Changes of c-fos, malondialdehyde and lactate in brain tissue after global cerebral ischemia under different brain temperatures.

Under global cerebral ischemia, the effect of different brain temperature on cerebral ischemic injury was studied. Male Sprague-Dawley rats were divided into normothermic (37-38°C) ischemia, mild hypothermic (31-32°C) ischemia, hyperthermic (41-42°C) ischemia and sham-operated groups. Global cerebral ischemia was established using the Pulsinelli four-vessel occlusion model and brain temperature was maintained at defined level for 60 min after 20-min ischemia. The expression of c-fos protein and the levels of malondialdehyde (MDA) and lactate in brain regions were detected by immunochemistry and spectrophotometrical methods, respectively. C-fos positive neurons were found in the hippocampus and cerebral cortex after cerebral ischemia reperfusion. Mild hypothermia increased the expression of c-fos protein in both areas, whereas hyperthermia decreased the expression of c-fos protein in the hippocampus at 24 h reperfusion, and the cerebral cortex at 48 h reperfusion when compared to normothermic conditions. In normothermic, mild hypothermic and hyperthermic ischemia groups, the levels of MDA and lactate in brain tissue were increased at 24, 48 and 72 h reperfusion following 20-min ischemia as compared with the sham-operated group (P<0.01). The levels of MDA and lactate in mild hypothermic group were significantly lower than those in normothermic group (P<0.01). It is suggested that brain temperature influences the translation of the immunoreactive protein product of c-fos after global cerebral ischemia, and MDA and lactate are also affected by hypothermia and hyperthermia.

Mitoxantrone exerts both cytotoxic and immunoregulatory effects on activated microglial cells.

Mitoxantrone (MX) is the most common immunosuppressive drug used in patients with rapidly worsening multiple sclerosis (MS), whose disease is not controlled by ß-interferon or glatiramer acetate. Although MX suppresses antigen-presenting cell (APC) and T-cell function in the periphery, its mechanism of action in the central nervous system (CNS) is not known. Given that MX can cross the disrupted blood-brain barrier, such as in MS patients, we in the present study have tested our hypothesis that MX in the CNS exerts cytotoxic and immunomodulatory effects on microglia, the major CNS-resident APCs that play a crucial role in MS pathogenesis. The cytotoxic effect of MX on microglial cells was determined by MTT and flow cytometry test, whereas the regulatory function was tested with enzyme-linked immunosorbent assay (ELISA) method. Indeed, we have found that MX induced microglial cell death in a dose-dependent manner, and the cell death was mainly from late apoptosis and necrosis. Further, MX induced significantly increased levels of interleukin (IL)-10 production of microglia, whereas IL-23p19 production/expression was significantly suppressed. Thus, our study for the first time demonstrates the immunosuppressive/regulatory effect of MX on microglia, which represents an important mechanism underlying the therapeutic effect of this drug on MS patients.

Sonic hedgehog protects cortical neurons against oxidative stress.

Oxidative stress is one of the most important pathological mechanisms in neurodegenerative diseases and ischemia. Recent studies have indicated that the sonic hedgehog (SHH) signaling pathway is involved in these diseases, but the underlying mechanisms remains elusive. Here we report that the SHH pathway was activated in primary cultured cortical neurons after exposure to hydrogen peroxide (H2O2). H2O2 treatment decreased the cell viability of neurons, and inhibition of endogenous SHH signaling exacerbated its neurotoxicity. Activation of SHH signaling protected neurons from H2O2-induced apoptosis and increased the cell viability while those effects were partially reversed by blocking SHH signals. Exogenous SHH increased the activities of Superoxide dismutase (SOD) and Glutathione peroxidase (GSH-PX) in H2O2-treated neurons and decreased production of Malondialdehyde (MDA). It also promoted expression of the anti-apoptotic gene Bcl-2 and inhibited expression of pro-apoptotic gene Bax. Activation of SHH signals upregulated both Neurotrophic factors vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF). Pretreatment with SHH inhibited the activation of ERK (extracellular signal-regulated kinases) signals induced by H2O2. Our findings demonstrate that activation of SHH signaling protects cortical neurons against oxidative stress and suggest a potential role of SHH for the clinic treatments of brain ischemia and neurodegenerative disorders.

A critical role of Sonic Hedgehog signaling in maintaining the tumorigenicity of neuroblastoma cells.

Accumulated evidence suggests a major role for the activation of the Sonic Hedgehog (SHH) signaling pathway in the development of neural crest stem cells that give rise to the sympathetic nervous system. We therefore investigated the involvement of SHH signaling in the pathogenesis of neuroblastoma, a common childhood malignant tumor of the sympathetic nervous system. Human neuroblastoma cell lines and a majority of primary neuroblastoma specimens showed high-level expression of the pathway targets and components, indicating persistent activation of the SHH pathway. All of the neuroblastoma cell lines we examined expressed significant levels of SHH ligand, suggesting an autocrine, ligand-dependent activation of the SHH pathway in neuroblastoma cells. Inhibition of SHH signaling by cyclopamine induced apoptosis and blocked proliferation in all major types of neuroblastoma cells, and abrogated the tumorigenicity of neuroblastoma cells. Moreover, the knockdown of GLI2 in neuroblastoma BE (2)-C and SK-N-DZ cell lines resulted in the inhibition of colony formation. Our study has revealed a molecular mechanism for the persistent activation of the SHH pathway which promotes the development of neuroblastoma, and suggests a new approach for the treatment of this childhood malignant tumor. (Cancer Sci 2009; 100: 1848-1855).

Effect of neuregulin on apoptosis and expressions of STAT3 and GFAP in rats following cerebral ischemic reperfusion.

The aim is to investigate the effect of neuregulin-1beta (NRG-1beta) on the neuronal apoptosis and the expressions of signal transducer and activator of transcription (STAT3) and glial fibrillary acidic protein (GFAP) in rats following cerebral ischemia/reperfusion. The animal models of middle cerebral artery occlusion/reperfusion (MCAO/R) were established by an intraluminal filament method from left external-internal carotid artery in 100 cases of adult healthy male Wister rats. NRG-1beta was administered from the internal carotid artery (ICA) into MCA in the treatment group. The neuronal apoptosis was detected by terminal deoxynucleotidyl transference-mediated biotinylated deoxyuridine triphosphate nick-end labeling technique. The expression alternations of STAT3 and GFAP proteins were determined by fluorescent labeling analysis and Western blotting assay. Ischemic cerebral injury could induce neuronal apoptosis. Furthermore, with the duration of ischemia, the amount of apoptotic cells increased in the control group. These apoptotic cells distributed in various brain regions, especially the cortex, striatum, and hippocampus, while only a small amount of apoptotic cells could be observed in the treatment group, and there were significant differences compared with that in the control group (P < 0.01). The expressions of STAT3 and GFAP proteins in brain tissue gradually increased in the control group with the duration of ischemia. And NRG-1beta could elevate the expressional level of STAT3 and GFAP proteins in contrast to the control group (P < 0.05). NRG-1beta may play a neuroprotective role in cerebral ischemic insult by activating JAK/STAT signal transduction pathway, promoting the astrocyte gumnosis and regulating the anti-apoptosis mechanism in neurocytes.

Effects of neuregulin on expression of MMP-9 and NSE in brain of ischemia/reperfusion rat.

The aim is to investigate the effects of neuregulin-1beta (NRG-1beta) on expression of matrix metalloproteinase-9 (MMP-9) and neuron-specific enolase (NSE) in brain tissue in rats following cerebral ischemia/reperfusion. One hundred and fifty adult healthy male Wistar rats were used in the present study. Ten of them were randomized into a sham-operation group (n = 10) and the rest suffered surgery operation of middle cerebral artery occlusion/reperfusion with intraluminal monofilament suture from the left external-internal carotid artery. As a result, 100 rats of successful models were randomly divided into a control group (n = 50) and a treatment group (n = 50). Rats in the treatment group were injected 1.5% NRG-1beta at a dosage of 0.3 microg/kg from the stump of the left external carotid artery into the internal carotid artery. The expressions of MMP-9 and NSE proteins were determined by immunohistochemical, immunofluorescent double labeling, and Western blot assay. Ischemia/reperfusion induced morphological changes of brain tissue, including neurocyte shrinkage, chromatin condensation, nuclei fragment, and gliacyte and endothelial cell swelling. NRG-1beta obviously reduced and delayed the cerebral damage. With the duration of ischemia, the expression of MMP-9 gradually increased in the control group. NRG-1beta decreased the level of MMP-9 compared with that in the control group (P < 0.01). NSE immunoreaction transiently elevated at the early stage of cerebral ischemia insult, and then gradually decreased in the control group. The administration of NRG-1beta significantly increased the level of NSE, and thus delayed the time and the degree of neuron damage. There were statistical differences in contrast to the control group (P < 0.01). There was no relationship between the expressions of the two proteins. MMP-9 might aim at various target cells at different stages and contribute to the inflammatory reaction after cerebral ischemia-reperfusion insult. NRG-1beta inhibits the activation of MMP-9 and development of inflammation, enhances the activity of NSE, improves the microenvironment of neuron survivals, and delays the phase of irreversible neuron necrosis. Therefore, NRG-1beta may play a neuroprotective role in cerebral ischemia/reperfusion.

Activation of sonic hedgehog signaling pathway in olfactory neuroblastoma.

OBJECTIVES: Sonic hedgehog (Shh) signaling pathway is associated with tumor development; however, the role of Shh signaling in the development of olfactory neuroblastoma (ONB) is unknown. This study aimed to investigate the relationship between the regulation of Shh signaling and the pathogenesis of ONB. METHODS: The expression of Shh signaling components was characterized by immunohistochemistry in human non-tumor olfactory epithelium and ONB specimens, and by RT-PCR and immunoblotting in human ONB cell lines. The impact of the treatment with cyclopamine (a selective inhibitor of the Shh pathway) and/or exogenous Shh on ONB cell proliferation, cycle and apoptosis was examined by MTT, soft agar colony formation and flow cytometry assays, respectively. The influence of Shh signaling on the expression of Shh signaling components and cell cycle-related regulators was determined by immunoblotting and quantitative RT-PCR, respectively. RESULTS: The expression of Pacthed1, Gli1 and Gli2 was detected in 70, 70, and 65% of human ONB specimens, respectively, and in proportion of ONB cell lines, but not in non-tumor olfactory epithelium. Treatment with cyclopamine inhibited the proliferation and colony formation of ONB cells, induced ONB cell cycle arrest and apoptosis, and down-regulated the expression of Pacthed1, Gli1 and cyclin D1, but up-regulated p21 expression in vitro. These regulatory effects of cyclopamine were partially or completely erased by exogenous Shh. CONCLUSION: These data suggest that the Shh signaling pathway is crucial for the growth of ONB.

[Effects of transcranial magnetic stimulation on recovery of neural functions and changes of synaptic interface and dendritic structure in the contralateral brain area after cerebral infarction: experiment with rats].

OBJECTIVE: To evaluate the effects of transcranial magnetic stimulation (TMS) on the brain plasticity and its role in functional outcome in cerebral infarction. METHODS: Twenty male SD rats underwent suture of the unilateral middle cerebral artery (MCA) so as to establish focal cerebral infarction models and then were randomly divided into 2 equal groups: model group, to be reared in the original living state, and TMS group, given in addition TMS treatment 1 day after infarction 2 times per day and 30 pulses per time for 4 weeks. Twenty-eight days after the rats were killed. Four rats from each group underwent microscopy of the brain to measure the dendritic structure of the pyramidal cells quantitatively. Other 4 rats from each group underwent electron microscopy of the brain to measure the parameters of synaptic interface in the sensorimotor cortex. Neural function scoring was conducted 24 hours after the establishment of model and before being killed. RESULTS: There was no significant difference in the neural function 24 h after the establishment of models, however, 28 days after the score of neural function of the TMA group was 0.58 +/- 0.49, significantly lower than that of the model group (0.92 +/- 0.28, P < 0.05). The total dendritic length, number of dendritic branching points, and dendritic density in layer V pyramidal cells within the undamaged motor cortex of the TMS group were 898 microm +/- 127 microm, 6.6 +/- 1.5, and 0.75/microm +/- 0.19/microm, all significantly higher than those of the model group (788 microm +/- 112 microm, 5.8 +/- 1.5, and 0.60/microm +/- 0.16/microm, P < 0.05 or < 0.01). Electron microscopy showed that the synaptic curvatures and post-synaptic density of the TMS group were 1.06 +/- 0.08 and 64 +/- 13 respectively, both significantly higher than those of the model group (1.02 +/- 0.06 and 54 +/- 12 nm respectively, P < 0.05 and P < 0.01), and the synapse cleft width of the TMS group was 19.5 +/- 2.1, significantly narrower than that of the model group (23.3 +/- 2.3, P < 0.01). CONCLUSION: TMS promotes the improvement of neural functions of the rats with cerebral ischemia by the potential mechanism that TMS strengthen the compensatory roles of the synaptic interface and dendritic structure in the undamaged sensorimotor cortex area and increase synaptic plasticity.

Corilagin Protects Against HSV1 Encephalitis Through Inhibiting the TLR2 Signaling Pathways In Vivo and In Vitro.

In this study, we tried to explore the molecular mechanism that Corilagin protected against herpes simplex virus-1 encephalitis through inhibiting the TLR2 signaling pathways in vivo and in vitro. As a result, Corilagin significantly prevented increase in the levels of TLR2 and its downstream mediators following Malp2 or HSV-1 challenge. On the other hand, in spite of TLR2 knockdown, Corilagin could still significantly suppress the expression of P38 and NEMO, phosphor-P38, and nuclear factor kappa B. The mRNA and protein expression of TLR2 and its downstream mediators in the brain tissue were also significantly lowered in mice treated with Corilagin. In addition, Corilagin inhibited expression of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 protein. In conclusion, Corilagin shows the potential to protect against HSV-1-induced encephalitis, and the beneficial effects may be mediated by inhibiting TLR2 signaling pathways.

Involvement of TLR2 and TLR9 in the anti-inflammatory effects of chlorogenic acid in HSV-1-infected microglia.

AIMS: There is no effective medication to date for herpes simplex virus encephalitis (HSE). In this study, we investigated the anti-inflammatory effect of chlorogenic acid (CGA) on herpes simplex virus (HSV)-1-induced responses in BV2 microglia. MAIN METHODS: The cellular model was established with BV2 cells stimulated by HSV-1 and then treated with CGA at different concentrations. Cell viability was assayed by the MTT assay. The mRNA expression of Toll-like receptor (TLR)-2, TLR9 and myeloid differentiation factor88 (Myd88) was assayed by real-time quantitative PCR, and the protein expression was assayed by flow cytometry or Western blotting. Tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 were measured by ELISA as well as real-time quantitative PCR. Nuclear NF-κB p65 protein was assayed by Western blotting. KEY FINDINGS: The cell survival rate was significantly improved after CGA treatment, and CGA prevented increases in TLR2, TLR9 and Myd88 following HSV-1 challenge in BV2 cells both at the mRNA and protein levels. Moreover, CGA could attenuate HSV-induced TNF-α and IL-6 release into the supernatant. The mRNA levels of TNF-α and IL-6 were also significantly inhibited by CGA. The expression of NF-κB p65 increased significantly in the nucleus in HSV-1-stimulated microglia but could be reduced by CGA. SIGNIFICANCE: CGA inhibits the inflammatory reaction in HSE via the suppression of TLR2/TLR9-Myd88 signaling pathways. CGA may serve as an anti-inflammatory agent and provide a new strategy for treating HSE.

[Effects of three-stage rehabilitation treatment on acute cerebrovascular diseases: a prospective, randomized, controlled, multicenter study].

OBJECTIVE: To evaluate the effect of tertiary rehabilitation treatment on acute cerebrovascular diseases. METHODS: Fifteen tertiary rehabilitation networks were set up throughout the country. 1078 patients with acute cerebrovascular diseases were randomly divided into 2 groups: rehabilitation group and control group, out of which 19 patients died, 157 dropped out, and 7 successive evaluations were completed in 902 patients that. 439 of the remaining 902 patients in the rehabilitation group, 266 males and 173 females, aged 61 +/- 11, 278 cases with cerebral infarction and 161 with cerebral hemorrhage, received routine treatment and early rehabilitation for 28 days in the ward of neurology, and then went home and received community rehabilitation for 6 months or underwent specialized reinforcement training for 2 months and after that went home and received community rehabilitation for 4 months. The 463 patients in the control group, 281 males and 182 females, aged 60 +/- 11, 291 of which with cerebral infarction and 172 with cerebral hemorrhage, received only routine treatment and early rehabilitation for 28 days in the ward of neurology, and then went home to conduct rehabilitation training by themselves or their family members for 6 months. Evaluation was conducted 7 times, with National Institutes of Health Stroke Scale (NIHSS), Fugl-Meyer motor function scale, Barthel index, SF-36 scale, Lowenstein occupational therapy cognitive assessment (LOTCA), Westen aphasia battery, Hamilton depression scale, and modified Ashworth spasm scale, one week after the onset and by the ends of 1, 2, 3, 4, 5, and 6 months after the onset respectively. RESULTS: The scores of clinical neurological impairment, Fugl-Meyer scores, SF-36 scores, incidence of PSD, and modified Ashworth scores (for upper and lower limbs) were lower, and LOTCA scores and Barthel indexes were higher at different time points in the rehabilitation group than in the control group; and the differences were statistically significant since the 2nd month after the onset. By the end of the 6th month, the patients of the rehabilitation group basically re-achieved the ability of self-care in daily activities with a Barthel index of 84 +/- 33. The patients of the control group also recovered to a certain degree, however, to a smaller extent in comparison with the rehabilitation group. CONCLUSION: Tertiary rehabilitation treatment of cerebrovascular diseases is effective in improving motor function, ability of daily living activities, and quality of life and reducing the incidence rates of secondary complications.

[Protective effect of ginkgo biloba extract on cerebral ischemia/reperfusion injury in rats].

OBJECTIVE: To study the effect of Ginkgo biloba extract (GbE) on dynamic equilibrium of free radicals and amino-acids in cortex of rats with cerebral ischemia/reperfusion (I/R) injury and its influence and characteristics to intracellular free calcium concentration ([Ca2+]i) in primary cultured hippocampal neuron of rats. METHODS: Amino-acids were quantified by high performance liquid chromatographic (HPLC) analysis. Concentration of MDA and GSH-Px were determined by thiobarbituric acid (TBA) technique. SOD was assayed through xanthine method, and microfluoremetric technique was used to assay the change of [Ca2+]i and its characteristics. RESULTS: Compared with the non-treated groups, at all time points (3 hrs after ischemia, 1 and 2 hrs after I/R separately), in the GbE treated groups, the levels of Glu, Asp and MDA were lower and SOD and GSH-Px were higher (P < 0.01 or P < 0.05), the GABA and Gly levels were lower in groups treated with middle (10 mg/kg) or high dosage (15 mg/kg) of GbE (P < 0.05). Compared with the group treated with small dosage GbE (5 mg/kg), Glu, Asp and MDA were lower and GABA, Gly, SOD and GSH-Px were higher in the groups treated with middle or high dosage of GbE (P < 0.05), while the difference in the latter two groups was insignificant. Level of [Ca2+]i in cultured neurons treated with 1 x 10(-5) mol/L glutamate combined 25 micrograms/ml GbE for 20s was lower with lower peak value and longer time for reaching the peak than that in neurons treated with 1 x 10(-5) mol/L glutamate alone. Besides, the time of decline phase was also shorter in the former, so the flatform stage was prolonged. The response was recovered by re-applying of glutamate after [Ca2+]i back to base line. CONCLUSION: GbE can protect damaged neurons through keeping the balance of inhibitory/excitatory amino-acids, enhancing free radicals scavengers system, and inhibiting the effect of glutamate to [Ca2+]i.

[Development and clinical application of the EMG information management system].

This paper introduces an EMG multi-gateway analysis diagnosis and information management system. The clinical applications show that this system has higher efficiency and standard report contents, and easy statistical analysis. And it also offers EMG standard figure, normal value data, nerve and muscle select scheme etc, for reference.

Passive movement improves the learning and memory function of rats with cerebral infarction by inhibiting neuron cell apoptosis.

Passive movement has been found to improve evidently ischemic stroke patients' impaired capacity of learning and memory, but the optimal time window of initiating the therapy and the underlying mechanism are not fully understood. In this study, the effect of passive movement at different time windows on learning and memory of rats with cerebral infarction was detected. The results showed that the expression of caspase-3 and escape latency in the passive movement group were all considerably lower than those in the model group (P < 0.05), while the expression of Bcl-2 mRNA was significantly higher than those in the model group (P < 0.05). Moreover, we found that there were most significant changes of escape latency and expressions of Bcl-2 mRNA and caspase-3 when the therapy started at 24 h after focal cerebral infarction. These results suggest that passive movement is able to contribute to the recovery of learning and memory of rats with cerebral infarction, which is partially mediated by inhibiting neuron cell apoptosis, and the optimal therapeutic time is at 24 h after cerebral infarction.

Astrocyte-derived sonic hedgehog contributes to angiogenesis in brain microvascular endothelial cells via RhoA/ROCK pathway after oxygen-glucose deprivation.

The human adult brain possesses intriguing plasticity, including neurogenesis and angiogenesis, which may be mediated by the activated sonic hedgehog (Shh). By employing a coculture system, brain microvascular endothelial cells (BMECs) cocultured with astrocytes, which were incubated under oxygen-glucose deprivation (OGD) condition, we tested the hypothesis that Shh secreted by OGD-activated astrocytes promotes cerebral angiogenesis following ischemia. The results of this study demonstrated that Shh was mainly secreted by astrocytes and the secretion was significantly upregulated after OGD. The proliferation, migration, and tube formation of BMECs cocultured with astrocytes after OGD were significantly enhanced, but cyclopamine (a Shh antagonist) or 5E1 (an antibody of Shh) reversed the change. Furthermore, silencing Ras homolog gene family, member A (RhoA) of BMECs by RNAi and blocking Rho-dependent kinase (ROCK) by Y27632, a specific antagonist of ROCK, suppressed the upregulation of proliferation, migration, and tube formation of BMECs after OGD. These findings suggested that Shh derived from activated astrocytes stimulated RhoA/ROCK pathway in BMECs after OGD, which might be involved in angiogenesis in vitro.

S-methylisothiourea induces apoptosis of herpes simplex virus-1-infected microglial cells.

Our study showed that S-methylisothiourea (SMT) had anti-inflammatory effects in treating herpes simplex encephalitis in mice, and SMT also induced apoptosis of herpes simplex virus (HSV-1)-infected microglial cells. Both animal and cell models were employed in this study. Both models included the following five groups: a normal control group, a virus group (HSV-1 infected), an SMT group (HSV-1-infected + SMT (0.1 mg/10 g)), a dexamethasone group (HSV-1 infected + dexamethasone (2 µg/10 g)), and an APS group (HSV-1-infected + APS (0.8 mg/10 g)). ELISA was used to measure tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-10, and Greiss method was used for measuring nitric oxide (NO) secretion. HE staining was performed for detecting changes in mice brain. Flow cytometry assay for caspase-3, caspase-8, caspase-9, and caspase-12 expressions was also carried out to assess apoptosis. Expressions of TNF-α, IL-1ß, and NO were significantly elevated after stimulation of microglial cells with HSV-1. Following SMT intervention, TNF-α, IL-1ß, and NO levels were significantly decreased. The inflammatory changes in HSV-1-infected murine brain tissues were also reduced. SMT induction of apoptosis of HSV-stimulated microglia seemed to be through three pathways: the death receptor, mitochondrially gated, and endoplasmic reticulum. SMT can reduce HSV-induced inflammatory insult to the brain. Its mechanism of action is most probably due to the induction of microglial cell apoptosis.

Effects of immediate and delayed mild hypothermia on endogenous antioxidant enzymes and energy metabolites following global cerebral ischemia.

BACKGROUND: The optimal time window for the administration of hypothermia following cerebral ischemia has been studied for decades, with disparity outcomes. In this study, the efficacy of mild brain hypothermia beginning at different time intervals on brain endogenous antioxidant enzyme and energy metabolites was investigated in a model of global cerebral ischemia. METHODS: Forty-eight male Sprague-Dawley rats were divided into a sham-operated group, a normothermia (37°C - 38°C) ischemic group and a mild hypothermic (31°C - 32°C) ischemia groups. Rats in the last group were subdivided into four groups: 240 minutes of hypothermia, 30 minutes of normothermia plus 210 minutes of hypothermia, 60 minutes of normothermia plus 180 minutes of hypothermia and 90 minutes of normothermia plus 150 minutes of hypothermia (n = 8). Global cerebral ischemia was established using the Pulsinelli four-vessel occlusion model for 20 minutes and mild hypothermia was applied after 20 minutes of ischemia. Brain tissue was collected following 20 minutes of cerebral ischemia and 240 minutes of reperfusion, and used to measure the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), reduced glutathione (GSH) and adenosine triphosphate (ATP). RESULTS: Mild hypothermia that was started within 0 to 60 minutes delayed the consumption of SOD, GSH-Px, GSH, and ATP (P < 0.05 or P < 0.01) in ischemic tissue, as compared to a normothermic ischemia group. In contrast, mild hypothermia beginning at 90 minutes had little effect on the levels of SOD, GSH-Px, GSH, and ATP (P > 0.05). CONCLUSIONS: Postischemic mild brain hypothermia can significantly delay the consumption of endogenous antioxidant enzymes and energy metabolites, which are critical to the process of cerebral protection by mild hypothermia. These results show that mild hypothermia limits ischemic injury if started within 60 minutes, but loses its protective effects when delayed until 90 minutes following cerebral ischemia.

Favorable effects of MMP-9 knockdown in murine herpes simplex encephalitis using small interfering RNA.

BACKGROUND AND PURPOSE: The prognosis of herpes simplex encephalitis (HSE) remains poor despite available antiviral treatment. Matrix metalloproteinase-9 (MMP-9) is currently considered to play a major role in promoting cerebrovascular complications which contribute to the high mortality and morbidity of HSE. We hypothesize that temporally knockdown MMP-9 expression in early phase of HSE might be an effective treatment strategy. METHODS: The animal models of herpes simplex encephalitis were established by intracerebrally inoculated herpes simplex virus type 1 (HSV-1) in mice. Mice were inoculated intracerebrally with MMP-9 targeting siRNA (MMP-9 siRNA). MMP-9 expression was assessed by RT-PCR and western blot analysis at 3 and 7 days after HSV-1 infected. The blood-brain barrier (BBB) permeability was quantitated by Evans blue dye extravasations and brain water content. Immunohistochemistry method was adopted to analyse the expression of AQP4 protein. Quantitative real-time PCR analysis was used to detect cytokines expression. Neurological score was quantified using an established neurological scale at 7 days after HSE. RESULTS: Using synthetic small interfering RNA, we found a single intracerebral injection of siRNA targeting murine MMP-9 mRNA (MMP-9 siRNA) silenced MMP-9 expression and reduced it to normal level at day 7 post-infection. The improvement in neurological function and increased cumulative survival reflected the functional consequence of this therapy. MMP-9 knockdown mice also displayed less uptake of Evans blue and reduced brain water content compared with control siRNA-treated group. Also the HSV-1-induced upregulation of proinflammatory cytokines was significantly diminished in MMP-9 siRNA-treated mice. In addition, aquaporin-4 expression in perivascular decreased in MMP-9 siRNA-treated mice and might contribute to the protection of blood-brain barrier. DISCUSSION: This compelling evidence suggests that MMP-9 is a key pathogenic factor within HSE, and local injection of synthetic siRNA in the brain could knock down MMP-9 expression in acute phase of HSE, reduce brain edema and improves mice neurological function and increase cumulative survival.

Effect of Corilagin on anti-inflammation in HSV-1 encephalitis and HSV-1 infected microglias.

The aim of this explore is to study the anti-inflammatory effect of Corilagin in herpes simplex virus (HSV)-1 infected microglial cells and HSV-1 infected mouse brain. The cellular model was set with microglial cells stimulated by HSV-1 and divided respectively, into virus, astragalus polysaccharides (APS), Dexamethasone and Corilagin group. A normal control group consisting of uninfected microglial cells was also included. ELISA for measuring TNF-alpha, IL-1beta and IL-10 and Greiss method for detecting NO secretion in supernatant, flow cytometry assay for examining apoptosis rate, expression of caspase-3, caspase-8, caspase-9 and caspase-12, and western-blot for measuring protein expression of cytochrome c were performed. The animal model was set up using Balb/c male mice that were intracranially inoculated with HSV-1. Animals were then divided in groups as described for the cellular model. Here, too a normal control group was included. HE staining was used to assay pathological changes in brain. As results, after Corilagin intervention, the release of TNF-alpha, IL-1beta and NO from HSV-stimulated migroglia cells was significantly inhibited. Furthermore, Corilagin induced apoptosis of HSV-stimulated microglia through all the 3 known apoptotic pathways. The animal model treated with Corilagin also displayed significant decrease of herpes simplex encephalitis induced brain pathological changes. In conclusion, Corilagin has the potential to reduce HSV-1-induced inflammatory insult to the brain, and its mode of action is through the induction of apoptosis of microglias and reduction of cytokines production.

Cell cycle inhibitor enhances the resolution of HSV-1-induced proinflammatory response in murine microglial cells.

BACKGROUND AND PURPOSE: Herpes simplex encephalitis remains one of the most devastating intracranial infections despite available antiviral treatment, with sequelae secondary to a persistent inflammatory response. Recently, cyclin-dependent kinases (CDKs) have been found to act as cellular targets for antiviral drugs. However, the pharmacological effects of CDK inhibitors on glial cell function in herpes simplex encephalitis have not been elucidated. The aim of this work was to determine the influence of olomoucine on microglial activation during the inflammatory response after herpes simplex virus 1(HSV-1) infection. METHODS: Microglial cells were treated with various concentrations of olomoucine after HSV-1 infection. The expression change of cyclin D1 and myeloid cell leukemia 1 (Mcl-1) in microglia were detected by Western blot analysis. Flow cytometry was used to assess the apoptosis ratio of microglial cells among the groups of control, HSV-1 infected and olomoucine treated with or without zVAD-fmk. ELISA was adopted to analyse cytokines in the supernatant. We used semiquantitative reverse transcription polymerase chain reaction to detect HSV glycoprotein D gene. RESULTS: The following are the results of this work: (1) olomoucine reduced HSV-1-induced proliferation associated cyclin D1 expression; (2) olomoucine also induced microglial cells apoptosis early at 24 hours post-infection and inhibited the release of proinflammatory cytokine and chemokine, including tumor necrosis factor alpha and monocyte chemoattractant protein 1; and (3) olomoucine-induced apoptosis was caspase-dependent, and it also reduced the antiapoptotic protein Mcl-1. DISCUSSION: Our conclusion is that microglial cells are targets for olomoucine and that modulation of glial response and inflammation may be an appendant mechanism of CDK inhibitor-mediated neuroprotection in HSV-1 encephalitis.