Blocking toll-like receptor 2 and 4 signaling during a stressor prevents stress-induced priming of neuroinflammatory responses to a subsequent immune challenge

Acute and chronic stressors sensitize or prime the neuroinflammatory response to a subsequent peripheral or central immunologic challenge. However, the neuroimmune process(es) by which stressors prime or sensitize subsequent neuroinflammatory responses remains unclear. Prior evidence suggested that toll-like receptors (TLRs) might be involved in the mediation of primed neuroinflammatory responses, but the role of TLRs during a stressor has never been directly tested. Here, a novel TLR2 and TLR4 antagonist, OxPAPC, was used to probe the contribution of TLRs in the stress sensitization phenomenon. OxPAPC has not previously been administered to the brain, and so its action in blocking TLR2 and TLR4 action in brain was first verified. Administration of OxPAPC into the CNS prior to stress prevented the stress-induced potentiation of hippocampal pro-inflammatory response to a subsequent peripheral LPS challenge occurring 24 h later. In addition, in vivo administration of OxPAPC prior to stress prevented the sensitized pro-inflammatory response from isolated microglia following administration of LPS ex vivo, further implicating microglia as a key neuroimmune substrate that mediates stress-induced sensitized neuroinflammation.     

Psychological stress exacerbates primary vaginal herpes simplex virus type 1 (HSV-1) infection by impairing both innate and adaptive immune responses

Chronic psychological stress is generally immunosuppressive and contributes to an increase in herpes simplex virus (HSV) pathogenicity. We have previously shown that mice experiencing stress at the time of intranasal HSV infection have increased levels of infectious virus in their nasal cavity, as compared to control mice that were not subjected to stress. We have extended our studies to determine the effects of stress at another clinically-relevant mucosal site by examining the immune response to and pathogenesis of vaginal HSV infection. Mice experiencing psychological stress during vaginal HSV infection exhibited an increase in both vaginal viral titers and the pathology associated with this HSV infection. We demonstrate that these observations result from the failure of both the innate and HSV-specific adaptive immune responses. At 2 days post-infection, NK cell numbers were significantly decreased in mice experiencing restraint stress. Studies examining the adaptive immune response revealed a decrease in the number of HSV-specific CD8⁺ T cells in not only the vaginal tissue itself but also the draining iliac lymph nodes (ILN). Furthermore, the number of functional cells, in terms of both their degranulation and interferon-γ production, in the ILN of stressed mice was decreased as compared to non-stressed mice. We conclude that psychological stress, through its suppression of both innate and adaptive immune responses, may be an important factor in the ability to control vaginal HSV infection.     

Variation in the purinergic P2RX(7) receptor gene and schizophrenia

INTRODUCTION: The purinergic receptor gene P2RX(7) is located in a major linkage hotspot for schizophrenia and bipolar disorders, 12q21-33. It has previously been associated with bipolar disorder but has never been analysed in relation to schizophrenia, although it is involved in several neuronal processes associated with schizophrenia. METHODS: Nine functionally characterised variants in P2RX(7) were genotyped in 389 patients diagnosed with schizophrenia, each matched on sex, birth-year and month with two healthy controls. RESULTS: We did not find association between P2RX(7) and schizophrenia and stratification on gender did not change this result. The high ethnic and diagnostic homogeneity of the sample adds credibility to this finding. CONCLUSION: P2XR(7) was not associated with schizophrenia in this study.     

Association of the trace amine associated receptor 6 (TAAR6) gene with schizophrenia and bipolar disorder in a Korean case control sample

Trace amines and their receptors may be implicated in the pathogenesis of psychiatric disorders. Previous studies have reported association of the trace amine associated receptor 6 (TAAR6) gene with susceptibility to schizophrenia and bipolar disorder but results have not been consistent. The purpose of this study was to examine these associations in Korean patients and also to test for association of TAAR6 with susceptibility to major depressive disorder (MDD). A case control sample consisting of 281 patients with schizophrenia, 190 patients with bipolar disorder, 187 patients with MDD and 288 psychiatrically healthy control subjects, was examined. Patients with schizoaffective disorder were not included in any of the psychiatric samples. Five single nucleotide polymorphisms (SNPs: rs4305745; rs8192625; rs7452939; rs6903874 and rs6937506) were genotyped in the TAAR6 gene and in the 3' regulatory region, using pyrosequencing. SNP rs6903874 was significantly associated with schizophrenia (p = 0.012) and bipolar disorder (p = 0.004). A three SNP haplotype consisting of alleles GCT from SNPs rs7452939, rs6903874 and rs6937506, respectively, was significantly over-represented in patients with schizophrenia (p = 0.0003) and bipolar disorder (p = 0.00002). A second three SNP haplotype (GTT) derived from the same SNPs was significantly under-represented in patients with bipolar disorder (p = 0.001). The GTT haplotype associations withstand the most rigorous corrections for multiple testing. These findings strongly support association of the TAAR6 gene with susceptibility to both schizophrenia and bipolar disorder in Korean patients. Further studies are needed to confirm these findings in this and other populations and to identify functional variants in TAAR6 that may be implicated in pathogenesis.     

Association of specific haplotypes of neurotrophic tyrosine kinase receptor 2 gene (NTRK2) with vulnerability to nicotine dependence in African-Americans and European-Americans.

BACKGROUND: The gene encoding neurotrophic tyrosine kinase receptor 2 (NTRK2) has been localized to a region on chromosome 9q22-q23 that showed a "suggestive" linkage to nicotine dependence (ND) in our previous linkage analyses. However, no association of NTRK2 with ND has been identified. METHODS: Family-based association analyses of 2037 participants (1366 African Americans [AA], 671 European Americans [EA]) representing 602 nuclear families were performed to evaluate association of nine single nucleotide polymorphisms (SNPs) within NTRK2 with ND. RESULTS: Individual SNP-based association analysis indicated that in the EA sample, SNPs rs1659400 and rs1187272 were significantly associated with at least one adjusted ND measure. Haplotype analysis revealed that even after Bonferroni correction, the haplotype T-T-A of rs1659400-rs1187272-rs1122530 had a highly significant positive association, with adjusted ND measures in the EA sample (max Z = 3.78; p = .0001, frequency 59.9%). We further identified a major haplotype, T-G-C-A-A (26%), formed by rs993315-rs736744-rs920776-rs4075274-rs729560, which showed a significant positive association (max Z = 2.97, p = .003) with adjusted ND measures in the AA sample. CONCLUSIONS: These results strongly suggest that NTRK2 is a susceptibility gene for ND. These findings imply that NTRK2 plays a role in the etiology of ND and represents an important biological candidate for further investigation.     

Role of 5-HT(2) receptor subtypes in depletion of 5-HT induced by p-chloroamphetamine in the mouse frontal cortex

A serotonin (5-hydroxytryptamine, 5-HT)-releasing drug, p-chloroamphetamine elicited decreases in 5-HT levels in the mouse frontal cortex. 5-HT reduction elicited by p-chloroamphetamine was inhibited by the 5-HT(2A/2B/2C) receptor antagonist, LY 53857 and the 5-HT(2A) receptor antagonist, ketanserin. However, the 5-HT(2B/2C) receptor antagonist, SB 206553, enhanced it. LY 53857 and ketanserin can inhibit hyperthermia elicited by p-chloroamphetamine, although SB 206553 enhances it. The effects of the 5-HT(2) receptor antagonists on neurotoxicity are very similar to those on hyperthermia. Since hyperthermia facilitates neurotoxicity induced by amphetamine analogue, these 5-HT(2) receptor antagonists may modify 5-HT depletion induced by p-chloroamphetamine through responses to body temperature.     

髓样细胞触发受体 2与阿尔茨海默病关系的研究进展

阿尔茨海默病(AD)是老年期痴呆最常见的类型,是一种复杂的、不可逆的神经退行性疾病.其特征性的病理改变是细胞外出现β淀粉样蛋白(Aβ)沉积形成的淀粉样斑块(SP)、细胞内过度磷酸化的tau蛋白形成的纤维缠结及显著的突触丢失、脑萎缩和神经元细胞的死亡.     

Effects of D2 dopamine receptor agonist and antagonist on brain activity in the rat assessed by functional magnetic resonance imaging

The effects of D₂ dopamine receptor agonist, bromocriptine (BROMO), and antagonist, haloperidol (HPD), on brain activity were investigated in rats by functional magnetic resonance imaging. T2^*-weighted signal intensity was increased in the hypothalamus at 120 min after acute administration of BROMO, and in the ventral posterior and dorsomedial nuclei of the thalamus from 30 to 120 min. In contrast, the signal intensity was decreased in the caudate–putamen at 30 min after acute administration of HPD, in the hypothalamus from 30 to 60 min, and in the perirhinal cortex at 30 min. After chronic (2 weeks) HPD treatment, acute administration of HPD decreased signal intensity in the caudate–putamen at 60 min, in the hypothalamus at 30 min, the perirhinal cortex from 2 to 120 min, the dorsomedial and ventral posterior nuclei of the thalamus from 2 to 120 min, and the medial nucleus of the amygdala from 60 to 120 min. These results suggest that (1) the D₂ receptor agonist increased the activity of the thalamic nuclei and the hypothalamus, while the D₂ receptor antagonist suppressed brain activity in the regions where D₂ receptors were present, (2) the suppression of brain activity in the thalamic nuclei and the perirhinal cortex by acute HPD administration was enhanced by chronic HPD treatment, and (3) the effects of antipsychotic drugs on the thalamus, amygdala, and perirhinal cortex may be related to their therapeutic efficacy, since clinical improvement in schizophrenic patients appears several days after the start of HPD treatment.     

Novel P2Y12 adenosine diphosphate receptor antagonists for inhibition of platelet aggregation (II): pharmacodynamic and pharmacokinetic characterization

Antiplatelet drugs are used to prevent aberrant platelet activation in pathophysiologic conditions such as myocardial infarction and ischemic stroke. The key role that ADP plays in this process has led to the development of antiplatelet drugs that target the P2Y12 receptor. The aim of this study was to characterize the pharmacodynamic (PD) and pharmacokinetic (PK) properties of the novel P2Y12 receptor antagonists, BX 667 and BX 048. BX 667 blocks ADP-induced platelet aggregation in human, dog and rat blood (IC50=97, 317 and 3000 nM respectively). BX 667 had nominal effects on collagen-induced aggregation and weakly inhibited arachidonic acid-induced aggregation. BX 667 has an active metabolite, BX 048, that also potently inhibits ADP-induced aggregation (IC50=290 nM) in human blood. BX 667 was shown to have high oral bioavailability in both dog and rat unlike BX 048. Administration of BX 667 resulted in a rapid and sustained inhibition of platelet aggregation where the extent and duration of platelet inhibition was directly proportional to circulating plasma levels. This report describes the PK/PD properties of BX 667 showing that it has the properties required for a potential antiplatelet therapeutic agent.     

Gene expression of cyclooxygenase-1 and Ca(2+)-independent phospholipase A(2) is altered in rat hippocampus during normal aging

Brain aging is associated with inflammatory changes. However, data on how the brain arachidonic acid (AA) metabolism is altered as a function of age are limited and discrepant. AA is released from membrane phospholipids by phospholipase A(2) (PLA(2)) and then further metabolized to bioactive prostaglandins and thromboxanes by cyclooxygenases (COX)-1 and -2. We examined the phospholipase A(2) (PLA(2))/COX-mediated AA metabolic pathway in the hippocampus and cerebral cortex of 4-, 12-, 24- and 30-month-old rats. A two-fold increase in brain thromboxane B(2) level in 24 and 30 months was accompanied by increased hippocampal COX-1 mRNA levels at 12, 24, and 30 months. COX-2 mRNA expression was significantly decreased only at 30 months. Hippocampal Ca(2+)-independent iPLA(2) mRNA levels were decreased at 24 and 30 months without any change in Ca(2+)-dependent PLA(2) expression. In the cerebral cortex, mRNA levels of COX and PLA(2) were not significantly changed. The specific changes in the AA cascade observed in the hippocampus may alter phospholipids homeostasis and possibly increase the susceptibility of the aging brain to neuroinflammation.     

Effects of prostaglandin E2 and cyclooxygenase inhibitors on clustering and level of nicotinic acetylcholine receptor in mouse myotubes co-cultured with spinal cord explant

The clustering and level of nicotinic acetylcholine receptor (n-AChR) in cultured mouse myotubes are negatively controlled by endogenous phospholipase A2 (PLA2) (Kimura et al., Int. J. Devl. Neurosci. 5, 127-133, 1987). The effects of PLA2-related metabolites, prostaglandins, leukotrienes and platelet-activating factor (PAF) were investigated using fluorescein isothiocyanate-alpha-bungarotoxin. Peak and total fluorescence within a cluster were used as indices of clustering and level of n-AChR, respectively. Prostaglandin E2 (PGE2, 1-10 microM) decreased both indices in a concentration-dependent manner. Aspirin and indomethacin, cyclooxygenase inhibitors, increased the indices at 1.0 microM and 10-30 nM, and decreased them at higher concentrations of 10-30 microM and 0.1-1 microM, respectively. Prostaglandin F2 alpha (PGF2 alpha, 1-10 microM), nordihydroguaiaretic acid (30 microM), a lipoxygenase inhibitor, and PAF (10 microM) had no effect. These results suggest that the control of endogenous PLA2 on the clustering and level of n-AChR is due to PGE2, but not to PGF2 alpha, leukotrienes or PAF.     

The role of dopamine transporter (SLC6A3) and dopamine D2 receptor/ankyrin repeat and kinase domain containing 1 (DRD2/ANKK1) gene polymorphisms in personality traits

Variations in personality traits are caused by interactions between multiple genes of small effect and environmental factors. To date, gender- and ethnicity-specific variations in personality have been established. In the present study, we aimed to test: 1) the effects of four polymorphisms of dopamine system genes: ANKK1/DRD2 Taq1A, DRD2 rs6275, SLC6A3 40-bp VNTR and rs27072, on personality traits; 2) whether these effects differ between men and women and between Russians and Tatars. A sample of 652 healthy individuals (222 men and 430 women) of Caucasian origin (233 Russians and 419 Tatars) from Russia was subjected to personality traits assessment with Eysenck Personality Inventory (EPI) and Temperament and Character Inventory-125 (TCI-125). The associations between each personality trait and polymorphisms were assessed with regression models adjusted for gender and ethnicity. There were significant effects of ANKK1/DRD2 Taq1A on Neuroticism (p = 0.016) and of SLC6A3 rs27072 on Persistence (p = 0.021) in both genders. The association between ANKK1/DRD2 Taq1A A2/A2-genotype and higher Novelty Seeking and lower Reward Dependence was shown in men only (p for gender interaction = 0.018). In women only, there was a significant association between SLC6A3 10R*G-haplotype and higher Persistence (p = 0.002). Our findings provide evidence for a modifying effect of gender on the associations between dopamine system genes and approach-related traits (in men) and Persistence (in women).     

I(4), a new synthetic sulfonylurea compound, inhibits the action of TXA(2)in vivo and in vitro on platelets and aorta vascular smooth muscle

Introduction

1-[4-[2-(4-Bromobenzene-sulfonamino)ethyl]phenylsulfonyl]-3-(trans-4-methylcy-clohexyl)urea(I₄, CAS865483-06-3); a totally synthetic new sulfonylurea compound, combining the hypoglycemic active structure of Glimepiride (CAS 93479-97-1) and anti-TXA₂ receptor (TP) active structure of BM-531(CAS 284464-46-6), was designed and synthesized. Its effects on TXA₂ synthesis and TP have not been reported yet.

Aim

To study the inhibitory effects of I₄ and its mechanisms of action on TXA₂ and TP.

Methods

Platelet aggregation studies were performed on human platelet, rat whole blood platelet and rabbit platelet, platelets aggregation was induced by TP agonist U-46619(stable analog of TXA₂, CAS 56985-40-1). Plasma TXB₂ and 6-keto-prostaglandin F_1α (6-keto-PGF_1α) were used as markers to determine the effect of I₄ on thromboxane synthesis. Fluo-3-AM was used to measure the cytosolic Ca^2 + concentrations ([Ca^2 +]_i) in rabbit platelet. Aorta rings with and without endothelium were prepared and aorta contraction was induced by U-46619. A model of type 2 diabetes mellitus was established by intraperitoneal injection of low dose of streptozocin to rats fed a high-calorie diet. Both normal rats and type 2 diabetic rats were used to assay the inhibitory effect of I₄ on platelet aggregation induced by U-46619.

Results

I₄ exhibited a higher inhibitory potency than Glimepiride on U-46619 induced platelet aggregation in vitro and in vivo. I₄ increased the ratio of plasma PGI₂/TXA₂ and decreased [Ca^2 +]_i release from platelet internal stores. In addition, I₄ presented a vasorelaxant activity on isolated rat aorta contraction induced by U-46619.Oral administration of I₄ (1 ~ 10 mg/kg) markedly and dose-dependently inhibited platelet aggregation in both normal rats and type 2 diabetic rats.

Conclusion

I₄ significantly inhibited platelet aggregation induced by U-46619 in vitro and in vivo, and rat aorta contraction. It probably acts by partly blocking TXA₂ action, decreasing the platelet intracellular Ca^2 +, and increasing the PGI₂/TXA₂ ratio.     

Acute effects of 3,4-methylenedioxymethamphetamine on striatal single-unit activity and behavior in freely moving rats: differential involvement of dopamine D(1) and D(2) receptors

3,4-Methylenedioxymethamphetamine (MDMA) is a widely abused amphetamine derivative that increases dopamine (DA) and serotonin release via a reverse transport mechanism. Changes in the activity of striatal neurons in response to increased DA transmission may shape the behavioral patterns associated with amphetamine-like stimulants. To determine how the striatum participates in MDMA-induced locomotor activation, we recorded the activity of >100 single units in the striatum of freely moving rats in response to a dose that increased motor activation (5.0 mg/kg). MDMA had a predominantly excitatory effect on neuronal activity that was positively correlated with the magnitude of locomotor activation. Categorizing neurons according to baseline locomotor responsiveness revealed that MDMA excited significantly more neurons showing movement-related increases in activity compared to units that were non-movement-related or associated with movement-related decreases in activity. Further analysis revealed that the drug-induced striatal activation was not simply secondary to the behavioral change, indicating a primary action of MDMA on striatal motor circuits. Prior administration of SCH-23390 (0.2 mg/kg), a D(1) antagonist, resulted in a late onset of MDMA-induced locomotion, which correlated positively with delayed neuronal excitations. Conversely, prior administration of eticlopride (0.2 mg/kg), a D(2) antagonist, completely abolished MDMA-induced locomotion, which paralleled its blockade of MDMA-induced excitatory neuronal responses. Our results highlight the importance of striatal neuronal activity in shaping the behavioral response to MDMA, and suggest that DA D(1) and D(2) receptors have distinct functional roles in the expression of MDMA-induced striatal and locomotor activation.     

Somato-dendritic distribution of 5-HT(1A) and 5-HT(1B) autoreceptors in the BDNF- and cAMP-differentiated RN46A serotoninergic raphe cell line

The rapid differentiating effects of brain-derived neurotrophic factor (BDNF) or dibutyryl-cAMP (dBcAMP) were characterized on RN46A, a rat raphe-derived neuronal cell line. After BDNF treatment, RN46A cells were serotonin-immunopositive and bipolar, and expressed the microtubule-associated-protein 2 (Map2). After dBcAMP treatment, the cells often became multipolar, bearing very long processes strongly immunopositive for serotonin and Map2. Under both conditions, the expression and distribution of 5-HT(1A) and 5-HT(1B) autoreceptors remained identical. 5-HT(1A) and Map2 immunolabelings were superimposable, as expected of their somato-dendritic targeting. Surprisingly, the distribution of 5-HT(1B) immunoreactivity was similar, in contrast with its usual localization in axons and nerve terminals in the brain. In conclusion, both BDNF and cAMP-differentiated RN46A cells towards a neuronal serotoninergic-like phenotype without the typical differential targeting of the 5-HT(1) autoreceptors, an interesting model to study the molecular mechanisms ensuing the targeting of 5-HT(1) autoreceptors to somas and dendrites.     

Catalase prevents elevation of [Ca(2+)](i) induced by alcohol in cultured canine cerebral vascular smooth muscle cells: Possible relationship to alcohol-induced stroke and brain pathology

Several studies have suggested that alcohol-induced brain injury is associated with generation of reactive oxygen species (ROS). The recent findings, that antioxidants (Vitamin E and pyrrolidine dithiocarbamate (PDTC)) prevent intracellular Ca(2+) ([Ca(2+)](i)) overload in cerebral vascular smooth muscle cells, induced by alcohol, demonstrate indirectly that ROS formation is related to cerebral vascular injury. The present experiments were designed to test the hypothesis that catalase, an hydrogen peroxide (H(2)O(2)) scavenging enzyme, can prevent or ameliorate alcohol-induced elevation of [Ca(2+)](i). Preincubation of cultured canine cerebral vascular smooth muscle cells with catalase (20-1000 units/ml) didn't produce any apparent changes from controls in resting levels of [Ca(2+)](i) after 1-3 days. Exposure of the cerebral vascular cells to culture media containing 10-100mM ethanol resulted in significant rises in [Ca(2+)](i) (p<0.01). Although exposure of these cells to a low concentration of catalase (20 units/ml) failed to prevent the increased level of [Ca(2+)](i) induced by ethanol, concomitant addition of higher concentrations of catalase (100-1000 units/ml) and ethanol (10-100mM) inhibited or ameliorated the rises of [Ca(2+)](i) induced by ethanol either at 24h or at 3 days, in a concentration-dependent manner. Catalase, in the range of 100-200 units/ml, inhibited approximately 50% of the [Ca(2+)](i) increases caused by ethanol in the first 24h. Catalase at a concentration of 1000 units/ml inhibited completely excessive [Ca(2+)](i) accumulation. The present results when viewed in light of other recently published data suggest that H(2)O(2) generation may be one of the earliest events triggered by alcohol in alcohol-induced brain-vascular damage, neurobehavioral actions and stroke.     

Effect of denervation of the locus coeruleus projections by DSP-4 treatment on [3H]-raclopride binding to dopamine D(2) receptors and D(2) receptor-G protein interaction in the rat striatum

Changes in the control of dopaminergic neurotransmission by noradrenergic locus coeruleus (LC) projections has been implicated in such disorders as depression, drug addiction, and Parkinson's disease. In the present study, the effect of DSP-4, a neurotoxin highly selective for LC projections, on D(2) receptor abundance as assessed by [3H]-raclopride binding in the striatum was studied in rats after administration in doses of 10 and 50 mg/kg either 3 days or 1 month before decapitation. Three days after DSP-4 the levels of noradrenaline in the frontal cortex were dose-dependently reduced; after 1 month, noradrenaline levels were lowered only by the higher dose. DOPAC levels were dose-dependently reduced in the frontal cortex and striatum 3 days but not 1 month after DSP-4 treatment. Cortical 5-HIAA levels were reduced 3 days but not 1 month after DSP-4. The apparent number of D(2) receptor binding sites in the striatum was higher 1 month after either dose of DSP-4. DSP-4 treatment had no effect on [3H]-raclopride binding affinity, the ability of dopamine (DA) to compete with [3H]-raclopride binding and to activate [35S]GTPgammaS binding or on the binding affinities of GDP and [35S]GTPgammaS for corresponding G proteins 1 month after administration of the neurotoxin. These data suggest that after administration of DSP-4, short-term reduction in DA and 5-HT metabolism occurs. Subsequently, an upregulation of D(2) receptor binding sites develops in the striatum even after a minor denervation of the LC projections. Thus, alterations in the LC projection systems elicit lasting adaptive changes in DA-ergic neurotransmission that can serve as a substrate for psychiatric disorders.     

Effects of LF 16-0687 Ms,a bradykinin B(2) receptor antagonist,on brain edema formation and tissue damage in a rat model of temporary focal cerebral ischemia

Bradykinin, an endogenous nonapeptide produced by activation of the kallikrein–kinin system, promotes neuronal tissue damage as well as disturbances in blood–brain barrier function through activation of B₂ receptors. LF 16-0687 Ms, a non-peptide competitive bradykinin B₂ receptor antagonist, was recently found to decrease brain swelling in various models of traumatic brain injury. We have investigated the influence of LF 16-0687 Ms on the edema formation, neurological outcome, and infarct size in temporary focal cerebral ischemia in rats. Sprague–Dawley rats were subjected to MCA occlusion for 90 min by an intraluminal filament. Local CBF was bilaterally recorded by laser Doppler flowmetry. Study I: animals were assigned to one of three treatment arms (n=11 each): (a) vehicle, (b) LF 16-0687 Ms (12.0 mg/kg per day), or (c) LF 16-0687 Ms (36.0 mg/kg per day) given repetitively s.c. over 3 days. The neurological recovery was examined daily. The infarct volume was assessed histologically 7 days after ischemia. Study II: brain swelling and bilateral hemispheric water content were determined at 48 h post ischemia in eight rats, subjected to the low dose regimen as described above, and in eight vehicle-treated control animals. All treated animals showed tendency to exhibit improved neurological recovery throughout the observation period as compared to the vehicle-treated controls, while this improvement was only significant within the low dose group from postischemic days 3 to 4. Low dose LF 16-0687 Ms significantly attenuated the total and cortical infarct volume by 50 and 80%, respectively. Furthermore, postischemic swelling (−62%) and increase in water content of the infarcted brain hemisphere (−60.5%) was significantly inhibited. The present findings provide strong evidence for an involvement of bradykinin-mediated secondary brain damage following from focal cerebral ischemia. Accordingly, specific inhibition of bradykinin B₂ receptors by LF 16-0687 Ms attenuated postischemic brain swelling, improved the functional neurological recovery, and limited ischemic tissue damage, raising its potential for clinical evaluation in patients with acute stroke.     

Neuroprotective effect of ginkgolide K on glutamate-induced cytotoxicity in PC 12 cells via inhibition of ROS generation and Ca(2+) influx

Glutamate is considered to be responsible for the pathogenesis of cerebral ischemia disease. [Ca²⁺]_i influx and reactive oxygen species (ROS) production are considered to be involved in glutamate-induced apoptosis process. In this study, we investigated the neuroprotective effects of ginkgolide K in the glutamate-induced rat's adrenal pheochromocytoma cell line (PC 12 cells) and the possible mechanism. Glutamate cytotoxicity in PC 12 cells was accompanied by an increment of malondialdehyde (MDA) content and lactate dehydrogenase (LDH) release, as well as Ca²⁺ influx, bax/bcl-2 ratio, cytochrome c release, caspase-3 protein and ROS generation, and reduction of cell viability and mitochondrial membrane potential (MMP). Moreover, treatment with glutamate alone resulted in decrease activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activity. However, pretreatment with ginkgolide K significantly reduced MDA content, LDH release, as well as Ca²⁺ influx, cytochrome c release, bax/bcl-2 ratio, caspase-3 protein and ROS production, and attenuated the decrease of cells viability and MMP. In addition, ginkgolide K remarkedly up-regulated SOD and GSH-PX activities. All these findings indicated that ginkgolide K protected PC12 cells against glutamate-induced apoptosis by inhibiting Ca²⁺ influx and ROS production. Therefore, the present study supports the notion that ginkgolide K may be a promising neuroprotective agent for the treatment of cerebral ischemia disease.