The Effect of Ascorbic Acid on Hepatic Ischaemia-Reperfusion Injury in Wistar Rats: An Experimental Study.

Liver ischaemia-reperfusion (IR) during hepatic surgeries can lead to liver cell death via oxidative stress and the activation of immune cells, the release of cytokines, and damage-associated molecular patterns. Ascorbic acid has been shown to confer potential protective effects against IR injury, mainly due to its antioxidant properties. This study evaluated the effect of ascorbic acid infusion at different time points during hepatic IR in rats. Thirty-six male Wistar rats were divided into control and experimental groups that received the same total ascorbic acid dose at three different infusion times: before ischaemia, before reperfusion, or before both ischaemia and reperfusion. All of the animals experienced hepatic IR injury. We measured the hepatic enzymes, cytokines, and portal blood flow. Animals receiving ascorbic acid before both ischaemia and reperfusion had lower liver enzyme levels, reduced inflammation, and better portal venous flow than other animals. Divided doses of ascorbic acid before IR may be beneficial for reducing liver injury associated with IR.

Anatomic and Surgical Aspects of Total Pancreatectomy in Swine.

BACKGROUND: The swine is a valuable model for preclinical research and surgical technique training. Induction of Type I diabetes is achieved by total pancreatectomy, therefore these animals may be used in several research studies, including islet transplantation field. Given the lack of information in the literature, the purpose of this work is to describe anatomic aspects of swine pancreas, the total pancreatectomy surgical technique, intra- and postoperative complications and the autopsy results. MATERIAL AND METHODS: Five hybrid male pigs, 20-35 kg, submitted to total pancreatectomy with duodenum, bile duct, and spleen preservation. Postoperatively, daily clinical assessment and capillary blood glucose collection were performed. At the end of the 30-day period or in the occurrence of serious clinical complications, euthanasia and autopsy were performed. RESULTS: The average duration of surgery was 128 minutes, without intraoperative deaths or anesthesia induction failures. The median survival was 6.6 days. Postoperative complications were weight loss (3), emesis (2), constipation (2), abdominal distension (2), diarrhea (1), and loss of appetite (1). All animals were euthanized due to serious complications. Two animals presented surgical complications (duodenal necrosis with gastroparesis and internal hernia with intestinal necrosis). The other 3 animals presented serious clinical complications related to exocrine pancreatic insufficiency due to deficiency of pancreatic enzymes. Glycemic values above 200 mg/dL were found on the first postoperative day and above 300 mg/dL on the seventh day in all animals. CONCLUSION: A model of total pancreatectomy with duodenum, spleen, and bile duct preservation in pigs was established. All animals became diabetic, however, animals without postoperative complications were euthanized due to serious complications related to pancreas exocrine insufficiency.

Study of laser fluorescence spectroscopy in livers of rats with hypothermic ischemia.

PURPOSE: After partial hepatectomy (PH), the remaining liver (RL) undergoes regenerative response proportional to the host. Limited literature exists on hepatic viability after tissue injury during hypothermic preservation. Spectroscopy measures cellular fluorescence and is explored for tissue characterization and parameter investigation. This study aimed to assess fluorescence analysis (spectroscopy) in evaluating liver viability and its relationship with hepatic tissue regeneration 24 hours after PH. Additionally, we analyzed liver regeneration in RL after 70% partial hepatectomy under hypothermic conditions with laser irradiation. METHODS: Fifty-six Wistar rats were divided into four groups: total non-perfused liver (control), total perfused liver, partial hepatectomy "in situ", and partial hepatectomy "ex situ". Tissue analysis was performed at 0 and 24 hours using spectroscopy with laser devices emitting at 532 (green) and 405 nm (violet). RESULTS: Spectroscopy identified tissue viability based on consistent results with Ki67 staining. The fluorescence spectra and Ki67 analysis displayed similar patterns, linking proliferative activity and absorption intensity. CONCLUSIONS: Fluorescence spectroscopy proves to be promising for real-time analysis of cellular activity and viability. Metabolic activity was observed in groups of live animals and hypothermically preserved samples, indicating cellular function even under blood deprivation and hypothermic conditions.

PRECLINICAL MODELS OF LIVER CÂNCER.

•In this review, we described different murine models of carcinogenesis: classic models, new transgenic and combined models, that reproduce the key points for HCC and CCA genesis allowing a better understanding of its genetic physiopathological, and environmental abnormalities. •Each model has its advantages, disadvantages, similarities, and differences with the corresponding human disease and should be chosen according to the specificity of the study. Ultimately, those models can also be used for testing new anticancer therapeutic approaches. •Cholangiocarcinoma has been highlighted, with an increase in prevalence. This review has an important role in understanding the pathophysiology and the development of new drugs. Background - This manuscript provides an overview of liver carcinogenesis in murine models of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Objective - A review through MEDLINE and EMBASE was performed to assess articles until August 2022.Methods - Search was conducted of the entire electronic databases and the keywords used was HCC, CCA, carcinogenesis, animal models and liver. Articles exclusion was based on the lack of close relation to the subject. Carcinogenesis models of HCC include HCC induced by senescence in transgenic animals, HCC diet-induced, HCC induced by chemotoxicagents, xenograft, oncogenes, and HCC in transgenic animals inoculated with B and C virus. The models of CCA include the use of dimethylnitrosamine (DMN), diethylnitrosamine (DEN), thioacetamide (TAA), and carbon tetrachloride (CCl4). CCA murine models may also be induced by: CCA cells, genetic manipulation, Smad4, PTEN and p53 knockout, xenograft, and DEN-left median bile duct ligation. Results - In this review, we described different murine models of carcinogenesis that reproduce the key points for HCC and CCA genesis allowing a better understanding of its genetic, physiopathological, and environmental abnormalities. Conclusion - Each model has its advantages, disadvantages, similarities, and differences with the corresponding human disease and should be chosen according to the specificity of the study. Ultimately, those models can also be used for testing new anticancer therapeutic approaches.

Modelos pré-clínicos de câncer hepático / Preclinical models of liver câncer

ABSTRACT Background: This manuscript provides an overview of liver carcinogenesis in murine models of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Objective: A review through MEDLINE and EMBASE was performed to assess articles until August 2022. Methods: Search was conducted of the entire electronic databases and the keywords used was HCC, CCA, carcinogenesis, animal models and liver. Articles exclusion was based on the lack of close relation to the subject. Carcinogenesis models of HCC include HCC induced by senescence in transgenic animals, HCC diet-induced, HCC induced by chemotoxicagents, xenograft, oncogenes, and HCC in transgenic animals inoculated with B and C virus. The models of CCA include the use of dimethylnitrosamine (DMN), diethylnitrosamine (DEN), thioacetamide (TAA), and carbon tetrachloride (CCl4). CCA murine models may also be induced by: CCA cells, genetic manipulation, Smad4, PTEN and p53 knockout, xenograft, and DEN-left median bile duct ligation. Results: In this review, we described different murine models of carcinogenesis that reproduce the key points for HCC and CCA genesis allowing a better understanding of its genetic, physiopathological, and environmental abnormalities. Conclusion: Each model has its advantages, disadvantages, similarities, and differences with the corresponding human disease and should be chosen according to the specificity of the study. Ultimately, those models can also be used for testing new anticancer therapeutic approaches.

RESUMO Contexto: Este manuscrito fornece uma visão geral da carcinogênese hepática em modelos murinos de carcinoma hepatocelular (CHC) e colangiocarcinoma (CCA). Objetivo: Realizar uma revisão de artigos científicos até agosto de 2022 utilizando as bases de dados MEDLINE e EMBASE. Métodos: A busca foi realizada em todas as bases de dados eletrônicas e as palavras-chave usadas foram CHC, CCA, carcinogenesis, modelos animais e fígado. A exclusão dos artigos baseou-se na falta de estreita relação com o assunto. Os modelos de carcinogênese do CHC incluíram: CHC induzido por senescência em animais transgênicos, CHC induzido por dieta, CHC induzido por agentes quimiotóxicos, xenoenxerto, oncogenes e CHC em animais transgênicos inoculados com vírus B e C. Os modelos de CCA incluíram: o uso de dimetilnitrosamina (DMN), dietilnitrosamina (DEN), tioacetamida (TAA) e tetracloreto de carbono (CCl4). Os modelos murinos de CCA induzidos por incluir: células de CCA, manipulação genética, animais nocaute para Smad4, PTEN e p53, xenoenxerto e ligadura do ducto biliar mediano esquerdo. Resultados: Nesta revisão, descrevemos diferentes modelos murinos de carcinogênese que reproduzem os pontos-chave para a gênese do CHC e do CCA, permitindo uma melhor compreensão de suas anormalidades genéticas, fisiopatológicas e ambientais. Conclusão: Cada modelo tem suas vantagens, desvantagens, semelhanças e diferenças com a doença humana correspondente e deve ser escolhido de acordo com a especificidade do estudo. Em última análise, esses modelos também podem ser utilizados para testar novas abordagens terapêuticas anticancerígenas.

Multivisceral transplantation of pelvic organs in rats.

Background: Multivisceral transplantation of pelvic organs would be a potential treatment for severe pelvic floor dysfunction with fecal and urinary incontinence, extensive perineal trauma, or congenital disorders. Here, we describe the microsurgical technique of multivisceral transplantation of pelvic organs, including the pelvic floor, in rats. Donor operation: We performed a perineal (including the genitalia, anus, muscles, and ligaments) and abdominal incision. The dissection progressed near the pelvic ring, dividing ligaments, muscles, external iliac vessels, and pudendal nerves, allowing pelvic floor mobilization. The aorta and vena cava were isolated distally, preserving the internal iliac and gonadal vessels. The graft containing the skin, muscles, ligaments, bladder, ureter, rectum, anus and vagina, uterus and ovarian (female), or penile, testis and its ducts (male) was removed en bloc, flushed, and cold-stored. Recipient operation: The infrarenal aorta and vena cava were isolated and donor/recipient aorta-aorta and cava-cava end-to-side microanastomoses were performed. After pelvic floor and viscera removal, we performed microanastomoses between the donor and the recipient ureter, and the rectum and pudenda nerves. The pelvic floor was repositioned in its original position (orthotopic model) or the abdominal wall (heterotopic model). We sacrificed the animals 2 h after surgery. Results: We performed seven orthotopic and four heterotopic transplantations. One animal from the orthotopic model and one from the heterotopic model died because of technical failure. Six orthotopic and three heterotopic recipients survived up to 2 h after transplantation. Conclusion: The microsurgical technique for pelvic floor transplantation in rats is feasible, achieving an early survival rate of 81.82%.

Study of laser fluorescence spectroscopy in livers of rats with hypothermic ischemia

Purpose: After partial hepatectomy (PH), the remaining liver (RL) undergoes regenerative response proportional to the host. Limited literature exists on hepatic viability after tissue injury during hypothermic preservation. Spectroscopy measures cellular fluorescence and is explored for tissue characterization and parameter investigation. This study aimed to assess fluorescence analysis (spectroscopy) in evaluating liver viability and its relationship with hepatic tissue regeneration 24 hours after PH. Additionally, we analyzed liver regeneration in RL after 70% partial hepatectomy under hypothermic conditions with laser irradiation. Methods: Fifty-six Wistar rats were divided into four groups: total non-perfused liver (control), total perfused liver, partial hepatectomy "in situ", and partial hepatectomy "ex situ". Tissue analysis was performed at 0 and 24 hours using spectroscopy with laser devices emitting at 532 (green) and 405 nm (violet). Results: Spectroscopy identified tissue viability based on consistent results with Ki67 staining. The fluorescence spectra and Ki67 analysis displayed similar patterns, linking proliferative activity and absorption intensity. Conclusions: Fluorescence spectroscopy proves to be promising for real-time analysis of cellular activity and viability. Metabolic activity was observed in groups of live animals and hypothermically preserved samples, indicating cellular function even under blood deprivation and hypothermic conditions.

Hexafluoroisopropanol decreases liver ischemia-reperfusion injury by downregulation of high mobility group box-1 protein.

Liver ischemia-reperfusion (IR) injury is associated with poor outcome after liver transplantation and liver resections. Hexafluoroisopropanol (HFIP) is a tri-fluorinated metabolites of volatile anesthetics and has modulatory effects on inflammation that have been observed mainly in cell culture experiments. In this survey, we investigated the effects of HFIP in a rat model of normothermic hepatic ischemia-reperfusion injury. Twenty-four male Wistar rats were randomized into three groups: (1) control in which animals were submitted to 30 min of partial liver ischemia with resection of non-ischemic liver lobes immediate after reperfusion, (2) pre-ischemia (PI) group in which animals received intravenous HFIP (67 mg/kg) 5 min before liver ischemia, and (3) pre-reperfusion (PR) group in which animals received intravenous HFIP (67 mg/kg) 5 min before reperfusion. Four hours after reperfusion, all animals were euthanized for sample collection. Aspartate and alanine transaminases, glucose, and high mobility group box-1 (HMGB-1) protein concentrations showed a significant decreased, and malondialdehyde was increased in the PR group compared with control and PI groups. Interleukin 6 (IL-6) was increased in the PI group compared with control and PR groups. IL-10 and -12 were increased in the PR and PI groups, respectively, when compared with the control group. Glucose decreased in the PR when compared with the control group. Post-conditioning with HFIP led to a decrease in hepatocellular injury and was associated with a downregulation of HMGB-1. The HFIP resulted in a better control of inflammatory response to ischemia-reperfusion even without causing a reduction in oxidative stress.

Small-for-Size Syndrome: Systemic Review in a Porcine Experimental Model.

BACKGROUND: The small-for-size syndrome (SFSS) is characterized by prolonged hyperbilirubinemia, coagulopathy, and/or encephalopathy caused by a small liver graft that cannot sustain the metabolic demands of the recipient after a partial liver transplant (PLT). Models of PLT in pigs are excellent for studying this syndrome. This review aimed to identify the different porcine models of SFSS in the literature and compare their technical aspects and therapeutics methods focused on portal inflow modulation (PIM). METHODS: We performed a systematic review of the porcine experimental model and SFSS. The MEDLINE-PubMed, EMBASE, Cochrane Library, LILACS, and SciELO databases were electronically searched and updated until June 20, 2021. The MeSH terms used were ''ORGAN SIZE'' AND ''LIVER TRANSPLANTATION". RESULTS: Thirteen SFSS porcine models were reported. Four were performed with portocaval shunt to PIM and 3 with mesocaval shunt to PIM. A few studies focused on clinical therapeutics to PIM; a study described somatostatin infusion to avoid SFSS. Initially, studies on PIM showed its potentially beneficial effects without mentioning the minimum portal flow that permits liver regeneration. However, an excessive portal diversion could be detrimental to this process. CONCLUSIONS: The use of porcine models on SFSS resulted in a better understanding of its pathophysiology and led to the establishment of various types of portal modulation, surgical techniques with different complexities, and pharmaceutical strategies such as somatostatin, making clear that without reducing the portal vein pressure the outcomes are poor. With the improvement of these techniques, SFSS can be avoided.

Relationship of Anatomic Variations and Arterial Reconstruction of the Hepatic Artery: Prevalence and Effect on Orthotopic Liver Transplantation.

BACKGROUND: Identifying anatomic variations of the hepatic artery is essential in liver transplantation. The artery supply is crucial for the procedure's success, and, in some cases of anatomic variations, they need reconstruction. Hepatic artery thrombosis is a severe vascular complication. This study evaluated the prevalence of anatomic variations and correlated arterial reconstructions with hepatic artery thrombosis. METHODS: We performed a retrospective analysis of medical records, adult patients undergoing liver transplant, donor's arterial anatomy, arterial reconstructions, and thrombosis after transplant from January 2019 to December 2020. RESULTS: Among 226 cases, 71% had normal anatomy. All these patients met Michel's classification subtypes, of which 161 (71%) were class I, which is the most common. The second most common variation was class II, with 25 donors (11%), followed by class III, with 17 donors (7.5%). Anatomic artery variations were a risk factor for hepatic artery thrombosis development (odds ratio [OR], 7.2; 95% confidence interval [CI], 2.1-22.5; P = .002). In the same way, the artery reconstruction was associated with hepatic artery thrombosis arising with postoperative time (OR, 18.0; 95% CI, 4.9-57.5; P < .001). Global hepatic artery thrombosis occurred in 11 cases (4.87%). CONCLUSION: Anatomic hepatic artery variations are frequent and do not make liver transplant unfeasible. However, variations that require reconstruction may raise the risk of thrombosis.

Innovative Technique Avoiding Bleeding After Reperfusion in Living Donor Liver Transplant Using a Modified Right Lobe Graft.

BACKGROUND: Living donor liver transplant (LDLT) is a valuable therapeutic option for overcoming the deceased donor shortage. Modified right lobe graft (MRLG) keeps the middle hepatic vein (MHV) trunk with the remnant liver to improve donor safety. Hemostasis in the MHV tributary reconstruction can be tricky; surgical stitches and energy coagulation are ineffective. Fibrin glues are excellent vascular sealants but are poor in maintaining hemostasis in an active hemorrhage or preventing resection surface-related complications after liver resection. We propose applying fibrin sealant during back table graft preparation to seal the hepatic edge and MHV reconstruction to avoid bleeding after graft revascularization. METHODS: Our retrospective cohort study included all adult patients undergoing LDLT between August 2017 and December 2021. During the back table procedure, we performed the reconstruction of the inferior right hepatic vein and/or MHV tributaries from segment 5 (V5) and segment 8 (V8) using a vein harvested from a nonrelated deceased donor. Before initiating the hepatic graft implantation, we applied fibrin sealant in the resected parenchyma, especially in the V5 and V8 anastomosis, to seal the hepatic edge and hepatic vein reconstruction. RESULTS: No bleeding was identified in the hepatic edge, and blood product transfusion was unnecessary for any recipients after reperfusion. CONCLUSION: In LDLT using MRLG with MHV reconstruction, the fibrin sealant, when applied on the raw hepatic surface, and vascular reconstruction during back table graft preparation avoided bleeding after graft revascularization.

Experimental model of portal hypertension and esophagogastric varices in minipigs: pressure and endoscopic pilot study.

INTRODUCTION: Portal hypertension still represents an important health problem worldwide. In the search for knowledge regarding this syndrome, experimental studies with animal models have proven to be useful to point the direction to be taken in future randomized clinical trials. PURPOSE: To validate the experimental model of portal hypertension and esophagogastric varices in a medium-sized animal. METHODS: This study included five minipigs br1. Midline laparotomy with dissection of the portal vein and production of a calibrated stenosis of this vein was performed. Measurement of pressure in the portal venous and digestive endoscopic were performed before and five weeks after the production of a stenosis. RESULTS: All animals were 8 months old, average weight of 17 ± 2.5 kg. The mean pressure of the portal vein immediately before the partial ligation of the portal vein was 8.9 + 1.6 mm Hg, with 26.6 + 5.4 mm Hg in the second measurement five weeks later (p < 0.05). No gastroesophageal varices or hypertensive portal gastropathy were seen at endoscopy procedures in our sample at any time in the study. CONCLUSION: Portal vein ligation in minipigs has been validated in the production of portal hypertension, but not in the formation of esophageal varices.

Experimental model of portal hypertension and esophagogastric varices in minipigs: pressure and endoscopic pilot study

Introduction: Portal hypertension still represents an important health problem worldwide. In the search for knowledge regarding this syndrome, experimental studies with animal models have proven to be useful to point the direction to be taken in future randomized clinical trials. Purpose: To validate the experimental model of portal hypertension and esophagogastric varices in a medium-sized animal. Methods: This study included five minipigs br1. Midline laparotomy with dissection of the portal vein and production of a calibrated stenosis of this vein was performed. Measurement of pressure in the portal venous and digestive endoscopic were performed before and five weeks after the production of a stenosis. Results: All animals were 8 months old, average weight of 17 ± 2.5 kg. The mean pressure of the portal vein immediately before the partial ligation of the portal vein was 8.9 + 1.6 mm Hg, with 26.6 + 5.4 mm Hg in the second measurement five weeks later (p < 0.05). No gastroesophageal varices or hypertensive portal gastropathy were seen at endoscopy procedures in our sample at any time in the study. Conclusion: Portal vein ligation in minipigs has been validated in the production of portal hypertension, but not in the formation of esophageal varices.

Creatinine-lactate score predicts mortality in non-acetaminophen-induced acute liver failure in patients listed for liver transplantation.

BACKGROUND: The aim of this study was to analyze prognostic indicators of in-hospital mortality among patients listed for urgent liver transplantation (LT) for non-acetaminophen (APAP)-induced acute liver failure (ALF). METHODS: ALF patients listed for LT according to the King's College Criteria were retrospectively reviewed. Variables were recorded from medical records and electronic databases (HCMED and RedCap). RESULTS: The study included 100 patients, of which 69 were subject to LT and 31 died while waiting for LT. Patients were 35.5 ± 14.73 years old, and 78% were females. The main etiologies were virus (17%), drug-induced (32%), autoimmune (15%), and indeterminate hepatitis (31%). The prioritization-to-LT time interval was 1.5 days (0-9). The non-LT patients showed higher lactate (8.71 ± 5.36 vs. 4.48 ± 3.33 mmol/L), creatinine (229 ± 207 vs. 137 ± 136 µm/L), MELD (44 ± 8 vs. 38 ± 8), and BiLE scores (15.8 ± 5.5 vs. 10.3 ± 4.1) compared to LT patients (p < 0.05). Multiple logistic regression analysis identified creatinine and lactate as independent prognostic factors, and a creatinine-lactate (CL) score was developed. ROC analysis showed that creatinine, lactate, MELD, BiLE, and CL scores had considerable specificity (71-88%), but only BiLE, lactate, and CL presented high sensitivities (70%, 80%, and 87% respectively). AUCs were 0.696 for creatinine, 0.763 for lactate, 0.697 for MELD, 0.814 for BiLE, and 0.835 for CL. CONCLUSIONS: CL and BiLE scores predict mortality with more accuracy than MELD in patients with ALF during prioritization time. Creatinine and lactate are independent prognostic factors for mortality.

Hypertonic saline solution decreases oxidative stress in liver hypothermic ischemia.

BACKGROUND: Liver ischemia reperfusion injury is still an unsolved problem in liver surgery and transplantation. In this setting, hypothermia is the gold standard method for liver preservation for transplantation. Hypertonic saline solution reduces inflammatory response with better hemodynamic recovery in several situations involving ischemia reperfusion injury. Here, we investigated the effect of hypertonic saline solution in hypothermic liver submitted to ischemia reperfusion injury. METHODS: Fifty male rats were divided into 5 groups: SHAM, WI (animals submitted to 40 minutes of partial warm liver ischemia and reperfusion), HI (animals submitted to 40 minutes hypothermic ischemia), HSPI (animals submitted to hypothermic ischemia and treated with 7.5% hypertonic saline solution preischemia), and HSPR (animals submitted to hypothermic ischemia and treated with hypertonic saline solution previously to liver reperfusion). Four hours after reperfusion, the animals were euthanized to collect liver and blood samples. RESULTS: Aspartate aminotransferase and alanine aminotransferase, histologic score, and hepatocellular necrosis were significantly decreased in animals submitted to hypothermia compared with the warm ischemia group. Malondialdehyde was significantly decreased in hypothermic groups with a further decrease when hypertonic saline solution was administrated preischemia. Hypothermic groups also showed decreased interleukin-6, interleukin-10, and tumor necrosis factor-α concentrations and better recovery of bicarbonate, base excess, lactate, and glucose blood concentrations. Moreover, hypertonic saline solution preischemia was more effective at controlling serum potassium concentrations. CONCLUSION: Hypertonic saline solution before hypothermic hepatic ischemia decreases hepatocellular oxidative stress, cytokine concentrations, and promotes better recovery of acid-base disorders secondary to liver ischemia reperfusion.