| Objective:Hypoxic-ischemic brain damage is caused by asphyxia in perinatal, and a significant cause of neurological morbidity in infants. In recent years, some studies showed stromal cell derived factor-1(SDF-1) had some protective effect on hypoxic-ischemic brain damage. This experiment have prepared neonatal hypoxic-ischemic brain damage(HIBD) mice model. Through intraperitoneal injection of exogenous recombinant murine SDF-1α,①Detect the degree of brain encephalatrophy by the weight of hemisphere;②The general morphological change of the brain was observed;③The expression of SDF-1 gene in the brain were tested by the method of the Real-Time fluorescent quantitation PCR(RT-Q PCR);④The expression of SDF-1 in the brain were tested with immunohistochemisty;⑤The expression of caspase-3 in the brain were tested by the method of the western blot The protective effect of SDF-1 on hypoxic-ischemic brain damage and its possible molecular mechanism were investigated, our experiment may provide an effective way as clinical therapy for neonatal HIBD.Methods:Seven-day-old mice were randomly divided into three groups: Sham-operated group, NS group, and SDF-1 treatment group, n=30 in each group. Then each group was randomly divided into three subgroups (n=10 in each subgroup) based on different time points at 24h,3d and 7d after HIBD. The NS group and SDF-1 treatment group animal model was constructed according to Rice method, SDF-1 (2.0μg/d) were intraperitoneal injected 30min after HIBD each day for 7 days, Only saline was applied in sham group and NS group. Observation behavior of mice: the turnover ability and dextro-rotatory ability when their tails were gripped were observed before and after hypoxic-ischemia respectively. All mice were decapitated at different time points and brain tissues were collected. Then general morphological change of the brain was observed. And the brains of 7d group were used for the detection of the degree of encephalatrophy first. The expression of SDF-1 gene was examined by RT-PCR methods. The expression of SDF-1 protein in hippohamus was tested by immunohistochemistry. And the expressions of caspase-3 in cerebral hippothmaus were detected by Western Blot method at different time points.Results:1. Behavior change of the experimental miceBefore the experiment all neonatal mice were normal. After experiment sham group were still normal. While out of 60 mice with HI insult,19 mice couldn't turn themselves over,46 mice became dextro-rotatory when their tails were gripped, another 16 mice exhibited both behavior problems.2. General examination of the brainThe sham operation group showed no pathological change. The ligated hemisphere of NS group were badly damaged. They showed obvious pallor and edema at 24h. The volume was larger than the contralateral side, and showed congestion, liquefaction necrosis at 3d, there was obvious encephalatrophy at 7d. While pathological changes of the SDF-1 treatment group mice was significantly reduced compared with same time points NS group.3. Comparison of encephalatrophyThe appearance of sham-operation group was normal, there was no encephalatrophy. The encephalatrophy of the NS group was the lowest [(29.95±2.05)%], showing a significant difference against the sham-operation group[(1.27±0.01)%] and the SDF-1 treatment group[(12.20±1.78)%] (P<0.05); While there were also a significant difference between the SDF-1 therapy group and the sham group (P<0.05)4. Expression of SDF-1 in the brainSDF-1 was weakly expressed in the brain tissue of sham-operation group mice, but increased obviously in the NS group. After HI insult, the expression of SDF-1 began to increase at 24h, and reached a peak at 3d, then gradually decreased, but still maintained the high level at 7d, which had a significant difference against the sham group (P<0.05); In the SDF-1 treatment group, SDF-1 was contionus highly expressed, which had a significant difference against the NS group (P<0.05)5. Caspase-3 protein expression in the brainThere was only weakly expression in the sham group. The expression of caspase-3 in NS group reached a peak after HIBD 24h, then gradually decreased, it was still higher than sham group till 7d after HIBD, while the level of caspase-3 in SDF-1 treatment group was decreased significantly at different time points (P<0.05)Conclusion:1. The HIBD model was successfully established.2. The expression of SDF-1 in the brain tissue of neonatal mice after hypoxic-ischemic brain injury was significantly increased, first gradually increased, then decreased gradually.3. Exogenous SDF-1 can pass the blood brain barrier, protect the brain tissue, and stimulate the expression of SDF-1 mRNA in the lesioned region.4. Exogenous SDF-1 treament can alleviate encephalatrophy following HI brain injury.5. SDF-1 can down-regulate the expression of caspase-3 of brain tissues. It may inhibit neuronal apoptosis and provide a protective effect for hypoxia-ischemia induced neuronal injury of neonatal mice. Further, our conclusions may provide new path for clinical treatment of newborns with HIBD. |
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