The Changes Of Excitability Neurotransmitter In Brain Cortex After Hepatic Portal Occlusion Reperfusion And The Effect Of Limb Ischemic Preconditioning

PerfaceHepatic portal occlusion(HPO) is a important measure aid to reduce bleedingduring operation of liver transplantation and hepatectomy. Extended ischemic episodesin anhepatic phase followed by reperfusion are inevitably during operation and it is thesource of all pathophysiological change. The ischemia-reperfusion(IR) injury inducedremote organs dysfunction such as acute lung injury or cardiac insufficiency evenMOSF. The organ damage excluding liver which resulted from hepatic transplantationis one of the key points at present researches. It was reported that the morbidity ofmental functional disorder posttransplantation upto 8%—47%. But the mechanisms ofneurological complications after transplantation are still not clear. The piror study ourteam completed had shown that hepatic IR injury could damage tight junction in BBBand increase permeability and induced cerebral injury.In this research we used the Pringle measure to install hepatic portalocclusion(HPO) model without bypass in rats and observed the subsequent pathologicalchanges in the brain, searched the possible mechanisms at the levels of cell, protein andgene. Moreever, we apply a LIP model for a pretreatment to protect brain after HPOreperfusion. It is expected to reduce the neurological complications. The study includedthree parts: 1. The alteration of cortex and serum S100βprotein undergoing hepaticportal occlusion reperfusion in rats. 2. The ischemic-reperfusion injury following hepatic portal occlusion increase brain glutamic acid and aminosuccinic acid andNMDAR mRNA expression in rats. 3. Limb ischemia preconditioning amelioratesbrain glutamic acid and aminosuccinic acid and NMDAR expression increaseundergone hapetic portal occlusion.Materials and methods1. Animals: healthy male wistar rats weighting 200—250g were used in this study.2. Kits and drugs: S100βELISA Kits, The standard preparation of Glu and Aspwere SIGMA company product. The RNAout kit. AMV reverse transcriptase, dNTPs,oligo-dTA, PCR marker and RNAase inhibitor purchase by TaKaRa kits. Primer weredesigned by Primer 5.0 and synthesized by BORED company. Anti NMDAR2Bmonoclonal antibody was Biosouce company product.3. Equipment: DATEX monitor, PHILIPS CM-10 transmission electronmicroscope, KODAK ID gel imaging analytical system, 8800 type uhrathin slice,PTC-100 PCR amplication device, BIORAD powerpac 200 electrophoresis device,HERMLEZ 383K high speed centrifuger, OLYMPUS 1000 microscropic camera.SHIMADZU SCL-10AVP high performance liquid chromatogram system4. Methods: Rats anesthetized by isoflurane inhalation and followed heparin(500U/kg) intravenous injection. Animals noninvasive arteria caudilis blood pressurewere monitored. Disinfection and followed median incision for the perihepatic ligamentdeligation to block collateral circulation. MAP was kept above 60 mmHg according tothe hemodynamics parameters during operation. Rectum temperature were kept37-38℃.Experimental designsRats were randomly allocated into three groups. Group Sham, rats were onlyanesthesized and surgery of median incision to deligate the perihepatic ligament; GroupHPO, according to the Pringle method, hepatic portal occlusion(HPO) by block portalvein, hepatic artery and common bile duct at hepatoduodenal ligament for liver ischemia 30 min and then followed reperfusion. Group LIP according to the zhanglianyuan and Kuntscher and Petrishchev method, hind limb ischemia 10 min followed30 min reperfusion, and then followed HPO treat as described above. All animals werekilled by the end of reperfusion 6h, 12h or 24h. The tissue samples were stored in liquidnitrogen.Observations of parameters and methods1. The hymodynamic.2. Blood-gas analysis test the pH,[K⁺] and [Ca²⁺] in portal vein blood.3. HPLC measure the content ofglumatic acid and aspartic acid in cortex.4. RT-PCR method test the mRNA expression. of NMDAR subunit NR1 and NR2B5. Intestine MPO measurement test PMN infiltration level.6. Western blot test the expression of NMDAR subunit NR2B in cortex.7. ELISA test the S100βprotein concentration in serum and content in cortex tissue.8. Morphology study by electron microscope and light microscopeResults1. The alteration of cortex and serum S100βprotein after hepaticportal occlusion reperfusion in rats(1) hymodynamic: MAP all above 60mmHg in all cases included in study.(2) Compared with Sham group, HPO group portal vein blood pH reduced and[K⁺] enhanced significanty respectiveiy, and [Ca²⁺] decreased and last to reperfusion12h.(3) light microscope show that hepatic damage was less serious than intestine andbrain cortex edema in HPO group. All those in Sham group were normal.(4) Electron microscope show cortex gliacyte damage in HPO group, such asgliacyte degeneration, a few chondriosome degeneration. All those in Sham group werenormal.(5) Compared with Sham, tissue S100βprotein content increased significantly in HPO group. But S100βprotein serum concentration were not difference between twogroups.2. The ischemia-reperfusion injury after hepatic portal occlusionincrease brain glutamic acid and aminosuccinic acid and NMDAR mRNAexpression in rats(1) HPLC measure showed that the content of cortex glutaminic acid(Glu)andaspartic acid(Asp) in Group HPO elevated significantly after reperfusion comparedwith Group Sham, the peaks were in 6h.(2) RT-PCR analysis discovered that the expression of NMDARmRNA increasedsignificantly, the subunit NR1mRNA expression is higher on reperfusion 6h and last to12h, the peak of NR2BmRNA expression is on reperfusion 6h, but they did not furtherincrease after reperfusion 24h.3. Limb ischemia preconditioning ameliorates brain glutamic acid andaminosuccinic acid and NMDAR mRNA expression increase undergonehapetic portal occlusion.(1) Model survival rate in LIP group was higher than that in Group HPO (P＜0.05)(2) Morphology study show hapetic and intestine were improved by LIPpretreated, electron microscope also demonstrate the cortex were same trend.(3) LIP group reduced intestine PMN infiltration undergone HPO model, butmight not restore to Sham group level.(4) Glu in LIP group were less than HPO group and closed to the Sham group.(5) NMDAR2B protein expression after HPO were enhanced. Compare withHPO group, NMDAR2B protein in LIP group were relative less but higher than Shamgroup level. (all P＜0.05). Conclusions1. HPO followed reperfusion injury not only included hepatic IR injury but alsointestine congestive reperfusion injury, but the gut damage were serious than liver.2. There were reversibilitive subclinical cerebral injury after HPO reperfusioninjury undergone present model.3. Cortex S100βprotein content and serum S100βprotein were not dependabilityafter HPO. The increase of cortex S100βprotein content last to reperfusion 12h and itimplicated the protective reaction by the CNS gliocyte when faced to stress. But thehigher concenstration S100βprotein would lead to damage.4. HPO could switch on the Glu and Asp release and inhibit reabsorption inneurapophysis space and lead to EAAs accumulation. At the same time, upreguratedNMDARmRNA and NR2B protein expression. It is possibility that HPO reperfusioninjury induced cerebral injury throught EAAs-NMDAR pathway.5. LIP pretreatment could ameliorate EAAs-NMDAR activity after HPOreperfusion and it can relieve CNS damages induced by HPO 30min followedreperfusion.