Protective Effect Of X-linked Inhibitor Of Apoptosis Protein (XIAP) In Hypoxic-ischemic Neonatal Mice Brain

Neonatal hypoxic-ishchemic brain damage (HIBD) is a major cause of central nervous system injury in infants. The mechanism behind HIBD is still unclear and there is no effective treatment so far. Elucidating the mechanism and looking for a effective therapy of HIBD is focused by the researchers both at home and at abroad. Recent study shows that neuronal apoptosis plays a key role in HIBD. At the heart of the apoptosis pathway is a group of cysteine proteases termed caspases. While inhibitor of apoptosis protein (lAPs) family is the unique intrinsic apoptosis inhibitor through inhibiting caspase activation in mammals. Among lAPs, X-chromosome-linked inhibitor of apoptosis (XIAP) is the most potent and versatile regulator of apoptosis which blocks not only the activation of the initiator caspases (caspase-9) but also the effector caspase (caspase-3, -7). To investigate the protective effect of the XIAP in HIBD, 9-day-old C57BL/6 wild type mice and C576176 mice over-expressing the XIAP were subjected to hypoxia and ischemia (HI). The degree of injury was assessed by measuring the volume of tissue loss and by a neuropathological scoring system. Caspases activity was measured.Objects:(1) XIAP over-expressing mice (XIAP group): 9-day-old C578L/6 mice over-expressing XIAP of either sex (n=79) were randomly divided into 3h (n=12),24h (n=12), 72h (n=ll), and 5d (n=26) post- HI respectively. XIAP over-expression mice of 9d (n=9) and 12d (n=9) were taken as normal control group.(2)Wild type mice: 9-day-old wild type C₅₇BL/6 mice of either sex (n=82) were randomly divided into 3h (n=ll), 24h (n=10), 72h (n=9), and 5d (n=31) post- HI respectively. Wild type C₅₇BL/6 mice of 9d (n=ll) and 12d (n=9) were taken as normal control group.Methods:(1) The preparation of the mice HIBD model: 9-day-old mice were anesthetized with 3-4% halothane and the left common carotid artery was ligated by using the anatomic microscope. Pups were allowed to go back to the mother and rest for Ih then placed in a humidified 36癈 gas mixture (10 ?0.01% O2 in nitrogen) for 60min. There were no hypoxic and operation in normal control group pups.(2) Microtubule-associatedprotein-2 (MAP-2) immunohistochemistry staining: 5 days after HI, pups were perfused with histofix and brains were taken out and processed routinely. After dehydration with graded ethanol and xylene, the brains were paraffin-embedded and cut into 5 or 10 um coronal sections were stained every 50th section for MAP-2.(3) The determination of caspases activity: The pups were decapitated at 3h, 24h, 72h after HI and at 9d, 12d after birth .The cortex was separated and divided into left and right hemisphere and then homogenized by hand in homogenate buffer. The supernatant was got after tissue homogenate was centrifuged for 15 min at 9200g at 4 The caspases activity of supernatant was assayed by spectrofluorometer.(4) XIAP and XIAP associated factor 1 (XAF-1) double immunofluorescent staining: To investigate the distribution and the relationship of XIAP and XAF-1 in neurons.(5) Western blot: To show the expression of XIAP protein in the brain between XIAP group and wild type group.(6) Evaluation of brain infarction Volume: Using Micro Image software to measure the MAP2 negative area and positive area by taken every 50th sections of5 um. The infarction volume was calculated by the formula.(7) Neuropathological score of brain injury: The extent of the brain injury was evaluated by the neuropathological scoring system with the MAP-2 and HE staining.(S)Statistic analysis: All the data were showed as X盨D and were tested with unpaired T test. P<0.05 was regarded as a standard of the statistic significant. All the results were run on the statView software.Results:(1) Western Blot displayed XIAP protein is a single band with apparent molecular 57kDa.Half quantitative analysis showed XIAP protein content in the XIAP over-expressing mice was obviously higher than it was in wild type mice.(2) Neuropathological sco...