PEDF-34 attenuates neurological deficit and suppresses astrocyte-dependent neuroinflammation by modulating astrocyte polarization via 67LR/JNK/STAT1 signaling pathway after subarachnoid hemorrhage in rats.

BACKGROUND: Reactive astrocytes participate in various pathophysiology after subarachnoid hemorrhage (SAH), including neuroinflammation, glymphatic-lymphatic system dysfunction, brain edema, BBB disruption, and cell death. Astrocytes transform into two new reactive phenotypes with changed morphology, altered gene expression, and secretion profiles, termed detrimental A1 and beneficial A2. This study investigates the effect of 67LR activation by PEDF-34, a PEDF peptide, on neuroinflammation and astrocyte polarization after the experimental SAH. METHODS: A total of 318 male adult Sprague-Dawley rats were used in experiments in vivo, of which 272 rats were subjected to the endovascular perforation model of SAH and 46 rats underwent sham surgery. 67LR agonist (PEDF-34) was administrated intranasally 1 h after SAH. 67LR-specific inhibitor (NSC-47924) and STAT1 transcriptional activator (2-NP) were injected intracerebroventricularly 48 h before SAH. Short- and long-term neurological tests, brain water content, immunostaining, Nissl staining, western blot, and ELISA assay were performed. In experiments in vitro, primary astrocyte culture with hemoglobin (Hb) stimulation was used to mimic SAH. The expression of the PEDF-34/67LR signaling pathway and neuro-inflammatory cytokines were assessed using Western blot, ELISA, and immunohistochemistry assays both in vivo and in vitro. RESULTS: Endogenous PEDF and 67LR expressions were significantly reduced at 6 h after SAH. 67LR was expressed in astrocytes and neurons. Intranasal administration of PEDF-34 significantly reduced brain water content, pro-inflammatory cytokines, and short-term and long-term neurological deficits after SAH. The ratio of p-JNK/JNK and p-STAT1/STAT1 and the expression of CFB and C3 (A1 astrocytes marker), significantly decreased after PEDF-34 treatment, along with fewer expression of TNF-α and IL-1ß at 24 h after SAH. However, 2-NP (STAT1 transcriptional activator) and NSC-47924 (67LR inhibitor) reversed the protective effects of PEDF-34 in vivo and in vitro by promoting A1 astrocyte polarization with increased inflammatory cytokines. CONCLUSION: PEDF-34 activated 67LR, attenuating neuroinflammation and inhibiting astrocyte A1 polarization partly via the JNK/STAT1 pathway, suggesting that PEDF-34 might be a potential treatment for SAH patients.

Fractalkine Enhances Hematoma Resolution and Improves Neurological Function via CX3CR1/AMPK/PPARγ Pathway After GMH.

BACKGROUND: Hematoma clearance has been a proposed therapeutic strategy for hemorrhagic stroke. This study investigated the impact of CX3CR1 (CX3C chemokine receptor 1) activation mediated by r-FKN (recombinant fractalkine) on hematoma resolution, neuroinflammation, and the underlying mechanisms involving AMPK (AMP-activated protein kinase)/PPARγ (peroxisome proliferator-activated receptor gamma) pathway after experimental germinal matrix hemorrhage (GMH). METHODS: A total of 313 postnatal day 7 Sprague Dawley rat pups were used. GMH was induced using bacterial collagenase by a stereotactically guided infusion. r-FKN was administered intranasally at 1, 25, and 49 hours after GMH for short-term neurological evaluation. Long-term neurobehavioral tests (water maze, rotarod, and foot-fault test) were performed 24 to 28 days after GMH with the treatment of r-FKN once daily for 7 days. To elucidate the underlying mechanism, CX3CR1 CRISPR, or selective CX3CR1 inhibitor AZD8797, was administered intracerebroventricularly 24 hours preinduction of GMH. Selective inhibition of AMPK/PPARγ signaling in microglia via intracerebroventricularly delivery of liposome-encapsulated specific AMPK (Lipo-Dorsomorphin), PPARγ (Lipo-GW9662) inhibitor. Western blot, Immunofluorescence staining, Nissl staining, Hemoglobin assay, and ELISA assay were performed. RESULTS: The brain expression of FKN and CX3CR1 were elevated after GMH. FKN was expressed on both neurons and microglia, whereas CX3CR1 was mainly expressed on microglia after GMH. Intranasal administration of r-FKN improved the short- and long-term neurobehavioral deficits and promoted M2 microglia polarization, thereby attenuating neuroinflammation and enhancing hematoma clearance, which was accompanied by an increased ratio of p-AMPK (phosphorylation of AMPK)/AMPK, Nrf2 (nuclear factor erythroid 2-related factor 2), PPARγ, CD36 (cluster of differentiation 36), CD163 (hemoglobin scavenger receptor), CD206 (the mannose receptor), and IL (interleukin)-10 expression, and decreased CD68 (cluster of differentiation 68), IL-1ß, and TNF (tumor necrosis factor) α expression. The administration of CX3CR1 CRISPR or CX3CR1 inhibitor (AZD8797) abolished the protective effect of FKN. Furthermore, selective inhibition of microglial AMPK/PPARγ signaling abrogated the anti-inflammation effects of r-FKN after GMH. CONCLUSIONS: CX3CR1 activation by r-FKN promoted hematoma resolution, attenuated neuroinflammation, and neurological deficits partially through the AMPK/PPARγ signaling pathway, which promoted M1/M2 microglial polarization. Activating CX3CR1 by r-FKN may provide a promising therapeutic approach for treating patients with GMH.

Activation of GPR40 attenuates neuroinflammation and improves neurological function via PAK4/CREB/KDM6B pathway in an experimental GMH rat model.

BACKGROUND: Germinal matrix hemorrhage (GMH) is defined by the rupture of immature blood vessels in the germinal matrix, where subsequent hemorrhage enters the subependymal zone and the cerebral lateral ventricles. The consequent blood clot has been identified as the causative factor of secondary brain injury, which triggers a series of complex parallel and sequential harmful mechanisms, including neuroinflammation. The orphan G-protein-coupled receptor 40 (GPR40), a free fatty acid (FFA) receptor 1, has been shown to exert anti-inflammatory effects when activated and improved outcomes in animal models of stroke. We aimed to investigate the anti-inflammatory effects of GPR40 and its underlying mechanisms after GMH. METHODS: GMH model was induced in 7-day-old rat pups by an intraparenchymal injection of bacterial collagenase. GPR40 agonist, GW9508, was administered intranasally 1 h, 25 h, and 49 h after GMH induction. CRISPR targeting GPR40, PAK4, and KDM6B were administered through intracerebroventricular injection 48 h before GMH induction. Neurologic scores, microglia polarization, and brain morphology were evaluated by negative geotaxis, right reflex, rotarod test, foot fault test, Morris water maze, immunofluorescence staining, Western blots, and nissl staining respectfully. RESULTS: The results demonstrated that GW9508 improved neurological and morphological outcomes after GMH in the short (24 h, 48 h, 72h) and long-term (days 21-27). However, the neuroprotective effects of treatment were abolished by GW1100, a selective GPR40 antagonist. GW9508 treatment increased populations of M2 microglia and decreased M1 microglia in periventricular areas 24 h after GMH induction. GW9508 upregulated the phosphorylation of PAK4, CREB, and protein level of KDM6B, CD206, IL-10, which was also met with the downregulation of inflammatory markers IL-1ß and TNF-α. The mechanism study demonstrated that the knockdown of GPR40, PAK4, and KDM6B reversed the neuroprotective effects brought on by GW9508. This evidence suggests that GPR40/PAK4/CREB/KDM6B signaling pathway in microglia plays a role in the attenuation of neuroinflammation after GMH. CONCLUSIONS: In conclusion, the present study demonstrates that the activation of GPR40 attenuated GMH-induced neuroinflammation through the activation of the PAK4/CREB/KDM6B signaling pathway, and M2 microglia may be a major mediator of this effect. Thus, GPR40 may serve as a potential target in the reduction of the inflammatory response following GMH, thereby improving neurological outcomes in the short- and long-term.

A comprehensive review of therapeutic targets that induce microglia/macrophage-mediated hematoma resolution after germinal matrix hemorrhage.

Currently, there is no effective treatment for germinal matrix hemorrhage and intraventricular hemorrhage (GMH-IVH), a common and often fatal stroke subtype in premature infants. Secondary brain injury after GMH-IVH is known to involve blood clots that contribute to inflammation and neurological deficits. Furthermore, the subsequent blood clots disrupt normal cerebrospinal fluid circulation and absorption after GMH-IVH, contributing to posthemorrhagic hydrocephalus (PHH). Clinically, GMH-IVH severity is graded on a I to IV scale: Grade I is confined to the germinal matrix, grade II includes intraventricular hemorrhage, grade III includes intraventricular hemorrhage with extension into dilated ventricles, and grade IV includes intraventricular hemorrhage with extension into dilated ventricles as well as parenchymal hemorrhaging. GMH-IVH hematoma volume is the best prognostic indicator, where patients with higher grades have worsened outcomes. Various preclinical studies have shown that rapid hematoma resolution quickly ameliorates inflammation and improves neurological outcomes. Current experimental evidence identifies alternatively activated microglia as playing a pivotal role in hematoma clearance. In this review, we discuss the pathophysiology of GMH-IVH in the development of PHH, microglia/macrophage's role in the neonatal CNS, and established/potential therapeutic targets that enhance M2 microglia/macrophage phagocytosis of blood clots after GMH-IVH.

Posthemorrhagic hydrocephalus development after germinal matrix hemorrhage: Established mechanisms and proposed pathways.

In addition to being the leading cause of morbidity and mortality in premature infants, germinal matrix hemorrhage (GMH) is also the leading cause of acquired infantile hydrocephalus. The pathophysiology of posthemorrhagic hydrocephalus (PHH) development after GMH is complex and vaguely understood, although evidence suggests fibrosis and gliosis in the periventricular and subarachnoid spaces disrupts normal cerebrospinal fluid (CSF) dynamics. Theories explaining general hydrocephalus etiology have substantially evolved from the original bulk flow theory developed by Dr. Dandy over a century ago. Current clinical and experimental evidence supports a new hydrodynamic theory for hydrocephalus development involving redistribution of vascular pulsations and disruption of Starling forces in the brain microcirculation. In this review, we discuss CSF flow dynamics, history and development of theoretical hydrocephalus pathophysiology, and GMH epidemiology and etiology as it relates to PHH development. We highlight known mechanisms and propose new avenues that will further elucidate GMH pathophysiology, specifically related to hydrocephalus.

Bliverdin reductase-A improves neurological function in a germinal matrix hemorrhage rat model.

Germinal matrix hemorrhage is induced by stereotaxic injection of collagenase into the germinal matrix of P7 Sprague-Dawley rats. Hemoglobin assay, western blot, immunofluorescence and neurobehavioral tests were used to test the effects of BLVRA on hematoma resolution and anti-inflammatory response. We showed that BLVRA triggered a signaling cascade that ameliorated post-hemorrhagic neurological deficits in both short-term and long-term neurobehavioral tests in a GMH rat model. Specifically, BLVRA inhibited toll-like receptor 4 (TLR4) expression by translocating to the nucleus in an endothelial nitric oxide (eNOS)/nitric oxide (NO)-dependent manner. BLVRA also induced the upregulation of CD36 scavenger receptor level in microglia/microphages, of which the prominent role is to enhance hematoma resolution. However, the beneficial effects of BLVRA were abolished with the knockdown of eNOS, indicating that the eNOS/NO system is an important downstream factor of BLVRA. Our results demonstrate a mechanism of BLVRA modulating hematoma resolution and suppressing inflammation through eNOS/NO/TLR4 pathway in the GMH rat model.

PPARγ-induced upregulation of CD36 enhances hematoma resolution and attenuates long-term neurological deficits after germinal matrix hemorrhage in neonatal rats.

Germinal matrix hemorrhage remains the leading cause of morbidity and mortality in preterm infants in the United States with little progress made in its clinical management. Survivors are often afflicted with long-term neurological sequelae, including cerebral palsy, mental retardation, hydrocephalus, and psychiatric disorders. Blood clots disrupting normal cerebrospinal fluid circulation and absorption after germinal matrix hemorrhage are thought to be important contributors towards post-hemorrhagic hydrocephalus development. We evaluated if upregulating CD36 scavenger receptor expression in microglia and macrophages through PPARγ stimulation, which was effective in experimental adult cerebral hemorrhage models and is being evaluated clinically, will enhance hematoma resolution and ameliorate long-term brain sequelae using a neonatal rat germinal matrix hemorrhage model. PPARγ stimulation (15d-PGJ2) increased short-term PPARγ and CD36 expression levels as well as enhanced hematoma resolution, which was reversed by a PPARγ antagonist (GW9662) and CD36 siRNA. PPARγ stimulation (15d-PGJ2) also reduced long-term white matter loss and post-hemorrhagic ventricular dilation as well as improved neurofunctional outcomes, which were reversed by a PPARγ antagonist (GW9662). PPARγ-induced upregulation of CD36 in macrophages and microglia is, therefore, critical for enhancing hematoma resolution and ameliorating long-term brain sequelae.

Protease-activated receptor 1 and 4 signal inhibition reduces preterm neonatal hemorrhagic brain injury.

BACKGROUND AND PURPOSE: This study examines the role of thrombin's protease-activated receptor (PAR)-1, PAR-4 in mediating cyclooxygenase-2 and mammalian target of rapamycin after germinal matrix hemorrhage. METHODS: Germinal matrix hemorrhage was induced by intraparenchymal infusion of bacterial collagenase into the right ganglionic eminence of P7 rat pups. Animals were treated with PAR-1, PAR-4, cyclooxygenase-2, or mammalian target of rapamycin inhibitors by 1 hour, and ≤5 days. RESULTS: We found increased thrombin activity 6 to 24 hours after germinal matrix hemorrhage, and PAR-1, PAR-4, inhibition normalized cyclooxygenase-2, and mammalian target of rapamycin by 72 hours. Early treatment with NS398 or rapamycin substantially improved long-term outcomes in juvenile animals. CONCLUSIONS: Suppressing early PAR signal transduction, and postnatal NS398 or rapamycin treatment, may help reduce germinal matrix hemorrhage severity in susceptible preterm infants.

Acute and delayed deferoxamine treatment attenuates long-term sequelae after germinal matrix hemorrhage in neonatal rats.

BACKGROUND AND PURPOSE: This study investigated if acute and delayed deferoxamine treatment attenuates long-term sequelae after germinal matrix hemorrhage (GMH). METHODS: Bacterial collagenase (0.3 U) was infused intraparenchymally into the right hemispheric ganglionic eminence in P7 rat pups to induce GMH. GMH animals received either deferoxamine or vehicle twice a day for 7 consecutive days. Deferoxamine administration was initiated at either 1 hour or 72 hours post-GMH. Long-term neurocognitive deficits and motor coordination were assessed using Morris water maze, rotarod, and foot fault tests between day 21 to 28 post-GMH. At 28 days post-GMH, brain morphology was assessed and extracellular matrix protein (fibronectin and vitronectin) expression was determined. RESULTS: Acute and delayed deferoxamine treatment improved long-term motor and cognitive function at 21 to 28 days post-GMH. Attenuated neurofunction was paralleled with improved overall brain morphology at 28 days post-GMH, reducing white matter loss, basal ganglia loss, posthemorrhagic ventricular dilation, and cortical loss. GMH resulted in significantly increased expression of fibronectin and vitronectin, which was reversed by acute and delayed deferoxamine treatment. CONCLUSIONS: Acute and delayed deferoxamine administration ameliorated long-term sequelae after GMH.

Mitochondrial ferritin upregulation reduced oxidative stress and blood-brain-barrier disruption by maintaining cellular iron homeostasis in a neonatal rat model of germinal matrix hemorrhage.

Germinal matrix hemorrhage (GMH) is a devasting neurological disease in premature newborns. After GMH, brain iron overload associated with hemoglobin degradation contributed to oxidative stress, causing disruption of the already vulnerable blood-brain barrier (BBB). Mitochondrial ferritin (FTMT), a novel mitochondrial outer membrane protein, is crucial in maintaining cellular iron homeostasis. We aimed to investigate the effect of FTMT upregulation on oxidative stress and BBB disruption associated with brain iron overload in rats. A total of 222 Sprague-Dawley neonatal rat pups (7 days old) were used to establish a collagenase-induced GMH model and an iron-overload model of intracerebral FeCl2 injection. Deferiprone was administered via gastric lavage 1 h after GMH and given daily until euthanasia. FTMT CRISPR Knockout and adenovirus (Ad)-FTMT were administered intracerebroventricularly 48 h before GMH and FeCl2 injection, respectively. Neurobehavioral tests, immunofluorescence, Western blot, Malondialdehyde measurement, and brain water content were performed to evaluate neurobehavior deficits, oxidative stress, and BBB disruption, respectively. The results demonstrated that brain expressions of iron exporter Ferroportin (FPN) and antioxidant glutathione peroxidase 4 (GPX4) as well as BBB tight junction proteins including Claudin-5 and Zona Occulta (ZO)-1 were found to be decreased at 72 h after GMH. FTMT agonist Deferiprone attenuated oxidative stress and preserved BBB tight junction proteins after GMH. These effects were partially reversed by FTMT CRISPR Knockout. Iron overload by FeCl2 injection resulted in oxidative stress and BBB disruption, which were improved by Ad-FTMT mediated FTMT overexpression. Collectively, FTMT upregulation is neuroprotective against brain injury associated with iron overload. Deferiprone reduced oxidative stress and BBB disruption by maintaining cellular iron homeostasis partially by the upregulating of FTMT after GMH. Deferiprone may be an effective treatment for patients with GMH.

Isoflurane post-treatment ameliorates GMH-induced brain injury in neonatal rats.

BACKGROUND AND PURPOSE: This study investigated whether isoflurane ameliorates neurological sequelae after germinal matrix hemorrhage (GMH) through activation of the cytoprotective sphingosine kinase/sphingosine-1-phosphate receptor/Akt pathway. METHODS: GMH was induced in P7 rat pups by intraparenchymal infusion of bacterial collagenase (0.3 U) into the right hemispheric germinal matrix. GMH animals received 2% isoflurane either once 1 hour after surgery or every 12 hours for 3 days. Isoflurane treatment was then combined with sphingosine-1-phosphate receptor-1/2 antagonist VPC23019 or sphingosine kinase 1/2 antagonist N,N-dimethylsphingosine. RESULTS: Brain protein expression of sphingosine kinase-1 and phosphorylated Akt were significantly increased after isoflurane post-treatment, and cleaved caspase-3 was decreased at 24 hours after surgery, which was reversed by the antagonists. Isoflurane significantly reduced posthemorrhagic ventricular dilation and improved motor, but not cognitive, functions in GMH animals 3 weeks after surgery; no improvements were observed after VPC23019 administration. CONCLUSIONS: Isoflurane post-treatment improved the neurological sequelae after GMH possibly by activation of the sphingosine kinase/Akt pathway.

Annexin A1 upregulates hematoma resolution via the FPR2/p-ERK(1/2)/DUSP1/CD36 signaling pathway after germinal matrix hemorrhage.

Germinal matrix hemorrhage (GMH) is one of the leading causes of morbidity and mortality in preterm infants in the United States, with little progress made in its clinical management. Blood clots disrupting normal cerebrospinal fluid circulation and absorption after germinal matrix hemorrhage are key contributors towards post-hemorrhagic hydrocephalus development. n-formyl peptide receptor 2 (FPR2), a G-protein-coupled receptor, has been associated with the activation of p-ERK1/2, which in turn promotes the transcription of the DUSP1 gene, which may play a role in CD36 signaling. CD36 scavenger, a transmembrane glycoprotein, plays an essential role in microglia phagocytic blood clot clearance after GMH. FPR2's role in blood clot clearance after hemorrhagic stroke is unknown. We hypothesize that FPR2 activation by FPR2 agonist Annexin A1 (AnxA1) will enhance hematoma resolution via the upregulation of the CD36 signaling pathway, thereby improving short- and long-term neurological outcomes. Bacterial collagenase (0.3 U) was infused intraparenchymally into the right hemispheric ganglionic eminence in P7 rat pups to induce GMH. AnxA1 and FPR2 Inhibitor (Boc2) were given at 1-h post-GMH via intranasal administration. FPR2 CRISPR was given 48-h prior to GMH induction. Short-term neurological deficits were assessed using negative geotaxis test. Hematoma volume was assessed using hemoglobin assay. Protein expression was assessed using western blots. Long-term neurocognitive deficits and motor coordination were assessed using Morris water maze, rotarod, and foot fault tests. We have demonstrated that AnxA1 treatment enhances hematoma resolution and improved short and long-term outcomes. Lastly, FPR2 agonist AnxA1 treatment resulted in the upregulation of the FPR2/p-ERK(1/2)/DUSP1/CD36 signaling pathway.

Secukinumab attenuates neuroinflammation and neurobehavior defect via PKCß/ERK/NF-κB pathway in a rat model of GMH.

AIMS: Germinal matrix hemorrhage (GMH) is a disastrous clinical event for newborns. Neuroinflammation plays an important role in the development of neurological deficits after GMH. The purpose of this study is to investigate the anti-inflammatory role of secukinumab after GMH and its underlying mechanisms involving PKCß/ERK/NF-κB signaling pathway. METHODS: A total of 154 Sprague-Dawley P7 rat pups were used. GMH was induced by intraparenchymal injection of bacterial collagenase. Secukinumab was administered intranasally post-GMH. PKCß activator PMA and p-ERK activator Ceramide C6 were administered intracerebroventricularly at 24 h prior to GMH induction, respectively. Neurobehavioral tests, western blot and immunohistochemistry were used to evaluate the efficacy of Secukinumab in both short-term and long-term studies. RESULTS: Endogenous IL-17A, IL-17RA, PKCß and p-ERK were increased after GMH. Secukinumab treatment improved short- and long-term neurological outcomes, reduced the synthesis of MPO and Iba-1 in the perihematoma area, and inhibited the synthesis of proinflammatory factors, such as NF-κB, IL-1ß, TNF-α and IL-6. Additionally, PMA and ceramide C6 abolished the beneficial effects of Secukinumab. CONCLUSION: Secukinumab treatment suppressed neuroinflammation and attenuated neurological deficits after GMH, which was mediated through the downregulation of the PKCß/ERK/NF-κB pathway. Secukinumab treatment may provide a promising therapeutic strategy for GMH patients.

IL-20R Activation via rIL-19 Enhances Hematoma Resolution through the IL-20R1/ERK/Nrf2 Pathway in an Experimental GMH Rat Pup Model.

AIMS: Blood clots play the primary role in neurological deficits after germinal matrix hemorrhage (GMH). Previous studies have shown a beneficial effect in blood clot clearance after hemorrhagic stroke. The purpose of this study is to investigate interleukin-19's role in hematoma clearance after GMH and its underlying mechanism of IL-20R1/ERK/Nrf2 signaling pathway. METHODS: A total of 240 Sprague-Dawley P7 rat pups were used. GMH was induced by intraparenchymal injection of bacterial collagenase. rIL-19 was administered intranasally 1 hour post-GMH. IL-20R1 CRISPR was administered intracerebroventricularly, or Nrf2 antagonist ML385 was administered intraperitoneally 48 hours and 1 hour before GMH induction, respectively. Neurobehavior, Western blot, immunohistochemistry, histology, and hemoglobin assay were used to evaluate treatment regiments in the short- and long-term. RESULTS: Endogenous IL-19, IL-20R1, IL-20R2, and scavenger receptor CD163 were increased after GMH. rIL-19 treatment improved neurological deficits, reduced hematoma volume and hemoglobin content, reduced ventriculomegaly, and attenuated cortical thickness loss. Additionally, treatment increased ERK, Nrf2, and CD163 expression, whereas IL-20R1 CRISPR-knockdown plasmid and ML385 inhibited the effects of rIL-19 on CD163 expression. CONCLUSION: rIL-19 treatment improved hematoma clearance and attenuated neurological deficits induced by GMH, which was mediated through the upregulation of the IL-20R1/ERK/Nrf2 pathways. rIL-19 treatment may provide a promising therapeutic strategy for the GMH patient population.

Anti-inflammation conferred by stimulation of CD200R1 via Dok1 pathway in rat microglia after germinal matrix hemorrhage.

CD200 has been reported to be neuroprotective in neurodegenerative diseases. However, the potential protective effects of CD200 in germinal matrix hemorrhage (GMH) have not been investigated. We examined the anti-inflammatory mechanisms of CD200 after GMH. A total of 167 seven-day-old rat pups were used. The time-dependent effect of GMH on the levels of CD200 and CD200 Receptor 1 (CD200R1) was evaluated by western blot. CD200R1 was localized by immunohistochemistry. The short-term (24 h) and long-term (28 days) outcomes were evaluated after CD200 fusion protein (CD200Fc) treatment by neurobehavioral assessment. CD200 small interfering RNA (siRNA) and downstream of tyrosine kinase 1 (Dok1) siRNA were injected intracerebroventricularly. Western blot was employed to study the mechanisms of CD200 and CD200R1. GMH induced significant developmental delay and caused impairment in both cognitive and motor functions in rat pups. CD200Fc ameliorated GMH-induced damage. CD200Fc increased expression of Dok1 and decreased IL-1beta and TNF-alpha levels. CD200R1 siRNA and Dok1 siRNA abolished the beneficial effects of CD200Fc, as demonstrated by enhanced expression levels of IL-1beta and TNF-alpha. CD200Fc inhibited GMH-induced inflammation and this effect may be mediated by CD200R1/Dok1 pathway. Thus, CD200Fc may serve as a potential treatment to ameliorate brain injury for GMH patients.

Delayed Recanalization Promotes Functional Recovery in Rats Following Permanent Middle Cerebral Artery Occlusion.

Most large vessel stroke patients have permanent occlusion, for which there are no current treatment options. Recent case studies have indicated delayed recanalization, that is recanalization outside of the 6-h treatment window, may lead to improved outcome. We hypothesized that delayed recanalization will restore cerebral blood flow, leading to improved function in rats. Male SD rats were subjected to pMCAO or sham surgery. Delayed recanalization was performed on either day 3, 7, or 14 after pMCAO in a subset of animals. Cerebral blood flow was monitored during suture insertion, during recanalization, and then at sacrifice. Neurological function was evaluated for 1 week after delayed recanalization and at 4 weeks post-ictus. After sacrifice, cerebral morphology was measured. Compared to no treatment, delayed recanalization restored cerebral blood flow, leading to sensorimotor recovery, improved learning and memory, reduced infarct volume, and increased neural stem/progenitor cells within the infarction. The data indicate that earlier delayed recanalization leads to better functional and histological recovery. Yet, even restoring cerebral blood flow 14 days after pMCAO allows for rats to regain sensorimotor function. This exploratory study suggests that delayed recanalization may be a viable option for treatment of permanent large vessel stroke.

α7 Nicotinic Acetylcholine Receptor Stimulation Attenuates Neuroinflammation through JAK2-STAT3 Activation in Murine Models of Intracerebral Hemorrhage.

Accounting for high mortality and morbidity rates, intracerebral hemorrhage (ICH) remains one of the most detrimental stroke subtypes lacking a specific therapy. Neuroinflammation contributes to ICH-induced brain injury and is associated with unfavorable outcomes. This study aimed to evaluate whether α7 nicotinic acetylcholine receptor (α7nAChR) stimulation ameliorates neuroinflammation after ICH. Male CD-1 mice and Sprague-Dawley were subjected to intracerebral injection of autologous blood or bacterial collagenase. ICH animals received either α7nAChR agonist PHA-543613 alone or combined with α7nAChR antagonist methyllycaconitine (MLA) or Janus kinase 2 (JAK2) antagonist AG490. Neurobehavioral deficits were evaluated at 24 hours, 72 hours, and 10 weeks after ICH induction. Perihematomal expressions of JAK2, signal transducer and activator of transcription 3 (STAT3), tumor necrosis factor-α (TNF-α), and myeloperoxidase (MPO) were quantified via Western blot. Histologic volumetric analysis of brain tissues was conducted after 10 weeks following ICH induction. PHA-543613 improved short-term neurobehavioral (sensorimotor) deficits and increased activated perihematomal JAK2 and STAT3 expressions while decreasing TNF-α and MPO expressions after ICH. MLA reversed these treatment effects. PHA-543613 also improved long-term neurobehavioral (sensorimotor, learning, and memory) deficits and ameliorated brain atrophy after ICH. These treatment effects were reduced by AG490. α7nAChR stimulation reduced neuroinflammation via activation of the JAK2-STAT3 pathway, thereby ameliorating the short- and long-term sequelae after ICH.

Dabigatran ameliorates post-haemorrhagic hydrocephalus development after germinal matrix haemorrhage in neonatal rat pups.

We aim to determine if direct thrombin inhibition by dabigatran will improve long-term brain morphological and neurofunctional outcomes and if potential therapeutic effects are dependent upon reduced PAR-1 stimulation and consequent mTOR activation. Germinal matrix haemorrhage was induced by stereotaxically injecting 0.3 U type VII-S collagenase into the germinal matrix of P7 rat pups. Animals were divided into five groups: sham, vehicle (5% DMSO), dabigatran intraperitoneal, dabigatran intraperitoneal + TFLLR-NH2 (PAR-1 agonist) intranasal, SCH79797 (PAR-1 antagonist) intraperitoneal, and dabigatran intranasal. Neurofunctional outcomes were determined by Morris water maze, rotarod, and foot fault evaluations at three weeks. Brain morphological outcomes were determined by histological Nissl staining at four weeks. Expression levels of p-mTOR/p-p70s6k at three days and vitronectin/fibronectin at 28 days were quantified. Intranasal and intraperitoneal dabigatran promoted long-term neurofunctional recovery, improved brain morphological outcomes, and reduced intracranial pressure at four weeks after GMH. PAR-1 stimulation tended to reverse dabigatran's effects on post-haemorrhagic hydrocephalus development. Dabigatran also reduced expression of short-term p-mTOR and long-term extracellular matrix proteins, which tended to be reversed by PAR-1 agonist co-administration. PAR-1 inhibition alone, however, did not achieve the same therapeutic effects as dabigatran administration.

Modulating the Immune Response Towards a Neuroregenerative Peri-injury Milieu After Cerebral Hemorrhage.

Cerebral hemorrhages account for 15-20 % of stroke sub-types and have very poor prognoses. The mortality rate for cerebral hemorrhage patients is between 40 and 50 %, of which at least half of the deaths occur within the first 2 days, and 75 % of survivors are incapable of living independently after 1 year. Current emergency interventions involve lowering blood pressure and reducing intracranial pressure by controlled ventilations or, in the worst case scenarios, surgical intervention. Some hemostatic and coagulatherapeutic interventions are being investigated, although a few that were promising in experimental studies have failed in clinical trials. No significant immunomodulatory intervention, however, exists for clinical management of cerebral hemorrhage. The inflammatory response following cerebral hemorrhage is particularly harmful in the acute stage because blood-brain barrier disruption is amplified and surrounding tissue is destroyed by secreted proteases and reactive oxygen species from infiltrated leukocytes. In this review, we discuss both the destructive and regenerative roles the immune response play following cerebral hemorrhage and focus on microglia, macrophages, and T-lymphocytes as the primary agents directing the response. Microglia, macrophages, and T-lymphocytes each have sub-types that significantly influence the over-arching immune response towards either a pro-inflammatory, destructive, or an anti-inflammatory, regenerative, state. Both pre-clinical and clinical studies of cerebral hemorrhages that selectively target these immune cells are reviewed and we suggest immunomodulatory therapies that reduce inflammation, while augmenting neural repair, will improve overall cerebral hemorrhage outcomes.

Erythropoietin inhibits HIF-1α expression via upregulation of PHD-2 transcription and translation in an in vitro model of hypoxia-ischemia.

Hypoxia inducible factor (HIF)-1α is the central transcriptional factor for the regulation of oxygen-associated genes in response to hypoxia. Erythropoietin (EPO), a hematopoietic growth factor, increases oxygen availability during hypoxia/ischemia and is associated with neuroprotection following hypoxia-ischemia in laboratory models of stroke. However, EPO has failed to translate in a clinical setting. Thus, it is critical to elucidate the key players in EPO-induced neuroprotection. Our preliminary studies have shown that EPO, as a downstream gene of HIF, inhibits HIF-1α in a dose-dependent manner in an in vitro model of hypoxia-ischemia. This study is designed to elucidate the primary mediator of EPO-induced HIF-1α inhibition and subsequent cell survival/neuroprotection. Oxygen and glucose deprivation (OGD) of nerve growth factor-differentiated rat pheochromocytoma (PC-12) cells were used to model hypoxia-ischemia in an in vitro environment. The profile of HIF-1α, HIF-2α and prolyl hydroxylase domain 2 (PHD-2) expression; HIF-1α and prolyl hydroxylase (PHD-2) mRNA levels; matrix metalloproteinase (MMP)-9; and cell death was evaluated in the presence and absence of either EPO or PHD-2 inhibitor during OGD. Our findings showed that EPO treatment resulted in an increase in PHD-2 transcription and translation, inhibition of HIF-1α expression, reactive oxygen species formation, and MMP-9 activity, resulting in increased cell survival after OGD. We also observed that EPO-induced cell survival/neuroprotection was reversed by siRNA silencing of PHD-2. This led to the conclusion that PHD-2 is a key mediator of EPO-induced HIF-1α inhibition and subsequent neuroprotection in an in vitro model of hypoxia-ischemia.