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Background: Stroke is the primary cause of chronic disability in the elderly, as there are no neurorestorative treatments for those who do not qualify for recanalization therapy. Experimental evidence in stroke animals suggests that transplantation of bone marrow–derived human mesenchymal stem cells (hMSCs) holds promise, but hMSC transplantation has not been systematically tested in aged animals. We tested the hypothesis that poststroke hMSC transplantation improves stroke recovery in aged mice by promoting brain repair. Methods: Permanent focal cerebral ischemia was induced in 20-month-old C57BL/6 male mice by distal middle cerebral artery occlusion. Bone marrow–derived hMSCs were expanded in vitro and then administrated intravenously into mice (1×10 6 cells in PBS) 24 hours after distal middle cerebral artery occlusion. Sensorimotor and cognitive functions, brain atrophy, and brain repair processes (neurogenesis, angiogenesis, oligodendrogenesis) were assessed for up to 56 days after stroke. Results: Poststroke hMSC transplantation did not mitigate brain atrophy or improve neuronal survival at 56 days after distal middle cerebral artery occlusion. However, hMSC-treated mice displayed superior neurobehavioral performances in the open field, rotarod, adhesive removal, novel object, and Morris water maze tests compared with PBS-treated controls. hMSCs promoted white matter integrity and enhanced angiogenesis and oligodendrogenesis—but not neurogenesis—in the stroke brain. Positive correlations between neurobehavioral performance and brain repair profiles or white matter integrity were observed in stroke mice. Conclusions: Poststroke hMSC transplantation improves long-term stroke recovery in aged mice, likely via mechanisms involving enhanced microvascular regeneration and white matter restoration.

Traumatic brain injury (TBI) is commonly followed by long-term cognitive deficits that severely impact the quality of life in survivors. Recent studies suggest that microglial/macrophage (Mi/MΦ) polarization could have multidimensional impacts on post-TBI neurological outcomes. Here, we report that repetitive intranasal delivery of interleukin-4 (IL-4) nanoparticles for 4 weeks after controlled cortical impact improved hippocampus-dependent spatial and non-spatial cognitive functions in adult C57BL6 mice, as assessed by a battery of neurobehavioral tests for up to 5 weeks after TBI. IL-4-elicited enhancement of cognitive functions was associated with improvements in the integrity of the hippocampus at the functional ( e.g., long-term potentiation) and structural levels (CA3 neuronal loss, diffusion tensor imaging of white matter tracts, etc.). Mechanistically, IL-4 increased the expression of PPARγ and arginase-1 within Mi/MΦ, thereby driving microglia toward a global inflammation-resolving phenotype. Notably, IL-4 failed to shift microglial phenotype after TBI in Mi/MΦ-specific PPARγ knockout (mKO) mice, indicating an obligatory role for PPARγ in IL-4-induced Mi/MΦ polarization. Accordingly, post-TBI treatment with IL-4 failed to improve hippocampal integrity or cognitive functions in PPARγ mKO mice. These results demonstrate that administration of exogenous IL-4 nanoparticles stimulates PPARγ-dependent beneficial Mi/MΦ responses, and improves hippocampal function after TBI.

As an integral part of the innate immune system of the brain, resident microglia must react rapidly to the onset of brain injury and neurological disease. These dynamic cells then continue to shift their phenotype along a multidimensional continuum with overlapping pro- and anti-inflammatory states, allowing them to adapt to microenvironmental changes during the progression of brain disorders. However, the ability of microglia to shift phenotype through nimble molecular, structural, and functional changes comes at a cost, as the extreme pro-inflammatory states may prevent these professional phagocytes from clearing toxic debris and secreting tissue-repairing neurotrophic factors. Evolution has strongly favored heterogeneity in microglia in both the spatial and temporal dimensions-they can assume diverse roles in different brain regions, throughout the course of brain development and aging, and during the spatiotemporal progression of brain injuries and neurological diseases. Age and sex differences add further diversity to microglia functional status under physiological and pathological conditions. This article reviews recent advances in our knowledge of microglia with emphases on molecular mediators of phenotype shifts and functional diversity. We describe microglia-targeted therapeutic opportunities, including pharmacologic modulation of phenotype and repopulation of the brain with fresh microglia. With the advent of powerful new tools, research on microglia has recently accelerated in pace and may translate into potential therapeutics against brain injury and neurological disease.

Long-term neurological recovery after severe traumatic brain injury (TBI) is strongly linked to the repair and functional restoration of injured white matter. Emerging evidence suggests that the anti-inflammatory cytokine interleukin-4 (IL-4) plays an important role in promoting white matter integrity after cerebral ischemic injury. Here, we report that delayed intranasal delivery of nanoparticle-packed IL-4 boosted sensorimotor neurological recovery in a murine model of controlled cortical impact, as assessed by a battery of neurobehavioral tests for up to five weeks. Post-injury IL-4 treatment failed to reduce macroscopic brain lesions after TBI, but preserved the structural and functional integrity of white matter, at least in part through oligodendrogenesis. IL-4 directly facilitated the differentiation of oligodendrocyte progenitor cells (OPCs) into mature myelin-producing oligodendrocytes in primary cultures, an effect that was attenuated by selective PPARγ inhibition. IL-4 treatment after TBI in vivo also failed to stimulate oligodendrogenesis or improve white matter integrity in OPC-specific PPARγ conditional knockout (cKO) mice. Accordingly, IL-4-afforded improvements in sensorimotor neurological recovery after TBI were markedly impaired in the PPARγ cKO mice compared to wildtype controls. These results support IL-4 as a potential novel neurorestorative therapy to improve white matter functionality and mitigate the long-term neurological consequences of TBI.

Background and Purpose— Microglia/macrophages (Mi/MΦ) can profoundly influence stroke outcomes by acquiring functionally dominant phenotypes (proinflammatory or anti-inflammatory; deleterious or salutary). Identification of the molecular mechanisms that dictate the functional status of Mi/MΦ after brain ischemia/reperfusion may reveal novel therapeutic targets for stroke. We hypothesized that activation of TAK1 (transforming growth factor beta-activated kinase 1), a key MAP3K upstream of multiple inflammation-regulating pathways, drives Mi/MΦ toward a proinflammatory phenotype and potentiates ischemia/reperfusion brain injury. Methods— Young adult mice were subjected to 1 hour of middle cerebral artery occlusion (MCAO) followed by reperfusion. TAK1 was targeted by tamoxifen-induced Mi/MΦ-specific knockout or administration of a selective inhibitor 5Z-7-Oxozeaenol after MCAO. Neurobehavioral deficits and long-term gray matter and white matter injury were assessed up to 35 days after MCAO. Mi/MΦ functional status and brain inflammatory profiles were assessed 3 days after MCAO by RNA-seq, flow cytometry, and immunohistochemistry. Results— TAK1 Mi/MΦ-specific knockout markedly ameliorated neurological deficits in the rotarod and cylinder tests for at least 35 days after MCAO. Mechanistically, RNA-seq of purified brain Mi/MΦ demonstrated that proinflammatory genes and their predicted biological functions were downregulated or inhibited in microglia and macrophages from TAK1 Mi/MΦ-specific knockout mice versus WT mice 3 days after MCAO. Consistent with the anti-inflammatory phenotype of Mi/MΦ-specific knockout, oxozeaenol treatment mitigated neuroinflammation 3 days after MCAO, manifested by less Iba1 + /CD16 + proinflammatory Mi/MΦ and suppressed brain invasion of various peripheral immune cells. Oxozeaenol treatment beginning 2 hours after MCAO improved long-term sensorimotor and cognitive functions in the foot fault, rotarod, and water maze tests. Furthermore, Oxozeaenol promoted both gray matter and white matter integrity 35 days after MCAO. Conclusions— TAK1 promotes ischemia/reperfusion-induced inflammation, brain injury, and maladaptive behavior by enhancing proinflammatory and deleterious Mi/MΦ responses. Therefore, TAK1 inhibition is a promising therapy to improve long-term stroke outcomes.

Traumatic brain injury (TBI) triggers a plethora of inflammatory events in the brain that aggravate secondary injury and impede tissue repair. Resident microglia (Mi) and blood-borne infiltrating macrophages (MΦ) are major players of inflammatory responses in the post-TBI brain and possess high functional heterogeneity. However, the plasticity of these cells has yet to be exploited to develop therapies that can mitigate brain inflammation and improve the outcome after TBI. This study investigated the transcription factor STAT1 as a key determinant of proinflammatory Mi/MΦ responses and aimed to develop STAT1 as a novel therapeutic target for TBI using a controlled cortical impact model of TBI on adult male mice. TBI induced robust upregulation of STAT1 in the brain at the subacute injury stage, which occurred primarily in Mi/MΦ. Intraperitoneal administration of fludarabine, a selective STAT1 inhibitor, markedly alleviated proinflammatory Mi/MΦ responses and brain inflammation burden after TBI. Such phenotype-modulating effects of fludarabine on post-TBI Mi/MΦ were reproduced by tamoxifen-induced, selective KO of STAT1 in Mi/MΦ (STAT1 mKO). By propelling Mi/MΦ away from a detrimental proinflammatory phenotype, STAT1 mKO was sufficient to reduce long-term neurologic deficits and brain lesion size after TBI. Importantly, short-term fludarabine treatment after TBI elicited long-lasting improvement of TBI outcomes, but this effect was lost on STAT1 mKO mice. Together, our study provided the first line of evidence that STAT1 causatively determines the proinflammatory phenotype of brain Mi/MΦ after TBI. We also showed promising preclinical data supporting the use of fludarabine as a novel immunomodulating therapy to TBI. SIGNIFICANCE STATEMENT The functional phenotype of microglia and macrophages (Mi/MΦ) critically influences brain inflammation and the outcome after traumatic brain injury (TBI); however, no therapies have been developed to modulate Mi/MΦ functions to treat TBI. Here we report, for the first time, that the transcription factor STAT1 is a key mediator of proinflammatory Mi/MΦ responses in the post-TBI brain, the specific deletion of which ameliorates neuroinflammation and improves long-term functional recovery after TBI. We also show excellent efficacy of a selective STAT1 inhibitor fludarabine against TBI-induced functional deficits and brain injury using a mouse model, presenting STAT1 as a promising therapeutic target for TBI.

Introduction: Identification of key molecules that control conversion of resting microglia/macrophages (Mi/MΦ) to a detrimental or beneficial phenotype may help develop novel therapies to ischemic stroke. The transcription factor STAT1 contributes to acute (< 24h) neuronal death after brain ischemia/reperfusion (I/R), but its role in Mi/MΦ and impact on long term stroke outcome remain unknown. Hypothesis: Activation of STAT1 dictates proinflammatory responses of Mi/MΦ at subacute stage (3-5 d) after I/R. Selective deletion of STAT1 reprograms Mi/MΦ into an inflammation-resolving phenotype and improves long term stroke outcome. Methods: We generated mice with tamoxifen-induced, Mi/MΦ-specific knockout (mKO) of STAT1. Brain injury, inflammation and various behavioral deficits were assessed after 1 h MCAO and 1-35 d reperfusion. Mi/MΦ phenotype was examined in vivo (FACS followed by RNA-seq) and in vitro (primary microglia). Results: STAT1 was activated (phosphorylated) in Mi/MΦ 3 d after I/R in WT but not in STAT1 mKO mice. STAT1 mKO did not alter 24-h infarct size (TTC; p>0.05, n=8), but attenuated Mi/MΦ release of HMGB1 and increased arginase 1-producing Mi/MΦ 3d after I/R (immunostain, n=6), suggesting boosted inflammation-resolving responses of Mi/MΦ. RNA-seq further revealed downregulated proinflammatory genes and a concomitant elevation of phagocytosis-related genes in STAT1 mKO Mi/MΦ 3d after I/R (n=3, FDR Conclusions: Mi/MΦ-targeted STAT1 KO does not offer acute neuroprotection but primes Mi/MΦ toward an inflammation-resolving phenotype, thereby facilitating long term functional recovery after stroke.

Immunomodulation holds therapeutic promise against brain injuries, but leveraging this approach requires a precise understanding of mechanisms. We report that CD8+CD122+CD49dlo T regulatory-like cells (CD8+ TRLs) are among the earliest lymphocytes to infiltrate mouse brains after ischemic stroke and temper inflammation; they also confer neuroprotection. TRL depletion worsened stroke outcomes, an effect reversed by CD8+ TRL reconstitution. The CXCR3/CXCL10 axis served as the brain-homing mechanism for CD8+ TRLs. Upon brain entry, CD8+ TRLs were reprogrammed to upregulate leukemia inhibitory factor (LIF) receptor, epidermal growth factor-like transforming growth factor (ETGF), and interleukin 10 (IL-10). LIF/LIF receptor interactions induced ETGF and IL-10 production in CD8+ TRLs. While IL-10 induction was important for the antiinflammatory effects of CD8+ TRLs, ETGF provided direct neuroprotection. Poststroke intravenous transfer of CD8+ TRLs reduced infarction, promoting long-term neurological recovery in young males or aged mice of both sexes. Thus, these unique CD8+ TRLs serve as early responders to rally defenses against stroke, offering fresh perspectives for clinical translation.

Blood monocytes/macrophages infiltrate the brain after ischemic stroke and critically influence brain injury and regeneration. We investigated stroke-induced transcriptomic changes of monocytes/macrophages by RNA sequencing profiling, using a mouse model of permanent focal cerebral ischemia. Compared to non-ischemic conditions, brain ischemia induced only moderate genomic changes in blood monocytes, but triggered robust genomic reprogramming in monocytes/macrophages invading the brain. Surprisingly, functional enrichment analysis of the transcriptome of brain macrophages revealed significant overrepresentation of biological processes linked to neurovascular remodeling, such as angiogenesis and axonal regeneration, as early as five days after stroke, suggesting a previously underappreciated role for macrophages in initiating post-stroke brain repair. Upstream Regulator analysis predicted peroxisome proliferator-activated receptor gamma (PPARγ) as a master regulator driving the transcriptional reprogramming in post-stroke brain macrophages. Importantly, myeloid cell-specific PPARγ knockout (mKO) mice demonstrated lower post-stroke angiogenesis and neurogenesis than wild-type mice, which correlated significantly with the exacerbation of post-stroke neurological deficits in mKO mice. Collectively, our findings reveal a novel repair-enhancing transcriptome in brain macrophages during post-stroke neurovascular remodeling. As a master switch controlling genomic reprogramming, PPARγ is a rational therapeutic target for promoting and maintaining beneficial macrophage functions, facilitating neurorestoration, and improving long-term functional recovery after ischemic stroke.

Aims Blood‐borne monocytes/macrophages infiltrate the brain in massive numbers after ischemic stroke, but their impact on poststroke brain injury and recovery remains elusive. This study examined the transcriptomic changes in monocytes/macrophages after ischemic stroke and the functional implications of these changes, particularly with regards to the contribution of these cells to the phagocytic clearance of dead/dying cells (efferocytosis) in the poststroke brain. Methods We performed whole‐genome RNA sequencing on the monocyte/macrophage population sorted from mouse brain and peripheral blood 5 days after permanent focal cerebral ischemia. In addition, the spatial and temporal profiles of macrophage efferocytosis were examined in vivo by immunohistochemistry 3‐7 days after brain ischemia. Results Robust transcriptomic changes occurred in monocytes/macrophages upon infiltrating the poststroke brain. Functional enrichment analysis revealed a transcriptome of brain macrophages that strongly favored efferocytic activity. A large number of efferocytosis‐related genes were upregulated in brain macrophages, the products of which are essential components involved in various steps of efferocytosis, such as chemotaxis, recognition of dead cells, engulfment, and processing of phagosomes. The efferocytic activity of brain macrophages were verified by immunohistochemistry, wherein Iba1‐labeled microglia/macrophages effectively cleared apoptotic neurons in the infarct during the subacute stage after brain ischemia. We also identified PPARγ and STAT6 as potential upstream regulators that shaped this proefferocytic and inflammation‐resolving transcriptome of macrophages in the poststroke brain. Conclusion Macrophages play a crucial role in the phagocytic clearance of dead neurons after ischemic stroke and promote the resolution of inflammation in the brain. Molecular therapies that enhance macrophage efferocytic capability may be promising treatments for ischemic stroke by facilitating inflammation resolution, brain repair, and recovery of neurological functions.

Post-stroke treatment with omega-3 polyunsaturated fatty acids (n-3 PUFAs) may be a promising therapy in young animals but this has not been tested in aged subjects, a population at most risk of ischemic stroke. Herein we examined the therapeutic efficacy of n-3 PUFAs after distal middle cerebral artery occlusion (dMCAO) in young (10–12 weeks old) and aged (18 months old) mice. Post-ischemic mice were randomly assigned to 4 groups that received: 1) regular food with low content of n-3 PUFAs, 2) intraperitoneal docosahexaenoic acid (DHA, a major component of n-3 PUFAs) injections, 3) Fish oil (FO, containing high concentration of n-3 PUFAs) dietary supplement, or 4) combined treatment with DHA and FO dietary supplement. Long-term neurorestoration induced by n-3 PUFA post-stroke administration and its underlying mechanism(s) were analyzed up to 35 days after dMCAO. Aged mice showed more severe neurological deficits than young mice after dMCAO with histological lesions extended to the striatum. Notably, post-stroke treatment with combined DHA injections and FO dietary supplementation was more effective in reducing brain injury and improving sensorimotor function in aged mice than either treatment alone, albeit to a lesser extent than in the young mice. Unlike the improvement in spatial cognitive function observed in young mice, the combined treatment regimen failed to improve cognitive function in aged mice. The reduction in stroke-induced neurological deficits with n-3 PUFA post-treatment was associated with enhanced angiogenesis, oligodendrogenesis, neuron survival and white matter restoration. Together, these results indicate that the neurological benefits of n-3 PUFA administration after stroke extend to older animals and are associated with improved neuronal survival and brain remodeling, therefore suggesting that post-stroke administration of n-3 PUFAs is a viable clinically relevant treatment option against stroke.

White matter injury is a crucial component of human stroke, but it has often been neglected in preclinical studies. Most human stroke is associated with one or more comorbidities, including aging, hypertension, diabetes and metabolic syndrome including hyperlipidemia. The purpose of this review is to examine how age and hypertension impact stroke-induced white matter injury as well as white matter repair in both human stroke and preclinical models. It is essential that comorbidities be examined in preclinical trials as they may impact translatability to the clinic. In addition, understanding how comorbidities impact white matter injury and repair may provide new therapeutic opportunities for patients with those conditions.

Intracerebral hemorrhage (ICH) is a devastating form of stroke affecting millions of people worldwide. Parenchymal hematoma triggers a series of reactions leading to primary and secondary brain injuries and permanent neurological deficits. Microglia and macrophages carry out hematoma clearance, thereby facilitating functional recovery after ICH. Here, we elucidate a pivotal role for the interleukin (IL)-4)/signal transducer and activator of transcription 6 (STAT6) axis in promoting long-term recovery in both blood- and collagenase-injection mouse models of ICH, through modulation of microglia/macrophage functions. In both ICH models, STAT6 was activated in microglia/macrophages (i.e., enhanced expression of phospho-STAT6 in Iba1(+) cells). Intranasal delivery of IL-4 nanoparticles after ICH hastened STAT6 activation and facilitated hematoma resolution. IL-4 treatment improved long-term functional recovery in young and aged male and young female mice. In contrast, STAT6 knockout (KO) mice exhibited worse outcomes than WT mice in both ICH models and were less responsive to IL-4 treatment. The construction of bone marrow chimera mice demonstrated that STAT6 KO in either the CNS or periphery exacerbated ICH outcomes. STAT6 KO impaired the capacity of phagocytes to engulf red blood cells in the ICH brain and in primary cultures. Transcriptional analyses identified lower level of IL-1 receptor-like 1 (ST2) expression in microglia/macrophages of STAT6 KO mice after ICH. ST2 KO diminished the beneficial effects of IL-4 after ICH. Collectively, these data confirm the importance of IL-4/STAT6/ST2 signaling in hematoma resolution and functional recovery after ICH. Intranasal IL-4 treatment warrants further investigation as a clinically feasible therapy for ICH.

TGFβ-activated kinase 1 (TAK1) is a master regulator that drives multiple cell death and proinflammatory signaling pathways, making it a promising therapeutic target to treat ischemic stroke. However, whether targeting TAK1 could improve stroke outcomes has never been tested in female subjects, hindering its potential translation into clinical use. Here we examined the therapeutic effect of 5Z-7-Oxozeaenol (OZ), a selective TAK1 inhibitor, in ovariectomized female mice after middle cerebral artery occlusion (MCAO). OZ significantly reduced neuronal cell death and axonal injury at the acute stage and mitigated neuroinflammation at the subacute stage after MCAO in ovariectomized female mice. Consistent with RNA sequencing analysis that TAK1 activation contributed to microglia/macrophage-mediated inflammatory responses in the post-stroke brain, inhibition of TAK1 with OZ caused phenotypic shift of microglia/macrophages toward an inflammation-resolving state. Furthermore, microglia/macrophage-specific TAK1 knockout (TAK1 mKO) reproduced OZ's effects, causally confirming the role of TAK1 in determining proinflammatory microglial/macrophage responses in post-stroke females. Post-stroke treatment with OZ for 5 days effectively promoted long-term neurological recovery and the integrity of both gray matter and white matter in female mice. Together, the TAK1 inhibitor OZ elicits long-lasting improvement of stroke outcomes in female mice, at least partially through enhancing beneficial microglial/macrophage responses and inflammation resolution. Given its therapeutic efficacy on both male and female rodents, TAK1 inhibitor is worth further investigation as a valid treatment to ischemic stroke.

As an integral part of the innate immune system of the brain, resident microglia must react rapidly to the onset of brain injury and neurological disease. These dynamic cells then continue to shift their phenotype along a multidimensional continuum with overlapping pro- and anti-inflammatory states, allowing them to adapt to microenvironmental changes during the progression of brain disorders. However, the ability of microglia to shift phenotype through nimble molecular, structural, and functional changes comes at a cost, as the extreme pro-inflammatory states may prevent these professional phagocytes from clearing toxic debris and secreting tissue-repairing neurotrophic factors. Evolution has strongly favored heterogeneity in microglia in both the spatial and temporal dimensions-they can assume diverse roles in different brain regions, throughout the course of brain development and aging, and during the spatiotemporal progression of brain injuries and neurological diseases. Age and sex differences add further diversity to microglia functional status under physiological and pathological conditions. This article reviews recent advances in our knowledge of microglia with emphases on molecular mediators of phenotype shifts and functional diversity. We describe microglia-targeted therapeutic opportunities, including pharmacologic modulation of phenotype and repopulation of the brain with fresh microglia. With the advent of powerful new tools, research on microglia has recently accelerated in pace and may translate into potential therapeutics against brain injury and neurological disease.

Senescence-associated alterations in microglia may have profound impact on cerebral homeostasis and stroke outcomes. However, the lack of a transcriptome-wide comparison between young and aged microglia in the context of ischemia limits our understanding of aging-related mechanisms. Herein, we performed RNA sequencing analysis of microglia purified from cerebral hemispheres of young adult (10-week-old) and aged (18-month-old) mice five days after distal middle cerebral artery occlusion or after sham operation. Considerable transcriptional differences were observed between young and aged microglia in healthy brains, indicating heightened chronic inflammation in aged microglia. Following stroke, the overall transcriptional activation was more robust (>13-fold in the number of genes upregulated) in young microglia than in aged microglia. Gene clusters with functional implications in immune inflammatory responses, immune cell chemotaxis, tissue remodeling, and cell-cell interactions were markedly activated in microglia of young but not aged stroke mice. Consistent with the genomic profiling predictions, post-stroke cerebral infiltration of peripheral immune cells was markedly decreased in aged mice compared to young mice. Moreover, post-ischemic aged microglia demonstrated reduced interaction with neighboring neurons and diminished polarity toward the infarct lesion. These alterations in microglial gene response and behavior may contribute to aging-driven vulnerability and poorer recovery after ischemic stroke.

Age-dependent alterations in microglia behavior have been implicated in neurodegeneration and CNS injuries. Here, we compared the transcriptional profiles of young versus aged microglia during stroke recovery. CD45intermediateCD11b+ microglia were FACS-isolated from the brains of young (10-week-old) and aged (18-month-old) male mice with sham operation or 14 days after distal middle cerebral artery occlusion and subjected to RNA-sequencing analysis. Functional groups enriched in young microglia are indicative of upregulation in cell movement, cell interactions, inflammatory responses and angiogenesis, while aged microglia exhibited a reduction or no change in these features. We confirmed reduced chemoattractive capacities of aged microglia toward ischemic brain tissue in organotypic slide co-cultures, and delayed accumulation of aged microglia around dead neurons injected into the striatum in vivo. In addition, aging is associated with an overall failure to increase the expression of microglial genes involved in cell–cell interactions, such as CXCL10. Finally, impaired upregulation of pro-angiogenic genes in aged microglia was associated with a decline in neovascularization in aged mice compared to young mice after distal middle cerebral artery occlusion. This study provides a new resource to understand the mechanisms underlying microglial alterations in the aged brain milieu and sheds light on new strategies to improve microglial functions in aged stroke victims.

Oxidative DNA damage (ODD) after cerebral ischemia/reperfusion injury occurs rapidly and has been implicated in the patholgenesis of ischemic injury. Ischemic ODD in the brain are primarly characterized as apurinic/apyrimidinic (AP) sites and single strand breaks (SSB). Following ODD, key enzymes in the base excision-repair (BER) pathway including AP endonuclease-1 (APE1) and the DNA polymerase-beta (PolyB) are critical to repair AP sites and SSB, and to mitigate cellular damage. Global knockout of either APE1 or PolyB leads to embryonic lethality. We have previously demonstrated that conditional deletion of APE1 in the adult heightens sensitivity to ischemic injury. PolyB is a highly inducible gene, and its expression is increased after sublethal brain injury, yet the contribution of PolyB to ischemic brain recovery remains unknown. PolyB conditional knockout (cKO) mice were generated by subjecting PolyB-CAG-CreER mice to tamoxifen administration for 5 days. Transient middle cerebral artery occlusion (tMCAO) was induced for 45 min following STAIR guidelines, and behavioral recovery and grey/white matter injury were assessed by over 35 days after ischemia. Cortical action field potentials (CAFP) and compound action potentials (CAP) were used to assess functional integrity of grey and white matter. PolyB cKO mice were significantly impaired up to 35 days after ischemic injury compared to WT mice, displaying exacerbated neuronal tissue loss, extensive demyelination in the CC/EC, and diminished CAFP and CAP compared to WT mice. In addition, accumulation of AP sites and SSBs were evident in the ischemic PolyB cKO. These results were compared to to APE1 cKO ischemic outcomes. PolyB cKO was associated with PARP1 activation, NAD + depletion, necrotic neuronal cell death, and neuronal release of HMGB1, and PJ34, a specific PARP-1 inhibitor, significantly improved stroke outcomes in PolyB cKO mice. Along with APE1, PolyB plays an essential role in ischemic brain recovery in both grey and white matter. However, the molecular mechanisms between exacerbation of injury in ischemic neurons by PolyB vs APE1 may be distinct. DNA base excision repair pathway may potentially serve as a therapeutic target for long-term neurological recovery after stroke.

Introduction: Blood monocytes/macrophages (MΦ) infiltrate the brain in massive numbers after ischemic stroke, but their functional roles in the post-stroke brain remain elusive. Methods: We sorted CD11b + CD45 high monocytes/MΦ from mouse blood and brain 5d after distal MCAO (dMCAO) and investigated their transcriptomic profiles by RNA-seq (n=3/group). Results: Robust genomic changes occurred in MΦ invading the post-dMCAO brain compared to their counterparts in the blood (3196 differentially expressed genes with >2 fold changes; FDR-15 ) that drives the transcriptional reprogramming in post-stroke brain MΦ and dictates their functional phenotype. Accordingly, tamoxifen-induced, myeloid cell-specific PPARγ knockout mice demonstrated less post-stroke angiogenesis (BrdU+CD31 staining) and neurogenesis (BrdU+NeuN) than WT mice (n=9/group, p Conclusions: Our study reveals a novel repair-enhancing transcriptome in brain MΦ during post-stroke neurovascular remodeling. As a master molecule controlling genomic reprogramming, PPARγ is a rational therapeutic target for promoting beneficial MΦ functions, and facilitating neurorestoration and long term functional recovery after ischemic stroke.

Introduction: Microglia/macrophages (Mi/MΦ) profoundly influence stroke outcomes, but the molecular mechanisms dictating their functional phenotype after brain ischemia/reperfusion (I/R) are poorly understood. We hypothesized that activation of TAK1, a key MAPKKK upstream of multiple inflammation-regulating pathways, drives Mi/MΦ towards a proinflammatory phenotype and potentiates I/R brain injury. Methods: TAK1 was targeted in mice by tamoxifen-induced, Mi/MΦ-specific knockout (mKO) or a relatively selective inhibitor 5Z-7-oxozeaenol (OZ; 2.5 mg/kg i.p.). Sensorimotor/cognitive deficits, grey/white matter injury and brain inflammatory profiles were assessed after 1 h MCAO and reperfusion. Results: TAK1 mKO markedly ameliorated neurological deficits in the rotarod, cylinder and water maze tests (plow ) and MΦ (CD45 high ) cells revealed that multiple genes participating in pro-inflammatory responses were downregulated in both Mi and MΦ from mKO mice versus WT mice 3 d after I/R. Consistent with the phenotype of mKO, OZ mitigated neuroinflammation 3 d after I/R, reflected by less Iba1 + /CD16 + proinflammatory Mi/MΦ and less brain invasion of peripheral immune cells (neutrophils, MΦ, T cells). OZ treatment beginning 2 h after I/R effectively improved long term (35 d) sensorimotor and cognitive functions in the foot fault, rotarod and water maze tests (p Conclusions: TAK1 promotes I/R-induced inflammation, brain injury, and maladaptive behavior by enhancing neurotoxic Mi/MΦ responses. TAK1 inhibition is a promising therapy to improve long term stroke outcomes in both sexes.

Supplemental material, sj-pdf-1-jcb-10.1177_0271678X20941393 for Interleukin-4 improves white matter integrity and functional recovery after murine traumatic brain injury via oligodendroglial PPARγ by Hongjian Pu, Xuan Zheng, Xiaoyan Jiang, Hongfeng Mu, Fei Xu, Wen Zhu, Qing Ye, Yunneng Jizhang, T Kevin Hitchens, Yejie Shi, Xiaoming Hu, Rehana K Leak, C Edward Dixon, Michael VL Bennett and Jun Chen in Journal of Cerebral Blood Flow & Metabolism

Supplemental material, sj-pdf-1-jcb-10.1177_0271678X20925655 for Genome-wide transcriptomic analysis of microglia reveals impaired responses in aged mice after cerebral ischemia by Ligen Shi, Marcelo Rocha, Wenting Zhang, Ming Jiang, Sicheng Li, Qing Ye, Sulaiman H Hassan, Liqiang Liu, Maya N Adair, Jing Xu, Jianhua Luo, Xiaoming Hu, Lawrence R Wechsler, Jun Chen and Yejie Shi in Journal of Cerebral Blood Flow & Metabolism

Supplemental material, JCB902542 Supplemental Material6 for Transcriptomic and functional studies reveal undermined chemotactic and angiostimulatory properties of aged microglia during stroke recovery by Lu Jiang, Hongfeng Mu, Fei Xu, Di Xie, Wei Su, Jing Xu, Zeyu Sun, Silvia Liu, Jianhua Luo, Yejie Shi, Rehana K Leak, Lawrence R Wechsler, Jun Chen and Xiaoming Hu in Journal of Cerebral Blood Flow & Metabolism

Regulatory T cells (Tregs) are known to protect against ischemic stroke. However, the low frequency of Tregs restricts their clinical utility. This study investigated whether expanding the number of Tregsin vivowith the IL-2/IL-2 antibody complex (IL-2/IL-2Ab) could improve stroke outcomes and further elaborated the mechanisms of protection in male mice. C57BL/6 mice received IL-2/IL-2Ab or isotype IgG (IsoAb) intraperitoneally for 3 d before (pretreatment) or starting 2 h after (posttreatment) 60 min middle cerebral artery occlusion (MCAO). IL-2/IL-2Ab selectively increased the number of Tregs in the blood, spleen, and lymph nodes. The IL-2/IL-2Ab treatment significantly reduced infarct volume, inhibited neuroinflammation, and improved sensorimotor functions, as manifested by rotarod test and foot fault test, compared with IsoAb-treated stroke mice. Treg depletion was then achieved by diphtheria toxin (DT) injection into transgenic mice expressing the DT receptor under the control of the Foxp3 promoter (DTR mice). The depletion of Tregs completely eliminated IL-2/IL-2Ab-afforded neuroprotection. Interestingly, adoptive transfer of Tregs collected from IL-2/IL-2Ab-treated mice demonstrated more potent neuroprotection than an equal number of Tregs prepared from IsoAb-treated mice, suggesting that IL-2/IL-2Ab not only elevated Treg numbers, but also boosted their functions. Mechanistically, IL-2/IL-2Ab promoted the expression of CD39 and CD73 in expanded Tregs. CD73 deficiency diminished the protective effect of IL-2/IL-2Ab-stimulated Tregs in stroke mice. The results show that IL-2/IL-2Ab expands Tregsin vivoand boosts their immunomodulatory function. The activation of CD39/CD73 signaling in Tregs may participate as a potential mechanism underlying IL-2/IL-2Ab-afforded neuroprotection against ischemic brain injury.SIGNIFICANCE STATEMENTRegulatory T cells (Tregs) are known to protect against ischemic stroke. However, the low frequency of Tregs restricts their clinical utility. This study reported that systemic administration of the IL-2/IL-2 antibody complex (IL-2/IL-2Ab) robustly and selectively expanded the number of Tregs after stroke. IL-2/IL-2Ab pretreatment or posttreatment significantly improved stroke outcomes in a rodent model of ischemic stroke. We further discovered that IL-2/IL-2Ab not only elevated Treg numbers, but also boosted their functions and enhanced the expression of CD39 and CD73. Using CD73-deficient mice, we confirmed the importance of CD73 in the protective effect of IL-2/IL-2Ab-stimulated Tregs in stroke mice. These results shed light on IL-2/IL-2Ab as a clinically feasible immune therapy to boost endogenous Treg responses and ameliorate ischemic brain injury.

p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 8.0px Helvetica; color: #2f2a2b} span.s1 {font: 5.5px Helvetica} Na1/H1 exchanger (NHE1) activation is required for multiple microglial functions. We investigated effects of selective deletion of microglial Nhe1 in Cx3cr1-CreER;Nhe1f/f mice on neuroinflammation and tissue repair after ischemic stroke. Infarct volume was similar in corn oil or tamoxifen (Tam)- treated mice at 48 hr and 14 days post-stroke. However, the Tam-treated mice showed significantly higher survival rate and faster neurological function recovery during day 1–14 post-stroke. Deletion of microglial Nhe1 prevented the elevation of CD11b1/CD45low-med microglia in the ischemic hemisphere at day 3 post-stroke, but stimulated expression of Ym1, CD68, TGF-b, IL-10, decreased expression of CD86 and IL-1b, and reduced GFAP1 reactive astrocytes. Moreover, at day 14 post-stroke, enhanced white matter myelination was detected in the microglial Nhe1 deleted mice. In comparison, neuronal Nhe1-null mice (the CamKII-Cre1/2;Nhe1f/f mice) showed a significant reduction in both acute and subacute infarct volume, along with increased survival rate and moderate neurological function recovery. However, these neuronal Nhe1-null mice did not exhibit reduced activation of CD11b1/CD45low-med microglia or CD11b1/CD45hi macrophages in the ischemic brains, and they exhibited no reductions in white matter lesions. Taken together, this study demonstrated that deletion of microglial and neuronal Nhe1 had differential effects on ischemic brain damage. Microglial NHE1 is involved in pro-inflammatory responses during post-stroke brain tissue repair. In contrast, neuronal NHE1 activation is directly associated with the acute ischemic neuronal injury but not inflammation. Our study reveals that NHE1 protein is a potential therapeutic target critical for differential regulation of ischemic neuronal injury, demyelination and tissue repair.

Brain ischemic preconditioning (IPC) with mild ischemic episodes is well known to protect the brain against subsequent ischemic challenges. However, the underlying mechanisms are poorly understood. Here we demonstrate the critical role of the master redox transcription factor, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), in IPC-mediated neuroprotection and blood-brain barrier (BBB) preservation. We report that IPC causes generation of endogenous lipid electrophiles, including 4-hydroxy-2-nonenal (4-HNE), which release Nrf2 from inhibition by Keap1 (via Keap1-C288) and inhibition by glycogen synthase kinase 3β (via GSK3β-C199). Nrf2 then induces expression of its target genes, including a new target, cadherin 5, a key component of adherens junctions of the BBB. These effects culminate in mitigation of BBB leakage and of neurological deficits after stroke. Collectively, these studies are the first to demonstrate that IPC protects the BBB against ischemic injury by generation of endogenous electrophiles and activation of the Nrf2 pathway through inhibition of Keap1- and GSK3β-dependent Nrf2 degradation. Keywords: Stroke, Neuroprotection, Ischemic tolerance, Electrophile, Lipid peroxidation, VE-cadherin, Conditioning