The Cleveland Medical Gazette, Vol. 1, No. 5, March 1886

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.

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.