Ultrastructural and temporal changes of the microvascular basement membrane and astrocyte interface following focal cerebral ischemia.

Microvascular integrity is lost during cerebral ischemia. Detachment of the microvascular basement membrane (BM) from the astrocyte, as well as degradation of the BM, is responsible for the loss of microvascular integrity. However, their ultrastructural and temporal changes during cerebral ischemia are not well known. Male Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion (MCAO) for 1, 4, 8, 12, 16, 20, and 48 hr. By using transmission electron microscopy, the proportion of intact BM-astrocyte contacts and electron densities of the BM were measured from five randomly selected microvessels in the ischemic basal ganglia. Their temporal changes and associations with activities of the matrix metalloproteinases (MMPs) were investigated. The intact portion of the BM-astrocyte contacts was decreased significantly within 4 hr and was rarely observed at 48 hr after MCAO. Decreases in the electron density and degradation of the BM were significant 12 hr after MCAO. The intact BM-astrocyte contacts and the mean BM density showed a significant positive correlation (r = 0.784, P < 0.001). MMP-9 activity was correlated negatively with the intact BM-astrocyte contacts (r = -0.711, P < 0.001) and with the BM density (r = -0.538, P = 0.0016). The increase in MMP-9 coincided temporally with the loss of the BM-astrocyte contacts and a decrease in the BM density. Ultrastructural alterations occurring in the microvascular BM and its contacts with astrocyte endfeet were temporally associated in cerebral ischemia. Time courses of their alterations should be considered in the treatment targeted to the microvascular BM and its contact with astrocytes.ope

Microvascular Response Following Focal Cerebral Ischemia in Diabetic Rats Treated with Tissue Plasminogen Activator

Background: Diabetes mellitus (DM) or hyperglycemia is known to be associated with increased risk of intracranial hemor-rhage in acute stroke patients who are treated with IV tissue-type plasminogen activator (tPA). However, the underlying mechanism remains unknown. We tested the hypothesis that different expression of matrix metalloproteinase (MMP) and occludin in DM play a role in increased risk of hemorrhages in DM by using a diabetic rat model. Methods: DM was induc-ed in Spargue Dawley rats by using injection of streptozotocin (60 mg/kg) and harvesting for 20 weeks. Focal cerebral ischemia was induced by occlusion of the middle cerebral artery occlusion (MCAO) for 2 hours and then reperfusion for 18 hours by using a nylon thread. tPA (10 mg/kg) or normal saline was infused via the femoral vein. Spectrophotometry, western blot and zymography were performed to measure the amount of hemorrhages, levels of occludin (tight junction protein) and activity of matrix metalloproteinase (MMP)-2 and MMP-9, respectively. Results: Mortality was significantly higher in diabetic rats. In surviving rats, hemorrhagic amounts and levels of cerebral occludin were not different between diabetic and nondiabetic rats, and tPA-infused and normal saline-infused rats. Although MMP-9 were higher in ischemic groups (P=0.001), their activities were not changed in diabetic or tPA-treated rats. Conclusion: Occludin and MMP may not play a major role in increasing the risk of hemorrhage in diabetic rats.ope

Neutrophil Recruitment in Arterial Thrombus and Characteristics of Stroke Patients with Neutrophil-Rich Thrombus

Purpose: Neutrophils contribute to thrombosis. However, there is limited information on the temporal course of neutrophil recruitment in thrombosis, the contribution of neutrophils to thrombus growth, and the characteristics of stroke patients with neutrophil-rich thrombi. Materials and methods: After inducing carotid artery thrombosis in Institute of Cancer Research mice using ferric chloride, aged thrombi were produced by ligating the distal portion of the carotid artery in mice for 0.5, 1, 2, 3, 6, or 24 h. For thrombus analysis in stroke patients, we used registry data and thrombi that were obtained during intra-arterial thrombectomy. Immunohistochemistry was performed to determine thrombus composition. Results: In the thrombi of 70 mice, Ly6G positive cell counts (neutrophils) and histone H3-positive cell counts increased in a time-dependent manner (both p<0.001). Ly6G-positive cell count was strongly correlated with histone H3-positive cell counts (r=0.910, p<0.001), but not with thrombus size (p=0.320). In 75 stroke patients, atrial fibrillation and cardioembolism were more frequent in the higher neutrophil group (32/37, 86.5%) than in the lower neutrophil group (19/38, 50%) (p=0.002). The median erythrocyte fraction was higher [52.0 (interquartile range 39.9-57.8)] in the higher neutrophil group than in the lower neutrophil group [40.3 (interquartile range 23.5-53.2)]. The fraction of neutrophils was positively correlated with that of erythrocytes (R=0.35, p=0.002). Conclusion: Neutrophils were recruited and increased in arterial thrombosis in a time-dependent manner; however, they were not associated with the growth of formed thrombi. Neutrophil fractions in the thrombi of stroke patients appeared to be associated with atrial fibrillation and erythrocyte fraction.ope

Effects of Interleukin-17A on the Early Stages of Arterial Thrombosis in Mice

Purpose: Interleukin (IL)-17A has been suggested to play a role in the growth and organization of thrombi. We examined whether IL-17A plays a role in the early stages of thrombosis and whether there are sex differences in the effects of IL-17A. Materials and methods: We performed a blinded, randomized, placebo-controlled study to compare time to thrombotic occlusion and sex differences therein between mice treated with IL-17A and those treated with saline using a ferric chloride-induced model. We also assessed thrombus histology, blood coagulation, and plasma levels of coagulation factors. Results: Time to occlusion values did not differ between the IL-17A group and the control group (94.6±86.9 sec vs. 121.0±84.4 sec, p=0.238). However, it was significantly shorter in the IL-17A group of female mice (74.6±57.2 sec vs. 130.0±76.2 sec, p=0.032). In rotational thromboelastometry, the IL-17A group exhibited increased maximum clot firmness (71.3±4.5 mm vs. 66.7±4.7 mm, p=0.038) and greater amplitude at 30 min (69.7±5.2 mm vs. 64.5±5.3 mm, p=0.040) than the control group. In Western blotting, the IL-17A group showed higher levels of coagulation factor XIII (2.2±1.5 vs. 1.0±0.9, p=0.008), monocyte chemoattractant protein-1 (1.6±0.6 vs. 1.0±0.4, p=0.023), and tissue factor (1.5±0.6 vs. 1.0±0.5, p=0.003). Conclusion: IL-17A plays a role in the initial st ages of arterial thrombosis in mice. Coagulation factors and monocyte chemoattractant protein-1 may be associated with IL-17A-mediated thrombosis.ope

Elevation of the Gut Microbiota Metabolite Trimethylamine N-Oxide Predicts Stroke Outcome

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Thrombolytic Effects of the Snake Venom Disintegrin Saxatilin Determined by Novel Assessment Methods: A FeCl3-Induced Thrombosis Model in Mice.

Saxatilin, a novel disintegrin purified and cloned from the venom of the Korean snake Gloydius saxatilis, strongly inhibits activation and aggregation of platelets. Glycoprotein (GP) IIb/IIIa receptor antagonists can resolve thrombus, so saxatilin might also have thrombolytic effects. We investigated the thrombolytic effects of saxatilin in mice using a ferric chloride-induced carotid arterial thrombosis model. Thrombotic occlusion and thrombus resolution were evaluated quantitatively by measuring blood flow in the carotid artery with an ultrasonic flow meter and calculating the degree of flow restoration on a minute-by-minute basis; results were confirmed by histological examination. Saxatilin dissolved thrombi in a dose-dependent manner. Saxatilin at 5 mg/kg restored blood flow to baseline levels. As saxatilin dose increased, time to recanalization decreased. A bolus injection of 10% of a complete dose with continuous infusion of the remaining dose for 60 minutes resulted in effective recanalization without reocclusion. The thrombolytic effect of saxatilin was also demonstrated in vitro using platelet aggregometry by administering saxatilin in preformed thrombi. Bleeding complications were observed in 2 of 71 mice that received saxatilin. Fibrin/fibrinogen zymography and platelet aggregometry studies indicated that saxatilin does not have fibrinolytic activity, but exerted its action on platelets. Integrin-binding assays showed that saxatilin inhibited multiple integrins, specifically α2bβ3 (GP IIb/IIIa), α5β1, αvβ3, αvβ1, and αvβ5, which act on platelet adhesion/aggregation. Saxatilin inhibited multiple integrins by acting on platelets, and was safe and effective in resolving thrombi in mice.ope

Automated Composition Analysis of Thrombus from Endovascular Treatment in Acute Ischemic Stroke Using Computer Vision

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Effects of Mesenchymal Stem Cell Treatment on the Expression of Matrix Metalloproteinases and Angiogenesis during Ischemic Stroke Recovery

BACKGROUND: The efficacy of mesenchymal stem cell (MSC) transplantation in ischemic stroke might depend on the timing of administration. We investigated the optimal time point of MSC transplantation. After MSC treatment, we also investigated the expression of matrix metalloproteinases (MMPs), which play a role in vascular and tissue remodeling. METHODS: Human bone marrow-derived MSCs (2 × 10(6), passage 5) were administrated intravenously after permanent middle cerebral artery occlusion (MCAO) was induced in male Sprague-Dawley rats. First, we determined the time point of MSC transplantation that led to maximal neurological recovery at 1 h, 1 day, and 3 days after MCAO. Next, we measured activity of MMP-2 and MMP-9, neurological recovery, infarction volume, and vascular density after transplanting MSCs at the time that led to maximal neurological recovery. RESULTS: Among the MSC-transplanted rats, those of the MSC 1-hour group showed maximal recovery in the rotarod test (P = 0.023) and the Longa score (P = 0.018). MMP-2 activity at 1 day after MCAO in the MSC 1-hour group was significantly higher than that in the control group (P = 0.002), but MMP-9 activity was not distinct. The MSC 1-hour group also showed smaller infarction volume and higher vascular density than did the control group. CONCLUSIONS: In a permanent model of rodent MCAO, very early transplantation of human MSCs (1 h after MCAO) produced greater neurological recovery and decreased infraction volume. The elevation of MMP-2 activity and the increase in vascular density after MSC treatment suggest that MSCs might help promote angiogenesis and lead to neurological improvement during the recovery phase after ischemic stroke.ope

새로운 혈전용해제 후보물질로서 삭사틸린의 혈전용해효과

Dept. of Medical Science/박사Saxatilin, a novel disintegrin purified and cloned from Korean snake (Gloydius saxatilis) venom, strongly inhibits aggregation and activation of platelets by inhibiting multiple integrins such as α2bβ3 (GP IIb/IIIa), α5β1, and αvβ3. The thrombolytic effect of saxatilin in mice was investigated using the ferric chloride-induced carotid arterial thrombosis model. Thrombotic occlusion and resolution of the thrombus were assessed by measuring blood flow in the carotid artery with an ultrasonic flow meter and confirmed by histological observations. Thrombolytic patterns of rt-PA, u-PA, and abciximab were investigated and compared to the thrombolytic pattern of saxatilin. The thrombolytic effectiveness of saxatilin on the aged thrombus were also assessed and compared with the effectiveness of rt-PA. Saxatilin dissolved the thrombus in a dose-dependent manner. Blood flow was notably restored at a dose of 2.5 mg/kg, significantly restored at a dose of 3.75 mg/kg, and nearly reached the baseline level at a dose of 5 mg/kg. As the dose of saxatilin increased, the time to recanalization decreased. Among several regimens tested, a bolus injection of 10% of the dose and continuous infusion of the remaining dose for one hour resulted in effective recanalization without reocclusion. Thrombolytic therapy by saxatilin showed a stronger efficacy, a shorter time to effective recanalization, and a lower frequency of reocclusion than by rt-PA, u-PA, and abciximab. The effectiveness of thrombolytic therapy by both saxatilin and rt-PA decreased as thrombus age increased. Bleeding complications were observed in two of 51 mice that received saxatilin. Fibrin/fibrinogen zymography and platelet aggregometry studies indicated that saxatilin exerts its action on platelets. Saxatilin, which inhibits multiple integrins acting on platelets, was safe and effective in resolving ferric chloride-induced thrombus in mice.ope

Economic burden of treating asthma in Korea

보건정책관리과/석사[한글] 이 연구는 건강보험 청구자료를 이용하여 2003년 한 해 동안 우리나라의 천식치료를 위해 발생하는 천식환자 1인당 연간 의료이용정도 및 비용, 그리고, 우리나라 전체 천식환자의 연간 사회경제적 비용규모를 추계하였다.2003년도 우리나라 만 1세 이상 인구 47,437,668명 중 699,603명이 연간 2회 이상 천식관련 의료이용을 하여 2003년 1년간 천식치료를 받은 환자의 유병률은 1.47%로 추계되었다. 이상 천식환자들은 천식치료를 위하여 연간 1인당 외래방문은 7.56회, 입원은 0.02회, 입원에 따른 재원일수는 0.10일, 응급실방문을 0.01회 이용하였다. 연간 총 급여진료비로 약 1,218억원을 사용한 것으로 추계되었다. 이는 2003년 우리나라 인구 전체의 총 급여진료비 13.8조원의 약 0.88%에 해당된다. 이중 약제비가 약 578억원(47.48%)으로 가장 많았고, 외래방문은 약 576억원(47.27%), 입원은 약 57억원(4.74%), 응급실방문은 약 6억원(0.51%)이 소요된 것으로 추계되었다.연간 1인당 총 급여진료비는 외래방문으로 82,345원, 입원으로 8,249원, 응급실방문으로 890원, 총약제비로 82,707원을 사용하였다. 의료이용 당 단위 급여진료비는 외래방문 1회에 10,897원, 입원 1회에 484,269원, 재원일수 1일에 83,408원, 응급실 1회 방문에 68,339원 이었다. 우리나라 천식환자의 천식치료에 따른 비급여진료비는 연간 약 535억원인 것으로 추계되었으며, 의료이용에 따른 교통비용은 약 144억원, 천식으로 인한 생산성 손실액은 약 901억원이었다. 2003년도 천식으로 인한 우리사회의 총 비용은 급여진료비와 비급여진료비를 합한 직접의료비가 약 1,754억원(62.65%), 교통비 약 144억원(5.15%), 생산성 손실액 약 901억원(32.20%)으로 총 2,800억원으로 추계되었다. 이는 2003년 우리나라 GDP(724조원)의 약 0.039% 인 것으로 추계되었다.향후 이 연구결과는 천식 관리 전략의 비용효과성 검토와 천식예방 및 정책수립을 위한 기초 자료로써 활용될 것으로 기대된다. 또한, 건강보험 청구자료를 이용한 질병부담연구의 방법론을 제시하는 사례연구로 활용될 것으로 기대된다. [영문]The present study estimated the total utilization and cost per asthma patient per year and the yearly socioeconomic cost of asthma patients in Korea during year 2003 using the National Health Insurance (NHI) Claims data.In 2003, 699,603 out of 47,437,668 Korean people aged one year used medical service two or more times, so the estimated prevalence of patients who received asthma treatment in 2003 was 1.4%. Each of these asthma patients had 7.56 times of outpatient visit, 0.02 times of hospital admission, 0.10 inpatient day and 0.01 times of emergency department visit on the average during the year. The total amount of insurance？]covered medical costs for the asthma patients during the year was estimated to be around 121.8 billion won. This is equivalent to 0.88% of the total amount of insurance？]covered medical costs for the whole population (13.8 trillion won). Of the amount, 57.8 billion won (47.48%) was pharmaceutical costs, 57.6 billion won (47.27%) was outpatient visit costs, 5.7 billion won (4.74%) was hospital admission costs, and 0.6 billion won (0.51%) was emergency department visit costs.The amount of insurance？]covered medical costs per patient was 82,345 won for outpatient visit, 8,249 won for hospital admission, 890 won for emergency department visit and 82,707 won as pharmaceutical costs. The amount of insurance？]covered medical costs per unit service was 10,897 won for each outpatient visit, 484,269 won for each hospital admission, 83,408 won for each inpatient day and 68,339 won for each emergence department visit. The total amount of non？]insurance？]covered medical costs for asthma patients？ asthma treatment was estimated to be around 53.5 billion won, the amount of transportation costs to use medical services around 14.4 billion won, and the loss of productivity caused by asthma around 90.1 billion won. The total amount of socioeconomic costs for asthma in 2003 was 175.4 billion won (62.65%) for the sum of insurance？]covered medical costs and non？]insurance？]covered medical costs, 14.4 billion won (5.1%) for transportation costs and 90.1 billion won for the loss of productivity, so a total of 280 billion won. This is around 0.039% of GDP (724 trillion won) in Korea in 2003.The results of this study are expected to be used as basic data for assessing the cost？]effectiveness of asthma management strategies and making policies for asthma prevention. In addition, this study can be used as a case suggesting a methodology of disease load researches using the National Health Insurance (NHI) Claims data.restrictio