ORIGINAL ARTICLES.
TWO DISPUTED POINTS IN THE HISTOLOGY OF THE SUBMAXILLARY GLAND. MEMBRANA PROPRIA—NERVE-ENDINGS (WITH PLATE.)[[1]]
BY CHR. SIHLER, M. D., PH. D., CLEVELAND, OHIO.
Formerly Fellow of the Johns Hopkins University.
The submaxillary gland is of importance not only for its own sake, but because its anatomical connections and situation are such that it can be subjected to physiological experiment, and a number of important results have been reached which I may discuss at some future time.
[1]. Read before the Cuyahoga County Medical Society, January 7, 1886.
The results of my work are not only contradictory to the authorities in histology, but also do not harmonize with the requirements of prevailing physiological theories. Bringing this before you does not mean that I ask you to accept either facts or conclusions. I am fully aware of the difficulty of such work, the doubtfulness of the facts and the liability to error in the conclusion. But it is just possible that some of the younger members may fare as I did—have not all their time occupied by practice—and if I enlist the interest of any in this most important region I shall feel happy.
A number of years have passed since I undertook this question, and the work on the nerve-endings on muscle, which I had the pleasure to communicate to this society a few months ago, was undertaken more as a study, to make myself familiar with analogous structures, than that I expected to find anything new.
METHOD.
In the investigations on the nerve-endings I used the submaxillary of the half-grown cat, the calf, the ox and the puppy. The method followed was in the main that of Beale. In the case of the cat I injected the whole animal from the aorta with Turnbull’s blue, dissected out the gland, duct, and the nerves entering it. After breaking up the gland into pieces by aid of a needle, from the size of a bean to a pea, I placed it in a dish with a light cover, containing Beale’s carmine (carmine dissolved in ammonia and glycerine). I am in the habit of using a stronger solution than Beale’s. I suspect that the carmine I used in some of these stainings was adulterated with eosin, and that possibly this may have been of advantage. In some of my stainings I used a fluid prepared from cochineal; used ammonia in dissolving the coloring matter, and then added carmine besides. It is of the utmost importance to have no excess of ammonia present, otherwise the staining will be slow and imperfect. I have been staining with this method for years, yet I cannot say why the results differ so much. Some time ago I stained a frog for the nerve-endings in muscle and obtained the most beautiful results, but in the number of stainings I have made since (trying to follow the same method) I have not been by far as successful as then. In breaking up the gland I do not always separate all the pieces, but try to remove the connective tissue holding together the small lobules with the dull end of a needle, and then throw the coherent mass into the stain. In this way I procured a very perfectly injected and beautifully stained submaxillary of a half-grown cat, from which I made a number of valuable specimens. The material may remain in the staining fluid for weeks, and may be examined every two or three days to note how the staining is advancing. When the masses stained are large, or the whole gland is subjected to staining, of course the outer parts are more deeply stained than the inner ones, but it is at times convenient to have material of different depths of staining. After the process has continued long enough—the nuclei at least should be very distinctly colored—the material is transferred into a fluid containing glycerine five parts, water and alcohol each two parts, acetic acid one part. Here it may remain about twenty-four hours, and finally it is to be preserved in a similar mixture containing but a trace of acetic acid. I hold acetic acid of varying strength diluted with glycerine in high esteem in such investigation. It does two things, removes the superfluous stain and softens and clears up the connective tissue. Thus treated, the material is ready for examination.
The tissues thus prepared may be hardened in alcohol and sections cut, but this will not aid much in the investigation of the questions that interest us. For this purpose teasing and compression with the cover glass are mainly to be relied upon. It is to be commended to isolate one of the little lobules the gland is composed of, because thus we certainly have ready for examination all the elements making up the gland. The little root which connects the lobule with the rest of the gland will consist of the duct, vessel and nerves supplying the lobule. Such a lobule is broken up with needles and by compression between slides. All these manipulations are to be carried on in glycerine. When the fragments are small enough they are examined with lower powers. The ducts in well injected specimens can be recognized by the rich supply of vessels, the nerve-trunks by the arrangement of the nuclei.
After examining larger fragments with lower powers, smaller ones are selected, subjected to pressure with cover glass, and examined with higher powers. By compression with cover glass, pushing from side to side, one can isolate almost any structure. One can also then make sure whether a fine fibre is really in connection with any other structure, or only lying above or beneath the same.
The staining with the carmine will generally not attack the fine nerve fibres. To show these up I have used aniline blue dissolved in water. By these means I have brought out very plainly the nerve fibres, in nerve trunks, of considerable size, as well as those along vessels. I took the material prepared as described, broke up a little piece in fragments of the size of mustard seeds, and left them twelve hours in the blue fluid in a watch glass. Glycerine, if necessary, slightly acidulated, will remove the superfluous stain. I was very much pleased with the action of the aniline blue.
Notwithstanding its physiological importance there is among the authorities as yet no uniformity of opinion on the histology of this gland, save that it belongs to the racemose glands, and is made up of epithelial cells.
The question whether there is present a special membrana propria, enveloping the glandular epithelium, as Pflüger teaches, or a network of partly coalesced connective tissue cells, as Kölliker supposes, may not seem very important, but certainly such points must be cleared up before such questions as to how the nerves end can satisfactorily be answered.
This question, the importance of which will be admitted by all, is also a disputed one. In Stricker’s handbook Pflüger has, in the article on the submaxillary gland, given a detailed account of the mode of nerve-ending in this gland. According to his investigations it is of various kinds.
(1.) The medullary fibre approaches an alveolus penetrates this membrane, the axis cylinder breaks up into innumerable fine fibres, and these pass into the body of the gland cell. (2) Or the nerve passes into a pale cell provided with numerous processes, which in their turn form connections with the secreting cells.
These results of Pflüger have, however, not been accepted by all investigators. Thus Kölliker says on this question:
“The investigations on the nerve-endings in the salivary glands are evidently far from being concluded, nevertheless so much can be gathered from the work done that the nerve fibres are in more intimate relations with the glandular elements than has been surmised heretofore. What these relations may be I cannot say, for notwithstanding a very careful investigation of this gland it has not been possible for me to reach views definite and not open to doubt, although frequently enough I have seen fibres and threads of various kinds apparently come in contact with salivary cells. Further, regarding the drawings and descriptions of Pflüger, I must confess, having at the same time nothing but the highest esteem of this investigator, as well as of the care and accuracy he employed in this question, that these do not seem altogether convincing to me.”
MEMBRANA PROPRIA.
I find that the gland cells are enclosed by a membrane (agreeing altogether with Pflüger) which I look upon as decidedly homogeneous (histologically speaking, of course), not showing any structure; by no means being merely a reticulum of connective tissue corpuscles, as Kölliker suggests.
Further: This membrane is provided with nuclei of an oval form (generally), reminding me of the nuclei supplying the nerve sheaths.
Finally: This membrana propria sends out from the alveolar walls, processes, ensheathing—how complete I cannot say—the gland cells. I cannot otherwise explain the glistening lines, which we see in examining sections VI extending from the alveolar wall between the cells, resembling altogether the sections of the alveolar walls. To investigate this matter more accurately, I have stained sections with carmine, also with log-wood, and have found that the alveolar wall and these lines stain the same way. Further breaking up such a section by manipulation with cover-glass, one can obtain fragments of these processes, thus demonstrating that these lines are not merely optical illusions.
The nuclei of this membrana propria are situated on its inner surface and could be distinguished in the gland of the cat, mentioned above, not only by their more oval, oblong or elliptical form, from the more roundish nuclei of the gland cells, but just as plainly by the lighter red or pinkish staining which they take on the nuclei of the gland cells being stained a deeper red.
Compressing very small fragments of gland-substance (always of course in glycerine), by means of the cover-glass, beating and pushing it from side to side, thus bursting the alveolus and tearing the membrane and isolating it from the contained gland cells, one can prepare for examination greater or smaller pieces of this membrane. There are by such means obtainable fragments with all sorts of processes and fringes, short and long, many and few, which undoubtedly may remind one of “connective tissue cells.” (See fig. 4.) More convincing than these are membraneous pieces, which may be obtained of various sizes. (See figs. 5, 6.) I have a drawing of such a fragment, which, if enlarged as figs. 1 and 2, would cover over one-quarter of the page, and to which a number of nuclei, more than six, are attached. As a rule, this membrane will, by the manipulations with the cover-glass, not be spread out, but rather folded up, huddled together inclosing some of the gland cells, forming an indescribable nest.
I do not know if it will be necessary to mention in this connection that I consider these nuclei identical with the half-moon cells of Gianuzzi, and cannot help expressing my surprise at the physiological importance assigned to them; for they have been considered playing the role of mother cells, giving origin to glandular epithelium formed to take the place of cells used up in the process of secretion. The dark staining, which in section they seem to assume as well as the semilunar form, will be discussed later. Of course I look upon these nuclei as belonging to flat epithelial cells, as we find them lining nerve sheaths, and serous membranes, and consider them of the same physiological importance as the nuclei of such endothelial cells. A similar ensheathing membrane, or membrana propria, I find in the ducts; but here I have not been able to distinguish processes passing in between the cells. If present they must be exceedingly delicate, for it is not difficult to press out groups of cells from the ducts so that empty pieces (short ones of course) of such can be examined.
While the membrana propria of the ducts can be separated rather easily from the inclosed cells, it is different from the surrounding network of capillaries. It seems impossible to remove the capillaries from the duct-walls; while, in the gland-structure proper, among the alveoli, the separation of the capillary from an alveolus, can with little difficulty often be accomplished, so that a piece of capillary can be examined without any other structures lying above or beneath it.
BLOODVESSELS.
It was somewhat surprising to me, and not a little interesting, to find that the ducts have a decidedly richer blood supply than the gland substance proper, the meshes of the capillaries covering the ducts very closely indeed.
I will also call attention to the fact that, along the terminal portions of the ducts, no arteries or veins can be seen; and what is still more important, no free nerve-fibres can be discovered. Following the ducts outwards, as these attain larger dimensions, and especially where they give off side branches, we find arteries and veins accompanying them, and there will also be found along-side of them fine nerve-branches. So much is certain, that in that region, where arteries and veins are not to be seen, there are also no separate nerve twigs, and that here the union or coalescence of nerve and tissue which the nerves are to influence must have taken place. If one considers the fine meshes of the capillary network enveloping the ducts, and keeps in mind the abundance of nuclei belonging to the capillary, the membrana propria and the duct cells proper, he can imagine how difficult it must be to follow the course of the nerves through and among all these structures. That it has been impossible for me to separate capillary and membrana propria, has been mentioned.
NERVES.
If one were to ask the physiologist: What do you expect us to find in our anatomical investigations on the nerve-endings in the submaxillary gland, he would undoubtedly answer: “You will find an arrangement analogous, if not similar, to that in the striped muscle; the nerve-fibre will be seen to approach the alveolus, penetrate its covering, and the protoplasm of the nerve and the gland-cell will be found to come in contact, if not to coalesce.” And that is what has been found by Pflüger.
It has been my endeavor for some time to find something similar to the descriptions of Pflüger, but so far in vain, although many an alveolus has been carefully examined, flattened out and slowly crushed, so that each nucleus could be diagnosed, but never have I found anything which would remind me of the drawings by Pflüger in Stricker’s ‘Histological Handbook.’
In this matter I reasoned thus: If there are any such structures as specialized nerve-endings present in the glandular elements, we might look for an analogy either to the striped muscle fibres or to the capillaries, in both of which nerve-endings are known to be present. In the former case we have the so-called end-plate—the nerve fibre ends in a multi-nucleated mass—but as the muscle fibre, standing for a number of cells, is so different a structure from the gland-cells, we might, with greater probability, expect some such arrangement as we see on the capillaries. Here we see fine fibrils, provided with nuclei, exceeding the fibres two to four times in thickness, entwining the capillary, thus coming in contact with each of the cells composing the capillary wall. In the (submaxillary) gland there are similar structures to be influenced; at least in the capillary as well as in the gland, we have cells lying side by side to be influenced by nerves—hence we might expect similar structures.
Although the books in general have very little to say about the nerve supply of the capillaries, yet it is a fact, and can well be demonstrated, e. g., in the frog’s muscle, that such is the arrangement. As a rule, the fibril cannot so readily be detected, but the nuclei belonging to it are not difficult to recognize. A still more convenient and instructive locality is the base of the frog’s mouth, where everything is one plane, and where the nerve fibres can be followed to their ultimate ramifications.
Let us now see what evidence of nerve-endings we find in or on the alveoli of the gland, examining one, or more accurately, a part of one (the round eminence). We shall see what fig. 1 presents. We see there two sets of nuclei; one kind oval, longish, stained lighter than the rest, of a pink hue (fig. 1, m), and these a further examination will demonstrate to be the nuclei belonging to the membrana propria, which have also been described before. Besides these nuclei, fewer in number, there is another kind, making up the bulk of the nuclei, more rounded in form (fig. 1, g), and of a deep-red color, which, in their turn, will prove to be the nuclei of the gland-cells; and these two are the only elements or structures that I have been able to demonstrate in a glandular alveolus.
At one time I thought I had found in or on the alveoli the evidence of a network of fine nerves. Quite frequently one can observe longish, even spindle-shaped, often deeply stained, nuclei, which resemble very much those belonging to the fine nerve fibres. (Fig. 2, n, such nuclei are figured.) But so far, whenever I examined such a spindle-shaped nucleus more carefully, I found that it proved to be one of the nuclei belonging to the membrana propria, already described, and the form, as well as its dark color, was due to the fact that it had been examined edgewise. If, by manipulation, such a nucleus was turned over and compressed to present a flat surface, it would have an oval outline and be of a light-red color. Fig. 3 (a, b, c), shows the same nucleus as it appeared when seen edgewise, when seen from the flat and when seen in a position midway between these. Thus is also to be explained the demilune: If the section would strike a nucleus so that it would be seen only edgewise—especially if the membrana propria has a chance to roll up slightly—we should then have a deeply stained body, somewhat half-moon-shaped, the demilunes of the books.
So far, nothing but the nuclei have been mentioned, and the observation might very appropriately be made, that there might exist some exceedingly fine and delicate nerve fibrils without nuclei. Such a system of structures would, of course, be almost impossible to demonstrate with any degree of certainty. I have not, of course, sufficient reason to deny such an arrangement, and would not consider it improbable if we had not, in the capillaries of the frog’s muscle (and other structures) evidence certainly not in favor of such an assumption. The fine nerve fibrils supplying the capillaries are richly supplied with nuclei, although the cells which build up the walls of the capillary are by far more insignificant elements than the secreting cells of the gland. Certain it is that as yet there is no evidence of the existence of such fine non-nucleated fibrils, arranged either in the form of a network or a bundle of fine branches.
The examination of the alveoli not leading to any results as to nerve-endings—no structures being discovered there that might be called nervous—I attacked the problem from the other side. I took up the nerve trunks accompanying the ducts, and tried how far I could follow their divisions and branches, and can say that I have spent a great deal of time on this point. In speakings of the bloodvessels, the fact was mentioned that, where arteries and veins of any size run along-side of the duct, these are accompanied by nerve branches, i. e., bundles of nerve fibres, inclosed in a sheath, and these—at least in the cat, the calf and the ox—are made up (excepting, possibly, the very large trunks) of fine nerve fibres. Thicker or medullary nerves I have not been able to discover, even in branches of considerable size, consisting of, say six or eight fibres, and giving off smaller twigs containing one to three fibres. Fig. 7 shows such a nerve-trunk with side branches; neither in the main stem nor in the offshoots are there any medullary nerves discoverable.
If this observation is correct, we see that what has to be traced further, is not the more easily recognized medullary fibre, but exceedingly fine fibrils carrying in certain intervals nuclei, the only distinguishing feature. It will be admitted also (first) that it must be a matter of considerable difficulty to follow such indifferent looking structures, as delicate fibres. And (secondly), one cannot expect to find medullary nerves approach the alveoli, penetrate the membrane, come in connection with the secretory cells, etc., etc, as has been done by Pflüger. If the question should be asked how far I have been able to follow these nerves, I should say: Small twigs consisting of one to three fine fibrils, enclosed in a sheath branch off from a trunk, not much larger, and apply themselves to the membrana propria of the duct; their sheaths in all probability coalescing with that membrane. Here they have been seen to apply themselves to some bloodvessel and are now lost out of sight, being hid by all the many structures mentioned above, the nuclei of membrane, capillary, duct cells, and the fibre cannot so readily be distinguished from the membrana propria of the duct upon which it rests. But I do not wish to be understood that because the nerves have been lost out of sight, along-side of vessels, that they have their “ending” there, inasmuch as the network of bloodvessels is such a close one that the nerves cannot but help coming in contact with them. Fig. 8 shows such a fine nerve-trunk. By the aid of aniline blue the fine fibrils have been made quite plain. Such fine fibrils can by the same means be demonstrated in much larger trunks, as shown in fig. 7, and it can be shown that even such large trunks consist of nothing but these fine fibrils.
I have also seen, exceptionally however, fine nerve twigs run into the alveoli directly, but the nerves here were of the same nature as the ones described going to the duct. Fig. 9 shows a fine fibril passing on to an alveolus and running probably on to a vessel; the nerve fibre resting upon gland cells, also stained, could not with absolute certainty be traced to the vessel. There is a small break shown in the drawing.
As intimated, especially if the cells of the duct are still in situ, it is impossible to follow the fine nerve-fibres any further. By pressing out the cells from the ducts, one can obtain portion of the duct, with the capillaries belonging to them, but they are short pieces, and the nerve fibril is not easily discoverable.
But there remains one fact to be mentioned, which may throw light on the question, as to how the nerves end and which in my opinion might and does clear up the difficulty.
Examining the capillaries supplying the gland alveoli, one is able to find nuclei, not belonging to the capillaries themselves. This statement can be made for two reasons. In the first place, the majority of these nuclei have not the form of the nuclei of the capillary, and in the second place these nuclei are situated on the outside of the capillary wall. Whoever has examined the capillaries of the frog’s muscle knows that just such or the same nuclei belong to nerve-fibres, supplying the capillary wall. In the mammal of course these nuclei are smaller and not so conspicuous, nor can we procure pieces of capilliary of any length (the structures under examination here, not being in one plane.) Thus we cannot expect to have matters so plain here and easy of demonstration. After finding these nuclei, it was of course my desire to find if they were united by fibres. It must in this matter be kept in mind, that if there are such fibres present to which these nuclei belong, these may be expected to be in the most intimate relation with the capilliary wall, if one is to influence the other—and therefore not of ready and easy demonstration.
I have, however, succeeded in showing in a number of instances that such fibres exist. Figs. 10 and 11 show such a fibre uniting several nuclei. The shortness of these fragments makes a more perfect demonstration almost impossible.
If this observation is correct, we find on the capillaries the same (only finer) fibres provided with nuclei, which we have lost out of sight following the branching out of the nerves.
There is also some evidence of fine fibres in the membrana propria of the ducts, inasmuch as we find here nuclei along-side of the capillaries, these not being the nuclei of the membrane, but resembling altogether the nuclei of the nerve fibres.
Again, as stated above, I have not been able to find any such evidence of nerves in connection with the gland cells themselves. In case, however, such an arrangement or something similar existed, why should it have been impossible for me to find evidences of such as well here on the gland cells as on the capillaries; and should they not be more readily found in the gland than in the capillary, inasmuch as we have larger structures to deal with here in the gland.
The conclusion then regarding the nerve-endings to which my work has led me are these:
There were no special nerve-endings found in connection with the gland cells themselves. We find, however, upon the capillaries evidence of the same kind of fine nucleated fibrils, which we find to be present in the fine nerve twigs, as far as we can follow them in their course towards their final distribution. To explain the action of nerves on the process of secretion we would have to assume either that the nerves accompanying the capillaries also influence the gland cells, or that the glands are stimulated to activity by some changes in the condition of the capillary wall furnishing a greater amount of oxygen and nutritive fluids.
The results are partly negative, partly positive. The latter I wish to emphasize, believing it worthy of attention, as it may throw light on the physiology of the capillary wall, which seems to be altogether too much neglected. The negative I have offered, knowing well enough that such negative results must be accepted as final with much hesitation.
To any one feeling inclined to undertake investigation on this gland, I would make a few suggestions:
(1) Make sections through gland hardened in bichromate of potash, or alcohol, and stain with staining fluids.
(2) Compare in material prepared as above described, the vascular supply of duct and gland substance proper.
(3) Compare gland and duct cells.
(4) Find the nuclei of the membrana propria and compare the same with the nuclei of the gland cells.
(5) See if you can examine the same nucleus from different sides. Do you think that the demilune of books and the nucleus of the membrana propria might be the same thing?
(6) Prepare pieces of the membrana propria covering the alveoli. What is your opinion—is there a membrane or only a reticulum of connective tissue cells?
(7) Prepare pieces of membrana propria of duct.
(8) Can you find the nuclei of the duct, and other nuclei on the exterior of the capillaries?
(9) Can you find pieces of capillaries belonging to the alveoli, and do you find any nuclei on them?
(10) Examine the wall of the duct in injected and uninjected material, both stained.
(11) Follow the bloodvessels—can you find nerves on the walls of arteries?
(12) Of what structure do you find the root of a little lobule to consist?
(13) How do the nerve trunks differ from the fibrous envelope holding together all these structures?
(14) Differentiate between the nuclei of the nerve fibres and the nuclei of the nerve sheath.
(15) Examine nerve branches of various thicknesses—what do you find the nature of the nerve fibres to be? Do not forget the aniline blue and try other staining fluids.
(16) Can you find evidence in the alveoli of other structures besides capillary, membrana propria and salivary cells?
(17) How far have you succeeded in following the fine nerve twigs?
(18) By all means try different methods, different tissue, and compare the results. See what you can discover by simply teasing material treated with Muller’s fluid, and other stained and treated as I have described.
(19) Follow the course of the arteries, and note where the larger vessels pass into the capillaries.