Effect Of Hemichannel Opening On Hippocampus Neuron Viability In Ischemia And Reperfusion

Ischemic stroke, a severe disease with high incidence, high death rate and high mutilation rate, is increasingly threatening human health nowadays. It stems from a transient or permanent reduction in cerebral blood supply, which initiates cascade reactions, including over-activity of excitatory amino acid, intracellular calcium overload, production of free radical, inflammatory reactions and initiation of apoptosis and so on, which eventually lead to cerebral damage. Among them, dysregulation of cellular calcium homeostasis, viewed as a key event during ischemic damage, always attract wide interest to study on its mechanism. Recently, active hemichannels beyond gap junctions were introduced to such studies, and evidence of their opening in ischemic neurons has been reported, which is supposed to provide a new clue for exploring the mechanism of neuron ischemic damage.In present study, we observed a leakage of an inactive fluorescent dye for live cells, calcein green, from neuron cytoplasma to extracellular space during oxygen-glucose deprivation (OGD) in vitro, which was recorded through laser scanning confocal microscopy (LSCM). Our results offered support to the newly proposed opening of hemichannels in ischemic neurons. Besides, we analyzed the temporal feature of hemichannel opening induced by OGD, and found that the incidence of hemichannels opening was low during the early stage of OGD, and then increased significantly.Furthermore, we carried out exploratory study on the status of hemichannels during reperfusion, which has not been reported yet. Our results showed an even greater leakage rate during reperfusion, especially during the early time of reperfusion, which indicated a dramatic amount of hemichannels opening.Then we studied the effect of opening of hemichannels on the viability of neurons suffered from ischemia-reperfusion insult. It turned out that the specific inhibitor of hemichannels lowered the degree of ischemia-reperfusion damage with significance, indicating that the opening of hemichannels contributes to the damage to some extent. At last, we studied the effect of inhibition of hemichannels on intracellular calcium homeostasis, using LSCM and Ca²⁺ fluorescent prober. We saw effective attenuation of calcium overload during ischemia and reperfusion by applying hemichannel inhibitor. In addition, we compared major calcium channels on plasma membrane, which were supposed to open, in terms of contribution to the loss of calcium homeostasis during ischemia. We found the effect of hemichannels' opening on intracellular calcium homeostasis may only next to that of NMDA receptors, but significantly above that of voltage-operated Ca²⁺ channels, and comparatively increasing with time.