Image_2_ErbB4 Preserves Blood-Brain Barrier Integrity via the YAP/PIK3CB Pathway After Subarachnoid Hemorrhage in Rats.TIF

<p>Studies have suggested that blood-brain barrier (BBB) disruption contributes to the pathogenesis of early brain injury after subarachnoid haemorrhage (SAH). Activation of the receptor tyrosine kinase ErbB4 can cause intramembrane proteolysis and release a soluble intracellular domain (ICD) that modulates transcription in the nucleus. This study was carried out to investigate the potential roles of ErbB4 in preserving BBB integrity after experimental SAH, as well as the underlying mechanisms of its protective effects. Endovascular perforation was used to prepare a rat SAH model. The SAH grade, neurological score, brain edema and BBB permeability were evaluated after surgery. Immunohistochemistry was used to determine the localization of ErbB4 and yes-associated protein (YAP). ErbB4 activator Nrg1 isoform β1 (Nrg1β1), Specific ErbB4 siRNA, YAP siRNA and PIK3CB specific inhibitor TGX 221 were used to manipulate the proposed pathway. The expression levels of ErbB4 ICD and YAP were markly increased after SAH. Double immunohistochemistry labeling showed that ErbB4 and YAP were expressed in endothelial cells and neurons. Activation of ErbB4 by Nrg1β1 (dosage 150 ng/kg) treatment promoted the neurobehavioral deficit, alleviated the brain water content and reduced albumin leakage 24 and 72 h after SAH. ErbB4 activation significantly promoted YAP and PIK3CB activity and increased the expression of tight junction proteins Occludin and Claudin-5. Depletion of ErbB4 aggravated neurological impairment and BBB disruption after SAH. The beneficial effects of ErbB4 activation were abolished by YAP small-interfering RNA and specific PIK3CB inhibitor. Activation of ErbB4 improved neurological performance after SAH through the YAP/PIK3CB signaling pathway, this neuroprotective effects may associated with BBB maintenance.</p

HIF-1 mediates hypoxia-induced inflammation.

<p>(A and B) Microglia were treated with 3 µM 2ME2, 1 µM YC-1, or the indicated combinations for 6 hours. The protein levels of IL-8 and TNF-α were determined. (C) Detection of IL-8 and TNF-α by ELISA in microglia transfected with siRNA Con or siRNA HIF-1α (24 h). Experiments performed in triplicate showed consistent results. Data are presented as the mean±SD of three independent experiments. * <i>P</i><0.05.</p

Hypoxia induced cell death and inflammation of microglia cells.

<p>(A) To assess cell death in vitro, microglia with the treatment for the indicated time were subjected to PI staining, and analyzed by flow cytometry. The percentage of cells with PI-positive relative to total cell number at each treatment is shown. (B) The effect of hypoxia on the viability of microglia cells. Microglia was treated with the indicated time. Cell viability was assessed using MTT. (C, D, E and F) microglia were treated with the indicated time. The mRNA and protein levels of IL-8 and TNF-α were determined. Experiments performed in triplicate showed consistent results. Data are presented as the mean ±SD of three independent experiments. * <i>P</i><0.05.</p

HIF-1α mediates microglia autophagy induced by hypoxia.

<p>(A) Microglia cells were transfected with siRNAs targeting HIF-1α (100 nM each) for 24 hrs following hypoxia treatment for 16 hrs, and then the protein levels of the target were evaluated by western blot. (B and C) Confocal microscopy and electron microscopy detected autophagosomes. (D) Microglia cells were transfected with siRNA HIF-1α, after treated with hypoxia for 16 hrs. Flow cytometry detected acridine orange positive cell. The asterisks denote significant differences from controls (* <i>P</i><0.05). Experiments performed in triplicate showed consistent results.</p

Intracranial pressure and histology.

<p>Relative ventricle area (ventricle area / brain area) (A) in SAH animals with normal ICP (≤ 10 mmHg) or elevated ICP (> 10 mmHg). Representative micrographs of coronal Nissl stained brain sections in a SAH animal with normal ICP (B) and a SAH animal with ICP elevation (C). Mean is shown. ** p<0.01.</p

Time course of intracranial pressure.

<p>Intracranial pressure (ICP) in mmHg at three different time points in SHAM or SAH animals. Repeated-measures two-way ANOVA showed a significantly different time course of ICP in SHAM or SAH animals (p<0.05). * p<0.05 for the post-hoc comparison of SHAM vs. SAH at day 21 (Bonferroni-corrected).</p

Image_1_ErbB4 Preserves Blood-Brain Barrier Integrity via the YAP/PIK3CB Pathway After Subarachnoid Hemorrhage in Rats.TIF

<p>Studies have suggested that blood-brain barrier (BBB) disruption contributes to the pathogenesis of early brain injury after subarachnoid haemorrhage (SAH). Activation of the receptor tyrosine kinase ErbB4 can cause intramembrane proteolysis and release a soluble intracellular domain (ICD) that modulates transcription in the nucleus. This study was carried out to investigate the potential roles of ErbB4 in preserving BBB integrity after experimental SAH, as well as the underlying mechanisms of its protective effects. Endovascular perforation was used to prepare a rat SAH model. The SAH grade, neurological score, brain edema and BBB permeability were evaluated after surgery. Immunohistochemistry was used to determine the localization of ErbB4 and yes-associated protein (YAP). ErbB4 activator Nrg1 isoform β1 (Nrg1β1), Specific ErbB4 siRNA, YAP siRNA and PIK3CB specific inhibitor TGX 221 were used to manipulate the proposed pathway. The expression levels of ErbB4 ICD and YAP were markly increased after SAH. Double immunohistochemistry labeling showed that ErbB4 and YAP were expressed in endothelial cells and neurons. Activation of ErbB4 by Nrg1β1 (dosage 150 ng/kg) treatment promoted the neurobehavioral deficit, alleviated the brain water content and reduced albumin leakage 24 and 72 h after SAH. ErbB4 activation significantly promoted YAP and PIK3CB activity and increased the expression of tight junction proteins Occludin and Claudin-5. Depletion of ErbB4 aggravated neurological impairment and BBB disruption after SAH. The beneficial effects of ErbB4 activation were abolished by YAP small-interfering RNA and specific PIK3CB inhibitor. Activation of ErbB4 improved neurological performance after SAH through the YAP/PIK3CB signaling pathway, this neuroprotective effects may associated with BBB maintenance.</p

HIF-1 mediates hypoxia-induced cell death.

<p>(A) Microglia cells were treated with hypoxia for 0, 2, 4, 16, 24 and 48 h. The kinetics of HIF-1α induction was assayed by qRT-PCR. (B) Measurement of HIF-1α in microglia with the indicated treatments by western blot assay. (C and D) microglia were treated with 3 µM 2ME2, 1 µM YC-1, or the indicated combinations for 6 hours, and the cells were cultured with hypoxia for 16 hrs. The percentage of dead cells was determined using the MTT assay or cell death assay. (E and F) Detection of the inhibition efficiency of siRNAs against HIF-1α. Microglia cells were transfected with siRNAs targeting HIF-1α (100 nM each) for 24 h, and the protein and RNA levels of the target was evaluated by western blot and q-PCR assays. (G and H) The effect of cell death on hypoxia transfected with siRNA control and HIF-1α in microglia (24 h). The % PI positive cells have been normalized to 100% in the control. Experiments performed in triplicate showed consistent results. Data are presented as the mean ± SD of three independent experiments. * <i>P</i><0.05.</p

Autophagy is induced in microglia after hypoxia treatment.

<p>(A) Hypoxia induced complete autophagic flux in microglia. The cells were treated with hypoxia for the indicated time in the presence of Baf A1 (10 nM). Control represents the normal oxygen conditions. (B) Microglia cells were transfected with a plasmid expressing GFP-MAP1LC3B. After 24 hrs, the cells were exposed to hypoxia for 6 hrs. Cells were visualised by confocal microscopy immediately after fixation. The number of GFP-MAP1LC3B puncta in each cell was counted. (C) Ultrastructural changes in hypoxia-treated microglia. The control shows samples without hypoxia treatment. Closed arrows indicate autophagosomes. (D and E) Microglia were treated with hypoxia for 6 hrs and stained with 1 mg/ml acridine orange or 50 mM MDC for 15 min. After incubation, cells were immediately analysed by flow cytometry. The bar chart demonstrates an increase in mean fluorescent intensity. The asterisks denote significant differences from controls (* <i>P</i><0.05). Experiments performed in triplicate showed consistent results.</p

Correlation between behavior and intracranial pressure.

<p>Correlation analyses between the results of T-maze test at day 21 after surgery and intracranial pressure (ICP) in SHAM and SAH animals. Number of alterations (A) and mean latency to decision in seconds (B). Spearman’s rho and p-values are given.</p