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.

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.

Role of SCH79797 in maintaining vascular integrity in rat model of subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Plasma thrombin concentration is increased after subarachnoid hemorrhage (SAH). However, the role of thrombin receptor (protease-activated receptor-1 [PAR-1]) in endothelial barrier disruption has not been studied. The aims of this study were to investigate the role of PAR-1 in orchestrating vascular permeability and to assess the potential therapeutics of a PAR-1 antagonist, SCH79797, through maintaining vascular integrity. METHODS: SCH79797 was injected intraperitoneally into male Sprauge-Dawley rats undergoing SAH by endovascular perforation. Assessment was conducted at 24 hours after SAH for brain water content, Evans blue content, and neurobehavioral testing. To explore the role of PAR-1 activation and the specific mechanism of SCH79797's effect after SAH, Western blot, immunoprecipitation, and immunofluorescence of hippocampus tissue were performed. A p21-activated kinase-1 (PAK1) inhibitor, IPA-3, was used to explore the underlying protective mechanism of SCH79797. RESULTS: At 24 hours after SAH, animals treated with SCH79797 demonstrated a reduction in brain water content, Evans blue content, and neurobehavioral deficits. SCH79797 also attenuated PAR-1 expression and maintained the level of vascular endothelial-cadherin, an important component of adherens junctions. Downstream to PAR-1, c-Src-dependent activation of p21-activated kinase-1 led to an increased serine/threonine phosphorylation of vascular endothelial-cadherin; immunoprecipitation results revealed an enhanced binding of phosphorylated vascular endothelial-cadherin with endocytosis orchestrator ß-arrestin-2. These pathological states were suppressed after SCH79797 treatment. CONCLUSIONS: PAR-1 activation after SAH increases microvascular permeability, at least, partly through a PAR-1-c-Src-p21-activated kinase-1-vascular endothelial-cadherin phosphorylation pathway. Through suppressing PAR-1 activity, SCH79797 plays a protective role in maintaining microvascular integrity after SAH.

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.

The protective effect of Cerebralcare Granule® on brain edema, cerebral microcirculatory disturbance, and neuron injury in a focal cerebral ischemia rat model.

OBJECTIVE: The purpose of the present study was to explore the protective effects of CG on rat cerebral injury after focal cerebral I /R. METHODS: Male Sprague-Dawley rats were subjected to right middle cerebral artery occlusion for 60 minutes followed by reperfusion for 60 minutes or 24 hours. CG (0.4 or 0.8 g/kg) was administrated 90 minutes before ischemia. Brian edema was evaluated by Evan's blue dye extravasations and brain water content, leukocyte adhesion, and albumin leakage were determined with an upright fluorescence microscope, and neuron damage was assessed by 2,3,5-triphenyltetrazolium chloride staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and immunohistochemistry of caspase-3, p53, p53 upregulated modulator of apoptosis. RESULTS: Focal cerebral I/R elicited a prominent brain edema, an increase in leukocyte adhesion, and albumin leakage, as well as neuron damage. All the insults after focal cerebral I/R were significantly attenuated by pretreatment with CG. CONCLUSIONS: Pretreatment with CG significantly reduced focal cerebral I/R-induced brain edema, cerebral microcirculatory disturbance, and neuron damage, suggesting the potential of CG as a prophylactic strategy for patients in danger of stroke.

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.

Isoflurane enhanced hemorrhagic transformation by impairing antioxidant enzymes in hyperglycemic rats with middle cerebral artery occlusion.

BACKGROUND AND PURPOSE: Because the potential neuroprotective effect of isoflurane is controversial, we attempted to study whether isoflurane after treatment provides neuroprotection in a rat model of hyperglycemia-induced ischemic hemorrhagic transformation. METHODS: Rats received an injection of 50% dextrose (6 mL/kg intraperitoneally) and had a middle cerebral artery occlusion 30 minutes later. Four groups were included: sham-operated, ischemia/reperfusion, isoflurane treatment, and vehicle groups. In the treatment group, after 2 hours of ischemia, 2% isoflurane was administered at the onset of reperfusion. We measured the level of blood glucose at 0, 2.5, 4.5, and 6.5 hours after dextrose injection. Infarct and hemorrhagic volumes, neurological scores, oxidative stress (malondialdehyde, 4-hydroxy-2-nonenal, and nitrotyrosine) and the activities of superoxide dismutase and catalase were measured at 24 hours after ischemia. RESULTS: Isoflurane had no effects on blood glucose, it failed to reduce infarct, hemorrhage volume, and brain edema, and it enhanced neurobehavioral deficits when compared with the ischemia/reperfusion group at 24 hours after middle cerebral artery occlusion. On the contrary, isoflurane exacerbated these parameters compared with the vehicle group. In addition, it increased the expressions of malondialdehyde, 4-hydroxy-2-nonenal, and nitrotyrosine, and it decreased the activities of superoxide dismutase and catalase compared to the vehicle group. CONCLUSIONS: Isoflurane after treatment worsened physiological and neurological outcomes in this ischemia hyperglycemia-induced hemorrhagic transformation possibly by impairing the antioxidant defense system.

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.

Suppression of hypoxia-inducible factor-1alpha and its downstream genes reduces acute hyperglycemia-enhanced hemorrhagic transformation in a rat model of cerebral ischemia.

We evaluated a role of hypoxia-inducible factor-1alpha (HIF-1alpha) and its downstream genes in acute hyperglycemia-induced hemorrhagic transformation in a rat model of focal cerebral ischemia. Male Sprague-Dawley rats weighing 280-300 g (n = 105) were divided into sham, 90 min middle cerebral artery occlusion (MCAO), MCAO plus HIF-1alpha inhibitors, 2-methoxyestradiol (2ME2) or 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), groups. Rats received an injection of 50% dextrose (6 ml/kg intraperitoneally) at 15 min before MCAO. HIF-1alpha inhibitors were administered at the onset of reperfusion. The animals were examined for neurological deficits and sacrificed at 6, 12, 24, and 72 hr following MCAO. The cerebral tissues were collected for histology, zymography, and Western blot analysis. The expression of HIF-1alpha was increased in ischemic brain tissues after MCAO and reduced by HIF-1alpha inhibitors. In addition, 2ME2 reduced the expression of vascular endothelial growth factor (VEGF) and the elevation of active matrix metalloproteinase-2 and -9 (MMP-2/MMP-9) in the ipsilateral hemisphere. Both 2ME2 and YC-1 reduced infarct volume and ameliorated neurological deficits. However, only 2ME2 attenuated hemorrhagic transformation in the ischemic territory. In conclusion, the inhibition of HIF-1alpha and its downstream genes attenuates hemorrhagic conversion of cerebral infarction and ameliorates neurological deficits after focal cerebral ischemia.

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.

Early inhibition of HIF-1alpha with small interfering RNA reduces ischemic-reperfused brain injury in rats.

Hypoxia-inducible factor-1 (HIF-1) plays an essential role in cerebral ischemia as a proapoptotic factor. We hypothesized that HIF-1alpha siRNA can protect the brain from ischemic damage by inhibiting HIF-1alpha induced apoptotic pathway at the RNA level in a rat focal ischemic model. Results showed that treatment with HIF-1alpha siRNA reduced the infarct volume, decreased mortality, improved neurological deficits and reduced Evans blue extravasation. The expression of HIF-1alpha mRNA (Real-Time PCR) and protein were significantly silenced and the immunohistochemistry and Western blot revealed the suppression of HIF-1alpha, VEGF, p53 and Caspase-3. Double fluorescence labeling showed HIF-1alpha positive immunoreactive materials were partly colocalized with NeuN, p53 and Caspase-3 in the injured cerebral cortex. This study showed that HIF-1alpha siRNA may protect the ischemic-reperfused neurons in vivo via inhibition of HIF-1alpha, its downstream VEGF and other apoptotic-related proteins such as p53 and Caspase-3 and may have potentials for the early treatment of ischemic cerebral stroke.

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.

Pifithrin-alpha reduces cerebral vasospasm by attenuating apoptosis of endothelial cells in a subarachnoid haemorrhage model of rat.

BACKGROUND: The mechanism of cerebral vasospasm following subarachnoid haemorrhage (SAH) is not understood. Here, we hypothesized that apoptosis of endothelial cells induced by p53 and its target gene em dash p53 upregulated modulator of apoptosis (PUMA) played an important role in development of cerebral vasospasm. We also observed the effects of a p53 inhibitor, pifithrin-alpha (PFT-alpha), on reducing the expression of p53 and PUMA, consequently decreasing the apoptosis of endothelial cells and alleviating cerebral vasospasm. METHODS: Male Sprague-Dawley rats weighing 300-350 g were randomly divided into five groups: a control group (sham surgery), a SAH group, a SAH+dimethyl sulfoxide (DMSO) group, a SAH + PFT-alpha (0.2 mg/kg) group and a SAH + PFT-alpha (2.0 mg/kg) group. PFT-alpha was injected intraperitoneally immediately after SAH. Rats were sacrificed 24 hours after SAH. Western blot and immunohistochemical staining were used to detect the levels of p53, PUMA and caspase-3 protein. In addition, mortality and neurological scores were assessed for each group. Statistical significance was assured by analysis of variance performed in one way ANOVA followed by the Tukey test. The neurological and mortality scores were analyzed by Dunn's method and Fisher exact test, respectively. RESULTS: After SAH, Western blot and immunohistochemical staining showed the levels of p53, PUMA and caspase-3 in the endothelial cells and the numbers of TdT mediated dUTP nick end labelling (TUNEL) positive endothelial cells were all significantly increased in the basilar arteries (P<0.05), but significantly reduced by PFT-alpha (P<0.05). These changes were accompanied by increasing diameters and declining wall thickness of basilar arteries (P<0.05), as well as reduced mortality and neurological deficits of the rats (P<0.05). CONCLUSIONS: PFT-alpha could protect cerebral vessels from development of vasospasm and improve neurological outcome as well as reduce the mortality via suppressing apoptosis induced by p53 in the endothelial cells of cerebral vessels.

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.

Effect of dexamethasone on peroxisome proliferator activated receptor-gamma mRNA expression in 3T3-L1 adipocytes with the human recombinant adiponectin.

BACKGROUND: The fat derived protein adiponectin plays an important role in the regulation of glucose metabolism. The aim of this study was to provide the experimental basis for further investigating on adiponectin (ADPN) function. Its eukaryotic recombinant was constructed and expressed in precursor cells of 3T3-L1 adipocytes. The effects of dexamethasone on peroxisome proliferator activated receptor-gamma (PPAR-gamma) mRNA expression in 3T3-L1 cells with human recombinant adiponectin were assessed. METHODS: The recombinant plasmid pMD18-T-hADPN and eukaryotic expression vector pcDNA3.1(+) were digested by two restrictive endonucleases and adiponectin and linear pcDNA3.1(+) were obtained. Then, they were ligated and translated into JM109. The recombinant pcDNA3.1(+)-hADPN so obtained was identified by digestion by restrictive endonuclease and nucleotide sequencing. The 3T3-L1 precursor cells were transfected using SuperFect Transfection Reagent (Qiagen). Furthermore, 3T3-L1 cells with human recombinant adiponectin incubated with dexamethasone (0.5 mmol/L) for 24 hours, cells were collected and total RNA was extracted. The PPAR-gamma mRNA expression was quantified by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: After eukaryotic recombinant was digested by Hind III and EcoR I, fragments of 800 bp and 5.4 kb were identified by nucleotide sequence scanning and consistent with theoretical values. Electrophoretogram of RT-PCR in 3T3-L1 precursors showed only one band in front of 250 bp, which was consistent with theoretical value 234 bp. In the 3T3-L1 cells, 3T3-L1 cells with plasmid and 3T3-L1 cells human recombinant adiponectin, treatment with dexamethasone (0.5 mmol/L) decreased PPAR-gamma mRNA expression compared to untreated controls (P < 0.01). Effect of dexamethasone on PPAR-gamma mRNA expression in 3T3-L1 cells was reversed by stably transfected human recombinant adiponectin. CONCLUSION: The 3T3-L1 cells stably transfected human recombinant adiponectin had increased PPAR-gamma mRNA expression. Dexamethasone suppressed PPAR-gamma mRNA expression in the 3T3-L1 cells. Effect of dexamethasone on PPAR-gamma mRNA expression in 3T3-L1 cells was reversed by stably transfected human recombinant adiponectin.

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.

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.

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.