Bile Acid Overload Induced by Bile Duct and Portal Vein Ligation Improves Survival after Staged Hepatectomy in Rats.

OBJECTIVE: Compared to portal vein ligation (PVL), simultaneous bile duct and portal vein ligation (BPL) can significantly enhance hypertrophy of the intact liver. This study aimed to investigate whether BPL could improve survival after extended hepatectomy independently of an increased remnant liver. METHODS: We adopted rat models of 90% BPL or 90% PVL. To investigate the role of bile acids (BAs) the BA pools in the PVL and BPL groups were altered by the diet. Staged resection preserving 10% of the estimated liver weight was performed 3 days after BPL; PVL; or sham operation. Histology, canalicular network (CN) continuity; and hepatocyte polarity were evaluated. RESULTS: At 3 days after BPL; PVL; or sham operation when the volumetric difference of the intended liver remained insignificant, the survival rates after extended hepatectomy were 86.7%, 47%, and 23.3%, respectively (P<0.01). BPL induced faster restoration of canalicular integrity along with an intensive but transient BA overload. Staged hepatectomy after BPL shortened the duration of the bile CN disturbance and limited BA retention. Decreasing the BA pools in the rats that underwent BPL could compromise these effects, whereas increasing the BA pools of rats that underwent PVL could induce similar effects. The changes in CN restoration were associated with activation of LKB1. CONCLUSION: In addition to increasing the future remnant liver, BPL shortened the duration of the spatial disturbance of the CN and could significantly improve the tolerance of the hypertrophied liver to staged resection. BPL may be a safe and efficient future option for patients with an insufficient remnant liver.

Preserving hepatic artery flow during portal triad blood occlusion improves regeneration of the remnant liver in rats with obstructive jaundice following partial hepatectomy.

In certain cases, major hepatectomy is essential and inevitable in patients with hilar cholangiocarcinoma and obstructive jaundice (OJ). The current study was designed to evaluate effects of a novel method of portal blood occlusion, where the portal vein was occluded (OPV) and the hepatic artery flow was preserved in rats with OJ that underwent partial hepatectomy. OJ was induced in rats by ligation of the common bile duct for 7 days. Subsequently, OJ rats underwent hepatectomy removing 76% of the liver following occlusion of the portal triad (OPT), OPV or without portal blood occlusion. Liver blood flow (LBF), liver damage and regeneration were assessed. The safety limit for the duration of liver ischemia was 20 min for OPT and 40 min for OPV in rats with OJ. OPT and OPV methods resulted in significantly decreased microvascular LBF in rats with OJ from 529.53±91.55 laser speckle perfusion units (LSPU) in the control to 136.89±32.32 and 183.99±49.25 LSPU, respectively. Liver damage was assessed analyzing levels of serum alanine transaminase and direct bilirubin, determining interleukin-1ß and tumor necrosis factor-α expression and histological examination. It was demonstrated that liver damage and caspase-3 and -9 expression in the liver were substantially reduced in the OPV group compared with the OPT group. In addition, the OPV method significantly improved liver regeneration in OJ rats, as indicated by increased rates of liver regeneration and expression of proliferating cell nuclear antigen and Ki-67 compared with the OPT group. Therefore, the OPV method may prolong the duration of portal blood occlusion, reduce liver injury and improve liver regeneration by preserving hepatic arterial flow during portal blood control in rats with OJ undergoing partial hepatectomy. The current study describes a novel technique, which may be applied in liver surgery in patients with complex jaundice.

In situ splitting after selective partial portal vein ligation or simultaneous hepatic artery ligation promotes liver regeneration.

This study seeks to compare the impact of selective partial portal vein ligation (PPVL) or the combination of simultaneous hepatic artery ligation (PPVAL) with in situ splitting (ISS) on liver regeneration and injury. Rats were randomized into three groups; namely: selective PVL, PPVL + ISS and PPVAL + ISS. The changes in hepatic hemodynamics, liver regeneration and hepatocytic injury were examined. Blood flow to the left portal branch and the microcirculation of the left median lobe after PPVL or PPVAL was significantly reduced. Liver regeneration of PPVAL + ISS group was more pronounced than that in the PPVL + ISS and PVL groups at 48 and 72 hours as well as 7 d postoperatively. The serum biochemical markers and histopathological examination demonstrated reduced levels of liver injury in the PPVL + ISS group. Injury to hepatocytes was more pronounced with PPVAL + ISS than PVL. HGF, TNF-α and IL-6 expression in the regenerated lobes in both PPVAL + ISS and PPVL + ISS groups increased significantly when compared to the PVL group. We demonstrated that both PPVL + ISS and PPVAL + ISS were effective and feasible means of inducing remnant liver hypertrophy and could serve as a rapid clinical application for qualified patients.

Laser speckle contrast imaging and Oxygen to See for assessing microcirculatory liver blood flow changes following different volumes of hepatectomy.

OBJECTIVE: Portal hyperperfusion after extended hepatectomy or small-for-size liver transplantation may induce organ dysfunction and failure. This study was designed to monitor and characterize the hepatic microcirculatory perfusion following different volumes of hepatectomy in rats by using laser speckle contrast image (LSCI) and Oxygen to See (O2C), a spectrometric device. METHODS: The microcirculatory liver blood flow of the rats that underwent 68%, 85% and 90% hepatectomy (68PH, 85PH and 90PH) was monitored with LSCI and O2C before and following the hepatectomy. The portal venous flow (PVF) and hepatic arterial flow (HAF) were measured with an ultrasonic flowmeter. Liver regeneration, liver injury, histologic evaluation and gene expression were also assessed at 12h, 24h, 3d and 7d post hepatectomy. RESULTS: All the 68PH and 85PH rats survived, and 57% of the 90PH rats survived. After hepatectomy, both PVF and HAF decreased transiently, with the PVF of the 85PH and 90PH rats significantly lower than that of the 68PH rats. In contrast, the PVF and HAF per gram of liver weight were greatly increased after liver resection and were proportional to the volume of resected liver. Correspondingly, the microcirculatory liver blood flow of the 68PH, 85PH and 90PH rats, as assessed by both LSCI and O2C, were increased after hepatectomy, and the 90PH group was significantly higher than the 68PH and 85PH groups. The hyperperfusion continued for approximately 3days and returned to baseline following the completion of liver regeneration. The liver venous oxygen saturation of the three groups decreased immediately after hepatectomy and returned to baseline from 24h after hepatectomy. The 90PH rats also showed delayed liver regeneration and the most severe liver injury, as reflected by increased serum ALT, AST and TBIL levels, hepatocellular vacuolization, and inflammatory and endothelial constriction gene expressions (TNF-α, IL-1ß, MIP-1α, ET-1 and TM-1). CONCLUSION: Hepatic microcirculation hyperperfusion resulting from major and extended liver resection could be assessed by LSCI and O2C methods. The 90PH in rats led to extraordinary sinusoidal hyperperfusion, severe endothelial injury and liver failure. Monitoring the changes of hepatic microcirculation perfusion following extended hepatectomy or small-for-size liver transplantation may help to analyze the extent of hyperperfusion.

Preserving low perfusion during surgical liver blood inflow control prevents hepatic microcirculatory dysfunction and irreversible hepatocyte injury in rats.

Hepatic ischaemia/reperfusion (I/R) injury is of primary concern during liver surgery. We propose a new approach for preserving low liver blood perfusion during hepatectomy either by occlusion of the portal vein (OPV) while preserving hepatic artery flow or occlusion of the hepatic artery while limiting portal vein (LPV) flow to reduce I/R injury. The effects of this approach on liver I/R injury were investigated. Rats were randomly assigned into 4 groups: sham operation, occlusion of the portal triad (OPT), OPV and LPV. The 7-day survival rate was significantly improved in the OPV and LPV groups compared with the OPT group. Microcirculatory liver blood flow recovered rapidly after reperfusion in the OPV and LPV groups but decreased further in the OPT group. The OPV and LPV groups also showed much lower ALT and AST levels, Suzuki scores, inflammatory gene expression levels, and parenchymal necrosis compared with the OPT group. An imbalance between the expression of vasoconstriction and vasodilation genes was observed in the OPT group but not in the OPV or LPV group. Therefore, preserving low liver blood perfusion by either the OPV or LPV methods during liver surgery is very effective for preventing hepatic microcirculatory dysfunction and hepatocyte injury.

mTOR-Dependent Suppression of Remnant Liver Regeneration in Liver Failure After Massive Liver Resection in Rats.

BACKGROUND AND AIMS: Massive hepatectomy often leads to fatal liver failure because of a small remnant liver volume. The aim of this study was to investigate the potential mechanisms leading to liver failure. METHODS: Sprague-Dawley rats had performed a sham operation, 85 % partial hepatectomy (PH) or 90 % PH, and all had free access to water with or without supplemented glucose. Liver function and survival were evaluated. Liver parenchymal injury was assessed by evaluating hepatic pathology, blood biochemistry, and apoptotic and necrotic alterations. The regeneration response was assessed by the weight gain of the remnant liver, hepatocyte proliferation markers, and regeneration-related molecules. RESULTS: The 90 % hepatectomy resulted in a significantly lower survival rate and impaired liver function; however, no significant more serious liver parenchymal injuries were detected. TNF-α, HGF, myc and IL-6 were either similarly expressed or overexpressed; however, the increase in remnant liver weight, mitotic index, and the presence of Ki-67 and PCNA were significantly lower in the 90 %-hepatectomized rats. mTOR, p70S6K and 4EBP1 were not activated in the remnant liver after a 90 % hepatectomy as obviously as those after an 85 % hepatectomy, which was concomitant with the higher expression of phospho-AMPK and a lower intrahepatic ATP level. Glucose treatment significantly improved the survival rate of 90 %-hepatectomized rats. CONCLUSIONS: Suppression of remnant liver regeneration was observed in the 90 % PH and contributed to fatal liver failure. This suppressed liver regenerative capacity was related to the inhibited activation of mTOR signaling.

Establishment of a stable mouse model of brain death by the method of the gradually increasing intracranial pressure.

OBJECTIVE: To establish a stable and modified mouse model of brain death (BD) and to share our experiences in BD induction and maintenance. METHODS: Totally 35 C57BL/6 male mice were randomized into BD group (n=25) or sham control group (n=10). BD was induced by inserting a 2F Fogarty catheter connected to a syringe pump after trepanation of the left frontoparietal area and injecting volume at the speed of 6 µl/min until spontaneous respiration ceased. BD was diagnosed by electroencephalogram, apnea testing,as well as testing of brain stem reflexes. Mechanical ventilation was performed by orotracheal intubation. Right carotid artery was intubated by a PE-10 cannula for the continuous monitoring of mean blood pressure (MAP) and heart rate (HR). The right external jugular vein was catheterized for volume resuscitation.The sham control group underwent the same procedure with catheter insertion but without balloon inflation.Livers were removed and fixed in paraffin to evaluate the histological alterations with the light microscopy. RESULTS: Mouse models of BD were successfully established about 20 minutes after balloon inflation, and the mean balloon volume at the time of BD was (105.77 ± 21.57)µl. The MAP and HR rapidly increased on occurrence of BD and the peak value was (128.28 ± 17.16) mmHg and (434.16 ± 55.75) beat/min, respectively, which were significant higher than those in the sham control group at the same time point (P=0.000). During the 4-hour follow-up time, MAP and HR in 72% (18/25) of BD animals remained haemodynamically stable. No animal died due to anesthesia and surgical operation.Hepatic tissues in BD mice showed mild focal ischemic damages (cellular edema, congestion, and inflammatory infiltration), which were slighter and fewer in sham control group. CONCLUSION: The mouse model of BD was successfully established with lower surgical difficulty and can be performed in a standardized, reproducible and successful way.

Effects of combined anisodamine and neostigmine treatment on the inflammatory response and liver regeneration of obstructive jaundice rats after hepatectomy.

BACKGROUND: Cholestasis is associated with high rates of morbidity and mortality in patients undergoing major liver resection. This study aimed to evaluate the effects of a combined anisodamine and neostigmine (Ani+Neo) treatment on the inflammatory response and liver regeneration in rats with obstructive jaundice (OJ) after partial hepatectomy. MATERIALS AND METHODS: OJ was induced in the rats by bile duct ligation. After 7 days biliary drainage and partial hepatectomy were performed. These rats were assigned to a saline group or an Ani+Neo treatment group. The expressions of inflammatory mediators, liver regeneration, and liver damage were assessed at 48 h after hepatectomy. RESULTS: The mRNA levels of TNF-α, IL-1ß, IL-6, MCP-1, and MIP-1α, in the remnant livers, and the serum levels of TNF-α and IL-1ß were substantially reduced in the Ani+Neo group compared with saline group (P<0.05). The Ani+Neo treatment obviously promoted liver regeneration as indicated by the liver weights and Ki-67 labeling index (P<0.05). The serum albumin and γ-GT levels and liver neutrophil infiltration also significantly improved in the Ani+Neo group (P<0.05) compared with the saline group. CONCLUSIONS: These results demonstrate that the combined anisodamine and neostigmine treatment is able to improve the liver regeneration in rats with OJ by substantially alleviating the inflammatory response.

[Transient hepatic venous occlusion induced liver hemodynamic change and reperfusion injury in rats].

OBJECTIVE: To observe the hemodynamic change and reperfusion injury cause by transient hepatic venous occlusion and transient hepatic inflow occlusion in rats. METHODS: The rat liver was divided into 3 different areas: the ischemia reperfusion (IR) area: the inflow of the right superior lobe was clamped for half an hour; the non-isolated lobe congestive reperfusion (NIL-CR) area: the outflow of the right median lobe was clamped for half an hour; and the isolated lobe congestive reperfusion (IL-CR) area: the outflow of the left lobe was clamped for half an hour. The flux value and the oxygen saturation of microcirculation were monitored before at clamping for 30 minutes, and on 1 day, 3 days ,and 7 days after reperfusion. The hepatic damage and Suzuki's score were evaluated. RESULTS: After clamping for 30 minutes, the flux value in the IR area was significantly higher than in NIL-CR area (P<0.01) and IL-CR area (P<0.01), the oxygen saturation in the IR area was significantly higher than in NIL-CR area (P<0.01) and IL-CR area (P<0.05). Compared with IR area, both NIL-CR area and IL-CR area were found having more severe liver damage in terms of Suzuki's score in early postoperative period (at clamping for 30 minutes and on 1 day, P<0.01). However, there was no significant difference between NIL-CR area and IL-CR area in flux value, oxygen saturation, and Suzuki's score (P>0.05). CONCLUSIONS: Hepatic venous occlusion can more effectively decrease the blood perfrusion and oxygen saturation; thus, compared to the IR, CR can result in more severe liver damage. The presence of normal liver tissue around the congestion area can not influence liver damage in transient hepatic venous occlusion.

The monitoring of microvascular liver blood flow changes during ischemia and reperfusion using laser speckle contrast imaging.

OBJECTIVE: The recovery of microvascular liver blood flow (LBF) after ischemia is an important determinant of the degree of hepatocellular injury. Laser speckle contrast imaging (LSCI) was recently suggested to be a suitable instrument for monitoring the LBF. This study was designed to evaluate LSCI in monitoring the LBF changes during liver ischemia and reperfusion (IR). METHODS: A rat model with 120-min ischemia and 60-min reperfusion to 90% of the liver (entire liver except the caudate lobe, which was kept as portal blood bypass) was used. The LBF of the sham operation (SO) group and the IR group was measured with LSCI at the following time points: before ischemia (Baseline), 5 min after the start of ischemia (I-5 min), 5 min before the end of ischemia (I-115 min) and 5 and 60 min after the start of reperfusion (R-5 min and R-60 min). The reproducibility among different rats or repeated measurements, the liver histopathology, the liver biological zero (BZ) and the influence of liver movement on the LSCI measurements were investigated. RESULTS: The entire exposed liver surface after laparotomy was suitable for full-view LSCI imaging. Establishing many circular or oval regions of interest (ROIs) on the LSCI flux image was a simple and convenient method for calculating and comparing the LBF of different ROIs and different liver lobes. There was good-to-moderate intra-individual and inter-individual reproducibility for the LSCI measurements of the LBF in the rats of the SO group. In the IR group, the total blood inflow occlusion resulted in a notable drop of the LBF from the baseline (P<0.05) that remained for the 120 min of ischemia. The LBF decreased further after the reperfusion (P<0.05), reflecting the IR-induced liver microcirculation dysfunction. The histopathological examination revealed severe hepatic sinus congestion and damaged hepatocytes in the IR group. The no flow BZ and liver movement contributed to the LBF values. CONCLUSIONS: LSCI technology is a simple, convenient and accurate method for the real-time monitoring of microvascular LBF changes during ischemia and reperfusion, regardless of the contribution of biological zero and liver movement. This finding suggests the possible application of LSCI for monitoring the microvascular LBF changes intraoperatively.