Intracellular calcium homeostasis disorder and mitochondrial dysfunction are involved in many acute and chronic brain diseases including ischemic brain injury. mitochondrial function as evidenced by reduced reactive oxygen Zosuquidar species (ROS) generation and cytochrome c release as well as prevented loss of mitochondrial membrane potential (MMP). Moreover mdivi A and mdivi B significantly suppressed mitochondrial Ca2+ uptake but had no effect on cytoplasmic Ca2+ after OGD injury. The results of calcium imaging and immunofluorescence staining showed that Drp-1 inhibitors attenuated endoplasmic reticulum (ER) Ca2+ release and prevented ER morphological changes induced by OGD. These results demonstrate that Drp-1 inhibitors protect against ischemic neuronal injury through inhibiting mitochondrial Ca2+ uptake from the ER store and attenuating mitochondrial dysfunction. gene dynamin-related protein 1 (Drp-1) is considered to be a key molecular in regulating mitochondrial fission [6]. Drp-1 activation leads to abnormalities in mitochondrial structure and function inhibits ATP generation and activates pro-apoptotic signaling cascades [6]. A recent study showed that embryos of Drp-1 knockout mouse died on days 11 to 12 [7] and experiments using pharmacological inhibitors seem to be an ideal strategy. In the present study small molecule inhibitors were used to investigate Drp-1 dependent mitochondrial death pathways in oxygen-glucose deprivation (OGD) in PC12 cells. We also examined the changes of intra-cellular calcium homeostasis to address the potential underlying mechanisms. 2 2.1 Effects of Drp-1 Inhibitors on Mitochondrial Dynamic Proteins Cultured PC12 cells were treated with mdivi A or mdivi B in different concentrations (25 50 and 100 μM) to examine the possible toxic effects of mdivi compounds at higher concentrations. As shown in Figure 1A the cell viability was decreased by mdivi A (100 μM) and mdivi B (100 μM) whereas mdivi compounds at low concentrations (25 or 50 μM) had no effect on cell viability. These results were confirmed by lactate dehydrogenase (LDH) release assay (Figure 1B). Furthermore western blot was used to detect Zosuquidar the expression of mitochondrial dynamic proteins (Figure 1C). Both mdivi A and mdivi B significantly increased the expression of optic atrophy type 1 (Opa1) and mitofusin 1 (Mfn1) two fusion related mitochondrial dynamic proteins and decreased the expression of Drp-1 (Figure 1D). All these data indicated that mdivi A and mdivi B at 50 μM differentially regulated mitochondrial dynamics-related proteins but had no toxic effects in PC12 cells. Figure 1. Effects of Drp-1 inhibitors on mitochondrial dynamic proteins. PC12 cells were treated with mdivi A or mdivi B at different concentrations (25 50 or 100 μM) for 24 h. Cell viability was measured with the WST assay (A); and cytotoxicity was measured … 2.2 Drp-1 Inhibitors Reduce Ischemic Toxicity in PC12 Cells Cultured PC12 cells were pretreated with mdivi A or mdivi B in different concentrations (25 50 and 100 μM) for 30 min before OGD and cell viability was measured at 24 h after reoxygenation. It was found that the cell Zosuquidar viability increased with the concentrations of mdivi A and mdivi B added although 100 μM mdivi A or mdivi B was not effective compared with OGD injured cells (Figure 2A). LDH assay also showed that pretreatment with mdivi A and mdivi B (25 and 50 μM) induced a significant decrease in LDH release after OGD insult (Figure 2B). Moreover terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was used to determine the effects of mdivi A and mdivi B on OGD-induced apoptotic cell death (Figure 2C). As shown in Figure 2D the OGD-induced increase of TUNEL-positive cells Bmpr2 was significantly decreased by mdivi A and mdivi B pretreatment indicating the anti-apoptotic activity Zosuquidar of Drp-1 inhibition. Figure 2. Drp-1 inhibitors reduce ischemic toxicity in PC12 cells. PC12 cells were pretreated with mdivi A or mdivi B at different concentrations (25 50 or 100 μM) for 30 min before oxygen-glucose deprivation (OGD) injury. Cell viability was measured with … 2.3 Drp-1 Inhibitors Attenuate Mitochondrial Dysfunction In order to investigate the effects of Drp-1 inhibitors on mitochondrial Zosuquidar dysfunction PC12 cells were pretreated with 50 μM mdivi A or 50 μM mdivi B based on the results mentioned above. Exposure to OGD insults resulted in an increase in intracellular ROS generation at both 6 and Zosuquidar 12 h after OGD initiation and pretreatment with mdivi A or mdivi B significantly reduced the ROS overproduction (Figure 3A). As shown in.