Identification of B.1.346 Lineage of SARS-CoV-2 in Japan: Genomic Evidence of Re-entry of Clade 20C.

SARS-CoV-2 whole-genome sequencing of samples from COVID-19 patients is useful for informing infection control. Datasets of these genomes assembled from multiple hospitals can give critical clues to regional or national trends in infection. Herein, we report a lineage summary based on data collected from hospitals located in the Tokyo metropolitan area. We performed SARS-CoV-2 whole-genome sequencing of specimens from 198 patients with COVID-19 at 13 collaborating hospitals located in the Kanto region. Phylogenetic analysis and fingerprinting of the nucleotide substitutions were performed to differentiate and classify the viral lineages. More than 90% of the identified strains belonged to Clade 20B, which has been prevalent in European countries since March 2020. Only two lineages (B.1.1.284 and B.1.1.214) were found to be predominant in Japan. However, one sample from a COVID-19 patient admitted to a hospital in the Kanto region in November 2020 belonged to the B.1.346 lineage of Clade 20C, which has been prevalent in the western United States since November 2020. The patient had no history of overseas travel or any known contact with anyone who had travelled abroad. Consequently, the Clade 20C strain belonging to the B.1.346 lineage appeared likely to have been imported from the western United States to Japan across the strict quarantine barrier. B.1.1.284 and B.1.1.214 lineages were found to be predominant in the Kanto region, but a single case of the B.1.346 lineage of clade 20C, probably imported from the western United States, was also identified. These results illustrate that a decentralized network of hospitals offers significant advantages as a highly responsive system for monitoring regional molecular epidemiologic trends.

Head positioning in suspected patients with acute stroke from prehospital to emergency department settings: a systematic review and meta-analysis.

AIM: This study aimed to clarify whether the lying-flat position from prehospital to emergency department settings more effectively improves neurological outcomes of patients suspected with acute stroke over the sitting-up position. METHODS: We searched PubMed, the Cochrane Central Register of Controlled Trials, and Igaku Chuo Zasshi for published randomized controlled trials until September 2019. The study population included patients suspected with acute stroke from prehospital to emergency department settings. We compared outcomes between the lying-flat position and sitting-up position groups. The critical outcome was the modified Rankin Scale score at 90 days, and important composite outcomes were 90-day mortality, pneumonia recurrence, and recurrent ischemic stroke. The certainty of evidence of the outcome level was compared using the Grading of Recommendations Assessment, Development, and Evaluation approach. RESULTS: In total, 881 studies were identified from the databases, and two randomized controlled trials were included in the analysis. The pooled risk ratio of 90-day modified Rankin Scale score was not statistically significant (risk ratio 0.86; 95% confidence interval [CI] 0.56-1.32) between the lying-flat position and sitting-up position groups. When comparing the 90-day mortality, pneumonia occurrence, and recurrent ischemic stroke, no significant differences were observed between the two groups. Risk ratio was 1.00 (95% CI 0.87-1.14), 0.90 (95% CI 0.74-1.11), and 0.81 (95% CI 0.14-4.64) for 90-day mortality, pneumonia occurrence, and recurrent ischemic stroke, respectively. CONCLUSION: This study suggests that the lying-flat position is not more effective than the sitting-up position in terms of 90-day modified Rankin Scale score in patients suspected with acute stroke.

Identification of B.1.346 lineage of SARS-CoV-2 in Japan: Genomic evidence of re-entry of Clade 20C

ObjectivesWhole SARS-CoV-2 genome sequencing from COVID-19 patients is useful for infection control and regional trends evaluation. We report a lineage data collected from hospitals in the Kanto region of Japan. MethodsWe performed whole genome sequencing in specimens of 198 COVID-19 patients at 13 collaborating hospitals in the Kanto region. Phylogenetic analysis and fingerprinting of the nucleotide substitutions underwent to differentiate and classify the viral lineages. ResultsMore than 90% of the strains belonged to Clade 20B and two lineages (B.1.1.284 and B.1.1.214) have been detected predominantly in the Kanto region. However, one sample from a COVID-19 patient in November 2020, belonged to the B.1.346 lineage of Clade 20C, which has been prevalent in western United States. The patient had no history of overseas travel and no contact with anyone who had travelled abroad, suggesting that this strain appeared likely to have been imported from western United States, across the strict quarantine barrier. ConclusionB.1.1.284 and B.1.1.214 have been identified predominantly in the Kanto region and B.1.346 of clade 20C in one patient was probably imported from western United States. These results illustrate that a decentralized network of hospitals can be significantly advantageous for monitoring regional molecular epidemiologic trends. Highlights{middle dot} Whole SARS-CoV-2 genome sequencing is useful for infection control {middle dot} B.1.1.284 and B.1.1.214 have been identified predominantly in the Kanto region {middle dot} B.1.346 of Clade 20C was detected in one COVID-19 patient in November {middle dot} Molecular genomic data sharing provides benefits to public health against COVID-19

Screening for impaired cognitive domains in a large Parkinson's disease population and its application to the diagnostic procedure for Parkinson's disease dementia.

BACKGROUND: Dementia is a new focus of research on improved treatment for Parkinson's disease (PD). In 2007, a screening tool for PD dementia (PD-D) was developed by the Movement Disorder Society (Level I testing), which still requires verification by a large population study. METHODS: We conducted a cross-sectional and multicenter study including 13 institutions administering the Mini-Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA) to 304 PD patients (mean age: 70.6 ± 8.3 years; mean Hoehn and Yahr stage: 2.7 ± 0.7). RESULTS: In all, 34.5% of the patients had MMSE scores <26; 94.3% of these patients had impairments in ≥2 cognitive domains and met the criteria for probable PD-D by Level I testing. Executive dysfunction combined with attention and memory impairment was most common (51.4%). In the Level I subtests of executive function, the score for phonemic fluency declined by <50% in patients with high MoCA scores (24-30 points) and lacked specificity for PD-D. No patient had visuospatial impairment (measured by the pentagon copying subtest) alone, and the score for pentagon copying stayed at ≥70% even in patients with low MMSE scores (12-25 points), therefore lacking sensitivity for PD-D. CONCLUSIONS: Level I testing with administration of the MMSE and MoCA is a practical and efficient screening tool for PD-D. However, the phonemic fluency and pentagon copying tests should be replaced by more specific/sensitive ones when screening for PD-D.

CHADS2 and CHA2DS2-VASc scores as bleeding risk indices for patients with atrial fibrillation: the Bleeding with Antithrombotic Therapy Study.

The CHADS2 and CHA2DS2-VASc scores, that is, ischemic stroke risk indices for patients having atrial fibrillation (AF), may also be useful as bleeding risk indices. Japanese patients with AF, who routinely took oral antithrombotic agents were enrolled from a prospective, multicenter study. The CHADS2 and CHA2DS2-VASc scores were assessed based on information at entry. Scores of 0, 1 and ⩾2 were defined as the low, intermediate and high ischemic risk categories, respectively, for each index. Of 1221 patients, 873 took warfarin, 114 took antiplatelet agents and 234 took both. The annual incidence of ischemic stroke was 0.76% in the low-risk category, 1.46% in the intermediate-risk category and 2.90% in the high-risk category by CHADS2 scores, and 1.44, 0.42 and 2.50%, respectively, by CHA2DS2-VASc scores. The annual incidence of major bleeding in each category was 1.52, 2.19 and 2.25% by CHADS2, and 1.44, 1.69 and 2.24% by CHA2DS2-VASc. After multivariate adjustment, the CHADS2 was associated with ischemia (odds ratio 1.76, 95% confidence interval 1.03-3.38 per 1-category increase) and the CHA2DS2-VASc tended to be associated with ischemia (2.18, 0.89-8.43). On the other hand, associations of the indices with bleeding were weak. In conclusion, bleeding risk increased gradually as the CHADS2 and CHA2DS2-VASc scores increased in Japanese antithrombotic users, although the statistical impact was rather weak compared with their predictive power for ischemic stroke.

Clinical features and varieties of non-motor fluctuations in Parkinson's disease: a Japanese multicenter study.

OBJECTIVE: This multicenter cross-sectional study aimed to investigate the clinical features and varieties of non-motor fluctuation in Parkinson's disease (PD). METHODS: To identify motor and non-motor fluctuation, we employed the wearing-off questionnaire of 19 symptoms (WOQ-19) in 464 PD patients. We compared the frequency of levodopa-related fluctuation as identified by the WOQ-19 with recognition by neurologists. We compared patients with both motor and non-motor fluctuations with those who only had motor fluctuations. Non-motor fluctuations were separated into psychiatric, autonomic, and sensory categories for further analysis. RESULTS: The patients' average age was 70.8 ± 8.4 years (mean ± SD) and disease duration was 6.6 ± 5.0 years. The frequency of motor fluctuations was 69% and for non-motor fluctuation 40%. Fifty-three percent of patients with motor fluctuations also had non-motor fluctuations, whereas 93% of patients with non-motor fluctuations also had motor fluctuations. The WOQ-19 showed a sensitivity of 82% but a specificity of only 40%. The patients with both non-motor and motor fluctuations exhibited more severe motor symptoms, more non-motor symptoms and higher levodopa daily doses (p < 0.05). Patients had significantly higher fluctuation rates if they had psychiatric (49%) and sensory (45%) symptoms than patients with autonomic symptoms (32%, p < 0.01). Forty-eight percent of patients with non-motor fluctuations exhibited more than one type of non-motor fluctuation. CONCLUSION: Forty percent of PD patients presented with non-motor fluctuations, and almost half of these exhibited more than one type. Appropriate recognition of levodopa-related fluctuations, both motor and non-motor, can lead to treatment modifications in PD patients.

REM sleep behavior disorder in Japanese patients with Parkinson's disease: a multicenter study using the REM sleep behavior disorder screening questionnaire.

REM sleep behavior disorder (RBD) is known to be observed more frequently in patients with an α-synucleinopathy such as Parkinson's disease (PD) than in the general population. The precise prevalence of RBD in Japanese PD patients is not known. Therefore, we investigated the prevalence and the clinical characteristics of patients with RBD in a large population of Japanese patients with PD. We investigated various clinical features and employed the Japanese version of the RBD screening questionnaire on 469 non-demented Japanese PD patients in this multicenter study. Probable or possible RBD was detected in 146 patients (31.1%) and was significantly associated with longer PD duration, higher Hoehn and Yahr stage, higher Unified Parkinson's Disease Rating Scale part III subscale (7 items), more motor fluctuations, and a higher levodopa-equivalent daily dose (p < 0.01). As to the major autonomic dysfunctions, severe constipation was significantly more frequent in PD patients with RBD than in those without it (p < 0.01). The RBD symptoms of 53 patients (39.0%) preceded the onset of PD motor symptoms. The median interval from the onset of RBD symptoms to PD motor symptoms was 17.5 years, and 3 patients had intervals of over 50 years. This large-scale multicenter study revealed that RBD is a frequent non-motor symptom in Japanese patients with PD, which may precede the onset of motor symptoms. Moreover, RBD that increases with the duration and severity of PD may be associated with autonomic dysfunction.

Camptocormia in Japanese patients with Parkinson's disease: a multicenter study.

OBJECTIVE: The aim of this work was to investigate the prevalence of camptocormia and the clinical characteristics of patients with camptocormia in a large population of PD patients. BACKGROUND: Although camptocormia has been recognized as a prominent phenomenon in PD, the previous epidemiological reports were limited, especially in terms of sample size. METHODS: We evaluated 531 PD patients (disease duration: 7.0 ± 5.5 years, mean ± standard deviation). We examined their clinical features and the prevalence of camptocormia. RESULTS: Camptocormia was detected in 22 patients (4.1%) and found in patients who were older and had more severe motor symptoms and a higher levodopa (L-dopa) dose (P < 0.05), compared to the patients without camptocormia. Patients with camptocormia showed significantly higher frequencies of autonomic symptoms, such as constipation and urinary incontinence (P < 0.05). CONCLUSIONS: Camptocormia is uncommon in PD and is associated with disease severity, higher L-dopa dose and higher frequencies of autonomic symptoms.

Blood pressure levels and bleeding events during antithrombotic therapy: the Bleeding with Antithrombotic Therapy (BAT) Study.

BACKGROUND AND PURPOSE: A prospective, multicenter, observational cohort study was conducted to clarify the association between major bleeding events and blood pressure (BP) levels during follow-up before development of bleeding events in antithrombotic users. METHODS: A total of 4009 patients taking oral antithrombotic agents for cardiovascular or cerebrovascular diseases (2728 men, 69+/-10 years old) were followed. Changes in systolic and diastolic BPs between entry and the last clinic visit before intracranial hemorrhage (ICH) or extracranial hemorrhage were assessed. RESULTS: Over a median follow-up of 19 months, ICH developed in 31 patients and extracranial hemorrhage developed in 77. Entry BP levels were similar among patients with ICH, those with extracranial hemorrhage, and those without hemorrhagic events. Both systolic BP and diastolic BP were relatively high during follow-up as compared with the levels at entry in patients with ICH, whereas they showed plateaus in patients with extracranial hemorrhage and patients without hemorrhagic events. Average systolic BP levels between 1 and 6 months (hazard ratio, 1.45; 95% CI, 1.08 to 1.92 per 10-mm Hg increase) and between 7 and 12 months (hazard ratio, 1.47; 95% CI, 1.05 to 2.01) as well as average diastolic BP levels between 7 and 12 months (hazard ratio, 2.05; 95% CI, 1.15 to 3.62) were independently associated with development of ICH after adjustment for established ICH predictors. The optimal cutoff BP level to predict impending risk of ICH was >or=130/81 mm Hg using receiver operating characteristic curve analysis. CONCLUSIONS: An increase in BP levels during antithrombotic medication was positively associated with development of ICH, suggesting the importance of adequate BP control for avoiding ICH. BP levels did not appear to be associated with extracranial hemorrhage.

Antithrombotic therapy influences location, enlargement, and mortality from intracerebral hemorrhage. The Bleeding with Antithrombotic Therapy (BAT) Retrospective Study.

BACKGROUND: To determine whether the use of oral antithrombotic agents before the onset of intracerebral hemorrhage (ICH) affects hematoma features and early patient outcome. METHODS: A retrospective, multicenter study involving 1,006 consecutive Japanese patients (607 men, 67 +/- 12 years of age) hospitalized within 24 h after the onset of nontraumatic ICH was conducted. RESULTS: One hundred and eighty patients were taking oral antiplatelet agents (17.9%, AP group), 67 were taking warfarin (6.7%, W group), and 21 were taking both (2.1%, W + AP group). After adjustment for age, sex, and known confounders, the taking of each kind of antithrombotic therapy was independently related to cerebellar hemorrhage; the odds ratios (OR) and 95% CI, with patients taking no antithrombotic agents as the reference group, were 2.31 (1.23-4.32) for the AP group, 2.90 (1.26-6.63) for the W group, and 3.43 (1.02-11.59) for the W + AP group. Similarly, the taking of each kind of antithrombotic therapy was independently related to hematoma enlargement within the initial 24 h (OR and 95% CI: AP group, 1.92, 1.10-3.34; W group, 4.80, 2.12-10.87; W + AP group, 4.94, 1.31-18.61) and mortality at 3 weeks post-ICH (OR and 95% CI: AP group, 2.70, 1.56-4.68; W group, 2.50, 1.05-5.96; W + AP group, 9.41, 2.78-31.88). CONCLUSIONS: Prior medication with antiplatelet agents, warfarin, or both was predictive of cerebellar hemorrhage, hematoma enlargement, and early death in Japanese ICH patients.

Appropriate data cleaning methods for genome-wide association study.

Genome-wide association studies (GWAS) using a large number of single nucleotide polymorphisms (SNPs) have successfully been applied to identify genetic variants of common diseases. However, genotyping using the new array technologies is often associated with spurious results that could unfavorably affect analyses of GWAS. Consequently, data cleaning is of paramount importance in excluding spurious genotyping results. In this study, we investigated the criteria required for the appropriate cleaning of 389 unrelated healthy Japanese samples analyzed using the GeneChip Human Mapping 500K Array Set for GWAS. The samples were randomly subdivided into two groups, and the allele frequencies in the groups were compared for individual SNPs as a quasi-case-control study. Then, observed results were filtered by four parameters (SNP call rate, confidence score obtained using the Bayesian Robust Linear Model with Mahalanobis genotype-calling algorithm, Hardy-Weinberg equilibrium, and minor allele frequency) and assessed for deviation from the null hypothesis. We found that appropriate data cleaning could be achieved using these four parameters. Our findings offer an avenue for obtaining appropriate data from GWAS.

Dual antithrombotic therapy increases severe bleeding events in patients with stroke and cardiovascular disease: a prospective, multicenter, observational study.

BACKGROUND AND PURPOSE: We sought to determine the incidence and severity of bleeding events in patients with stroke and cardiovascular diseases who were taking oral antithrombotic agents in Japan, where the incidence of hemorrhagic stroke is higher than in Western countries. METHODS: A prospective, multicenter, observational study was conducted; 4009 patients who were taking oral antithrombotic agents for stroke and cardiovascular diseases were enrolled. The patients were classified into 4 groups according to their antithrombotic treatment: the single antiplatelet agent group (47.2%); the dual antiplatelet agent group (8.7%); the warfarin group (32.4%); and the warfarin plus antiplatelet agent group (11.7%). The primary end point was life-threatening or major bleeding according to the MATCH trial definition. RESULTS: During a median follow-up of 19 months, there were 57 life-threatening and 51 major bleeding events, including 31 intracranial hemorrhages. The annual incidence of the primary end point was 1.21% in the single antiplatelet agent group, 2.00% in the dual antiplatelet agent group, 2.06% in the warfarin group, and 3.56% in the warfarin plus antiplatelet agent group (P<0.001). After adjustment for baseline characteristics, adding an antiplatelet agent to warfarin increased the risk of the primary end point (relative risk=1.76; 95% CI, 1.05 to 2.95), and adding another antiplatelet agent to single antiplatelet agent therapy increased the secondary end point of any bleeding, including minor events (relative risk=1.37; 95% CI, 1.07 to 1.76). CONCLUSIONS: The incidence of bleeding events during antithrombotic therapy in Japan was similar to that reported for Western countries, although the trials used different study designs. Dual antithrombotic therapy was independently related to an increased risk of bleeding events.

The effect of weekends and holidays on stroke outcome in acute stroke units.

BACKGROUND AND PURPOSE: In almost all acute stroke units in Japan, staffing level is lower on weekends and holidays and rehabilitative services are provided only on weekdays. We sought to investigate the effects of low-volume care early after stroke resulting from weekends and holidays on the outcome of stroke. METHODS: Patients with completed stroke within 72 h of onset were prospectively registered by 10 acute stroke units in Japan. Main outcome measures were favorable outcomes as indicated by a score of 0-1 on the modified Rankin scale (mRS01) on their 21st hospital day and at discharge and case fatality during the hospital stay. Cox proportional hazardsmodels were used to identify the effects of weekday admission and a weekday ratio (a number of weekdays / total length of hospital stay, or 21 days if hospitalization was longer than 21 days) on the main outcome measures. RESULTS: In a total of 1,134 patients, Cox proportional hazards regression analyses demonstrated that the weekday admission was significantly associated with mRS01 at discharge (hazard ratio, HR: 1.385, 95% CI: 1.087-1.764) and case fatality (HR: 0.477, 95% CI: 0.285-0.798). In 858 patients with rehabilitative therapy, the weekday ratio was significantly associated with mRS01 at discharge (p = 0.014). Compared with the lowest tertile of weekday ratio (<66.6%), the highest tertile (>71.4%) was significantly positively associated with mRS01 at discharge (HR: 1.524, 95% CI: 1.053-2.206; p < 0.026). CONCLUSIONS: Weekday admission was an independent negative predictor of case fatality and a positive predictor of favorable outcome (mRS01) at discharge from acute stroke units. In patients with rehabilitative therapy, a reduction in the weekday ratio was also associated with unfavorable outcome, probably due to a reduction in multidisciplinary care.

Hospital cost of ischemic stroke and intracerebral hemorrhage in Japanese stroke centers.

BACKGROUND: In Japan, the healthcare expenditure has increased to 8.0% of the gross domestic products in 2001. Stroke care is costly. OBJECTIVE: To examine hospital costs and clinical outcomes of ischemic stroke (IS) and intracerebral hemorrhage (ICH) in Japanese stroke centers. DESIGN: A prospective non-interventional multi-center study. SETTING: Ten Japanese stroke centers. STUDY PERIOD: Fourteen months between October 2000 and December 2001. PATIENTS: Patients were those who were consecutively hospitalized with acute IS or ICH within 72 h of onset, excluding subarachnoid hemorrhage. Stroke was defined as focal neurological deficits lasting more than 24 h and the relevant lesions were to be confirmed by brain CT and/or MRI. METHODS: We examined demography, in-hospital cares, length of hospital stay, clinical outcomes at discharge, and direct hospital medical costs including physician's fees. The hospital medical cost data were collected from official hospital medical cost charts for reimbursement to the healthcare insurance systems. RESULTS: There were a total of 1113 patients with a mean age of 70 years, of whom 913 (82%) patients had an IS and 200 (18%) patients had an ICH. The 317 patients (28%) experienced a recurrent stroke. Patients with ICH had the higher baseline stroke severity, resulting in longer hospitalization (39 days for IS and 46 days for ICH; P<0.001), lower independence rate at discharge (55 and 40%; P<0.001), higher mortality rate (5 and 10%; P=0.03), and higher medical costs (US dollar 8662 and US dollar 10,260; P<0.001) than those with IS. Patients with recurrent stroke had significantly older age, higher stroke severity, and lower independence rate at discharge than those with first-ever stroke. The length of stay, in-hospital mortality, and hospital medical costs were similar among first-ever and recurrent strokes. In subtype of IS, patients with cardioembolic stroke had the worst neurological deficits, poorest outcomes, and highest medical costs. The hospital costs had a greatest association with length of stay. CONCLUSIONS: Stroke care is costly in Japan. ICH is more likely to impose substantial physical and economic burden than IS. Because the cares of both first-ever and recurrent stroke were costly, primary and secondary prevention of stroke is important on the healthcare aspects.

FK506 abrogates delayed neuronal death via suppression of nitric oxide production in rats.

BACKGROUND AND PURPOSE: The mechanism of the neuroprotective effect of FK506 in relation to nitric oxide (NO) production has not been clarified in vivo. We have investigated the effect of FK506 on ischemia-induced NO production in association with the pathogenesis of delayed neuronal death (DND) in rats. METHODS: In vivo microdialysis was performed in the hippocampus of male Sprague-Dawley rats (250-350 g). Dialysate samples were collected every 3 min. In the ischemia group (n=16), global ischemia was induced for 21 min and reperfusion was achieved. In the FK506 treatment group (n=25), FK506 (1 mg/kg, i.v.) was administered 21 min prior to the onset of global ischemia. Sham operations were done (n=15). The levels of NO(2)(-) in the dialysate samples were determined by the Griess reaction. The animals were decapitated 7 days after ischemia. Coronal brain sections were stained with hematoxylin and eosin. RESULTS: In the ischemia group, the NO(2)(-) level significantly increased during ischemia. In the FK506 treatment group, there was no significant change in the NO(2)(-) level during ischemia. In histological examinations, FK506 treatment showed a neuroprotective effect against DND. CONCLUSIONS: The effect of FK506 inhibiting NO production contributes to the neuro-protective effect of FK506 on DND in the hippocampus.

Subcellular localization of a promoter and an inhibitor of apoptosis (Smac/DIABLO and XIAP) during brain ischemia/reperfusion.

We explored the expression of Smac/DIABLO, a newly identified mitochondrial apoptogenic molecule, and X-linked inhibitor of apoptosis protein (XIAP) in the brain subjected to ischemia/reperfusion. Transient focal ischemia was produced for 1 hour in mice. We observed only a negligible amount of Smac/DIABLO in both mitochondria and cytosol in the normal state. The mitochondrial expression level of Smac/DIABLO increased after 2-11 h reperfusion. There was increased Smac/DIABLO expression in the cytosol after 5 h reperfusion, implying the translocation of Smac/DIABLO into the cytosol. The subcellular localization of XIAP became more extensive within the cells during reperfusion, as compared with the normal state. Our results imply that Smac/DIABLO and XIAP are implicated in the pathophysiological mechanisms of reperfusion injury.

Temporal profiles of the subcellular localization of Bim, a BH3-only protein, during middle cerebral artery occlusion in mice.

BH3-only proteins are a subfamily of proapoptotic Bcl-2 proteins that act upstream of the mitochondrially mediated cell death pathway, and their association with the pathogenesis of brain ischemia remains largely unknown. The authors explored the temporal profiles of the expression levels and subcellular localization of BH3-only proteins in permanent middle cerebral artery occlusion (MCAO) by Western blot analysis. They observed an increased mitochondrial distribution of Bim at 3 to 6 hours of MCAO that appeared unrelated to transcriptional upregulation, as assessed by semiquantitative reverse transcription-polymerase chain reaction. At 3 to 6 hours of MCAO, Bim immunoreactivity was enhanced in neurons and oligodendrocytes in the ischemic regions. The increased mitochondrial localization of Bim coincided with a marked cytochrome c release and preceded the peak of caspase-9 activation. The authors observed an association of Bim with the dynein intermediate chain, a major component of the dynein motor complex, in the brain using a coimmunoprecipitation assay. Cerebral ischemia induced a time-dependent significant decrease in dynein expression, which started at 3 hours of MCAO. The authors deduced that the liberation of Bim from the dynein motor complex is a likely mechanism for the increased mitochondrial localization of Bim. During MCAO, Bad did not show any change in phosphorylation state or subcellular localization.

Substrate-dependence of reduction of MTT: a tetrazolium dye differs in cultured astroglia and neurons.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction is widely used to evaluate cell proliferation and viability. MTT reduction is interpreted to be indicative of cellular metabolic activity, and the site of reduction includes both mitochondrial and cytosolic redox reactions. Astrocytes are believed to rely mainly on glycolysis for ATP generation, whereas neurons are considered to depend more on oxidative metabolism. The present study, therefore, tested the substrate-preference of glucose and its metabolites for MTT reduction in cultures of rat type 1 astroglia and neurons.MTT specific activity of astroglia was much higher than that of neurons. Astroglial MTT reducing activity in glucose-free medium or 2mM glucose with iodoacetate (5mM) was completely blocked. In glucose-depleted medium, 2mM lactate, pyruvate, malate, or acetate elicited minimal increases in MTT reduction by astroglia. In contrast, MTT reducing activity in neurons was enhanced two-fold by pyruvate and the reducing activity of lactate was equivalent to that of glucose, while malate had a small and acetate had no effect on MTT reduction. These results indicate that these two cell types differ markedly in their substrate-preferences for MTT reduction. In astroglia, MTT reduction reflects mainly cytosolic redox activity and is dependent on glyceraldehyde-3-phosphate dehydrogenase. In neurons, pyruvate dehydrogenase supports MTT reduction more effectively than glucose or lactate, even though both of these substrates can produce NADH and pyruvate.