Toshihisa Sumii

Effects of Matrix Metalloproteinase-9 Gene Knock-Out on the Proteolysis of Blood–Brain Barrier and White Matter Components after Cerebral Ischemia

Deleterious processes of extracellular proteolysis may contribute to the progression of tissue damage after acute brain injury. We recently showed that matrix metalloproteinase-9 (MMP-9) knock-out mice were protected against ischemic and traumatic brain injury. In this study, we examined the mechanisms involved by focusing on relevant MMP-9 substrates in blood–brain barrier, matrix, and white matter. MMP-9 knock-out and wild-type mice were subjected to transient focal ischemia. MMP-9 levels increased after ischemia in wild-type brain, with expression primarily present in vascular endothelium. Western blots showed that the blood–brain barrier-associated protein and MMP-9 substrate zonae occludens-1 was degraded after ischemia, but this was reduced in knock-out mice. There were no detectable changes in another blood–brain barrier-associated protein, occludin. Correspondingly, blood–brain barrier disruption assessed via Evans Blue leakage was significantly attenuated in MMP-9 knock-out mice compared with wild types. In white matter, ischemic degradation of the MMP-9 substrate myelin basic protein was significantly reduced in knock-out mice compared with wild types, whereas there was no degradation of other myelin proteins that are not MMP substrates (proteolipid protein and DM20). There were no detectable changes in the ubiquitous structural protein actin or the extracellular matrix protein laminin. Finally, 24 hr lesion volumes were significantly reduced in knock-out mice compared with wild types. These data demonstrate that the protective effects of MMP-9 gene knock-out after transient focal ischemia may be mediated by reduced proteolytic degradation of critical blood–brain barrier and white matter components.

Blood-Brain Barrier Disruption and Matrix Metalloproteinase-9 Expression During Reperfusion Injury Mechanical Versus Embolic Focal Ischemia in Spontaneously Hypertensive Rats

Background and Purpose— Most experimental models of cerebral ischemia use mechanical methods of occlusion and reperfusion. However, differences between mechanical reperfusion versus clot thrombolysis may influence reperfusion injury profiles. In this study we compared blood flow recovery, blood-brain barrier (BBB) permeability, and matrix metalloproteinase-9 (MMP-9) expression in cortex after mechanical versus thrombolytic reperfusion in rat focal ischemia. Methods— Male spontaneously hypertensive rats were used. Mechanical ischemia/reperfusion was achieved with the use of an intraluminal filament to occlude the middle cerebral artery for 2 hours. Thrombolytic reperfusion was achieved by administering tissue plasminogen activator at 2 hours after embolic focal ischemia. Regional cortical blood flow was monitored by laser-Doppler flowmetry. BBB permeability in cortex was measured by Evans blue dye leakage. Cortical MMP-9 levels were assessed with zymography and immunohistochemistry. Results— Blood flow recovery during mechanical reperfusion was complete in both central and peripheral areas of ischemic cortex. However, after thrombolysis, reperfusion was incomplete, with moderate recovery in the periphery only. BBB permeability was mainly increased in the central regions of the ischemic cortex after mechanical reperfusion but was increased in both central and peripheral areas after thrombolysis. Overall, MMP-9 levels were higher after embolic versus mechanical ischemia/reperfusion, even though ischemic injury was similar in both models at 24 hours. Conclusions— There are significant differences in the profiles of blood flow recovery, BBB leakage, and MMP-9 upregulation in mechanical versus thrombolytic reperfusion after focal ischemia.

Effects of Normobaric Hyperoxia in a Rat Model of Focal Cerebral Ischemia—Reperfusion

Recent studies suggest that normobaric hyperoxia can be beneficial, if administered during transient stroke. However, increased oxygenation theoretically may increase oxygen free-radical injury, particularly during reperfusion. In the present study, the authors assessed the benefit and risks of hyperoxia during focal cerebral ischemia and reperfusion. Rats were subjected to hyperoxia (Fio2 100%) or normoxia (Fio2 30%) during 2-hour filament occlusion and 1-hour reperfusion of the middle cerebral artery. At 24 hours, the hyperoxia group showed 70% (total) and 92% (cortical) reduction in infarct volumes as compared to the normoxia group. Levels of oxidative stress were evaluated using three indirect methods. First, since oxygen free radicals increase blood—brain barrier (BBB) damage, Evans blue dye extravasation was quantified to assess BBB damage. Second, the expression of heme oxygenase-1 (HO-1), a heat shock protein inducible by oxidative stress, was assessed using Western blot techniques. Third, an immunoblot technique (“OxyBlot”) was used to assess levels of protein carbonyl formation as a marker of oxidative stress—induced protein denaturation. At 24 hours, Evans blue dye extravasation per average lesion volume was similar between groups. There were no significant differences in HO-1 induction and protein carbonyl formation between groups, in the ipsilateral or contralateral hemispheres, at 6 hours and at 24 hours. These results indicate that hyperoxia treatment during focal cerebral ischemia—reperfusion is neuroprotective, and does not increase oxidative stress.

Hemoglobin-Induced Cytotoxicity in Rat Cerebral Cortical Neurons Caspase Activation and Oxidative Stress

Background and Purpose— Apoptotic-like pathways may contribute to brain cell death after intracerebral hemorrhage. In this study, we used a simplified in vitro model of hemoglobin neurotoxicity to map the caspase cascades involved and to document the role of oxidative stress. Methods— Primary neuronal cultures were obtained from rat cerebral cortex and exposed to hemoglobin to induce cell death. Cytotoxicity was assessed via measurements of mitochondrial viability (MTT assay) and lactate dehydrogenase (LDH assay). Activation of caspase-3, -8, and -9 was measured by Western blot and enzyme activity assays. Various caspase inhibitors (zVADfmk, zDEVDfmk, zIETDfmk, and zLEHDfmk) were tested for neuroprotective efficacy. The role of oxidative stress was assessed with the use of U83836E as a potent scavenger of free radicals. Results— Exposure of primary cortical neurons to hemoglobin induced a dose- and time-dependent cytotoxicity. Western blots showed upregulation of cleaved caspase-3. Enzyme assays showed an increase in caspase-9–like and caspase-3–like activity. However, caspase inhibition did not result in neuroprotection. In contrast, the free radical scavenger U83836E significantly reduced hemoglobin-induced neuronal death. Combination treatment with both U83836E and the broad spectrum caspase inhibitor zVADfmk did not yield additional protection. Conclusions— Upstream and downstream caspases were upregulated after hemoglobin-induced neurotoxicity in vitro, but only an antioxidant approach with a potent free radical scavenger significantly improved neuronal survival. These data suggest that in addition to the activation of caspase cascades, parallel pathways of oxidative stress may predominate in this model of hemoglobin neurotoxicity.

Mitogen-Activated Protein Kinase Inhibition in Traumatic Brain Injury: In Vitro and In Vivo Effects

The authors provide the first in vitro and in vivo evidence that perturbations in mitogen-activated protein kinase (MAPK) signal-transduction pathways are involved in the pathophysiology of traumatic brain injury. In primary rat cortical cultures, mechanical trauma induced a rapid and selective phosphorylation of the extracellular signal-regulated kinase (ERK) and p38 kinase, whereas there was no detectable change in the c-jun N-terminal kinase (JNK) pathway. Treatment with PD98059, which inhibits MAPK/ERK 1/2, the upstream activator of ERK, significantly increased cell survival in vitro. The p38 kinase and JNK inhibitor SB203580 had no protective effect. Similar results were obtained in vivo using a controlled cortical impact model of traumatic injury in mouse brain. Rapid and selective upregulation occurred in ERK and p38 pathways with no detectable changes in JNK. Confocal immunohistochemistry showed that phospho-ERK colocalized with the neuronal nuclei marker but not the astrocytic marker glial fibrillary acidic protein. Inhibition of the ERK pathway with PD98059 resulted in a significant reduction of cortical lesion volumes 7 days after trauma. The p38 kinase and JNK inhibitor SB203580 had no detectable beneficial effect. These data indicate that critical perturbations in MAPK pathways mediate cerebral damage after acute injury, and further suggest that ERK is a novel therapeutic target in traumatic brain injury.

Matrix metalloproteinase 2 gene knockout has no effect on acute brain injury after focal ischemia.

Matrix metalloproteinases (MMPs) may contribute to tissue damage after cerebral ischemia. In this study, wildtype and MMP-2 knockout mice were subjected to permanent and transient (2 h) occlusions of the middle cerebral artery. Gelatin zymography showed that MMP-9 levels were increased in all brains after ischemia. MMP-2 levels did not show a significant increase in wildtype mice, and were not detectable in knockout mice. Laser doppler flowmetry demonstrated equivalent ischemic reductions in perfusion in wildtype and knockout mice. In both permanent and transient occlusion paradigms, there were no statistically significant differences between wildtype and knockout mice in terms of 24 h ischemic lesion volumes. These data suggest that MMP-2 does not contribute to acute tissue damage in this model of focal ischemia.

Protective effects of statins involving both eNOS and tPA in focal cerebral ischemia

Previous studies have shown that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) protect the brain against ischemic injury by upregulating endothelial nitric oxide synthase (eNOS). Here, we tested the hypothesis that statins provide additional beneficial effects by also upregulating endogenous tissue plasminogen activator (tPA) and enhancing clot lysis in a mouse model of embolic focal ischemia. Heterologous blood clots (0.2 mm) were injected into the distal internal carotid artery to occlude blood flow in the middle cerebral artery territory after long-term (14 days) simvastatin, atorvastatin or vehicle treatment. Ischemic lesion volume, neurologic deficits, as well as residual blood clots were measured at 22 h. Reverse transcription-polymerase chain reaction assessed mRNA levels of eNOS, tPA, and the endogenous plasminogen activator inhibitor PAI-1. Ischemic lesion volumes and neurologic deficits were significantly reduced in wild-type mice by both simvastatin and atorvastatin. Statins increased eNOS and tPA mRNA levels but did not change mRNA levels of PAI-1. In eNOS knockout mice, atorvastatin reduced the volume of ischemic tissue and improved neurologic outcomes after arterial occlusion by blood clot emboli. In contrast, statins did not have protective effects in tPA knockout mice after embolic focal ischemia, but only in a filament model where focal ischemia was achieved via mechanical occlusion. These results suggest that statins protect against stroke by multiple mechanisms involving both eNOS and tPA. The involvement of each pathway may be revealed depending on the choice of experimental stroke model.

Infarct prediction and treatment assessment with MRI-based algorithms in experimental stroke models.

There is increasing interest in using algorithms combining multiple magnetic resonance imaging (MRI) modalities to predict tissue infarction in acute human stroke. We developed and tested a voxel-based generalized linear model (GLM) algorithm to predict tissue infarction in an animal stroke model in order to directly compare predicted outcome with the tissues histologic outcome, and to evaluate the potential for assessing therapeutic efficacy using these multiparametric algorithms. With acute MRI acquired after unilateral embolic stroke in rats (n = 8), a GLM was developed and used to predict infarction on a voxel-wise basis for saline (n = 6) and recombinant tissue plasminogen activator (rt-PA) treatment (n = 7) arms of a trial of delayed thrombolytic therapy in rats. Pretreatment predicted outcome compared with post-treatment histology was highly accurate in saline-treated rats (0.92±0.05). Accuracy was significantly reduced (P=0.04) in rt-PA-treated animals (0.86±0.08), although no significant difference was detected when comparing histologic lesion volumes. Animals that reperfused had significantly lower (P < 0.01) GLM-predicted infarction risk (0.73±0.03) than nonreperfused animals (0.81 ± 0.05), possibly reflecting less severe initial ischemic injury and therefore tissue likely more amenable to therapy. Our results show that acute MRI-based algorithms can predict tissue infarction with high accuracy in animals not receiving thrombolytic therapy. Furthermore, alterations in disease progression due to treatment were more sensitively monitored with our voxel-based analysis techniques than with volumetric approaches. Our study shows that predictive algorithms are promising metrics for diagnosis, prognosis and therapeutic evaluation after acute stroke that can translate readily from preclinical to clinical settings.

Antiactin-Targeted Immunoliposomes Ameliorate Tissue Plasminogen Activator-Induced Hemorrhage after Focal Embolic Stroke

Thrombolytic stroke therapy with tissue plasminogen activator (tPA) is limited by serious risks of intracerebral hemorrhage. In this study, the authors show that a novel antiactin-targeted immunoliposome significantly reduced tPA-induced hemorrhage in an established rat model of embolic focal stroke. Spontaneously hypertensive rats were subjected to focal ischemia using homologous blood clot emboli. Delayed administration of tPA (10 mg/kg, 6 hours after ischemia) induced intracerebral hemorrhage at 24 hours. In control rats treated with tPA plus vehicle, hemorrhage volumes were 9.0 ± 2.4 uL (n = 7). In rats treated with tPA plus antiactin immunoliposomes, hemorrhage volumes were significantly reduced to 4.8 ± 2.7 uL (n = 8, P < 0.05). No significant effects were seen when rats were treated with tPA plus a nontargeted liposome (7.8 ± 2.1 uL, n = 9). Fluorescent immunohistochemistry showed that rhodamine-labeled targeted liposomes colocalized with vascular structures in ischemic brain that stained positive for endothelial barrier antigen, a marker of cerebral endothelial cells. These data suggest that immunoliposomes may ameliorate vascular membrane damage and reduce hemorrhagic transformation after thrombolytic therapy in cerebral ischemia.

Protective effects of pamiteplase, a modified t-PA, in a rat model of embolic stroke

The effects of alteplase (tissue plasminogen activator, t-PA) and pamiteplase (a modified t-PA with longer half-life and increased potency) were compared in a clinically relevant model of embolic stroke. Rats were treated with pamiteplase (0.5 mg/kg or 1 mg/kg bolus), alteplase (10 mg/kg infusion) or normal saline. Pamiteplase (1 mg/kg) was as effective as alteplase in reducing 24 h brain infarct volumes, neurological deficit scores and residual clot grades. Cerebral blood flow recovery at 30 min after thrombolytic treatment was partial and did not correlate with 24 h infarct volumes or neurological deficits. However, there was good correlation between 24 h residual clot grades and infarct volumes, suggesting a delayed timeframe for pamiteplase- and alteplase-induced reperfusion.