G-CSF suppresses edema formation and reduces interleukin-1beta expression after cerebral ischemia in mice

Granulocyte-colony stimulating factor (G-CSF) is reported to be neuroprotective after transient cerebral ischemia with respect to decreasing lesion volume and enhancing functional recovery. We investigated whether G-CSF is neuroprotective after permanent ischemia and the possible mechanisms underlying this neuroprotection. Mice underwent permanent or 60-minute middle cerebral artery occlusion (MCAO) and received G-CSF (50 microg/kg) or vehicle at the onset or 1 hour post-MCAO. Forty-eight hours after transient MCAO, structural magnetic resonance imaging revealed a significant reduction (50%) in the amount of edematous tissue present in G-CSF-treated mice (p < 0.05). G-CSF treatment also prevented a significant increase in ipsilateral brain water content that was present in vehicle-treated mice after transient (p < 0.05) and permanent (p < 0.001) MCAO. Forty-eight hours after permanent MCAO, G-CSF decreased (50%) the cortical lesion volume (p < 0.05). Using real-time polymerase chain reaction, we found that G-CSF treatment significantly suppressed (p < 0.05) the injury-induced upregulation of IL-1beta mRNA while having no effect on TNFalpha and NOS-2 mRNA expression. This suggests that part of the neuroprotection may be attributed to the ability of G-CSF to reduce the inflammatory response.     

Deficiency of PAR4 attenuates cerebral ischemia/reperfusion injury in mice

Stroke is the third leading cause of death in the USA. Antithrombotic therapy targeting platelet activation is one of the treatments for ischemic stroke. Here we investigate the role of one of the thrombin receptors, protease-activated receptor 4 (PAR4), in a mouse transient middle cerebral artery occlusion (MCAO) model. After a 60 min MCAO and 23 h reperfusion, leukocyte and platelet rolling and adhesion on cerebral venules, blood–brain barrier (BBB) permeability, and cerebral edema were compared in PAR4-deficient mice and wild-type mice. Cerebral infarction volume and neuronal death were also measured. PAR4−/− mice had more than an 80% reduction of infarct volume and significantly improved neurologic and motor function compared with wild-type mice after MCAO. Furthermore, deficiency of PAR4 significantly inhibits the rolling and adhesion of both platelets and leukocytes after MCAO. BBB disruption and cerebral edema were also attenuated in PAR4−/− mice compared with wild-type animals. The results of this investigation indicate that deficiency of PAR4 protects mice from cerebral ischemia/reperfusion (I/R) injury, partially through inhibition of platelet activation and attenuation of microvascular inflammation.     

Expression of nitric oxide synthase (NOS)-2 following permanent focal ischemia and the role of nitric oxide in infarct generation in male, female and NOS-2 gene-deficient mice

Considerable evidence implicates nitric oxide (NO) in the pathological events following cerebral ischemia and, depending on the enzyme/cell source, NO is considered to be either damaging or protective. As a role for the enzyme nitric oxide synthase (NOS)-2 in permanent focal ischemia is not clear, we examined its expression following permanent middle cerebral artery occlusion in mice. At 24 h after occlusion, NOS-2 was expressed in cells infiltrating the infarct, while at later times, there was also expression in astrocytes around the infarct. To reveal a role for NO derived from this source, we compared infarct size in male and female mice with littermates in which the NOS-2 gene was disrupted. No differences were found between gender and genotype at 24 h. At 72 h, the infarct was increased in male mice, but not in females or in either gender with the gene disruption. These results suggest that NOS-2 plays a role in the later development of the infarct in male mice. Female mice are protected either against the damaging effects of NO, or because NOS-2 expression/activity is modulated by steroids.     

Sex differences in neuroprotection provided by inhibition of TRPM2 channels following experimental stroke

The calcium-permeable transient receptor potential M2 (TRPM2) ion channel is activated following oxidative stress and has been implicated in ischemic damage; however, little experimental evidence exists linking TRPM2 channel activation to damage following cerebral ischemia. We directly assessed the involvement of TRPM2 channels in ischemic brain injury using pharmacological inhibitors and short-hairpin RNA (shRNA)-mediated knockdown of TRPM2 expression. Each of the four TRPM2 inhibitors tested provided significant protection to male neurons following in vitro ischemia (oxygen–glucose deprivation, OGD), while having no effect in female neurons. Similarly, TRPM2 knockdown by TRPM2 shRNA resulted in significantly reduced neuronal cell death following OGD only in male neurons. The TRPM2 inhibitor clotrimazole reduced infarct volume in male mice, while having no effect on female infarct volume. Finally, intrastriatal injection of lentivirus expressing shRNA against TRPM2 resulted in significantly smaller striatal infarcts only in male mice following middle cerebral artery occlusion, having no significant effect in female mice. Data presented in the current study demonstrate that TRPM2 inhibition and knockdown preferentially protects male neurons and brain against ischemia in vitro and in vivo, indicating that TRPM2 inhibitors may provide a new therapeutic approach to the treatment of stroke in men.     

Neuronal protective role of PBEF in a mouse model of cerebral ischemia

Pre-B-cell colony-enhancing factor (PBEF) (also known as nicotinamide phosphoribosyltransferase) is a rate-limiting enzyme in the salvage pathway for mammalian biosynthesis of nicotinamide adenine dinucleotide (NAD⁺). By synthesizing NAD⁺, PBEF functions to maintain an energy supply that has critical roles in cell survival. Cerebral ischemia is a major neural disorder with a high percentage of mortality and disability. Ischemia leads to energy depletion and eventually neuronal death and brain damage. This study investigated the role of PBEF in cerebral ischemia using a photothrombosis mouse model. Using immunostaining, we initially determined that PBEF is highly expressed in neurons, but not in glial cells in the mouse brain. To study the role of PBEF in ischemia in vivo, we used PBEF knockout heterozygous (Pbef+/−) mice. We showed that these mice have lower PBEF expression and NAD⁺ level than do wild-type (WT) mice. When subjected to photothrombosis, Pbef+/− mice have significantly larger infarct volume than do age-matched WT mice at 24 hours after ischemia. Higher density of degenerating neurons was detected in the penumbra of Pbef+/− mice than in WT mice using Fluoro-Jade B staining. Our study shows that PBEF has a neuronal protective role in cerebral ischemia presumably through enhanced energy metabolism.     

NCX3 knockout mice exhibit increased hippocampal CA1 and CA2 neuronal damage compared to wild-type mice following global cerebral ischemia

There is uncertainty as to whether the plasma membrane Na(+)/Ca(2+)exchanger (NCX) has a neuroprotective or neurodamaging role following cerebral ischemia. To address this issue we compared hippocampal neuronal injury in NCX3 knockout mice (Ncx3(-/-)) and wild-type mice (Ncx3(+/+)) following global cerebral ischemia. Using a bilateral common carotid artery occlusion (BCCAO) model of global ischemia we subjected NCX3 knockout and wild-type mice to 17 and 15 minutes of ischemia. Following the 17 minute period of ischemia, wild-type mice exhibited approximately 80% CA1 neuronal loss and approximately 40% CA2 neuronal loss. In contrast, NCX3 knockout mice displayed >95% CA1 neuronal loss and approximately 95% CA2 neuronal loss. Following the 15 minute period of ischemia, wild-type mice did not exhibit any significant hippocampal neuronal loss. In contrast, NCX3 knockout mice displayed approximately 45% CA1 neuronal loss and approximately 25% CA2 neuronal loss. The results clearly demonstrate that mice deficient in the NCX3 protein are more susceptible to global cerebral ischemia than wild-type mice. Our findings suggest NCX3 has a positive role in maintaining neuronal intracellular calcium homeostasis following ischemia, and that when exchanger function is compromised neurons are more susceptible to calcium deregulation and cell death.     

The interaction between tumor necrosis factor-like weak inducer of apoptosis and its receptor fibroblast growth factor-inducible 14 promotes the recruitment of neutrophils into the ischemic brain

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) are expressed in endothelial cells and perivascular astrocytes. Here, we show that TWEAK induces a dose-dependent increase in the expression of the chemokine monocyte chemoattractant protein-1 (MCP-1) in astrocytes, and that this effect is mediated by its interaction with Fn14 via nuclear factor-κB pathway activation. Exposure to oxygen-glucose deprivation (OGD) conditions increases TWEAK and Fn14 mRNA expression in wild-type (Wt) astrocytic cultures. Likewise, incubation under OGD conditions induces the expression of MCP-1 in Wt astrocytes but not in astrocytes deficient on either TWEAK (TWEAK^−/−) or Fn14 (Fn14^−/−). We also found that TWEAK induces the passage of neutrophils to the abluminal side of an in vitro model of the blood–brain barrier. Our earlier studies indicate that cerebral ischemia increases the expression of TWEAK and Fn14 in the endothelial cell-basement membrane-astrocyte interface. Here, we report that middle cerebral artery occlusion increases the expression of MCP-1 and the recruitment of neutrophils into the ischemic tissue in Wt but not in TWEAK^−/− or Fn14^−/− mice. These novel results indicate that during cerebral ischemia, the interaction between TWEAK and Fn14 leads to the recruitment of leukocytes into the ischemic tissue.     

Macrophage migration inhibitory factor promotes cell death and aggravates neurologic deficits after experimental stroke

Multiple mechanisms contribute to tissue demise and functional recovery after stroke. We studied the involvement of macrophage migration inhibitory factor (MIF) in cell death and development of neurologic deficits after experimental stroke. Macrophage migration inhibitory factor is upregulated in the brain after cerebral ischemia, and disruption of the Mif gene in mice leads to a smaller infarct volume and better sensory-motor function after transient middle cerebral artery occlusion (tMCAo). In mice subjected to tMCAo, we found that MIF accumulates in neurons of the peri-infarct region, particularly in cortical parvalbumin-positive interneurons. Likewise, in cultured cortical neurons exposed to oxygen and glucose deprivation, MIF levels increase, and inhibition of MIF by (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) protects against cell death. Deletion of MIF in Mif^−/− mice does not affect interleukin-1β protein levels in the brain and serum after tMCAo. Furthermore, disruption of the Mif gene in mice does not affect CD68, but it is associated with higher galectin-3 immunoreactivity in the brain after tMCAo, suggesting that MIF affects the molecular/cellular composition of the macrophages/microglia response after experimental stroke. We conclude that MIF promotes neuronal death and aggravates neurologic deficits after experimental stroke, which implicates MIF in the pathogenesis of neuronal injury after stroke.     

Alterations in nitric oxide and endothelin-1 bioactivity underlie cerebrovascular dysfunction in ApoE-deficient mice

Hypercholesterolemia is associated with decreased nitric oxide (NO) bioavailability and endothelial dysfunction, a phenomenon thought to have a major role in the altered cerebral blood flow evident in stroke. Therefore, strategies that increase endothelial NO production have potential utility. Vascular reactivity of the middle cerebral artery (MCA) from C57BL/6J wild-type (WT) mice, apolipoprotein-E knockout (ApoE^−/−) mice, and mice treated with the phosphodiesterase inhibitor cilostazol (100 mg/kg) was analyzed using the tension myograph. Contractile responses to endothelin-1 were significantly enhanced in MCA from ApoE^−/− mice compared with WT mice (P<0.01), an effect absent in cilostazol-treated ApoE^−/− mice. Acetylcholine-induced relaxation (which is entirely NO-dependent) was significantly impaired in MCA of ApoE^−/− mice compared with WT mice (P<0.05), again an effect prevented by cilostazol treatment. Endothelial NOS phosphorylation at Ser¹¹⁷⁹ was decreased in the aorta of ApoE^−/− mice compared with WT mice (P<0.05), an effect normalized by cilostazol. Taken together, our data suggest that the endothelial dysfunction observed in MCA associated with hypercholesterolemia is prevented by cilostazol, an effect likely due to the increase in eNOS phosphorylation and, therefore, activity.     

A new role for downstream Toll-like receptor signaling in mediating immediate early gene expression during focal cerebral ischemia

To better understand the role of downstream Toll-like receptor (TLR) signaling during acute cerebral ischemia, we performed cDNA microarrays, on brain RNA, and cytokine arrays, on serum, from wild type (WT), MyD88−/− and TRIF-mutant mice, at baseline and following permanent middle cerebral artery occlusion (pMCAO). The acute stress response pathway was among the top pathways identified by Ingenuity Pathway Analysis of microarray data. We used real-time polymerase chain reaction to confirm the expression of four immediate early genes; EGR1, EGR2, ARC, Nurr77, in this pathway, and insulin degrading enzyme (IDE). Compared to WT, baseline immediate early gene expression was increased up to10-fold in MyD88−/− and TRIF-mutant mice. However, following pMCAO, immediate early gene expression remained unchanged, from this elevated baseline in these mice, but increased up to 12-fold in WT. Furthermore, expression of IDE, which also degrades β-amyloid, decreased significantly only in TRIF-mutant mice. Finally, sE-Selectin, sICAM, sVCAM-1, and MMP-9 levels were significantly decreased only in MyD88−/− compared with WT mice. We thus report a new role for downstream TLR signaling in immediate early gene expression during acute cerebral ischemia. We also show that the TRIF pathway regulates IDE expression; a major enzyme that clears β-amyloid from the brain.     

Scavenger receptor class-A has a central role in cerebral ischemia–reperfusion injury

The innate immune response is involved in the pathophysiology of cerebral ischemia–reperfusion (I/R) injury. Recent evidence suggests that scavenger receptors have a role in the induction of innate immunity. In this study, we examined the role of scavenger receptor A (SR-A) in focal cerebral I/R injury. Both SR-A^−/− mice (n=10) and age-matched wild-type (WT) mice (n=9) were subjected to focal cerebral ischemia (60 minutes), followed by reperfusion (for 24 hours). Infarct size was determined by TTC (triphenyltetrazolium chloride) staining. The morphology of neurons in the brain sections was examined by Nissl''s staining. Activation of intracellular signaling was analyzed by western blot. Cerebral infarct size in SR-A^−/− mice was significantly reduced by 63.9% compared with WT mice after cerebral I/R. In SR-A^−/− mice, there was less neuronal damage in the hippocampus compared with WT mice. Levels of FasL, Fas, FADD, caspase-3 activity, and terminal deoynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling-positive apoptotic cells were significantly increased in WT mice after cerebral I/R, but not in SR-A^−/− mice. Cerebral I/R increased nuclear factor-κB activation in WT mice, but not in SR-A^−/− mice. These data suggest that SR-A has a central role in cerebral I/R injury and that suppression of SR-A may be a useful approach for ameliorating brain injury in stroke patients.     

A detrimental role for nitric oxide synthase-2 in the pathology resulting from acute cerebral injury

Nitric oxide (NO) synthesized from the inducible isoform of nitric oxide synthase (NOS-2) has been suggested to play both beneficial and deleterious roles in various neuropathologies. To define the role of nitric oxide in traumatic brain injury, we subjected male mice lacking a functional NOS-2 gene (NOS-2-/-) and their wild-type littermates (NOS-2+/+) to mild or severe aseptic cryogenic cerebral injury. Expression of NOS-2 mRNA and protein was observed in NOS-2+/+ animals following injury. Lesion volume (as measured by histology and brain imaging) and neurological outcome (using motor and cognitive behavioral paradigms) were assessed at various times after injury. While magnetic resonance imaging revealed the extent of edema of the 2 genotypes to be similar, histology showed a reduced (32%) lesion volume in severely injured NOS-2-/- compared with NOS-2+/+ mice. In addition, NOS-2-/- mice showed significant improvements in both contralateral sensorimotor deficits (grid test: p = 0.011) and cognitive function (Morris water maze: p = 0.009) after severe injury compared to their wild-type littermates. This indicates that lesion volume is reduced and neurological recovery is improved after acute traumatic injury in mice lacking a functional NOS-2 gene, and strongly suggests that the post-trauma production of NO from this source contributes to neuropathology.     

Knockout of silent information regulator 2 (SIRT2) preserves neurological function after experimental stroke in mice

Sirtuin-2 (Sirt2) is a member of the NAD⁺-dependent protein deacetylase family. Various members of the sirtuin class have been found to be involved in processes related to longevity, regulation of inflammation, and neuroprotection. Induction of Sirt2 mRNA was found in the whole hemisphere after experimental stroke in a recent screening approach. Moreover, Sirt2 protein is highly expressed in myelin-rich brain regions after stroke. To examine the effects of Sirt2 on ischemic stroke, we induced transient focal cerebral ischemia in adult male Sirt2-knockout and wild-type mice. Two stroke models with different occlusion times were applied: a severe ischemia (45 minutes of middle cerebral artery occlusion (MCAO)) and a mild one (15 minutes of MCAO), which was used to focus on subcortical infarcts. Neurological deficit was determined at 48 hours after 45 minutes of MCAO, and up to 7 days after induction of 15 minutes of cerebral ischemia. In contrast to recent data on Sirt1, Sirt2^−/− mice showed less neurological deficits in both models of experimental stroke, with the strongest manifestation after 48 hours of reperfusion. However, we did not observe a significant difference of stroke volumes or inflammatory cell count between Sirt2-deficient and wild-type mice. Thus we postulate that Sirt2 mediates myelin-dependent neuronal dysfunction during the early phase after ischemic stroke.     

SK2 channels are neuroprotective for ischemia-induced neuronal cell death

In mouse hippocampal CA1 pyramidal neurons, the activity of synaptic small-conductance Ca²⁺-activated K⁺ channels type 2 (SK2 channels) provides a negative feedback on N-methyl--aspartate receptors (NMDARs), reestablishing Mg²⁺ block that reduces Ca²⁺ influx. The well-established role of NMDARs in ischemia-induced excitotoxicity led us to test the neuroprotective effect of modulating SK2 channel activity following cerebral ischemia induced by cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Administration of the SK channel positive modulator, 1-ethyl-benzimidazolinone (1-EBIO), significantly reduced CA1 neuron cell death and improved CA/CPR-induced cognitive outcome. Electrophysiological recordings showed that CA/CPR-induced ischemia caused delayed and sustained reduction of synaptic SK channel activity, and immunoelectron microscopy showed that this is associated with internalization of synaptic SK2 channels, which was prevented by 1-EBIO treatment. These results suggest that increasing SK2 channel activity, or preventing ischemia-induced loss of synaptic SK2 channels, are promising and novel approaches to neuroprotection following cerebral ischemia.     

Loss of TREM2 Confers Resilience to Synaptic and Cognitive Impairment in Aged Mice

Triggering receptor expressed on myeloid cells 2 (TREM2), a receptor exclusively expressed by microglia in the brain, modulates microglial immune homeostasis. Human genetic studies have shown that the loss-of-function mutations in TREM2 signaling are strongly associated with an elevated risk of age-related neurodegenerative diseases including Alzheimer''s disease (AD). Numerous studies have investigated the impact of TREM2 deficiency in the pathogenic process of AD. However, the role of TREM2 in shaping neuronal and cognitive function during normal aging is underexplored. In the present study, we employed behavioral, electrophysiological, and biochemical approaches to assess cognitive and synaptic function in male and female young and aged TREM2-deficient (Trem2−/−) mice compared with age-matched, sex-matched, and genetic background-matched wild-type (WT) C57BL/6J controls. Young Trem2−/− mice exhibited normal cognitive function and synaptic plasticity but had increased dendritic spine density compared with young WT. Unexpectedly, aged Trem2−/− mice showed superior cognitive performance compared with aged WT controls. Consistent with the behavioral data, aged Trem2−/− mice displayed significantly enhanced hippocampal long-term potentiation (LTP) and increased dendritic spine density and synaptic markers compared with aged WT mice. Taken together, these findings suggest that loss of TREM2 affects the neuronal structure and confers resilience to age-related synaptic and cognitive impairment during non-pathogenic aging.SIGNIFICANCE STATEMENT Microglia are innate immune cells of the brain that orchestrates neurodevelopment, synaptic function, and immune response to environmental stimuli. Microglial triggering receptor expressed on myeloid cells 2 (TREM2) signaling plays pivotal roles in regulating these functions and loss of TREM2 signaling leads to increased risk of developing age-related neurologic disorders. However, the neurologic role of TREM2 in normal aging is poorly understood. The results of the present study unveil the positive impacts of TREM2 deficiency on cognitive and synaptic function during aging and suggest that TREM2 may exert detrimental effects on neuronal function. The possibility of age-related negative impacts from TREM2 is critically important since TREM2 has emerged as a major therapeutic target for Alzheimer''s dementia.     

Neuron-specific deletion of presenilin enhancer2 causes progressive astrogliosis and age-related neurodegeneration in the cortex independent of the Notch signaling

IntroductionPresenilin enhancer2 (Pen‐2) is an essential subunit of γ‐secretase, which is a key protease responsible for the cleavage of amyloid precursor protein (APP) and Notch. Mutations on Pen‐2 cause familial Alzheimer disease (AD). However, it remains unknown whether Pen‐2 regulates neuronal survival and neuroinflammation in the adult brain.MethodsForebrain neuron‐specific Pen‐2 conditional knockout (Pen‐2 cKO) mice were generated for this study. Pen‐2 cKO mice expressing Notch1 intracellular domain (NICD) conditionally in cortical neurons were also generated.ResultsLoss of Pen‐2 causes astrogliosis followed by age‐dependent cortical atrophy and neuronal loss. Loss of Pen‐2 results in microgliosis and enhanced inflammatory responses in the cortex. Expression of NICD in Pen‐2 cKO cortices ameliorates neither neurodegeneration nor neuroinflammation.ConclusionsPen‐2 is required for neuronal survival in the adult cerebral cortex. The Notch signaling may not be involved in neurodegeneration caused by loss of Pen‐2.     

Neuronal estrogen receptor-�� mediates neuroprotection by 17��-estradiol

17β-Estradiol (E₂) was shown to exert neuroprotective effects both in in vitro and in vivo models of stroke. Although these effects of E₂ are known to require estrogen receptor-α (ERα), the cellular target of estrogen-mediated neuroprotection remains unknown. Using cell type-specific ER mutant mice in an in vivo model of stroke, we specifically investigated the role of ERα in neuronal cells versus its role in the microglia in the mediation of neuroprotection by estrogens. We generated and analyzed two different tissue-specific knockout mouse lines lacking ERα either in cells of myeloid lineage, including microglia, or in the neurons of the forebrain. Both E₂-treated and E₂-untreated mutant and control mice were subjected to a permanent middle cerebral artery occlusion for 48 h, and the infarct volume was quantified. Although the infarct volume of E₂-treated female myeloid-specific ERα knockout mice was similar to that of E₂-treated control mice, both male and female neuron-specific ERα mutant mice had larger infarcts than did control mice after E₂ treatment. We conclude that neuronal ERα in female and male mice mediates neuroprotective estrogen effects in an in vivo mouse model of stroke, whereas microglial ERα is dispensable.     

Inhibition of microRNA-181 reduces forebrain ischemia-induced neuronal loss

MicroRNA (miRNA), miR-181a, is enriched in the brain, and inhibition of miR-181a reduced astrocyte death in vitro and infarct volume after stroke in vivo. This study investigated the role of miR-181a in neuronal injury in vitro and hippocampal neuronal loss in vivo after forebrain ischemia. miR-181a levels were altered by transfection with mimic or antagomir. N2a cells subjected to serum deprivation and oxidative stress showed less cell death when miR-181a was reduced and increased death when miR-181a increased; protection was associated with increased Bcl-2 protein. In contrast, transfected primary neurons did not show altered levels of cell death when miR-181a levels changed. Naive male rats and rats stereotactically infused with miR-181a antagomir or control were subjected to forebrain ischemia and cornus ammonis (CA)1 neuronal survival and protein levels were assessed. Forebrain ischemia increased miR-181a expression and decreased Bcl-2 protein in the hippocampal CA1 region. miR-181a antagomir reduced miR-181a levels, reduced CA1 neuronal loss, increased Bcl-2 protein, and significantly prevented the decrease of glutamate transporter 1. Thus, miR-181a antagomir reduced evidence of astrocyte dysfunction and increased CA1 neuronal survival. miR-181a inhibition is thus a potential target in the setting of forebrain or global cerebral ischemia as well as focal ischemia.     

Astrocytic P2Y1 receptor is involved in the regulation of cytokine/chemokine transcription and cerebral damage in a rat model of cerebral ischemia

After brain ischemia, significant amounts of adenosine 5′-triphosphate are released or leaked from damaged cells, thus activating purinergic receptors in the central nervous system. A number of P2X/P2Y receptors have been implicated in ischemic conditions, but to date the P2Y₁ receptor (P2Y₁R) has not been implicated in cerebral ischemia. In this study, we found that the astrocytic P2Y₁R, via phosphorylated-RelA (p-RelA), has a negative effect during cerebral ischemia/reperfusion. Intracerebroventricular administration of the P2Y₁R agonist, MRS 2365, led to an increase in cerebral infarct volume 72 hours after transient middle cerebral artery occlusion (tMCAO). Administration of the P2Y₁R antagonist, MRS 2179, significantly decreased infarct volume and led to recovered motor coordination. The effects of MRS 2179 occurred within 24 hours of tMCAO, and also markedly reduced the expression of p-RelA and interleukin-6, tumor necrosis factor-α, monocyte chemotactic protein-1/chemokine (C-C motif) ligand 2 (CCL2), and interferon-inducible protein-10/chemokine (C-X-C motif) ligand 10 (CXCL10) mRNA. P2Y₁R and p-RelA were colocalized in glial fibrillary acidic protein-positive astrocytes, and an increase in infarct volume after MRS 2365 treatment was inhibited by the nuclear factor (NF)-κB inhibitor ammonium pyrrolidine dithiocarbamate. These results provide evidence that the P2Y₁R expressed in cortical astrocytes may help regulate the cytokine/chemokine response after tMCAO/reperfusion through a p-RelA-mediated NF-κB pathway.