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Liver fibrosis in heterozygous WHHL rabbits fed cholesterol and fats; a new animal model
Intemtionul
Hepatology International Hepatology Communications 3 (1995) 310-315
ELSEVIER
communications
Liver fibrosis in heterozygous WHHL rabbits fed cholesterol and fats; a new animal model I&e Simonsen*a, Thomas Horna, Alicja Mortensenb, Birgit Fischer Hansenc,J#rgenFischer Hansend aDepartment of Pathology. Herlev Hospiial, DK-2730 Herlev, Denmark bInstitute of Toxicology, National Food Agency, DK-2860 Sgborg. Denmark CDepartment of Pathology, Hwdovre Hospital, DK-2650 Hvidovre. Denmark ‘Department of Cardiology, Hvidovre Hospital, DK-26.50 Hvidovre, Denmark
Received 6 October 1994; accepted18 January 1995
Abstract
Heterozygous WHHL rabbits were fed a cholesterol enriched diet with added vegetable oil or marine oil for 14 weeks. All rabbits developed varying degreesof liver fibrosis, which was most severein the cholesteroYmarine oil fed group. In all groups the pattern of fibrosis was very similar to that seen in human alcoholics. The advantages and disadvantages of this new animal model are discussed. Kpywor& Heterozygous WHHL rabbits; Cholesterol rich diets; Vegetable oil; Marine oil; Liver fibrosis
1. Introduction
Several experimental animal models exist for the study of liver fibrosis/cirrhosis [ 11. None reproduces exactly the disease in man, and all have their advantages and disadvantages. This paper describes a new rabbit model, which in many ways is clearly superior to most other animal models. The homozygous Watanabe heritable hyperlipidemic (WHHL) rabbit is a wellknown animal for studying atherosclerosis, which it develops spontaneously [2]. The
l
Corresponding author, Drosselvej 18, DK-2000 Frederiksberg, Denmark. Tel.: +45 38 335691.
0928-4346!95/$09.50 SSDI
0 1995 Elsexier Science Ireland Ltd. All rights reserved
0928-4346(95)00199-S
K. Simonsen et al. /International
Hepatology Communications 3 (1995) 310-315
311
heterozygous WHHL rabbit develops only minimal spontaneous atherosclerosis at an age of approximately 2 years. The development of atherosclerosis in younger heterozygotes is conditioned by addition of cholesterol to the diet. The present study was designed with the purpose of examining the morphology of the experimental atherosclerosis and the influence on this of different types of fat in heterozygous WHHL rabbits [3]. Totally unexpectedly, considerable liver fibrosis was detected in some of the rabbits, and the pattern of fibrosis was very much like that found in human alcoholics [41. 2. Materials and methods 2.1. Experimental design
Twenty-two heterozygous WHHL rabbits, 8-9.5 months old with plasma cholesterol levels of 0.95 mmol/l f 0.53 (mean f S.D.) were randomized in three groups: group I consisted of eight rabbits (three females and five males) and groups II and III of seven rabbits each (four females and three males). For 14 weeks the rabbits in group I were fed a cholesterol enriched standard diet. For the same period of time the rabbits in group II were fed a cholesterol enriched diet with added vegetable oil and the rabbits in group III were fed a cholesterol enriched diet with added marine oil. During the first 5 weeks the dose of cholesterol was increased by 0.2% and the fats by 2% per week up to 1% and lo%, respectively. The dose of fats in group II and III was reduced to 5% from the sixth week. The animals were fed 100 g diet daily and had free access to tap water. Additionally, nine rabbits kept on a standard diet without cholesterol or fats were monitored until at least 19 months of age and their livers were examined as an untreated control. The body weight was recorded weekly and the feed intake daily. At the end of the experiment the animals were euthanized by an intravenous injection of 5% phenobarbital solution. Samples for histological examination were taken from various organs. Sections from the liver were stained with HE, vGH, MST, PAS/D and elastin. Furthermore sections were stained immunohistochemically using monoclonal ubiquitin (mouse anti-human, Zymed Laboratories Inc.). The histological examination was performed blindly. The pattern of fibrosis was registered and the degree graded as none, slight, moderate and severe. The steatosis was graded as none, questionable, slight, moderate and severe and the size of the fat vacuoles was noted. Furthermore special attention was given regarding inflammation (in parenchyma and in portal tracts) and to the presence of megamitochondria and Mallory-like bodies. 2.2. Diets
Three different powdered diets from Altromin, Lage, Germany were used: the standard diet and two concentrated diets containing the nutrients of the standard diet allowing for addition of 5% and 10% by weight of fat, respectively. The diets were used to produce pellets with 0 and 1.11% (w/w) cholesterol (Sigma C 8503). The vegetable oil contained 0.6% n-3 polyunsaturated fatty acids (PUFA), 32.9% n-6 PUFA, 30.6% saturated fatty acids (SFA) and 35.5% monounsaturated fatty acids
312
K. Simonsen
et al. /International
Hepatology
Communications
3 (1995)
310-315
(MUFA). The marine oil contained 16.6% n-3 PUFA, 17.7% n-6 PUFA, 27.9% SFA and 35.2% MUFA.
3.Results The mean body weight was 3.66 kg f 0.45 (S.D.), 3.65 kg f 0.31 and 3.78 kg f 0.28 at the beginning of the study and 3.36 kg f 0.33,3.28 kg f 0.26 and 3.22 kg i 0.08 at the termination in groups I, II and III respectively. No significant difference in body weight was found between the groups during the experiment. The mean body weight was 3.66 kg f 0.45 (S.D.), 3.65 kg f 0.31 and 3.78 kg f 0.28 at the beginning of the study and 3.36 kg f 0.33,3.28 kg f 0.26 and 3.22 kg * 0.08 at the termination in groups I, II and III, respectively. No significant difference in body weight was found between the groups during the experiment. The mean daily feed intake was 95 g i 4 (S.D.), 87 g f 9 and 75 g f 15 in groups I, II and III, respectively. During the experiment the mean feed intake was from the histological investigation), and one female in group II was euthanized after 11 weeks. The liver from this female and the liver from the female from group III which died unexpectedly in week 12 (the cause of death was myocardial infarction) were included in the histological examination. The cholesterol feeding caused pronounced hypercholesterolemia which was significantly enhanced by the added oils. The results of the changes in the blood lipids, as well as the results of the histological examination of the various organs besides the livers, have been published elsewhere [3]. 3.1. Pathology
The untreated controls showed histologically normal livers with no signs of tibrosis and none or questionable steatosis. Group I (cholesterol fed) developed questionable to slight steatosis, mostly microvesicular. There was none to slight perivenular fibrosis in Rappaports zone 3 (Fig. la). In some of the animals there were Mallory-like bodies (Fig. 2), but no inflammatory changes. The results of the immunohistochemical staining of these Mallory-like bodies were inconclusive in all three groups. Cholestasis or megamitochondria were not present. No periportal fibrosis was seen. Group II (cholesterol and vegetable oil fed) showed changes similar to the rabbits in group I, but the steatosis and the fibrosis were more pronounced. The pattern of fibrosis was similar to the one described below, although not as severe (Fig. lb). Here also there were few Mallory-like bodies, but no inflammation. Periportal fibrosis was slight. Group III (cholesterol and marine oil fed) all developed severe steatosis (both micro- and macrovesicular) and they all developed severe fibrosis. Constant findings were severe pericellullar fibrosis, particularly in Rappaports zone 3, and in some areas as bridging fibrosis primarily between central veins and portal tracts but between two central veins as well. In some of the rabbits the fibrosis was so severe that it mimicked cirrhosis (Fig. lc). Few Mallory-like bodies were seen here as well, and in one rabbit there was additional cholestasis. Megamitochondria were not seen. Portal tracts were enlarged to varying degrees and revealed infiltration with mononuclear cells.
K. Simonsen et al. /International
Hepatology Communications 3 (1995) 330-315
Fig. 1. All three illustrations represent Reticulin stained sections. Original magnification x 67. (a) GI.oup I; a Iilight 1perisinusoidal fibrosis of zone 3 is found. Terminal hepatic venule: V. Portal tract: P. (b) GI*oup 11; a xate fibrosis, perisinusoidal and pericellullar, is seen in zone 3, whereas periportal , lib Irotris is sligkIt. Terminal hepatic venule: V. Portal tract: P. (c) Group III; severe fibrosis with bridgit ‘g t jet\Neen indb qidual zone 3 areas and a nodular parenchymal pattern is seen. Terminal hepatic venuh :: v
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K. Simonsen
et al. /International
Hepatology
Communications
3 (1995)
310-315
Fig. 2. H and E section. Original magnification x 400. Group I; zone 3 hepatocytes some of which contain Mallory-like bodies (arrows).
4. Discussion The heterozygous WHHL rabbit fed cholesterol with added oils is a new animal model for studying fibrogenesis in the liver. It has several advantages, one being the development of a pattern of fibrosis very similar to that observed in human alcoholics for the group having cholesterol-rich diet with added marine oil [4]. Pericellullar fibrosis in a chickenwire fashion and bridging fibrosis between central veins and portal tracts as well as between two central veins were prominent. This finding seems to be constant and reproducible as all the rabbits in this group had similar changes in their livers. Furthermore they presented severe steatosis and occasional Mallory-like bodies, which were negative in an immunohistochemical staining with ubiquitin antibody. Other advantages are that the changes can be provoked in a relatively short time and that the diets can be given in pellets instead of having to administrate the diet/toxins in a less natural way. These features are in contrast to many other animal models [l]. Finally, this animal model is a relatively cheap model compared to some of the better known animal models such as the baboon [5]. A disadvantage of the model is the fact that a few animals had to be removed because of continuously decreased food intake. Rats fed fish oil have been shown to develop focal fibrosis and rats fed fish oil with ethanol have been shown to develop less fatty accumulation but increased inflammation and necrosis when compared to rats fed corn oil with ethanol [6]. Fur-
K. Simonsen et al. /international
Hepatology Communications 3 (1995) 310-315
315
thermore there is evidence which indicates that fish oil increases lipid peroxidation in the liver, which might cause fatty accumulation and fibrosis [7]. To our knowledge tibrosis as severe as described and as closely mimicking that seen in alcoholic liver disease in humans has not been described in an experimental animal model fed a marine oil added cholesterol-rich diet. Many areas concerning the initiating of and the pathways in liver fibrogenesis are still not well understood. One of the major questions to be answered is the possible reversibility or irreversibility of the fibrotic changes. To investigate this the above described model might be suitable if one included a larger number of animals. Furthermore it would be of interest to study the possible relevance of drawing parallels to humans considering the extensive use of commercially produced marine oils. Acknowledgements The authors wish to thank Walter Schmidtsdorff, Technical Laboratory, Ministry of Fisheries and Carl-Erik Hay, Danish Technical University for providing the marine and vegetable oils. References 111 Tsukamato H, Matsuoka M, French SW. Experimental models of hepatic fibrosis: A review. Semin PI 131 141 151
161 [71
Liver Dis 1990; IO: 56-65. Hansen BF, Mortensen A, Hansen JF, Ibsen P, Frandsen H, Nordestgaard BG. Atherosclerosis in Watanabe heritable hyperlipidaemic rabbits. Evaluation by macroscopic, microscopic and biochemical methods and comparison of atherosclerosis variables. APMIS 1994; 102: 177-90. Mortensen A, Meyer 0, Hansen BF et al. Changes in blood lipids and aorta in heterozygous WHHL rabbits fed cholesterol and fats. In: Abstracts from 62nd EAS Congress, 1993. Christoffersen P, Junge J. The spectrum of alcohol induced liver disease. Histological features. APMIS Suppl 1991; 23: 40-5. Rubin E, Lieber CS. Fatty liver, alcoholic hepatitis and cirrhosis produced by alcohol in primates. N Engl J Med 1974; 290: 128-35. French SW. Nutrition in the pathogenesis of alcoholic liver disease. Alcohol Alcohol 1993; 28: 97-109. Morimoto M, Zem MA, Hagbjdrk A-L, Ingelman-Sundberg M, French SW. Fish oil, alcohol and liver pathology: Role of cytochrome P450 2El. Proc Sot Exp Biol Med 1994; 207: 197-205.
Hepatology International Hepatology Communications 3 (1995) 310-315
ELSEVIER
communications
Liver fibrosis in heterozygous WHHL rabbits fed cholesterol and fats; a new animal model I&e Simonsen*a, Thomas Horna, Alicja Mortensenb, Birgit Fischer Hansenc,J#rgenFischer Hansend aDepartment of Pathology. Herlev Hospiial, DK-2730 Herlev, Denmark bInstitute of Toxicology, National Food Agency, DK-2860 Sgborg. Denmark CDepartment of Pathology, Hwdovre Hospital, DK-2650 Hvidovre. Denmark ‘Department of Cardiology, Hvidovre Hospital, DK-26.50 Hvidovre, Denmark
Received 6 October 1994; accepted18 January 1995
Abstract
Heterozygous WHHL rabbits were fed a cholesterol enriched diet with added vegetable oil or marine oil for 14 weeks. All rabbits developed varying degreesof liver fibrosis, which was most severein the cholesteroYmarine oil fed group. In all groups the pattern of fibrosis was very similar to that seen in human alcoholics. The advantages and disadvantages of this new animal model are discussed. Kpywor& Heterozygous WHHL rabbits; Cholesterol rich diets; Vegetable oil; Marine oil; Liver fibrosis
1. Introduction
Several experimental animal models exist for the study of liver fibrosis/cirrhosis [ 11. None reproduces exactly the disease in man, and all have their advantages and disadvantages. This paper describes a new rabbit model, which in many ways is clearly superior to most other animal models. The homozygous Watanabe heritable hyperlipidemic (WHHL) rabbit is a wellknown animal for studying atherosclerosis, which it develops spontaneously [2]. The
l
Corresponding author, Drosselvej 18, DK-2000 Frederiksberg, Denmark. Tel.: +45 38 335691.
0928-4346!95/$09.50 SSDI
0 1995 Elsexier Science Ireland Ltd. All rights reserved
0928-4346(95)00199-S
K. Simonsen et al. /International
Hepatology Communications 3 (1995) 310-315
311
heterozygous WHHL rabbit develops only minimal spontaneous atherosclerosis at an age of approximately 2 years. The development of atherosclerosis in younger heterozygotes is conditioned by addition of cholesterol to the diet. The present study was designed with the purpose of examining the morphology of the experimental atherosclerosis and the influence on this of different types of fat in heterozygous WHHL rabbits [3]. Totally unexpectedly, considerable liver fibrosis was detected in some of the rabbits, and the pattern of fibrosis was very much like that found in human alcoholics [41. 2. Materials and methods 2.1. Experimental design
Twenty-two heterozygous WHHL rabbits, 8-9.5 months old with plasma cholesterol levels of 0.95 mmol/l f 0.53 (mean f S.D.) were randomized in three groups: group I consisted of eight rabbits (three females and five males) and groups II and III of seven rabbits each (four females and three males). For 14 weeks the rabbits in group I were fed a cholesterol enriched standard diet. For the same period of time the rabbits in group II were fed a cholesterol enriched diet with added vegetable oil and the rabbits in group III were fed a cholesterol enriched diet with added marine oil. During the first 5 weeks the dose of cholesterol was increased by 0.2% and the fats by 2% per week up to 1% and lo%, respectively. The dose of fats in group II and III was reduced to 5% from the sixth week. The animals were fed 100 g diet daily and had free access to tap water. Additionally, nine rabbits kept on a standard diet without cholesterol or fats were monitored until at least 19 months of age and their livers were examined as an untreated control. The body weight was recorded weekly and the feed intake daily. At the end of the experiment the animals were euthanized by an intravenous injection of 5% phenobarbital solution. Samples for histological examination were taken from various organs. Sections from the liver were stained with HE, vGH, MST, PAS/D and elastin. Furthermore sections were stained immunohistochemically using monoclonal ubiquitin (mouse anti-human, Zymed Laboratories Inc.). The histological examination was performed blindly. The pattern of fibrosis was registered and the degree graded as none, slight, moderate and severe. The steatosis was graded as none, questionable, slight, moderate and severe and the size of the fat vacuoles was noted. Furthermore special attention was given regarding inflammation (in parenchyma and in portal tracts) and to the presence of megamitochondria and Mallory-like bodies. 2.2. Diets
Three different powdered diets from Altromin, Lage, Germany were used: the standard diet and two concentrated diets containing the nutrients of the standard diet allowing for addition of 5% and 10% by weight of fat, respectively. The diets were used to produce pellets with 0 and 1.11% (w/w) cholesterol (Sigma C 8503). The vegetable oil contained 0.6% n-3 polyunsaturated fatty acids (PUFA), 32.9% n-6 PUFA, 30.6% saturated fatty acids (SFA) and 35.5% monounsaturated fatty acids
312
K. Simonsen
et al. /International
Hepatology
Communications
3 (1995)
310-315
(MUFA). The marine oil contained 16.6% n-3 PUFA, 17.7% n-6 PUFA, 27.9% SFA and 35.2% MUFA.
3.Results The mean body weight was 3.66 kg f 0.45 (S.D.), 3.65 kg f 0.31 and 3.78 kg f 0.28 at the beginning of the study and 3.36 kg f 0.33,3.28 kg f 0.26 and 3.22 kg i 0.08 at the termination in groups I, II and III respectively. No significant difference in body weight was found between the groups during the experiment. The mean body weight was 3.66 kg f 0.45 (S.D.), 3.65 kg f 0.31 and 3.78 kg f 0.28 at the beginning of the study and 3.36 kg f 0.33,3.28 kg f 0.26 and 3.22 kg * 0.08 at the termination in groups I, II and III, respectively. No significant difference in body weight was found between the groups during the experiment. The mean daily feed intake was 95 g i 4 (S.D.), 87 g f 9 and 75 g f 15 in groups I, II and III, respectively. During the experiment the mean feed intake was from the histological investigation), and one female in group II was euthanized after 11 weeks. The liver from this female and the liver from the female from group III which died unexpectedly in week 12 (the cause of death was myocardial infarction) were included in the histological examination. The cholesterol feeding caused pronounced hypercholesterolemia which was significantly enhanced by the added oils. The results of the changes in the blood lipids, as well as the results of the histological examination of the various organs besides the livers, have been published elsewhere [3]. 3.1. Pathology
The untreated controls showed histologically normal livers with no signs of tibrosis and none or questionable steatosis. Group I (cholesterol fed) developed questionable to slight steatosis, mostly microvesicular. There was none to slight perivenular fibrosis in Rappaports zone 3 (Fig. la). In some of the animals there were Mallory-like bodies (Fig. 2), but no inflammatory changes. The results of the immunohistochemical staining of these Mallory-like bodies were inconclusive in all three groups. Cholestasis or megamitochondria were not present. No periportal fibrosis was seen. Group II (cholesterol and vegetable oil fed) showed changes similar to the rabbits in group I, but the steatosis and the fibrosis were more pronounced. The pattern of fibrosis was similar to the one described below, although not as severe (Fig. lb). Here also there were few Mallory-like bodies, but no inflammation. Periportal fibrosis was slight. Group III (cholesterol and marine oil fed) all developed severe steatosis (both micro- and macrovesicular) and they all developed severe fibrosis. Constant findings were severe pericellullar fibrosis, particularly in Rappaports zone 3, and in some areas as bridging fibrosis primarily between central veins and portal tracts but between two central veins as well. In some of the rabbits the fibrosis was so severe that it mimicked cirrhosis (Fig. lc). Few Mallory-like bodies were seen here as well, and in one rabbit there was additional cholestasis. Megamitochondria were not seen. Portal tracts were enlarged to varying degrees and revealed infiltration with mononuclear cells.
K. Simonsen et al. /International
Hepatology Communications 3 (1995) 330-315
Fig. 1. All three illustrations represent Reticulin stained sections. Original magnification x 67. (a) GI.oup I; a Iilight 1perisinusoidal fibrosis of zone 3 is found. Terminal hepatic venule: V. Portal tract: P. (b) GI*oup 11; a xate fibrosis, perisinusoidal and pericellullar, is seen in zone 3, whereas periportal , lib Irotris is sligkIt. Terminal hepatic venule: V. Portal tract: P. (c) Group III; severe fibrosis with bridgit ‘g t jet\Neen indb qidual zone 3 areas and a nodular parenchymal pattern is seen. Terminal hepatic venuh :: v
314
K. Simonsen
et al. /International
Hepatology
Communications
3 (1995)
310-315
Fig. 2. H and E section. Original magnification x 400. Group I; zone 3 hepatocytes some of which contain Mallory-like bodies (arrows).
4. Discussion The heterozygous WHHL rabbit fed cholesterol with added oils is a new animal model for studying fibrogenesis in the liver. It has several advantages, one being the development of a pattern of fibrosis very similar to that observed in human alcoholics for the group having cholesterol-rich diet with added marine oil [4]. Pericellullar fibrosis in a chickenwire fashion and bridging fibrosis between central veins and portal tracts as well as between two central veins were prominent. This finding seems to be constant and reproducible as all the rabbits in this group had similar changes in their livers. Furthermore they presented severe steatosis and occasional Mallory-like bodies, which were negative in an immunohistochemical staining with ubiquitin antibody. Other advantages are that the changes can be provoked in a relatively short time and that the diets can be given in pellets instead of having to administrate the diet/toxins in a less natural way. These features are in contrast to many other animal models [l]. Finally, this animal model is a relatively cheap model compared to some of the better known animal models such as the baboon [5]. A disadvantage of the model is the fact that a few animals had to be removed because of continuously decreased food intake. Rats fed fish oil have been shown to develop focal fibrosis and rats fed fish oil with ethanol have been shown to develop less fatty accumulation but increased inflammation and necrosis when compared to rats fed corn oil with ethanol [6]. Fur-
K. Simonsen et al. /international
Hepatology Communications 3 (1995) 310-315
315
thermore there is evidence which indicates that fish oil increases lipid peroxidation in the liver, which might cause fatty accumulation and fibrosis [7]. To our knowledge tibrosis as severe as described and as closely mimicking that seen in alcoholic liver disease in humans has not been described in an experimental animal model fed a marine oil added cholesterol-rich diet. Many areas concerning the initiating of and the pathways in liver fibrogenesis are still not well understood. One of the major questions to be answered is the possible reversibility or irreversibility of the fibrotic changes. To investigate this the above described model might be suitable if one included a larger number of animals. Furthermore it would be of interest to study the possible relevance of drawing parallels to humans considering the extensive use of commercially produced marine oils. Acknowledgements The authors wish to thank Walter Schmidtsdorff, Technical Laboratory, Ministry of Fisheries and Carl-Erik Hay, Danish Technical University for providing the marine and vegetable oils. References 111 Tsukamato H, Matsuoka M, French SW. Experimental models of hepatic fibrosis: A review. Semin PI 131 141 151
161 [71
Liver Dis 1990; IO: 56-65. Hansen BF, Mortensen A, Hansen JF, Ibsen P, Frandsen H, Nordestgaard BG. Atherosclerosis in Watanabe heritable hyperlipidaemic rabbits. Evaluation by macroscopic, microscopic and biochemical methods and comparison of atherosclerosis variables. APMIS 1994; 102: 177-90. Mortensen A, Meyer 0, Hansen BF et al. Changes in blood lipids and aorta in heterozygous WHHL rabbits fed cholesterol and fats. In: Abstracts from 62nd EAS Congress, 1993. Christoffersen P, Junge J. The spectrum of alcohol induced liver disease. Histological features. APMIS Suppl 1991; 23: 40-5. Rubin E, Lieber CS. Fatty liver, alcoholic hepatitis and cirrhosis produced by alcohol in primates. N Engl J Med 1974; 290: 128-35. French SW. Nutrition in the pathogenesis of alcoholic liver disease. Alcohol Alcohol 1993; 28: 97-109. Morimoto M, Zem MA, Hagbjdrk A-L, Ingelman-Sundberg M, French SW. Fish oil, alcohol and liver pathology: Role of cytochrome P450 2El. Proc Sot Exp Biol Med 1994; 207: 197-205.