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The effect of trypan blue on the hepatotoxicity of carbon tetrachloride in the rat
EXPERIMENTAL
AND
MOLECULAR
PATHOLOGY
16, 115-128
(1969)
The Effect of Trypan Blue on the Hepatotoxicity Carbon Tetrachloride in the Rat’ MARY PETRELLI Department
of Pathology,
AND RICHARD J.
Cleveland Metropolitan University School of Medicine, Received
October
of
STENGER"
Gene& Hospital, and Cleveland, Ohio 44109
Case
Western
Reserve
28,1968
The toxic effects of carbon tetrachloride (CCL) on the liver have been studied extensively (Cameron and Karunaratne, 1936; Dawkins, 1963; Recknagel, 1967; Stenger, 1966a; Wigglesworth, 1964). Investigators generally hold that this toxicity develops within the liver parenchymal cells (McLean and McLean, 1966; Slater, 1966; Recknagel, 1967). So far, the role played by the sinusoidal lining cells in the liver’s response to CCL has received little attention. Of course, as part of the reticula-endothelial system, the liver sinusoidal lining cells have well known functions in relation to phagocytosis of foreign materials (Biozzi et al., 1953, 1955; Fisher, 1966, 1967; Hampton, 1958; Howard, 1959; Rebuck et al., 1960; Thorbecke et al., 1960). Recently, however, it was noted that prior carbon loading of the sinusoidal lining cells greatly protects the liver from the toxic effects of CCL (Stenger et al., 1968). The present experiment has demonstrated that prior loading of these cells with trypan blue affords a similar protection. MATERIALS
AND METHODS
Thirty-six female rata (Holtzman), weighing 230-260 gm and maintained on stock diet ad libitum, were divided into three groups: Group A: Four animals were given 3 ml of 2.5% suspension of trypan blue (Allied Chemical CI 23850) in physiological saline by tail vein; two animals received 3 ml of saline alone. All animals were killed 24 hours later. Group B: Eight animals were given trypan blue and eight animals saline, as in Group A. After 24 hours, all were given intraperitoneally 0.5 ml of CCL, as a 50% solution in olive oil, and they were killed 24 hours later. Group C: Seven animals were given trypan blue and seven saline, as in Group A. After 24 hours, they were given CCL, as in Group B; but these rats were killed 48 hours later. The animals were decapitated with a guillotine and exsanguinated. Immediately, representative blocks were taken from the liver and fixed in cold 1 This investigation was supported, in part, by a Public Health Service Research Grant (AM-08416) from the National Institute of Arthritis and Metabolic Diseases; and, in part, by a Public Health Service Graduate Pathology Training Grant (5Tl-GM-1122) and a Research Career Program Award (K3-GM-22,575) from the National Institute of General Medical Sciences. ‘Present address: New York Medical College, Fifth Avenue at 106th Street, New York, New York 10029. 115
116
PETRELLI
AND
STENGER
(4%) 10% formalin solution, buffered to pH 7.0. Paraffin sections were cut at 5 ~1 and stained with hematoxylin and eosin, periodic acid-&hi& and periodic acid-Schiff after diastase digestion. For electron microscopy, segments of liver were fixed in 4% glutaraldehyde, phosphate buffered to pH 7.3 and held at 4°C for 2 hours. Subsequently, they were washed in the phosphate buffer for 1% hour and postfrxed for 2 hours in 1% osmium tetroxide, phosphate buffered to pH 7.3 (Millonig, 1961a) and maintained at 4°C. Dehydration was accomplished with graded acetone solutions and then the tissues were infiltrated with and embedded in Araldite (Stenger, 1963). Blocks were cured for 2-4 weeks at 50°C. Sections were obtained with glass knives and a Porter-Blum ultramicrotome. The sections were stained with lead hydroxide (Millonig, 1961b) and examined with an RCA-EMU-3 F electron microscope. RESULTS Group A Light
microscopy.
histologically normal. In the trypan-blue mal and contained
In the saline-injected
control
animals,
the livers were
injected animals, the liver parenchymal cells were norno perceptible dye. The sinusoids in the centrilobular
FIG. 1. Central portion of a liver lobule from a rat injected with trypan blue alone. The endothelial cells of the central vein are swollen. Adjacent to the vein are several small aggregates of macrophages. The centrilobular sinusoids are dilated and partially filled with hypertrophied sinusoidal lining cells. x 560.
EFFECT
OF TRYPAN
BLUE
AND
CCL
ON
LIVER
117
FIG. 2. Portion of a hepatic parenchymal cell from a rat injected with trypan blue alone. The nucleus, RER and mitochondria are normal. The glycogen areas contain numerous clusters of dense glycogen particles, interspersed with slightly dilated vesicles of SER (arrows). The Golgi zone (G) consists of parallel smooth cisternae, focally dilated by a content of small, moderately dense globules. x 18,000.
areas were dilated and, throughout, the lobules were lined by large protuberant Kupffer cells or flat, but prominent sinusoidal lining cells (Fig. 1). Both cell types contained variable amounts of trypan blue. Central veins showed swelling of the endothelium and small subendothelial accumulations of macrophages. Medium sized portal zones were edematous and sparsely infiltrated by macrophages, plasma cells, and monocytes. Electron microscopy. In the saline-injected animals, the liver cells showed no changes and were comparable to previously described normal liver cells (Bruni and Porter, 1965; Fawcett, 1966; Rouiller and Jezequel, 1963). The sinusoids and sinusoidal lining cells were morphologically similar to those described by others (Novikoff and Essner, 1960; Schaffner et al., 1963; Schmidt, 1960; Steiner, 1961). There were two types of sinusoidal lining cells, one consisting of flat, endothelium-like cells and the other appearing as large, phagocytic cells (Kupffer cells). Lying in sinusoidal recesses between adjacent liver cells was a third cell type, which contained abundant lipid, as described by Yamagishi (1959). In the trypan-blue injected animals, the parenchymal cells were also essentially normal. They contained clusters of particulate glycogen (Revel et al., 1960), disposed in relation to slightly prominent vesicular and tubular profiles of smooth endoplasmic reticulum (SER) (Fig. 2). The rough endo-
118
FIG. 3. Portion RER, mitochondria, widely distributed. the slightly prominent
PETRELLI
AND
STENGER
of a hepatic parenchymal cell from a rat injected with trypan blue alone. The and microhodies are normal. Small clusters of dense glycogen particles are A few residual bodies (Rb) with dense osmiophilic content are scattered near Golgi zones (G). x 18,OW.
plasmic reticulum (RER) consisted of parallel layers of narrow cisternae studded with ribosomes (Figs. 2 and 3). Mitochondria, microbodies, and single membrane limited lysosomes containing osmiophilic globules (residual bodies) were normal in distribution and appearance (Figs. 2 and 3). The Golgi zones were well-defined (Figs. 2 and 3). Liver cell nuclei were unaltered (Figs. 2 and 3). The sinusoidal lining cells, however, were larger than those of the saline controls (Figs. 4 and 5). This enlargement was primarily due to the presence of numerous phagosomes, which were delimited by single membranes and contained either coarse lamellar or finely reticulated materials (Fig. 4). The lining cells were further characterized by prominent Golgi zones, a well-developed RER, multiple small mitochondria, frequent pinocytotic vacuoles, and nuclei of irregular contour (Figs. 4 and 5). At their periphery, the sinusoidal lining cells of the trypan-blue injected rata displayed either wide pseudopodia or numerous slender villus-like processes (Figs. 4 and 5). Group B Light microscopy. The saline-treated, CC&-injected animals exhibited wide zones of liver necrosis, disposed about central veins and involving as much as % of each lobule. The necrotic zones were characterized by shrunken, eosinophilic, largely anuclear, partially fragmented parenchymal cells, interspersed with infiltrating polymorphonuclear leukocytes. The cells in the
PETRELLI
AND
STENGER
FIG. 5. Portions of several hypertrophied sinusoidal lining cells from a rat injected with trypan blue alone. The cells are enlarged by phagosomes of variable size and content. The cytoplasm contains prominent Golgi zones (G), as well as both narrow and dilated RER cistemae. Near the plasma membrane, the cells show multiple pinocytotic vesicles and vacuoles (arrows). Along the left margin is a small segment of a neighboring parenchymal cell. x 21,400.
FIG. 6. Liver from a saline-injected rat, killed 24 hours after CCla administration. Necrosis is evident in the central 11 of the lobule. The necrotic parenchymal cells are essentially anuclear, and inflammatory cells have infiltrated the necrotic zone. Surviving hepatic cells at the periphery of the lobule appear finely vacuolated due to fat accumulation. x 150. FIG. 7. Liver from a trypan-blue injected rat, killed 24 hours after CC14 administration. In the immediate vicinity of the central vein, there are a few necrotic cells, which have excited a focal inflammatory reaction. A small number of centrilobular liver cells show ballooning and hydropic change. Elsewhere, the parenchymal cells are normal, except for fine vacuolization, reflecting lipid accumulation. x 150. 121
FIG. 8. Sinusoidal lining cell from a trypan blue injected rat, killed 48 hours after CCL administration. The cell is enlarged primarily due to the presence of many, membrane-limited phagosomes, which vary in both size and shape. The phagosomes have a heterogeneous content, including, in one instance, a partially degraded, but recognizable mitochondrion (open arrow). The cytoplasm contains numerous free ribosomes, a well developed RER, many small mitochondria, and the edge of a Golgi zone (G). At its periphery, the cell exhibits many pinocytotic vacuoles (thin arrows) and numerous slender or blunt cytoplasmic projections. This cell is overlapped on two sides by tenuous processes of unidentified cells. At the upper right and the extreme lower left are small segments of the flanking hepatic parenchymal cells. x 18,000. 122
EFFECT
OF
TRYPAN
BLUE
AND
Ccl,
ON
LIVER
periportal areas were intact but showed moderate to severe fat accumulation (Fig. 6). Triaditis was seen in the larger portal zones. Glycogen content of surviving parenchymal cells was moderate. Even after diastase digestion, Kupffer cells contained abundant PAS positive material. In contrast, the trypan-blue, CCL-injected animals revealed a minimal degree of liver necrosis around the central veins (Fig. 7). Scattered parenchymal cells in the central zones showed ballooning and hydropic change, and adjacent to these were small collections of histiocytes and macrophages. The intact liver cells showed mild to moderate fat accumulation. Medium-sized portal zones exhibited a triaditis and proliferation of histiocytes (Fig. 7). Glycogen was abundant in all cells except the hydropic and necrotic cells. In the Kupffer cells, the PAS diastase preparations showed a larger amount of PAS positive material than was seen in the control animals. Electron microscopy. In the saline-treated, CCL-injected animals, the changes were extensive and similar to those that have been described previously (Ashworth et al., 1963; Bassi, 1960; Recknagel, 1967; Stenger, 1966a). The changes included dilatation of the RER and SER, loss of glycogen, and degenerative changes of mitochondria, with loss of cristae and appearance of dense aggregates (Krishnan and Stenger, 1966; Reynolds, 1964, 1965). Lipid accumulation and increased numbers of autophagic vacuoles and residual bodies (lysosomes) were also present. Sinusoidal lining cells, however, showed no degenerative changes, but contained increased numbers of phagosomes. In the trypan-blue treated, CCL-injected animals, although less extensive, the changes in the parenchymal cells were qualitatively similar to those found in the saline controls. Sinusoidal lining cells were large and showed abundant RER and multiple small mitochondria. They also contained a complex group of phagosomes, some with dark osmiophilic material, others with laminated vesicles and degenerating mitochondria (Fig. 8). Group C
The saline-treated, CClr-injected animals exhibited centrilobular residues of liver cell necrosis and collections of histiocytes containing diastase resistant, PAS positive material. The surviving liver parenchyma showed widespread mitotic activity and moderate to severe fat accumulation. Glycogen content, except in areas of necrosis, was abundant. In the trypan-blue treated, CCL-injected animals, regeneration had occurred and was almost complete, with little evidence of necrotic residues. Histiocytes, however, persisted around the central veins. Surviving liver cells exhibited abundant glycogen and moderate amounts of lipid. Mitotic activity was moderate. The histiocytes and Kupffer cells contained PAS positive material, even after diastase digestion. Electron microscopy. The qualitative changes in both saline and trypanblue treated, CClr-injected animals were essentially similar to those in group B. Light
microscopy.
124
PETRELLI
AND STENGER
DISCUSSION Intravenous administration of trypan blue afforded a marked protection from the hepatotoxicity of CCL, when animals were challenged 24 hours after the dye injection. At both 24 and 48 hours after CCL administration, the livers of trypan-blue injected rats showed far less necrosis and inflammatory reaction than was found in saline-injected controls. The reasons for this result are obscure. The injection of trypan blue alone caused centrilobular dilatation of liver sinusoids, endothelial swelling in central veins, and adjacent aggregations of macrophages (Fig. 1). Scott (1963, 1968) observed proliferative and inflammatory alterations in the pulmonary arterial tree after trypan blue administration. These changes would indicate that trypan blue has an adverse effect on blood vessels. It is not clear, however, whether such changes in the blood vasculature are accompanied by alterations in blood flow. Nor is it known to what extent hepatic circulation might be affected by the trypan blue induced hypertrophy of the liver sinusoidal lining cells. Concerning the latter, it has been noted that injections of biologic ink resulted in greater enlargement of hepatic sinusoidal lining cells than was effected by trypan blue, yet the extent of liver protection from CCL after such carbon loading (Stenger et al., 1968) was not appreciably different from that observed in the present experiments. The question of changes in hepatic blood flow after trypan blue administration will not be resolved until direct measurements are made. (1954) It might be pertinent to note, however, that Shore and Zilversmit measured blood flow after carbon loading of the liver and found no significant change in hepatic circulation. In view of this, it would seem unlikely that alterations in hepatic blood flow could completely explain the marked differences in hepatotoxicity reported here. In the present study, at 24 hours after injection of trypan blue alone, there was no evidence, either by light or electron microscopy, that the dye had even entered the hepatic parenchymal cells. Morgan et al. (1966) also failed to demonstrate trypan blue in the liver parenchyma. Beck et al. (1967), however, have identified trypan blue in visceral yolk sac epithelium, 24 hours after exposure. In this instance, the dye was concentrated in deep digestive vacuoles (heterolysosomes) and its presence was associated with an inhibition of selected hydrolytic enzymes. More to the point, Mjrsliwski and Michalik (1966) have observed morphologic liver changes indicative of hepatotoxicity at 12 hours after trypan blue injection. It seems apparent, therefore, that trypan blue can enter cells, perhaps even liver ceils, and that such entry may be associated with intracellular damage. It is conceivable that, in the present investigation, the dye gained access to the hepatocytes and caused subcellular changes at early intervals, but it then must be inferred that these changes were transient and that complete morphologic restitution of hepatocellular architecture had taken place by 24 hours after the dye injection. This would be analogous to the transient effects of neutral red dye on the liver parenchyma, as reported by Alousi et al. (1968).
EFFECT
OF TRYPAN
BLUE
AND
CCL
ON
LIVER
125
It is widely held that CCL activation and consequent toxicity develop entirely within the parenchymal cells of the liver (McLean and McLean, 1966; Slater, 1966). As shown here, however, there was no difference, at least in structural terms, between the parenchymal cells of the trypan-blue injected rats and those of the saline-treated controls, at the time of CC14 challenge. If the parenchymal cell were the only important factor in Ccl, hepatotoxicity, an equivalent response would have been expected in the trypan-blue and saline-treated rats. As noted above, this was not the case. Attention was directed, therefore, to structural differences that might account for the results obtained. One obvious difference was that noted in the liver sinusoidal lining cells, which were notably hypertrophied in the trypan blue injected rats and essentially normal in the saline-treated animals. It is here proposed that this difference might be related to the observed divergent response to CC% administration. Whether the protective effect of prior dye treatment is related simply to the increased cytoplasmic mass of the sinusoidal lining cells or to a specific metabolic process is, at present, unknown. It is generally accepted that phagocytosis is the primary function of the liver sinusoidal lining cells. In the present investigation, this function was manifest after trypan blue injection (Figs. 4 and 5) and prominent after CC14 administration (Fig. 8). In the former, the phagocytosis was related to dye ingestion and was accompanied by other cytoplasmic changes, including an increase of RER and enlargement of the Golgi zones. After Ccl,, the increased numbers of phagosomes were considered to reflect phagocytosis of parenchymal cell debris, in accord with previous suggestions (Popper et al., 1960; Schaffner et al., 1963; Stenger, 1966b). More recently additional functions have been ascribed to the hepatic sinusoidal lining cells. Cohen et al. (1960), Schaffner and Popper (1962), and Steiner (1961) have attributed to these cells a role in antibody formation. Schaffner et al. (1963) have proposed a role in hepatic fibrogenesis and Ackerman et al. (1961), a role in erythropoiesis, at least in embryonic liver. Snell (1960) has indicated that the sinusoidal lining cells respond to drugs and that this capability influences their phagocytic activity. Finally, as discussed above, the results of the present investigation raise the possibility that sinusoidal lining cells might also function in the handling of toxic agents. SUMMARY Rats were given intravenous injections of either physiologic saline or trypan blue dye suspended in saline. After 24 hours, the livers of these animals were sampled for light and electron microscopic study. The livers of the saline treated rats showed no morphologic alterations. The livers of the trypan blue injected animals exhibited no structural abnormalities in the hepatic parenchymal cells, but their sinusoidal lining cells were hypertrophied, mainly due to the appearance of numerous phagosomes, increased quantities of rough endoplasmic reticulum, and prominent Golgi zones. Similarly prepared rats were given intraperitoneal injections of CCL, 24 hours after the intravenous administration of saline or trypan blue. The livers of these animals were sampled, 24 and 48 hours after the toxic challenge. At both intervals, the livers of the rata treated with trypan blue and CCL displayed far less necrosis and inflammatory reaction than was found in the animals given saline and CCL. It was considered unlikely that this difference could be completely ex-
126
PETRELLI
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STENGER
plained by changes in hepatic blood flow. It was also demonstrated that, at 24 hours after administration of trypan blue, there were no hepatocellular morphologic alterations that might account for the divergent response to Ccl,. It was proposed, therefore, that the hypertrophied sinusoidal lining cells of the trypan blue treated rats might be related to the observed protection from CCh hepatotoxicity. ACKNOWLEDGMENTS The authors are greatly indebted to Drs. J. D. Reid, J. Wiener, structive criticism and advice in the preparation of the manuscript, for his excellent technical assistance.
and M. A. Alousi for their conand to Mr. J. N. Williamson
REFERENCES ACKERMAN, G. A., GRASSO, J. A., and KNOW-F, R. A. (1961). Erythropoiesis in the mammalian embryonic liver as revealed by electron microscopy. Lab. Invest. 10,787-796. ALOLJS~, M. A., STENGER, R. J., and MORGAN, W. S. (1968). The fine structure of pancreatic acinar and hepatic parenchymal cells after neutral red dye injection. Exptl. Mol. Pathol. 9, 97109. Hepatic cell degeneration. ASHWORTH, C. T., LUIFIEL, F. J., SANDERS, E., and ARNOLD, N. (1963). Correlation of fine structural with chemical and histochemical changes in hepatic cell injury produced by carbon tetrachloride in rata. Arch. Pathol. 75, 212-225. B~ssr, M. (1969). Electron microscopy of rat liver after carbon tetrachloride poisoning. Expt.
Cell Res. 20,313-323. BECK, F., LLOYD, J. B., and GRIFFITHS, A. (1967). Lysceomal enzyme inhibition by trypan blue: A theory of teratogenesis. Science 157,118&1182. BIOZZI, G., BENACERRAF, B., and HALPERN, B. N. (1953). Quantitative study of the granulopectic activity of the reticula-endothelial system. II. A study of the kinetics of the granulopectic activity of the R.E.S. in relation to the dose of carbon injected; relationship between the weight of the organs and their activity. Brit. J. Exptl. Pathol. 34,44-457. BIOZZI, G., BENACERRAF, F., and HALPERN, B. N. (1955). The effect of Sahn. typhi and its endotoxin on the phagocytic activity of the reticula-endothelial system in mice. Brit. J. Ezptl.
Pathol. 36,226-235. BRUNI, C., and PORTER, K. R. (1965). The fme structure of the parenchymal cell of the rat liver. I. General observations. Am. J. Pathol. 46,691-755. CAMERON, G. R., and KARUNARATNE, W. A. E. (1936). Carbon tetrachloride cirrhosis in to liver regeneration. J. Puthol. Bacterial. 42,1-21. COHEN, S., OHTA, G., SINGER, E. J., and POPPER, H. (1969). Immunocytochemical study of globulin in liver in hepatitis and p&necrotic cirrhosis. J. Exptl. Med. 111, 285-294. DAWKINS, M. J. R. (1963). Carbon tetrachloride poisoning in the liver of the newborn
normal relation gamma rat.
J.
Pathol. Bacterial. 85,189-196. FAWCETT, D. W. (1966). “An Atlas of Fine Structure. The Cell, Its Organelles and Inclusions.” Saunders, Philadelphia, Pennsylvania. FISHER, S. (1966). Stimulation of splenic antigen uptake and of antibody response in mice by India ink or other “blockading” agents. Immunology 11,127-136. FISHER, S. (1967). Localization of radioiodinated endotoxin in organs of mice and rabbits: Effect of thorotrast, trypan blue, endotoxin and carbon administered intravenously. Nature 213, 511-512. HAMPTON, J. C. (1958). An electron microscope study of the hepatic uptake and excretion of submicroscopic particles injected into the blood stream and into the bile duct. Acta Anat. 32,
262-291. HOWARD, J. G. (1959). Activation of the reticula-endothelial cells of the mouse liver by bacterial lipopolysaccharide. J. Pathol. Bacterial. 78,465-470. KRISHNAN, N. and STENGER, R. J. (1966). Effects of starvation on the hepatotoxicity of carbon tetrachloride. A light and electron microscopic study. Am. J. Pathol. 49,239-255. MCLEAN, A. E. M., and MCLEAN, E. K. (1966). The effect of diet and l,l,l-tri-chloro-2,2-bis-
EFFECT
OF TRYPAN
BLUE AND CCL ON LIVER
127
(p-chlorophenyl) ethaue (DDT) on microsomal hydroxylating enzymes and on sensitivity of rats to carbon tetrachloride poisoning. Rio&em. J. 100,564~571. MK.LONIG, G. (1961a). The advantages of a phosphate buffer for 0~04 solutions in fixation. J. Appl. Phys. 32,1637. MILLONIG, G. (1961b). A modified procedure for lead staining of thin sections. J. Biophys. Biothem. Cytol. 11,736-739. MORGAN, W. S., FERNANDO,J., and ALOUSI, M. A. (1966). Studies of the biological activity of neutral red. Exptl. Mol. Pathol. 5,491X503. MY~LIWSKI, A., and MICHALIK, T. (1966). Histochemiczne badania kwasow nukleinowych i glikogenu w komorkach watrobowych szczurbw poddanych dzialaniu blekitu trypanu i blekitu Evansa,Folia Morphol. 25,61-67. NOV~OFF, A. B., and ESSNER,E. (1960). The Liver Cell. Some new approaches to its study. Am. J. Med. 29, 102-131. POPPER, H., PARONFITO, F., and BARKA, T. (1960). PAS-positive structures of nonglycogenic character in normal and abnormal liver. Arch. Pathol. 70,300-313. REBUCK, J. W., BOYD, C. B., and RIDDLE, J. M. (1960). Skin windows and the action of the reticuloendothelial system in man. Ann. N. Y. Acad. Sci. 88,30-42. RECKNAGEL,R. 0. (1967). Carbon tetrachloride hepatotoxicity. Pharmacol. Reu. 19,145-208. REVEL, J. P., NAPOLITANO, L., and FAWCETT, D. W. (1966). Identification of glycogen in electron micrographs of thin tissue sections. J. Biophys. Biochem. Cytol. 8,575-589. REYNOLDS, E. S. (1964). Liver parenchymal cell injury. II. Cytochemical events concerned with mitdchondrial dysfunction following poisoning with carbon tetrachloride. Lab. Invest. 13, 14571470. REYNOLDS,
E. S. (1965). Liver parenchymal cell injury. III. The nature of calcium-associated electron-opaque masses in rat liver mitochondria following poisoning with carbon tetrachloride. J. Cell. Biol. 25,53-75. ROUILLER, CH., aqd JI~ZEQUEL, A.-M. (1963). Electron microscopy of the liver. In “The Liver. Morphology, Biochemistry, Physiology.” (Ch. Rouiller, ed.) Vol. 1, pp. 195-264. Academic Press, New York. SCHAFFNEFI, F., and POPPER, H. (1962). A phagocytic and protein-forming mesenchymal cell in human cirrhosis. Nature 196.684-685. SCHAFFNER, F., BARKA, T., and POPPER, H. (1963). Hepatic mesenchymal cell reaction in liver disease. Exptt. Mol. Pathol. 2,419-441. SCHMIDT, F. C. (1960). Elektronenmikroskopische Untersuchungen an den Sinusoid-Wandzellen (Kupffersche Sternzellen) der weissen Maus. Anat. Anz. 108,376-387. Scorr, G. B. D. (1963). The effects of trypan blue on the formation of thrombi in the generalized Shwartzman reaction. Brit. J. Exptl. Pathol. 44,317-325. Scorn, G. B. D. (1968). Trypan blue and the generalized Shwartzman reaction. The nature and formation of fibrinoid material in the pulmonary arteries. Brit. J. Erptl. Pathol. 49, 251-256. SHORE, M. L., and ZILVERSMIT, D. B. (1954). Effect of India ink on bromsulfalein excretion, phagocytosis and circulation in the liver. Am. J. Physiol. 177,436440. SLATER, T. F. (1966). Necrogenic action of carbon tetrachloride in the rat: a speculative mechanism based on activation. Nature 209,36-40. SNELL, J. F. (1960). The reticuloendothelial system: I. Chemical methods of stimulation of the reticuloendothelial system. Ann. N. Y. Acad. Sci. 88,56-77. STEINER, J. W. (1961). Investigations of allergic liver injury. I. Light, fluorescent and electron microscopic study of the effects of soluble immune aggregates. Am. J. Pathol. 38,411-436. STENGER, R. J. (1963). Hepatic parenchymal cell alterations after long-term carbon tetrachloride administration. A light and electron microscopic study. Am. J. Pathol. 43,867-895. STENGER, R. J. (1966a). Ultrastructural alterations within hepatic parenchymal cells after carbon tetrachloride poisoning. fn “Methods and Achievements in Experimental Pathology” (E. Bajusz and G. Jasmin, eds.), Vol. 1, pp. 677-700. Karger, Basel. STENGER, R. J. (1966b). Hepatic sinusoids in carbon tetrachloride-induced cirrhosis. An electron microscopic study. Arch. Pathol. 81,439-447. STENGER, R. J., WILLIAMSON, J. N., and JOHNSON, E. A. (1968). The effect of reticuloendothelial
128 blockade
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carbon tetrachloride hepatotoxicity. A light and electron microscopic study. 52, lla (Abst.). THORBECKE, G. J., MAURER, P. H., and BENACERRAF, B. (1960). The affinity of the reticuloendothelial system for various modified serum proteins. &it. J. En@. Path. 41, 190-197. WIGGLESWORTH, J. S. (1964). The use of the osmium-ethyl gallate technique in the study of carbon tetrachloride liver injury. J. Pathol. Bacterial. 87,333-339. YAMAGISHI, M. (1959). Electron microscope studies on the fine structure of the sinusoidal wall and fat-storing cells of rabbit livers. Arch. Histol. hp. 18, 223-261.
Am. J. Pathol.
AND
MOLECULAR
PATHOLOGY
16, 115-128
(1969)
The Effect of Trypan Blue on the Hepatotoxicity Carbon Tetrachloride in the Rat’ MARY PETRELLI Department
of Pathology,
AND RICHARD J.
Cleveland Metropolitan University School of Medicine, Received
October
of
STENGER"
Gene& Hospital, and Cleveland, Ohio 44109
Case
Western
Reserve
28,1968
The toxic effects of carbon tetrachloride (CCL) on the liver have been studied extensively (Cameron and Karunaratne, 1936; Dawkins, 1963; Recknagel, 1967; Stenger, 1966a; Wigglesworth, 1964). Investigators generally hold that this toxicity develops within the liver parenchymal cells (McLean and McLean, 1966; Slater, 1966; Recknagel, 1967). So far, the role played by the sinusoidal lining cells in the liver’s response to CCL has received little attention. Of course, as part of the reticula-endothelial system, the liver sinusoidal lining cells have well known functions in relation to phagocytosis of foreign materials (Biozzi et al., 1953, 1955; Fisher, 1966, 1967; Hampton, 1958; Howard, 1959; Rebuck et al., 1960; Thorbecke et al., 1960). Recently, however, it was noted that prior carbon loading of the sinusoidal lining cells greatly protects the liver from the toxic effects of CCL (Stenger et al., 1968). The present experiment has demonstrated that prior loading of these cells with trypan blue affords a similar protection. MATERIALS
AND METHODS
Thirty-six female rata (Holtzman), weighing 230-260 gm and maintained on stock diet ad libitum, were divided into three groups: Group A: Four animals were given 3 ml of 2.5% suspension of trypan blue (Allied Chemical CI 23850) in physiological saline by tail vein; two animals received 3 ml of saline alone. All animals were killed 24 hours later. Group B: Eight animals were given trypan blue and eight animals saline, as in Group A. After 24 hours, all were given intraperitoneally 0.5 ml of CCL, as a 50% solution in olive oil, and they were killed 24 hours later. Group C: Seven animals were given trypan blue and seven saline, as in Group A. After 24 hours, they were given CCL, as in Group B; but these rats were killed 48 hours later. The animals were decapitated with a guillotine and exsanguinated. Immediately, representative blocks were taken from the liver and fixed in cold 1 This investigation was supported, in part, by a Public Health Service Research Grant (AM-08416) from the National Institute of Arthritis and Metabolic Diseases; and, in part, by a Public Health Service Graduate Pathology Training Grant (5Tl-GM-1122) and a Research Career Program Award (K3-GM-22,575) from the National Institute of General Medical Sciences. ‘Present address: New York Medical College, Fifth Avenue at 106th Street, New York, New York 10029. 115
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AND
STENGER
(4%) 10% formalin solution, buffered to pH 7.0. Paraffin sections were cut at 5 ~1 and stained with hematoxylin and eosin, periodic acid-&hi& and periodic acid-Schiff after diastase digestion. For electron microscopy, segments of liver were fixed in 4% glutaraldehyde, phosphate buffered to pH 7.3 and held at 4°C for 2 hours. Subsequently, they were washed in the phosphate buffer for 1% hour and postfrxed for 2 hours in 1% osmium tetroxide, phosphate buffered to pH 7.3 (Millonig, 1961a) and maintained at 4°C. Dehydration was accomplished with graded acetone solutions and then the tissues were infiltrated with and embedded in Araldite (Stenger, 1963). Blocks were cured for 2-4 weeks at 50°C. Sections were obtained with glass knives and a Porter-Blum ultramicrotome. The sections were stained with lead hydroxide (Millonig, 1961b) and examined with an RCA-EMU-3 F electron microscope. RESULTS Group A Light
microscopy.
histologically normal. In the trypan-blue mal and contained
In the saline-injected
control
animals,
the livers were
injected animals, the liver parenchymal cells were norno perceptible dye. The sinusoids in the centrilobular
FIG. 1. Central portion of a liver lobule from a rat injected with trypan blue alone. The endothelial cells of the central vein are swollen. Adjacent to the vein are several small aggregates of macrophages. The centrilobular sinusoids are dilated and partially filled with hypertrophied sinusoidal lining cells. x 560.
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FIG. 2. Portion of a hepatic parenchymal cell from a rat injected with trypan blue alone. The nucleus, RER and mitochondria are normal. The glycogen areas contain numerous clusters of dense glycogen particles, interspersed with slightly dilated vesicles of SER (arrows). The Golgi zone (G) consists of parallel smooth cisternae, focally dilated by a content of small, moderately dense globules. x 18,000.
areas were dilated and, throughout, the lobules were lined by large protuberant Kupffer cells or flat, but prominent sinusoidal lining cells (Fig. 1). Both cell types contained variable amounts of trypan blue. Central veins showed swelling of the endothelium and small subendothelial accumulations of macrophages. Medium sized portal zones were edematous and sparsely infiltrated by macrophages, plasma cells, and monocytes. Electron microscopy. In the saline-injected animals, the liver cells showed no changes and were comparable to previously described normal liver cells (Bruni and Porter, 1965; Fawcett, 1966; Rouiller and Jezequel, 1963). The sinusoids and sinusoidal lining cells were morphologically similar to those described by others (Novikoff and Essner, 1960; Schaffner et al., 1963; Schmidt, 1960; Steiner, 1961). There were two types of sinusoidal lining cells, one consisting of flat, endothelium-like cells and the other appearing as large, phagocytic cells (Kupffer cells). Lying in sinusoidal recesses between adjacent liver cells was a third cell type, which contained abundant lipid, as described by Yamagishi (1959). In the trypan-blue injected animals, the parenchymal cells were also essentially normal. They contained clusters of particulate glycogen (Revel et al., 1960), disposed in relation to slightly prominent vesicular and tubular profiles of smooth endoplasmic reticulum (SER) (Fig. 2). The rough endo-
118
FIG. 3. Portion RER, mitochondria, widely distributed. the slightly prominent
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of a hepatic parenchymal cell from a rat injected with trypan blue alone. The and microhodies are normal. Small clusters of dense glycogen particles are A few residual bodies (Rb) with dense osmiophilic content are scattered near Golgi zones (G). x 18,OW.
plasmic reticulum (RER) consisted of parallel layers of narrow cisternae studded with ribosomes (Figs. 2 and 3). Mitochondria, microbodies, and single membrane limited lysosomes containing osmiophilic globules (residual bodies) were normal in distribution and appearance (Figs. 2 and 3). The Golgi zones were well-defined (Figs. 2 and 3). Liver cell nuclei were unaltered (Figs. 2 and 3). The sinusoidal lining cells, however, were larger than those of the saline controls (Figs. 4 and 5). This enlargement was primarily due to the presence of numerous phagosomes, which were delimited by single membranes and contained either coarse lamellar or finely reticulated materials (Fig. 4). The lining cells were further characterized by prominent Golgi zones, a well-developed RER, multiple small mitochondria, frequent pinocytotic vacuoles, and nuclei of irregular contour (Figs. 4 and 5). At their periphery, the sinusoidal lining cells of the trypan-blue injected rata displayed either wide pseudopodia or numerous slender villus-like processes (Figs. 4 and 5). Group B Light microscopy. The saline-treated, CC&-injected animals exhibited wide zones of liver necrosis, disposed about central veins and involving as much as % of each lobule. The necrotic zones were characterized by shrunken, eosinophilic, largely anuclear, partially fragmented parenchymal cells, interspersed with infiltrating polymorphonuclear leukocytes. The cells in the
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FIG. 5. Portions of several hypertrophied sinusoidal lining cells from a rat injected with trypan blue alone. The cells are enlarged by phagosomes of variable size and content. The cytoplasm contains prominent Golgi zones (G), as well as both narrow and dilated RER cistemae. Near the plasma membrane, the cells show multiple pinocytotic vesicles and vacuoles (arrows). Along the left margin is a small segment of a neighboring parenchymal cell. x 21,400.
FIG. 6. Liver from a saline-injected rat, killed 24 hours after CCla administration. Necrosis is evident in the central 11 of the lobule. The necrotic parenchymal cells are essentially anuclear, and inflammatory cells have infiltrated the necrotic zone. Surviving hepatic cells at the periphery of the lobule appear finely vacuolated due to fat accumulation. x 150. FIG. 7. Liver from a trypan-blue injected rat, killed 24 hours after CC14 administration. In the immediate vicinity of the central vein, there are a few necrotic cells, which have excited a focal inflammatory reaction. A small number of centrilobular liver cells show ballooning and hydropic change. Elsewhere, the parenchymal cells are normal, except for fine vacuolization, reflecting lipid accumulation. x 150. 121
FIG. 8. Sinusoidal lining cell from a trypan blue injected rat, killed 48 hours after CCL administration. The cell is enlarged primarily due to the presence of many, membrane-limited phagosomes, which vary in both size and shape. The phagosomes have a heterogeneous content, including, in one instance, a partially degraded, but recognizable mitochondrion (open arrow). The cytoplasm contains numerous free ribosomes, a well developed RER, many small mitochondria, and the edge of a Golgi zone (G). At its periphery, the cell exhibits many pinocytotic vacuoles (thin arrows) and numerous slender or blunt cytoplasmic projections. This cell is overlapped on two sides by tenuous processes of unidentified cells. At the upper right and the extreme lower left are small segments of the flanking hepatic parenchymal cells. x 18,000. 122
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periportal areas were intact but showed moderate to severe fat accumulation (Fig. 6). Triaditis was seen in the larger portal zones. Glycogen content of surviving parenchymal cells was moderate. Even after diastase digestion, Kupffer cells contained abundant PAS positive material. In contrast, the trypan-blue, CCL-injected animals revealed a minimal degree of liver necrosis around the central veins (Fig. 7). Scattered parenchymal cells in the central zones showed ballooning and hydropic change, and adjacent to these were small collections of histiocytes and macrophages. The intact liver cells showed mild to moderate fat accumulation. Medium-sized portal zones exhibited a triaditis and proliferation of histiocytes (Fig. 7). Glycogen was abundant in all cells except the hydropic and necrotic cells. In the Kupffer cells, the PAS diastase preparations showed a larger amount of PAS positive material than was seen in the control animals. Electron microscopy. In the saline-treated, CCL-injected animals, the changes were extensive and similar to those that have been described previously (Ashworth et al., 1963; Bassi, 1960; Recknagel, 1967; Stenger, 1966a). The changes included dilatation of the RER and SER, loss of glycogen, and degenerative changes of mitochondria, with loss of cristae and appearance of dense aggregates (Krishnan and Stenger, 1966; Reynolds, 1964, 1965). Lipid accumulation and increased numbers of autophagic vacuoles and residual bodies (lysosomes) were also present. Sinusoidal lining cells, however, showed no degenerative changes, but contained increased numbers of phagosomes. In the trypan-blue treated, CCL-injected animals, although less extensive, the changes in the parenchymal cells were qualitatively similar to those found in the saline controls. Sinusoidal lining cells were large and showed abundant RER and multiple small mitochondria. They also contained a complex group of phagosomes, some with dark osmiophilic material, others with laminated vesicles and degenerating mitochondria (Fig. 8). Group C
The saline-treated, CClr-injected animals exhibited centrilobular residues of liver cell necrosis and collections of histiocytes containing diastase resistant, PAS positive material. The surviving liver parenchyma showed widespread mitotic activity and moderate to severe fat accumulation. Glycogen content, except in areas of necrosis, was abundant. In the trypan-blue treated, CCL-injected animals, regeneration had occurred and was almost complete, with little evidence of necrotic residues. Histiocytes, however, persisted around the central veins. Surviving liver cells exhibited abundant glycogen and moderate amounts of lipid. Mitotic activity was moderate. The histiocytes and Kupffer cells contained PAS positive material, even after diastase digestion. Electron microscopy. The qualitative changes in both saline and trypanblue treated, CClr-injected animals were essentially similar to those in group B. Light
microscopy.
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DISCUSSION Intravenous administration of trypan blue afforded a marked protection from the hepatotoxicity of CCL, when animals were challenged 24 hours after the dye injection. At both 24 and 48 hours after CCL administration, the livers of trypan-blue injected rats showed far less necrosis and inflammatory reaction than was found in saline-injected controls. The reasons for this result are obscure. The injection of trypan blue alone caused centrilobular dilatation of liver sinusoids, endothelial swelling in central veins, and adjacent aggregations of macrophages (Fig. 1). Scott (1963, 1968) observed proliferative and inflammatory alterations in the pulmonary arterial tree after trypan blue administration. These changes would indicate that trypan blue has an adverse effect on blood vessels. It is not clear, however, whether such changes in the blood vasculature are accompanied by alterations in blood flow. Nor is it known to what extent hepatic circulation might be affected by the trypan blue induced hypertrophy of the liver sinusoidal lining cells. Concerning the latter, it has been noted that injections of biologic ink resulted in greater enlargement of hepatic sinusoidal lining cells than was effected by trypan blue, yet the extent of liver protection from CCL after such carbon loading (Stenger et al., 1968) was not appreciably different from that observed in the present experiments. The question of changes in hepatic blood flow after trypan blue administration will not be resolved until direct measurements are made. (1954) It might be pertinent to note, however, that Shore and Zilversmit measured blood flow after carbon loading of the liver and found no significant change in hepatic circulation. In view of this, it would seem unlikely that alterations in hepatic blood flow could completely explain the marked differences in hepatotoxicity reported here. In the present study, at 24 hours after injection of trypan blue alone, there was no evidence, either by light or electron microscopy, that the dye had even entered the hepatic parenchymal cells. Morgan et al. (1966) also failed to demonstrate trypan blue in the liver parenchyma. Beck et al. (1967), however, have identified trypan blue in visceral yolk sac epithelium, 24 hours after exposure. In this instance, the dye was concentrated in deep digestive vacuoles (heterolysosomes) and its presence was associated with an inhibition of selected hydrolytic enzymes. More to the point, Mjrsliwski and Michalik (1966) have observed morphologic liver changes indicative of hepatotoxicity at 12 hours after trypan blue injection. It seems apparent, therefore, that trypan blue can enter cells, perhaps even liver ceils, and that such entry may be associated with intracellular damage. It is conceivable that, in the present investigation, the dye gained access to the hepatocytes and caused subcellular changes at early intervals, but it then must be inferred that these changes were transient and that complete morphologic restitution of hepatocellular architecture had taken place by 24 hours after the dye injection. This would be analogous to the transient effects of neutral red dye on the liver parenchyma, as reported by Alousi et al. (1968).
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It is widely held that CCL activation and consequent toxicity develop entirely within the parenchymal cells of the liver (McLean and McLean, 1966; Slater, 1966). As shown here, however, there was no difference, at least in structural terms, between the parenchymal cells of the trypan-blue injected rats and those of the saline-treated controls, at the time of CC14 challenge. If the parenchymal cell were the only important factor in Ccl, hepatotoxicity, an equivalent response would have been expected in the trypan-blue and saline-treated rats. As noted above, this was not the case. Attention was directed, therefore, to structural differences that might account for the results obtained. One obvious difference was that noted in the liver sinusoidal lining cells, which were notably hypertrophied in the trypan blue injected rats and essentially normal in the saline-treated animals. It is here proposed that this difference might be related to the observed divergent response to CC% administration. Whether the protective effect of prior dye treatment is related simply to the increased cytoplasmic mass of the sinusoidal lining cells or to a specific metabolic process is, at present, unknown. It is generally accepted that phagocytosis is the primary function of the liver sinusoidal lining cells. In the present investigation, this function was manifest after trypan blue injection (Figs. 4 and 5) and prominent after CC14 administration (Fig. 8). In the former, the phagocytosis was related to dye ingestion and was accompanied by other cytoplasmic changes, including an increase of RER and enlargement of the Golgi zones. After Ccl,, the increased numbers of phagosomes were considered to reflect phagocytosis of parenchymal cell debris, in accord with previous suggestions (Popper et al., 1960; Schaffner et al., 1963; Stenger, 1966b). More recently additional functions have been ascribed to the hepatic sinusoidal lining cells. Cohen et al. (1960), Schaffner and Popper (1962), and Steiner (1961) have attributed to these cells a role in antibody formation. Schaffner et al. (1963) have proposed a role in hepatic fibrogenesis and Ackerman et al. (1961), a role in erythropoiesis, at least in embryonic liver. Snell (1960) has indicated that the sinusoidal lining cells respond to drugs and that this capability influences their phagocytic activity. Finally, as discussed above, the results of the present investigation raise the possibility that sinusoidal lining cells might also function in the handling of toxic agents. SUMMARY Rats were given intravenous injections of either physiologic saline or trypan blue dye suspended in saline. After 24 hours, the livers of these animals were sampled for light and electron microscopic study. The livers of the saline treated rats showed no morphologic alterations. The livers of the trypan blue injected animals exhibited no structural abnormalities in the hepatic parenchymal cells, but their sinusoidal lining cells were hypertrophied, mainly due to the appearance of numerous phagosomes, increased quantities of rough endoplasmic reticulum, and prominent Golgi zones. Similarly prepared rats were given intraperitoneal injections of CCL, 24 hours after the intravenous administration of saline or trypan blue. The livers of these animals were sampled, 24 and 48 hours after the toxic challenge. At both intervals, the livers of the rata treated with trypan blue and CCL displayed far less necrosis and inflammatory reaction than was found in the animals given saline and CCL. It was considered unlikely that this difference could be completely ex-
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plained by changes in hepatic blood flow. It was also demonstrated that, at 24 hours after administration of trypan blue, there were no hepatocellular morphologic alterations that might account for the divergent response to Ccl,. It was proposed, therefore, that the hypertrophied sinusoidal lining cells of the trypan blue treated rats might be related to the observed protection from CCh hepatotoxicity. ACKNOWLEDGMENTS The authors are greatly indebted to Drs. J. D. Reid, J. Wiener, structive criticism and advice in the preparation of the manuscript, for his excellent technical assistance.
and M. A. Alousi for their conand to Mr. J. N. Williamson
REFERENCES ACKERMAN, G. A., GRASSO, J. A., and KNOW-F, R. A. (1961). Erythropoiesis in the mammalian embryonic liver as revealed by electron microscopy. Lab. Invest. 10,787-796. ALOLJS~, M. A., STENGER, R. J., and MORGAN, W. S. (1968). The fine structure of pancreatic acinar and hepatic parenchymal cells after neutral red dye injection. Exptl. Mol. Pathol. 9, 97109. Hepatic cell degeneration. ASHWORTH, C. T., LUIFIEL, F. J., SANDERS, E., and ARNOLD, N. (1963). Correlation of fine structural with chemical and histochemical changes in hepatic cell injury produced by carbon tetrachloride in rata. Arch. Pathol. 75, 212-225. B~ssr, M. (1969). Electron microscopy of rat liver after carbon tetrachloride poisoning. Expt.
Cell Res. 20,313-323. BECK, F., LLOYD, J. B., and GRIFFITHS, A. (1967). Lysceomal enzyme inhibition by trypan blue: A theory of teratogenesis. Science 157,118&1182. BIOZZI, G., BENACERRAF, B., and HALPERN, B. N. (1953). Quantitative study of the granulopectic activity of the reticula-endothelial system. II. A study of the kinetics of the granulopectic activity of the R.E.S. in relation to the dose of carbon injected; relationship between the weight of the organs and their activity. Brit. J. Exptl. Pathol. 34,44-457. BIOZZI, G., BENACERRAF, F., and HALPERN, B. N. (1955). The effect of Sahn. typhi and its endotoxin on the phagocytic activity of the reticula-endothelial system in mice. Brit. J. Ezptl.
Pathol. 36,226-235. BRUNI, C., and PORTER, K. R. (1965). The fme structure of the parenchymal cell of the rat liver. I. General observations. Am. J. Pathol. 46,691-755. CAMERON, G. R., and KARUNARATNE, W. A. E. (1936). Carbon tetrachloride cirrhosis in to liver regeneration. J. Puthol. Bacterial. 42,1-21. COHEN, S., OHTA, G., SINGER, E. J., and POPPER, H. (1969). Immunocytochemical study of globulin in liver in hepatitis and p&necrotic cirrhosis. J. Exptl. Med. 111, 285-294. DAWKINS, M. J. R. (1963). Carbon tetrachloride poisoning in the liver of the newborn
normal relation gamma rat.
J.
Pathol. Bacterial. 85,189-196. FAWCETT, D. W. (1966). “An Atlas of Fine Structure. The Cell, Its Organelles and Inclusions.” Saunders, Philadelphia, Pennsylvania. FISHER, S. (1966). Stimulation of splenic antigen uptake and of antibody response in mice by India ink or other “blockading” agents. Immunology 11,127-136. FISHER, S. (1967). Localization of radioiodinated endotoxin in organs of mice and rabbits: Effect of thorotrast, trypan blue, endotoxin and carbon administered intravenously. Nature 213, 511-512. HAMPTON, J. C. (1958). An electron microscope study of the hepatic uptake and excretion of submicroscopic particles injected into the blood stream and into the bile duct. Acta Anat. 32,
262-291. HOWARD, J. G. (1959). Activation of the reticula-endothelial cells of the mouse liver by bacterial lipopolysaccharide. J. Pathol. Bacterial. 78,465-470. KRISHNAN, N. and STENGER, R. J. (1966). Effects of starvation on the hepatotoxicity of carbon tetrachloride. A light and electron microscopic study. Am. J. Pathol. 49,239-255. MCLEAN, A. E. M., and MCLEAN, E. K. (1966). The effect of diet and l,l,l-tri-chloro-2,2-bis-
EFFECT
OF TRYPAN
BLUE AND CCL ON LIVER
127
(p-chlorophenyl) ethaue (DDT) on microsomal hydroxylating enzymes and on sensitivity of rats to carbon tetrachloride poisoning. Rio&em. J. 100,564~571. MK.LONIG, G. (1961a). The advantages of a phosphate buffer for 0~04 solutions in fixation. J. Appl. Phys. 32,1637. MILLONIG, G. (1961b). A modified procedure for lead staining of thin sections. J. Biophys. Biothem. Cytol. 11,736-739. MORGAN, W. S., FERNANDO,J., and ALOUSI, M. A. (1966). Studies of the biological activity of neutral red. Exptl. Mol. Pathol. 5,491X503. MY~LIWSKI, A., and MICHALIK, T. (1966). Histochemiczne badania kwasow nukleinowych i glikogenu w komorkach watrobowych szczurbw poddanych dzialaniu blekitu trypanu i blekitu Evansa,Folia Morphol. 25,61-67. NOV~OFF, A. B., and ESSNER,E. (1960). The Liver Cell. Some new approaches to its study. Am. J. Med. 29, 102-131. POPPER, H., PARONFITO, F., and BARKA, T. (1960). PAS-positive structures of nonglycogenic character in normal and abnormal liver. Arch. Pathol. 70,300-313. REBUCK, J. W., BOYD, C. B., and RIDDLE, J. M. (1960). Skin windows and the action of the reticuloendothelial system in man. Ann. N. Y. Acad. Sci. 88,30-42. RECKNAGEL,R. 0. (1967). Carbon tetrachloride hepatotoxicity. Pharmacol. Reu. 19,145-208. REVEL, J. P., NAPOLITANO, L., and FAWCETT, D. W. (1966). Identification of glycogen in electron micrographs of thin tissue sections. J. Biophys. Biochem. Cytol. 8,575-589. REYNOLDS, E. S. (1964). Liver parenchymal cell injury. II. Cytochemical events concerned with mitdchondrial dysfunction following poisoning with carbon tetrachloride. Lab. Invest. 13, 14571470. REYNOLDS,
E. S. (1965). Liver parenchymal cell injury. III. The nature of calcium-associated electron-opaque masses in rat liver mitochondria following poisoning with carbon tetrachloride. J. Cell. Biol. 25,53-75. ROUILLER, CH., aqd JI~ZEQUEL, A.-M. (1963). Electron microscopy of the liver. In “The Liver. Morphology, Biochemistry, Physiology.” (Ch. Rouiller, ed.) Vol. 1, pp. 195-264. Academic Press, New York. SCHAFFNEFI, F., and POPPER, H. (1962). A phagocytic and protein-forming mesenchymal cell in human cirrhosis. Nature 196.684-685. SCHAFFNER, F., BARKA, T., and POPPER, H. (1963). Hepatic mesenchymal cell reaction in liver disease. Exptt. Mol. Pathol. 2,419-441. SCHMIDT, F. C. (1960). Elektronenmikroskopische Untersuchungen an den Sinusoid-Wandzellen (Kupffersche Sternzellen) der weissen Maus. Anat. Anz. 108,376-387. Scorr, G. B. D. (1963). The effects of trypan blue on the formation of thrombi in the generalized Shwartzman reaction. Brit. J. Exptl. Pathol. 44,317-325. Scorn, G. B. D. (1968). Trypan blue and the generalized Shwartzman reaction. The nature and formation of fibrinoid material in the pulmonary arteries. Brit. J. Erptl. Pathol. 49, 251-256. SHORE, M. L., and ZILVERSMIT, D. B. (1954). Effect of India ink on bromsulfalein excretion, phagocytosis and circulation in the liver. Am. J. Physiol. 177,436440. SLATER, T. F. (1966). Necrogenic action of carbon tetrachloride in the rat: a speculative mechanism based on activation. Nature 209,36-40. SNELL, J. F. (1960). The reticuloendothelial system: I. Chemical methods of stimulation of the reticuloendothelial system. Ann. N. Y. Acad. Sci. 88,56-77. STEINER, J. W. (1961). Investigations of allergic liver injury. I. Light, fluorescent and electron microscopic study of the effects of soluble immune aggregates. Am. J. Pathol. 38,411-436. STENGER, R. J. (1963). Hepatic parenchymal cell alterations after long-term carbon tetrachloride administration. A light and electron microscopic study. Am. J. Pathol. 43,867-895. STENGER, R. J. (1966a). Ultrastructural alterations within hepatic parenchymal cells after carbon tetrachloride poisoning. fn “Methods and Achievements in Experimental Pathology” (E. Bajusz and G. Jasmin, eds.), Vol. 1, pp. 677-700. Karger, Basel. STENGER, R. J. (1966b). Hepatic sinusoids in carbon tetrachloride-induced cirrhosis. An electron microscopic study. Arch. Pathol. 81,439-447. STENGER, R. J., WILLIAMSON, J. N., and JOHNSON, E. A. (1968). The effect of reticuloendothelial
128 blockade
PETRELLI upon
AND
STENGER
carbon tetrachloride hepatotoxicity. A light and electron microscopic study. 52, lla (Abst.). THORBECKE, G. J., MAURER, P. H., and BENACERRAF, B. (1960). The affinity of the reticuloendothelial system for various modified serum proteins. &it. J. En@. Path. 41, 190-197. WIGGLESWORTH, J. S. (1964). The use of the osmium-ethyl gallate technique in the study of carbon tetrachloride liver injury. J. Pathol. Bacterial. 87,333-339. YAMAGISHI, M. (1959). Electron microscope studies on the fine structure of the sinusoidal wall and fat-storing cells of rabbit livers. Arch. Histol. hp. 18, 223-261.
Am. J. Pathol.