Transient receptor potential M2 channel in the hypothalamic preoptic area and its impact on thermoregulation during menopause.

BACKGROUND: Decreased estrogen levels can cause abnormal thermosensitivity of the preoptic area (POA) in the hypothalamus during menopause, which may cause hot flashes. Thermosensitive transient receptors (ThermoTRPs) affect the thermosensitivity of neurons. It is worth exploring whether ThermoTRPs change under low estrogen state and participate in the abnormal thermoregulation of POA. METHODS: Adult female Sprague-Dawley rats were randomly divided into sham operation (SHAM), ovariectomy (OVX) and estrogen treatment after ovariectomy (OVX+E) groups. Under 10 â„ƒ, 18 â„ƒ, 25 â„ƒ, 37 â„ƒ and 45 â„ƒ incubations, their skin temperature was monitored and the expression of TRPA1, TRPM8, TRPM2, and TRPV1 in POA were investigated. RESULTS: The skin temperature of ovariectomized rats changed faster and more dramatically under different incubation temperatures. The results at mRNA level show that only the expression of TRPM2 decreased in POA of OVX group compared with the other two groups at 25 â„ƒ, TRPA1 expression in POA of the three groups increased at 10 â„ƒ, TRPM8 increased at 10 â„ƒ and 18 â„ƒ, TRPV1 increased at 10 â„ƒ and 45 â„ƒ, while the expression of TRPM2 decreased at 10 â„ƒ and 18 â„ƒ and increased at 37 â„ƒ and 45 â„ƒ. In all these cases, the magnitudes of the changes were less in the OVX group relative to the other two groups. The further immunohistochemical and Western blot results of TRPM2 and the activated TRPM2 positive cells labeled by c-Fos were consistent with the results of mRNA level. CONCLUSIONS: The expression and thermosensitivity of TRPM2 in POA changed greatly under different incubation temperatures, but the changes in ovariectomized rats were less. This may be the key factor triggering thermoregulation dysfunction under low estrogen and may cause hot flashes.

Transcriptomic changes in the hypothalamus of ovariectomized mice: Data from RNA-seq analysis.

BACKGROUND: Menopausal symptoms can affect the physical and mental health of females and are often related to abnormal function of the hypothalamus. In this study, we evaluated changes in the hypothalamus transcriptome in ovariectomized mice to identify key mRNAs, and systematically elucidated the possible molecular mechanisms underlying the menopausal syndrome to provide a theoretical basis for clinical diagnosis and treatment. METHODS: Forty-six adult female C57BL/6 J mice were randomly divided into SHAM and OVX groups, 23 mice per group. Eight weeks after the procedure, differentially expressed genes (DEGs) in the hypothalamus were identified through RNA-sequencing. DEGs were analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) analyses. Key DEGs were then evaluated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemical staining. RESULTS: Compared with SHAM group, 7295 genes were upregulated, and 8979 genes were downregulated in the hypothalamus of OVX mice with a fold change of 1.5 (log2 fold change ≥0.585). GO and KEGG analyses suggested these key genes were involved in thermoregulation, food intake, glucose and lipid metabolism, cardiovascular regulation, biological rhythm, and endocrine regulation. CONCLUSIONS: Differential expression of genes in the hypothalamus of OVX mice involved in thermoregulation, eating, sleeping, homeostasis, and endocrine regulation 8 weeks after ovariectomy suggest potential roles in the pathogenesis of climacteric syndrome.

The perceptions of anatomy teachers for different majors during the COVID-19 pandemic: a national Chinese survey.

During the spring semester of 2020, medical school anatomists in China were forced by the COVID-19 pandemic to transition from face-to-face educators or part-time online educators to full-time online educators. This nationwide survey was conducted to assess online anatomy education during the pandemic for medical students from nonclinical medicine and clinical medicine majors at medical schools in China via WeChat. The total of 356 responders included 293 responders from clinical medicine and 63 respondents from nonclinical medicine majors (i.e., 21 from preventive medicine, 13 from stomatology, and 29 from traditional Chinese medicine). The survey results showed that several aspects of online anatomy education were quite similar in clinical and nonclinical majors' classes, including theoretical and practical sessions, active learning, assessments and evaluations. However, there were statistically significant differences in class size, implementation of active learning activities prior to the pandemic, and the evaluation of the effectiveness of online learning during the pandemic, between clinical and nonclinical medicine majors. These results indicated that, compared with teachers of anatomy courses in clinical medicine, teachers of nonclinical medicine majors using online learning in medical schools in China had relatively poor preparation for online learning in response to the unforeseen pandemic.

Changes in TRPV1 expression in the POA of ovariectomized rats regulated by NE-dependent α2-ADR may be involved in hot flashes.

BACKGROUND: Hot flashes (HF) caused by low estrogen in menopause result from changes in thermoregulatory processes in the hypothalamic preoptic area (POA). In the POA, transient receptor potential vanilloid 1 (TRPV1) participates in heat dissipation processes. Studies suggest that TRPV1 expression may be regulated by norepinephrine (NE)-activated α2-adrenergic receptors (α2-ADR) in the dorsal root ganglia. The goal of this study was to investigate the relationship between NE-regulated TRPV1 expression in the POA of ovariectomized rats and the development of HF. METHODS: Ninety female adult Sprague-Dawley rats were divided into three groups: SHAM, OVX and E2 (n = 30 per group). The numbers of TRPV1- and α2-ADR-positive cells and the expression of TRPV1 and α2-ADR in the POA of each group were determined using immunohistochemical staining after 4 weeks of estrogen treatment. Western blotting was used to detect the expression of TRPV1 and α2-ADR in the POA tissue, and NE content in the POA tissue was detected using high-performance liquid chromatography. In addition, the coexpression of TRPV1 and α2-ADR in POA neurons was investigated using immunofluorescent staining. RESULTS: In the POA of ovariectomized rats, the number of TRPV1-positive cells and TRPV1 expression increased while NE content decreased. Concomitantly, the number of α2-ADR-positive cells and α2-ADR expression decreased. Estrogen treatment reversed these changes in the POA of ovariectomized rats. In addition, we found that TRPV1 and α2-ADR were coexpressed in POA neurons. CONCLUSIONS: Under low-estrogen conditions, NE-activated α2-ADR regulated TRPV1 expression in the POA, and increased expression of TRPV1 may be an important factor for triggering HF.

Salvinorin A attenuates early brain injury through PI3K/Akt pathway after subarachnoid hemorrhage in rat.

Early brain injury (EBI) refers to the direct injury to the brain during the first 72 h after subarachnoid hemorrhage (SAH), which is one of the major causes for the poor clinical outcome after SAH. In this study, we investigated the effect and the related mechanism of Salvinorin A (SA), a selective kappa opioid receptor agonist, on EBI after SAH. SA was administered by intraperitoneal injection at 24 h, 48 h and 72 h after SAH. The volume of lateral ventricle was measured by magnetic resonance imaging (MRI). The neuronal morphological changes and the apoptotic level in CA1 area of hippocampus were observed by Nissl and TUNEL staining respectively. Protein expression of p-PI3K, p-Akt, p-IKKα/ß, p-NF-κB, FoxO1, Bim, Bax and Cleaved-caspase-3 was measured to explore the potential mechanism. We found that SA alleviated the neuronal morphological changes and apoptosis in CA1 area of hippocampus. The mechanism might be related to the increased protein expression of p-PI3K/p-Akt, which accompanied by decreased expression of p-IKKα/ß, p-NF-κB, FoxO1, Bim, Bax and Cleaved-caspase-3 in the hippocampus. Thus, therapeutic interventions of SA targeting the PI3K/Akt pathway might be a novel approach to ameliorate EBI via reducing the apoptosis and inflammation after SAH.

Team-based learning: assessing the impact on anatomy teaching in People's Republic of China.

OBJECTIVES: In this study, the effects of team-based learning (TBL) method on the anatomy course for students in People's Republic of China were assessed. METHODS: The students were randomly divided into the traditional lecture-based teaching group (traditional learning [TL] group, 99 students) and TBL teaching group (98 students). The TBL method required the students to prepare the assigned content in advance and discuss some specific topics in small groups. The test scores and questionnaire were applied to evaluate the effects of the two methods. RESULTS: The students in TBL group had higher examination scores (81.70±8.53 vs 74.41±8.27, F[1,195] =124.6, p<0.01). The ratios of students with excellent (13.27% vs 9.09%, χ2[1] =4.00, p=0.041) and good scores (25.51% vs 16.16%, χ2[1] =4.85, p=0.027) were markedly increased in the TBL than the TL group, and the ratio of students who had just managed to pass was decreased (17.34% vs 32.33% in TL group, χ2[1] =5.91, p=0.015). The students in TBL group significantly achieved some improvement in mutual communication ability (χ2[1] =7.54, p=0.006), expression ability (χ2[1] =4.930, p=0.026), generalization capacity (χ2[1] =4.08, p=0.043), cooperative ability cultivation (χ2[1] =5.04, p=0.024), knowledge extension (χ2[1] =4.50, p=0.034), and enthusiasm mobilization (χ2[1] =4.27, p=0.039). CONCLUSION: TBL could improve not only the test scores of the students, but also their study enthusiasm, initiative learning ability, communication ability, and team awareness.

In vivo observation of cerebral microcirculation after experimental subarachnoid hemorrhage in mice.

Acute brain injury caused by subarachnoid hemorrhage is the major cause of poor prognosis. The pathology of subarachnoid hemorrhage likely involves major morphological changes in the microcirculation. However, previous studies primarily used fixed tissue or delayed injury models. Therefore, in the present study, we used in vivo imaging to observe the dynamic changes in cerebral microcirculation after subarachnoid hemorrhage. Subarachnoid hemorrhage was induced by perforation of the bifurcation of the middle cerebral and anterior cerebral arteries in male C57/BL6 mice. The diameter of pial arterioles and venules was measured by in vivo fluorescence microscopy at different time points within 180 minutes after subarachnoid hemorrhage. Cerebral blood flow was examined and leukocyte adhesion/albumin extravasation was determined at different time points before and after subarachnoid hemorrhage. Cerebral pial microcirculation was abnormal and cerebral blood flow was reduced after subarachnoid hemorrhage. Acute vasoconstriction occurred predominantly in the arterioles instead of the venules. A progressive increase in the number of adherent leukocytes in venules and substantial albumin extravasation were observed between 10 and 180 minutes after subarachnoid hemorrhage. These results show that major changes in microcirculation occur in the early stage of subarachnoid hemorrhage. Our findings may promote the development of novel therapeutic strategies for the early treatment of subarachnoid hemorrhage.

Salvinorin A ameliorates cerebral vasospasm through activation of endothelial nitric oxide synthase in a rat model of subarachnoid hemorrhage.

OBJECTIVE: This study aimed to demonstrate the potential of salvinorin A (SA) for cerebral vasospasm after subarachnoid hemorrhage (SAH) and investigate mechanisms of therapeutic effect using rat SAH model. METHODS: Salvinorin A was injected intraperitoneally, and the neurobehavioral changes were observed at 12 hours, 24 hours, 48 hours, and 72 hours after SAH. Basilar artery was observed by magnetic resonance imaging (MRI). The inner diameter and thickness of basilar artery were measured. The morphological changes and the apoptosis in CA1 area of hippocampus were detected. Endothelin-1 (ET-1) and nitric oxide (NO) levels were detected by ELISA kit. The protein expression of endothelial NO synthase (eNOS) and aquaporin-4 (AQP-4) was determined by Western blot for potential mechanism exploration. RESULTS: Salvinorin A administration could relieve neurological deficits, decrease the neuronal apoptosis, and alleviate the morphological changes in CA1 area of hippocampus. SA alleviated CVS by increasing diameter and decreasing thickness of basilar artery, and such changes were accompanied by the decreased concentration of ET-1 and increased level of NO. Meanwhile, SA increased the expression of eNOS and decreased the expression of AQP-4 protein in the basilar artery and hippocampus. CONCLUSIONS: Salvinorin A attenuated CVS and alleviated brain injury after SAH via increasing expression of eNOS and NO content, and decreasing ET-1 concentration and AQP-4 protein expression.

Protective effects of oxymatrine against arsenic trioxide-induced liver injury.

Oxymatrine, a quinolizidine natural drug extracted from Sophora japonica, has been reported to have neuroprotective effect and cardioprotective effect. However, the protective effect of oxymatrine on arsenic trioxide (As2O3)-induced liver injury has not been reported. In the present study, we investigated the protective effects of oxymatrine on As2O3-induced liver injury in rats. Male Wistar rats were administrated 3mg/kg As2O3 intravenous injection on alternate days for 4 days. Oxymatrine was given 1 h before As2O3 treatment. The results showed that oxymatrine inhibited As2O3-induced hepatic pathological damage, liver ROS level and MDA level in a dose-dependent manner. As2O3 decreased the antioxidant enzymes SOD, GPX, and CAT activity and the decrease was inhibited by treatment of oxymatrine. Furthermore, oxymatrine attenuated the retention of arsenic in liver tissues and improved the expression of Nrf2 and HO-1. In conclusion, our results suggested that oxymatrine protected against As2O3-induced oxidative damage by activating Nrf2/HO-1 signaling pathway.

Knockout of programmed cell death 5 (PDCD5) gene attenuates neuron injury after middle cerebral artery occlusion in mice.

Loss of von Hippel-Lindau tumor suppressor protein (VHL) or hypoxia results in nuclear relocalization of PDCD5 and subsequent mouse double minute 2 homolog (Mdm2) degradation. Thus, VHL may involved in the PDCD5 mediated apoptosis and autophagy after MCAO. In the present study, using PDCD5 knockout (PDCD5-/-) mice, we aimed to demonstrate that knockout of PDCD5 gene could protect the brain from ischemic injury by inhibiting the PDCD5-VHL pathway. 24h post MCAO surgery, PDCD5 gene knockout mice presented obvious improved brain blood flow, improved neurological behavior and decreased cerebral infarction compared with wild type mice. The levels of apoptotic and autophagic proteins were increased both in wild type and PDCD5 knockout mice, whereas they were more pronounced in the wild type mice. We observed decrease in the expression of VHL in wild type mice after MCAO. Reduced expression of VHL may result in increased expression of hypoxia-inducible factor 1α(HIF-1α) and (BCL2/adenovirus E1B 19kDa protein-interacting protein 3) BNIP3. However, mice lacking PDCD5 had no changes in the expression of VHL and have slighter increases in the expression of HIF-1α and BNIP3, suggesting that PDCD5 may regulate apoptosis and autophagy through VHL-HIF-1α-BNIP3 pathway.

Protection against Experimental Stroke by Ganglioside GM1 Is Associated with the Inhibition of Autophagy.

UNLABELLED: Ganglioside GM1, which is particularly abundant in the central nervous system (CNS), is closely associated with the protection against several CNS disorders. However, controversial findings have been reported on the role of GM1 following ischemic stroke. In the present study, using a rat middle cerebral artery occlusion (MCAO) model, we investigated whether GM1 can protect against ischemic brain injury and whether it targets the autophagy pathway. GM1 was delivered to Sprague-Dawley male rats at 3 doses (25 mg/kg, 50 mg/kg, 100 mg/kg) by intraperitoneal injection soon after reperfusion and then once daily for 2 days. The same volume of saline was given as a control. Tat-Beclin-1, a specific autophagy inducer, was administered by intraperitoneal injection at 24 and 48 hours post-MCAO. Infarction volume, mortality and neurological function were assessed at 72 hours after ischemic insult. Immunofluorescence and Western blotting were performed to determine the expression of autophagy-related proteins P62, LC3 and Beclin-1 in the penumbra area. No significant changes in mortality and physiological variables (heart rate, blood glucose levels and arterial blood gases) were observed between the different groups. However, MCAO resulted in enhanced conversion of LC3-I into LC3-II, P62 degradation, high levels of Beclin-1, a large area infarction (26.3±3.6%) and serious neurobehavioral deficits. GM1 (50 mg/kg) treatment significantly reduced the autophagy activation, neurobehavioral dysfunctions, and infarction volume (from 26.3% to 19.5%) without causing significant adverse side effects. However, this biological function could be abolished by Tat-Beclin-1. IN CONCLUSION: GM1 demonstrated safe and robust neuroprotective effects that are associated with the inhibition of autophagy following experimental stroke.

Effects and Mechanism of Action of Inducible Nitric Oxide Synthase on Apoptosis in a Rat Model of Cerebral Ischemia-Reperfusion Injury.

Inducible nitric oxide synthase (iNOS) is a key enzyme in regulating nitric oxide (NO) synthesis under stress, and NO has varying ability to regulate apoptosis. The aim of this study was to investigate the effects and possible mechanism of action of iNOS on neuronal apoptosis in a rat model of cerebral focal ischemia and reperfusion injury in rats treated with S-methylisothiourea sulfate (SMT), a high-selective inhibitor of iNOS. Seventy-two male Sprague-Dawley (SD) rats were randomly divided into three groups: the sham, middle cerebral artery occlusion (MCAO) + vehicle, and MCAO + SMT groups. Neurobehavioral deficits, infarct zone size, and cortical neuron morphology were evaluated through the modified Garcia scores, 2,3,5-triphenyltetrazolium chloride (TTC), and Nissl staining, respectively. Brain tissues and serum samples were collected at 72 hr post-reperfusion for immunohistochemical analysis, Western blotting, Terminal deoxynucleotidyl transferase-mediated dUTP-biotin Nick End Labeling assay (TUNEL) staining, and enzyme assays. The study found that inhibition of iNOS significantly attenuated the severity of the pathological changes observed as a result of ischemia-reperfusion injury: SMT reduced NO content as well as total nitric oxide synthase (tNOS) and iNOS activities in both ischemic cerebral hemisphere and serum, improved neurobehavioral scores, reduced mortality, reduced the infarct volume ratio, attenuated morphological changes in cortical neurons, decreased the rate of apoptosis (TUNEL and caspase-3-positive), and increased phospho (p)-AKT expression in ischemic penumbra. These results suggested that inhibition of iNOS might reduce the severity of ischemia-reperfusion injury by inhibiting neuronal apoptosis via maintaining p-AKT activity.

The role of rhynchophylline in alleviating early brain injury following subarachnoid hemorrhage in rats.

Rhynchophylline (Rhy) has been demonstrated protective effects on some neurological diseases. However, the roles of Rhy in the subarachnoid hemorrhage (SAH) are still to be cleared. In the present study, the effects of Rhy on attenuation of early brain injury (EBI) after SAH have been evaluated. The adult male Sprague-Dawley rats (280-300g) were used to establish the SAH models using endovascular perforation method. Rhy was administered by intraperitoneal injection immediately following SAH. Brain edema was assessed by magnetic resonance imaging (MRI) at 24h after SAH. Neurological deficits, brain water content, malondialdehyde (MDA) concentration, myeloperoxidase (MPO) activity and reactive oxygen species (ROS) content in hippocampus were also evaluated. Immunofluorescence and western blot were used to explore the underlying protective mechanism of Rhy. The results showed that, following 10mg/kg Rhy treatment, the brain edema and neurological deficits, and blood-brain barrier (BBB) disruption were significantly attenuated at 24h after SAH. Additionally, in hippocampus, MDA concentration, MPO activity and ROS content were markedly decreased. Meanwhile, the levels of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase (NQO-1) were increased, while the expressions of p-p53, cleaved-caspase-3 and tumor necrosis factor-α (TNF-α) were significantly decreased. Our results indicated that Rhy could attenuate early brain injury by reducing inflammation and apoptosis in hippocampus after SAH.

The Effects of Methylene Blue on Autophagy and Apoptosis in MRI-Defined Normal Tissue, Ischemic Penumbra and Ischemic Core.

Methylene blue (MB) USP, which has energy-enhancing and antioxidant properties, is currently used to treat methemoglobinemia and cyanide poisoning in humans. We recently showed that MB administration reduces infarct volume and behavioral deficits in rat models of ischemic stroke and traumatic brain injury. This study reports the underlying molecular mechanisms of MB neuroprotection following transient ischemic stroke in rats. Rats were subjected to transient (60-mins) ischemic stroke. Multimodal MRI during the acute phase and at 24 hrs were used to define three regions of interest (ROIs): i) the perfusion-diffusion mismatch salvaged by reperfusion, ii) the perfusion-diffusion mismatch not salvaged by reperfusion, and iii) the ischemic core. The tissues from these ROIs were extracted for western blot analyses of autophagic and apoptotic markers. The major findings were: 1) MB treatment reduced infarct volume and behavioral deficits, 2) MB improved cerebral blood flow to the perfusion-diffusion mismatch tissue after reperfusion and minimized harmful hyperperfusion 24 hrs after stroke, 3) MB inhibited apoptosis and enhanced autophagy in the perfusion-diffusion mismatch, 4) MB inhibited apoptotic signaling cascades (p53-Bax-Bcl2-Caspase3), and 5) MB enhanced autophagic signaling cascades (p53-AMPK-TSC2-mTOR). MB induced neuroprotection, at least in part, by enhancing autophagy and reducing apoptosis in the perfusion-diffusion mismatch tissue following ischemic stroke.

Post-hypoxic and ischemic neuroprotection of BMP-7 in the cerebral cortex and caudate-putamen tissue of rat.

Previous reports have indicated that exogenous bone morphogenetic protein-7 (BMP-7) has a neuroprotective effect after cerebral ischemia injury and promotes motor function recovery, but the appropriate BMP-7 concentration and time course are unclear. Here, we assessed endogenous BMP-7 expression in hypoxia and ischemia-damaged brain tissues and investigated the effects of different BMP-7 concentrations in pre- and post-hypoxic primary rat neurons. The results showed that BMP-7 expression was significantly higher in the ischemic hemisphere. The expressions of BMP-7 and caspase-3 were localized in the cytoplasm of the primary cerebral cortical and caudate-putamen neurons 24h after hypoxia/reoxygenation. After BMP-7 treatment, the number of caspase-3 positive neurons began to decrease with increasing BMP-7 concentrations up to 80ng/ml, but not beyond. Although the numbers of caspase-3-positive neurons between pre- and post-hypoxia/reoxygenation were not significantly different, more dendrites were observed in the groups treated prior to hypoxia/reoxygenation. These results suggest that increased BMP-7 expression can be induced in the cerebral cortex and caudate-putamen both in vivo and in vitro in hypoxic-ischemic states. The neuroprotective mechanism of BMP-7 may include apoptosis suppression, and its effect was enhanced from 40 to 80ng/ml. Pre-hypoxic BMP-7 treatment may be useful to stimulate dendrite sprouting in non-injured neurons.

G-CSF ameliorates neuronal apoptosis through GSK-3ß inhibition in neonatal hypoxia-ischemia in rats.

Granulocyte-colony stimulating factor (G-CSF), a growth factor, has known neuroprotective effects in a variety of experimental brain injury models. Herein we show that G-CSF administration attenuates neuronal apoptosis after neonatal hypoxia-ischemia (HI) via glycogen synthase kinase-3ß (GSK-3ß) inhibition. Ten day old Sprague-Dawley rat pups (n=157) were subjected to unilateral carotid artery ligation followed by 2.5h of hypoxia or sham surgery. HI animals received control siRNA, GSK-3ß siRNA (4 µL/pup), G-CSF (50 µg/kg), G-CSF combined with 0.1 or 0.4 nM G-CSF receptor (G-CSFR) siRNA, phosphatidylinositol 3-kinase (PI3K) inhibitor Wortmannin (86 ng/pup), or DMSO (vehicle for Wortmannin). Pups were euthanized 48 h post-HI to quantify brain infarct volume. G-CSFR, activated Akt (p-Akt), activated GSK-3ß (p-GSK-3ß), Cleaved Caspase-3 (CC3), Bcl-2, and Bax were quantified using Western blot analysis and the localizations of each was visualized via immunofluorescence staining. Neuronal cell death was determined using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Our results showed p-GSK-3ß increased after HI until its peak at 48 h post-ictus, and both GSK-3ß siRNA and G-CSF administration reduced p-GSK-3ß expression, as well as infarct volume. p-GSK-3ß and CC3 were generally co-localized in neurons. Furthermore, G-CSF increased p-Akt expression and the Bcl-2/Bax ratio and also decreased p-GSK-3ß and CC3 expression levels in the ipsilateral hemisphere, which were all reversed by G-CSFR siRNA, Wortmannin, and GSK-3ß siRNA. In conclusion, G-CSF attenuated caspase activation and reduced brain injury by inhibiting GSK-3ß activity after experimental HI in rat pups. This neuroprotective effect was abolished by both G-CSFR siRNA and Wortmannin.

Voltage-dependent anion channels (VDACs) promote mitophagy to protect neuron from death in an early brain injury following a subarachnoid hemorrhage in rats.

The term mitophagy is coined to describe the selective removal of mitochondria by autophagy but the process itself is still contentious, especially in the early period following subarachnoid hemorrhage (SAH). In the present study, we investigated the role of mitophagy following 48h after SAH injury in rats. Specifically evaluating whether mitophagy, through voltage dependant anion channels (VDACs) interacting with microtubule-associated protein 1 light chain 3, could orchestrate the induction of apoptotic and necrotic cell death in neurons, a VDAC1siRNA and an activitor Rapamycian (RAPA), were engaged. One hundred and twelve male Sprague-Dawley rats were randomly divided into 4 groups: Sham, SAH, SAH+VDAC1siRNA, and SAH+RAPA. Outcomes measured included mortality rate, brain edema, BBB disruption, and neurobehavioral testing. We also used western blotting techniques to analyze the expressions of key mitophagic/autophagic proteins and pro-apoptotic protein such as ROS, VDAC1, LC-3II and Caspase-3. Rapamycin treatment significantly improved the mortality rate, cerebral edema, and neurobehavioral deficits; apoptotic and necrotic cell death in neurons were reduced by Rapamycin following SAH injury. However, VDAC1siRNA worsened the brain injury following SAH. Immunohistochemical staining and western blot analysis demonstrated a decreased expression of VDAC1, LC3II, and an increase of ROS and Caspase-3 followed by VDAC1siRNA administration. In conclusion, mitophagy induced by VDAC1 following SAH injury may in fact play a significant role in neuroprotection, the mechanism which may be through the attenuation of the apoptosic and necrosic molecular pathways. This translates a preservation of functional integrity and an improvement in mortality.

Role of NADPH oxidase in total salvianolic acid injection attenuating ischemia-reperfusion impaired cerebral microcirculation and neurons: implication of AMPK/Akt/PKC.

OBJECTIVE: TSI is a new drug derived from Chinese medicine for treatment of ischemic stroke in China. The aim of this study was to verify the therapeutic effect of TSI in a rat model of MCAO, and further explore the mechanism for its effect. METHODS: Male Sprague-Dawley rats were subjected to right MCAO for 60 minutes followed by reperfusion. TSI (1.67 mg/kg) was administrated before reperfusion via femoral vein injection. Twenty-four hours after reperfusion, the fluorescence intensity of DHR 123 in, leukocyte adhesion to and albumin leakage from the cerebral venules were observed. Neurological scores, TTC staining, brain water content, Nissl staining, TUNEL staining, and MDA content were assessed. Bcl-2/Bax, cleaved caspase-3, NADPH oxidase subunits p47(phox)/p67(phox)/gp91(phox), and AMPK/Akt/PKC were analyzed by Western blot. RESULTS: TSI attenuated I/R-induced microcirculatory disturbance and neuron damage, activated AMPK, inhibited NADPH oxidase subunits membrane translocation, suppressed Akt phosphorylation, and PKC translocation. CONCLUSIONS: TSI attenuates I/R-induced brain injury in rats, supporting its clinic use for treatment of acute ischemic stroke. The role of TSI may benefit from its antioxidant activity, which is most likely implemented via inactivation of NADPH oxidase through a signaling pathway implicating AMPK/Akt/PKC.

The ameliorating effects of long-term electroacupuncture on cardiovascular remodeling in spontaneously hypertensive rats.

BACKGROUND: The purpose of this study was to investigate the inhibitory effects of long-term electroacupuncture at BaiHui (DU20) and ZuSanLi (ST36) on cardiovascular remodeling in spontaneously hypertensive rats (SHR) and underlying mechanisms. METHODS: 6-weeks-old SHR or Wistar male rats were randomly, divided into 6 groups: the control group (SHR/Wistar), the non-acupoint electroacupuncture stimulation group (SHR-NAP/Wistar-NAP) and the electroacupuncture stimulation at DU20 and ST36 group (SHR-AP/Wistar-AP), 24 rats in each group. Rats were treated with or without electroacupuncture at DU20 and ST36, once every other day for a period of 8 weeks. The mean arterial pressure (MAP) was measured once every 2 weeks. By the end of the 8th week, the left ventricular structure and function were assessed by echocardiography. The content of angiotensin II (Ang II), endothelin-1 (ET-1) and nitric oxide (NO) in the plasma was determined using enzyme-linked immunosorbent assay. Histological studies on the heart and the ascending aorta were performed. The expression of angiotensin II type 1 receptor (AT1R), endothelin-1 type A receptor (ETAR), eNOS and iNOS in rat myocardium and ascending aorta was investigated by Western blotting. RESULTS: The MAP in SHR increased linearly over the observation period and significantly reduced following electroacupuncture as compared with sham control SHR rats, while no difference in MAP was observed in Wistar rats between electroacupuncture and sham control. The aortic wall thickness, cardiac hypertrophy and increased collagen level in SHR were attenuated by long term electroacupuncture. The content of Ang II, ET-1 in the plasma decreased, but the content of NO increased after electroacupuncture stimulation in SHR. Long term electroacupuncture significantly inhibited the expression of AT1R, ETAR and iNOS, whereas increased eNOS expression, in myocardium and ascending aorta of SHR. CONCLUSIONS: The long term electroacupuncture stimulation at DU20 and ST36 relieves the increased MAP and cardiovascular abnormality in both structure and function in SHR, this beneficial action is most likely mediated via modulation of AT1R-AT1R-ET-1-ETAR and NOS/NO pathway.

Autophagic effect of programmed cell death 5 (PDCD5) after focal cerebral ischemic reperfusion injury in rats.

Former studies indicated that programmed cell death 5 (PDCD5) protein could accelerate the process of apoptosis in response to some stimuli in various kinds of cells via the intrinsic or extrinsic pathway. In this study, we aimed to demonstrate for the first time that protein level of PDCD5 are related to autophagic activity after focal ischemic brain injury in rats. One hundred and twenty-five Sprague-Dawley rats (male) were randomly divided into the following groups: Sham operated, Middle Cerebral Artery Occlusion/Reperfusion (MCAO), MCAO+Control siRNA and MCAO+PDCD5 siRNA. Outcome measurements include neurobehavioral outcomes, brain infarct volume, brain water content, BBB disruption, MRI and double fluorescence labeling. Western blot and histopathophysiological techniques were used to measure the expression of PDCD5 and some pro-autophagic proteins such as Beclin 1 and the LC3-II/LC3-I ratio. The study found that decreased PDCD5 expression via intracerebroventricular injection of PDCD5 siRNA significantly improved the neurobehavioral outcome, reduced the infarct ratio, cerebral edema and BBB disruption. These results were associated with decreased expression of Beclin 1 and the LC3-II/LC3-I ratio in the penumbra area. Rapamycin, an inducer of autophagy, partially weakened the effect of PDCD5 siRNA. In conclusion, this study suggested that PDCD5 was a key regulator of autophagy that might play an important role following MCAO injury.