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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Transforming growth factor α (TGF-α) has been reported to play important roles in neurogenesis and angiogenesis in the injured brain. The present study characterizes a novel role for TGFα in oligodendrocyte lineage cell survival and white matter integrity after ischemic stroke. Three days after transient (60 min) middle cerebral artery occlusion (tMCAO), TGFα expression was significantly increased in microglia/macrophages and neurons. Expression of the receptor of TGFα—epidermal growth factor receptor (EGFR)—was increased in glial cells after ischemia, including in oligodendrocyte lineage cells. TGFα knockout enlarged brain infarct volumes and exacerbated cell death in oligodendrocyte precursor cells (OPCs) and oligodendrocytes three days after tMCAO. TGFα-deficient mice displayed long-term exacerbation of sensorimotor deficits after tMCAO, and these functional impairments were accompanied by loss of white matter integrity and impaired oligodendrocyte replacement. In vitro studies confirmed that 5 or 10 ng/mL TGFα directly protected OPCs and oligodendrocytes against oxygen and glucose deprivation (OGD)-induced cell death, but exerted no effects on OPC differentiation. Further studies identified STAT3 as a key transcription factor mediating the effects of TGFα on OPCs and oligodendrocytes. In conclusion, TGFα provides potent oligodendrocyte protection against cerebral ischemia, thereby maintaining white matter integrity and improving neurological recovery after stroke. [ABSTRACT FROM AUTHOR]

Ischemic injury can be alleviated by the judicious use of hypothermia. However, the optimal regimens and the temporal kinetics of post-stroke neurovascular responses to hypothermic intervention have not been systematically studied. These gaps slow the clinical translation of hypothermia as an anti-stroke therapy. Here, we characterized the effects of transient selective brain hypothermia (TSBH) from the hyperacute to chronic stages of focal ischemia/reperfusion brain injury induced by transient middle cerebral artery occlusion in mice. A simple cooling device was used to induce TSBH during cerebral ischemia. This treatment reduced mortality from 31.8% to 0% and improved neurological outcomes for at least 35 days post-injury. TSBH mitigated blood–brain barrier leakage during the hyperacute and acute injury stages (1–23 h post-reperfusion). This early protection of the blood–brain barrier was associated with anti-inflammatory phenotypic polarization of microglia/macrophages, reduced production of pro-inflammatory cytokines, and less brain infiltration of neutrophils and macrophages during the subacute injury stage (three days post-reperfusion). TSBH elicited enduring protective effects on both grey and white matter for at least 35 days post-injury and preserved the long-term electrophysiological function of fiber tracts. In conclusion, TSBH ameliorates ischemia/reperfusion injury in the neurovascular unit from hyperacute to chronic injury stages after experimental stroke. [ABSTRACT FROM AUTHOR]

Recent advances in stroke reperfusion therapies have led to remarkable improvement in clinical outcomes, but many patients remain severely disabled, due in part to the lack of effective neuroprotective strategies. In this review, we show that 95% of published preclinical studies on "neuroprotectants" (1990–2018) reported positive outcomes in animal models of ischemic stroke, while none translated to successful Phase III trials. There are many complex reasons for this failure in translational research, including that the majority of clinical trials did not test early delivery of neuroprotectants in combination with successful reperfusion. In contrast to the clinical trials, >80% of recent preclinical studies examined the neuroprotectant in animal models of transient ischemia with complete reperfusion. Furthermore, only a small fraction of preclinical studies included long-term functional assessments, aged animals of both genders, and models with stroke comorbidities. Recent clinical trials demonstrate that 70%–80% of patients treated with endovascular thrombectomy achieve successful reperfusion. These successes revive the opportunity to retest previously failed approaches, including cocktail drugs that target multiple injury phases and different cell types. It is our hope that neurovascular protectants can be retested in future stroke research studies with specific criteria outlined in this review to increase translational successes. [ABSTRACT FROM AUTHOR]

Human performance, endurance, and resilience have biological limits that are genetically and epigenetically predetermined but perhaps not yet optimized. There are few systematic, rigorous studies on how to raise these limits and reach the true maxima. Achieving this goal might accelerate translation of the theoretical concepts of conditioning, hormesis, and stress adaptation into technological advancements. In 2017, an Air Force-sponsored conference was held at the University of Massachusetts for discipline experts to display data showing that the amplitude and duration of biological performance might be magnified and to discuss whether there might be harmful consequences of exceeding typical maxima. The charge of the workshop was "to examine and discuss and, if possible, recommend approaches to control and exploit endogenous defense mechanisms to enhance the structure and function of biological tissues." The goal of this white paper is to fulfill and extend this workshop charge. First, a few of the established methods to exploit endogenous defense mechanisms are described, based on workshop presentations. Next, the white paper accomplishes the following goals to provide: (1) synthesis and critical analysis of concepts across some of the published work on endogenous defenses, (2) generation of new ideas on augmenting biological performance and resilience, and (3) specific recommendations for researchers to not only examine a wider range of stimulus doses but to also systematically modify the temporal dimension in stimulus inputs (timing, number, frequency, and duration of exposures) and in measurement outputs (interval until assay end point, and lifespan). Thus, a path forward is proposed for researchers hoping to optimize protocols that support human health and longevity, whether in civilians, soldiers, athletes, or the elderly patients. The long-term goal of these specific recommendations is to accelerate the discovery of practical methods to conquer what were once considered intractable constraints on performance maxima. [ABSTRACT FROM AUTHOR]

Nicotinamide phosphoribosyltransferase (NAMPT) has been implicated in neuroprotection against ischemic brain injury, but the mechanism underlying its protective effect remains largely unknown. To further examine the protective effect of NAMPT against ischemic stroke and its potential mechanism of action, we generated a novel neuron-specific NAMPT transgenic mouse line. Transgenic mice and wild-type littermates were subjected to transient occlusion of the middle cerebral artery (MCAO) for 60 minutes. Neuron-specific NAMPT overexpression significantly reduced infarct volume by 65% (P=0.018) and improved long-term neurologic outcomes (P≤0.05) compared with littermates. Interestingly, neuronal overexpression of NAMPT increased the area of myelinated fibers in the striatum and corpus callosum, indicating that NAMPT protects against white matter injury. The mechanism of protection appeared to be through extracellular release of NAMPT. First, NAMPT was secreted into the extracellular medium by primary cortical neurons exposed to ischemia-like oxygen-glucose deprivation (OGD) in vitro. Second, conditioned medium from NAMPT-overexpressing neurons exposed to OGD protected cultured oligodendrocytes from OGD. Third, the protective effects of conditioned medium were abolished by antibody-mediated NAMPT depletion, strongly suggesting that the protective effect is mediated by the extracellular NAMPT released into in the medium. These data suggest a novel neuroprotective role for secreted NAMPT in the protection of white matter after ischemic injury. [ABSTRACT FROM AUTHOR]

Although severe stress can elicit toxicity, mild stress often elicits adaptations. Here we review the literature on stress-induced adaptations versus stress sensitization in models of neurodegenerative diseases. We also describe our recent findings that chronic proteotoxic stress can elicit adaptations if the dose is low but that high-dose proteotoxic stress sensitizes cells to subsequent challenges. In these experiments, long-term, low-dose proteasome inhibition elicited protection in a superoxide dismutase-dependent manner. In contrast, acute, high-dose proteotoxic stress sensitized cells to subsequent proteotoxic challenges by eliciting catastrophic loss of glutathione. However, even in the latter model of synergistic toxicity, several defensive proteins were upregulated by severe proteotoxicity. This led us to wonder whether high-dose proteotoxic stress can elicit protection against subsequent challenges in astrocytes, a cell type well known for their resilience. In support of this new hypothesis, we found that the astrocytes that survived severe proteotoxicity became harder to kill. The adaptive mechanism was glutathione dependent. If these findings can be generalized to the human brain, similar endogenous adaptations may help explain why neurodegenerative diseases are so delayed in appearance and so slow to progress. In contrast, sensitization to severe stress may explain why defenses eventually collapse in vulnerable neurons. [ABSTRACT FROM AUTHOR]

Mononuclear phagocytes are a population of multi-phenotypic cells and have dual roles in brain destruction/reconstruction. The phenotype-specific roles of microglia/macrophages in traumatic brain injury (TBI) are, however, poorly characterized. In the present study, TBI was induced in mice by a controlled cortical impact (CCI) and animals were killed at 1 to 14 days post injury. Real-time polymerase chain reaction (RT-PCR) and immunofluorescence staining for M1 and M2 markers were performed to characterize phenotypic changes of microglia/macrophages in both gray and white matter. We found that the number of M1-like phagocytes increased in cortex, striatum and corpus callosum (CC) during the first week and remained elevated until at least 14 days after TBI. In contrast, M2-like microglia/macrophages peaked at 5 days, but decreased rapidly thereafter. Notably, the severity of white matter injury (WMI), manifested by immunohistochemical staining for neurofilament SMI-32, was strongly correlated with the number of M1-like phagocytes. In vitro experiments using a conditioned medium transfer system confirmed that M1 microglia-conditioned media exacerbated oxygen glucose deprivation-induced oligodendrocyte death. Our results indicate that microglia/macrophages respond dynamically to TBI, experiencing a transient M2 phenotype followed by a shift to the M1 phenotype. The M1 phenotypic shift may propel WMI progression and represents a rational target for TBI treatment. [ABSTRACT FROM AUTHOR]

Dietary supplementation with omega-3 (ω-3) fatty acids is a safe, economical mean of preventive medicine that has shown protection against several neurologic disorders. The present study tested the hypothesis that this method is protective against controlled cortical impact (CCI). Indeed, mice fed with ω-3 polyunsaturated fatty acid (PUFA)-enriched diet for 2 months exhibited attenuated short and long-term behavioral deficits due to CCI. Although ω-3 PUFAs did not decrease cortical lesion volume, these fatty acids did protect against hippocampal neuronal loss after CCI and reduced pro-inflammatory response. Interestingly, ω-3 PUFAs prevented the loss of myelin basic protein (MPB), preserved the integrity of the myelin sheath, and maintained the nerve fiber conductivity in the CCI model. ω-3 PUFAs also directly protected oligodendrocyte cultures from excitotoxicity and blunted the microglial activation-induced death of oligodendrocytes in microglia/oligodendrocyte cocultures. In sum, ω-3 PUFAs elicit multifaceted protection against behavioral dysfunction, hippocampal neuronal loss, inflammation, and loss of myelination and impulse conductivity. The present report is the first demonstration that ω-3 PUFAs protect against white matter injury in vivo and in vitro. The protective impact of ω-3 PUFAs supports the clinical use of this dietary supplement as a prophylaxis against traumatic brain injury and other nervous system disorders. [ABSTRACT FROM AUTHOR]

Mitochondria play a central role in cell fate after stressors such as ischemic brain injury. The convergence of intracellular signaling pathways on mitochondria and their release of critical factors are now recognized as a default conduit to cell death or survival. Besides the individual processes that converge on or emanate from mitochondria, a mitochondrial organellar response to changes in the cellular environment has recently been described. Whereas mitochondria have previously been perceived as a major center for cellular signaling, one can postulate that the organelle's dynamics themselves affect cell survival. This brief perspective review puts forward the concept that disruptions in mitochondrial dynamics-biogenesis, clearance, and fission/fusion events-may underlie neural diseases and thus could be targeted as neuroprotective strategies in the context of ischemic injury. To do so, we present a general overview of the current understanding of mitochondrial dynamics and regulation. We then review emerging studies that correlate mitochondrial biogenesis, mitophagy, and fission/fusion events with neurologic disease and recovery. An overview of the system as it is currently understood is presented, and current assessment strategies and their limitations are discussed. [ABSTRACT FROM AUTHOR]

Ischemic stroke is a devastating condition lacking effective therapies. A promising approach to attenuate ischemic injury is mild hypothermia. Recent studies show that adenosine nucleotides can induce hypothermia in mice. The purpose of the present study was to test the hypothesis that adenosine 5′-triphosphate (ATP) induces mild hypothermia in rats and reduces ischemic brain injury. We found that intraperitoneal injections of ATP decreased core body temperature in a dose-dependent manner; the dose appropriate for mild hypothermia was 2 g/kg. When ATP-induced hypothermia was applied to stroke induced by middle cerebral artery occlusion, however, a neuroprotective effect was not observed. Instead, the infarct volume grew even larger in ATP-treated rats. This was accompanied by an increased rate of seizure events, hemorrhagic transformation, and higher mortality. Continuous monitoring of physiologic parameters revealed that ATP reduced heartbeat rate and blood pressure. ATP also increased blood glucose, accompanied by severe acidosis and hypocalcemia. Western blotting showed that ATP decreased levels of both phospho-Akt and total-Akt in the cortex. Our results reveal that, despite inducing hypothermia, ATP is not appropriate for protecting the brain against stroke. Instead, we show for the first time that ATP treatment is associated with exaggerated ischemic outcomes and dangerous systemic side effects. [ABSTRACT FROM AUTHOR]

Single-cell RNA sequencing (scRNA-seq) is a high-throughput transcriptomic approach with the power to identify rare cells, discover new cellular subclusters, and describe novel genes. scRNA-seq can simultaneously reveal dynamic shifts in cellular phenotypes and heterogeneities in cellular subtypes. Since the publication of the first protocol on scRNA-seq in 2009, this evolving technology has continued to improve, through the use of cell-specific barcodes, adoption of droplet-based systems, and development of advanced computational methods. Despite induction of the cellular stress response during the tissue dissociation process, scRNA-seq remains a popular technology, and commercially available scRNA-seq methods have been applied to the brain. Recent advances in spatial transcriptomics now allow the researcher to capture the positional context of transcriptional activity, strengthening our knowledge of cellular organization and cell-cell interactions in spatially intact tissues. A combination of spatial transcriptomic data with proteomic, metabolomic, or chromatin accessibility data is a promising direction for future research. Herein, we provide an overview of the workflow, data analyses methods, and pros and cons of scRNA-seq technology. We also summarize the latest achievements of scRNA-seq in stroke and acute traumatic brain injury, and describe future applications of scRNA-seq and spatial transcriptomics. [ABSTRACT FROM AUTHOR]