batimastat and Brain-Ischemia

Hippocampal neuronal death following transient global ischemia in the mouse takes days to occur, providing a potential timeframe for therapeutic intervention. Since matrix metalloproteinase-3 (MMP-3) enhances inflammation and tissue inhibitor of metalloproteinases-3 (TIMP-3) promotes apoptosis in ischemia, we hypothesized that they are involved in neuronal death secondary to transient global ischemia. Timp-3 knockout (T3KO) and wild type (T3WT) mice underwent 30 min bilateral carotid artery occlusion (BCAO), which causes hippocampal neuronal death 7 days after reperfusion. Mice lacking the Timp-3 gene have significantly less astrocytosis, microglial reactivity, MMP-3 activity and neuronal cell death. In addition, T3KO mice had decreased tumor necrosis factor (TNF) receptor-1 (TNFR1) expression and increased TNF-alpha converting enzyme (TACE) activity. Mmp-3 KO mice with a similar BCAO showed significantly fewer microglial cells, reduced TNF-alpha expression, and less neuronal death than the Mmp-3 WT. To see if TIMP-3 and MMP-3 cell death pathways were independent, we blocked MMPs with the broad-spectrum MMP inhibitor, BB-94, on days 3 through 6 of reperfusion in T3WT and T3KO mice. BB-94 rescued hippocampal neurons at 7 days in both T3WT and T3KO mice, but significantly fewer neurons died in T3KO mice treated with BB-94. Our results indicate a novel additive role for TIMP-3 and MMP-3 in delayed neuronal death, and show that delayed treatment with MMP inhibitors can be used to reduce hippocampal death.

Topics: ADAM Proteins; ADAM17 Protein; Animals; Apoptosis; Brain Ischemia; Disease Models, Animal; Encephalitis; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Gliosis; Hippocampus; Matrix Metalloproteinase 3; Matrix Metalloproteinase Inhibitors; Mice; Mice, Inbred C57BL; Mice, Knockout; Nerve Degeneration; Neuroprotective Agents; Phenylalanine; Receptors, Tumor Necrosis Factor, Type I; Reperfusion Injury; Thiophenes; Tissue Inhibitor of Metalloproteinase-3; Tumor Necrosis Factor-alpha

Intravenous recombinant tissue plasminogen activator (rtPA) can be beneficial in ischemic stroke despite an increased risk of hemorrhage and potential neurotoxic effects. We hypothesized that rtPA-mediated adverse effects depend on the timing of reperfusion and injury to the blood-brain barrier (BBB).. Male Wistar rats had middle cerebral artery occlusion (MCAO) by intraluminal thread placement. Intervals of ischemia/reperfusion, respectively, in hours were 0/18, 1.5/16.5, 3/15, 6/12, 18/0, and 6/1. Animals received either rtPA or saline for 1 hour at the time of reperfusion or, for the 18/0 trial, starting 1 hour after MCAO. Outcome parameters were mortality, matrix metalloproteinase-2 and -9 (MMP-2 and -9) concentrations, tissue hemoglobin, and brain water content. We analyzed the permeability of the BBB by using the brain 14C[sucrose] uptake method. Effects of the MMP inhibitor BB-94 on the BBB without rtPA treatment and on mortality with rtPA were tested in animals with 6/1 and 6/12, respectively.. In delayed reperfusion (6/12), rtPA increased mortality from 17% to 83% (P<0.01) without significantly affecting other outcome parameters. In 6/1, sucrose uptake in the ischemic hemisphere was markedly increased (8.80+/-1.14% vs 2.15+/-0.26%; P<0.01). This uptake was reduced by treatment with BB-94 (3.95+/-1.48%, P<0.01). Furthermore, BB-94 reduced rtPA-mediated mortality in 6/12 to 33% (P<0.05).. rtPA-mediated mortality in delayed reperfusion is associated with early opening of the BBB. Closure of the BBB with BB-94 given before rtPA treatment reduced mortality, suggesting that treatment with MMP inhibitors might reduce the risk associated with thrombolysis.

Topics: Animals; Blood-Brain Barrier; Brain; Brain Ischemia; Disease Models, Animal; Drug Administration Schedule; Enzyme Inhibitors; Infarction, Middle Cerebral Artery; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Phenylalanine; Rats; Rats, Wistar; Recombinant Proteins; Reperfusion; Sucrose; Survival Rate; Thiophenes; Time Factors; Tissue Plasminogen Activator; Treatment Outcome; Water

Thrombolytic therapy with tissue plasminogen activator (tPA) for acute ischemic stroke remains complicated by risks of hemorrhagic transformation. In this study we used a previously established quantitative rat model of tPA-associated hemorrhage to test the hypothesis that matrix metalloproteinases (MMPs) are involved.. Spontaneously hypertensive rats were subjected to embolic focal ischemia by placing homologous blood clots into the middle cerebral artery. Three groups of rats were studied: (1) untreated controls that received saline at 6 hours after ischemia; (2) rats that received tPA alone (10 mg/kg at 6 hours after ischemia); and (3) rats that received tPA plus the broad-spectrum MMP inhibitor BB-94 (50 mg/kg of BB-94 before ischemia and at 3 and 6 hours after ischemia plus tPA at 6 hours). Gelatin zymography was used to quantify MMP levels. A hemoglobin spectrophotometry method was used to quantify cerebral hemorrhage. Ischemic lesions were measured at 24 hours with tetrazolium staining.. At 6, 12, and 24 hours, pro-MMP-9 and cleaved MMP-9 were upregulated in ischemic brain. At 12 hours, tPA-treated rats showed significantly higher levels of pro-MMP-9 and cleaved MMP-9 than untreated controls. By 24 hours, all rats showed evidence of hemorrhagic transformation in the ischemic territory. Rats treated with BB-94 and tPA showed significantly reduced hemorrhage volumes compared with those that received tPA alone. There was no effect on infarct size.. These results indicate that (1) tPA treatment increases levels of MMP-9 after embolic focal cerebral ischemia, (2) MMPs are involved in the mechanism of tPA-associated hemorrhage, and (3) combination therapies with MMP inhibitors may be useful for decreasing the risk and severity of this dreaded complication of thrombolytic therapy.

Topics: Animals; Blood Flow Velocity; Brain; Brain Ischemia; Cerebral Hemorrhage; Cerebrovascular Circulation; Disease Models, Animal; Enzyme Inhibitors; Intracranial Embolism; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Phenylalanine; Rats; Rats, Inbred SHR; Sodium, Dietary; Survival Rate; Thiophenes; Thrombolytic Therapy; Tissue Plasminogen Activator

It has been shown recently that matrix metalloproteinases (MMPs) are elevated after cerebral ischemia. In the current study, we investigated the pathophysiologic role for MMP-9 (gelatinase B, EC.3.4.24.35) in a mouse model of permanent focal cerebral ischemia, using a combination of genetic and pharmacologic approaches. Zymography and Western blot analysis demonstrated that MMP-9 protein levels were rapidly up-regulated in brain after ischemic onset. Reverse transcription polymerase chain reaction showed increased transcription of MMP-9. There were no differences in systemic hemodynamic parameters and gross cerebrovascular anatomy between wild type mice and mutant mice with a targeted knockout of the MMP-9 gene. After induction of focal ischemia, similar reductions in cerebral blood flow were obtained. In the MMP-9 knockout mice, ischemic lesion volumes were significantly reduced compared with wild type littermates in male and female mice. In normal wild type mice, the broad spectrum MMP inhibitor BB-94 (batimastat) also significantly reduced ischemic lesion size. However, BB-94 had no detectable protective effect when administered to MMP-9 knockout mice subjected to focal cerebral ischemia. These data demonstrate that MMP-9 plays a deleterious role in the development of brain injury after focal ischemia.

Topics: Animals; Brain; Brain Ischemia; Male; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Mice; Mice, Inbred Strains; Mice, Knockout; Neuroprotective Agents; Phenylalanine; Protease Inhibitors; Stroke; Thiophenes; Transcription, Genetic