| Currently, livers used in liver transplantation are mainly derived from donation of patients suffering from brain death. However, experimental and clinical research found that compared to living donor, brain death donor livers after liver transplantation were associated with worse ischemia-reperfusion injury, higher rates of acute rejection, initial liver dysfunction and primary graft nonfunction. Therefore,many animal models of brain death were established to study the influence of brain death on the potential donor organs and the possible treatment measures to improve the quality of donor organs. Brain death is defined as the irreversible loss of brain and brain stem function, which could cause complex pathophysiological changes associated with hemodynamic instability, hormone dysrugulation, systemicinflammation and immune activation, complement and coagulation system activation.The apoptosis related genes are changed and the apoptosis is increased in the liver of brain death donor. These adverse factors of brain death seriously affect the quality of the donor liver, and its specific molecular mechanism is still not clear. So, studying the changes of genes in the liver of brain death could provide novel target genes for preventing the hepatic injury induced by brain death.Heme oxygenase 1(HO-1) also termed as heat shock protein 32(HSP32), is the rate-limiting enzyme in heme degradation, and has the anti-inflammatory and anti-apoptotic effects. Brain death upregulated the expression of HO-1 and HSP70 in kidneys. Induction of HO-1 by cobalt protoporphyrin(Co PP) in brain death donors could significantly improve the survival rate of renal allograft at 90 days after kidney transplantation from 20% to 50%, and reduce the infiltration of lymphocytes and monocytes. Whether brain death influences the expression of heat shock proteins in the liver, and whether HO-1 inducer(Co PP) can reduce the liver injury induced by brain death and the underlying molecular mechanism are still unclear.This study is divided into three parts: building a rat model of brain death,analysis of the gene expression in liver of brain death rat and investigating the molecular mechanism of the effect of Co PP on liver injury induced by brain death.We expect to provide new insights and methods for improving the quality of liver derived from brian death donor, so as to provide more available livers from brain death donors for clinical liver transplantation.Part one:Establishment of A Rat Model of Brain Death by Gradually Intermittent Increasing Intracranial PressureObjectiveTo establish a stable and reliable rat model of brain death for studying the effects of brain death on the quality of donor liver.MethodsSprague-Dawley(S-D) rats were anesthetized by intra-peritoneal injection of pentobarbital sodium(6 mg/100 g body weight). Brain death was induced bygradually increasing intra-cranial pressure by slow infla-tion of a No. 3 Fogarty catheter balloon inserted into the epidural space with the tip pointing caudally through a 2 × 2 mm burr hole in the frontomedial part of the skull. Brain death was confirmed by resting electroencephalography, apnoea, deep coma, and the absence of corneal reflexes. Rats were infused with 10% hydroxyethyl starch via the tail vein to maintain mean arterial pressure above 80 mm Hg. Sham-operated rats underwent the same surgical procedures without BD induction. There were 20 rats in brain death group and 10 rats in sham control group. Monitor the arterial blood pressure, heart rate, end tidal CO2, infused fluid volume and urine volume. Serum ALT and AST were measured with a standard clinical automatic analyzer. Histological alterations of livers were observed by light microscopy.Results90% of brian death rats were maintained hemodynamically stable(MAP>80mm Hg) for 6 hours. The preparation time was 70 + 10 min before the brain death induction, the time of brain death induction was 34±6min, the balloon volume at the time of brain death was 136±24μl. Hydroxyethyl starch input volume, urine volume, serum ALT and AST in brain death group were respectively 36.46±17.85 ml,32.77±18.44 ml, 70.17±15.87U/L, 201±23.31U/L, and were all significantly higher than sham control group(all P <0.05). Hepatic tissue in brain death rats showed obvious congestion, cellular edema and swelling, hepatic cord widened, hepatic sinusoids narrowed, scattered punctate necrosis. Liver tissue morphology is normal in sham control group rats.Part two: The Changes of Gene Expression In Livers of Brain Death RatsObjectiveTo explore the changes of gene expression in livers of brain death rats, which provides experimental basis for the study of molecular mechanism of liver injury induced by brain death.MethodsSamples were collected at 0h, 1h, 2h, 4h and 6 h after induction of brain death or sham operation(6 rats per time point). The expression of 41012 genes in livers collected at 6h after induction of brain death or sham operation were analyzed using Agilent whole rat genome oligo microarray. The expression of HO-1, HSP27, HSP70,Mcl-1, Bcl-2, BNIP3, Caspase-3, HIF-1α, HIF-2α, HIF-3α, ARNT and Glut-1 in livers collected at 0h, 1h, 2h, 4h and 6 h after induction of brain death were detected by Real-time PCR. The HO-1 protein in livers collected after induction of brain death was detected by western blotting and immunohistochemical staining.Results149 genes were upregulated in brain death livers(>10-fold difference in brain death samples at 6 hour). 136 genes were downregulated in brain death livers(<1/10-fold difference in brain death samples at 6 hour). The expression of HO-1m RNA and protein were significantly upregulated in livers collected at 0h, 1h, 2h, 4h and 6 h after induction of brain death. The HO-1 protein was expressed in cytoplasm and high expression in hepatocytes around the central vein of the liver of brain death rat. The expression of BNIP3 and Caspase-3 m RNA were significantly upregulated in livers collected at 0h, 2h, 4h and 6 h after induction of brain death.Part three: The Molecular Machenism of Co PP on Liver Injury Induced by Brain DeathObjectiveTo investigate the effect of HO-1 inducer-Co PP on liver injury and its molecular mechanism in rats of brain death.Methods24 rats were randomly allocated to four groups:(S group) rats undergoing sham operation,(CS group) rats undergoing Co PP administration(5 mg/kg i.p.) 24 h before the sham operation,(B group) rats undergoing BD and(CBgroup) rats undergoing Co PP administration(5 mg/kg i.p.) 24 h before the induction of BD. Samples were collected at 6h after induction of BD or sham operation. Serum ALT and AST were measured with a standard clinicalautomatic analyzer. The expression of Bcl-2, Bax,Mcl-1, CHOP, Caspase-12, cytosolic Cytochrome c and cleaved-Caspase-3 proteins in livers of each group rats were detected by western blotting. Apoptotic cells in liver tissue of each group rats were detected by TUNEL.ResultsCo PP significantly enhanced the expression of HO-1 protein in livers of pretreated rats. In the CB group compared with the B group, the increased hepatic expression of HO-1 correlated with a significant decrease in serum ALT and AST levels, fewer apoptotic cells in liver, increased hepatic expression of Mcl-1 and Bcl-2,and decreased hepatic expression of Bax, CHOP, Caspase-12, cytosolic cytochrome c and cleaved caspase-3.Conclusions1. A stable, reliable and reproducible rat model of brain death has been established,which provides the model foundation for the further study of the effect of brain death on the donor organs.2. This is the first analysis of global gene expression in livers of brain death rats,providing new target genes for preventing liver injury induced by brain death. The high expression of HO-1 in liver of brain death may be related to the protective mechanism induced by brain death associated stress.3. HO-1 inducer-Co PP can significantly enhance HO-1 protein expression, and reduce apoptosis in rat liver of brain death via modulating the key proteins of mitochondrial and endoplasmic reticulum apoptosis pathway. HO-1 will be potential target for improving the quality of liver from brain death donor. |
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