Lianjun Guo

Enhanced activity of GABA receptors inhibits glutamate release induced by focal cerebral ischemia in rat striatum

Cerebral ischemia causes an excess release of glutamate, which can injure neurons. The striatum is one of the important regions vulnerable to hypoxia and ischemia. Using push-pull perfusion technique, we investigated the regulatory role of gamma-aminobutyric acid (GABA) and its receptors in modifying the amount of glutamate in rat striatum with ischemia. Perfusion with exogenous GABA (1 mM) inhibited cerebral ischemia-induced glutamate release by as much as 47%. We further characterized relative roles of subtype receptors of GABA on glutamate release by using pharmacological tools. While baclofen (500 microM), a GABA(B) receptor agonist, suppressed ischemia-induced glutamate release by 52%, GABA(B) receptor antagonist saclofen (500 microM) failed to produce a significant increase of glutamate release. The GABA(A) receptor agonist muscimol (500 microM) also reduced by 38% the release of glutamate induced by cerebral ischemia but the GABA(A) receptor antagonist bicuculline (500 microM) had very little effect. The present study demonstrates that the excessive release of glutamate or the overly activated glutamate receptor, triggered by cerebral ischemia, can be down-regulated by exogenous GABA or by increased activity of GABA receptors, especially the presynaptic GABA(B) receptors, which might be one of the important mechanisms to protect against striatum neuronal damage from over stimulation by excessive glutamate during ischemia.

Neuroprotective Potential of Fasudil Mesylate in Brain Ischemia-Reperfusion Injury of Rats

We previously reported that inhibition of Rho-kinase (ROCK) by hydroxyl fasudil improves cognitive deficit and neuronal damage in rats with chronic cerebral ischemia (Huang et al., Cell Mol Neurobiol 28:757–768, 2008). In this study, fasudil mesylate (FM) was investigated for its neuroprotective potential in rats with ischemia following middle cerebral artery occlusion (MCAO) and reperfusion. The effect of fasudil mesylate was also studied in rat brain cortical and hippocampal slices treated with oxygen-glucose deprivation (OGD) injury. Gross anatomy showed that cerebral infarct size, measured with 2,3,5-triphenyltetrazolium chloride (TTC) staining, was significantly smaller in the FM-treated than in the non-FM-treated ischemic rats. In the brain regions vulnerable to ischemia of ischemic rats, fasudil mesylate was also found to significantly restore the enzyme protein expression level of endothelial nitric oxide synthase (eNOS), which was decreased in ischemia. However, it remarkably reduced the protein synthesis of inducible nitric oxide synthase (iNOS) that was induced by ischemia and reperfusion. In rat brain slices treated with OGD injury, fasudil mesylate increased the neuronal cell viability by 40% for cortex and by 61% for hippocampus, respectively. Finally, in the presence of OGD and fasudil mesylate, superoxide dismutase (SOD) activity was increased by 50% for cortex and by 58% for hippocampus, compared to OGD only group. In conclusion, our in vivo study showed that fasudil mesylate not only decreased neurological deficit but also reduced cerebral infarct size, possibly and at least partially by augmenting eNOS protein expression and inhibiting iNOS protein expression after ischemia-reperfusion.

Betulinic acid protects against cerebral ischemia–reperfusion injury in mice by reducing oxidative and nitrosative stress

Increased production of reactive oxygen and nitrogen species following cerebral ischemia-reperfusion is a major cause for neuronal injury. In hypercholesterolemic apolipoprotein E knockout (ApoE-KO) mice, 2h of middle cerebral artery (MCA) occlusion followed by 22h of reperfusion led to an enhanced expression of NADPH oxidase subunits (NOX2, NOX4 and p22phox) and isoforms of nitric oxide synthase (neuronal nNOS and inducible iNOS) in the ischemic hemisphere compared with the non-ischemic contralateral hemisphere. This was associated with elevated levels of 3-nitrotyrosine, an indicator of peroxynitrite-mediated oxidative protein modification. Pre-treatment with betulinic acid (50mg/kg/day for 7days via gavage) prior MCA occlusion prevented the ischemia reperfusion-induced upregulation of NOX2, nNOS and iNOS. In parallel, betulinic acid reduced the levels of 3-nitrotyrosine. In addition, treatment with betulinic acid enhanced the expression of endothelial eNOS in the non-ischemic hemispheres. Finally, betulinic acid reduced infarct volume and ameliorated the neurological deficit in this mouse stroke model. In conclusion, betulinic acid protects against cerebral ischemia-reperfusion injury in mice. This is likely to result from a reduction of oxidative stress (by downregulation of NOX2) and nitrosative stress (by reduction of nNOS and iNOS), and an enhancement of blood flow (by upregulation of eNOS).

DDPH: Improving cognitive deficits beyond its α1-adrenoceptor antagonism in chronic cerebral hypoperfused rats

DDPH (1-(2, 6-dimethylphenoxy)-2-(3, 4-dimethoxyphenylethylamino) propane hydrochloride), a candidate drug known to be an alpha(1)-adrenoceptor antagonist, can efficiently penetrate through blood brain barrier and inhibit the contraction of vascular smooth muscle in the brain. In rats with chronic cerebral hypoperfusion after permanent bilateral carotid artery ligation, we found that DDPH treatment at 6 or 12 mg/kg per day for 30 days significantly reversed pathological changes such as glial cell proliferation and nuclei shrinkage and reduced neuronal cell loss. In vivo electrophysiological studies revealed that DDPH increased long-term potentiation that was inhibited in these animals. In water maze tests, the percentage of time spent in the target quadrant (Q3) for ischemic rats (20.17+/-2.87%) was much shorter than that for the sham rats (45.39+/-3.68%), but DDPH at 12 mg/kg increased the time (39.58+/-3.77%) spent in Q3 in ischemic rats by 96.23%. These data suggested that DDPH improved the learning and memory performance significantly in rats with ischemia induced by bilateral carotid artery ligation. DDPH also lowered the levels of malondialdehyde (MDA), which was increased in the hypoperfused rats, and enhanced the activities of superoxide dismutase (SOD) and glutathione peroxidase, which were decreased in these rats. Further more, immunohistochemistry, RT-PCR assays and Western blot study demonstrated that DDPH attenuated the decreased expression of NMDAR2B (NR2B) in cortex and hippocampal CA1 region of the rats after bilateral carotid artery ligation. Our results suggest that DDPH may have favorable effects for the subjects in cerebrovascular insufficiency state following ischemic stroke.

Astrocytic toll-like receptor 3 is associated with ischemic preconditioning- induced protection against brain ischemia in rodents

Background Cerebral ischemic preconditioning (IPC) protects brain against ischemic injury. Activation of Toll-like receptor 3 (TLR3) signaling can induce neuroprotective mediators, but whether astrocytic TLR3 signaling is involved in IPC-induced ischemic tolerance is not known. Methods IPC was modeled in mice with three brief episodes of bilateral carotid occlusion. In vitro, IPC was modeled in astrocytes by 1-h oxygen-glucose deprivation (OGD). Injury and components of the TLR3 signaling pathway were measured after a subsequent protracted ischemic event. A neutralizing antibody against TLR3 was used to evaluate the role of TLR3 signaling in ischemic tolerance. Results IPC in vivo reduced brain damage from permanent middle cerebral artery occlusion in mice and increased expression of TLR3 in cortical astrocytes. IPC also reduced damage in isolated astrocytes after 12-h OGD. In astrocytes, IPC or 12-h OGD alone increased TLR3 expression, and 12-h OGD alone increased expression of phosphorylated NFκB (pNFκB). However, IPC or 12-h OGD alone did not alter the expression of Toll/interleukin receptor domain-containing adaptor-inducing IFNβ (TRIF) or phosphorylated interferon regulatory factor 3 (pIRF3). Exposure to IPC before OGD increased TRIF and pIRF3 expression but decreased pNFκB expression. Analysis of cytokines showed that 12-h OGD alone increased IFNβ and IL-6 secretion; 12-h OGD preceded by IPC further increased IFNβ secretion but decreased IL-6 secretion. Preconditioning with TLR3 ligand Poly I:C increased pIRF3 expression and protected astrocytes against ischemic injury; however, cells treated with a neutralizing antibody against TLR3 lacked the IPC- and Poly I:C-induced ischemic protection and augmentation of IFNβ. Conclusions The results suggest that IPC-induced ischemic tolerance is mediated by astrocytic TLR3 signaling. This reprogramming of TLR3 signaling by IPC in astrocytes may play an important role in suppression of the post-ischemic inflammatory response and thereby protect against ischemic damage. The mechanism may be via activation of the TLR3/TRIF/IRF3 signaling pathway.

Inhibition of intracellular Ca2+ release by a Rho-kinase inhibitor for the treatment of ischemic damage in primary cultured rat hippocampal neurons

The effects of hydroxy fasudil, a specific Rho-kinase inhibitor, on behavior and brain neuronal activity in animal studies have been described previously. However, whether a Rho-kinase inhibitor can directly protect neurons against ischemic damage and the molecular mechanisms underlying these effects are poorly understood. The present work was designed to investigate the effect of hydroxy fasudil against oxygen-glucose deprivation (OGD) induced acute neuronal injury and the underlying mechanisms in vitro. Pretreatment with hydroxy fasudil at 5 and 10 microM could concentration-dependently improve cell viability and decrease Lactate dehydrogenase (LDH) level in extracellular solution of neurons suffered from OGD either in Ca(2+)-containing or Ca(2+)-free culture medium. Moreover, we found that abnormal elevation of extracellular glutamate (Glu) level induced by OGD was markedly repressed by hydroxy fasudil as measured by high performance liquid chromatography (HPLC). Using Fura-2 based calcium imaging techniques, we further demonstrated that preincubation with hydroxy fasudil suppressed the increase of [Ca(2+)](i) induced by 50 microM Glu and 20 microM ATP, but had no effect on the increase of [Ca(2+)](i) induced by 50 mM KCl. These data demonstrated that the neuroprotective effect of hydroxy fasudil was attributed to repressing Glu excitotoxicity and ischemic induced calcium overload by inhibiting Ca(2+) release from Ca(2+) stores rather than by inhibiting Ca(2+) influx via receptor-operated or voltage-dependent calcium channel.

BEHAVIORAL ALTERATIONS ASSOCIATED WITH A DOWN REGULATION OF HCN1 mRNA IN HIPPOCAMPAL CORNUS AMMON 1 REGION AND NEOCORTEX AFTER CHRONIC INCOMPLETE GLOBAL CEREBRAL ISCHEMIA IN RATS

It has been suggested that hyperpolarization-activated cyclic-nucleotide-gated cation non-selective channel (HCN) 1 is primarily expressed in the hippocampus and can be regulated in many pathological settings. However, little is known about its change under ischemic conditions. In the present study, we performed neurophysiological recordings of sham-operated and chronic ischemic rats with hypoperfusion during the resolution of the neurological deficits respectively. In situ hybridization methods and reverse transcriptase-polymerase chain reaction (RT-PCR) assays were used to investigate whether and how HCN1 mRNA may be altered in global incomplete chronic cerebral ischemic rat model. Our results suggested that attenuated spatial learning and memory function of rats shown by longer escape latency, shorter time spent in the target quadrant and impaired long-term potentiation (LTP) after chronic cerebral ischemia. In the in situ hybridization cytochemistry experiment, HCN1 mRNA declined to 52.00% and 46.00% of the control values in the cornus ammon 1 (CA1) regions of hippocampus and neocortex separately after chronic cerebral ischemia. HCN1 mRNA in the hippocampal CA1 region and neocortex was markedly down regulated by ischemia, reaching 48.90% and 45.80% of the control values respectively in the semi-quantitative RT-PCR experiment. The phenomenon opened new insights for further investigation of the physiological and pathological significances of HCN1 in chronic incomplete global cerebral ischemia.

Baclofen mediates neuroprotection on hippocampal CA1 pyramidal cells through the regulation of autophagy under chronic cerebral hypoperfusion.

GABA receptors play an important role in ischemic brain injury. Studies have indicated that autophagy is closely related to neurodegenerative diseases. However, during chronic cerebral hypoperfusion, the changes of autophagy in the hippocampal CA1 area, the correlation between GABA receptors and autophagy, and their influences on hippocampal neuronal apoptosis have not been well established. Here, we found that chronic cerebral hypoperfusion resulted in rat hippocampal atrophy, neuronal apoptosis, enhancement and redistribution of autophagy, down-regulation of Bcl-2/Bax ratio, elevation of cleaved caspase-3 levels, reduction of surface expression of GABAA receptor α1 subunit and an increase in surface and mitochondrial expression of connexin 43 (CX43) and CX36. Chronic administration of GABAB receptors agonist baclofen significantly alleviated neuronal damage. Meanwhile, baclofen could up-regulate the ratio of Bcl-2/Bax and increase the activation of Akt, GSK-3β and ERK which suppressed cytodestructive autophagy. The study also provided evidence that baclofen could attenuate the decrease in surface expression of GABAA receptor α1 subunit, and down-regulate surface and mitochondrial expression of CX43 and CX36, which might enhance protective autophagy. The current findings suggested that, under chronic cerebral hypoperfusion, the effects of GABAB receptors activation on autophagy regulation could reverse neuronal damage.

Flupirtine attenuates chronic restraint stress-induced cognitive deficits and hippocampal apoptosis in male mice

Chronic restraint stress (CRS) causes hippocampal neurodegeneration and hippocampus-dependent cognitive deficits. Flupirtine represents neuroprotective effects and we have previously shown that flupirtine can protect against memory impairment induced by acute stress. The present study aimed to investigate whether flupirtine could alleviate spatial learning and memory impairment and hippocampal apoptosis induced by CRS. CRS mice were restrained in well-ventilated Plexiglass tubes for 6h daily beginning from 10:00 to 16:00 for 21 consecutive days. Mice were injected with flupirtine (10mg/kg and 25mg/kg) or vehicle (10% DMSO) 30min before restraint stress for 21 days. After stressor cessation, the spatial learning and memory, dendritic spine density, injured neurons and the levels of Bcl-2, Bax, p-Akt, p-GSK-3β, p-Erk1/2 and synaptophysin of hippocampal tissues were examined. Our results showed that flupirtine significantly prevented spatial learning and memory impairment induced by CRS in the Morris water maze. In addition, flupirtine (10mg/kg and 25mg/kg) treatment alleviated neuronal apoptosis and the reduction of dendritic spine density and synaptophysin expression in the hippocampal CA1 region of CRS mice. Furthermore, flupirtine (10mg/kg and 25mg/kg) treatment significantly decreased the expression of Bax and increased the p-Akt and p-GSK-3β, and flupirtine (25mg/kg) treatment up-regulated the p-Erk1/2 in the hippocampus of CRS mice. These results suggested that flupirtine exerted protective effects on the CRS-induced cognitive impairment and hippocampal neuronal apoptosis, which is possibly associated with the activation of Akt/GSK-3β and Erk1/2 signaling pathways.

Toll-like receptor 3 agonist Poly I:C protects against simulated cerebral ischemia in vitro and in vivo

Aim:To examine the neuroprotective effects of the Toll-like receptor 3 (TLR3) agonist Poly I:C in acute ischemic models in vitro and in vivo.Methods:Primary astrocyte cultures subjected to oxygen-glucose deprivation (OGD) were used as an in vitro simulated ischemic model. Poly I:C was administrated 2 h before OGD. Cell toxicity was measured using MTT assay and LDH leakage assay. The levels of TNFα, IL-6 and interferon-β (IFNβ) in the media were measured using ELISA. Toll/interleukin receptor domain-containing adaptor-inducing IFNβ (TRIF) protein levels were detected using Western blot analysis. A mouse middle cerebral artery occlusion (MCAO) model was u sed for in vivo study. The animals were administered Poly I:C (0.3 mg/kg, im) 2 h before MCAO, and examined with neurological deficit scoring and TTC staining. The levels of TNFα and IL-6 in ischemic brain were measured using ELISA.Results:Pretreatment with Poly I:C (10 and 20 μg/mL) markedly attenuated OGD-induced astrocyte injury, and significantly raised the cell viability and reduced the LDH leakage. Poly I:C significantly upregulated TRIF expression accompanied by increased downstream IFNβ production. Moreover, Poly I:C significantly suppressed the pro-inflammatory cytokines TNFα and IL-6 production. In mice subjected to MCAO, administration of Poly I:C significantly attenuated the neurological deficits, reduced infarction volume, and suppressed the increased levels of TNFα and IL-6 in the ischemic striatum and cortex.Conclusion:Poly I:C pretreatment exerts neuroprotective and anti-inflammatory effects in the simulated cerebral ischemia models, and the neuroprotection is at least in part due to the activation of the TLR3-TRIF pathway.