Activation of GPR39 with TC-G 1008 attenuates neuroinflammation via SIRT1/PGC-1α/Nrf2 pathway post-neonatal hypoxic-ischemic injury in rats.

BACKGROUND: Hypoxic-ischemic encephalopathy (HIE) is a severe anoxic brain injury that leads to premature mortality or long-term disabilities in infants. Neuroinflammation is a vital contributor to the pathogenic cascade post-HIE and a mediator to secondary neuronal death. As a plasma membrane G-protein-coupled receptor, GPR39, exhibits anti-inflammatory activity in several diseases. This study aimed to explore the neuroprotective function of GPR39 through inhibition of inflammation post-hypoxic-ischemic (HI) injury and to elaborate the contribution of sirtuin 1(SIRT1)/peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α)/nuclear factor, erythroid 2 like 2(Nrf2) in G-protein-coupled receptor 39 (GPR39)-mediated protection. METHODS: A total of 206 10-day-old Sprague Dawley rat pups were subjected to HIE or sham surgery. TC-G 1008 was administered intranasally at 1 h, 25 h, 49 h, and 73 h post-HIE induction. SIRT1 inhibitor EX527, GPR39 CRISPR, and PGC-1α CRISPR were administered to elucidate the underlying mechanisms. Brain infarct area, short-term and long-term neurobehavioral tests, Nissl staining, western blot, and immunofluorescence staining were performed post-HIE. RESULTS: The expression of GPR39 and pathway-related proteins, SIRT1, PGC-1α and Nrf2 were increased in a time-dependent manner, peaking at 24 h or 48-h post-HIE. Intranasal administration of TC-G 1008 reduced the percent infarcted area and improved short-term and long-term neurological deficits. Moreover, TC-G 1008 treatment significantly increased the expression of SIRT1, PGC-1α and Nrf2, but downregulated the expressions of IL-6, IL-1ß, and TNF-α. GPR39 CRISPR EX527 and PGC-1α CRISPR abolished GPR39's neuroprotective effects post-HIE. CONCLUSIONS: TC-G 1008 attenuated neuroinflammation in part via the SIRT1/PGC-1α/Nrf2 pathway in a neonatal rat model of HIE. TC-G 1008 may be a novel therapeutic target for treatment post-neonatal HIE injury.

Activation of MC1R with BMS-470539 attenuates neuroinflammation via cAMP/PKA/Nurr1 pathway after neonatal hypoxic-ischemic brain injury in rats.

BACKGROUND: Microglia-mediated neuroinflammation plays a crucial role in the pathogenesis of hypoxic-ischemic (HI)-induced brain injury. Activation of melanocortin-1 receptor (MC1R) has been shown to exert anti-inflammatory and neuroprotective effects in several neurological diseases. In the present study, we have explored the role of MC1R activation on neuroinflammation and the potential underlying mechanisms after neonatal hypoxic-ischemic brain injury in rats. METHODS: A total of 169 post-natal day 10 unsexed rat pups were used. HI was induced by right common carotid artery ligation followed by 2.5 h of hypoxia. BMS-470539, a specific selective MC1R agonist, was administered intranasally at 1 h after HI induction. To elucidate the potential underlying mechanism, MC1R CRISPR KO plasmid or Nurr1 CRISPR KO plasmid was administered via intracerebroventricular injection at 48 h before HI induction. Percent brain infarct area, short- and long-term neurobehavioral tests, Nissl staining, immunofluorescence staining, and Western blot were conducted. RESULTS: The expression levels of MC1R and Nurr1 increased over time post-HI. MC1R and Nurr1 were expressed on microglia at 48 h post-HI. Activation of MC1R with BMS-470539 significantly reduced the percent infarct area, brain atrophy, and inflammation, and improved short- and long-term neurological deficits at 48 h and 28 days post-HI. MC1R activation increased the expression of CD206 (a microglial M2 marker) and reduced the expression of MPO. Moreover, activation of MC1R with BMS-470539 significantly increased the expression levels of MC1R, cAMP, p-PKA, and Nurr1, while downregulating the expression of pro-inflammatory cytokines (TNFα, IL-6, and IL-1ß) at 48 h post-HI. However, knockout of MC1R or Nurr1 by specific CRISPR reversed the neuroprotective effects of MC1R activation post-HI. CONCLUSIONS: Our study demonstrated that activation of MC1R with BMS-470539 attenuated neuroinflammation, and improved neurological deficits after neonatal hypoxic-ischemic brain injury in rats. Such anti-inflammatory and neuroprotective effects were mediated, at least in part, via the cAMP/PKA/Nurr1 signaling pathway. Therefore, MC1R activation might be a promising therapeutic target for infants with hypoxic-ischemic encephalopathy (HIE).

GW0742 activates miR-17-5p and inhibits TXNIP/NLRP3-mediated inflammation after hypoxic-ischaemic injury in rats and in PC12 cells.

This study aimed to investigate the effects of PPAR-ß/δ receptor agonist GW0742 on neuroinflammation in a rat model of hypoxia-ischaemia (HI) and in PC12 cells in OGD model. HI was induced by ligating the common carotid artery and inducing hypoxia for 150 minutes. Immunofluorescence was used for quantification of microglia activation and for determining cellular localization of PPAR-ß/δ. Expression of proteins was measured by Western blot. Activation of miR-17-5p by GW0742 was assessed in PC12 cells by Dual-Luciferase Reporter Gene Assay. The endogenous expression of TXNIP, NLRP3, cleaved caspase-1 and IL-1ß was increased after HI. GW0742 treatment significantly reduced the number of activated pro-inflammatory microglia in ipsilateral hemisphere after HI. Mechanistically, GW0742 significantly decreased the expression of TXNIP, NLRP3, IL-6 and TNF-α. Either PPAR-ß/δ antagonist GSK3787, miR-17-5p inhibitor, or TXNIP CRISPR activation abolished the anti-inflammatory effects of GW0742. Activation of PPAR-ß/δ by GW0742 activated miR-17-5p expression in PC12 cells and increased cell viability after OGD, which was accompanied by decreased expression of TXNIP and reduced secretion of IL-1ß and TNF-α. In conclusion, GW0742 may be a promising neurotherapeutic for the management of HI patients.

IRE1α inhibition attenuates neuronal pyroptosis via miR-125/NLRP1 pathway in a neonatal hypoxic-ischemic encephalopathy rat model.

BACKGROUND: Inhibition of inositol-requiring enzyme-1 alpha (IRE1α), one of the sensor signaling proteins associated with endoplasmic reticulum (ER) stress, has been shown to alleviate brain injury and improve neurological behavior in a neonatal hypoxic-ischemic encephalopathy (HIE) rat model. However, there is no information about the role of IRE1α inhibitor as well as its molecular mechanisms in preventing neuronal pyroptosis induced by NLRP1 (NOD-, LRR- and pyrin domain-containing 1) inflammasome. In the present study, we hypothesized that IRE1α can degrade microRNA-125-b-2-3p (miR-125-b-2-3p) and activate NLRP1/caspased-1 pathway, and subsequently promote neuronal pyroptosis in HIE rat model. METHODS: Ten-day old unsexed rat pups were subjected to hypoxia-ischemia (HI) injury, and the inhibitor of IRE1α, STF083010, was administered intranasally at 1 h after HI induction. AntimiR-125 or NLRP1 activation CRISPR was administered by intracerebroventricular (i.c.v) injection at 24 h before HI induction. Immunofluorescence staining, western blot analysis, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), brain infarct volume measurement, neurological function tests, and Fluoro-Jade C staining were performed. RESULTS: Endogenous phosphorylated IRE1α (p-IRE1α), NLRP1, cleaved caspase-1, interleukin-1ß (IL-1ß), and interleukin-18 (IL-18) were increased and miR-125-b-2-3p was decreased in HIE rat model. STF083010 administration significantly upregulated the expression of miR-125-b-2-3p, reduced the infarct volume, improved neurobehavioral outcomes and downregulated the protein expression of NLRP1, cleaved caspase-1, IL-1ß and IL-18. The protective effects of STF083010 were reversed by antimiR-125 or NLRP1 activation CRISPR. CONCLUSIONS: IRE1α inhibitor, STF083010, reduced neuronal pyroptosis at least in part via miR-125/NLRP1/caspase-1 signaling pathway after HI.

Rh-CSF1 attenuates neuroinflammation via the CSF1R/PLCG2/PKCε pathway in a rat model of neonatal HIE.

BACKGROUND: Hypoxic-ischemic encephalopathy (HIE) is a life-threatening cerebrovascular disease. Neuroinflammation plays an important role in the pathogenesis of HIE, in which microglia are key cellular mediators in the regulation of neuroinflammatory processes. Colony-stimulating factor 1 (CSF1), a specific endogenous ligand of CSF1 receptor (CSF1R), is crucial in microglial growth, differentiation, and proliferation. Recent studies showed that the activation of CSF1R with CSF1 exerted anti-inflammatory effects in a variety of nervous system diseases. This study aimed to investigate the anti-inflammatory effects of recombinant human CSF1 (rh-CSF1) and the underlying mechanisms in a rat model of HIE. METHODS: A total of 202 10-day old Sprague Dawley rat pups were used. HI was induced by the right common carotid artery ligation with subsequent exposure of 2.5-h hypoxia. At 1 h and 24 h after HI induction, exogenous rh-CSF1 was administered intranasally. To explore the underlying mechanism, CSF1R inhibitor, BLZ945, and phospholipase C-gamma 2 (PLCG2) inhibitor, U73122, were injected intraperitoneally at 1 h before HI induction, respectively. Brain infarct area, brain water content, neurobehavioral tests, western blot, and immunofluorescence staining were performed. RESULTS: The expressions of endogenous CSF1, CSF1R, PLCG2, protein kinase C epsilon type (PKCε), and cAMP response element-binding protein (CREB) were gradually increased after HIE. Rh-CSF1 significantly improved the neurological deficits at 48 h and 4 weeks after HI, which was accompanied by a reduction in the brain infarct area, brain edema, brain atrophy, and neuroinflammation. Moreover, activation of CSF1R by rh-CSF1 significantly increased the expressions of p-PLCG2, p-PKCε, and p-CREB, but inhibited the activation of neutrophil infiltration, and downregulated the expressions of IL-1ß and TNF-α. Inhibition of CSF1R and PLCG2 abolished these neuroprotective effects of rh-CSF1 after HI. CONCLUSIONS: Our findings demonstrated that the activation of CSF1R by rh-CSF1 attenuated neuroinflammation and improved neurological deficits after HI. The anti-inflammatory effects of rh-CSF1 partially acted through activating the CSF1R/PLCG2/PKCε/CREB signaling pathway after HI. These results suggest that rh-CSF1 may serve as a potential therapeutic approach to ameliorate injury in HIE patients.

The characteristics of the ancient cell death suppressor, TMBIM6, and its related signaling pathways after endoplasmic reticulum stress.

Activation of the unfolded protein response in combination with generation of reactive oxygen species, from cytochrome P450 members and NADPH-P450 reductases, are two major consequences of Endoplasmic Reticulum (ER) stress that cause oxidative damage and cell death. Herein, we reviewed the role of Bax Inhibitor-1 (BI-1), an evolutionarily conserved protein encoded by the Transmembrane Bax inhibitor Motif Containing 6 gene, in protection from ER stress. As BI-1 has multimodal properties that can target a wide array of pathophysiological consequences after injury, our main objective was to explore BI-1's protective role in ER stress and its potential signaling pathways.

Viral-mediated gene delivery of TMBIM6 protects the neonatal brain via disruption of NPR-CYP complex coupled with upregulation of Nrf-2 post-HI.

BACKGROUND: Oxidative stress, inflammation, and endoplasmic reticulum (ER) stress play a major role in the pathogenesis of neonatal hypoxic-ischemic (HI) injury. ER stress results in the accumulation of unfolded proteins that trigger the NADPH-P450 reductase (NPR) and the microsomal monooxygenase system which is composed of cytochrome P450 members (CYP) generating reactive oxygen species (ROS) as well as the release of inflammatory cytokines. We explored the role of Bax Inhibitor-1 (BI-1) protein, encoded by the Transmembrane Bax inhibitor Motif Containing 6 (TMBIM6) gene, in protection from ER stress after HI brain injury. BI-1 may attenuate ER stress-induced ROS production and release of inflammatory mediators via (1) disruption of the NPR-CYP complex and (2) upregulation of Nrf-2, a redox-sensitive transcription factor, thus promoting an increase in anti-oxidant enzymes to inhibit ROS production. The main objective of our study is to evaluate BI-1's inhibitory effects on ROS production and inflammation by overexpressing BI-1 in 10-day-old rat pups. METHODS: Ten-day-old (P10) unsexed Sprague-Dawley rat pups underwent right common carotid artery ligation, followed by 1.5 h of hypoxia. To overexpress BI-1, rat pups were intracerebroventricularly (icv) injected at 48 h pre-HI with the human adenoviral vector-TMBIM6 (Ad-TMBIM6). BI-1 and Nrf-2 silencing were achieved by icv injection at 48 h pre-HI using siRNA to elucidate the potential mechanism. Percent infarcted area, immunofluorescent staining, DHE staining, western blot, and long-term neurobehavior assessments were performed. RESULTS: Overexpression of BI-1 significantly reduced the percent infarcted area and improved long-term neurobehavioral outcomes. BI-1's mediated protection was observed to be via inhibition of P4502E1, a major contributor to ROS generation and upregulation of pNrf-2 and HO-1, which correlated with a decrease in ROS and inflammatory markers. This effect was reversed when BI-1 or Nrf-2 were inhibited. CONCLUSIONS: Overexpression of BI-1 increased the production of antioxidant enzymes and attenuated inflammation by destabilizing the complex responsible for ROS production. BI-1's multimodal role in inhibiting P4502E1, together with upregulating Nrf-2, makes it a promising therapeutic target.

IRE1α inhibition decreased TXNIP/NLRP3 inflammasome activation through miR-17-5p after neonatal hypoxic-ischemic brain injury in rats.

BACKGROUND: The endoplasmic reticulum (ER) is responsible for the control of correct protein folding and protein function which is crucial for cell survival. However, under pathological conditions, such as hypoxia-ischemia (HI), there is an accumulation of unfolded proteins thereby triggering the unfolded protein response (UPR) and causing ER stress which is associated with activation of several stress sensor signaling pathways, one of them being the inositol requiring enzyme-1 alpha (IRE1α) signaling pathway. The UPR is regarded as a potential contributor to neuronal cell death and inflammation after HI. In the present study, we sought to investigate whether microRNA-17 (miR-17), a potential IRE1α ribonuclease (RNase) substrate, arbitrates downregulation of thioredoxin-interacting protein (TXNIP) and consequent NLRP3 inflammasome activation in the immature brain after HI injury and whether inhibition of IRE1α may attenuate inflammation via miR-17/TXNIP regulation. METHODS: Postnatal day 10 rat pups (n = 287) were subjected to unilateral carotid artery ligation followed by 2.5 h of hypoxia (8% O2). STF-083010, an IRE1α RNase inhibitor, was intranasally delivered at 1 h post-HI or followed by an additional one administration per day for 2 days. MiR-17-5p mimic or anti-miR-17-5p inhibitor was injected intracerebroventricularly at 48 h before HI. Infarct volume and body weight were used to evaluate the short-term effects while brain weight, gross and microscopic brain tissue morphologies, and neurobehavioral tests were conducted for the long-term evaluation. Western blots, immunofluorescence staining, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), and co-immunoprecipitation (Co-IP) were used for mechanism studies. RESULTS: Endogenous phosphorylated IRE1α expression was significantly increased after HI. Intranasal administration of STF-083010 alleviated brain injury and improved neurological behavior. MiR-17-5p expression was reduced after HI, and this decrease was attenuated by STF-083010 treatment. MiR-17-5p mimic administration ameliorated TXNIP expression, NLRP3 inflammasome activation, caspase-1 cleavage, and IL-1ß production, as well as brain infarct volume. Conversely, anti-miR-17-5p inhibitor reversed IRE1α inhibition-induced decrease in TXNIP expression and inflammasome activation, as well as exacerbated brain injury after HI. CONCLUSIONS: IRE1a-induced UPR pathway may contribute to inflammatory activation and brain injury following neonatal HI. IRE1a activation, through decay of miR-17-5p, elevated TXNIP expression to activate NLRP3 inflammasome and aggravated brain damage.

Chemerin suppresses neuroinflammation and improves neurological recovery via CaMKK2/AMPK/Nrf2 pathway after germinal matrix hemorrhage in neonatal rats.

Chemerin, an adipokine, has been reported to reduce the production of pro-inflammatory cytokines and neutrophil infiltration. This study investigated the role of Chemerin and its natural receptor, ChemR23, as well as its downstream mediator calmodulin-dependent protein kinase kinase 2 (CAMKK2)/adenosine monophosphate-activated protein kinase (AMPK) /Nuclear factor erythroid 2-related factor 2 (Nrf2) following germinal matrix hemorrhage (GMH) in neonatal rats, with a specific focus on inflammation. GMH was induced by intraparenchymal injection of bacterial collagenase (0.3U) in P7 rat pups. The results demonstrated that human recombinant Chemerin (rh-Chemerin) improved neurological and morphological outcomes after GMH. Rh-Chemerin promoted accumulation and proliferation of M2 microglia in periventricular regions at 72 h. Rh-Chemerin increased phosphorylation of CAMKK2, AMPK and expression of Nrf2, and decreased IL-1beta, IL-6 and TNF-alpha levels. Selective inhibition of ChemR23/CAMKK2/AMPK signaling in microglia via intracerebroventricular delivery of liposome-encapsulated specific ChemR23 (Lipo-alpha-NETA), CAMKK2 (Lipo-STO-609) and AMPK (Lipo-Dorsomorphin) inhibitor increased the expression levels of IL-1beta, IL-6 and TNF- alpha, demonstrating that ChemR23/CAMKK2/AMPK signaling in microglia suppressed inflammatory response after GMH. Cumulatively, these data showed that rh-Chemerin ameliorated GMH-induced inflammatory response by promoting ChemR23/CAMKK2/AMPK/Nrf2 pathway, and M2 microglia may be a major mediator of this effect. Thus, rh-Chemerin can serve as a potential agent to reduce the inflammatory response following GMH.

Sestrin2 induced by hypoxia inducible factor1 alpha protects the blood-brain barrier via inhibiting VEGF after severe hypoxic-ischemic injury in neonatal rats.

OBJECTIVE: Hypoxic ischemic (HI) encephalopathy remains the leading cause of perinatal brain injury resulting in long term disabilities. Stabilization of blood brain barrier (BBB) after HI is an important target, therefore, in this study we aim to determine the role of sestrin2, a stress inducible protein which is elevated after various insults, on BBB stabilization after moderate and severe HI injuries. METHODS: Rat pups underwent common carotid artery ligation followed by either 150min (severe model) or 100min (moderate model) of hypoxia. 1h post HI, rats were intranasally administered with recombinant human sestrin2 (rh-sestrin2) and sacrificed for infarct area, brain water content, righting reflex and geotaxis reflex. Sestrin2 was silenced using siRNA and an activator/inhibitor of hypoxia inducible factor1α (HIF1α) was used to examine their roles on BBB permeability. RESULTS: Rats subjected to severe HI exhibited larger infarct area and higher sestrin2 expression compared to rats in the moderate HI group. rh-sestrin2 attenuated brain infarct and edema, while silencing sestrin2 reversed these protective effects after severe HI. HIF1α induced sestrin2 activation in severe HI but not in moderate HI groups. A HIF1a agonist was shown to increase permeability of the BBB via vascular endothelial growth factor (VEGF) after moderate HI. However, after severe HI, HIF1α activated both VEGF and sestrin2. But HIF1α dependent sestrin2 activation was the predominant pathway after severe HI which inhibited VEGF and attenuated BBB permeability. CONCLUSIONS: rh-sestrin2 attenuated BBB permeability via upregulation of endogenous sestrin2 which was induced by HIF1α after severe HI. However, HIF1α's effects as a prodeath or prosurvival signal were influenced by the severity of HI injury.

Cannabinoid Receptor Type 2 Agonist Attenuates Acute Neurogenic Pulmonary Edema by Preventing Neutrophil Migration after Subarachnoid Hemorrhage in Rats.

We evaluated whether JWH133, a selective cannabinoid type 2 receptor (CB2R) agonist, prevented neurogenic pulmonary edema (NPE) after subarachnoid hemorrhage (SAH) by attenuating inflammation. Adult male rats were assigned to six groups: sham-operated, SAH with vehicle, SAH with JWH133 (0.3, 1.0, or 3.0 mg/kg) treatment 1 h after surgery, and SAH with JWH133 (1.0 mg/kg) at 1 h with a selective CB2R antagonist, SR144528 (3.0 mg/kg). The perforation model of SAH was performed and pulmonary wet-to-dry weight ratio was evaluated 24 and 72 h after surgery. Western blot analyses and immunohistochemistry were evaluated 24 h after surgery. JWH133 (1.0 mg/kg) significantly and most strongly improved lung edema 24 h after SAH. SR144528 administration significantly reversed the effects of JWH133 (1.0 mg/kg). SAH-induced increasing levels of myeloperoxidase (MPO) and decreasing levels of a tight junction (TJ) protein, junctional adhesion molecule (JAM)-A, were ameliorated by JWH133 (1.0 mg/kg) administration 24 h after SAH. Immunohistochemical assessment also confirmed substantial leukocyte infiltration in the outside of vessels in SAH, which were attenuated by JWH133 (1.0 mg/kg) injection. CB2R agonist ameliorated lung permeability by inhibiting leukocyte trafficking and protecting tight junction proteins in the lung of NPE after SAH.

Subarachnoid Hemorrhage-Triggered Acute Hypotension Is Associated with Left Ventricular Cardiomyocyte Apoptosis in a Rat Model.

Whether hypotension that occurs due to neurogenic stunned myocardium after subarachnoid hemorrhage (SAH) is associated with cardiomyocyte apoptotic cell death remains unknown. In this study, 18 male rats were subjected to sham or the endovascular perforation model of SAH surgery. Based on the mean arterial pressure (MAP) after SAH, rats were separated into SAH with hypotension (SAH hypotension) or SAH with blood pressure preservation (SAH BP preservation) groups. All animals were euthanized 2 h after the surgical procedure. Hearts were removed and separated transversely into base and apex parts, then Western blot analyses and immunohistochemistry were performed only in the apex part. One rat died as a result of severe SAH and two rats with mild SAH were excluded. We analyzed data from 15 rats that were divided into three groups: sham, SAH hypotension, and SAH BP preservation (n = 5, each). There was a significantly higher cleaved caspase-3/caspase-3 ratio in the SAH hypotension group compared with sham and the SAH BP preservation group. Cardiomyocyte apoptosis was demonstrated in the SAH rats. This is the first experimental report that describes SAH-induced neurogenic stunned myocardium with ensuing hypotension may result from the acute apoptotic cardiomyocyte cell death in the left ventricle.

Intranasal IGF-1 Reduced Rat Pup Germinal Matrix Hemorrhage.

Germinal matrix hemorrhage (GMH) is the most devastating neurological problem of premature infants. Current treatment strategies are ineffective and brain injury is unpreventable. Insulin-like growth factor 1 (IGF-1) is an endogenous protein shown to have multiple neuroprotective properties. We therefore hypothesized that IGF-1 would reduce brain injury after GMH. Neonatal rats (P7 age) received stereotactic collagenase into the right ganglionic eminence. The following groups were studied: (1) sham, (2) GMH + vehicle, (3) GMH + intranasal IGF-1. Three days later, the animals were evaluated using the righting-reflex (early neurobehavior), Evans blue dye leakage (blood-brain barrier (BBB) permeability), brain water content (edema), and hemoglobin assay (extent of bleeding). Three weeks later, juvenile rats were tested using a water maze (delayed neurobehavior), and then were sacrificed on day 28 for assessment of hydrocephalus (ventricular size). Intranasal IGF-1 treated animals had improved neurological function, and amelioration of BBB permeability, edema, and re-bleeding. IGF-1 may play a part in protective brain signaling following GMH, and our observed protective effect may offer new promise for treatment targeting this vulnerable patient population.

Intranasal Osteopontin for Rodent Germinal Matrix Hemorrhage.

Germinal matrix hemorrhage (GMH) is the most common and devastating neurological problem of premature infants. Current treatment is largely ineffective and GMH has been nonpreventable. Osteopontin (OPN) is an endogenous protein that has been shown to be neuroprotective, however, it has not been tested in GMH. P7 neonatal rats were subjected to stereotactic ganglionic eminence collagenase infusion. Groups were as follows: (1) sham, (2) GMH + vehicle, (3) GMH + intranasal OPN. Seventy-two hours later, the animals were evaluated using righting reflex, blood-brain barrier (BBB) permeability by Evans blue dye leakage, brain water content, and hemoglobin assay. Intranasal OPN improved outcomes after GMH by attenuation of brain swelling, BBB function, re-bleeding, and neurological outcomes. OPN may play an important role in enhancing neuroprotective brain signaling following GMH. These observed effects may offer novel possibilities for therapy in this patient population.

Osteopontin-Rac1 on Blood-Brain Barrier Stability Following Rodent Neonatal Hypoxia-Ischemia.

Osteopontin (OPN) is a neuroprotective molecule that is upregulated following rodent neonatal hypoxic-ischemic (nHI) brain injury. Because Rac1 is a regulator of blood-brain barrier (BBB) stability, we hypothesized a role for this in OPN signaling. nHI was induced by unilateral ligation of the right carotid artery followed by hypoxia (8 % oxygen for 2 h) in P10 Sprague-Dawley rat pups. Intranasal (iN) OPN was administered at 1 h post-nHI. Groups consisted of: (1) Sham, (2) Vehicle, (3) OPN, and (4) OPN + Rac1 inhibitor (NSC23766). Evans blue dye extravasation (BBB permeability) was quantified 24 h post-nHI, and brain edema at 48 h. Increased BBB permeability and brain edema following nHI was ameliorated in the OPN treatment group. However, those rat pups receiving OPN co-treatment with the Rac1 inhibitor experienced no improvement compared with vehicle. OPN protects the BBB following nHI, and this was reversed by Rac1 inhibitor (NSC23766).

Changes in Brain Swelling and Infarction Volume over Four Days After Hypoxia Ischemia in Neonatal Rats.

The leading cause of morbidity and mortality in infants is hypoxia-ischemia (HI). The current therapies for HI have limited success, in part due to a lack of understanding of HI pathophysiology and underlying mechanisms. Herein, a neonatal rat model of HI was used to examine the changes in brain swelling and infarct volume over 4 days after HI. Forty-four P10 rat pups were sacrificed at 2, 3, or 4 days post-HI. After sacrifice, the brains were removed, sliced, and stained with TTC (2,3,5-triphenyl-2H-tetrazolium chloride). Images of TTC-stained brains were used for measurement of the ipsilateral hemisphere brain volumes and infarct volumes, calculated using standard equations. The hemispheric brain volumes of HI animals in all groups was lower than that of sham animals and decreased as the post-HI sacrifice time increased. The infarct volume of HI animals was larger than that of sham animals. Infarct volumes tended to decrease over the days post-HI. The change in infarct volume is likely the result of a combination of brain growth and repair mechanisms. However, changes in the hemispheric brain volume may include tissue growth and repair mechanism, so also may be a limitation of the current algorithm used for calculating ipsilateral hemisphere brain volume.

Delayed hyperbaric oxygen therapy promotes neurogenesis through reactive oxygen species/hypoxia-inducible factor-1α/ß-catenin pathway in middle cerebral artery occlusion rats.

BACKGROUND AND PURPOSE: Hyperbaric oxygen (HBO) has been reported to be neuroprotective and to improve neurofunctional outcomes in acute stroke. However, it is not clear whether delayed HBO enhances endogenous neurogenesis and promotes neurofunctional recovery. The aim of this study is to evaluate the effects of delayed HBO therapy on neurogenesis and its potential mechanisms. METHODS: One hundred eleven male Sprague-Dawley rats that survived for 7 days from 2 hours of middle cerebral artery occlusion and reperfusion were used. Delayed and multiple HBO were administrated beginning at 7 days after middle cerebral artery occlusion and lasting for 42 days with 3 HBO-free intervals (5 days each). Motor sensory deficits were measured by foot-fault test, and learning and memory abilities were evaluated by Morris water maze. Neurogenesis was examined by double immunostaining of bromodeoxyuridine and doublecortin, bromodeoxyuridine and neuronal nuclei at day 42. For mechanism studies, inhibitors for reactive oxygen species (ROS), hypoxia-inducible factor (HIF)-1α, and ß-catenin were administrated, and the levels of ROS, HIF-1α, ß-catenin, lymphoid enhancer-binding factor-1, T-cell factor-1, neurogenin-1, doublecortin, and synapsin-1 were assessed by ELISA or Western blot at day 14. RESULTS: Delayed HBO treatment promoted neurogenesis and improved neurofunctional recovery at day 42, and the improvements were reversed by inhibition of ROS and HIF-1α. Delayed HBO significantly increased ROS and HIF-1α, and upregulated the expression of neurogenin-1, doublecortin, and synapsin-1. Inhibition of ROS and HIF-1α removed the effects of delayed HBO. CONCLUSIONS: Delayed HBO enhanced endogenous neurogenesis and improved neurofunctional recovery in the late-chronic phase of stroke possibly mediated by ROS/HIF-1α/ß-catenin pathway. Delayed HBO may serve as an alternative treatment to improve long-term recovery of stroke survivors.

Anti-neutrophil antibody enhances the neuroprotective effects of G-CSF by decreasing number of neutrophils in hypoxic ischemic neonatal rat model.

OBJECTIVES: Neonatal hypoxia ischemia (HI) is an injury that can lead to neurological impairments such as behavioral and learning disabilities. Granulocyte-colony stimulating factor (G-CSF) has been demonstrated to be neuroprotective in ischemic stroke however it has also been shown to induce neutrophilia, ultimately exacerbating neuronal injury. Our hypothesis is that coadministration of anti-neutrophil antibody (Ab) with G-CSF will decrease blood neutrophil counts thereby reducing infarct volume and improving neurological function post HI brain injury. METHODS: Rat pups were subjected to unilateral carotid artery ligation followed by 2.5h of hypoxia. Animals were randomly assigned to five groups: Sham (n=15), vehicle (HI, n=15), HI with G-CSF treatment (n=15), HI with G-CSF+Ab treatment (n=15), and HI with Ab treatment (n=15). Ab (325µg/kg) was administered intraperitoneally while G-CSF (50µg/kg) was administered subcutaneously 1h post HI followed by daily injections for 3 consecutive days. Animals were euthanized at 96h post HI for blood neutrophil counts and brain infarct volume measurements as well as at 5weeks for neurological function testing and brain weight measurements. Lung and spleen weights at both time points were further analyzed. RESULTS: The G-CSF treatment group showed tendencies to reduce infarct volume and improve neurological function while significantly increasing neutrophil counts. On the other hand, the G-CSF+Ab group significantly reduced infarct volume, improved neurological function and decreased neutrophil counts. The Ab alone group showed reversal of the neuroprotective effects of the G-CSF+Ab group. No significant differences were found in peripheral organ weights between groups. CONCLUSION: Our data suggest that coadministration of G-CSF with Ab not only prevented brain atrophy but also significantly improved neurological function by decreasing blood neutrophil counts. Hence the neuroprotective effects of G-CSF may be further enhanced if neutrophilia is avoided.

Delayed recanalization reduced neuronal apoptosis and neurological deficits by enhancing liver-derived trefoil factor 3-mediated neuroprotection via LINGO2/EGFR/Src signaling pathway after middle cerebral artery occlusion in rats.

Delayed recanalization at days or weeks beyond the therapeutic window was shown to improve functional outcomes in acute ischemic stroke (AIS) patients. However, the underlying mechanisms remain unclear. Previous preclinical study reported that trefoil factor 3 (TFF3) was secreted by liver after cerebral ischemia and acted a distant neuroprotective factor. Here, we investigated the liver-derived TFF3-mediated neuroprotective mechanism enhanced by delayed recanalization after AIS. A total of 327 male Sprague-Dawley rats and the model of middle cerebral artery occlusion (MCAO) with permanent occlusion (pMCAO) or with delayed recanalization at 3 d post-occlusion (rMCAO) were used. Partial hepatectomy was performed within 5 min after MCAO. Leucine-rich repeat and immunoglobulin-like domain-containing nogo receptor-interacting protein 2 (LINGO2) siRNA was administered intracerebroventricularly at 48 h after MCAO. Recombinant rat TFF3 (rr-TFF3, 30 µg/Kg) or recombinant rat epidermal growth factor (rr-EGF, 100 µg/Kg) was administered intranasally at 1 h after recanalization, and EGFR inhibitor Gefitinib (75 mg/Kg) was administered intranasally at 30 min before recanalization. The evaluation of outcomes included neurobehavior, ELISA, western blot and immunofluorescence staining. TFF3 in hepatocytes and serum were upregulated in a similar time-dependent manner after MCAO. Compared to pMCAO, delayed recanalization increased brain TFF3 levels and attenuated brain damage with the reduction in neuronal apoptosis, infarct volume and neurological deficits. Partial hepatectomy reduced TFF3 levels in serum and ipsilateral brain hemisphere, and abolished the benefits of delayed recanalization on neuronal apoptosis and neurobehavioral deficits in rMCAO rats. Intranasal rrTFF3 treatment reversed the changes associated with partial hepatectomy. Delayed recanalization after MCAO increased the co-immunoprecipitation of TFF3 and LINGO2, as well as expressions of p-EGFR, p-Src and Bcl-2 in the brain. LINGO2 siRNA knockdown or EGFR inhibitor reversed the effects of delayed recanalization on apoptosis and brain expressions of LINGO2, p-EGFR, p-Src and Bcl-2 in rMCAO rats. EGFR activator abolished the deleterious effects of LINGO2 siRNA. In conclusion, our investigation demonstrated for the first time that delayed recanalization may enhance the entry of liver-derived TFF3 into ischemic brain upon restoring blood flow after MCAO, which attenuated neuronal apoptosis and neurological deficits at least in part via activating LINGO2/EGFR/Src pathway.

Three Days Delayed Recanalization Improved Neurological Function in pMCAO Rats by Increasing M2 Microglia-Possible Involvement of the IL-4R/STAT6/PPARγ Pathway.

Current approved therapies for acute ischemic stroke have a restricted therapeutic time window. Delayed recanalization, which has been utilized clinically in patients who have missed the time window for administration, may be a promising alternative for stroke patients. However, the underlying molecular mechanisms remain undiscovered. Herein, we hypothesized that delayed recanalization would increase M2 microglial polarization through the IL-4R (interleukin-4 receptor)/STAT6 (signal transducer and activators of transcription 6)/PPARγ (peroxisome proliferator-activated receptor γ) pathway, subsequently promoting stroke recovery in rats. The permanent middle cerebral artery occlusion (pMCAO) model was induced via intravascular filament insertion. Recanalization was induced by withdrawing the filament at 3 days after MCAO (rMCAO). Interleukin (IL)-4 was administered intranasally at 3 days after pMCAO. AS1517499, a specific STAT6 inhibitor, was administered intranasally at 3 days after MCAO induction. Immunofluorescence staining, enzyme-linked immunosorbent assay (ELISA), western blot analysis, volumetric measurements of brain infarct, and neurological behavior tests were conducted. Delayed recanalization at 3 days after MCAO increased the polarization of M2 microglia, decreased inflammation, and improved neurological behavior. IL-4 treatment administered on the 3rd day after pMCAO increased M2 microglial polarization, improved neurological behavior, and reduced infarction volume of pMCAO rats. The inhibition of STAT6 decreased the level of p-STAT6 and PPARγ in rats treated with delayed recanalization. Delayed recanalization improved neurological function by increasing microglial M2 polarization, possibly involved with the IL-4R/STAT6/PPARγ pathway after MCAO in rats.