| Stroke is the second most common cause of death and the leading cause of long-term disability worldwide in adults. Because of its huge socioeconomic burden, basic and clinical researches on the pathogenic mechanisms and therapeutic strategies of brain stroke are remarkably increased. Stroke can be subdivided into two categories, ischemic and hemorrhagic stroke. Ischemic stroke, which results from a thrombotic occlusion of a cerebral artery (often middle cerebral artery, MCA) or its branches, accounts for approximately 70~80% of all strokes. The occlusion of an artery supplying a specific territory of the brain tissue results in cerebral ischemia. Brain injury produced by cerebral ischemia depends on the duration and severity of the cerebral blood flow (CBF) reduction. Ischemic brain injury develops from a complex series of pathophysiological events that evolve in time and space, which are involved in excitotoxicity, ion homeostasis disturbance, peri-infarct depolarizations, oxygen free radical injury, inflammatory reaction, blood-brain barrier (BBB) breakdown and apoptosis. Cerebral ischemia injury can be separated into three serial stages: acute stage (minutes to hours), subacute stage (hours to days) and chronic stage (days to months). Ischemic brain tissue can be separated into two regions: ischemic center region and peri-ischemia region. Cerebral blood flow is most severely reduced in a central region of brain (infarct core) and gradually increased in a graded fashion centrifugally from the core (peri-infarct zone or"ischemic penumbra"). Within the infarct core, necrotic cell death occurs within minutes. In the penumbral areas, collateral arterial blood flow can compensate the detrimental effects of ischemic stroke, then the degree of ischemia and the timing of reperfusion determine the outcome of individual cells. Cerebral ischemia injury possesses two major potential therapeutic strategies: thrombolytic therapy (TT) and neuroprotective therapy (NT). Thrombolysis might be effective for acute ischemic stroke within a 3 h therapeutic time window. However, only 1~8.5% of patients could receive thrombolytic therapy mainly due to the short time window for administration. In animal models of ischemic stroke, many potential neuroprotective strategies targeted at different harmful mechanisms in ischemic brain injury have been explored before. In the present study, we first explored the neuroprotective effect of cerebral ischemic preconditioning (IPC) in a rat model of permanent MCAO (pMCAO). Then, we further investigated the potential neuroprotective mechanisms underlying cerebral IPC against the pMCAO in rats.Part I: Exploration on the best ischemic preconditioning program from several transient cerebral ischemic preconditioning strategies against cerebral ischemia injury in rats.Objective: To explore the best cerebral IPC program against cerebral ischemia injury in rats.Methods: In the first group, to observe the histopathological characteristic produced by various IPC programs, adult male Sprague-Dawley (SD) rats underwent different cerebral IPC by occluding bilateral common carotid artery (CCA) involved repeatedly transient cerebral ischemia with a 15 min-intermission , which including sham IPC, IPC 1×5 min, IPC 2×5 min, IPC 3×5 min and IPC 1×15 min groups. Then, 2,3,5-triphenyltetrazolium chloride (TTC) staining, hematoxylin and eosin (HE) staining were performed 72 h after simple IPC. In the second group, to explore the best IPC program against ischemic brain injury, SD rats received various sublethal IPC programs, and then underwent lethal permanent middle cerebral artery occlusion (pMCAO) after 48 hours'reperfusion. Neurological deficit scores (NDS) and cerebral infarct volume were measured 24 h after lethal ischemia. Results: Neuronal injury in frontotemporal cortex and striatum were observed in cerebral IPC 1×15 min and IPC 3×5 min groups, which was not obvious in sham IPC, IPC 1×5 min or IPC 2×5 min group. All IPC groups but not sham IPC group significantly reduced cerebral infarct volume. Among the all groups, IPC 2×5 min group exerts the most enormous neuroprotection against the subsequent pMCAO in rats.Conclusion: Experimental results suggested that cerebral transient IPC possessed a neuroprotective effect in a rat model of permanent cerebral ischemia. And in the present study, the IPC 2×5 min might be the best program in all the designed IPC groups.Part II: Neuroprotective effect of IPC against pMCAO in rats: involvement of PI3-K/Akt and ERK1/2 signaling pathways.Objective: To study the pathological changes and time-couse expression of Akt and ERK1/2 signaling after pMCAO in rats and further investigate whether PI3-K/Akt and ERK1/2 signaling mediates the IPC's neuroprotection against pMCAO in rats.Methods: In the first group, adult male SD rats underwent simple pMCAO model. NDS, cerebral infarct volume, morphological characteristic, immunohistochemistry (IHC) and western blot were performed at 6, 12, 24, 48 and 72 h after simple pMCAO. In the second group, to investigate whether PI3-K/Akt and ERK1/2 signaling mediates the IPC's neuroprotection, LY294002 (Akt inhibitor) or PD98059 (ERK1/2 inhibitor) was injected intracerebroventricularly to inhibit PI3-K/Akt or ERK1/2, then SD rats underwent sublethal IPC 2×5 min and lethal pMCAO, NDS and cerebral infarct volume were measured 24 h after lethal pMCAO. Expression of Akt and ERK1/2 in ischemic rat brains were determined by western blot.Results: Neuronal injury or death were observed in ischemic rat brains after pMCAO. Ischemic brain tissue was separated into infarct core and peri-infarct zone. NDS and infarct volume were significantly increased after pMCAO, with a peak at 24 h. The protein expression of p-Akt was increased at 6-12 h, and p-ERK1/2 was increased only at 6 h after pMCAO, then both of them decreased at 24-72 h. Expression of t-Akt and t-ERK1/2 were not significantly different. IPC reduced ischemia-induced cerebral infarct volume, which was abolished by LY294002, PD98059 or their mix. Ischemia-induced low expression for p-Akt and p-ERK1/2 were reinforced by IPC, which were abolished by LY294002, PD98059 or their mix.Conclusion: These results suggested that p-Akt and p-ERK1/2 were activated at early stage and inhibited at delayed stage after cerebral ischemia. Moreover, PI3-K/Akt and ERK1/2 signaling pathways mediated the IPC's neuroprotection in rats of pMCAO.Part III: IPC provides neuroprotection against ischemic brain injury by inhibiting inflammatory reaction through PI3-K/Akt and ERK1/2 signaling pathways.Objective: To explore the spatio-temporal distribution of inflammatory reaction and the time-course expression of inflammatory mediators in rat brain after pMCAO and further investigate whether ischemic preconditioning reduced pMCAO-induced inflammatory reaction through PI3-K/Akt and ERK1/2 signaling pathways.Methods: In the first group, to explore the spatio-temporal distribution of inflammatory reaction after cerebral ischemia, adult male SD rats underwent simple pMCAO model for 6, 12, 24, 48, 72 h. Myeloperoxidase (MPO) assay was performed to indicate the inflammatory extent. Inflammatory mediators including neuclear factor-kappaB (NF-κB), cyclooxygenase-2 (COX-2) were determined by western blot, and serum tumor necrosis factor-α(TNF-α) was examined by ELISA. In the second group, SD rats received intracerebroventricular injection of LY294002 or PD98059, then underwent sublethal IPC 2×5 min and lethal pMCAO, MPO and the inflammatory mediators were measured 24 h after lethal pMCAO.Results: Inflammatory marker MPO was increased 6 h after pMCAO, with a peak at 24 h. Expression of NF-κB, COX-2 and TNF-αwere increased after pMCAO, peaked at 24 h, 12 h and 12 h respectively. Elevated inflammation and the inflammatory mediators were attenuated by IPC, however, both of which were abolished by LY294002 or PD98059 at 24 h after pMCAO.Conclusion: Experimental results suggest that cerebral ischemia resulted in an increase of inflammatory reaction, and the underlying inflammatory mediators were also increased in rats. Moreover, IPC provides neuroprotection against ischemic brain injury by inhibiting inflammatory reaction through PI3-K/Akt and ERK1/2 signaling pathway.Part IV: IPC provides neuroprotection against ischemic brain injury by inhibiting neuronal apoptosis through PI3-K/Akt and ERK1/2 signaling pathways.Objective: To explore the spatio-temporal distribution of neuronal apoptosis and the time-course release of mitochondrial cytochrome c and activation of caspase-3 in ischemic rat brain after pMCAO, and further investigate whether ischemic preconditioning reduced neuronal apoptosis through PI3-K/Akt and ERK1/2 signaling pathways.Methods: In the first group, to explore the spatio-temporal distribution of neuronal apoptosis after cerebral ischemia, adult male SD rats underwent simple pMCAO model for 6, 12, 24, 48, 72 h. Neuronal apoptosis was detected by the method of terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining. Mitochondrial cytochrome c, cytosolic cytochrome c and pro-caspase-3 were determined by western blot. In the second group, SD rats received intracerebroventricular injection of LY294002 or PD98059, then underwent sublethal IPC 2×5 min and lethal pMCAO, TUNEL and western blot were performed 72 h after lethal pMCAO.Results: TUNEL-positive cells were increased 6 h after pMCAO, with a peak at 72 h, and dominantly in peri-infarct zone. Expression of cytochrome c was decreased in mitochondria and increased in cytosol, and expression of pro-caspase-3 was decreased at 24-72 h. Neuronal apoptosis and expression of cytosolic cytochrome c were attenuated by IPC, and expression of mitochondrial cytochrome c and pro-caspase-3 were increased by IPC, however, all of which were abolished by LY294002 or PD98059 at 72 h after pMCAO.Conclusion: Experimental results suggest that cerebral ischemia induces neuronal apoptosis, the release of mitochondrial cytochrome c into cytosol and the activation of caspase-3. Moreover, IPC provides neuroprotection against ischemic brain injury by inhibiting neuronal apoptosis through PI3-K/Akt and ERK1/2 signaling pathways. |
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