Blood brain barrier (BBB) disruption occurs early enough to be within the thrombolytic time window, and this early ischemic BBB damage is closely associated with hemorrhagic transformation and thus emerging as a promising target for reducing the hemorrhagic complications of thrombolytic stroke therapy. However, the mechanisms underlying early ischemic BBB damage remain poorly understood. Here we investigated the early molecular events of ischemic BBB damage using in vitro oxygen-glucose deprivation (OGD) and in vivo rat middle cerebral artery occlusion (MCAO) models. Exposure of bEND3 monolayer to OGD for 2 h significantly increased its permeability to FITC-labeled dextran, and promoted the secretion of metalloproteinase-2 and 9 (MMP-2/9) and cytosolic translocation of caveolin-1 (Cav-1). This same OGD treatment also led to rapid degradation of tight junction protein occludin and dissociation of claudin-5 from the cytoskeleton, which contributed to OGD-induced endothelial barrier disruption. Using selective MMP-2/9 inhibitor SB-3CT or their neutralizing antibodies or Cav-1 siRNA, we found that MMP-2 was the major enzyme mediating OGD-induced occludin degradation, while Cav-1 was responsible for claudin-5 redistribution. The interaction between Cav-1 and claudin-5 was further confirmed by coimmunoprecipitation. Consistent with these in vitro findings, we observed fluorescence tracer extravasation, increased gelatinolytic activity and elevated interstitial MMP-2 levels in ischemic subcortical tissue after 2-h MCAO. Moreover, occludin protein loss and claudin-5 redistribution were detected in ischemic cerebromicrovessels. These data indicate that cerebral ischemia initiates two rapid parallel processes, MMP-2-mediated occludin degradation and Cav-1-mediated claudin-5 redistribution, to cause BBB disruption at early stroke stages relevant to acute thrombolysis.

Using in vitro oxygen-glucose deprivation (OGD) model, we have previously demonstrated that 2-h OGD induces rapid, caveolin-1-mediated dissociation of claudin-5 from the cellular cytoskeletal framework and quick endothelial barrier disruption. In this study, we further investigated the fate of translocated claudin-5 and the mechanisms by which OGD promotes caveolin-1 translocation. Exposure of bEND3 cells to 4-h OGD, but not 2-h OGD plus 2-h reoxygenation, resulted in claudin-5 degradation. Inhibition of autophagy or the fusion of autophagosome with lysosome, but not proteasome, blocked OGD-induced claudin-5 degradation. Moreover, knockdown of caveolin-1 with siRNA blocked OGD-induced claudin-5 degradation. Western blot analysis showed a transient colocalization of caveolin-1, claudin-5, and LC3B in autolysosome or lipid raft fractions at 2-h OGD. Of note, inhibiting autophagosome and lysosome fusion sustained the colocalization of caveolin-1, claudin-5, and LC3B throughout the 4-h OGD exposure. EPR spin trapping showed increased nitric oxide (NO) generation in 2-h OGD-treated cells, and inhibiting NO with its scavenger C-PTIO or inducible nitric oxide synthase (iNOS) inhibitor 1400W prevented OGD-induced caveolin-1 translocation and claudin-5 degradation. Taken together, our data provide a novel mechanism underlying endothelial barrier disruption under prolonged ischemic conditions, in which NO promotes caveolin-1-mediated delivery of claudin-5 to the autophagosome for autophagy-lysosome-dependent degradation.

Small vessel disease is associated with white-matter (WM) magnetic resonance imaging (MRI) hyperintensities (WMHs) in patients with vascular cognitive impairment (VCI) and subsequent damage to the WM. Although WM is vulnerable to hypoxic-ischemic injury and O 2 is critical in brain physiology, tissue O 2 level in the WM has not been measured and explored in vivo. We hypothesized that spontaneously hypertensive stroke-prone rat (SHR/SP) fed a Japanese permissive diet (JPD) and subjected to unilateral carotid artery occlusion (UCAO), a model to study VCI, would lead to reduced tissue oxygen (pO 2 ) in the deep WM. We tested this hypothesis by monitoring WM tissue pO 2 using in vivo electron paramagnetic resonance (EPR) oximetry in SHR/SP rats over weeks before and after JPD/UCAO. The SHR/SP rats experienced an increase in WM pO 2 from 9 to 12 weeks with a maximal 32% increase at week 12, followed by a dramatic decrease in WM pO 2 to near hypoxic conditions during weeks 13 to 16 after JPD/UCAO. The decreased WM pO 2 was accompanied with WM damage and hemorrhages surrounding microvessels. Our findings suggest that changes in WM pO 2 may contribute to WM damage in SHR/SP rat model, and that EPR oximetry can monitor brain pO 2 in the WM of small animals. Keywords: brain oxygen; EPR oximetry; vascular cognitive impairment; white matter INTRODUCTION Brain cells poorly tolerate hypoxia, and even short periods of low oxygen (O 2 ) can initiate molecular pathways that are lethal to cells in both the gray and white matter (WM). Deep WM is a common site of hypoxic/ischemic injury in the elderly where it is associated with cognitive decline, gait disturbances, and focal ischemia leading to vascular cognitive impairment (VCI). Magnetic resonance imaging (MRI) reveals white-matter hyperintensities (WMHs) in the elderly located in the periventricular and subcortical regions, and considered to be due to strokes and secondary small vessel disease. An alternative mechanism is that the deep WM is vulnerable to hypoxia for a variety of reasons. Studies in humans suggest that there is impairment in the ability of the deep WM to respond to increased demand by raising cerebral blood flow or O 2 . 1 Although glutamate mediated excitotoxicity, inflammatory cytokines, and protease activation result in oligodendrocyte death in acute ischemic injury, the pathophysiology of WM injury in chronic vascular disease has not been fully explored. 2 Small vessel disease is associated with WMHs in patients with VCI; damage to small vessels causes an inflammatory response with disruption in the blood-brain barrier (BBB) associated with expression of matrix metalloproteinases (MMPs) and subsequent damage to the WM. Vascular dementia patients show expression of MMPs in regions of loss of myelin. [3][4][5]

No abstract

Background: Cysteine oxidation of zinc finger proteins plays an important role in protein function. Results: Arsenic binding selectively sensitizes C3H1/C4 zinc finger proteins to oxidation by ROS. Conclusion: Selectivity in arsenic binding to zinc finger motifs determines target proteins for oxidation by ROS. Significance: This work provides an example of how an environmental insult may alter protein oxidation profiles and redox signaling.

Normobaric hyperoxia (NBO), which maintains penumbral oxygenation, reduces brain injury during cerebral ischemia, and minocycline, a tetracycline derivative, reduces reperfusion injury, including inflammation, apoptosis and matrix metalloproteinases (MMPs) activation. Since they have different mechanisms of action, we hypothesized that combining them would provide greater neuroprotection. To test the hypothesis, we evaluated the neuroprotective effects of the combination of NBO with minocycline. Male Sprague Dawley rats were exposed to NBO (95% O2) or normoxia (21% O2) during 90-min filament occlusion of the middle cerebral artery, followed by 48 hrs of reperfusion. Minocycline (3 mg/kg) or vehicle was intravenously administered to rats 15 min after reperfusion onset. Treatment with NBO and minocycline alone resulted in 36% and 30% reductions in infarction volume, respectively. When the two treatments were combined, there was a 68% reduction in infarction volume. The combination therapy also significantly reduced hemispheric swelling, which was absent with monotherapy. In agreement with its greater neuro-and vasoprotection, the combination therapy showed greater inhibitory effects on MMP-2/9 induction, occludin degradation, caspase-3 and -9 activation and apoptosis inducing factor (AIF) induction in ischemic brain tissue. Our results show that NBO plus minocycline effectively reduces brain injury in transient focal cerebral ischemia with protection due to inhibition on MMP-2/9-mediated occludin degradation and attenuation of caspase-dependent and independent apoptotic pathways.

No abstract

The mechanism of early blood–brain barrier (BBB) disruption after stroke has been intensively studied but still not fully understood. Here, we report that microRNA-30a (miR-30a) could mediate BBB damage using both cellular and animal models of ischemic stroke. In the experiments in vitro, inhibition of miR-30a decreased BBB permeability, prevented the degradation of tight junction proteins, and reduced intracellular free zinc in endothelial cells. We found that the zinc transporter ZnT4 was a direct target of negative regulation by miR-30a, and ZnT4/zinc signaling pathway contributed significantly to miR-30a-mediated BBB damage. Consistent with these in vitro findings, treatment with miR-30a inhibitor reduced zinc accumulation, increased the expression of ZnT4, and prevented the loss of tight junction proteins in microvessels of ischemic animals. Furthermore, inhibition of miR-30a, even at 90 min post onset of middle cerebral artery occlusion, prevented BBB damage, reduced infarct volume, and ameliorated neurological deficits. Together, our findings provide novel insights into the mechanisms of cerebral ischemia-induced BBB disruption and indicate miR-30a as a regulator of BBB function that can be an effective therapeutic target for ischemic stroke.