Matrix metalloproteinases (MMPs) disrupt the blood-brain barrier (BBB) during reperfusion. Occludin and claudins are recently described tight junction proteins (TJPs) that form the BBB. We hypothesized that the opening of the BBB was because of the degradation of TJPs by the MMPs. Spontaneously hypertensive rats had a 90 mins middle cerebral artery occlusion with reperfusion for 2, 3, or 24 h. Matrix metalloproteinases were measured by immunohistochemistry and in situ and gel zymography. Real-time polymerase chain reaction (PCR) measured mRNAs of MMP-2 and -9, furin, membrane-type MMP (MT1-MMP), occludin, and claudin-5. There was opening of the BBB in the piriform cortex after 3 h of reperfusion, and an MMP inhibitor, BB-1101 (30 mg/kg), prevented the opening. At 3 h, in situ zymograms showed gelatinase activity. Zymography and PCR showed greater increases in MMP-2 than in MMP-9. There were increased mRNA and immunohistochemistry for MT1-MMP and furin, which activate MMP-2. Claudin-5 and occludin mRNA expression decreased at 2 h in both hemispheres with fragments of both proteins seen on Western blot by 3 h on the ischemic side; treatment with BB-1101 reversed the degradation of the TJPs. Immunohistochemistry at 3 h showed fragmented TJPs within the endothelial cell clefts. By 24 h, in situ zymography showed gelatinase activity and gel zymography showed elevated levels of MMP-9. Disrupted TJPs previously seen in endothelial cells appeared in the surrounding astrocytes. Our results provide direct evidence that MMPs open the BBB by degrading TJPs and that an MMP inhibitor prevents degradation of the TJPs by MMPs.

BackgroundMinocycline reduces reperfusion injury by inhibiting matrix metalloproteinases (MMPs) and microglia activity after cerebral ischemia. Prior studies of minocycline investigated short-term neuroprotective effects during subacute stage of stroke; however, the late effects of minocycline against early reperfusion injury on neurovascular remodeling are less well studied. We have shown that spontaneous angiogenesis vessels in ischemic brain regions have high blood–brain barrier (BBB) permeability due to lack of major tight junction proteins (TJPs) in endothelial cells at three weeks. In the present study, we longitudinally investigated neurological outcome, neurovascular remodeling and microglia/macrophage alternative activation after spontaneous and minocycline-induced stroke recovery.MethodsAdult spontaneously hypertensive rats had a 90 minute transient middle cerebral artery occlusion. At the onset of reperfusion they received a single dose of minocycline (3 mg/kg intravenously) or a vehicle. They were studied at multiple time points up to four weeks with magnetic resonance imaging (MRI), immunohistochemistry and biochemistry.ResultsMinocycline significantly reduced the infarct size and prevented tissue loss in the ischemic hemispheres compared to vehicle-treated rats from two to four weeks as measured with MRI. Cerebral blood flow measured with arterial spin labeling (ASL) showed that minocycline improved perfusion. Dynamic contrast-enhanced MRI indicated that minocycline reduced BBB permeability accompanied with higher levels of TJPs measured with Western blot. Increased MMP-2 and −3 were detected at four weeks. Active microglia/macrophage, surrounding and within the peri-infarct areas, expressed YM1, a marker of M2 microglia/macrophage activation, at four weeks. These microglia/macrophage expressed both pro-inflammatory factors tumor necrosis factors-α (TNF-α) and interleukin-1β (IL-1β) and anti-inflammatory factors transforming growth factor-β (TGF-β) and interleukin-10 (IL-10). Treatment with minocycline significantly reduced levels of TNF-α and IL-1β, and increased levels of TGF-β, IL-10 and YM1.ConclusionsEarly minocycline treatment against reperfusion injury significantly promotes neurovascular remodeling during stroke recovery by reducing brain tissue loss, enhancing TJP expression in ischemic brains and facilitating neuroprotective phenotype alternative activation of microglia/macrophages.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-015-0245-4) contains supplementary material, which is available to authorized users.

Background and purpose Small vessel disease is the major cause of white matter injury in patients with vascular cognitive impairment. Matrix metalloproteinase (MMP)-mediated inflammation may be involved in the white matter damage with oligodendrocyte (Ol) death. Therefore, we used spontaneously hypertensive stroke-prone rats (SHR-SP) to study the role of neuroinflammation in white matter damage. Methods Permanent unilateral carotid artery occlusion (UCAO) was performed at 12-weeks of age in SHR-SP. Following surgery, rats were placed on a Japanese permissive diet (JPD) and received 1 % NaCl in drinking water. MRI, histology, biochemistry, and ELISA characterized white matter lesions and cognitive impairment was tested by Morris water maze (MWM). Results white matter damage was observed 4-5 weeks following UCAO/JPD. Immunoblotting showed marked reduction in myelin basic protein (MBP) and up regulation of immature Ols. Mature Ols underwent caspase-3-mediated apoptosis. MWM showed cognitive impairment. Abnormally appearing vessels were observed and surrounded by inflammatory-like cells. IgG extravasation and hemorrhage, indicating blood-brain barrier (BBB) disruption, was closely associated with MMP-9 expression. Lesions in white matter showed reactive astrocytosis and activated microglia that expressed tumor necrosis factor-α (TNF-α). MMP-3 and MMP-9 were significantly increased and MMP-2 reduced in astrocytes and Ol. Conclusion We found apoptosis of mature Ols with an increase in immature Ols. Increased MMP-3, MMP-9 and TNF-α were associated with myelin breakdown and BBB disruption. Neuroinflammation is an important factor in white matter damage and Ol death, and studies using this new model can be done to assess agents to block inflammation.

In cerebral ischemia, matrix metalloproteinases (MMPs) have a dual role by acutely disrupting tight junction proteins (TJPs) in the blood-brain barrier (BBB) and chronically promoting angiogenesis. Since TJP remodeling of the neurovascular unit (NVU) is important in recovery and early inhibition of MMPs is neuroprotective, we hypothesized that short-term MMP inhibition would reduce infarct size and promote angiogenesis after ischemia. Adult spontaneously hypertensive rats had a transient middle cerebral artery occlusion with reperfusion. At the onset of ischemia, they received a single dose of the MMP inhibitor, GM6001. They were studied at multiple times up to 4 weeks with immunohistochemistry, biochemistry, and magnetic resonance imaging (MRI). We observed newly formed vessels in peri-infarct regions at 3 weeks after reperfusion. Dynamic contrast-enhanced MRI showed BBB opening in new vessels. Along with the new vessels, pericytes expressed zonula occludens-1 (ZO-1) and MMP-3, astrocytes expressed ZO-1, occludin, and MMP-2, while endothelial cells expressed claudin-5. The GM6001, which reduced tissue loss at 3 to 4 weeks, significantly increased new vessel formation with expression of TJPs and MMPs. Our results show that pericytes and astrocytes act spatiotemporally, contributing to extraendothelial TJP formation, and that MMPs are involved in BBB restoration during recovery. Early MMP inhibition benefits neurovascular remodeling after stroke.

Degradation of the extracellular matrix by elevated matrix metalloproteinase (MMP) activity following ischemia/reperfusion is implicated in blood–brain barrier disruption and neuronal death. In contrast to their characterized extracellular roles, we previously reported that elevated intranuclear MMP-2 and -9 (gelatinase) activity degrades nuclear DNA repair proteins and promotes accumulation of oxidative DNA damage in neurons in rat brain at 3-h reperfusion after ischemic stroke. Here, we report that treatment with a broad-spectrum MMP inhibitor significantly reduced neuronal apoptosis in rat ischemic hemispheres at 48-h reperfusion after a 90-min middle cerebral artery occlusion (MCAO). Since extracellular gelatinases in brain tissue are known to be neurotoxic during acute stroke, the contribution of intranuclear MMP-2 and -9 activities in neurons to neuronal apoptosis has been unclear. To confirm and extend our in vivo observations, oxygen–glucose deprivation (OGD), an in vitro model of ischemia/reperfusion, was employed. Primary cortical neurons were subjected to 2-h OGD with reoxygenation. Increased intranuclear gelatinase activity was detected immediately after reoxygenation onset and was maximal at 24 h, while extracellular gelatinase levels remained unchanged. We detected elevated levels of both MMP-2 and -9 in neuronal nuclear extracts and gelatinase activity in neurons co-localized primarily with MMP-2. We found a marked decrease in PARP1, XRCC1, and OGG1, and decreased PARP1 activity. Pretreatment of neurons with selective MMP-2/9 inhibitor II significantly decreased gelatinase activity and downregulation of DNA repair enzymes, decreased accumulation of oxidative DNA damage, and promoted neuronal survival after OGD. Our results confirm the nuclear localization of gelatinases and their nuclear substrates observed in an animal stroke model, further supporting a novel role for intranuclear gelatinase activity in an intrinsic apoptotic pathway in neurons during acute stroke injury.