The Implant-Induced Foreign Body Response Is Limited by CD13-Dependent Regulation of Ubiquitination of Fusogenic Proteins.

Implanted medical devices, from artificial heart valves and arthroscopic joints to implantable sensors, often induce a foreign body response (FBR), a form of chronic inflammation resulting from the inflammatory reaction to a persistent foreign stimulus. The FBR is characterized by a subset of multinucleated giant cells (MGCs) formed by macrophage fusion, the foreign body giant cells (FBGCs), accompanied by inflammatory cytokines, matrix deposition, and eventually deleterious fibrotic implant encapsulation. Despite efforts to improve biocompatibility, implant-induced FBR persists, compromising the utility of devices and making efforts to control the FBR imperative for long-term function. Controlling macrophage fusion in FBGC formation presents a logical target to prevent implant failure, but the actual contribution of FBGCs to FBR-induced damage is controversial. CD13 is a molecular scaffold, and in vitro induction of CD13KO bone marrow progenitors generates many more MGCs than the wild type, suggesting that CD13 regulates macrophage fusion. In the mesh implant model of FBR, CD13KO mice produced significantly more peri-implant FBGCs with enhanced TGF-ß expression and increased collagen deposition versus the wild type. Prior to fusion, increased protrusion and microprotrusion formation accompanies hyperfusion in the absence of CD13. Expression of fusogenic proteins driving cell-cell fusion was aberrantly sustained at high levels in CD13KO MGCs, which we show is due to a novel CD13 function, to our knowledge, regulating ubiquitin/proteasomal protein degradation. We propose CD13 as a physiologic brake limiting aberrant macrophage fusion and the FBR, and it may be a novel therapeutic target to improve the success of implanted medical devices. Furthermore, our data directly implicate FBGCs in the detrimental fibrosis that characterizes the FBR.

Fluorescent liposomal nanocarriers for targeted drug delivery in ischemic stroke therapy.

Ischemic stroke causes acute CNS injury and long-term disability, with limited treatment options such as surgical clot removal or clot-busting drugs. Neuroprotective therapies are needed to protect vulnerable brain regions. The purinergic receptor P2X4 is activated during stroke and exacerbates post-stroke damage. The chemical compound 5-(3-Bromophenyl)-1,3-dihydro-2H-Benzofuro[3,2-e]-1,4-diazepin-2-one (5BDBD) inhibits P2X4 and has shown neuroprotective effects in rodents. However, it is difficult to formulate for systemic delivery to the CNS. The current manuscript reports for the first time, the synthesis and characterization of 5BDBD PEGylated liposomal formulations and evaluates their feasibility to treat stroke in a preclinical mice model. A PEGylated liposomal formulation of 5BDBD was synthesized and characterized, with encapsulation efficacy of >80%, and release over 48 hours. In vitro and in vivo experiments with Nile red encapsulation showed cytocompatibility and CNS infiltration of nanocarriers. Administered 4 or 28 hours after stroke onset, the nanoformulation provided significant neuroprotection, reducing infarct volume by ∼50% compared to controls. It outperformed orally-administered 5BDBD with a lower dose and shorter treatment duration, suggesting precise delivery by nanoformulation improves outcomes. The fluorescent nanoformulations may serve as a platform for delivering and tracking therapeutic agents for stroke treatment.

Transcriptomic analysis reveals novel age-independent immunomodulatory proteins as a mode of cerebroprotection in P2X4 receptor knockout mice after ischemic stroke.

Identification of new potential drug target proteins and their plausible mechanisms for stroke treatment is critically needed. We previously showed that genetic deletion and short-term pharmacological inhibition of P2X4, a purinergic receptor for adenosine triphosphate (ATP), provides acute cerebroprotection. However, potential mechanisms remain unknown. Therefore, we employed RNA-Seq technology to identify the gene expression profiles and pathway analysis followed by qPCR validation of differentially expressed genes (DEGs). This analysis identified roles of DEGs in certain biological processes responsible for P2X4R-dependent cerebroprotection after stroke. We subjected both young and aged male and female global P2X4 receptor knock out (P2X4RKO) and littermate WT (WT) mice to ischemic stroke. After three days, mice were sacrificed, and total RNA was isolated using Trizol and subjected to RNA-Seq and NanoString-mediated qPCR. DESeq2, Gene Ontology (GO), and Ingenuity Pathway Analysis (IPA) were used to identify gene expression profiles and biological pathways. We found 2246 DEGs in P2X4R KO vs. WT tissue after stroke. Out of these DEGs, 1920 genes were downregulated and 325 genes were upregulated in P2X4R KO. GO/IPA analysis of the top 300 DEGs suggests an enrichment of inflammation and extracellular matrix component genes. qPCR validation of the top 30 DEGs revealed downregulation of two common age-independent genes in P2X4R KO mice: Interleukin-6 (Il-6), an inflammatory cytokine, and Cytotoxic T Lymphocyte-Associated Protein 2 alpha (Ctla2a), an immunosuppressive factor. These data suggest that P2X4R-mediated cerebroprotection after stroke is initiated by attenuation of immune modulatory pathways in both young and aged mice of both sexes.

Transcriptomic analysis reveals novel age-independent immunomodulatory proteins as a mode of cerebroprotection in P2X4R KO mice after ischemic stroke.

Identification of new potential drug target proteins and their plausible mechanisms for stroke treatment is critically needed. We previously showed that genetic deletion and short-term pharmacological inhibition of P2X4R, a purinergic receptor for adenosine triphosphate ATP, provides acute cerebroprotection. However, potential mechanisms remain unknown. Therefore, we employed RNA-seq technology to identify the gene expression profiles, pathway analysis, and qPCR validation of differentially expressed genes (DEGs). This analysis identified roles of DEGs in certain biological processes responsible for P2X4R-dependent cerebroprotection after stroke. We subjected both young and aged male and female global P2X4 KO and littermate WT mice to ischemic stroke. After 3 days, mice were sacrificed, total RNA was isolated using Trizol, and subjected to RNA-seq and Nanostring-mediated qPCR. DESeq2, Gene Ontology (GO), and Ingenuity Pathway Analysis (IPA) were used to identify mRNA transcript expression profiles and biological pathways. We found 2246 DEGs in P2X4R KO vs WT tissue after stroke. Out of these DEGs, 1920 gene were downregulated, and 325 genes were upregulated in KO. GO/IPA analysis of the top 300 DEGs suggests an enrichment of inflammation and extracellular matrix component genes. qPCR validation of the top 30 DEGs revealed downregulation of two common age-independent genes in P2X4R KO mice: Interleukin-6 ( IL-6) , an inflammatory cytokine, and Cytotoxic T Lymphocyte-Associated Protein 2 alpha ( Ctla2a ), an immunosuppressive factor. These data suggest that P2X4R-mediated cerebroprotection after stroke is initiated by attenuation of immune modulatory pathways in both young and aged mice of both sexes.

Molecular mediators of angiogenesis and neurogenesis after ischemic stroke.

The mechanisms governing neurological and functional recovery after ischemic stroke are incompletely understood. Recent advances in knowledge of intrinsic repair processes of the CNS have so far translated into minimal improvement in outcomes for stroke victims. Better understanding of the processes underlying neurological recovery after stroke is necessary for development of novel therapeutic approaches. Angiogenesis and neurogenesis have emerged as central mechanisms of post-stroke recovery and potential targets for therapeutics. Frameworks have been developed for conceptualizing cerebral angiogenesis and neurogenesis at the tissue and cellular levels. These models highlight that angiogenesis and neurogenesis are linked to each other and to functional recovery. However, knowledge of the molecular framework linking angiogenesis and neurogenesis after stroke is limited. Studies of potential therapeutics typically focus on one mediator or pathway with minimal discussion of its role within these multifaceted biochemical processes. In this article, we briefly review the current understanding of the coupled processes of angiogenesis and neurogenesis after stroke. We then identify the molecular mediators and signaling pathways found in pre-clinical studies to upregulate both processes after stroke and contextualizes them within the current framework. This report thus contributes to a more-unified understanding of the molecular mediators governing angiogenesis and neurogenesis after stroke, which we hope will help guide the development of novel therapeutic approaches for stroke survivors.

Structure-Activity Relationship and Neuroprotective Activity of 1,5-Dihydro-2H-naphtho[1,2-b][1,4]diazepine-2,4(3H)-diones as P2X4 Receptor Antagonists.

We analyzed the P2X4 receptor structure-activity relationship of a known antagonist 5, a 1,5-dihydro-2H-naphtho[1,2-b][1,4]diazepine-2,4(3H)-dione. Following extensive modification of the reported synthetic route, 4-pyridyl 21u (MRS4719) and 6-methyl 22c (MRS4596) analogues were most potent at human (h) P2X4R (IC50 0.503 and 1.38 µM, respectively, and selective versus hP2X1R, hP2X2/3R, hP2X3R). Thus, the naphthalene 6-, but not 7-position was amenable to substitution, and an N-phenyl ring aza-scan identified 21u with 3-fold higher activity than 5. Compounds 21u and 22c showed neuroprotective and learning- and memory-enhancing activities in a mouse middle cerebral artery occlusion (MCAO) model of ischemic stroke, with potency of 21u > 22c. 21u dose-dependently reduced infarct volume and reduced brain atrophy at 3 and 35 days post-stroke, respectively. Relevant to clinical implication, 21u also reduced ATP-induced [Ca2+]i influx in primary human monocyte-derived macrophages. This study indicates the translational potential of P2X4R antagonists for treating ischemic stroke, including in aging populations.

A Model for Encephalomyosynangiosis Treatment after Middle Cerebral Artery Occlusion-Induced Stroke in Mice.

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The brain's ability to self-heal after ischemic stroke is limited by inadequate blood supply in the impacted area. Encephalomyosynangiosis (EMS) is a neurosurgical procedure that achieves angiogenesis in patients with moyamoya disease. It involves craniotomy with placement of a vascular temporalis muscle graft on the ischemic brain surface. EMS has never been studied in the setting of acute ischemic stroke in mice. The hypothesis driving this study is that EMS enhances cerebral angiogenesis at the cortical surface surrounding the muscle graft. The protocol shown here describes the procedure and provides initial data supporting the feasibility and efficacy of the EMS approach. In this protocol, after 60 min of transient middle cerebral artery occlusion (MCAo), mice were randomized to either MCAo or MCAo + EMS treatment. The EMS was performed 3-4 h after occlusion. The mice were sacrificed 7 or 21 days after MCAo or MCAo + EMS treatment. Temporalis graft viability was measured using nicotinamide adenine dinucleotide reduced-tetrazolium reductase assay. A mouse angiogenesis array quantified angiogenic and neuromodulating protein expression. Immunohistochemistry was used to visualize graft bonding with brain cortex and change in vessel density. The preliminary data here suggest that grafted muscle remained viable 21 days after EMS. Immunostaining showed successful graft implantation and increase in vessel density near the muscle graft, indicating increased angiogenesis. Data show that EMS increases fibroblast growth factor (FGF) and decreases osteopontin levels after stroke. Additionally, EMS after stroke did not increase mortality suggesting that protocol is safe and reliable. This novel procedure is effective and well-tolerated and has the potential to provide information of novel interventions for enhanced angiogenesis after acute ischemic stroke.

Nanoparticle Delivered Anti-miR-141-3p for Stroke Therapy.

Ischemic stroke and factors modifying ischemic stroke responses, such as social isolation, contribute to long-term disability worldwide. Several studies demonstrated that the aberrant levels of microRNAs contribute to ischemic stroke injury. In prior studies, we established that miR-141-3p increases after ischemic stroke and post-stroke isolation. Herein, we explored two different anti-miR oligonucleotides; peptide nucleic acid (PNAs) and phosphorothioates (PS) for ischemic stroke therapy. We used US FDA approved biocompatible poly (lactic-co-glycolic acid) (PLGA)-based nanoparticle formulations for delivery. The PNA and PS anti-miRs were encapsulated in PLGA nanoparticles by double emulsion solvent evaporation technique. All the formulated nanoparticles showed uniform morphology, size, distribution, and surface charge density. Nanoparticles also exhibited a controlled nucleic acid release profile for 48 h. Further, we performed in vivo studies in the mouse model of ischemic stroke. Ischemic stroke was induced by transient (60 min) occlusion of middle cerebral artery occlusion followed by a reperfusion for 48 or 72 h. We assessed the blood-brain barrier permeability of PLGA NPs containing fluorophore (TAMRA) anti-miR probe after systemic delivery. Confocal imaging shows uptake of fluorophore tagged anti-miR in the brain parenchyma. Next, we evaluated the therapeutic efficacy after systemic delivery of nanoparticles containing PNA and PS anti-miR-141-3p in mice after stroke. Post-treatment differentially reduced both miR-141-3p levels in brain tissue and infarct injury. We noted PNA-based anti-miR showed superior efficacy compared to PS-based anti-miR. Herein, we successfully established that nanoparticles encapsulating PNA or PS-based anti-miRs-141-3p probes could be used as a potential treatment for ischemic stroke.

Neuroprotective and neuro-rehabilitative effects of acute purinergic receptor P2X4 (P2X4R) blockade after ischemic stroke.

Stroke remains a leading cause of disability in the United States. Despite recent advances, interventions to reduce damage and enhance recovery after stroke are lacking. P2X4R, a receptor for adenosine triphosphate (ATP), regulates activation of myeloid immune cells (infiltrating monocytes/macrophages and brain-resident microglia) after stroke injury. However, over-stimulation of P2X4Rs due to excessive ATP release from dying or damaged neuronal cells can contribute to ischemic injury. Therefore, we pharmacologically inhibited P2X4R to limit the over-stimulated myeloid cell immune response and improve both acute and chronic stroke recovery. We subjected 8-12-week-old male and female wild type mice to a 60 min right middle cerebral artery occlusion (MCAo) followed by 3 or 30 days of reperfusion. We performed histological, RNA sequencing, behavioral (sensorimotor, anxiety, and depressive), and biochemical (Evans blue dye extravasation, western blot, quantitative PCR, and flow cytometry) analyses to determine the acute (3 days after MCAo) and chronic (30 days after MCAo) effects of P2X4R antagonist 5-BDBD (1 mg/kg P.O. daily x 3 days post 4 h of MCAo) treatment. 5-BDBD treatment significantly (p < .05) reduced infarct volume, neurological deficit (ND) score, levels of cytokine interleukin-1 beta (IL-1ß) and blood brain barrier (BBB) permeability in the 3-day group. Chronically, 5-BDBD treatment also conferred progressive recovery (p < .05) of motor balance and coordination using a rotarod test, as well as reduced anxiety-like behavior over 30 days. Interestingly, depressive-type behavior was not observed in mice treated with 5-BDBD for 3 days. In addition, flow cytometric analysis revealed that 5-BDBD treatment decreased the total number of infiltrated leukocytes, and among those infiltrated leukocytes, pro-inflammatory cells of myeloid origin were specifically reduced. 5-BDBD treatment reduced the cell surface expression of P2X4R in flow cytometry-sorted monocytes and microglia without reducing the total P2X4R level in brain tissue. In summary, acute P2X4R inhibition protects against ischemic injury at both acute and chronic time-points after stroke. Reduced numbers of infiltrating pro-inflammatory myeloid cells, decreased surface P2X4R expression, and reduced BBB disruption are likely its mechanism of neuroprotection and neuro-rehabilitation.

EMMPRIN/CD147 plays a detrimental role in clinical and experimental ischemic stroke.

BACKGROUND: Ischemic stroke is a devastating disease, often resulting in death or permanent neurological deficits. EMMPRIN/CD147 is a plasma membrane protein that induces the production of matrix metalloproteinases (MMPs), which contribute to secondary damage after stroke by disrupting the blood brain barrier (BBB) and facilitating peripheral leukocyte infiltration into the brain. RESULTS: CD147 surface expression increased significantly after stroke on infiltrating leukocytes, astrocytes and endothelial cells, but not on resident microglia. Inhibition of CD147 reduced MMP levels, decreased ischemic damage, and improved functional, cognitive and histological outcomes after experimental ischemic stroke in both young and aged mice. In stroke patients, high levels of serum CD147 24 hours after stroke predicted poor functional outcome at 12 months. Brain CD147 levels were correlated with MMP-9 and secondary hemorrhage in post-mortem samples from stroke patients. CONCLUSIONS: Acute inhibition of CD147 decreases levels of MMP-9, limits tissue loss, and improves long-term cognitive outcomes following experimental stroke in aged mice. High serum CD147 correlates with poor outcomes in stroke patients. This study identifies CD147 as a novel, clinically relevant target in ischemic stroke.

Micro RNA 181c-5p: A promising target for post-stroke recovery in socially isolated mice.

Dysregulation of microRNAs (miRNAs) has been tied to several neurological disorders, including ischemic stroke. It has also been established that social environments can modulate miRNA profiles. We have previously shown that post-stroke social isolation (SI) is linked to poor stroke outcomes and that miR-181c-5p emerged as one of few lead miRNAs that was downregulated in both stroke and SI. Therefore, in this study we examined the potential role of miR-181c-5p mimic in reversing the detrimental effects of post-stroke SI. Two to three-month-old C57BL/6 male mice were pair-housed (PH) for at least two weeks. After two weeks, mice underwent stroke survival surgery using middle cerebral artery occlusion (MCAO) and were randomly assigned to one of two housing conditions: stroke isolation (ST-ISO) or stroke pair-housing with a healthy partner (ST-PH). ST-ISO mice were randomized to receive either miR-181c-5p mimic or a scrambled RNA (7 mg/kg i.v./day×drug) control at 24 h and 48 h after stroke. The effects of miR-181c-5p mimic treatment were evaluated at 1, 3, and 7 days after stroke at histological, behavioral, and biochemical levels. Target genes of miR-181c-5p were then analyzed by qPCR using an RT2 Profiler qPCR Array of pre-coated miR-181c gene targets. Temporal profile expression data suggested that miR-181c-5p was significantly downregulated (p < 0.05 vs ST-PH) up to 7 days after post-stroke SI. MiR-181c-5p mimic treatment significantly increased miR-181c-5p expression in brain tissue and showed partial swift recovery in sensorimotor deficit. Target gene analysis identified downregulation of several calcium signaling-related genes, e.g., Cpne2 and Gria 1 & 2 after miR-181c-5p mimic treatment. In summary, present data suggests that miR-181c-5p is a potential target for post-stroke SI. Data also suggests that genes related to calcium and glutamate signaling might be involved in the beneficial effect of the miR-181c-5p mimic.

Role of bone marrow-derived macrophages (BMDMs) in neurovascular interactions during stroke.

Stroke is a leading cause of disability worldwide and hence remains a major medical concern. Besides several pathological features, such as excitotoxicity, peri-infarct depolarization, acidosis, reactive oxygen species generation, apoptosis, and necrosis, dysregulation of the immune system severely affects stroke outcomes. After stroke onset, microglia - the brain-resident macrophage immune cells - and peripheral immune cells affect stoke injury/recovery by releasing pro-inflammatory and/or anti-inflammatory cytokines depending on their microenvironment. These pro- or anti-inflammatory cytokines further affect integrity of the blood brain barrier (BBB) and modulate immune infiltration after stroke. Among peripheral immune cells, bone marrow-derived macrophages (BMDMs) play a critical role in stroke pathology which peaks between three and seven days post-stroke. BMDMs have been extensively studied for their role in exacerbation of stroke injury, however they have rarely been studied for their role in tissue repair. Nonetheless, these reparative roles are gaining attention since recent studies have shown either failure or worsening of long-term post-stroke recovery after blockade of peripheral immune infiltration. These diverse but paradoxical effects of infiltrating monocytes/macrophages encouraged us to summarize the latest findings in neuro-immune and immune-vascular interactions. This review highlights the multifaceted role of BMDMs in stroke onset and resolution, and emphasizes the significance of tapping the potential of these cells to gain better insight into disease progression and therapy.

CD200-CD200R1 inhibitory signaling prevents spontaneous bacterial infection and promotes resolution of neuroinflammation and recovery after stroke.

BACKGROUND: Ischemic stroke results in a robust inflammatory response within the central nervous system. As the immune-inhibitory CD200-CD200 receptor 1 (CD200R1) signaling axis is a known regulator of immune homeostasis, we hypothesized that it may play a role in post-stroke immune suppression after stroke. METHODS: In this study, we investigated the role of CD200R1-mediated signaling in stroke using CD200 receptor 1-deficient mice. Mice were subjected to a 60-min middle cerebral artery occlusion and evaluated at days 3 and 7, representing the respective peak and early resolution stages of neuroinflammation in this model of ischemic stroke. Infarct size and behavioral deficits were assessed at both time points. Central and peripheral cellular immune responses were measured using flow cytometry. Bacterial colonization was determined in lung tissue homogenates both after acute stroke and in an LPS model of systemic inflammation. RESULTS: In wild-type (WT) animals, CD200R1 was expressed on infiltrating monocytes and lymphocytes after stroke but was absent on microglia. Early after ischemia (72 h), CD200R1-knockout (KO) mice had significantly poorer survival rates and an enhanced susceptibility to spontaneous bacterial colonization of the respiratory tract compared to wild-type (WT) controls, despite no difference in infarct or neurological deficits. While the CNS inflammation was resolved by day 7 post-stroke in WT mice, brain-resident microglia and monocyte activation persisted in CD200R1-KO mice, accompanied by a delayed, augmented lymphocyte response. At this time point, CD200R1-KO mice displayed greater weight loss, more severe neurological deficits, and impaired motor function compared to WT. Systemically, CD200R1-KO mice exhibited signs of persistent infection including lymphopenia, T cell activation and memory conversion, and narrowing of the TCR repertoire. These findings were confirmed in a second model of acute neuroinflammation induced by systemic endotoxin challenge. CONCLUSION: This study defines an essential role of CD200-CD200R1 signaling in stroke. Loss of CD200R1 led to high mortality, increased rates of post-stroke infection, and enhanced entry of peripheral leukocytes into the brain after ischemia, with no increase in infarct size. This suggests that the loss of CD200 receptor leads to enhanced peripheral inflammation that is triggered by brain injury.

Evaluation of the Neuroprotective Effect of Sirt3 in Experimental Stroke.

Sirtuins (Sirt) are a family of NAD+ dependent histone deacetylase (HDAC) proteins implicated in aging, cell cycle regulation, and metabolism. These proteins are involved in the epigenetic modification of neuromodulatory proteins after strokevia acetylation/deacetylation. The specific role of Sirt3, a mitochondrial sirtuin, in post-stroke injury has been relatively unexplored. Using male Sirt3 knockout (KO) mice and wild-type littermates (WT), we show that Sirt3 KO mice show significant neuroprotection at 3 days after ischemia/reperfusion (I/R) or stroke injury. The deacetylation activity of Sirt3, measured as the amount of reduced acetylated lysine, was increased after stroke. Stroke-induced increases in liver kinase 1 (LKB1) activity were also reduced in KO mice at 3 days after stroke. On further investigation, we found that the levels of Sirt1, another important member of the Sirtuin family, were increased in the brains of Sirt3 KO mice after stroke. To determine the translational relevance of these findings, we then tested the effects of pharmacological inhibition of Sirt3. We found no benefit of Sirt3 inhibition despite clear evidence of deacetylation. Overall, these data suggest that Sirt3 KO mice show neuroprotection by a compensatory rise in Sirt1 rather than the loss of Sirt3 after stroke. Further analysis reveals that the beneficial effects of Sirt1 might be mediated by a decrease in LKB1 activity after stroke. Finally, our data clearly demonstrate the importance of using both pharmacological and genetic methods in pre-clinical stroke studies.

Inhibition of miR-141-3p Ameliorates the Negative Effects of Poststroke Social Isolation in Aged Mice.

BACKGROUND AND PURPOSE: Social isolation increases mortality and impairs recovery after stroke in clinical populations. These detrimental effects have been recapitulated in animal models, although the exact mechanism mediating these effects remains unclear. Dysregulation of microRNAs (miRNAs) occurs in both strokes as well as after social isolation, which trigger changes in many downstream genes. We hypothesized that miRNA regulation is involved in the detrimental effects of poststroke social isolation in aged animals. METHODS: We pair-housed 18-month-old C57BL/6 male mice for 2 weeks before a 60-minute right middle cerebral artery occlusion or sham surgery and then randomly assigned mice to isolation or continued pair housing immediately after surgery. We euthanized mice either at 3, 7, or 15 days after surgery and isolated the perilesional frontal cortex for whole microRNAome analysis. In an additional cohort, we treated mice 1 day after stroke onset with an in vivo-ready antagomiR-141 for 3 days. RESULTS: Using whole microRNAome analysis of 752 miRNAs, we identified miR-141-3p as a unique miRNA that was significantly upregulated in isolated mice in a time-dependent manner up to 2 weeks after stroke. Posttreatment with an antagomiR-141-3p reduced the postisolation-induced increase in miR-141-3p to levels almost equal to those of pair-housed stroke controls. This treatment significantly reduced mortality (by 21%) and normalized infarct volume and neurological scores in poststroke-isolated mice. Quantitative PCR analysis revealed a significant upregulation of Tgfßr1 (transforming growth factor beta receptor 1, a direct target of miR-141-3p) and Igf-1 (insulin-like growth factor 1) mRNA after treatment with antagomiR. Treatment also increased the expression of other pleiotropic cytokines such as Il-6 (interleukin 6) and Tnf-α (tumor necrosis factor-α), an indirect or secondary target) in brain tissue. CONCLUSIONS: miR-141-3p is increased with poststroke isolation. Inhibition of miR-141-3p improved mortality, neurological deficits, and decreased infarct volumes. Importantly, these therapeutic effects occurred in aged animals, the population most at risk for stroke and poststroke isolation.

Aging alters the immunological response to ischemic stroke.

The peripheral immune system plays a critical role in aging and in the response to brain injury. Emerging data suggest inflammatory responses are exacerbated in older animals following ischemic stroke; however, our understanding of these age-related changes is poor. In this work, we demonstrate marked differences in the composition of circulating and infiltrating leukocytes recruited to the ischemic brain of old male mice after stroke compared to young male mice. Blood neutrophilia and neutrophil invasion into the brain were increased in aged animals. Relative to infiltrating monocyte populations, brain-invading neutrophils had reduced phagocytic potential, and produced higher levels of reactive oxygen species and extracellular matrix-degrading enzymes (i.e., MMP-9), which were further exacerbated with age. Hemorrhagic transformation was more pronounced in aged versus young mice relative to infarct size. High numbers of myeloperoxidase-positive neutrophils were found in postmortem human brain samples of old (> 71 years) acute ischemic stroke subjects compared to non-ischemic controls. Many of these neutrophils were found in the brain parenchyma. A large proportion of these neutrophils expressed MMP-9 and positively correlated with hemorrhage and hyperemia. MMP-9 expression and hemorrhagic transformation after stroke increased with age. These changes in the myeloid response to stroke with age led us to hypothesize that the bone marrow response to stroke is altered with age, which could be important for the development of effective therapies targeting the immune response. We generated heterochronic bone marrow chimeras as a tool to determine the contribution of peripheral immune senescence to age- and stroke-induced inflammation. Old hosts that received young bone marrow (i.e., Young → Old) had attenuation of age-related reductions in bFGF and VEGF and showed improved locomotor activity and gait dynamics compared to isochronic (Old → Old) controls. Microglia in young heterochronic mice (Old → Young) developed a senescent-like phenotype. After stroke, aged animals reconstituted with young marrow had reduced behavioral deficits compared to isochronic controls, and had significantly fewer brain-infiltrating neutrophils. Increased rates of hemorrhagic transformation were seen in young mice reconstituted with aged bone marrow. This work suggests that age alters the immunological response to stroke, and that this can be reversed by manipulation of the peripheral immune cells in the bone marrow.

Deletion of the P2X4 receptor is neuroprotective acutely, but induces a depressive phenotype during recovery from ischemic stroke.

INTRODUCTION: Acute ischemic injury leads to severe neuronal loss. One of the key mechanisms responsible for this effect is inflammation, which is characterized by the activation of myeloid cells, including resident microglia and infiltrating monocytes/macrophages. P2X4 receptors (P2X4Rs) present on these immune cells modulate the inflammatory response. For example, excessive release of adenosine triphosphate during acute ischemic stroke triggers stimulation of P2X4Rs, leading to myeloid cell activation and proliferation and further exacerbating post-ischemic inflammation. In contrast, during recovery P2X4Rs activation on microglia leads to the release of brain-derived neurotrophic factor (BDNF), which alleviate depression, maintain synaptic plasticity and hasten post-stroke behavioral recovery. Therefore, we hypothesized that deletion of the P2X4R specifically from myeloid cells would have differential effects on acute versus chronic recovery following stroke. METHODS: We subjected global or myeloid-specific (MS) P2X4R knock-out (KO) mice and wild-type littermates of both sexes to right middle cerebral artery occlusion (60min). We performed histological, behavioral (sensorimotor and depressive), and biochemical (quantitative PCR and flow cytometry) analyses to determine the acute (three days after occlusion) and chronic (30days after occlusion) effects of receptor deletion. RESULTS: Global P2X4R deletion led to reduced infarct size in both sexes. In MS P2X4R KO mice, only females showed reduced infarct size, an effect that did not change with ovariectomy. MS P2X4R KO mice of both sexes showed swift recovery from sensorimotor deficits during acute recovery but exhibited a more pronounced post-stroke depressive behavior phenotype that was independent of infarct size. Quantitative PCR analysis of whole cell lysate as well as flow-sorted myeloid cells from the perilesional cortex showed increased cellular interleukin 1 beta (IL-1ß), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) mRNA levels but reduced plasma levels of these cytokines in MS P2X4R KO mice after stroke. The expression levels of BDNF and other depression-associated genes were reduced in MS P2X4R KO mice after stroke. CONCLUSIONS: P2X4R deletion protects against stroke acutely but predisposes to depression-like behavior chronically after stroke. Thus, a time-sensitive approach should be considered when targeting P2X4Rs after stroke.

Multiparity improves outcomes after cerebral ischemia in female mice despite features of increased metabovascular risk.

Females show a varying degree of ischemic sensitivity throughout their lifespan, which is not fully explained by hormonal or genetic factors. Epidemiological data suggest that sex-specific life experiences such as pregnancy increase stroke risk. This work evaluated the role of parity on stroke outcome. Age-matched virgin (i.e., nulliparous) and multiparous mice were subjected to 60 min of reversible middle cerebral artery occlusion and evaluated for infarct volume, behavioral recovery, and inflammation. Using an established mating paradigm, fetal microchimeric cells present in maternal mice were also tracked after parturition and stroke. Parity was associated with sedentary behavior, weight gain, and higher triglyceride and cholesterol levels. The multiparous brain exhibited features of immune suppression, with dampened baseline microglial activity. After acute stroke, multiparous mice had smaller infarcts, less glial activation, and less behavioral impairment in the critical recovery window of 72 h. Behavioral recovery was significantly better in multiparous females compared with nulliparous mice 1 mo after stroke. This recovery was accompanied by an increase in poststroke angiogenesis that was correlated with improved performance on sensorimotor and cognitive tests. Multiparous mice had higher levels of VEGF, both at baseline and after stroke. GFP+ fetal cells were detected in the blood and migrated to areas of tissue injury where they adopted endothelial morphology 30 d after injury. Reproductive experience has profound and complex effects on neurovascular health and disease. Inclusion of female mice with reproductive experience in preclinical studies may better reflect the life-long patterning of ischemic stroke risk in women.

Nano-particle delivery of brain derived neurotrophic factor after focal cerebral ischemia reduces tissue injury and enhances behavioral recovery.

BACKGROUND: Low levels of brain-derived neurotrophic factor (BDNF) are linked to delayed neurological recovery, depression, and cognitive impairment following stroke. Supplementation with BDNF reverses these effects. Unfortunately, systemically administered BDNF in its native form has minimal therapeutic value due to its poor blood brain barrier permeability and short serum half-life. In this study, a novel nano-particle polyion complex formulation of BDNF (nano-BDNF) was administered to mice after experimental ischemic stroke. METHODS: Male C57BL/6J (8-10weeks) mice were randomly assigned to receive nano-BDNF, native-BDNF, or saline treatment after being subjected to 60min of reversible middle cerebral artery occlusion (MCAo). Mice received the first dose at 3 (early treatment), 6 (intermediate treatment), or 12h (delayed treatment) following stroke onset; a second dose was given in all cohorts at 24h after stroke onset. Post-stroke outcome was evaluated by behavioral, histological, and molecular analysis for 15days after stroke. RESULTS: Early and intermediate nano-BDNF treatment led to a significant reduction in cerebral tissue loss. Delayed treatment led to improved memory/cognition, reduced post-stroke depressive phenotypes, and maintained myelin basic protein and brain BDNF levels, but had no effect on tissue atrophy. CONCLUSIONS: The results indicate that administration of a novel nano-particle formulation of BDNF leads to both neuroprotective and neuro-restorative effects after stroke.