Qingyi Ma

NLRP3 inflammasome contributes to inflammation after intracerebral hemorrhage.

The NLRP3 (NALP3, cryopyrin) inflammasome, a key component of the innate immune system, facilitates caspase‐1 and interleukin (IL)−1β processing, which amplifies the inflammatory response. Here, we investigated whether NLRP3 knockdown decreases neutrophil infiltration, reduces brain edema, and improves neurological function in an intracerebral hemorrhage (ICH) mouse model. We also determined whether mitochondrial reactive oxygen species (ROS) governed by mitochondrial permeability transition pores (mPTPs) would trigger NLRP3 inflammasome activation following ICH.

P2X7R/cryopyrin inflammasome axis inhibition reduces neuroinflammation after SAH.

Neuroinflammation contributes to the pathogenesis of early brain injury (EBI) after subarachnoid hemorrhage (SAH). Cytotoxic events following SAH, such as extracellular accumulation of adenosine triphosphate (ATP), may activate the P2X purinoceptor 7 (P2X7R)/cryopyrin inflammasome axis, thus inducing the proinflammatory cytokine IL-1β/IL-18 secretion. We therefore hypothesized that inhibition of P2X7R/cryopyrin inflammasome axis would ameliorate neuroinflammation after SAH. In the present study, SAH was induced by the endovascular perforation in rats. Small interfering RNAs (siRNAs) of P2X7R or cryopyrin were administered intracerebroventricularly 24h before SAH. Brilliant blue G (BBG), a non-competitive antagonist of P2X7R, was administered intraperitoneally 30min following SAH. Post-assessments including SAH severity score, neurobehavioral test, brain water content, Western blot and immunofluorescence, were performed. Administration of P2X7R and cryopyrin siRNA as well as pharmacologic blockade of P2X7R by BBG ameliorated neurological deficits and brain edema at 24h following SAH. Inhibition of P2X7R/cryopyrin inflammasome axis suppressed caspase-1 activation, which subsequently decreased maturation of IL-1β/IL-18. To investigate the link between P2X7R and cryopyrin inflammasome in vivo, Benzoylbenzoyl-ATP (BzATP), a P2X7R agonist, was given to lipopolysaccharide (LPS) primed naive rats with scramble or cryopyrin siRNAs. In LPS-primed naive rats, BzATP induced caspase-1 activation and mature IL-1β release were neutralized by cryopyrin siRNA. Thus, the P2X7R/cryopyrin inflammasome axis may contribute to neuroinflammation via activation of caspase-1 and thereafter mature IL-1β/IL-18 production following SAH. Therapeutic interventions targeting P2X7R/cryopyrin pathway may be a novel approach to ameliorate EBI following SAH.

PDGFR-α Inhibition Preserves Blood-Brain Barrier after Intracerebral Hemorrhage

Perihematomal edema results from disruption of the blood‐brain barrier (BBB) by key mediators, such as thrombin, following intracerebral hemorrhage (ICH). Platelet‐derived growth factor receptor alpha (PDGFR‐α), a tyrosine kinase receptor, was found in previous studies to play a role in orchestrating BBB impairment. In the present study, we investigated the role of PDGFR‐α following ICH‐induced brain injury in mice, specifically investigating its effect on BBB disruption.

Vascular Adhesion Protein-1 Inhibition Provides Antiinflammatory Protection after an Intracerebral Hemorrhagic Stroke in Mice:

The systemic immune response has a vital role in propagating the damage of an intracerebral hemorrhage (ICH). Vascular adhesion protein-1 (VAP-1), a semicarbazide (SCZ)-sensitive-amine-oxidase, was found in previous studies to have a role in migration of immune cells. In this study, we hypothesize that VAP-1 inhibition may decrease brain injury by attenuating the transmigration of immune cells to the injury site, and by doing so, reduce cerebral edema and improve neurobehavioral function in mice. Two VAP-1 inhibitors, LJP1586 and SCZ were given 1 hour after ICH induction by either collagenase or autologous blood injection. The VAP-1 siRNA, a VAP-1 gene silencer, and human recombinant AOC3 protein, a VAP-1 analogue, were delivered by intracerebroventricular injection. Postassessment included neurobehavioral testing, brain edema measurement, quantification of neutrophil infiltration and microglia/macrophage activation, and measurement of intercellular adhesion molecule-1 (ICAM-1), P-selectin, monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor-α (TNF-α) expression 24 hours after ICH. We found that LJP1586 and SCZ reduced brain edema and neurobehavioral deficits 24 hours after ICH induction. These two drugs were also found to decrease levels of ICAM-1, MCP-1, TNF-α, and inhibit neutrophilic infiltration and microglia/macrophage activation. We conclude that VAP-1 inhibition provided antiinflammation effect by reducing adhesion molecule expression and immune cell infiltration after ICH.

CHOP Silencing Reduces Acute Brain Injury in the Rat Model of Subarachnoid Hemorrhage

Background and Purpose— Endoplasmic reticulum stress triggers apoptotic cascades in neurons of the central nervous system after subarachnoid hemorrhage. The aim of this work was to study the mechanism of neuroprotection conferred by targeting cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) in the acute brain injury following subarachnoid hemorrhage. Methods— A total of 172 rats were used. Endovascular perforation induced subarachnoid hemorrhage. Two small interfering RNAs for CHOP were injected 24 hours before hemorrhage induction. At 24 or 72 hours, rats were neurologically evaluated and euthanized. The brains were recovered for molecular biology and histology studies. Results— Western blot analysis revealed effective silencing of CHOP associated with suppression of Bim-Caspase-3 apoptotic pathway. Moreover, the antiapoptotic Bcl2 was found upregulated with CHOP siRNA treatment. A reduced number of TUNEL-positive cells in the subcortex and in the hippocampus reflected histological protection. CHOP siRNA treatment ameliorated intracranial sequelae of and improved functional performance. Conclusions— We conclude that CHOP silencing alleviates early brain injury following subarachnoid hemorrhage via inhibiting apoptosis and that CHOP siRNA treatment has a clinical potential for patients with this type of hemorrhagic stroke.

Osteopontin Reduced Hypoxia–Ischemia Neonatal Brain Injury by Suppression of Apoptosis in a Rat Pup Model

Background and Purpose— Osteopontin (OPN) is neuroprotective in ischemic brain injuries in adult experimental models; therefore, we hypothesized that OPN would provide neuroprotection and improve long-term neurological function in the immature brain after hypoxic–ischemic (HI) injury. Methods— HI was induced by unilateral ligation of the right carotid artery followed by hypoxia (8% O2 for 2 hours) in postnatal Day 7 rats. OPN (0.03 &mgr;g or 0.1 &mgr;g) was injected intracerebroventricularly at 1 hour post-HI. Temporal expression of endogenous OPN was evaluated in the normal rat brain at the age of 0, 4, 7, 11, 14, and 21 days and in the ipsilateral hemisphere after HI. The effects of OPN were evaluated using 2-3-5-triphenyl tetrazolium chloride staining, apoptotic cell death assay, and cleaved caspase-3 expression. Neurological function was assessed by the Morris water maze test. Results— Endogenous OPN expression in the brain was the highest at the age of 0 day with continuous reduction until the age of 21 days during development. After HI injury, endogenous OPN expression was increased and peaked at 48 hours. Exogenous OPN decreased infarct volume and improved neurological outcomes 7 weeks after HI injury. OPN-induced neuroprotection was blocked by an integrin antagonist. Conclusions— OPN-induced neuroprotection was associated with cleaved-caspase-3 inhibition and antiapoptotic cell death. OPN treatment improved long-term neurological function against neonatal HI brain injury.

Isoflurane Enhanced Hemorrhagic Transformation by Impairing Antioxidant Enzymes in Hyperglycemic Rats With Middle Cerebral Artery Occlusion

Background and Purpose— Because the potential neuroprotective effect of isoflurane is controversial, we attempted to study whether isoflurane after treatment provides neuroprotection in a rat model of hyperglycemia-induced ischemic hemorrhagic transformation. Methods— Rats received an injection of 50% dextrose (6 mL/kg intraperitoneally) and had a middle cerebral artery occlusion 30 minutes later. Four groups were included: sham-operated, ischemia/reperfusion, isoflurane treatment, and vehicle groups. In the treatment group, after 2 hours of ischemia, 2% isoflurane was administered at the onset of reperfusion. We measured the level of blood glucose at 0, 2.5, 4.5, and 6.5 hours after dextrose injection. Infarct and hemorrhagic volumes, neurological scores, oxidative stress (malondialdehyde, 4-hydroxy-2-nonenal, and nitrotyrosine) and the activities of superoxide dismutase and catalase were measured at 24 hours after ischemia. Results— Isoflurane had no effects on blood glucose, it failed to reduce infarct, hemorrhage volume, and brain edema, and it enhanced neurobehavioral deficits when compared with the ischemia/reperfusion group at 24 hours after middle cerebral artery occlusion. On the contrary, isoflurane exacerbated these parameters compared with the vehicle group. In addition, it increased the expressions of malondialdehyde, 4-hydroxy-2-nonenal, and nitrotyrosine, and it decreased the activities of superoxide dismutase and catalase compared to the vehicle group. Conclusions— Isoflurane after treatment worsened physiological and neurological outcomes in this ischemia hyperglycemia-induced hemorrhagic transformation possibly by impairing the antioxidant defense system.

Role of SCH79797 in Maintaining Vascular Integrity in Rat Model of Subarachnoid Hemorrhage

Background and Purpose— Plasma thrombin concentration is increased after subarachnoid hemorrhage (SAH). However, the role of thrombin receptor (protease-activated receptor-1 [PAR-1]) in endothelial barrier disruption has not been studied. The aims of this study were to investigate the role of PAR-1 in orchestrating vascular permeability and to assess the potential therapeutics of a PAR-1 antagonist, SCH79797, through maintaining vascular integrity. Methods— SCH79797 was injected intraperitoneally into male Sprauge-Dawley rats undergoing SAH by endovascular perforation. Assessment was conducted at 24 hours after SAH for brain water content, Evans blue content, and neurobehavioral testing. To explore the role of PAR-1 activation and the specific mechanism of SCH79797’s effect after SAH, Western blot, immunoprecipitation, and immunofluorescence of hippocampus tissue were performed. A p21-activated kinase-1 (PAK1) inhibitor, IPA-3, was used to explore the underlying protective mechanism of SCH79797. Results— At 24 hours after SAH, animals treated with SCH79797 demonstrated a reduction in brain water content, Evans blue content, and neurobehavioral deficits. SCH79797 also attenuated PAR-1 expression and maintained the level of vascular endothelial-cadherin, an important component of adherens junctions. Downstream to PAR-1, c-Src–dependent activation of p21-activated kinase-1 led to an increased serine/threonine phosphorylation of vascular endothelial-cadherin; immunoprecipitation results revealed an enhanced binding of phosphorylated vascular endothelial-cadherin with endocytosis orchestrator &bgr;-arrestin-2. These pathological states were suppressed after SCH79797 treatment. Conclusions— PAR-1 activation after SAH increases microvascular permeability, at least, partly through a PAR-1-c-Src-p21-activated kinase-1-vascular endothelial-cadherin phosphorylation pathway. Through suppressing PAR-1 activity, SCH79797 plays a protective role in maintaining microvascular integrity after SAH.

Fibroblast growth factors preserve blood-brain barrier integrity through RhoA inhibition after intracerebral hemorrhage in mice.

Fibroblast growth factors (FGFs) maintain and promote vascular integrity; however whether FGFs protect the blood-brain barrier (BBB) after intracerebral hemorrhage (ICH) remains unexplored. In this present study, we hypothesized that exogenous FGF administration attenuates brain injury after ICH, specifically by preserving endothelial adherens junctions, therefore reducing vasogenic brain edema and attenuating neurofunctional deficits in mice subjected to experimental ICH. Acid fibroblast growth factor (FGF1) or basic fibroblast growth factor (FGF2) was administered intracerebroventricularly (ICV) at 0.5 h after intrastriatal injection of bacterial collagenase (cICH) or autologous whole blood (bICH). Fibroblast growth factor receptor (FGFR) inhibitor PD173074 and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 were additionally administered with FGF2. The selective Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) inhibitor Y27632 was independently administered at 0.5 h after cICH. Brain water content and neurofunctional deficits were evaluated at 24 and 72h after ICH induction. Evans blue extravasation as well as Western blot analysis for the quantification of activated FGFR, Akt, Ras-related C3 botulinum toxin substrate 1 (Rac1), Ras homolog gene family member A (RhoA) and adherens junction proteins (p120-catenin, β-catenin and VE-cadherin) were conducted at 72 h post-cICH. FGF treatment reduced perihematomal brain edema and improved neurofunctional deficits at 72 h after experimental ICH (p<0.05, compared to vehicle); however, FGFR and PI3K inhibition reversed these neuroprotective effects. Exogenous FGF2 increased activated FGFR, Akt, and Rac1 but reduced activated RhoA protein expression at 72 h after cICH (p<0.05, compared to vehicle), which was reversed by FGFR and PI3K inhibition. Y27632 treatment reduced brain injury at 72 h after cICH (p<0.05, compared to vehicle) and increased the expression of catenins (p120-catenin, β-catenin). In conclusion, our findings suggest that exogenous FGF treatment reduced RhoA activity via FGFR-induced activation of the PI3K-Akt-Rac1 signaling pathway, thus preserving BBB integrity, and therefore attenuating secondary brain injury after experimental ICH in mice.

Isoflurane posttreatment reduces brain injury after an intracerebral hemorrhagic stroke in mice.

BACKGROUND: Intracerebral hemorrhage (ICH) is a devastating stroke subtype affecting 120,000 Americans annually. Of those affected, 40%to 50% will die within the first 30 days, whereas the survivors are left with a lifetime of neurobehavioral disabilities. Recently, it has been shown that volatile anesthetics such as isoflurane can reduce brain injury after an ischemic stroke. As a result, in this study, we investigated the effects of isoflurane as a posttreatment therapeutic modality in ICH-injured mice. Specifically, we investigated whether isoflurane posttreatment can preserve the structural integrity of the brain by reducing apoptotic damage and, in turn, improve functional outcome by amelioration of brain edema and neurobehavioral deficits. METHODS: Male CD1 mice (n = 53) were divided into the following groups: sham (n = 14), ICH (n = 14), ICH treated with 1.5% isoflurane posttreatment for 1 hour (n = 15), and ICH treated with 1.5% isoflurane posttreatment for 2 hours (n = 10). The blood injection ICH model was adapted; this involved extracting autologous blood from the mouse tail and injecting it directly into the right basal ganglia. One hour after surgery, treated mice were placed in a glass chamber maintained at 37°C and infused with 1.5% isoflurane for 1 or 2 hours. At 24 hours postinjury, mice were assessed for neurobehavioral deficits using the Modified Garcia Score and then killed and assessed for brain water content. Double immunofluorescent staining was performed using neuronal marker MAP-2 and TUNEL under a fluorescent microscope to assess for apoptosis. RESULTS: Our results indicated that after 1-hour 1.5% isoflurane posttreatment, there was a significant reduction in brain edema, a decrease in apoptotic cell death, and a significant improvement in neurobehavioral deficits. CONCLUSIONS: Our results suggest that isoflurane may be an effective posttreatment therapeutic option for ICH because of its ability to reduce structural damage and subsequently preserve functional integrity.