Section 5: Muscular Tissue

Muscle fibers are elongated cells with distinctive shapes specialized for shortening or contraction. These contractile fibers provide the means of movement for minute body hairs, air in respiration, ingested food and liquid, reproductive cells, blood and lymph, and small and large parts of the body. Muscle permits appropriate responses to external and internal stimuli as well as every form of communication by the individual with the external environment.

Muscular contractions, which may be coarse or extremely refined and are graded between fast and slow, are controlled by the nervous system, which is devoted in large measure to these essential activities. Muscle fibers in vertebrates may be classified structurally as nonstriated, plain, or smooth; striated cardiac; or striated skeletal. A broadened classification, which considers function, follows: smooth, involuntary; striated cardiac, involuntary; and striated skeletal, voluntary. The structural/functional classification indicates whether the contractile activity is under intentional or autonomic control.

Another functional consideration is concerned with the ability of smooth and cardiac muscle to contract spontaneously in the absence of a nerve supply (myogenic contraction). The contractile activity of involuntary muscle is normally regulated by the autonomic (sympathetic and parasympathetic) nervous system. Striated skeletal muscle fibers are totally dependent upon the nervous system, however, for both their structural integrity and function. Each striated skeletal muscle fiber is supplied with a nerve fiber ending on a specialized region of the cell membrane or sarcolemma, the subneural region of the motor end plate. If the nerve supply to a skeletal muscle is interrupted, the component muscle fibers will atrophy rapidly (denervation atrophy). If muscle is worked, it increases in size and strength; if it is not used, it will also atrophy (disuse atrophy).

Smooth muscle fibers are generally small fusiform fibers that vary from about 15 to 200 µm in length and from 3 to 10 µm in diameter. Each muscle fiber possesses a single, elongated nucleus, which characteristically becomes shorter and broader and may coil when the muscle fiber contracts. Smooth muscle fibers may occur singly, as in the scrotum (tunica dartos); in small bundles or fascicles associated with hair follicles (arrector pili muscle); in well-defined, layered sheets that are coiled, as in muscular arteries, or arranged in two thick layers at right angles to each other, as in the intestines; or with an additional layer in an irregular pattern, as in the stomach, bladder, and uterus. Branched smooth muscle fibers can be found in the nipple of the mammary glands and in the enclocardium of the atrium of the heart.

Smooth muscle fibers usually contract slowly but are capable of sustained contractile activity. Most of the smooth muscle fibers of the gastrointestinal and genitourinary tracts are linked to each other by specialized surface membrane (sarcolemmal) contacts (gap junctions or nexus), which transmit electrical excitatory stimuli from cell to cell. The gap junction can be seen in the introductory plates of this book. This structural/functional arrangement permits large numbers of smooth muscle fibers to be activated sequentially by a minimal nerve supply. The excitatory nerve impulse is transmitted to a smooth muscle fiber, which conducts it over its surface, across the gap junction to another fiber, which passes it on, resulting thereby in a sustained and coordinated contraction (peristalsis) over long distances.

Cardiac muscle fibers are generally larger than smooth muscle fibers and appear cross-striated when stained or examined with polarized light. Cardiac muscle fibers are joined serially end to end and characteristically branch to unite with adjacent fibers. Cardiac muscle fibers form a functional but not a protoplasmic syncytium. The junctional site between fibers is called the intercalated disc. The intercalated disc is composed of two important components: the adhesion plate (desmosome) between adjacent cells and the gap junction (or nexus), which allows the electrical excitatory impulse to be transmitted from cell to cell in the same way as in smooth muscle, resulting in a synchronized coordinated contraction relaxation cycle essential to normal heart function. The branched cardiac fibers possess one or two nuclei, which are centrally located. The contractile substance of the cardiac fiber is organized into subunits called myofibrils, which are cross-striated. The cross striations will be discussed in relation to striated skeletal muscle. The myofibril characteristic of cardiac and skeletal muscle is not seen in smooth muscle, although the myofilaments of which the myofibril is composed are found in all three muscle fiber types. Myofilaments cannot be resolved by the light microscope, although Brücke (1858) postulated their existence based on polarization microscopy data analysis and Kölliker (1888) suggested that the hypothetical myofilaments were composed of the newly discovered protein myosin (Kühne, 1864).

Striated skeletal muscle fibers vary in length between 2 and 25 cm, depending upon the muscle. The diameter of a single muscle fiber is also variable but is usually between 10 and 100 µm. Normal mature striated skeletal muscle fibers are irregularly polygonal in shape (Bowman, 1840), whereas developing fibers are small and round; pathologic muscle fibers tend to be round or sharply angular and usually abnormally small. The multinucleated skeletal muscle fibers, unlike smooth and cardiac muscle fibers, are not structurally or functionally uniform. Two or more distinct muscle fiber types have been identified in man and other species by light and electron microscopy, histochemistry, and functionally. Characteristically, the nuclei of skeletal muscle fibers are located peripherally adjacent to the outer limiting membrane or sarcolemma. The usual (normal) position of skeletal muscle nuclei and the discovery of the sarcolemma is credited to Bowman (1840). The nuclei of developing muscle and of cardiac muscle are typically centrally located within the muscle fiber (Bowman, 1840).

In certain skeletal muscle fibers, namely the red or slow contracting muscle fibers, the nuclei may be found scattered throughout the sarcoplasm. Based upon structural/functional studies, living muscle fibers that appear red are designated, in man, as Type I muscle fibers. These muscle fibers contain many mitochondria (Kölliker, 1857), they store and utilize lipid (droplets) metabolically, and they are red in color and contract slowly. One type of red fiber requires only a single stimulus, whereas a second type requires multiple stimuli to initiate a contraction. The former is designated a slow twitch muscle fiber, and the latter as slow tonic muscle fiber. Lorenzini (1678) first noted color differences in muscles; some are red in color, others are white. Kühne (1850) analyzed the intrafiber pigment and reported its similarity to hemoglobin. Lankester (1871) noted that although red muscles were slow in contracting, they were the most active and strongest and capable of sustained contractile activity. He also contrasted red pigeon breast muscle with white chicken breast muscle and, with both birds, capacity for sustained flight. Gunther (1921) introduced the term myoglobin for the red (intrafiber) pigment. Most whole muscles are a mixture of red and white muscle fibers, which vary in number for any particular muscle; these are seen histologically in thin sections.

Muscle fibers that appear white contain few mitochondria, store and utilize glycogen (Bernard, 1855), are essentially devoid of myoglobin, and contract rapidly but fatigue quickly. They are normally larger than red fibers. In man, these are designated as Type II muscle fibers.

In cross section, skeletal muscle fibers are seen to be composed of numerous small aggregates (1 to 2 µm) of contractile substance, the myofibrils. Myofibrils are composed of myofilaments (Hall, Jakus, Schmidt, 1946). Huxley (1954) has shown two types of myofilaments: a thick, A band myofilament 1.6 µm in length; and a thin, 1 band myofilament 1.0 µm in length, which extends from the Z line into the A band for a variable distance.

In longitudinal section, the muscle fiber and the myofibril appear cross-striated (Leeuwenhoek, 1674). The darkly stained segments, 1.6 µm in length (Krause, 1868), is designated the A band (anisotropic band), which is a region of high refractive index and is birefringent when examined with a polarizing microscope (Brücke, 1858). Alternating with the A bands is a lightly staining region of variable length, the I band (isotropic band), which is a region of low refractive index (Brücke, 1858). The I band is bisected by a thin dark-staining Z line. When a muscle fiber contracts, the band pattern changes with the A bands moving toward each other and meeting at the Z line (Bowman, 1840); the I bands disappear, and the middle of the A band becomes dark. The band pattern changes can be precisely related to the movement of myofilaments in relation to each other. Additional structural/functional details of the contractile process will be considered in the legends of the plates of this section.

 

STRIATED MUSCLE
Embryonic tissue
cross section

Human, Helly's fluid, H. & E., 612 x.

Developing muscle fibers are seen in different stages of development. Embryonic muscle fibers are characterized by centrally placed nuclei and peripherally disposed myofibrils. These fibers grow in length and diameter by myoblast fusion. No nuclear divisional figures are ordinarily seen at this stage. As the muscle fiber matures, the nuclei become located primarily beneath the sarcolemma at the periphery of the fiber.

A congenital disorder of skeletal muscle seen in children (centrovacuolar myopathy) is characterized by a histologic picture identical with that seen in embryonic skeletal muscle.

 

STRIATED MUSCLE
Sarcolemma

Human, Zenker's fluid, Verhoeff & Van Gieson's stain, 612 x.

Sarcolemma: External limiting membrane of muscle fibers. Not ordinarily seen in light microscopic preparations. Seen here because of artifactual retraction of contractile elements. This artifact permitted Bowman, in 1840, to demonstrate the membrane and to name it the sarcolemma. The true sarcolemma, very much thinner than seen here, is responsible for the conduction and spread of electrical impulses from the motor end plate over the entire muscle surface, resulting in contractile activity. Electron microscopy has shown the sarcolemma to be 100 Å or 10 nm in thickness. The apparent increase in thickness rendering it visible in this preparation is due to adherent stainable sarcoplasm and, externally, to a thin basement membrane and associated reticular connective tissue fibers.

 

STRIATED MUSCLE
Polarization microscopy

Human, Helly's fluid, H. & E., 1416 x.

The names given to the two major transverse striations of skeletal and cardiac muscle are derived from the studies of Brücke* (1858). With routine light microscopic techniques, alternating dark and light bands are seen within striated muscle fibers . Polarization microscopy reverses the appearance of the dark band, which becomes bright, and the light band, which appears dark. The dark band of routine light microscopy, exhibiting birefringence with polarized light, is anisotropic and is called the A band. The light band of routine light microscopy is poorly refractile and relatively isotropic and is called the I band.

Muscle fibers: Showing cross striations formed by alternating segments of high and low refractive index resulting from their submicroscopic structure, which is revealed by electron microscopy.

A band: Anisotropic band.

I band: Isotropic band. Note the birefringence or anisotropy of the Z line in the center of the I band.

 

STRIATED MUSCLE
Transverse striations
longitudinal section

Human, Helly's fluid, phosphotungstic acid
hematoxylin stain, A. 612 x; B. 1416 x.

Phosphotungstic acid hematoxylin is a stain particularly suited for the demonstration of striations in skeletal muscle. Iron hematoxylin and Mallory-azan are also effectively used for this purpose. Note that at low magnification only the two major cross striations can be seen. The dark band is the A band, and the light band is the I band. Higher magnifications are usually required to see the light-staining area in the center of the A band, which is known as the H zone, and the thin, dark line bisecting the I band, which is named the Z line. The repeating structural unit between two Z lines is called a sarcomere.

 

STRIATED MUSCLE
Tongue

Human, Zenker's fluid, Verhoeff & Van Gieson stain, 1446 x.

I band: Isotropic band determined by polarization microscopy. Note the darker Z line bisecting the I band. Electron microscopy has shown that the I band contains thin filaments 50 Å or 5 nm in diameter and approximately 2 µm in length. The contractile protein actin is found in the thin I band filaments.

A band: Anisotropic band determined by polarization microscopy is bisected by the lighter H zone. The A band contains filaments of the protein myosin, which are 100 Å or 10 nm in diameter and 1.6. µm in length, as well as thin (actin) filaments extending from the I band into the A band. The A band filaments are composed of myosin molecules, which, through enzymatic activity (adenosine triphosphatase), release energy essential for the contractile process.

Z line: Bisects the I band. Z from the German word Zwischenscheibe, or in-between line.

H zone: Variable in width, it bisects the A band and vanishes in contraction as the thin I band filaments are drawn further into and through the middle of the A band. When seen, it contains only thick filaments. H for the German word Hell (bright) and also for the zone's discoverer, Hensen.*

Sarcomere: The contractile substance between two Z lines constitutes a convenient structural unit but not the precise functional or primary contractile unit of muscle fibers. See Plate 68.

Nucleus: Elongated and located beneath the sarcolemma. The sarcolemma is seen in Plate 64.

Collagen: Bundles of this connective tissue separate individual muscle fibers (endomysium), bind fascicles, or bundles, of muscle fibers (perimysium), and invest the entire muscle (epimysium). Through this tough and inelastic connective tissue, contractile forces are transmitted to bone and skin.

 

STRIATED MUSCLE
Relaxed and contracted
muscle fibers

Human; Helly's fluid;
A., C., phosphotungstic acid hematoxylin,
B. Mallory-azan,
D. H. & E.; 4416 x.

In this plate, the structural basis of skeletal muscle fiber contraction is shown.

A: Relaxed fiber showing distinct cross striations, the darker staining A band and the lighter staining I band. Note that the I band is bisected by a thin but deeply staining line (Z line), while the A band is bisected by a lightly staining line (H zone).

B: A fiber seen in the relaxed state except for a small segment of localized contraction. Note the change in the band pattern in this segment. Two adjacent A bands are in contact, and the I band has disappeared.

C: A fiber shown with both a relaxed and contracted segment. The A and I bands are clearly outlined in the relaxed segment but not in the contracted segment. In contraction, the I band becomes narrower and disappears. The A band does not normally become shorter except in extreme contraction. Contraction bands appear as a result of an increase in density and staining of the Z line.

D: A portion of a fully contracted muscle fiber is shown. The changes here are similar to those described in C for a contracted segment except that the normal distance between the thickened Z lines (contraction bands) is reduced, denoting extreme contraction.

 

The upper figure, showing two sarcomeres, accounts for the usual light microscopic appearance (i.e., the staining densities) of sarcomeric cross striations in relaxed (left), contracting (middle), and fully contracted (right) skeletal and cardiac muscle fibers.

In the lower figure, also showing two sarcomeres, the comparable electron microscopic, ultrastructural configuration is shown. The appearance of cross striations or bands by both light and electron microscopy have their basis in the relative position and resulting density of the two major sets of myofilaments that constitute the sarcomere, that is, thin (actin) filaments, emanating from the Z line, and thick (myosin) filaments, held in hexagonal register at the M line, and their relative interdigitation in relaxation and in shortening (contraction).

 

STRIATED MUSCLE
Lateral rectus cross section

Cat, glutaraldehyde-osmium fixation,
toluidine blue stain, 162 x.

Epimysium: Envelope primarily composed of collagenous connective tissue wrapping the entire muscle.

Perimysium: Connective tissue partitions between bundles, or fascicles, of muscle fibers.

White fibers (A fibers): Also known in the human as Type II fibers. These large fibers demonstrate pronounced myofibrillar ATPase activity and glycogen stores. These fibers are fast contracting.

Red fibers (B fibers): Also known in the human as Type I fibers. Characteristically smaller than white fibers, they contain numerous mitochondria and lipid stores. These fibers are slow contracting. .

Note the variation in fiber diameter. Normally, skeletal muscle fibers vary from 10 to 100 µm in diameter, depending upon muscle and species.

Nerve fibers: Somatic motor nerve fibers are distributed in the connective tissue septa of the muscle. These terminate on individual muscle fibers.

Capillaries: Widely distributed in the connective tissue septa (endomysium) between and around individual muscle fibers. Blood cells are seen within some capillaries.

 

STRIATED MUSCLE
Cremaster muscle myofibrils

Human, 10% formalin, H. & E., 1416 x.

A capillary containing a red blood cell is seen between muscle fibers. Muscle fibers are provided with a rich capillary network that supplies essential nutrients and oxygen and removes metabolic wastes.

Myofibrils: Subunits of each muscle fiber. Each myofibril is composed of myofilaments. Myofibrils vary in size, depending upon the number of myofilaments they contain. Myofilaments cannot be resolved by the light microscope.

Nucleus with nucleolus: The nucleus shown in this mature muscle fiber is characteristically located near the sarcolemma. In some skeletal muscles, particularly those that are slow contracting (Type I, Type B, or red), the nuclei may be found more centrally located within the muscle fiber.

 

STRIATED MUSCLE
Semitendinosus, cross section
Mitochondria; succinic dehydrogenase localization

Rat, frozen section, Tetrazolium method, 612 x.

Histochemical methods similar to the one used here, which is specific for mitochrondria, have been instrumental in distinguishing muscle fiber types in health and disease.

Red muscle fiber (B fiber): Rich in mitochondria and lipids, this type of fiber is slow contracting. Known in the human as Type I muscle fiber.

White muscle fiber (A fiber): Relatively poor in mitochondria and lipids, but rich in myofibrillar ATPase activity and glycogen, these fibers are fast contracting. Known in the human as Type II muscle fiber.

 

MOTOR END PLATE, SUBNEURAL APPARATUS
Intercostal muscle

Rat, Seligman acetylcholinesterase method, 612 x.

Enzyme location: The muscle sarcolemma that forms the primary and secondary clefts of the subneural apparatus is rich in acetylcholinesterase activity. Axon terminals (not seen in this preparation) lie within the primary synaptic cleft  and liberate acetylcholine when a nerve action potential, originating in the spinal cord, arrives at the nerve endings. Acetylcholine results in the depolarization of the muscle membrane and the appearance of a muscle action potential, which spreads over the muscle fiber leading to muscular contraction. The acetylcholine is hydrolyzed by the enzyme located on the sarcolemma beneath the nerve terminals, and the sarcolemma is repolarized in preparation for the next nerve impulse.

 

NEUROMUSCULAR SPINDLE
Cross section
A. tongue B. sartorius

A. Human, 10% formalin, H. & E., 435 x.
B. Frog, glutaraldehyde-osmium fixation, toluidine blue stain, 1416 x.

This plate shows some of the histological features of neuromuscular spindles as seen in the human tongue (A) and frog sartorius (B). Note that the neuromuscular spindle is surrounded by skeletal muscle fibers (extrafusal fibers). Each spindle contains several small muscle fibers (the intrafusal fibers), myelinated nerve fibers enclosed within a connective tissue capsule, which is pierced by the nerve fibers reaching the spindle. Nerve fibers of the spindle are both sensory and motor. Information conveyed from and to the muscle spindle is not consciously received but is important in reflex regulation of muscle tone. Intrafusal muscle fibers of the spindle receive axons of the gamma motor neurons in the spinal cord, whereas the extrafusal muscle fibers receive axons of the larger alpha motor neurons. .

 

CARDIAC MUSCLE

Human, Helly's fluid, Mallory's stain, 612 x.

The characteristically branched cardiac muscle fibers are separated by collagenous connective tissue. The differentiation of collagenous connective tissue and cardiac muscle is clearly seen with this stain. Note the capillary containing red cells. Compare capillary diameter (approximately 8 µm) with that of the cardiac muscle fiber.

 

CARDIAC MUSCLE
Longitudinal section

Human, Zenker's fluid, phosphotungstic acid hematoxylin stain, 162 x.

Muscle fibers: Each 9 to 22 µm in diameter, serially arranged in columns with short branches contacting adjacent fibers.

Branching fibers: Characteristic of cardiac muscle fibers. Each branching fiber limited by an intercalated disc constitutes a single muscle fiber. .

Striations: A and I bands. A bands are usually inconspicuous. Z lines are particularly prominent. Contractile apparatus and cross striations, although not usually stained well in cardiac muscle, are similar to those found in skeletal muscle (see Plates 65, 66 and 67).

Intercalated disc: Site of termination and junction of adjacent cardiac muscle fibers. Consists of snugly fit projections and indentations of adjacent cell membranes. Intercalated discs are the sites of transmission of excitatory impulses from cell to cell and provide firm attachment for contiguous fibers. The stain used in this preparation is particularly useful for demonstrating intercalated discs.

 

CARDIAC MUSCLE
Relaxed and contracted muscle fibers

Human, Helly's fluid, Mallory-azan stain, 1416 x.

This is a longitudinal section of cardiac muscle stained with Mallory-azan, which differentiates muscular tissue (red-brown) from collagenous connective tissue (blue).

Several muscle fibers are seen. A vesicular nucleus is seen in one. The muscle fibers are separated by narrow spaces containing delicate strands of collagen fibers. Each muscle fiber is formed of subunits, the myofibrils. To the extreme left of the figure, a muscle fiber in the relaxed state is shown. Note the distinct striations. Adjacent to this fiber is another relaxed fiber except for a small area of localized contraction. Note that the muscle striations are less distinct in the contracted area. in the middle of the plate is a contracted fiber in which the striation pattern is indistinct, although the Z lines are evident.

 

PURKINJE FIBERS

A. Sheep, Rossman's fixative, periodic acid-Schiff and hematoxylin stains, 162 x.
B. Human, 10% formalin, H. & E., 162 x.

In this figure, the contrast between ordinary cardiac muscle fibers and their specialized variety, the Purkinje fibers, is evident. Purkinje fibers are larger than ordinary cardiac muscle fibers and stain less intensely. Note the clear areas in the cytoplasm of Purkinje fibers in B. These represent areas from which glycogen was lost during the preparation of the tissue. By contrast, in the Purkinje fibers seen in A, the glycogen is preserved by the fixation method used. Note the subendocardial location of Purkinje fibers.

 

CARDIAC MUSCLE
Purkinje fibers cross section

Sheep, Bouin's fluid, H. & E., 162 x.

Purkinje fibers: Larger and paler than ordinary cardiac muscle fibers. Areas of clear sarcoplasm represent regions that normally contain glycogen as well as areas devoid of myofibrils. These fibers contain irregularly arranged thin myofibrils and nuclei.

 

CARDIAC MUSCLE
Purkinje fibers cross section

Sheep, Bouin's fluid, H. & E., 162 x.

Purkinje fibers: Larger and paler than ordinary cardiac muscle fibers. Areas of clear sarcoplasm represent regions that normally contain glycogen as well as areas devoid of myofibrils. These fibers contain irregularly arranged thin myofibrils and nuclei.

 

SMOOTH MUSCLE
Duodenum longitudinal section

Human, Helly's fluid, H. & E., 612 x.

Outer longitudinal layer of smooth muscle in the tunica muscularis of the duodenum. Muscle fibers are divided by connective tissue septa into bundles. Each muscle cell has a central nucleus and abundant sarcoplasm. The muscle fibers are long, slender, and spindle-shaped. Note that differentiation between smooth muscle cells and connective tissue fibers is difficult in this preparation because of the staining method used (H. & E.). Differentiation of smooth muscle .

 

SMOOTH MUSCLE
Duodenum cross section

Human, Helly's fluid, H. & E., 612 x.

Bundles of smooth muscle fibers separated by connective tissue septa. Each fiber is characterized by abundant sarcoplasm and central nucleus. Myofibrils are not seen in smooth muscle. Note variation in cross-sectional diameter, which can be accounted for on the basis of their spindle shape (as seen in longitudinal section).

 

SMOOTH MUSCLE
A. Longitudinal and circular (jejunum)
B. Small fascicle (skin)

Human, 10% formalin, H. & E., 162 x.

This plate shows smooth muscle fibers from two locations. In A, they are seen distributed as an outer longitudinal and an inner circular layer in the wall of the jejunum. These two layers are separated by connective tissue and by neurons and fibers of Auerbach's* autonomic plexus. In B, a smooth muscle fiber bundle of the arrector pili muscle is seen between bundles of connective tissue in the skin. Note the elongated nuclei and homogeneous cytoplasm. Arrector pili muscles originate in the papillary connective tissue and insert on hair follicles. Their contraction erects hairs in animals and produces "goose-flesh" in man. Note the proximity of the arrector pili muscle to a sebaceous gland.

 

 

Section 4: Blood

Blood is a connective tissue whose matrix is fluid. It is composed of: red corpuscles, white cells, platelets, and blood plasma. It is transported throughout the body within blood vessels, which is the subject of Section 8 of this atlas.

Red Blood Cells

Red blood cells are also known as erythrocytes or red blood corpuscles. In humans, mature red blood corpuscles do not contain a nucleus and are therefore incomplete cells. They are incapable of cell division or reproduction and self-maintenance and have very little metabolic activity. Red corpuscles are usually biconcave discs, but they are flexible and can bend and fold depending upon specific circumstances as they circulate throughout the body. The biconcave shape favors the rapid absorption and release of oxygen and carbon dioxide by providing a large surface/volume ratio. Absence of a nucleus provides additional room for the carrier protein hemoglobin, which also facilitates respiratory function.

Circulating red blood corpuscles average about 8.0 µm., whereas in dried blood smears, they are approximately 7.5 µm. In fixed and sectioned tissues, they may shrink further, but they can still be used as a rough 6 µm for internal size estimation of cells and other structures because of their widespread histological availability. In human males, there are about 5.5 million red blood corpuscles per mm³ of blood. In females, the number is about 5.0 million per mm³. It has been estimated that a 150-pound (68.2 kg) human has about 5 liters of blood.

Massed red blood corpuscles are red in color owing to the presence of the respiratory pigment hemoglobin. Mature red blood corpuscles are membrane bound and normally devoid of a nucleus, nucleolus, cell organelles, and inclusions. A small number (about 0.5 to 1.5 percent) of immature but circulating red blood corpuscles (reticulocytes) contain some ribonucleoprotein (RNA) in the form of ribosomes. Because of their RNA content, they can be stained with nuclear dyes such as brilliant cresyl blue; the RNA will appear as a reticular network, hence the name reticulocyte. When their circulating number exceeds 1 per cent, an increase in oxygen-carrying capacity is indicated owing, perhaps, to hemorrhage, a change in altitude above sea level, or pathologic changes in the vital capacity of the lungs. It is well established that the life span of red blood corpuscles Is approximately 120 days. This means that about 25 X 10¹⁰ corpuscles are replaced daily, a turnover rate of 2.5 million per second. Both damaged and normal but "worn-out" erythrocytes are removed from the vascular system by macrophages, which are found primarily in the liver, spleen, and bone marrow. Breakdown products of hemoglobin are used in the formation of bile (bilirubin), and iron is conserved and used in new red cell production.

Red corpuscles, filled with a self-synthesized protein/iron complex, hemoglobin, carry carbon dioxide to the lungs from cells and tissues where it is exchanged for oxygen. The oxygen-carrying corpuscles are passively carried in blood plasma within blood vessels. Both exchanges, in tissues and lung, take place at the capillary level; this will be considered later. The cycle of gaseous exchange is repeated about 200,000 times during the life of the corpuscle. Red blood corpuscles, normally devoid of nucleic acids (DNA, RNA), stain with acid dyes because of their content of strongly basic hemoglobin. They stain red with the widely used hematoxylin and eosin (H. & E.) and other stains. The red corpuscle may therefore also be called eosinophilic or an erythrocyte ("red cell").

Since red cells are normally only found within blood vessels, any extravascular red cells may be an artifact of tissue preparation or the result of disease or a vascular accident (stroke).

White Blood Cells

White blood cells or leucocytes ("white cells") are complete cells because they contain a nucleus and other vital organelles. Two distinct types are recognized: (1) the so-called agranular leucocytes include lymphocytes and monocytes. These "agranular" leucocytes do not have cell type-specific granules. They are, however, not devoid of granules (as their name implies) but may contain varying numbers of azurophilic granules. (2) The granular leucocytes include neutrophiles, eosinophils, and basophils, each of which have their own type-specific granules from which they derive their names. Thus, the agranular leucocytes may or may not have nonspecific granules whereas the granular leucocytes always contain type-specific granules except in the earliest stages of their development.

The relative proportion of leucocytes in normal adult human blood (per mm³) is as follows:

• Neutrophils-60 to 75 per cent, or 4200 to 5200/mm³
• Eosinophils-1 to 3 per cent, or 70 to 21O/mm³
• Basophils-0.5 to 1 per cent, 35 to 70/mm³
• Lymphocytes-20 to 45 per cent, or 1400 to 3150/mm³
• Monocytes-2 to 10 per cent, or 140 to 700/mm³

The average number of leucocytes in a normal adult varies between 5000 and 9000 per mm³. The number of white blood cells is increased (above 12,000) or decreased (below 5000) in disease states. An increase over the normal values is termed leucocytosis; a decrease is termed leucopenia. As examples, neutrophils are known to increase in number in bacterial (pus-forming) infections, eosinophils increase in allergic conditions and parasitic infections, and basophils may increase in certain inflammatory conditions of skin. Other diseases may result in changes in the number of more than one type of leucocyte.

The life span of white blood cells is considered to be shorter than that of red blood cells. The exact life span is, however, not known, because these cells normally leave the vascular system to enter tissue spaces to perform their special functions. Aging leucocytes are removed from the circulation by macrophages located in the liver and spleen. They may die and disintegrate in the connective tissue with remnants being phagocytized by histiocytes, or they may migrate through the epithelium of the gastrointestinal and respiratory tracts and be eliminated.

Some leucocytes can be recognized in tissue sections, but others are not seen to advantage by this method. A peripheral blood smear is the preferred method for identification of blood cell types. In this method, a drop of blood is spread thinly and evenly over a microscope slide. The thin layer of blood air- dries rapidly, is fixed with methanol, and stained with a Romanovsky stain. Romanovsky (1891) discovered that certain dye mixtures stained blood cell components in a way that permitted accurate determination or differential counts of the variety of cells in the circulating blood and bone marrow. Some white cell cytoplasmic components (primarily inactive DNA and RNA) stain blue with methylene blue (hence, they are called basophilic), some (primarily lysosomes and a variety of other hydrolytic enzymes) may bind the azures (dye products of methylene blue oxidation) and appear light purple; some (primarily hydrolases, which digest phagocytized materials such as antigen-anti body complexes) may bind eosin (hence, they are called eosinophilic or acidophilic), and some (primarily hydrolytic enzymes related to phagocytic function) may bind another dye complex, which produces a dusty-pink or violet color (and are called neutrophilic, in spite of the fact that the particles are not chemically neutral). In this atlas, we have elected to use Wright's stain, which is classified as a Romanovsky-type stain. Giemsa's stain is also widely used and will be similar in its staining characteristics. Leucocytes are relatively inactive while being passively carried in the blood stream, but, because they are capable of ameboid movement, they concentrate in sites of infection and are always found in sites of "potential infection" in tissues and organs; the particular vulnerability of the diges tive system has already been mentioned. Neutrophils and monocytes are the most phagocytic of the white blood cells; they ingest foreign particles, bacteria, and degenerating cells and cell fragments whether or not they can digest them. Monocytes are considered to be the most active phagocyte. Neutrophils provide the first line of defense against invading foreign bodies and organisms, and lymphocytes are believed to form antibodies, a function shared with plasma cells.

Agranulocytes

     Lymphocytes

Lymphocytes vary widely in size. Small lymphocytes are 7 to 10 µm in diameter, and large lymphocytes are approximately 14 to 20 µm in diameter, although intermediate sizes may be encountered. Larger lymphocytes are thought to be involved in humoral immunity, because they are activated by specific antigens; they differentiate into B lymphocytes and are formed in specific areas of the spleen and lymph node. Most (80 per cent) of the lymphocytes, however, are T lymphocytes, which are long- lived and are formed in different areas of the spleen and lymph node than are the B lymphocytes.

The nuclei of lymphocytes are usually round but may be slightly indented. Nuclear chromatin is clumped, inactive heterochromatin, which stains intensely with Wright's stain. The cytoplasm immediately adjacent to the nucleus is agranular and poorly stained and appears as a perinuclear halo. The thin rim of remaining cytoplasm is usually intensely basophilic but may stain variable shades of blue. Some lymphocytes possess a few azurophilic granules, but they are not evenly distributed.  Lymphocytes are produced in lymphold tissues, which are discussed in Section 9.

Monocytes

Monocytes are approximately 15 to 25 µm in diameter. The nuclei of monocytes are usually kidneyshaped, indented, or lobed. The cytoplasm of monocytes is gray-blue and contains azurophilic granules, which are generally evenly distributed. Vacuoles are often demonstrable in the cytoplasm. Monocytes frequently show evidence of ameboid movement and are voracious phagocytes. Monocytes are also produced in lymphoid organs

Granulocytes

The granulocytes include the neutrophils, eosinophils, and basophils. These cells are also known as polymorphonuclear cells because of their characteristic segmented nucleus. The three polymorphonuclear cell types are produced in the bone marrow.

Neutrophils

Neutrophils constitute 60 to 70 per cent of circulating white blood cells. They are 12 to 15 µm in diameter and possess a characteristic segmented nucleus with two to five lobes joined by fine strands of chromatin, hence the name polymorphonuclear neutrophils (PMN). The stainable heterochromatin is inactive DNA; there are no nucleoli. Immature "polymorphs" have a non-segmented oblong or rectangular nucleus; hence they are called bands. They are often bent and look like horseshoes, but they never bear this name. In females, the X chromosome may appear as a "drumstick-like" appendage on one of the lobes of the nucleus Neutrophils have abundant cytoplasm with two types of granules of different size and staining characteristics. When stained with Romanovsky-type stains, the cytoplasm appears a dusty-rose color because of cell type-specific granules that are near and below the resolving power of the light microscope (about 0.2 µm). The granules contain several enzymes: alkaline phosphatase, collagenase, and lysozyme. The second population of granules are not cell-specific. They are azurophilic, about 0.5 µm in diameter, and stain metachromatically (light purple or violet). These are primary lysosomes rich in enzymes. Although not seen with the light microscope, these cells have few mitochondria and utilize anaerobic pathways to degrade glycogen for their energy requirements. Neutrophils survive 1 to 4 days in tissues once they leave the blood stream. They traverse the connective tissues by ameboid movement and are the most active phagocytes of the three granulocytes. The azurophilic granules or lysosomes are capable of hydrolyzing bacteria, cellular debris, fungi, and viruses. Ameboid movement and, to a lesser degree, phagocytosis is seen in eosinophils and basophils.

Eosinophils

Eosinophils constitute 2 to 4 per cent of circulating white blood cells. The cell is 12 to 15 µm in diameter and usually has a bilobed nucleus. The cell is easily identified by the presence of many (about 250) large and refractile cell-specific granules. These stain red with Romanovsky-type stains. The granules stain with the dye eosin; hence, the name eosinophil, which means "eosin-loving." In the eosinophil, unlike the neutrophil, specific granules are primary lysosomes.

Basophils

Basophils constitute less than 1 per cent of the circulating white blood cells and usually require patient examination of a blood smear to locate, but they are worth the search when found. They are 12 to 15 µm in diameter but may be smaller. They possess an irregularly lobed nucleus most often obscured by the large, metachromatically basophilic granules; hence, the name basophil. The specific granules are irregular in size and shape and stain metachromatically owing to the presence of heparin. They also contain histamine.

Cell Type	Size (µm)	Number (mm^3)	Function
Neutrophil	12-15	300-700	Phagocytosis (cellular debris, bacteria, fungi, viruses, etc)
Eosinophil	12-15	120-400	Phagocytosis (antigen -antibody complexes), antiparasite agents
Basophil	12-15	30-100	Immediate hypersensitivity reaction

Platelets

Blood platelets are fragments of the cytoplasm of megakaryocytes. Platelets are small discs about 2 to 4 µm in diameter and number between 200,000 to 350,000 per mm³ of blood. In general, two to six blood platelets or thrombocytes are seen in an oil immersion field, but their distribution is variable and they may appear in large clumps. Their specific function is related to the clotting of blood both inside and outside blood vessels.

Blood Plasma

The fluid in which the blood cells reside (when within blood vessels) is called blood plasma. Plasma constitutes 55 per cent of whole blood, whereas the cellular components total 45 per cent in a normal hematocrit determination. Blood plasma contains gases, proteins, carbohydrates, amino acids, lipids, inorganic salts, enzymes, hormones, and antibodies (immunoglobulins). It is slightly alkaline. Blood plasma serves an important role in coagulation, temperature regulation, respiration, regulation of blood pH (as a buffer), and fluid balance. Hormones, absorbed nutrients, and metabolic wastes are carried in the plasma to sites of action, utilization, or elimination. When blood plasma clots, the remaining fluid is called blood serum.

Origin of Blood Cells

Since blood cells have a short life span, they must be constantly replaced in vast numbers. The term applied to this process is hematopoiesis and takes place in the bone marrow and lymphoid tissues of adults. In the embryo and fetus, various organs are active in hematopoiesis, including the yolk sac, liver, spleen, thymus, and lymph nodes, as well as bone marrow.

Erythropoiesis

Red blood corpuscles undergo their maturation within bone marrow, and several "stages" can be recognized  The earliest cells of this series have a large round nucleus, reticulated chromatin, and one or more small nucleoli. The cytoplasm is seen as a thin rim, which stains a royal blue color with Wright's stain. These cells, unfortunately, are called by several names of which you should be aware but not memorize. Determine the nomenclature preferred by your instructor and then underline the name to simplify the learning process; for example, rubriblast, proerythroblast, pronormoblast, or megaloblast. As the rubriblast matures, the nucleus becomes smaller, chromatin coarsens, and nucleoli become ill defined or disappear. The cytoplasm remains basophilic and stains blue. These cells are termed prorubricytes, basophilic erythroblasts, basophilic normoblasts, or early erythroblasts. The next recognizable stage involves further coarsening and reduction of nuclear size. Nucleoli are absent. Relatively, the cytoplasm appears to occupy more of the cell and is seen to contain a mixture of eosinophilic (red) and basophilic (blue) purplish cytoplasm. These cells are named rubricytes, polychromatophilic erythroblasts, normoblasts, intermediate erythroblasts, or intermediate normoblasts. The nucleus of the next stage is still smaller than the preceding stage and is a solid blue-black color. The nucleus is now non-functional and ready to be discarded. The cytoplasm is predominantly acidophilic with some residual basophilia. The hemoglobin, which is eosinophilic, dominates with only minimal amounts of residual ribonucleoprotein staining the cytoplasm a purplish tint. The nucleus is ejected from the cell in the next "stages" and the cytoplasm still retains a very slight purple tint, signifying the increased synthesis of hemoglobin. These cells are termed diffusely basophilic erythrocytes or polychromatophilic erythrocytes. In the final "stage,ff the cytoplasmic ribonucleoprotein disappears and the corpuscles appear as flexible biconcave discs, 6 to 8 µm in diameter, and reddish in color when stained with Romanovsky-type stains; in this atlas, with Wright's stain.

Granulocytic Series

Granular leucocytes )develop in the bone marrow from undifferentiated cells called myeloblasts. Myeloblasts are approximately 20 µm in diameter. The nucleus is round, stains a purple color, and contains two or more nucleoli. The cytoplasm is basophilic, and, when stained with Wright's stain, it appears agranular and pale blue. In the next recognizable "stage," the nucleus is reduced in size and the chromatin becomes more coarse and unevenly stained. This cell now contains the granules that stain variably from red to purple-blue and is designated a progranulocyte or a promyelocyte. A progranulocyte becomes a myelocyte when the granules become sufficiently differentiated in size, color, and shape to be positively identified as the specific granules of neutrophils, eosinophils, or basophils. The subsequent developmental "stages" are identical for the three types of granulocytes or polymorphonuclear cells.

The primary changes include a reduction in cell size and alterations in nuclear shape. The nucleus of the myelocyte tends to be slightly flattened. The chromatin becomes increasingly coarse, and nucleoli are usually indistinct or absent. The next stage, the metamyelocyte, contains an indented kidney-shaped nucleus. Additional folding results in a horseshoe-shaped nucleus, which stains deeply with basic dyes. The overall cell size continues to decrease. These cells are called "bands." The final developmental 11stageff results in a cell with a segmented or lobed nucleus, the lobes being united by narrow filaments or strands of chromatin. The cytoplasm contains the specific granules characteristic of the three types. These cells are called segmented granulocytes or polymorphonuclear granulocytes. The mature polymorphonuclear granulocyte is approximately 15 µm in diameter. 

All lymphocyte progenitor cells are believed to originate in the bone marrow. They leave the marrow to develop in the thymus to form T lymphocytes, which will leave the thymus to populate other lymphoid organs. Other bone marrow lymphocyte progenitor cells become B lymphocytes, which leave the marrow to populate other specific areas of lymphoid tissue. T and B lymphocytes cannot be distinguished by ordinary histologic methods. See Section 9.

 

WHITE BLOOD CELLS
Granulocytes

Human, air-dried blood smear, Wright's stain, 1416 x.

 

Erythrocyte: Usually biconcave and circular outline, devoid of a nucleus. Number in man varies between 5 and 5.5 million per cubic mm of blood. Erythrocytes carry oxygen from the lungs to the tissues and carbon dioxide from the tissues to the lungs.

Neutrophil: Compare sizes of the neutrophil and the erythrocyte. Lobulated nucleus, individual lobes connected by thin bridges. Cell type-specific cytoplasmic granules are small. Neutrophils constitute 40 to 75 per cent of the total white blood cell count. The number of neutrophils increases in inflammation, and they act as the first line of defense against invading pyogenic organisms.

Eosinophil: Nucleus bilobed. Cell type-specific cytoplasmic granules are large and uniform in size and stain intensely red with acid dyes. They constitute 1 to 3 per cent of total white count and increase in number in allergic states and in parasitic infections.

Basophil: The nucleus is large but less lobulated than other white blood cells. Cell type-specific cytoplasmic granules are large and variable in size and have a strong affinity for basic dyes. They constitute 0.5 to 1 per cent of white count and are believed to synthesize the heparin and histamine found in circulating blood.

 

LYMPHOCYTES
Small and large lymphocytes

Human, air-dried blood smear, Wright's stain, 1416 x.

 

Small lymphocytes: These are the most common type in normal blood. They have a large, dense, round nucleus and thin basophilic cytoplasm and are capable of ameboid movement and the production of antibodies.

Large lymphocytes: These are not very common in normal blood. The nucleus is indented, and cytoplasm is more abundant than in small lymphocytes. Azurophilic granules are frequently found in large lymphocytes; they are less commonly detected in small lymphocytes

 

MONOCYTES

Human, air-dried blood smear, Wright's stain, 4416 x.

 

Monocytes are the largest cells found in normal blood. The nucleus is centrally or peripherally located, indented, and ovoid or horseshoe-shaped; the nuclear chromatin is not as dense as that of lymphocytes. Cytoplasm is abundant and contains azurophilic granules, which are usually smaller than those seen in lymphocytes. Monocytes are voracious phagocytes. The monocyte seen on the extreme right shows pseudopodia extending from the cell body and contains a phagocytized red cell nucleus

Note the comparative size of erythrocytes and monocytes.

 

BONE MARROW
PERIPHERAL BLOOD

A. Human, air-dried marrow smear, Wright's stain, 1416 x.
B. Human, air-dried blood smear, brilliant cresyl blue, 1416 x.

 

Polychromatophilic erythroblasts (rubricytes): Derivatives of basophilic erythroblasts (prorubricytes). Dense nuclear chromatin with polychromatophilic cytoplasm owing to a declining RNA content and an increase in newly synthesized hemoglobin.

Reticulocytes: Immature red blood cells seen in the circulating blood. Clumping of ribosomes gives them a reticulated appearance.

Platelets: Also called thrombocytes, these are minute round or ovoid structures. They are important in blood coagulation and are derived from bone marrow megakaryocytes.

 

RED BONE MARROW
In situ

Human, Müller's fluid, H. & E., 50 x.

In this plate, a layer of compact bone surrounds the red bone marrow cavity.

Red or hemopoietic marrow is the characteristic variety of marrow until middle childhood. By late adolescence, most red marrow is replaced by fatty or yellow bone marrow. In adults, red marrow occurs in the sternum, ribs, vertebrae, heads of long bones, and cranium.

Red marrow is characterized by high cellularity and sinuses filling spaces between a delicate reticular supporting tissue. The cells seen are giant megakaryocytes and developing red and white blood cells.

 

BONE MARROW

Human, air-dried marrow smear, Wright's stain, 1416 x.

Rubriblast (proerythroblast, hernocytoblast, myeloblast): Stem cell of the erythroid series with a large rounded nucleus, basophilic cytoplasm.

Prorubricyte (basophilic erythroblast): This develops from the rubriblast. It is smaller than the stem cell, and the nucleus has coarser chromatin. RNA-rich cytoplasm is densely basophilic. Basophilia obscures hemoglobin content. It undergoes mitotic division, giving rise to rubricytes.

Rubricyte (polychromatophilic erythroblast): This is a product of mitotic division of prorubricytes and is smaller than the mother cell. Nuclear chromatin more compact. Cytoplasmic basophilia are less marked and hemoglobin content is greater than in the mother cell. Rubricytes have an affinity for both acid and basic dyes (because of their content of hemoglobin and RNA respectively), which determines their polychromatophilic staining characteristics.

Metarubricyte (normoblast): It arises by mitotic division of the rubricyte. The nucleus is small and pyknotic. Cytoplasm is distinctly aciclophilic owing to increased hemoglobin content.

Erythrocyte: Non-nucleated (nuclei of metarubricytes have been extruded) with circular outline. In side view, they appear dumbbell-shaped because of the biconcave nature of their surfaces. The number varies in man from 5 to 5.5 million per cubic mm. They carry oxygen from lungs to.tissue and carbon dioxide from tissue to lungs, and are filled with hemoglobin. immature stages in development (reticulocytes) have a diffusely basophilic cytoplasm because of the residual content of RNA.

Degenerating cell: Often found in bone marrow. They are remnants of damaged corpuscles, megakaryocytes, or myeloblasts. These are primarily artifacts of a marrow smear preparation.

 

BONE MARROW
Developing neutrophils

Human, air-dried marrow smear, Wright's stain, 1416 x.

 

Myeloblast: The stem cell of the leucocytic series with lightly basophilic cytoplasm. The nuclei are large and rounded. The chromatin is in the form of moderately coarse interconnected strands. They constitute 0.3 to 0.5 per cent of marrow cells. Myeloblasts increase in leukemia.

Progranulocyte: Also called promyelocyte. It arises and differentiates from myeloblasts. It has large cells; its nuclei are rounded with coarse chromatin. Cytoplasm is basophilic with some azurophilic granules. This cell type constitutes about 4 per cent of marrow cells.

Neutrophilic myelocyte: This arises from progranulocytes. It is smaller, has less basophilic cytoplasm containing differentiated granules and a nucleus with more compact chromatin.

Neutrophilic bands: These are immature neutrophils. The nuclei are horseshoe- or drumstick-shaped.

Neutrophilic metamyelocyte: It has a kidney-shaped nucleus and is not capable of division. It differentiates into mature neutrophilic myelocytes.

Neutrophil (segmented): This is a mature cell. Its nucleus is markedly lobulated. The lobules may be connected with a thin chromatin thread. Chromatin is compact, and there is abundant cytoplasm. Granules in the cytoplasm are small and may be inconspicuous.

 

BONE MARROW
Developing eosinophils

Human, air-dried marrow smear, Wright's stain, 1416 x.

Myeloblast: Stem cell of the leucocytic series. It has a rounded large nucleus and lightly basophilic agranular cytoplasm. .

Eosinophilic myelocyte: These develop from myeloblasts. Specific acidophilic granules appear in cytoplasm. The nucleus is rounded or oval. Chromatin of nucleus is coarser than in the myeloblast. This cell is capable of division.

Eosinophilic metamyelocyte: This cell is no longer capable of cell division. The nucleus is kidney- shaped or indented. Cytoplasm contains acidophilic granules.

Eosinophilic band: Immature or juvenile eosinophil. The nucleus is horseshoe- or drumstick-shaped, and there are eosinophilic granules in cytoplasm.

Segmented eosinophil: Mature eosinophil. The nucleus is lobulated and the lobes are connected with thin chromatin threads. There is abundant granular cytoplasm.

 

BONE MARROW
Developing basophils

Human, air-dried marrow smear, Wright's stain, 1416 x.

 

Basophilic metamyelocyte: Derived from basophilic myelocyte, which is not represented in this figure. Basophilic myelocytes are scarce and may not be seen in a single marrow smear preparation. It is believed that their granules are water soluble. This cell is no longer capable of cell division. The nucleus is oval to kidney-shaped. Cytoplasm has basophilic granules.

Basophilic band: An immature basophil with a horseshoe-shaped nucleus. There are basophilic granules in the cytoplasm.

 

 

BONE MARROW
Wandering cells

Human, air-dried marrow smear, Wright's stain, 1416 x.

 

Phagocytic histiocyte: Large cell. Irregular cell outline with many short cell processes (pseudopods). There is abundant cytoplasm containing phagocytized material. The nucleus is oval.

Monocyte: Large cell with a prominent eccentric nucleus. It has a highly ameboid cytoplasm containing various inclusions.

Lymphocyte: Spherical, dense nucleus with a thin, inconspicuous rim of basophilic cytoplasm

 

BONE MARROW

Human, air-dried marrow smear, Wright's stain, 1416 x.

Megakaryocyte: Giant cell characteristic of bone marrow, with a conspicuous multilobed nucleus. Cytoplasm contains fine granules. Pseudopodia extend from the cell surface and later detach to form the blood platelets. Blood platelets participate in the blood-clotting mechanism by contributing to the formation of thromboplastin, by "plugging" abnormal breaks in the endothelium of blood vessels, and by inducing the constriction of damaged blood vessels.

Red blood cells: Non-nucleated corpuscles having a circular or dumbbell-shaped appearance. Contain hemoglobin.

Lipocyte: Fat cells are constantly present in bone marrow. They have irregular outlines and are filled with lipid droplets.

Section 3: Connective Tissue

The connective tissues include a variety of cells, non-living cell products, and blood. A classification and a concise discussion of the various connective tissues follows.

 

 

1. Adult Connective Tissue

Connective Tissue Proper

The loose or areolar connective tissue is made up of many cell types and intercellular materials (matrix), which also comprise other connective tissues but in varying proportions. It is widely distributed in the body and is found most readily beneath the skin and superficial fascia (fatty connective tissue), separating muscles, in all potential spaces, and beneath the epithelial lining in the lamina propria of the digestive system. This web-like tissue binds cells and organs together but permits these cells and organs to move, as necessary, in relation to each other. Because it is composed of a large amount of amorphous ground substance (whose consistency varies from liquid to gel), it allows wandering cells to move around freely and other structures, such as blood vessels and nerve, to pass through it. This connective tissue is important, because of its cellular content, for defense against infection and the repair of damaged tissue                                    

Important cells found in the loose connective tissue include the following: Fibroblasts, which synthesize collagenous connective tissue fibers that are flexible but of great tensile strength; macrophages (or histiocytes) and monocytes, which ingest, digest, or "store" microscopic particles such as debris of dead cells; certain microorganisms; and other non-biodegradable matter. Capable of ameboid movement, these cells wander throughout the connective tissue and congregate in regions requiring their specialized function. Mast cells synthesize and release substances of physiological importance (e.g., heparin and histamine).

Heparin is a powerful anticoagulant of blood, whereas histamine increases the permeability of blood capillaries. Circulating eosinophils increase in number in parasitic infections and in hypersensitivity reactions, such as in hay fever and asthma. Factors within the specific granules of eosinophils are thought to function as anti-larval agents in helminthic infections; additional factors can be directed against histamine and other inflammatory agents. Lymphocytes and plasma cells also populate loose connective tissue and play a vital role in the defense mechanism by producing antibodies, the immunoglobulins of the blood. Eosinophils, lymphocytes, and plasma cells are particularly abundant in the lamina propria of the digestive system  and other potentially vulnerable areas of the body.

In the digestive system and elsewhere, the individual is separated from pathogenic organisms of the external environment by only a delicate single cell layer. This cell layer is essential for absorption, excretion, and gaseous exchange; it is therefore vital to maintain defensive cells just below the vulnerable surface. Fat cells may occur singly or in small or large numbers. When fat cells predominate, the tissue is called adipose tissue. One of the special connective tissues, adipose tissue, serves as a reservoir of energy and as a soft packing in potential spaces (e.g., axilla and ischiorectal fossa) and for organs. It also envelopes glands that undergo cyclic or functional variation in size and activity (e.g., mammary glands), surrounds mobile organs (e.g., eye and heart), and protects other organs (e.g., kidney), blood vessels, and nerve fiber bundles.

The more important intercellular components of the loose connective tissue include three kinds of fibers (collagenous, previously mentioned, and elastic and reticular) and amorphous ground substance. Collagenous and reticular fibers belong to the same class of protein, collagen, whereas elastic fibers are formed of elastin. There are many kinds of collagen, and it is the most abundant protein of the human body (about 30 percent of the dry weight). Ground substance is composed primarily of two classes of compounds: glycosaminoglycans and structural glycoproteins. The term glycosaminoglycan is replacing the older, but widely used, term mucopolysaccharide to denote a linear polysaccharide with characteristic repeating disaccharide units. The repeating units are usually a uronic acid and a hexosamine. The uronic acid may be glucuronic or iduronic acid, and the hexosamine may be glucosamine or galactosamine. The structural glycoproteins play an important role in cell interaction and in migration and adhesion of cells. Fibronectin, laminin, and chondronectin are three structural glycoproteins; these cannot, however, be distinguished by routine histological techniques. It might be useful to remember, as you review your microscope slides and the photomicrographs in this book, that collagenous fibers are acidophilic and therefore stain red with eosin, stain blue with Mallory's trichrome, and stain green with Masson's trichrome. Elastic fibers may or may not stain well with eosin or Masson's stain, and they may or may not stain (red or yellow) with Mallory's stain; however, old elastic fibers will stain better than younger elastic fibers. Elastic fibers do stain well with toluidine blue (often used to stain 1 µm plastic-embedded tissues) , aldehyde fuchsin orcein phosphotungstic acid hernatoxylin and Weigert's elastic tissue stain .Reticular fibers are argyrophilic and therefore stain with silver stains such as Wilder's method. Ground substance or basement membrane is stained by aldehyde fuchsin periodic acid-Schiff stain and toluidine blue

Dense connective tissue contains fewer cells, but, when they are present, they are similar in type to those found in loose connective tissue. Collagenous (Type 1) fibers predominate in this type of connective tissue. Dense connective tissue appears in two forms: dense irregular and dense regular connective tissue. The irregular type is found in the dermis of the skin, deep fascia surrounding and defining muscles, capsules of organs, and nerve sheaths. Dense regular connective tissue is found mainly in ligaments and tendons , which provide flexible but inelastic unions between bones and between bones and skeletal muscle. At low magnification, a tendon may be confused with striated muscle, because the fibers are axially arranged and the alignment of fibroblast nuclei resembles that found in striated muscle. At higher magnifications, however, the structural differences are easily recognized and a proper identification is readily accomplished. Other examples of dense regular connective tissue include most ligaments, aponeuroses, and the cornea of the eye

Connective tissues with special characteristics of structure and function include elastic, reticular, and pigmented types. Adipose tissue, which also belongs to this group, has already been mentioned and will be indelibly remembered by students of gross anatomy as the layer of tissue immediately below the skin. The fatty tissue is also known as superficial fascia or as the panniculus adiposus. Adipose tissue is of two types: white, "signet ring," unilocular fat , which is ubiquitous; and brown, multilocular, fat , which is uncommon in the adult human.

Elastic fibers in dense parallel bands (elastic tissue) can be found associated with the vertebral column (ligamenturn flava), the suspensory ligament of the penis, and vocal cords. The fascia of the lower anterior abdominal wall (Scarpa's fascia) is predominantly elastic tissue. Elastic fibers are obviously functionally important components of skin  and hollow organs, including elastic arteries of the vascular system , trachea and bronchi of the respiratory system, and others. Reticular fibers are composed of collagen (Type 3) and are very fine and highly branched. They form supporting networks around blood vessels and cells in some organs. They are in continuity with other collagenous fibers; they are also inelastic. These fibers are only revealed by special histological techniques . Reticular fibers are found in abundance in lymph nodes, blood-forming organs, spleen, liver, and elsewhere, but, as mentioned previously, they cannot be seen unless special methods using silver salts are used to reveal their presence. This type of connective tissue is associated with phagocytic reticular cells, for example, the reticular cells of lymph nodes  and Kupffer's cells of the liver . Pigment tissue is a cellular connective tissue rather than a fibrous non-living connective tissue and has many melanin-containing connective tissue cells. It is found principally in the choroid and iris of the eye

Cartilage

Cartilage is a non-vascular tissue containing fibrous connective tissue (collagen Type 2) embedded in an abundant and firm matrix. The cells that produce cartilage are called chondroblasts, and, in mature cartilage where the cells are housed in lacunae, they are termed chondrocytes. In early development, the greater part of the skeleton is cartilaginous, but, during later stages of development, the cartilage is remodeled and replaced by bone. The process is called endochondral ossification. Three types of cartilage are recognized: hyaline, elastic, and fibrocartilage. Hyaline cartilage is found at the ventral ends of ribs and in the nose, larynx, trachea, and articular surfaces of adjacent bones of movable joints. The matrix or ground substance of cartilage is strongly basophilic and stains metachromatically with toluidine blue and other similar basic dyes . It is the acidic sulfate groups of the proteoglycans comprising the ground substance that account for the staining reaction just noted. Metachromasia means that a cell or tissue takes on a color different from the dye solution with which it is stained.

Fibrocartilage has a limited distribution. It is found in the intervertebral discs, pubic symphysis, menisci and ligaments of the knee and other joints, and in the tendons of some muscles where they glide over bones (e.g., tendons of peroneus longus and tibialis posterior. Fibrocartilage is composed predominantly of collagenous (Type 1) fibers arranged in bundles, with cartilage cells surrounded by a sparse cartilage matrix between the fibrous bundles. Fibrocartilage has characteristics similar to both dense connective tissue and hyaline cartilage. It is always associated with dense connective tissue, and, because of its usual paucity of cartilage cells, there appears to be a gradual transition between the two types of connective tissue. Although cartilage cells are not abundant, they are arranged in scattered clusters in parallel arrays, reflecting the direction of stresses placed upon the tissue. Fibrocartilage has no identifiable perichondrium and differs in this regard from hyaline and elastic cartilage. Elastic cartilage is found in the external ear (pinna), auditory tube, epiglottis, and corniculate and cuneiform cartilages of the larynx . It is yellow and is more flexible and elastic than the other cartilage types owing to abundant branching elastic fibers in its matrix. Elastic fibers are often concentrated in the walls of lacunae, which house cartilage cells.

Bone

Bone is a tissue that forms the greatest part of the skeleton and is one of the hardest structures of the body. It is the rack upon which all the soft parts are suspended or attached. Only the dentin and enamel of teeth are harder. The skeleton is tough and slightly elastic, withstanding tension and compression. Bone differs from cartilage by having its collagenous connective tissue matrix impregnated with organic salts (primarily calcium phosphate and lesser amounts of calcium carbonate, calcium fluoride, magnesium phosphate, and sodium chloride). The osteoblasts, which form the osseous tissue , become encapsulated in lacunae but maintain contact with the vascular system via microscopic canaliculi . When they become encapsulated, they are referred to as osteocytes.

A characteristic feature of a cross section of the shaft (diaphysis) of a long bone is its organization in concentric rings around a central canal containing a blood vessel. This is called a Haversian system (osteon). Between neighboring Haversian systems are non-concentric lamellae, devoid of Haversian canals, termed interstitial lamellae. Vascular canals, called Volkmann's canals , traverse the long axis of the bone; they are always at right angles to Haversian canals. Their function is to link vascular canals of adjacent Haversian systems with each other and with the periosteal and endosteal blood vessels of the bone. The outer perimeter of a long bone, beneath the osteogenic connective tissue (called periosteum), is composed of circumferential lamellae, which also lack Haversian canals. This thick-walled hollow shaft of compact bone (the diaphysis) contains bone marrow. At the distal ends of long bones, where Haversian systems are not found, the bone appears spongy and is therefore called cancellous, or spongy, bone. The spongy appearance is misleading, because careful examination of the architecture reveals a highly organized trabecular system providing maximal structural support with minimal density of bony tissue. The epiphyses  at the ends of the diaphysis or shaft contain the spongy bone covered by a thin layer of compact bone. The cavities of the epiphyseal spongy bone are in contact with the bone marrow core of the diaphysis except during growth of long bones in young animals. Interposed between the epiphysis and the diaphysis is the cartilaginous epiphyseal plate. The epiphyseal plate is joined to the diaphysis by columns of cancellous bone; this region is known as the metaphysis.

When bone is formed in and replaces a cartilaginous "model," the process is termed endochondral ossification. Some parts of the skull develop from osteogenic mesenchymal connective tissue, however, without a cartilaginous "model" having been formed first. This is termed intramembranous ossification, and these bones are called membrane bones. In both instances, three types of cells are associated with bone formation, growth, and maintenance: osteoblastsosteocytes and osteoclasts .The osteoblasts produce osseous tissue (bone), become embedded in the matrix they manufacture, and are then renamed osteocytes, to reflect their change of status. They remain viable, because they have access to the vascular supply via microscopic canaliculi through which cellular processes extend to receive nutrients and oxygen. Osteoclasts actively resorb and remodel bone as required for growth; these are giant, multinuclear, phagocytic, and osteolytic cells.

Blood and Lymph

This type of connective tissue is peculiar because its matrix is liquid. The blood is carried in blood vessels and is moved throughout the body by the contractile power of the heart. Blood vessels and heart are discussed in Section 8 of this book. Lymph is found in lymph vessels but originates in extracellular spaces as extracellular fluid, which is normally extravasated from blood capillaries. The extracellular fluid, which enters the lymphatic system of vessels, will have mononuclear white blood cells added to it as the fluid is filtered through lymph nodes, which produce such cells. Lymph is returned to the blood stream near the right and left venous angles junction of the internal jugular and subclavian veins). The lymphatic system is discussed more fully in Section 9.

Embryonic Connective Tissue

Mesenchyme

Derived from embryonic mesoderm, mesenchyme  is the first connective tissue formed. The cells are widely spaced, with an abundance of intercellular matrix. The primitive mesenchymal cells differentiate into all the supporting tissues of the body. The cells derived from the mesenchyme include blood cells, megakaryocytes, endothelium, mesothelium, reticular cells, fibroblasts, mast cells, plasma cells, special phagocytic cells of the spleen and liver, cartilage cells, and bone cells as well as smooth muscle.

Mucoid Tissue

Widely distributed in the embryo as a loose connective tissue, mucoid tissue is composed of large stellate fibroblasts in an abundant intercellular substance, which is homogeneous and soft. In the umbilical cord it is known as Wharton's jelly.

AREOLAR CONNECTIVE TISSUE
Subcutaneous

Rat, 10% formalin, H. & E., 612 x.

Areolar connective tissue is so-named because of the many small areas or potential spaces that are seen within this tissue. It is the most widely encountered type of connective tissue and contains most of the connective tissue components.

Collogenous fibers: Coarse interlacing bundles of fibers that run in all directions in the connective tissue.

Elastic fibers: Slender network of branching fibers irregularly dispersed in the connective tissue. Smaller than the collagen fiber bundles.

Mast cell: A large cell with a small spherical nucleus and abundant cytoplasm containing coarse granules. Produces heparin and histamine. In some animals, 5-hydroxytryptamine (serotonin) is also produced by this cell.

Fibroblasts: Only nuclei are seen in this preparation. Nuclei are ovoid and larger than other connective tissue nuclei. Fibroblasts are the most common cell type found in areolar connective tissue. They synthesize and deposit collagen.

Lymphocyte: Only nuclei are seen in this preparation. Smaller than fibroblast nuclei, rounder and more deeply stained. They are not as abundant as fibroblasts.

 

MAST CELLS

A. Human, 10% formalin, H. & E., 162 x.
B. Rat, Helly's fluid, toluidine blue and erythrocin stains, 1416 x.
C. Rat, glutaraldehyde-osmium fixation, toluldine blue stain, 1416 x.

Mast cells are found in areolar connective tissue and along the course of small blood vessels. They have a spheroid nucleus and abundant cytoplasm. The cytoplasm is filled with coarse granules that stain red in H. & E. preparations (A), but stain metachromatically with toluidine blue and other basic aniline dyes (B and C). Granules may be so abundant as to obscure the nucleus.

In man, mast cells produce heparin, an anticoagulant substance that prevents blood clots. They also produce histamine, which increases the permeability of capillaries and influences the blood pressure.

In rats, mast cells also contain serotonin (5-hydroxytryptamine), which causes vasoconstriction and elevation of blood pressure.

Collagen and elastic fibers are scattered in the interstices between mast cells. Two fat cells stained black by osmium tetroxide fixation are seen in C.

 

PLASMA CELLS
Lamina propria
jejunum

 

Human, 10% formalin, H. & E., 612 x.

Plasma cells, although uncommon in loose connective tissue, are plentiful in the lamina propria of the digestive tract. Note the ovoid shape of the cell, the eccentric round or oval nucleus, and the intensely basophilic cytoplasm. The less densely stained area of the cytoplasm in juxtaposition to the nucleus contains the Golgi complex and centrioles. Nuclear chromatin is characteristically clumped around the periphery of the nucleus and produces, in negative image, a radial pattern resembling the spokes of a wheel. The basophilia of the cytoplasm is shown by electron microscopy to be due to an extensive system of membrane-bound ribonucleoprotein. These cells produce antibodies.

Note the bilobed nucleus characteristic of human eosinophils in loose connective tissue. Eosinophils reach the lamina propria from the blood capillaries. The coarse, intensely eosinophilic 

Compare the size of lymphocytes and plasma cells. Note that the nucleus of the lymphocyte fills most of the cell, with only a thin rim of basophilic cytoplasm around it. Some of the lymphocytes in the lamina propria migrate through the epithelium to the lumen, where they are eliminated.

 

MACROPHAGES
Lung
terminal bronchiole

Cat, 10% formalin, H. & E., 324 x.

This illustration shows a cross section of a terminal bronchiole with phagocytized material (black) in macrophages within the lumen of the bronchiole (airway). The pleating of the epithelial lining denotes a constricted bronchiole. Note the low columnar epithelial lining of the wall of the bronchiole and the smooth muscle bundle adjacent to the lining epithelium.

 

TENDON
Embryonic triceps muscle tendon

Human, glutaraldehyde-osmium fixation, toluidine blue stain, 1416 x.

Fibroblasts: Also known in mature tendons as tendon cells, or fibrocytes, they are the only cell type present. They are stellate in shape with cytoplasmic processes extending between and around the collagen bundles.

Collagen: In thick bundles or fascicles, separated by tendon cells and loose connective tissue. Collagenous connective tissue fibers are protein and synthesized by fibroblasts

 

COLLAGENOUS CONNECTIVE TISSUE
Tendo calcaneus (tendon of Achilles)
longitudinal section

Human, 10% formalin, H. & E., 162 x.

Tendons may be confused with striated muscle sections. This confusion can be avoided by examining the tissue at higher magnifications, where the presence or absence of cross striations will be diagnostic.

Collagen: Fibers oriented in one direction and in dense aggregates and bundles separated by a small amount of areolar connective tissue containing vessels and nerves. Collagen fibrils are flexible, have high tensile strength, and are inelastic. This large tendon links the gastrocnemius and soleus muscles with the calcaneus bone at the rear of the foot.

Fibrocyte nuclei: The predominant cell in this type of connective tissue (dense collagenous type) is oriented primarily in the longitudinal axis, in rows between the bundles of collagenous fibers.

 

LIGAMENT
Temporal bone and stapes

 

Cat, Müller's fluid, iron hematoxylin, 162 x.

Tympanic cavity: Air-containing space in the middle ear. Limited laterally by the tympanic membrane and medially by the osseous labyrinth. Although not illustrated here, the cavity contains the chorda tympani nerve, the auditory ossicles, and the small tendons of the stapedius and tensor tympani muscles, which are connected to the bony ossicles.

Stapes: One of the three auditory ossicles in the tympanic cavity. It was so-named by the Italian anatomist, Ingrassias, who described it in 1546. The name is derived from Latin (stare, to stand; pes, foot; the thing in which the foot stands) because of its resemblance to a stirrup. The ossicle consists of a head, neck, two limbs, or crura, and a base. The two crura are connected to the base. This figure shows only the anterior crus of the stapes and part of the base.

Anterior crus of stapes: Shorter and less curved than the posterior crus with which it is connected by the base.

Base of stapes: Also called foot plate of the stapes. Fixed to the margin of the fenestra vestibuli (ovalis) by the annular ligament. Also connects the two crura of the stapes.

Annular ligament: A ring of fibroelastic tissue that fixes the base of the stapes to the fenestra vestibuli but permits the stapes to rock or move in response to vibrations of the eardrum. The sound waves are transmitted to the stapes by the incus and malleus (the other two auditory ossicles).

Vestibule: Large bony cavity medial to the tympanic cavity. In its lateral wall is the fenestra vestibuli, to which the foot of the stapes is fixed.

Temporal bone: Surrounding the ear cavities and containing the vestibular and sound receptor organs (semicircular canals and the cochlea

 

ELASTIC MEMBRANES
COLLAGENOUS FIBERS
Aorta

 

Rat, Helly's fluid, H. & E., 162 x.

 The general method used in this preparation allows differentiation of the various vessel wall components only by the variations in the intensity of staining. The pronounced eosinophilia of the elastic fibers reflects their high content of basic amino acids. .

Elastic membranes: Abundant in the media of elastic arteries. The elastic fibers anastomose to form a fenestrated "membrane," which is circularly arranged in layers.

Smooth muscle nuclei: Smooth muscle fibers are located between the elastic fiber networks. Circularly disposed.

Collagen: In the adventitia, collagen forms a loose irregular connective tissue layer surrounding the blood vessel. Elastic fibers, not distinguishable by this method, are also found in this connective tissue coat.

 

ELASTIC MEMBRANES
COLLAGENOUS FIBERS
Aorta

Rat, Helly's fluid, Mallory's stain (A),
Mallory-azan stain (B), 162 x.

 Elastic membranes: Elastic membranes are a striking feature of the aorta. Located within the tunica media of the vessel wall, they serve as "shock absorbers." Elastic arteries are subject to the greatest and most rapid changes in blood pressure. The elastic membranes or laminae are separated from each other by smooth muscle fibers, fibroblasts, and collagenous and reticular connective tissue fibers. Note that the elastic laminae are unstained by the methods used here. 

Collagen: Primarily located external to the outermost elastic lamina, it stains a bright blue with Mallory and Mallory-azan stains. Note the collagenous connective tissue immediately adjacent to the elastic laminae. The methods used here selectively stain collagenous fibers

 

RETICULAR CELLS
Lymph node
subcapsular sinus

Rhesus monkey, Helly's fluid, H. & E., 612 x.

The subcapsular sinus of a lymph node is a lymph channel beneath the capsule of the node . The component elements of the sinus are seen in this figure: reticular cells and their processes, which form a meshwork. Reticular cells are "star-shaped," with lightly staining cytoplasm and processes that are in contact but not continuous with processes of adjacent cells. These stellate reticular cells and their processes form the reticular tissue meshwork of the node in which lymphocytes and free macrophages are found.

 

RETICULAR FIBERS
Liver

Rhesus monkey, Wilder's* method, 612 x.

Reticular fibers: Reticular fibers branch and anastomose in a delicate fibrous network delineating the sinusoids. They are of small diameter and are resistant to dyes, making them difficult to demonstrate except by special techniques such as the method used in this preparation.

Binucleate liver cells: These are polyhedral cells with centrally placed nuclei and prominent nucleoli. Occasionally, they are multinucleate.

Sinusoids: Sinusoids constitute the intralobular system of specialized vascular channels. They carry blood from interlobular branches of the portal vein centripetally to the central vein. They anastomose and separate adjacent hepatic cellular plates.

Central vein: This is located in the center of the hepatic lobule. It is the smallest radicle of hepatic veins and receives the contents of all the sinusoids of the hepatic lobule.

Red blood cells: Filling the central vein, they carry oxygen to the hepatic cells and remove carbon dioxide. The red blood cell is approximately 6 µm in diameter and can be used as a rough internal measure.

 

RETICULAR FIBERS
Duodenum smooth muscle

Rat, 10% formalin, Gomori silver, 435 x.

This is part of the muscular coat of the duodenum, stained with a special silver technique to demonstrate reticular fibers. Note the abundance of reticular fibers between muscle fibers. The latter are not stained by this method. The negative outlines of the transversely sectioned muscle fibers of the inner circular coat are well delineated by the stained reticular fibers.

 

BROWN FAT
Mediastinum

Rhesus monkey, Helly's fluid, H. & E., A. 162 x; B. 612 x.

Brown fat is an uncommon variety of human fat found in the upper back (interscapular region) of the body. Unlike the more common white fat, brown fat cells contain a number of small lipid droplets, hence the name multilocular fat. White fat cells, in contrast, contain a single lipid droplet

 

FAT CELLS
Panniculus adiposus

Human, 10% formalin, H. & E., 612 x.

The fat cell lipid is in the form of a single droplet, and these cells are described as unilocular. In brown fat .the lipid appears as small multiple droplets, and these cells are described as multilocular.

Fat cell cytoplasm: This appears as a thin rim at the periphery of the cell. Stored fat is the predominant component of cytoplasm.

Fat cell nucleus: The nucleus is flattened in the cytoplasm, permitting maximum storage of fat globules.

Fat globules: These appear as empty spaces because the fat has been dissolved out by solvents used in the preparation of tissues. Special fixatives and stains are needed to demonstrate lipid droplets .At body temperature, the fat is liquid.

 

HYALINE CARTILAGE
Trachea

 

A. Rat, Helly's fluid, Mallory's stain, 612 x.
B. Rat, Helly's fluid, toluidine blue and erythrosin, 612 x.

Perichondrium: A dense layer of irregular fibrous connective tissue that always invests hyaline cartilage except at the free surfaces of articular cartilage. Note how the perichondrium in A is better shown by a connective tissue stain (Mallory's) specific for collagen.

Chondrocytes: Two or more cartilage cells enclosed within smooth-walled spaces or lacunae. Note the centrally placed, large spherical nuclei (isogenous grouping). The vacuolation noted in the cytoplasm of chondrocytes is an artifact of processing resulting from poor preservation of fat droplets and glycogen. Note the rounded appearance of chondrocytes centrally located and the flattened appearance of cells near the perichondrium.

Matrix: Derived from a Latin word meaning womb, matrix is a place where something is formed and a medium enclosing other bodies. The matrix is composed of ground substance (protein polysaccharicle) and connective tissue fibers. It fills the space between chondrocytes. In B, note the metachromasia of the matrix stained with toluidine blue. The metachromasia is due to the high content of chondroitin sulfate.

Capsule: The walls of the lacunae are referred to as a capsule. The capsule is a condensation of the matrix surrounding the lacunae.

 

ELASTIC CARTILAGE
Epiglottis

Dog, Zenker's* fluid, H. & E., 162 x.

 

Cartilage cells: These cells are large and pleomorphic and are housed in lacunae of the matrix. One or more cells may be found in one lacuna.

Capsules: Condensed matrix surrounding cartilage cells. These capsules stain intensely and represent the most recent deposition of matrix.

Matrix: The matrix contains collagenous and elastic fibers embedded in a highly acidic ground substance consisting of chondroitin sulfate and protein.

Elastic fibers: Oriented in all directions within the matrix, elastic fibers give flexibility to the cartilage.

 

ENDOCHONDRAL OSSIFICATION
Finger phalanx

Human, 10% formalin, H. & E., 162 x.

Endochondral bone is a form of ossification in which an embryonal type of hyaline cartilage precedes the formation of bone. In this figure, note the change from the zone of reserve cartilage cells to the zone of hypertrophic cartilage cells. Lacunae are increased in size, and the interlacunar space is reduced in the region of the hypertrophic cells. Note the concomitant change in the perichondrium in which the inner cells change into osteogenic cells (osteoblasts) leading to the formation of the periosteal (perichondral) bone collar.

 

DEVELOPING LONG BONE

Rodent, 10% formalin, decalcified, H. & E., 22 x.

Detail from a later stage of bone formation in which a bony collar has formed and cartilage is undergoing various bone growth-related changes. The development of a bone collar initiates the hypertrophy of cartilage cells. This is followed by a vascular intrusion into the cartilaginous core bringing along with it osteoblasts and chondroclasts from the osteogenic connective tissue perichondrium. The osteogenic cells lay down osseous tissue internally and the bony collar expands, in the long axis distally, in both directions. The structural components of the cartilage at this stage have been identified as (1) so-called resting, epiphyseal, or articular cartilage, which is adjacent to (2) proliferating cartilage, which is characterized by mitotic figures and flattened chondrocytes, which synthesize and secrete matrix. The next zone (3) is named maturing cartilage, where matrix is also synthesized and presumably prepared for calcification. The cells enlarge and accumulate glycogen; then the cells become inactive and vacuolated and the nuclei pyknotic, after which they die. This region is known as (4) the zone of hypertrophy. When the lacunae housing chondrocytes break down in the transverse plane, they form spicules of cartilage, which become calcified, resulting in the formation of (5) the so-called primary spongiosa into which capillary loops advance. The advancing blood vessels bring in osteogenic cells, which continue the calcification process and development of bone.

The formation of a mature bone involves the following developmental stages: (1) intramembranous ossification, (2) endochondral ossification, (3) growth, (4) shaping or modeling to desired shape, and (5) remodeling mature bone over time.

 

FETAL LONG BONE

Rodent, Helly's fluid, Mallory-azan, 5.5 x.

At this stage of development, the upper epiphyseal cartilage has been invaded by blood vessels and mesenchyme prior to the formation of the epiphyseal ossification center.

The diaphyseal bony collar extends to the epiphyseal plate composed of calcified cartilage. The formation and remodeling of the diaphysis can be seen to be well advanced.

The process of bone formation will not be complete, and bone growth ended, until the epiphyseal plate disappears and the marrow cavity becomes continuous throughout the bone. The blood vessels of the diaphysis, epiphyses, and metaphyses will then intercommunicate.

 

MEMBRANE BONE
Mandible

Cat, Müller's* fluid, H. & E., A. 162 x; B. & C. 1416 x.

0steoclast: A multinucleated giant cell associated with areas of bone resorption. The surface adjacent to bone being resorbed has numerous cytoplasmic processes, giving the ruffled border appearance.

Osteoblasts: Responsible for bone matrix formation and present wherever osseous tissue is elaborated. In membranous bone, osteoblasts arise as differentiated mesenchymal cells.

 

ENDOCHONDRAL BONE
AND FIBROCARTILAGE
Tibia knee joint

Rat, 10% formalin, A., H. & E., 50 x;
B. Gomori's aldehyde fuchsin, 612 x.

Enclochondral bone formation is a process in which an embryonal type of hyaline cartilage precedes bone formation. In A, a stage in the endochondral ossification in a long bone (tibia) is shown. Note the cartilaginous epiphyseal plate that separates the epiphysis (above) from the diaphysis (below). The epiphyseal plate is the source of new cartilage, which is replaced by bone during growth in length. Note the zone of hypertrophic cartilage within the epiphyseal plate. This is a stage in endochondral bone formation preceding calcification. Islands of formed endochondral bone are seen above and below the epiphyseal plate in the epiphysis and diaphysis. Note the marrow cavity between plates of endochrondral bone in the diaphysis. This cavity is formed by resorption of endochondral bone.

In B, note the characteristic grouping of fibrocartilage cells and their arrangement in rows separated by dense collagenous connective tissue. Adjacent to fibrocartilage, note the hyaline cartilage cells.

 

BONE
Fibula

 

Human, ground bone, unstained, 612 x.

Haversian canal: Conducts blood vessels, lymphatics, and nerves through bone. Haversian canals surrounded by concentric lamellae of compact bone form the Haversian system. These canals are named after Clopton Havers, an English physician, who described them in his Osteologia Novia, published in London in 1691.

Blood pigment: From disintegrated blood elements in the vessels within the Haversian canals.

Lacunae and canalicull: The former are cell spaces that housed osteocytes, and the latter are channels extending out of the lacunae that accommodated cell processes of osteocytes.

 

BONE
Compact cross section

 

Human, ground bone, 162 x.

Haversian canals: These are abundant and characteristic of compact bone. Their course follows the main axis of long bone. They conduct blood vessels, lymphatics, and nerves throughout the bone. They branch and anastomose and become continuous with Volkmann's canals. Haversian* canals surrounded by concentric lamellae of compact bone form the Haversian system.

Volkmann's canals: These are cross connections between Haversian canals. They pierce the lamellae of two Haversian systems. They contain blood vessels, lymphatics, and nerves and supply the interstitial lamellae.

Interstitial lamellae: A set of bone lamellae that fill the spaces between Haversian systems. They vary in shape and orientation

 

MESENCHYME
Fetal pig

Pig, 10% formalin, H. & E., A. 88 x; B. 55 x; C. 220 x.

Mesenchymal connective tissue has a delicate spongy consistency and is composed of cells and a viscous matrix or ground substance containing few fibers.

Mesenchymal cells are characterized by oval elongate nuclei with prominent nucleoli and a mix of hetero- and euchromatin. These cells have little cytoplasm but many thin processes that appear to extend from the nucleus. Mesenchymal cells can differentiate into most of the adult connective tissue cell types, including: (1) fibroblasts, (2) chondroblasts, (3) osteoblasts, (4) odontoblasts, (5) reticular cells, and (6) adipocytes.

The matrix is composed of two classes of compounds: glycosaminoglycans and structural glycoproteins.

In this plate, note several of the mature cell types that have differentiated from mesenchymal cells. Examine the developing tooth in A and the developing hair follicles in B.

 

MUCOUS CONNECTIVE TISSUE
Umbilical cord

Rhesus monkey, Helly's fluid, H. & E., 162 x.

Mucous connective tissue is characteristically found in the umbilical cord. It also is transiently encountered as a stage in the differentiation of mesenchyme into connective tissue.

The distinctive cell of mucous connective tissue is a primitive fibroblast, which may be spindle-shaped or stellate. In H. & E. preparations, only nuclei of fibroblasts are evident. Fine collagenous fibrils aggregate in the ground substance, which is characteristically abundant and gelatinous.

 

 

Section 2: Epithelial Tissue

The layer of cells that covers the outer, and lines the inner, body surfaces is designated as epithelium. In general, many of these cells have a free surface, which is actually or potentially exposed to the external environment (skin, and the respiratory tract), or to a moist environment continuous with the external environment (digestive, reproductive, and urinary tracts). Other epithelial cells, comprising glands found in underlying connective tissue, are in continuity with the surface epithelium by epithelial duct cells. The glandular epithelium secretes diverse products, which are carried to the external surface. The products of these glands include sweat, bile, urine, reproductive cells and associated glandular secretions, mucus, milk, digestive enzymes, hydrochloric acid, and so on. Some epithelial cells have migrated away and have lost contact with the free surface. These cells form distinctive cellular masses, which are termed endocrine glands. The secretory products of these cellular masses are delivered into the vascular system to be carried to their specific sites of activity by the blood stream. The endocrine system will be considered in Section 15 of this atlas.

It is important to remember that everything that enters or leaves the body is either modified or synthesized by epithelial cells or has diffused or has been transported through this tissue. The various functions of epithelium include protection, secretion, excretion, digestion, absorption, lubrication, sensory reception, and reproduction. Such a diversity of functional activity depends upon structurally diverse cell types and cell groupings.

Epithelia are classified by histologists according to cell layering and cell shape. On this basis, three distinct types of epithelium are recognized: (1) simple, which is a single cell layer; (2) pseudostratified, which is a single cell layer but appears to have two or more layers; and (3) stratified, which is composed of several to many cell layers. Only the simple and stratified epithelia have important subgroupings, which are classified according to the shape of the cells that are exposed to the free surface. The simple epithelia are described as squamous (sheets of flattened cells), cuboidal and columnar (in which the cells are greater in height than width when seen in most sections-these, too, are actually five- or six-sided in cross section). The stratified epithelia include stratified squamous, in which the superficial cells on the free surface are flattened; stratified cuboidal, in which the superficial cells on the free surface are cuboidal; and stratified columnar, in which the superficial cells on the free surface are columnar.

From a functional point of view, the simple epithelia carry out the most diverse activities, which include absorption, excretion, synthesis, secretion, and sensory reception, whereas the stratified epithelia have protective functions, serve as conduits or ducts, and produce reproductive cells. In order to serve their distinctive functional roles, epithelial cells often display distinctive cell membrane or surface modifications and appendages.

The epithelial types shown in this section represent the morphological varieties of simple and stratified epithelia. The structural features of many other epithelial cell types and groupings are found in other sections of this atlas, where their functional role will be considered in the context of organ function.

A classification of epithelial cell types and some of their locations in the body follows.

 

1. Simple Epithelium

    

   1. Squamous
      Innermost lining of blood and lymph vessels and the heart (endothelium). Lining of the pleural, cardiac, and abdominal cavities. Initial segments of ducts of glands. Air sacs or alveoli of the respiratory system. Renal glomeruli and corpuscles. Kidney tubules (thin segment of loop of Henle of the nephron).
   2. Cuboidal
      "Germinal" epithelium covering the ovary. Ducts of many glands. Ciliary body of the eye.
   3. Columnar Stomach, intestines, and gallbladder of the digestive system. Small bronchi of the respiratory system.
      Uterine tubes. The secretory cells of many glands (endocrine and exocrine) vary from cuboidal to columnar. Size and shape may vary with the functional state (e.g., thyroid gland).
   4. Pseuclostratified
      Pharynx, trachea, and large bronchi. Male excurrent ducts (epididymis and vas deferens). Parts of the female and male urethra.
   5. Specialized
      Glands of intestinal tract, nasal cavity, bronchi, uterine tubes, and accessory sex glands.
   6. Pigmented
      Epithelium of retina.
   7. Neuroepithelium
      Receptor cells of taste, hearing, and balance.

    

2. Stratified Epithelium
   1. Stratified squamous
      Keratinized and non-keratinized epithelium of skin, palpebral conjunctivum, oral cavity, esophagus, and anus. Urethra near the external orifice. Vagina.
   2. Stratified cuboidal
      Ducts of sweat and sebaceous glands of the skin. Graafian follicles of ovary.
   3. Stratified columnar
      Pharynx, larynx, urethra, and portions of the excretory ducts of salivary and mammary glands.
   4. Transitional (urothelium)
      Renal calyces and pelvis, ureter, and urinary bladder.

Several specializations of epithelial cells found on their free or exposed surface include the brush or striated border of the absorbing cells of the intestine and kidney, motile cilia of the pseudostratified epithelium of the respiratory system, and non-motile stereocilia of the pseudostratified epithelium lining the epididymis. Specializations that structurally and functionally link adjacent cells together include the "terminal bars" illustrated in Plate and the "intercellular bridges" or clesmosomes associated with prickle cells found in stratified squamous epithelium

. Marked infoldings of the basal cell membrane, termed basal striations, are seen in certain active transport cells such as the proximal convoluted tubule cells of the kidney and ducts of certain glands  

Between the basal surface of epithelial cells and the underlying connective tissue is the basement membrane, which varies markedly from place to place and in certain disease states. This extracellular structure has been shown by electron microscopy to have several components that are produced by both epithelial cells and the underlying connective tissue fibroblasts

 

CUBOIDAL EPITHELIUM
Brush border, basal striations proximal tubules kidney

Rhesus monkey, Helly's fluid,
iron hematoxylin-orange G, 1416 x.

The cells of the proximal tubule with their apical and basal specializations have the capacity to reabsorb selectively and transport metabolically valuable substances from the glomerular filtrate (e.g., glucose and amino acids), returning them to the vascular system. They also transport and secrete other substances in the lumen of the proximal tubule to be eliminated in the urine.

Nucleus: Round and large with prominent nucleolus.

Brush border: On the luminal surface of the tubule cells. Consists of microvilli that vastly increase the cellular absorptive surface.

Basal striations: Consist of rod-shaped mitochondria contained within compartments formed by specialized infoldings of the basal cell membrane.

 

CUBOIDAL EPITHELIUM
Kidney medulla collecting ducts

Human, 10% formalin, H. & E., A. 85 x; B. and C. 216 x.

The cells commonly referred to as cuboidal are always found to be five- or six-sided when cut through their axial or cross-sectional plane. This can be seen most readily in sections of the digestive system and kidney where almost every section contains both longitudinal and transverse profiles of cuboidal and columnar epithelia

 

SIMPLE COLUMNAR EPITHELIUM
UNICELLULAR GLAND

 

Cat, Helly's fluid, Mallory's stain, 1416 x.

Absorbing cell: Single cell layer of tall columnar cells. Basal ovoid nucleus. Although the cells appear retangular in this section, they are actually five- or six-sided when they are cross-sectioned. When the underlying tissue folds or bends, these cells may have a pyramidal appearance

Nucleus: Ovoid. Situated in lower half of the columnar cell. The nuclei in tightly packed cells may appear elongated and staggered at different levels within the cell. This is readily seen in pseudostratified ciliated columnar

Brush border: Also know as the straited border. Made up of fine, closely packed microvilli that vastly increase the surface area of the cell. Characteristic of absorptive surfaces. Adequate absorption of digestive products is dependent upon this cell surface specialization of absorbing columnar epithelial cells.

Goblet cell: Unicellular mucous glands scattered among the tall columnar cells appear empty because mucin is extracted during tissue processing. These unicellular gland cells are a specialization of simple epithelium and serve a protective function for the principal epithelial cell type.

Basement membrane: Delicate in appearance but a firm support for the columnar cells

Lamina propria: Connective tissue stroma. Reticular framework containing a.variety of wandering cells as well as vascular and lymphatic channels. Cells commonly found in the lamina propria include lymphocytes, plasma cells, eosinophils, and mast cells.

 

PSEUDOSTRATIFIED COLUMNAR EPITHELIUM
WITH STEREOCILIA
Epididymal duct

Rhesus monkey, Helly's fluid, H. & E., 612 x.

The epididymal duct is lined by a pseudostratified columnar epithelium containing two types of cells: tall columnar cells bearing so-called stereocilia and rounded basal cells. The cells forming a pseudostratified epithelium deceptively appear to be stratified in two or more layers. The cells actually vary in height, but all are in contact with the basement membrane.

Columnar cells: Tall cells bearing stereocilia. These are non-motile processes of the columnar cells projecting into the lumen. Although they are called cilia, electron micrographs show that they lack the structural characteristics of cilia, and they resemble greatly elongated microvilli. In this figure, they are seen in both cross and longitudinal section. Nuclei of columnar cells are elongated and lie at different levels.

Basal cells: Rounded or triangular cells, lying against the basement membrane, form a discontinous layer around the duct.

 

BASEMENT MEMBRANE
Pseudostratified columnar ciliated epithelium and
goblet cells trachea

Rhesus monkey, Helly's fluid,
modified aldehyde fuchsin stain, 1416 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, provided by goblet cells, 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 mucous 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.

 

STRATIFIED GERMINAL EPITHELIUM

 

Rhesus monkey, Helly's fluid,
iron hematoxylin and orange G stains,
A. 162 x; B. 1416 x.

The germinal epithelium of the seminiferous tubules is composed of several layers of spermatogenic cells disposed between the basement membrane of the tubule and the lumen 

Primary spermatocyte: Largest germ cell. Nuclei are large and vesicular and have condensed chromatin. Chromatin may appear as elongated threads.

Maturing spermatozoa: Mature germinal cell consisting of a head and a tail. The heads are in close association with Sertoli* cells, and the tails project into the lumen of the seminiferous tubule. Condensed nuclei forming the heads of spermatozoa contain a single set of chromosomes. Spermatozoa are the source of testicular hyaluronidase, an enzyme that may play a role in fertilization.

Sertoli Cell: These are supporting cells of the testicular epithelium. Tall columnar cells extend from the basement membrane to the lumen. These cells possess ovoid nuclei with a prominent nucleolus (seen here). Cell borders are not distinguished with light microscopy. Spermatozoa develop in intimate relation with the apical cytoplasmic processes of Sertoli cells.

Basement membrane: Surrounds seminiferous tubules, and is augmented by outer layers of connective tissue.

 

STRATIFIED SQUAMOUS EPITHELIUM
A. Non-keratinized B. Keratinized

A. Human, 10% formalin, H. & E., 162 x.
B. Human, glutaraldehyde-osmium fixation, toluldine blue stain, 612 x.

Stratified squamous epithelium is made up of several layers of cells. The deepest layer is composed of cuboidal or low columnar cells, the middle layer of polygonal cells, and the superficial layers of flattened cells. The epithelium caps connective tissue papillae (lamina propria or dermis). The stratified squamous epithelium located internally (esophagus) is non-keratinized, whereas that located externally (skin) is keratinized (i.e., possesses a stratum corneum). The stratum corneum is made up of flattened non-viable, non-nucleated epithelial cells containing keratin

 

STRATIFIED COLUMNAR EPITHELIUM
Mucous gland duct tongue

 

Human, Zenker's fluid,
iron hematoxylin & carmine stain, 1416 x.

Columnar cell: Columnar cells form the superficial layer of the stratified columnar epithelium.

Basal cell: These cells are irregularly polyhedral and form the deep layers of this stratified epithelium.

Terminal bars: Darkly stained, thickened zone is the specific attachment site of the lateral surface of adjacent superficial columnar cells (so-called junctional complex from electron microscopic studies).

Collagen: A component of the connective tissue stroma.

Plasma cell: Eccentrically placed, prominent and structurally characteristic nucleus in an abundant basophilic cytoplasm. Plasma cells produce antibodies

 

TRANSITIONAL EPITHELIUM
Ureter

Human, Helly's fluid, H. & E., 612 x.

The term transitional epithelium does not imply that this epithelium is in actual transition from one type to another, but rather refers to the appearance of the cells, which changes as the organs with which they are associated are stretched or relaxed.

Transitional epithelium (uroepithellum): This stratified epithelium is found lining the urinary tract from the renal calyces to the urethra. It is in direct continuity with the simple epithelium of the ducts and collecting tubules of the kidney and the stratified squamous epithelium of the urethra. Superficial cells are cuboidal and large, and the basal cells are cuboidal to columnar. The surface cells of this epithelium vary in shape from squamous when stretched to columnar when contracted. Note the convex luminal border of the surface cells. These cells may be multinucleated and polyploid.

Lamina propria: Predominantly reticular and collagenous connective tissue fibers with some elastic fibers. The lamina propria contains many cells, including lymphocytes, plasma cells, eosinophils, and mast cells, in addition to blood capillaries and lymphatic vessels.

 

PIGMENT EPITHELIUM
Eye choroid layer

Rhesus monkey, Helly's fluid, H. & E., 612 x.

The choroid layer of the eye is a highly vascular and pigmented coat surrounding the retina. Shown in this figure is a part of the retina adjoining the choroid layer, as well as the major choroid layers. In the outermost layer of the retina, the following structures are seen:

Rods and cones: Neuroepithelial cells sensitive to light, arranged vertically and parallel. 

Pigment epithelium: Single layer of pigmented cuboidal epithelial cells firmly bound to the choroid layer. Contains melanin pigment. In retinal detachments, the pigment epithelium remains attached to the choroid. The two major layers of the choroid seen in this plate are the following:

Choriocapillary layer: Composed of a network of wide lumen capillaries disposed in one plane and separated by delicate connective tissue fibers. Note that pigmented cells are essentially lacking in this layer. This layer supplies nutrition to the cells of the outermost layers of the retina.

Choriovascular layer: Filled with pigmented cells (melanin) and large-sized vessels

 

GLANDULAR EPITHELIUM
Zymogen pancreatic acinar cells

 

Rhesus monkey;
A. Glutaraldehyde-osmium fixation; toluldine blue and periodic acid-Schiff* stains;
B. Helly's fluid, Gomori's chrome alum hematoxylin; 1416 x.

The configuration of pancreatic acinar cells is seen in these two preparations.

Note the pyramidal shape of the acinar cell, the basally located round nuclei with distinct nucleoli, and two discrete zones of the cytoplasm. The apical zone near the lumen contains zymogen granules; the basal zone is intensely basophilic and free of granules. Electron microscopy has shown that the intense basophilia of the basal zone is due to its rich content of ribonucleoprotein bound to membrane (endoplasmic reticulum). Protein synthesized by the rough encloplasmic reticulum is transported to the supranuclear Golgi apparatus where zymogen (secretory) granules are formed, which are in turn transported to the apical cytoplasm, where they are discharged by exocytosis into the acinar lumen

 

 

introduction

Through the process of cell division, differentiation, and specialization, four basic tissues arise from a single cell, the fertilized ovum. These tissues, the epithelial, connective, muscular, and nervous tissues, carry out all the diverse functions essential for life. The cells that constitute these basic tissues share certain common characteristics but also differ strikingly in their size, shape, organelle content, and function. General and special techniques have been developed by the biologist to visualize cellular structure and to establish functional correlates. The photomicrographs in this section were selected to reveal specific organelles and inclusions common to most cell types as demonstrated to advantage by a variety of preparative and staining methods. Cytological structural/functional correlations of the four basic tissues will be emphasized in subsequent sections that deal with the specialized cells that form the organs. Understanding cellular function depends upon the recognition of the role played by each component part of the cell. Examples follow.

In general, all cells possess

1. a cell membrane or plasmalemma;
2. one or more nuclei with nucleoli containing primarily deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), respectively;
3. cytoplasmic RNA;
4. a Golgi apparatus;
5. membranes in the form of vacuoles or saccules;
6. mitochondria; and

energy stored in the form of glycogen and lipid.

The plasmalemma, demonstrated by electron microscopy to be about 100 Angstroms (10 nm) in thickness, cannot be resolved, per se, by light microscopy, because the use of visible light as the illuminating source limits resolution to about 2750A (0.275 µm*). However, the plasmalemma together with associated connective tissue and surface polysaccharide coat may be stained and resolved as the cell boundary under certain conditions.  The nucleus is of special importance in understanding cell function. Because it is large enough for detailed examination by the light microscope when stained even by routine methods (such as hematoxylin and eosin, H. & E.), its varying functional states can be assessed. It has been demonstrated that active DNA does not stain with nuclear stains; the nucleus may thus appear empty except for a nucleolus, which will be stained. Inactive DNA is readily stained with hematoxylin, toluidine blue, and other similar basic dyes. Most nuclei contain varying amounts of functional (active) and nonfunctional (inactive) DNA. The stainable DNA may appear in clumps or may be in a reticulated pattern. The functional DNA is termed euchromatin, whereas the nonfunctional, or inactive, DNA is called heterochromatin. The nerve cell nucleus contains no stainable DNA, which indicates its active involvement in the metabolism of the cell. By contrast, the densely stained heterochromatin seen in the nucleus of the maturing red blood cell (or erythrocyte) signals the termination of nuclear involvement in the cytoplasmic synthesis of hemoglobin. Such nuclei are called pyknotic. In the case of the red blood cell, the useless heterochromatin is eventually ejected from the cell and phagocytized by macrophages . During cell division, the stainable, inactive DNA appears in the form of threads or rods called chromosomes .

The nucleus also contains one or more nucleoli, which stain routinely with one of the nuclear stains cited previously. The nucleolus consists principally of RNA and is the source of cytoplasmic RNA

The cytoplasm of most cells contains some RNA that may not be detectable by routine methods. In these instances, it is likely that the protein synthesis related to this RNA is mainly associated with the maintenance and repair of cellular structures or organelles. In certain instances, however, the cytoplasm contains a significant amount of RNA that is readily stained and can be directly related to some specific function, such as the elaboration of digestive enzymes . In certain nerve cells, cytoplasmic RNA appears as specific blue-staining (so-called basophilic) patches called Nissl bodies. In these two examples, the staining pattern is a permanent and recognizable feature of the normal cell. It has been detected by electron microscope that, in these cases, the RNA is bound to cytoplasmic membranes. In the developing red blood cell and muscle fiber, however, the RNA is not membrane-bound and gradually disappears when these cells become structurally and functionally mature.

The Golgi apparatus is well developed in cells actively engaged in protein synthesis and secretion, and its role is well understood in the enzyme-producing pancreatic acinar cell. Proteins synthesized through the interaction of nuclear, nucleolar, and cytoplasmic nucleic acids are first concentrated in the sacs of the Golgi apparatus in the form of granules or droplets. Except for protein glycosylation and conversions of proproteins, it is unlikely that the Golgi apparatus is directly involved enzymatically in synthetic activity of the cell and appears to be "packaging" the secretory product for transport to the extracellular space. Although this organelle was first convincingly demonstrated by Golgi in nerve cells, its precise role in these cells is not completely understood.

The cytoplasm of many mature cells contains little RNA, and, when these cells are stained with hematoxyln and eosin, the most widely used combination of stains, the cytoplasm binds the eosin and appears red. In these cells, functions other than protein synthesis predominate. The parietal cell of the stomach, which elaborates hydrochloric acid, is an example of such an eosinophilic (or acidophilic) cell . The cytoplasm of this cell contains numerous mitochondria and membranes, but little cytoplasmic RNA.

Mitochondria are found in all cells except the mature red blood cell. They vary in number, size, shape, and distribution, depending upon cell type and its specific energy requirements. Mitochondria are membranous sacs with membranous partitions to which enzymes may be tightly or loosely bound and which are themselves integral component parts of the organelle. This organelle produces the energy- rich and ubiquitous adenosine triphosphate necessary for synthetic and other cellular functions such as muscular contraction and active transport. Additional details will be found in the legends to Plates.

The substrates utilized by the mitochondrial enzymes in the elaboration of energy-yielding compounds include stored glycogen and lipid droplets. The structure of cells, tissues, and organs is examined by optical instruments with varying capacities for resolving constituents of differing sizes. In microscopic anatomy, it is not uncommon to use the naked eye, a microscope ocular, a light microscope with several lenses, and the electron microscope in such an investigation. For a variety of technical and economic reasons, electron microscopes are not usually placed in the hands of a beginning student; hence, direct experience with this instrument is primarily limited to demonstrations and the examination of electron micrographs of cells and tissues.

Most textbooks of microscopic anatomy or histology contain an abundant number of electron micrographs, and, with each new edition, they present fewer light micrographs of actual tissue sections in color. For this and other reasons, this book is devoted to illustrations of cells, tissues, and organs prepared for the light microscope.

Students of medicine, in particular, and of biology will find that the light microscope is the most important tool they will use, in spite of physical limitations in its resolving power. Advances in light microscope techniques, which use glutaraldehyde-osmium fixation, plastic embedding, and 1-µm (micron) sections, permit the highest resolution possible by this instrument.

The light microscope owes its essentiality to the vast experience gained by its use over the past 200 years. Its utility in the diagnosis of both normal and pathologic conditions is firmly established in practice. In addition, very large tissue sections may be examined (measured in centimeters), and a variety of special and selective stains may be used, enabling the study not only of cells and tissues but also of critically important large cell populations and their distribution in tissues and organs.

The electron microscope, in spite of its vastly improved resolution of exquisitely prepared tissue, is severely limited by the extremely small area of tissue (several millimeters) that can be examined, with the significant risk that important structures or structural alterations may never be seen. It is also very time-consuming to use it for surveying a significant amount of tissue adequately, and it is expensive and not widely used by practitioners of medicine and pathology except under special circumstances. Therefore, the judicious use of the light microscope is a primary prerequisite of electron microscopic study.

It is certain that future advances in electron- microscope technique will allow this instrument to perform a more important function in medical practice than it now does. Of course, its use in research on structure and function in health and disease is securely established as an important extension of the continuum of other optical systems. For the present, however, a firm background in structure, as revealed by the light microscope as well as the electron microscope, is essential to the training of the basic science researcher and to the future physician.

As mentioned earlier, with the exception of the accompanying plate, electron micrographs are not included in this atlas. It is believed that most textbooks of histology provide an abundant number of electron micrographs. The cellular organelles shown here by electron microscopy are cross referenced to light micrographs found elsewhere. With the exception of the lysosome, microbody, and intracellular membrane systems, every organelle and inclusion of the cell have been previously discovered by light microscopy, and their functions identified. Confirmation, details of organelle fine structure, and very important variations in intracellular and outer cell membrane ultrastructure required the resolving power of the electron microscope for their elucidation. For details, one should refer to various monographs and textbooks of histology, some of which are listed in the reference section.

Structure	Primary Function	Plate References for Corresponding Light Micrographs
Nucleus	Contains metabolically active and inactive deoxyribonucleic and ribonucleic acid	1, 2, 3, 4
Euchromatin	Active deoxyribonucleic acid	1, 2
Heterochromatin	Inactive deoxyribonucleic acid	1, 2
Nucleolus	Ribonucleic acid, source of cytoplasmic ribonucleic acid (ribosomes)	1, 6
Nuclear pores	Complex channels in the membrane surrounding the nuclear nucleic acid or chromatin. Important for nuclearcytoplasmic exchanges of ions, metabolites, and nuclear RNA	--
Centriole	Important in cell division. Modified in ciliated and sperm cells	29
Ribosomes	Free or unbound ribosomes found in cells undergoing differentiation. Synthesize protein for intracellular use	1, 5, 55, 57
Ribosome-studded reticulum	Abundant in glandular tissue and nerve. Form products for extracellular digestive and other functions	1, 5, 90, 213
Agranular reticulum	Important in metabolic and detoxifying functions and ion transport	--
Golgi apparatus	Important in concentration and packaging of secretory products such as protein, enzymes, and mucus	7, 29, 194
Mitochondria	The energy-producing organelle (adenosine triphosphate) and other metabolic functions	6, 72
Peroxisome (microbody)	Contains the enzyme uricase, D-amino acid oxidase, and catalase	--
Lysosome	Contains hydrolytic enzymes for use in intracellular metabolic functions	--
Antibody "granule"	Protein antibody synthesized by the ribosome-studded reticulum, condensed into a "granule" in the Golgi apparatus	23
Glycogen	Energy-yielding metabolite in the stored form	8, 78
Cell membrane	External limiting structure of the cells, which regulates influx and efflux of ions and metabolites. Essential in conduction of electrical impulses of muscle and nerve	15, 64
Desmosome	Attachment sites between epithelial cells	23, 137
Close or gap junction	Regulates intercellular interactions between epithelial cells, smooth and cardiac muscle. Site of electrical coupling of cells	76
Microvilli	Increase in absorbing surface of epithelial cells	16, 18, 19, 20
Bile canaliculus	Smallest of channels formed initially of hepatic cells from liver lobule, which transport bile