Early appearance of activated matrix metalloproteinase-9 and blood–brain barrier disruption in mice after focal cerebral ischemia and reperfusion

Blood–brain barrier (BBB) disruption is thought to play a critical role in the pathophysiology of ischemia/reperfusion. Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that can degrade all the components of the extracellular matrix when they are activated. Gelatinase A (MMP-2) and gelatinase B (MMP-9) are able to digest the endothelial basal lamina, which plays a major role in maintaining BBB impermeability. The present study examined the expression and activation of gelatinases before and after transient focal cerebral ischemia (FCI) in mice. Adult male CD1 mice were subjected to 60 min FCI and reperfusion. Zymography was performed from 1 to 23 h after reperfusion using the protein extraction method with detergent extraction and affinity-support purification. MMP-9 expression was also examined by both immunohistochemistry and Western blot analysis, and tissue inhibitors to metalloproteinase-1 was measured by reverse zymography. The BBB opening was evaluated by the Evans blue extravasation method. The 88-kDa activated MMP-9 was absent from the control specimens, while it appeared 3 h after transient ischemia by zymography. At this time point, the BBB permeability alteration was detected in the ischemic brain. Both pro-MMP-9 (96 kDa) and pro-MMP-2 (72 kDa) were seen in the control specimens, and were markedly increased after FCI. A significant induction of MMP-9 was confirmed by both immunohistochemistry and Western blot analysis. The early appearance of activated MMP-9, associated with evidence of BBB permeability alteration, suggests that activation of MMP-9 contributes to the early formation of vasogenic edema after transient FCI.     

Effects of berberine on hippocampal neuronal damage and matrix metalloproteinase-9 activity following transient global cerebral ischemia

Berberine, an isoquinoline alkaloid with a long history of use in Chinese medicine, has several important pharmacological effects. Several studies have revealed that berberine has neuroprotective and neuropsychiatric effects. However, there are few reports regarding the protective effect of berberine against neuronal damage following transient global cerebral ischemia. In this study, mice were subjected to 20 min of global brain ischemia and sacrificed 72 hr later. Berberine was administered for 7 days prior to ischemia and daily until sacrifice. Mice treated with berberine showed reduced matrix metalloproteinase-9 (MMP-9) activity. Berberine inhibited gelatinase activity directly in in situ zymography and reduced neuronal damage following global ischemia. Laminin expression and NeuN expression were markedly reduced in CA1 and CA2 areas after ischemia, and berberine reduced the laminin degradation and neuronal loss. In the TUNEL assay, damaged neurons were also apparent in the CA1 and CA2 areas, and berberine reduced TUNEL-positive cells. These data demonstrate that berberine, a plant alkaloid, may protect from hippocampal neuronal damage following transient global ischemia by reducing MMP-9 activity.     

Hypoxia-controlled matrix metalloproteinase-9 hyperexpression promotes behavioral recovery after ischemia

Matrix metalloproteinase-9(MMP-9) plays a beneficial role in the sub-acute phase after ischemic stroke.However,unrestrained MMP-9 may disrupt the blood-brain barrier(BBB),which has limited its use for the treatment of brain ischemia.In the present study,we constructed lentivirus mediated hypoxiacontrolled MMP-9 expression and explored its role after stroke.Hypoxia response element(HRE)was used to confine MMP-9 expression only to the hypoxic region of mouse brain after 120-min transient middle cerebral artery occlusion.Lentiviruses were injected into the peri-infarct area on day 7 after transient ischemia.We found hyperexpression of exogenous HRE-MMP-9 under the control of hypoxia,and its expression was mainly located in neurons and astrocytes without aggravation of BBB damage compared to the CMV group.Furthermore,mice in the HRE-MMP-9 group showed the best behavioral recovery compared with the normal saline,GFP,and SB-3CT groups.Therefore,hypoxia-controlled MMP-9 hyperexpression during the sub-acute phase of ischemia may provide a novel promising approach of gene therapy for stroke.     

Mast cells promote blood brain barrier breakdown and neutrophil infiltration in a mouse model of focal cerebral ischemia

Blood brain barrier (BBB) breakdown and neuroinflammation are key events in ischemic stroke morbidity and mortality. The present study investigated the effects of mast cell deficiency and stabilization on BBB breakdown and neutrophil infiltration in mice after transient middle cerebral artery occlusion (tMCAo). Adult male C57BL6/J wild type (WT) and mast cell-deficient (C57BL6/J Kit^Wsh/Wsh (Wsh)) mice underwent tMCAo and BBB breakdown, brain edema and neutrophil infiltration were examined after 4 hours of reperfusion. Blood brain barrier breakdown, brain edema, and neutrophil infiltration were significantly reduced in Wsh versus WT mice (P<0.05). These results were reproduced pharmacologically using mast cell stabilizer, cromoglycate. Wild-type mice administered cromoglycate intraventricularly exhibited reduced BBB breakdown, brain edema, and neutrophil infiltration versus vehicle (P<0.05). There was no effect of cromoglycate versus vehicle in Wsh mice, validating specificity of cromoglycate on brain mast cells. Proteomic analysis in Wsh versus WT indicated that effects may be via expression of endoglin, endothelin-1, and matrix metalloproteinase-9. Using an in vivo model of mast cell deficiency, this is the first study showing that mast cells promote BBB breakdown in focal ischemia in mice, and opens up future opportunities for using mice to identify specific mechanisms of mast cell-related BBB injury.     

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.     

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.     

Role of matrix metalloproteinases in apoptosis after transient focal cerebral ischemia in rats and mice

The involvement of matrix metalloproteinases (MMPs) in cerebral ischemia-induced apoptosis was investigated in a model of transient focal cerebral ischemia in rats treated intracerebroventricularly (i.c.v.) with 4-((3-(4-phenoxylphenoxy)propylsulfonyl)methyl)-tetrahydropyran-4-carboxylic acid N-hydroxy amide, a broad spectrum non-peptidic hydroxamic acid MMP inhibitor, and in MMP-9-deficient mice. Our results showed that MMP inhibition reduced DNA fragmentation by 51% (P < 0.001) and cerebral infarct by 60% (P < 0.05) after ischemia. This protection was concomitant with a 29% reduction of cytochrome c release into the cytosol (P < 0.005) and a 54% reduction of calpain-related alpha-spectrin degradation (P < 0.05), as well as with an 84% increase in the immunoreactive signal of the native form of poly(ADP) ribose polymerase (P < 0.01). By contrast, specific targeting of the mmp9 gene in mice did reduce cerebral damage by 34% (P < 0.05) but did not modify the apoptotic response after cerebral ischemia. However, i.c.v. injection of MMP-9-deficient mice with the same broad-spectrum inhibitor used in rats significantly reduced DNA degradation by 32% (P < 0.05) and contributed even further to the protection of the ischemic brain. Together, our pharmacological and genetic results indicate that MMPs other than MMP-9 are actively involved in cerebral ischemia-induced apoptosis.     

Early appearance of activated matrix metalloproteinase-9 after focal cerebral ischemia in mice: a possible role in blood-brain barrier dysfunction.

During cerebral ischemia blood-brain barrier (BBB) disruption is a critical event leading to vasogenic edema and secondary brain injury. Gelatinases A and B are matrix metalloproteinases (MMP) able to open the BBB. The current study analyzes by zymography the early gelatinases expression and activation during permanent ischemia in mice (n = 15). ProMMP-9 expression was significantly (P < 0.001) increased in ischemic regions compared with corresponding contralateral regions after 2 hours of ischemia (mean 694.7 arbitrary units [AU], SD +/- 238.4 versus mean 107.6 AU, SD +/- 15.6) and remained elevated until 24 hours (mean 745.7 AU, SD +/- 157.4). Moreover, activated MMP-9 was observed 4 hours after the initiation of ischemia. At the same time as the appearance of activated MMP-9, we detected by the Evan's blue extravasation method a clear increase of BBB permeability. Tissue inhibitor of metalloproteinase-1 was not modified during permanent ischemia at any time. The ProMMP-2 was significantly (P < 0.05) increased only after 24 hours of permanent ischemia (mean 213.2 AU, SD +/- 60.6 versus mean 94.6 AU, SD +/- 13.3), and no activated form was observed. The appearance of activated MMP-9 after 4 hours of ischemia in correlation with BBB permeability alterations suggests that MMP-9 may play an active role in early vasogenic edema development after stroke.     

Tyrosine phosphatase inhibition induces loss of blood-brain barrier integrity by matrix metalloproteinase-dependent and -independent pathways

Tight junctions between endothelial cells of brain capillaries form the structural basis of the blood-brain barrier (BBB), which controls the exchange of molecules between blood and CNS. Regulation of cellular barrier permeability is a vital and complex process involving intracellular signalling and rearrangement of tight junction proteins. We have analysed the impact of tyrosine phosphatase inhibition on tight junction proteins and endothelial barrier integrity in a primary cell culture model based on porcine brain capillary endothelial cells (PBCEC) that closely mimics the BBB in vitro. The tyrosine phosphatase inhibitor phenylarsine oxide (PAO) induced increased matrix metalloproteinase (MMP) activity, which was paralleled by severe disruption of cell-cell contacts and proteolysis of the tight junction protein occludin. ZO-1 and claudin-5 were not affected. Under these conditions, the transendothelial electrical resistance (TEER) was markedly reduced. PAO-induced occludin proteolysis could be prevented by different MMP inhibitors. Pervanadate (PV) reduced the TEER similar to PAO, but did not increase MMP activity. Cell-cell contacts of PV-treated cells appeared unaffected, and occludin proteolysis did not occur. Our results suggest that tyrosine phosphatase inhibition can influence barrier properties independent of, but also correlated to MMPs. Evidence is given for a role of MMPs in endothelial tight junction regulation at the BBB in particular and probably at tight junctions (TJs) in general.     

Correlation of aquaporin-4 expression to blood-brain barrier permeability in rats with focal cerebral ischemia

BACKGROUND: Ischemic cerebrovascular disease causes injury to the blood-brain barrier. The occurrence of brain edema is associated with aquaporin expression following cerebral ischemia/reperfusion. OBJECTIVE: To analyze the correlation of aquaporin-4 expression to brain edema and blood-brain barrier permeability in brain tissues of rat models of ischemia/reperfusion. DESIGN, TIME AND SETTING: The randomized control experiment was performed at the Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, China from December 2006 to October 2007. MATERIALS: A total of 112 adult, male, Sprague-Dawley rats, weighing 220-250 g, were used to establish rat models of middle cerebral artery occlusion and reperfusion by the suture method. Rabbit anti-aquaporin-4 (Santa Cruz, USA) and Evans blue (Sigma, USA) were used to analyze the tissue. METHODS: The rats were randomized into sham-operated (n = 16) and ischemia/reperfusion (n = 96) groups. There were 6 time points in the ischemia/reperfusion group, comprising 4, 6, 12, 24, 48, and 72 hours after reperfusion, with 16 rats for each time point. Rat models in the sham-operated group at 4 hours after surgery and rat models in the ischemia/reperfusion group at different time points were equally and randomly assigned into 4 different subgroups. MAIN OUTCOME MEASURES: Brain water content on the ischemic side and the control side was measured using the dry-wet weight method. Blood-brain barrier function was determined by Evans Blue. Aquaporin-4 expression surrounding the ischemic focus, as well as the correlation of aquaporin-4 expression with brain water content and Evans blue staining, were measured using immunohistochemistry and Western blot analysis. RESULTS: Brain water content on the ischemic side significantly increased at 12 hours after reperfusion, reached a peak at 48 hours, and was still high at 72 hours. Brain water content was greater on the ischemic hemispheres, compared with the control hemispheres at 6, 12, 24, 48, and 72 hours after reperfusion, as well as both hemispheres in the sham-operated group (P<0.05). Evans blue content significantly increased on the ischemic side at 4 hours after ischemi',dreperfusion, and reached a peak at 48 hours. Evans blue content was greater on the ischemic hemispheres, compared with the control hemispheres at various time points, as well as both hemispheres in the sham-operated group (P<0.05). Aquaporin-4-positive cells were detected in the cortex and hippocampus, surrounding the ischemic penumbra focus, at 4-6 hours after ischemia/reperfusion. The number of positive cells significantly increased at 12 hours and reached a peak at 48-72 hours. Aquaporin-4 was, however, weakly expressed in the control hemispheres and the sham-operated group. The absorbance ratio of aquaporin-4 to β-actin was greater at 12, 24, 48, and 72 hours following cerebral ischemia/reperfusion, compared with the sham-operated group (P<0.05). Aquaporin-4 expression positively correlated to brain water content and Evans blue staining following cerebral ischemia/reperfusion (r1 = 0.68, r2= 0.81, P<0.05). CONCLUSION: Aquaporin-4 is highly expressed in brain tissues, participates in the occurrence of ischemic brain edema, and is positively correlated to blood-brain barrier permeability following cerebral ischemia/reperfusion.     

orrelation of aquaporin-4 expression to blood-brain barrier permeability in rats with focal cerebral ischemia**★

BACKGROUND： Ischemic cerebrovascular disease causes injury to the blood-brain barrier. The occurrence of brain edema is associated with aquaporin expression following cerebral ischemia/reperfusion. OBJECTIVE： To analyze the correlation of aquaporin-4 expression to brain edema and blood-brain barrier permeability in brain tissues of rat models of ischemia/reperfusion. DESIGN, TIME AND SETTING： The randomized control experiment was performed at the Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, China from December 2006 to October 2007. MATERIALS： A total of 112 adult, male, Sprague-Dawley rats, weighing 220-250 g, were used to establish rat models of middle cerebral artery occlusion and reperfusion by the suture method. Rabbit anti-aquaporin-4 （Santa Cruz, USA） and Evans blue （Sigma, USA） were used to analyze the tissue. METHODS： The rats were randomized into sham-operated （n = 16） and ischemia/reperfusion （n = 96） groups. There were 6 time points in the ischemia/reperfusion group, comprising 4, 6, 12, 24, 48, and 72 hours after reperfusion, with 16 rats for each time point. Rat models in the sham-operated group at 4 hours after surgery and rat models in the ischemia/reperfusion group at different time points were equally and randomly assigned into 4 different subgroups. MAIN OUTCOME MEASURES： Brain water content on the ischemic side and the control side was measured using the dry-wet weight method. Blood-brain barrier function was determined by Evans Blue. Aquaporin-4 expression surrounding the ischemic focus, as well as the correlation of aquaporin-4 expression with brain water content and Evans blue staining, were measured using immunohistochemistry and Western blot analysis. RESULTS： Brain water content on the ischemic side significantly increased at 12 hours after reperfusion, reached a peak at 48 hours, and was still high at 72 hours. Brain water content was greater on the ischemic hemispheres, compared with the control hemispheres at 6, 12, 24, 48, an     

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.     

Hyperthermia enhances spectrin breakdown in transient focal cerebral ischemia

Calpain-mediated spectrin degradation is triggered by cerebral ischemia and, when persistent, is thought to signal irreversible neuronal injury. Hyperthermia superimposed upon cerebral ischemia may exacerbate the injury process. In this study, we compared the extent of spectrin degradation in the brains of rats subjected to 1 h of transient proximal middle cerebral artery (MCA) clip-occlusion performed under conditions of cranial normothermia (37°C) or mild cranial hyperthermia (39°C). Immunocytochemical localization of spectrin breakdown products was achieved by the use of a rabbit polyclonal antibody which reacted selectively with calpain-generated fragments of brain spectrin. The perfusion times studied were 1, 4 or 24 h. Following normothermic MCA occlusion, spectrin immunoreactivity was present only occasionally and only in scattered cortical neurons immediately upon reperfusion and 1 h later; all normothermic brains showed sparse immunoreactivity at 4 h of reperfusion; and no immunoreactivity was detected at 24 h. By contrast, following hyperthermic MCA occlusion, moderate-to-intense immunostaining was present in cortical pyramidal neurons even immediately upon reperfusion and persisted at 1 h of reperfusion. At 4 and 24 h, most brains exhibited dense immunoreactivity associated with morphologically shrunken neurons. Following 24 h survival, semi-thick plastic sections revealed intact neuropil and only selective neuronal necrosis in normothermic rats. By contrast, pan-necrosis was evident 24 h after the hyperthermic ischemic insult. These results indicate that mild cranial hyperthermia superimposed upon transient focal ischemia markedly enhances calpain activation and spectrin degradation; this process appears to be an important mechanism by which hyperthermia exacerbates ischemic injury.     

Time course of aquaporin expression after transient focal cerebral ischemia in mice

Cerebral edema contributes to morbidity and mortality in stroke. Aquaporins (AQPs)-1, -4, and -9 have been identified as the three main water channels in the brain. To clarify their role in water movement, we have compared their expression patterns with brain swelling after transient focal brain ischemia. There were two peaks of maximal hemispheric swelling at 1 hr and at 48 hr after ischemia, coinciding with two peaks of AQP4 expression. At 1 hr after occlusion, AQP4 expression was significantly increased on astrocyte endfeet in the core and in the border of the lesion. At 48 hr, AQP4 expression was increased in astrocytes in the border of the lesion over the whole cell. AQP9 showed a significant induction at 24 hr that increased gradually with time, without correlation with the swelling. The expression of AQP1 remained unchanged. These results suggest that AQP4, but not AQP1 or AQP9, may play an important role in water movement associated with the pathophysiology of edema after transient cerebral ischemia in the mouse.     

Temporal profile of tissue plasminogen activator (tPA) and inhibitor expression after transient focal cerebral ischemia

Altered tPA expression may influence the fate of neurons after cerebral ischemia. We determined the changes in tPA and plasminogen activator inhibitor (PAI-1) expression in adult rat brain after transient middle cerebral artery (MCA) occlusion. Immunohistology revealed tPA staining in ipsilateral but not contralateral cortex and striatum 6 h after occlusion. This pattern was maintained at 24 h. Staining data was supported by Western blot data which showed no tPA protein in contralateral cortex at 3 h but abundant protein in ipsilateral cortex which increased further at 6 h and 24 h. In contrast there was prominent PAI-1 immunostaining and protein expression in control tissue after MCA occlusion but it diminished progressively at 3, 6 and 24 h in the lesioned cortex.     

Expression and activation of matrix metalloproteinase-2 and -9 in rat brain after transient focal cerebral ischemia

Matrix metalloproteinases (MMPs) degrade the extracellular matrix and carry out key functions during development and after injury. By means of zymography, Western blot and immunohistochemistry, we studied MMP-2 (gelatinase A) and MMP-9 (gelatinase B) in rat brain after focal cerebral ischemia. The control rat brain showed constitutive MMP-2 and, to a lesser extent, MMP-9, which were mainly present as prozymogens. MMP-2 protein was located in the cell body of neurons, glia, and endothelium, whereas MMP-9 was associated to neurons and myelinated fibre tracts. Ischemia greatly increased MMP activation in two temporal waves, in the first one, MMP-9 protein was induced from 4 h to 4 days, and also a small and short-lasting increase in MMP-2 was detected at 4 h. The second wave showed a massive increase in MMP-2 protein expression and activation by day 4, which was compatible with abundant MMP-2 in reactive microglia/macrophages. Our results are compatible with progressive induction of MMP-9 proform, likely in neurons, shortly after ischemia. For MMP-2, the results suggest a discrete production immediately after reperfusion, while a very enhanced expression and activation of MMP-2 attributable to microglia/macrophages occurs on day 4, and it might contribute to the phagocytic action of these reactive cells.     

Increases in circulating matrix metalloproteinase-9 levels following fibrinolysis for acute pulmonary embolism

Introduction

Fibrinolyis is one of the first line therapies in high risk pulmonary embolism (PE) according to current guidelines. Previous studies showed that fibrinolytic therapy with tPA (tissue plasminogen activator, or alteplase) upregulates the concentrations of matrix metalloproteinases (MMPs) and contributes to hemorrhagic transformation after cardioembolic stroke. However, no previous study has described the circulating MMPs levels following fibrinolysis for acute PE.

Materials and Methods

We serially measured the circulating levels of MMPs (MMP-9 and MMP-2) and their endogenous inhibitors, the tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in alteplase and in streptokinase-treated patients with acute PE by gelatin zymography and by enzyme-linked immunosorbent assays, respectively.

Results

We found that therapy of PE streptokinase or with alteplase is associated increased pro-MMP-9, but not MMP-2, concentrations for up to 24 hours, whereas no significant changes were found in TIMP-1 or TIMP-2 concentrations. This alteration returned to normal 3 to 5 days after thrombolysis. This is the first study reporting on MMPs alterations following fibrinolysis for acute PE.

Conclusions

We found transient increases in circulating pro-MMP-9 levels following fibrinolysis for acute PE. Our findings support the hypothesis that increased MMP-9 levels may underlie the risk of intracerebral hemorrhage or other bleeding complication of thrombolysis for acute PE, and the use of MMP inhibitors may decrease such risk.