Topographic and Subcellular Anatomy of the Guinea Pig Gallbladder

Vol. 63, No. 5 Printed in U.S.A.

GASTROENTEROLOG Y

Copyright © !972 by The Willia ms & Wilkins Co.

TOPOGRAPHIC AND SUBCELLULAR ANATOMY OF THE GUINEA PIG GALLBLADDER JOHN

c. MUELLER,

M.D., ALBERT L. JONES, M .D. , AND JOHN A. LONG, PH.D.

Cell Biology Section, Veterans Administration Hospital, and Departments of Medicine and Anatomy, and the Scanning Electron Microscopy Laboratory, University of California, San Francisco, California

The normal guinea pig gallbladder was fixed in the filled and empty state, and examined with scanning and transmission electron microscopy and light microscopy. Striking differences in the epithelial morphology were noted and found to be dependent on the state of filling of the gallbladder. "Folds," " crests," and "bays," previously described, disappear when the gallbladder is fixed in its usual filled state. Fixation of the gallbladder in the filled state allowed the detection of cryptlike glands not easily appreciated in the empty, relaxed state. These glands contained cells which resembled intestinal goblet cells, were periodic acid-Schiff positive, and, with scanning electron microscopy, appeared to be discharging material. The lateral epithelial cell membJane, thought to be the site of active sodium chloride transport, contains microfolds which do not resemble microvilli as has been previously thought. With scanning electron microscopy, the labyrinthine canals that exist between the epithelial cells can be fully appreciated. This correlative approach to morphologic studies offers new insight into the structure of the gallbladder. Although there have been previous ultrastructural descriptions of gallbladder epithelium from several species, I-s includIng one of the guinea pig, 6 this is the first report correlating scanning and transmission electron microscopy with light microscopy and histochemistry. Numerous studies have emphasized Received December 6, 1971. Accepted June 1, 1972. Address requests for reprints to : Dr. John C. Mueller, Cell Biology Section (151E), Veterans Administration Hospital, 4150 Clement Street, San Francisco, California 94121. The research was supported in part by Grant RET R-48 of the Veterans Administration Hospital and National Institutes of Health Grant AM 13519. The authors wish to acknowledge the helpful comments of Dr. Marvin Sleisenger during the preparation of the manuscript, and the technical assistance of Miss Maria Maglio, Miss Joan Hahn, and Miss Yoshiko Hayashino.

the presence of folds in the epithelial surface of the gallbladder, and more recent reports, based on studies in the rabbit, have ascribed functional significance to these structures. 7 - 9 In most studies, the gallbladder is fixed empty and relaxed, a condition which poorly reflects its physiologic state, and in those where it is fixed distended the degree of fullness is not specified. In this study, we have confirmed and extended these earlier observations and have compared the guinea pig gallbladder fixed in a filled and an empty state. We found that fixing the gallbladder filled strikingly alters the inner surface topography and allows the visualization of structures such as gland orifices not readily observed m the empty relaxed tissue. These studies will serve as a basis for subsequent studies of gallbladders from guinea pigs fed lithogenic diets.

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Methods Male, albino guinea pigs (200 to 300 g) were anesthesized with sodium nembutal. Gallbladders were fixed in two ways in Karnovsky 's fixative, 10 modified by reduction of the paraformaldehyde and glutaraldehyde concentrations to 0.8 and 2.6%, respectively. The contents of some gallbladders were aspirated when the animals were killed and replaced in situ with an equal volume of fixative. Simultaneously, the outside of the gallbladder was bathed in fixative. After fixation had proceeded for 15 to 20 min, the gallbladders were removed from the animals, cut into two or more pieces along the long axis, gently pinned f1at in wax-filled petri dishes, washed and covered with fixative, and allowed to fix at 10 C for 24 hr. Also, gallbladders were fixed in the empty, relaxed state by removing them from the anesthesized animal, cutting them open longitudinally, and immersing them in fixative . After an initial fixation of 15 to 20 min, these gallbladders were gently pinned f1at in petri dishes and treated as the others. Two previous studies reporting morphologic observations of the actively transporting rabbit gallbladder used intraluminal fixative in their fixation technique. 8 • 9 However, as reported, the volume of intraluminal fixative was not related to observed luminal volumes prior to fixation. Tissues for scanning electron microscopy were left in the fixative for 24 hr, then placed in 16% glycerine for 24 hr, and then in 20% ethanol for 24 hr. They were then dehydrated through graded concentrations of ethanol to 100%. 11 The ethanol was then replaced by either Freon and dried by the critical point method described by Cohen et al. , 12 or by amyl acetate and then C0 2 , and dried by the critical point method of Anderson. 1 3 The dried specimens were gold-coated in a Kinney vacuum evaporator while being rotated and examined with the Cambridge S-4 scanning electron microscope . Tissues were also fixed for light and transmission electron microscopy and selected areas examined to elucidate observations made by scanning electron microscopy . Sections for the light microscope were prepared by staining 1 J1. sections of the Eponembedded material with a solution of 1% toluidine blue in 1% sodium borate. Other tissues were fixed in Bouin 's solution, embedded in paraffin, and stained by the periodic acidSchiff (PAS) technique. 14 For transmission electron microscopy, the

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tissues were postfixed in 2% osmium tetroxide, dehydrated, and embedded in Epon 812. Silver and pale gold sections were cut with diamond knives on a Porter Blum MT-2 microtome, stained with uranyl acetate and lead citrate, and examined with the Hitachi HS-8 or Philips 300 electron microscope.

Results Topography. The gallbladder mucosal surface, fixed and examined in the unfilled state, presents a strikingly folded surface when viewed with the scanning electron microscope (fig. 1). These long, heaped-up folds are closely approximated without any apparent repetitive pattern. Light microscopic examination of sections of the unfilled gallbladder reflects this folded configuration (fig. 2). The transected folds comprise the previously described "crests" and " folds and bays. " 7 - 9 Frequently, a large, thin-walled blood vessel is seen in the center of a fold. Higher magnification (fig. 3) of the unfilled gallbladder shows the individual epithelial cell to have a bulging luminal surface which is often not fully appreciated by conventional microscopy. The surface of the gallbladder fixed in the full state presents quite a different appearance (compare figs. 1 and 4) . The surface is much more nearly flat and little trace of the extensive folding of the empty gallbladder is seen except for the occasional low ridges which overlie large subepithelial blood vessels (compare figs . 4 and 5). This change was observed throughout the gallbladder. Sections examined with the light microscope (fig. 5) reflect this change in configuration. The individual epithelial cells (fig. 6) often have a much less protuberant luminal surface than is seen in the undistended gallbladder. Glands. Also noted in the scanning electron micrographs of the inner surface of the filled gallbladders were glandlike openings (figs. 4 and 6) which appear to be discharging material. The mucosa about the openings is slightly heaped up, giving the entire structure the appearance of a volcanic cinder cone. The openings

FIG. 1. A low power scanning electron micrograph of unfilled gallbladder mucosa. The extensive folding of the mucosa shows no consistent repetitive pattern ( x 120). This, and subsequent scanning electron micrographs were taken at 1000 lines per frame and a frame speed of 40 sec. 858

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FIG. 2. Light photomicrograph of a cross section of an unfilled gallbladder wall showing folds of mucosa overlying large thin-walled vessels ( x 320).

were more numerous toward the ductal end of the gallbladder but were present throughout the organ. There was some variation in the size of the opening ranging from a few to several cell widths across. There is no apparent difference in the surface morphology of the cells forming these pits when compared with the surrounding epithelium. These structures are seen in PAS-stained light microscopy sections (fig. 7), and they appear to lead to simple shallow glands lined with cells which are laden with an intensely PASpositive material in their apical regions. The PAS reactivity is not diminished by amylase digestion and it therefore is not due to glycogen. Transmission electron microscopy reveals these cells to be similar to the goblet cells seen in the intestine (fig. 8). They contain numerous membrane-limited droplets which resemble the mucin granules of goblet cells. Epithelial cells. The apical pole of epi-

thelial cells (figs. 9 and 10) contains mitochondria and vacuoles of various sizes. Vacuoles often lie quite close to the surface membranes and are filled with material of variable electron density. Light microscopy of PAS-stained sections reveals numerous PAS-positive granules in this region (fig. 7). The luminal surface of the epithelial cells is covered with microvilli (figs. 10 and 11) which are not as densely packed as they are on intestinal epithelial cells. Antennulae microvilares were seen in only a few sections which may reflect fixation changes or a true species difference. The difference in cell surface protrusion between the empty and full gallbladder is not as apparent with light microscopy and transmission electron microscopy as it is with scanning electron microscopy. Transmission electron microscopy shows numerous foldings of the lateral membranes of the epithelial cells. These

FIG. 3. Scanning electron micrograph of unfilled gallbladder mucosa showing bulging cell surfaces ( x 520). Inset is a higher magnification of individual cell surfaces covered with microvilli ( x 3200) . 860

FIG. 4. Scanning electron micrograph of the filled gallbladder mucosa. Note the replacement of folds with a few low ridges. Openings to glandlike crypts (arrows) can be seen ( x 310).

FIG. 5. Light photomicrograph of the filled gallbladder wall showing marked reduction in thickness of wall and absence of mucosal folding. Note the thin-walled vessel causing a slight protrusion of the mucosal surface ( x 320) . 861

FIG. 6. Detailed views of cryptlike glands seen in the filled gallbladder mucosa. Note material discharging from the gland on the right (left, x 1200; right, x 2200) .

FIG. 7. Light photomicrograph of a periodic acid-Schiff stained cross section of a glandlike epithelial structure. Note the periodic acid-Schiff positive material in the apical portion of the gland cells. Periodic acid-Schiff positive granules may also be seen in lesser quantity in the apical portion of adjacent epithelial cells ( x 500) .

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FIG. 8. A transmission electron micrograph of cells making up the glandlike structure. Note the numerous membrane-limited droplets. Microvilli are apparent on the apical cell membrane ( x 5500).

folds interdigitate with foldings of the adjacent membranes (fig. 12) and are connected by desmosomes. Viewed with the scanning electron microscope, the folds (fig. 12) are seen to be elaborately reticulated plates, not at all similar to the microvilli of the luminal surface of the cell (compare figs. 11 and 12).

radiographic techniques and they suggest a functional correspondence between the valleys and crests of the gallbladder epithelium and the crypts and free surfaces of other intestinal epithelium. Kaye and co-workers 8 suggest a difference in regard to fluid transport between folds and valleys. These authors' observations suggest that the fold is a morphologic and Discussion physiologic entity in the rabbit. This The striking difference seen in the mor- study in the guinea pig casts some doubt phology of the filled and empty gallblad- on the universality of this concept for, in der raises questions as to the proper the guinea pig, mucosal folds disappear description of the normal gallbladder as the gallbladder fills. The mouse and mucosa. The filled gallbladder is the more hamster show a similar disappearance of usual state, with partial emptying occur- folds in the filled gallbladder, the folds ring only a few times a day under active of Rhesus monkey and rabbit gallbladders smooth muscle contraction mediated diminish but remain with filling, and the through humoral and/or vagal stimulation. fold of the chicken gallbladder are interIt is therefore more logical to consider mediate (unpublished observations). In the morphology of the unstimulated filled fact, there is no undisputed evidence that gallbladder as the "normal" resting state tht) folds must recur in the same place each time the gallbladder empties, alof the organ. Kaye et al. 7 have studied cell replica- though it is possible that the subendothetion in the rabbit gallbladder using auto- lial blood vessels provide a basis for a

FIG. 9. Transmission electron micrograph of several gallbladder epithelial cells. Numerous vesicles of varying size are prominent in the apical region of the cell. Mitochondria are small and abundant. Note the elaborate interdigitation of lateral cell membranes ( x 8300). 864

FIG. 10. A detail of the luminal surface of the epithelial cell showing numerous vesicles and mitochondria close to the cell surface. Numerous microvilli with antennulae microvilares are apparent ( x 31, 100).

FIG. 11. A scanning electron micrograph of a small area of a gallbladder epithelial cell surface showing the abundant microvilli ( x 10,000).

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Vol. 63, No . 5

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FIG. 12. A scanning electron micrograph of the lateral surface of the epithelial cells. The apical surface (A) of some cells is seen in the foreground. Note the elaborate reticulated plates formed by the lateral cell membrane which are quite different from the microvilli covering the apical surface ( x 5000) . The inset is a transmission electron micrograph, showing for comparison a section of the lateral cell membranes of adjacent cells ( x 8800).

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LIVER PHYSIOLOGY AND DISEASE

fixed recurrence of folds in some cases. We detected no other relation between the folds and subendothelial structures which appeared to offer a basis for an invarying recurrence of folds. The lateral cell membrane of the gallbladder epithelial cell has received considerable attention as it is postulated to be the site of active neutral salt transport responsible for bile concentration in the gallbladder; 9 • 15 The elaborate foldings of the membrane seen with transmission electron · microscopy have been thought to represent "microvilliform projections," 16 "fingerlike evaginations," 9 or "elaborate digital processes" 17 seen in cross section. In the guinea pig, this is clearly not the case as scanning electron micrographs show these structures as reticulated plates or microfolds in the plasma membrane. In vitro studies of the rabbit gallbladder8· 9 show' increasing distention of intercellular spaces between ~djacent epithelial cells with increasing rates of active transport of solute. In many reported micrographs, 8 • 9 • 15 there is virtually no contact between opposing cell membranes. Desmosomes, mentioned to occur in rabbit epithelium in one study 8 and not in another, 9 are quite numerous in the guinea pig epithelium. They appear to ensure an elaborate labyrinth of interconnecting canals between the opposing cells which would provide the long, narrow channels required by the standing-gradient osmotic flow model postulated by Diamond and Bossert 18 much more completely than nonconnecting finger-like projections. It is possible that grossly distended intracellular spaces, seen in the in vitro rabbit gallbladder studies, reflect some degree of artifactual disruption due to manipulation and interruption of the blood supply as well as possible mechanical deformation of older cells. 9 The small cryptlike structures seen in the luminal surface of the filled gallbladder appear to lead to simple glands. Often an amorphous material is seen extruding from them. The PAS-stained preparations show the cells which make up these structures to contain large amounts of car-

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bohydrate material which is not glycogen. Similar structures are described by Tusques et al. 19 who reported simple glandular structures in the gallbladder epithelium of both the guinea pig and the sheep. Based on histochemical studies, they felt that the glandular cells contained glycoprotein. The relation of these structures to previously reported gallbladder epithelial structures is not entirely clear to us. They seem clearly not to be RokitanskyAschoff sinuses or Luschka ducts as described and differentiated in human gallbladders by Elfving, 20 and furthermore, we did not observe such structures in the guinea pig gallbladders. On the basis of structure and distribution, the glands we observed seemed most closely related to "neck glands" as described by Halpert 21 in the human gallbladder. The " neck glands", however, are described as being "tubuloalveolar," being limited to the neck region of the gallbladder and having small flat nuclei which appear crowded to the basal ends of the cells. Several authors 2 2 - 25 have studied the histochemical properties of the material found in granules in the apical portion of the epithelial cell of the gallbladder, and there is agreement that it is a glycoprotein. The studies of Tusques et al. 19 suggest that it is different from that found in the gland cells. The glycoproteins are of particular importance as they appear to participate in gallstone formation. 26 There is evidence in the rabbit that the epithelial cells increase their production of these complexes when exposed to a lithogenic diet. 27 Future studies correlating light microscopy, transmission and scanning electron microscopy, and histochemistry of gallbladders from animals fed lithogenic diets should more completely resolve this question. REFERENCES 1. Yamada E: The fine structure of the gallbladder

epithelium of the mouse. J Biophys Biochem Cytol 1:445-458, 1955 2. Johnson FR, McMinn RMH, Birchenough RF: The ultrastructure of the gallbladder epithelium of the dog. J Anat 96:477-487, 1962

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3. Evett RD, Higgins JA, Brown AL Jr: The fine structure of normal mucosa in human gallbladder. Gastroenterology 47 :49-60, 1964 4. Hayward AF: The fine structure of the gallbladder epithelium of the sheep. Z Zellforsch Mikrosk Anat 65:331-339, 1965 5. Hayward AF: An electron microscopic study of developing gallbladder epithelium in the rabbit. J Anat 100:245-259, 1966 6. Hayward AF: Electron microscopic observations on absorption in the epithelium of the guinea pig gallbladder. Z Zellforsch Mikrosk Anat 56:197202, 1962 7. Kaye GI, Maenza RM, Lane N: Cell replication in rabbit gallbladder. Gastroenterology 51:670680, 1966 8. Kaye GI, Wheller HO, Witlock RT, et al: Fluid transport in the rabbit gallbladder. J Cell Bioi 30:237-268, 1966 9. Tormey J McD, Diamond JM: The ultrastructural route of fluid transport in rabbit gallbladder. J Gen Physiol 50:2031-2060, 1967 10. Karnovsky MJ: Formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J Cell Bioi 27:137A, 1965 11. Nemanic MK, Pitelka DR: A scanning electron microscopic study of the lactating mammary gland. J Cell Bioi 48:410-415, 1971 12. Cohen AL, Garner GE, Marlow DP: A rapid critical point method using fluorocarbons ("Freons") as intermediate and transitional fluids. J Microscop 7:331-342, 1968 13. Anderson TF: Techniques for the preservation of three dimensional structure in preparing specimens for the electron microscope. Trans NY Acad Sci Ser II 13:130-134, 1951 14. Pearse AGE: Histochemistry, Theoretical and Applied. Boston, Little, Brown and Co, 1960, p 998 15. Dietschy JM: Recent developments in solute and water transport across the gallbladder epithelium. Gastroenterology 50:692-707, 1966

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16. Hayward AF: The structure of gallbladder epithelium. Int Rev Gen Exp Zoo! 3:205-239, 1968 17. Wheeler HO: Concentrating function of the gallbladder. Am J Med 51:588-595, 1971 18. Diamond JM, Bossert WH : Standing gradient osmotic flow. J Gen Physiol 50:2061-2083, 1967 19. Tusques J, Senelar R, Gingune Y, et al: Etude histochimique sur les vesicules biliaires de mouton et de cobaye: les mucopolysaccharides. Ann Histochim 9:269-276, 1964 20. Elfving G: Crypt and ducts in the gallbladder wall. Acta Pathol Microbiol Scand 49 (suppl 135): 1-451 1960 21. Halpert B: Morphological studies on the gallbladder. I. A note on the development and microscopic structure of the normal human gallbladder. Bull Johns Hopkins Hosp 40:390-408, 1927 22. Yamada K: Morphological and histochemical aspects of secretion in the gallbladder epithelium of the guinea pig. Anat Rec 114:117-124, 1962 23. Wolf-Heidegger G, Staubbi W, Hess R : Zur ultrastruktur und histochemie der gallenblasenschleimhaut des menschen und der katze. Acta Anat 62:606- 618, 1965 24. Lev R, Spicer SS : A histochemical comparison of human epithelial mucins in normal and hypersecretory states including pancreatic cystic fibrosis. Am J Pathol 46:23-47, 1965 25. Esterly JR, Spicer SS : Mucin histochemistry of human gallbladder: changes in adenocarcinoma, cystic fibrosis and cholecystitis. J Natl Cancer Inst 40:1-10, 1968 26. Bouchier IAD : Macromolecular material in bile and its relation to gallstone formation. South Afr Med J 40:735-738, 1966 27. Hayward AF, Froston JW, Bouchier IAD: Changes in the ultrastructure of gallbladder epithelium in rabbits with experimental gallstones. Gut 9:550-556, 1968