The amount of light entering the eye is regulated by the size of the pupil. Pupillary size is controlled by the action of the constrictor and dilator smooth muscles of the iris. The constrictor muscle is supplied by the parasympathetic nervous system, and the dilator by the sympathetic nervous system.

The receptive integrating layer is the retina, which is an extension of the brain, to which it is connected by the optic nerve. The rods and cones are the sensory retinal receptors . The rods are about 20 times as numerous as the cones. The rods and cones differ in their distribution along the retina. in humans, a modified region of the retina, the fovea, contains only cones and is adapted for high visual acuity. At all other points along the retina, rods greatly outnumber cones. Rods function best for peripheral vision and during dim light vision; cones function for central vision, during bright light vision, and in color discrimination. The outer segments of rods and cones contain the visual pigments, rhodopsin and iodopsin (cone opsin), respectively. Light falling on these pigments results in a series of chemical changes leading to depolarization of the receptor cell membrane (receptor or generator potential) and the formation of an action potential, which is then conducted to the brain.

Audition

The organ of hearing (the organ of Corti) is located in the scala media (cochlear duct) of the inner ear and is separated from the underlying scala tympani by the basilar membrane (see .Sound waves reaching the tympanic membrane will initiate vibrations that are transmitted through the bony ossicles of the middle ear to the oval window. Vibrations of the oval window are transmitted to the perilymph in the scala vestibuli and across the vestibular membrane to the endolymph of the cochlear duct .Such induced pulsations in the endolymph will displace the basilar membrane on which the organ of Corti lies and alter the relationship of the tectorial membrane, which overlies the organ of Corti, to the hairs of the hair cells. Thus, bending or stretching of the hairs acts as a stimulus to the hair cells, causing release of a chemical neurotransmitter, generation of a receptor potential, and subsequent development of an action potential in the peripheral processes of bipolar neurons of the spiral ganglion .The central processes of bipolar neurons constitute the auditory component of the eighth cranial nerve, which projects centrally to the cochlear nuclei. In man, the cochlea and the organ of Corti follow a spiral course of two and one half turns. The lower turns are wider than the apical turns. It is believed that the hair cells in the lower turns respond best to high frequency sounds, whereas those of the upper turns respond best to low-frequency sounds. Exposure to excessively loud sound as occurs in discos and around jet engines results in damage to hair cells in the lower turns of the cochlea (high-tone deafness).

Position and Movement (Vestibular Sensations)

The receptor organ of posture and equilibrium is a composite one located in the semicircular canals, the utricle, and the saccule of the inner ear. The utricle and saccule are located in the main cavity of the bony labyrinth, the vestibule; the semicircular canals, three in number, are extensions from the utricle. The dilated ends of the semicircular canals (ampullae) contain the cristae (see Plate 314), which constitute the neurosensory epithelium that responds to changes in rotational or angular acceleration. The apical processes of receptor cells are embedded in a dome-shaped, gelatinous protein- polysaccharide mass, the cupula . The cupula swings from side to side in response to currents in the endolymph bathing it. Movement of the cupula bends or deforms the hairs of receptor cells embedded within it and thus modifies the rate of impulse discharge from these receptor cells. Each crista is stimulated by movements occurring in the plane of its semicircular canal. The neuroepithelial component of the utricle and saccule provides information regarding static equilibrium and position of the head in space. The macula is similar in structure to the crista of the semicircular canals. The apical processes of receptor cells in the macula project into a gelatinous mass, the otolithic membrane. It is flat and contains numerous small crystalline bodies, the otoliths or otoconia, composed of calcium carbonate and protein. Gravitational pull acts on the otoconia on the surface of the macula, and the hair tufts of underlying neuroepithelial hair cells are thus stimulated. Stimuli from the vestibular sense organs travel by way of the peripheral processes of the bipolar neurons of the ganglion of Scarpa. The central processes form the vestibular component of the eighth nerve. Although we are normally not aware of the vestibular component of our sensory experience, this component is essential for the coordination of motor responses, eye movements, and posture.

 

OLFACTORY MUCOSA

Rodent, 10% formalin, H. & E., 218 x.

 

Olfactory epithelium: Thick pseudostratified epithelium containing (1) sustentacular (supporting) cells, (2) bipolar receptor neurons, and (3) basal cells. Cells are densely packed and difficult to differentiate in thick sections. The nuclei of these cells are layered (from the outside) in the order given above. No goblet cells are located in this region, but the area is flushed by seromucous glands located beneath the epithelium.

Glands of Bowman*: Located in the lamina propria. Branched tubuloalveolar glands that secrete a seromucus. The secretions keep the surface moist, facilitate solution of substances being smelled, and subsequently cleanse the olfactory receptors of the olfactory stimulus.

Olfactory nerve fibers: Non-myelinated axons of bipolar receptor neurons. Nerve bundles are located deep in the lamina propria.

 

OLFACTORY EPITHELIUM
Bipolar receptor neurons

Dog, 10% formalin, silver diaminohydroxide, 500 x.

 

In this plate, a special staining technique is used to demonstrate the olfactory receptor (bipolar) neurons in the olfactory mucosa.

The nuclei of the receptor cells are usually deeply located in the epithelium. From the nuclear region of the neuron, a delicate peripheral neural process reaches the exposed surface of the epithelium. At the opposite pole of the cell, an unmyelinated axon extends centrally. The axons of receptor cells are gathered together to form the olfactory nerve (cranial nerve 1). The olfactory nerve terminates in the olfactory bulb, where synaptic contacts are established with neurons, whose axons form the olfactory tract.

The olfactory bipolar neurons are located between the more numerous sustentacular cells

 

TASTE BUD
Tongue
vallate papilla

Human, Zenker's fluid,
phosphotungstic acid hematoxylin stain, 612 x.

 

Taste buds are located in the tongue epithelium of vallate (circumvallate) papillae, and occasionally in the fungiform papillae and in the surface epithelium around them. A few taste buds are also present in the palate and epiglottis.

Trench: Surrounds vallate papillae. Covered by non-keratinized stratified squamous epithelium.

Taste bud in stratified squamous epithelium: Barrel-shaped. Extends from the basement membrane to the free surface of the stratified squamous epithelium covering the papilla. Taste buds are the receptor organs of taste.

Taste pore: The opening of the taste canal at the surface of the epithelium.

Neuroepithelial and supporting cells: The two types of cells are packed within the concavity of the taste bud. The neuroepithelial cells are slender, dense, and spindle-shaped. They constitute the taste receptor cells. Supporting cells or sustentacular cells are stouter and lighter.

Lamina propria: Connective tissue core of the papilla.

 

CONJUNCTIVA AND CORNEA

 

Rhesus monkey, 10% formalin, H. & E., 162 x.

 

This plate includes part of the cornea (right) and the covering bulbar conjunctiva (left). The conjunctiva at this site is composed of the following elements.

Lamina propria: A loose, superficial fibroelastic connective tissue stroma, which becomes increasingly dense at deeper levels. Rich in blood vessels.

Stratified columnar epithelium: Eight to ten cells thick. Superficial layers near the cornea rich in goblet cells.

Corneal epithelium and stroma

 

CORNEA

Rhesus monkey, 10% formalin, H. & E., 612 x.

 

The cornea is the bulging front portion of the eye. It is nonvascular and transparent. Microscopically, five distinct layers can be recognized.

Corneal epithelium: Non-keratinized stratified squamous type of epithelium. Note that the basal layer of cells is columnar, and the most superficial are flattened.

Anterior limiting membrane: The second layer of the cornea was described by Sir William Bowman, an English surgeon, and therefore is called Bowman's membrane. This membrane appears homogeneous and structureless by light microscopy. By electron microscopy, it is shown to be composed of fine collagenous fibrils.

Substantia propria: Constitutes nine tenths of the thickness of the cornea. Composed of collagen fibrils, fibroblasts, and cementing substance. The fibrils are arranged in lamellae that run parallel to the surface of the cornea. The fibroblasts are flattened and lie between the fibrous lamellae. A mucopolysaccharicle cements the different lamellae and the collagenous fibrils within lamellae together. The metachromatic protein polysaccharide ground substance and the arrangement of fibrils within the substantia propria contribute to the transparency of the cornea.

Posterior limiting membrane: The posterior limiting membrane of the cornea was described by the French surgeon Descemet in 1758 and is known by his name. English anatomists state that it was first described by Benedict Duddell, an English oculist. This membrane appears homogeneous in the light microscope. Electron microscopy reveals a wide basement membrane made of atypical collagen.

Corneal endothelium: Low cuboidal epithelium. The term endothelium is a misnomer, since this epithelium is bathed by aqueous humor of the anterior chamber and not blood or lymph.  

 

 

EYE

Rhesus monkey, 10% formalin, H. & E., 50 x.

 

Ciliary body: A ring of muscle and vascular tissue, part of the vascular and pigmented tunic of the eye, which includes, in addition, the choroid and iris. The ciliary body is attached to the lens by the suspensory ligament. Contraction of muscle of the ciliary body results in lens accommodation.

Ciliary processes: Ridges of the ciliary body as it approaches the iris. Run in a meridional plane. Provide an anchor for the suspensory ligaments of the lens. Produce aqueous humor.

Suspensory ligament: Made up of delicate collagenous fibers that stretch between the lens capsule and ciliary processes from which the lens is suspended. The suspensory ligament is under tension when the eye is at rest and relaxes when the lens accommodates, as in near vision.

Lens: Transparent biconvex disc enclosed in a homogeneous elastic capsule and located behind the iris. Made up of concentric layers of lens fibers and a cement substance. Changes in configuration of the lens are important in accommodation. The lens, cornea, and the vitreous are the important refractive media of the eye.  

 

CILIARY MUSCLE AND CILIARY PROCESSES

Rhesus monkey, 10% formalin, H. & E., 612 x.

 

Ciliary processes: Ridges of the ciliary body as it approaches the iris run in a meridional plane. They provide an anchor for the suspensory ligaments of the lens.

Zonular fibers of suspensory ligament: Seen attached to the ciliary processes. Inelastic and radially arranged fibers from which the lens is suspended. Extend from ciliary processes to the lens capsule. When the eye is at rest, zonular fibers under tension from elastic fibers in the choroid stretch the lens. Tension in zonular fibers is reduced when ciliary muscles contract. This results in a change in the shape of the lens (accommodation). The lens becomes more spherical owing to its inherent elasticity.

Columnar epithelium: Columnar or cuboidal epithelium, which covers the ciliary processes. Indistinct cell borders. Elaborates aqueous humor.

Posterior chamber: The space between the iris and suspensory ligament of the lens. Contains aqueous humor.

Ciliary muscle: Smooth muscle fibers intermixed with melanocytes. Runs in three directions: circular, radial, and meridional. Circular fibers lie at the inner edge of the ciliary body. Contraction of ciliary muscles releases tension in suspensory ligament of the lens and of the lens capsule, thus allowing the lens to change shape to accommodate for near vision. The ciliary muscle is a continuation of the suprachoroid layer.

Melanocytes: Pigment-laden cells scattered in the connective tissue elements between muscle fibers.  

 

IRIS AND LENS

Rhesus monkey, 10% formalin, H. & E., 162 x.

 

The iris, like the ciliary body, is a continuation of the retina and choroid. It is attached to the ciliary body and extends in front of the lens. All the layers of the iris are shown in this figure except the endothelial cell layer, which outlines the anterior boundary of the iris. This is a thin and delicate layer, which is difficult to see in ordinary light microscopic preparations.

Anterior chamber: Located anterior to the iris and communicates with the posterior chamber through the pupil. Contains aqueous humor.

Anterior border layer: A condensation of the stroma of the iris. Formed principally of pigment cells containing a variable amount of yellowish-brown pigment. Thickness of this layer determines the color of the iris. It is thin in blue-eyed individuals and thick in brown-eyed individuals.

Stroma: Consists of loose connective tissue, in which are found a large number of blood vessels. Pigment connective tissue cells are scattered in this loose connective tissue stroma.

Constrictor smooth muscle fibers: Constricts the pupil. Supplied by the parasympathetic postganglionic neurons of the oculomotor nerve located in the ciliary ganglion.

Pigment epithelium: Continuation of the ciliary epithelium. Vast amounts of melanin pigment obscure cell boundaries and nuclei.

Posterior chamber: Between the iris and the lens. Communicates with anterior chamber through the pupil. Contains aqueous humor.

 

IRIS

Rhesus monkey, 10% formalin, H. & E., 612 x.

 

Pigmented epithelium: On the posterior surface of the iris. A layer of cuboidal cells whose outlines are masked by the heavy pigment.

Constrictor smooth muscle: The fibers are arranged circumferentially at the margin of the pupils. Contraction will constrict the pupil. Supplied by parasympathetic postganglionic neurons of the oculomotor nerve located in the ciliary ganglion.

Blood vessels: In the iris stroma. Have a thick adventitial layer forming a unique fibrous acellular wall.

Melanocytes: Spindle-shaped connective tissue cells, with long processes containing yellow-brown pigment, scattered in the loose stroma of the iris. Most commonly seen along the anterior border of the iris. The number of pigment cells varies with the individual's complexion.  

 

RETINA

Rhesus monkey, Helly's fluid, H. & E., 162 x.

 

Nerve fiber layer: Consists of non-myelinated axons of ganglion cells. They converge at the optic disc to form the optic nerve. Fibrous neuroglial cells are scattered among nerve fibers.

Ganglion cell layer: Composed of multipolar ganglion cells. Only nuclei are seen by this method of staining.

Inner plexiform layer: Also known as inner synaptic layer. Contains processes of amacrine cells, axons of bipolar cells, and dendrites of ganglion cells.

Inner nuclear layer: Contains the nuclei of bipolar neurons and association neurons (horizontal and amacrine cells), as well as the nuclei of supporting (Müller's*) cells.

Outer plexiform layer: Also known as outer synaptic layer. Contains axons of rod and cone cells, dendrites of bipolar cells, and processes of the horizontal cells.

Outer nuclear layer: Contains rod and cone cell bodies and nuclei. Cone nuclei are ovoid and limited to a single row . Rod nuclei are rounded and distributed in several layers.

Cones and rods: Light-sensitive end portions of rod and cone cells. Contain an unstable, light-sensitive substance (rhodopsin in rods and iodopsin in cones). Both pigments play an important role in the visual process. Rhodopsin and iodopsin consist of vitamin Al aldehyde conjugated to a specific protein (rod opsin and cone opsin).

Pigment epithelium: Single layer of pigmented cuboidal cells firmly bound to the choroid layer. Contains melanin pigment.

Choroid: Heavily pigmented layer of loose connective tissue characterized by extreme vascularity. Provides nourishment for the outer portions of the retina.

Sclera: Firm external coat of the eye. Composed of dense collagenous connective tissue. A narrow pigmented zone near its internal surface merges with the choroid. The tendons of the six extraocular striated muscles insert on the sclera.

 

RETINA
Fovea centralis

Rhesus monkey, Helly's fluid, H. & E., 612 x.

 

Outer plexiform layer: Contains axons of cone cells, dendrites of bipolar neurons, and processes of horizontal cells.

Outer nuclear layer: Nuclei of visual receptor cells. Note ovoid nuclei of the cones. Multiple rows of nuclei are characteristic of the fovea.

Outer limiting membrane: Sieve-like sheet. Region of junctional complexes between the outer ends of the tall supporting Müller cells and the adjoining photoreceptor cells. Not an actual membrane. The visual cells pass through perforations in this so-called membrane.

Cones: Light-sensitive end portions of cone cells. Respond to light of high intensity. Function is visual acuity and color perception. Compare cones in fovea with those seen in other regions of the retina (Plate 308).

Inner segments: Thicker proximal segments of the cones. Rich in mitochondria.

Outer segments: Slender distal segments of cones. Consist largely of stacked discs 0.014 µm thick. Contain iodopsin, an unstable, light-sensitive visual pigment, which chemically is composed of vitamin A, aldehyde conjugated to a specific protein (cone opsin).

Pigment epithelium: Single layer of pigmented cuboidal cells firmly bound to the choroid layer. Cells contain melanin pigment. The cytoplasmic processes of the pigment cells interdigitate with the outer segment of cones.  

 

EYE
Cone and rod vision

Rhesus monkey, 10% formalin, H. & E., 612 x.

 

This figure shows variation in retinal structure near the fovea centralis and at the periphery of the retina (ora serrata). In both sites, the layers of the retina are diminished. Absent are the ganglion cell layer, the inner plexiform layer, and the bipolar cell layer. The fovea constitutes the zone of greatest visual acuity. At the fovea, the photoreceptors are thin, slender elements (cones). They resemble rods more than cones.

. The thinning of the retina at the fovea reduces to a minimum tissue through which light passes, and hence improves visual acuity. Note ovoid nuclei of cones in the outer nuclear layer. Multiple rows of these nuclei are characteristic of this region. Typical cones, which predominate here, function for sharp vision and color perception. It is estimated that there are 6 to 7 million cones in the retina.

Near the ora serrata, rods increase in number and in thickness, and become shorter. The cones decrease in number and also become shorter at the periphery of the retina. Rods number approximately 100 million and function for night vision and black and white discrimination. Note the multiple rows of rounded rod nuclei in the outer nuclear layer. The pigmented epithelium layer is similar in both fovea and ora serrata. It is firmly bound to the choroid layer and contains melanin pigment.  

 

EYELID

Rhesus monkey, 10% formalin, H. & E., 50 x.

 

The eyelid is a fold of skin; superficially, the keratinized epidermis blends internally with a mucous membrane (the conjunctiva). These layers are supported by a dermal core of connective tissue in which striated and smooth muscle, glands, and hair follicles are located.

Eyelash follicle: A row of short stout hairs are found at the free margin of the lid. Penetrate deep into the dermis. Their follicles are similar to those found elsewhere in the body but lack the arrector pili smooth muscle.

Skin: Thin layer of epidermis continuous with the conjunctiva.

Fat lobule: Scattered in the connective tissue core of the eyelid.

Orbicular muscle: Skeletal muscle bundles that lower the eyelids.

Conjunctiva: Mucous membrane lining the inside of the eyelid. The epithelium is stratified columnar with goblet cells scattered among the superficial cells.

Meibomian gland: The tarsal glands of the eyelids, first noted by Casserius in 1609 and described by Heinrich Meibom, a German anatomist, in 1666. These are simple, branched alveolar sebaceous glands disposed in a plane perpendicular to the lid margin. The glandular alveoli are connected by short lateral ducts to a long central excretory duct lined with stratified squamous epithelium. The glands open at the inner free margin of the lid at the junction of the skin and conjunctiva. Secretion of the glands serves to lubricate the surface of the lids.  

 

LACRIMAL GLAND

Human, 10% formalin, H. & E., 126 x.

 

The lacrimal gland is composed of a number of compound tubuloalveolar serous glands. The serous acini are made up of tall cells with basal nuclei. The secretory product fills the apical cytoplasm. The acini are separated by a thin connective tissue stroma. The aqueous secretion (tears), after flushing the conjunctivalcorneal surfaces, drains into ducts that carry it, via the nasolacrimal duct, to the anterior portion of the inferior meatus of the nose.  

 

ORGAN OF CORTI

Cat, Müller's* fluid, H. & E., 50 x.

 

Temporal bone: The bone in which the cochlea is located.

Spiral ligament: A projection of thickened periosteum along the outer wall of the osseous canal of the cochlea.

Osseous spiral lamina: A bony shelf projecting from the modiolus across the osseous canal of the cochlea. Follows the spiral turns of the cochlea. Divides the osseous canal of the cochlea into an upper scala vestibuli and a lower scala tympani. The former is continuous with the oval window; the latter ends at the round window. The two scalae are continuous at the helicotrema.

Vestibular membrane: Also called Reissner's* membrane. A thin membrane extending from the upper surface of the limbus spiralis to the upper part of the spiral ligament. It forms the roof of the cochlear duct, which is a part of the membranous labyrinth and houses the organ of hearing.

Limbus spiralis: A thickening of periosteal connective tissue at the outer border of the osseous spiral lamina. The vestibular membrane attaches to the upper surface of the limbus.

Basilar membrane: Forms the base of the cochlear duct. Gives support to the organ of Corti.

Spiral ganglion: Ganglion of the bipolar cells. Peripheral processes contact the hair cells of the organ of Corti, and the longer central processes form the cochlear nerve.

Cochlear nerve: Formed by the central processes of the bipolar cells of the spiral ganglion, these nerve fibers, upon entering the brain stem, terminate in the dorsal and ventral cochlear nuclei

 

ORGAN OF CORTI
Cochlea

Guinea pig, 20% formalin, H. & E., 162 x.

 

This plate is a cross section of the bony cochlea and shows three compartments: the upper, scala vestibuli; the lower, scala tympani; and the middle, scala media. The scala vestibuli and scala tympani are perilymphatic spaces. The scala vestibuli reaches the inner surface of the oval window. The scala tympani reaches the inner surface of the round window. Both scalae communicate at the helicotrema at the apex of the cochlea. The scala media or cochlear duct is part of the endolymphatic system and contains the organ of Corti. The vestibular membrane or Reissner's membrane forms the roof of the cochlear duct. The basilar membrane forms the base of the cochlear duct and gives support to the organ of Corti.* The outer wall of the cochlear duct is made up of the spiral ligament, which is a thickening of the periosteum. The crest of the spiral ligament forms the spiral prominence. The part of the spiral ligament between the spiral prominence and the vestibular membrane is the stria vascularis. The epithelium here is thick and pseudostratified. The subepithelial connective tissue is rich in capillaries. The stria vascularis is believed to be active in the production of endolymph and the regulation of its ion content. The epithelium of the spiral prominence continues onto the basilar membrane. Cells here become cuboidal. Those cells continuing onto the pars pectinata of the basilar membrane are known as cells of Claudius.* In parts of the cochlea, polyhedral cells separate the Claudius cells and the basilar membrane and are known as cells of Boettcher.* The organ of Corti is composed of two types of cells, supporting and hair cells. The supporting cells are tall, slender cells extending from the basilar membrane to the free surface of the organ of Corti. The supporting cells include the outer and inner pillars, inner and outer phalangeal cells, and cells of Hensen.* The inner tunnel within the organ of Corti is bounded below by the basilar membrane and above by the outer and inner pillar cells. The outer phalangeal cells act as supporting elements for the three to four rows of outer hair cells. The cells of Hensen are located adjacent to the last row of outer phalangeal cells. They constitute the outer border of the organ of Corti. The hair cells are of two types: the inner and outer hair cells. The inner hair cells are in a single row. The outer hair cells form three rows lodged between the outer pillar and outer phalangeal cells. The tectorial membrane is in contact with the hairs of hair cells and transmits to them endolymph vibrations. The cochlear nerve is formed by central processes of the bipolar cells of the spiral ganglion. In the inner angle of the scala media, the periosteal connective tissue of the spiral lamina bulges into the scala media overhanging the internal spiral tunnel.

 

 

SEMICIRCULAR CANAL
Temporal bone

Cat, Müller's fluid, H. & E., 162 x.

 

Perilymphatic space: Intervenes between the osseous labyrinth and the membranous labyrinth. Traversed by fibrous trabeculae. Contains the fluid, perilymph.

Osseous tissue: Compact bone forming the osseous labyrinth. The surface facing the perilymph has a periosteum covered by mesenchymal tissue.

Endolymphatic space: The stimulation of the vestibular receptors (the crista ampullaris and the two maculae) depends upon the movement of endolymph, the fluid contained within the endolymphatic space in the semicircular canals, saccule, and utricle. Note the mesothelial lining enclosing the endolymphatic space.

 

  

SEMICIRCULAR CANAL
Crista ampullaris

Guinea pig, Müller's fluid, iron hematoxylin stain, 612 x.

 

Cupula: Gelatinous dome-shaped mass in which the hairs of the hair cells are embedded. Similar to the cells of the otolithic membrane but lacks otoconia. Movements of the cupula are transmitted to the hairs of the receptor cells.

Hair cells: Flask-shaped cells that occupy the outer part of the epithelium but do not reach the basement membrane. Nuclei close to the lumen. A tuft of hairs is found in the apex of each cell. Compare hair cells to the adjacent tall, columnar sustentacular cells with basal nuclei that reach the basement membrane.

Nerve fibers: Myelinated processes of the vestibular nerve lose their myelin close to the hair cells of the crista. Naked fibers ramify among the hair cells of the crista, forming a dense arborization.  

 

MACULA UTRICULI
Membranous labyrinth

Cat, Müller's fluid, iron hematoxylin, 162 x.

 

Sensory area of the utricle. The sensory epithelium is composed of two types of cells.

Hair cells: Flask-shaped. Nuclei occupy the upper part of the epithelial sheet. These receptors of the macula utriculi are concerned with the orientation of the body with regard to gravity. The receptor cells of the macula sacculi, however, appear to respond primarily to vibratory stimuli.

Supporting cells: Slender. Nuclei are lined up near the basement membrane of the epithelium.

The surface of the macula is covered by a gelatinous material, the otolithic membrane, through which hairs of the hair cells project. The upper surface of the membrane contains densely packed crystals of a calcium carbonate- protein mixture, the otoliths. The utricle and saccule, which are similar in appearance, constitute the "otolith organ."

Nerve fibers: Afferent components of the vestibular portion of the eighth cranial nerve, the vestibulocochlear nerve.

Temporal bone: Forming the osseous labyrinth.  

 

 

Section 15: Endocrine Glands

These cellular masses, designated as the ductless glands or glands of internal secretion, have during development lost their original connection with the epithelium of the free surface. Their secretions are called hormones. The gland cells produce specific chemical substances, which are secreted in a rich capillary bed and carried by the blood to another part, or parts, of the body where they have a distinctive function. The endocrine glands are essentially a vertebrate development, and any one of these hormonal substances has a similar action in all vertebrates with little or no species-specific function.

Endocrine glands may appear as distinct organs (e.g., the hypophysis and adrenal glands), may be found associated with exocrine glands (e.g., pancreatic islets and the interstitial cells of the testes), may appear as mixed endocrine glands (e.g., the thyroid and parathyroid glands), or may have cells so diffusely distributed that they are not usually considered as organs (e.g., argentaffin cells of the digestive system).

Some endocrine glands are essential for life; these include the adrenal cortex, pancreatic islets, and the parathyroid glands. The other endocrine glands, although not essential for life, determine to a great extent the quality of ones life and the ability to adapt to stress. The endocrine glands, separately and in conjunction with the nervous system, are coordinators of body functions that maintain the organism in a viable homeostatic state.

The structural/functional organization of the endocrine glands is diverse but distinctive. In general, all endocrine glands store their secretory products either within the cells of origin or within cellular follicles or sacs. The cells of the adrenal cortex contain minimal amounts of stored hormone, whereas in the pancreas and pituitary gland (hypophysis), secretory granules (if preserved) are usually evident. In the thyroid gland, the hormone is stored extracellularly in a pool surrounded by epithelial gland cells (a follicle). In this case, the release of the hormone into the blood stream involves the reabsorption and transfer of the hormone through the cells of origin into the extracellular space, where it enters the capillaries.

An essential feature of the endocrine glands is the manner in which the secretory activity is regulated by a feedback mechanism. As an example, the beta cell of the anterior lobe of the hypophysis secretes adrenocorticotropic hormone (ACTH), which stimulates the secretion of certain hormones from the adrenal cortex. As the level of adrenal cortical hormones rises in the blood stream, the secretion of ACTH is inhibited. Declining levels of the hormones of the adrenal cortex result in an increased secretion of ACTH by the pituitary. In this manner, appropriate levels of adrenal cortical hormones are maintained in the blood stream.

Specific details of the structure and function of the endocrine glands will be found in this section and, as appropriate, in the sections concerned with the digestive, urinary, male reproductive, female reproductive, and nervous systems.

 

HYPOPHYSIS

Human, Susa, Halmi's* AFT, 11 x.

 

Several features of the hypophysis can be seen in this low-power photomicrograph. Elements of the infundibulum or neural stalk, which develops from the floor of the diencephalon, and the derivatives of Rathke's* pouch, which arise from an outpocketing of ectoderm from the roof of the mouth of the embryo, are shown. The pouch derivatives include the pars distalis and pars intermedia. Note the cyst- like clefts between the pars distalis and the pars intermedia, which are developmental remnants of the primitive cavity of Rathke's pouch. Elements of the important hypophysealportal blood circuit can be seen in the pars tuberalis.

Even at this low magnification, clusters of basophils and eosinophils can be seen in the pars distalis, permitting their study at higher magnifications.

 

HYPOPHYSIS CEREBRI
Pituitary Gland

Human, Susa, AFT,
A. 32 x, B. 316 x, C. 316 x D. 82 x.

 

This plate is at higher magnification than the preceding one. This permits easier location and identification of various cell types that populate the pituitary gland. One may find three cell types grouped under two headings: (1) chromophils, which include eosinophils (two types in this preparation) and basophils, and (2) chromophobes.

it must be remembered that empirical histological methods cannot differentiate precisely cell types. This can now be done by immunocytochernistry, but this method effaces other structural detail; therefore, several methods are usually required. Students interested in the complexities of cell typing should consult recent original investigative references. Nevertheless, one can locate populations of cells whose functions can be ascribed because they bear consistent staining characteristics.

Eosinophils are of two types and are known to secrete growth hormone (somatotropin) and prolactin. Both cells are stainable with orange G, but not with the periodic acid-Schiff method.

Basophils fall into three types: one type produces follicle-stimulating hormone (FSH) and luteinizing hormone (LH); a second type produces thyrotropic hormone; and a third type produces adrenocorticotropic (ACTH) hormone. These cells do not stain with orange G but do stain with the periodic acid-Schiff method, reflecting the glycoprotein, rather than any acid, nature of the synthesized secretory product.

Some chromophobes may be supporting cells with long branching processes forming a network within the parenchyma of the organ. Other chromophobe cells are not numerous and may be either transitionally degranulated cells or may be considered as reserve cells.

The pituitary gland receives its blood supply from the internal carotid artery and the superior and inferior hypophyseal arteries. The superior artery becomes a primary capillary plexus with fenestrated capillaries. Neurosecretory neurons contain releasing or inhibitory factors and are intimately associated with the capillary plexuses. These capillary plexuses rejoin to form portal (vessels located between and joining two capillary beds) veins, which once again form capillary plexuses around cells of the adenohypophysis (pars distalis). It can be appreciated that this system is critically important to normal hypophyseal function.

Pituicyte nuclei: The pituicyte perikaryon is not seen. These cells constitute 25 to 30 percent of the posterior lobe of the hypophysis. Function is poorly understood. Pituicytes are related to the neurosecretory axons and they correspond to neuroglia of the brain.

Capillary: Capillaries receive hormones secreted from Herring* bodies for distribution throughout the body. The posterior lobe of the hypophysis is highly vascularized.

 

HYPOPHYSIS
Anterior, intermediate, and posterior lobes

Rat, 10% formalin,
Gomori's chrome alum hematoxylin and phloxine stains, 162 x.

 

The anterior lobe is made of anastomosing cords and plates separated by capillaries. Several trophic hormones are produced in this lobe: somatotropin, follicle-stimulating hormone, luteinizing hormone, luteotropic hormone (prolactin), thyrotropic hormone, and adrenocorticotropic hormone.

The intermediate lobe is sandwiched between the anterior and posterior lobes. It has colloid-filled cysts. it elaborates melanocyte-stimulating hormone in some species (amphibia). In man and other mammals, the hormone appears to influence melanin synthesis.

The posterior lobe is made of sheets of cells and is rich in neurosecretory material. The latter is concentrated around the sinusoids. Two hormones have been extracted from this neurosecretory material, antidiuretic hormone and oxytocin.  

 

PITUITARY GLAND
Anterior lobe

Human, 10% formalin, Masson's* stain, 612 x.

 

Chromophobe: Small, poorly staining cell. The cytoplasm is scanty and devoid of granules. Cell contours are rounded or polygonal. Also known as reserve or chief cell.

Acidophil: Larger than a chromophobe. Cytoplasm rich in granules, which stain with acid dyes. Since they also take basic dyes, a better name might be alpha cell. Secrete growth and lactogenic hormones. The lactogenic hormone- producing acidophils increase during pregnancy. Excessive use or production of growth hormone as occurs in pituitary tumors produces gigantism if it occurs before puberty and acromegaly if it occurs after puberty.

Basophil: Beta cell. Larger than the average acidophil and less heavily granulated. Secretes thyroid- stimulating hormone (TSH), adrenocorticotropic hormone (ACTH), follicle-stimulating hormone (FSH), and luteinizing hormone (LH).

Colloid: Secretion product. Found in center of some cords.

 

PITUITARY GLAND
Anterior lobe

Human, 10% formalin, Masson's stain, 612 x.

Colloid: Secretion product. Found in the center of some cords.

Chromophobe: Small, faintly staining and less numerous than other cells in the pituitary. Scanty cytoplasm devoid of granules. Tend to cluster near center of cords.

Eosinophils: Alpha cells. Larger than chromophobes. Cytoplasm filled with acidophilic granules. Source of growth and lactogenic hormones.

Basophil: Beta cell. Larger than the average eosinophil and less heavily granulated. Secretes thyroid- stimulating hormone, adrenocorticotropic hormone, follicle-stimulating hormone, and luteinizing hormone.

Red blood cells: Filling the sinusoids.  

 

PITUITARY GLAND
Posterior lobe Herring body

Human, Susa, AFT, 220 x.

 

Herring* body: Herring bodies represent dilated terminal portions of neurosecretory axons constituting the hypothalamohypophyseal tract. They are always in close proximity to capillaries. Within the dilatations of various sizes are neurosecretory granules, which stain blue in this preparation. The Herring body granules consist of hormone precursors for either oxytocin or vasopressin, plus a binding protein for each hormone and adenosine triphosphate (ATP). The neurosecretory material is synthesized in neurons located in the hypothalamus and transported down the axons of the hypothalamohypophyseal tract to reach the axon terminals (Herring bodies). Most of the vasopressin is thought to be synthesized in neurons located in the supraoptic nucleus and oxytocin in neurons of the paraventricular nucleus. Vasopressin (pitressin, 13-hypophamine) is an antidiuretic hormone and in pharmacologic doses causes smooth muscle to contract, particularly those in blood vessels. Oxytocin (pitocin, A-hypophamine) causes myometrial contractions at term and promotes milk release during lactation.

*Herring bodies also contain synaptic vesicles 40 to 60 nm in diameter that are similar in structure to those found in cholinergic synapses. Their function here is not known.  

 

THYROID GLAND

Human, 10% formalin, H. & E., 162 x.

 

Follicles: Structural units of the thyroid gland. Note variations in shape (rounded or tubular) and size (0.05 to 0.5 mm in diameter). Close packing with a thin reticular network between adjacent follicles. Single layer of cells forms a hollow sphere. Nucleus centrally or basally placed.

Colloid: Found in the lumen of follicles. Chemical composition is a glycoprotein-iodine complex (thyroglobulin). Of the several iodinated compounds found in the gland the 3,5,3-triiodothyronine is hormonally the most active. The follicles release about 100 mg of hormone daily. Normal thyroid function is essential for the normal growth, development, and well-being of man and animals. Hypofunction of the thyroid in infants results in cretinism, characterized by dwarfism, mental deficiency, slow heart rate, muscular weakness, and gastrointestinal disturbances. Thyroid hormone given to infants at an early stage of cretinism can alleviate the symptoms. In adults, hypothyroidism results in muscular weakness, mental slowing, cold intolerance, and reflex and skin changes. When the hormone is produced in excess (hyperthyroidism), excessive appetite and thirst, weight loss, rapid respiration, sweating, muscular weakness, tremor, and an increase in heart rate (tachycardia) follow. Emotional disturbance and nervousness are also common symptoms.

Blood vessels: The thyroid is richly supplied with blood vessels, which are intimately associated with the follicles.  

 

THYROID GLAND

Human, 10% formalin,
A. H. & E.; B. periodic acid-Schiff and hematoxylin stains, 612 x.

 

Follicles: Structural units of the thyroid gland supported and separated by connective tissue. Note variation in size. A single layer of cells forms the follicle. Shape of cells reflects functional activity. Cells in these follicles are cuboidal with central, rounded nuclei, indicating normal activity.

In A, the colloid in the lumen of the follicle is not stained. In B, the colloid is specifically stained red with the periodic acid-Schiff method because of the chemical composition of colloid, which is a glycoprotein-iodine complex (thyroglobulin).  

 

PARAFOLLICULAR CELLS (C CELLS)
Thyroid gland

Rhesus monkey, glutaraldehyde, plastic section, H. & E.,
A. 119 x; B. & C. 169 x.

 

Parafollicular cells may be found intimately associated with thyroid follicles or as isolated or interstitial clusters of cells. They are not readily found in routine thicker sections of the thyroid gland. The photornicrographs above are from 1.5 µm plastic sections.

Although parafollicular cells (also known as light cells) appear, at the light microscopic level, to be in intimate contact with thyroid colloid, they are, in fact, separated from colloid by thin intervening processes of adjacent thyroid follicular cells.

Parafollicular cells synthesize and secrete the hormone calcitonin. This hormone lowers blood calcium levels by inhibiting bone resorption. The secretion of calcitonin results from elevation of blood calcium concentration above normal levels.  

 

PARATHYROID GLAND

A. Rhesus monkey, glutaraldehyde, H. & E., 47 x;
B. Human, 10% formalin, H. & E., 47 x.

 

Two cell types are found in the parathyroid gland. The most abundant type is the chief (or principal) cell, which secretes parathyroid hormone. Chief cells have a prominent nucleus, and a cytoplasm that stains variably and may be light or dark depending upon its secretory activity.

The second type, oxyphilic (acidophilic or eosinophilic) cells, occurs in small clumps and in fewer numbers. These cells usually have small densely staining heterochromatin and an oxyphilic cytoplasm whose perimeter is usually well defined. Oxyphilic cells usually increase in number with age but their specific function is unknown.

The parathyroids are essential for life. They control blood calcium and phosphate levels. A significant decrease in blood calcium results in tetany, abnormal twitching, and muscle spasms, caused by changes in excitability at the neuromuscular junction, and death.

Dietary addition of calcium and especially administration of parathyroid hormone relieves the abnormal spasms, preventing death of the organism.

Parathyroid hormone secretion is apparently regulated by blood calcium levels only in hypoparathyroidism, phosphate levels increase and calcium levels decrease. in hyperparathyroidism, blood phosphate is low and blood calcium levels are increased. Abnormal levels of calcium may result in abnormal deposition of calcium in the kidneys and muscle. Abnormally increased blood levels of calcium occur at the expense of bone, which may fracture as a result. Calcium removal from bone is related to osteoclastic activity, which is the site of action of parathyroid hormone.

The small parathyroid glands (4 to 5 mm in diameter) vary in number from three to six and are usually found on the posterior surface of the thyroid gland.

In the lower left corner, a few follicles of the thyroid gland can be seen.

 

PARATHYROID GLAND

Human, 10% formalin, H. & E., 162 x.

 

Parathyroid glands are essential for life. in the absence of parathyroid hormone, there is a pronounced decrease in blood calcium resulting in tetany, the intense, involuntary spasm of skeletal muscle.

Oxyphil cells: Occur in groups or nests among chief cells. Larger than chief cells, cytoplasm acidophilic. Oxyphils increase with age and are not found in all mammals. Function remains unknown.

Chief or principal cells: Much more numerous than oxyphil cells and functionally important. Nucleus round and centrally located. Cytoplasm homogeneous. Arranged in cords or plates separated by vascular channels. These cells produce parathyroid hormone, which is important in calcium metabolism.

Thyroid follicle: Seen adjacent to the capsule of the parathyroid gland

 

PANCREAS
Islet of Langerhans

Rhesus monkey, Helly's fluid, H. & E., 612 x.

 

Islet: Pale-staining area demarcated by arrows. Contains irregular clumps of cells. Separated from the acinar cells by a thin partition of reticular tissue. This endocrine gland elaborates the hormones insulin and glucagon, which are secreted into the rich capillary bed and carried via the portal system of veins into and through the hepatic lobules before reaching the general circulation. Insulin and glucagon constitute an important system for the regulation of blood glucose levels. This endocrine organ is essential for life. Dysfunction results in diabetes mellitus, a common disorder of man, characterized in its uncontrolled and most severe form by polyuria (frequent urination), polydipsia (excessive water intake), hyperglycernia (elevated glucose level in blood), glycosuria (sugar in the urine), ketonuria (ketones in the urine), acidosis (inability to buffer the blood at pH 7.2 to 7.4), wasting of the body and, if not treated, coma and death.

Acinar cells: Darker staining, irregular cells. Rich in cytoplasmic ribonucleic acid (RNA). Elaborate digestive enzymes, which are carried to, and are active in, the duodenum. Pancreatic enzymes break down partially digested food from the stomach into simple compounds. Carbohydrates, fats, and proteins are hydrolyzed by the enzymes amylase, lipase, and various proteases (including the nucleoproteases) secreted by the acinar cells.  

 

PANCREAS
Islet of Langerhans

Human, Helly's fluid, Mallory-azan stain, 612 x.

 

The islets of Langerhans were described by Paul Langerhans, a German physician, anatomist, and pathologist, in 1869. Although in routine histological preparations all of the islet cells appear to be similar, special methods reveal three types, alpha, beta, and delta.

Delta cell: Few in number compared to alpha or beta cells. Significance not well understood. Cytoplasm stains blue with Mallory-azan.

Beta cell: More numerous than alpha or delta cells. Produce insulin. Insulin increases cellular uptake of glucose and its conversion to glycogen. Beta cells may occur outside the islets. Granules are diffusely scattered in cytoplasm.

Pancreatic duct: Found interlobular connective tissue, lined by cuboidal to columnar epithelium. Size varies Collagen: In the interlobular connective tissue. Stains dark blue with Mallory-azan.

Alpha cells (not seen in this preparation) secrete the hormone glucagon, which effects the breakdown of liver glycogen and elevates the blood glucose level.  

 

ADRENAL GLAND

Rhesus monkey, Helly's fluid, H. & E., 50 x.

 

Capsule: A tough fibroelastic covering of the adrenal gland.

Cortex: Three concentric zones, each with a different cell arrangement, are shown. The adrenal cortex is essential to life. It controls the electrolyte and water distribution in the body and maintains proper carbohydrate balance.

Zona glomerulosa: Outermost narrow zone of the adrenal cortex. Deeply staining, densely packed nuclei. Elaborates aldosterone.

Zona fasciculata: The broadest zone of the adrenal cortex. Cells are arranged in long cords. Elaborates cortisol.

Zona reticularis: Innermost layer of adrenal cortex. Cells are arranged in irregular cords separated by sinusoids (clear spaces) giving the appearance of a meshwork. Stains deeper than zona fasciculata.

Medulla: Irregularly arranged mass of cells in cords. Highly vascularized. Rich in chrornaffin substance. Produces epinephrine and norepinephrine.  

 

SUPRARENAL (ADRENAL) GLAND
Chromaffin reaction

Human, Zenker's fluid, H. & E., 22 x.

 

This low-power micrograph illustrates the structural organization of the suprarenal gland. There are two major subdivisions: cortex and medulla.

The suprarenal cortex exhibits three distinctly different structural layers: (1) zona glomerulosa, (2) zona fasciculata, and (3) zona reticularis.

The zona glomerulosa synthesizes and secretes mineralocorticoids, primarily aldosterone, which maintains sodium and potassium (electrolyte) and water balance. Zona fasciculata, and probably zona reticularis, synthesize and secrete cortisone, cortisol, and glucocorticoids, which are concerned with the regulation of carbohydrate, protein, and fat metabolism. Small amounts of androgen and estrogen are also secreted by zona fasciculata and zona reticularis. The cortical cells do not store their steroid hormones.

The medulla constitutes the core of the gland. In this preparation, the medulla has a distinctly brown coloration. Zenker's* fixative contains potassium dichromate, which oxidizes catecholamines to a brown (melanin-like) color. The affinity of adrenal medullary cells for chromium salts results in what is called a chrornaffin reaction;. the cells that produce the reaction are called chromaffin cells or chromaffin granule-containing cells. Chromaffin granules contain either epinephrine or norepinephrine, a binding protein, and dopamine B-hydroxylase.

 

ADRENAL GLAND
ortex and medulla

Rhesus monkey, Zenker's fluid, H. & E., 612 x.

 

This plate illustrates the different zones of the adrenal gland, from the capsule to the medulla.

Capsule: A tough fibroelastic covering with delicate trabeculae extending into the substance of the gland.

Zona glomerulosa: Outermost narrow zone of the adrenal cortex. Cells arranged in ovoid groups without a significant lumen. Component cells are columnar with spherical, deeply staining nuclei. Richly supplied with blood. The zona glomerulosa secretes hormones concerned primarily with mineral metabolism. The mineralocorticoids are deoxycorticosterone and aldosterone.

Sinusoid: Sinusoids arise from multiple arterioles in the capsule. Course between cell cords.

Zona fasciculata: The middle and broadest zone of the adrenal cortex. Cells are regularly arranged in parallel cords, one to two cells thick. Component cells are cuboidal, frequently containing two vesicular nuclei. Vacuoles seen in some cells represent dissolved lipid droplets. Cholesterol is chiefly present in this zone. Cell cords are surrounded by sinusoids. Secretes the glucocorticoids, cortisone and cortisol.

Zona reticularis: Innermost layer of the adrenal cortex. Cells are arranged in irregular cords, are smaller than those of the zona fasciculata, and stain darker. Nuclei stain deeply. Sinusoids separate cell cords. Secretes the same glucocorticoids as the zona fasciculata. These hormones participate in carbohydrate, protein, and fat metabolism.

Demarcation zone: Shows zone of transition from zona reticularis to medulla.

Medulla: Polyhedral cells arranged in anastomosing cords. Prominent nuclei. Contain chromaffin granules, precursors of epinephrine and norepinephrine. Richly supplied with blood.

The adrenal cortex is essential for life, and, through its hormones, is involved in numerous body functions. These activities include the maintenance of water and electrolyte balance, carbohydrate metabolism, and the normal functioning of connective tissue cells. Destruction or removal of the cortex results in Addison's* disease unless cortical hormones are given to the patient.

The adrenal medulla is not essential for life. The hormones of the adrenal medulla influence the metabolic rate and cardiovascular function and induce lipolysis and the release of fatty acids from adipose tissue, and opiate-like pepticles, including leu- and met-enkephalin. Medullary cells store their hormones in the form of granules.

During normal activity only a small amount of catecholamine is secreted, but this is greatly increased during stressful periods of intense emotional reaction. It is believed that epinephrine and norepinephrine are secreted by two different types of cells. The epinephrine secreting cells are primarily located around the distal ends of sinusoids draining the adrenal cortex, whereas norepinephrine-secreting cells are associated with capillaries of the direct arterial supply of the medulla. All adrenal medullary cells are innervated by cholinergic nerve terminals of preganglionic sympathetic neurons.

Chromaffin cells are also found in paraganglia, which are collections of catecholamine-secreting cells associated with autonomic ganglia.

 

CHROMAFFIN CELLS
Heart coronary sulcus

Human, Helly's* fluid, Mallory*-azan stain, 612 x.

 

Scattered islands of chromaffin cells are found in the subepicardial connective tissue of the coronary sinus. Note relation to capillaries into which it is believed they pour their secretion of catecholamine. Chromaffin cells are usually closely associated with sympathetic nerve fibers and ganglion cells. The brownish coloration is due to oxidation of chromaffin granules by potassium dichromate in the fixative used in this preparation. Because of their structural similarity to cells of the adrenal medulla, it is assumed that chromaffin cell secretion augments action of the sympathetic nervous system by elevating blood sugar, increasing heart rate, raising blood pressure, and generally preparing the organism for emergency situations ("flight or fight").

 

PINEAL GLAND

Human, 10% formalin, H. & E., 162 x.

 

The pineal gland is made up of plates of cells separated by septa. Two cell types are recognized by special techniques, the more common parenchymal cell and the neuroglial (astrocyte-like) supporting cell. The latter is found between clusters of parenchymal cells. The two cell types cannot be distinguished in ordinary H. & E. preparations. Concretions or brain sand characterize the pineal gland and increase with age. They have a mineralized organic matrix and, at high magnification, appear lamellated.  

 

 

Section 14: Male Reproductive System

The male reproductive organs include the primary sex glands, the testes, the various excretory ducts, the accessory glands, and the penis.

The testes, contained within serous cavities within the scrotum, are compound tubular glands serving both exocrine and endocrine functions. The exocrine function is the formation of the mature male germ cells (the spermatozoa), and the endocrine function is the production of the male sex hormone, testosterone. The testes are ovoid glands approximately 4.5 cm in length and are covered by a thick capsule (tunica albuginea) composed primarily of collagenous connective tissue with some elastic fibers. The tunica albuginea thickens posteriorly to form the mediastinum testis, which is an area where ducts, blood vessels, and nerves leave or enter. From the mediastinum, thin, incomplete, and branching fibrous septa and associated blood vessels radiate into the testis and divide it into about 250 lobules. These incomplete pyramidal spaces contain the seminiferous tubules surrounded by a stroma rich in vascular and cellular elements. Of particular interest in the stroma are the interstitial cells (of Leydig), which secrete the male sex hormone, testosterone. These cells lie within the intertubular or interstitial space, in close proximity to vascular and lymph vascular channels.

The lymphatic endothelium, fibroblasts, and, in many mammalian species, several layers of epithelioid cells possessing many of the fine structural features of smooth muscle ensheath the seminiferous tubules and constitute their tunica propria (boundary) tissue. Apical to the basement membrane lies the seminiferous epithelium, which consists of cells of the germ line that will by means of meiotic divisions and a complex process of cytodifferentiation give rise to the haploid male gamete, the spermatozoon. The production of sperm by the epithelium proceeds in an orderly, cyclic fashion from the base of the epithelium' to its apex, as well as in a coordinated, wave-like manner along the length of the tubule. The mitotic and meiotic divisions in the more basal aspects of the epithelium displace the developing sex cells progressively farther away from the basement membrane, with the more mature sex cells occupying, therefore, the most apical (luminal) position.

Cytoplasmic processes of a second population of cells, termed Sertoli cells (also sustentacular or nurse cells), extend from the basement membrane to the most apical portion of the seminiferous epithelium. Sertoli cells are somatic cells that establish an intimate morphological and physiological relationship with each of the developing germ cells, from the earliest, most primitive gonial cell at the basal lamina, to the apically situated mature spermatids ready to be shed into the tubular lumen. Because of the cyclic nature of the process of spermatogenesis occurring in all seminiferous tubules and the microtome knife's random entry into this cycle, the student must be reconciled to studying many sections of seminiferous tubules in order to appreciate and correctly identify the named germ cells of the epithelium and their place in the process of spermatogenesis.

As in other renewing epithelia with which the student is already familiar, the most primitive, regenerative cells lie at the basement membrane of the epithelium. In the testis, these cells are termed the spermatogonia (and are diploid). They divide mitotically and renew the gonial stem cell population. However, a subpopulation of spermatogonia is programmed to enter meiotic prophase and thus become primary spermatocytes. As daughter cells of spermatogonia, the primary spermatocytes reside just apical to the layer of spermatogonia. These cells, the largest germ cells, are recognized by their distinct meiotic nucleus, as well as by their position near the spermatogonia at the base of the epithelium. Primary spermatocytes undergo first meiotic division, giving rise to secondary spermatocytes; these cells occupy a place within the seminiferous epithelium just apical to the primary spermatocytes. The secondaries are short-lived (and thus not frequently observed within sections of the germinal epithelium); secondaries complete second meiotic division and give rise to round spermatids, which occupy an adluminal site within the epithelium. A complex process of cytodifferentiation ensues, whereby these round cells are transformed into elongate spermatids and, finally, spermatozoa.

There exists no "free space" within the seminiferous epithelium, all interstices being filled with Sertoli cell cytoplasm. Because of the attenuated nature of the Sertoli cell processes within the seminiferous epithelium, it is not possible to distinguish cell-to-cell boundaries between Sertoli cells. For this reason, the Sertoli cells were considered prior to the advent of electron microscopy to be syncytial. Indeed, the occluding tight junctions between Sertoli cells are now recognized as the morphological basis for the "blood-testis" (blood epithelial) barrier. Because of the placement of the tight junctions just apical to the gonial stem cells, the seminiferous epithelium is segregated into a basal compartment, open to the interstitium, and an apical compartment containing the meiotic cell line (considered non-self by the body's immune surveillance system). Thus, the haploid spermatids develop within an immunologically privileged compartment maintained by the Sertoli-Sertoli cell occluding junctions.

Within the seminiferous tubules (also termed tubuli contorti in recognition of the convoluted course that they describe with in lobules), sperm are transported along their lengths by means of a remarkably high fluid flux across the epithelium. Upon reaching the epididymis, the spermatozoa undergo additional physiological maturation (capacitation) and gain fertilizing capacity and motility; there, also, the testicular fluid is resorbed.

Leading from the seminiferous tubules are the tubuli recti or straight tubules lined only with columnar cells apparently derived from Sertoli cells. The straight tubules open into the rete testis, a network of irregular, anastomosing channels lined with a simple cuboidal or columnar epithelium, which may possess a single flagellum. The efferent ductules (10 to 15 in man) emerge from the rete testis and join to form a portion of the head of the epididymis. Histologically, each efferent duct presents a stellate luminal profile, reflecting an epithelium in which ciliated columnar cells alternate with non-ciliated cuboidal cells. The epididymal duct in man is a highly coiled tube about 5 m in length. It is lined with a pseudostratified columnar epithelium with tall columnar cells and rounded basal cells. The columnar cells possess long non-motile stereocilia. The ductus deferens is continuous with the epididymis. The lumen of the ductus deferens increases in size, and the wall thickens as it extends distally. Near the prostate gland, the ductus is enlarged to form the ampulla; immediately thereafter, it is joined by the duct from the seminal vesicle. The duct formed by this confluens, the ejaculatory duct, courses through the substance of the prostate gland and empties into the prostatic urethra. It is lined with a pseudostratified or simple columnar epithelium.

Three major accessory sex glands are associated with the male reproductive system: the seminal vesicles, the prostate gland, and the bulbourethral (Cowper's) glands. The seminal vesicles develop as outgrowths of the ductus deferens. Each is a glandular sac honey-combed by thin branching folds of the mucosa lined with a pseudostratified columnar or cuboidal epithelium. The prostate gland encircles the urethra adjacent to the neck of the bladder and is formed of 30 to 50 tubuloalveolar glands grouped into lobes. The glandular epithelium consists of simple cuboidal or columnar cells. Prostatic concretions (corpora amylacea) are prominent constituents of the alveoli. The bulbourethral glands are compound tubuloalveolar glands that secrete a clear, viscous mucoid product. The secretory epithelium is cuboidal to columnar. The ducts of the gland enter the cavernous urethra. The secretions of each of these glands are important physiological components of semen.

The penis serves as an outlet for urine and semen and as a copulatory organ. The penis is made up of three cylinders of erectile tissue. These include the corpus spongiosum, which enlarges distally into the glans penis; parallel with, and dorsal to, the corpus spongiosum are the paired corpora cavernosa, which extend distally to the glans. The corpora cavernosa are united distally by a median partition, the pectiniform septum. All three structures are surrounded by a thick fibrous tunica albuginea and a subcutaneous connective tissue layer covered by a thin skin.

Some names associated with the male reproductive system follow: Cowper was a seventeenthcentury English anatomist, von Leydig, a nineteenth-century German anatomist, and Sertoli, a nineteenthcentury Italian histologist.

 

TESTIS

Human, 10% formalin, H. & E., 3.4 x.

 

The integrity of the delicate, tubular parenchyma of the male gonad is maintained by the robust connective tissue capsule of white fibrous connective tissue, the tunica albuginea, and the septa, which project interiorly, dividing the organ into lobules. The lobules contain the seminiferous tubules, lined with the specialized seminiferous epithelium, which gives rise to the male gametes, the spermatozoa. In addition, a posterior connective tissue mass (the mediastinum) projects to the interior of the organ and provides an avenue for the egress of fluid and spermatozoa from the seminiferous tubules. Irregular, epithelium-lined, anastomosing channels form a network, the rete, within the mediastinum. From these channels, the ductuli efferentes arise, pierce the tunica albuginea, and subsequently form, in man, the first portion of the epididymis.

Neither the seminiferous tubules nor the intratesticular duct system have significant muscular coats, or motile cilia by which to move the tubular contents. This underscores the role of fluid flux in moving the spermatozoa through the initial portion of the excurrent duct system. The ductuli efferentes are the sole portion of the male duct system that bears motile cilia on the lining epithelial cells.

The testis is invested on its anterior and lateral aspects by a serous envelope, the tunica vaginalis, which is a peritoneal vestige carried with the testis in its descent to the scrotum. The testis is cushioned by and glides within this serous envelope, relieving compression, to which the organ is exquisitely sensitive.

 

TESTIS
Periphery

Monkey, glutaraldehyde; 1.5 µm plastic-embedded section, H. & E., 84 x

 

The testis is enclosed by a robust dense white fibrous connective tissue capsule, the tunica albuginea, designated for its glistening white appearance in the unfixed state. Vascular elements pierce the tunica albuginea and travel within septa that are continuous with the intertubular connective tissue of the organ; posteriorly, the tunica albuginea is specialized as the mediastinum testis. Deep to the tunica, note the many profiles of seminiferous tubules, which are lined with the complex seminiferous epithelium. The epithelium is seen to better advantage in later plates at higher magnification.

 

TESTIS

Human, 10% formalin, H. & E., 162 x.

 

This is a section of the testis showing the seminiferous tubules separated by interstitial connective tissue. The seminiferous tubules are lined by a stratified epithelium, the germinal epithelium, composed primarily of sex cells with some supporting cells. The epithelium rests on a basement membrane that varies in thickness with age. The lumina of the seminiferous tubules contain mature sex cells (spermatozoa). Seminiferous tubules are separated by an interstitial stroma made up of loose connective tissue and containing the interstitial cells of Leydig. These large ovoid cells that occur in groups secrete testosterone, the male sex hormone.

 

SEMINIFEROUS TUBULE

Rhesus monkey, Helly's fluid, iron hematoxylin and orange G stains, 612 x.

 

In the seminiferous tubule, spermatogenic cells are arranged in orderly layers between the basement membrane and the lumen.

Spermatogonia: Located directly above the basement membrane. Spherical nucleus. Spermatogonia are the germ cells from which spermatozoa ultimately arise. They are the only sex cells present before onset of puberty. They contain 23 pairs of chromosomes.

Primary spermatocytes: Lie in the next layer, deep to the spermatogonia. Largest germ cells. Nuclei are large and vesicular with condensed chromatin. Chromatin may appear as elongated spiremes, i.e., irregularly disposed chromatin filaments. Primary spermatocytes divide by meiosis. Meiosis is nuclear division in which the diploid chromosome number (23 pairs) is halved to the haploid number (23 single set) in the formation of sex cells.

Round spermatids: Adjacent to the lumen. Small in size. Spermatids constitute the last stage in the transformation to spermatozoa.

Mature spermatids: Heads are located near Sertoli cells, and tails project into the lumen. Heads are transformed nuclei of round spermatids. Will be released into lumen as spermatozoa.

Sertoli cells: Supporting cells of the testicular epithelium, which were first described by the Italian physiologist Enrico Sertoli in 1865. Tall columnar cells extend from the basement membrane to the lumen. Nucleus ovoid in shape with prominent nucleolus. Cell borders are difficult to outline in this preparation.

The process of spermatogenesis from spermatogonia to mature spermatozoa requires about 64 days in man.

 

TESTIS
Seminiferous epithelium

Monkey, glutaraldehyde, 1.5 µm plastic section, H. & E., 216 x.

 

Plastic-embedded, 1.5 µm, sections reveal to particular advantage the cell types constituting the seminiferous epithelium. The longitudinal section of a tubule in the center of the field contains many such representative cell types. Note the spermatogonia on the basement membrane. The largest of the germ cells, the primary spermatocytes with their characteristic meiotic prophase nuclei, occupy the region of the epithelium just apical to the basal layer of spermatogonia. The infrequently seen secondary spermatocytes are intermediate in size and placement within the epithelium between primary spermatocytes and spermatids.

Most apically situated within the epithelium are the spermatids, which possess either the smallest round nuclei of any cell of the epithelium or elongate, condensed nuclei, depending upon their stage of maturation toward mature spermatozoa.

The eosinophilic cytoplasmic remnants resulting from the differentiation of round spermatids to elongate spermatids are seen at the luminal surface and are termed residual bodies. Sustentacular cells, the Sertoli cells, phagocytize much of this residual cytoplasm. The nuclei of Sertoli cells are identified by their distinctive urn shape and prominent nucleolus. Note also how clusters of spermatids develop synchronously, enveloped within the apical extensions of Sertoli cell cytoplasm.

Adjacent seminiferous tubules share a common lamina propria; an area of interstitial connective tissue is shown where three adjacent tubules meet. Leydig cells are commonly found in such areas but are not present in this particular section.  

 

TESTIS
Seminiferous epithelium

Monkey, glutaraldehyde, 1.5 µm plastic section, H. & E., 216 x.

 

The process of spermatogenesis is best appreciated from the study of the various stages represented in adjacent sections of a seminiferous tubule. Occasionally, however, a tubule is cut in fortuitous tangential section in such a manner that the processes occurring along its length can be appreciated. Such is the case in this figure, in which, toward the upper left of the section, primary spermatocytes are seen; these are the daughter cells of the basally situated spermatogonia. Note their distinctive meiotic prophase nuclei. Division of primary spermatocytes (meiosis 1) gives rise to daughter cells, the secondary spermatocytes. These, in turn, complete the second meiotic division, giving rise to haploid daughter cells, the round spermatids (upper right). Over time, the round spermatids complete cytodifferentiation and become mature, elongate spermatids. These cells lie most apically within the epithelium, among the cytoplasmic residual bodies. Mature spermatids shed into the tubular lumen are termed spermatozoa. Other tubules (such as the one in the lower right portion of the figure) may be cross-sectioned tangentially such that the plane of section passes predominantly through one cell population of the seminiferous epithelium. This accounts for the absence of a luminal profile in this tubule and the predominance of round spermatids.

 

INTERSTITIAL CELLS
Testis

Human, 10% formalin, H. & E., 612 x.

 

This figure shows parts of two seminiferous tubules separated by a connective tissue sheath. Within this connective tissue sheath are embedded large ovoid cells, the interstitial cells of Leydig. These occur in groups, have a rounded, large eccentric nucleus with a prominent nucleolus and a vacuolated cytoplasm, which results from the loss of lipid droplets and crystals during tissue processing. The interstitial cells are the source of testosterone, the male sex hormone, whose functions include the development and maintenance of secondary sex characteristics and the structure and function of the male accessory organs, the development of psychosexual behavior (in part) in the mature male, a role in protein metabolism, and the regulation of the output of the pituitary gonadotropic hormone, interstitial cell-stimulating hormone (ICSH). The seminiferous tubules shown reveal part of their contents, spermatogonia, spermatids, and Sertoli cells

 

TESTIS
Straight tubules and rete testis

Human, 10% formalin, H. & E., 50 x.

 

The seminiferous tubules of the testis (tubuli contorti) open into the straight tubules (tubuli recti). The latter are short, straight tubules lined with a single layer of tall columnar Sertoli cells. The straight tubules empty into a system of irregular, anastomosing epithelium-lined cavernous spaces, the rete testis, located in the dense connective tissue of the mediastinum.  

 

EPIDIDYMIS

Human, 10% formalin, H. & E., 54 x.

 

The epididymis, a highly coiled duct approximately 6 m in length in man, is firmly adherent to the gonad. in histological preparations, therefore, it is not unusual to observe sections of both testis and epididymis. The duct is divided anatomically into three major portions (head, body, and tail) and by histological criteria, several further subdivisions may be recognized. Because of the tight coiling of the duct, histological sections invariably reveal many tubular profiles in different planes of section.

The portion of the epididymis shown here is from the distal portion of the head, in which the epithelial lining of the duct is a tall pseudostratified columnar epithelium, lacking goblet cells. The tall columnar cells bear prominent (non-motile) stereocilia on their apical surfaces, facilitating the resorption of testicular tubular fluid in which the spermatozoa are transported to the organ. A thin coat of smooth muscle surrounds the duct and is readily differentiated from the loose connective tissue surrounding the coils of the duct. A packed mass of non-motile sperm is seen within the lumen of the epididymis. In this preparation, striated muscle fibers belonging to the cremaster muscle are seen investing the organ. This muscle of the spermatic cord enables the gonad to be retracted within the scrotum toward the, abdomen.

 

EPIDIDYMIS

Human, 10% formalin, H. & E., 345 x.

 

 the tall pseudostratified columnar epithelium is demonstrated to advantage. The luminal cells bear remarkable numbers of highly developed stereocilia on their apical surfaces. The lumen of the duct contains a packed mass of spermatozoa. Sperm gain motility and fertilizing capacity during their passage through the epididymis.

 

 

VAS DEFERENS
Cross section

Dog, 10% formalin, H. & E.,
A. 50 x., B. 162 x., C. & D. 612 x.

 

The ductus deferens (A) is the most prominent component of the spermatidicord and is readily palpated through the scrotal skin. Within the cord, the vas lies in company with a plexus of veins, the pampiniform plexus (so-named for the resemblance of the tortuous veins to tendrils of a vine), branches of the spermatic artery and nerves of the spermatic plexus, and the cremaster muscle.

The mucosa of the vas deferens consists of a prominent pseudostratified columnar epithelium bearing conspicuous stereocilia (B and C and is underlaid by a lamina propria, which abuts the robust muscular components of the organ without a defined submucosal layer. The musculature of the organ is organized into sparse inner longitudinal, prominent middle circular, and well-defined outer longitudinal layers. The vas is surrounded by an adventitia throughout its length. The organ's overall topography, together with the tendency of its mucosa to be thrown into deep folds upon fixation, causes this organ to be readily mistaken for other muscular ducts of the body (i.e., ureter and oviduct). Careful examination of the lining epithelium will confirm identification of each; in addition, the vas may exhibit a packed mass of luminal sperm (D). At the time of ejaculation, the vas rhythmically contracts, conveying spermatozoa stored within the terminal portion of the epididymis and along the length of the vas itself to the ejaculatory ducts. Electron-microscopic studies of the vas have confirmed that its fine structure differs significantly along its length and that the description of the vas as a passive conduit for sperm is inadequate. The scrotal ductus deferens (frequently referred to clinically as the vas) is the common site of ligation or excision of a portion of the organ in the surgical procedure of vasectomy.  

 

SEMINAL VESICLE

Monkey, 10% formalin, H. & E., 21 x.

 

The seminal vesicles arise as tortuous diverticula of the vas deferens and therefore present histologically as multiple sections through a tubular structure. The wall consists of adventitia, muscularis, and elaborate mucosa, thrown into several orders of branching folds. The folds, like villi of the intestinal tract, are underlaid by a tunica propria of loose connective tissue. Secretions produced by the epithelial cells are delivered to a central luminal channel*, the extent of which is largely obscured by the tortuosity of the tube. Lobulation of the organ by septa from the adventitia is incomplete and inconspicuous

 

SEMINAL VESICLE

Human, 10% formalin, H. & E., 162 x.

 

The seminal vesicle is a diverticulum of the adjacent ductus deferens with a remarkably folded mucosal lining of pseudostratified cuboidal or columnar epithelium projecting into the lumen. The lumen frequently contains acidophilic rounded secretion masses. Underlying the epithelial lining is a thin supporting sheath of connective tissue, the lamina propria, which extends into the mucosal folds. Beneath the lamina propria is a coat of smooth muscle fibers consisting of an inner circular layer and an outer longitudinal layer.  

 

SEMINAL VESICLE

Monkey, 10% formalin, H. & E., 269 x.

 

The complex ramifications of the mucosa are shown; the folds are lined by a columnar to pseudostratified columnar epithelium. Epithelial development is androgen-dependent. The height of the epithelium in this specimen suggests ample hormonal stimulation and active secretion. Post-mortem migration of spermatozoa (asterisks) into the recesses and pockets formed within the mucosal lining gave rise to the earlier erroneous interpretation that the seminal vesicle serves as a reservoir or storage depot for sperm. The gland is in fact a major secretory accessory sex gland, the secretions of which, in man, contribute the majority of the seminal volume.  

 

PROSTATE GLAND

Human, 10% formalln, H. & E., 162 x.

 

Stroma: Abundant and continuous with the gland capsule, it constitutes one third to one fourth of the gland volume and is composed of fibroelastic connective tissue intermixed with smooth muscle fibers. Glands are embedded in the stroma.

Tubuloalveolar glands: Irregular, large lumen, widely spaced tubules with alveolar extensions, which vary greatly in shape and size. Epithelial lining in tissue sections is simple cuboidal to columnar in shape, depending upon physiological state.

Prostatic concretions: Corpora amylacea, acidophilic condensed secretions of prostatic glands. They may be lamellated and increase in number with advancing age. Source of prostatic calculi.

The prostate is located at the origin of the urethra (which it surrounds), adjacent to the urinary bladder. The prostate secretes a thin, opalescent, slightly acid fluid, which contains several enzymes, including diastase and proteases, and citric acid. The smaller prostatic concretions are found in the prostatic fluid.

 

PENIS
Cross section

Human, 10% formalin, carmine stain, 2 x.

 

The penis is formed primarily of three cylindrical masses of erectile tissue. Note the paired corpora cavernosa and the ventrally placed corpus spongiosum (corpus spongiosum urethrae) containing the urethra. A dense collagenous tissue capsule, the tunica albuginea, surrounds the corpora cavernosa. This capsule fuses in the midline to form the pectinate septum, which is thickest and most complete near the root of the penis. The tunica albuginea of the corpus spongiosum is thin. Each corpus consists of a network of cavernous vascular sinuses lined with endothelium, separated by fibromuscular trabeculae composed of connective tissue and smooth muscle fibers.

The three corpora are encompassed by a common, loose connective tissue fascia rich in elastic fibers and a thin skin. Note the dorsal vessels (arteries and veins) of the penis, located in the fascia, which are part of the complicated blood supply of this organ.

 

PENIS
Corpus cavernosum

Human, 10% formalin, H. & E., 162 x.

 

The erectile tissue of the corpus cavernosum of the penis is composed of cavernous spaces separated by fibromuscular septa or trabeculae. The latter are extensions of the tunica albuginea, the fibrous coat that surrounds the corpus. The cavernous spaces are filled with blood, and the engorgement of these spaces results in the erection of the penis. Note the central (deep) artery, which traverses the corpus cavernosum. This artery gives rise to the spiraling helicine arterioles that open into the sinuses. The central artery is the principal vessel for filling the sinuses during erection.

Adjacent to the central artery, note the nerve cut in cross section. The penis is richly supplied with spinal, sympathetic, and parasympathetic fibers. The autonomic fibers innervate the smooth muscle in the arterial wall and trabeculae.  

 

 

Section 13: Female Reproductive System

The female reproductive system includes the ovaries, the uterine tubes, uterus, vagina, and external genitalia. The ovaries perform both an exocrine function by producing ova and an endocrine function by producing estrogen and progesterone.

The ovaries are ovoid structures, approximately 3 cm in length, lying on each side of the uterus within the pelvis. The ovary consists of a cortical zone composed of a specialized stroma, which contains follicles with ova. In the mature functional ovary, many follicles are quiescent, whereas others exhibit a wide range of histomorphology, depending upon their stage of maturation or regression. The medulla consists primarily of connective tissue and an extremely rich vascular supply.

Immature ova or oocytes are spherical cells, about 30 µm in diameter; when fully mature, they have increased in size to about 120 µm and are designated ova. The nucleus of an oocyte is large and vesicular and contains a prominent nucleolus. The cytoplasm is rich in nutritive material, the yolk.

The events of meiosis and the maturation stages of the female gamete parallel those events occurring in spermatogenesis discussed in Section 14, entitled Male Reproductive System. During human fetal development, the primordial germ cells migrate to and are incorporated within the developing ovary and are termed oogonia. The oogonia multiply by mitosis, but early in fetal life, they enter meiosis. However, the meiotic events are arrested by a mechanism not understood in prophase (diplotene stage) of the first meiotic division. These cells, about 40 µm in diameter and termed primary oocytes, are enclosed within a single layer of squamous cells, forming a primordial follicle. The primordial follicles in each human fetal ovary number more than 200,000 and decline in number until very few or none remain at about the 50th year.

The transition from an inactive primordial follicle to a growing and maturing primary follicle involves changes in the oocyte, the follicular cells, and the adjacent connective tissue. As the oocyte enlarges, the single layer of follicular cells increases in size through mitotic division and gives rise to cells (granulosa cells) that eventually form a stratified epithelium termed the granulosa. A distinctive feature of the multilaminar follicle is the elaboration of a highly refractile zona pellucida interposed between the oocyte and granulosa cells; the zona is secreted by both the egg and surrounding follicular cells. Concomitant with the development of the granulosa cells, a sheath of stromal cells (theca folliculi) develops around the follicle and subsequently forms two layers. The inner layer exhibits a well- developed capillary plexus and secretory cells and is termed the theca interna. The cells of the theca interna are believed to secrete androstenedione, which is subsequently converted to estradiol by the granulosa cells. Secondary follicles can be identified when they are about 0.2 mm in diameter and are recognized by the presence of irregular spaces among the granulosa cells filled with a clear liquid (liquor folliculi), which increases with continued growth of the follicle. Eventually, the oocyte comes to be eccentrically placed within the follicle upon a pedestal of follicular cells, the cumulus oophorus. The oocyte is intimately surrounded by a crown of follicular cells, the corona radiata. The cumulus projects into a single large fluid-filled space, the antrum, formed from the coalescence of the smaller spaces noted previously.

Even after the primary oocyte has reached full size, the follicle may continue to enlarge until it reaches approximately 10 mm in diameter. Follicles that have matured to maximal size, exhibit a large antrum, and extend through the entire thickness of the cortex are termed graafian follicles*. just prior to ovulation, a bulge on the surface of the ovary (the stigma) marks the site where ovulation will occur. The growth of a primordial follicle to full maturity takes about 10 to 14 days. The thecae folliculi, particularly the theca interna, reach their highest development in relation to the mature follicle.

At mid-menstrual cycle (approximately day 14), the surge of pituitary luteinizing hormone (LH) induces ovulation. At this time, the primary oocyte's first meiotic division occurs, resulting in the formation of the first polar body and the secondary oocyte. In the human female, the secondary oocyte completes its second meiotic division at the time of fertilization, and the male and female haploid genomes fuse in the formation of the zygote.

Following ovulation and discharge of the liquor folliculi and the oocyte within its cumulus mass, the walls of the follicle collapse and the granulosa cell lining becomes folded. Rupture of blood vessels in the theca interna is associated with bleeding into the partially collapsed follicle, and a clot is formed. The cells of the granulosa layer and the theca interna undergo transformation and are renamed granulosa lutein and theca lutein cells, respectively. These changes in the follicle following ovulation result in a new but transitory organ, the corpus luteum (yellow body, for its appearance in fresh specimens). The corpus luteum secretes the hormone progesterone. If the ovulated oocyte fails to be fertilized, the corpus luteum remains functional for only about 14 days and then regresses and is reduced eventually to a scar within the ovary termed the corpus albicans (white body). In the event of fertilization, the corpus luteurn enlarges and persists as a functional endocrine gland throughout most of the pregnancy but begins to involute after the sixth month. Its ultimate fate after the termination of pregnancy is to become a large corpus albicans.

Most follicles never develop into mature follicles, since that number is limited to about 400 (or 1 of every 1000 follicles) during the reproductive span of the human female. The process by which follicles degenerate and disappear is little understood and is termed follicular atresia. This process can begin at any stage of follicular development. The smallest follicles leave no trace of their dissolution, but the larger follicles may leave a remnant of the zona pellucida as a persistent marker within the ovary. In larger secondary follicles, the earliest signs of atresia include the loosening and shedding of the granulosa cells, the invasion of the granulosa layers by vascular tissue and wandering cells, and the collapse or partial collapse of the follicle.

At the time of ovulation, the oocyte is shed upon the surface of the ovary, from which it must be transported to the interior of the ovarian (fallopian) tube. The oviduct possesses a highly specialized, flared terminal portion, the infundibulum, which bears long, frond-like extensions of the mucosa (termed fimbriae), which sweep over the surface of the ovary; the ovulated oocyte within its cumulus mass is transported by means of ciliary action along the surface of the fimbriae toward the ostium, or opening of the oviduct. The ostium leads to the second portion of the oviduct, the ampulla, which is the duct's dilated mid-portion where fertilization usually occurs. A constricted isthmic portion joins the ampulla to the uterine wall; the length of the oviduct that passes through the wall of the uterus is termed the intramural portion of the organ. The epithelium lining the oviduct is principally simple columnar; many of the lining cells are ciliated. Transport of the cumulus and oocyte within the oviduct is facilitated through vigorous peristaltic action of the oviduct; the muscularis is composed of two layers of smooth muscle, which become progressively more well developed as the uterine tube approaches the uterus.

The uterus lies within the pelvis in relation to the bladder anteriorly and the rectum posteriorly and is a hollow pear-shaped organ that opens into the vagina. The uterus is composed of a mucosa, given the special name of endometrium; a muscularis termed the myometrium; and a serosa, or perimetrium. The uterine mucosa undergoes cyclic changes, which are synchronized with ovarian secretory activity. The surface epithelium is simple columnar with patches of ciliated columnar cells. Uterine glands, lined with a similar columnar epithelium, open to the surface and secrete mucus. The endometrial stroma has a framework consisting of reticular fibers and stromal cells. Lymphocytes and granular leucocytes are also found in the stroma. The endometrium is composed of two parts, the superficial functionalis, which undergoes changes during the menstrual cycle and is shed during menstruation, and the basalis, which does not undergo cyclic changes and remains intact during menstruation.

The myometrium is a thick coat containing smooth muscle and abundant connective tissues. The smooth muscle of the uterus, in response to female sex hormones, undergoes cyclic variation in length and diameter and in functional activity. Three layers of smooth muscle are recognized: an inner longitudinal layer, a middle circular and oblique layer, and an outer longitudinal layer.

The following cyclic changes occur in the uterine endometrium during an idealized 28-day menstrual cycle: (1) The proliferative or estrogenic phase extends from about day 4 to day 14 of the cycle. This period involves re-epithelialization of the denuded endometrial surface and growth in thickness of the endometrium and glands. The glands are initially straight but begin to coil toward the end of this phase. Estrogen is the dominant hormonal influence during this phase. (2) The secretory, progestational, or luteal phase constitutes days 15 to 28 of the cycle. During this period, the uterine glands become highly coiled and , irregularly sacculated in the middle of the endometrium. The glandular epithelium secretes a mucoid fluid rich in glycogen. The endometrium becomes edematous and may reach a thickness of 5 mm.

Progesterone is the dominant hormonal influence during this phase. On day 27 or 28, the uterus enters the ischemic phase, during which the arterial supply constricts intermittently. At this point, glandular secretion is interrupted. (3) The menstrual phase involves the extravasation of blood and the detachment of patches of hemorrhagic endometrium until the entire functionalis is sloughed. The basal layer remains intact during this phase and is the source of the regenerating functional layer during the ensuing proliferative phase. The menstrual phase lasts from days 1 to 4 or 5.

The outlet of the uterus is the vagina, a fibromuscular sheath lined with thick stratified squamous epithelium. The underlying lamina propria is also thick and contains numerous lymphocytes and other wandering cells that invade the epithelium. The muscularis is irregularly arranged in two layers: an inner circular or spiral layer and an outer longitudinal layer. The vagina does not possess a muscularis mucosae or glands in the lamina propria. The adventitia is a dense collagenous tissue that merges with the adventitia of the bladder and rectum and is highly vascular.

In pregnancy, an important but temporary organ, the placenta, is formed in the uterus. The placenta is derived from both maternal (uterine) and fetal components. The maternal component is the endometrium; the fetal contribution consists of the chorionic plate and its branching villi, which are of two types: (1) the anchoring villi, which extend from the chorionic plate to the decidua basalis; and (2) the free villi, formed by branches from the anchoring villa. A villus has a fibromuscular core with fetal blood vessels and is covered by an epithelium termed the trophoblast. The trophoblast is arranged in two layers: (1) an outer layer without cell boundaries, the syncytiotrophoblast; and (2) an inner cuboidal cell layer, the cytotrophoblast. In the later stages of pregnancy, the cellular layer disappears. The source of human chorionic gonadotropin (HCG) and other placental hormones is the syncytiotrophoblast.

The fetal and maternal blood do not mix within the placenta. In the human, the fetal blood is contained entirely within small vessels and capillaries, whereas the maternal blood within the placenta leaves arterial vessels and gains free access to the space surrounding the fetal villi. Thus, what is termed the maternal blood lake is formed. It is across this interface of trophoblastic epithelium and maternal blood that oxygen, carbon dioxide, nutrients, and waste products are exchanged. Maternal blood is drained by veins from the intervillous spaces of the placenta.

 

OVARY
Overview

 

Monkey, glutaraldehyde, 1.5 µm, plastic section, H. & E., 47.5 x.

 

In this low magnification micrograph of primate ovary, the differentiation of the organ into cortical and medullary regions is seen. The highly vascular medulla is overlaid by a cortex in which various stages of follicular maturation may be identified. For example, the primordial follicles are most numerous and lie peripheral to growing follicles. Mature follicles (not shown here), which eventually ovulate, will have overgrown the entire width of the cortex. Follicles that have initiated growth but that have regressed are indicated by arrows and are termed atretic follicles.

The outer connective tissue investment, the tunica albuginea, is covered with a thin epithelium of peritoneal origin, the so-called germinal epithelium, named for its earlier, erroneously conceived role of seeding the ovary with germ cells.

 

Rabbit, 10% formalin, H. & E., 162 x.

 

Germinal epithelium: Forms the surface layer of the ovary and consists of a specialized peritoneal mesothelium. Misnomer, since there is no convincing evidence that it is the source of germ cells.

Stroma: Connective tissue stroma, richly cellular and compact. Stromal cells are spindle-shaped with elongated nuclei. The ovarian follicles are scattered within the stroma.

Primordial follicle: Consists of an immature ovum surrounded by a single layer of low cuboidal epithelium.

Growing follicle: At the initiation of follicular growth, the follicle cells assume a cuboidal shape, divide, and become multilayered.

Zona pellucida: Thick layer surrounds the growing ovum. Rich in polysaccharides.

Oocyte: Immature ovum containing the somatic number (diploid) of chromosomes. Will undergo meiosis to produce mature ovum.

Nucleus: The nucleus of an oocyte is large and contains a prominent nucleolus.

 

OVARY
Cortex

Monkey, glutaraldehyde, plastic section, H. & E., 68 x.

 

An overview of the ovarian cortex is shown. Note that the surface of the ovary is epithelium-lined ("germinal epithelium," simple squamous, detail not discernible at this magnification). The underlying discrete layer of connective tissue represents the tunica albuginea, which is devoid of follicles. Immediately deep to the tunica albuginea, numerous primordial follicles are present, as is a follicle that has begun to grow (primary follicle). The eosinophilic zona pellucida (arrow) is a prominent feature of such follicles. Other follicles that have begun to grow and mature, but that have regressed at various stages of growth, are indicated by asterisks, and are termed atretic follicles.

 

OVARY
Cortical region

Rhesus monkey, Helly's fluid, H. & E., 50 x.

Primordial follicles: Consist of an immature ovum surrounded by a single layer of low cuboidal or flattened epithelium (follicular or granulosa cells). Note the distribution in peripheral layers of the cortex.

Maturing follicle: Note the multilayered follicular cells, the increased size, the eccentric position of the developing ovum, and the prominent connective tissue capsule. Maturing follicles occupy deeper zones of the cortex. Note the vesicular nucleus and the deeply staining small nucleolus. The oocyte is pushed to the side of the follicle by the accumulation of follicular fluid.

Atretic follicle: Follicles that do not reach maturity degenerate and are called atretic follicles. The nucleus becomes pyknotic and later fragments. Follicular cells also degenerate. Atretic follicles are later resorbed and are replaced by a connective tissue stroma. The zona pellucida of an atretic follicle stains deeply and may persist by itself in the stroma.

Corpus albicans: A hyaline scar resulting from the degeneration of a corpus luteum of ovulation.

 

OVARY
Theca interna
growing follicle

Rabbit, 10% formalin, H. & E., 612 x.

 

This plate shows a rather advanced stage,in the maturation of a primary ovarian follicle. Note the increase in size of the oocyte and the follicular cells in comparison to that seen in early primary follicles .The nucleus of the oocyte is large and has a sparse reticulated chromatin network. The cytoplasm is granular. The zona pellucida separates the oocyte from the follicular cells. This prominent layer is rich in polysaccharides and is believed to be elaborated by both oocyte and follicular cells. It persists even after the degeneration of the oocyte during atresia of the follicle (.The follicular cells have formed a stratified epithelial layer at this stage of growth. Mitotic figures are frequent, indicating continued active proliferation. Accumulations of densely staining material are seen among follicular cells. These are vacuoles of Call-Exner*. They are believed to represent droplets within the cytoplasm of follicular cells. They stain positively with PAS and may be the precursors of follicular fluid. The stroma around the follicular epithelium is composed of an inner theca interna and an outer theca externa. A basement membrane separates the follicular cells from the theca interna; no distinct boundary exists between the thecae interna and externa. The cells of the theca interna have epithelioid characteristics and are believed to elaborate androstenedione. Note the ovoid or round nuclei of the theca interna. The theca externa is composed of spindle-shaped cells and is more fibrous. Both thecae are connective tissue derivatives.

 

CORPUS LUTEUM

Human, 10% formalin, H. & E., 162 x.

 

The corpus luteum (yellow body) is a stage in the transformation of an ovarian follicle following ovulation.

Granulosa lutein cells: Larger in size, more centrally located; nuclei less densely stained and cytoplasm more abundant. They are transformed cells of the stratum granulosum of the ovarian follicle.

Theca lutein cells: Smaller, have less cytoplasm, are more peripherally located, and nuclei stain more densely. They are transformed cells of the theca interna of the ovarian follicle.

Both types of cells are epithelioid and produce steroids. The corpus luteum secretes both estrogens and progestins. Progesterone induces changes in the uterine endometrium (secretory phase), in preparation for the implantation of a fertilized ovum, and inhibits spontaneous contractions of the smooth muscle of the uterus so that gestation can be maintained. The vacuoles seen in some cells are due to the lipid droplets dissolved during processing of tissue.

Ovarian stroma: Connective tissue stroma, remnant of theca externa of the ovarian follicle. Sends fine septa into the parenchyma

 

OVARY

Rabbit, 10% formalin, H. & E., 162 x.

 

Corpus albicans: A hyaline scar resulting from the degeneration of the corpus luteum.

Arterioles: Arteries enter the medulla of the ovary at the hilum and spiral their way through to the cortex.

Venules: Rich plexus of veins accompanies the arteries and leaves the ovary at the hilum.

Stroma: Compact connective tissue. Spindle-shaped cells with elongated nuclei and fine reticular connective tissue fibers.

 

UTERUS
Endometrium
A. early postmenstrual, B. proliferative phase,
C. secretory phase

Human, 10% formalin, H. & E., 50 x.

 

The uterine wall undergoes four phases during the menstrual cycle excluding menstruation. These are the (1) resurfacing, (2) proliferative, (3) secretory, and (4) ischemic phases.

The first three phases are shown in this plate. In A, the resurfacing phase, corresponding to days 5 and 6 of the cycle, is shown. During this stage, remnants of the glands in the basal zone of the mucosa proliferate and migrate to cover the raw surface of the endometrium denuded from its mucosa by menstrual flow. The thick myometrium is shown. This is a massive coat of smooth muscle fibers arranged in three concentric layers.

In B, the proliferative or follicular phase of the menstrual cycle, which lasts from day 7 to day 14 of the cycle, is shown. During this stage, the mucosal glands become longer and assume a curved or coiled configuration. The stroma between glands also increases by proliferation of connective tissue cells. The proliferative phase is induced by estrogen.

In C, the third or secretory phase, corresponding to days 15 to 27 of the menstrual cycle, is shown. This is also known as the progravid or luteal phase. During this stage, glands stop proliferating and begin to distend and secrete abundantly. in the middle region of the mucosa, saccular outpouchings of the glands are seen. The changes observed in this stage are induced by progesterone following estrogen priming.

 

MAMMARY GLAND

A. Human, in pregnancy, 10% formalin, 22 x.
B. Monkey, late pregnancy, 10% formalin, 36 x.
C. Human, resting, 10% formalin, 36 x.

 

The mammary glands are compound tubuloalveolar glands derived from the integument; they are responsive to reproductive and other hormones, and therefore undergo marked developmental, aging, and physiological changes during the course of the reproductive life of the mammalian female. Of great clinical import is the sobering statistic that cancer of the breast (usually adenocarcinoma of the ductal lining) is the most common cancer in women.

This plate illustrates distinctive differences in the histomorphology, breast tissue exhibits, depending upon physiologic state. During the development of the gland at pregnancy (A), secretory units (alveoli) bud from the ducts, and the glandular parenchyma enlarges in mass at the expense of the surrounding connective tissue. The duct system is best visualized in a gland that is "resting" (C), that is, one that is neither a gland of pregnancy nor of full lactation. Note in C how rudimentary secretory units, the alveoli (arrow), lie at the terminal arborizations of the alveolar duct system. The lobation of the gland is not well appreciated in histological section, a lobe being defined as that region of the breast drained by one lactiferous duct. The lobes are subdivided into lobules, the smallest order of which are seen here in each figure, separated by connective tissue partitions. Note the fortuitous section in A, revealing the continuity of the alveolar and higher-order system of ducts.

During pregnancy (A), the expanded alveolar component of the breast is evidenced by the highly basophilic cytoplasm of the alveolar cells, reflecting the initial synthesis of a product abundant in lactoprotein and low in lipid content (i.e., colostrum). The alveoli, however, do not reach their full degree of development until post-parturn and active lactation. Note that in the gland from late pregnancy (prelactating, B), the alveoli have expanded both in extent throughout the lobules and in size. Some alveoli are distended but exhibit regular walls and little secretory product. in contrast, the alveoli of the fully developed lactating gland typically are distended and saccular in appearance and contain a lightly eosinophilic secretory product .Such alveoli are lined by a cuboidal or flattened epithelium. The alveoli and lobules are separated by sparse connective tissue, the mass of the entire gland being largely taken up with secretory alveoli and associated ducts. Like the sweat glands of the integument, the alveoli of the breast are enfolded by a network of stellate myoepithelial cells, which contract in response to oxytocin, thus facilitating the milk ejection mechanism.

The student is cautioned that the mammary gland as a whole exhibits considerable normal variation in its histological development and, in addition, undergoes cyclic changes in synchrony with the menstrual cycle. Considerable latitude is encountered even within lobules with respect to activity and distention of alveoli (e.g., B). Therefore, variation from the "ideal" description is to be expected in your laboratory study.

 

MAMMARY GLAND, LACTATING

Monkey, 10% formalin, H. & E., 36 x.

 

A low power view of a section of actively lactating breast post parturn. The surface of the mammary gland is covered by a thin epithelium. The underlying dermis contains the follicles of fine surface hairs; the areola (not shown) is devoid of hairs. Alveoli are dilated and sacculated and contain a light- staining secretion product. With the degree of dilation shown by the secretory units, the smaller ducts are not distinguishable from the secretory units. Profiles of higher-order ducts are seen in the lower portion of the field.

 

MAMMARY GLAND, LACTATING

Monkey, 10% formalin, H. & E., 113 x.

 

Note the dilation and sacculation of the secretory alveoli, lined by a squamous to cuboidal epithelium. The alveoli contain a pale-staining eosinophilic secretory product and occasional macrophages (arrows).

 

MAMMARY GLAND

Monkey, 10% formalin, H. & E., 6.5 x.

The lactiferous ducts (galactophores) empty independently upon the surface of the nipple, imparting a serrated, pitted apprearance to the nipple surface. The openings of the ducts are lined with stratified squamous epithelium, keratinizing variety, which is continuous with that of the skin. The ductal orifices contain a keratinous cell debris. At the base of the nipple, the lactiferous ducts expand into large lactiferous sinuses; these, in turn, give rise to the lobular ductal system of the gland. This specimen is from the first trimester of pregnancy, and the mammary tissue is not well developed. During suckling, it is not uncommon for a lactiferous duct to become blocked and painfully engorged.

 

VAGINA

Human, 10% formalin, H. & E., 162 x.

 

This is a section of part of the wall of the vagina showing the mucosa. Note the stratified squamous non- keratinized epithelium. The lamina propria consists of loose connective tissue and is rich in lymphocytes. Occasional lymph nodules (not seen here) are found. The vaginal epithelium undergoes cyclic changes during the menstrual cycle.

 

PLACENTA

Rhesus monkey, 10% formalin, H. & E., 3.5 x.

 

The components of the placenta are seen at low magnification in this plate. The maternal contribution is the decidua basalis. This is the part of the endometrial mucosa lying beneath the embryo between the embryo and the myometrium. The fetal contribution is the chorionic plate and its villi. The chorionic plate is a portion of the chorionic sac about the embryo. Chorionic villi arise from the chorionic plate and lie in the spaces through which maternal blood circulates. Many villi are floating; others attach to the decidua basalis as anchoring elements. Villi receive their blood from the umbilical arteries and drain into the umbilical vein. Outside the chorionic plate, note the amnion that lines the amniotic cavity containing the umbilical cord. The embryo is not seen.

 

PLACENTA

Human, Bouin's-Halmi's AFT, A. 121 x; B. 357 x.

 

Fetal components of the placenta include the chorionic plate and the villi that originate from it (A); a high-magnification view of the epithelial lining of these structures is shown in B. In A, note that the epithelium-lined chorionic plate gives rise to villi, which project into the maternal blood space. Fetal mesenchyme constitutes the core of the villi; fetal blood vessels are present in the plate, and a rich capillary network is formed within the villi, analogous to the histophysiological arrangement of vessels within intestinal villi, which are also specialized to optimize absorptive capacity. Note that the capillaries in B contain fetal erythrocytes (stained green), which contain nuclei. The villi are lined by two layers of embryonic trophoblast. The inner cytotrophoblast consists of cells that give rise to the outer syncytial layer, the syncytiotroph o blast. Gradually, during the latter half of pregnancy, the cytotrophoblast is largely incorporated within the syncytial layer; therefore, specimens obtained at delivery will demonstrate only the syncytial layer. This histological feature can assume importance to the forensic pathologist in medicolegal examination of placental specimens, as well as to inexperienced histologists seeking a layer that no longer exists in the most common source of placental tissues, the afterbirth.

 

PLACENTA

A. Human, 10% formalin, H. & E., 89 x.
B. Human, Bouin's-Halmi's AFT, 107 x.

 

The maternal component of the placenta is shown in A; the endometrium of pregnancy is termed the decidua, and the stromal cells, which differentiate during pregnancy, are termed decidual cells. They are shown in profusion in B. Decidual cells are polygonal, possess a large, vesicular nucleus, and demonstrate the electron- microscopic characteristics of secretory cells; they are imputed to be the source of placental prolactin. Note the finger-like incursion of the maternal blood space into the substance of the decidua. This space is lined with trophoblastic epithelium

 

UMBILICAL CORD
Human

Human, 10% formalin, H. & E., 8.5 x.

 

The umbilical cord in transverse section reveals two umbilical arteries and a single umbilical vein embedded in mucous connective tissue (Wharton's* jelly). Also present is the endodermal remnant of the allantois, which extends the length of the cord in the human. The cord is covered by a simple epithelium of amniotic derivation, which becomes stratified in late gestation. The umbilical arteries are atypical of those of the remainder of the body in that they carry oxygen-poor blood, lack an internal elastic lamina, and have considerable metachromatic ground substance within their muscular tunic. They also lack an external elastic lamina, and the mucous connective tissue replaces the adventitia found in other arteries. Likewise, the umbilical vein exhibits an unusually thick muscularis, with intermingling circular, longitudinal, and obliquely disposed smooth muscle fibers. Furthermore, an internal elastic lamina is present, which serves to distinguish the vein from the accompanying arteries. Mucous connective tissue, a form of loose connective tissue, is characterized by its copious ground substance, rich in sulfated proteoglycan, which embeds a profusion of collagenous fibers. The cells of mucoid connective tissue are primitive fibroblasts, stellate in outline, which are not revealed with routine preparative techniques; their nuclei, however, are seen.

 

 

Section 12: Urinary System

The urinary system consists of the kidneys, ureters, urinary bladder, and urethra.

The volume and composition of body fluids are maintained remarkably constant regardless of fluid and solute intake. The regulation of body fluid and the constancy of the internal environment is the primary role of the kidneys.

Each kidney resembles the bean of the same name. At the center of the concave side, where the ureters and veins issue and arterial vessels and nerves usually enter, is the hilus. The kidneys are covered by a dense collagenous capsule called the renal capsule. The organ has a cortex and an inner medulla. The cortical area is clearly defined from the inverted conical or pyramid-shaped medulla. The base and sides are surrounded by cortical tissue, and the apex protrudes into the renal calyces. These protrusions are called renal papillae, whose surface is perforated by openings of numerous papillary ducts (of Bellini) and is thus called the area cribrosa.

From the base of the medullary pyramid, several hundred elongated parallel arrays of tubules enter the cortex. These so-called medullary rays consist of individual units composed of a straight collecting duct surrounded by many parallel tubular parts of the nephron. A renal lobule is defined as a single medullary ray and the cortical tissue that surrounds it. The cortical tissue in the area between the medullary pyramids constitutes the renal columns of Bertin. The cortex is punctuated by many renal or Malpighian corpuscles. The renal cortex consists of nephrons and blood vessels.

The human kidney has about four million filtering units called nephrons. The nephron consists of four parts: (1) renal corpuscle (capillary loops surrounded by an epithelial cover), (2) proximal convoluted tubule, (3) thin and thick limbs of Henle, and (4) distal convoluted tubule. The components of the nephron are completely covered by a basal lamina. The renal corpuscle is composed of two parts: a "tuft" of capillaries and a double-walled epithelial capsule called Bowman's capsule. The interior of Bowman's capsule (Bowman's space) is continuous with the proximal convoluted tubule.

Every minute, approximately one fourth of the cardiac output of blood enters the kidneys, and all the circulating blood passes through the kidneys every 5 minutes. The blood flows through the glomerular capillaries under 45 mm Hg pressure, and about one fifth of the plasma water leaves the capillaries and enters Bowman's space and the proximal tubules. The blood remaining in the capillaries flows into the efferent arterioles, which break up into capillary peritubular networks closely applied to renal tubules.

Plasma water in Bowman's space is termed the glomerular filtrate and normally contains all the major ions, amino acids, glucose, urea, and other substances in approximately the same concentration that exists in the blood plasma. However, this ultrafiltrate does not normally contain red blood corpuscles or significant amounts of protein. Both water and solutes are reabsorbed across the renal tubule cells to the fluid-filled interstitial space and into the peritubular capillaries to reenter the blood stream. This is called tubular reabsorption. Other substances leave the peritubular capillaries, enter the interstitial fluid, and are transported across the tubule cells, where they enter the tubule to be added to the glomerular filtrate. This is called tubular secretion.

The proximal convoluted tubule reabsorbs the filtered glucose and amino acids and some of the sodium, chloride, and bicarbonate ions. This active process, enhanced by the numerous microvilli that increase the surface area of proximal tubule cells, results in an osmotic gradient, which causes the passive reabsorption of water. The content of the proximal tubule is isotonic with respect to the blood plasma. The distal straight portion of the proximal tubule is in continuity with the thin limb of the loop of Henle, which extends into the kidney medulla and returns as the thick limb to the cortex. The content of the descending thin limb becomes increasingly hypertonic as salt (active chloride pump) is actively transported from the ascending limb into the interstitial space surrounding the descending thin limb. Chloride, together with sodium ions, passively enters the descending limbs, as water is drawn to the interstitium. Unlike the descending limb, the ascending limb of the loop of Henle is impermeable to water, and the content of the ascending limb of the tubule becomes increasingly hypotonic as salt is actively transported from it. Sodium ions are actively reabsorbed, and water is passively reabsorbed in the distal tubule.

The ascending thin limb of the loop is in continuity with the distal tubule. The distal tubule and the collecting duct are engaged primarily in the regulation of acid-base and potassium ion balance in the blood. Sodium ions are conserved (under the influence of the hormone, aldosterone) and exchanged for hydrogen or potassium ion, or both. The distal convoluted tubule cells do not have numerous microvilli but rather are characterized by infoldings of their basal cell membrane forming compartments containing mitochondria. These result in the appearance in light microscopy of so-called basal striations. These cellular architectural modifications facilitate active transport between the interstitial fluid and the lumen of the distal tubule.

The final concentration of urine, by passive reabsorption of water to form a hypertonic urine, occurs in the collecting ducts as they pass through the region of increasing osmolality in the medulla produced by the thin loop of Henle. This is facilitated by the action of antidiuretic hormone on the membrane permeability of the cells lining the collecting ducts. The urine leaves the kidney via papillary ducts, enters the ureter, and is stored'in the urinary bladder. Contraction of the urinary bladder forces the urine into the urethra to be eliminated from the body. A single kidney produces roughly 63 ml of glomerular filtrate per minute and reabsorbs 62 ml. Hence, 1 ml is drained into the bladder as urine, resulting in an output of 1440 ml per 24 hours.

The kidneys are also the site of synthesis of the hormone renin. Adjacent to the renal corpuscle and distal convoluted tubule, the internal elastic lamina of the afferent arteriole disappears and the smooth muscle cells become structurally modified (epithelioid in appearance) and synthesize and store the hormone renin in the form of granules. These granules stain intensely with periodic acid-Schiff method and also following Zenker's fixation with several common histologic dyes. These cells are called juxtaglomerular (JG) cells. The distal tubule cells immediately adjacent to JG cells are also modified, appearing with tightly packed nuclei, and the area is called the macula densa. The macula densa and JG cells are jointly called the juxtaglomerular apparatus. In addition to the juxtaglomerular apparatus, there are some pale staining cells of unknown function associated with afferent arterioles that are variously given several names, including extraglomerular mesangial, lacis, or polkissen cells. Anything that results in a reduction of blood flow to the kidney, such as an aneurysm or significant blood loss, will activate the secretion of stored renin. Renin acts on a plasma protein termed angiotensinogen, resulting in the formation of an inactive decapeptide, angiotensin I. In the presence of a converting enzyme (found in high concentration in lung endothelium), angiotensin I becomes an octapeptide termed angiotensin II. Angiotensin II is a powerful vasoconstrictor. It also causes enhanced secretion of the adrenal cortical hormone, aldosterone, which acts on the cells of distal tubules to increase the reabsorption of sodium and chloride ions. This, in effect, increases blood pressure by increasing the volume of extravascular fluid. Chronic deficiency in blood flow to the kidneys can result in a serious, life-threatening hypertension.

Some names associated with the urinary system follow: Bellini was a seventeenth -century Italian anatomist; Bertin, an eighteen th-century French anatomist, Malpighi, a seventeen th-century Italian anatomist, Henle, a nineteenth-century German anatomist; Bowman, a nineteenth-century English anatomist; Schiff, a twentieth-century German chemist in Florence, and Zenker, a nineteenth-century German pathologist

 

KIDNEY AND ADRENAL GLAND
Fetal

Rhesus monkey, 10% formalin, H. & E., 6 x.

 

In this plate, the gross histologic features of the adrenal gland and the kidney are seen. Note the location of the adrenal gland, riding the superior pole of the kidney. Within the kidney, note the hilum where the ureter is seen in cross section and where vessels are seen entering and leaving the kidney. The divisions of the kidney into cortex and medulla are clearly seen. Note the peripheral location of the cortex and the cortical columns that dip between the medullary pyramids. The latter are pyramid-shaped medullary structures, with the base of the pyramid resting against the peripheral cortex and the apex toward the hilum. Between the pyramids, note the interlobar vessels. The interlobar artery is a branch of the renal artery. The interlobar veins form the renal vein. in the marginal zone, between the cortex and base of the pyramids, course the arcuate arteries and veins, which are tributaries of the interlobar vessels.

In the adrenal gland, note the subdivisions into cortex and medulla

 

KIDNEY
Cortex

Rhesus monkey, 10% formalin, H. & E., 50 x.

In this plate, the various constituents of kidney cortex are shown. The cortex is composed of radiating columns of straight renal tubules (medullary rays), alternating with regions containing glomeruli and convoluted renal tubules (cortical labyrinth). Other names for these two divisions are pars radiata or the processes of Ferrein* for the medullary rays, and the pars convoluta for the cortical labyrinth. The cortical labyrinths contain glomeruli, proximal and distal convoluted tubules, and the arched collecting tubules. The medullary rays contain the straight portions of proximal tubules (medullary segments), the thick segments of ascending arms of Henle's* loops, and the straight collecting tubules.

 

KIDNEY
Vascular system cortex

Cat, vascular injection (gelatin), carmine, 50 x.

This is an injected specimen to demonstrate some aspects of vascular supply of the kidney. The interlobular arteries are branches of the arcuate arteries. The latter are located in the zone separating the cortex from the base of the medullary pyramids. The interlobular arteries ascend perpendicularly to the surface of the kidney and provide numerous short lateral branches (afferent arterioles) that enter one or more renal corpuscles (glomeruli). The interlobular arteries terminate at the periphery of the cortex as afferent arterioles, and each supplies a glomerulus. From every glomerulus, an efferent arteriole leaves and divides into a system of capillaries called the peritubular plexus around the tubules of the cortex. The injected carmine gelatin illustrates the larger size of the lumen of afferent arterioles. This relative size difference presumably increases the glomerular filtration pressure.

 

KIDNEY
Cortex

Rhesus monkey, Zenker's fluid, H. & E., 612 x.

 

Proximal tubule: Lumen continuous with that of glomerular capsule (Bowman's space). Large cuboidal cells, abundant eosinophilic cytoplasm, and large spheroidal nuclei. Brush border.

Neck: The first part of the proximal tubule leading away from the glomerulus. Narrow and straight.

Distal tubule: Wider lumen, shorter cells, without brush border.

Glomerulus: A tuft of winding capillaries surrounded by epithelial cells and few connective tissue cells.

Arteriole: Afferent arteriole breaks up into many capillary loops inside the glomerulus. From these capillaries, the efferent arteriole is formed.

Capillary: Surrounds the renal tubules. These capillaries are branches of the efferent arteriole

 

KIDNEY

Cat, glutaraldehyde-osmium fixation, toluidine blue stain, 1416 x.

Proximal tubule: Single layer of cuboidal cells with irregular surface. Rounded nucleus. Granular cytoplasm (see also Plate 16).

Capillary: Within the renal corpuscle. Filled with red blood cells.

Podocytes: The name given to cells of the visceral epithelium of Bowman's capsule. They have many foot- like processes (podia) resting upon the basement membrane covering the capillaries.

Bowman's space: Space between the parietal and visceral layers of Bowman's capsule in continuity with the lumen of the proximal tubules. .

Renal corpuscle: Includes Bowman's capsule plus the glomerulus formed of capillaries. Also known as Malpighian body

 

KIDNEY
Cortex

Rhesus monkey, Zenker's fluid, Mallory's stain, 612 x.

 

Bowman's capsule: Squamous epithelium forming a double-walled cup surrounding the glomerular capillaries. The portion of the wall applied to the capillaries is termed the visceral epithelium . The visceral epithelium is separated from the outer wall by Bowman's space .The outer wall is continuous with the proximal tubule, and Bowman's space is continuous with the lumen of the proximal tubule at the urinary pole of the capsule.

Vascular pole: The point of entry of the afferent arteriole into Bowman's capsule. The arteriole immediately forms the tuft of glomerular capillaries and the point of origin of the efferent arteriole from glomerular capillaries, which leaves Bowman's capsule.

Afferent arteriole: Carries blood to the glomerular capillaries.

Efferent arteriole: The arterial vessel that carries blood away from the glomerular capillaries. Uniquely, in the kidney, a capillary bed is interposed between arterial vessels. The efferent arteriole leads to the cortical intertubular capillary network

Juxtaglomerular cells: Myoepithelioid cells replace typical smooth fibers in the wall of the afferent arteriole as it approaches the glomerulus. These cells secrete a hypertensive factor, renin. .

Proximal tubule: Single layer of cuboidal cells with an irregular brush border.

Distal tubule: Cuboidal cells without a brush border, which stain less intensely than the proximal tubule cells.

 

KIDNEY
Cortex

Rhesus monkey, Zenker's fluid, Mallory's stain, 612 x.

 

Afferent arteriole: Seen here in cross section. Its proximity to the glomerulus (not seen here), which it serves, is indicated by the presence of cells containing conspicuous granules, which replace smooth muscle fibers normally found in the wall of arterioles.

Juxtaglomerular cells: Rich in cytoplasmic granules containing renin. Renin secreted into the blood is known to play a role in the formation of a hypertensive substance known as angiotensin II.

Glomerulus: Tuft of capillaries having their origin from the afferent arteriole and surrounded by Bowman's capsule. The glomerulus, together with Bowman's capsule, constitutes the renal corpuscle.

Proximal convoluted tubule: Outlet of Bowman's capsule approximately 14 mm long with many small loops near the renal corpuscle. It ultimately straightens and runs toward the medulla in the medullary rays.

Distal convoluted tubule: This portion of the renal tubule has many short loops in close association with the proximal convoluted tubule and the glomerulus. It is about one third the length of the proximal tubule. The distal tubule is continuous with the collecting tubules.

Macula densa: Specialized region of the distal convoluted tubule with tightly packed tubule cells in contact with the afferent arteriole. The bases of the cells of the macula densa are consistently found in intimate association with the juxtaglomerular cells in the wall of the afferent arteriole. Because this structural relationship suggests a functional relationship (supported experimentally), the macula densa and the juxtaglomerular cells together are referred to as the juxtaglomerular apparatus.

 

KIDNEY
Juxtaglomerular cells

Rhesus monkey, Zenker's fluid, Mallory's stain, 1416 x.

 

Afferent arteriole: Terminal branch of the interlobular artery entering the glomerulus. The renal afferent arterioles are volume receptors and are sensitive to changes in perfusion (blood) pressure.

Juxtaglomerular cells: Granular variety of myoepithelioid cells in the wall of the afferent arteriole. Replace the typical smooth muscle cells of the tunica media of the artery. A decrease in afferent arterial volume secondary to low perfusion pressure results in the release of renin. Renin is an enzyme that is released into the blood and acts upon blood proteins to produce a potent vasoconstrictor, angiotensin, which can, under abnormal conditions, elevate blood pressure to dangerous levels. Hypertension of renal origin in humans can be cured by removal of the diseased or ischemic kidney. Renin also affects blood volume and osmolarity by initiating a chain of events leading to the release of the hormone aldosterone by the cells of the zona glomerulosa of the adrenal cortex. Aldosterone acts upon the renal tubules to enhance sodium reabsorption. A second system unrelated to the kidney (the hypothalamus of the brain and the posterior lobe of the pituitary, neurohypophysis), also regulates the volume and osmolarity of the extracellular fluid of the body.

Macula densa: A group of specialized cells of the straight portion of the distal tubule, in contact with the afferent arteriole and contiguous with the juxtaglomerular cells. The cells are taller, thinner, and tightly packed compared to other distal tubule cells. These cells are functionally related to the juxtaglomerular cells, although their exact role is undefined. The macula densa marks the origin of the convoluted portion of the distal tubule.

Lumen of Bowman's capsule: Located between the parietal and visceral epithelial layers. Receives the ultrafiltrate of blood plasma circulating through the glomerular capillaries. The glomerular filtrate traverses the glomerular endothelium, the basal lamina (basement membrane), and the visceral epithelium to reach Bowman's space. Bowman's space is continuous with the lumen of the proximal convoluted tubule.

Proximal convoluted tubule: Deeply staining cuboidal cells surrounded by a thin basal lamina (basement membrane).

 

KIDNEY
Medulla

Rhesus monkey, Zenker's fluid, Mallory's stain, 612 x.

 

Collecting tubule: Cuboidal or columnar lining. Nuclei round and dark. Cytoplasm clear with distinct cell outlines.

Capillary: Filled with red cells.

Ascending limb of Henle's loop: Lined by cuboidal cells.

Thin segments of Henle's loop: Cells flattened, single layer, nuclei bulge into lumen.

 

KIDNEY
Papilla, area cribrosa, minor calyx

Rhesus monkey, Helly's fluid, H. & E., 162 x.

 

Papillary ducts (of Bellini): Named after Lorenz Bellini, the Italian anatomist, who described them in 1662. Arise by convergence of collecting tubules in the medulla near the pelvis. These ducts have large lumina and open at the area cribrosa at the apex of the papilla. Note the tall columnar epithelium lining the ducts. Cytoplasm of epithelial cells is clear; nuclei are dark and basally located. The tops of cells tend to bulge into the lumen.

Minor calyx: Subdivision of a major calyx in the pelvis of the kidney. The minor calyx is an infolded tube forming a double-walled cup. The inner wall of the calyx fits over the papilla of a pyramid. The transitional epithelium of the minor calyx is continuous with the columnar epithelium of the papillary ducts. The lamina propria is made up mostly of collagenous connective tissue and lacks papillae.

Area cribrosa: The sieve-like appearance of the papilla is produced by the large number of collecting tubules passing through it.

 

URETER

Human, 10% formalin, H. & E., 35 x.

 

In cross section, the ureter typically exhibits a "festooned" appearance, reflecting contraction of the thick surrounding muscular layer and the consequent development of deep mucosal folds, consisting of the transitional epithelium and underlying lamina propria. There is no submucosa-the tissue of the lamina propria blends with the epimysial connective tissue of the muscularis. As in other urinary passages, the muscular layers are defined as inner longitudinal and outer circular (the converse of the gastrointestinal tract). However, these are not consistently clearly demarcated in histological section owing to incomplete separation of layers and intermingling of muscle fibers. The well-developed muscular layer generates the vigorous peristaltic wave, which propels urine from the renal pelvis to the urinary bladder. At the site of entry of the ureters into the bladder (the intramural portion of the ureter), the circular muscle layer disappears, and the ureters run an oblique course through the bladder wall. This anatomical arrangement, together with the closing of a fold of the bladder mucosa over the orifice of the ureter by hydrostatic backpressure of the bladder contents, provides an effective "physiological valve," which prevents backflow (reflux) of urine from the bladder to the ureter, and subsequently to the kidney pelvis. These anatomical features ensure against the kidney's being subject to hydrostatic pressure and tissue damage (hydronephrosis) upon voiding, and prevents reflux of bladder urine and transmission of infection to the upper urinary tract.

 

URINARY BLADDER

Human, 10% formalin, H. & E., 22 x

 

The component parts of the wall of the urinary bladder are shown at low power. The innermost layer, the mucosa, consists of transitional epithelium, underlaid by a prominent lamina propria. The folds in the mucosa are seen in three dimension to be extensive longitudinal ridges and represent one means by which the bladder wall accommodates to distention. The thickness of the epithelium is also reduced upon stretching, as is the height of the luminal epithelial cells (dome, or umbrella cells, see Plate 24 for details of the epithelium). Thus, the overall topography of sections of the bladder will always depend upon the state of distention and turgidity of the organ at the time of fixation. The muscular layer of the bladder is generally recognized as consisting of three layers: (1) inner longitudinal, (2) middle circular or spiral; and (3) outer longitudinal. The definition of these as discrete layers is rendered difficult by the intertwining of muscle bundles and fascicles from adjacent layers, and by variation in the thickness of the respective layers in different parts of the organ (e.g., the formation of the internal sphincter by the internal longitudinal layer in the region of the trigone). This histological feature is in contradistinction to the well-defined muscle layers evident in preparations of the gastrointestinal tract. Such a muscular arrangement does, however, facilitate the occlusion of the bladder lumen upon voiding, and the avoidance of residual urine in the bladder, which is recognized as predisposing to bladder infections. The organ is covered by a connective tissue adventitia, except on its superior aspect, which bears a serosa of reflected peritoneum. Present within this layer are blood vessels, nerves, and underlying adipose tissue.

 

URETHRA
Female

Human, 10% formalin, H. & E., 162 x.

 

This is a section of female urethra showing the mucosa. The lining epithelium here is stratified squamous. The type of epithelium lining the urethra is variable at different sites. It is transitional near the urinary bladder and stratified squamous throughout most of its extent except for interrupted segments of stratified columnar or pseudostratified epithelium. The lamina propria of loose connective tissue lacks papillae.

 

URETHRA
Cavernous portion
penis

Human, 10% formalin, H. & E., 162 x.

 

This plate shows the histology of the Cavernous portion of the male urethra. This portion of the urethra extends throughout the penis to open at the end of the glans. Note the stratified columnar epithelium mucosal lining intermixed with stratified squamous epithelium. The latter type of epithelium is found in interrupted areas throughout the extent of the urethra and is the only epithelial type found at the external opening of the urethra.

Note the deep recesses of the mucosal surface known as lacunae of Morgagni* Isolated intraepithelial mucous gland cells are seen interspersed between the stratified columnar cells lining the lacunae.

The lamina propria is made up of loose connective tissue rich in elastic fibers.

 

 

Section 11: Respiratory System

The respiratory system is composed of conducting and respiratory passages, which serve several functions. The primary function of this system is the exchange of oxygen and carbon dioxide; secondary functions are phonation and olfaction.

The conducting portion of the respiratory system includes the nasal cavity, paranasal sinuses, nasopharynx, larynx, trachea, and bronchi. The conducting portion, from the nostrils to the lungs, warms, humidifies, and filters the air. Mucus secreted by goblet cells (approximately 1 liter/day) entraps particulate matter, and the cilia of the pseudostratified columnar epithelium lining carry a continuous carpet of secreted mucus toward the pharynx for elimination from the respiratory system. The familiar "smoker's cough" results from paralysis of ciliary action by nicotine and other products of tobacco smoking with resultant stasis and accumulation of mucus in the lower respiratory system; this, in turn, triggers the cough reflex in an effort to clear the distal airway. Bronchitis is a frequent sequel to such ciliary impairment.

The trachea is a relatively rigid tube about 11 cm in length and 2.5 cm in diameter in adults. At the inferior border of the superior mediastinum of the thorax, the trachea bifurcates into two primary bronchi, which are structurally similar to the trachea. The mucosa of these tubes is supported by incomplete cartilaginous rings. Three layers of the wall are distinguishable. (1) The mucosa is composed of a ciliated pseudostratified columnar epithelium and numerous goblet cells, both of which rest on a prominent, thick basement membrane (thickest in the body). A thin lamina propria composed of collagenous, elastic, and reticular fibers may harbor accumulations of lymphocytes, which play an important role in safeguarding the body from inhaled pathogenic organisms. There is no muscularis mucosae. (2) The submucosa contains seromucous glands, located primarily at the interspaces between the cartilaginous rings, and some fat cells. (3) The adventitia contains the cartilaginous rings interconnected by connective tissue. Each ring is composed of hyaline cartilage, appears in the form of the letter C or Y, and is open posteriorly. The open ends are connected by fibroelastic tissue and a band of smooth muscle (trachealis muscle). This soft tissue band on the posterior surface of the trachea, which faces the esophagus, is capable of yielding to esophageal dilation resulting from the passage of food or liquid. The cartilaginous rings mechanically hold the airway open but also give it flexibility. By preventing the collapse of the conducting pathway, respiration is not impeded  ).

The lungs are contained within the paired pleural cavities in the thorax. The lungs lie free within the pleural sac but are firmly attached at the hilus to the mediastinum, where a primary bronchus and the pulmonary vessels are located.

The designated subdivisions of the respiratory tree distal to the bronchus are the bronchiole, respiratory bronchiole, alveolar duct, alveolar sac, and alveolus.

The epithelium of the bronchus is pseudostratified columnar ciliated, with numerous goblet cells. The subsequent branching and reduction in size of the bronchi result in a change in the epithelium to simple columnar ciliated, with abundant goblet cells. The lamina propria is encircled by a smooth muscle layer, which, when contracted, gives the tube a folded appearance. The connective tissue outside the muscle contains seromucous glands. Solitary lymph nodules may be present in the mucosa and in the connective tissue around the cartilage. As the bronchi undergo progressive reduction in size through dichotomous branching, the cartilage support is eventually lost, at which time the passageway is termed a bronchiole.

As a smaller subdivision of the conducting tube, the bronchiole varies in size from about 0.5 to 1 mm in diameter. The ciliated epithelium is columnar and diminishes to a cuboidal form as the branching to successively smaller bronchioles continues. The muscle layer becomes the dominant structure and is composed of smooth muscle and elastic fibers. Within the adventitia, there is a reduction and then elimination of glands and lymph nodules. The mucosa of the smaller bronchioles may be highly folded owing to loss of firm supporting structures.Distal to the smallest bronchiole (terminal bronchiole) is the respiratory bronchiole. These crucially important segments of the lung mark the transition from the conducting to the respiratory passages, where oxygen and carbon dioxide are exchanged. The respiratory passages include (1) respiratory bronchioles, (2) alveolar ducts, (3) alveolar sacs, and (4) alveoli.

The epithelium of the respiratory bronchiole is primarily cuboidal and may be ciliated. Goblet cells are absent. The epithelium is supported by a thin collagenous layer in which smooth muscle and some elastic fibers are found. Alveoli appear as small pockets that interrupt the main wall. Alveoli become more numerous distally. The respiratory bronchiole branches to form alveolar ducts. These thin- walled, fibroelastic tubes are lined with a squamous epithelium and possess alveoli that appear as outpockets of the main wall. The main wall of the duct between alveoli contains smooth muscle. The terminal portion of the respiratory duct (atrium) gives rise to the alveolar sacs, composed of a variable number of alveoli that appear as small compartments opening into the alveolar sac. The alveoli are the smallest and most numerous subdivisions of the respiratory system. The interalveolar septum often contains 10 to 15 µm openings between neighboring alveoli that function to equalize air pressure in adjoining alveoli.

The alveolar wall is lined by a very thin (as thin as 25 nm) squamous epithelium (so-called Type I cells) covered with a thin film of fluid rich in hydrophilic phospholipid. This coating (pulmonary surfactant) is produced by Type II cells (also known as great alveolar cells or septal cells). Surfactant aids in keeping the alveoli open by reducing the surface tension of the moist interface between opposing alveolar surfaces, and thus reducing the inspiratory work required in breathing. Hyaline membrane disease in newborns has been correlated with insufficient pulmonary surfactant; neonates with this disorder have great difficulty in opening and expanding alveoli so that oxygen and carbon dioxide exchange can take place. The respiratory epithelium is composed largely of Type I cells (97 per cent), with the remainder being Type II cells. These cell types both rest on a basal lamina, which is, in turn, intimately associated with capillaries of the pulmonary vascular system. The extremely thin wall and rich capillary bed favor the transfer of oxygen to the red blood corpuscles and the release and transfer of carbon dioxide to the alveolar airway. The airway contains macrophages, which are monocytes derived from bone marrow. These so-called dust cells phagocytize debris that is found on alveolar surfaces and are important in the defense by the body against pathogens. Dust cells, laden with phagocytized material, are carried on a moving carpet of mucus to the pharynx, where they are swallowed and destroyed.

Several important arrangements are essential to respiratory function. The lungs are enclosed in the pleural cavities, which enlarge as the thorax expands during inspiration. This results in a negative pressure within the respiratory tree and air enters the system. During the filling of the lungs, the rich elastic network throughout the system becomes stretched, expanding and elongating the alveolar ducts and drawing oxygen-rich air into, and mixing it with, the carbon dioxide- rich air. During expiration, air is expelled from the system by elastic recoil as the thoracic cavity decreases in size. During muscular exercise, skeletal muscle assists in this process.

If the pleural cavities are exposed to air from the outside through an artificial opening in the thoracic wall (pneumothorax), the lungs will collapse and will not fill during inspiration owing to the equalization of the air pressure inside and outside the lungs.

Loss of elasticity and breakdown of interalveolar septa gives rise to emphysema, also often a sequel to long-term smoking. Smoking may cause a wide variety of pulmonary disorders quite apart from the well-documented correlation with the incidence of lung cancer.

 

LARYNX
Vocal folds

Cat, Bouin's, AFT, 5.5 x.

 

The larynx is a tubular organ whose framework consists of several cartilages and elastic membranes. Its mucous membrane is continuous with the pharynx cranially and the trachea posteriorly.

Vocal folds are formed from the mucous membrane; they vibrate as air from the lungs traverses them, thus generating sound. The activity of the vocal folds is regulated by nerve and muscle acting on the membrane through, primarily, the arytenoid cartilages (seen here in two parts on both sides, owing to sectioning). Important muscles seen in this section include the posterior cricoarytenoid, which is the only muscle to open the airway (rima glottis); the lateral cricoarytenoid, which is one of several muscles that closes the airway; and the vocalis muscle, which acts on the vocal process and fovea of the arytenoid cartilage to relax the vocal ligaments.

The esophagus is seen in its normal flattened condition. The lining epithelium is non-keratinized stratified squamous epithelium. The same epithelium covers the vocal folds, but cranially and posteriorly it is respiratory epithelium, i.e., pseudostratified ciliated columnar epithelium.

 

PSEUDOSTRATIFIED CILIATED EPITHELIUM
Basement membrane
trachea

Rhesus monkey, SUSA, AFT, 484 x

 

Epithelium: Pseudostratified ciliated columnar epithelium. The term pseudostratified refers to the appearance of the epithelium in section. Although the cells appear to be stratified because the nuclei are found in several layers, the basal portions of all cells are actually in contact with the basement membrane.

Cilia: These motile structures carry a carpet of mucus, which collects inhaled debris and takes it to the pharynx where it is either coughed out or swallowed.

Goblet cells: These non-ciliated mucus-secreting cells are seen in various stages of mucus synthesis and discharge.

Basement membrane: This common structure is thickest in the trachea, but wandering cells of the immune system can be found traversing the membrane. Other cells of the immune system are also seen at various levels of the epithelium.

Lamina propria: The lamina propria of the trachea is thin but contains small blood vessels and collagenous and elastic fibers.

 

CILIATED PSEUDOSTRATIFIED COLUMNAR EPITHELIUM
Trachea

Human, Helly's fluid, periodic acid-Schiff and hematoxylin stain, 1416 x.

Goblet cell: Mucin-secreting, non-ciliated columnar cell. The secretion of these cells collects inhaled particulate materials.

Ciliated cells: Tall columnar cells with central nucleus; form the ciliated pseudostratified epithelium of the trachea. Cilia extend into the lumen of the trachea and sweep particulate material away on a carpet of mucus from the respiratory alveoli and out of the respiratory tree.

Basement membrane: Unusually thick and prominent. Composed of reticular fibers and protein polysaccharides. The apparent thickness of the basement membrane in this location is attributed to closely applied elastic fibers.

Lamina propria: Thin, fibrous layer with abundant elastic fibers.

 

TRACHEA

Rat, Helly's fluid, H. & E., 612 x.

 

This is a section of the wall of the trachea showing the ciliated pseudostratified columnar epithelial lining, the fibrous lamina propria, and a small segment of hyaline cartilage with its perichondrium. The cartilage forms an irregular C- or Y-shaped ring around the trachea. The free ends are joined by a band of smooth muscle and face dorsally adjacent to the esophagus.

 

BRONCHUS AND BRONCHIOLE

Cat, 10% formalin, H. & E. stain, 162 x.

 

In A, part of a bronchus can be seen. Bronchi are lined by pseudostratified columnar epithelium with goblet cells. The thickness and layering of the epithelium decreases gradually with the decrease in size of bronchi. A smooth muscle layer encircles a thin connective tissue lamina propria. In contrast to the trachea, the smooth muscle of the bronchus is arranged in interlacing spirals around the bronchus. Between the smooth muscle layer and the cartilage is the submucosa, which may contain seromucous glands, not seen in this preparation. The hyaline cartilage is arranged in discontinuous plates around the bronchus.

In B, a bronchiole is seen in the midst of respiratory tissue. The epithelium is simple columnar ciliated. The lamina propria is replaced by the muscle layer that encircles the bronchiole. in man, cartilage and glands are characteristically present until bronchioles decrease in size to approximately 0.5 mm in diameter.

 

BRONCHIOLE

Cat, 10% formalin, H. & E., 162 x.

 

This is a cross section of a bronchiole surrounded by respiratory tissue. Note the low columnar epithelial lining, the prominence of smooth muscle fibers in the lamina propria, and the absence of cartilaginous plates and glands. Macrophages filled with black carbon particles are seen in the lumen of the bronchiole. The elastic spongework of respiratory tissue surrounding the bronchioles prevents their collapse during inspiration. Every inspiratory movement exerts a pull on the wall of the bronchiole protecting it from collapse, hence there is no need for cartilage rings or plates.

 

LUNG
Respiratory bronchioles

Cat, 10% formalin, H. & E., 50 x.

 

Refer to Figure 11A which can be used in conjunction with this plate in order to follow the structural changes that occur from the respiratory bronchiole to alveolar ducts to alveolar sacs where gaseous exchange takes place.

Respiratory bronchioles: A branch of the terminal bronchiole. Alveoli arise as outpouchings of the bronchiole. Pulmonary arteries and veins are found adjacent to the bronchiole, but only capillaries extend beyond the respiratory bronchiole.

Alveolar ducts: Arise by branching of respiratory bronchioles; walls made up of alveolar sacs and alveoli.

Alveolar sacs: Cluster of alveoli that open into the lumen of the alveolar ducts. Composed of squamous epithelial cells, basement membrane, and capillaries. Site of respiratory exchange.

Visceral pleura: A serous membrane that intimately covers the surface of the lung.

 

RESPIRATORY BRONCHIOLE, DUCT, AND ALVEOLI

Rat, glutaraldehyde-osmium fixation, toluidine
blue stain, A. 50 x, B. and C. 162 x.

 

Refer to Figure 11A which can be used in conjunction with this plate in order to follow the structural changes that occur from the respiratory bronchiole to alveolar ducts to alveolar sacs where gaseous exchange takes place.

Respiratory bronchiole: A branch of the terminal bronchiole. Epithelium low columnar to low cuboidal. Cilia present in the larger tubes only. Thin supporting wall of collagenous and elastic fibers and smooth muscle. Differs from the terminal bronchiole in having alveoli as outpouchings of its wall. Accompanying arterioles and venules are seen in the wall of the bronchiole. Note that large arterioles and venules are not seen at a level below the respiratory bronchiole.

Alveolar duct: Arises by branching of respiratory bronchioles; wall made up of alveolar sacs and alveoli. Lining epithelium is reduced to flattened cells with occasional cuboidal cells.

Alveolar sacs: Cluster of alveoli that open into the lumen of the alveolar duct. Individual alveoli are lined by thin squamous cells (Pneumocyte 1) and cuboidal cells that bulge into the alveolus (Pneumocyte II). The latter cell is responsible for the production of surfactant, which maintains the configuration and stability of the alveolus and plays a role in fluid transport across the alveolocapillary membrane. Site of respiratory exchange.

 

ALVEOLAR DUCT AND ALVEOLAR SACS

Rat, glutaraldehyde-osmium fixation,
toluidine blue stain, A. 162 x, B. 612 x.

Refer to Figure 11A which can be used in conjunction with this plate in order to follow the structural changes that occur from the respiratory bronchiole to alveolar ducts to alveolar sacs where gaseous exchange takes place.

Alveolar duct: A branch of the respiratory bronchiole. The duct is composed of alveolar sacs and alveoli.

Alveolar sacs: Cluster of alveoli opening into the main lumen of the sac. Individual alveoli are lined by a thin squamous (Pneumocyte type I) and cuboidal epithelium (Pneumocyte type II). A closely applied capillary network is separated from the epithelium by a thin basement membrane. It is across this trilaminar wall that oxygen, carbon dioxide and other inspired gases are exchanged in respiration.

 

ALVEOLUS

Rat, glutaraldehyde-osmium fixation, toluidine blue stain, 1416 x.

Refer to Figure 11A which can be used in conjunction with this plate in order to follow the structural changes that occur from the respiratory bronchiole to alveolar ducts to alveolar sacs where gaseous exchange takes place.

The partition between adjacent alveoli is shown in this plate. Note that capillaries form a major part of the partition, the very thin alveolar wall and the alveolar cells. The alveolar wall has been shown by electron microscopy to be composed of an epithelial cell (Type I or Type II), basement membrane (basal lamina), and an endothelial cell. The Type II alveolar cells (also known as great alveolar cells or septal cells) are cuboidal in shape and appear to have empty vacuoles in their cytoplasm. The vacuoles, as shown by electron microscopy, frequently contain osmiophilic bodies with concentric lamellae (cytosomes). Cytosomes are believed to contribute to the pulmonary surfactant that coats the alveolar epithelium in order to reduce the surface tension and keep the alveoli from collapsing. Type I, squamous alveolar cells constitute 97 per cent of the respiratory epithelium at the alveolar level. The remaining 3 per cent are Type II, great alveolar cells, which produce the surfactant. Through the alveolar wall, gaseous exchange takes place between blood and air.