The aim of this study was to determine whether leukemia inhibitory factor (LIF) exerts its neuroprotective effects through signal transduction of the transcription factor myeloid zinc finger-1 (MZF-1). According to the hypothesis of this study, MZF-1 mediates LIF-induced neuroprotective signaling during ELVO through increased expression and transcriptional activity. To determine the in vivo role of MZF-1 in LIF-induced neuroprotection, we used Genomatix software was used to MZF-1 sites in the promoter region of the rat superoxide dismutase 3 (SOD3) gene. Stroke was induced via middle cerebral artery occlusion, and animals were administered PBS or 125 μg/kg LIF at 6, 24, and 48 h after the injury. MZF-1 binding activity was measured using electrophoretic mobility shift assay (EMSA) and its expression/localization were determined using western blot and immunohistochemical analysis. To determine whether MZF-1 relays LIF-induced neuroprotection in vitro, primary cultured neurons were subjected to oxygen-glucose deprivation (OGD) after treatment with PBS or LIF. MZF-1 expression was measured in vitro using real time PCR and immunohistochemical staining. Transfection with siRNA was used to determine whether LIF protected cultured neurons against OGD after silencing MZF-1 expression. Four MZF-1 binding sites were identified by Genomatix, and EMSA confirmed in vivo binding activity in brain after MCAO. LIF significantly increased MZF-1 protein levels compared to PBS treatment at 72 h post-MCAO. In vivo nuclear localization of MZF-1 as well as co-localization of SOD3 and MZF-1 was observed in the cortical neurons of LIF-treated rats. Primary cultured neurons treated with LIF had significantly higher levels of MZF-1 mRNA and protein after LIF treatment compared to neurons treated with PBS. Finally, knockdown MZF-1 using siRNA counteracted the neuroprotective effects of LIF in vitro. These data demonstrate that LIF-mediated neuroprotection is dependent upon MZF-1 activity. Furthermore, these findings identify a novel neuroprotective pathway that employs MZF-1, a transcription factor associated with hematopoietic gene expression.

The migration of peripheral immune cells and splenocytes to the ischemic brain is one of the major causes of delayed neuroinflammation after permanent large vessel stroke. Other groups have demonstrated that leukemia inhibitory factor (LIF), a cytokine that promotes neural cell survival through upregulation of antioxidant enzymes, promotes an anti-inflammatory phenotype in several types of immune cells. The goal of this study was to determine whether LIF treatment modulates the peripheral immune response after stroke.

BACKGROUND AND PURPOSE:Acid/base and electrolytes could provide clinically valuable information about cerebral infarct core and penumbra. We evaluated associations between acid/base and electrolyte changes and outcomes in 2 rat models of stroke, permanent, and transient middle cerebral artery occlusion. METHODS:Three-month old Sprague-Dawley rats underwent permanent or transient middle cerebral artery occlusion. Pre- and post-middle cerebral artery occlusion venous samples for permanent and transient models provided pH, carbon dioxide, oxygen, glucose, and electrolyte values of ionized calcium, potassium, and sodium. Multiple regression determined predictors of infarct volume from these values, and Kaplan-Meier curve analyzed morality between permanent and transient middle cerebral artery occlusion models. RESULTS:Analysis indicated significant differences in the blood gas and electrolytes between pre- to post-middle cerebral artery occlusion. A decrease in pH and sodium with increases in carbon dioxide, potassium, ionized calcium, and glucose changes were found in both middle cerebral artery occlusion models; while hematocrit and hemoglobin were significant in the transient model. pH and ionized calcium were predictors of infarct volume in the permanent model, as changes in pH and ionized calcium decreased, infarct volume increased. CONCLUSIONS:There are acute changes in acid/base balance and electrolytes during stroke in transient and permanent rodent models. Additionally, we found pH and ionized calcium changes predicted stroke volume in the permanent middle cerebral artery occlusion model. These preliminary findings are novel, and warrant further exploration in human conditions.

Background and Purpose: Acid/base and electrolytes could provide clinically valuable information about cerebral infarct core and penumbra. We evaluated associations between acid/base and electrolyte changes and outcomes in 2 rat models of stroke, permanent, and transient middle cerebral artery occlusion. Methods: Three-month old Sprague-Dawley rats underwent permanent or transient middle cerebral artery occlusion. Pre- and post-middle cerebral artery occlusion venous samples for permanent and transient models provided pH, carbon dioxide, oxygen, glucose, and electrolyte values of ionized calcium, potassium, and sodium. Multiple regression determined predictors of infarct volume from these values, and Kaplan-Meier curve analyzed morality between permanent and transient middle cerebral artery occlusion models. Results: Analysis indicated significant differences in the blood gas and electrolytes between pre- to post-middle cerebral artery occlusion. A decrease in pH and sodium with increases in carbon dioxide, potassium, ionized calcium, and glucose changes were found in both middle cerebral artery occlusion models; while hematocrit and hemoglobin were significant in the transient model. pH and ionized calcium were predictors of infarct volume in the permanent model, as changes in pH and ionized calcium decreased, infarct volume increased. Conclusions: There are acute changes in acid/base balance and electrolytes during stroke in transient and permanent rodent models. Additionally, we found pH and ionized calcium changes predicted stroke volume in the permanent middle cerebral artery occlusion model. These preliminary findings are novel, and warrant further exploration in human conditions.

ObjectiveTo determine if leukemia inhibitory factor (LIF) administration after permanent focal stroke decreases post‐stroke neuroinflammation.MethodsFocal ischemia was induced in 3 month old male and 18 month old male/female Sprague‐Dawley using the middle cerebral artery occlusion (MCAO) procedure. Animals were treated with PBS or LIF at 6, 24, and 48 h after MCAO (125 μg/kg). Animals were euthanized at at 72 h post‐MCAO. Protein levels of CD11b, IL‐12 p40, IFNγ, and IP‐10 were measured in splenic tissue using immunoblotting and/or ELISA. Isolectin staining and immunoblotting were used to detect levels of CD11b+ macrophages/microglia in the brain. Murine bone marrow derived macrophages (BMDMs) stimulated with LPS (50 ng/ml) and IFNγ (20 ng/ml) in the presence or absense of LIF and IL‐12 p40/IL‐10 levels were detected in the supernatant.ResultsLIF treatment significantly increased the average spleen weight at 72 h after MCAO compared to PBS‐treated rats (p<0.05) and decreased splenic LIFR expression compared to sham operated (p<0.01) and PBS‐treated (p = 0.0682) rats. LIF‐treated rats showed significantly decreased levels of IL‐12 p40 (p <0.05) and IFNγ (p = 0.0644) in splenic tissue at 72 h after MCAO compared PBS‐treated animals. LIF treatment also counteracted the significant increase in splenic IP‐10 expression observed in the PBS‐treated rats (*p<0.01 compared to sham‐operated rats). CD11b levels were significantly lower in the spleens of LIF‐treated (p<0.001) and PBS‐treated (p<0.01) rats compared to sham rats. However, LIF treated significantly decreased normalized CD11b protein levels (p<0.05) and decreased isolectin reactivity in the brain at 72 h post‐MCAO compared to PBS‐treated animals. BMDMs treated with LIF prior to IFNγ and LPS stimulation released significantly lower levels of IL‐12 p40 and significantly higher levels of IL‐10 (p ≤ 0.05) compared to BMDMs treated with PBS prior to LPS+IFNγ stimulation.ConclusionLIF confers anti‐inflammatory signaling after stroke through modulation of the IL‐12 p40/IFNγ/IP‐10 axis in splenocytes and decreasing the infiltration of CD11b+ monocytes/macrophages into the ischemic brain.Support or Funding InformationProject Number: 5R01NS091146‐03 (NINDS; PI: Pennypacker)Project Number: 55R01NS091582‐03 (NINDS; PI: Gensel)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Objective: To determine whether the anti-inflammatory effects of leukemia inhibitory factor (LIF) are altered in aged male and female rats.

Leukemia inhibitory factor (LIF) has been shown to protect oligodendrocytes from ischemia by upregulating endogenous antioxidants. The goal of this study was to determine whether LIF protects neurons during stroke by upregulating superoxide dismutase 3 (SOD3). Animals were administered phosphate-buffered saline (PBS) or 125 μg/kg LIF at 6, 24, and 48 h after middle cerebral artery occlusion or sham surgery. Neurons were isolated from rat pups on embryonic day 18 and used between 7 and 15 days in culture. Cells were treated with LIF and/or 10 μM Akt inhibitor IV with PBS and 0.1 % DMSO acting as vehicle controls. Neurons transfected with scrambled or SOD3 small interfering RNA (siRNA) were subjected to 24-h ischemia after PBS or LIF treatment. LIF significantly increased superoxide dismutase activity and SOD3 expression in ipsilateral brain tissue compared to PBS. Following 24-h ischemia, LIF reduced cell death and increased SOD3 messenger RNA (mRNA) in vitro compared to PBS. Adding Akt inhibitor IV with LIF counteracted the decrease in cell death. Partially silencing the expression of SOD3 using siRNA prior to LIF treatment counteracted the protective effect of LIF-alone PBS treatment. These results indicate that LIF protects neurons in vivo and in vitro via upregulation of SOD3.

ObjectiveThe objective of this study is to determine whether leukemia inhibitory factor (LIF) induces neuroprotection through the transcription factors myeloid zinc finger‐1 (MZF‐1) and specificity protein 1 (Sp1).MethodsAfter middle cerebral artery occlusion or sham surgery, male Sprague‐Dawley rats were injected with PBS or LIF (125 μg/kg) (n=4 per group). Rats were euthanized 72 h after injury. Western blotting was used to measure MZF‐1 and Sp1 protein expression in brain tissue and spleen tissue. Genomatix was used to identify MZF‐1 and Sp1 binding sites in the promoter of superoxide dismutase 3 (SOD3), a LIF‐inducible gene. Gel shift assays were used to confirm binding of these transcription factors in brain nuclear extracts. Fluorescent immunohistochemistry was used to visualize localization of Sp1, MZF‐1, and SOD3. For in vitro studies, cultured rat neurons were isolated at embryonic day 18 and transfected with scrambled or MZF‐1 siRNA (n=3 per group). Neurons were treated with PBS or 200 ng/mL LIF prior to 24 h in vitro ischemia induced by oxygen glucose deprivation. Lactate dehydrogenase (LDH) release was measured to assess neuronal death. SOD3, Sp1, and MZF‐1 mRNA levels were measured with real‐time PCR. MZF‐1 protein levels were quantified in cultured neurons with immunocytochemistry.ResultsLIF treatment did not significantly alter MZF‐1 or Sp1 expression in the brain at 72 h after MCAO. However, LIF significantly increased expression of MZF‐1 in the spleen at 72 h post‐MCAO compared to the PBS treatment. Four MZF‐1 binding sites and two Sp1 binding sites were identified in the rat SOD3 promoter using Genomatix, and confirmed using gel shift assays. LIF treatment caused nuclear accumulation of MZF‐1 at 72 h after MCAO, while Sp1 and MZF‐1 co‐localized with SOD3 at this time point. In cultured neurons, LIF treatment prior to 24 h in vitro ischemia significantly increased the percentage of MZF‐1‐positive neurons compared to PBS treatment (p<0.01). Real time PCR confirmed the increase in MZF‐1 mRNA LIF‐treated neurons compared to PBS‐treated neurons (p<0.05). In these same samples, LIF increased SOD3 mRNA after 24 h ischemia compared to PBS. Sp1 mRNA was not significantly altered by LIF treatment after 24 h ischemia. However, transfection of cultured neurons with MZF‐1 and Sp1 siRNA counteracted the significant decrease in LDH release observed after LIF treatment alone (p<0.05).ConclusionsLIF induces neuroprotection against ischemia through the transcription factors MZF‐1 and Sp1. Moreover, the LIF‐mediated increase in MZF‐1 activity occurs through increased gene and protein expression in cultured neurons as well as splenic tissueSupport or Funding InformationFunding for experiments in this study was provided for by the National Institute for Neurological Disorders and Stroke (project numbers 1R56NS091146‐01, 7R01NS091146‐02, and 1R01NS091146‐01Al),

Objective: To identify how leukemia inhibitory factor (LIF) regulates antioxidant neuroprotective and anti-inflammatory signaling through the expression and trafficking of its receptor (LIFR). Hypothesis: LIF treatment after stroke confers neuroprotection by increasing protein expression and membrane localization of LIFR in neural cells and splenocytes. Methods: Genomatix software was used to identify binding sites for the LIF-dependent transcription factors specificity protein 1 (Sp1) and myeloid zinc finger-1 (MZF-1) in the LIFR promoter. Male Sprague-Dawley rats underwent middle cerebral artery occlusion or sham surgery and injected with PBS or LIF (125 μg/kg) (n=8 per group) at 6, 24 and 48 h post-MCAO. Levels of LIFR, MZF-1, and Sp1 were measured using western blotting. Immunohistochemistry was used to determine localization of Sp1, LIF receptor, MZF-1, and superoxide dismutase 3, a LIF-inducible enzyme. Results: LIF (1.494 OD ± 0.161) significantly increased brain LIFR levels in ipsilateral tissue at 72 h after stroke compared to sham surgery (0.299 OD ± 0.060) and PBS treatment (0.399 OD ± 0.154) (0.0281 OD ± 0.011, p<0.01). Splenic LIFR levels decreased significantly after LIF (0.170 OD ± 0.010) treatment compared to PBS (0.228 OD ± 0.285, p<0.05) and sham rats (0.329 OD ± 0.031, P<0.001). LIFR was localized to neuronal nuclei but translocated to the cell membrane after injury. After LIF treatment, MZF-1 and Sp1 co-localized with superoxide dismutase 3 in cortical neurons. LIF significantly increased MZF-1, but not Sp1, in spleen tissue (0.158 OD ± 0.038) compared to PBS (0.109 OD ± 0.044) and sham (0.090 OD ± 0.018). Conclusions: Injury increases membrane localization of LIFR in neurons while LIF increases its receptor’s expression and Sp1/MZF-1 in stroke-injured neurons. As a part of its anti-inflammatory action, LIF causes downregulation of LIFR in the spleen after stroke.

Ischemic stroke produces a complex injury profile that is characterized by multiple, successive waves of tissue injury. The acute phase primarily involves energy failure and excitotoxic injury, which are the hallmarks of most neurodegenerative diseases and set the stage for delayed cell death. The delayed phase includes progressive cell death facilitated, in part, by a broad category of neuroinflammatory responses that perpetuates tissue injury through amplified cytokine signaling and the induction of cell death pathways. Among the potential therapeutic targets, cytokines have long been under scrutiny by researchers and clinicians by virtue of their profound effects on tissue injury and remodeling. The overlapping roles and dualistic nature of cytokines has proven challenging in elucidating their net effects within the ischemic brain. Due to the complex regulation of cytokines, their multiplicity of roles, and the various components of cerebral infarct expansion, much work is needed to decipher which key signals should be targeted. In addition to identifying putative pro- and anti-inflammatory cytokines for therapeutic interventions, the timing of intervention will likely be critical in the success of any therapeutic that acts to modulate cytokine signaling within the ischemic microenvironment.

Objective: To determine whether leukemia inhibitory factor (LIF) upregulates superoxide dismutase 3 (SOD3), a neuroprotective antioxidant enzyme, through the transcription factor myeloid zinc finger-1 (MZF-1). Hypothesis: MZF-1 facilitates LIF-mediated neuroprotection by increasing transcription of the SOD3 gene in neurons. Methods: After middle cerebral artery occlusion or sham surgery, male Sprague-Dawley rats were injected with PBS or LIF (125 μg/kg) (n=4 per group). Rats were euthanized 72 h after injury and western blotting was used to measure MZF-1 protein expression in brain tissue. For in vitro studies, rat neurons were transfected with scrambled or MZF-1 siRNA (n=3 per group). Neurons were treated with PBS or 200 ng/mL LIF prior to 24 h in vitro ischemia induced by oxygen glucose deprivation. Lactate dehydrogenase (LDH) release was measured to assess neuronal death. MZF-1 levels were quantified in cultured neurons with immunocytochemistry. SOD3 and MZF-1 mRNA levels were measured with real-time PCR. Results: LIF (0.938 OD units ± 0.170), but not PBS (0.562 OD units ± 0.223), significantly increased MZF-1 protein expression in ipsilateral tissue 72 h after stroke compared to sham surgery (0.411 OD units ± 0.039, p<0.05). LIF treatment prior to 24 h in vitro ischemia significantly increased the percentage of MZF-1-positive neurons (52.17 % ± 0.93) compared to PBS 44.84 % ± 1.11, p<0.01). PCR results confirmed the increase in MZF-1 mRNA (1.89 fold change ± 0.33) in LIF-treated neurons compared to PBS-treated neurons (1.00 fold change ± 0.23, p<0.05). Moreover, LIF increased SOD3 mRNA after 24 h ischemia (1.35 fold change ± 0.03) compared to PBS (1.00 fold change ± 0.13, p<0.05). LIF decreased LDH release (363,967 ± 68,557 neuronal units) compared to PBS (559,856 ± 60,555 neuronal units, p<0.05) among neurons transfected with scrambled siRNA. However, MZF-1 siRNA attenuated the neuroprotective effect of LIF (690,633 neuronal units ± 19,167; p<0.05). Conclusions: MZF-1 plays a fundamental role in a novel neuroprotective pathway by enhancing SOD3 expression and provides insight regarding protection by antioxidant enzymes during stroke.

Cerebral edema after stroke is associated with poor neurological outcomes. Current therapies are limited to osmotic agents, such as hypertonic saline (HS), which reduce intracranial pressure. Although studies have demonstrated edema reductions following HS, tissue survival has not been thoroughly examined. Additionally, the efficacy of promising pharmacological agents has not been evaluated for synergy with osmotic agents. Conivaptan is an FDA-approved vasopressin receptor antagonist that may exert both osmotic and anti-inflammatory effects. In this study, rats were subjected to middle cerebral artery occlusion prior to treatment with 5 % HS bolus +5 % HS maintenance (HS), conivaptan alone (Con), conivaptan +5 % HS maintenance (Con + HS), or conivaptan +5 % HS bolus +5 % maintenance (Con + HSb). Treatments were initiated at six (Early) or 24 h (Late) following stroke and rats were euthanized at 48 h to evaluate infarct volume, brain edema, and microglia/macrophage activation. Infarct volume and brain edema in the Early HS, Early Con, and Late HS groups were significantly reduced compared with controls. Interestingly, only the Early Con group demonstrated reduced microglia/macrophage activation. These data suggest an anti-inflammatory mechanism for conivaptan and provide support for a multipronged approach combining osmotic agents with compounds that inhibit the neuroinflammatory response to stroke.

The consumption of flavan-3-ol-containing foods, including (-)-epicatechin (EC), has been linked to lower incidence of cardiovascular disease and stroke. We previously demonstrated nuclear transcription factor erythroid 2p45-related factor-2 (Nrf2) -dependent EC efficacy in reducing stroke-induced deficits in 2-mo-old mice; yet stroke is primarily a disease of the elderly. Because neuroinflammation, oxidative stress, and vascular dysfunction are hallmarks of aging, we tested whether Nrf2 mediates EC efficacy in aging mice through modulation of glial responses and blood brain barrier permeability. First, we compared anastomosis in naïve wild-type and C57BL/6 Nrf2(-/-) mice to identify potential differences in cerebrovascular architecture. Data showed no significant differences in the number of anastomoses or mean intersection points, indicating similar gross vascular physiology. To assess efficacy and mechanisms of protection, wild-type or Nrf2(-/-) mice were administered the minimum effective EC dose established in our previous studies before the permanent distal middle cerebral artery occlusion. Similar to previous results with young mice, 12-mo-old wild types also showed significant reductions in infarct volume (41.01 ± 29.57%) and improved performance in removing adhesive tape relative to vehicle-treated controls, whereas a trend toward protection was observed in Nrf2(-/-). However, EC did not reduce immunoreactivity for the microglia/macrophage marker anti-ionized calcium-binding adapter molecule 1, suggesting that dampened activation/recruitment did not account for EC protection. Furthermore, there were no differences in mouse IgG extravasation or spontaneous hemorrhage between EC-treated groups. These data demonstrate that EC protection occurs independent of microglia/macrophage modulation or blood brain barrier preservation, suggesting that the glial cell responses in young mice are compensatory to another, and potentially novel, protective mechanism.

Objective: To determine the molecular mechanisms by which leukemia inhibitory factor (LIF) protects neurons during permanent middle cerebral artery occlusion (MCAO). Hypothesis: LIF protects neurons from ischemic oxidative injury through Akt-mediated increases in superoxide dismutase 3 (SOD3) expression and total SOD activity. Methods: Male Sprague-Dawley rats injected with vehicle (PBS) or LIF (125 μg/kg) were euthanized 24, 48, and 72 h post-MCAO or sham surgery for assessment of SOD activity and SOD3 expression. Rat cortical neurons were subjected to 24 h oxygen glucose deprivation or normoxia following treatment with vehicle , 50, 200, or 1000 ng/mL LIF (n=3 per group). In a third experiment, neurons were treated with vehicle, 50 ng/mL LIF,10 μM Akt inhibitor, or 50 ng/mL LIF+10 μM Akt inhibitor (n=3 per group) prior to oxygen glucose deprivation. Lactate dehydrogenase levels in media were measured to assess neuronal death. We used immunocytochemistry to assess expression of SOD3, phospho-Akt (Ser473), and myeloid zinc finger-1 in neurons. Results: LIF significantly increased brain SOD activity (2.085 ± 0.476 U/mg lysate; n=5) compared to vehicle (0.919 ± 0.285 U/mg lysate; n=7) 72 h post-MCAO (p<0.05). LIF significantly increased SOD3 protein expression in the brain (3.707 ± 0.541 units; n=3) compared to vehicle (1.401 ± 0.825 units; n=3) at 72 h post-MCAO (p<0.05). Co-localization of phospho-Akt and SOD3 occurred 24 h post-MCAO. Additionally, 50 ng/mL LIF (0.789 ± 0.018 U/mL media; n=3) significantly decreased lactate dehydrogenase levels in vitro compared to vehicle (1.000 ± 0.023 U/mL media; n=3) (p<0.0001). Co-incubation of 50/mL ng LIF with 10 μM Akt inhibitor (1.070 ±0.106 U/mL media; n=3) reversed the protective effect of LIF (0.705 ± 0.050 U/mL media; n=3) (p<0.01). Increases in SOD3, phospho-Akt, and myeloid zinc finger-1 staining after oxygen glucose deprivation and LIF were abolished upon Akt inhibition. Conclusion: Our data demonstrates that LIF-mediated protection against ischemia in vivo and in vitro results from Akt-dependent increases in SOD3 expression and SOD activity.

Leonardo CC, Mendes M, Ahmad AS, Doré S. Efficacy of prophylactic flavan-3-ol in permanent focal ischemia in 12-mo-old mice. Am J Physiol Heart Circ Physiol 308: H583–H591, 2015. First published January 9, 2015; doi:10.1152/ajpheart.00239.2014.—The consumption of flavan-3-ol-containing foods, including ( )-epicatechin (EC), has been linked to lower incidence of cardiovascular disease and stroke. We previously demonstrated nuclear transcription factor erythroid 2p45-related factor-2 (Nrf2) -dependent EC efficacy in reducing stroke-induced deficits in 2-mo-old mice; yet stroke is primarily a disease of the elderly. Because neuroinflammation, oxidative stress, and vascular dysfunction are hallmarks of aging, we tested whether Nrf2 mediates EC efficacy in aging mice through modulation of glial responses and blood brain barrier permeability. First, we compared anastomosis in naïve wild-type and C57BL/6 Nrf2 / mice to identify potential differences in cerebrovascular architecture. Data showed no significant differences in the number of anastomoses or mean intersection points, indicating similar gross vascular physiology. To assess efficacy and mechanisms of protection, wild-type or Nrf2 / mice were administered the minimum effective EC dose established in our previous studies before the permanent distal middle cerebral artery occlusion. Similar to previous results with young mice, 12-mo-old wild types also showed significant reductions in infarct volume (41.01 29.57%) and improved performance in removing adhesive tape relative to vehicle-treated controls, whereas a trend toward protection was observed in Nrf2 / . However, EC did not reduce immunoreactivity for the microglia/macrophage marker anti-ionized calcium-binding adapter molecule 1, suggesting that dampened activation/recruitment did not account for EC protection. Furthermore, there were no differences in mouse IgG extravasation or spontaneous hemorrhage between EC-treated groups. These data demonstrate that EC protection occurs independent of microglia/macrophage modulation or blood brain barrier preservation, suggesting that the glial cell responses in young mice are compensatory to another, and potentially novel, protective mechanism.

Background: Spontaneous intracerebral hemorrhage (ICH) is the deadliest and most debilitating form of stroke with a first year mortality rate as high as 50% to 60%. Role of prostaglandin E2 (PGE2) receptor EP1 has been extensively studies in ischemic stroke; however, the precise role of this receptor in intracerebral hemorrhage-induced brain injury is unknown. Therefore, in this study we determined the role of the EP1 receptor in collagenase-induced hemorrhagic stroke. Methods: ICH was induced randomly in 2.0-2.5 month old male C57BL/6 wildtype (WT) and EP1 knockout (EP1-/-) mice by intrastriatal injection of collagenase. Functional outcomes including neurologic deficits, rotarod performance, open field activity, and adhesive removal performance were evaluated at 72h post ICH. Hematoma volume, and cell survival and death, were assessed using cresyl violet and Fluoro-Jade staining respectively. Microglial activation was estimated using Iba1 immunoreactivity. Phagocytosis was assessed with fluorescently-labeled microspheres injected into the site of the hematoma and at the end of the survival time Iba1 immunoreactivity was used to label cells with microspheres. Values are expressed in (mean±SEM) and the number of cells/field was provided by averaging four different regions surrounding the hematoma. Results: Following 72h post injury, EP1-/- mice showed worsened outcomes compared to the WT mice. The results revealed elevated neurological and sensorimotor deficits and exacerbated hematoma volume. Fluoro-Jade staining showed significantly increased numbers of degenerating neurons and reduced neuronal survival in EP1-/- as compared with the WT mice. The in vivo phagocytic behavior of microglial cells in WT and EP1-/- suggested that the number of microspheres incorporated into Iba1-positive cells was 145.4±15.4% greater in WT than in EP1-/- mice. Conclusion: These results suggest that, the deletion of PGE2 EP1 receptor results in augmented hemorrhagic brain injury partially through reduced phagocytosis.[Supported by NIH R01 funds (SD)]

Human umbilical cord blood (HUCB) cells have shown efficacy in rodent models of focal ischemia and in vitro systems that recapitulate stroke conditions. One potential mechanism of protection is through secretion of soluble factors that protect neurons and oligodendrocytes (OLs) from oxidative stress. To overcome practical issues with cellular therapies, identification of soluble factors released by HUCB and other stem cells may pave the way for treatment modalities that are safer for a larger percentage of stroke patients. Among these soluble factors is leukemia inhibitory factor (LIF), a cytokine that exerts pleiotropic effects on cell survival. Here, data show that LIF effectively reduced infarct volume, reduced white matter injury and improved functional outcomes when administered to rats following permanent middle cerebral artery occlusion. To further explore downstream signaling, primary oligodendrocyte cultures were exposed to oxygen-glucose deprivation to mimic stroke conditions. LIF significantly reduced lactate dehydrogenase release from OLs, reduced superoxide dismutase activity and induced peroxiredoxin 4 (Prdx4) transcript. Additionally, the protective and antioxidant capacity of LIF was negated by both Akt inhibition and co-incubation with Prdx4-neutralising antibodies, establishing a role for the Akt signaling pathway and Prdx4-mediated antioxidation in LIF protection.

Background: The consumption of flavanol containing foods, including (-)-epicatechin, has been linked to lower incidence of cardiovascular disease and stroke. We have previously demonstrated that epicatechin prophylaxis reduces stroke-induced anatomical and functional deficits in young, healthy mice; yet clinical stroke is primarily a disease of the elderly. Since neuroinflammation and vascular dysfunction are associated with aging, we aimed to test whether epicatechin is also protective in aging mice subjected to experimental stroke, and if so, whether this results from reduced glial cell activation and blood brain barrier permeability. Methods: Twelve-month-old wildtype and C57BL/6 Nrf2 knockout mice were administered 15mg/kg epicatechin, the minimum effective dose in young, healthy mice, prior to permanent distal middle cerebral artery occlusion. Mice were evaluated for functional recovery at one day post-stroke using the Adhesive Removal Test. Infarct volume, gliosis, and blood brain barrier permeability estimates were conducted seven days following stroke. Additionally, we compared anastomosis in wildtype and Nrf2 knockout mice and assessed hemorrhage frequency in studies using four- and 12-month-old mice. Results: Consistent with previous results in young mice, 12-month-old wildtype mice pretreated with epicatechin showed significant reductions in infarct volume and latency to remove adhesive tape relative to vehicle-treated controls, while Nrf2 knockout mice were not protected by epicatechin. Interestingly, epicatechin did not reduce Iba1 immunoreactivity or mouse IgG extravasation at seven days post-stroke. Similarly, there were no significant differences in anastomosis or spontaneous hemorrhage between wildtype and Nrf2 knockouts, indicating that cerebral vascular physiology is similar and gross vascular integrity was unaffected by treatment. Conclusion: Thus, although epicatechin prophylaxis reduces infarct volume and functional deficits, it does not exert sustained effects on gliosis or cerebrovascular integrity in aging mice.

Background: Epidemiological studies indicate that flavanol consumption reduces the propensity to develop cerebrovascular disease. Available data suggest actions on multiple pro-inflammatory pathways, yet it remains unclear which pathways mediate functional recovery after stroke. Our goal is to begin identifying the mechanisms by which the flavanol (-)-epicatechin (EC) improves anatomical and functional outcomes. Based upon data from initial dose-response experiments, ongoing studies are investigating hypothesized protective pathways involving matrix metalloproteinase-mediated blood brain barrier protection and Nrf2 transcriptional activation. Methods: Male, 8-10wk old C57BL/6 mice were pretreated with EC 90m prior to permanent distal middle cerebral artery occlusion. Vehicle or EC was administered by oral gavage to mimic dietary consumption. Mice were evaluated 1, 4 and 7d post-stroke for performance on various sensorimotor tasks prior to histological assessments. Results: Initial experiments demonstrated that mice treated with 15mg/kg EC showed reduced latency to remove adhesive tape at 1d compared to vehicle controls (n=12, p<0.01). Similarly, immunoreactivity for the microglia/macrophage marker Iba1 was increased in the ipsilateral hemispheres of mice 7d after treatment with vehicle (p<0.01), whereas pretreatment with 15mg/kg blocked this effect (n=4). Mice treated with 15mg/kg also showed a trend toward reduced infarct volume relative to vehicle controls (n=5-9 per group). In subsequent reduced dosing studies, vehicle-treated mice again showed deficiencies in removing adhesive tape at 1d (n=8, p<0.01). Remarkably, mice treated with 15, 10 or 5mg/kg EC showed no deficits. Similarly, vehicle control mice showed grip strength impairments up to 7d (n=8, p<0.05) that were absent in all groups of EC-treated mice. Conclusions: Preventative administration of EC promotes functional recovery in mice subjected to experimental stroke. Investigations are underway to determine the pathways mediated by EC following administration at these therapeutic doses. Together, these data will provide insights into the potential for (-)-epicatechin as a clinical therapeutic.