Saori Kohara

Post-ischemic delayed expression of hepatocyte growth factor and c-Met in mouse brain following focal cerebral ischemia.

We investigated long-term changes in the expression of protein and mRNA of hepatocyte growth factor (HGF) and its receptor c-Met in mouse brain after permanent occlusion of the middle cerebral artery, by using immunohistochemistry and quantitative reverse transcription-polymerase chain reaction. HGF-immunopositive cells were observed in the periinfarct region from 4 days after occlusion, peaking at 14-28 days. The area containing HGF-immunopositive cells continued to expand until 28 days after occlusion. c-Met-immunopositive cells were observed exclusively at the periinfarct region at 7 and 14 days after occlusion. At 28 days after occlusion, there were many c-Met-immunopositive cells in the widespread periinfarct region. Triple immunohistochemical staining by using confocal laser scanning microscopy (CLSM) demonstrated that most of the HGF-immunopositive cells were localized to reactive astrocytes. The c-Met-immunopositive cells were also localized to reactive astrocytes. HGF mRNA was upregulated exclusively in the periinfarct region at 14 days. c-Met mRNA was upregulated in the periinfarct region from as late as 28 days after occlusion. Thus, HGF and c-Met show delayed expression in the periinfarct region at both protein and mRNA levels after induction of ischemia. Because HGF was recently shown to play critical roles in angiogenesis and neurotrophic activities, the temporal profiles of their expression may imply the involvement of HGF in the process of post-ischemic brain tissue repair.

Quercetin, a natural flavonoid, attenuates vacuolar formation in the optic tract in rat chronic cerebral hypoperfusion model

Epidemiological studies suggest that the intake of flavonoids is inversely associated with risk of cardiovascular diseases and stroke, but there is no evidence showing the effect of flavonoids on vascular dementia. Because quercetin, a natural flavonoid, is known to scavenge free radicals, we investigated whether quercetin attenuates white matter damage in rats with chronic cerebral hypoperfusion, as a model of vascular dementia. Chronic hypoperfusion was induced by ligation of the bilateral carotid arteries in male Wistar rats, which received vehicle alone, 100 mg/kg quercetin, or 200 mg/kg quercetin intraperitoneally at 4-day intervals for 8 weeks after operation. Sham-operated rats were also studied. The area of vacuoles in the optic tract observed after hematoxylin and eosin staining was significantly reduced in the 200 mg/kg quercetin-treated hypoperfusion group versus the vehicle-treated hypoperfusion group (1.7+/-0.2% versus 3.9+/-0.3%; P<0.05). The present results are consistent with the idea that chronic treatment with quercetin could be protective against at least a part of ischemic white matter damage.

Activated platelets in transient global amnesia and TIA

To assess whether platelets are activated in transient global amnesia (TGA) and TIA, blood samples were analyzed by fluorescence-activated cytoscan using antibodies specific for platelet fibrinogen receptor (PAC1) and P-selectin (CD62P). Samples from TIA contained high levels of CD62P compared with age-matched control subjects, whereas those from TGA did not. The authors suggest that activated platelets are involved in brain ischemia, whereas ischemia appears not to be associated with most TGA.

Effect of single nucleotide polymorphisms of the prostacyclin receptor gene on platelet activation in Japanese healthy subjects and patients with cerebral infarction

Cerebral infarction (CI) is a complex multifactorial disorder that is thought to result from the interaction of various environmental factors and an individuals genetic make-up, including genes associated with platelet activation. In order to clarify whether single nucleotide polymorphisms (SNPs) of the prostacyclin receptor (IP) gene affects platelet activation in ischemic stroke, we investigated the relationship between platelet function and genetic polymorphism of the coding sequence of the IP gene in 64 Japanese patients with CI and 54 healthy subjects. We determined the entire nucleotide sequence of the IP gene in healthy Japanese subjects, and found that an adenine (A) to cytosine (C) substitution at base 984 (A984C) in exon 3 is the most frequent SNP. Using flow cytometry, the power-transformed mean percentage of PAC-1-positive platelets, [PAC-1](1/3), was significantly higher in healthy subjects with the C/C genotype than in healthy subjects with the A/A genotype (p ≤ 0.05), although there was no significant difference in patients with CI between these two genotypes. Furthermore, we genotyped 158 control patients and 106 patients with CI. The homozygous C/C genotype was more frequently found in the CI group (46.2%) than in the healthy control group (17.1%; p < 0.001). The present report is the first to show an association between the A984C polymorphism of the IP gene and platelet activation in Japanese subjects. This polymorphism may be clinically significant in disorders in which prostacyclin plays a key role, such as CI.

Cilostazol protects against microvascular brain injury in a rat model of type 2 diabetes.

Cilostazol, a pluripotent phosphodiesterase III-specific inhibitor with anti-platelet and vasculogenic effects, is useful for preventing recurrent brain vascular events, particularly in stroke patients with diabetes mellitus (DM). However, it is unclear whether cilostazol affects autoregulatory responses in small cerebral arteries. Thus, we investigated the effect of cilostazol on diabetic brain vasculopathy in a model of type II DM using male OLETF rats. OLETF rats were treated with either cilostazol (CG) or vehicle (VG) and subjected to microangiography with monochromatic synchrotron radiation to investigate middle cerebral artery (MCA) vasoreactivity following an injection of acetylcholine (Ach). Ach administration led to MCA diameter contraction in the VG, but MCA dilation in the CG. We also evaluated morphological changes in the small intracranial vessels and found that in the CG, the endothelial cell structure in the small artery was not destroyed. Moreover, protein levels of phosphorylated endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) were higher in each evaluated brain region in CG rats vs. VG rats. Our results suggest that cilostazol could potentially improve autoregulatory responses in the small cerebral arteries by increasing eNOS phosphorylation and VEGF expression in DM, and thus, may act as a neurovascular protectant.

Platelet Aggregates Detected by a Conventional Hematology Analyzer Method Is a Risk Factor for Stroke or a Predictive Factor in Patients With Chronic-Stage Cerebral Infarction

Assessment of platelet activation and/or function is important for primary and secondary prevention of vascular events. To test the hypothesis that determination of platelet aggregation in patients with chronic-stage cerebral infarction (CI) provides a simple measure of risk for ischemic stroke, we used a conventional hematology analyzer to detect aggregates and to assess the efficacy of antiplatelet agents in preventing spontaneous aggregate formation. Platelet aggregates were measured in citrated blood from 142 magnetic resonance imaging confirmed CI patients and 97 controls without CI (nonstroke). Aggregates were detected in 1 of 36 (2.8%) nonstroke subjects without risk factors, but in 24 of 52 (46.2%) nonstroke subjects with risk factors (odds ratio [OR], 3.32; 95% confidence interval [CI], 1.10-10.00), in 21 of 35 (60.0%) nonstroke subjects with a predictive factor (carotid artery intima-media thickness [IMT] >1.1 mm) (OR, 9.13; 95% CI, 2.70-30.48), and in 31 of 63 (49.2%) CI patients who had not received antiplatelet therapy (OR, 2.16; 95% CI, 1.12-4.17). After adjusting for other risk factors, the appearance of platelet aggregates was correlated only with IMT ≥1.1 mm. The rate of appearance of platelet aggregates was 0.33-fold lower in patients on antiplatelet therapy compared with those not on antiplatelet therapy (24.1%; 19 of 79 CI patients). Patients with platelet aggregates in the blood are considered at high risk for ischemic stroke, because the appearance of aggregates is associated with increased IMT. Our method is suitable for screening platelet function in high-risk patients and for examining the efficacy of antiplatelet agents.

Dual Therapy with Aspirin and Cilostazol May Improve Platelet Aggregation in Noncardioembolic Stroke Patients: A Pilot Study

Objective Some previous studies have found clinical benefit of dual antiplatelet therapy with aspirin and cilostazol for prevention of secondary stroke, but the physiological mechanism involved remains unknown. We aimed to clarify the effects of aspirin/cilostazol therapy on the platelet and endothelial functions of patients with acute noncardioembolic ischemic stroke, in comparison to patients who were treated with aspirin alone. Methods The present randomized prospective pilot study enrolled 24 patients within a week after the onset of noncardioembolic ischemic stroke. The patients were randomly allocated to receive aspirin (100 mg/day) (A group; 11 patients) or cilostazol (200 mg/day) plus aspirin (100 mg/day) (CA group; 13 patients). We measured platelet aggregation, platelet activation, and the thrombomodulin (TM), highly sensitive C-reactive protein (hs-CRP), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and von Willebrand (vWF) antigen levels and vWF activity over a 4-week period after enrollment. Results There was no significant difference in the platelet functions of the A and CA groups. However, the platelet aggregation induced by adenosine diphosphate (ADP) was decreased at 2 and 4 weeks (p<0.05) after treatment in comparison to the pre-treatment values in the CA group, but not in the A group. Platelet activation, and the hs-CRP, TM, ICAM-1, VCAM-1 and vWF values did not significantly decrease after treatment in either group. Conclusion Although there were no significant differences in platelet aggregation, platelet activation or the endothelial biomarker levels of the A and CA groups, dual therapy with aspirin and cilostazol inhibited platelet aggregation in comparison to the pre-treatment values, similarly to patients who received aspirin alone. This may suggest the clinical usefulness of dual therapy with aspirin and cilostazol in the treatment of patients with noncardioembolic ischemic stroke.

123I-Meta-iodobenzylguanidine (MIBG) myocardial scintigraphy in patients showing scans without evidence of dopaminergic deficits (SWEDDs)

OBJECTIVE Scans without evidence of dopaminergic deficits (SWEDDs) in dopamine transporter single-photon emission computed tomography (DAT-SPECT) are found in 3.6-19.6% of patients with clinically suspected Parkinsons disease (PD). We investigated whether combined use of 123I-meta-iodobenzylguanidine (MIBG) myocardial scintigraphy would be helpful to differentiate PD among SWEDDs patients. PATIENTS AND METHODS 145 patients with clinically suspected PD underwent both DAT-SPECT and MIBG myocardial scintigraphy. Striatal binding ratio (SBR) of DAT-SPECT and heart-to-mediastinal (H/M) ratio and washout rate (WR) of MIBG myocardial scintigraphy were calculated. RESULTS Among 18 SWEDDs patients (12.4%), 11 were finally diagnosed with PD based on follow-up for at least two years after the DAT-SPECT and MIGB myocardial scintigraphy scans. Among the latter group, 8 patients showed an H/M ratio of less than 2.2, and 9 showed WR above 30%. CONCLUSION Our results indicate that the combination of low H/M ratio and high WR of MIBG myocardial scintigraphy of SWEDDs patients may be helpful for detection of PD patients.

Effect of atorvastatin co‐treatment on inhibition of platelet activation by clopidogrel in patients with ischemic stroke

Clopidogrel is used to achieve sustained platelet inhibition in ischemic stroke patients (1). 3-Hydroxy-3methylglutaryl-CoA reductase inhibitors also reduce the stroke events (2). We hypothesized that an interaction between clopidogrel and 3-Hydroxy-3methylglutaryl-CoA reductase inhibitors would decrease P2Y12 receptor and inhibit platelet activation by acting on lipid rafts (3,4). To test this idea, we evaluated the effect of coadministration of atorvastatin on the inhibition of platelet activation in ischemic stroke patients receiving clopidogrel. Twenty ischemic stroke patients (13 atherothrombotic, 7 lacunar; mean age 68 ± 12, 65% male) receiving clopidogrel with dyslipidemia were recruited. Written informed consent was obtained from all patients, and the study was approved by Tokai University Ethics Committee. Blood samples were taken before, and at twoweeks and two-months after the administration of 10-mg/day atorvastatin. PAC1, CD62P, and WBC-platelet complex were quantified by means of three-color flow cytometry, according to our previous reports (5–8). Coadministration of atorvastatin for two-months significantly reduced LDLcholesterol from 143 ± 26 to 92 ± 20 mg/dl (P < 0·01). No significant difference in PAC-1-positive platelets beyond the cutoff value was observed (Fig. 1a). However, the percentage of CD62Ppositive platelets beyond the cutoff value was significantly reduced by coadministration of atorvastatin for two-months, compared with clopidogrel alone (Fig. 1b, P < 0·05). The percentage of WBCplatelet complex beyond the cutoff value was also significantly reduced by coadministration of atorvastatin for twoweeks and two-months, compared with clopidogrel alone (Fig. 1c, P < 0·05). The suppression of CD62P may suggest that the translocation of P2Y12 receptors away from lipid rafts, due to reduction of lipid rafts by atorvastatin (4), inhibits platelet activation by suppressing adenylate cyclase activation via Gi (9). Further, WBC-platelet complex is associated with expression of P-selectin (CD62P), which mediates adhesion of platelets to leukocytes (10). Our results indicate that coadministration of atorvastatin in ischemic stroke patients receiving clopidogrel further enhances the inhibition of platelet activation.

Estimation of the perioperative management of stroke patients receiving antiplatelet therapy

外科的手術あるいは内視鏡検査前などにおける抗血小板薬休薬の至適期間を,血小板凝集の面から検討した.手術・検査のために抗血小板薬を一時中止した患者22例(アスピリン9例,チクロピジン13例)を対象とし,休薬前,休薬3日,7日,14日目に比濁法にて血小板最大凝集率を求めた.その結果,アスピリン群では休薬前に対し休薬3日後より有意に凝集能が亢進し(14.0±8.5% vs 48.1±31.2%; p<0.05),チクロピジン群では休薬14日後に初めて有意差(19.5±11.4%vs58.4±30.8%; P<0.05)を認めた.確実な目処もなく日常的に行われている休薬期間を最小限にするという観点から血小板凝集能を指標とした場合,アスピリンは術前3日,チクロピジンは2週間が望ましいと結論された.