STUDIES O F VACUOLES I N THE COLLOID OF THYROID FOLLICLES I N LIVING MICE ROY G . WILLIAMS Department of Anatomy, Univepsity of Pennsylvania, Philadelphia T W O FlGURE,S 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, 263 264 ROY G. WILLIAMS 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- 265 VACUOLES I N COLLOID O F THYROID F O L L I C L E S 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 -a43 I t I 4 5 A B 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. 266 ROY G. WILLIAMS 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 VACUOLES I N COLLOID O F T H Y B O I D F O L L I C L E S 267 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. 268 ROY C . WILLIAMS 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 seconds. VACUOLES IK COLLOID O F THYROID FOLLICLES 269 DISCUSSION 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). 270 ROY G. W I L L I A M S SUMMAHY AND CONCLUSIONS 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. LTTERATUltE CITED 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.