Activation And Regulation Of Cerebrovascular Nuclear Factor-κB After Subarachnoid Hemorrhage

Spontaneous subarachnoid hemorrhage (SAH; all the following SAHs in this thesis referrs to spontaneous SAH) is mainly caused by the rupture of intracranial aneurysms. SAH is a common cause for death, disability in clinic, which accounts for 5% of all stroke patients. In general population, the incidence of SAH is about 8/1, 000,00 people. Despite considerable advances in diagnostic, surgical, and anesthetic techniques and perioperative management, the outcome of patients with SAH remains unsatisfied. Previous studies showed that the mortality of SAH is 45%, About one third of the survivors have a higher disability rate, who can not take care of themselves in daily life. Beside the abrupt onset lack of effective treatment is another major reason for high mortality and disability rate in SAH patients. The limitation in the understanding of SAH pathophysiology accounts for the lack of treatment.Cerebral vasospasm is regarded as the main cause of death and disability in SAH patients. Cerebral vasospasm can affect all cerebral arteries but principally the large arteries located around the ruptured aneurysm. At times the arterial narrowing can result in dramatic multifocal cerebral ischemia, a chief cause of unfavorable clinical outcomes after SAH. Despite the efforts of the medical and scientific communities, cerebral vasospasm remains a fearsome syndrome that can be prevented but rarely cured, which may due to the limitation in knowledge about the pathophysiology of cerebral vasospasm.Increasing evidence indicated that inflammation might play a critical role in the development of cerebral vasospasm. Clinical and experimental studies have shown the increased levels of the cytokines and endothelial adhesion molecules in the cerebrospinal fluid (CSF) and serum after SAH. Nuclear factor-κB (NF-κB) is a group of inducible dimeric transcription factors. They are composed of DNA-binding proteins (Rel) that recognize a common sequence motif on the NF-KB-regulated genes. It has been demonstrated that NF-κB plays a key role in regulating the expression of a wide variety of genes involved in the inflammatory responses. NF-κB could activate the genes coding cytokines, chemokines, and adhesion molecules. Therefore, the inflammatory responses in many diseases are modulated by NF-κB pathway. It was reported that NF-κB pathway could be activated in the cerebral ischemia and traumatic brain injury. Hence, NF-κB might be very important in the development of cerebral vasospasm and might be a therapeutic target for cerebral vasospasm regarding inflammation. The previous studies have demonstrated that in animal SAH model. However, there are no systemic studies on the effects of the main spasmogen of cerebral vasospasm after SAH-oxyhemoglobin (OxyHb)-on the activation of NF-κB in the cerebrovascular smooth muscle cells. Hence, one part of this thesis is studying the effect of different concentrations of OxyHb on DNA binding acitivity in the cultured vascular smooth muscle cells (VSMCs). Erythropoietin (EPO) can attenuate cerebral vasospasm after SAH both in clinical and in laboratory studies. However, the mechanisms remain obscure. Based on the first part of this thesis, the regulating of NF-κB by EPO and its effects on the cerebral vasospasm in SAH rabbit model were demonstrated.In conclusion, this study demonstrated the activation of NF-κB in cultured VSMCs by OxyHb. It was confirmed that the NF-κB was actived in smooth muscle cells by OxyHb and activation of NF-κB plays a role in development of cerebral vasospasm. And then, the study on the regulation of NF-κB by EPO in SAH model was conducted. It was showed that EPO can down-regulate the activation of NF-κB after SAH and attenuate cerebral vasospasm ultimately.PartⅠ:Effect of OxyHb on the DNA-binding activity of NF-κB in cultured vascular smooth muscle cellsOBJECTIVE:To investigate the effect of OxyHb on the cell density and DNA-binding activity of NF-κB in the cultured vascular smooth muscle cells(VSMCs). To test the hypothesis that OxyHb can effect the morphology and can activate NF-κB, both of which can induce cerebral vasospasm.METHODS:The cultured VSMCs were treated with different concentration from 1 to 100μmol/L of OxyHb. The cell density was measured. DNA-binding activity of NF-κB was detected using electrophoretic mobility shift assay (EMSA).RESULTS:Morphological alterations occurred in the VSMCs after treatment with OxyHb. The concentration of OxyHb at 100μmol/L produced a significant decrease in cell density at 24 hour, and 10μmol/L at 72 hour. DNA-binding activity of NF-κB was increased after treatment with OxyHb, and peaked at 10 and 50μmol/L.CONCLUSION:Alteration of NF-κB DNA-binding activity occurred in the VSMCs treating with OxyHb, which suggests that NF-κB pathway in the cerebrovascular smooth muscle cells may be involved in the pathogenesis of cerebral vasospasm.PartⅡ:Effects of EPO on cerebral vasospasm and regulation of NF-kB in the rabbit basilar artery after SAHObjective:To investigate the efficacy of EPO in delayed cerebral vasospasm following SAH and its regulatory effect on the espression of NF-κB in rabbits.Methods:A total of 60 male New Zealand white rabbits were selected, and they were randomly allocated into control, SAH,and EPO groups (n= 20 in each group). Delayed cerebral vasospasm was induced by using a SAH model of injecting autologous blood into the cisterna magna. The dose of EPO was 1000 IU/(kg.8h), and ti was injected intraperitoneally for 5 days. The animals were killed 5 days after the model established. Their basilar arteries were obtained and the crosssection area was detected by HE staining. The NF-κB binding activity was detected by electrophoretic mobility shift assay (EMSA).Results:①The lumen's cross-sectional area of basilar arterial in the control, SAH and EPO groups were 0.420±0.008,0.225±0.025 and 0.353±0.085 mm2, respectively. There was significant difference between the SAH and control groups (P < 0.01). and between the EPO and the SAH groups(P< 0.01).There was also significant difference between the EPO and control groups(P< 0.05).②The gray values of NF-κB activity in the 3 groups were 1.180±0.079,9.350±0.691 and 6.220±0.583, respectively. There were significant difference between the SAH and control groups, and between the EPO, control and SAH groups (P< 0.01).Conclusion:Cerebrovascular NF-κB was activated after SAH in rabbit double-hemorrhage SAH model. EPO can attenuate the degree of delayed cerebral vasospasm after SAH by inhibiting the activation of NF-κB in cerebral arteries after SAH. This series of studies demonstrated the role of the main spasmogen for cerebral vasospasm, OxyHb, in activation of NF-κB, The results indicated that OxyHb can induce cerebral vasospasm by upregulation of NF-κB. On the other hand, it was proved that EPO can inhibit the activation of NF-κB and can attenuate the degree of cerebral vasospasm ultimately in rabbit double-hemorrhage SAH model, which showed the role of NF-κB in development of cerebral vasospasm and the beneficial effects of EPO on cerebral vasospasm. This series of studies make it possible to investigate the further mechanism about the relationship between cerebral vasospasm and NF-κB.