Brain Injury Induced By Hepatic Ischemia Reperfusion And The Mechanisms

IntroductionSince the first success case of liver transplantation in the beginning the 1960s' , hepatic transplantation already has 40 years history. Along with the improvements of the surgical skill and the treatment of immune repulsion, and the understanding about the pathological effects of ischemia reperfusion, the success ratio of the liver transplantation has been increased gradually. But the complications of postoperative still unceasingly occurred and which was attached importance day by day. The remote organ damage which resulted from hepatic transplantation is one of the key points at present researches. The rate of cerebral complication of postoperative liver transplantation was 8 to 47% , average about 20%. The cerebral complication was mainly occurred at the early time of postoperative. The success of transplantation and patients healing was influenced seriously.The neurological complications after transplantation could be attributed to several reasons such as metabolism disorder, surgical injury, stress, ischemia reperfusion injury, the brain damage caused by late - stage hepatic disease, postoperative rejection and the use of immunosuppressive agents. But the mechanisms of neurological complications after transplantation are still not clear.One primary pathophysiological change after liver transplantation is the hepatic ischemia - reperfusion injury, which leads to not only the injury to the liv-er, but also injuries to distal organs - the lung, the kidney and the intestines by activating the reticuloendothelial system and the release of inflammatory mediators. There is no report concerning whether this process could cause pathologic changes to the brain tissue and whether it is responsible for the neurological complications after transplantation. In this research we made the model of total hepatic ischemia - reperfusion in rats, observed the subsequent pathological changes in the brain, searched the possible mechanisms at the levels of cell, molecular and gene; we also looked for the evidence of subclinical brain injury in patients who underwent liver transplantation and analyzed the possible explanations , in hope of providing the theoretical support for intraoperative brain protection and finding a way to lower the neurological complications after transplantation.This research consists of the following three aspects and four parts: first, the pathologic and structural changes in the brain tissue of rats after hepatic ischemia reperfusion; second, the discussion of the mechanisms of brain pathologic and structural changes in big mice after hepatic ischemia reperfusion: 1 the function of iNOS mRNA expression on brain injury after hepatic ischemia reperfusion, 2 the function of tight junction on the change of permeability of blood brain barrier after hepatic ischemia reperfusion; third, the changes of S - 100 protein concentration in serum of the patients and the discussion of its possible reasons.Materials and MethodsMaterials1. Animals: healthy male wistar rats weighting 240 - 280g were used in this studyClinical cases; liver transplantation patients without neurosystem disease.2. Kit and drugs: kits of MDA, SOD, NO, S100 - p and IL - lp; Nitroty-rosine monoclonal antibody, anti - zo - 1 monoclonal antibody, anti - occludin monoclonal antibody, primers of iNOS, ZO - 1, occluding and p - actin; PCR mark, Takara kit.3. Equipment: self-made rotary pump, Siemens 730 monitor; Phillips CM -10 transmission electron microscope; KODAKID gel imaging analytical system; 8800 type ultrathin slicer; PTC - 100 PCR amplication device, BIO -RAD powerpac 200 electrophoresis device; Bio - Rad Seimidry transfer system; HERMLEZ 383 K high speed centrifuger; OlympuslOOO microscropic camera.Methods1. The part of animal study1. 1 Animal modelThe model of total hepatic ischemia reperfusion under spleen vein - thigh vein bypass was made according to the method described by Suzuki.1. 2 Experimental designsThirty two rats were randomly allocated into four groups with 8 animals in each group. Group S, rats were only anesthesia, surgery, and its spleen was excised ; groupIR3, hepatic pedicle was clamped for 40 min, and liver reperfusion for 3 hours; group IR6, hepatic pedicle was clamped for 40 min, and liver reperfusion for 6 hours; groupIR24, hepatic pedicle was clamped for 40 min, and liver reperfusion for 24 hours.1. 3 Observation of parameters and method1.3.1 The hymodynamic changer during the period of clamping and pos-treperfusion;1.3.2 The pathology of hepatic and brain in group IR6, and the changes of brain ultrastructura were observed;1.3.3 The concentration of MDA, NO in serum and MDA, SOD, NO in brain were detected;1.3.4 The permission of blood brain barrier was assessed with the change of EB consentration in brain;1.3.5 The protein of NT, ZO - 1 and occludin in brain expressed with Western blot;1.3.6 mRNA of iNOS and occludin was expressed with RT - PCR.2. The part of clinical observation2. 1 case collection: patients of late - stage hepatic disease and with original liver transplantation.2.2 Observation of parameters and method; The MAP, HR, CO, CVP and blood -air analysis were recorded at preoperative, preclamp, anhepatic 10 min, 30min and 60min, after reperfusion lOmin, 90min and the end of operative. The concentration of S - 100fJ and IL - lfjin serum was also tested at preoperative, the end of anhepatic, reperfusion lh, postoperative 24h and 48h.Results1. Pathological and ultrastructura change in the brain:1. 1 The hymodynamic changer during the period of clamping and pos-treperfusion; There was stable hymodynamic in S group. Compared with S group and preclamp, MAP in IR group decreased significantly, which was maintained at 60 - 80 mmHg during ischemia ( p < 0. 05 ). There was an increase in HR and decrease in MAP for a while in IR group at the beginning of reperfusion.1. 2 light microscopic changes; liver tissue; Stasis in liver sinuses, local and flaky necrosis, infiltration of neutrocyte could be seen at 6 hour after reperfusion. Brain tissue; the cortex got loose and swollen at 6 hour after reperfusion, the neurological cells lost their clear layers; space began to appear around the vessels and the cells; the cells of the cortex and the hippocampus showed changes of pyknosis, and appeared the shape of triangle and polygon, with dense neucleus and blurred nucleolus.1. 3 Electron microscope changes; there is brain tissue swelling in the IR6 group, the neuroglial cell and the tissue around the capillary is much more severe, which forming the broad zone. The sparse electron of the capillary basal membrane, and the degeneration of endothelial cell was also observed.2. The mechanisms of ultrastructura change in the brain tissue induced by hepatic ischemia reperfusion.2. 1 Effects of the expression of iNOS - mRNA in brain injury induced by hepatic ischemia reperfusionCompared with group S, content of NO in serum and in brain tissue increased significantly at 6 hours at reperfusion, and at 24 hours, still higher slightly than group S in serum (p > 0. 05) but significantly in brain (p <0.01); The expression of protein NT in the brain tissue was markedly up - regulated compared with that in control group at 6, 24 hours after reperfusion (p <0. 05 or p <0. 01 respectively) ; The iNOS mRNA also revealed significantly greater expression in the experiment group than that in group S at 6 hours after hepatic ischemia reperfusion (p <0.05) and still had iNOS mRNA expression to some extent but showed no significant effects at 24 hours.2. 2 Role of tight junction in the change of permeability of blood brain barrier induced by hepatic ischemia reperfusionCompared with group S or IR3, the content of EB in the brain tissue increased significantly at 6 hours and the high level lasted until 24 hours after reperfusion. At the same time, the occludin expression were significantly reduced and characterized by |3 band at 6 hours , but both a and ï¿¡ band were down - regulation at 24 hours. ZO - 1 expression in each experiment group were gradually lower than that in groups S(p <0. 05) , as well as a lower expression of occluding mRNA which decreased gradually and decreased strikingly at 24 hours after reperfusion when compared to the group IR3 or IR6.3. Change about the serum concentration of S - 100 (3 in patients who undergoing liver transplantation and its mechanismAt preoperative, serum S - 100 (3 level was in normal range and had no difference between patients, but increased at 1 hour after postreperfusion and, at 24 hours, was still higher than that at preoperative. The highest level was 1.28 g ml . Then it decreased gradually to the level of the end of anhepatic period at 48h of postoperative. IL - 1(3 increased to the highest level at 24 hours after postreperfusion and then decreased gradually; At the first 10 minutes after vena cava clamped, MAP decreased and HR increased significantly, and then MAP remained in normal level adjusted by drugs, HR decreased to a certain extent but were still higher than that before clamped. Meanwhile, cardiac output (CO) decreased significantly; At the first 10 minutes after reperfusion, HR reversed but MAP decreased again, central venous pressure increased slightly, CO were still lower than that before clamped; All those were recovery to the level at preoperative after treated by blood vessel active drugs, hydragogue, etc. Partial pressure of carbon dioxide (Paco2 ) increased obviously during earlier period af-ter postxeperfusion, though sodium bicarbonate had been used during last period of anhepatic phase and earlier period of reperfusion, the value of PH still decreased (i. e. acidemia) and increased to normal level before the end of operation ; The level of ion remained in normal range on the whole through adjusted carefully. Results detected by correlation analysis showed that the change of S -100(3 had no apparent correlation with CO or IL - lp(r = -0. 327, r =0. 248, p>0.05).Conclusions1. The hepatic ischemia reperfusion can cause the damage in both brain and liver tissue.2. The increased expression of iNOS - mRNA in brain after hepatic ischemia reperfusion, produces great amount of NO, which lead to brain injury.3. The permeability of blood brain barrier is increased after hepatic ischemia reperfusion4. The decreased expression of occluding - mRNA, and the protein of oc-culdin and zo -1 is one of the biomolecular fundamendations that caused the increase of the permeability of blood brain barrier after hepatic ischemia reperfusion5. The increase of the permeability of blood brain barrier is a subclinical change that appeared after the reperfusion of the donor liver. It is possibly resulted from the hepatic ischemia reperfusion.