Glial growth factor 2 treatment alleviates ischemia and reperfusion-damaged integrity of the blood-brain barrier through decreasing Mfsd2a/caveolin-1-mediated transcellular and Pdlim5/YAP/TAZ-mediated paracellular permeability.

The impairment of blood-brain barrier (BBB) integrity is the pathological basis of hemorrhage transformation and vasogenic edema following thrombolysis and endovascular therapy. There is no approved drug in the clinic to reduce BBB damage after acute ischemic stroke (AIS). Glial growth factor 2 (GGF2), a recombinant version of neuregulin-1ß that can stimulates glial cell proliferation and differentiation, has been shown to alleviate free radical release from activated microglial cells. We previously found that activated microglia and proinflammatory factors could disrupt BBB after AIS. In this study we investigated the effects of GGF2 on AIS-induced BBB damage as well as the underlying mechanisms. Mouse middle cerebral artery occlusion model was established: mice received a 90-min ischemia and 22.5 h reperfusion (I/R), and were treated with GGF2 (2.5, 12.5, 50 ng/kg, i.v.) before the reperfusion. We showed that GGF2 treatment dose-dependently decreased I/R-induced BBB damage detected by Evans blue (EB) and immunoglobulin G (IgG) leakage, and tight junction protein occludin degradation. In addition, we found that GGF2 dose-dependently reversed AIS-induced upregulation of vesicular transcytosis increase, caveolin-1 (Cav-1) as well as downregulation of major facilitator superfamily domain containing 2a (Mfsd2a). Moreover, GGF2 decreased I/R-induced upregulation of PDZ and LIM domain protein 5 (Pdlim5), an adaptor protein that played an important role in BBB damage after AIS. In addition, GGF2 significantly alleviated I/R-induced reduction of YAP and TAZ, microglial cell activation and upregulation of inflammatory factors. Together, these results demonstrate that GGF2 treatment alleviates the I/R-compromised integrity of BBB by inhibiting Mfsd2a/Cav-1-mediated transcellular permeability and Pdlim5/YAP/TAZ-mediated paracellular permeability.

Role of NADPH Oxidase-Induced Hypoxia-Induced Factor-1α Increase in Blood-Brain Barrier Disruption after 2-Hour Focal Ischemic Stroke in Rat.

We recently showed that inhibition of hypoxia-induced factor-1α (HIF-1α) decreased acute ischemic stroke-induced blood-brain barrier (BBB) damage. However, factors that induce the upregulation of HIF-1α expression remain unclear. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase played a critical role in reperfusion-induced BBB damage after stroke. However, the role of NADPH oxidase in BBB injury during the acute ischemia stage remains unclear. This study is aimed at investigating the role of NADPH oxidase in BBB injury and the expression of HIF-1α after acute ischemic stroke. A sutured middle cerebral artery occlusion (MCAO) model was used to mimic ischemic stroke in rats. Our results show that the inhibition of NADPH oxidase by apocynin can significantly reduce the BBB damage caused by 2 h ischemic stroke accompanied by reducing the degradation of tight junction protein occludin. In addition, treatment with apocynin significantly decreased the upregulation of HIF-1α induced by 2 h MCAO. More importantly, apocynin could also inhibit the MMP-2 upregulation. Of note, HIF-1α was not colocalized with a bigger blood vessel. Taken together, our results showed that inhibition of NADPH oxidase-mediated HIF-1α upregulation reduced BBB damage accompanied by downregulating MMP-2 expression and occludin degradation after 2 h ischemia stroke. These results explored the mechanism of BBB damage after acute ischemic stroke and may help reduce the associated cerebral hemorrhage transformation after thrombolysis and endovascular treatment after ischemic stroke.

Dl-NBP (Dl-3-N-Butylphthalide) Treatment Promotes Neurological Functional Recovery Accompanied by the Upregulation of White Matter Integrity and HIF-1α/VEGF/Notch/Dll4 Expression.

Dl-3-n-butylphthalide (dl-NBP) was approved by the FDA of China for the treatment of acute ischemic stroke. Dl-NBP has been shown to promote neurological functional recovery and enhance white matter integrity using an endothelin-1-induced focal permanent cerebral ischemia model, which could mimic those patients who have no opportunity to receive either tissue plasminogen activator (tPA) thrombolysis or endovascular therapy. However, it is not clear whether dl-NBP could promote neurological functional recovery in a focal transient cerebral ischemia model, which could mimic those patients who have the opportunity to receive either tPA thrombolysis or endovascular therapy. In this study, using a model of middle cerebral artery occlusion in mice, we aim to explore the effect of two-week dl-NBP treatment on neurological functional recovery after ischemic stroke as well as its underlying mechanism. Our results showed that dl-NBP treatment promoted functional recovery assessed by neurological scores and an adhesive remove test, and this improved the integrity of white matter after 60-min ischemia and 14-day reperfusion. In addition, dl-NBP increased the number of RECA-1 positive vessels and enhanced the expression of the tight junction protein occludin. More importantly, dl-NBP also promoted the expression of hypoxia-induced factor-1α, the vascular endothelial growth factor, Notch, and delta-like ligand 4. In conclusion, our study provides evidence that dl-NBP treatment could also promote functional recovery after focal transient ischemia stroke, and this recovery is associated with upregulated white matter integrity, microvessels, and the tight junction protein occludin. Our results suggested that, in future, dl-NBP may also be applied in clinic to promote functional recovery during the later phase of focal transient ischemic stroke.

Inhibition of HIF-1α Reduced Blood Brain Barrier Damage by Regulating MMP-2 and VEGF During Acute Cerebral Ischemia.

Increase of blood brain barrier (BBB) permeability after acute ischemia stroke is a predictor to intracerebral hemorrhage transformation (HT) for tissue plasminogen activator (tPA) thrombolysis and post-endovascular treatment. Previous studies showed that 2-h ischemia induced damage of BBB integrity and matrix metalloproteinase-2 (MMP-2) made major contribution to this disruption. A recent study showed that blocking ß2-adrenergic receptor (ß2-AR) alleviated ischemia-induced BBB injury by reducing hypoxia-inducible factor-1 alpha (HIF-1α) level. In this study, we sought to investigate the interaction of HIF-1α with MMP-2 and vascular endothelial growth factor (VEGF) in BBB injury after acute ischemia stroke. Rat suture middle cerebral artery occlusion (MCAO) model was used to mimic ischemia condition. Our results showed that ischemia produced BBB damage and MMP-2/9 upregulation was colocalized with Rhodamine-dextran leakage. Pretreatment with YC-1, a HIF-1α inhibitor, alleviated 2-h ischemia-induced BBB injury significantly accompanied by decrease of MMP-2 upregulation. In addition, YC-1 also prevented VEGF-induced BBB damage. Of note, VEGF was shown to be colocalized with neurons but not astrocytes. Taken together, BBB damage was reduced by inhibition of interaction of HIF-1α with MMP-2 and VEGF during acute cerebral ischemia. These findings provide mechanisms underlying BBB damage after acute ischemia stroke and may help reduce thrombolysis- and post-endovascular treatment-related cerebral hemorrhage.

The degradation of mixed lineage kinase domain-like protein promotes neuroprotection after ischemic brain injury.

Mixed lineage kinase domain-like (MLKL) protein was recently found to play a critical role in necrotic cell death. To explore its role in neurological diseases, we measured MLKL protein expression after ischemia injury in a mouse model. We found that MLKL expression significantly increased 12 h after ischemia/reperfusion (I/R) injury with peak levels at 48 h. Inhibition of MLKL by intraperitoneal administration of NSA significantly reduced infarct volume and improved neurological deficits after 75 min of ischemia and 24 h of reperfusion. Further, we found NSA reduced MLKL levels via the ubiquitination proteasome pathway, but not by inhibiting RNA transcription. Interestingly, NSA administration increased cleaved PARP-1 levels, indicating the protective effects of MLKL inhibition is not related to apoptosis. These findings suggest MLKL is a new therapeutic target for neurological pathologies like stroke. Therefore, promoting degradation of MLKL may be a novel avenue to reduce necrotic cell death after ischemic brain injury.

Combined use of spatial restraint stress and middle cerebral artery occlusion is a novel model of post-stroke depression in mice.

Post stroke depression (PSD) is one of the most common complications of ischemic stroke. At present, the underlying mechanisms are unclear, largely because there are no reliable, valid and reproducible animal models of PSD. Here we report a novel animal model of PSD that displays consistent and reliable clinical features of hemiplegic stroke. The animal model encompasses a combination of the middle cerebral artery occlusion (MCAO) and spatial restraint stress. We found that a 60-minute MCAO followed by spatial restraint stress for 2 h daily for 2 to 4 weeks from the fourth day after MCAO induced PSD-like depressive phenotypes in mice. Importantly, the mice showed exacerbated deficits of neurological functions and decreased body weights, which were accompanied with reduced levels of brain derived neurotrophic factor and neurotransmitters including serotonin and dopamine. In addition, we identified increased levels of serum cortisol in our PSD mice. Finally, we found that mice with PSD were responsive to the tri-cyclic antidepressant imipramine as evidenced by their attenuated depressive behaviors, increased body weights, recovered brain serotonin levels, and decreased serum cortisol levels. This mouse model replicates multiple features of human post-stroke depression and thus provides a new model for the investigation of PSD.

Cystatin C is an Independent Risk Factor and Therapeutic Target for Acute Ischemic Stroke.

Cystatin C (CysC) has been used as a renal function indicator and a strong predictor of cardiovascular events. In this study, we determined the prognostic value of serum CysC in patients with acute ischemic stroke (AIS) and examined its protective role on ischemic brain injury. First-ever stroke patients (601 cases) and control subjects (325 cases) were recruited. Serum CysC level in patients with AIS were significantly higher than that in controls. Multivariate logistic regression analyses showed that elevated CysC is an independent risk factor of AIS; the odds ratio (95 % confidence interval) was 9.80 (3.12-30.83). The integrated population was divided into quintiles according to serum CysC. Compared with the first quintile, the odds ratios of risk for AIS in fourth quintile and fifth quintile were 1.92 (1.08-3.40) and 2.88 (1.49-5.54), respectively. Serum CysC was not associated with neurological deficits and the location of ischemic area; however, serum CysC in patients decreased after one-week therapy. This was consistent with CysC expression after ischemia/reperfusion injury in a mouse focal ischemia/reperfusion injury model. Exogenous CysC exerted neuroprotective effects by reducing infarct volume in this animal stroke model. Therefore, serum CysC is highly associated with AIS and is an independent prediction marker for AIS. Since our findings demonstrated the protection of exogenous CysC on ischemic brain injury, CysC is a novel and promising therapeutic target for AIS.