Metabolic control of excessive extracellular nucleotide accumulation by CD39/ecto-nucleotidase-1: implications for ischemic vascular diseases

Platelets are responsible for maintaining vascular integrity. In thrombocytopenic states, vascular permeability and fragility increase, presumably due to the absence of this platelet function. Chemical or physical injury to a blood vessel induces platelet activation and platelet recruitment. This is beneficial for the arrest of bleeding (hemostasis), but when an atherosclerotic plaque is ulcerated or fissured, it becomes an agonist for vascular occlusion (thrombosis). Experiments in the late 1980s cumulatively indicated that endothelial cell CD39-an ecto-ADPase-reduced platelet reactivity to most agonists, even in the absence of prostacyclin or nitric oxide. As discussed herein, CD39 rapidly and preferentially metabolizes ATP and ADP released from activated platelets to AMP, thereby drastically reducing or even abolishing platelet aggregation and recruitment. Since ADP is the final common agonist for platelet recruitment and thrombus formation, this finding highlights the significance of CD39. A recombinant, soluble form of human CD39, solCD39, has enzymatic and biological properties identical to the full-length form of the molecule and strongly inhibits human platelet aggregation induced by ADP, collagen, arachidonate, or TRAP (thrombin receptor agonist peptide). In sympathetic nerve endings isolated from guinea pig hearts, where neuronal ATP enhances norepinephrine exocytosis, solCD39 markedly attenuated norepinephrine release. This suggests that NTPDase (nucleoside triphosphate diphosphohydrolase) could exert a cardioprotective action by reducing ATP-mediated norepinephrine release, thereby offering a novel therapeutic approach to myocardial ischemia and its consequences. In a murine model of stroke, driven by excessive platelet recruitment, solCD39 reduced the sequelae of stroke, without an increase in intracerebral hemorrhage. CD39 null mice, generated by deletion of apyrase-conserved regions 2 to 4, exhibited a decrease in postischemic perfusion and an increase in cerebral infarct volume when compared with controls. "Reconstitution" of CD39 null mice with solCD39 reversed these changes. We hypothesize that solCD39 has potential as a novel therapeutic agent for thrombotic diatheses.     

Reduced microvascular thrombosis and improved outcome in acute murine stroke by inhibiting GP IIb/IIIa receptor-mediated platelet aggregation.

Treatment options in acute stroke are limited by a dearth of safe and effective regimens for recanalization of an occluded cerebrovascular tributary, as well as by the fact that patients present only after the occlusive event is established. We hypothesized that even if the site of major arterial occlusion is recanalized after stroke, microvascular thrombosis continues to occur at distal sites, reducing postischemic flow and contributing to ongoing neuronal death. To test this hypothesis, and to show that microvascular thrombosis occurs as an ongoing, dynamic process after the onset of stroke, we tested the effects of a potent antiplatelet agent given both before and after the onset of middle cerebral arterial (MCA) occlusion in a murine model of stroke. After 45 min of MCA occlusion and 23 h of reperfusion, fibrin accumulates in the ipsilateral cerebral hemisphere, based upon immunoblotting, and localizes to microvascular lumena, based upon immunostaining. In concordance with these data, there is a nearly threefold increase in the ipsilateral accumulation of 111In-labeled platelets in mice subjected to stroke compared with mice not subjected to stroke. When a novel inhibitor of the glycoprotein IIb/IIIa receptor (SDZ GPI 562) was administered immediately before MCA occlusion, platelet accumulation was reduced 48%, and fibrin accumulation was reduced by 47% by immunoblot densitometry. GPI 562 exhibited a dose-dependent reduction of cerebral infarct volumes measured by triphenyltetrazolium chloride staining, as well as improvement in postischemic cerebral blood flow, measured by laser doppler. GPI 562 caused a dose-dependent increase in tail vein bleeding time, but intracerebral hemorrhage (ICH) was not significantly increased at therapeutic doses; however, there was an increase in ICH at the highest doses tested. When given immediately after withdrawal of the MCA occluding suture, GPI 562 was shown to reduce cerebral infarct volumes by 70%. These data support the hypothesis that in ischemic regions of brain, microvascular thrombi continue to accumulate even after recanalization of the MCA, contributing to postischemic hypoperfusion and ongoing neuronal damage.     

Role of extracellular ATP metabolism in regulation of platelet reactivity

Extracellular adenosine triphosphate (ATP) regulates platelet reactivity by way of direct action on platelet purinergic receptors or by hydrolysis to adenosine diphosphate (ADP). Subsequent metabolism of ATP and ADP to adenosine monophosphate (AMP) and adenosine inhibits platelet aggregation. Endothelial cell membrane-bound ecto-ATP/ADPase (CD39, E-NTPDase1) is thought to be the main regulator of platelet responsiveness. However, the findings in studies of CD39-knockout mice imply that nucleotidase(s) in plasma regulates circulating adenine nucleotides levels. Understanding extracellular ATP metabolism by CD39 and plasma nucleotidases is therefore important. In this study, alpha-phosphorus 32- and gamma-phosphorus 32-labeled ATP were rapidly metabolized directly to AMP and pyrophosphate in human plasma at pH 7.4, suggesting the presence of pyrophosphatase/phosphodiesterase-like activity. A specific phosphodiesterase substrate, p-nitrophenol-5'-TMP (p-Nph-5'-TMP), was readily hydrolyzed in human plasma. The antiaggregatory action of beta,gamma-methylene-ATP (AMPPCP) (5 micromol/L) was blocked by DMPX, an adenosine-receptor antagonist, suggesting that in plasma, AMPPCP was metabolized to AMP and adenosine. Recombinant soluble CD39 (solCD39) was used to assess the role of CD39 in ATP metabolism. As little as 0.25 microg/mL of solCD39 inhibited ADP-induced platelet aggregation. However, in the presence of ADP-free ATP (10 micromol/L), solCD39 induced platelet aggregation in a dose-dependent manner. Because AMPPCP could not substitute for ATP in solCD39-stimulated platelet aggregation, it is likely that ADP formation from ATP was required. Endogenous CD39 may thus have a hemostatic function by promoting ADP formation from released ATP, in addition to its antiaggregatory properties. A plasma nucleotidase hydrolyzes ATP directly to AMP. This prevents ADP accumulation and generates adenosine, a potent, locally acting inhibitor of platelet reactivity. The presence of both endothelial CD39 and plasma nucleotidase appears to be important in the maintenance of normal hemostasis and prevention of excessive platelet responsiveness.     

Cerebral protection in homozygous null ICAM-1 mice after middle cerebral artery occlusion. Role of neutrophil adhesion in the pathogenesis of stroke.

Acute neutrophil (PMN) recruitment to postischemic cardiac or pulmonary tissue has deleterious effects in the early reperfusion period, but the mechanisms and effects of neutrophil influx in the pathogenesis of evolving stroke remain controversial. To investigate whether PMNs contribute to adverse neurologic sequelae and mortality after stroke, and to study the potential role of the leukocyte adhesion molecule intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of stroke, we used a murine model of transient focal cerebral ischemia consisting of intraluminal middle cerebral artery occlusion for 45 min followed by 22 h of reperfusion. PMN accumulation, monitored by deposition of 111In-labeled PMNs in postischemic cerebral tissue, was increased 2.5-fold in the ipsilateral (infarcted) hemisphere compared with the contralateral (noninfarcted) hemisphere (P < 0.01). Mice immunodepleted of neutrophils before surgery demonstrated a 3.0-fold reduction in infarct volumes (P < 0.001), based on triphenyltetrazolium chloride staining of serial cerebral sections, improved ipsilateral cortical cerebral blood flow (measured by laser Doppler), and reduced neurological deficit compared with controls. In wild-type mice subjected to 45 min of ischemia followed by 22 h of reperfusion, ICAM-1 mRNA was increased in the ipsilateral hemisphere, with immunohistochemistry localizing increased ICAM-1 expression on cerebral microvascular endothelium. The role of ICAM-1 expression in stroke was investigated in homozygous null ICAM-1 mice (ICAM-1 -/-) in comparison with wild-type controls (ICAM-1 +/+). ICAM-1 -/- mice demonstrated a 3.7-fold reduction in infarct volume (P < 0.005), a 35% increase in survival (P < 0.05), and reduced neurologic deficit compared with ICAM-1 +/+ controls. Cerebral blood flow to the infarcted hemisphere was 3.1-fold greater in ICAM-1 -/- mice compared with ICAM-1 +/+ controls (P < 0.01), suggesting an important role for ICAM-1 in the genesis of postischemic cerebral no-reflow. Because PMN-depleted and ICAM-1-deficient mice are relatively resistant to cerebral ischemia-reperfusion injury, these studies suggest an important role for ICAM-1-mediated PMN adhesion in the pathophysiology of evolving stroke.     

Heterologous cell–cell interactions: thromboregulation, cerebroprotection and cardioprotection by CD39 (NTPDase-1)

Summary. Blood platelets maintain vascular integrity and promote primary and secondary hemostasis following interruption of vessel continuity. Biochemical or physical damage to the coronary, carotid or peripheral arteries is followed by excessive platelet activation and recruitment culminating in vascular occlusion and tissue ischemia. Currently inadequate therapeutic approaches to stroke and coronary artery disease are a public health issue. Following our demonstration of neutrophil leukotriene production from arachidonate released from activated aspirin‐treated platelets, we studied interactions between platelets and other blood cells, leading to concepts of transcellular metabolism and thromboregulation. Thrombosis has a proinflammatory component whereby biologically active substances are synthesized by interactions between different cell types that could not individually synthesize the product(s). Endothelial cells control platelet reactivity via three biochemical systems—autacoids leading to production of prostacyclin and nitric oxide, and endothelial ecto‐ADPase/CD39/NTPDase‐1. The autacoids are fluid‐phase reactants, not produced by tissues in the basal state. They are only synthesized intracellularly and released upon interactions of cells with an agonist. When released, autacoids exert fleeting actions in the immediate milieu, and are rapidly inactivated. CD39 is an integral component of the endothelial cell surface and is substrate‐activated. It maintains vascular fluidity in the complete absence of prostacyclin and nitric oxide, indicating that they are ancillary components of hemostasis. Therapeutic implications for the autacoids have not been compelling because of their transient, local and fleeting action, and limited potency. Conversely, CD39, acting solely on the platelet releasate, is efficacious in three different animal models. It metabolically neutralizes a prothrombotic platelet releasate via deletion of ADP—the major recruiting agent responsible for formation of an occlusive thrombus. In addition, solCD39 reduced ATP‐ and ischemia‐induced norepinephrine release in the heart. This reduction can prevent fatal arrhythmia. Moreover, solCD39 ameliorated the sequelae of stroke in CD39 null mice. CD39 represents the next generation of cardioprotective and cerebroprotective molecules.     

Self-regulation of inflammatory cell trafficking in mice by the leukocyte surface apyrase CD39

Leukocyte and platelet accumulation at sites of cerebral ischemia exacerbate cerebral damage. The ectoenzyme CD39 on the plasmalemma of endothelial cells metabolizes ADP to suppress platelet accumulation in the ischemic brain. However, the role of leukocyte surface CD39 in regulating monocyte and neutrophil trafficking in this setting is not known. Here we have demonstrated in mice what we believe to be a novel mechanism by which CD39 on monocytes and neutrophils regulates their own sequestration into ischemic cerebral tissue, by catabolizing nucleotides released by injured cells, thereby inhibiting their chemotaxis, adhesion, and transmigration. Bone marrow reconstitution and provision of an apyrase, an enzyme that hydrolyzes nucleoside tri- and diphosphates, each normalized ischemic leukosequestration and cerebral infarction in CD39-deficient mice. Leukocytes purified from Cd39^–/– mice had a markedly diminished capacity to phosphohydrolyze adenine nucleotides and regulate platelet reactivity, suggesting that leukocyte ectoapyrases modulate the ambient vascular nucleotide milieu. Dissipation of ATP by CD39 reduced P2X₇ receptor stimulation and thereby suppressed baseline leukocyte α_Mβ₂-integrin expression. As α_Mβ₂-integrin blockade reversed the postischemic, inflammatory phenotype of Cd39^–/– mice, these data suggest that phosphohydrolytic activity on the leukocyte surface suppresses cell-cell interactions that would otherwise promote thrombosis or inflammation. These studies indicate that CD39 on both endothelial cells and leukocytes reduces inflammatory cell trafficking and platelet reactivity, with a consequent reduction in tissue injury following cerebral ischemic challenge.     

脑梗死和脑出血患者凝血和血小板状态的研究

目的　探讨脑梗死 ,脑出血患者凝血及血小板方面的变化情况。方法　脑梗死患者 4 0例 ,脑出血患者30例 ,正常对照组 35例 ,患者在发病后 72小时内抽肘静脉血抗凝。患者和对照组均未接受任何止血、凝血药物。分析脑梗死患者与健康人 ,脑出血患者与健康人 ,脑梗死患者与脑出血患者 ,大面积脑梗死患者与腔隙性脑梗死患者在凝血酶原时间 (PT)、活化部分凝血酶原时间 (APTT)、凝血酶时间 (TT)、纤维蛋白原 (FIB)、血小板计数 (PLT)、血小板平均体积 (MPV) 6项指标上有无统计学意义。结果　急性脑梗死组与健康人比较 ,FIB和MPV均明显高于对照组(P <0 .0 1)。高血压性脑出血组与健康人比较 ,FIB和MPV均明显高于对照组 (P <0 .0 1)。急性脑梗死组与高血压性脑出血组比较 ,PT ,APTT ,TT ,FIB ,PLT ,MPV 6项指标均无统计学意义 (P >0 .0 5 )。大面积脑梗死组与腔隙性脑梗死组比较 ,PT ,APTT ,TT ,FIB ,PLT ,MPV 6项指标均无统计学意义 (P >0 .0 5 )。结论　血浆纤维蛋白原升高和血小板体积增大是脑梗死的重要危险因素 ,也是脑出血的重要危险因素。脑梗死与脑出血有共同的发病基础。较大血管闭塞引起的脑梗死与腔隙性脑梗死在凝血与血小板状态方面无统计学意义。     

Targeted deletion of ectonucleoside triphosphate diphosphohydrolase 1/CD39 leads to desensitization of pre- and postsynaptic purinergic P2 receptors

We previously reported that ATP coreleased with norepinephrine from cardiac sympathetic nerves activates presynaptic P2X purinoceptors (P2XR), thereby enhancing norepinephrine exocytosis. Blockade of ectonucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1/CD39) potentiates norepinephrine exocytosis, whereas recombinant soluble CD39 (solCD39) in-hibits it. This suggested that CD39 gene (Entpd1) deletion would enhance purinergic and adrenergic signaling by preserving ATP and its norepinephrine-releasing activity. However, we found that the neurogenic contractile response of vasa deferentia from Entpd1-null (CD39(-/-)) mice was attenuated and accompanied by reduced activity of pre- and postsynaptic P2XR, whereas contractile responses to K(+) or norepinephrine remained intact. In addition, the magnitude of ATP and norepinephrine exocytosis from cardiac synaptosomes was decreased in CD39(-/-) mice. Inhibition of E-NTPDase1/CD39, or solCD39 administration, did not affect the attenuated contractile response of vasa deferentia from CD39(-/-) mice. Notably, Entpd1 deletion and pharmacological P2XR desensitization in control mice similarly attenuated vasa deferentia responses. Thus, excessive and prolonged ATP exposure resulting from CD39 deletion desensitizes pre- and postjunctional P2XR at the sympathetic neuromuscular junction. This diminishes purinergic activity directly and adrenergic activity indirectly. It remains to be determined whether this desensitization results from receptor internalization, changes in receptor conformation or phosphorylation. Shutdown of ATP signaling in CD39(-/-) mice may represent a defense mechanism for the prevention of purinergic overstimulation. Our findings emphasize the cardioprotective role of neuronal CD39: by reducing presynaptic facilitatory effects of neurotransmitter ATP, CD39 attenuates norepinephrine release and its dysfunctional consequences. Moreover, by virtue of its antithrombotic action CD39 can potentially prevent the transition from myocardial ischemia to infarction.     

Targeted inhibition of intrinsic coagulation limits cerebral injury in stroke without increasing intracerebral hemorrhage.

Agents that restore vascular patency in stroke also increase the risk of intracerebral hemorrhage (ICH). As Factor IXa is a key intermediary in the intrinsic pathway of coagulation, targeted inhibition of Factor IXa-dependent coagulation might inhibit microvascular thrombosis in stroke without impairing extrinsic hemostatic mechanisms that limit ICH. A competitive inhibitor of native Factor IXa for assembly into the intrinsic Factor X activation complex, Factor IXai, was prepared by covalent modification of the Factor IXa active site. In a modified cephalin clotting time assay, in vivo administration of Factor IXai caused a dose-dependent increase in time to clot formation (3.6-fold increase at the 300 micrograms/kg dose compared with vehicle-treated control animals, P < 0.05). Mice given Factor IXai and subjected to middle cerebral artery occlusion and reperfusion demonstrated reduced microvascular fibrin accumulation by immunoblotting and immunostaining, reduced 111In-labeled platelet deposition (42% decrease, P < 0.05), increased cerebral perfusion (2.6-fold increase in ipsilateral blood flow by laser doppler, P < 0.05), and smaller cerebral infarcts than vehicle-treated controls (70% reduction, P < 0.05) based on triphenyl tetrazolium chloride staining of serial cerebral sections. At therapeutically effective doses, Factor IXai was not associated with increased ICH, as opposed to tissue plasminogen activator (tPA) or heparin, both of which significantly increased ICH. Factor IXai was cerebroprotective even when given after the onset of stroke, indicating that microvascular thrombosis continues to evolve (and may be inhibited) even after primary occlusion of a major cerebrovascular tributary.     

Beneficial effects of a new 20-hydroxyeicosatetraenoic acid synthesis inhibitor, TS-011 [N-(3-chloro-4-morpholin-4-yl) phenyl-N'-hydroxyimido formamide], on hemorrhagic and ischemic stroke

The present study characterized the effects of TS-011 [N-(3-chloro-4-morpholin-4-yl) phenyl-N'-hydroxyimido formamide], a new selective inhibitor of the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE), on the metabolism of arachidonic acid by human and rat renal microsomes and the inhibitory effects of this compound on hepatic cytochrome P450 enzymes involved in drug metabolism. The effects of TS-011 on the fall in cerebral blood flow following subarachnoid hemorrhage (SAH) and in reducing infarct size in ischemic stroke models were also examined since 20-HETE may contribute to the development of cerebral vasospasm. TS-011 inhibited the synthesis of 20-HETE by human renal microsomes and recombinant CYP4A11 and 4F2, 4F3A, and 4F3B enzymes with IC50 values around 10 to 50 nM. It had no effect on the activities of CYP1A, 2C9, 2C19, 2D6, or 3A4 enzymes. TS-011 inhibited the synthesis of 20-HETE by rat renal microsomes with an IC50 of 9.19 nM, and it had no effect on epoxygenase activity at a concentration of 100 microM. TS-011 (0.01-1 mg/kg i.v.) reversed the fall in cerebral blood flow and the increase in 20-HETE levels in the cerebrospinal fluid of rats after SAH. TS-011 also reduced the infarct volume by 35% following transient ischemic stroke and in intracerebral hemorrhage in rats. Injection of 20-HETE (8 or 12 mg/kg) into the carotid artery produced an infarct similar to that seen in the ischemic stroke model. These studies indicate that blockade of the synthesis of 20-HETE with TS-011 opposes cerebral vasospasm following SAH and reduces infarct size in ischemic models of stroke.     

Combinatorial effect of probucol and cilostazol in focal ischemic mice with hypercholesterolemia

Hypercholesterolemia may increase stroke risk by accelerating atherosclerosis, narrowing the luminal diameter in cerebral vessels, and disrupting both vascular endothelial and smooth muscle function. In the present study, we investigated the beneficial effects of combinatorial therapy with probucol and cilostazol on focal cerebral ischemia with hypercholesterolemia. Apolipoprotein E (ApoE) knockout (KO) mice were fed a high-fat diet with or without 0.5% probucol and/or 0.2% cilostazol for 10 weeks. Probucol alone and probucol and cilostazol significantly decreased total, low-density lipoprotein, and high-density lipoprotein cholesterol, whereas cilostazol did not affect the plasma cholesterol levels in ApoE KO mice. Administration of probucol alone and cilostazol alone significantly decreased atherosclerotic lesion area in the aorta, with a significant decrease evident using combinatorial administration. Middle cerebral artery occlusion resulted in significantly larger infarct volumes in ApoE KO mice fed 10 weeks of high-fat diet compared with those in ApoE KO mice fed a regular diet. The infarct volume was reduced significantly using probucol alone or cilostazol alone and even was reduced significantly by their combinatorial administration. Consistent with a larger infarct size, the combinatorial therapy prominently improved neurological function. The combinatorial administration increased cerebral blood flow during ischemia. Expression of endothelial nitric oxide synthase and adiponectin in the cortex were decreased by high-fat diet but were elevated by combinatorial treatment. Adiponectin expression colocalized within the cerebral vascular endothelium. The data suggest that the combination of probucol and cilostazol prevents cerebrovascular damage in focal cerebral ischemic mice with hypercholesterolemia by up-regulation of endothelial nitric oxide synthase and adiponectin.     

定向微创血肿引流术对脑出血患者脑供血的影响

目的：观察脑出血患者行微创血肿抽吸引流术前后TCD参数的变化,研究该手术对脑灌注的影响。方法：动态监测66例发病24小时内入院的脑出血患者的血压,并于入院时及入院后第2、4、6、12天行TCD监测,其中36例入院24小时内接受血肿抽吸引流术（手术组）,30例行内科常规治疗（对照组）。结果：两组患者治疗过程中血压逐渐下降,手术组血压下降幅度较对照组大,第12天收缩压差异显著（P〈0.01）。双侧大脑中动脉（MCA）流速呈先降后升,双侧动脉指数（PI）值呈先升后降。手术组第6天MCA流速即开始升高,PI值降低,且在第12天两组间差异显著（P〈0.05）;对照组MCA流速于第6天内呈下降改变,而第12天开始升高,PI值改变与之相反。手术组平均住院日较对照组短（P〈0.01）。结论：微创血肿引流术可显著性改善脑出血患者的脑灌注。     

Thromboregulatory manifestations in human CD39 transgenic mice and the implications for thrombotic disease and transplantation

Extracellular nucleotides play an important role in thrombosis and inflammation, triggering a range of effects such as platelet activation and recruitment, endothelial cell activation, and vasoconstriction. CD39, the major vascular nucleoside triphosphate diphosphohydrolase (NTPDase), converts ATP and ADP to AMP, which is further degraded to the antithrombotic and anti-inflammatory mediator adenosine. Deletion of CD39 renders mice exquisitely sensitive to vascular injury, and CD39-null cardiac xenografts show reduced survival. Conversely, upregulation of CD39 by somatic gene transfer or administration of soluble NTPDases has major benefits in models of transplantation and inflammation. In this study we examined the consequences of transgenic expression of human CD39 (hCD39) in mice. Importantly, these mice displayed no overt spontaneous bleeding tendency under normal circumstances. The hCD39 transgenic mice did, however, exhibit impaired platelet aggregation, prolonged bleeding times, and resistance to systemic thromboembolism. Donor hearts transgenic for hCD39 were substantially protected from thrombosis and survived longer in a mouse cardiac transplant model of vascular rejection. These thromboregulatory manifestations in hCD39 transgenic mice suggest important therapeutic potential in clinical vascular disease and in the control of serious thrombotic events that compromise the survival of porcine xenografts in primates.     

凝血纤溶功能变化对急性自发性脑出血出血量和神经功能缺损评分及预后的影响

目的 探讨自发性脑出血患者凝血、纤溶功能的变化对出血量、脑损伤严重程度及预后的影响.方法 30例急性脑出血患者为脑出血组,另选30例为对照组,两组患者具有相同基础疾病,分别测定血浆纤维蛋白原(Fg)、抗凝血酶-Ⅲ(AT-Ⅲ)活性、血小板四项计数和D-二聚体(DD).入院时脑出血组予以神经功能缺损评分,病后3个月行改良 Rankin 量表评分.观察各指标对出血量、神经功能缺损评分及预后的影响.结果 ①脑出血组Fg、AT-Ⅲ、血小板四项计数和DD于发病后48 h显著变化,与对照组比较差异均有统计学意义(P均<0.01).②AT-Ⅲ与出血量、神经功能缺损评分及预后有负相关关系(P均<0.05).③Fg与出血量及预后有正相关关系(P均<0.05).④DD与出血量、神经功能缺损评分及预后有正相关关系(P均<0.05).⑤血小板四项计数与出血量、神经功能缺损评分及预后无明显线性相关关系 (P均>0.05).结论 早期检测凝血纤溶功能敏感指标对脑出血患者脑损伤判断具有重要的临床价值.     

不同时间窗溶解大鼠大脑中动脉血栓与梗死体积、行为学改变及脑出血的相关性

目的研究不同时间窗尿激酶溶解大鼠大脑中动脉血栓后脑梗死体积及脑出血情况。方法应用肾血管性高血压大鼠(RHRSP),用光化学法制成一侧大脑中动脉闭塞(MCAO)模型,在血栓形成后不同时间应用尿激酶静脉溶栓。结果溶栓治疗组在MCAO后0.5、1、2、3h应用尿激酶静脉溶栓,MCAO后0.5h和1h组比MCAO组体积小(P<0.05),而MCAO后2h组与MCAO组梗死体积无差别(P>0.05)。溶栓治疗组溶栓复流后0.5、1、2、3h脑出血分别为0/6(0%)、1/8(14.29%)、2/7(28.57%)和3/7(42.86%)。结论大鼠MCA血栓形成尿激酶静脉溶栓复流后,能减小脑梗死体积,此模型的溶栓复流时间窗<2h。脑梗死溶栓治疗后脑出血与治疗时间有密切的关系。     

CD133 Identifies a Human Bone Marrow Stem/Progenitor Cell Sub-population With a Repertoire of Secreted Factors That Protect Against Stroke

The reparative properties of bone marrow stromal cells (BMSCs) have been attributed in part to the paracrine action of secreted factors. We isolated typical human BMSCs by plastic adherence and compared them with BMSC sub-populations isolated by magnetic-activated cell sorting against CD133 (CD133-derived BMSCs, CD133BMSCs) or CD271 [p75 low-affinity nerve growth factor receptor (p75LNGFR), p75BMSCs]. Microarray assays of expressed genes, and enzyme-linked immunosorbent assays (ELISAs) of selected growth factors and cytokines secreted under normoxic and hypoxic conditions demonstrated that the three transit-amplifying progenitor cell populations were distinct from one another. CD133BMSC-conditioned medium (CdM) was superior to p75BMSC CdM in protecting neural progenitor cells against cell death during growth factor/nutrient withdrawal. Intracardiac (arterial) administration of concentrated CD133BMSC CdM provided neuroprotection and significantly reduced cortical infarct volumes in mice following cerebral ischemia. In support of the paracrine hypothesis for BMSC action, intra-arterial infusion of CD133BMSC CdM provided significantly greater protection against stroke compared with the effects of CD133BMSC (cell) administration. CdM from CD133BMSCs also provided superior protection against stroke compared with that conferred by CdM from p75BMSCs or typically isolated BMSCs. CD133 identifies a sub-population of nonhematopoietic stem/progenitor cells from adult human bone marrow, and CD133BMSC CdM may provide neuroprotection for patients with stroke.     

高血压脑出血患者血小板CD62p和CD42b的表达及其与脑水肿形成的关系

目的　研究高血压脑出血患者血小板CD6 2p和CD4 2b的表达及其与脑水肿形成的关系。方法　采用流式细胞术检测脑出血患者血小板CD6 2p和CD4 2b的表达 ,并与脑水肿进行相关性分析。结果　脑出血患者血小板CD6 2p和CD4 2b的表达量与脑水肿体积有相关性 (rCD4 2b=- 0 4 89,P <0 0 5 ;rCD6 2p=0 6 4 6 ,P <0 0 1)。结论　高血压脑出血患者出现血小板活化现象 ,血小板的活化有可能是促进脑水肿形成的病理机制之一     

脑出血患者血小板CD42b,CD62p的表达与脑水肿形成的相关性

目的分析比较脑出血患者与健康人血小板CD42b,CD62p的表达差异及急性期脑出血患者血小板CD42b,CD62p的表达量与脑水肿体积的相关性,探讨急性期脑出血患者血小板表面糖蛋白CD42b,CD62p的表达及其与血肿周围脑水肿形成的关系。方法选自2002-01/08华中科技大学同济医学院附属同济医院神经科住院治疗的脑出血患者20例。健康者选自2002-01/08华中科技大学同济医学院附属同济医院神经科住院患者家属,共15例,利用流式细胞术(FCM)测定20例脑出血患者和15例健康人血小板CD42b和CD62p的表达,根据头颅CT测量脑出血患者血肿周围脑水肿带的大小,并对脑出血患者血小板CD42b和CD62p的表达与血肿周围脑水肿带的大小进行相关性分析。结果与健康人犤(9.7±1.1)%,(81.6±3.1)%犦比较,急性期脑出血患者血小板CD42b的表达(74.2±6.4)%减少,CD62p的表达(4.7±4.8)%增加(tCD42b=4.328,P<0.01;tCD62p=4.017,P<0.01);急性期脑出血患者血小板CD42b和CD62p表达量与血肿周围脑水肿带的大小有相关性(rCD42b=-0.489,P<0.05;rCD62p=0.646,P<0.01)。结论脑出血急性期血小板活化,活化的血小板参与了血肿周围脑水肿形成这一病理损伤过程。     

The ASPECTS template is weighted in favor of the striatocapsular region

The extent of cerebral infarction correlates with increased risk of intracerebral hemorrhage (ICH) following recombinant tissue plasminogen activator (rt-PA) administration. The Alberta Stroke Program Early CT Score (ASPECTS) is a widely used, validated method which assesses involvement of 10 selected regions of the MCA territory. An ASPECTS score >7 is associated with a higher risk of ICH following thrombolysis than lower scores. To understand the internal structure of the ASPECTS template better, we estimated the infarct volume corresponding to each region. We hypothesized that, in the ASPECTS scoring system, the striatocapsular region is weighted disproportionally. Four experienced radiologists rated individual ASPECTS regions on subacute CT images (day 5-day 10) of 19 patients with MCA territory stroke. Infarct volume was determined from manual segmentation of infarcts on CT images. Linear regression was used to estimate the regional volume associated with each ASPECTS region. The ASPECTS regions are weighted unequally with the striatocapsular region accounting for 21% of the MCA territory infarct volume. Together, the 10 ASPECTS regions account for approximately 51% of the maximum MCA infarct territory volume. These findings should provide impetus for research to develop a scoring system explicitly based on regional hemorrhage risk as an aid to selecting patients for thrombolysis.     

Effects of estrogen on platelet reactivity after transient forebrain ischemia in rats

Estrogen's prothrombotic effects are of increasing concern, particularly in stroke risk and recovery. Using an ischemic rodent model, the authors sought to determine (a) if estrogen replacement increases postischemic platelet reactivity, (b) if changes in estrogen status alter intraplatelet endothelial nitric oxide synthase (eNOS) synthesis, and (c) if estrogen-mediated effects on platelets alter cerebral blood flow during reperfusion. Intact (I), ovariectomized (OVX), and OVX + 17 beta-estradiol (E50) rats were subjected to 30 min of forebrain ischemia and 60 min of reperfusion. Using the platelet activation marker P-selectin, postischemic platelet reactivity was quantified by flow cytometry. In a separate cohort (I, OVX, E50), the authors quantified platelet eNOS by Western blot. Another cohort (OVX, E50) was subjected to ischemia/reperfusion, and cerebral blood flow was determined using the iodoantipyrine technique. Collagen-stimulated platelet P-selectin expression was increased in the OVX rats at 60 min of reperfusion, and this effect was reversed by estrogen treatment. No differences in platelet eNOS expression were detected among groups. Cerebral blood flow at 60 min reperfusion was comparable between the OVX and the E50 rats. The authors conclude that during reper-fusion, estrogen deficiency increases postischemic platelet sensitivity to stimuli in estrogen-deficient rats. Estrogen treatment mitigates effects of estrogen loss on platelets, but this early effect is apparently not caused by intraplatelet eNOS depression. Neither estrogen deficiency nor estrogen treatment changes early postischemic regional brain blood flow. In this rodent global cerebral ischemic model, physiologic doses of estrogen are not deleterious to platelet reactivity and may initially reduce postischemic platelet reactivity.