Ryanodine receptor inhibitor dantrolene reduces hypoxic-ischemic brain injury in neonatal mice.

Ryanodine receptors (RyR) located on the membrane of the endoplasmic reticulum (ER), are a potent regulator of intracellular calcium levels upon activation. Dysregulated Ca2+ homeostasis is characteristic of hypoxic-ischemic (HI) brain injury and ultimately leads to neurodegeneration. RyRs have thereby been implicated in the Ca2+ imbalance that occurs during and after HI. In this study, we investigated the effects of RyR antagonist, dantrolene, on HI brain injury in neonatal mice. We found that administration of dantrolene (i.p.) on postnatal day 7 mice reduced the infarction volume and morphological damage induced by HI, and improved functional recovery as assessed by neurobehavioral testing. The neuroprotective effect of dantrolene was further demonstrated in neuronal cell culture in vitro, where dantrolene significantly reduced oxygen-glucose deprivation (OGD)-induced cell death. Fura-2 calcium imaging confirmed that dantrolene reduced the intracellular calcium level in cultured cortical neurons in vitro. Finally, Western blot analysis showed that dantrolene treatment reduced cleaved caspase-3 and -9 apoptotic proteins, and elevated pro-survival protein kinase C (PKC) protein levels. Taken together, our results demonstrate that dantrolene exerts neuroprotective effects against neonatal HI brain injury. This suggests that RyRs play a role in mediating the ionic imbalance induced by HI and therefore represent a potential target for drug development.

[Therapeutic effects of moxibustion on neonatal mice with hypoxia-ischemia brain injury].

Objective: To investigate the effects of moxibustion on the behavioral performance, brain morphological structure of mice with hypoxia-ischemia brain injury and to explore its mechanisms. Methods: One hundred and six ICR mice were randomly divided into three groups, sham group (n=23), model group (n=46) and moxibustion-treated group (n=37). Neonatal hypoxic-ischemia brain injury was induced by ligation of common carotid artery followed by hypoxia (8% oxygen, 100 min), and pups in the moxibustion-treated group were administered suspended moxibustion on the Dazhui points (GV14) at a height of approximately 2 cm over a hairless area of the skin once a day for 4 days (i.e. at 2, 24, 48 and 72 hours after hypoxia-ischemia procedure). Behavioral tests were used to evaluate behavioral performance. HE staining was used to observe brain morphological structure. Western blot was used to detect the expression of SOD2 protein, and spectrophotometry was used to determine the content of MDA in the ipsilateral brain. Results: Mouse pups in sham group showed that the behavioral performance was normal, the brain tissue cells were densely and neatly arranged, the expression of SOD2 and the level of MDA in the brain tissues were normal. Compared with sham group, mouse pups in the HI model group exhibited a significant longer latency to complete the righting reflex, geotaxis reflex, cliff avoidance (P＜0.05) and a marked shorter latency to complete the grip test (P＜0.05); and the HI model group had dramatic brain morphological changes showing missing regions, decreased expression of SOD2 protein (P＜0.05) and increased level of MDA in the brain. Compared with HI model group, mouse pups in the moxibustion-treated group exhibited a significant shorter latency to complete the righting reflex, geotaxis reflex, cliff avoidance test (P＜0.05) and a marked longer latency to complete the grip test (P＜0.05); and the moxibustion-treated group had less brain morphological changes, increased expression of SOD2 protein (P＜0.05) and decreased level of MDA in the brain (P＜0.05) . Conclusion: Moxibustion could improve behavioral performance and attenuate hypoxia-ischemia brain injury, which might be related to increasing the expression of SOD2 protein and decreasing the content of MDA, thus enhancing the anti-oxidative ability.

[Effect of moxibustion on learning and memory in neonatal hypoxic-ischemia encephalopathy mice].

OBJECTIVE: To observe the effect of moxibustion on the expression of phosphorylated calcium/calmodulin-dependent protein kinase Ⅱα(pCaMKⅡα) and neuronal nuclei (NeuN) and the ability of learning and memory in the neonatal mice model of hypoxic-ischemia encephalopathy(HIE), so as to explore its mechanism underlying improvement of learning and memory. METHODS: ICR mice (aged 7 days) were randomly divided into sham operation, model and moxibustion groups. HIE model was induced by ligation of the right common carotid artery combined with hypoxia in a closed transparent chamber. Mice in the moxibustion group were treated with gentle moxibustion at "Dazhui"(GV14) for 35 min，once daily for 3 consecutive days. The pathological changes of brain tissues were observed with the naked eyes and under microscope after H.E. staining, respectively. The expressions of pCaMKⅡα and NeuN in the ischemic penumbra were examined by immunofluorescent staining, and the learning and memory ablility was tested with Morris maze. RESULTS: No infarcts were found in the brain tissue of the mice in the sham operation group. Compared with the sham operation group, mice in the model group had infarcts and the expression of pCaMKⅡα and NeuN in the ischemic penumbra was significantly reduced (P<0.01), and the latency to find a platform was significantly prolonged in Morris maze test (P<0.01). After moxibustion, in comparison with the model group showed that, small areas of infarction were seen in the right hemisphere of the moxibustion group, and the expressions of pCaMKⅡα, NeuN increased significantly (P<0.01), and the latency to find a platform was significantly shortened (P<0.01). CONCLUSION: Moxibustion can improve the ability of learning and memory in the neonatal mice with HIE, which might be related to alleviating brain injury and increasing the expression of pCaMKⅡα in neurons of ischemic brain tissues.

Comparison of the anti-apoptotic effects of 15- and 35-minute suspended moxibustion after focal cerebral ischemia/reperfusion injury.

Heat-sensitive suspended moxibustion has a neuroprotective effect against focal cerebral ischemia/reperfusion injury, but the underlying mechanisms remain unclear. The duration of heat-sensitive suspended moxibustion (usually from 30 minutes to 1 hour) is longer than traditional suspended moxibustion (usually 15 minutes). However, the effects of 15- and 35-minute suspended moxibustion in rats with cerebral ischemia/reperfusion injury are poorly understood. In this study, we performed 15- or 35-minute suspended moxibustion at acupoint Dazhui (GV14) in an adult rat model of focal cerebral ischemia/reperfusion injury. Infarct volume was evaluated with the 2,3,5-triphenyltetrazolium chloride assay. Histopathological changes and neuronal apoptosis at the injury site were assessed by hematoxylin-eosin staining and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Caspase-9 and caspase-3 expression at the injury site was detected using immunofluorescent staining. Bax and Bcl-2 expression at the injury site was assessed using western blot assay. In the 35-minute moxibustion group, infarct volume was decreased, neuronal apoptosis was reduced, caspase-9, caspase-3 and Bax expression was lower, and Bcl-2 expression was increased, compared with the 15-minute moxibustion group. Our findings show that 35-minute moxibustion has a greater anti-apoptotic effect than 15-minute moxibustion after focal cerebral ischemia/reperfusion injury.

Neuroprotective Effects of a PSD-95 Inhibitor in Neonatal Hypoxic-Ischemic Brain Injury.

The postsynaptic density-95 inhibitor NA-1 uncouples NMDA glutamate receptors from downstream neurotoxic signaling pathways without affecting normal glutamate receptor function. NA-1 attenuates NMDA receptor-mediated neuronal cell death after stroke in multiple models and species. However, its efficacy in providing neuroprotection in models of neonatal hypoxic-ischemic brain injury has not yet been tested. In this study, a modified version of the Rice-Vannucci method for the induction of neonatal hypoxic-ischemic brain injury was performed on postnatal day 7 mouse pups. Animals received a single dose of NA-1 intraperitoneally either before or after right common carotid artery occlusion. All experiments were performed in a blinded manner. Infarct volumes were measured 1 and 7 days after the injury, while behavioral tests were conducted 1, 3, and 7 days after injury. Administration of NA-1 before right common carotid artery occlusion or immediately after ischemia significantly reduced infarct volume and improved neurobehavioral outcomes 1, 3, and 7 days post-injury. The neuroprotection and improvement in neurobehavioral outcomes conferred by NA-1 in this mouse neonatal hypoxic-ischemic injury model imply that NA-1 will be effective in reducing neonatal stroke damage and thus could potentially serve as a therapeutic drug for prevention or treatment of neonatal stroke.

TRPM7 inhibitor carvacrol protects brain from neonatal hypoxic-ischemic injury.

BACKGROUND: Our previous study found that suppression of TRPM7 reduced neuronal death in adult rat ischemic brain injury. It was reported that carvacrol blocked TRPM7 and attenuated brain injury in an adult rat MCAO model. The effects of carvacrol on neonatal stroke remain unknown. This study investigated the effects of carvacrol on neuronal injury and behavioral impairment after hypoxia-ischemia in neonatal mice and the potential signaling pathway underlying these effects. RESULTS: Carvacrol inhibited TRPM7 current in HEK293 cells over-expressing TRPM7 and TRPM7-like current in hippocampal neurons in a dose-dependent manner. Carvacrol (>200 µM) reduced OGD-induced neuronal injury in cortical neurons. 24 hours after HI, TRPM7 protein level in the ipsilateral hemisphere was significantly higher than in the contralateral hemisphere. Carvacrol (30 and 50 mg/kg) pre-treatment reduced brain infarct volume 24 hours after HI in a dose-dependent manner. Carvacrol pre-treatment also improved neurobehavioral outcomes. Furthermore, animals pre-treated with carvacrol had fewer TUNEL-positive cells in the brain compared to vehicle-treated animals 3 days after HI. Carvacrol pre-treatment also increased Bcl-2/Bax and p-Akt/t-Akt protein ratios and decreased cleaved caspase-3 protein expression 24 hours after HI. CONCLUSIONS: Carvacrol pre-treatment protects against neonatal hypoxic-ischemic brain injury by reducing brain infarct volume, promoting pro-survival signaling and inhibiting pro-apoptotic signaling, as well as improving behavioral outcomes. The neuroprotective effect may be mediated by the inhibition of TRPM7 channel function. Carvacrol is a potential drug development target for the treatment of neonatal stroke.

Neuronal K(ATP) channels mediate hypoxic preconditioning and reduce subsequent neonatal hypoxic-ischemic brain injury.

Neonatal hypoxic-ischemic brain injury and its related illness hypoxic-ischemic encephalopathy (HIE) are major causes of nervous system damage and neurological morbidity in children. Hypoxic preconditioning (HPC) is known to be neuroprotective in cerebral ischemic brain injury. K(ATP) channels are involved in ischemic preconditioning in the heart; however the involvement of neuronal K(ATP) channels in HPC in the brain has not been fully investigated. In this study, we investigated the role of HPC in hypoxia-ischemia (HI)-induced brain injury in postnatal seven-day-old (P7) CD1 mouse pups. Specifically, TTC (2,3,5-triphenyltetrazolium chloride) staining was used to assess the infarct volume, TUNEL (Terminal deoxynucleotidyl transferase mediated dUTP nick end-labeling) to detect apoptotic cells, Western blots to evaluate protein level, and patch-clamp recordings to measure K(ATP) channel current activities. Behavioral tests were performed to assess the functional recovery after hypoxic-ischemic insults. We found that hypoxic preconditioning reduced infarct volume, decreased the number of TUNEL-positive cells, and improved neurobehavioral functional recovery in neonatal mice following hypoxic-ischemic insults. Pre-treatment with a K(ATP) channel blocker, tolbutamide, inhibited hypoxic preconditioning-induced neuroprotection and augmented neurodegeneration following hypoxic-ischemic injury. Pre-treatment with a K(ATP) channel opener, diazoxide, reduced infarct volume and mimicked hypoxic preconditioning-induced neuroprotection. Hypoxic preconditioning induced upregulation of the protein level of the Kir6.2 isoform and enhanced current activities of K(ATP) channels. Hypoxic preconditioning restored the HI-reduced PKC and pAkt levels, and reduced caspase-3 level, while tolbutamide inhibited the effects of hypoxic preconditioning. We conclude that K(ATP) channels are involved in hypoxic preconditioning-induced neuroprotection in neonatal hypoxic-ischemic brain injury. K(ATP) channel openers may therefore have therapeutic effects in neonatal hypoxic-ischemic brain injury.

Marine compound xyloketal B reduces neonatal hypoxic-ischemic brain injury.

Neonatal hypoxic-ischemic encephalopathy causes neurodegeneration and brain injury, leading to sensorimotor dysfunction. Xyloketal B is a novel marine compound isolated from a mangrove fungus Xylaria species (no. 2508) with unique antioxidant effects. In this study, we investigated the effects and mechanism of xyloketal B on oxygen-glucose deprivation-induced neuronal cell death in mouse primary cortical culture and on hypoxic-ischemic brain injury in neonatal mice in vivo. We found that xyloketal B reduced anoxia-induced neuronal cell death in vitro, as well as infarct volume in neonatal hypoxic-ischemic brain injury model in vivo. Furthermore, xyloketal B improved functional behavioral recovery of the animals following hypoxic-ischemic insult. In addition, xyloketal B significantly decreased calcium entry, reduced the number of TUNEL-positive cells, reduced the levels of cleaved caspase-3 and Bax proteins, and increased the level of Bcl-2 protein after the hypoxic-ischemic injury. Our findings indicate that xyloketal B is effective in models of hypoxia-ischemia and thus has potential as a treatment for hypoxic-ischemic brain injury.

Heat-sensitive moxibustion attenuates the inflammation after focal cerebral ischemia/ reperfusion injury.

Heat-sensitive moxibustion has neuroprotective effects against focal cerebral ischemia/reperfusion injury, however its mechanism of action remains unclear. In this study, rat models of focal cerebral ischemia/reperfusion injury were treated with suspended moxibustion at acupoint Dazhui (DU14) for 35 minutes. Results showed that suspended moxibustion decreased infarct volume, reduced cortical myeloperoxidase activity, and suppressed serum levels of proinflammatory cytokines in rats with focal cerebral ischemia/reperfusion injury. Our experimental findings indicated that heat-sensitive moxibustion can attenuate inflammation and promote repair after focal cerebral ischemia/reperfusion injury.

[Effect of c-myb on hCG-induced testosterone secretion in isolated rat Leydig cells].

The present study was carried out to investigate the effect of antisense c-myb oligodeoxynucleotides (ODN) on hCG-induced testosterone secretion in isolated rat Leydig cells. The effects of cAMP, Ca(2+) and cycloheximide (CYX) on c-Myb protein expression and testosterone secretion were also observed. The results showed that antisense c-myb ODN inhibited hCG-induced testosterone secretion of isolated rat Leydig cells in a dose-dependent manner. At the same time, integral optical density immunostaining of Myb in Leydig cells was also remarkably reduced. Nonsense tat ODN had no effect on Leydig cells. Further experiments showed that dbcAMP (100 micromol/L) obviously increased hCG-induced testosterone secretion and integral optical density (IOD) immunostaining of Myb in Leydig cells. Verapamil (10 micromol/L), a Ca(2+) channel blocker, and cycloheximide (50 microg/ml), a protein synthesis inhibitor, reduced the immunostaining of c-Myb, and also lowered hCG-induced testosterone secretion in isolated rat Leydig cells. The results indicate that c-myb closely correlates with hCG-induced testosterone secretion, and that cAMP and Ca(2+)-dependent pathway participates in the expression of protooncogene.