[The protective effect of mild hypothermia pretreatment against injury to primary cultured cortical neurons induced of rat by glutamate].

OBJECTIVE: To investigate the effect of mild hypothermia preconditioning against ischemia/reperfusion (I/R) injury of cultured primary cortical rats neurons, and to compare the protective effect of mild hypothermia only and with its combination with drugs.

METHODS: Cortical neurons of neonatal Sprague-Dawley (SD) rat within 24 hours after birth were harvested and cultured in vitro. On the 3rd day, the cells were cultured in a medium containing 2.5 mg/L cytosine arabinoside to inhibit the growth of glial cells and fibroblast. Having cultured for 6 days they were randomly divided into blank control group, glutamate damaged group (cultured with 200 μmol/L glutamate for 0.5 hour after washing), mild hypothermia preconditioning group (cultured under 33.5 centigrade for 24 hours before injury induced by glutamate), mild hypothermia combining with edaravone preconditioning group, and the hypothermia combining with propofol preconditioning group (medium containing 100 μmol/L edaravone and 3 mg/L propofol). They were cultured under 33.5 centigrade for 24 hours before injury induced by glutamate. After 24 hours of culturing in various medium, apoptosis ratio was observed by flow cytometry. Cell surviving rate was determined with methyl thiazolyl triazolium (MTT), c-fos protein expression was assayed, and morphologic change of cells with hematoxylin-eosin (HE) staining under the microscope, and ultrastructure changes were observed after uranyl acetate and lead citrate staining under transmission electron microscope.

RESULTS: The apoptosis ratio and c-fos protein in glutamate damaged group were significantly higher than those in blank control group [apoptosis ratio: (9.85±0.76)% vs. (0.94±0.20)%, c-fos: 6.96±0.75 ng/L vs. 1.65±0.59 ng/L, both P<0.01], the cell surviving rate was significantly lower than that in blank control group [(0.20±0.02)% vs. (0.97±0.03)%, P<0.01]. Mild hypothermia preconditioning reversed surviving rate, apoptosis ratio and c-fos protein, and the effect of hypothermia combining with propofol preconditioning was obviously better [cell surviving rate: (0.80±0.04)% vs. (0.20±0.02)%, apoptosis ratio: (2.26±0.54)% vs. (9.85±0.76)%, c-fos: 2.98±0.46 ng/L vs. 6.96±0.75 ng/L, all P<0.01]. The morphology of cortical neurons in blank control group was normal. Most of the cells in glutamate damaged group showed bluish black cytoplasm with pyknic nuclei, with crumpled axons and of them were fractured, and cell number was obviously decreased. In each pre-conditional group, decrease in cell number was unconspicuous, and only a few cells showed apoptosis. Under transmission electron microscope, it was found that cell membrane, mitochondria and rough endoplasmic reticulum were intact in blank control group, but with reduction in organelles, severely swollen mitochondria, even with formation of vacuole or pyknosis, serious dilation of rough endoplasmic reticulum, with loss of cristae with loss of vacuoles or pyknosis, and marked dilatation of internal reticular endoplasm, and loss of cristae with vacuolation and chromatin were observed under electron microscope in glutamate damaged group. Compared with the glutamate damaged group, these pathologic changes were markedly alleviated in protected groups.

CONCLUSIONS: Mild hypothermia preconditioning can inhibit glutamate-induced injury to cortical neurons. The protective effect of mild hypothermia combined with propofol is better.