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.”