Intensive Ambulance-Delivered Blood-Pressure Reduction in Hyperacute Stroke.

BACKGROUND: Treatment of acute stroke, before a distinction can be made between ischemic and hemorrhagic types, is challenging. Whether very early blood-pressure control in the ambulance improves outcomes among patients with undifferentiated acute stroke is uncertain. METHODS: We randomly assigned patients with suspected acute stroke that caused a motor deficit and with elevated systolic blood pressure (≥150 mm Hg), who were assessed in the ambulance within 2 hours after the onset of symptoms, to receive immediate treatment to lower the systolic blood pressure (target range, 130 to 140 mm Hg) (intervention group) or usual blood-pressure management (usual-care group). The primary efficacy outcome was functional status as assessed by the score on the modified Rankin scale (range, 0 [no symptoms] to 6 [death]) at 90 days after randomization. The primary safety outcome was any serious adverse event. RESULTS: A total of 2404 patients (mean age, 70 years) in China underwent randomization and provided consent for the trial: 1205 in the intervention group and 1199 in the usual-care group. The median time between symptom onset and randomization was 61 minutes (interquartile range, 41 to 93), and the mean blood pressure at randomization was 178/98 mm Hg. Stroke was subsequently confirmed by imaging in 2240 patients, of whom 1041 (46.5%) had a hemorrhagic stroke. At the time of patients' arrival at the hospital, the mean systolic blood pressure in the intervention group was 159 mm Hg, as compared with 170 mm Hg in the usual-care group. Overall, there was no difference in functional outcome between the two groups (common odds ratio, 1.00; 95% confidence interval [CI], 0.87 to 1.15), and the incidence of serious adverse events was similar in the two groups. Prehospital reduction of blood pressure was associated with a decrease in the odds of a poor functional outcome among patients with hemorrhagic stroke (common odds ratio, 0.75; 95% CI, 0.60 to 0.92) but an increase among patients with cerebral ischemia (common odds ratio, 1.30; 95% CI, 1.06 to 1.60). CONCLUSIONS: In this trial, prehospital blood-pressure reduction did not improve functional outcomes in a cohort of patients with undifferentiated acute stroke, of whom 46.5% subsequently received a diagnosis of hemorrhagic stroke. (Funded by the National Health and Medical Research Council of Australia and others; INTERACT4 ClinicalTrials.gov number, NCT03790800; Chinese Trial Registry number, ChiCTR1900020534.).

Diphenyleneiodonium chloride synergizes with diazoxide to enhance protection against amyloid ß induced neurotoxicity.

We examined synergistic effects of inhibiting reactive oxygen species generated from the mitochondria and from nicotinamide adenine dinucleotide phosphate oxidase on neurotoxicity. Primary hippocampal neurons were exposed to amyloid ß, and the cells were treated with diazoxide or/and diphenyleneiodonium chloride. We found that the cell viability was decreased significantly after exposure to amyloid ß for 72 h with higer reactive oxygen species and malondialdehyde levels, higher caspase-3 and cleaved caspase-3 levels and lower B-cell lymphoma 2 (Bcl-2) level. Both diazoxide and diphenyleneiodonium increased cell viability by inhibiting the increase in reactive oxygen species and caspase-3 activity as well as the decrease in Bcl-2 induced by amyloid ß. The combination of diazoxide and diphenyleneiodonium exhibited better protective effects compared to a single treatment. In conclusion, the activation of a mitochondrial potassium channel in combination with the inhibitor of nicotinamide adenine dinucleotide phosphate oxidase exhibit synergistic protective effects against amyloid ß neurotoxicity.

Effects of NMDAR Antagonist on the Regulation of P-MARCKS Protein to Aß1-42 Oligomers Induced Neurotoxicity.

Alzheimer's disease (AD) is a well-known neurodegenerative disease. Deposition of ß-amyloid protein (Aß) oligomers plays a crucial role in the disease progression. Previous studies showed that toxicity induced by Aß oligomers in cultured neurons and adult rat brain was partially mediated by activation of glutamatergic N-methyl-D-aspartate receptors (NMDAR). Additionally, memantine, a noncompetitive NMDAR antagonist, can significantly improve cognitive functions in some AD patients. However, little is currently known about the potential role of NMDAR antagonist on the regulation of P-MARCKS protein to Aß1-42 oligomers induced neurotoxicity. The protective effect and mechanism of NMDAR antagonist on primary neurons exposed to Aß1-42 oligomers were investigated in the study. We have defined that the Aß1-42 treatment decreased cell viability and increased apoptosis. Moreover, Aß1-42 oligomers exposure increased P-MARCKS and PIP2 expressions, while decreased SYP expression. However, NMDAR antagonist pretreatment ameliorates Aß1-42 oligomers induced neuronal apoptosis and partially reverses the expression of P-MARCKS, PIP2 and SYP. In conclusion, NMDAR antagonist may ameliorate neurotoxicity induced by Aß1-42 oligomers through reducing neuronal apoptosis and protecting synaptic plasticity in rat primary neurons. The mechanism involved may be mediated by the variation of protein P-MARCKS.

Circulating FABP4 (Fatty Acid-Binding Protein 4) Is a Novel Prognostic Biomarker in Patients With Acute Ischemic Stroke.

BACKGROUND AND PURPOSE: FABP4 (fatty acid-binding protein 4) is an intracellular lipid chaperone involved in coordination of lipid transportation and atherogenesis. This study aimed at observing the effect of FABP4 on the 3-month outcomes in Chinese patients with acute ischemic stroke. METHODS: In a prospective multicenter observational study, serum concentrations of FABP4 were on admission measured in plasma of 737 consecutive patients with acute ischemic stroke. Serum concentrations of FABP4, National Institutes of Health Stroke Scale score, and conventional risk factors were evaluated to determine their value to predict functional outcome and mortality within 3 months. RESULTS: During follow-up, an unfavorable functional outcome was found in 260 patients (35.3%), and 94 patients (12.8%) died. In multivariate models comparing the third and fourth quartiles to the first quartile of FABP4, the concentrations of FABP4 were associated with poor functional outcome and mortality. Compared with the reference category (Q1-Q3), the concentrations of FABP4 in Q4 had a relative risk of 4.77 (95% confidence interval [CI], 2.02-8.15; P<0.001) for poor functional outcome and mortality (odds ratio, 6.15; 95% CI, 3.43-12.68) after adjusting for other significant outcome predictors in univariate logistic regression analysis. Receiver-operating characteristic curves to predict poor functional outcome and mortality demonstrated areas under the curve of FABP4 of 0.78 (95% CI, 0.75-0.82) and 0.83 (95% CI, 0.79-0.88), which improved the prognostic accuracy of National Institutes of Health Stroke Scale score with combined areas under the curve of 0.83 (95% CI, 0.76-0.89; P<0.01) and 0.86 (95% CI, 0.81-0.92), respectively. CONCLUSIONS: Data show that FABP4 is a novel independent prognostic marker improving the currently used risk stratification of stroke patients.

Association of the p22phox polymorphism C242T with the risk of late-onset Alzheimer's disease in a northern Han Chinese population.

The C242T polymorphism of the CYBA gene that encodes p22phox, a component of nicotinamide adenine dinucleotide phosphate oxidase, has been found to modulate reactive oxygen species (ROS) production. Oxidative stress is thought to play a pivotal role in the pathophysiology of Alzheimer's disease (AD), which is manifested as increased availability of ROS because of an imbalanced redox state. Therefore, the aim of this study was to investigate potential associations of the p22phox C242T polymorphism with the risk of late-onset AD (LOAD) in a northern Han Chinese population. Patients with LOAD (n = 276) and 320 control subjects were recruited for the study. Polymerase chain reaction-restriction fragment length polymorphism was used to detect the genotypes. No significant differences were found between LOAD and p22phox C242T polymorphism, but a significant association was obtained in the genotype and allele distributions of p22phox C242T between LOAD patients and controls in apolipoprotein E (ApoE) ϵ4 carriers. These results suggested that p22phox C242T polymorphism has a possible role in changing the genetic susceptibility to LOAD in ApoE ϵ4 carriers of this northern Han Chinese population.

Diazoxide pretreatment prevents Aß1-42 induced oxidative stress in cholinergic neurons via alleviating NOX2 expression.

The aggregation and accumulation of amyloid-ß (Aß) plays a significant role in the pathogenesis of Alzheimer's disease. Aß is known to increase free radical production in neuronal cells, leading to oxidative stress and cell death. Diazoxide (DZ), a highly selective drug capable of opening mitochondrial ATP-sensitive potassium channels, has neuroprotective effects against neuronal cell death. However, the mechanism through which DZ protects cholinergic neurons against Aß-induced oxidative injury is still unclear. The present study was designed to investigate the effects of DZ pretreatment against Aß1-42 induced oxidative damage and cytotoxicity. Through measures of DZ effects on Aß1-42 induced cellular damage, reactive oxygen species (ROS) and MDA generation and expressions of gp91phox and p47phox in cholinergic neurons, new insights into the neuroprotective mechanisms can be derived. Aß1-42 significantly decreased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide levels and increased ROS and MDA production; all effects were attenuated by pretreatment with DZ or diphenyleneiodonium chloride (a NOX2 inhibitor). Pretreatment with DZ also attenuated the upregulation of NOX2 subunits (gp91phox and p47phox) induced by Aß1-42. Since NOX2 is one of the main sources of free radicals, these results suggest that DZ can counteract Aß1-42 induced oxidative stress and associated cell death by reducing the level of ROS and MDA, in part, by alleviating NOX2 expression.

High PSQI score is associated with the development of dyskinesia in Parkinson's disease.

Dyskinesia is one of the most disabling motor complications in Parkinson's Disease (PD). Sleep is crucial to keep neural circuit homeostasis, and PD patients often suffer from sleep disturbance. However, few prospective studies have been conducted to investigate the association of sleep quality with dyskinesia in PD. The objective of the current study is to investigate the association between sleep quality and dyskinesia and build a prediction model for dyskinesia in PD. We prospectively followed a group of PD patients without dyskinesia at baseline for a maximum of 36 months. Univariable and multivariable Cox regression with stepwise variable selection was used to investigate risk factors for dyskinesia. The performance of the model was assessed by the time-dependent area under the receiver-operating characteristic curve (AUC). At the end of follow-up, 32.8% of patients developed dyskinesia. Patients with bad sleep quality had a significantly higher proportion of dyskinesia compared with those with good sleep quality (48.1% vs. 20.6%, p = 0.023). Multivariable Cox regression selected duration of PD, sleep quality, cognition, mood, and levodopa dose. Notably, high Pittsburgh sleep quality index (PSQI) score was independently associated with an increased risk of dyskinesia (HR = 2.96, 95% CI 1.05-8.35, p = 0.041). The model achieved a good discriminative ability, with the highest AUC being 0.83 at 35 months. Our results indicated that high PSQI score may increase the risk of developing dyskinesia in PD, implying that therapeutic intervention targeting improving sleep quality may be a promising approach to prevent or delay the development of dyskinesia in PD.

The effect of ApoE ε 4 on clinical and structural MRI markers in prodromal Alzheimer's disease.

BACKGROUND: Apolipoprotein E (ApoE) ε 4 has been identified as the strongest genetic risk factor for Alzheimer's disease (AD). However, the importance of ApoE ε 4 on clinical and biological heterogeneity of AD is still to be determined, particularly at the prodromal stage. Here, we evaluate the association of ApoE ε 4 with clinical cognition and neuroimaging regions in mild cognitive impairment (MCI) participants based on the AT (N) system, which is increasingly essential for developing a precise assessment of AD. METHODS: We stratified 178 A+T+MCI participants (prodromal AD) into ApoE ε 4 (+) and ApoE ε 4 (-) according to ApoE genotype from the Alzheimer's Disease Neuroimaging Initiative (ADNI). We determined Aß-positivity (A+) by the standardized uptake values ratios (SUVR) means of florbetapir-PET-AV45 (the cut-off value of 1.1) and fibrillar tau-positivity (T+) by cerebrospinal fluid (CSF) phosphorylated-tau at threonine 181 position (p-Tau) (cut-off value of 23 pg/mL). We evaluated the effect of ApoE ε 4 status on cognitive conditions and brain atrophy from structural magnetic resonance imaging (MRI) scans. A multivariate analysis of variance was used to compare the differences of cognitive scores and brain atrophy from structural MRI regions of interest (ROIs) between both groups. Furthermore, we performed a linear regression model to assess the correlation between signature ROIs of structural MRI and cognitive scores in the prodromal AD participants. RESULTS: ApoE ε 4 (+) prodromal AD participants had lower levels of CSF Aß 1-42, higher levels of t-Tau, more memory and global cognitive impairment, and faster decline of global cognition, compared to ApoE ε 4 (-) prodromal AD. ApoE ε 4 (+) prodromal AD participants had a thinner cortical thickness of bilateral entorhinal, smaller subcortical volume of the left amygdala, bilateral hippocampus, and left ventral diencephalon (DC) relative to ApoE ε 4 (-) prodromal AD. Furthermore, the cortical thickness average of bilateral entorhinal was highly correlated with memory and global cognition. CONCLUSIONS: ApoE ε 4 status in prodromal AD participants has an important effect on clinical cognitive domains. After ascertaining the ApoE ε 4 status, specific MRI regions can be correlated to the cognitive domain and will be helpful for precise assessment in prodromal AD.

Diazoxide reverses the enhanced expression of KATP subunits in cholinergic neurons caused by exposure to Aß1₋42.

The ATP-sensitive potassium channel (KATP) play a crucial role in coupling metabolic energy to the cell membrane potential, ß-amyloid peptide (Aß) neurotoxicity has been associated with cellular oxidative stress and metabolic impairment. Whether there is an interaction between KATP and Aß or not? The expression of KATP subunits was to be investigated after the cultured primary rat basal forebrain cholinergic neurons being exposed to Aß1₋42. The subunits of KATP: Kir6.1, Kir6.2, SUR1 and SUR2 expressing change was observed by double Immunofluorescence and immunoblotting in cultured cholinergic neurons from different groups: treatment with Aß1₋42 (group Aß1₋42), pretreatment with diazoxide and then exposure to Aß1₋42 (group diazoxide + Aß1₋42), and the control (group control). The results showed that in response to the treatment with Aß1₋42 (2 µmol/L) for 24 h, the expression of Kir6.1 and SUR2 were significantly up-regulated, while this change can be partly reversed by pretreatment with diazoxide (1 mmol/L) for 1 h. There were significant increases in all KATP subunits expression levels after exposure to Aß1₋42 for 72 h. However, the up-regulation of Kir6.1, Kir6.2 and SUR2 except SUR1 can be partly reversed by pretreatment with diazoxide (1 mmol/L) for 1 h. It is concluded that exposure to Aß1₋42 for different time (24 and 72 h) induced differential regulation of KATP subunits expression in cultured primary rat basal forebrain cholinergic neurons. The change in composition of KATP may contribute to the dysfunction of KATP and membrane excitability disturbance. The effect of diazoxide on KATP subunits expression may explain, in part, the resistance of diazoxide to the toxicity of Aß1₋42.

Cellular and behavioral effects of 5-HT1A receptor agonist 8-OH-DPAT in a rat model of levodopa-induced motor complications.

5-HT1A autoreceptor stimulation can act to attenuate supraphysiological swings in extracellular dopamine levels following long-term levodopa treatment and may be useful in the treatment and prevention of the motor complications. The purpose of this study was to investigate cellular and behavioral effects of 5-HT1A receptor agonist 8-OH-DPAT in a rat model of levodopa-induced motor complications. Two sets of experiments were performed. First, animals were treated with levodopa (50 mg/kg with benserazide 12.5 mg/kg, twice daily), intraperitoneally (i.p.) for 22 days. On day 23, animals received either 8-OH-DPAT (1 mg/kg, i.p.) or 8-OH-DPAT plus WAY-100635 (0.1 mg/kg, i.p) or vehicle with each levodopa dose. In the second set, animals were treated either with levodopa (50 mg/kg, i.p.) plus 8-OH-DPAT (1 mg/kg, i.p.) or levodopa (50 mg/kg, i.p.) plus vehicle, administered twice daily for 22 consecutive days. Our study showed that 8-OH-DPAT plus levodopa both prolonged the duration of the motor response and reduced peak turning. 8-OH-DPAT plus levodopa also decreased the frequency of failures to levodopa. Co-administration of WAY-100635, a 5-HT1A receptor antagonist, with 8-OH-DPAT eliminated the effect of 8-OH-DPAT on motor complications indicating that the observed 8-OH-DPAT responses were probably mediated at the 5-HT1A autoreceptor. Moreover, 8-OH-DPAT plus levodopa significantly reduced hyperphosphorylation of GluR1 at serine 845, which was closely associated with levodopa-induced motor complications.

Repetitive transcranial magnetic stimulation for treatment of lactacystin-induced Parkinsonian rat model.

The dysfunction of ubiquitin-proteasome system is an important pathogenesis in the neurodegenerative process of Parkinson's disease. Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive and potential method in treating Parkinson's disease. To investigate whether rTMS has neuroprotective effects in parkinsonian rat model induced by ubiquitin-proteasome system impairment, we gave rTMS daily for 4 weeks to proteasome inhibitor, lactacystin-induced parkinsonian rat model. Rotational behavior test demonstrated that rTMS obviously reduced apomorphine-induced turning number in parkinsonian rats. rTMS could significantly alleviate the loss of tyrosine hydroxylase-positive dopaminergic neurons in lactacystin-lesioned substantia nigra and prevent the loss of striatal dopamine levels. Furthermore, rTMS also reduced the levels of apoptotic protein (cleaved caspase-3) and inflammatory factors (cyclooxygenase-2 and tumor necrosis factor alpha) in lesioned substantia nigra. These results suggest that rTMS can protect nigral dopaminergic neurons against the ubiquitin-proteasome system impairment-induced degeneration by anti-apoptotic and anti-inflammatory molecular mechanism.

Copeptin and NT-proBNP for prediction of all-cause and cardiovascular death in ischemic stroke.

OBJECTIVE: To evaluate long-term mortality in patients with acute ischemic stroke (AIS) by exploring the correlation between death and plasma concentrations of copeptin and N-terminal pro-B-type natriuretic peptide (NT-proBNP) in a cohort study. METHODS: In a prospective, multicenter observational study of 4,215 patients with AIS, copeptin and NT-proBNP levels were measured with a standardized method when patients were admitted to hospital. The primary endpoint was all-cause mortality or cardiovascular disease (CVD) mortality within 1 year. RESULTS: During a follow-up period, 906 patients (20.1%, 95% confidence interval [CI] 18.9-21.2) died, including 589 cases of CVD mortality (13.1%, 95% CI 12.1-14.0). With the use of a multivariate analysis, both markers were found to have prognostic value in the same model (CVD mortality: odds ratio [OR] for fourth quartile of copeptin and NT-proBNP 1.68 and 2.58, 95% CI 1.22-2.49 and 1.76-4.05, respectively; all-cause mortality: OR for fourth quartile of copeptin and NT-proBNP 1.48 and 2.47, 95% CI 1.22-2.03 and 1.68-3.95, respectively). In a receiver operating characteristics analysis of CVD mortality, the area under the curve varied from 0.80 to 0.83 (95% CI 0.79-0.87) when the index of NT-proBNP was added and increased to 0.86 (95% CI 0.83-0.90) when both markers were added. CONCLUSIONS: Copeptin and NT-proBNP may be useful independent prognostic markers of all-cause or CVD mortality in Chinese patients with AIS.

Dihydroartemisinin transiently activates the JNK/SAPK signaling pathway in endothelial cells.

Artemisinin and its derivatives are well-known anti-malaria drugs and in the early stages of research for cancer treatment. Dihydroartemisinin (DHA), a more water-soluble derivative of artemisinin, has demonstrated strong anti-angiogenic activity. The purpose of the present study was to investigate the underlying molecular mechanisms of the effect of DHA on angiogenesis. Human umbilical vein endothelial cells (HUVECs) treated with DHA were examined for apoptosis and activation of the c-Jun N-terminal kinase (JNK) signaling pathway, one of the major mitogen-activated protein kinase cascades. It was observed that 20 µM DHA induces transient activation of JNK in HUVECs. DHA also elevates the expression of cyclooxygenase-2 and matrix metalloproteinase-13, which is abolished by treatment with the JNK inhibitor SP600125. Although DHA persistently increases inhibitor of κB-α protein and thus inhibits nuclear factor-κB signaling, it does not affect apoptosis or caspase 3/9 activities in HUVECs. The present study provides key information for understanding the effects of DHA on endothelial cells, which is required for investigating its potential for clinic application as a chemotherapeutic agent.

Involvement of proinflammatory factors, apoptosis, caspase-3 activation and Ca2+ disturbance in microglia activation-mediated dopaminergic cell degeneration.

Increasing evidences suggest that activated microglia may contribute to neurodegeneration in Parkinson's disease (PD). In the present study, primary ventral mesencephalic (VM) cultures from E14 rats and PC12 cells were utilized as in vitro models to examine the mechanism underlying microglia activation mediated dopaminergic neurodegeneration. Using lipopolysaccharide (LPS) (1-100 ng/ml) as a tool, we observed that microglia activation-mediated a selective dopaminergic neurodegeneration in VM neuron-glia cultures, which was supported by the further study showing that conditioned medium (CM) from microglia-enriched cultures treated with LPS (10-100 ng/ml) decreased PC12 cell viability. The results from antibody neutralization, NO inhibition and superoxide neutralization suggested that the dopaminergic cell death was due to the production of microglia-derived proinflammatory factors (TNF-alpha, NO and superoxide), among which reactive oxygen species (ROS) might outweigh proinflammatory cytokines. Apoptosis assay on PC12 cells and primary dopaminergic neurons showed that apoptosis was a mechanism for both microglia activation-mediated dopaminergic cell death. Through Western blot and immunocytochemistry, we found that caspase-3 activation was involved in both dopaminergic cell injuries. Finally, the results from laser scanning confocal microscope demonstrated that PC12 cell intracellular free Ca(2+) ([Ca(2+)](i)) increased early after CM treatment. [Ca(2+)](i) increase involved influx of calcium from the extracellular milieu and release from intracellular stores and participated in the CM-induced PC12 cell apoptosis. Further investigations indicated that TNF-alpha, IL-1beta, NO and superoxide contributed at different degrees to CM-induced [Ca(2+)](i) increase and apoptosis in PC12 cells. Using primary VM cultures and PC12 cells, our study shows the roles of proinflammatory factors, apoptosis, caspase-3 activation and Ca(2+) disturbance in microglia activation-mediated dopaminergic cell degeneration. Understanding the mechanism for microglia activation-mediated dopaminergic neurodegeneration may contribute to the development of new neuroprotective strategies against PD.

Genistein protects dopaminergic neurons by inhibiting microglial activation.

Inflammation participates in the pathogenesis and progression of Parkinson's disease, in which microglia play a key role. Inhibition of microglia activation has been shown to attenuate inflammation-mediated dopaminergic neurodegeneration. In this study, we found that genistein, the primary soybean isoflavone, concentration-dependently attenuated the lipopolysaccharide-induced decrease in dopamine uptake and loss of tyrosine hydroxylase-immunoreactive neurons in rat mesencephalic neuron-glia cultures. Genistein also inhibited lipopolysaccharide-induced microglia activation and production of tumor necrosis factor-alpha, nitric oxide and superoxide in mesencephalic neuron-glia cultures and microglia-enriched cultures. Our results indicate that genistein may protect dopaminergic neurons from lipopolysaccharide-induced injury and its effective inhibition of microglia activation may be one of the mechanisms.

Postsynaptic density protein 95-regulated NR2B tyrosine phosphorylation and interactions of Fyn with NR2B in levodopa-induced dyskinesia rat models.

CONTEXT: Abnormality in interactions between N-methyl-d-aspartate (NMDA) receptor and its signaling molecules occurs in the lesioned striatum in Parkinson's disease (PD) and levodopa-induced dyskinesia (LID). It was reported that Fyn-mediated NR2B tyrosine phosphorylation, can enhance NMDA receptor function. Postsynaptic density protein 95 (PSD-95), one of the synapse-associated proteins, regulates interactions between receptor and downstream-signaling molecules. In light of the relationship between PSD-95, NR2B, and Fyn kinases, does PSD-95 contribute to the overactivity of NMDA receptor function induced by dopaminergic treatment? To further prove the possibility, the effects of regulating the PSD-95 expression on the augmented NR2B tyrosine phosphorylation and on the interactions of Fyn and NR2B in LID rat models were evaluated. METHODS: In the present study, parkinsonian rat models were established by injecting 6-hydroxydopamine. Subsequently, valid PD rats were treated with levodopa (50 mg/kg/day with benserazide 12.5 mg/kg/day, twice daily) intraperitoneally for 22 days to create LID rat models. Then, the effect of pretreatment with an intrastriatal injection of the PSD-95mRNA antisense oligonucleotides (PSD-95 ASO) on the rotational response to levodopa challenge was assessed. The effects of pretreatment with an intrastriatal injection of PSD-95 ASO on the augmented NR2B tyrosine phosphorylation and interactions of Fyn with NR2B in the LID rat models were detected by immunoblotting and immunoprecipitation. RESULTS: Levodopa administration twice daily for 22 days to parkinsonian rats shortened the rotational duration and increased the peak turning responses. The altered rotational responses were attenuated by PSD-95 ASO pretreatment. Meanwhile, PSD-95 ASO pretreatment decreased the level of PSD-95 protein expression and reduced both the augmented NR2B tyrosine phosphorylation and interactions of Fyn with NR2B triggered during the levodopa administration in the lesioned striatum of PD rats. However, the protein levels of Fyn and NR2B showed no difference under the above conditions. CONCLUSION: The data demonstrate that the inhibition of PSD-95 protein expression suppressed the interactions of Fyn with NR2B and NR2B tyrosine phosphorylation and subsequently downregulated NMDA receptor overactivation, thus providing benefit for the therapy of LID. Therefore, PSD-95 is important for overactivity of NMDA receptor function due to facilitating NR2B tyrosine phosphorylation dependent on Fyn kinase by regulating interactions of Fyn with NR2B under the pathological conditions of LID.

Effects of diazoxide on Aß1-42-induced expression of the NR2B subunit in cultured cholinergic neurons.

The accumulation of amyloid-ß protein (Aß) is significant in the pathogenesis of Alzheimer's disease. Several previous studies indicate that the NR2B­containing N­methyl­D­aspartate receptors are critically involved in the Aß mediated disruption of neuronal function. Diazoxide (DZ), a highly selective drug capable of opening mitochondrial ATP­sensitive potassium channels, has neuroprotective effects against neuronal cell death. However, the mechanism by which DZ protects cholinergic neurons against Aß­induced cytotoxicity remains to be elucidated. The present study was designed to investigate the effects of DZ pretreatment against Aß1­42­induced expression of NR2B in order to gain novel insights into the neuroprotective mechanisms. Following exposure to Aß1­42 for 24 h, the expression of the NR2B subunit remained unchanged compared with the control group. However, a significant increase in the expression of the NR2B subunit was observed following treatment with Aß1­42 for 72 h (P<0.05); and the upregulation of the expression of the NR2B subunit was reversed by pretreatment with DZ (P<0.05). These results suggested that DZ may counteract Aß1­42­mediated cytotoxicity by alleviating the expression of NR2B.

Diazoxide and N omega-nitro-L-arginine counteracted A beta 1-42-induced cytotoxicity.

K(+) channel openers can activate K channels and have been shown to protect cultured neurons against excitotoxicity. Our study showed that diazoxide, a K(+) channel opener, could counteract the effects of A beta(1-42) and protect cells from A beta(1-42)-induced the increasing of mitochondrial membrane potential and the associated increase in intracellular reactive oxygen species levels; an inducible nitric oxide synthase inhibitor, N omega-nitro-L-arginine could protect cells from A beta(1-42)-induced the increasing of both mitochondrial membrane potential and intracellular reactive oxygen species levels. A 24 h exposure to A beta(1-42) did not result in apoptosis, suggesting that the increase in both mitochondrial membrane potential and reactive oxygen species levels preceded cell apoptosis or death.

Diazoxide preconditioning antagonizes cytotoxicity induced by epileptic seizures.

Diazoxide, an activator of mitochondrial ATP-sensitive potassium channels, can protect neurons and astrocytes against oxidative stress and apoptosis. In this study, we established a cellular model of epilepsy by culturing hippocampal neurons in magnesium-free medium, and used this to investigate effects of diazoxide preconditioning on the expression of inwardly rectifying potassium channel (Kir) subunits of the ATP-sensitive potassium. We found that neuronal viability was significantly reduced in the epileptic cells, whereas it was enhanced by diazoxide preconditioning. Double immunofluorescence and western blot showed a significant increase in the expression of Kir6.1 and Kir6.2 in epileptic cells, especially at 72 hours after seizures. Diazoxide pretreatment completely reversed this effect at 24 hours after seizures. In addition, Kir6.1 expression was significantly upregulated compared with Kir6.2 in hippocampal neurons after seizures. These findings indicate that diazoxide pretreatment may counteract epileptiform discharge-induced cytotoxicity by suppressing the expression of Kir subunits.

Diazoxide and cyclosporin A protect primary cholinergic neurons against beta-amyloid (1-42)-induced cytotoxicity.

OBJECTIVE: Activation of mitochondrial (MitoKATP) channels was found to protect against anoxic and chemical stress in brain. This present study sought to investigate the ability of diazoxide and cyclosporin A to antagonize cytotoxicity induced by beta-amyloid peptide (A-beta1-42) in cultured rat primary basal forebrain cholinergic neurons. METHODS: Cytotoxicity was induced by A-beta1-42 (2 µM) in the presence of either diazoxide (500 µM), a selective opener of the mitochondrial ATP-sensitive potassium channel (MitoKATP), or cyclosporin A (20 µM), an inhibitor of the mitochondrial permeability transition pore (MTP), or the combination of both the reagents. We determined cell morphology and cell viability using MTT assay and expression levels of anti-apoptotic protein (Bcl-2), pro-apoptotic proteins (Bax, cytochrome C, caspase-3 and cleaved caspase-3) using Western blotting at 24 hours and 72 hours. RESULTS: Cell viability decreased markedly after exposure to A-beta1-42 for 72 hours with a decrease in the expression of Bcl-2 protein and cytochrome C and an increase in the caspase-3 and cleaved caspase-3 levels. Both diazoxide and cyclosporin A exerted significant protective effects on cell viability by ameliorating the decrease in Bcl-2 and the increase in cytochrome c and caspase-3 activity induced by A-beta1-42. The combination of both the reagents had a greater protective effect than either one alone. CONCLUSIONS: The present research demonstrates that activation of MitoKATP channels independently or in combination with inhibitors of the MTP can elicit a protective effect against primary cholinergic neuron cytotoxicity induced by A-beta1-42. These findings suggest new mitochondrial targets for the development of therapeutic agents against A-beta-induced cytotoxicity.