An ultrasound and histomorphological analysis of experimental liver cirrhosis in rats

992 Brazilian Journal of Medical and Biological Research (2008) 41: 992-999 ISSN 0100-879X J.V. Dias et al. An ultrasound and histomorphological analysis of experimental liver cirrhosis in rats J.V. Dias1*, B.D. Paredes2*, L.F.Q. Mesquita2, A.B. Carvalho2, E.O. Kozlowski3, A.S. Lessa1, C.M. Takiya4, C.M.C. Resende1, H.S.M. Coelho1, A.C. Campos-de-Carvalho2, G.F.M. Rezende1 and R.C.S. Goldenberg2 1Departamento de Clínica Médica, Faculdade de Medicina, 2Instituto de Biofísica Carlos Chagas Filho, 3Instituto de Bioquímica Médica, 4Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil Correspondence to: R.C.S. Goldenberg, Instituto de Biofísica Carlos Chagas Filho, Centro de Ciências da Saúde, Av. Carlos Chagas Filho, 373, Bloco G, Sala G2-053, 21941-902 Rio de Janeiro, RJ, Brasil Fax: +55-21-2280-8193. E-mail: We investigated whether liver injury by dual exposure to ethanol and carbon tetrachloride (EtOH + CCl4) for 15 weeks would persist after hepatotoxic agents were removed (EtOH + CCl4/8wR). After 15 weeks of hepatic injury with ethanol (5.5%, m/v) and carbon tetrachloride (0.05, mL/kg, ip), 5 of 11 female Wistar rats were sacrificed. The other 6 rats were maintained for an additional 8 weeks without hepatotoxic agents. Ultrasonography showed increased liver echogenicity and dilation of portal vein caliber in both groups (EtOH + CCl4: 0.22 ± 0.01 cm, P < 0.001; EtOH + CCl4/8wR: 0.21 ± 0.02 cm, P < 0.01) vs control (0.16 ± 0.02 cm). Histopathology showed regenerative nodules in both experimental groups. Histomorphometry revealed increased fibrosis content in both groups (EtOH + CCl4: 12.6 ± 2.64%, P < 0.001; EtOH + CCl4/8wR: 10.4 ± 1.36%, P < 0.05) vs control (2.2 ± 1.21%). Collagen types I and III were increased in groups EtOH + CCl4 (collagen I: 2.5 ± 1.3%, P < 0.01; collagen III: 1.3 ± 0.2%, P < 0.05) and EtOH + CCl4/8wR (collagen I: 1.8 ± 0.06%, P < 0.05; collagen III: 1.5 ± 0.8%, P < 0.01) vs control (collagen I: 0.38 ± 0.11%; collagen III: 0.25 ± 0.06%). Tissue transglutaminase increased in both groups (EtOH + CCl4: 66.4 ± 8%, P < 0.01; EtOH + CCl4/8wR: 58.8 ± 21%, P < 0.01) vs control (7.9 ± 0.8%). Cirrhosis caused by the association of CCl4-EtOH remained for at least 8 weeks after removal of these hepatotoxic agents. Ultrasound images can be a useful tool to evaluate advanced hepatic alterations. Key words: Rat; Cirrhosis; Collagen; Ultrasound; CCl4; Ethanol Research supported by FAPERJ (#E-26/170.315/2006), CNPq (#460408/2006-5), and CAPES-MEC (#31001017-108D4). *These authors contributed equally to this study. Received January 22, 2008. Accepted November 3, 2008 Introduction Hepatic fibrosis is an early finding in chronic liver injury characterized by failure of degradation and excessive synthesis of extracellular matrix components, mostly collagen types I and III. Persistence of the insult disrupts normal hepatic architecture, leading to development of Braz J Med Biol Res 41(11) 2008 regenerative nodules and vascular dysfunction, configuring the cirrhosis stage (1,2). Hepatic cirrhosis was described as an irreversible disease, which results from the incapacity of the wounded liver to remodel fibrosis (3,4). However, experimental studies with animals (5,6) and humans (7-9) have recently refuted this concept. Whether advanced cirrhosis undergoes remodeling to a near nor- www.bjournal.com.br Morphological analysis of hepatic cirrhosis mal liver architecture is controversial (10). Animal models of hepatic cirrhosis use carbon tetrachloride (CCl4) extensively as a hepatotoxic agent to evaluate liver injury and recovery (10-15). Spontaneous remodeling of extracellular matrix has been reported after cessation of CCl4 intoxication leading to macronodular cirrhosis (14-16). This spontaneous recovery might limit application of these experimental models in chronic fibrosis studies and make long-term investigations of liver cirrhosis difficult when the hepatotoxic agent is interrupted. The association of CCl4 with ethanol potentiates toxicity of the former, since it increases activation of cytochrome P450, the enzymatic complex responsible for CCl4 metabolism (17). This reaction leads to production of oxidative radicals that cause lipid peroxidation and, consequently, hepatotoxic injury (7). Ethanol also induces synthesis of type I collagen fibrils (18,19), inhibition of hepatocyte proliferation (20-22), exacerbation of activity in hepatic stellate cells (20,23), and increased expression of tissue transglutaminase (tTG) (24). Since a mechanism that renders matrix resistant to degradation results from the formation of ε-(γ-glutamyl) lysine cross-links by tissue transglutaminase (5,25,26), use of the hepatotoxic agents in combination could be more effective in maintaining the fibrotic pattern. The purpose of the present research was to determine if liver injury by exposure to both ethanol and CCl4 for 15 weeks would persist after the hepatotoxic agents were interrupted. Using a non-invasive diagnostic method, the ultrasound, and comparing it with the “gold standard” histological analysis for diagnosis of hepatic cirrhosis, we show that ultrasonographic images can be used to follow-up the morphological changes associated with liver injury in an experimental model of cirrhosis in rats. Furthermore, our results demonstrate that the association of ethanol and CCl4 resulted in cirrhosis, which was sustained for at least 8 weeks after insult withdrawal. Material and Methods Animals This investigation conforms to “The Guide for Care and Use of Laboratory Animals” [DHHS Publication No. (NIH) 85-23, revised 1996, Office of Science and Health Reports, Bethesda, MD] and was approved by the Ethics Committee of Instituto de Biofísica Carlos Chagas Filho (protocol #021). Female Wistar rats were obtained from the Instituto de Biofísica Carlos Chagas Filho (IBCCF, Rio de Janeiro, RJ, Brazil). Animals were housed at controlled temperature (23°C) with daily exposure to a 12:12-light-dark cycle. www.bjournal.com.br 993 Chronic liver injury model Cirrhosis was induced in 11 female Wistar rats (3 to 4 months of age, weighing 150-200 g) with injections of a 20% solution of CCl4 (1:5 in olive oil, dose of 0.05 mL/kg) intraperitoneally (ip) three times a week on alternate days for 15 weeks. In response to the need to develop an animal model with an alcohol consumption of clinical relevance, while maintaining dietary control, an alcohol liquid diet in accordance with the AIN-93 guidelines (27) was administered for 15 weeks. Prior to CCl4 administration, an adaptation phase was carried out with a non-alcoholic liquid diet (control diet) administered for 1 week followed by a second week of the ethanol diet (EtOH + CCl4). Control diet ingredients were identical to those used in the alcoholic diet, except that ethanol was replaced by water in the same volume. Rats were given ad libitum access to liquid diets. After 15 weeks, 5 animals we (...truncated)