Experiment On The Change Of Calcium And Injury In Cultured Mice Cortex Neurons Under Ischemia-reperfusion Conditioning

Ischemic and the following reperfusional injury in neurons accompanied by the increase of intracellular calcium are the two major causes for ischemic brain damage. Researchers are interested in the change of intracellular calcium in neurons for it may be the result of different agents or the final common road to ischemic brain damage by which various agents manifest their neuronal toxicity through rising calcium. Although the enhancement of intracellular calcium is not thought to be necessary for ischemic brain damage, it properly is one of the important activator for neuron ischemic injury. In our experiments, confocal laser scanning microscope, transmission electron microscope, fluorescence microscope, and stereomicroscope were utilized. With Ca"1"1" channel antagonist being interferencing factor, immuno-fluorescence, terminal deoxynucleotidyl transferase mediated nick end labeling, confocal laser scanning microscope, MTT assay and transmission electron microscope were adopted to investigate the change of intracellular calcium, neuron viability and-6-neuron ultrastructure. Therefore, we provided a simple model and some basic facts for the research on the mechanism of ischemic brain damage.We established neuron ischemic and ischemia-reperfusion model in vitro via neuron primary culture. Dividing the identified mature neurons as normal, ischemia-reperfusion group, treatment group, we observed the change of the intracellular calcium, cell viability, ultrastructure, and apoptosis of different groups. The results were showed as the following:(1) The purity of the neurons we cultured is about 83.4+2.0%, above the level required for primary neuronal culture (>80%).(2) At 30min, Ih, 2h after ischemia-reperfusion, the intracellular calcium concentration in neuron was higher than that in treatment group (P<0.01), while the A value(MTT assay) was much lower.(3) Neuronal damage developed at 30min, Ih, 2h, 3h following ischemia-reperfusion at the level of ultrastructure. At the early stage, the cell swelled for the intumescence of mitochondrion, Golgi apparatus, and endoplasmic reticulum, cell processes shrinked, microvillus disappeared, the cell edge smoothened, followed by vacuolation, karyopyknosis, and the appearance of autophagolysosome and apoptotic body. The later stage was dominanted by naked nucleus, karyorrhexis, and karyolysis, and it is interesting that damage was less in Flunarizine treatment group.(4) The number of apoptotic neurons continuously increased-7-at 30min, Ih, 2h after ischemia-reperfusion, ultimated at 2h. Viewed under stereomicroscopy, cell body swelled, took a round or oval shape, processes shortened or disappeared. The number of apoptotic cells is remarkably higher than that of Flunarizine group accordingly(P<0.01).The following is the conclusion of our experiments:(1) Pathological change of neurons ischemia-like conditioned and reperfused in vitro is similar to that of in vivo, thus providing an ideal model for the research on ischemic brain injury.(2) The concentration of intracellular calcium in neurons raised continuously at different time points (SOmin, Ih, 2h) after ischemia-reperfusion and the cell viability reduced with the reperfusion time passed on. Flunarizine demonstrated remarkably inhibitory effects for the up-lift of calcium level at each time point, and some protect effects on the reperfusion damage.(3) Neuronal damage aggravated following 30min, Ih, 2h, 3h of ischemia-reperfusion, and the major phenotype displayed is the change of ultrastructure. Flunarizine can attenuate this change to some extent.(4) The number of apoptotic neurons continuously increased after 30min, Ih, 2h of ischemia-reperfusion, ultimated at 2h. Flunarizine has strong inhibitory effects on the apoptosis of ischemia-reperfused neurons.