Role of mitochondrial fission in hypoxia-reoxygenation injury to hippocampal neurons of rats

Abstract

Objective To evaluate the role of mitochondrial fission in hypoxia-reoxygenation (H/R) injury to hippocampal neurons of rats. Methods Primarily cultured hippocampal neurons obtained from newborn Wistar rats were randomly divided into 5 groups (n=60 each) using a random number table: normal group (N group), vehicle group (V group), H/R group, H/R + vehicle group (H/R + V group), and mitochondrial division inhibitor group (group M). The cells were cultured in normal culture medium in group N. Dimethyl sulfoxide (DMSO) was added to the culture medium with the final concentration <0.1%, and the cells were incubated for 40 min in group V. The cells were subjected to 6 h hypoxia, followed by 20 h reoxygenation in H/R, H/R+ V and M groups.DMSO was added to the culture medium with the final concentration <0.1% at 40 min before hypoxia in group H/R+ V.In group M, mitochondrial division inhibitor Mdivi-1 50 mmol/L (dissolved in DMSO, DMSO concentration <0.1%) was added to the culture medium at 40 min prior to hypoxia.Mito Tracker staining was used to examine mitochondrial morphology.Western blot was used to measure the expression of mitochondrial dynamin-related protein 1 (Drp1), mitofusin 2 (Mfn2), peroxisome proliferator activated receptor γ coactivator 1α (PGC-1α), nuclear respiratory factor-1 (NRF-1), and mitochondrial transcription factor A (TFAM). Multifunctional microplate reader and fluorescent microscope were used to detect the mitochondrial reactive oxygen species (ROS) level.The flow cytometer was used to detect the apoptosis in hippocampal neurons.Apoptosis rate was calculated. Results Compared with group N, the expression of Drp1, PGC-1α, NRF-1 and TFAM was significantly up-regulated, the ROS content and apoptosis rate were increased, and the expression of Mfn2 was down-regulated in group H/R (P<0.05). Compared with group H/R, the expression of Drp1 was significantly down-regulated, the ROS content and apoptosis rate were decreased, and the expression of Mfn2, PGC-1α, NRF-1 and TFAM was up-regulated in group M (P<0.05). Conclusion Mitochondrial fission is involved in H/R injury to hippocampal neurons of rats. Key words: Mitochondria; Hippocampus; Neurons; Cell hpoxia; Oxygen