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The use of an operating microscope in rat liver surgery makes it possible to obtain new experimental models and improve the already existing macrosurgical models. Thus, microsurgery could be a very valuable technique to improve experimental models of hepatic insufficiency. In the current review, we present the microsurgical techniques most frequently used in the rat, such as the portacaval shunt, the extrahepatic biliary tract resection, partial and total hepatectomies and heterotopic and orthotopic liver transplantation. Hence, reducing surgical complications allows for perfecting the resulting experimental models. Thus, liver atrophy related to portacaval shunt, prehepatic portal hypertension secondary to partial portal vein ligation, cholestasis by resection of the extrahepatic biliary tract, hepatic regeneration after partial hepatectomies, acute liver failure associated with subtotal or total hepatectomy and finally complications derived from preservation or rejection in orthotopic and heterotopic liver transplantation can be studied in more standardized experimental models. The results obtained are therefore more reliable and facilitates the flow of knowledge from the bench to the bedside. Some of these microsurgical techniques, because of their simplicity, can be performed by researchers without any prior surgical training. Other more complex microsurgical techniques require in-depth surgical training. These techniques are ideal for achieving a complete surgical training and more select microsurgical models for hepatology research. [ABSTRACT FROM AUTHOR]

Partial portal vein ligation is the experimental model most frequently used to study prehepatic portal hypertension. Different systemic and splanchnic biochemical and histological alterations in short-term (28–45 days) and long-term (12–14 months) evolutive phases which has been described in this experimental model suggest the existence of different pathophysiological mechanisms involved in their production. The enteropathy produced could develop in three phases: an early or acute phase with vasomotor hemodynamic alterations (ischemia-reperfusion associated with intestinal hyperemia, edema and oxidative stress); an intermediate phase with immunological alterations (mesenteric lymphadenopathy, increased mucosal infiltration by mast cells and the hepato-intestinal release of pro- and anti-inflammatory mediators); and a late or chronic phase with intestinal remodeling (vascular and epithelial). The alterations which are produced in these three evolutive phases make it possible to propose an inflammatory etiopathogeny for hypertensive portal enteropathy. [ABSTRACT FROM AUTHOR]

A simplified technique of end-to-side portacaval shunt in the rat is described, consisting in using a microsuture with a looped end. By using this technique, combined with two-step portal vein venotomy, the portal vein and caval vein can be brought closer together in a single movement, with no need for a knot at the start of the shunt. As a result, this modified technique makes it easier and reduces the time required to perform the shunt, without any rise in associated mortality. [ABSTRACT FROM AUTHOR]

One approach to assess neuroendocrine response to portal hypertension in short-term portal vein-stenosed rats consists in studying metabolic and functional activity patterns in adrenal glands using mitochondrial enzyme cytochrome c oxidase (COX) as a histochemical marker. Male Wistar rats were divided into two groups: a control group (Group I; n = 8), in which the animals did not undergo any operative intervention, and a triple calibrated portal vein stenosis group (TPVS) (Group II; n = 7). The sections of suprarenal glands were histochemically stained for COX and the optical densitometry was measured by a computer image analyzer attached to a microscope. In TPVS rats, COX activity in the adrenal gland cortex is lower than in control rats and affects the fascicular (52.30, 47.16–60.98, vs. 67.12, 60.31–73.89, p = .002), glomerular (49.68, 46.19–53.56 vs. 70.47, 64.64–73.51, p [ABSTRACT FROM AUTHOR]

Abstract: Objective: To describe a modified technique of end-to-side portacaval shunt in the rat in order to simplify this relevant microsurgical model of liver disease. Method: A loop in the distal end of the suture makes it easier both, the beginning and the end, of the portacaval anastomoses. Conclusion: This surgical innovation decreases the technical difficulty of the portacaval shunt in the rat without adding complications, like portal hypertension related to anastomoses stenosis or hemorrhage. [Copyright &y& Elsevier]

Abstract: Mast cells are involved in various liver diseases and appear to play a broader pathogenic role than originally thought. They may participate in the splanchnic alterations related to a porto-systemic shunt. To verify this hypothesis we studied the serum and hepatic histological changes in rats four weeks after an end-to-side portacaval shunt. In this experimental model of chronic liver insufficiency we also assessed the mucosal mast cells (MMC) and connective tissue mast cells (CTMC) in the liver, mesenteric lymph nodes and small intestine, as well as the serum levels of rat mast cell protease-II (RMCP-II). The results show liver and testes atrophy, with hypoalbuminemia (p =0.0001), hyperbilirubinemia (p =0.0001) and increase in aspartate aminotransferase (p =0.004) and alanine aminotransferase (p =0.0001). Hepatic histopathology demonstrates hepatocytic necrosis and apoptosis, portal inflammation, biliary proliferation, steatosis and fibrosis. There is a decrease of MMCs and CTMCs in the liver, while in the ileum CTMCs increase and MMCs decrease. These results suggest the involvement of mast cells in the pathophysiological splanchnic impairments in this experimental model. In particular, the decreased number of liver mast cells may be associated with the hepatic atrophy. If this is the case, we propose that the disruption of the hepato-intestinal axis after a portocaval shunt in the rat could inhibit the ability of the liver to developing an appropriate repair response mediated by mast cells. [Copyright &y& Elsevier]

Abstract: Portosystemic shunt surgery is the best procedure to prevent recurrent bleeding of esophageal varices, but carries a high risk of postoperative inflammatory complications, including hepatic encephalopathy. Thus, portosystemic shunting procedures could induce a systemic inflammatory response with multiple organ dysfunction syndrome, including hepatic encephalopathy. To verify this hypothesis we used male Wistar rats at 6weeks of postoperative evolution: Control (CR; n =14), Sham-operated (SO; n =8) and rats with end-to-side portacaval shunt (PCS; n =15). TNF-α, IL-1β and IL-10 were assayed by ELISA techniques, the expression of the endothelial constitutive nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), constitutive and inducible heme-oxygenase (HO-1 and HO-2) were assayed by Western-blot. mRNA levels of HO-1, HO-2, TNF-α, IL-1β and IL-10 were quantified by reverse transcriptase polymerase chain reaction amplification (RT-PCR) in the small bowel, liver, spleen and lungs. Portacaval shunting in the rat produces an interorgan imbalance of pro- and anti-inflammatory mediators. TNF-α mRNA expression is decreased in the liver (0.69±0.28, p <0.05). The hepatic production of IL-Iβ (204.13±71.90pg/100g; p <0.001) and IL-10 (4505.47±337.97pg/100g; p <0.001) is also decreased. However, the intestinal pro-inflammatory (TNF-α: 1471.86±153.62pg/100g, p <0.001; IL-1β: 48.35±9.84pg/100g, p <0.001 and iNOS: 0.59±0.01, p <0.01) and anti-inflammatory (IL-10: 1503.39±53.5pg/100g, p <0.001 and HO-1: 2.23±0.16, p <0.001) mediators are increased. Total portacaval shunting in the rat induces impairments of pro- and anti-inflammatory mediators in the splanchnic–lung axis that could be associated with a multiple organ dysfunction syndrome. Therefore, the complications after portosystemic shunts could be integrated into a systemic inflammatory response of possible intestinal origin. [Copyright &y& Elsevier]

Abstract: Patients with liver malfunction often suffer from hepatic encephalopathy, a neurological complication which can affect attention and cognition. Diverse experimental models have been used to study brain alterations that may be responsible for hepatic encephalopathy symptoms. The aim of the study was to determine whether cognitive impairment found in cirrhosis could be due to disturbance of acetylcholinesterase activity. Acetylcholinesterase activity was assessed in the brains of Wistar rats with thioacetamide-induced cirrhosis. The cirrhotic group displayed up-regulation of acetylcholinesterase levels in the entorrhinal cortex, anterodorsal and anteroventral thalamus and accumbens, whereas down-regulation was found in the CA1, CA3 and dentate gyrus of the hippocampus. Our results indicate that the experimental model of hepatic encephalopathy by chronic administration of thioacetamide presents alterations of acetylcholinesterase activity in brain limbic system regions, which play a role in attention and memory. [Copyright &y& Elsevier]

Background: Long-term extrahepatic cholestasis in the rat induces ductular proliferation and fibrosis in the liver, portal hypertension, splenomegaly, portosystemic collateral circulation, and ascites. These splanchnic alterations could have an inflammatory pathophysiology. Material and Methods: We measured serum levels of hepatobiliary injury markers and the acute phase proteins, alpha-1-major acid protein (α1-MAP) and alpha-1-acid glycoprotein (α1-GPA) in rats 6 wk after microsurgical extrahepatic cholestasis. We also assayed Th1 (TNF-α and IL-1β) and Th2 (IL-4 and IL-10) cytokine levels in the liver, ileum, spleen, and mesenteric lymph complex by enzyme-linked immunosorbent assay (ELISA) techniques. Liver fibrosis was measured by Sirius red stain and by using an image system computer-assisted method and mast cell liver infiltration by Giemsa stain. Results: The cholestatic rats showed an increase (P <0.001) in serum levels of bile acids, total and direct bilirubin, AST, ALT, AST/ALT index, γ-GT, alkaline phosphatase, α1- MAP, α1-GPA, and LDH (P <0.05) in relation to sham-operated rats. TNF-α, IL-1β, IL-4, and IL-10 increased in the ileum (P <0.01) and mesenteric lymph complex (P <0.001), and decreased in the liver (P <0.001). A marked bile proliferation associated with fibrosis (P <0.001) and mast cell infiltration was also shown in the liver of cholestatic rats. Conclusion: The splanchnic redistribution of cytokines, with an increase of Th1 and Th2 production in the small bowel and in the mesenteric lymph complex, supports the key role of inflammatory mechanisms in rats with secondary biliary fibrosis. [Copyright &y& Elsevier]

Summary: It is currently believed that portal hypertension induces an inflammatory response in which mast cells may be involved. The aim of this study was to verify the involvement of the intestinal submucosal and mesenteric lymph node mast cells in the splanchnic inflammatory response related to portal hypertension. Mast cell infiltration in the intestine (duodenum, jejunum, ileum, caecum and distal colon) and in the mesenteric lymph node complex (MLC) was measured using a stereological method in sham-operated rats (SO; n=12), in two experimental models of portal hypertension, chronic (triple partial portal vein ligation, TPVL; n=12) and transient (microsurgical partial hepatectomy; n=12) and in rats in which the MLC was resected (n=12). The small and large bowel submucosal infiltration increases in MLC-resected rats (p=0.0001), in TPVL rats (p=0.0001) and in rats with partial hepatectomy (p=0.0001). An extensive mast cell infiltration in the MLC (p=0.0001) was found in TPVL rats and in rats with partial hepatectomy (347.40±45.25 and 351.92±99.28/mm3, respectively) in relation to sham-operated rats (135.27±30.28/mm3). We conclude that mast cells could be involved in the splanchnic alterations developed in the surgical experimental models of portal hypertension studied. [Copyright &y& Elsevier]

Abstract: People with hepatic insufficiency can develop hepatic encephalopathy (HE), a complex neuropsychological syndrome covering a wide range of neurological and cognitive and motor alterations. The cognitive deficits include disturbances in intellectual functions such as memory and learning. In spite of its high prevalence in western societies, the causes of HE have not yet been clearly established. For this reason, experimental models of HE are used to study this condition. In this work, two experimental models were used, one Type B HE (portacaval shunt) and the other Type C HE (cirrhosis by intoxication with thioacetamide), to evaluate its effect on two tasks of associative learning: two-way active avoidance and step-through passive avoidance. The results show an impediment both in acquisition and retention of active avoidance in both models of HE. However, in passive avoidance, only the rats with portacaval shunt presented a memory deficit for the aversive event. In our opinion, these results can be explained by alterations in the neurotransmission system presented by animals with hepatic insufficiency, which are mainly caused by a rise in cerebral histamine and a dysfunction of the glutamatergic system. [Copyright &y& Elsevier]

Abstract: Hepatic encephalopathy (HE) is a frequent neurological complication observed in patients with liver malfunction. Previous studies have shown memory impairment in these patients. In order to investigate brain substrates of spatial working memory impairment in chronic HE, neuronal expression of c-Fos protein was studied in an experimental model of cirrhosis. Control and cirrhotic rats were trained on a spatial working memory task in the Morris water maze (MWM). Differences between groups were found in the working memory task. Cirrhotic rats were unable to locate the platform in the retention trial. Neuronal activation, measured by c-Fos protein, was compared between groups. No differences were found in c-Fos expression of control and cirrhotic rats that were not tested in the MWM. Working memory task produced increase in c-Fos positive cells in dorsal hippocampus, CA1 and CA3, and prefrontal cortex in control group compared to thioacetamide group or naïve, which only swam in the maze during a similar time. These findings suggest that cirrhotic rats show spatial working memory impairment that could be linked to dysfunction in neuronal activity in prefrontal cortex and hippocampus. [Copyright &y& Elsevier]

Abstract: Brain tissue of patients diagnosed with hepatic encephalopathy exhibits cellular morphological changes that could be associated with memory impairment. The mammillary nuclei, located in the posterior part of the hypothalamus, are important for spatial memory formation. This work aimed to assess spatial reference memory and cellular changes in the mammillary nuclei of cirrhotic rats. Spatial reference memory of Wistar rats with thioacetamide-induced cirrhosis was assessed in the Morris water maze. Total cell number of neurons and glial cells and volume of the mammillary nuclei were quantified by stereology. Neuronal and astrocytic nuclear volume in mammillary nuclei and CA1 dorsal hippocampal subfield were assessed by nucleator probe. Cirrhotic rats showed an impaired spatial reference memory in comparison with control animals. Total number of neurons and glial cells were unaltered. In the medial mammillary nucleus (MMn), glial fibrillary acidic protein-immunoreactive astrocytes decreased in the cirrhotic group while the lateral part was unaffected. The medial part of the MMn was larger in the cirrhotic group. The cirrhotic rats showed morphometric cellular changes characterised by an increased neuronal and astrocytic nuclear volume in all the mammillary nuclei and CA1 hippocampal region. These findings suggest that cirrhotic rats show spatial memory impairment that could be linked to astrocytes and neuronal impairment in mammillary nuclei and hippocampus. [Copyright &y& Elsevier]

Abstract: An inflammatory etiopathogeny can be suggested in portal hypertensive enteropathy since infiltration of the intestinal wall by mononuclear cells has been described in this condition. This work was carried out with the intention of shedding light on this matter. Male Wistar rats were divided into 4 control groups and 4 groups with partial portal vein ligation at 1, 2, 3 and 15 months. TNF-α, IL-1β and IL-10 were quantified in liver and ileum by ELISA. CO and NO were measured in splanchnic and systemic vein by spectrophotometry and Griess reaction, respectively. Expression of constitutive and inducible isoforms of NO and HO were assayed by Western blot in liver and ileum. An increased hepatic release of proinflammatory mediators (TNF-α, IL-1β and NO) associated with intestinal release of anti-inflammatory mediators (IL-10, CO) occurs in an early evolutive phase (1 month) of experimental portal hypertension. On the contrary, in the long-term (15 months), the increase in the intestinal release of proinflammatory mediators (TNF-α, IL-1β) is associated with an increase in the hepatic release of anti-inflammatory mediators (IL-10, CO). These results suggest that experimental prehepatic portal hypertension presents changes in the serum and tissular (liver and small bowel) concentrations of mediators which are considered as pro- and anti-inflammatory. [Copyright &y& Elsevier]