| Objective: Cerebrovascular disease (CVD) is commonly and frequently encountered, endangering the people's life and work because of its high mortality and serious disability. Cerebral ischemia accounts for seventy percents of CVD. The mechanism of brain edema formation and secondary neuron injury are not very clear now. Up to now, there is no one kind of mechanisms to explain the damage theorem after cerebral ischemia. Researchers consider that severeal mechanisms participate the process of cerebral infarction: metabolic block, calcium overload, cell apoptosis, the toxic action of excitatory amino acids and free radicle, the nerve damage mediated by nitrogen monoxidum, et al. Hemorrhagic infarction, as named hemorrhagic transformation, is a frequent outcome of ischemic cerebral infarction, which is especially severe after thrombolytic and anticoagulant therapy. However, its specific mechanism ate not very clear. Neuroglobin (Ngb) is a newly discovered protein localized in neurons of the central and peripheral nervous systems in vertebrates. It functions to bind, store, and facilitate the utilization of oxygen in neuronal cells. Recent studies suggest that it may modulate hypoxic and ischemic injury. Ngb can also be induced by hemin in a dose-dependent manner. Binding assay in vitro confirmed the interaction between Ngb and Na~+, K~+-ATPase beta 2 subunit (NKA1b2). It is suggested that hypoxia-induced Ngb could inhibit the Na~+, K~+-ATPase activity to certain extent to protect the neuron from hypoxic injury. The purposes of this study are to study the expression of neuroglobin after middle cerebral artery occlusion (MCAO) and hemorrhagic infarction (HI) in rats and the effect of hemin on ischemic brain tissues.Methods: 200 Sprague-Dawley rats were randomly divided into sham-operated group, MCAO group, HI group and hemin treatment group. Three later groups were divided into 4 subgroups respectively according to 3-, 6-, 12-, and 24h after the animal modal formation. All the rats were tested for neurological behavior and then were sacrificed. The brain water content was calculated as the percentage changes between wet weight and dry weight using the following formula:(WW-DW)/WW×100%. The infarcted tissue volume, neuropathologic changes and immunocytochemical expression of neuroglobin (Ngb) were examined. Ngb mRNA, Na~+, K~+ -ATPaseα1,α3 isoforms mRNA expression were determined by RT-PCR. The method of spectrophotometry was applied to measure the activities of Na~+, K~+-ATPase.Results:1 The content of brain water among different groups: Brain water content peaked at 24 h (85.36±1.23) % in MCAO group and at 12 h (85.28±0.64) % in HI group. In contrast to the MCAO and HI rats, at 24 h the content of brain water in hemin treatment groups was higher than those of the sham group (P<0.01) and lower than the MCAO(P<0.01).2 The infracted volume: At 6 h after ischemic, the infracted tissue volume begin to enlarge and peaked at 24 h. The hemin treatment group shown a significant small volume compared to the MCAO and HI groups (P<0.01). It did not show much obvious changes between the latter groups.3 Changes of Na~+, K~+-ATPase activity: The ouabain- sensitive ATPase activities in the MCAO rats were begin to updown from 3 h to 24 h. It was significantly lower at 3 h (2.809±0.346) U/mgprot in HI rats than those of the MCAO rats (4.565±0.776) U/mgprot, hemin treatment (5.539±1.366) U/mgprot and sham-operated rats (5.612±0.615) U/mgprot (P<0.01). In contrast to the sham-operated rats, the hemin treatment rats did not show much obvious changes.4 Morphometric evaluation of ischemic neuronal damage: There were few inflammatory cells infiltration in the brain of sham group and earlier contral tissue of MCAO, HI and hemin treatment groups. Inflammatory cells became apparent in the ischemic tissue at and 6 h. After then, morphological changes of the neurons became evident in the cerebral cortex, major parts of the cortical showed significant cell edema, but only a few neurons showed irreversible cell necrosis. After 12 h, the cell edema was dramatically and a large amount of neurons showed irreversible cell necrosis. 5 Changes of Ngb expression in the brain: The number of Ngb-IR cells was significantly decreased in MCAO and HI rats than the hemin treatment group. There was a short time over-expression in earlier HI rats. With the time of ischemic, few expression of Ngb could be founded. At 24 h less intense staining was observed in all the groups.6 Expression of Ngb, Na~+, K~+-ATPaseα1,α3 mRNA: We found a significant difference of Ngb mRNA after 6 h in the ischemic groups (P<0.01). After 6 h, a lower level of Ngb mRNA to be shown, while much higher than the MCAO rats. There was a short time over-expression in earlier HI rats (0.518±0.034) and then down-gredulated quickly (0.439±0.114). There was no difference in Na~+, K~+-ATPaseα1 mRNA among MCAO, HI and hemin treatment groups, while a lower level compared to sham-operated rats (P<0.05, P<0.01). For the Na+, K+-ATPaseα3 mRNA, after 6 h a significant higher level in the ischemic cortex of hemin treatment group in contrast to the MCAO rats (P<0.01). After 24 h, the level of Na~+, K~+-ATPaseα3 mRNA was lower as well as the MCAO rats (P<0.05) .Conclusion: The Ngb can be induced by hemin that may probably be beneficial to the protection of focal cerebral ischemia through inducing the expression of Ngb. The change of Na~+, K~+-ATPaseα3 inform expression was similar with the activity of Na~+, K~+-ATPase which may be because of theα3 inform was more sensitive to the ischemic. There was a over-expression of Ngb in HI rats, while the brain edema was gravity and activity of Na~+, K~+-ATPase was lower. |
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