Book - A textbook of histology, including microscopic technic (1910) General Histology 2

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Böhm AA. and M. Von Davidoff. (translated Huber GC.) A textbook of histology, including microscopic technic. (1910) Second Edn. W. B. Saunders Company, Philadelphia and London.

A Textbook of Histology (1910): Introduction To Microscopic Technic | General Histology | I. The Cell | II. Tissues | Special Histology | I. Blood And Blood-Forming Organs, Heart, Blood-Vessels, And Lymph- Vessels | II. Circulatory System | III. Digestive Organs | IV. Organs Of Respiration | V. Genito-Urinary Organs | VI. The Skin and its Appendages | VII. The Central Nervous System | VIII. Eye | IX. Organ of Hearing | X. Organ of Smell | Illustrations - Online Histology
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General Histology

II. The Tissues

The first few generations of cells which result from the segmentation of the fertilized ovum have no pronounced characteristics. They are embryonic cells of rounded form, and are known as biastomeres. As they increase in number they become smaller and of polygonal shape, owing to the pressure to which they are subjected. From the mass of blastomeres, known as the morula mass, there are formed, under various processes described under the name of gastrulation, two layers of cells, the so-called primary germ layers, of which the outer is the ectoderm, the inner the entoderm. To the primary germ layers is added still a third layer, called the mcsodcnn ; it is derived from both the ectoderm and entoderm, but principally from the latter. From these three layers of cells, known as the primary blastodermic layers, are developed all the tissues, each layer developing into certain tissues that are distinct for this layer. ' In their further development and differentiation the cells of the blastodermic layers undergo a change in shape and structure characteristic for each tissue, and there is developed an intercellular substance varying greatly in amount and character in the several tissues. In the tissues developed from the ectoderm and entoderm the cellular elements give character to the tissue, while the intercellular substance is present in small quantity ; in the majority of the tissues developed from the mesoderm, the intercellular substance is abundant, while the cellular elements form a less conspicuous portion.

The tissues derived from the ectoderm are :

The epidermis of the skin, with the epidermal appendages and glands ; the epithelium lining the mouth, with the salivary glands and the enamel of the teeth ; the epithelium and glands of the nasal tract and the cavities opening into it ; the lens of the eye and retina, and the epithelium of the membranous labyrinth of the ear ; and finally, the entire nervous system, central and peripheral.

From the entoderm :

The epithelium lining the digestive tract, and all glands in connection with it, including the liver and pancreas ; the epithelium of the respiratory tract and its glands ; the epithelium of the bladder and urethra (in the male, only the prostatic portion, the remainder being of ectodermal origin).

The cells of the mesoderm are early differentiated into three groups (Minot, 99) :

(a) Mesothelium. The mesothelial cells retain the character of epithelial cells. They form the lining of the pleural, pericardial, and peritoneal cavities, and give origin to the epithelium of the urogenital organs (with the exception of the bladder and urethra), and striated and heart muscle tissue.

(b) McscncJiyinc, from which are derived all the fibrous connective tissues, cartilage, and bone, involuntary muscle tissue, the spleen, lymph-glands, and bone-marrow ; and cells of an epithelioid character, lining the blood and lymph-vessels and lymph-spaces, known as cndotliclial cells.

(c) Mesamcboid cells, comprising all red and white blood-cells.

It would be extremely difficult to attempt a classification of tissues according to their histogenesis, as identical tissue elements owe their origin to different germinal layers. The classification adopted by us is based rather on the structure of the tissues in their adult stage.

We distinguish :

A. Epithelial tissues with their derivatives.

B. Connective tissues ; adipose tissue ; supporting tissues (cartilage, bone).

C. Muscular tissue.

D. Nervous tissue.

E. Blood and lymph.


A. Epithelial Tissues

Epithelial tissues are nonvascular, and composed almost wholly of epithelial cells, united into continuous membranes by a substance known as intercellular cement. They serve to protect exposed surfaces, and perform the functions of absorption, secretion, and excretion.

The epithelia are developed from all of the three layers of the blastoderm.

They secrete the cement-substance found between their contiguous surfaces. This takes the form of thin lamellae between the cells, gluing them firmly together. In certain regions the epithelial cells develop short lateral processes (prickles), which meet like structures from neighboring cells, thus forming intercellular bridges. Between these bridges are intercellular spaces filled with lymph-plasma for the nourishment of the cells. Epithelia do not, as a rule, possess processes of 'any length. However, it would appear that the basement membranes, situated beneath the epithelia, consist chiefly of processes from the basal portion of the cells. Some authors ascribe to them a connective-tissue origin, a theory which conflicts with the fact that such membranes are present in the em.bryo before connective tissue, as such, has been developed (jnembrana priina, Hensen, 76).


The free surfaces of epithelia often support cuticular structures which are to be regarded as the products of the cells. The cuticulae of neighboring cells fuse to form a cuticular membrane or marginal zone which can be detached in pieces of considerable size (cuticula). In longitudinal sections the cuticula show, in many cases, a striation which would seem to indicate that they are composed of a large number of rod-like processes cemented together by a substance possessing a different refractive index. The cell-body is also striated for more than half its length, corresponding to the rods of the marginal zone. In the region of the nucleus at the basal portion the striation disappears, the cell here consisting of granular protoplasm of a more indifferent character.


Since one surface of each epithelial layer lies free, .and is consequently exposed to other conditions than the inner surface, certain differences are noticed between the two ends of each cell. The cells may develop cuticular structures as above stated. In other cases motile processes (cilia) are developed on their exposed surface, which move in a definite direction in the medium surrounding them, and by means of this motion sweep away foreign bodies. It is not strange that the free surface of the epithelia, exposed as it is to stimulation from without, should develop special structures for the reception of sensations (sense cells).


On the other hand, the inner or basal surfaces of the cells usually retain a more indifferent character, and serve for the attachment of the cells and the conveyance of their nourishment. For this reason the nuclei of such cells are usually situated near the basal surface.


From the above it is seen that the two ends of the epithelial cell undergo varying processes of differentiation, the outer being adapted more to the animal, the inner more to the vegetative functions. This differentiation has recently been known as the polarity of the cell. This polarity appears to be retained even when the cell loses its epithelial character and assumes other functions (Rabl, 90).


With few exceptions, blood- and lymph-vessels do not penetrate into the epithelia, but the latter are richly supplied with nerves. The finer morphology of the epithelia will be described in the chapters on the different organs in Part II.

Epithelia are classified according to the shape and relation of the epithelial cells.


We give the following classification :

1. Simple epithelia (with or without cilia).

(a) Squamous epithelium.

(b) Cubic epithelium.

(c) Columnar epithelium.

(d} Pseudostratified columnar epithelium.

2. Stratified epithelia (with or without cilia).

(a) Stratified squamous epithelium, with superficial flattened cells (without cilia). (ft) Transitional epithelium. (<r) Stratified columnar epithelium, with superficial columnar cells (with or without cilia).

3. Glandular epithelium.

4. Neuro-epithelium.


1. Simple Epithelium

In simple epithelia the cells lie in a single continuous layer. Simple epithelia are very widely distributed. They line almost the entire alimentary tract, the smaller respiratory passages and air sacs, the majority of the gland ducts, the oviducts and uterus, and the central canal of the spinal cord and ventricles of the brain.




Fig. 38. Isolated cells of squamous epithelium (surface cells of the stratified squamous epithelium lining the mouth): a, a, Cells presenting under surface ; b, cell with two nuclei.


Fig. 39. Surface view of squamous epithelium from skin of a frog; X 400.


(a) Simple Squamous Epithelium. In simple squamous epithelium the cells are flattened. Their contiguous surfaces appear regular, forming, when seen from above, a mosaic. The nuclei lie, as a rule, in the middle of the cell, and if the latter be very much flattened, the position of the nucleus is made prominent by a bulging of the cell at this point. It occurs in the alveoli of the lung.

(b) Simple Cubic Epithelium. Epithelial cells of this type differ from the above only in that they are somewhat higher. They appear as short polygonal prisms. Their outlines are, as a rule, not irregular, but form straight lines. Cubic epithelium occurs in the smaller and smallest bronchioles of the lungs, in certain portions of the 'uriniferous tubules and their collecting ducts, in the smaller ducts of salivary and mucous glands, liver, pancreas, etc.

(c] Simple Columnar Epithelium. In this type the cells take the form of prisms or pyramids of varying length. Cuticular structures are especially well developed. Columnar epithelium occurs in the entire intestinal tract from the cardiac end of the stomach to the anus, in certain portions of the kidney, etc.


Fig. 40. Simple columnar epithelium from the small intestine of man : a, Isolated cells ; b, surface view ; c, longitudinal section.

Simple ciliated columnar epithelium is found in the oviduct and uterus, central canal of the spinal cord, and smaller bronchi.

(d] Pseudostratified Columnar Epithelium. This type is one in which all the cells rest on a basement membrane, but they are so placed that the nuclei come to lie in different planes. Thus, in a longitudinal section the nuclei are seen to be placed in several rows.

The development of this type from the simpler forms occurs when the cells are too crowded to retain their normal breadth. As a result, they become pyramidal, alternate cells resting their bases or apices on the basement membrane. As the nucleus is usually situated at the broader portion of the cell, the result is that there are two rows of nuclei simulating a stratified epithelium. Occasionally there are spindle-shaped cells wedged in between the pyramidal cells, and as the broad portion of these cells is midway between the basement membrane and external surface, a third row of nuclei is seen midway between the other two. Such epithelia usually possess cilia (portions of the respiratory passages).



Fig. 41. Diagram of pseudostratified columnar epithelium.


2. Stratified Epithelium

Should the increase of the cells forming the last type of simple epithelium proceed to such an extent that all the cells no longer rest on the basement membrane, an epithelium is formed having distinct layers of cells a stratified epithelium. It is clear that all the cells of a stratified epithelium can not be equally well nourished by the blood-supply from the vessels in the highly vascular connective tissue beneath. The middle and outer layers of cells accordingly suffer. The deeper layers are much better nourished, and as a consequence their cells increase much more rapidly than those above ; they push outward, replacing the superficial cells as fast as they die or are thrown off. The proliferation of cells in a stratified epithelium occurs, therefore, chiefly in its basal layers.


Fig. 42. Schematic diagram of stratified pavement epithelium.


(a) Stratified Squamous Epithelium. Stratified squamous epithelium with superficial flattened cells forms the epidermis with its continuations into the body, as, for instance, the walls of the oral cavity and the esophagus, the epithelium of the conjunctiva, the vagina, the external auditory canal, and the external sheath of the hair follicles.

The cells of the basal layer are here mostly cubic-cylindric. Then follow, according to the situation of the epithelium, one or more layers of polyhedral cells, which become gradually flattened toward the surface, the outermost layers consisting of thin plate-like cells.

In stratified squamous epithelia, where the outer cells become horny (as in the skin), the stratification is still more specialized. Here layers are inserted in which the horny or chitinous substance is gradually formed, although the cells do not become chitinous until the superficial layers are reached.

Especially characteristic of stratified squamous epithelium is the arrangement of the connective tissue on which this epithelium rests. There are cone-like projections, known as papilla, arising from the connective tissue beneath the epithelium, projecting into the latter in such a way that on cross-section the junction of the two tissues appears as a wave-like line. These papillae not only serve to fasten the epithelium more firmly to the connective tissue below, but influence very favorably the nourishment of the former by allowing a greater number of its basal cells to approximate the under lying blood-capillaries. The pyramidal extensions of the epithelium between the papilla are designated interpapillary epithelial processes. In regions where the stratified squamous epithelium consists of many layers, the prickle cells, intercellular bridges, and the intercellular spaces are especially well developed. These spaces facilitate the passage of the lymph-plasma to the more superficial layers of cells.



Fig. 43- Cross - section of stratified squamous epithelium from the esophagus of man.


(b] Transitional Epithelium. Transitional epithelium is a stratified epithelium occurring in the pelvis of the kidney, the ureters, bladder, and the posterior portion of the male urethra. It is composed of four to six layers of cells and rests on a connective tissue free from papillae. In sections the cells of the deeper layers appear to be of irregularly columnar, cubic or triangular shape. The cells forming the superficial layer are large, somewhat flattened cells, with convex free surfaces, often possessing two, sometimes three, nuclei. They cover a number of the cells of the layer just beneath them, their under surfaces being pitted to receive the upper ends of the deeper cells. In teased preparations the cells of the deeper layers appear very irregular, often showing ridges or variously shaped processes. (See Fig. 44.)


Fig. 44. Isolated transitional epithelial cells from the bladder of man : a, b, f, d, Large surface cells, <;and d presenting the pitted undersurface ; <:, variously shaped cells from the deeper layers.


Fig. 45 Cross-section of transitional epithelium from the bladder of a young child.


(c] Stratified Columnar Epithelium. In this type the superficial layer consists of columnar cells, the basal ends of which are usually somewhat pointed, or may branch. The deeper cells, which may be arranged in one or more layers, are of irregular, triangular, polyhedral, or spindle shape. It is found in the larger gland ducts, olfactory mucous membrane, palpebral conjunctiva, portions of the male urethra and the vas deferens, and in certain regions of the larynx.


The ciliated variety of this epithelium differs from the foregoing in that the superficial columnar cells are provided with cilia. Stratified ciliated columnar epithelium is found in the respiratory portion of the nose, larynx, trachea, and larger bronchi, in the Eustachian tube, epididymis, and a portion of the vas deferens.


Fig. 46. Schematic diagram of stratified columnar epithelium.

Fig. 47. Ciliated cells from the bronchus of the dog, the left cell with two nuclei ; X 600.


All epithelial cells are probably joined together by short processes forming intercellular bridges, the lymph supplying them with nourishment circulating in the intercellular spaces thus formed. Toward the surface, these intercellular spaces are roofed over, thus preventing the escape of the fluid. When seen from the surface, epithelia treated by certain methods (iron-hematoxylin) show the cells joined together by very minute, clearly defined and continuous cement-lines. Bonnet has called them terminal ledges or bars (Schlussleisten). The function of this structure would seem to consist in its power to prevent the escape of lymph from the surface, and the penetration of micro-organisms (M. Heidenhain, 92 ; Bonnet, 95).



Fig. 48. Cross-section of stratified ciliated columnar epithelium from the trachea of a rabbit.


3. Glandular Epithelium

Glandular epithelium is composed of epithelial cells differentiated so as to possess the power of elaborating certain compounds or substances which are finally given off from the cells in the form of secretions. Those substances which form the essential constituents of such secretions appear in the protoplasm of the majority of glandular cells, in the intervals of secretory activity, in the form of smaller and larger granules which may be discharged from the cells in granular form or may be changed into homogeneous, viscid substances before leaving or on leaving the cells. Glandular epithelium appears in the form of isolated glandular cells, scattered here and there among other epithelial cells, in certain types of epithelium, or as smaller or larger aggregations of glandular cells, possessing definite and typical arrangement and associated with other tissues connective tissue, blood- and lymph-vessels, nerve tissue to form structures or organs known as secreting glands.


Fig. 49 Goblet cells from the bronchus of a dog. The middle cell still possesses its cilia ; that to the right has already emptied its mucous contents (collapsed goblet cell) ; X DO0


Fig. 50. A mucus-secreting cell (goblet cell), showing secretory granules, situated between two epithelial cells. From the epithelium of the large intestine of man.

Unicellular Glands

Isolated glandular cells, which we may know as unicellular glands, are frequently met with in the epithelium of the intestinal canal and respiratory organs, where, owing to their shape, they are known as goblet cells, or, again, as mucus secreting cells, since their secretion is mucus. Such cells are in the ordinary preparation distinguished from the neighboring cells by the fact that their free ends appear clearer and are more vesicular, while their basal portions, containing the nuclei, are narrow and pointed. Closer examination generally reveals a fine protoplasmic network in the clear portion of the cell, the interspaces of which are filled with the mucus. (See Fig. 49.)

The secretion is, however, elaborated in the cell-protoplasm in the form of rather coarse granules, which may be as large as I y 2 ft to 2 fj.. These granules are found in a hyaline substance, from which they are probably formed, which substance is found in the interspaces of a protoplasmic network with relatively wide meshes (Langley). The granules as they develop and enlarge distend the free portions of the cells. They are eventually extruded from the cells, probably in the form of granules, as granules identical with those found in the cells are found in the lumina of intestinal glands in well-fixed material. After the extrusion of the secretion the cell collapses, and may again assume a secretory function by the elaboration of new granules. (See Fig. 50.)


Multicellular glands originate by the metamorphosis of a number of adjacent cells into glandular cells. This is usually accompanied by a more or less marked dipping down of the epithelial layer into the underlying connective tissue. The glandular cells are generally arranged in a single layer, and rest on a delicate membrane, known as the basement membrane (membrana propria); outside of this there is found fibrous connective tissue, containing the terminal ramifications of capillaries and lymph-spaces and of nerve-fibers. The simplest form of such an invagination is a cylindrical tube or a small sac (known as an alveolus) lined entirely by glandular cells. A further differentiation may take place in that all the invaginated cells do not assume a secretoiy function, those at the upper portion of the tube or sac forming the lining membrane of an excretory duct. The originally uniform tube or sac is thus differentiated into a duct and a secretory portion. Multicellular glands may lie entirely within the epithelium, and are then known as intra-epithelial glands, in contrast to the extra-epithelial or ordinary type, the greater part of which lies imbedded in the underlying connective tissue. Glands of the former type have been studied in amphibian larvae, and, according to Sigmund Mayer, occur also in the epididymis, conjunctiva, etc.', of mammals.

General Consideration of the Structure and Classification of Glands. Since glandular tissue is composed almost wholly of epithelial cells, it may not be out of place to consider at this time the classification of glands. The fuller consideration of these structures will, however, be deferred to a later time. In the classification here given we have been guided by that presented by Maziarski, in an observation on the structure and classification of glands, based on a series of reconstructions with the Born waxplate method and comprising nearly all the important glandular structures of the human body. In brief, it may be stated that the variation in glandular types affects principally the secretory portions of glands, while the excretoiy ducts are more or less uniform. Glands are classified, according to their shape, into tubular and alveolar glands; each of these types is further divided into simple and branched tubular, and simple and branched alveolar glands. In certain glands tubules and alveoli unite to form the secretory portion of a compound tubular liver. Chrome-silver preparation ; X 12 gland. Lingual gland of the rabbit. Chrome-silver preparation ; X 2I 5 portion; such glands are known as tubulo-alveolar glands. They may also be simple or branched.



Fig. 51. Simple tubular glands. Lieberkuhn's glands from the large intestine of man. Sublimate fixation ; X 9


Fig. 52. Excretory ducts Fig. 53. Lumina of the secreting portion and lumina of the secretory of a reticulated tubular gland; from the human

Tubular Glands

In a tubular gland the secreting portion consists of a longer or shorter tubule, which may be relatively straight or variously twisted or coiled, one end of which ends blindly while the other end opens on a free surface or into a duct. The blind ends of the tubules of tubular glands often present more or less well-marked enlargements. Simple tubular glands consist of a single tubule, which may be lined throughout by secretory epithelium or may be differentiated into a portion lined by secretory epithelium and a portion lined by a non-secretory epithelium forming a duct. An increase of the secretory surface of tubular glands is obtained in one of the following ways:

  1. Coiled Tubular Gland. The secreting portion of the tubule may be coiled up into a compact mass ;
  2. Simple Branched Tubular Glands. Several tubules, which may be either branched or unbranched, and which may vary greatly in length, may unite in one duct, which carries to the surface the secretion of all the tubules connected with it ;
  3. Compound Branched Tubular Glands. Glands of this type consist of a varying number of simple branched tubular glands, the ducts of which unite to form a common duct (Fig. 52);
  4. Reticulated Tubular Glands. In certain of the branched tubular glands the secreting tubules anastomose with each other, forming a reticrdated gland (Fig. 53) ;
  5. Tubulo-alveolar Gland. The secreting surface of tubular glands may be further increased by the formation of small and variously shaped protuberances or saccules, known as alveoli, which may be situated at the end or on the sides of the tubules, and are lined by secretory epithelium and empty the secretion formed in them into the tubules with which they are connected. We have thus, in addition to the several types of tubular glands above mentioned : Simple tubulo-alveolar glands, simple branched tubulo-alveolar glands, and compound branched tubulo-alveolar glands.


Fig. 54. Schematic diagram of glandular classification : I, Simple tubular gland; 2, simple tubular gland with coiled secreting portion ; 3, 4, 5, types of simple branched tubular glands; 6, compound branched tubular gland; 7, 8, types of simple branched tubulo-alveolar glands ; 9, simple alveolar gland; IO, II, types of simple branched alveolar glands ; 12, compound branched alveolar gland ; 13, type of follicular gland ; 14, reticular gland, the shaded portions representing anastomosing tubules.

Alveolar Glands

In the alveolar glands the secreting compartments have the form of variously shaped vesicles or saccules, known as alveoli, lined by secretory epithelium, which communicate with narrow tubules of varying length and lined by nonsecretory epithelium, which form the ducts. Alveolar glands are classified as :

1. Simple alveolar glands, consisting of a single alveolus which communicates with the surface by means of a narrow duct.

2. Simple Branched Alveolar Glands. In this type a varying number of alveoli are united through their respective ducts to a larger duct which reaches the surface.

3. Compound Branched Alveolar Glands. Glands of this type consist of a varying number of simple branched alveolar glands united by a common duct.

4. Follicular Glands. Glands of this type may be classed under alveolar glands, since they consist of numerous closed alveoli or follicles, of round, oval, or even irregular shape, which do not communicate with a duct system.

The main features of this classification of glands are portrayed in the accompanying diagram (Fig. 54).

According to the above description multicellular glands may be classified as follows :

Tubular glands.

Tubulo-alveolar glands.

Alveolar glands.


1. Simple tubular glands: crypts of Lieberkiihn, the majority of the sweat glands.

2. Simple branched tubular glands: fundus glands of stomach, the majority of the pyloric glands, uterine glands.

3. Compound branched tubular glands : kidneys, testis, lachry mal glands, serous glands of mucous membranes.

4. Reticulated tubular glands : Liver (fully developed in mammals).

1. Simple tubulo-alveolar glands: certain of the pyloric glands.

2. Simple branched tubulo-alveolar glands : Littre's glands, cer tain of the sweat glands, and modified sweat glands (circumanal and axillary glands, ceruminous glands, ciliary glands).

3. Compound branched tubulo-alveolar glands : many mucous glands, Brunner's glands, prostate, lung.

1. Simple alveolar glands : the smallest sebaceous glands, the skin glands of amphibia.

2. Simple branched alveolar glands : sebaceous glands, Meibo mian glands.

3. Compound branched alveolar glands : pancreas, mammary gland, serous salivary glands in the latter, however, a portion of the duct system possesses secretory function (Maziarski).

4. Follicular glands : ovary, hypophysis, thyroid (according to Streiff, certain of the closed follicles of the thyroid have a tubular form, others show secondary alveolar enlargements on the primary follicles).


The secretory epithelium of the various types of glands rests upon a thin membrane (membrana propria), which has, according to some authors, a connective-tissue origin, while, according to others, it is the product of the glandular cells themselves. In some cases it appears structureless, in others a cellular structure can be distinguished ; in the latter case the cells are flattened, with very much flattened nuclei, and show irregular outlines. Macroscopically, compound glands present a more or less lobular structure, the separate lobules being held together by connective tissue. In the immediate neighborhood of the gland and its larger lobes, the connective tissue is thickened to form the so-called tunica albtiginea or capsule. In this fibrous-tissue sheath are found numerous blood-vessels which penetrate between the lobes and lobules of the gland and form a dense capillary network about the tubules and alveoli immediately beneath the membrana propria. Nerve-fibers are also plentiful. f

Remarks on the Process of Secretion. The gland-cell varies in its microscopic appearance according to its functional condition. In the great majority of the glandular epithelial cells the essential constituents of the secretion are stored in the cell in the form of secretory granules, in others in vacuoles which are filled with the secretion. The secretory process varies. In one case the cell remains intact throughout the process (salivary glands) ; in another a portion of each cell is used up in the production of the secretion, only the basal portion containing the nucleus being preserved. When this occurs, the upper part of the cell is reconstructed from the remaining basal portion, and the cell is ready to renew the process (mammary glands). In a third type the whole cell is destroyed, and is replaced by an entirely new cell (sebaceous glands).


4. Neuro-Epithelium

In certain of the organs of special sense (inner ear and taste-buds) the epithelial cells about which the nerves terminate undergo a high degree of specialization. This differentiation is more apparent in the outer portions of these cells, resulting in the formation of one or several stiff, hair-like processes, which appear especially receptive to stimuli. Such cells are known as ncuro- epithelial cells. In the epithelia in which they occur they are surrounded by supporting or sustentacular cells.


5. Mesothelium and Endothelium

The pleural, pericardial, and peritoneal cavities are lined by a single layer of flattened epithelioid cells which develop from the mesothelium lining the primitive body cavity (celom). For this reason, as has been suggested by Minot (90), the term mesothelium may with propriety be applied to this layer in its developed condition. In silver nitrate preparations, in which the boundaries of these cells are brought to view, they appear as much flattened cells, resembling those of squamous epithelium, with faintly granular protoplasm, possessing flattened, oval, or nearly round nuclei. These cells are of polyhedral shape, and appear to be united into a single layer by a small amount of intercellular cement substance. The borders of these cells may be quite regular or slightly wavy (Fig. 55); more often they are serrated (Figs. 56, 57). According to Kolossow, who has investigated these cells by means of special methods devised by him, the mesothelial cells are said to be made up of two quite distinct portions: a superficial, homogeneous cellplate, beneath which is found a finely granular protoplasm containing the nucleus. These two portions are intimately united to form a single cell. The outlines of the superficial cell-plates are figured in the accompanying illustrations. The protoplasmic portion of one cell unites with that of contiguous cells by means of protoplasmic branches, between which are found intercellular spaces. These intercellular spaces are here and there indicated in silver nitrate preparations, forming what are known as stigmata and stomata, which are looked upon by certain writers as representing openings between the mesothelials cells through which fluids and solid particles may pass into underlying lymph spaces. They are, however, now generally regarded as artefacts.


Fig. 55. Mesothelium from pericardium of rabbit. Silver nitrate preparation, stained in hematoxylin.


Fig. 56. Mesothelium from mesentery of rabbit.


Fig. 57. Mesothelium from peritoneum of frog; X 4


Endothelial cells are differentiated mesenchymal cells. They line the blood- and lymph-vessels and lymph-spaces (arachnoidal ant. synovial spaces, anterior chamber of the eye, bursae, and tendon sheaths). Endothelial cells are in structure like those of the mesothelium. In blood- and lymph-vessels they are of irregular, oblong shape, with serrated borders. The boundaries of these cells are clearly brought out by silver nitrate.


Fig. 58. Mesothelium covering posterior abdominal wall of frog. Stained with silver nitrate and hematoxylin.


Fig. 59. Endothelial cells from small artery of the mesentery of a rabbit. Stained with silver nitrate and hematoxylin.

Technic

Epithelium may be examined in a fresh condition. The simplest method consists in placing some saliva under a cover-glass and examining it with a moderate power. In it will be found a number of isolated squamous epithelial cells, suspended in the saliva singly and in groups. The cells that are cornified still show the nucleus and a small granular area of protoplasm.


In order to examine isolated epithelial cells of organs, it is necessary to treat the epithelial shreds or whole epithelial layers with the so-called isolating or maceration fluids. These are : ( i ) Iodized serum; (2) very dilute osmic acid (0.1% 100.5%); (3) very weak chromic acid solution (about 1:5000 of water) ; (4) 0.5% or i% solution of ammonium or potassium bichromate ; and, above all, the one-third alcohol recommended by Ranvier (28 vols. absolute alcohol, 72 vols. distilled water). The mixture recommended by Soulier (91), consisting of sulphocyanid of potassium or ammonium, and the mixture of Ripart and Petit serve the same purpose. All these solutions are used by allowing a quantity of the isolation fluid to act upon a small fresh piece of epithelium for from twelve to twenty-four hours, according to the temperature of the medium and quality of the tissue. As soon as the isolation fluid has done its work, it is easy to complete the isolation of the cells by shaking the specimen or teasing it with needles. Separation of the elements may be accomplished either in the isolation solution itself or in a so-called indifferent fluid, or in gum-glycerin. The macerated preparation may be stained in a hematoxylin or carmin solution before teasing and mounting in gum-glycerin.

The movement of the cilia can be observed in mammalian tissues by scraping the epithelium from the trachea with a scalpel and examining it in an indifferent fluid. As the ciliated epithelium of mammals is very delicate and sensitive, specimens with a longer duration of ciliary movement are more desirable. They can be obtained by using the mucous membrane from the palate of a frog (examine in normal salt solution). Particularly large epithelial cells, as well as very long cilia, are found on the gill -plates of mussels or oysters.

In order to study the relations of mesothelial and endothelial cells, the silver method is the most satisfactory. The outlines of the mesothelial cells may be clearly brought out by placing pieces of the pericardium, central tendon of the diaphragm, or the mesentery in a o. 75 % to i fc solution of silver nitrate. Before placing in this solution, they should be rinsed in distilled water in order to remove any adherent foreign bodies, such as blood-corpuscles, etc. In this solution they remain until opaque, which occurs in from ten to fifteen minutes. They are then again rinsed with distilled water, in which they are exposed to sunlight until they begin to assume a brownish-red color. Once again they are washed with distilled water, and either placed in glycerin, in which they may be mounted, or dehydrated and mounted in Canada balsam, according to the usual methods. The margins of the cells subjected to this treatment will appear black.

Endothelial cells may be demonstrated after the following method : A small mammal (rat, Guinea-pig, rabbit, or cat) is narcotized. Before the heart's action is completely arrested, the thorax is opened and the heart incised. As soon as the blood stops flowing, a cannula is inserted and tied in the thoracic aorta a short distance above the diaphragm, and 50 to 80 c.c. of a \Jc aqueous solution of silver nitrate injected through the cannula. About fifteen minutes after the injection of the silver nitrate solution, there is injected through the same cannula 100 to 150 c.c. of a 4% solution of formalin (formalin 10 parts, distilled water 90 parts). The abdominal cavity is then opened, loops of the intestine with the attached mesentery removed and placed in a 4% solution of formalin, in which the tissue is exposed to the sunlight. As soon as the reduction of the silver nitrate has taken place, which is easily recognized by the reddishbrown color assumed by the tissues, the mesentery is divided into small pieces, dehydrated first in 95%, then in absolute alcohol, cleared in oil of bergamot, and mounted in balsam. As a rule, the mesothelial cells covering the two surfaces of the mesentery, and the endothelial cells lining the arteries, veins, and capillaries are clearly outlined by the reduced silver nitrate.

If desired, the tissue may be further stained in hematoxylin (we have used Bohmer's hematoxylin solution) or in a carmin solution after dehydration in 95% alcohol, after which they are dehydrated, cleared, and mounted in balsam. In preparations made after this method the endothelial cells are outlined by fine lines of dark brown or black color.

Silver nitrate may also be dissolved in a 2% to 2,% solution of nitric acid, in osmic acid, and various other fluids. Stratified epithelia can also be impregnated with silver nitrate, but only after prolonged immersion. They are exposed to sunlight after sectioning on the freezing microtome, or after hardening and imbedding, followed by sectioning. After the reduction of the silver the sections are dehydrated and mounted in balsam.

Kolossow has devised the following excellent method for demonstrating intercellular bridges : Fine membranes, or even minute fragments of previously fixed tissues, are placed for about a quarter of an hour in a 0.5% to \Jc osmic acid (or in a mixture composed of 50 c.c. absolute alcohol, 50 c.c. distilled water, 2 c.c. concentrated nitric acid, and i to 2 gm. osmic acid) and then into a 10% aqueous solution of tannin for five minutes, or into a developer consisting of the following : water, 450 c.c. ; 85% alcohol, 100 c.c. ; glycerin, 50 c.c. ; purified tannin, 30 gm., and pyrogallic acid, 30 gm. In the latter case they are subsequently rinsed in a weak solution of osmic acid, washed with distilled water, and then carried over into alcohol.

There are, of course, special methods of fixing and subsequently examining epithelial structures ; these, and the methods of examining gland tissue, will be discussed in the chapters devoted to the various organs.

B. The Connective Tissues

In the connective tissues, the intercellular substance gives character to the tissue, the cellular elements forming a less conspicuous portion. In their fully developed condition some of the members of the connective-tissue group are only slightly altered from embryonic connective tissue. In other members there are developed, in less or greater number, fibers, known as connective-tissue fibers, thus forming reticular connective tissue and the looser and denser forms of fibrous connective tissue. A more marked condensation of the intercellular substance is observed in cartilage; and in bone and dentin a still greater degree of density is obtained by the deposition of calcareous salts in the intercellular matrix. In the different types of connective tissue the cellular elements are morphologically very similar. The role played by the connective tissues in the economy of the body is largely passive, depending on their


THE CONNECTIVE TISSUES.


97


Cell process.


Nucleus.


physical properties. Bone and cartilage serve as supporting tissues ; the looser fibrous tissues for binding and holding the organs and parts of organs firmly in place. The denser fibrous connective tissues come into play where strength and pliability are desired, as in ligaments, or else are used in the transmission of muscular force, as in tendons. Another important characteristic of connective tissue is that its various members are capable of undergoing transformation into wholly different types ; bone, for instance, being developed from fibrous connective tissue and from cartilage. Certain structures are represented by different members of the connective-tissue group in the different classes of vertebrates. In certain fishes the skeleton is cartilaginous, and in certain birds the leg tendons are formed of osseous tissue, etc. The connective tissues receive their nutrition from the lymph. In the denser connective tissues this permeates the tissues through clefts or spaces in the groundsubstance, in which the connective-tissue cells are found and which are united by means of fine canals into a canalicular system. In the looser fibrous tissues and in mucous connective tissue the system of lymph-channels is not present; here the lymph seems to pass through the ground-substance.

Certain connective-tissue cells have the function of producing fat. In various parts of the body, masses of fat tissue are formed as a protection to various organs

and as a reserve material upon which the body can call when necessary. This type can hardly be considered a separate class of connective tissues, as it can be demonstrated that it is merely modified connective tissue, and can occur wherever the latter is found. Finally, certain elements of the middle germinal layer are capable of producing colored substances known as pigments. To this class belong the pigment cells and the red blood-corpuscles.

All the members of this group are developed from the mesenchyme, an embryonic tissue developed early in embryonic life from the middle germ layer or mesoderm. In its early development the mesenchyme is probably composed of individual cells. As development advances the protoplasm of these cells increases, and is united by means of the protoplasmic branches formed by the cells to form a protoplasmic complex known as a syncytium. The further 7



Fig. 60. Mesenchymatous tissue from the subcutis of a duck embryo ; X ^5


9 8


THE TISSUES.


development and differentiation of the syncytium has been described in full by F. P. Mall, whose account is here followed. As soon as the syncytium is formed its protoplasm grows rapidly, and appears in large bands with spaces between them, and with relatively few nuclei. In its further development the protoplasm of the syncytium differentiates into a fibrillar part, which forms the main portion of the syncytium the exoplasm and a granular part which surrounds



Fig. 61. Schematic diagram given to show the development of the different types of connective tissue from the mesenchyma : a, Mesenchymal cells, certain of which are separate, others are joined by protoplasmic branches; b, syncytium with large strands of protoplasm and relatively few nuclei ; c, reticular tissue, the dark fibers are elastic fibers ; d, white fibrous tissue ; e, cartilage; f, membranous bone.


the nucleus the endoplasm. The fibrils of the exoplasm are very delicate and anastomose freely. Probably in all the members of the connective-tissue group, the so-called intercellular substance fibers, matrix of cartilage and bone is developed in or from the exoplasm, while the cellular elements are differentiated from the nuclei and endoplasm. The main features of the development of the different types of connective tissue are portrayed, in part schemati


THE CONNECTIVE TISSUES.


99


cally, in fig. 61, combined from a number of figures illustrating F. P. Mall's article dealing with this subject.

The fibers of white fibrous tissue develop in the exoplasm, while the endoplasm containing the nuclei rests on the bundles. In cartilage the ground-substance or matrix is deposited into the exoplasm of the syncytium, the endoplasm and nuclei forming the cartilage cells. In bone, the bone substance or matrix is developed from the exoplasm, either by a transformation of it or by a deposition in it, while the endoplasm increases and the nuclei enlarge to form the bone-forming cells, the osteoblasts. The reticulum of reticular connective tissue is developed directly from the exoplasm of the syncytium, while the nuclei and endoplasm are converted into cells which rest upon the reticulum fibrils.

The following kinds of connective tissue are recognized: (i) mucous connective tissue, (2) reticular connective tissue, (3) fibrous connective tissue, (4) adipose. tissue, (5) cartilage, (6) bone.



Fig. 62. White fibrils and small bundles of white fibrils from teased preparation of a fresh tendon from the tail of a rat.


Fig. 62^. Elastic fibers from the ligamentum nuchse of the ox, teased fresh ; X 500. At a the fiber is curved in a characteristic manner.


The fibrous connective tissues are composed of a ground-substance or matrix in which are imbedded the cellular elements and two kinds of connective-tissue fibers, namely, white and elastic fibers. As the character of the fibrous connective tissue depends largely on the arrangement of the fibers and on the relative proportion of the white and elastic fibers, these will be considered prior to a description of the several types of fibrous connective tissue.

White Fibers. White fibrous connective tissue consists of exceedingly fine homogeneous fibrillae, cemented by a small amount of an interfibrillar cement substance into bundles varying in size. In the bundles these fibrillae have a parallel course, although the bundles are often slightly wavy. The fibrillae of white fibrous connective tissue vary in size from 0.25 to I //, and neither branch nor anastomose. They become transparent and swollen when treated with acetic acid, are not at all or only very slowly digested by pancreatin, and yield gelatin on boiling.


TOO THE TISSUES.

Elastic Fibers. These are homogeneous, highly refractive, distinctly contoured fibers, varying in size from I // to 6 //, and in somd animals are even larger. They branch and anastomose, and are not cemented into bundles. When extended, they appear straight ; when relaxed, they show broad, bold curves, or are arranged in the form of a spiral. The broken ends of the fibers are bent in the form of a hook. F. P. Mall has shown that elastic fibers are composed of two distinct substances an outer delicate sheath which does not stain in magenta, and an interior substance which is intensely colored in this stain. The interior substance is highly refractive. Elastic fibers are not affected by acetic acid, but are readily digested in pancreatin and less readily in pepsin. They yield elastin on boiling.

J. MUCOUS CONNECTIVE TISSUE.

Mucous connective tissue is a purely embryonal type, and scarcely represented in the adult human body. It consists of branched, anastomosing cells imbedded in a ground-substance which gives a reaction for mucus and contains a varying number of white fibrous tissue fibers which are developed from a syncytial protoplasm. The latter as well as the mucous matrix are, directly or indirectly, the products of the cells. During the development of the embryo this tissue is found in large quantities in the umbilical cord' and is here known as Whartoris jelly. Mucous connective tissue is merely another name for embryonic connective tissue and is found as such wherever connective tissue develops. In the adult it occurs in the posterior chamber of the eye as the vitreous humor.

2. RETICULAR CONNECTIVE TISSUE.

Reticular connective tissue is a fibrous connective tissue in which the intercellular substance has disappeared. The tissue is often described as being composed of anastomosing branched cells, arranged in the form of a network with open spaces. The observations of Ranvier and Bizzozero, and more recently those of Mall, have shown that the framework of reticular tissue is composed of very fine fibrils or bundles of fibrils. These interlace in all planes to form a most intricate network, surrounding spaces of varying size and shape. According to F. P. Mall, the fibrils of reticular tissue differ chemically from both the white and elastic fibers, although their composition has not been fully determined. Like white fibrous tissue, reticular tissue is not digested by pancreatin, but, unlike white fibrous tissue, it does not appear to yield gelatin upon boiling in water, but a mixture of gelatin and reticulin, a substance identified by Siegfried.

The cells of reticular connective tissue, which are flattened and often variously branched, lie on the reticular network, being often wrapped about the bundles of fibrils. Unless they are removed, the reticulum has the appearance of a network composed of branched and anastomosing cells.

Reticular connective tissue is found in adenoid tissue and lymphglands, in the spleen, and in the mucous membrane of the intestinal canal, and in these locations the meshes of the reticulum are filled with lymph-cells and other cellular elements, which, unless removed, obscure the reticulum. Connective -tissue fibrils giving the same reaction as those found in the adenoid reticulum are found associ



Fig. 63. Reticular fibers from a thin section of a lymph-gland, digested on the slide in pancreatin and stained in iron-lac-hematoxylin.

ated with white and elastic fibers in the liver, kidneys, in the lung, and in many other tissues. In bone-marrow a reticulum is found, in the meshes of which are the cellular elements of this tissue.


3. Fibrous Connective Tissue

Fibrous connective tissue can be divided morphologically into two groups : In one the bundles of fibers cross and interlace in all directions, forming a network with meshes of varying size formless or areolar connective tissue. In the other the bundles of fibers are parallel to each other, as in tendon and many of the aponeuroses and ligaments, or less regularly arranged, yet very densely woven, as in fascias, the dura mater, and the firm, fibrous capsules of some of the organs.

(a) In areolar connective tissue the bundles of white fibers, which vary greatly in size and which often divide and anastomose with portions of other branching bundles, intercross and interlace in all directions. If the bundles of fibers are numerous, the interlacement is more compact, thus forming a dense areolar connective tissue ; if less numerous, the network is more open, as in loose areolar connective tissue. Elastic fibers are always found in areolar connective tissue, though in varying quantity. They anastomose to form a network with large, irregular meshes, and run on or between the bundles of white fibers. The meshes between the bundles of fibers, and the minute spaces between the fibrils in these bundles, are occupied by a semifluid, homogeneous substance known as the ground-substance, or matrix. The fibrous elements of areolar connective tissue are, therefore, imbedded in this ground-substance. In dense areolar connective tissue the fibrous elements appear to have nearly displaced the ground-substance. In the ground-substance are found irregular, branched spaces, cell-spaces, in which lie the cellular elements of this connective tissue. These spaces anastomose by means of their branches, thus forming part of a



Fig. 64. Reticular connective tissue from lymph-gland of man ; X 2 %- Brush preparation.


system of spaces and small channels, known as the lymph canalicitlar system. These spaces and channels permeate the groundsubstance in all directions, and serve to convey lymph to the tissue elements. The cell-spaces and their anastomosing branches can be demonstrated by immersing areolar connective tissue (preferably from a young animal), spread out in a thin layer, in a solution of silver nitrate ( I f ) until the tissue becomes opaque. If then the tissue is exposed to

sunlight, the silver is reduced in the ground-substance, giving it a brown color, while the cell-spaces remain unstained. The groundsubstance of areolar connective tissue contains mucin.



Fig. 65. Areolar connective tissue from the subcutaneous tissue of a rat. Elastic fibers not shown.


The cellular elements of areolar connective tissue, which, as above stated, are found in the cell-spaces, are either fixed connective-tissue cells or wandering or migratory cells. The former are again divided, according to their shape and structure, into true connective-tissue cells or corpuscles, plasma cells, mast-cells, and pigment-cells.



Fig. 66. Cell - spaces in the groundsubstance of areolar connective tissue (subcutaneous) of a young rat. Stained in silver nitrate.


Fig. 67. Three connective-tissue cells from the pia mater of a dog. Stained in methylene-blue (intra vitani).


The connective-tissue cells or corpuscles are flattened, variously shaped cells of irregular form, usually having many branches. The protoplasm is free from granules ; the nucleus, situated in the thicker portion of the cell-body and of oval shape, shows a nuclear network and one or several nucleoli. The cells assume the shape of the space that they occupy and nearly fill. The branches of neighboring cells often anastomose through the fine channels uniting the cell-spaces.



Fig. 68. Two pigment cells found on the capsule of a sympathetic ganglion of a frog.


Plasma cells (Unna) vary in size and shape according to the space which they occupy. They may be round, oval, or /spindleshaped, and measure from 6 M to 10 /A The nucleus is round or oval. They are characterized by the fact that their protoplasm stains intensely in basic aniline dyes, often of a color differing from that of the solution used. According to some observers, the plasma cells are thought to be developed / from the connective-tissue cells,

v, ^^bn while others regard tnem as de , rived from the white blood-cells

/^Tl^'A (lymphocytes). They are found u:

Nucleus. t-'-i -I -I " \J^fJj

'--\ / %. various mucous membranes and m.

lymphoid tissues generally.

Mast-cells (Ehrlich) are relatively large cells of round, oval, or irregular shape, the protoplasm of which contains relatively large granules which stain chiefly in basic aniline dyes, which granules Fig. 69. Leucocyte of a frog with are often found in such numbers pseudopodia. The cell has included a that they COVCr Up the nucleus, bacterium which is in process of diges- , o-^nnlps arf > Drained bv a tion. (After Metschnikoff, from O. Hertwig, 93, II.) number of basic aniline dyes, often of a color differing from that of the stain used. They are found generally in mucous membranes, generally near the vessels, in the skin, in involuntary muscle, and in the bone-marrow.


Pigment cells are branched connective-tissue cells, in the protoplasm of which are found brown or nearly black granules. In man they occur in the choroid and iris and in the dermis. In the lower animals they have, however, a much wider distribution, and in the frog and other amphibia they are very large and irregular. These cells have the power of withdrawing their processes and, to a limited degree, of changing their location (dermis).

The wandering or migratory cells are described in this connection not because they form one of the structural elements of areolar connective tissue, but because they are always associated with it. They are lymph- or white blood-cells, which have left the lymph- or blood-vessels and have migrated into the lymph canalicular system. They possess ameboid movement, and wander from place to place, and are the phagocytes of Metschnikoff. They seem to be intrusted with the removal of substances either superfluous or detrimental to the body (as bacteria). These are either digested or rendered harmless. The wandering cells even transport substances thus taken up to some other region of the body, where they are deposited.

In the peritoneum and other serous membranes the network formed by the fibrous tissue lies in one plane, and does not branch and intercross in all directions, as where areolar tissue is found in larger quantity. (Fig. 70.)

(<) Tendons, aponeuroses, and ligaments represent the densest variety of fibrous connective tissue, and are composed almost wholly of white fibrous tissue. This is found in the form of relatively large bundles of white fibrils, having a parallel or nearly parallel course. In tendons these bundles are known as primary tendon bundles or tendon fasciculi. The fibrils of white fibrous connective tissue forming the fasciculi are cemented together by an interfibrillar cement substance. Here and there the fasciculi branch at very acute angles and anastomose with other fasciculi. The fa'sciculi are grouped into larger or smaller bundles, the secondary tendon bundles, which are surrounded by a thin layer of areolar connective tissue, and in part covered by endothelial cells. Between the tendon fasciculi there is found a ground-substance, interfasdcular ground-substance, identical with the ground-substance in areolar connective tissue. In this there are cell-spaces occupied by the tendon cells, morphologically similar to the branched cells of areolar connective tissue. The tendon cells are arranged in rows between the tendon fasciculi. They have an irregular, oblong body, containing a nearly round or oval nucleus. Two, three, or even more winglike processes (lamellae) come from the cell-body and pass between the tendon fasciculi. In cross-section the tendon cells have a stellate shape.


Fig. 70. Fibrous connective tissue (areolar) from the great omentum of the rabbit ; X400.


The secondary tendon bundles are grouped to form the tendon, and the whole is surrounded and held together by a layer of areolar connective tissue, called the peritendineum. From this, septa pass in between the secondary tendon bundles, forming the internal peritendineum. The blood- and lymph-vessels and the nerve-fibers reach the interior of the tendon through the external and internal peritendineum. developed gland capsules, differs from that of the formed connective tissues above described, in that the fasciculi are not so regularly arranged, but branch and anastomose and intercross in several planes.



Fig. 71. Longitudinal section of tendon ; Fig. 7 2 Cross-section of secondary X 270. tendon bundle from tail of a rat.


(c) Elastic Fibrous Tissue. In certain connective tissues the elastic fibers predominate greatly over the fibers of white fibrous connective tissue. These are spoken of as elastic fibrous tissues and their structural peculiarities warrant the making of a special subgroup.

The ligamentum nuchae of the ox consists almost exclusively of elastic fibers, many of which attain a size of about io//. The elastic fibers branch and anastomose, retaining, however, a generally parallel course. They are separated by a small amount of areolar connective tissue, in which a connective-tissue cell is here and there found, and are grouped into bundles surrounded by thin layers of areolar connective tissue ; the whole ligament receives an investment of this tissue. In cross-sections of the ligamentum nuchae, the larger elastic fibers have an angular outline ; the smaller ones are more regularly round or oval. (Fig. 74.) In man the ligamenta subflava, between the laminae of adjacent vertebrae, are elastic ligaments.

In certain structures (arteries and veins), the elastic tissue is arranged in the form of membranes. It is generally stated that such membranes are composed of flat, ribbon-like fibers or bands of elastic tissue arranged in the form of a network, with larger or smaller openings ; thus the term fenestrated membranes. F. P. Mall has reached the conclusion that such membranes are composed of three layers an upper and a lower thin transparent layer in which no openings are found and which are identical with the sheaths of elastic fibers described by this observer, and a central layer, containing openings, and staining deeply in magenta. This substance is identical with the central substance of elastic fibers.


Fig. 73. Tendon cells from the tail of a rat. Stained in methyleneblue (intra vitam}.


Fig. 74. Cross-section of ligamentum nucha; of ox.


4. Adipose Tissue

In certain well-defined regions of the body occur typical groups of fixed connective-tissue cells which always change into fat-cells (fat organs,Toldt). Connective-tissue cells in various other portions of the body may also change into fat-cells, but in this case the fat, as such, sometimes disappears, allowing the cells to resume their original connective-tissue type, only again to appear and a second time change the character of the tissue. The formation of fat is very gradual. Very fine fat globules are deposited in the cell ; these coalesce to form larger ones, until finally the cell is almost entirely filled with a large globule (vid. also H. Rabl, 96). As the fat globule grows larger and larger, the protoplasm of the cell, together with its nucleus, is crowded to the periphery. The protoplasm then appears as a thin layer just within the clear cellular membrane. The nucleus becomes flattened by pressure, until in profile view it has the appearance of a long, flat body. In regions in which large masses of fatcells are developed, they are seen to be gathered into rounded groups of various sizes (fat lobules) separated by strands of connective tissue. Such lobules have, as was first pointed out by Toldt, a typical and very rich blood-supply from the time that they are recognized as fat organs in the embryo. A small artery courses through the center of the fat lobule, breaking up into capillaries which form a network around the fat cells. The capillaries unite to form several veins which are situated at the periphery of the lobule. Where fat cells develop from connective-tissue cells, even though these are present in considerable number this typic arrangement of the blood-vessels is wanting.




Fig. 75 - Scheme of a fat-cell.



Microscopically, fat is easily recognized by its peculiar glistening appearance (by direct light). It has a specific reaction to certain reagents. It becomes black on treatment with osmic acid, and is stained red by Sudan III and blue in cyanin.

5. Cartilage

Cartilage is readily distinguished from other connective tissues by its ground-substance or matrix, intercellular substance, which yields chondrin on boiling. Three varieties are found in higher vertebrates: (i) hyaline cartilage; (2) elastic cartilage; (3) white fibro-cartilage or connective-tissue cartilage.

The simplest type is hyaline cartilage, so named because of its homogeneous and transparent ground-substance, which, however, in reality consists of fibrils and an interfibrillar substance, the two having essentially the same refractive index. In this groundsubstance are found the cartilage cells, occupying spaces known as lacunae. The spaces or lacuna:; are surrounded by a narrow zone of ground-substance, which does not stain as does the ground substance and which refracts the light more strongly. This zone is generally known as the capsule of the cartilage cells. As previously stated, the matrix or ground-substance, develops in the exoplasm of the protoplasmic syncytium from which cartilage has its origin, while the endoplasm and nuclei form the cartilage cells. Cartilage cells, as such, are of various shapes, and have no typical appearance. They are usually scattered irregularly throughout the matrix, but are often arranged in groups of two, three, four, or even more cells. At the periphery of cartilage, either where it borders upon a cavity (articular cavity) or where it joins the perichondrium, the cells are arranged in several rows parallel to the surface of the tissue. Cartilage cells often contain glycogen, either in the form of drops or diffused throughout their protoplasm.



Fig. 76. Hyaline cartilage (costal cartilage of the ox). Alcohol preparation ; X 300. The cells are seen inclosed in their capsules. In the figure a are represented frequent but by no means characteristic radiate structures.


Cartilage grows by intussusception, and an appositional growth, although in a lesser degree, also takes place. It occurs where the cartilage borders upon its connective -tissue sheath or perichondrium, a vascular, fibrous-tissue membrane composed of white and elastic fibers, which covers the cartilage except where it forms a joint surface. The relations of the cartilage and perichondrium are extremely intimate. Fibers are seen passing from the perichondrium into the cartilaginous matrix, and the connective-tissue cells appear to change directly into cartilage-cells.



Fig. 77. From a section through the cranial cartilage of a squid (after M. Fiirbringer, from Bergh).


Certain observers (Wolters, Spronk, and others) have described a system ofcanaliculi in the ground substance, which are said to unite the lacunae and are thought to serve as channels for the passage of lymph. Such structures are, however, not generally recognized. It is an interesting fact, however, that the cartilage of certain invertebrate animals, the cephalopoda, shows cells with anastomosing processes. (Fig. 77.) In this case the cartilage-cell is similar to a bone-cell, thus theoretically allowing of the possibility of the metamorphosis of the elements of cartilage into those of bone (M. Fiirbringer).


Hyaline cartilage occurs as articular cartilage, covering joint surfaces, as costal cartilage and in the nose, larynx, trachea, and bronchi. All bones except those of the vault of the skull and the majority of the bones of the face are preformed in hyaline cartilage.


Fig. 78. Insertion of the ligamentum teres into the head of the femur. section ; X 650.



In white fibrocartilage (Fig. 78) there are from the beginning, even in precartilage, fibrous strands in the ground-substance. They preponderate over the matrix and, as a rule, have a parallel direction. White fibrocartilage is found in the intervertebral and interarticular disks, the symphysis pubis, and in the insertion of the ligamentum teres ; it deepens the cavity of ball-and-socket joints, and lines the tendon grooves.

In some places elastic fibers are found imbedded in hyaline cartilage -fibro-elastic cartilage. The elastic fibers send off at acute angles finer or coarser threads which interlace to form a delicate or dense network which permeates the hyaline matrix (Fig. 79), passing over into the corresponding elements of the perichondrium. Elastic cartilage is found in the external ear, the cartilage of the Eustachian tube, the epiglottis, a portion of the arytenoid cartilages, and the cartilages of Wrisberg and Santorini.


Fig. 79- Elastic cartilage from the external ear of man; X 7 o - a > Fine elastic network in the immediate neighborhood of a capsule.


The ground-substance of cartilage undergoes changes as age advances. In certain cartilages there is observed a fibrillar formation, in the ground-substance between the cells. The fibers are coarse and differ from white fibrous or yellow elastic fibers. This change is observed in laryngeal cartilages as early as the twentieth year, and is sometimes designated as an asbestos-like alteration of cartilage. Calcification occurs in many cartilages laryngeal, tracheal, costal and consists of the deposition in the ground-substance of fine granules of carbonate of lime, first in the immediate vicinity of the cartilage cells. Calcification is observed as early as the twentieth year in the laryngeal cartilages. Ossification may be regarded as a normal occurrence in many cartilages. It begins with an ingrowth of blood-vessels from the perichondrium into the matrix. These vessels are surrounded by connective tissue. Around such locations ossification occurs. Chievitz has shown that the laryngeal cartilages begin to ossify in men at about the twentieth year, and in women at about the thirtieth year; and the tracheal cartilage in men about the fortieth year, and in women about the sixtieth year.

To obtain chondrin, a piece of cartilage matrix is placed in a tube containing water. This is hermetically closed and heated to 120 C, after which it is opened and the fluid filtered and treated with alcohol. A precipitate of chondrin is the result. This substance is insoluble in cold water, alcohol, and ether, but soluble in hot water, although, on cooling, it gelatinizes. In contrast to gelatin, chondrin is precipitated by acetic acid. This precipitate does not redissolve in an excess of this acid but disappears in an excess of certain mineral acids.


6. Bone

(a) Structure of Bone. Bone nearly always develops from a connective-tissue foundation, even where it occurs in places formerly occupied by cartilage.

The inorganic substance of bone is deposited in or between the fibers of connective tissue, while the cells of the latter are transformed into bone-cells.

As in connective tissue, so also in bone, the ground-substance is fibrous. Between the fibers remain uncalcified cells, bone-cells. each of which rests in a cavity of the matrix lacuna.

Primarily, bone consists of a single thin lamella, its later complicated structure being produced by the formation of new lamellae in apposition to the first. During its development the bone becomes vascularized, and the vessels are inclosed in especially formed canals known as vascular or Haversian canals.

The bone-cells have processes that probably anastomose, and that lie in special canals known as bone canaliculi. Whether, in man, all the processes of bone-cells anastomose is still an open question.

The appearance presented by a transverse section of the shaft of a long bone is as follows : In the center is a large marrow cavity, and at the periphery the bone is covered by a dense connectivetissue membrane, the periosteum. In the new-born and in young individuals the periosteum is composed of three layers an outer layer, consisting mainly of rather coarse, white fibrous-tissue bundles that blend with the surrounding connective tissue ; a middle fibro-elastic layer, in which the elastic tissue greatly predominates ; and an inner layer, the osteogenetic layer, vascular and rich in cellular elements, containing only a few smaller bundles of white fibrous tissue. In the adult the osteogenetic layer has practically disappeared, leaving only here and there a few of the cells of the layer, while the fibro-elastic layer is correspondingly thicker (Schulz, 96). A large number of Haversian canals containing blood-vessels, seen mostly in transverse section, are found in compact bone-substance.

Lamellae of bone are plainly visible throughout the ground-substance, and are arranged in the following general systems :

First, there is a set of bone lamellae running parallel to the external surface of the bone, while another set is similarly arranged around the marrow cavity. These are the so-called fundamental, or outer and inner circumferential lamella (known also as periosteal and marrow lamella}. Around the Haversian canals are the concentrically arranged lamellae, forming systems of Haversian or concentric lamella. Besides the systems already mentioned, there are found interstitial or ground lamella wedged in between the Haversian or concentric systems of lamellae. Some authors group the interstitial lamellae with the systems of fundamental lamellae.




Fig. 80. Longitudinal section through a lamellar system.


Figs. 81 and 82. Lamellae seen from the surface; X 460 (after v. Ebner 75). a, Primitive fibrils and fibril-bundles ; c, bone-corpuscles with bone-cells ; d, bone canaliculi.



Lying scattered between the lamellae are found spaces known as bone corpuscles (Virchow) or lacuna. These are present in all the lamellar systems. It is very probable that all the lacunae are in more or less direct communication with each other by means of fine canals called canaliculi ( I . I fi. to 1 . 8 // in diameter). It can be demonstrated without difficulty that the lacunae of a single lamellar system communicate not only with each other, but also with those of 8 adjacent systems. In the lamellae adjoining the periosteum and matrow cavity the canaliculi end respectively in the subperiosteal tissue and in the marrow cavity. The canaliculi of the Haversian lamellae empty into the Haversian canals.


Fig. 83. Segment of a transversely ground section from the shaft of a long bone, showing all the lamellar systems. Metacarpus of man ; X 5


The lamellae of bone are compose'd of fine white fibrous-tissue fibrils, embedded in a ground-substance, in which they are arranged in layers, superimposed in such a way that the fibrils in the several layers cross at about a right angle, forming an angle of 45 with the long axis of the Haversian canal. It is as yet undecided whether the mineral salts (phosphate and carbonate of lime, sodium chlorid, magnesium salts, etc.) are deposited in the ground-substance (v. Ebner) or in the fibrillae (Kolliker). The lacunae (13;* to 3 1 // long, 6 // to 15 //-wide, and 4 // to 9 // thick) have, in common with the canaliculi, walls which present a greater resistance to the action of strong acids than the rest of the solid bone-substance. In each lacuna there is found a bone-cell, the nucleated body of which practically fills the lacuna, while its processes extend out into the canaliculi.

The Haversian canals contain blood-vessels, either an artery or a vein or both. Between the vessels and the walls of the canals are perivascular spaces bounded by endothelial cells, resting on the adventitious coats of the vessels and the sides of the canals. Into these spaces empty the canaliculi of the Haversian system. Lymphspaces beneath the periosteum and at the periphery of the marrow cavity communicate directly with the canaliculi of the circumferential systems.



Fig. 84. Portion of a transversely ground disc from the shaft of a human femur; X40Q.


All the lacunae and canaliculi should be thought of as filled by lymph plasma which circulates throughout, bathing the bone-cells and their processes. The formed elements of the lymph are probably too large to force their way through the very small canaliculi. The plasma current probably flows from the periosteal and marrow regions toward the Haversian canals.

Between the lamellae are bundles of fibers (some of which are calcified), which can be demonstrated by heating the bone, or in decalcified preparations on staining by certain methods. These are the so-called fibers of Sharpey ; in the adult they contain elastic fibers.

In the circumferential lamellae are found canals, not surrounded by concentric lamellae, which convey blood-vessels from the periosteum to the Haversian canals. These are called Volkmanrf s* canals.

The structure of bone-marrow will be discussed with the bloodforming organs.

() Development of Bone. Nearly all the bones of the adult body are, in the earlier stages of embryonic life, preformed in embryonic cartilage. As development proceeds, this embryonic cartilage assumes the character of hyaline cartilage, its cells becoming vesicular, and probably disappearing. In the* matrix, 1 however, there are formed spaces that are soon occupied by cells and vessels which grow in from a fibrous-tissue membrane (the future periosteum) surrounding the cartilage fundaments of the bones. These cells deposit a bone matrix in the cartilage spaces. Bone developed in this manner is known as endochondral or intracartilaginous bone. In certain bones namely, those of the vault of the skull and nearly all the bones of the face there is no preformation in cartilage, these bones being developed from a connective -tissue foundation. They are known as intramcmbranous bones. As will become evident upon further discussion of the subject, the formation of fibrous-tissue bone (intramembranous) is not confined to bones not preformed in cartilage. In bones preformed in cartilage, fibrous-tissue bone develops from the connective-tissue membrane surrounding the cartilage fundaments, the two types of bone-development going on simultaneously in such bones. Attention may further be drawn to the fact that nearly all endochondral bone is absorbed, so that the greater portion of all adult bone, even that preformed in cartilage, is developed from a foundation of fibrous tissue. The two modes of ossification endochondral or intracartilaginous and intramembranous even though appearing simultaneously in the majority of bones, will, for the sake of clearness, be discussed separately.

I. Endochondral Bone=developrnent. The cartilage that forms the fundaments of the bones preformed in cartilage has at first the appearance of embryonic cartilage, consisting largely of cells with a small amount of intercellular matrix. These fundaments are surrounded by a fibrocellular membrane the perichondrium. Ossification is initiated by certain structural changes in the embryonic cartilage, in one or several circumscribed areas, known as centers of ossification. In the long bones a center of ossification appears in the middle of the future diaphysis. In this region the intercellular matrix increases in amount and the cells in size ; thus the embryonic cartilage assumes the character of hyaline cartilage. This is followed by a further increase in the size of the cartilage-cells, at the expense of the thinner partitions of matrix separating neighboring cells, while at the same time lime granules are deposited in the matrix remaining. During this stage the cells appear first vesicular, distending their capsules, then shrunken, only partly filling the enlarged lacunae. They stain less deeply, and their nuclei show degenerative changes. The center of ossification, in the middle of which these changes are most pronounced, is surrounded by a zone in which these structural changes are not so far advanced and which has the appearance at its periphery of hyaline cartilage.

Simultaneously with these changes in the cartilage, a thin layer of bone is deposited by the perichondrium (in a manner to be described under the head of intramembranous bone-development) and the perichondrium becomes the periosteum. This in the meantime has differentiated ino two layers an outer, consisting largely of fibrous tissue with few cellular elements, and an inner, the osteogenetic layer, vascular and rich in cellular elements and containing few fibrous-tissue. fibers.

Ossification in the cartilage begins after the above-described structural changes have taken place at the center of ossification. Its commencement is marked by a growing into the cartilage of one or several buds or tufts of tissue derived principally from the osteogenetic layer of the periosteum. As the periosteal buds grow into the cartilage, some of the septa of matrix separating the altered cartilage-cells disappear, and the cells become free and probably degenerate. In this way the cartilage at the center of ossistructural changes have taken place at the center of ossification. Its commencement is marked by a growing into the cartilage of one or several buds or tufts of tissue derived principally from the osteogenetic layer of the periosteum. As the periosteal buds grow into the cartilage, some of the septa of matrix separating the altered cartilage-cells disappear, and the cells become free and probably degenerate. In this way the cartilage at the center of ossification becomes hollowed out, and there are formed irregular anastomosing spaces, primary marrow spaces, separated by partitions or trabeculae of calcified cartilage matrix. Into these primary marrow spaces grow the periosteal buds, consisting of small bloodvessels, cells, and some few connective-tissue fibers, forming embryonic marrow, tissue. Some of the cells which have thus grown into the primary marrow spaces arrange themselves in layers on the trabeculae of calcified matrix, which they envelop with a layer of osseous matrix formed by them. The cells thus engaged in the formation of osseous tissue are known as osteoblasts.



Fig. 85. Longitudinal section through a long bone (phalanx) of a lizard embryo. The primary bone lamella originating from the periosteum is broken through by the periosteal bud. Connected with the bud is a periosteal blood-vessel containing red bloodcorpuscles.


Fig. 86. Longitudinal section of the proximal end of a long bone (sheep embryo) ; X30.


Ossification proceeds from the center of ossification toward the extremities of the diaphysis (in a long bone), and is always preceded as at the center of ossification, by the characteristic structural changes above described. Beginning at the center of ossification and proceeding toward either extremity of the diaphysis, the enlarged and .vesicular cartilage-cells will be observed to be arranged in quite regular columns, separated by septa or trabeculse of calcified cartilage matrix. The cells thus arranged in columns show the degenerative changes above described. They are shrunken and flattened, and their nuclei, when seen, stain less deeply than the nuclei of normal cartilage-cells. Beyond this zone of columns of altered cartilage-cells are found smaller or larger groups of less changed cartilage-cells, and beyond this zone, hyaline cartilage.

The arrangement of the cartilagecells in the columns above mentioned is, according to Schiefferdecker, mainly due to two factors the current of lymph plasma which flows from the center of ossification toward the two extremities of the cartilage fundament, and the mutual pressure exerted by the groups of cartilage-cells in their growth and proliferation. Ossification proceeds from the center of the diaphysis toward its two extremities by a growth of osteoblasts and small vessels into the columns of cartilage-cells. Here, also, these degenerate, leaving in their stead irregular, oblong, anastomosing spaces, separated by septa and trabeculae of calcified cartilage matrix on which the osteoblasts arrange themselves in layers, and which they envelop in osseous tissue. In a longitudinal section of a long bone, preformed in cartilage, the various steps of endochondral t>one-development may, therefore, be observed by viewing the preparation from either end to the center of the diaphysis, as may be seen in figures 86, 87. The former represents the appearance as seen under low magnification, the latter a small portion of such a section from the area of ossification, more highly magnified.

Adjoining the primary marrow spaces is vesicular cartilage and columns and groups of cartilage-cells and finally hyaline cartilage.



Fig. 87. Longitudinal section through area of ossification from long bone of human embryo.


In the upper portion of figure 87 is observed a zone composed of groups of cartilage-cells, adjoining this a zone composed of columns of vesicular and shrunken cartilage-cells, the nuclei of which are indistinctly seen. These columns are separated by septa and trabeculae of calcified matrix. This zone is followed by one in which the cartilage-cells have disappeared, leaving spaces into which the osteoblasts and small blood-vessels have grown. In certain parts of the figure, the osteoblasts are arranged in a layer on the trabeculae of calcified cartilage, some of which are enveloped in a layer of osseous matrix, less deeply shaded than the darker cartilage remnants.

As the development of endochondral bone proceeds from the center of ossification toward the extremities of the diaphysis in the manner described, the primary marrow spaces at the center of ossification are enlarged, a result of an absorption of many of the smaller osseous trabeculae and the remnants of calcified cartilage matrix enclosed by them. In this process are concerned certain large and, for the most part, polynuclear cells, which are differentiated from the embryonic marrow. These are the osteoclasts (bone breakers) of Kolliker (73). They are 43 JJL to 91 // long and 30 ju. to 40 // broad, and have the function of absorbing the bone. The spaces which they hollow out during the beginning of the process appear as small cavities or indentations, containing osteoclasts either single or in groups, and are known as Htnvship's lacunce. All bone absorption goes hand in hand with their appearance. At the same time, the osseous trabeculae not absorbed become thickened by a deposition of new layers of osseous tissue (by osteoblasts), during which process some of the osteoblasts are enclosed in the newly formed bone and are thus converted into bone-cells. In this way there is formed at the center of ossification a primary or embryonic spongy or cancellous bone, surrounding secondary marrow spaces or Havcrsian spaces, filled with embryonic marrow. This process of the formation of embryonic cancellous bone follows the primary ossification from the center of ossification toward the extremities of the diaphysis. It should be further stated, that long before the developing bone has attained its full size indeed, before the end of embryonic life the embryonic cancellous bone is also absorbed through the agency of osteoclasts. The Haversian spaces are thus converted into one large cavity, which forms a portion of the future marrow cavity of the shaft of the fully developed bone. The absorption of the embryonic cancellous bone begins at the center of ossification and extends toward the ends of the diaphysis.

Some time after the beginning of the process of bone development at the center of ossification of the diaphysis, centers of ossification appear in the epiphyses, the manner of the development of bone being here the same as in the diaphysis. Several periosteal buds grow into each center of ossification, filling the irregular spaces formed by the breaking down of the degenerated cartilage-cells. Osteoblasts are arranged in rows on the trabeculae of cartilage thus formed, which they envelop in osseous tissue. As development proceeds, the primary osseous tissue is converted into embryonic cancellous bone as above described.

In the development of the epiphyses, as in the development of the smaller irregular bones, the formation of bone proceeds from the center or centers of ossification in all directions, and not only in a direction parallel to the long axis of the bone as described for the diaphysis. The epiphyses grow, therefore, in thickness as well as in length, by endochondral bone-development.

There remains between the osseous tissue developed in the diaphysis and that in the epiphyses, at each end of the diaphysis, a zone of hyaline cartilage in which ossification is for a long time delayed ; this is to permit the longitudinal growth of the bone. These layers of cartilage constitute the epiphyseal cartilages. Here the periosteum (perichondrium) is thickened and forms a raised ring around the cartilage. As it penetrates some distance into the substance of the cartilage, the latter is correspondingly indented. (Fig. 86.) The impression thus formed appears in a longitudinal section of the bone as an indentation, the ossification groove (encoche d 'ossification , Ranvier, 89). That portion of the perichondrium filling the latter is called the ossification ridge. The relation of the elements of the perichondrium to the cartilage in the region of the groove just described is an extremely intimate one, both tissues, perichondrium and cartilage, merging into each other almost imperceptibly. It is a generally accepted theory that so long as the longitudinal growth of the bone persists, new cartilage is constantly formed at these points by the perichondrium. In the further production of bone this newly developed cartilage passes through the preliminary changes necessary before the actual commencement of ossification i. e., it goes through the stages of vesicular cartilage and the f}rmation of columns of cartilage-cells, in place of which, later, the osteoblasts and primary marrow cavities develop.

By the development of new cartilage elements from the encoche tre longitudinal growth of the bone is made possible ; at the same tine, those portions of the cartilage thus used up in the process of ossification are immediately replaced. (Fig. 88.)

The following brief summary of the several stages of endochondral bone-development may be of service to the student :

1. The embryonic cartilage develops into hyaline cartilage, beginning at the centers of ossification.

2. The cartilage-cells enlarge and become vesicular. In the diaphysis of long bones such cells are arranged in quite regular colunns, while in the epiphyses and irregular bones this arrangement is not so apparent.

3 Calcification of the matrix ensues ; the cartilage-cells disappear (degenerate) ; primary marrow spaces develop.

4. Ingrowth of periosteal buds. The osteoblasts are arranged in layers on the trabeculae of calcified cartilage, which they envelop with osseous tissue.

5. Osteoclasts cause the absorption of many of the smaller osseous trabeculae ; others become thickened by a deposition of new layers of osseous tissue. Osteoblasts are enclosed in bonetissue and become bone-cells. In this way there is formed embryonic cancellous bone, bounding Haversian spaces inclosing embryonic marrow.

6. In the diaphysis, the greater portion of the embryonic cancellous bone is also absorbed (by osteoclasts) ; the Haversian spaces unite to form a part of the marrow space of the shaft of the bone.

2. Intramembranous Bone. This, the simpler type of ossification, occurs in bone developed from a connective-tissue foundation, and is exemplified in the formation of the bones of the cranial vault and the greater number of the bones of the face, aid also in bone developed from the periosteum (perichondrium) sirrounding the cartilage fundaments of endochondral bone. All fibrous-tissue bone is developed in the same way.




Fig. 88. Longitudinal section through epiphysis of arm bone of sheep embryo ; X I; * a, b, Primary marrow spaces and bone lamellae of the diaphysis.


The intramembranous bone-development begins by an apprcximation and more regular arrangement of the osteoblasts of the osteogenetic layer of the periosteum about small fibrous-thsue bundles. The osteoblasts then become engaged in the formation of the osseous tissue which envelops the fibrous-tissue bundles. In this way a spongy bone with large meshes is formed, consisting of irregular osseous trabeculae, surrounding primary marrow spaces. These latter are filled by embryonic marrow and blood-vesses developed from the tissue elements of the periosteum not engaged in the formation of bone.


Intramembranous bone first appears in the form of a thin lamella of bone, which increases in size and thickness by the formation of trabeculae about the edges and surfaces of that previously formed and in the manner above described. A layer of intramembranous bone thus surrounds the endochondral bone in bones preformed in hyaline cartilage. The two modes of ossification may, therefore, be observed in either a cross or a longitudinal section of a developing bone preformed in hyaline cartilage. In such preparations the endochondral bone can be readily distinguished from the intramembranous bone by reason of the fact that remnants of calcified cartilage matrix may be observed in the osseous trabeculae of the former. It will be remembered that these osseous trabeculse develop about the calcified cartilage matrix remaining after the disappearance of the cartilage-cells. In figure 90, which shows a cross-section of a bone from the leg of a human embryo, these facts are clearly shown. A study of this figure shows the endochondral bone, with the remnants of the cartilage matrix (shaded more deeply) inclosed in osseous tissue, making up the greater portion of the section and surrounded by the intramembranous bone.




Fig. 89. Section through the lower jaw of an embryo sheep (decalcified with picric acid) ; X 3- At a and immediately below are seen the fibers of a primitive marrow cavity lying close together and engaged in the formation of the ground- substance of the bone, while the cells of the marrow cavity, with their processes, arrange themselves on either side of the newly formed lamella and functionate as osteoblasts.


In figure 91, more highly magnified, the relations of endochondral to intramembranous bone and the details of their mode of development are shown ; also the structure of the periosteum.

As was stated in the previous section, soon after the formation of the endochondral bone, this is again absorbed ; the process of endochondral bone-formation and absorption extending from the center of ossification toward the ends of the diaphysis. Before the absorption of the endochondral bone, the intramembranous bone has attained an appreciable thickness and surrounds the marrow cavity formed on the absorption of the endochondral bone. Before,



Fig. 9- Cross-section of developing bone from leg of human embryo, showing endochondral and intramembranous bone-development.

however, the marrow cavity can attain its full dimensions, much of the intramembranous bone must also undergo absorption. While intramembranous bone is being developed from the periosteum and thus added to the outer surface of that already formed, osteoclasts are constantly engaged in its removal from the inner surface of the intramembranous bone. The marrow cavity is thus enlarged, the process continuing until the shaft attains its full size.

The compact bone of the shaft is developed from the primary spongy intramembranous bone after the following manner : The primary marrow spaces are enlarged by an absorption, through the agency of osteoclasts, of many of the smaller trabeculae of osseous tissue and by a partial absorption of the larger ones, the primary marrow spaces thus becoming secondary marrow spaces, or Haversian spaces. The osteoblasts now arrange themselves in layers about the walls of the Haversian spaces and deposit lamella after lamella of bone matrix, concentrically arranged, until the large Haversian spaces have been reduced to Haversian canals. During this process many of the osteoblasts become inclosed in bone matrix, forming bone-cells and the blood-vessels of the Haversian spaces remain as the vessels found in the Haversian canals. The spongy intramembranous bone not absorbed at the commencement of the formation of the system of concentric lamellae, remains between the concentric systems as interstitial lamellae. The circumferential lamellae are those last formed by the periosteum. Calcificaation of the osseous matrix takes place after its formation by the osteoblasts.



Fig. 91. From a cross-section of a shaft (tibia of a sheep) ; X 55- I n l ^ e lower part of the figure is endochondral bone- formation (the black cords are the remains of the cartilaginous matrix) ; in the upper portion is bone developed from the periosteum.



From what has been stated it may be seen that the shafts of the long bones and bones not preformed in cartilage develop by the process of intramembranous bone-formation, while the cancellous bone in the ends of the diaphysis and in the epiphyses is endochondral bone. Further, that long bones grow in length by endochondral bone-development, and in thickness by the formation of intramembranous bone. In the development of the smaller irregular bones, both processes may be engaged ; the resulting bone can not, however, be so clearly defined.


Technic

Ranvier's Method. One of the methods for examining connectivetissue cells and fibers is that recommended by Ranvier (89) ; it is as follows : The skin of a recently killed dog or rabbit is carefully raised, and a o. i / aqueous solution of nitrate of silver injected subcutaneously by means of a glass syringe. The result is an edematous swelling in which the connectivetissue cells and fibers (the latter somewhat stretched) come into immediate contact with the fixing fluid and are consequently preserved in their original condition. In about three-quarters of an hour the whole elevation should be cut out (it will not now collapse) and small fragments placed upon a slide and carefully teased. Isolated connective-tissue cells with processes of different shapes, having the most varied relations to those from adjacent cells, are seen. The fibers themselves either consist of several fibrils, or, if thicker, are often surrounded by a spirally encircling fibril. By this method numerous elastic fibers and fat-cells are also brought out. If a drop of picrocarmin be added to such a teased preparation and the whole allowed to remain for twelve hours in a moist chamber, and formic glycerin (a solution of i part formic acid in 100 parts glycerin) be then substituted for twenty-four hours, the following instructive picture is obtained : All nuclei are colored red, the white fibrous connective-tissue fibers pink, the fibrils encircling the latter brownishred, and the elastic fibers canary yellow. The peripheral protoplasm of the fat-cells is particularly well preserved, a condition hardly obtainable by any other method.

Connective tissue with a parallel arrangement of its fibers is best studied in tendon, those in the tails of rats and mice being particularly well adapted to this purpose. If one of the distal vertebrae of the tail be loosened and pulled away from its neighbor, the attached tendons will become separated from the muscles at the root of the tail and appear as thin glistening threads. These are easily teased on a slide into fibers and fibrils. Such preparations are also useful in studying the action of reagents (see below) .

The substance resembling mucin which cements the fibrillae together is soluble in lime-water and baryta-water a circumstance made use of and recommended by Rollet (72, II) as a method for the isolation of connective-tissue fibrils. In necrotic tissue the fibers show a degeneration into fibrils (Ranvier, 89).

If connective tissue be heated in water or dilute acids to 120 C., and the fluid then filtered, a solution is obtained from which collagen can be precipitated by means of alcohol. This is insoluble in cold water, alcohol, and ether, but is soluble in hot water and when dissolved in the latter and cooled, becomes transformed into a gelatinous substance. Unlike mucin and chondrin this substance does not precipitate on the addition of acetic and mineral acids. Tannic acid and corrosive sublimate will cause precipitation, as also in the case of chondrin, but not with mucin (vid. also Hoppe-Seyler).

Elastic tissue may be obtained by treating connective tissue with potassium hydrate solution, and if the alveoli of the lungs be treated for some time with this reagent, very small elastic fibers can be obtained. By this means the connective-tissue fibers are dissolved, but not the elastic fibers. Particularly coarse fibers are found in the ligamenta subflava.

According to Kiihne, connective and elastic tissues are differently affected by trypsin digestion /. >., alkaline glycerin -pancreas extract at 35 C. white fibrous connective tissue being resolved into fibrils, while elastic tissue is entirely dissolved.

To F. P. Mall also belongs the credit for a few data, which we insert, as to the different reactions which various connective-tissue substances show when treated by the same reagents.

When a tendon is boiled it becomes shorter, but if it be fixed before boiling, there is no change. Adenoid reticulum shrinks when boiled, but after a short time swells, and finally dissolves. Both tendon and adenoid reticulum shrink at 70 C. If, however, they be first treated with a 0.5% solution of osmic acid, the shrinkage will not take place until 95 C. is reached. If the reticulum or the tendon has become shrunken through heat, they are easily digested with pancreatin, and putrefy very readily. Tendon fibers do not become swollen in glacial acetic acid, either concentrated or m strengths of 0.05% or less, but in strengths of 0.5% to 25% they swell, and if placed in a 25% solution they will dissolve in twenty-four nours. They also swell in hydrochloric acid in strengths of 0.1% to 6%. In strengths of 6% to 25% the fibers remain unchanged for some time, and only dissolve in a concentrated solution of this acid. Reticulated tissue, on the other hand, swells in a 3% hydrochloric acid solution, but remains unchanged in strengths of 3% to 10%. It dissolves in twenty-four hours in solutions of 25% and over. After treatment with a dilute solution of acid, tendon dissolves more rapidly on boiling than does reticular tissue.

Tendon exposed to the action of the gastric juice of a dog does not dissolve more rapidly than elastic tissue ; but if placed in an artificial solution of gastric juice, tendon dissolves first, then reticular tissue, and finally elastic fibers. Pancreatin affects neither tendon nor reticulated tissue, but if boiled, both tissues are easily digested by its action. If taken out of the body, neither tendon nor reticulum will become affected by putrefaction. In the body, however, and especially at high temperatures (37 C.), both tissues are decomposed within a few days.


Elastic fibers remain unchanged in acetic acid, and even when boiled in a 20% solution they only become slightly brittle. They are, however, rapidly destroyed by concentrated hydrochloric acid, although in a 10% solution at ordinary temperature no change is seen. In a 50% solution the fiber is dissolved in seven days, and in a concentrated solution in two days. The inner substance of the fiber is first attacked, then the membrane. To demonstrate this membrane, the fibers are boiled several times in concentrated hydrochloric acid and the whole then poured into cold water. Occasionally, a longitudinal striation of the membrane is seen, indicating a fibrillar structure. Concentrated solutions of potassium hydrate disintegrate the fibers in a few days ; weak solutions, more slowly. A i c /o solution of potassium hydrate requires months to produce the effect ; a 2 % solution, one month ; a 5 % , three days ; a 10%, one day ; and 20% to 40%, only a few hours. A weak solution of potassium hydrate, even when brought to the boiling-point, does not dissolve elastic fibers, nor does it cause them to become brittle. If, however, they be boiled in a 5% or 10% solution of potassium hydrate, the membranes of the fibers will be isolated. A cold 20% solution has the same effect in one or two days. Pepsin induces a disintegration of the contents of the fiber, leaving the membranes intact.

To demonstrate the inner substance of elastic fibers and their membranes, magenta red has been recommended (a small granule is added to 50 c.c. glycerin and 50 c.c. water). By this method the internal substance is colored red while the sheath remains colorless.

Orcein, Unna's Method. Make a solution consisting of Grubler's orcein i part, hydrochloric acid i part, absolute alcohol 100 parts. The sections are stained in a porcelain dish. The stain is heated over a flame or in an oven until the stain becomes quite thick. Rinse thoroughly in alcohol, clear in xylol, and mount. Elastic fibers stain a dark brown, white fibrous tissue a light brown.

Fuchsin-resorcin Elastic Fibers Stain (Weigert). A solution containing i % of basic fuchsin and 2 % of resorcin is made and brought to boiling. To 200 c.c. of this solution there is added 25 c.c. of liquor ferri sesquichlorati (Germ. Pharm.). Boil for about five minutes, stirring the meanwhile. Filter on cooling, and place the filter paper and the precipitate collected in a porcelain dish and add 200 c.c. of 95% alcohol and bring to boiling. Filter on cooling and add to the nitrate 4 c.c. of hydrochloric acid and enough alcohol to bring it up to 200 c.c. Stain sections for about one hour. Sections are then washed in alcohol or acidulated alcohol, or, better still, in alcohol to which a few crystals of picric acid have been added. Clear in xylol and mount. Elastic fibers are stained dark blue or bluish-black if washed in picric alcohol.

Differential Stain for Connective-tissue Fibrilla? and Reticulum (Mallory). Fix tissues in corrosive sublimate or in Zenker's solution. (Tissues fixed by other methods may also be used, although the results are not quite so satisfactory, if the sections are immersed for fifteen to thirty minutes in a saturated corrosive sublimate solution just before staining. ) The sections, which may be cut in celloidin or paraffin, are stained for one to three minutes in a ^% aqueous solution of acid fuchsin, rinsed in water, and placed in a i % aqueous solution of phosphomolybdic acid for five to ten minutes, and then washed in two changes of water. They are now stained in the following solution for two to twenty minutes: Griibler's aniline blue soluble in water, 0.5 gm. ; Griibler's orange G, 2 gm. ; oxalic acid, 2 gm. ; distilled water, 100 c.c. After staining, the sections are washed in water and dehydrated in 95% alcohol, blotted on the slide, and cleared in xylol and mounted in xylol balsam. The connective-tissue fibers and reticulum stain blue.

Dr. Sabin's modification of this method deserves mention. Fix in Zenker's fluid, cut in paraffin, and fix sections to the slide with the water method. After removing the paraffin, stain sections in y 1 ^^ acid fuchsin until red, and without washing fix in a saturated aqueous solution of phosphomolybdic acid diluted ten times for about ten minutes. Wash in 95% alcohol and stain for a very short time in the following solution : Griibler's aniline blue soluble in water, i gm. ; orange G, 2 gm. ; oxalic acid, 2 gm. ; boiling water, 100 c.c. Wash in alcohol, blot on the slide, clear in xylol and mount in xylol balsam.

Digestion Method for Demonstrating the Connective-tissue Framework of Organs and Tissues (Mall, Spalteholz, Hoehl, Flint). For bringing out the framework of white fibrous and reticular fibers of organs and tissues digestion by means of trypsin may be recommended. For the account here given we follow Flint. The tissues are fixed in graded alcohol, corrosive acetic, or Van Gehuchten's chloroformacetic-alcohol mixture. After complete dehydration, small pieces of tissue, not to exceed 3 mm. in thickness, are placed in paper cups and dropped into a Soxhlet apparatus and extracted with ether for a period of six to eight days in order to free the tissue of the fat. After the fat has been removed, the tissues are brought into water, through graded alcohol, and then digested in pancreatin. (Griibler's pancreatin is recommended ; that of Park, Davis & Co. may be used.) The -pancreatin solution to be used is made by adding as much pancreatin as can be taken up on the end of an ordinary scalpel handle to 100 c.c. of a 0.5% solution of bicarbonate of soda. This solution is changed every fortyeight hours. To prevent putrefaction enough chloroform is added to cover the bottom of the dish. The digestion is continued until the cellular element has been removed five to ten days. It is often necessary to repeat the fat extraction and digestion several times. After the cellular elements have been removed the tissue is thoroughly washed in flowing water, and may then be mounted in glycerine and studied with a stereoscopic microscope, or it may be dehydrated and imbedded in celloidin and sectioned. Such sections may then be stained in fuchsin and thoroughly washed in alcohol ; this removes the stain from the celloidin, leaving only the connective tissue stained.

Slide Digestion. The method may also be applied for digesting tissues on the slide. Fix as above described, imbed in paraffin, and cut very thin sections which are fixed to the slide by the water method. Remove the paraffin and place the sections from alcohol into the Soxhlet apparatus, where they are extracted with ether for a number of hours. Bring the sections through graded alcohol into water, in which they remain several hours. The sections are now digested in the above-mentioned pancreatin solution for several hours to several days, or until the cellular elements have been removed. Wash carefully in water. The remaining connective tissue may now be stained in iron-lac hematoxylin or in an aqueous solution of toluidin blue or in an aqueous solution of fuchsin. Dehydrate, clear, and mount.


Fresh adipose tissues can be obtained in lobules and in small groups of cells from the mesenteries of small animals. As a rule, the highly refractive fat globule hides from view the nucleus and protoplasm of the cell. The latter structures can be brought out by the subcutaneous injection of silver nitrate solution, this forming the edematous elevation previously described. Fresh fat is soluble in ether and chloroform, especially if the latter be heated. Strong sulphuric acid does not dissolve fat. The stains made from the root of the henna plant color fat red (the color disappearing in ethereal oils). Quinolin-blue, dissolved in dilute alcohol, stains fat a dark blue. If a 40% potassium hydrate solution be then added, everything will become decolorized except the fat. The most important reagent for demonstrating adipose tissue is osmic acid (and its mixtures). Small pieces of adipose tissue are treated for twenty-four hours with a 0.5% to i% osmic acid solution ; if mixtures containing osmic acid be used, the specimens are generally immersed for a somewhat longer period. The pieces are then washed with water, and should not be placed directly into alcohol of full strength, as all the structures would then become intensely black (Flemming, 89), but carried into alcohols of ascending strength. When treated in this way the globules of fat take a more intense stain than the other tissues, which, nevertheless, are blackened to some extent. Fat that has been subjected to osmic acid treatment dissolves readily in turpentine, xylol, toluol, ether, and creosote, with difficulty in oil of cloves, and not at all in chloroform. Such preparations are best carried from chloroform into paraffin. Fat that has been stained with osmic acid can be decolorized by nascent chlorin. The specimens are placed in a jar of alcohol in which crystals of potassium chlorid have been previously placed. Hydrochloric acid is then added (to i%) and the vessel tightly sealed (P. Mayer, 81).

L. Daddi has recently recommended Sudan III as a stain for fat. This reagent can be applied in two ways : ( i ) Either the animals are fed with the coloring matter for some days, in which case all the fat will be colored red, or (2) either fresh or fixed pieces of tissue or sections are stained. Fixation before staining must be done in media that do not dissolve fat, as, for instance, Miiller's fluid. A saturated alcoholic solution of the stain is used and allowed to act from five to ten minutes. The specimen is then washed with alcohol and mounted in glycerin. The author's experiments with Sudan have been very satisfactory.

Thin lamellae of fresh cartilage are examined after separating them from the soft parts and placing them in indifferent fluids. Cartilage removed from the hyposternum or episternum or scapula of a frog is especially adapted for examination. Larger pieces of uncalcified cartilage may be used if cut into sufficiently thin sections with a razor moistened with an indifferent fluid. Under the microscope such sections show a finely punctated background with capsules containing cartilage -cells, provided the latter have not fallen out in the process of cutting, in which case lacunas will be observed.

Osmic acid and corrosive sublimate are by far the best fixing agents for cartilage. If the cartilage be calcified, it is fixed for some time in picric acid, which at the same time acts as a decalcifying agent. Although alcohol fixes cartilage fairly well, it causes shrinkage of the cells. The ground substance may be specifically colored by certain reagents, safranin producing an orange and hematoxylin a blue stain.

On treating cartilage by certain methods, systems of lines appear in its ground substance, possibly indicating a canalicular system in the cartilage. In order to make these structures visible, Wolters recommends staining thin sections for twenty -four hours in a dilute solution of Delafield's hematoxylin (violet blue). They are then treated with a concentrated alcoholic solution of picric acid.

The capsules are seen to best advantage if small pieces of cartilage are treated with a 1% solution of gold chlorid.

Connective-tissue and elastic fibers in cartilage are easily demonstrated by staining the specimens with picrocarmin. The connective-tissue fibers are colored a pale pink, the elastic fibers yellow. The latter may also be stained with a i / aqueous solution of acid fuchsin.

If a section of fresh cartilage be placed in a weak solution of iodo-iodid of potassium (Lugol's solution), glycogen can sometimes be seen in the cartilage-cells, stained a peculiar mahogany brown. If elastic fibers be present, they also are stained brown, but of a different shade.

Thin bone lamellae, such as occur in the walls of the ethmoidal cells, can be cleaned of all the soft parts and examined without further manipulation. If larger bones are scraped with a sharp knife, pieces suitable for microscopic examination are sometimes obtained.

Microscopic Preparation of Undecalcified Bone. A long bone is thoroughly freed from fat and other soft parts by allowing it to macerate, after which it is thoroughly washed and dried, thus freeing it from its organic material. Then, by means of two parallel cuts with a saw, as thin a disc as possible is cut out. The section is now ground still thinner, either between two hones or upon a piece of glass covered with emery. One surface of the bone is then polished and fastened by means of heated Canada balsam to a thick square plate of glass with the polished side toward the glass. Care should be taken that no air-bubbles are inclosed between the section and the glass. As soon as the specimen is firmly adherent, the other side is ground upon the emery plate or hone, during which manipulation the glass to which the bone has been fastened is held between the fingers. As soon as the section is sufficiently thin and transparent, it is polished. In order to remove the Canada balsam and powdered bone from the section, the glass and bone are dried and placed in some solvent of Canada balsam, such as xylol. This loosens the specimen from the glass, after which it is immersed in absolute alcohol, thoroughly washed, and dried in the air. On examining the bone through the microscope, its lacunae will appear black on a colorless background. The reason is, that the air has taken the place of the evaporated alcohol and the spaces appear black by direct light. Sections thus prepared may be permanently mounted as follows : Small pieces of dry Canada balsam are placed both upon a slide and a coverglass and warmed until they have become fluid, then allowed to cool until a thin film forms over the balsam ; the bone disc is then placed upon the balsam on the slide and quickly covered with the cover-glass. A firm pressure will evenly distribute the balsam, and if the whole has been done with sufficient rapidity the air will have been caught in the open spaces of the bone before the Canada balsam has had a chance to enter these spaces.


Other substances may be used to demonstrate the spaces in bone. Ranvier (75) recommends the following method: A few c.c. of a concentrated alcoholic solution of anilin blue (which is soluble in alcohol and not soluble in water and sodium chlorid solution) are placed in an evaporating dish containing the dry bone. The solution is very carefully evaporated, as the alcohol may otherwise ignite. The specimen, which will soon be covered on both surfaces by a blue powder, is taken out and ground upon a rough glass plate until thoroughly clean. While being polished the bone should be kept moist by a solution of sodium chlorid. On heating in the evaporating dish, the air is driven from the spaces and replaced by the anilin blue. As already stated, anilin blue is insoluble in sodium chlorid solution, and it therefore remains unaffected by the latter during the process of grinding and cleaning. Hence it remains in the lacunse and canaliculi of the bone, which then appear blue. The specimen may either be mounted in glycerin-sodium chlorid and the edge of the cover-glass sealed with varnish, or the section may be washed for a short time in water (in order to remove the sodium chlorid), dried, and finally mounted in Canada balsam as directed.


A method adapted to the study of the hard and soft parts together is that first used by von Koch in studying corals. The specimen is first fixed, and if it be a long bone, the marrow cavity should first be opened to permit the fixing agent to come in contact with all parts of the tissue. After fixing, the bone is stained and then placed in absolute alcohol, and when completely dehydrated the pieces are placed in chloroform, then in a thin solution of Canada balsam in chloroform, and finally put into an oven kept at a temperature of about 50 C. for from three to four months. By this means the pieces are completely penetrated by the Canada balsam, and as the latter becomes very hard on cooling, the sections may be afterward ground without difficulty. Long as this procedure may seem, it is still the one which enables us to see the soft and hard parts of bone in a relationship the least changed by manipulation.

In bone, as also in cartilage, there sometimes occur amorphous as well as crystalline deposits of lime-salts. Upon the addition of acetic acid the carbonate of calcium gives off bubbles ; upon the addition of sulphuric acid, short, thin needles will be formed crystals of gypsum. Hematoxylin stains the lime-salts blue, with the exception of the oxalate of lime. Alkaline solution of purpurin stains calcium carbonate red. Caustic potash does not affect lime.

In order to study the organic constituents of bone, it must first be decalcified and thus rendered suitable for sectioning /. e. , the lime-salts must first be removed, and that without destroying the cellular elements of the bone. The process of decalcification consists in substituting the acids of the decalcifying fluids for those of the bone salts. As a consequence^ new combinations are formed, soluble in water or in an excess of the decalcifying acids themselves.

The decalcifying fluids most frequently used are : (a) Hydrochloric acid (i% aqueous solution), used in quantities amounting to about fifty times the volume of the specimen. The solution is changed daily, and the bone remains immersed until it is soft enough to be cut. This stage is reached when a needle can be introduced with no resistance.

(<) An aqueous solution of nitric acid in strengths of 3% to 10%, according to the delicacy of the specimen, and of a specific gravity of 1.4. Instead of water, 70% alcohol may be used as a solvent for the acid. Thoma has recommended for this purpose a solution consisting of i vol. nitric acid of a specific gravity of 1.3, and 5 vols. alcohol. This fluid is changed daily and decalcifies small objects in a few days. The specimens are then washed several times in 70$ alcohol to remove as much as possible of the acid. 95% alcohol, with the addition of a little precipitated calcium carbonate, has been recommended for washing sections that have been treated by Thoma' s method. After from eight to fourteen days the specimens are again washed with clear 95% alcohol.

(c) The process of decalcification recommended by v. Ebner (75) is of considerable value, as it also reveals the fibrillar structure of the bone lamellae. A cold saturated solution of sodium chlorid is diluted with 2 vols. of water, and 2^> of hydrochloric acid added. This fluid decalcifies very slowly, and must either be changed daily or a small quantity of hydrochloric acid occasionally added. As soon as the specimen is thoroughly decalcified, it is washed with a half-saturated solution of sodium chlorid. A little ammonia is now added from time to time until the reaction of the fluid and bone is neutral.

(*/) Very small pieces that contain very little lime-salts, as, for instance, bones in an e.mbryonal condition where calcification has only just begun, can be deprived of their lime-salts by means of acid fixing solutions like Flemming's fluid, chromic acid, picric acid, etc.

(e) Bone should be first fixed in some one of the fixing fluids and then decalcified.

Schmorl's Method for Demonstrating the Bone Corpuscles and their Processes in Decalcified Preparations. The tissues are fixed in Miiller's fluid or in Miiller's fluid with formalin, decalcified in V. Ebner's fluid, and imbedded in celloidin. The sections are stained in either of the following thionin solutions: concentrated 50% alcoholic thionin solution, 10 c.c.; i per cent, carbolic acid water, 90 c.c.; or concentrated 50% alcoholic thionin solution, 10 c.c.; distilled water, 100 c.c.; liquor ammonite, 10 drops. Bring sections from water into trie stain, in which they remain from five to ten minutes or longer. Rinse sections in water, and place them in a saturated aqueous solution of picric acid for one to two minutes or longer. Rinse in water and wash in 70% alcohol until no more stain is given off. Dehydrate in alcohol, clear in xylol, and mount in balsam. The bone corpuscles and processes are stained brownish-black, the ground substance yellow, the cells redviolet.

Schmorl' s method for staining the boundary -sheaths of the bone corpuscle: Harden, decalcify, imbed, and stain as in the preceding method. After staining wash in water for two minutes or longer ; rinse in alcohol for one-half minute, and again rinse in water and place the sections in a saturated aqueous solution of phosphomolybdic os phosphotungstic acid for three minutes or longer ; wash in water which needs to be changed frequently for ten minutes. The sections are now placed for three to five minutes in a 10% aqueous solution of liquor ammoniae, after which they are washed in 90% alcohol, dehydrated, cleared in xylol, and mounted. The boundary-sheaths are stained bluish-black, the bone cells dark blue, and the bone substance light blue.

Fibers of Sharpey. Sections treated by Ranvier's method show the perforating fibers of Sharpey as bright, sharply defined ribbons, appearing as streaks or circles, according to the section made (longitudinal or transverse). If decalcified specimens be first rendered transparent by glacial acetic acid, and then immersed for a minute in a concentrated aqueous solution of indigocarmin, washed with water, and then mounted in glycerin or Canada balsam, the fibers of Sharpey will appear red and the remaining structures blue. Thin sections of bone can be deprived of their organic elements by bringing them for from one-half a minute to a minute into a platinum crucible at a red heat. In such preparations calcified Sharpey's fibers may be seen (Kolliker, 86).

Virchow's bone corpuscles may be isolated in the following manner : Very thin fragments or discs of bone are immersed for some hours in concentrated nitric acid. They are then placed on a slide and covered with a cover-glass ; pressure with a needle upon the latter will isolate the lacunae, and occasionally also their numerous processes, the canaliculi.

C Muscular Tissue

Almost all the muscles of vertebrates have their origin from the middle germinal layer. In the simplest type the protoplasm of the formative cell changes into contractile muscle substance, the cell in the meantime undergoing a change in shape (unstriped muscle-cell). In other cases contractile fibrils are formed which are separated by the remains of the undifferentiated protoplasm (striped muscle-cells). In this case the cells either increase very little in length and possess only a single nucleus (heart muscle), or they grow considerably longer and develop many nuclei (voluntary skeletal and skin muscles).

A peculiarity of muscle-substance is that it contracts in only one direction, while undifferentiated protoplasm contracts in all directions.

J. NONSTRIATED MUSCLE-CELLS.

The smooth, unstriped, or nonstriated muscle-cells belong to involuntary muscle, and are found in the walls of the intestine, trachea, and bronchi, genito- urinary apparatus, blood-vessels, in certain glands, and also in connection with the hair follicles of the skin. The involuntary muscle-cells are spindle-shaped cells, which are 40-200 // long and 3-8 // broad. The longest are found in the pregnant uterus, where they attain a length of 500 p. At the thickened middle portion of the cell is a long rod-like nucleus, typic of this class of cells. Nonstriated muscle-cells are doubly refractive anisotropic. The cell substance is longitudinally striated, the striation being due to relatively coarse fibrils situated in the outer portion of the cell substance (M. Heidenhain, Schaper, Benda). These fibrils have a longitudinal course, and probably run the entire length of the cell ; whether they branch and anastomose must be regarded as an open question. In the interior of the cell substance there are found much finer fibrils, which branch and anastomose. Between the fibrils there is found a homogeneous substance, which we may know as the sarcoplasm, in which granules are often seen, situated at the poles of the nuclei. It is generally stated that nonstriated muscle-cells are united into membranes and bundles by a small amount of intercellular cement substance which may be darkened by silver nitrate. Recent investigations have, however, revealed the fact that nonstriated muscle-cells are encased in delicate connective membranes, which membranes unite to form compartment-like spaces, of fusiform shape, in which the muscle-cells are found. These membranes are not to be regarded as cell-membranes sarcolemma since one membrane serves the sheath for two contiguous


Nucleus.


Protoplasm. -


as



muscle-cells (Schaffer, v. Lenhossek, Henneberg). The existence of such membranes is clearly shown in involuntary muscle tissue subjected to trypsin digestion. In such preparation stained in iron- lac -hematoxylin it may be observed that the membranes are not complete, but are fenestrated, showing a varying number of round or oval openings (Henneberg). The membranes are also clearly shown in tissue fixed in corrosive sublimate and stained in Mallory's differential connective-tissue stain, the membranes showing as delicate blue lines while the muscle-cells are stained of a red or orange color. (See Fig. 92.) According to certain observers (Kultschitzky, Barfurth), nonstriated muscle-cells are thought to be joined by intercellular, protoplasmic bridges. It may, however, be clearly shown that such intercellular bridges are artifacts, due to peripheral vacuolization and to shrinkage of the muscle-cells (Schaffer, v. Lenhossek, Henneberg). What


Fig. 92. Nonstriated muscle from the intestine of a cat. X 3a, Isolated muscle-cell ; b, from cross-section of nonstriated muscle, stained after Mallory's differential connective- tissue stain. Observe the apparent difference in size of the cross-cut cells; four of the cells show ( nuclei ; the black lines separating the cells represent the connective-tissue membranes. c, Crosssections ;of the connective-tissue membranes separating involuntary muscle-cells ; d, an area showing so-called intercellular bridges; they are attached to the connective tissue membranes surrounding the cells (Mallory's differential connectivetissue stain).


136


THE TISSUES.


has been described as intercellular bridges may readily be seen in corrosive sublimate preparation, stained in Mallory's differential connective-tissue stain, especially in portions of the preparation not well fixed. In such preparation the so-called intercellular bridges end at the connective membranes separating cells, to which they are attached but which they do not penetrate. Nonstriated musclecells develop from the mesenchyme. (Exceptions to this statement appear to be found in the nonstriated muscular tissue of the iris [Szili] and in the sweat-glands, where the muscular tissue appears to be developed from ectodermal cells.) The nuclei of the mesenchymal cells elongate and become rod-shaped, with oval ends, while the cells become spindle-shaped, the protoplasm staining somewhat more deeply than that of the surrounding mesenchymal cells. Further details as to the development of nonstriated muscular tissue are lacking.

2. STRIPED MUSCLE-FIBERS.

Soon after the segmentation of the mesoderm begins, certain cells of the mesoblastic somites or myotomes commence the formation of muscle-substance in their interior, a process which is accompanied by increase in the number of nuclei, the formation of a membrane, a lengthening of the cells, and the appearance of fibrils in the peripheral protoplasm of the cells.


^K Free ending.


Nucleus.



Fig- 93- Cross-section of striated muscle-fibers : I, Of man ; 2, of the frog. The relations of the nuclei to the muscle-substance and sarcolemma are clearly visible ; X 670.


Fig. 94. Muscle-fiber from one of the ocular muscles of a rabbit, showing its free end ;

X.I75


Voluntary or striated muscle-cells are large, highly differentiated, polynuclear cells, which may attain a length of 12 cm., with a width of 10 100 fj.. They are consequently known as muscle-fibers. Their free ends are usually pointed ; the ends attached to tendon rounded (Fig. 94).


MUSCULAR TISSUE. 137

Each striated muscle-fiber consists of a delicate membrane, the sarcolemma, a muscle protoplasm, in which are recognized very fine fibrils and a semifluid interfibrillar substance (the sarcoplasm) and the muscle nuclei. The sarcolemma is a very delicate, transparent, and apparently structureless membrane, which resists strong acetic acid, even after boiling for a long time. If we examine in an indifferent fluid fresh muscle-fibers, the contents of which have been broken without rupturing the sarcolemma, we may see this sheath as a fine glistening line. (Fig. 95.)

The fibrils of the muscle-protoplasm constitute the contractile part of the muscle-fiber. They are exceedingly fine and extend the entire length of the muscle-fiber. These fibrils are, however, not of the same composition throughout, but are made up of segments which show different physical properties and stain differently. The structure of the fibrils may be expressed in the form of a diagram (Fig. 96) giving the more recently expressed views of the structure of these fibrils. The fibrils present alternating darker and lighter segments, which taken together give the striation which is so char


Fig. 95. Striated muscle-fiber of frog, showing sarcolemma.

acteristic of striated muscle. The darker segments are slightly longer, are doubly refracting, anisotropic, and in general stain more deeply than do the lighter segments, which are slightly shorter and are singly refracting, isotropic. The darker segments, known as the transverse discs, or Briicker's lines, are indicated in the diagram by the letter Q ; the lighter segments, known as the intermediate discs of Krause, are indicated by the letter j. In the intermediate discs of Krause there is found a dark line, which is doubly refractive, which is known as Krause's membrane (z) (Grundmembran), and which, according to certain observers (M. Heidenhain, J. B. MacCallum), is continuous through the fibril bundles, as will be stated more fully later. This membrane divides disc j into two equal parts. The transverse disc (Q) is likewise divided into equal parts by a narrow, isotropic band, known as the median disc of Hensen, and designated by the letter H. In the median discs of Hensen H there is found a thin membrane, known as the median membrane of M. Heidenhain, and designated as M, which, like the membrane of Krause, is continuous through the fibril bundles, uniting


138


THE TISSUES.


the fibrils (M. Heidenhain). By grouping the unequally refracting substances (or unequally staining substances) a fibril may be divided into successive portions or protoplasmic metameres which may be termed sarcomeres (Schafer) and which are bounded by the membrane of Krause (z). In such a sarcomere or muscle-casket we may recognize, beginning with Krause's membrane, z, an isotropic intermediary disc, j ; an anisotropic, transverse disc, Q, divided by a less refracting Hensen's disc, H, into two equal parts, Hensen's disc showing the median membrane of Heidenhain, M ; again an iso


Fig. 96. Diagram of the structure of the fibrils of a striated muscle-fiber. The light spaces between the fibrils may represent the sarcoplasm.



Fig- 97- Diagrams of the transverse striation in the muscle of an arthropod ; to the right with the objective above, to the left with the objective below its normal focal distance (after Rollett, 85): Q, Transverse disc; h, median disc (Hensen) ; E, terminal disc (Merkel); A 7 , accessory disc (Engelmann) ; J, isotropic substance.


tropic intermedian disc, j, and Krause's membrane, z. Krause's membrane, as above stated, is continuous across the small bundles into which the fibrils are grouped, and is also attached to the sarcolemma (M. Heidenhain, J. B. MacCallum). This is shown to the left in Fig. 96, where the sarcolemma appears festooned, with Krause's membrane attached, thus indicating clearly the sarcomeres. One of the best objects for the study of transverse striation is the muscle of some of the arthropods (beetles). In the striated


MUSCULAR TISSUE.


139


muscle of beetles and other arthropods there is, however, a further division into isotropic and anisotropic substance. Here it will be noticed that the disc j is separated by an anisotropic disc, known as the accessory disc of Engelman, and designated by the letter N, into an isotropic disc j, next to the anisotropic transverse disc Q, and an isotropic disc, known as Merkel's terminal disc, and designated by the letter E, situated next to Krause's membrane (z). (See lower portion of Fig. 96.) The muscle fibrils present a different appearance when focused high than they do when focused low, as may be seen from the diagram given in Fig. 97; those parts which appear light on high focusing appear dark on deep focusing.



Sarcoplasm.


Cohnheim's area.


Sarcolemma.


Sarcoplasm.


Cohnheim's area.



Sarcolemma.


Fig. 98. Transverse section through striated muscle-fibers of a rabbit. I and 3, from a muscle of the lower extremity ; 2, from a lingual muscle ; X 9- I n 2 Cohnheim's fields are distinct; in I, less clearly shown ; in 3, the muscle-fibrils are more evenly distributed.


It has recently been suggested by J. B. MacCallum that Krause's membrane with the primitive fibrils form a continuous network in the muscle-fiber, the meshes of which would be fairly regular, the fibrils of such a network which run parallel to the long axis of the muscle-fiber being larger than the cross fibrils. Such a network is not to be confused with a network which may be brought out on staining striated muscle-fibers with gold chlorid, which network is due, in part at least, to a staining of the sarcoplasm.

The ultimate fibrils are grouped into small bundles (0.3-0. 5 // in


140


THE TISSUES.


diameter), forming tint fibril bundles or muscle-cohimns of Kolliker. In the muscle-columns the fibrils are so placed that the larger segments fall respectively in the same plane. (See Fig. 96.) The same disposition of the fibrils prevails in all the numerous muscle-columns forming a muscle-fiber, and all the muscle-columns bear such a relation to each other that the larger segments of the fibrils fall in the same plane. The semifluid, interfibrillar substance, the sarcoplasm, penetrates between the fibrils of the muscle-columns and separates these from each other and from the sarcolemma. In fresh preparations the substance forming the fibrils appears somewhat darker and dimmer, while the sarcoplasm appears clearer. The sarcoplasm is found in greater abundance between the muscle-columns than between the fibrils in the columns. The sarcoplasm between the muscle-columns appears in the form of narrower or broader lines, parallel to the long axis of the muscle-fibers, giving the cross -striated muscle-fiber also a longitudinal striation. The sarcoplasm between the musclecolumns is seen to best advantage in cross-sections of the musclefiber. Here it appears in the form of a network inclosing the muscle-columns. Thus, we have in a cross-section slightly darker areas, the cross-sections of the muscle-columns, known as Cohnheim's fields or areas, separated by the network of sarcoplasm. (Fig. 98.)



Fig. 99. From a striated muscle of man ; obtained by teasing ; X ' 2O - ^> -Amedian disc lying in the transverse disc Q; z, the membrane of Krause borders above and below on the light isotropic discs.


Fig. 100. From a cross-section through the trapezius muscle of man, showing dark fibers rich in protoplasm, and light fibers containing very little protoplasm (after Schaffer, 93, II) : d, Dark fibers ; a, light fibers ; b and c, transitional fibers from light to dark.


In figure 99 is shown a portion of a striated muscle-fiber of man very highly magnified. The larger and darker transverse disc (0 formed by the larger segments of fibrils is divided by a light line (H), Hensen's median disc; the clearer band, largely isotropic substance, is divided by a dark line, the membrane of Krause, z.

After a prolonged treatment with 98 % alcohol the muscle-fibers


MUSCULAR TISSUE.


141


of the water-beetle (Hydrophilns piceus] can be made to separate into transverse discs (Rollet, 85). One of these discs would correspond to the segment Q, and it is very probable that this is the portion which has long been known under the name of Bowman's disc. Other reagents, as weak chromic acid, cause a separation of the muscle-substance into longitudinal fibrils. In this case the discs Q are split up longitudinally into a number of very small columns which were at one time regarded as the primary elements of the fiber and termed by Bowman sarcoiis elements.

In adult skeletal and skin muscle-fibers of mammalia the positions of the nuclei vary. There are muscles in which the nuclei are imbedded in the sarcoplasm between the muscle-columns (so-called red muscles, as the semitendinosus of the rabbit) ; in other muscles they lie immediately beneath the sarcolemma (white muscles, as the semimembranosus of the rabbit ; Ranvier, 89). In the striated muscle-fibers of the lower vertebrates and of mammalian embryos the nuclei lie between the fibrillae, or muscle-columns. The red musclefibers are rich in sarcoplasm, and the fibrils are grouped in wellmarked and large muscle-columns surrounded by sarcoplasm which often contains granules of various sizes, the interstitial granules of Kolliker, often especially abundant at the poles of the nuclei. The white muscle-fibers have a relatively small quantity of sarcoplasm. In cross-sections of the light fibers the fibrils show as fine points, not distinctly grouped, and surrounded by the homogeneous sarcoplasm. Both varieties occur in almost every human muscle, and the relative number of each varies greatly in the different muscles (Schaffer, 93, II, Fig. 100).

Muscles with transversely striated fibers are, with the exception of those of the heart, subject to the will of the individual, and are characterized by a rapid contraction in which the anisotropic substance increases in size at the cost of the isotropic discs ; the former appears to play the chief role. Besides morphologic differences, the red and white muscle-fibers appear to possess differences of a physiologic character, in that the contraction in the red



Fig. 101. Branched, striated muscle-fiber from the tongue ot a frog.

variety is slower than that in the white (Ranvier, 80). Only the striated muscles of the esophagus, the external cremaster, and a few others, as well as the somewhat differently constructed muscles of the heart, are involuntary.


142


THE TISSUES.



p&. c



Fig. 102. Cross-section of rectus abdominis of child, as seen under low magnification.


Muscle.



i t > - //./ i'i j PUflrw

fHHiii

, ti&Jr!'*. r<<i ('( <f< (


Tendon.


V V,l V ' ^ ' U ' ( ( I >),

Stil'ii'i^/f^^i


Fig. 103, Part of a longitudinal section through the line of junction between muscle and tendon; X 1 5- At the line where the tendon-fibrils join the sarcolemma (a), the nuclei of the muscle are very numerous. Sublimate preparation.


MUSCULAR TISSUE. 143

Transversely striated muscle-fibers are usually unbranched. The muscle-fibers of the tongue and of the ocular muscles do, however, show occasionally communicating branches ; the same are but very rarely seen in other muscles. In regions where striated muscle-fibers terminate under the epithelium, as in the tongue and in the skin of the face, the end of the fiber terminating under the epithelium is often very much branched ; the cross-striation and nuclei may be observed in the finest branches. (Fig. 101.)

Each muscle-fiber is surrounded by a thin connective-tissue envelope, the endomysium, which binds them into primary and secondary bundles, the muscle-fasciculi. These are surrounded by a denser sheath of similar character, the perimysiuui. The muscle is made up of numerous fasciculi, all bound together by a thicker connective-tissue covering, the epimysium. (Fig 102.)

Blood-vessels are very numerous in transversely striated mus



Contractile

substance.


Nucleus.


Fig. 104. Fig. 105.

Longitudinal and cross-section of muscle-fibers from the human myocardium, hardened in alcohol ; X 640. The muscle-cells in the longitudinal section are not sharply defined from each other, and appear as polynuclear fibers blending with each other. Between them lie, here and there, connective-tissue nuclei.

cular tissue. One or several arteries enter each muscle and form superficial and deeper plexuses by, anastomosis. In these plexuses the arteries are accompanied by veins. On reaching the perimysium the arteries give off terminal branches which run transversely over the muscle fasciculi, at quite regular intervals. From these branches precapillaries and capillaries are given off which have a course which is in general parallel to the muscle-fibers ; these capillaries anastomose frequently and collect to form small veins, which are situated between the terminal arterial branches, the terminal arterial and venous branches thus alternating in such a way that one venous branch is situated between two arterial branches or vice versa. The veins, even the smallest, are provided with valves (Spalteholz).


144 THE TISSUES.

At its junction with tendon the muscle-fiber with its sarcolemma is rounded off into a blunt point, the fibrils of the tendon being cemented to the sarcolemma.

The longitudinal growth of muscle-fibers takes place principally at the distal ends of the fibers, at which point their nuclei are numerous. (Fig. 103, at .) Schaffer (93, II) has recently suggested that there is a formative tissue between the tendon and muscle-substance, from which, on the one hand, muscle-fibers are developed, and, OR the other hand, connective-tissue fibrils and cells are formed.

As recent investigations have shown, the development of muscle continues throughout the life of the individual. Muscular tissue is consequently to be regarded as in a perpetual stage of transition, the destruction and compensatory reproduction of its elements going on hand in hand. Its destruction is ushered in by a process which can be compared to a physiologic contraction. Nodes or thickened rings are formed, and at these points the muscle-substance separates into fragments with or without nuclei (sarcolytes), which are then absorbed, in most cases without phagocytic aid. This loss of substance is replaced by new elements developed from the free sarcoplasm, which is characterized by rapid growth and increase in the number of its nuclei. The result is that new elements are formed which have been called myoblasts. The process by which myoblasts are changed into the finished muscle-fibers is exemplified in the embryonal type of development of the tissue.

Development of Voluntary Muscle-fibers. The striated, voluntary muscular tissue, as above stated, develops from the myotomes, segmentally arranged differentiated portions of the mesoderm. In the myotomes are developed round or oval cells known as myoblasts, which proliferate by mitotic cell division. According to the observations of certain observers, the myoblasts elongate and become spindle-shaped, while the nuclei proliferate, without an accompanying division of the cell body, to form the muscle-fibers, which may thus be regarded as polynuclear cells developing from a single cell. Other observers, notably Godlewsky, state that only relatively few of the muscle-fibers develop in this way, the majority being formed by a fusion of myoblasts, forming a syncytium, a muscle-fiber being thus a syncytial structure developed from a varying number of myoblasts.

The contractile fibrils are differentiated from the protoplasm of the differentiating myoblasts. When first seen, they present a uniform structure, and only later can a differentiation into isotropic and anisotropic substance be recognized. The discs Q and j appear first ; the other parts of the sarcomeres somewhat later. The first formed fibrils divide longitudinally to give rise to new fibrils. Embryonic striated muscle tissue, even after striation of the fibers may be observed, forms a very compact tissue with only narrow interspaces between the cells. In the further development of this tissue certain of the embryonic muscle-fibers undergo degeneration


MUSCULAR TISSUE. 145

(Bardeen, Godlewski), and mesenchymal tissue, blood-vessels, and nerve-fibers make their appearance between the developing musclefibers.

CARDIAC MUSCLE.

Cardiac muscle or heart muscle is striated muscle, but differs physiologically and structurally from voluntary striated muscle. It resembles involuntary muscle in that it is not subject to the will. Heart muscle after fixation with many reagents used in the laboratories, and when treated with macerating fluids, or subjected to the action of silver nitrate, appears to consist of irregularly shaped oblong cells, cemented end-to-end to form heart muscle-fibers; such fibers appear to anastomose by means of side processes possessed by the cells. A number of recent investigators, notably v. Ebner and M. Heidenhain, have, however, shown that what has been regarded as cement lines uniting cells are to be otherwise interpreted, since they are known to bound nonnucleated areas of heart muscle, and since the contractile fibrils possessed by heart muscle pass through such lines without interruption. It would appear, therefore, that heart muscle must be regarded as a syncytium in which no distinct and separate cells occur, but rather of a complex plexus of branching and anastomosing fibers which differ in size and shape. Heart muscle-fibers consist, as was shown for voluntary striated muscle-fibers, of contractile, primitive fibrils, which are grouped into fibril bundles or muscle columns, between which there is found undifferentiated protoplasm, the sarcoplasm. They are surrounded by a sarcolemma, which differs, however, from the sarcolemma of voluntary muscle-fibers in not being so well developed. The primitive fibrils present the same structure as described for similar fibrils of voluntary muscle, each sarcomere consisting of Krause's membrane, z; two intermediary discs, j; the transverse disc, Q, bisected by Hensen's median disc, H, which in turn contains the median membrane of Heidenhain, M. (See Fig. 96.) Krause's membranes (z) and the median membranes (M) extend across the fibril bundles ; the former are attached to the sarcolemma (M. Heidenhain). The primitive fibrils are grouped into fibril bundles or muscle columns, which in cross-sections are often band-shaped and are placed radially with reference to the center of the heart muscle-fibers. The sarcoplasm is present in relatively larger quantity than in voluntary striated muscle, especially between the fibril bundles, giving the fibers a distinct longitudinal striation. The primitive fibrils pass uninterruptedly through the anastomoses between the fibers. The nuclei, which are round or oval and possess a distinct chromatin network, are situated near the center of the fibers, occurring at irregular intervals, and are surrounded by an axial core of undifferentiated protoplasm, in which are found granules which stain in basic stains, also fat droplets, and, especially in older individuals, pigment granules. The structures which have been regarded as intercellular


146


THE TISSUES.


cement lines may be especially stained in certain anilin stains. In such preparations it may be seen that they often do not extend through an entire fiber, are frequently irregular, often presenting the appearance of steps, and now and then involve only one or two fibril 'bundles. They are frequently seen to bound portions of a muscle-fiber which are nonnucleated. They are looked upon by M. Heidenhain as representing growth areas. See Fig. 106, in which such intercalated growth areas (cement lines ?) are represented darker than the remaining structures.

Heart muscle-fibers are surrounded by delicate connective-tissue sheaths, very much as described for nonstriated muscle tissue. These are well shown in tissue fixed in corrosive sublimate and stained after Mallory's differential connective-tissue stain. The



f& l^ s


b




Fig. 106. Longitudinal section of heart-muscle of a grown individual, fixed in corrosive sublimate and stained in hematein : a, Intercalated disc (so-called cement line); b, nucleus of heart muscle-fiber ; c, red blood-corpuscles ; d, nucleus of blood capillary.

fibers are grouped into bundles or fasciculi which are surrounded by internal perimysium.

Development of Heart Muscle-tissue. Heart muscle-tissue develops from the mesenchyme, and shows from the beginning a syncytial structure, in that the cells are united by protoplasmic branches (von Ebner, M. Heidenhain, Godlewsky). As development proceeds, the interspaces between the cells become smaller and the protoplasmic bridges larger and more prominent, forming a distinct syncytium, through which the nuclei are scattered. In this syncytial protoplasm are developed the contractile fibrils, which may be traced uninterruptedly for long distances. These fibrils show at first a uniform structure, and later differentiate into isotropic and anisotropic discs, Q and j discs appearing first as in voluntary striated


MUSCULAR TISSUE. 147

muscle, and later the other parts of the sarcomeres (Godlewsky). It may be stated that, according to J. B. MacCallum (and other observers), the heart-muscle develops from spindle-shaped cells lying close together in the protoplasm of which there is found a fairly regular network. As development proceeds, fibrils or fibril bundles which run parallel to the long axis of the cells make their appearance at the nodal points of this network.

The muscle-cells of the so-called fibers of Purkinje lie immediately beneath the endocardium, and are remarkable in that their protoplasm is only partially formed of transversely striated substance, and that only at their periphery. Such cells are found in great numbers in some animals (sheep), but rarely in man. Heart muscle has a rich blood supply, which will be considered more fully when the heart is discussed as an organ.

For the nerve-endings in smooth and striated muscle-fibers see the chapter on Nervous Tissues.

TECHNIC.

Fresh, striated muscle-fibers may be isolated by teasing them in an indifferent fluid. After a short time the sarcolemma may separate as a very fine membrane. If a freshly teased muscle be placed in a cold saturated solution of ammonium carbonate, the sarcolemma will become detached in places within five minutes (Solger, 89, III).

Striated muscle-fibers may be examined in an extended condition by placing an extremity in such a position as to stretch certain groups of muscles. A subcutaneous injection of 0.25-0.5 c.c. of a i$> osmic acid solution is then made. The acid penetrates between the fibers and fixes them. Pieces of muscle are then cut out and washed in distilled water. Teased fibers, even if not stained, will show the striation plainly if mounted in glycerin. Muscles thrown into a state of tetanic contraction by electric stimulation may also be fixed in this state and later examined.

Cross-sections of muscles, extended and fixed in osmic acid, also show the relation of the fibrils to the sarcoplasm (Cohnheim's fields). A remarkable quantity of sarcoplasm in proportion to the number of fibrils is seen, for instance, in the muscles which move the dorsal fin of hippocampus ; among the mammalia a similar condition is found in the pectoral muscles of the bat (Rollett, 89).

In the muscles of all adult vertebrates (except the mammalia) the nuclei lie between the fibrils. In young mammalia they also have this position, but in the adult animals only the nuclei of red muscles are found between the fibrillse ; in all other muscles the nuclei are under the sarcolemma.

The fibrillar structure of muscle-fibers can be seen by teasing old alcoholic preparations, or tissue treated with weak chromic acid (0.1%) or one of its salts.

In alcoholic preparations of mammalian muscle, the crossstriation is clearly seen, and is intensified by staining with hematoxylin. This stain colors everything anisotropic in the muscle, but does not affect the remaining structures. Similar results may be obtained with other


148 THE TISSUES.

stains, such as basic anilin dyes, but not with the same precision as with hematoxylin.

A certain species of beetle (Hydrophilus'} is admirably adapted for the study of the finer details of striation. The beetle is first wiped dry and then immersed alive in 93% alcohol. On examining in dilute glycerin after from twenty-four to forty-eight hours, the substance of its muscles will show disintegration into Bowman's discs (vid. p. 141). The latter swell up in acids and are finally dissolved, as may be seen, by adding a drop of formic acid to a specimen prepared as above (Rollet, 85).

In order to study the relation of muscle to tendon, small muscles with their tendons are put into a 35% potassium hydrate solution for a quarter of an hour, after which the specimen is placed upon a slide and teased at the line of junction of the two tissues. This will separate the muscle-fibers from their respective tendon-fibrils (Weismann).

Similar results may be obtained by immersing a frog in water at a temperature of 55 C., in which the animal soon dies with muscles perfectly rigid. As soon as the water begins to cool (one-quarter hour) the frog is removed and a small piece of its muscle cut out and teased in water on a slide (Ranvier).

Cardiac muscle-cells are isolated by maceration for twenty-four hours in a 20% solution of fuming nitric acid (potassium hydrate with a specific gravity of 1.3 will do the same in one-half or one hour). The margins of the cells may be brought more clearly into view by placing pieces of heart muscle for twenty-four hours in a 0.5% aqueous solution of silver nitrate and then cutting into sections.

Isolated fibers of Purkinje are obtained by immersing pieces of endocardium (0.5 mm. in size) in 33% alcohol and then teasing them on a slide. The sheep's heart is especially well adapted for this purpose.

Nonstriated muscle-fibers are isolated in the same way as heart muscle. In thin cross -sections (under 5 p. in thickness) of intestinal muscle, preferably of a cat, fixed in osmic acid, the intercellular bridges may be seen here and there between the fibers.


D. The Nervous Tissues

The entire nervous system, peripheral as well as central, is composed of cells possessing one or many processes. These cells develop early in embryonic life from certain ectodermal cells (neuroblasts] of the neural canal, which is formed by a dorsal invagination of the ectoderm. The neuroblasts soon develop processes, many of them in loco, others only after wandering from the neural canal.

The processes of the nerve-cells are of two kinds : (i) unbranched processes having a nearly uniform diameter throughout, with lateral offshoots known as collateral branches ; these, as we shall see, generally form the central part of a nerve-fiber, and are known as neuraxes (Deiters 1 processes, axis-cylinder processes, neurites, neuraxones or axones) ; and (2) processes which branch soon after leaving the cell-body and break up into many smaller branches ; these are the dcndrites, dendrons, or protoplasmic branches. In the spinal ganglia and the homologous cranial ganglia these morphologic differences in the processes are not observed, the neuraxis and the dendrites of each presenting essentially the same structure.

To the entire nerve -cell, cell-body and processes the term neurone (Waldeyer, 91) has been applied; ncnra (Rauber), or neurodendron (Kolliker, 93).

The neuraxes of many neurones attain great length. Those of some of the neurones, the cell-bodies of which are situated in the lower part of the spinal cord, extend to the foot. In other regions neuraxes nearly as long are to be found, and in the majority of neurones the neuraxes terminate some distance from the cell-body. It is therefore manifestly impossible in the majority of cases to see a neurone in its entirety. Usually, only a portion of one can be studied in any one preparation. Consequently, the more detailed description which follows will deal with the neurone in this fragmentary manner. The cell-bodies of the neurones, to which the term "nerve-cells" or "ganglion cells" is usually restricted, the dendrites and neuraxes, often forming parts of nerve-fibers, and their mode of terminating, will receive separate consideration.


Nerve-Cells, Or Ganglion Cells ; The Cell-Bodies Of Neurones

The cell-bodies of neurones are usually large. The bodies of the motor neurones of the human spinal cord measure 75 to 150 //, their nuclei 45 //, and their nucleoli 1 5 /u. The smallest nerve-cells, the neurones of the granular layer of the cerebellum, are 4 to 9 fi in diameter. The protoplasm of nerve-cells shows a distinct fibrillar structure and the fibrils may be followed into the processes. (Fig. 107.) Their nuclei are also large, with very little chromatin, but as a rule are supplied with a large nucleolus.

After treatment by certain special methods, the protoplasm of the ganglion cells shows granules or groups of granules which show special affinity to certain stains, consequently known as chromatophile granules ; these are densely grouped around the nucleus, so that the cell-body shows an inner darker and an outer lighter portion. These chromatophile granules, also spoken of as tigroid granules or as the tigroid substance (v. Lenhossek), as a rule are not arranged in concentric layers, but lie mostly in groups, giving to the protoplasm a mottled or reticular appearance. In the cells of the anterior horns (man, ox, rabbit) the granules join to form flakes, which are also more numerous in the region of the nucleus. In all cases the granules or flakes are continued into the dendrites of the cell. Here they change their shape into long pointed rods, with here and there nodules, which are probably the chief causes of the varicosities so often seen in dendrites (Golgi's method). The cell usually has a clear, nongranular peripheral border (not a membrane), and in the case of large cells there is a similar area around the nucleus, the inner border of which belongs to the nuclear membrane. H. Held has found that the chromatophile granules are brought out by treatment with alcohol and acid fixing fluids, but not in alkaline or neutral. They appear, according to the treatment, as fine or coarse granules. They can not be seen in fresh nerve-cells. He consequently regards them as artefacts precipitations of the protoplasm due to reagents (vid. A. Fischer). At its junction with the cell the neuraxis spreads out into a cone which is entirely free from granules, and apparently fitted into a depression in the granular substance of the cell (implantation cone or axone hillock). The shape, number, and size of the tigroid granules vary with the physiologic activity of neurones. They practically disappear from the neurones in certain diseased conditions or after the administration of poisons which affect more particularly nerve-cells ; also after extreme fatigue.

The cellular substance between the chromatophile granules consists also of very fine, highly refractive granules, which appear to be arranged in a reticulum surrounding the chromatophile granules (vid. Nissl, 94, and v. Lenhossek, 95), and the recent observations of Apathy and Bethe make it very probable that in the intergranular substance of the protoplasm of the nerve-cell there exist veiy fine fibrils which may be traced into the processes of the cell, and from the branches of one neurone to and into the branches of other neurones without interruption. It requires, however, further observation before more positive statements may be made concerning them.


Fig. 107. Bipolar ganglion cell from the ganglion acusticum of a teleost (longitudinal section). The medullary sheath of the neuraxis and dendrite is continued over the ganglion cell ; X 800.


Besides the granules above mentioned, and which are revealed by special methods, there are found in the protoplasm of many of the larger nerve-cells pigment granules of a yellow or brown color which stain black with osmic acid.

The dendrites are usually relatively thick at their origin, but gradually, as a result of repeated divisions, taper until their widely distributed arborescent endings appear as minute threads of widely different shapes. When treated by certain methods, they present uneven surfaces studded with varicosities and nodules, in contradistinction to the neuraxes, which are smooth and straight. Their terminal branches end either in points or in small terminal thickenings. The groups of terminal end-branches of a dendrite (also of a neuraxis) are known as telodendria (Rauber), or end-branches. The branches of the dendrites form a dense feltwork, which, together with the cell-bodies of the neurones and with other elements to be described later, constitute the gray substance (gray matter) of the brain and spinal cord.

All neurones, with possibly a few exceptions, possess only a single neuraxis. Neurones without a neuraxis have never been found in vertebrates. The neuraxis usually arises from a coneshaped extension of the cell-body free from chromatophile granules, the implantation cone or axone hillock, more rarely from the base of one of its dendrites, or from a dendrite at some distance from the cellbody. Its most important characteristics are its smooth and regular contour and its uniform diameter. At some distance from the cellbody, usually near its termination, now and then in its course, a neuraxis may divide into two equal parts. Golgi (94) called attention to the fact that the neuraxes of certain neurones (Purkinje's cells in the cerebellum, pyramidal cells of the cerebral cortex, and certain cells of the spinal cord) give off lateral processes, the collateral branches.



Fig. 108. Chromatophile granules of a ganglion cell from the Gasserian ganglion of a teleost : a, Nucleus ; b, implantation


Fig. 109. Nerve-cell from the anterior horn of the spinal cord of an ox, showing coarse chromatophile flakes.


Two types of cell are recognized according to the disposition of their neuraxes : In the first the neuraxis is continued as a nervefiber ; in the second and rarer type it does not long preserve its independence, nor is it continued as a nerve-fiber, but soon breaks up into a complicated arborization, the neuropodia of Kolliker (93). The latter type of cell occurs in the cortex of the cerebrum and cerebellum and in the gray matter of the spinal cord. The cells of the two types can be simply described as having long (type I) or short, branched neuraxes (type II). The neuraxes of the cells of type I possess the collateral branches which end in small branching tufts.

In its simplest form, a neurone consists of a cell-body and a neuraxis with its telodendrion. In more complicated types one or several dendrites may be present, as also collaterals from the neuraxis, and in rare cases even several neuraxes. According to the number of its processes, a ganglion cell is known as unipolar, bipolar, or multipolar.


Fig. HO. Motor neurones from the anterior horn of the spinal cord of a new-born cat.

Chrome-silver method.

Although neurones present a great variety of morphologic differences, large and variously shaped cell-bodies or small ones scarcely larger than the nucleus ; large and numerous dendrites or



Fig. in. A nerve-cell with branched dendrites (Purkinje's cell), from the cerebellar cortex of a rabbit ; chrome-silver method ; X I2 5 few and less conspicuous ones, and although these various forms are widely distributed and intermingled in the different parts of the nervous system, yet in many regions there are found nerve-cells of fixed and characteristic morphologic appearance, which would enable a determination of their source. A few of the most characteristic types are here figured and may receive brief consideration. In the anterior horn of the spinal cord are found large multipolar neurones (motor neurones), with numerous dendrites, which terminate after repeated branching in the neighborhood of the cell-body, while the neuraxis with its collateral branches proceeds from the cell-body and becomes a part of a nerve-fiber. (Fig. 1 10.)

In the cerebellum are found large neurones, discovered by Purkinje, and known as Purkinje's cells, with flask-shaped cell-body, from the lower portion of which arises a neuraxis with collateral branches,



Fig. 112. Pyramidal cell from the cerebral cortex of man ; chrome-silver method : a, b, c, Branches of a dendrite.

from the upper portion one or two very large and typic dendrites the smaller branches of which are beset with irregular granules. (Fig. in.)

In the cortex of the cerebrum occur large neurones, each with a cell-body the shape of a pyramid (pyramidal cell of the cerebral cortex), from the apex of which arises one large dendrite, and from angles at the base, or from the sides of the cell-body, several smaller dendrites. The neuraxis arises from the base directly or from one of the basal dendrites. (Fig. 112.)


In figure 113 is shown a neurone with relatively small cell-body and short dendrites, from the granular layer of the human cerebellum.

The function of the dendrites has given rise to considerable discussion. Golgi and his school regard them as the nutrient roots of the cell, a theory which is opposed by Ramon y Cajal (93, 1 ), van Gehuchten (93, I), and Retzius (92, II). According to the latter, all the processes of the nerve-cell are analogous structures ; they pass out from a sensitive element, and probably have a correspondingly uniform function.

In the spinal ganglia and the homologous cranial ganglia, are grouped the cell-bodies of neurones (peripheral sensory neurones, peripheral centripetal neurones) which differ in many respects from those above described. In the peripheral sensory neurones the neuraxes and dendrites have essentially the same structure, both forming part of a nerve-fiber. From a relatively large, nearly round, oval, or pear-shaped cell-body there arises a single process, which, at a variable distance from the cell-body, divides into two branches forming a right or obtuse angle with the single process (T-shaped or Y-shaped division of Ranvier, 78). Both of these branches form the central axis of a nerve-fiber ; one of the branches passing as a nerve-fiber to the spinal cord or brain, as the case may be ; the other forming a nerve-fiber which passes to the periphery. (Figs. 1 14 and 115.)



Fig. 113. Nerve-cell with dendrites ending in claw-like telodendria ; from the granular layer of the human cerebellum ; chrome-silver method ; X l Io


Fig. 114. Ganglion cell with a process dividing at a (T-shaped process); from a spinal ganglion of the frog ; X 2 3



The ganglion cells of the spinal ganglia and homodynamic structures of the brain are therefore apparently unipolar cells, but, as Ranvier has shown, their processes are subject to a T-shaped or Y-shaped division. The branches going to the periphery are regarded as dendrites, the others as neuraxes. As to the significance to be attached to the single process, the theory of v. Lenhossek



Fig. 115. Ganglion cell from the Gasserian ganglion of a rabbit ; stained in methylene blue (intra vitam).

(94, I) that it represents an elongated portion of the cell, and that therefore the origin of the dendrite and that of the neuraxis are in this case close together, is very plausible. In the embryo these ganglion cells are at first bipolar, a process arising from each end, of a spindle-shaped cell ; as development proceeds, the two processes approach each other and ultimately arise from a drawn-out portion of the cell - body, the single process. (Fig. 116.)

The sympathetic ganglia are composed mainly of the cellbodies and dendrites (also some structures to be mentioned later) of neurones of the sympathetic nervous system. In nearly all vertebrates, and with but few exceptions in any one ganglion, these neurones are multipolar and resemble morphologically the multipolar ganglion cells of the anterior horn of the spinal cord, though they are somewhat smaller. In the cell-body there may be observed fine chromatophile granules and a large nucleus and nucleolus. From the cell-body there proceed a varying number of dendrites which branch and rebranch and terminate, as a rule, near the cellbody, forming plexuses in the ganglia. The neuraxis arises either directly from the cell-body from an implantation cone, or from one of the dendrites at a variable distance from the cell-body. (Fig. 1 17.) In nearly all ganglia a few unipolar or bipolar cells are to be found. In the sympathetic nervous system of amphibia the sympathetic neurones are unipolar ; the single process present is the neuraxis.



Fig. 116 Three ganglion cells from a spinal ganglion of a rabbit embryo. The cells are still bipolar. Their processes come together in later stages, and finally form the T-shaped structure seen in the adult animal ; chrome - silver method ;



A most important result of the more recent investigations on the nervous system is the theory of the independence of the neurone. Each neurone develops from a single cell (neuroblast), and functionates as an independent cell under physiologic and pathologic conditions. Only very rarely has any direct connection between two neighboring neurones been demonstrated, so rarely that the scattered observations at hand do not vitiate the above statement. Recent investigations have, however, shown that, while a neurone is a distinct anatomic unit, it is always found associated with other neurones. Nowhere in the body of a vertebrate does one find a neurone completely disconnected from other neurones. This association of one neurone with one or several other neurones is always effected by a close contiguity existing between the telodendria (end-branches) of the neuraxis of one neurone with the cell-body or dendrites of one or several other neurones. The telodendrion of the neuraxis of one neurone may form a feltwork inclosing the cellbody of one or several neurones, forming structures known as terminal baskets or end-baskets, or the end ramifications of the neuraxis of a neurone may come in very close proximity to the end-branches of the dendrites of one or several neurones. By this contiguity of the telodendria of the neuraxis of one neurone with the cell-bodies or the dendrites of other neurones, they are, without losing their identity, linked into chains, so that a physiologic continuity exists between them. In such neurone chains the dendrites are regarded as cellulipetal, transmitting the stimulus to the cell ; the neuraxes as cellulifugal, transmitting the impulse imparted by the cell to the motor nerve-endings or central organs (Kolliker, 93). The entire nervous system may therefore be said to be made up of such neurone chains, the complexity of which varies greatly according to the number of neurones which enter into their construction. This subject will be considered more fully in a chapter on the nervous system.



Fig. 117. Neurone from inferior cervical sympathetic ganglion of a rabbit ; methylene blue stain.



THE NERVE-FIBERS.

The neuraxes of the cells of type I, and the dendrites of the peripheral sensory neurones (spinal ganglia and homologous cranial ganglia), form the chief elements in all the nerve-fibers. In the nerve-fibers they possess a distinctly fibrillar structure. The fibrils composing them, the axis-fibrils, are imbedded in a semifluid substance, the neuroplasm (Kupffer, 83, II) the whole being surrounded by a very delicate membrane, the axolemma. In the nervefibers, the axis-fibrils and the neuroplasm form axial cords which are surrounded by a special membrane or membranes, the presence or absence of which serves as a basis for a classification of nervefibers. Two kinds are distinguished, medullated and nonmedullated nerve fibers.

In medullated nerve-fibers, the axial cords (neuraxes of cells of type I, and dendrites of spinal ganglion cells) are surrounded by a highly refractive substance very similar to fat, which is blackened

in osmic acid, the so-called medullary or my din sheath. In a fresh condition this sheath is homogeneous, but soon changes and presents segments separated from each other by clear fissures. These segments vary in size and are known as " Schmidt- Lantermann-Kuhnt's segments." On boiling in ether or alcohol the entire medullary sheath of a nerve-fiber does not dissolve, but a portion is left in the shape of a fine network which is not affected by exposure to the action of trypsin. From the latter circumstance it has been thought that this network consists of a substance very similar to horn, and is therefore known as neurokeratin (horn-sheath, Ewald and Kiihne). On burning isolated neurokeratin, an odor exactly like that of burning horn is given off. It is thought that the meshes of this neurokeratin network contain the highly refractive substance similar to fat, composing the greater portion of the medullary sheath. The medullary sheath is interrupted at intervals of from 80 to 900 //, the constrictions thus formed being known as the nodes of Ranvier. The smaller the fiber, the less the distance between the nodes. In a fiber with a diameter of 2 p. the internodal segments are usually about 90 p in length.



Fig. 118. Longitudinal section through a nerve-fiber from the sciatic nerve of a frog; X830.



In peripheral nerves the medullary sheath is in its turn surrounded by a clear, structureless membrane, the neurilemma or sheath of Schwann. Nerve-fibers contain here and there relatively long, oval nuclei (neurilemma-nuclei) which are surrounded by a small quantity of protoplasm, and are situated in small excavations between the neurilemma and the medullary sheath. In the higher vertebrates a single nucleus is found midway between each two nodes ; in the lower vertebrates (fishes) several scattered nuclei (5-16) may be found in each internodal segment. At the nodes, where the medullary sheath is interrupted, the neurilemma is thickened and contracted down 'to the axial cord (contraction-ring).



Fig. 119. Transverse section through the sciatic nerve of a frog ; X 820. At a and b is a diagonal fissure between two Lantermann's segments ; as a result, the medullary sheath here appears double. (Compare Fig. 118.)


Just beneath the contraction-ring, Ranvier found that the axiscylinder presents a slight, biconic swelling (retirement bicbnique). Thus the sheath of Schwann represents a continuous tube throughout the length of the fiber in contrast to the medullary sheath. In the nerve-fibers of the spinal cord and brain there is no neurilemma, although the medullary sheath is present.

In the fresh nerve-fiber the axial cord fills the space (axial space) within the medullary sheath, and appears transparent. After treatment with many fixing fluids the neuroplasm coagulates and shrinks, no longer filling the entire axial space, but appears in the latter as a wavy cord composed of an apparently homogeneous mass, the fibrillae of which are no longer recognizable. Such pictures, which formerly were supposed to represent the normal condition of the nerve-fibers, gave rise to the conception of an axis-cylinder (vid. Technic). That which is known as an axis -cylinder is therefore, in reality, the changed contents of the axial space. It may be stated, however, that the term axis-cylinder is still much used, since the methods commonly employed in the investigation of the

nervous system do not preserve the axial cord in its integrity, but nearly always result in the formation of an axis-cylinder. Consequently, although we shall make use of the term, its limitations are to be kept in mind.

Medullated nerve fibers vary greatly in diameter, but whether this points to a corresponding variation in function has not been fully decided. Fine fibers possess a diameter of 2 4//, those of medium size 49 //, and large fibers 920 fj. (Kolliker, 93). A division of medullated fibers during their course through a nerve is relatively rare. The greater number of fibers pass unbranched from their central origin to the periphery, and only when in the neighborhood of their terminal arborization do they begin to divide. A point of division is always marked by a node of Ranvier.




Fig. I2O. Medullated nerve-fibers from a rabbit, varying in thickness and showing internodal segments of different lengths. In the fiber at the left the neurilemma has become slightly separated from the underlying structures in the region of the nucleus ; X I 4


Fig. 121. Remak's fibers (nonmedullated fibers) from the pneumogastric nerve of a rabbit ;


The segmental structure of nerve-fibers would seem to give the impression that they are formed by a number of cells fused end to end. After what has been said with regard to ganglion cells and their processes, this can be the case only so far as the nerve-sheaths are concerned. According to this theory, the formative cells of the latter gather in chains along the neuraxes or dendrites, forming a mantle around them, and in the adult nerve-fibers taking the shape of the segments or internodes just described (His, 87 ; Boveri, 85). The points at which the sheath-cells are joined would then correspond to the nodes of Ranvier. Other investigators have concluded that the whole nerve-fiber is developed from a terminal apposition of ectodermal cells. In this case not only the sheaths of the fibers but also the corresponding portions of the nerve processes are formed by them (Kupffer, 90). In both theories the neurilemma corresponds to the cell-membrane ; in the former the neurilemma nucleus corresponds to that of the sheath-forming cell, in the latter to that of the formative cell of the whole nerve segment. It should be noticed that, according to the second theory, a fiber segment is the product of a single cell, while according to the first it is evolved from at least two cells (ganglion cell (process) and sheath-forming cell). The former theory is now very generally accepted.

The nonmedullated nerve-fibers, Remak 's fibers, possess no medullary sheath ; the axial cord shows nuclei which can be regarded as belonging to a thin neurilemma. The majority of the neuraxes of the neurones of the sympathetic nervous system are of this structure, although small medullated nerve-fibers (the neuraxes of sympathetic neurones) are found in certain regions.

All nerve-fibers, medullated as well as nonmedullated, in the central and peripheral nervous systems lose the sheaths here described before terminating ; the axis-cylinders (axial cords) ending without special covering (naked axis-cylinders). These terminal branches are, in fixed and stained preparations, beset with small thickenings varicosities which vary greatly in size and shape. Nerve-fibers presenting such appearances are spoken of as varicosed fibers. The varicose enlargements may be regarded as small masses of neuroplasm ; the fine uniting threads, as representing the axial fibrils.

In the peripheral nervous system the nerve-fibers are grouped to form nerve-trunks. The nerve-fibers, as has been stated and as will be seen from the diagram (Fig. 122) on the next page, are the neuraxes of neurones, the cell-bodies of which are situated in the spinal cord or brain and in the sympathetic ganglia, and the dendrites of peripheral sensory neurones, the cell-bodies of which are found in the spinal and homologous cranial ganglia.

In the nerve-trunks the nerve-fibers are gathered into bundles termed funiculi. The nerve-fibers constituting such a bundle are separated by a small amount of fibro-elastic tissue, containing here and there connective-tissue cells, the endoneurium. This is continuous with a dense, lamellated fibrous sheath surrounding each funiculus, the perineurium. Between the lamellae of this sheath are lymphspaces, communicating with the lymph-clefts found between the nerve-fibers of the funiculi ; consequently, the lamellae are covered by a layer of endothelial cells. In the larger funiculi, septa of fibrous connective tissue pass from the perineurial sheath into the funiculi, dividing them into compartments varying in shape and size ; these are spoken of as compound funiculi. The funiculi of a nervetrunk are bound together by an investing sheath of loose fibro-elastic tissue, continuous with the perineurial sheaths, which penetrates between the funiculi, and which contains fat-cells, blood-vessels, and lymph-vessels ; the latter are in communication with the lymphspaces of the perineurial sheaths.


Fig. 122. Diagram to show the composition of a peripheral nerve-trunk.



Fig. 123. Part of a cross-section through a peripheral nerve treated with alcohol. The small circles represent the cross-sections of medullated nerve-fibers ; the axis-cylinders show as points in their centers. The nerve is separated by connective tissue into large and small bundles funiculi ; X 75II


When a nerve-trunk divides, the connective-tissue sheaths above mentioned are continued on to the branches, and this even to the smallest offshoots. Thus, single fibers even possess a connectivetissue sheath, Henle's sheath, which consists of a few connectivetissue fibers and of flattened cells.


PERIPHERAL NERVE TERMINATIONS.

According to the character of the peripheral organs in which the telodendria of nerve-fibers (neuraxes of type I cells and dendrites of spinal ganglion cells) occur, the nerve-fibers are known as motor and sensory nerve-fibers, the terminations as motor and sensory nerve-endings.

Motor Nerve-endings (the Telodendria of Nerve-fibers Ending in Muscle Tissue). The motor nerve -endings in striated, voluntary muscle tissue will first be considered. The motor nerve-endings in voluntary muscle tissue are the endings of neurones (peripheral motor neurones), the cell-bodies of which are situated in the ventral horns of the spinal cord and in the medulla. The neuraxes of these cells leave the cerebrospinal axis as medullated nerve-fibers (motor fibers) which, after branching, end in the muscle-fibers in the so-called motor endings. In figure 124 is represented, by way of diagram, a complete peripheral motor neurone. Each motor nerve - fiber branches repeatedly before terminating, although this branching does not often take place until near the termination of the nervefiber. Kolliker estimates that in the sternoradialis of the frog, each motor fiber innervates about twenty muscle-fibers ; but whether this number may be regarded as the average number of muscle-fibers receiving their motor nerve-supply from one motor neurone can not be stated with any degree of certainty at the present time.

Each motor ending represents the termination of one of the terminal medullated branches of a motor nerve-fiber. The neuraxis of this fiber passes under the sarcolemma and terminates in a telodendrion (end-brush) in an accumulation of sarcoplasm, in which are found numerous muscle nuclei, forming a more or less distinct elevation on the side of the muscle-fiber, Doyere's elevation. The medullary sheath accompanies the nerve-fiber until it passes under the sarcolemma, when it stops abruptly. The neurilemma of the nerve-fiber becomes continuous with the sarcolemma of the musclefiber at the place where the neuraxis passes under the sarcolemma. Henle's sheath continues over the motor ending as a thin sheath, containing here and there flattened nuclei, the telolemma nuclei.

With the majority of the reagents used to bring to view the motor endings, notably chlorid of gold, the sarcoplasm, in which


Fig. 124. Diagram of peripheral motor neurone.


the telodendrion of the nerve-fiber is found, has a granular appearance, and is consequently differentiated from the remaining sarcoplasm of the muscle-fiber. To this the term granular sole plate has been applied, the nuclei contained therein being known as sole nuclei, the whole ending as the motor end-plate. If the above interpretation of the structure of the motor nerve-ending is correct, there would seem to be no reason why the sarcoplasm in which the telodendria occur should be considered other than the sarcoplasm of the muscle-fiber, the nuclei as muscle-nuclei ; the terms motor endplate, granular sole plate, and sole nuclei would therefore seem unnecessary and misleading. It may be stated in this connection that Bardeen has recently shown that in teased muscle-tissue subjected to trypsin digestion the muscle substance may be removed from the fiber leaving the sarcolemma and on its inner surface a portion of the nerve -ending, with the neurolemma continuous with the sarcolemma. He has also shown that the motor ending is in part differentiated in connection with developing muscle-fibers before a sarcolemma can be shown on such fibers. In figures 126 to 130 are shown motor nerve-endings from several vertebrates as seen when stained with gold chlorid.

The mass of sarcoplasm in which the neu raxes terminate as above described is about 40 to 60 p long, 40 /abroad, and 6 to 10 /* thick ; these dimensions vary greatly, however ; they may be greater or less than the averages here given.

In amphibia the motor nerve-endings are not so localized as in the majority of vertebrates, as above described, but are spread over a relatively greater surface of the muscle-fiber, and there is no distinct accumulation of the sarcoplasm, and the muscle-nuclei are



Fig. 125. Motor nerve-ending in voluntary muscle of rabbit, stained in methyleneblue (intra vitam) (Huber, DeWitt, "Jour. Comp. Neurol.," vol. vn) : A, Surface view ; B, longitudinal section through motor ending ; C, cross-section : a, a, a, neuraxes of nerve-fibers ; s,s,s, sarcolemma ; /,/, neurilemma ; d, Doyere's elevation; mn, muscle nuclei ; tn, telolemma nucleus.

relatively less numerous. The telodendrion of the nerve-fiber is, however, under the sarcolemma, between it and the contractile substance of the muscle-fiber. (Fig. 131.)

Usually only one motor ending is found on each striated musclefiber. This may be situated near the center of the muscle-fiber or at a variable distance from the center, nearer one or the other of its extremities. Now and then two nerve-endings are found on one muscle-fiber, in which case the nerve-endings are found in close proximity.





Figs. 126-130. Motor endings in striated voluntary muscles.

Fig. 1 26, from Pseudopus Pallasii; X l6- Fig. 127, from Lncerta viridis ; X 160. Figs. 128 and 129, from a guinea-pig; X 7- Fig. 130, from a hedge-hog; X 1200. As a consequence of the treatment the arborescence is shrunken and interrupted in its continuity. In Figs. 126 and 127 the end plate is considerably larger than in 128 and 129. In Fig. 126 it is in connection with two nerve-branches. Fig. 130 shows a section through an end-plate. The latter is bounded externally by a sharply denned line, which can be traced along the surface of the muscle-fiber. This is to be regarded as the sarcolemma.


Heart muscle and nominated muscle receive their motor nervesupply from neurones of the sympathetic nervous system. The cell-bodies of these neurones are situated in sympathetic ganglia ; the neuraxes, the majority of which form nonmedullated nervefibers, branch repeatedly, forming primary and secondary plexuses which surround the larger or smaller bundles of heart muscle-fibers or involuntary muscle-cells. From these plexuses, naked, varicosed axis-cylinders, or small bundles of such, penetrate between the heart muscle-fibers or involuntary muscle-cells, also forming



Fig. 131. Motor nerve-ending in striated voluntary muscle of a frog ; methyleneblue stain (intra vitani) (Huber, DeWitt) : A, Surface view ; , cross- section ; s, s, sarcolemma ; /, neurilemma.



Fig. 132. Motor nerve-ending on heart muscle-cells of cat ; methylene-blue stain (Huber, De Witt).


Fig. 133. Motor nerve-ending on involuntary nonstriated muscle-cell from intestine of cat ; methylene-blue stain (Huber, De Witt).


plexuses. The fine fibers of this terminal plexus give off from place to place small, lateral twigs, which end on the muscle-fiber and muscle-cells. In heart muscle these lateral twigs may end in one or two small granules, or in a small cluster of such granules (Fig. 132); in involuntary, nonstriated muscle the ending is very simple, the small lateral twigs terminating in one or two small granules. (Fig. 133.)

Sensory Nerve-endings. The sensory nerve-endings are, in their essentials, the peripheral telodendria of dendrites of peripheral sensory neurones. The cell-bodies of such neurones, as has been stated, are found in the spinal and homologous cranial ganglia. Of the two branches arising from the single process possessed by each peripheral sensory neurone, the one going to the periphery is regarded as the dendrite and forms the axis-cylinder of a medullated nerve-fiber, such nerve-fibers constituting the sensory nerves of the peripheral nerve-trunks. A peripheral sensory neurone may therefore be diagramed as in figure 134. The statement was made above that the essential portion of a sensory nerve-ending is a telodendrion (end-brush) or several telodendria of the dendrite of a peripheral sensory neurone. The character of a sensory nerve ending depends, therefore, on the complexity of this end-brush and on its relation to the other tissue elements which take part in the formation of the sensory nerve-endings. Bearing this in mind, the




Fig. 134. Diagram of a peripheral sensory neurone.



Fig- 135- Termination of sensory nerve-fibers in the mucosa and epithelium of the urethra of cat; methylene-blue preparation (Huber, "Jour. Comp. Neurol.," vol. x).

following classification of such nerve-endings can be made :

1. Free Sensory Nerve-endings. In these the telodendrion is not inclosed in an investing capsule which forms a structural part of the ending.

2. Encapsulated Endings. In which the telodendrion or several telodendria are surrounded by an investing capsule which separates them more or less completely from the surrounding tissue.


1 . Free sensory nerve=endings are found in all epithelial tissues and in fibrous connective tissue of certain regions. A sensory nerve-fiber terminating in such an ending usually proceeds without branching to near its place of termination, where, while yet a medullated fiber, it branches and rebranches a number of times, always at the nodes of Ranvier, the resultant branches diverging at various angles. If the free sensory endings are in epithelial tissue, these larger medullated branches are situated in tne connectivetissue mucosa under the epithelium. From these larger medullated branches, are given off smaller ones, also medullated, which may divide further, and which pass up toward the epithelium, and near its under surface divide into nonmedullated branches. Nonmedullated branches are also given off from the medullated ones as they approach the epithelium, leaving the parent fibers at the nodes of Ranvier. Many of the nonmedullated branches thus formed, after coursing a variable distance under the epithelium, enter it and break up into numerous very small branches, which, after repeated division, terminate between the epithelial cells in small nodules or discs of variable size and configuration. The small branches resulting from a division of one of the larger nonmedullated Branches constitute one of the terminal telodendria or end-branches of the dendrites of peripheral sensory neurones terminating in free sensory nerve-endings. In fibrous connective tissue the same genera] arrangement of the branches prevails. In figure 135 is shown the peripheral distribution of the dendrite of a peripheral sensory neurone terminating in a free sensory nerve-ending.


2. Encapsulated Sensory Nerve-endings. These nerve-endings may be divided into two quite distinct groups, such as have a relatively thin fibrous-tissue capsule, containing mainly telodendria of the nerve or nerves terminating therein, and such as have a distinctly lamellated, fibrous tissue capsule, usually investing, besides the nerve-termination, other tissue elements. To the former group belong three types of sensory nerve - endings, which, owing to their similarity of structure, may be described together.


Fig. 136. End-bulb of Krause

These are the end-bulbs of Krause, f rom conjunctiva of man ; methylene Meissner's tactile corpuscles, and blue stain (Dogiel, "Arch. f. mik. the genital corpuscles. They have Anat ">" voh XXXVI1 )' all been investigated recently by


Dogiel, and the account here given follows closely his description.

End-bulbs of Krause.

Under this designation there are described a variety of endings which vary slightly in size and shape. They are found in the conjunctiva and edge of the cornea, in the lips and lining of the oral cavity, in the glans penis and clitoris, and probably also in other parts of the dermis. In form they are round, oval, or pear-shaped. Their size varies from 0.02 to 0.03 mm. long and from 0.015 to 0.025 mm. broad for the smaller ones, and from 0.045 to o. 10 mm. long and from 0.02 to 0.08 mm. broad for the larger ones. They have a relatively thin capsule in which nuclei are quite numerous. One, two, or three medullated nerves go to each end-bulb. These may lose their medullary sheath at the capsule or at a variable distance from it. The naked axis-cylinders, soon after entering the capsule, divide into two, three, or four branches, which form several circular or spiral turns in the same or in opposite directions. These fibers then divide into varicose branches, which undergo further division, the resulting branches interlacing to form a bundle of variously tangled fibers which may be loosely or tightly woven.

Between the nerve-fibers and their branches, within the capsule,

there is found a semifluid substance, which is granular in fixed preparations.

Meissner 's Corpuscles.

These corpuscles are found in man in the subepidermal connective tissue of the hand and foot and outer surface of the forearm, in the nipple, border of the eyelids, lips, glans penis and clitoris. They are most numerous in the palmar surface of the distal phalanx of the fingers. They are oval in shape, sometimes somewhat irregular, and vary in size, being from 45 fj. to 50 fj. broad and from no // to 1 80 fj. long. They possess a thin connective-tissue capsule, in which are found round or oval nuclei, some t of which have an oblique position to the axis of the corpuscle. One medullated nerve ends in the smaller corpuscles, two or three or even more in the larger ones. After piercing the capsule, the medullated nerves lose their medullary sheaths, the naked axis-cylinders making a variable number of circular or spiral turns, some of which are parallel, others crossing at various angles. These larger branches are all beset with large, spindle-shaped, round, or pear-shaped varicosities. The larger branches, after making the windings mentioned, break up into many varicose branches, which interlace and form a most complex network. One usually finds one or several larger naked axiscylinders, which pass up through the axis of the spiral of fibers thus formed ; these give off branches which contribute to the spiral formation.



Fig. 137. Meissner"s tactile corpuscle ; methylene-blue stain (Dogiel, "Internal. Monatsschr. f. Anat. u. Fhys.," vol. IX).


Genital Corpuscles.

These corpuscles are found in the deeper part of the mucosa of the glans penis and the prepuce of the male and the clitoris and neighboring structures of the female. Their shape varies ; they may be round, oval, egg- or pear shaped, or even slightly lobulated. Their size varies from 0.04 to o. 10 mm. in breadth and from 0.06 to 0.40 mm. in length. They are surrounded by a relatively thick fibrous capsule, consisting of from three to eight quite distinct lamellae, between which irregular flattened cells with round or oval nuclei are found. Within this capsule, there is found a core, which seems to consist of a semifluid substance, slightly granular in fixed preparations, the nature of which is not fully known. The number of sensory nerves going to each corpuscle varies from one to two for the smaller ones, and from eight to ten for the larger corpuscles. The medullated nerves, after entering the corpuscle, divide dichotomously, the resultant branches assuming a circular or spiral course, and interlacing in various ways, within the capsule. After a few turns, the medullated branches lose their medullary sheaths and undergo further division, often dividing repeatedly. The nonmedullated nerves resulting from these divisions, the majority of which are varicose, form a most complicated network, the whole nerve network presenting a structure which resembles a tangle of fine threads. In the meshes of this network is found the semifluid substance of the core. Now and then some of the larger fibers of the network leave the corpuscle and terminate in neighboring corpuscles, or pass to the epithelium, where they end between the cells.



Fig. 138. Genital corpuscle from the glans penis of man ; methylene-blue stain corpuscle from the glans penis of man ; (Dogiel, "Arch. f. mik. Anat.," vol. XLl).



These three sensory nerve-endings end-bulbs of Krause, Meissner's tactile corpuscles, genital corpuscles are, as Dogiel has stated, very similar in structure. Each has a thin connective -tissue capsule, surrounding a core, consisting of a semifluid substance, concerning which our knowledge is as yet imperfect. One or several medullated nerves go to each corpuscle, which, after losing their medullary sheaths, divide and subdivide into numerous fine varicose branches, which are variously interwoven, forming a more or less dense plexus of interlacing and, according to Dogiel, anastomosing fibers. The chief differences are those of form and size, and of position with reference to the epithelium. Of the three forms of endings, the genital corpuscle is the largest, and occupies the deepest position in the subepithelial connective tissue ; Meissner's corpuscle is intermediate in size, and is found immediately under the epithelium ; while the end-bulbs of Krause are the smallest of these three forms of sensory endings and may be found in the papillae or in the deeper connective tissue.



Fig. 139. Cylindric end-bulb of Krause from intermuscular fibrous tissue septum of cat; methylene-blue stain.


A somewhat smaller nerve-ending of long, oval, or cylindric form, known as the cylindric end-bulb of Krause, is found in various parts of the skin and oral mucous membrane, in striated muscle and in tendinous tissue. These corpuscles consist of a thin nucleated capsule, investing a semifluid core. The nerve-fiber, after losing its medullary sheath and fibrous sheath (the latter becomes continuous with the capsule), passes through the core, generally without branching, as a naked axis-cylinder, terminating at its end, usually in a small bulb. (Fig. 139.)

The majority of the sensory nerve-endings with well-developed lamellated capsules are relatively large structures. We shall consider especially the Vater-Pacinian corpuscles, the neuromuscular end-organs, and the neurotendinous end-organs.

Vater-Pacinian Corpuscles. These corpuscles are of oval shape and vary much in size, the largest being about o. 10 of an inch long and 0.04 of an inch broad. The greater portion of the corpuscle is made up of a series of concentric lamellae, varying in number from twenty to sixty. These lamellae are made up of



Fig. 140. Vater-Pacinian corpuscle from the mesentery of a cat; X 45- The figure shows a general view of the corpuscle, a, Axis-cylinder in the core ; ik, core ; tnn, medullated nerve-fibers entering the core (" Atlas and Epitome of Human Histology," Sobotta).

white fibrous tissue fibers, rather loosely woven, between which is found a small amount of lymph, containing usually a few leucocytes. The lamellae are covered on both surfaces by a layer of endothelial cells (Schwalbe). Between two consecutive lamellae there is found an interlamellar space, also containing lymph. The axis of the corpuscle is occupied by a core, consisting of a semifluid, granular substance, in the periphery of which oval nuclei are said to be found. Usually one large medullated nerve-fiber goes to each corpuscle. The fibrous tissue sheath of this nerve-fiber becomes continuous with the outer lamellae of the capsule. The medullary sheath accompanies the axis-cylinder through the concentric lamellae until the core is reached, where it disappears. The naked axiscylinder usually passes through the core to its distal end, where it divides into three, four, or five branches which terminate in large, irregular end-discs. The axis-cylinder may, however, divide soon after it enters the core into two or three or even four branches, these passing to the distal end of the core before terminating in the end-discs above mentioned. Both Retzius and Sala state that the naked axis-cylinders, after entering the core, give off numerous short side branches, terminating in small knobs, which remind these observers of the fine side branches or thorns seen on the dendrites of Purkinje's cells and of the pyramidal cells of the cortex, when stained after the Golgi method. In company with the large nerve-fibers here mentioned, Sala has described other nerve-fibers, quite independent of them and much finer, which after entering the corpuscle divide repeatedly, the resulting fibers forming a plexus around the central fiber. A small arteriole enters the corpuscle with the nerve-fiber, dividing into capillary branches found between the lamellae of the capsule.

The Vater-Pacinian corpuscles have a wide distribution. They are numerous in the deeper parts of the dermis of the hand and foot, and also near the joints, especially on the flexor side. They have been found in the periosteum of certain bones and in tendons and intermuscular septa, and even in muscles. They are further found in the epineurial sheaths of certain nerve-trunks and near



Fig. 141. Pacinian corpuscles from mesorectum of kitten : A, Showing the fine I/ranches on central nerve-fiber ; B, the network of fine nerve-fibers about the central fiber; methylene-blue preparation (Sala, "Anat. Anzeiger," vol xvi).

large vessels. They are numerous in the peritoneum and mesentery, pleura and pericardium. In the mesentery of the cat, where these nerve-endings are large and numerous, they are readily seen with the unaided eye as small, pearly bodies.

In the bill and tongue of water birds, especially of the duck, are found nerve-endings, known as the corpuscles of Herbst, which resemble the Vater-Pacinian corpuscles ; they differ from the latter in having cubic cells in the core. (Fig. 142.)

Neuromuscular Nerve End-organs. These nerve end-organs consist of a small bundle of muscle-fibers, surrounded by an investing capsule, within which one or several sensory nerves terminate. They are spindle-shaped structures varying in length from 0.75 to 4 mm., and in breadth, where widest, from 80 to 200 ju (Sherrington, 94). In them there is recognized a proximal polar region, an equatorial region, and a distal polar region. The muscle-fibers of this nerve-ending, known as the intrafusal fiber s t which may vary in number from 3 or 4 to 20 or even more, are much smaller than the ordinary voluntary muscle-fibers and differ from them structurally, and result from a division of one or several muscle-fibers of the red variety. In the proximal polar region the intrafusal fibers present an appearance which is similar to that of voluntary musclefibers of the red variety ; in the equatorial region they possess rela



Fig. 142. Corpuscle of Herbst from bill of duck; X o.

tively few muscle-fibrils and are rich in sarcoplasm and the musclenuclei are numerous ; the striation is here indistinct. In the distal polar region the intrafusal fibers are again more distinctly striated and, a short distance beyond the end-organ, become greatly reduced in size, and terminate as very small fibers, still showing, however, a cross-striation. In figure 143 is shown a single intrafusal musclefiber. Owing to the length of such a fiber it was necessary to represent it in several segments.

The intrafusal muscle-fibers are surrounded by a capsule consisting of from four to eight concentric layers of white fibrous tissue. At the proximal end this capsule is continuous with the connective tissue found between the muscle-fibers endo- and perimysium. It attains its greatest diameter in the equatorial region of the nerve end-organ, and becomes narrower again at its distal end, where it may end in tendon or become continuous with the connective tissue




Fig. 143. Intrafusal muscle-fiber from neuromuscular nerve end-organ of rabbit : A, From proximal polar region ; B } equatorial region ; C, distal polar region.

of the muscle. Immediately surrounding the intrafusal fibers is found another connective -tissue sheath known as the axial sheath, and between this and the capsule there is found a lymph-space bridged over by trabeculse of fibrous tissue, to which the name periaxial lymph-space has been given. (Fig. 144.)

By degenerating the motor nerves going to a muscle, Sherrington determined that the nerve-fibers ending in the neuromuscular nerve end-organs were sensory in character. The manner of termination in these end-organs of the nerve-fibers ending therein has been studied by Kerschner, Kolliker, Ruffini, Huber and DeWitt, Dogiel, and others. One or several (three or four) large medullated nerves, surrounded by a sheath of Henle, terminate in each neuromuscular end organ. As these nerves enter the capsule, the sheath of Henle blends with the capsule. The medullated nerve-fibers now and then divide before reaching the nerve end-organs, and divide several times as they pass through the capsule, periaxial space, and axial sheath. Within the axial sheath, the medullary sheath is lost, and the naked axis-cylinders terminate in one or several ribbon - like branches which are wound circularly or spirally about the intrafusal fibers (annulospiral ending) or they may terminate in a number of larger branches which again divide, these ending in irregular, round, oval, or pear-shaped discs {flower-like endings), which are also on the intrafusal fibers. These flower-like endings are usually at the ends of the annulospiral fibers. In the smaller endorgans only one area of nerve-termination has been observed ; in the larger, two, three, or even four such areas may be found.





Fig. 144. Cross-section of a neuromuscular nerve end-organ from interosseous (foot) muscle of man ; fixed in formalin and stained in hematoxylin and eosin.


Fig. 145. Neuromuscular nerve end-organ from the intrinsic plantar muscles of dog ; from teased preparation of tissue stained in methylene-blue. The figure shows the intrafusal muscle-fibers, the nerve-fibers and their terminations ; the capsule and the sheath of Henle are not shown (Huber and DeWitt, "Jour. Comp. Neurol.," vol. vn).



Neuromuscular nerve end-organs are found in nearly all skeletal muscles (not in the extrinsic eye muscles nor in the intrinsic muscles of the tongue), but they are especially numerous in the small muscles of the hand and foot. They are found in amphibia, reptilia, birds, and mammalia, presenting the same general structure, although the ultimate termination of the nerve-fibers varies somewhat in the different classes of vertebrates.

Neurotendinous Nerve End -organ (Golgi Tendon Spindle). In 1880 Golgi drew attention to a new nerve end-organ found in tendon, describing quite fully its general structure and less fully the nerve termination found therein. These, nerve end-organs are spindle-shaped structures, which in man vary in length from 1.28 mm. to 1.42 mm., and in breadth from 0.17 mm. to 0.25 mm. (Kolliker). Ciaccio mentions a neurotendinous nerve endorgan found in a woman, which was 2 Fig. 146. Neurotendinous o r 3 mm. long. A capsule consisting of nerve end-organ from rabbit; teased from 2 to 6 fibrous tissue lamella^, and preparation of tissue stained in broadest at the equatorial part of the methylene-blue (Huber and DeWitt, "Jour. Comp. Neural.," vol. x). end-organ, surrounds a number of intrafusal tendon fasciculi. The capsule is continuous at the proximal and distal ends of the end-organ with the internal peritendineum of the tendon in which it is found. The number of the intrafusal tendon fasciculi varies from eight to fifteen or even more. They are smaller than the ordinary tendon fasciculi, from which they originate by division, and structurally resemble embryonic tendon, in that they stain more deeply and present many more nuclei than fully developed tendon. The intrafusal tendon fasciculi are surrounded by an axial sheath of fibrous tissue, between which and the capsule there is found a periaxial lymph-space.


Fig. 147. Cross-section of neurotendinous nerve end-organ of rabbit ; from tissue stained in methylene-blue : m, Muscle-fibers ; t, tendon ; t, capsule of neurotendinous end-organ ; m n, medullated nerve-fiber (Huber and DeWitt, " Jour, of Comp. Neurol.," vol. X).

The termination of the nerve-fibers ending in these end-organs has been studied by Golgi, Cattaneo, Kerschner, Kolliker, Pansini, Ciaccio, Huber and DeWitt. One, two, or three large medullated nerve-fibers, surrounded by a sheath of Henle, end in each endorgan ; as they pass through the capsule, the sheath of Henle blends with the capsule. The medullated nerve-fibers before entering the capsule usually branch several times, branching further within the capsule and axial sheath. Before the resultant branches terminate on the intrafusal tendon fasciculi, the medullary sheath is lost, the naked axis-cylinder further dividing into two, three, or four branches, each of which runs along on the intrafusal fasciculi, giving off numerous short, irregular side branches, which partly enclasp the tendon fasciculi and end in irregular end-discs. Some of the terminal branches pass between the smaller fibrous tissue bundles of the fasciculi, ending between them.

In these end-organs, the larger nerve-branches are found near the center of the bundle of intrafusal tendon fasciculi, the terminal branches and the end-discs nearer their periphery. The neurotendinous nerve end-organs are widely distributed, being found in all tendons although not equally numerous in all. Like the neuromuscular nerve end-organs, they are especially numerous in the small tendons of the hand and foot. Sensory nerve end-organs, which resemble in structure the neurotendinous end-organs here described, though somewhat smaller than these, have been found in the tendons of the extrinsic eye-muscles.

In this brief account of the mode of ending of the telodendria of the dendrites of peripheral sensory neurones (sensoiy nerve-fibers) it has not been possible to discuss any but the more typical varieties of sensory nerve -endings. Other nerve -endings will be considered in connection with the several organs to be treated later. For a fuller discussion of this subject, the reader is referred to special works and monographs.

Technic

Fresh medullated nerve-fibers, when teased in an indifferent fluid, show the peculiar luster of the medullary sheath, and also the nodes of Ranvier, the neurilemma with its nuclei, and the segments of Lantermann. At the cut ends of the fibers, the typical coagulation of their medullary portions is seen in the form of drops of myelin. All these structures can also be seen after using i oJ osmic acid. A nerve (not too thick) is placed in a i % aqueous osmic acid solution, then washed for a few hours in distilled water, and finally carried over into absolute alcohol. After dehydration, small pieces are cleared with oil of cloves and the fibers teased apart upon a slide. The medullary sheath is stained black and hides the axial space, the nodes are clear, the neurilemma is sometimes seen as a light membrane, and the nuclei of the fibers are of a lenticular shape, and stained brown.

The nodes of Ranvier may also be demonstrated by means of silver nitrate solution. Fresh nerve-fibers are either teased in distilled water to which a trace of i% silver nitrate solution has been added (the nodes of Ranvier appear after a short time as small crosses), or whole nerves are placed for twenty-four hours in a 0.5% aqueous solution of silver nitrate, washed for a short time with water, hardened in alcohol, after which they are imbedded in paraffin and cut longitudinally. Exposure to light will soon bring out the ' ' crosses of Ranvier ' ' at the nodes. The appearance of these crosses is due to the fact that the silver nitrate solution first penetrates at the nodes of Ranvier, and then passes by capillary attraction along the axial cord for some distance. After the reduction of the silver, the cruciform figures appear colored black. Occasionally, a peculiar transverse striation is seen in the longitudinal portions of the crosses. These are known as Frommann's lines. Their origin and significance have not as yet been satisfactorily explained.

To demonstrate the fibrils of the axial cord a piece of a small nerve is stretched on a match or toothpick and fixed for four hours in a 0.5% osmic acid solution, after which it is washed in water for the same length of time and immersed in 90% alcohol for twenty-four hours. The preparation is now stained for another twenty-four hours in a saturated aqueous solution of fuchsin S and then placed for three days in absolute alcohol. Finally, the nerve is passed as rapidly as possible through toluol, toluol- paraffin, and then imbedded in paraffin. The proper orientation of the specimen is of the greatest importance, as is also the cutting of thin sections. In a longitudinal section red fibrils of almost uniform thickness and evenly distributed throughout the axial space Axis-cylin- are seen lying in the colorless neuroplasm, and parallel to the long axis of the nerve-fiber. In cross-section the axial fibrils appear as evenly distributed dots. Attention must be called to the fact that the fibrils are not equally well stained in all cases (Kupffer, 83, II ; compare also Jacobi and Joseph).



Fig. 148. Ranvier's crosses from sciatic nerve of rabbit treated with silver nitrate solution ; X * 2O - Frommann' s lines can be seen in a few fibers.


Fig. 149. Medullated nerve-fiber from sciatic nerve of frog. In two places the medullary sheath has been pulled away by teasing, showing the "naked axis-cylinder" ; X 2I2



When the fiber is less carefully treated, the fibrils fuse with the neuroplasm to form the ' ' axis-cylinder ' ' of authors. As the appearance of the latter is due to a shrinkage of the contents of the axial space, it is easy to understand that one reagent may have a greater effect in this respect than another. The thinnest axis-cylinders are produced by chromic acid and its salts, while thicker ones are seen in nerve-fibers fixed in alcohol. These variations are best seen in cross-sections, in which the axis-cylinders sometimes appear as round dots and again as stellate figures. The latter are due to pressure on the shrinking axial cord by the unevenly coagulated medullary sheath. As the medullary sheath in such preparations crumbles away in many places, large areas of the axis-cylinder may often be isolated by teasing (Fig. 149).

Sensory and motor nerve-endings may be stained after gold chlorid and chrome-silver methods (see methods of impregnation, page 47), or after the infra vitam methylene-blue method suggested by Ehrlich and variously modified by other investigators.

If freshly teased fibers be treated with glacial acetic acid, the axis-cylinders swell up and issue from the ends of the fibers in irregular masses showing fine longitudinal striation (Kolliker, 93). The structures of the axial space dissolve in i% hydrochloric acid, as well as in a 10% solution of sodium chlorid (Halliburton).

For the isolation of ganglion cells, 33% alcohol, o.i to 0.5% chromic acid, or \ C J solution of potassium bichromate may be used.

Small pieces of the spinal cord and brain containing ganglion cells are treated with a small quantity of one of the above solutions for one or two weeks. After this interval the preparations may be teased and the isolated ganglion cells stained on a slide and mounted in glycerin. They may even be fixed in situ by injecting a i % solution of osmic acid or 33% alcohol into the areas of the brain or spinal cord containing ganglion cells. The region thus treated is then cut out and teased.

The nonmedullated or "Remak's fibers" are obtained by teasing a sympathetic nerve, or, better, a piece of the vagus previously treated with osmic acid. Between the blackened medullated fibers of the pneumogastric are seen numerous unstained fibers of Remak.

The fibers of the olfactory nerve are stained brown by osmic acid.

Ehrlich' s methylene-blue method consists in an infra vitam staining of ganglion cells, nerve-fibers, and nerve-endings. The method is much more applicable to the staining of peripheral ganglia (spinal and sympathetic ganglia), peripheral nerves, and nerve-endings than to staining the elements of the central nervous system, although the latter may also be stained by means of this method.

Two methods for bringing the stain in contact with the nerve-tissues are now in use : ( i ) injecting the methylene-blue solution into the living tissues through the blood-vessels ; (2) adding a few drops of the stain to small pieces of perfectly fresh tissues removed from the body. The solution used for injecting tne tissues is prepared as follows : i gm. of methyl ene-blue 1 is mixed in a small flask with 100 c.c. of normal salt solution and heated over a flame until the solution becomes hot. It is then allowed to cool ; when filtered, it is ready for use. A cannula is tied into the main artery of the part in which it is desired to stain the nerve elements, and sufficient of the foregoing methylene-blue solution injected to give the part a decidedly blue color. After the injection the part to be studied remains undisturbed for about one-half hour, after which time small, or at least thin, pieces of the tissue to be studied are removed to a slide moistened in normal salt solution, and exposed to the air. The tissues remain on the slide until the nerve-cells, nerve-fibers, or nerveendings seem satisfactorily stained. After placing the tissues on the slide, they are examined under the microscope (without covering with a coverglass) every two or three minutes, until such examination shows blue color in the neuraxes of the nerve-fibers and their terminations, or in the nerve-cells, if there be any in the tissues examined. Care should be tak^n not to miss the time when the staining has reached its full development, as the blue color usually fades again and only inferior preparations are obtained.




Fig. 150. A ganglion cell from anterior horn of the spinal cord of calf ; teased preparation ; X 140. By this method only the coarsest ramifications of the dendrites are preserved ; the rest are torn off.


Tissues thus stained may be fixed by one of two methods (or modifications of these methods), the selection of the method depending somewhat on the results desired. If it is desired to gain preparations giving the general course of nerves, the formation of nerve-plexuses, the relations of afferent and efferent nerves to the nerve-cells in ganglia, or the general arrangement of the terminal branches of nerve-fibers in nerve endorgans, the tissues are placed in a saturated aqueous solution of ammonium picrate (Dogiel) in which the blue color of the tissues is in a short time changed to a purplish color. In this solution the tissues remain for from twelve to twenty-four hours, and are then transferred to a mixture consisting of equal parts of a saturated aqueous solution of ammonium picrate and glycerin, in which they remain another twenty-four hours ; they may, however, without detriment remain in the mixture several days. The tissues are then mounted in this ammonium picrate -glycerin mixture.

If, on the other hand, it is desired to section tissues stained infra vitam in methylene-blue, the following method, slightly modified from that given by Bethe, is suggested. The following fixative is prepared : Ammonium molybdate, i gm.; distilled water, 10 c.c.; hydrochloric acid, i drop. The solution is prepared by grinding the ammonium molybdate to a fine powder, removing it to a flask, and adding the required quantity of water. The flask is now heated until the ammonium molybdate is entirely dissolved, when the hydrochloric acid is added. Before using this fixative it is necessary to cool it to 2-5 C. It is, therefore, well to prepare it before" the injection is made, and surround it with an ice mixture. In this fixative the tissues remain for from twelve to twenty-four hours. After the first six to eight hours it is not necessary to keep the fixative below ordinary room-temperature. After fixation the tissues are washed for an hour in distilled water. They are then hardened and dehydrated in absolute alcohol. It is advisable to hasten this step as much as possible, though not at the risk of imperfect dehydration. 1 Methylenblau, rectificiert nach Ehrlich, Griibler, Leipzig.


The tissues are then transferred to xylol and imbedded in paraffin, sectioned, fixed to the slide or cover-glass with albumin fixative, and may be double stained in alum-carmin or alum-cochineal. After staining in either of these stains, the sections are thoroughly dehydrated and cleared in oil ofbergamot. The oil is washed off with xylol and the sections are mounted in Canada balsam.

In staining nerve-fibers with methylene-blue by local application of the stain to the tissues, the tissues to be studied are removed from an animal which has just been killed, divided in small pieces, and placed on a slide moistened with normal salt solution. A few drops of a -%-$% to -Y5% solution of methylene-blue in normal salt solution are added from time to time sufficient to keep the tissues moistened by the solution, but not enough to cover them. The preparations are examined from time to time, under the microscope, to see whether the nerve elements are stained. The length of time required for staining by this method varies. Sometimes the nerve elements are stained in half an hour ; again, it may require two and one-half hours ; on an average, about one hour. As soon as the tissues seem well stained they are fixed as previously directed. Dogiel has found that sympathetic ganglia and sensory nerve-fibers of the heart removed from the human body several hours after death may be stained by means of the foregoing method.

In order to obviate the necessity for the low temperature of the previous method, Bethe (96) has recommended the following procedure : According to the method of Smirnow and Dogiel, he first employs as a preliminary fixing agent a concentrated aqueous solution of ammonium picrate. In this he places the tissue, previously treated with methyleneblue, for from ten to fifteen minutes. Without further washing the larger objects are immersed in a mixture composed of ammonium molybdate (or sodium phosphomolybdate) i gm., distilled water 20 c.c., and pure hydrochloric acid i drop. The following mixtures may also be employed for the same purpose : ammonium molybdate (or sodium phosphomolybdate) i gm. , distilled water 10 c.c., 2% solution of chromic acid 10 c.c., and hydrochloric acid i drop ; or, for very thin gross specimens or sections, ammonium molybdate (or sodium phosphomolybdate) i gm., distilled water, 10 c.c., 0.5% osmic acid 10 c.c., and hydrochloric acid i drop. Small objects are permitted to remain no longer than from three quarters of an hour to one hour in either of the first two mixtures, and not more than from four to twelve hours in the third. After fixing, the specimens are washed with water, carried over into alcohol, then into xylol, and finally imbedded in paraffin. Subsequent staining with alum-carmin, alum -cochineal, or one of the neutral anilin dyes gives good results.

A very promising method recommended by Meyer (95) consists in injecting subcutaneously about 20 c.c. of normal salt solution containing from i% to 4% of methylene-blue into a young rabbit, and repeating the operation in one to two hours. Within the next two hours the animal usually dies and the central nervous organs are then removed and small pieces fixed according to Bethe' s method.

The method of Chr. Sihler may be recommended for demonstrating the nerve-endings in striated muscle : Muscle bundles of the thickness of a goose quill are first placed for eighteen hours in a solution composed of acetic acid i vol., glycerin i vol., and i% solution of chloral hydrate 6 vols., and then teased in pure glycerin. Afterward they are placed in a mixture of Ehrlich's hematoxylin i vol. , glycerin i vol. , and i c /c chloral hydrate solution 6 vols. , in which the specimens are allowed to remain for from three to ten days. The pieces are now placed in glycerin acidified with acetic acid (solution No. i), in which the color becomes differentiated, the nerves and nerve-endings in the muscles and vessels being deeply stained, while the remaining portion of the specimen becomes decolorized. After having stained with No. 2, the pieces may be preserved in pure glycerin, to be treated later with acetic acid (solution No. i).

These methods are most successful in reptilia and mammalia, more difficult in the other classes of vertebrate animals.


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A Textbook of Histology (1910): Introduction To Microscopic Technic | General Histology | I. The Cell | II. Tissues | Special Histology | I. Blood And Blood-Forming Organs, Heart, Blood-Vessels, And Lymph- Vessels | II. Circulatory System | III. Digestive Organs | IV. Organs Of Respiration | V. Genito-Urinary Organs | VI. The Skin and its Appendages | VII. The Central Nervous System | VIII. Eye | IX. Organ of Hearing | X. Organ of Smell | Illustrations - Online Histology

Reference: Böhm AA. and M. Von Davidoff. (translated Huber GC.) A textbook of histology, including microscopic technic. (1910) Second Edn. W. B. Saunders Company, Philadelphia and London.


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