Takeo Abumiya

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-µm 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.

Rapid Loss of Microvascular Integrin Expression During Focal Brain Ischemia Reflects Neuron Injury

The integrity of cerebral microvessels requires the close apposition of the endothelium to the astrocyte endfeet. Integrins α1β1 and α6β4 are cellular matrix receptors that may contribute to cerebral microvascular integrity. It has been hypothesized that focal ischemia alters integrin expression in a characteristic time-dependent manner consistent with neuron injury. The effects of middle cerebral artery occlusion (MCAO) and various periods of reperfusion on microvasclar integrin α1β1 and α6β4 expression were examined in the basal ganglia of 17 primates. Integrin subunits α1 and β1 colocalized with the endothelial cell antigen CD31 in nonischemic microvessels and with glial fibrillary acidic protein on astrocyte fibers. Rapid, simultaneous, and significant disappearance of both integrin α1 and β1 subunits and integrin α6β4 occurred by 2 hours MCAO, which was greatest in the region of neuron injury (ischemic core, Ic), and progressively less in the peripheral (Ip) and nonischemic regions (N). Transcription of subunit β1 mRNA on microvessels increased significantly in the Ic/Ip border and in multiple circular subregions within Ic. Microvascular integrin α1β1 and integrin α6β4 expression are rapidly and coordinately lost in Ic after MCAO. With loss of integrin α1β1, multiple regions of microvascular β1 mRNA up-regulation within Ic suggest that microvessel responses to focal ischemia are dynamic, and that multiple cores, not a single core, are generated. These changes imply that microvascular integrity is modified in a heterogeneous, but ordered pattern.

Integrin αIIbβ3 Inhibitor Preserves Microvascular Patency in Experimental Acute Focal Cerebral Ischemia

Background and Purpose—Platelets become activated and accumulate in brain microvessels of the ischemic microvascular bed after experimental focal cerebral ischemia. The binding of glycoprotein IIb/IIIa (integrin αIIbβ3) on platelets to fibrinogen is the terminal step in platelet adhesion and aggregation. This study tests the hypothesis that inhibition of platelet-fibrin(ogen) interactions may prevent microvascular occlusion after experimental middle cerebral artery occlusion (MCA:O). Methods—TP9201 is a novel Arg-Gly-Asp (RGD)-containing integrin αIIbβ3 inhibitor. Microvascular patency after 3-hour MCA:O and 1-hour reperfusion within the ischemic and nonischemic basal ganglia was compared in adolescent male baboons who received high-dose TP9201 (group A: IC80 in heparin, n=4), low-dose TP9201 (group B: IC30 in heparin, n=4), or no treatment (group C: n=4) before MCA:O. Results—After MCA:O, microvascular patency decreased significantly in group C. However, in the ischemic zones of groups A and B compared w...

Activated Microvessels Express Vascular Endothelial Growth Factor and Integrin αvβ3 During Focal Cerebral Ischemia

Both vascular endothelial growth factor (VEGF) and integrin αvβ3 play roles in angiogenesis. In noncerebral vascular systems, VEGF can induce endothelial integrin αvβ3 expression. However, it is unknown whether VEGF, like integrin αvβ3, appears in the initial response of microvessels to focal brain ischemia. Their coordinate expression in microvessels of the basal ganglia after middle cerebral artery occlusion (MCAO) in the nonhuman primate model was examined quantitatively. Cells incorporating deoxyuridine triphosphate (dUTP+) by the polymerase I reaction at 1 hour (n = 3), 2 hours (n = 3), and 7 days (n = 4) after MCAO defined the ischemic core (Ic) and peripheral regions. Both VEGF and integrin αvβ3 were expressed by activated noncapillary (7.5- to 30.0-μm diameter) microvessels in the Ic region at 1 and 2 hours after MCAO. At 7 days after MCAO, the number of VEGF+, integrin αvβ3+, or proliferating cell nuclear antigen-positive microvessels had decreased within the Ic region. The expressions of VEGF, integrin αvβ3, and proliferating cell nuclear antigen were highly correlated on the same microvessels using hierarchical log-linear statistical models. Also, VEGF and subunit αv messenger ribonucleic acids were coexpressed on selected microvessels. Here, noncapillary microvessels are activated specifically early during a focal cerebral ischemic insult and rapidly express VEGF and integrin αvβ3 together.

Activation systems for latent matrix metalloproteinase-2 are upregulated immediately after focal cerebral ischemia

During focal cerebral ischemia, matrix metalloproteinase-2 (MMP-2) can contribute to the loss of microvessel integrity within ischemic regions by degrading the basal lamina. MMP-2 is secreted in latent form (pro-MMP-2), but the activation of pro-MMP-2 in the ischemic territory has not been shown. Immunohistochemical and in situ hybridization studies of the expression of the direct activators of MMP-2, MT1-MMP and MT3-MMP, and the indirect activation system tissue plasminogen activator, urokinase (u-PA), its receptor (u-PAR), and its inhibitor PAI-1 after middle cerebral artery occlusion/reperfusion were undertaken in basal ganglia samples from 26 adolescent male baboons. The expressions of all three MMPs, u-PA, u-PAR, and PA1-1, but not tissue plasminogen activator, were increased from 1 hour after middle cerebral artery occlusion in the ischemic core. mRNA transcripts confirmed the increases in latent MMP-2, u-PA, u-PAR, and PAI-1 antigen very early after middle cerebral artery occlusion. The expression patterns are consistent with secretion of pro-MMP-2 and its activators in the ischemic core, perhaps from separate cell compartments. The rapid and coordinate appearance of pro-MMP-2 and its activation apparatus suggest that in the primate striatum this protease may participate in matrix injury during focal cerebral ischemia.

Aggravation of hemorrhagic transformation by early intraarterial infusion of low-dose vascular endothelial growth factor after transient focal cerebral ischemia in rats

Vascular endothelial growth factor (VEGF) is a unique growth factor associated with angiogenesis, vascular permeability, and neuroprotection. The aim of this study was to observe the effects of early intraarterial infusion of low-dose VEGF on ischemia/reperfusion injury after transient focal cerebral ischemia in rats. Male Sprague-Dawley rats were subjected to 2 h of focal ischemia by middle cerebral artery occlusion. After the 2 h ischemia, the rats were infused with 0.3 microg/kg of VEGF (n = 15), or the vehicle as a control (n = 15), via the reperfused internal carotid artery. The brains were collected after a 1 h, 6 h, or 72 h reperfused period. Severity of ischemic cellular injury, serum extravasation, hemorrhagic transformation, and matrix metalloproteinase (MMP)-2 and -9 expressions were compared between the VEGF-treated and control groups. No significant difference in the extent of ischemic cellular injury and serum extravasation was observed between the two groups. However, vessel numbers with hemorrhagic transformation were significantly greater in the VEGF-treated group than in the control group after the 72 h reperfusion (9.4 +/- 1.6 versus 2.6 +/- 1.5; P = 0.028). The severity of hemorrhagic transformation was not correlated with the extent of ischemic cellular injury or serum extravasation. MMP-2 and -9 expressions were not enhanced in the VEGF-treated group compared with the control group. These results suggest that exogenous VEGF administered intravascularly at a very early point in reperfusion aggravates hemorrhagic transformation. The aggravated hemorrhagic transformation does not seem to depend on the enlargement of ischemic cellular injury, serum extravasation, or overexpressions of MMP-2 and -9.

Unique Profile of Spreading Depression in a Primate Model

Spreading depression (SD) is considered to play a role in pathologic conditions of humans such as in the evolution of ischemic brain injury and migraine aura. Because many studies have demonstrated spreading hypoperfusion in patients with migraine and persistent hypoperfusion in nonprimate animal models of SD, these changes in cerebral blood flow (CBF) were regarded as an epiphenomenon of SD. However, there is no direct evidence of the occurrence of SD in primates. The authors attempted to elicit SD by applying 3.3 mol/L potassium chloride to the cerebral cortex of nine male cynomolgus monkeys. The CBF was monitored by positron emission tomography in five animals. Propagated direct-current shifts were found by the two neighboring microelectrodes only in one animal. The direct-current wave propagated at a speed of 4 mm/min and its amplitude was 20 mV, being consistent with the SD findings. Except in one animal with 6 SD episodes, SD waves were recorded infrequently at the rostral site (none in three animals, once in three, and twice in two). Focal hyperemia accompanied SD. Neither spreading hypoperfusion nor persistent hypoperfusion was found. These unique features of SD in primates raise a doubt as to whether the role of SD in nonprimate animals is the same as that in stroke and migraine in humans.

Cyclooxygenase-2 Expression Associated With Spreading Depression in a Primate Model

The authors previously provided evidence that spreading depression (SD) can be evoked in primates. Cyclooxygenase-2 (COX-2) expression has been found to increase in the rodent cortex undergoing SD, and the authors sought to determine whether this association exists in primate brain. In the present study, neuronal COX-2 expression was induced during SD in the primate cortex. The mean expression ratio of COX-2 messenger RNA in animals with SD was significantly higher than that measured in controls (1.69 vs. 0.5; P = 0.02). Induction of COX-2 in these animals was also detected by human microarray analysis. Results show that, as in rodents, neuronal COX-2 is induced in the primate cortex in response to SD.

Brain ischemia and spreading depression in a primate model

In order to investigate the pathophysiology of ischemic stroke, we developed a primate thromboembolic stroke model and examined serial changes in cerebral blood flow (CBF) and cerebral metabolic rate of glucose (CMRglc) using positron emission tomography (PET). In addition, we attempted to elicit spreading depression (SD), which is essential in the evolution of infarcts in small animal models, by chemical stimulation in primates.

Microvascular Integrin-Matrix Responses to Focal Cerebral Ischemia Are Rapid and Reflect Neuron Injury

In the ischemic basal ganglia, early changes in the microvascular basal lamina, cellular integrin expression, and matrix metalloproteinase (MMP) release occur in association with severe neuron injury. These changes are temporally and spatially related. Studies indicate that focal ischemia reduces microvascular expression of endothelial cell integrin α 1 β 1 and astrocyte integrin α 6 β 4 in a manner related to the severity of the neuron injury, and that MMP-2 upregulation is directly related to neuron injury. Further investigations are required to support the proposition that integrin-matrix responses by endothelial cells and astrocytes to focal cerebral ischemia, which together decrease microvascular integrity, also affect neuron survival.