| BackgroundHypoxic-ischemic brain damage induced by perinatal asphyxia is the commonclinical disease and one of the most common risk factors for neonatal mortality and permanent neurodevelopmental disability, because of the complicated pathogenesis, so far there are no powerful measures to the postinjury treatment. Therefore, further study to HIBD pathogenesis and cure is a especially closed attention to perinatology nowadays in the world. In the past few years , along with the further study to HIBD pathogenesis, there also appear new treatment ways one after another.Topiramate nowadays is widely used in clinical as an new antiepileptic agent .following the deeply study, TPM protection effect to brain injured after asphyxia also be confirmed already. In 2006 year, Noh MR and others through animal experiment presumed the TPM brain protection mechanism have some main spots below: (1) locking up voltage dependence Na2+ or/and Ca2+ passage, (2) activating GABA initiated Cl- inflowing, (3) stopping KA/AMPA glutamic acid receptor, (4) suppressing carbonic anhydrase enzymatic active, (5) lessening mitochondria damage; In internal, minority experiment finding indicate TPM can relieve brain tissue sodium cumulation after hypoxia-ischemia; lessen encephaledema; decrease neuron decease ; actively suppress oxygen free radical form and toxicity, possessing good prevention and cure effect to cerebral ischemia impaired. But with regard to the TPM administration time,dosage,style and concrete mechanism, there are still indefinite and the research are also little. The glial cell line-derived neurotrophic factor (GDNF) is a new subgroup member of transforming growing factorβsuperfamily, that already to be discovered in multiple nerve cell and nerve correlated cell cultivation. As the new member of neurotrophic factor, the GDNF play multiple effect in neurotrophy. More and more evidence indicate it can promote post trauma neuron repair and regenerate. Concerning the specificity neuron protection ways to nenatal HIBD therapy, neurotrophic factor is an important aspect, the correlated research also show extensive attention. In cerebral cortical neuronal degeneration experiment model established by excitatory amino acids culture in vitro, the culture fluid can obviously lessen glumatic acid toxicity; protect undamaged cell; make the damaged cell functional recovery; cut down cell mortality; relieve cerebral cortical neuron degeneration and decease, moreover show right dose-effect relationship. The GDNF expression is various in different period and present time series regular pattern after brain injury. The 7-days neonatal rat brain development is similar to neonates, also the condition caused hypoxia-ischemia. People generally apply 7-days neonate rat to establish HIBD model and carry out associated research , as well as the research between GDNF and HIBD.The problems mentioned above formed the objectives. Through setting up hypoxia-ischemia animal model to observe brain histopathology change,GDNF dynamic expression level influence between different. Topiramate administration group, then compare to control group, further to probe the therapy time window and regulation mechanism of TPM in brain protection. The deep study of such problems not only conduce to illuminate therapy mechanism of TPM in brain protection, but also obtain some degree probe to TPM therapy time window, offer drastic theory experiment proof for TPM opportunity and mechanism to cure perinatal period HIBD.Objectives1. To observe the GDNF dynamic expression change of neonate rat after HIBD.2. To observe the influence to GDNF dynamic expression between different time TPM administration group for HIBD neonate rat.3. To observe the TPM therapy time window in brain protection for HIBD neonate rat.4. Further to observe the mechanism of TPM in brain protection.Methods1. One hundred and fifty-two newborn Wistar rats aged 7 days were randomly divided to sham operation group, pure hypoxic-ischemic group and Topiramate administration group. The later again was divided into pretreatment, post-treatment, and delayed treatment groups according to different time administration.2. Establish neonate rats HIBD animal modals.3. Administration and management approach:(1) Topiramate administration group: dissolving Topiramate in water for injection to form 2% solution, the first dose is 150 mg/kg by gavage tube, for quickly achieving blood peak density, afterward every time administration is 100mg/kg. The pretreatment group received TPM immediately before and after exposure to hypoxia, and then at 12 h intervals over 5 days, The post-treatment group received TPM immediately and again 2 h after termination of hypoxia, and then at 12h intervals for 5 days, To address the clinical effectiveness of delayed -treatment, we examined the therapeutic time window by the two successive administrations of TPM 2 and 4h after the end of hypoxia and then 10 administrations with 12 h intervals for 5 days.(2) The sham operation group and pure hypoxic-ischemic group was received an equivalent volume of saline solution according to the same schedule for saline-treated group.(3) Every group put to death one fourth animal after hypoxia-ischemia 12 hour, 2 days, 3 days, 5days. (The sham operation group is 6 and the other group 8 every time)4. Apply HE drum dyeing, immunochemistry and Western-blotting experiment methods.Results1. Morphology findingsThere was 90 percent ipsilateral hemisphere of pure hypoxic-ischemic group present liquation and infarction , pathology change including on the hemisphere ipsilateral to the carotid artery coagulation included pallor, atrophy, and tissue loss in the striatum, hippocampus, cortex, and thalamus. In the most severely damaged brains, most of the ipsilateral hemisphere was liquefied, and few anatomic landmarks were recognizable. The brain tissue necrosis was also obvious in delayed treatment group, however, there was a dramatic reduction in the incidence of brain damage pretreatment and post-treatment group.2. HE dyeing findingsThe sham operation group tissue structure was clear, cell outline distinct, nuclear mediate and flecky body at nuclear periphery; the cortex and striatum in ipsilateral hemisphere of pure hypoxic-ischemic group appeared slight pathological change after HIBD 12 hours, the 2 day was most severe, showing large zone of necrosis in cortex, striatum, hippocampus and thalamus; the 3 day begin to relieve and emerge gliocyte proliferation; the 5 day we not found rupture cell on the whole and appear great glial scar.Meanwhile, in pretreatment and post-treatment group, pathological changes above-mentioned position unobvious, the 2day showing up light affection , displaying very few cell spaces widen and cellular nucleus condense, the3,5day we not found clear infection area; but there was no obvious brain damage lessen in delayed treatment group3. Immunochemistry findingsThe sham operation group almost no apparent GDNF positive cells expression, the individual sparsely distributed in the whole cerebral cortex, striatum, hippocampus and symmetry two sides, also not found dynamic change each time spot; GDNF positive cells expression state of non-ischemia side in pure hypoxic-ischemic and TPM treatment group was similar to the sham operation group. the ischemia cortex GDNF begin to express after HIBD 12 hours, further to rise at the 2nd day and reach the peak in the 3rd day, return to normal at the 5th day on the whole, merely thimbleful GDNF positive cells expression; at the same time, in pretreatment and post-treatment group, it was thus evident GDNF positive cells expression after HIBD 12 hours, further to rise at the 2nd day and reach the peak in the 3th day, although somewhat reduction at the 5th day, still more expression, and obviously super than pure hypoxic-ischemic group each time( P < 0.05), dyeing also deep; but the expression increase in delayed treatment group have no statistical significance to pure hypoxic-ischemic group( P >0.05).4. Western blotting findingsThere were all GDNF expression strap both pure hypoxic-ischemic and TPM treatment group, moreover, the pretreatment and post-treatment group obviously super than pure hypoxic-ischemic group, we applied biogel electrophoresis image analytical system (AlphaImager 2200) to scan and calculate, through calculating protein expression relative score of each group as relative amount, processing statistical treatment, we found the pretreatment and post-treatment group obviously super than pure hypoxic-ischemic group, had statistical significance (P < 0.05) , the pretreatment again super than post-treatment group , also had statistical significance(P<0.05), but the delayed treatment group had no statistical significant to pure hypoxic-ischemic group( P>0.05).Conclusion1. Topiramate has obviously cerebral protective effects not only by relieving cerebral cell edema, cerebral cell liquescence and cerebral cell necrosis caused by ischemia, but also reducing cerebral injury.2. The expression of GDNF have some determinate time series rule, reaching the peak at third days after HIBD, whereas Topiramate treatment can enhance GDNF expression.3. One mechanism of Topiramate brain protection action by making the glial cell excrete GDNF.4. The therapeutic potential window of Topiramate appears to be relatively narrow (<2 h),and the prophylactic treatment was more effective than the rescue treatment... |
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