The Neuroprotective Effects And Mechanisms Of L-type Calcium Channel Agonist On Ischemic Neuronal Death

Stroke,a cerebrovascular accident,is the third leading cause of death in the Western world.The disease remains a major cause of disability in the United States. In practice,stroke refers to an umbrella of conditions caused by the occlusion of or hemorrhage from blood vessels supplying the brain.Most often,blood flow is compromised within the territory of an occluded blood vessel.Less commonly,stroke results from the absence of blood flow to the entire brain due to cardiac arrest.In all instances,stroke ultimately involves death and dysfunction of brain cells,and neurological deficits that reflect the location and size of the compromised brain area.There are at least three hypotheses of fundamental mechanisms leading to cell death during ischemic brain injury:excitotoxicity and ionic imbalance, oxidative/nitrosative stress,and apoptototic cell death.These mechanisms demonstrate overlapping and redundant features.They mediate injury within neurons, glia and vascular elements,and at the subcellular level,they impair the functions of mitochondria,nuclei,cell membranes,endoplasmic reticula and lysosomes.Cell bodies and their processes and synaptic endings are all at risk,and cell death might proceed by mechanisms promoting rupture,lysis,phagocytosis or involution and shrinkage.Till now,however,the mechanism of cell death during ischemic brain injury is not clear,and the effective treatment is still lacking.Therefore,it is very important to study the mechanisms underlying neuronal death and explore new therapeutic targets.The theory of calcium overload has always been depicted as the major mechanism of ischemic neuronal death,and many studies have shown that the concentration of intracellular calcium was increased in the early period of ischemia and reperfusion.At present,during the process of apoptosis,the role of Ca²⁺ homeostasis is not clear.In sympathetic neurons,the concentration of intracellular calcium for inhibition of apoptosis is much lower（180-240 nM） than that for toxicity with overloading Ca²⁺ levels.For example,glutamate toxicity can raise the concentration of intracellular calcium greater than 5μM.Therefore,the hypothesis of "calcium set point" calls that there are three different concentrations of intracellular calcium.With a lower concentration of intracellular calcium,neurons will have a risk of apoptosis:at medium concentration of intracellular calcium,neurons will easily survive and cytotoxicity with the high calcium concentration will induce easy neuronal death.After ischemia and reperfusion,hippocampal CA1 neurons showed a delayed neuronal death,DND.and the CA3 neurons were almost not affected.Recently one of our researches showed that in animal models of forebrain or global ischemia and reperfusion,the currents mediated by L-type calcium channel was decreased,24 h after ischemia/reperfusion,in the CA1 region of the hippocampus but not in CA3.It suggested that the dysfunction of L type calcium channels may be involved in the mechanism of DND in the hippocampal CA1 region.In this study,we established a model for transient global cerebral ischemia via four-vessel occlusion,a modified Pulsinelli method in vivo.Also by the method of oxygen glucose deprivation,we established a hypoxia model in cultured primary hippocampal neurons.We observed the neuroprotective effects of L type calcium channel agonists following ischemic neuronal death and the signal transduction mechanism.With 180～200 g male Wistar rats,through improved Pulsinelli four-vessel occlusion method,we established a transient global cerebral ischemia model,and buried an osmotic pump in the lateral ventricle by a three-dimensional position reference.BayK 8644 was delivered by the osmotic pump.The results showed that compared to the control ischemic group,12 h and 24 h after ischemia/reperfusion, 0.2mM of BayK 8644 significantly reduced neuronal death（P＜0.05）.Treatment with 0.1mM of BayK 8644 at 12 h after ischemia/reperfusion could not produce neuroprotection（P＞0.05）.In hippocampal CA1 region,the number of neurons that survived is 35.94±11.8 / mm² in the control ischemic group.In groups treated with 0.2mM of BayK 8644 at 12 h and 24h after ischemia/reperfusion,the number survived neurons in the hippocampal CA1 region was 145.12±21.0 / mm²,and 120.94±27.9 / mm² respectively.In the group treated with 0.1 mM of BayK 8644 at 12 after ischemia/reperfusion,the number was 58.45±25.1 / mm².At the same time, we adopted Morris water maze to detect the effect of BayK 8644 on spatial learning and memory in rats after ischemia/reperfusion.Results showed that compared to the control ischemic group,those treated with 0.2mM of BayK 8644 at 12h after ischemia/reperfusion had an improved spatial learning and memory,and this is not due to an improve in exercise capacity.These results suggest that in vivo BayK 8644 has a protective effect on neurons following ischemia in hippocampal CA1 region.Next,we studied whether the protective target is neurons,in primary hippocampal 12 days cultured neurons,which were deprived of oxygen and glucose for 4 h followed by culture for 24 h at normoxic conditions.More than 30 percent of neuronal death was induced by the model of hypoxia/reoxygenation.Administration of BayK 8644（0.5μM） at 1h.3h,6h,12h after reoxygenation,respectively,could protect the neuronal death induced by hypoxia/reoxygenation（P＜0.05）.（42±0.8%versus 21±1%,24±2%,22±1%, 19±0.7%）.However,to further demonstrate that the neuroprotective effect of BayK 8644 was mediated by an activation of the L-type calcium channel,in cultured hippocampal neurons,treated with glucose/oxygen deprivation,nifedipine or nimodipine and BayK8644 was coadministered.The neuroprotective effect of BayK 8644 was blocked.The number of dead cells was 15±1%versus 40±1%,39±1% with BayK8644 administered singly to that of coadministration of nifedipine or nimodipine（10μM） and BayK8644.Many reports have found that the signal transduction mediated by MAPK has a relationship with cell survival and apoptosis.The cascade reactions of ERK / MAPK are involved in the growth factors of mitogen-regulation,cell proliferation and differentiation.Some stress stimulants,such as ultraviolet radiation.X-ray,heat shock,osmotic shock and some of the proinflammatory cytokines such as TNF （tumor necrosis factor）,IL-1（interleukin-1）,will enhance the activity of P38 and JNK kinase.So,this study further detected the changes in the activities of P38,JNK,ERK kinase,after oxygen glucose deprivation,by Western Blot in cultured hippocampal neurons.The results showed that 30 min alter reoxygenation,the phosphorylation of P38 and JNK were significantly enhanced,and were restored to normal 1 h after reoxygenation.The phosphorylation of ERK remained decreased throughout the 24 h time period after reoxygenation.Western Blot showed that the phosphorylation of ERK kinase can be increased by treatment with BayK 8644（0.5μM） at 12 h after reoxygenation.These results suggest that the neuroprotective effect of the agonists of the L-type calcium channel,BayK8644,on ischemic neuronal death might be due to an enhancement in the activity of ERK kinase.So in the cultured hippocampal oxygen glucose deprived model of neurons,BayK8644（0.5μM） and the MEK inhibitor U0126（10μM） were coadministered at 12 h after reoxygenation.The results show that the neuroprotective effect of BayK8644 on ischemic neuronal death was completely blocked by this treatment.（P＜0.05）.（19±0.7%versus 46±2%）.In conclusion,administration of L-type calcium channel agonist BayK8644 at 12h of reperfution protects ischemia-induced neuronal death in CA1 hippocampus in part by ERK activation.Moreover,Behavioral test in Morris water maze suggests that the survived CA1 neurons remain normal function.