| ObjectiveFull - term infants with hypoxic - ischemic encephalopathy ( HIE) may die or survive with long - term neurological deficits. It was proved that there existed TUNEL positive cells in ipsilateral cortex, hippocampus, thalamus and striatum following unilateral cerebral hypoxia - ischemia in the neonatal rat. Recent evidence demonstrates that apoptosis involves the activation of caspases , a unique family of structurally related, high conserved , aspartate - specific, cysteine proteases that are necessary to carry out the signal for apoptotic cell death. Since this discovery, more than 14 new caspases have been identified. Activation of Caspase - 3 appears to be a event in initiating PCD. Once it was activated , the apoptosis was destined. Caspase - 3 appears to be an important down stream e-vent after HI in the immature brain, which could be inhibited afterwards. It was the main evidence of differentiation of aopotosis and necrosis whether or not it was activated. Caspase - 3, the caspase that has the highest homology to ced -3, the key step in commiting a cell to PCD. There is evidence that Caspase -3 is expressed, cleaved, and activated after cerebral ischemia. Bel - xL is one of the Bel - 2 family members, and it can protect neurons from apoptosis. The up -regulation of Bel -xL can inhibit the activation of Caspase -3 and followed cascade.The aim is to find the timepoint of neuronal apoptosis , the expression ofCaspase -3mRNA, Caspase - 3 , Bel - xLmRNA, Bel - xL and the effect of bFGF on brain after HIBD.MethodsThe hypoxic - ischemic brain damage ( HIBD) model was produced in the traditional model of neonatal HI which subjected 7 - day - old Wistar rats to the left common carotid artery ligation followed by a hypoxic (8% oxygen, 92%nitrogen) episode of 1. 5 hours. The rat pups were sacrificed in different time-points after HI compared with normal and sham operated rats. Contents as following ; Part one: The timepoint of neuronal apoptosis after HI injuries was examined in neonatal rat using Hematoxylin and Eosin(H and E) staining, transmission electron microscopy, terminal deoxynucleotidyl transferase mediated dUTP - biotin nick - end labeling (TUNEL) staining and flowcytometry ( FCM). Part two: The expression of Caspase -3mRNA, Caspase -3 protein, Bel -xLmRNA and Bel - xL protein in brain of normal neonatal rats and the HIBD model rats were assessed using reverse transcription polymerase chain reaction (RT-PCR) and immuonhistochemistry, respectively. RT-PCR; cDNA was synthesed using reverse transcription ,and then followed polymerase chain reaction. The PCR products (412 for Caspase - 3, 343 for Bel - xL and 528 for GAPDH) were separated on 2% agarose gels , stained with EB, and photographed under UV light. The pictures were scanned, and the bands were quantified using the software CIS - 700D gel image system. The relative amount of Caspase -3mRNA and Bel - xL were calculated after normalization to GAPDH to compensate for errors introduced during the preparation of RNA. Immunohis-tochemical procedures: the brains were paraffin - embedded and cut into 6 - jxm coronal sections. Antigen recovery was performed by boiling the sections in l0mmol sodium citrate buffer(PH 6.0) for 10 min. SABC was applied and incubated for 20 minutes at 37 . Five rats brain tissue slices (2 slices per brain) was observed under 400x lightmicroscopy . The staining density of positive cells was transfomed into quantity using MetaMorph/DP10/Bx 51 Imager System Ver-sion 4.6, and thus we could calculated their average opital density values. Part three: We observed the persistent protective mechanism of bFGF on HIBD and the effect of different methods of bFGF on the expressions of Caspase - 3 protein , Bel - xL protein and DNA fragmentation. The HIBD model was performed according to Rice. The rats were divided into 6 groups: bFGF group 1 (4u g-1, one time daily ,total 3 weeks) , bFGF group2 (4u g-1, one time daily ,the first and the third week) , bFGF group3 (4u g-1,one time daily ,the first week only) ,...
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