Matrix metalloproteinase induction by EMMPRIN in experimental focal cerebral ischemia

Focal cerebral ischemia leads to the gradual disruption of the extracellular matrix. A key role in the turnover of the extracellular matrix is played by the system of matrix metalloproteinases (MMPs). In this study we describe changes of the MMP inducer protein (EMMPRIN) following experimental cerebral ischemia (induced for 3 h and followed by 24 h reperfusion, suture model) in rats. Extracellular EMMPRIN was measured by Western blot of the ischemic and nonischemic basal ganglia and cortex separately. Compared with the contralateral nonischemic area, the ischemic hemisphere showed a significant increase in EMMPRIN: basal ganglia, 158% +/- 4% (P < 0.05); cortex, 128% +/- 25% (P < 0.05). Immunohistochemistry was used to localize EMMPRIN on cerebral microvessels. EMMPRIN-positive microvascular structures were quantified by automatic morphometric video-imaging analysis and a significant increase in the number of cerebral microvessels staining positive for EMMPRIN in the ischemic basal ganglia was shown. The significant loss of microvascular basal lamina antigen collagen type IV in ischemic cortex and basal ganglia was calculated by Western blot. Measured by gelatin zymography, we demonstrated an MMP-2 and MMP-9 increase in the ischemic brain regions (P < 0.05). For the first time the MMP activation system EMMPRIN was shown to be relevant in cerebral ischemia. These results raise the possibility that the increased expression of EMMPRIN, the increase in MMPs and the damage of the basal lamina following cerebral ischemia are connected and part of a network of related changes.     

Pravastatin reduces microvascular basal lamina damage following focal cerebral ischemia and reperfusion

Transient ischemia has been shown to damage the basal lamina of the cerebral microvasculature. Other studies proved statins to be beneficial to non-cerebral microvessels. The aim of this study was to determine whether pravastatin pretreatment ameliorates microvascular basal lamina damage following transient ischemia. Using the suture model, we subjected 15 rats to focal ischemia (3 h) and reperfusion (24 h). Rats received pravastatin (20 mg/kg/day) or saline for 4 weeks prior to the experiment. The outcome was determined by a behavior test and the infarct size. Collagen type IV, a marker for an intact basal lamina, and hemoglobin extravasation were measured by Western blot analysis. A ratio (in percentage) between ischemic and contralateral hemispheres was calculated. Pravastatin pretreatment resulted in a significantly better neurological outcome and reduced infarct size (15 +/- 0.5 and 59 +/- 10 mm(3), respectively) compared with controls (12.25 +/- 0.4 and 167 +/- 13 mm(3), respectively, P < 0.01 for both). In controls, loss of collagen type IV was seen in the basal ganglia and in the cortex (43 +/- 4 and 64 +/- 5%, respectively). Pravastatin prevented significant collagen loss (basal ganglia: 106 +/- 17%; cortex: 112 +/- 14%, P < 0.01 for both) and significantly reduced the hemoglobin extravasation compared with controls in the basal ganglia (198 +/- 49 vs. 553 +/- 47%, P < 0.01). Pravastatin pretreatment resulted in a reduction of microvascular basal lamina damage and hemoglobin extravasation following transient ischemia. Pravastatin seems to protect the cerebral microvascular system.     

Recombinant human tissue plasminogen activator protects the basal lamina in experimental focal cerebral ischemia

While recombinant tissue plasminogen activator (rt-PA) is successfully used in human ischemic stroke, it may also cause hemorrhagic complications. Animal experiments have shown that hemorrhages are related to microvascular basal lamina damage. We investigated the effects of different doses of rt-PA on the brain microvasculature. Experimental cerebral ischemia in rats was induced for 3 h and followed by 24 h reperfusion (suture model). Each group of rats (n = 6) received either treatment (0.9, 9, or 18 mg rt-PA/kg body weight) or saline (control group) at the end of ischemia. The loss of microvascular basal lamina antigen collagen type IV was measured by Western blot of the ischemic and non-ischemic basal ganglia and cortex. Compared with the contralateral non-ischemic area, collagen type IV was significantly reduced in the ischemic area: (basal ganglia/cortex) 43% +/- 9% / 64% +/- 4 %. Low/moderate doses of rt-PA had a protective effect: 0.9 mg 79% +/- 3% / 89% +/- 6%, 9 mg 72% +/- 9%/ 81% +/- 12% (p < 0.05). Higher doses of rt-PA (18 mg) had a similar effect as seen in untreated controls: 57% +/- 11% / 59% +/- 9% (p < 0.05, Anova). MMP-9 and MMP-2, measured by gelatine zymography, steadily increased over higher doses of rt-PA: MMP-9 (basal ganglia/cortex): control 115% +/- 4% / 123% +/- 3% compared with 18 mg rt-PA 146% +/- 5%/ 162% +/- 6% (p < 0.05) and MMP-2: control 109% +/- 4%/ 116% +/- 5% and 18 mg rt-PA 222% +/- 15%/ 252% +/- 2% (p < 0.05). Low to moderate doses of rt-PA protect the microvascular basal lamina, whereas high doses of rt-PA have the opposite effect, probably due to increased coactivation of MMP-2 and MMP-9.     

Microvascular Basal lamina damage after embolic stroke in the rat: relationship to cerebral blood flow.

Microvascular basal lamina damage has been demonstrated after balloon occlusion of the middle cerebral artery in the nonhuman primate and after intravascular filament occlusion in the rat. The aim of the present study was to investigate in the rat whether microvascular damage can be found in the stroke model of intracarotid clot injection as early as 3 hours after clot injection and whether microvascular damage relates to the level of regional cerebral blood flow (rCBF). Microvascular densities and total stained microvascular areas were determined by immunohistochemistry of collagen type IV in cortex and basal ganglia and automatic video-imaging analysis. rCBF was measured by autoradiography in the same brain areas. Compared with the corresponding areas in the nonischemic hemisphere, a significant loss of microvascular density (-16%) and total stained microvascular areas (-10%) was observed in these areas. The reduction of microvascular basal lamina staining was comparable in all animals and was not related to the value of rCBF when measured 3 hours after onset of embolic stroke. In conclusion, microvascular damage occurs as soon as 3 hours after intracarotid clot injection, even in brain areas in which rCBF has returned to normal values.     

Influence of the duration of ischemia and reperfusion on infarct volume and microvascular damage in mice

OBJECTIVES: Focal cerebral ischemia is responsible for alterations of vascular permeability, and the loss of microvascular integrity is a primary source of subsequent hemorrhages. We evaluated the influence of different durations of ischemia and reperfusion on infarction size and microvascular damage after focal cerebral ischemia in the mouse.METHODS: C57BL/6 mice (n=39) were subjected to focal cerebral ischemia (I) and reperfusion (R). Consecutive brain sections were analysed for infarction volumes (Nissl-staining) and for collagen type IV (immunohistochemistry and western blot).RESULTS: Infarction size (percentage of the infarction volume versus ipsilateral hemisphere) increased with total time of ischemia and reperfusion: 19+/-2% (I3R0), 30+/-2% (I3R3), 36+/-4% (I3R12), 41+/-4% (I1R24), 45+/-6% (I2R24) and 58+/-2% (I3R24). The ischemic hemispheres showed a significant progressive reduction of collagen type IV positive vessels (ischemic versus non-ischemic contralateral area): 90+/-3% (I3R0), 88+/-1% (I3R3), 82+/-3% (I3R12), 85+/-3% (I1R24), 79+/-3% (I2R24), 72+/-2% (I3R24).CONCLUSIONS: Both prolonged ischemia and reperfusion lead to an increased infarction volume, as well as progressive microvascular damage.     

Recombinant tissue plasminogen activator attenuates basal lamina antigen loss after experimental focal cerebral ischemia

BACKGROUND AND PURPOSE: The use of recombinant tissue plasminogen activator (rt-PA) is a proven therapy in acute stroke. Main concerns are based on hemorrhagic complications, which are connected with microvascular integrity loss. The aim of this study was to evaluate microvascular changes after various doses of rt-PA. METHODS AND RESULTS: Focal cerebral ischemia for 3 hours was induced using the suture model in rats and followed by 24 hours of reperfusion. Six rats received either saline, 0.9, 9, or 18 mg rtPA/kg body weight at the end of ischemia. By immunostaining of collagen type IV the density of microvessels and the total stained area in the basal ganglia and cortex was measured. Comparison of the ischemic with the non-ischemic hemisphere showed significantly less reduction of the number of microvessels in rats treated with low-dose rt-PA than in the other groups: controls 17 +/- 3% (basal ganglia), 12 +/- 7% (cortex); 0.9 mg rt-PA, 18 +/- 3%, 10 +/- 4%; 9 mg, 21 +/- 4%, 13 +/- 7%; 18 mg, 22 +/- 4%, 15 +/- 8%. A similar effect was observed on the total stained area: control 25 +/- 4% (basal ganglia), 14 +/- 7% (cortex); 0.9 mg rt-PA, 23 +/- 2%, 7 +/- 4%; 9 mg, 28 +/- 4%, 15 +/- 4%; 18 mg, 29 +/- 4%, 17 +/- 5%, p<0.001. The significant reduction of the area of infarction after low and moderate doses of rt-PA was visualized with an MAP2-antibody, and the volume was calculated by 3-D reconstruction: control, 165.2 mm 3 +/- 21%; 0.9 mg rt-PA, 102.6 mm 3 +/- 16%; 9 mg, 101.2 mm 3 +/- 17%; 18 mg, 133.0 mm 3 +/- 24%; p < 0.001. CONCLUSIONS: Rats exposed to low-dose rt-PA preserved basal lamina structures, and showed smaller infarct sizes. The protective effect of low-dose rt-PA might be due to an increased microvascular patency rate.     

Plasminogen activation in focal cerebral ischemia and reperfusion.

In focal cerebral ischemia the plasminogen-plasmin system plays a role in the fibrinolysis of vessel-occluding clots and also in the proteolysis of extracellular matrix components, which potentially contributes to brain edema and bleeding complications. The authors investigated the plasminogen activation after middle cerebral artery occlusion with and without reperfusion (reperfusion intervals 9 and 24 hours) in rats by histologic zymography and compared areas of increased plasminogen activation to areas of structural injury, which were detected immunohistochemically. After 3 hours of ischemia, increased plasminogen activation was observed in the ischemic hemisphere. The affected area measured 5.2%+/-8.5% and 19.4%+/-30.1% of the total basal ganglia and cortex area, respectively. Reperfusion for 9 hours after 3 hours of ischemia led to a significant expansion of plasminogen activation in the basal ganglia (68.8%+/-42.2%, P < 0.05) but not in the cortex (43.0%+/-34.6%, P = 0.394). In the basal ganglia, areas of increased plasminogen activation were related to areas of structural injury (r = 0.873, P < 0.001). No such correlation was found in the cortex (r = 0.299, P = 0.228). In this study, increased plasminogen activation was demonstrated early in focal cerebral ischemia. This activation may promote early secondary edema formation and also secondary hemorrhage after ischemic stroke.     

Plasminogen activation in experimental permanent focal cerebral ischemia

BACKGROUND: Previous experimental work using in situ zymography has shown very early increased plasminogen activation in ischemic regions after 3 h of ischemia with and without reperfusion. The objective of the present study was to evaluate the time course and extent of plasminogen activation in long-term permanent focal cerebral ischemia. MATERIAL AND METHODS: The middle cerebral artery in male Fisher rats was irreversibly occluded by electrocoagulation. Duration of ischemia was 48, 72, and 168 h. Occlusion was controlled in vivo by MRI at day 2. Plasminogen activation was detected by in situ zymography of 10 microm cryosections with an overlay containing plasminogen and the plasmin substrate caseine. Areas of plasminogen activation were compared to structural lesions (immunohistochemical loss of microtubule-associated protein 2; MAP 2). RESULTS: Compared to controls, increased plasminogen activation was observed in the basal ganglia and the cortex of the ischemic hemisphere after 48, 72, and 168 h (affected area of basal ganglia: 44.5+/-21.9, 70.1+/-2.3 and 66.6+/-2.8%, respectively; affected area of cortex: 63.4+/-9.8, 67.7+/-0.7 and 64.0+/-3.7%, respectively). The duration of ischemia had no significant influence on the extent of plasminogen activation. Areas of increased plasminogen activation significantly overlapped with and exceeded areas of MAP 2 loss (P<0.005). DISCUSSION: Permanent focal cerebral ischemia leads to increased plasminogen activation in ischemic regions. This plasminogen activation remains elevated at persistent levels over days. It may contribute to extracellular matrix (ECM) disruption, secondary hemorrhage, and brain edema in subacute stages of ischemic stroke.     

Effects of tissue plasminogen activator on cerebral microvessels of rats during focal cerebral ischemia and reperfusion

The time window in the treatment of ischemic stroke with tissue plasminogen activator (tPA) is narrow, arbitrarily within 3 hours after the onset of symptom. Hemorrhagic transformation resulting from cerebral ischemia may be related to damage of the microvascular basal lamina of the brain, which may in turn cause microvascular fibrin deposition and aggravate cerebral ischemia. Here, we investigated the effect of tPA on the microvascular tissue changes during cerebral ischemia/reperfusion. Sprague-Dawley rats were subjected to focal cerebral ischemia by ligation of the right middle cerebral artery and bilateral common carotid arteries for 90 minutes. Sixty minutes after the onset of ischemia, escalated dosages of tPA from 2.5 to 10 mg/kg or saline were intravenously infused for 60 minutes. Twenty-four hours after reperfusion, the animals were allowed to be killed for examination. Low dosage of tPA (2.5-7.5 mg/kg) reduced post-ischemic brain infarction, suppressed metalloproteinase 2 (MMP-2) activity and restored blood-brain barrier (BBB) integrity. In contrast, high dose of tPA (10 mg/kg) aggravated brain infarction, increased MMP-2 activity and exacerbated BBB disruption. Cerebral ischemia/reperfusion decreased the immunoreactivity of both collagen type IV- and laminin-positive microvessels, whereas the low dosage of tPA (2.5-7.5 mg/kg) attenuated the reduction. When these molecules in whole cortical tissues were analysed, tPA dosage-dependently decreased the total content of collagen type IV, laminin and fibronectin. Although the detailed mechanisms regarding the action of tPA are yet to be investigated, our findings demonstrate that the detrimental effect of tPA was mediated, at least in part, through the destruction of the basal lamina in the cerebral microvessels by activating MMP-2.     

Tumor necrosis factor-alpha neutralization reduced cerebral edema through inhibition of matrix metalloproteinase production after transient focal cerebral ischemia.

After focal cerebral ischemia, tumor necrosis factor-alpha deteriorates cerebral edema and survival rate. Therefore, tumor necrosis factor-alpha neutralization could reduce cerebral microvascular permeability in acute cerebral ischemia. Left middle cerebral artery occlusion for 120 mins followed by reperfusion was performed with the thread method under halothane anesthesia in Sprague-Dawley rats. Antirat tumor necrosis factor-alpha neutralizing monoclonal antibody with a rat IgG Fc portion (15 mg/kg) was infused intravenously right after reperfusion. Stroke index score, infarct volume, cerebral specific gravity, and the endogenous expression of tumor necrosis factor-alpha, matrix metalloproteinase (MMP)-2, MMP-9, and membrane type 1-MMP in the brain tissue were quantified in the ischemic and matched contralateral nonischemic hemisphere. In the antitumor necrosis factor-alpha neutralizing antibody-treated rats, infarct volume was significantly reduced (P=0.014, n=7; respectively), and cerebral specific gravity was dramatically increased in the cortex and caudate putamen (P<0.001, n=7; respectively) in association with a reduction in MMP-9 and membrane type 1-MMP upregulation. Tumor necrosis factor-alpha in the brain tissue was significantly elevated in the ischemic hemisphere 6 h after reperfusion in the nonspecific IgG-treated rats (P=0.021, n=7) and was decreased in the antitumor necrosis factor-alpha neutralizing antibody-treated rats (P=0.001, n=7). Postreperfusion treatment with antirat tumor necrosis factor-alpha neutralizing antibody reduced brain infarct volume and cerebral edema, which is likely mediated by a reduction in MMP upregulation.     

Recombinant tissue plasminogen activator attenuates basal lamina antigen loss after experimental focal cerebral ischemia

Abstract

Background and purpose: The use of recombinant tissue plasminogen activator (rt-PA) is a proven therapy in acute stroke. Main concerns are based on hemorrhagic complications, which are connected with microvascular integrity loss. The aim of this study was to evaluate microvascular changes after various doses of rt-PA.

Methods and results: Focal cerebral ischemia for 3 hours was induced using the suture model in rats and followed by 24 hours of reperfusion. Six rats received either saline, 0.9, 9, or 18 mg rtPA/kg body weight at the end of ischemia. By immunostaining of collagen type IV the density of microvessels and the total stained area in the basal ganglia and cortex was measured. Comparison of the ischemic with the non-ischemic hemisphere showed significantly less reduction of the number of microvessels in rats treated with low-dose rt-PA than in the other groups: controls 17 ± 3% (basal ganglia), 12 ± 7% (cortex); 0.9 mg rt-PA, 18 ± 3%, 10 ± 4%; 9 mg, 21 ± 4%, 13 ± 7%; 18 mg, 22 ± 4%, 15 ± 8%. A similar effect was observed on the total stained area: control 25 ± 4% (basal ganglia), 14 ± 7% (cortex); 0.9 mg rt-PA, 23 ± 2%, 7 ± 4%; 9 mg, 28 ± 4%, 15 ± 4%; 18 mg, 29 ± 4%, 17 ± 5%, p<0.001. The significant reduction of the area of infarction after low and moderate doses of rt-PA was visualized with an MAP2-antibody, and the volume was calculated by 3-D reconstruction: control, 165.2 mm 3 ± 21%; 0.9 mg rt-PA, 102.6 mm 3 ± 16%; 9 mg, 101.2 mm 3 ± 17%; 18 mg, 133.0 mm 3 ± 24%; p < 0.001.

Conclusions: Rats exposed to low-dose rt-PA preserved basal lamina structures, and showed smaller infarct sizes. The protective effect of low-dose rt-PA might be due to an increased microvascular patency rate.     

Increased intracellular calpain detection in experimental focal cerebral ischemia

Calpains are intracellular proteinases whose proteolytic activity is directed mainly against the cytoskeleton and regulatory proteins. We studied the presence of calpain by immunohistochemistry in a rat model of reversible focal cerebral ischemia (3 h) at various times of reperfusion. The numbers of calpain-positive cells on the ischemic side were compared with the non-ischemic side. In controls only 2 +/- 1% cells were positive, whereas the cortex of the ischemic vs the non-ischemic side showed 88 +/- 3% vs 13 +/- 4% calpain-positive cells (p < 0.001), and the basal ganglia 47 +/- 3% vs 13 +/- 4% (p < 0.01) after 3 h ischemia and 24 h reperfusion. This is the first demonstration of elevated intracellular levels of calpains in areas of cerebral ischemia. Longer reperfusion resulted in an increase in calpain positivity.     

脑缺血再灌流脑微血管损害及u-PA表达的实验研究

目的探讨脑缺血再灌流后继发的脑水肿、出血的发生机制。方法应用光镜、透射电镜、免疫组织化学、显微镜－计算机图像分析等技术，观察大鼠局部脑缺血２小时再灌流不同时间，脑微血管结构、Ⅳ型胶原抗原及尿激酶型纤溶酶原激活物（ｕ－ＰＡ）表达。结果局部脑缺血再灌流２４小时，缺血侧ＭＣＡ区脑微血管外细胞间质水肿最严重，基底膜节段性溶解、缺损，有红细胞漏出，微血管壁及管外细胞间质ｕ－ＰＡ大量表达达高峰，同时微血管基底膜Ⅳ型胶原抗原减少。随再灌流时间延长，微血管基底膜损害加重，Ⅳ型胶原抗原逐渐消失，ｕ－ＰＡ表达减少。结论脑缺血再灌流后脑微血管结构损害是导致脑水肿、出血的主要病理基础，而脑微血管壁和管外细胞间质ｕ－ＰＡ表达可能是引起微血管损害的主要机制之一。     

Matrix metalloproteinases increase very early during experimental focal cerebral ischemia.

Microvascular integrity is lost during focal cerebral ischemia. The degradation of the basal lamina and extracellular matrix are, in part, responsible for the loss of vascular integrity. Matrix metalloproteinases (MMPs) may play a primary role in basal lamina degradation. By using a sensitive modification of gelatin zymography, the authors investigated the activity of MMP-2 and MMP-9 in frozen 10-microm sections of ischemic and nonischemic basal ganglia and plasma samples of 27 non-human primates after middle cerebral artery occlusion/reperfusion (MCAO/R) for various periods. The gelatinolytic activities were compared with parallel cell dUTP incorporation in the ischemic zones of adjacent sections. In the brain, the integrated density of MMP-2 increased significantly by 1 hour after MCAO and was persistently elevated thereafter. Matrix metalloproteinase-2 expression was highly correlated with the extent of neuron injury and the number of injured neurons (r = 0.9763, SE = 0.004, 2P < 0.0008). Matrix metalloproteinase-9 expression only was significantly increased in subjects with hemorrhagic transformation. In plasma, only MMP-9 increased transiently at 2 hours of MCAO. These findings highlight the early potential role of MMP-2 in the degradation of basal lamina leading to neuronal injury, and an association of MMP-9 with hemorrhagic transformation after focal cerebral ischemia.     

Exercise pre-conditioning ameliorates blood-brain barrier dysfunction in stroke by enhancing basal lamina

OBJECTIVE: We investigated whether exercise pre-conditioning ameliorates stroke-induced blood-brain barrier (BBB) dysfunction by strengthening basal lamina. METHODS: Adult male Sprague-Dawley rats were subjected to a 30 minute exercise program on a treadmill each day for 3 weeks. Stroke was induced by a 2 hour middle cerebral artery (MCA) occlusion using an intraluminal filament in the exercised and non-exercised groups. BBB dysfunction was then determined by brain edema. Expression of collagen IV, the major component of basal lamina essential for maintenance of the endothelial permeability barrier, was quantitatively detected by Western blot and immunocytochemistry. Ex vivo techniques were used to compare collagen IV-labeled vessels in response to ischemic insult. RESULTS: Brain edema was significantly (p<0.05) reduced after stroke in the exercised group. Western blot analysis indicated that exercise pre-conditioning enhanced collagen IV expression and reduced the loss after stroke. Immunocytochemistry demonstrated that collagen IV-positive vessels were significantly (p<0.01) increased in exercised rats. In ex vivo study, after exercised brain was incubated with ischemic brain tissue, a significantly (p<0.01) higher expression of collagen IV in cortex and striatum was observed compared to non-exercised brain following the same treatment. The ex vivo study also revealed that matrix metalloproteinase (MMP)-9 plays a key role in exercise-strengthened collagen IV expression against ischemia/reperfusion injury. DISCUSSION: Our results indicate that exercise pre-conditioning improved BBB function and enhanced basal lamina, which involved MMP-9.     

Dynamic platelet accumulation at the site of the occluded middle cerebral artery and in downstream microvessels is associated with loss of microvascular integrity after embolic middle cerebral artery occlusion

Information is lacking regarding dynamic platelet accumulation at the site of the occluded middle cerebral artery (MCA) and the relationship between platelet aggregation in downstream cerebral microvessels and loss of perfusion and vascular integrity of these microvessels. In the present study, we employed a model of embolic MCA occlusion in the rat to simultaneously measure temporal and spatial profiles of platelet accumulation at the site of the embolus occluding the MCA and within downstream cerebral microvessels. We also measured the integrity of microvessels and matrix metalloproteinase (MMP) activity in ischemic brain. Rats (n=36) were subjected to embolic MCA occlusion. Immunohistochemistry was used to detect microvascular integrity, plasminogen activator inhibitor 1 (PAI-1) and the deposition of fibrin. SDS-PAGE zymography was used to measure MMP2 and MMP9 activities. Accumulation of platelets and increases in PAI-1 immunoreactivity at the site of the embolus occluding the MCA were detected 1 h (n=7) and 4 h (n=7) after ischemia, respectively, and numbers of GPIIb/IIIa immunoreactive downstream cerebral microvessels increased significantly (209+/-59; n=7; P<0.05) 4 h after ischemia, suggesting dynamic platelet aggregation. A significant (n=7; P<0.01) diffuse loss of type IV collagen immunoreactivity in microvessels was temporally associated with platelet GPIIb/IIIa immunoreactivity within the vessels. Triple immunostaining revealed that microvessels containing platelet aggregates exhibited loss of type IV collagen immunoreactivity and both intra- and extra-vascular fibrin deposition, suggesting that intravascular platelet aggregation is associated with decreases in the integrity of the microvascular basal lamina and blood-brain barrier leakage. A significant increase (P<0.05) in MMP9 was detected at 4 h (n=3) and 24 h (n=3) after ischemia but levels of MMP2 were not significantly changed in ischemic brain. Our data suggest that dynamic platelet aggregation in ischemic brain may contribute to time-dependent resistance to fibrinolysis. In addition, platelet deposition and increased MMP9 coincided with degradation of type IV collagen and loss of vascular integrity. These data suggest an important role for post-occlusive distal platelet deposition in the pathophysiology of stroke.     

Reperfusion activates metalloproteinases that contribute to neurovascular injury

In this study, we examine the effects of reperfusion on the activation of matrix metalloproteinase (MMP) and assess the relationship between MMP activation during reperfusion and neurovascular injury. Ischemia was produced using suture-induced middle cerebral artery occlusion in rats. The MMP activation was examined with in situ and gel zymography. Injury to cerebral endothelial cells and basal lamina was assessed using endothelial barrier antigen (EBA) and collagen IV immunohistochemistry. Injury to neurons and glial cells was assessed using Cresyl violet staining. These were examined at 3 h after reperfusion (8 h after initiation of ischemia) and compared with permanent ischemia at the same time points to assess the effects of reperfusion. A broad-spectrum MMP inhibitor, AHA (p-aminobenzoyl-Gly-Pro-D-Leu-D-Ala-hydroxamate, 50 mg/kg intravenously) was administered 30 min before reperfusion to assess the roles of MMPs in activating gelatinolytic enzymes and in reperfusion-induced injury. We found that reperfusion accelerated and potentiated MMP-9 and MMP-2 activation and injury to EBA and collagen IV immunopositive microvasculature and to neurons and glial cells in ischemic cortex and striatum relative to permanent ischemia. Administering AHA 30 min before reperfusion decreased MMP-9 activation and neurovascular injury in ischemic cerebral cortex.     

MRI predicts area of increased plasminogen activation in permanent focal cerebral ischemia

To determine if MRI can predict intracerebral plasminogen activation after focal cerebral ischemia (FCI), ischemic regions detected by MRI after 48 h of permanent FCI in rats were compared with areas of increased plasminogen activation, defined by histological zymography after 72 h of ischemia. The overlap between areas of MRI alterations (64.5% +/- 5.4% of total ischemic hemisphere) and areas with increased plasminogen activation (62.2% +/- 3.6%) was significant for the hemisphere (p < 0.001), the cortex (p < 0.05), and the basal ganglia (p < 0.05). Thus, MRI can predict the extent of increased plasminogen activation, which may play a role in BBB-mediated post-ischemic brain edema and secondary hemorrhage.     

Doxycycline inhibits MMPs via modulation of plasminogen activators in focal cerebral ischemia

Tetracyclines inhibit matrix metalloproteinases (MMPs) and reduce infarction volume following cerebral ischemia. In this thesis an involvement of urokinase could be proven. Cerebral ischemia in rats was induced for 3 h followed by 24 h reperfusion (suture model). Each 6 animals received orally either doxycycline or water. Doxycycline treatment began 10 days before ischemia. MMP-2 and MMP-9 were substantially decreased. The possibility of involvement of the endogenous MMP inhibitors in the MMP inhibiting mechanisms was excluded. The plasminogen activator uPA was significantly decreased by doxycycline indicating an MMP inhibiting mechanism including the plasminogen/plasmin system. In the doxycycline group, this resulted in a decreased damage to the cerebral microvessels and less loss of the basal lamina antigen collagen type IV. Hemoglobin extravasation was also significantly reduced. Our results suggest that doxycycline may have a potential use as an anti-ischemic compound since it provides microvascular protection by inhibiting the plasminogen system.     

脑缺血再灌注时bcl-2蛋白的表达及药物干预作用的研究

探讨ｂｃｌ－２在脑缺血再灌注时脑损伤过程的作用,以及川芎嗪和尼莫通对其影响。方法：采用免疫组化法检测大鼠缺血再灌注不同时间内ｂｃｌ－２蛋白表达的水平,以及川芎嗪和尼莫通干预后ｂｃｌ－２蛋白表达的变化。结果（１）脑缺血再灌注时缺血侧皮层和基底节区均有ｂｃｌ－２阳性表达,其阳性表达率随再灌注时间不同而不同,于缺血２再灌注３ｈ阳性表达达高峰。（２）川芎嗪,尼莫通干预组脑缺血再灌注时层和基底节区ｂｃｌ－２