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The endothelial lining of cerebral microvessels is damaged relatively early after cerebral ischemia/reperfusion (I/R) injury and mediates blood-brain barrier (BBB) disruption, neurovascular injury, and long-term neurological deficits. I/R induces BBB leakage within 1 h due to subtle structural alterations in endothelial cells (ECs), including reorganization of the actin cytoskeleton and subcellular redistribution of junctional proteins. Herein, we show that the protein peroxiredoxin-4 (Prx4) is an endogenous protectant against endothelial dysfunction and BBB damage in a murine I/R model. We observed a transient upregulation of Prx4 in brain ECs 6 h after I/R in wild-type (WT) mice, whereas tamoxifen-induced, selective knockout of Prx4 from endothelial cells (eKO) mice dramatically raised vulnerability to I/R. Specifically, eKO mice displayed more BBB damage than WT mice within 1 to 24 h after I/R and worse long-term neurological deficits and focal brain atrophy by 35 d. Conversely, endothelium-targeted transgenic (eTG) mice overexpressing Prx4 were resistant to I/R-induced early BBB damage and had better long-term functional outcomes. As demonstrated in cultures of human brain endothelial cells and in animal models of I/R, Prx4 suppresses actin polymerization and stress fiber formation in brain ECs, at least in part by inhibiting phosphorylation/ activation of myosin light chain. The latter cascade prevents redistribution of junctional proteins and BBB leakage under conditions of Prx4 repletion. Prx4 also tempers microvascular inflammation and infiltration of destructive neutrophils and proinflammatory macrophages into the brain parenchyma after I/R. Thus, the evidence supports an indispensable role for endothelial Prx4 in safeguarding the BBB and promoting functional recovery after I/R brain injury. [ABSTRACT FROM AUTHOR]

Aging compromises the repair and regrowth of brain vasculature and white matter during stroke recovery, but the underlying mechanisms remain elusive. To understand how aging jeopardizes brain tissue repair after stroke, we performed single-cell transcripto mic profiling of young adult and aged mouse brains at acute (3 d) and chronic (14 d) stages after ischemic injury, focusing a priori on the expression of angiogenesis- and oligodendrogenesis-related genes. We identified unique subsets of endothelial cells (ECs) and oligodendrocyte (OL) progenitors in proangiogenesis and pro-oligodendrogenesis phenotypic states 3 d after stroke in young mice. However, this early prorepair transcrip-tomic reprogramming was negligible in aged stroke mice, consistent with the impairment of angiogenesis and oligodendrogenesis observed during the chronic injury stages after ischemia. In the stroke brain, microglia and macrophages (MG/MΦ) may drive angiogenesis and oligodendrogenesis through a paracrine mechanism. However, this reparative cell--cell cross talk between MG/MΦ and ECs or OLs is impeded in aged brains. In support of these findings, permanent depletion of MG/MΦ via antagonism of the colony-stimulating factor 1 receptor resulted in remarkably poor neurological recovery and loss ofpoststroke angiogenesis and oligodendrogenesis. Finally, transplantation of MG/MΦ from young, but not aged, mouse brains into the cerebral cortices of aged stroke mice partially restored angiogenesis and oligodendrogenesis and rejuvenated sensorimotor function and spatial learning and memory. Together, these data reveal fundamental mechanisms underlying the age-related decay in brain repair and highlight MG/MΦ as effective targets for promoting stroke recovery. [ABSTRACT FROM AUTHOR]

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 bloodand 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. [ABSTRACT FROM AUTHOR]

Emerging evidence suggests that tissue plasminogen activator (tPA), currently the only FDA-approved medication for ischemic stroke, exerts important biological actions on the CNS besides its well-known thrombolytic effect. In this study, we investigated the role of tPA on primary neurons in culture and on brain recovery and plasticity after ischemic stroke in mice. Treatment with recombinant tPA stimulated axonal growth in culture, an effect independent of its protease activity and achieved through epidermal growth factor receptor (EGFR) signaling. After permanent focal cerebral ischemia, tPA knockout mice developed more severe sensorimotor and cognitive deficits and greater axonal and myelin injury than wildtype mice, suggesting that endogenously expressed tPA promotes longterm neurological recovery after stroke. In tPA knockoutmice, intranasal administration of recombinant tPA protein 6 hours poststroke and 7 more times at 2 d intervals mitigated white matter injury, improved axonal conduction, and enhanced neurological recovery. Consistent with the proaxonal growth effects observed in vitro, exogenous tPA delivery increased poststroke axonal sprouting of corticobulbar and corticospinal tracts, which might have contributed to restoration of neurological functions. Notably, recombinant mutant tPA-S478A lacking protease activity (but retaining the EGF-like domain) was as effective aswild-type tPA in rescuing neurological functions in tPA knockout stroke mice. These findings demonstrate that tPA improves long-term functional outcomes in a clinically relevant stroke model, likely by promoting brain plasticity through EGFR signaling. Therefore, treatment with the protease-dead recombinant tPA-S478A holds particular promise as a neurorestorative therapy, as the risk for triggering intracranial hemorrhage is eliminated and tPA-S478A can be delivered intranasally hours after stroke. [ABSTRACT FROM AUTHOR]

Recombinant tissue plasminogen activator (tPA) is a Food and Drug Administration-approved thrombolytic treatment for ischemic stroke. tPA is also naturally expressed in glial and neuronal cells of the brain, where it promotes axon outgrowth and synaptic plasticity. However, there are conflicting reports of harmful versus neuroprotective effects of tPA in acute brain injury models. Furthermore, its impact on white matter integrity in preclinical traumatic brain injury (TBI) has not been thoroughly explored, although white matter disruption is a better predictor of long-term clinical outcomes than focal lesion volumes. Here we show that the absence of endogenous tPA in knockout mice impedes long-term recovery of white matter and neurological function after TBI. tPA-knockout mice exhibited greater asymmetries in forepaw use, poorer sensorimotor balance and coordination, and inferior spatial learning and memory up to 35 d after TBI. White matter damage was also more prominent in tPA knockouts, as shown by diffusion tensor imaging, histological criteria, and electrophysiological assessments of axon conduction properties. Replenishment of tPA through intranasal application of the recombinant protein in tPA-knockout mice enhanced neurological function, the structural and functional integrity of white matter, and postinjury compensatory sprouting in corticofugal projections. tPA also promoted neurite outgrowth in vitro, partly through the epidermal growth factor receptor. Both endogenous and exogenous tPA protected against white matter injury after TBI without increasing intracerebral hemorrhage volumes. These results unveil a previously unappreciated role for tPA in the protection and/or repair of white matter and long-term functional recovery after TBI. [ABSTRACT FROM AUTHOR]

The damage borne by the endothelial cells (ECs) forming the blood– brain barrier (BBB) during ischemic stroke and other neurological conditions disrupts the structure and function of the neurovascular unit and contributes to poor patient outcomes. We recently reported that structural aberrations in brain microvascular ECs—namely, uncontrolled actin polymerization and subsequent disassembly of junctional proteins, are a possible cause of the early onset BBB breach that arises within 30–60 min of reperfusion after transient focal ischemia. Here, we investigated the role of heat shock protein 27 (HSP27) as a direct inhibitor of actin polymerization and protectant against BBB disruption after ischemia/reperfusion (I/R). Using in vivo and in vitro models, we found that targeted overexpression of HSP27 specifically within ECs—but not within neurons—ameliorated BBB impairment 1–24 h after I/R. Mechanistically, HSP27 suppressed I/R-induced aberrant actin polymerization, stress fiber formation, and junctional protein translocation in brain microvascular ECs, independent of its protective actions against cell death. By preserving BBB integrity after I/R, EC-targeted HSP27 overexpression attenuated the infiltration of potentially destructive neutrophils and macrophages into brain parenchyma, thereby improving long-term stroke outcome. Notably, early poststroke administration of HSP27 attached to a cell-penetrating transduction domain (TAT-HSP27) rapidly elevated HSP27 levels in brain microvessels and ameliorated I/R-induced BBB disruption and subsequent neurological deficits. Thus, the present study demonstrates that HSP27 can function at the EC level to preserve BBB integrity after I/R brain injury. HSP27 may be a therapeutic agent for ischemic stroke and other neurological conditions involving BBB breakdown. [ABSTRACT FROM AUTHOR]

A major hallmark of oxidative DNA damage after stroke is the induction of apurinic/apyrimidinic (AP) sites and strand breaks. To mitigate cell loss after oxidative DNA damage, ischemic cells rapidly engage the base excision-repair proteins, such as the AP site-repairing enzyme AP endonuclease-1 (APE1), also named redox effector factor-1 (Ref-1). Although forced overexpression of APE1 is known to protect against oxidative stress-induced neurodegeneration, there is no concrete evidence demonstrating a role for endogenous APE1 in the long-term recovery of gray and white matter following ischemic injury. To address this gap, we generated, to our knowledge, the first APE1 conditional knockout (cKO) mouse line under control of tamoxifen-dependent Cre recombinase. Using a well-established model of transient focal cerebral ischemia (tFCI), we show that induced deletion of APE1 dramatically enlarged infarct volume and impaired the recovery of sensorimotor and cognitive deficits. APE1 cKO markedly increased postischemic neuronal and oligodendrocyte degeneration, demonstrating that endogenous APE1 preserves both gray and white matter after tFCI. Because white matter repair is instrumental in behavioral recovery after stroke, we also examined the impact of APE1 cKO on demyelination and axonal conduction and discovered that APE1 cKO aggravated myelin loss and impaired neuronal communication following tFCI. Furthermore, APE1 cKO increased AP sites and activated the prodeath signaling proteins, PUMA and PARP1, after tFCI in topographically distinct manners. Our findings provide evidence that endogenous APE1 protects against ischemic infarction in both gray and white matter and facilitates the functional recovery of the central nervous system after mild stroke injury. [ABSTRACT FROM AUTHOR]