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Effect of antirat hepatic serum on rats--a histopathological study.
In patients with cirrhosis of liver and chronic active hepatitis particularly the lupoid variety auto-antibodies to cytoplasm[9],[13] smooth muscles[14] and against bile canaliculi[5] have been demonstrated. These autoantibodies are neither organ specific nor species specific. A search fox organ specific autoantibodies in the liver diseases has had limited success. Experimental liver injury has been produced by liver specific lipoprotein,[10] by homologous liver protein incorporated with incomplete Freund's adjuvant[4] and by large doses of antihepatic serum.[2] However, no changes in the liver have been reported in the past after administration of either large or small doses of antihepatic serum over a period of months. The present study was undertaken to see the effect of small doses of antihepatic serum over a period of one month, as well as large doses administered for short duration on various organs of rats.
The experiments were performed on 66 adult albino rats of both sexes, each weighing 200-250 gin, purchased from local dealer and 7 albino rabbits (U.K. strain) of both sexes, weighing 2.5-3.0 kg procured from Central Drug Research Institute, Lucknow. Animals were kept separately and given standard diet. Preparation of antigen. Antigen was prepared from aseptically removed and washed liver, kidneys, lungs and heart by a modified technique.12 The modification was that the minced tissues were mixed with 10 volumes of phosphate buffered saline in case of heart, lung and kidneys, whereas no solution was added in case of liver, and then homogenised in a glass tissue homogeniser for 20-30 minutes. The paste in case of liver was diluted with phosphate buffered saline to make 20 per cent suspension. The final coarse homogenates were cultured on different media to test the sterility. If no growth was found, these were stored in screw capped bottles at -20°C to -10°C. Production of antisera Half ml of 20 per cent suspension of rat's liver antigen mixed with 0.5 ml of complete Freund's adjuvant was injected intraperitoneally (ip.) into rabbits 1 to 5, followed by injection of pure antigen 1.0 ml; 1.5 ml; 2.0 ml; 3.0 ml and 4.5 ml on fourth, seventh, tenth, thirteenth and sixteenth days respectively. Rabbits numbered 6 and 7 served as control. Antibody titer Animals were rested for 10-15 days after the last injection, followed by bleeding from ear veins, on day 0, day 21 and after 1 month and 2 months. Antibody titre was determined in all samples by precipitation test.15 When antibody titer was more than 1:1000, rabbits were bled to death. Serum was separated and tested for sterility by culturing on MacConkey's, blood agar. Sera were pooled and stored at -20°C. Administration of antisera to rats Sera were inactivated before use by heating at 56°C for 30 minutes and treated with one volume of rat's red blood cells by contact for 10 minutes at room temperature to minimise haemolysins. Twenty albino rats (Group 'A') were injected 2.0 ml of antiliver serum i.p. in a single dose. Sixteen albino rats (Group 'B') were given 0.5 ml of antiliver serum i.p. on alternate days for one month. Five albino were given control sera from nonimmunised animals (Group C) and other 5 animals were kept as untreated controls. Pathological procedures At appropriate intervals after one or more injections of serum, the rats were sacrificed. Various organs viz. kidneys, liver, heart, lung and brain were removed. Organs were examined grossly, sections were prepared and stained by routine haematoxylin and eosin staining technique.
Antibody litre Anti-liver serum from rabbit 1 had a precipitin titre of 1:5000 against liver antigen whereas other rabbits' sera had a titre of 1: 2000 at the time of sacrifice. All the sera showed strong cross-reactivity with kidney antigen (1:500 to 1:1000 titre) and weak insignificant reaction with heart and lung antigen. No gross change was observed in any of the rat's organs studied in two sets of experiments. Microscopic changes in group 'A' [Table - 1]: Of 20 rats, 3 died immediately after receiving the single dose and were not included in the study. Nine rats killed after 48 hours and 8 rats killed after 96 hours of after single dose injection showed essentially similar features with slight variation in severity of liver cell necrosis which was more pronounced in rats sacrified after only 48 hours. There was widening of portal region, severe chronic inflammatory cell infiltration mainly in portal region, with involvement of adjacent parenchyma and death of single hepatocyte or group of hepatocytes. There was relatively more fibrous tissue in the portal tract in one animal after 96 hours. The band like necrosis of liver cells was also evident besides infiltration by lymphocytes extending from one portal area to other. Moderate Kupffer cell hyperplasia was also seen [Fig. 1]. In some areas, scattered small granulomatous lesions consisting of areas of necrosed liver cells, infiltrated by mononuclear cells, lymphocytes and eosinophils were seen [Fig. 2]. There was also moderate periportal, central and sinusoidal infiltration by lymphocytes, mononuclear cells and eosinophilic granulocytes. Microscopic changes in group 'B': Sixteen albino rats of this group were sacrificed after 10, 15, 21 and 30 days in batches of four each by which time they had received 5, 7, 10 and 15 injections of antiliver serum respectively. As depicted in [Table - 1] in all the rats there was mild to moderate lymphocytic and mononuclear cell infiltration in portal, central and sinusoidal areas. In one rat after 30 days, there was marked infiltration by eosinophilic granulocytes at portal area. No change specific for cirrhosis was seen. No change was observed in brain, heart, lungs and kidneys of group A or B animals, nor was there any change in any organs in control groups C and D.
Autoimmune liver damage has been experimentally produced by injection into the portal circulation of antigen and antibody complexes prepared in vitro by injection of heterologous substances following initial damage by carbon tetrachloride or allyl alcohol.[7] Liver homogenates, liver fractions and antiliver antibodies have all been used to produce hepatic lesions.[2] Many workers have suggested that the mechanisms of liver damage is via deposition of immune complexes and this may be facilitated by previous injection.[7] The present work investigates the effect of heterologous antiliver antibodies (produced in rabbits) on the rat liver. The maximum titre of antiliver antibodies was 1:5000 and the latter showed cross reactive antibodies against kidneys, heart and lungs. Antiliver antibody titre upto 1:2000 and precipitation with rat's kidney have been reported in the past.[2] The liver of animals sacrificed 48 and 96 hours after a single large dose of heterologous serum revealed parenchymatous degeneration, focal necrosis, mononuclear cell infiltration, Kupffer cell hyperplasia and periductal and perivascular mononuclear cell infiltration. Estes[4] and Barmera et al[1] who injected butanol soluble fraction of homologous liver homogenate also reported similar lesions. We also found portal mononuclear inflammatory cells infiltration and involvement of periportal parenchyma resembling piecemeal necrosis. This has also been reported by Paronetto et al.[6] Barmera et al[1] could produce similar lesions by passage of sensitized lymphocytes implicating a role of lymphocyte mediated mechanisms in such liver lesions. The overall histological picture that was obtained in this experiment, closely resembled chronic active hepatitis in human beings but did not show any scarring. However, Gall[3] produced lesion similar to cirrhosis with heterologous serum. Paronetto and Popper[8] produced chronic liver injury resulting into cirrhosis by heterologous sera and related the lesions to the localisation of immune complexes in the portal tracts indicating that vascular and connective tissue structure of the liver were the primary sites of injury. However, prolonged administration of the serum did not cause any lesion except lymphocytic and eosinophilic infiltration in the portal, central and sinusoidal areas in the liver. This suggests that the liver is capable of rapid recovery following the early lesions and then developes tolerance to subsequent doses.[2] From the above data, it appears that large doses of heterologous sera result in lesions of chronic active hepatitis which could be attributed to liver damage by immune complexes in antibody excess. We feel that this is a good animal model to further explore the pathogenic mechanism in chronic liver disease of autoimmune type.
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