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The neuroprotective role of propofol in transient global and focal cerebral ischemia reperfusion (I/R) animal model has recently been highlighted. However, no studies have conducted to explore the relationship between mitochondrial fission/fusion and I/R injury under the intervention of propofol. Moreover, neuroprotective mechanism of propofol is yet unclear. Culturing primary hippocampal cells were subjected to oxygen-glucose deprivation and re-oxygenation (OGD/R) model, as a model of cerebral I/R in vitro. Methods CCK-8 assay was used to test the effect of propofol on cell viability. We examined the effect of propofol on mitochondrial ultrastructure and mitochondrial fission evoked by OGD/R with transmission electron microscopy and immunofluorescence assay. To investigate possible neuroprotective mechanisms, the authors then examined whether propofol could inhibit calcium-overload, calcineurin (CaN) activation and the phosphorylation of dynamin-related protein 1 (Drp1) during the period of OGD/R, as well as the combination of Drp1-ser 637 and fission 1 (Fis1) protein by immunofluorescence assay, ELISA and double-labeling immunofluorescence analysis. Finally, the expression of Drp1-ser 637 and Fis1, apoptosis inducing factor (AIF) and cytochrome C (Cyt C) were detected by western blot. When added in culture media during OGD period, propofol (0.1μM-50μM) could alleviate neurons injury and protect mitochondrial ultrastructure, meanwhile inhibit mitochondrial fission. Furthermore, the concentration of intracellular free Ca2+, CaN activition and the phosphorylation of Drp1-ser637 were suppressed, as well as the translocation and combination of Drp1-ser 637 and Fis1. The authors also found that the expression of Cyt C, AIF, Drp1-ser637 and Fis1 were down-regulated. Notably, high dose of propofol (100μM-200μM) were confirmed to decrease the survival of neurons based on results of cell viability. Propofol could inhibit mitochondrial fission and mitochondrial apoptotic pathway evoked by OGD/R in rat hippocampal neurons, which may be via depressing calcium-overload., Competing Interests: The authors have declared that no competing interests exist.

Mitophagy plays an important role in mitochondrial quality control and cell survival during the process of ischemia/reperfusion (I/R) injury. Mitochondrial calcium uniporter (MCU) is the most important channel responsible for Ca(2+) influx into mitochondria and Ca(2+) signal plays a potential role in modulating mitophagy. However, the effect of MCU on mitophagy during the process of I/R injury remains unknown. This study constructed an in vitro I/R model by subjecting oxygen and glucose deprivation/reperfusion (OGD/RP) model to SH-SY5Y cells to mimic the cerebral I/R injury and aimed to explore the exact effect of MCU on I/R induced mitophagy. The results showed that OGD/RP induced autophagy and mitophagy in SH-SY5Y cells. Ru360, the inhibitor of MCU, improved mitochondrial morphology and fuctional stability as well as cell viability, significantly reduced OGD/RP induced mitophagy as evidenced by the decrease in Beclin-1 and the increase in Tom20 and P62 expression. Whereas spermine, the agonist of MCU, had no significant impact on the expression of those mitophagy related proteins compared with OGD/RP group. This study indicates that inhibition of MCU can inhibit excessive mitophagy and protect the neurocytes from I/R injury., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Objectives: To explore the effects of calcium-sensing receptors (CaSR) on apoptosis in rat hippocampus during hypoxia/reoxygenation (H/R)., Methods: After rat hippocampus was isolated, the cultures were subjected to H/R, and meanwhile gadolinium chloride (GdCl3, agonist of CaSR) and NPS 2390 (antagonists of CaSR) were added to reperfusion solution. The number of hippocampal neuron, cell viability and apoptosis rate were determined by inverted microscope, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and flow cytometer (FCM), respectively. Besides, caspase-3, Bax, cytochrome C (Cyt-c), extracellular signal-regulated protein kinase (ERK) 1/2, pERK1/2, P38 and pP38 were analyzed by Western blotting., Results: The hippocampal neuron number and cell viability were significantly decreased during H/R, and were further significantly reduced when co-treatment with CaSR agonist GdCl3. But the effects of GdCl3 were attenuated by NPS-2390. Whereas, apoptosis rate, the expression level of caspase-3, Bax and Cyt-c were all significantly increased under H/R condition, and was further significantly increased by GdCl3, but were reversed by NPS-2390 (P < 0.05). Moreover, there were no significant differences in expression of ERK1/2, P38 and pP38 among different groups. However, the expression of pERK1/2 was significantly increased during H/R, but was significantly reduced by NPS 2390 (P < 0.05)., Conclusion: The results suggest that CaSR might play significant roles in the induction of hippocampus apoptosis in rat during H/R through phosphorylation of ERK1/2.

The mitochondrial calcium uniporter (MCU) transports free Ca(2+) into the mitochondrial matrix, maintaining Ca(2+) homeostasis, thus regulates the mitochondrial morphology. Previous studies have indicated that there was closely crosstalk between MCU and mitochondrial fission during the process of ischemia/reperfusion injury. This study constructed a hypoxia reoxygenation model using primary hippocampus neurons to mimic the cerebral ischemia/reperfusion injury and aims to explore the exactly effect of MCU on the mitochondrial fission during the process of ischemia/reperfusion injury and so as the mechanisms. Our results found that the inhibitor of the MCU, Ru360, decreased mitochondrial Ca(2+) concentration, suppressed the expression of mitochondrial fission protein Drp1, MIEF1 and Fis1, and thus improved mitochondrial morphology significantly. Whereas spermine, the agonist of the MCU, had no significant impact compared to the I/R group. This study demonstrated that the MCU regulates the process of mitochondrial fission by controlling the Ca(2+) transport, directly upregulating mitochondrial fission proteins Drp1, Fis1 and indirectly reversing the MIEF1-induced mitochondrial fusion. It also provides new targets for brain protection during ischemia/reperfusion injury., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Apoptosis is one of the major mechanisms of neuronal injury during ischemic-reperfusion (I/R). Mitochondrial division inhibitor (mdivi-1) is a selective inhibitor of mitochondrial fission protein Drp1. The previous experiments support that mdivi-1 reduce I/R injury in the heart model of rat, but the neuroprotective effect of the mdivi-1 is not yet clearly defined at the cellular levels in brain. In our present study, we estimated a brain model of I/R injury in vitro by subjecting oxygen and glucose deprivation (OGD) followed by reoxygenation to the cultured rat primary hippocampal cells, which aimed to find the neuroprotective mechanism of mdivi-1. The cell was pretreated with mdivi-1 for 40 minutes and then ischemia for 6 hours followed by reperfusion for 20 hours. The redox state, cell apoptosis, and expression of Drp1, Bcl-2, Bax, and cytochrome C proteins were measured. The data showed that administration of mdivi-1 at the doses of 50 μM significantly reduced oxidative stress, attenuated cell apoptosis, upregulated Bcl-2 expression, and downregulated Drp1, Bax, and cytochrome C expression. The results suggested that mdivi-1 protected brain from OGD reperfusion injury, which through suppressing the ROS initiated mitochondrial pathway., (Copyright © 2014 National Stroke Association. Published by Elsevier Inc. All rights reserved.)

Background: It is reported that ischemic penumbra is a dynamic process, in which irreversible necrosis in the center of ischemia propagates to the neighboring tissue over time. Recent research has indicated that mitochondrial adenosine triphosphate (ATP)-sensitive potassium channels (mitoKATP) opener diazoxide plays an important role in cerebral protection; however, the role of mitochondrial calcium uniporter (MCU) in the effect of diazoxide on penumbra and infarct core remains unclear., Methods: Adult male Wistar rats were randomly divided into 5 groups: the Sham group, the ischemia-reperfusion (I/R) group, the diazoxide and ischemia-reperfusion (Dzx + I/R) group, the diazoxide and spermine and ischemia-reperfusion (Dzx + Sper + I/R) group, and the spermine and ischemia-reperfusion (Sper + I/R) group. The animals were exposed to 24-hour reperfusion after 2-hour ischemia. Diazoxide and spermine were administrated at 30 minutes or 10 minutes before the beginning of ischemia or reperfusion, respectively. After 24-hour reperfusion, animals were given neurologic performance tests and when overdosed with general anesthesia their brains were excised for infarct volume, apoptosis, and immunohistochemical., Results: Rats in the Dzx + I/R group displayed improved neurologic deficits, decreased infarct volume in cortex but not in subcortex, and apoptosis (evidenced by decreased terminal deoxynucleotidyl transferase-mediated dUTP nick end lebeling-positive percentage and the immunohistochemistry of cytochrome c) in cortex caused by ischemia/reperfusion. Rats in the Dzx + Sper + I/R group displayed worse neurologic deficits, larger infarct volume in cortex but not in subcortex, and more apoptosis both in penumbra and infarct core of cortex than those in the Dzx + I/R group., Conclusions: Results in our study suggested that diazoxide improved neurologic deficits, decreased infarct volume in cortex but not in subcortex, and apoptosis in cortex against ischemia/reperfusion injury is mediated by spermine., (Copyright © 2014 National Stroke Association. Published by Elsevier Inc. All rights reserved.)

Background: Recent research has indicated that mitochondrial adenosine triphosphate-sensitive potassium channels play an important role in cerebral protection, which involves in attenuating the calcium of mitochondria. However, the effect of diazoxide on cerebral ischemia-reperfusion and the role of spermine, the agonist of mitochondrial calcium uniporter (MCU), remain unknown., Objective: We investigated the effect of MCU opener spermine on diazoxide against focal cerebral ischemia-reperfusion injury in rats., Methods: Adult male Wistar rats were randomly divided into 5 groups: the Sham group, the I/R group, the Dzx + I/R group, the Dzx + Sper + I/R group, and the Sper + I/R group. Rats were exposed to 2-hour ischemia and 24-hour reperfusion. Diazoxide were administrated 30 minutes before ischemia, and spermine were given 10 minutes before reperfusion. Rats in the Sham group did not experience the process of ischemia-reperfusion. After 24-hour reperfusion, rats were given neurological performance tests, overdosed with general anesthesia, and then their brains were excised for infarct volume, pathological changes, and biochemical evaluation and analysis., Results: Rats in the Dzx + I/R group displayed improved neurological deficits and decreased infarct volume and oxidative stress (evidenced by decreased nitric oxide and malondialdehyde but increased antioxidant enzymes [eg, glutathione peroxide and superoxide dismutase]) caused by ischemia-reperfusion. The beneficial effects of diazoxide were significantly attenuated by spermine treatment. Rats in the Sper + I/R group displayed worse neurological deficits, larger infarct volume and more oxidative stress, and less antioxidant enzymes than those in the Dzx + I/R., Conclusions: Our results suggested that diazoxide, which improved neurological deficits and decreased infarct volume and oxidative stress against ischemia-reperfusion injury, is mediated by spermine., (Copyright © 2014 National Stroke Association. Published by Elsevier Inc. All rights reserved.)

Mitochondrial division inhibitor (mdivi-1) is a derivative of quinazolinone that acts as a selective inhibitor of a mitochondrial fission protein Drp1. A previous study demonstrated that as a selective inhibitor of Drp1, mdivi-1 has a protective effect in an experimental model of heart ischemia/reperfusion injury. In this study, we investigated the protective effects of mdivi-1 on cerebral ischemia/reperfusion injury in a middle cerebral artery occlusion mouse model. We found that mdivi-1 (1.2mg/kg) significantly reduced cerebral damage induced by ischemia/reperfusion. This neuroprotective effect was dose-dependent. Mdivi-1 treatment blocked apoptotic cell death in cerebral ischemia/reperfusion injury, and significantly decreased the expression of Drp1 and Cytochrome C. These results suggest that mdivi-1 exerts neuroprotective effects against nerve injury after cerebral ischemia/reperfusion, and the underlying mechanism may be through the prevention of Cytochrome C release and suppression of the mitochondrial apoptosis pathway., (Crown Copyright © 2013. Published by Elsevier Ireland Ltd. All rights reserved.)

Objective To investigate the protective effect of sevoflurane postconditioning against cerebral ischemia/reperfusion (I/R) injury in rats and its mechanism of action. Methods A total of 80 specific pathogen-free healthy adult male Sprague-Dawley rats were selected and randomly divided into sham-operation group (S group), cerebral I/R group (I/R group), cerebral I/R+sevoflurane postconditioning group (ISP group), and cerebral I/R+sevoflurane postconditioning+nuclear factor ery-throid 2-related factor 2 (Nrf2) inhibitor group (ISPB group), with 20 rats in each group. All rats except those in the S group were used to establish a rat model of cerebral I/R injury using the suture method for occlusion of the middle cerebral artery for 2 h, followed by reperfusion for 24 h, and for the rats in the S group, threading was performed below the middle cerebral artery without ligation. The rats in the ISP group were given inhalation of 3% sevoflurane immediately after reperfusion for 30 min, and those in the ISPB group were given intraperitoneal injection of the Nrf2 inhibitor brusatol (2 mg/kg) at 30 min before ischemia in addition to the treatment in the ISP group. After successful modeling, neurological deficit score was used to eval-uate the degree of neurological impairment. Left ventricular blood samples and pathological sections of brain tissue were obtained, and 2,3,5-triphenyltetrazolium chloride staining was used to determine the percentage of cerebral infarct volume; enzyme-linked immunosorbent assay was used to measure the serum levels of inflammatory factors (interleukin-1β [IL-1β] and tumor necrosis factor-α [TNF-α]) and oxidative stress-related factors (malondialdehyde [MDA] and superoxide dismutase [SOD]); Western blotting was used to mea-sure the expression of apoptosis-related proteins (B-cell lymphoma-2 [Bcl-2], Bcl-2 related x [Bax], and Caspase-3) and Nrf2 signaling pathway-related proteins (Nrf2 and heme oxygenase-1 [HO-1]) in brain tissue; immunofluorescence assay was used to measure the expression of Nrf2 inside and outside the nucleus of brain tissue cells. Results Compared with the I/R group, the ISP group had significant reductions in neurological deficit score, the percentage of cerebral infarct volume, the serum levels of IL-1β, TNF-α, and MDA, and the levels of Bax and Caspase-3 in brain tissue (t=5.76-18.39,P

Objective To investigate the protective effect of remimazolam on cerebral nerves in a rat model of cerebral ischemia/reperfusion injury and its mechanism. Methods A total of 50 male Sprague-Dawley rats were randomly divided into sham-operation group (Sham group), cerebral ischemia/reperfusion group (I/R group), I/R+remimazolam (RE) group, I/R+RE+LY294002 (LY) group, and I/R+LY group. Neurological deficit score was used to evaluate the degree of neurological dysfunction. After arterial blood samples and brain tissue pathological sections were collected from the rats in each group, 2,3,5-trip-henyltetrazolium chloride staining was performed for the brain tissue sections to calculate cerebral infarct volume ratio; ELISA was used to measure the serum levels of interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α); Western blotting was used to measure the relative expression levels of protein kinase B (AKT), phosphorylated AKT (p-AKT), nuclear factor-kappa B p65 (NF-κB p65), and phosphorylated NF-κB p65 (p-NF-κB p65) in brain tissue, and then immunofluorescence assay was used to measure the expression level of p-NF-κB p65 inside and outside the nucleus of cells in brain tissue. Results There were significant differences between these groups in cerebral infarct volume ratio, neurological deficit score, the serum levels of IL-6, IL-1β, and TNF-α, the relative expression levels of p-AKT and p-NF-κB p65, p-AKT/AKT ratio, and p-NF-κB p65/NF-κB p65 ratio (F=72.41-654.90,P

Ischemia/reperfusion cerebral injury can cause serious damage to nerve cells. The injured organelles are cleared by autophagy eventually, which is critical for cell survival. Dexmedetomidine is neuroprotective in various ischemia/reperfusion models. Mitochondrial calcium uniporter (MCU) is the most important channel of mitochondrial Ca2+ influx into mitochondria, where Ca2+ has a potential effect on mitochondrial autophagy. However, the role of MCU in the changes of mitophagy and autophagy caused by dexmedetomidine is unknown. In this study, we constructed an in vitro I/R model by subjecting the oxygen and glucose deprivation/reperfusion model to SH-SY5Y cells to mimic the cerebral I/R injury. We found that postconditioning with dexmedetomidine and 3-methyladenine (3MA, an autophagy inhibitor) increased the cell survival meanwhile reduced the production of autophagic vesicles and the expression of LC3 and Beclin 1. This process also increased the expression of BCL-2, P62, and TOM20. After applied with spermine (MCU-specific agonist), the expression of autophagy proteins by dexmedetomidine was reversed, and the same changes were also observed in immunofluorescence. The results of our study suggested that dexmedetomidine can inhibit MCU and reduce excessive mitophagy and autophagy for conferring protection against I/R injury. [ABSTRACT FROM AUTHOR]

The mitochondrial calcium uniporter (MCU) transports Ca from the cytoplasm to the mitochondrial matrix and thus maintains Ca homeostasis. Previous studies have reported that inhibition of MCU by ruthenium red (RR) protects the brain from ischemia/reperfusion (I/R) injury and that mitochondrial fission plays an important role in I/R injury. However, it is still not known whether MCU affects mitochondrial fission. In the present study, treatment with RR was found to decrease the concentration of free calcium in the mitochondria, calcineurin enzyme activity and dynamin-related protein 1 expression, and treatment with spermine was found to have the opposite effect in organisms subjected to occlusion of the middle cerebral artery lasting 2 h followed by 24 h reperfusion. These results indicate that MCU may be related to mitochondrial fission via modulating mitochondrial Ca uptake and this relationship between MCU and mitochondrial fission may protect the brain from I/R injury. [ABSTRACT FROM AUTHOR]

The article presents a study which investigates the impact of agonist spermine and mitochondrial calcium uniporter inhibitor ruthenium red on cerebral edema in rat models with cerebral ischemia reperfusion injury. The study uses the suture method to induce left middle cerebral artery occlusion (MCAO) in rats. Results show that preventive administration of the mitochondrial calcium uniporter inhibitor ruthenium red can reduce immunoglobulin G (IgG) expression and aquaporin 4 (AQP4) .