The Effect of Therapeutic Mild Hypothermia on Brain Microvascular Endothelial Cells During Ischemia–Reperfusion Injury
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To determine the cerebral protective effects of mild hypothermia (MH) on cerebral microcirculation.
We established ischemia–reperfusion (I/R) injury and MH treatment models with rat brain microvascular endothelial cells (RBMECs) in vitro and examined the apoptotic changes. The cultured RBMECs were randomly divided into the control group, I/R group, and MH group, which was further divided into two subgroups: intra-ischemia hypothermia (IIH) and post-ischemia hypothermia (PIH). Cell morphological changes were assessed using fluorescence microscopy. Apoptotic rates were obtained by flow cytometry. Expressions of caspase-3, Bax, and Bcl-2 were analyzed by Western blot.
I/R injury in vitro induced apoptosis of RBMECs. The apoptotic rates in the control group, I/R group, and MH group were 0.13, 19.04, and 13.13%, respectively (P < 0.01). Compared with the I/R group, the MH group showed a significant decrease in the number of apoptotic cells, mainly in stage I apoptotic cells (P < 0.0083). The caspase-3 and Bax expressions were significantly enhanced (P < 0.05) in RBMECs after I/R injury, while substantial decreases in Bcl-2 expression were noted (P < 0.05). Following MH intervention, the increase in caspase-3 and Bax expression was suppressed (P < 0.05), while Bcl-2 expression significantly increased. The apoptotic rates or protein expressions between the two subgroups were not different significantly (P > 0.05).
These results indicate that MH could inhibit RBMEC apoptosis by preventing pro-apoptotic cells and early apoptotic cells from progressing to intermediate and advanced stages. This may be due to the effect of MH on I/R-induced apoptotic gene expression changes.
KeywordsCardiopulmonary resuscitation Cerebral microcirculation Brain microvascular endothelial cells Ischemia–reperfusion injury Mild hypothermia Apoptosis Timing of hypothermia
This work was supported by the National Natural Science Foundation of China (NSFC: 81201446, https://isis.nsfc.gov.cn). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors declare no conflicts of interest.
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