a micro-manipulative study of blood capillaries

A MICRO-MANIPULATIVE STUDY O F BLOOD CAPILLARIES

BENJAMIN W. ZWEIFACH Washington Square College, New Pork University

INTRODUCTION

Within recent years nothing conclusive has been demonstrated regarding the reactions of the different structural elements that go to make up the living blood capillaries and the cells surrounding them. The historical evidence still leaves the mechanism responsible for capillary contraction and relaxation in doubt, with contradictory evidence being offered with respect to the mechanism of contraction in the capillary vessels of related forms.

This paper deals chiefly with experiments on the rich capillary network in the intestinal mesentery of the frog where the Rouget cell type of capillary contractility has been claimed to exist. By means of the micro-manipulative technique, it was possible to pass through the mesothelial layer with a microneedle, to stimulate individual cells on and around the capillaries and to manipulate or dislodge them under the oil immersion powers of the microscope. Previously (Krogh, '2O), mechanical stimulation had been induced by means of hairs manipulated by hand under comparatively low magnifications. Even under the best conditions, this procedure never brought the hair into direct contact with the capillary wall. Electrical and chemical means have also been used to stimulate the cells. These latter methods left much to be desired since their effect could not be localized.

83

84 BENJAMIN W. ZWEIFACH

Vimtrup ( '22), combining histological technique with observations on the tail-fin capillaries of larval Triton puncta- tus and the nictitating membrane of the frog, was convinced that contractions started a t points where certain cells lie on the capillary wall. By supravital methylene blue staining, he identified these cells as the peri-capillary cells observed by Rouget (1873), and concluded that they were responsible for the contraction of the capillary. Experimental data claim- ing to have demonstrated muscular peri-capillary cells in the living system was also offered by Tannenberg ( '25). Work- ing with the mesenteric capillaries of the rabbit, he found spur-like closures occurring near the junction points of the vessels. These he attributed to the peri-capillary cells (which took the methylene blue stain) acting as valves. Schaly ( '26), using fixing and staining methods on a great number of different vertebrate tissues, showed the presence of these peri- capillary cells to be of an almost universal occurrence on all types of capillaries.

Clark and Clark ('25) observed that the Rouget cells in amphibian larvae were derived from wandering, stellate, connective tissue cells which flattened out on the capillary endothelium. They noted that the capillary was able to contract before the Rouget cells had developed. Larvae were placed in a chloretone solution and it was observed that the capillaries contracted more often between the Rouget cells than at those points where these cells were located, and that, while contracting, the.capi1Iary wall would pull away from a Rouget cell. They concluded that the endothelium possesses the property of active contractility. Federighi ( '28) found two types of contraction in the blood vessels of Nereis, viz., a peristaltic wave of contraction of the endothelium (independ- ent of the central nervous system), and a local, non-trans- missible type of contraction obtained by direct chemical, mechanical or electrical stimulation.

Bensley and Vimtrup ('28) repeated the earlier experiments of Vimtrup ( '22) on the excised nictitating membrane of the frog and concluded that the constrictions of the capil-

MICRO-MANIPULATION O F CAPILLARIES 85

lary wall were due to contractions of the encircling cells. Us- ing Janus green, they stained elements in these cells which they believed to be myofibrils. Rogers (’32) stained peri- capillary cells in the rabbit’s mesentery with Janus green and could find no evidence for the presence of myofibrils in these cells. He also found no change in capillary outline as a result of peri-capillary cell stimulation (electrical).

The Rouget cell has been demonstrated encircling mammalian capillaries (Schaly, ’26). More recently, Sandison (’31, ’32) and Clark and Clark ( ’32), using the transparent chamber method in the rabbit’s ear, found capillary contraction to be rare and negligible in its effect upon the circulation. They attributed the differences in amount of capillary flow to periodic contractions of the arteries and arterioles.

The papers of the Clarks and of Sandison tend to show that, at least in mammals, capillary contraction is negligible, despite the presence of the so-called ‘Rouget cells ’ encircling these capillaries. The evidence cited by the Clarks and by Sandison did not disprove the possible action of adventitial cells as contractile elements in special cases. Whether the adventitial cells in amphibia are contractile, as in Vimtrup’s experiments, or whether the cells are not a factor in the contraction phase, as claimed by the Clarks, is not at all clear. I n the lower forms, such as Nereis, the endothelium is definitely the contractile element.

The present investigation was carried on under the super- vision of Prof. Robert Chambers. The author takes this opportunity to thank him for his helpful criticisms and con- stant advice throughout the work.

METHODS AND TECHNIQUE

The micro-manipulative technique was used to study the effect of localized mechanical stimulation upon the various cellular elements visible in the capillary wall of the mesentery, tongue and nictitating membrane of the frog.

86 BEN JAMIN W. ZWEIFACH

I n preparing the material, special precautions were taken to prevent bleeding. A frog was immobilized by carefully crushing the forebrain with artery forceps. The skin and abdominal wall were cut open by electro-cautery. An intestinal loop was then drawn out and carefully spread over a small cork horseshoe of sufficient height to permit the introduction of microneedles from below. The inner sides of the ‘horseshoe’ were lined with strips of moistened blotting paper. The mesentery was covered with a glass slip and kept moistened with Ringer’s solution. The cork horseshoe roofed by the mesentery and the cover-slip served as a moist chamber, through the open end of which t,he microneedles were introduced. The mesentery, adhering to the under surface of the glass cover slip, gave an even, resistant surface against which to work. In order to reach the capillaries in the mesentery the upturned tips of the microneedles were passed through the mesothelial layer. This restricted side to side motions of the needle but permitted easy probing and gave sufficient play for dissecting cells. Such preparations, with the body of the frog encased in wet cloths, maintained normal circulation for at least 8 to 10 hours.

The capillary vessels of the dorsal surface of the tongue were used in preference to those of the ventral surface which has a thick epithelium. In order to i7se this surface for the preparations, the frog was tied down on its back; the tongue was spread over the cork horseshoe and covered with a glass slip. The nictitating membrane was also used. This was excised and placed in a hanging drop in the regular Chambers’ micro-dissecting moist chamber. Observations and manipulations of the vessels in the nictitating membrane were made within 10 to 15 minutes after removal from the body.

The method used was mechanical stimulation with the tips of the microneedles. By this means cells could be individually stimulated. No previous experimenter could control his reacting elements to so fine a degree as is possible with the micro-manipulative technique.

All observations, including the micro-manipulative opera- tions, were made a t a magnification of 450 to 1,700 diameters,

MICRO-MANIPULATION OF CAPILLARIES 87

EXPERIMENTAL

1. Pre- afid postcapillary structures A . Along the smallest arterioles and the precapillary

branches, the muscular coat loses its continuity and separates into bundles of about 3 to 5 muscle cells. I n the precapillaries the individual muscle cells are spaced at greater in- tervals than in the arterioles. The discontinuous muscular coat stops and is never found on the capillaries. The true capillaries anastomose frequently and are approximately all of the same diameter. The comparatively short postcapillary vessels have a connective tissue coat. This thickens gradually in the small venules to three or four times that of the endothelial wall before the muscle cells appear. This is in direct contrast with the arterial end where a sudden disappearance of the muscle cells causes a sharp demarcation. Somewhat similar observations have been recently described by Sandison in the mammalian blood vessels.

B. Cells vitally staiBed with methyleme blue. Three or four drops of 0.1 per cent Ehrlich’s methylene blue in Ringer’s solution were deposited on the mesentery and a cover slip added. The methylene blue was talien up immediately by some of the cells adjoining the capillaries, on the arteriole walls, and in the connective tissue. The different types of peri-capillary cells always took up the stain within 5 to 10 minutes. If no additional methylene blue was added and the preparation was allowed to stand, these cells soon lost their coloration, However, if three or four applications of the dye were made, the muscle cells on the arterioles, precapillaries and venules also took up the stain. At this stage the peri-capillary cells were very heavily stained, whereas the muscle cells on the other vessels were only beginning to take up the stain. A few minutes later the muscle cells took on a heavy granuIar nuclear stain, with a pale bluish cytoplasm. Thus, there seems to be a distinct difference in the staining time of the typical muscle cells and of the peri- capillary cells.

THE .41UATONICAL RECORD, VOL. 59, NO. 1, AND SUPPLEYEHT


Throughout the numerous observations on the arterioles and capillaries, no gradual change of a muscle cell into a transitional type of cell was ever found. Along the arterioles, the closely spaced muscle cells are spindle shaped and encircle the vessels at right angles to the long axis. They are sepa- rated from the endothelial wall by a thin layer of connective tissue. I n the smaller arterioles, where the muscle layer is discontinuous, the muscle cells, now with sufficient space between them, flatten out and tend to be broader and more elliptical (fig. 1). I n the precapillaries, the muscle cells are further apart, but they always keep the typical spindle shape.

These typical muscle cells on the precapillary abruptly terminate when a true capillary branch is given off (fig. 2) . The various cells which now appear adjoining the capillaries have irregular side processes, and are lightly stained in contrast to the heavily stained, typical, muscle cells. They closely resemble cells in the surrounding areolar tissue. The muscle cells not only take longer to stain but when once stained they retain their coloration for a long time, never losing their stain completely, while the lightly stained peri-capillary cells lose their coloration much sooner. The muscle cells reappear in the venule as typical spindle-shaped cells, lying a t varying angles on the wall (fig. 3 ) .

I was unable to confirm the observations of Vimtrup who identified by this method of staining the peri-capillary Rouget cells with the muscle cells on arterioles and precapillaries.

The arterioles and venules react readily to mechanical stimulation. By bringing the microneedle under an arteriole and prodding it, a constriction of the vessel was induced. There is a short latent period (1 to 2 seconds), followed by a slow, even contraction of the wall. Suddenly within 1 second its lumen is obliterated. This reaction always spreads in the opposite direction to that of the blood flow and often involves one or two large neighboring arterioles. The contraction persists for 2 to 3 minutes. Relaxation passes through three distinct phases. First the lumen of the vessel opens very slightly,

C. Stirnulatiom of arterioles and uemules.

89 MICRO-MANIPULATION OF CAPILLARIES

with an oscillatory movement of the contained blood cor- puscles. This is followed by a further gradual opening of the lumen allowing the blood to flow. When the blood has

3

Fig. 1

Fig.2

Transversely arranged muscle cells in the arteriole of the frog, vitally

Breaking up of muscle sheet on the larger arterioles into bundles with Abrupt

No transitional types

Irregularly arranged typical muscle cells in the wall of small venules,

stained with methylene blue.

several typical muscle cells. ending of muscle cells a t precapillary-capillary junction. of muscle cells visible. X 450.

vitally stained with methylene blue.

X 1380.

Single muscle cells on the precapillaries.

Fig. 3 X 1380.

been flowing for about 5 seconds, the vessel suddenly opens to its initial diameter. It is interesting to note that after four or five stimuli and the ensuing contraction-relaxation phases, the vessel becomes fatigued and the reactions become less and less marked. I f , however, the stimulus is applied

90 BENJAMIN W. ZWEIFACE

in a different region of the vessel, the typical contraction ensues.

A localized constriction can be induced by carefully bringing the microneedle to one side of the arteriole and, with the point of the needle, stimulating a single muscle cell. There is a latent period of approximately 1 second and then the contraction of the muscle cell slowly constricts the wall. This constriction may persist for about 3 minutes. If a partially contracted vessel is stimulated at the point where a typical muscle cell encircles it, this muscle cell will relax and allow the wall to dilate. Thus, a local dilatation of the arteriole wall could be induced.

The venules can be stimulated in a similar manner. The venule vessel, however, instead of contracting as a whole, is constricted only in certain regions where the muscle cells adhere to the wall. This gives the vessel a varicose appearance. Localized constrictions and relaxations can also be induced.

I n addition, if partially contracted venules o r arterioles are rubbed lengthwise with a microneedle, dilatation of these vessels takes place.

TI. T h e capillaries

A. E f f ec t of t h e connective t issue. Capillaries may be bent sharply in several places by bands of connective tissue fibers, which prevent local dilatation of the wall at these points. A bulging out of the capillary wall between such fibers gives the vesseI a varicose appearance. If the microneedles are then introduced to exert a force opposite to that of these fibers, the pressure on the wall is released and the previously restricted points then dilate, so that the varicose appearance is lost.

The capillaries also have numerous connective tissue fibers adhering to their outer surface. Consequently, when these capillaries contract, local regions of the wall remain expanded (fig. 4). This also gives the vessel a varicose appearance.

91 MICRO-MANIPULATION O F CAPILLARIES

B. Encircling tines. Occasionally associated with the nuclear thickenings of the capillary wall could be seen thin, fiber-like lines encircling the capillary in many regions. Very often these lines were traced to the endothelial nuclear thickenings, but never could be traced to adventitial cell nuclei. In the contracted capillaries, where some of the endothelial cells were partially contracted and others were in an expanded state, these lines were unusually prominent. In the shorter, cross connecting, curved branches, the general outline of the capillary was very irregular, and it was here that the encircling lines showed up most prominently.

Fig. 4 (a) Partially contracted capillary vessel with varicose appearanw due t o the connective tissue fibers holding back portions of the wall. Rubbing a microneedle along the length of the vessel causes (b) a slight dilatation and smooth contour of the wall. X 450.

C. Cells on the inner surface of the endothelial wall. Flat- tened cells, closely resembling endothelial nuclear swellings, were seen on the inner surface of the endothelium. These move very slowly (movement is apparent only when observed over a period of 60 to 90 minutes). The cells are elliptical in outline and about half the size of the leucocytes in the blood stream. Prodding the wall where these cells are adhering loosens them and causes them to drop off into the blood stream (figs. 5, 6). This phenomenon is especially noticeable in preparations with a sluggish circulation or in mesentery spreads that have been exposed for 9 to 10 hours.


D. The peri-capillary cells. Four types of tissues were used in these experiments; the intestinal wall, the mesentery, the tongue and the nictitating membrane. In the tongue and the intestinal wall the capillaries, embedded in muscular tissue,

5

Fig. 5 Prodding the endothelial wall in region of what appears t o be an endothelial nucleus loosens flattened cell on the inner surface of vessel into blood stream. r is red blood cell. X 1380.

Stimulation causes the cell to be detached into the blood stream.

Fig.6 Similar cell flattened over a fold in the endothelium. x 1380.

have very few peri-capillary cells encircling them. In contrast, the vessels in the nictitating membrane and the intestinal mesentery lie in a considerable amount of connective tissue, and here the capillaries have numerous peri-capillary cells adhering to them.


D(a). Types of cells adhering to and touching the capillary wall. 1. These thin, mononucleate, triangular-shaped cells, with a clear inconspicuous nucleus, have a faintly granu- lated cytoplasm containing small oil-like droplets (fig. 7) . Methylene blue stains the cytoplasmic granules. The nucleus stains only after death. The cells can be observed to move very slowly. Mononucleate and often binucleate star-shaped cells, which are probably of the same type as those described above, also were seen. These cells, usually found near the capilIary wall but rarely adhering to it, have thin thread-like processes touching the wall.

2. These are narrow and very elongate, rapidly moving cells with numerous heavy cytoplasmic granules and a transparent, elliptical nucleus. The cytoplasm takes a heavy methylene blue stain, showing deep purplish granules (fig. 8).

3. Mononucleated cells with a large ovoid nucleus adhere closely to the capillary wall. The nucleus is usually found to one side of the cell. The clear glassy cytoplasm is irregularly spread out on the capillary wall. The nucleus quickly takes an intense methylene blue stain (fig. 9). 4. Large cells (2 to 3 times the size of the endothelial cells)

with irregularly shaped nuclei were observed. The cytoplasm is homogeneous and transparent, making it difficult to trace the cell outlines. The nucleus alone is lightly stained by methylene blue, as shown in figure 10. These cells are found in large numbers along the course of the capillaries and, frequently, in groups or strands throughout the connective tissue. They were not observed to move.

5. Adventitail cells of the type described by Marchand were observed on the capillary wall. They are long, spindle-shaped cells, with large elliptical nuclei, and a granular cytoplasm which stains greenish with methylene blue (fig. 11). In dilated vessels the cytoplasmic granules are arranged fre - quently in a linear alignment (probably due to lines of stress, since pushing the cell with the side of the microneedle dis- rupts this arrangement). These numerously distributed cells stain faintly with methylene blue and soon fades out.

12

Figs. 7, 8, 9 Three types of peri-capillary cells adhering closely t o the surface of the vessel. Stained with methylene blue. X 1380.

Figs. 10,11,12 Three other types of cells adjoining the capillary wall, vitally stained with methylene blue. Figure 10 shows the nucleus alone, the cell outline being difficult t o trace. X 1380.

The Rouget cell: (a) Focusing on a cross section of the cell to show its relation. ship to the endothelial wall. Endothelial nucleus shown a t the left. (b) A more dorsal aspect of the same cell. Vital staining with methylene blue.

Fig. 13

X 2300.

94


6. A type of cell, resembling the preceding adventitial cell but which is smaller and less numerous, was seen on the capillary wall (fig. 12). The cells are about three-fourths the size of the endothelial cells, and have an opaque cytoplasm which stains heavily with methylene blue.

7 . There remains one other type of peri-capillary cell which resembles the description of the Rouget cell morphologically. These cells are somewhat larger than the endothelial nuclei, are very granular, and adhere closely to the endothelial wall (fig. 13 a and b). They usually have an elliptical outline, although frequently exhibiting side processes which resemble short pseudopodia rather than long, thin, cytoplasmic processes. They take up methylene blue readily and, within 10 to 15 minutes, assume a dark granular color. These cells were not observed to wander.

On cell types 1, 2 , 3 : The cells reacted by an instantaneous withdrawal of their processes, accompanied by a rounding up and an increased opacity of the cell, as shown in figures 14, 15, 16. No effect upon the underlying endothelium was ever found. These cells could be picked off from the capillary wall without affecting the endothelium.

On cell types 4, 5,6 : These cells showed no visible response to mechanical prodding.

On cell type 7 , the ‘Rouget cell’: There thus remained one other cell type which, as Vimtrup has claimed, should, by its contractility, affect the diameter of the capillary lumen. These cells are flattened out on the capillary wall and are difficult to dislodge never being loosened entirely by simple prodding. When prodded, they changed shape, rounded somewhat and slowly withdrew their processes, although they were still attached to the capillary wall (fig. 17). The pull exerted by the reacting cell had no effect on the endothelium and no accompanying endothelial constriction was ever noted over a period of 5 t o 10 minutes. In the above experiments, to stimulate the peri-capillary cell alone, the microneedle was brought up from the side at an angle to the wall and was

D(b). Effect of mechanical stimulation.


drawn lightly over the cell surface. Caution was taken not to touch the endothelium.

Both the peri-capillary cell and the capillary wall could be stimulated by rubbing the needle along the length of the

I4

Figs. 14,15,16 Stimulation of different cells whose processes touch the capil- No

Fig. 17 Careful stimulation of Rouget cell alone with tip of microneedle.

lary wall. effect upon the endothelium.

Only cell reacts. Capillary wall is unaffected. X 1380.

They withdraw their processes and move away from the wall. X 1380.

cell and including the endothelial wall in its path (fig. 18 a). This procedure caused both to react at the same time (fig. 18 b). The cell rounded up, became thicker and more opaque, while the underlying endothelium constricted down. Now, by further manipulation of the peri-capillary cell alone with the point of the needle, the cell slowly flattened out again.

97 MICRO-MANIPULATION O F CAPILLARIES

However, as seen in figure 18 c, the endothelial wall constriction remained unaffected.

I n the nictitating membrane of the frog, where the adhering cells are large, stimulation caused the cells to assume various irregular shapes but had no effect upon the capillary diameter at those points.

To insure the complete separation of the responses of the peri-capillary cell from those of the endothelial wall, experi-

Fig. 18 (a) Stimulating both the endothelium and Rouget cell simultaneously. Endothelium constricts and wrinkles, while the Ronget cell tends t o round up. (b) If the broadened Rouget cell is now stimulated alone, the cell slowly flattens out. (c) The endothelial constriction remains. X 1380.

ments of the following type were performed. With a sharp microneedle the peri-capillary cells were pulled off from the wall without affecting or injuring the endothelium (fig. 19). Removal was much more difficult than had been the case in the previously mentioned cell types. Unless removed completely from the capillary wall, they flattened out again when released and slowly assumed their former position. If the naked endothelium (after removal of the peri-capillary cell) was then stimulated, the capillary wall responded by a localized constriction.


An endothelial constriction was produced by stimulating the endothelial wall definitely beyond the region where a peri- capillary cell was adhering to it. The wall wrinkled in the stimulated region except where the peri-capillary cell ad- hered. When the cell was removed with a microneedle, the part of the wall originally underlying it also wrinkled. This suggests that the cell may act as a supporting element. This fact also demonstrates a striking difference between the Rouget cell and other peri-capillary cells. When the endothelial wall is stimulated in the region where any of the other peri-capillary cells are adhering to it, the ensuing constric-

Fig. 19 The Rouget cell being pulled off from the wall with the t ip of a fine microneedle without affecting the underlying endothelium. The naked exposed endothelium can then be stimulated, (d) giving rise t o a typical endothelial constriction. X 1380.

tion of the wall carries the adhering cell down with it. In the case of the Rouget cell, the constriction is retarded by the presence of this more resistant peri-capillary cell on the endothelial surface.

E . Endothelial wall. (a) Silver nitrate staining of endothelium in the living mesentery. A method to demonstrate the capillary endothelial cells without interfering with the streaming blood flow was developed. With a micropipette a fresh 10 per cent solution of silver nitrate was introduced into the mesentery at a distance of about 40 to 50 p from the capillary wall. The advancing outline of the edge of the silver nitrate drop could be watched issuing from the mouth


of the micropipette. As soon as the edge of the spreading drop came into contact with the capillary wall, the micropipette was withdrawn. The outlines of the endothelial cells appeared first as a series of fine granules deposited in a linear fashion. Within 1 minute the endothelial cell outlines ap- appeared as solid black lines. The blood flow, which was momentarily arrested when the silver nitrate came into contact with the endothelial wall, soon resumed its streaming. I n dilated capillaries the cells were broader and more uniformly outlined, while in contracted vessels they were narrower and raggedly outlined. At the branching points of the capillaries, the cells are usually broader and irregularly arranged.

Within 5 minutes after applying the silver nitrate, the endothelial nuclei, which were normally visible only on the sides of capillaries, began to show up. By clearly outlining the endothelial tube and the endothelial nuclei, it was much easier to differentiate them from the adjoining peri-capillary cells.

The endothelial nuclei faded in 15 minutes, and, after 20 minutes, the endothelial cell outlines also faded. Often, if too great an amount of silver nitrate was injected into the tissue, the stained capillaries soon went into stasis. Instead of recovering, these regions assumed a brown coloration with only the heavy black endothelial outlines showing.

E (b). Stimulation of the endothelium. Effect of light: I f a mesentery preparation, in which the capillaries were constricted, was allowed to remain for 10 to 15 minutes with the microscope illumination striking it, capillary dilatation always took place. The dilatation did not always produce a uniform outline, since certain regions of the vessel remained partially constricted. These constricted parts had peri-capillary cells adhering to the outer surface of the endothelium. By stimulating these cells, they partially withdrew from the capillary wall. The underlying constricted endothelium remained so for about 1 minute, and then slowly dilated to the same extent as the neighboring cells.


Manipulations with needles : The endothelium is definitely contractile. The prodding of any endothelial nucleus chosen a t random usually brought about a constriction of the capillary wall in that region (fig. 20). Some of the nuclei reacted more readily than others-that is to say, in curved and wrinkled capillaries, the nuclei reacted more readily to mechanical stimulation (figs. 21, 22). In capillaries with more uniform contours, repeated proddings are necessary to bring about contractions in these cells. Immediately after stimulation, the endothelium in the vicinity began to wrinkle, and within 15 to 20 seconds had bulged into the lumen of the vessel. The reaction was strictly localized and has never been observed to spread.

If the endothelial wall was pushed inward with the broad side of the microneedle, the elasticity of the wall caused it to resume its former shape with no visible contraction. This is a passive phenomenon, without an actual stimulation of the endothelium. However, if a cell was gently pushed inward with the tip of a microneedle a t the bulging endothelial nucleus, a definite constriction was obtained (fig. 23). As the needle was withdrawn, the wall relaxed with it, except at the point where the needle was in actual contact with it. Here the endothelial wall constricted, showing fine ridges or folds on its surface.

The unequal, separate stimulation of different endothelial nuclei produced an irregularly outlined appearance of the vessel. Uniform stimulation, obtained by rubbing the needle along the length of the vessel, gave the contracted capillary a smoother contour. Contractions, involving whole lengths of a capillary, completely closed the lumen of the vessel only at its ends. Along the mid-length, constriction was only partial, beginning a t the time of stimulation and slowly con- tinuing thereafter, but never to the same extent as at the ends of the vessels. This was due to the fact that the capillary branches are given off at an angle from the precapillary source, and hence have an irregular arrangement of the endothelial cells in this region. When the endothelial cells in this


junction region contracted, their arrangement produced a marked constriction of the wall. A somewhat analogous situ- ation is found at the arteriole-precapillary junction. Here the junction of the two vessels was always surrounded by two or three muscle cells which seemed to be connected with the I a

20 jii

/-- ,."-

c - a

4- - 6 21

Fig. 20 Mechanical stimulation of the endothelial nucleus causes a localized constriction. Endothelium wrinkles, showing folds in its surface. Endothelial nucleus bulges into lumen of vessel.

By rubbing the tip of a microneedle against the capillary endothelium, a constriction in the stimulated area was induced.

Stimulating endothelial nucleus in the bend of a capillary causes a constriction to appear with endothelial bands or lines showing.

X 1380. Fig. 2 1

Fig. 22 X 450.

X 1380.


muscle layer surrounding the arterioles. When the arteriole was mechanically stimulated, it reacted by a severe contraction. This contraction spread to the two or three muscle cells surrounding the junction point, closing the region. The contraction did not spread beyond this point. Thus, only one end of the vessel was closed, while the remainder of the precapillary was still open.

a

NeGk

withdrawn CS

+

Fig. 23 Pushing the capillary wall inward with the tip of the microneedle. The wall relaxes, but remains constricted and shows folds in

X 1380. Remove needle. its surface at the point where the needle had been in contact with it.

When the contracted capillaries were gently stimulated by rubbing a microneedle along their length, a dilatation of the vessel took place. In some cases only a partial dilatation resulted, while in others the tremendous increase in capillary diameter was accompanied by a condition of stasis in that vessel. Both the contracted and dilated capillaries, therefore, reacted to mechanical stimulation, with contracted capillaries requiring gentle stimulation, while dilated capillaries needed a series of repeated stimuli.


DISCUSSION

The adherents to the ‘Rouget cell’ hypothesis have the work of two investigators to fall back upon. Vimtrup’s (’22) evidence was demonstrated chiefly upon the excised nictitating membrane of the frog. It was necessary for him to stimulate the tissue for 2 to 3 minutes before any capillary contractiori took place. This, in itself, was an abnormal procedure. Dur- ing the contraction phase, induced by electrical stimulation, the tissues became more opaque and the various elements were difficult to distinguish from one another. This again did not permit accurate observations to be made. Electrical stimulation is not localized. Hence, both the adventitial cells and the endothelium would be stimulated simultaneously. Whether one or both the stimulated elements were responsible for the ensuing contraction could not be demonstrated con- clusively by this means. Tannenberg (’25) also claims to have demonstrated muscular peri-capillary cells in the mesentery of the rabbit.

The data offered in the present investigation shows no evidence for the existence of a contractile peri-capillary cell, and supports the view that the endothelium is the contractile element in the capillaries of the frog. The identification of the cellular elements stimulated was confirmed by histological methods.

The methylene blue evidence indicated a distinct difference between the typical muscle cells stained on the arteriole, precapillary wall, the venule wall, and those cells encircling the capillary wall. The cells were morphologically different, took up the stain at different times, held their coloration f o r different periods and stained with different intensities. There was no gradual change in the character of the muscle cells found on the arterioles and precapillaries. The only change observed was the breaking up of the continuous muscle sheet around the larger vessels, first into separate bundles of cells, and finally into separate closely spaced cells. With the branching off of the capillary, the muscle cells abruptly dis- appeared. In the arteriole and precapillary wall typical

THE ANATOMICAL RECORD, VOL. 59, N O . 1, A N D SUPPLEMENT

104 BENJAMIN W. ZWEIFACEE

muscle cells, lying at right angles to the long axis of the vessel, were brought out by the methylene blue stain. These were of an elongated elliptical shape and had a prominent, granular nucleus. The cells never exhibited cytoplasmic side-processes. Numerous, different, lightly stained cells encircled the capillary wall. There seemed to be no definite arrangement of these peri-capillary cells, and no morphological difference between those cells adhering to the capillary wall and the connective tissue cells in the vicinity.

Still another type of cell was found adhering to the inner surface of the endothelial wall. It was almost impossible to distinguish these cells from endothelial nuclei and from some of the peri-capillary cells adhering closely to the capillary wall. Careful observation at 1500 x magnifications showed that these cells had a more granular cytoplasmic content, were slightly larger than the endothelial nuclei, and moved slowly. The cells can easily be confused with the endothelial nuclei. Sandison mentioned that the endothelial nuclei wander from time to time and could be seen changing position in the capillary wall. It is possible that he was observing these cells which wander very slowly. It is also possible that these were the cells that previously had been observed to migrate into the blood stream, simulating the division of endothelial cells to give rise to free cells in the blood stream. It was only after mechanical prodding with the microneedle that their true nature could be brought out.

There are three possible types of cells whose nuclei appear prominently in the capillary wall, namely, the actual endothelial cells, connective tissue cells lying on the capillary wall, and cells adhering to the inner wall of the capillary. Simple observation alone could not always distinguish these types from one another.

Mechanical stimulation of the arterioles and venules produced a diffuse contraction of these vessels. The contractions always spread in the opposite direction to that of the blood flow. Localized constrictions and dilatations of these vessels could be induced by stimulation of single muscle cells.


Mechanical stimulation of every type of peri-capillary cell brought about no visible effect on the capillary wall. The cellular elements vitally stained by methylene blue were closely investigated as to structure, ramifications and char- acteristic locations. They were mechanically prodded, pulled, and irritated with the microneedles, both in the stained and unstained specimens. In no case did their stimulation produce an effect upon the underlying endothelial region. In instances where the cells were successfully removed from the capillary wall, the underlying endothelium maintained its normal tonicity and did not show signs of collapse or injury. Follow- ing this, stimulation of the naked endothelium gave rise to a constriction of the capillary wall in that region.

This evidence shows that the peri-capillary cells (including the so-called ‘Rouget cells’) are not muscular in nature. Stimulation of these cells produced no accompanying constriction of the underlying capillary wall. However, stimulation of the typical muscle cells on the arterioles, precapillaries and venules produced a marked constriction of the underlying wall. The only difference between the so-called ‘Rouget cell’ and the peri-capillary cells of other types is that in many instances the Rouget cell acts as a supporting element in the capillary wall.

The endothelial cells could be readily stimulated by prodding their nuclear thickenings whereupon the cell would contract. Each endothelial cell was capable of contracting inde- pendently of its neighboring cells. The stimulated cell drew its ends closer to the nuclear thickening, which bulged into the lumen.

The endothelial wall has a definite tonicity. Stimulation of the vessels that were comparatively straight (smoothly outlined), and with a normal rate of flow in them, did not always produce a constriction. Marked stimulation, or repeated gentle stimulation, induced a constriction of the vessel in that region. I n vessels that were partially constricted and had an irregular outline, stimulation brought about a marked dilatation. In contracting, the outline of the capillary is in-


fluenced by fibers, adhering to its outer surface, which hold back certain regions of the vessel and give rise to a varicose appearance.

Constriction was not the only response to mechanical stimulation. If contracted capillaries were gently stimulated with a microneedle, they dilated. This reaction was brought about quite readily. On the other hand, dilated vessels reacted only to repeated stimulation. Their walls had lost most of their tonicity and were much more susceptible to mechanical injury than were contracted vessels.

CONCLUSIONS

The conclusions to be drawn from the preceding experimental data are that in the frog the endothelium itself acts as the contractile element bringing about changes in the capillary diameter, with its functional activities being somewhat obscured by the presence of numerous connective tissue cells adjoining its outer surface, by the restricting connective tissue strands preventing unimpaired relaxations and dilatations, by the irregular arrangement of the endothelial cells themselves, and by the curved, tortuous course that these capillary vessels take. N o adjoining structures were found to be factors in the functional relaxation and contraction of the capillaries.

SUMMARY

1. The capillaries observed and manipulated were those in the tongue, the nictitating membrane, the mesentery, and the intestinal wall of the frog.

2. I n the mesentery and intestinal wall, the layer of smooth muscle cells encircling the arterioles and precapillaries abruptly ends at the precapillary-capillary junction, and reap- pears on the venule after it has been surrounded by a connective tissue layer about three to four times the thickness of the endothelium.


3. Upon mechanical stimulation of the arterioles, precapillaries and venules, a contraction is induced which spreads in the opposite direction to that of the blood flow. Localized stimulation of single muscle cells causes a localized constriction of the underlying wall. 4. No peri-capillary cell was ever found which resembled

morphologically the typical muscle cells, or which showed similar staining reactions to those cells. All of the peri- capillary cells closely resemble the cells in the surrounding connective tissue.

5 . The so-called endothelial lines or bands are due to folds in the endothelium proper. Occasionally, the processes of adventitial cells are found lying in these folds but they play no part in their formation.

6. Prodding peri-capillary cells of any type with the microneedle causes a rounding up of these cells, with no effect on the capillary wall, unless the capillary endothelium is also touched. The cells can also be picked off from the wall with the tip of a fine microneedle without injuring the endothelium.

7. Cells are found adhering to the inner surface of the capillary that are difficult to distinguish from endothelial nuclear swellings. They are set loose into the blood stream when the overlying endothelial wall is prodded.

8. Mechanical stimulation of the endothelium, especially at the nuclear nodes, causes a localized constriction of the capillary wall with the appearance of ridges or folds on the surface, and accompanied by a thickening and bulging of the endothelial nucleus into the lumen of the vessel.

9. A widespread and gentle mechanical stimulus along the length of a contracted capillary brings about a dilatation of the vessel in that region, while a number of repeated mechanical stimuli along the length of a dilated capillary is necessary to produce a contraction of the vessel.


LITERATURE CITED

BENSLJCY, R. R., AND B. VIMTRUP On the nature of Rouget cells in the capillary. Anat. Rec., vol. 39, p. 37.

CLARK, E. R., AND E. L. CLARK Relation of the Rouget oells t o capillary contraction. Am. J. Anat., vol. 35, p. 265.

1932 Observations on living preformed vessels as seen in the transparent chamber inserted into a rabbit’s ear. Am. J. Anat., vol. 49, p. 441.

1928

1925

FEDERIOHI, H. 1928 Blood vessels of annelids. J. Exp. Zool., vol. 50, p. 257. KROQH, A. 1920 Studies on the capillarimotor mechanism. I. Reaction to

stimuli and innervation of the blood vessels in the tongue of the frog. J. Physiol., vol. 53, p. 399.

1929 Anatomy and physiology of the capillaries. Yale University Press.

MARCHAND, F. Ueber die Contractilitiit der Capillaren und die Adventitia- zellen. Munch. med. Wochenschr., Bd. 70, S. 385.

ROGERS, J. B. 1932 Observations on the peri-capillary cells of the rabbit mesentery. Anat. Rec., vol. 54, p. 1.

ROUQET, C. MBmoire sur le developpement, la structure, et les proprietes physiologiques des capillaires sanguins et lymphatiques. Arch. de physiol. norm. et pathol., Paris, T. 5, p. 603.

1879 Sur le contractilite des capillaires sanguins. Arch. de physiol. norm. et pathol., Paris, T. 88, p. 916.

Observations on the circulating blood cells, adventitial cells (Rouget and muscle cells), endothelium, and macrophages in the transparent chamber of the rabbit’s ear. Anat. Rec., vol. 50, p. 355.

1932 Contraction of the blood vessels and observations on circulation in the transparent chamber of the rabbit’s ear. Anat. Rec., vol. 54, p. 105.

1926 Over het Voorkomnen van de Cellen van Rouget op den Wand van de Capillairen in het Oog van der Mensch. Dissertation, Groningen, p. 1.

TANNENBERQ, J. 1925 Ueber die Capillartatigkeit. Zeitsch. f . allg. Path. u. path. Anat., 36, Erg. heft. 374.

VIMTRUP, B. 1922 Beitrage zur Anatomie der Capillaren. I. Ueber contractile Elemente in der Gefasswand der Blutcapillaren. Zeitsch. f. Anat. u. Entwickelungsg., Bd. 65, S. 150.

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SANDISON, J. C. 1931

SCHALY, G.

a micro-manipulative study of blood capillaries

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