Chunxia Luo

Norrin Protected Blood–Brain Barrier Via Frizzled-4/β-Catenin Pathway After Subarachnoid Hemorrhage in Rats

Background and Purpose— Norrin and its receptor Frizzled-4 have important roles in the blood–brain barrier development. This study is to investigate a potential role and mechanism of Norrin/Frizzled-4 on protecting blood–brain barrier integrity after subarachnoid hemorrhage (SAH). Methods— One hundred and seventy-eight male Sprague–Dawley rats were used. SAH model was induced by endovascular perforation. Frizzled-4 small interfering RNA was injected intracerebroventricularly 48 hours before SAH. Norrin was administrated intracerebroventricularly 3 hours after SAH. SAH grade, neurological scores, brain water content, Evans blue extravasation, western blots, and immunofluorescence were used to study the mechanisms of Norrin and its receptor regulation protein TSPAN12, as well as neurological outcome. Results— Endogenous Norrin and TSPAN12 expression were increased after SAH, and Norrin was colocalized with astrocytes marker glial fibrillary acidic protein in cortex. Exogenous Norrin treatment significantly alleviated neurobehavioral dysfunction, reduced brain water content and Evans blue extravasation, promoted &bgr;-catenin nuclear translocation, and increased Occludin, VE-Cadherin, and ZO-1 expressions. These effects were abolished by Frizzled-4 small interfering RNA pretreated before SAH. Conclusions— Norrin protected blood–brain barrier integrity and improved neurological outcome after SAH, and the action of Norrin appeared mediated by Frizzled-4 receptor activation, which promoted &bgr;-catenin nuclear translocation, which then enhanced Occludin, VE-Cadherin, and ZO-1 expression. Norrin might have potential to protect blood–brain barrier after SAH.

Role of HCN Channels in Neuronal Hyperexcitability after Subarachnoid Hemorrhage in Rats

Disruption of ionic homeostasis and neuronal hyperexcitability contribute to early brain injury after subarachnoid hemorrhage (SAH). The hyperpolarization-activated/cyclic nucleotide (HCN)-gated channels play critical role in the regulation of neuronal excitability in hippocampus CA1 region and neocortex, in which the abnormal neuronal activities are more readily provoked. This study was to investigate the interactions between HCN channels and hyperneuronal activity after experimental SAH. The present results from whole-cell recordings in rat brain slices indicated that (1) perfusion of hemoglobin (Hb)-containing artificial CSF produced neuronal hyperexcitability and inhibited HCN currents in CA1 pyramidal neurons, (2) nitric oxide/Spermine (NO/Sp), a controlled releaser of nitric oxide, attenuated neuronal excitability and enhanced HCN currents in CA1 pyramidal neurons, while l-nitroarginine (l-NNA), an inhibitor of nitric oxide synthase, reduced the HCN currents; and (3) the inhibitory action of Hb on HCN currents was reversed by application of NO/Sp, which also reduced neuronal hyperexcitability; conversely, l-NNA enhanced inhibitory action of Hb on HCN currents. Additionally, Hb perfusion scavenged the production of nitric oxide and decreased the expression of HCN1 subunits in CA1 region. In the rat SAH model, the expression of HCN1, both at mRNA and protein level, decreased in hippocampus CA1 region at 24 h and more pronounced at 72 h after SAH. These observations demonstrated a reduction of HCN channels expression after SAH and Hb reduced HCN currents in hippocampus CA1 pyramidal neurons. Inhibition of HCN channels by Hb may be a novel pathway for inducing the hyperneuronal excitability after SAH.

Adenosine A3 receptor agonist reduces early brain injury in subarachnoid haemorrhage.

Inflammation plays an important role in the pathogenesis of early brain injury after subarachnoid haemorrhage. Adenosine A3 receptor (A3R) activation produces anti-inflammatory effects. In this study, the effects of a selective A3R agonist, 2-chloro-N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (CL-IB-MECA), on early brain injury and inflammatory response after subarachnoid haemorrhage were studied. Our results showed that mortality, neurological impairment and brain oedema were significantly attenuated after the administration of CL-IB-MECA. Moreover, treatment with CL-IB-MECA inhibited microglial activation and reduced the expression of proinflammatory cytokines including tumour necrosis factor-α and interleukin-1β. These data suggest that activation of A3R provides a neuroprotective effect against brain injury after subarachnoid haemorrhage, and that these effects may be associated with the anti-inflammatory properties of A3R.

Administration of a PTEN inhibitor BPV(pic) attenuates early brain injury via modulating AMPA receptor subunits after subarachnoid hemorrhage in rats.

The aim of this study was to investigate whether the phosphatase and tensin homolog deleted on chromosome ten (PTEN) inhibitor dipotassium bisperoxo(pyridine-2-carboxyl) oxovanadate (BPV(pic)) attenuates early brain injury by modulating α-amino-3-hydroxy-5-methyl-4-isoxa-zolep-propionate (AMPA) receptor subunits after subarachnoid hemorrhage (SAH). A standard intravascular perforation model was used to produce the experimental SAH in Sprague-Dawley rats. BPV(pic) treatment (0.2mg/kg) was evaluated for effects on neurological score, brain water content, Evans blue extravasation, hippocampal neuronal death and AMPA receptor subunits alterations after SAH. We found that BPV(pic) is effective in attenuating BBB disruption, lowering edema, reducing hippocampal neural death and improving neurological outcomes. In addition, the AMPA receptor subunit GluR1 protein expression at cytomembrane was downregulated, whereas the expression of GluR2 and GluR3 was upregulated after BPV(pic) treatment. Our results suggest that PTEN inhibited by BPV(pic) plays a neuroprotective role in SAH pathophysiology, possibly by alterations in glutamate AMPA receptor subunits.

Hemoglobin induced NO/cGMP suppression Deteriorate Microcirculation via Pericyte Phenotype Transformation after Subarachnoid Hemorrhage in Rats

Subarachnoid hemorrhage (SAH) usually results from ruptured aneurysm, but how leaked hemoglobin regulates the microcirculation in the pathophysiology of early brain injury after SAH is still unclear. In the present study, we sought to investigate the role and possible mechanism of hemoglobin induced pericyte phenotype transformation in the regulation of microcirculation after SAH. Endovascular perforation SAH rat model, brain slices and cultured pericytes were used, and intervened with endothelial nitric oxide synthase (eNOS) antagonist L-NNA and its agonist scutellarin, hemoglobin, DETA/NO (nitric oxide(NO) donor), PITO (NO scavenger), 8-Br-cGMP (cGMP analog). We found modulating eNOS regulated pericyte α-SMA phenotype transformation, microcirculation, and neurological function in SAH rats. Modulating eNOS also affected eNOS expression, eNOS activity and NO availability after SAH. In addition, we showed hemoglobins penetrated into brain parenchyma after SAH. And hemoglobins significantly reduced the microvessel diameters at pericyte sites, due to the effects of hemoglobin inducing α-SMA expressions in cultured pericytes and brain slices via inhibiting NO/cGMP pathway. In conclusion, pericyte α-SMA phenotype mediates acute microvessel constriction after SAH possibly by hemoglobin suppressing NO/cGMP signaling pathway. Therefore, by targeting the eNOS and pericyte α-SMA phenotype, our present data may shed new light on the management of SAH patients.

Intraventricular administration of urokinase as a novel therapeutic approach for communicating hydrocephalus

&NA; Fibrosis of the subarachnoid space (SAS) after infection, inflammation, or hemorrhage can impair cerebrospinal fluid absorption and circulation, causing diffuse ventricular dilatation. In the present study, we tested the hypothesis that urokinase (also known as urokinase‐type plasminogen activator [uPA]), a fibrinolytic agent, attenuates fibrosis and ventriculomegaly in a rat model of kaolin‐induced communicating hydrocephalus and thus may have potential as a therapy for these conditions. Thirty microliters of sterile 25% kaolin suspension was injected into the basal cisterns of adult Sprague–Dawley rats to induce hydrocephalus, and 2 intraventricular injections of either uPA or vehicle (saline) were administered immediately and 3 days thereafter. Ventricular volumes were measured by magnetic resonance imaging (MRI) on days 3, 14, and 28 after kaolin injection. Fibrosis and reactive astrogliosis were evaluated on day 28 by immunofluorescence and Western blotting. Neurocognitive features were tested using the Morris water maze from days 23 to 28. MRI analysis demonstrated that kaolin administration successfully induced hydrocephalus in rats and that uPA treatment significantly attenuated ventricular enlargement. In addition, uPA inhibited the deposition of laminin and fibronectin, extracellular matrix molecules, in the SAS, attenuated gliosis, and improved learning and memory in kaolin‐treated rats. Therefore, we concluded that uPA prevents the development of kaolin‐induced communicating hydrocephalus by preventing the development of subarachnoid fibrosis and by eliciting improvements in neurocognition. The results of this study indicate that uPA may be a novel clinical therapy for communicating hydrocephalus.

PKGIα Inhibits the Proliferation of Cerebral Arterial Smooth Muscle Cell Induced by Oxyhemoglobin After Subarachnoid Hemorrhage1

The purpose of the present study was to observe the proliferation of cerebral arterial smooth muscle cell (CASMC) induced by oxyhemoglobin (Oxyhb) and interfered by Adenovirus-mediate-PKGI (Ad-PKGI), and to investigate the potential regulative role of the PKGI gene in the molecule mechanism of cerebral vasospasm (CVS) after Subarachnoid hemorrhage (SAH). Tissue-sticking method was used for primary cultured rat CASMCs. Semi-quantitative reverse transcription and polymerase chain reaction (RT-PCR) and western blot were used to examine the PKGI mRNA and protein expressions after CASMC were transfected by Ad-PKG. The proliferation of CASMCs was determined by MTT assay and 3H-TdR incorporation. Ad-PKGI could be transfected into CASMCS and highly express. Oxyhemoglobin could stimulate the proliferation of CASMC; the value of 3H-TdR incorporation and the absorbance value of MTT increased and could block up after CASMC was transfected by Ad-PKG. The results suggested that the PKG signaling pathway might play an important role in CVS after SAH, and the PKG gene might be a target point of gene therapy.