Studies on the Role of Nuclear Factor-κB in Cerebral Stroke

Abstract

Stroke is a cerebrovascular accident that occurs due to substantially reduced cerebral blood flow. Cerebral stroke, which can result from interruption in blood supply to the brain even for a short duration, underlines the vulnerability of the central nervous system to glucose and oxygen deficiency. Such an interruption in blood supply can occur due to presence of a thrombus or an embolus clogging the blood vessel (ischemic stroke) or due to bleeding (haemorrhagic stroke). Stroke is the leading cause of death and disability as its symptoms are general and easily misdiagnosed and no suitable treatment is commonly available. The only FDA approved drug, tissue plasminogen activator, is to be administered within three hours of onset of stroke. Moreover, prolonged ischemic episodes result in irreversible tissue damage and few patients recover fully. NF-κB is a transcription factor that participates in the signalling pathways involved in development, immunity, survival, inflammation and apoptosis. It is composed of a dimeric combination of any of the several NF-κB proteins which, on activation, translocate into the nucleus and in most cases, activate gene transcription after binding to specific kappa B sequences on promoters of specific genes. As inflammation and cell death are central in ischemic pathophysiology, the role of NF-κB was probed in I/R mediated injury. Initial studies demonstrated the successful induction of the transient focal cerebral ischemia in rat model of MCAO as assessed by development of ND and progressively spreading infarction with increasing reperfusion periods, which was delineated by TTC staining of ischemic rat brain sections. NF-κB activation in I/R injury was subsequently investigated by studying nuclear translocation of NF-κB proteins, viz., p65, p50 and c-rel and the changes in the expression of IκBα as well as its phosphorylation status. An increase in the nuclear p65 in the striatum and increased phosphorylation of IκBα along with an increase in the ratio of phospho-IκBα and IκBα indicate activation of NF-κB in course of reperfusion after the ischemic insult. NF-κB activation was further confirmed by the results of p65 DNA binding activity that showed an increase in DNA binding induced by I/R injury. Also, PDTC, an NF-κB inhibitor and an antioxidant, reduced the infarct size at 24 hr. post reperfusion when administered at the onset of reperfusion. Therefore, to further confirm activation of NF-κB in course of ischemic injury as well as to determine its role, specific peptide inhibitors of NF-κB were used in the study. Pre-treatment of the inhibitor peptide SN50 resulted in decrease in phopho-JNK, a mediator in apoptotic signaling, in the striatum. Furthermore, a decreasing trend in the number of cells exhibiting DNA fragmentation was observed by TUNEL staining in cortex and striatum of SN50 treated rats. The NF-κB inhibitor peptide NBD did not affect the phosphorylation of JNK or cleaved spectrin levels significantly. However, the number of cells possessing fragmented DNA decreased significantly in cortex and striatum of IKK-NBD treated rats besides a decrease in the proinflammatory cytokine IL-1β in IKK-NBD treated rats. The peptide also reduced the blood brain barrier permeability following I/R injury. These findings indicate that cerebral NF-κB inhibition by IKK-NBD provides significant neuroprotection after I/R injury. Therefore, this protective effect, observed in terms of reduced cell death as inferred by DNA fragmentation, may be mediated through modulation of multiple signaling mechanisms including apoptotic/necrotic and inflammatory pathways. Another possible inference that can be drawn from this study is that inhibition of NF-κB probably has no significant effect on the generation of oxidative stress. Free radical generation during ischemia is not altered by IKK-NBD; moreover, SOD2 expression is not affected by SN50 or IKK-NBD. Hyperglycaemia is a strong predisposing factor for stroke. Therefore, the involvement of NF-κB in ischemic pathophysiology in hyperglycaemic rats was studied. Initial experiments indicated increased I/R induced damage in hyperglycaemic rats as compared to that occurring in normoglycaemic rats. This is evidenced by increased ND and early onset of infarction as well as more pronounced infarction in hyperglycaemic rats. NF-κB activation assessed in hyperglycaemic ischemic rats revealed decrease in NF-κB p65 in the nucleus as well as in the cytoplasm, indicating that the overall pattern of NF-κB p65 expression was decreased. The NF-κB inhibitor IκBα was also decreased in hyperglycaemic rats. But p65 DNA binding was not affected in these rats. The relevance of NF-κB in hyperglycaemic transient focal ischemic injury was further investigated by using different NF-κB inhibitors. However, pretreatment with the NF- κB peptide inhibitors SN50 and IKK-NBD as well as by the proteasomal inhibitor mg132 did not result in significant alteration in DNA fragmentation in the hyperglycaemic ischemic rats as assessed by TUNEL staining. Therefore, these results indicate that NF-κB may not have a prominent role in I/R mediated cerebral damage in hyperglycaemic rats.