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Studies of vacuoles in the colloid of thyroid follicles in living mice.

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Department of Anatomy, Univepsity of Pennsylvania, Philadelphia
I n stained microscopic sections of fixed thyroid gland it is
common to find vacuoles at the periphery of the colloid or
distributed at random in the body of the colloid. Opinion is
divided on the significance of these. Vacuoles appear from
time to time in the colloid of living thyroid follicles transplanted into transparent chambers installed in the rabbits
ear (Williams, '39) and one might therefore suppose that they
represent a normal part of the histological picture. They are
frequently so considered although some interpret them in
sections as shrinkage artifacts. I n seeking t o extend observations on living thyroid follicles to other animals than the
rabbit and in other ways than by transplantation I obtained
data which indicated that certain vacuoles are not found in
living intact mouse glands free from injury but do occur
following experimental procedures.
The method used in this study was based on the fact that
the isthmus of the thyroid gland in small mammals is very
thin and can be illuminated through the trachea by some of
the various light-conducting substances now available, eg.,
Lucite, after the fashion of Knisely ( '36). The procedure will
be described in detail in another place. Essentially it is as
follows: The trachea of a white mouse under Nembutal anaesthesia is exposed and a glass cannula inserted therein through
an incision made as low in the suprasternal notch as possible.
This acts as a tracheotomy tube, permitting the animal to
breathe during the observations. The isthinus of the gland,
which is frequently only one layer of follicles thick, is covered
with paraffin oil. The first ring of the trachea is then incised.
Into this opening the tip of the illuminator is introduced and
disposed directly under the isthmus. A 6-volt Bausch and
Lomb lamp supplies adequate light for a magnification of
X 1000, the light being passed through 3 inches of water and
2.5 inches of oil. An 8 mm. apochromatic objective and 15 x
compensating ocular, magnification X 300, is adequate for
most studies. A special long working distance oil immersion
objective may be used but this precludes all manipulative
procedures and is used only occasionally. When certain precautions a s to temperature and moisture are taken such a
preparation will last for 1 2 hours, or perhaps longer, without
evidence of damage to blood vessels or follicles. It is necessary to repeat the dose of Nembutal about every 3 hours.
Should there be any objection to the use of a drug as an
anaesthetic agent, recourse may be had to Swenson's ( ' 2 5 )
method of cerebral compression to render the animal unconscious. Micromanipulations are carried out by an excellent
straight thrust machine with three-way adjustment which was
perfected by Dr. A. N. Richards for canalyzing individual
renal glomeruli and which he kindly loaned me.
When so prepared individual thyroid follicles with their
original nerves and blood vessels intact and uninjured can be
studied microscopically for several hours. The morphology
of the isthmus varies from animal to animal. I n all specimens
the follicles appear sharp in optical section but in a few the
arrangement of parts is so favorable that individual cells of a
follicle can be examined in detail in three dimensions. The
true and false capsules are left intact as further protection t o
the gland. These capsules are very thin and do not interfere
with microscopic studies but they, together with the respiratory movements, have so Par prevented satisfactory photomicrographs.
When first observed and thereafter in the absence of experimental procedures and if temperature and humidity remained
proper, only occasional vacuoles were seen in the colloid, irre-
spective of whether the colloid was increasing o r decreasing
o r remaining stationary. Most follicles had none, but a few
follicles in every animal studied (fifty animals were used)
contained each a single vacuole free in the colloid. These
vacuoles were uniformly round and about the size of a white
blood cell. They could be moved around in the colloid by a
micro-needle inserted into the lumen. When depressed to the
deepest p a r t of the colloid they slowly rose to the most superficial part. When compressed against the thyroid cells they
Fig. 1 Camera lucida tracings of living thyroid follicles illustrating, 1. A racuolc
found normally in the colloid. The circles a t the right represent its gradual diminution in size and eventual disappearance when punctured. 2. A common distributiou
of vacuoles following exposure of the gland t o some abnormal procedure. 3. Vacu.
oles occurring in tho colloid of a follicle (B) adjacent t o one (A) receiving
injection of water. 4. The black represents a column of colloid withdrawn from
a follicle, the needle being left in the follicle. A break occurred in the column,
represented b y the white spot. This vacuole was incorporated in the colloid without
change in position of the highest meniscus in the same manner as the vacuole in
no. 1 disappeared. It suggests t h a t the colloid may undergo vacuolation without
cellular activity. 5 . Illustrates, A, a vacuole produced in a cell following introduction of a needle and B, extrusion of the vacuole into the colloid. The changes
in the apical membrane produced by the latter process were not observed. X 350.
were distorted but could not be broken in two. They returned
to a round shape when the pressure was removed. I n some
cases the pressure caused them to disappear. When punctured with a sharp needle such a vacuole rapidly diminished
i n size and disappeared, leaving no trace in the colloid
(fig. 1, no. 1). Occasionally they disappeared spontaneously
in the same manner as when punctured.
Attempts were inade to draw one of these vacuoles into a
capillary tube. This could not be done even with the largest
tube (10 p outside diameter) which could be successfully introduced into the follicle. Such a n effort resulted i n distortion
of the vacuoles and occasionally caused their disappearance.
Frequently when colloid was being drawn into a tube the
column of colloid became broken as shown in figure 1, no. 4.
The space intervening was incorporated in the column of
colloid without change in position of the highest meniscus in
the same manner as the vacuoles illustrated in figure 1, no. 1
disappeared in the colloid.
If a gentle stream of cool air is directed across the gland,
or injections of water, Ringer's solution o r certain dyes are
made into the colloid, or if one makes observations some time
after cessation of heart action, vacuoles then appear in the
colloid (fig. 1, no. 2). The process of formation was not observed. 'Cf7hen few in number they were located at the periphery of the colloid next the cells but were not, apparently,
attached to them and all those which were tested could be
freely moved around with a micro-needle. They varied in
number in different follicles froiii four or five to so many they
c~oulclnot be counted. A t times they were distributed only a t
the periphery of the colloid, while at other times the lumen was
completely filled with them. TTheri punctured with a needle
they disappeared abruptly and left no trace in the colloid.
Occasionally they disappeared spontaneously. Those which
appeared after death of the animal were seen about 2 hours
after cessation of heart beat. Prolonged observation of the
dead aninla1 indicated that after 3 hours these vacuoles disappeared arid did not recur. After the stoppage of blood flom
all cytological details became obscured, the follicular wall
diminished in thickness and the colloid became more fluid.
Although this vacuolation was a common finding after the
above procedures, not all follicles reacted in this manner and
there were no constant factors as regards the size, shape or
location of a follicle or consistency of colloid which mnuld
enable one to predict in what follicle they would or would
not form.
Fixed scctioiis were made of the specimens examined in the
living state and cornpared with scctioiis made from glands
which had not been studied in the living. Many follicles from
the experimental animals showed vacuoles of a type not seen
in the controls and which a r e believed to be the ones seen
after various experiniental procedures in the living animal
(fig. 2). I f that is SO then study of tlie sections indicates that
Fig. 2 Staiiied sections from a thyroid g l m d which liad been subjected to slight
cooling in the living animal for 4 hours. It illustrates types of vacuolation not
seen norinally. Fixation, Zenker-forinol. Stain, iron liaematoxylin ; 6 p sections.
A, vacuolatioii similar to no. 5 , figure 1; B and C, vacuolatioii similar to no. 2,
figuie 1. B suggests acute liquefaction of the cytoplasm with cellular components
in tho iacuole. C is the next srction of tlie series after R. I n the section following
C, not shoivii, the vacuoles were gone and the cclls near the vacuoles resembled
those i n C . E aiid C illustrate a localized response involving s e v r l a l cells. I), E
and F illustrate a more general peripheral colloid vacuolation. These are siinihr
t o tlic ~ a c i i o l e sin no. 2, figure 1. The intense staining of the colloid in A, B and
C is tlic coiunioii reaction in normal mice, although in a few follicles the colloid
stains gray with iron haeimtoxylin. After cooliiig of the gland or a variety of
other experimental procedures many follicles have a gray staining colloid and
fewer have a black colloid. Original magnification x 5.50. x 367.
they iiiay be produced in most cases by an acute liquefaction
of the cytoplasm followed by extrusion of it into the follicular
lumen arid in others by changes in tlie colloid independent of
cellular activity.
A sharp glass needle was introduced about half way into
tlie wall of a follicle arid left in place f o r a time. The apex of
the cell involved was clearly visible. Within this apex a vacuole formed and was discharged into the lumen (fig.1, no. 5).
This droplet was small, higlily ref ractile, and when punctured
disappeared quickly.
I f water is injected into a follicle, cells of adjacent follicles
a r c a t times affected but oiily at the places where the follicles
nieet. The effect is cliaruc rizcd by the appearance of‘ small
droplets at the distal ends of the cells (fig. 1, no. 3 ) . These
become iiicorporatccl in tlie colloid.
Attempts were made to produce vacuoles artificially. The
tip of a 1:iicro-pipette, outside diameter 5 p, was introduced
just tlirocgli the wall in a number of nicdiurn sized follicles
ltnown t o be in various stages of activity. Colloid in different
amounts from 20 C U . ~to 500 cu.p was drawn into the tube a t
various rates, but no vacuoles could he produced at the periphery of the colloid. The only result u7as a reduction in the size
of the follicle. I n other follicles water, Ringer’s solution and
colloid fixmi adjacent follicles were in turn injected in relatively large amounts, in others in small amounts, and then
drawn off a t various rates but no vacuoles formed. Water or
Ringer’s solution or colloid from another follicle could not be
so injected a s to form a vacuole. They mixed immediately
with the colloid. Paraffin oil will form a droplet and does not
mix wit11 the colloid-remaining unchanged throughout the
period of observation. A i r can bc injected in any quantity
desired. I t is absorbed quickly. An air filled vacuole has R
refractility entirely different from anything seen normally
in living follicles. The disappearance of such a vacuole occurs
in a rnamic~rsiriiilar to those first described, although at. a much
reduced tempo-a small air vacuole disappearing in approximately 5 minutcs, zvhile the others disperse in less than 5
The foregoing observations indicate that extensive vacuolatioii of the colloid is not present normally in the thyroid glands
of living white mice. That it occurs to different degrees i n
various follicles and not at all in others following sonic
manipulative procedure or after death indicates an important
difference o r differences between follicles, a condition which
has long been recognized. There is no evidence here as to what
those differences are. Spectroscopic analysis of pure samples
of colloid from individual follicles is being done and may
afford some evidence a s to the nature of those differences.
There were two ways in which vacuoles formed. I n one
changes probably occurred in the physical state of the colloid
since there were no obvious cell involvements and in the other
there was acute liquefaction of the cytoplasm and extrusion
of it into the colloid. The first method being coninion after
death and the second during life. The latter is similar to the
response seen in rabbits following large doses of sodium iodide
( MTilliams, '39). The acute liquefaction of the cytoplasm and
its formation into droplets which a r e in turn incorporated in
the colloid seems to be ail exaggeration of what occurs normally in the gland, namely, secretion into the lumen.
I n sectioned mouse thyroid gland if there is obvious retraction of the colloid away from the cells it is likely that this
represents shrinkage of the colloid but when the periphery of
the colloid is serrated o r vacuolated it may be assumed that
the vacuoles mere filled with fluid of a different consistency
than tho rest of the colloid, but that none of these vacuoles
was present in the normal, undisturbed state, they being an
expression of cellular 01' colloid reaction to abnormal procedures, chemical or manipulative.
The formation of vacuoles in follicles adjacent to one receiving injection of water suggests that follicles are not necessarily independently functioning units receiving their stimuli
from the blood stream and possibly from nerves, but may be
influenced directly by the activity of surrounding follicles.
This has a n obvious bearing on the formation of adenomata
in the gland. Other pheiioniena suggesting this possibility
were seen in rabbit folliclcs ( IVilliams, '39).
Follicles in the isthmus of the thyroid glands of fifty living
mice were studied microscopically by transillumination
through the trachea. Under nornial conditions n o vacuoles
were present in the colloid except for a few distributed here
arid there, o ~ i cto a follicle. These a r e considered t o be the
ones seen in sections deep in the colloid. Their origin was not
observed. Because of their scarcity and because follicular
changes generally occur in their absence they a r e considered
of no special importance.
Followi~iga variety of rxpcrimental procedures or after
death of the animal vacuoles appear a t the periphery of the
colloid i n some follicles, in others throughout the colloid, while
other folliclcs a r e unchanged. Most of these vacuoles a r e produced by colloid liquefaction of tlic cytoplasm and extrusion
of it into the lumen. I n some cases, especially after death,
they may he produced mitliout cellular activity by a separation of thc colloid into two phases. They contain a thin, watery
material vhich loolrs a i d behaves quite differently from the
contents of the few vacuoles seen in normal glands. They a r e
generally produced only by certain of the cells of a follicle.
Since they do not OCCIII' normally in the mouse, they a r e considered to be i n most cases an abnormal secretory response to
a n unusual situation. Their presence is an index of cellular
and colloid cliff erencc of uncertain significance.
Thyi*oidfollicles may be affected by adjacent follicles without mediation of the nerve o r blood supply.
No evidence lias been found indicating that vacuoles occur
in the colloid of rabbit or mouse thyroids a s the result of
retraction of cells which have absorbed colloid.
KNISELY,M E L V I N 1%. 1936 A method of illuminating living structurrs f o r microscopic study. Anat. Ref., vol. 64, pp. 4 9 9 4 2 4 .
S W E N S ~E.NA.
192.5 The use of ccrc4,r;tl aiiemia in experiincntal rmbryological
studies upon mammals. Anat. Rer., rol. 30, pp. 147-151.
WILLIAMS, ROY G. 1939 Further olwvvations on tlrr microscopic appearance
and brharior of living thjroid follicles in the rabbit. J. Morph., vol. 63,
pp. 17-51.
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living, colloid, mice, thyroid, studies, vacuoles, follicle
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