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Recent research highlights the function of regulatory T cells (Tregs) in white matter integrity in CNS diseases. Approaches that expand the number of Tregs have been utilized to improve stroke recovery. However, it remains unclear if Treg augmentation preserves white matter integrity early after stroke or promotes white matter repair. This study evaluates the effect of Treg augmentation on white matter injury and repair after stroke. Adult male C57/BL6 mice randomly received Treg or splenocyte (2 million, iv) transfer 2 h after transient (60 min) middle cerebral artery occlusion (tMCAO). Immunostaining showed improved white matter recovery after tMCAO in Treg-treated mice compared to mice received splenocytes. In another group of mice, IL-2/IL-2 antibody complexes (IL-2/IL-2Ab) or isotype IgG were administered (i.p) for 3 consecutive days starting 6 h after tMCAO, and repeated on day 10, 20 and 30. The IL-2/IL-2Ab treatment boosted the number of Tregs in blood and spleen and increased Treg infiltration into the ischemic brain. Longitudinal in vivo and ex vivo diffusion tensor imaging analysis revealed an increase in fractional anisotropy 28d and 35d, but not 14d, after stroke in IL-2/IL-2Ab-treated mice compared to isotype-treated mice, suggesting a delayed improvement in white matter integrity. IL-2/IL-2Ab also improved sensorimotor functions (rotarod test and adhesive removal test) 35d after stroke. There were correlations between white matter integrity and behavior performance. Immunostaining confirmed the beneficial effects of IL-2/IL-2Ab on white matter structures 35d after tMCAO. IL-2/IL-2Ab treatment starting as late as 5d after stroke still improved white matter integrity 21d after tMCAO, suggesting long-term salutary effects of Tregs on the late-stage tissue repair. We also found that IL-2/IL-2Ab treatment reduced the number of dead/dying OPCs and oligodendrocytes in the brain 3d after tMCAO. To confirm the direct effect of Tregs on remyelination, Tregs were cocultured with lysophosphatidyl choline (LPC)-treated organotypic cerebella. LPC exposure for 17 h induced demyelination in organotypic cultures, followed by gradual spontaneous remyelination upon removal of LPC. Co-culture with Tregs accelerated remyelination in organotypic cultures 7d after LPC. In conclusion, Boosting the number of Tregs protects oligodendrocyte lineage cells early after stroke and promotes long-term white matter repair and functional recovery. IL-2/IL-2Ab represents a feasible approach of Treg expansion for stroke treatment., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

SPAK inhibitor ZT-1a was previously shown to be neuroprotective in murine ischemic stroke models. In this study, we further examined the efficacy of four ZT-1a derivatives (ZT-1c, -1d, -1g and -1h) on reducing stroke-induced sensorimotor function impairment and brain lesions. Vehicle control (Veh) or ZT-1 derivatives were administered via osmotic pump to adult C57BL/6J mice during 3-21 h post-stroke. Neurological behavior of these mice was assessed at days 1, 3, 5, and 7 post-stroke and MRI T2WI and DTI analysis was subsequently conducted in ex vivo brains. Veh-treated stroke mice displayed sensorimotor function deficits compared to Sham mice. In contrast, mice receiving ZT-1a derivatives displayed significantly lower neurological deficits at days 3-7 post-stroke (p < 0.05), with ZT-1a, ZT-1c and ZT-1d showing greater impact than ZT-1h and ZT-1g. ZT-1a treatment was the most effective in reducing brain lesion volume on T2WI and in preserving NeuN + neurons (p < 0.01), followed by ZT-1d > -1c > -1g > -1h. The Veh-treated stroke mice displayed white matter tissue injury, reflected by reduced fractional anisotropy (FA) or axial diffusivity (AD) values in external capsule, internal capsule and hippocampus. In contrast, only ZT-1a-as well as ZT-1c-treated stroke mice exhibited significantly higher FA and AD values. These findings demonstrate that post-stroke administration of SPAK inhibitor ZT-1a and its derivatives (ZT-1c and ZT-1d) is effective in protecting gray and white matter tissues in ischemic brains, showing a potential for ischemic stroke therapy development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationship that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

Differential microglial inflammatory responses play a role in regulation of differentiation and maturation of oligodendrocytes (OLs) in brain white matter. How microglia-OL crosstalk is altered by traumatic brain injury (TBI) and its impact on axonal myelination and neurological function impairment remain poorly understood. In this study, we investigated roles of a Na + /H + exchanger (NHE1), an essential microglial pH regulatory protein, in microglial proinflammatory activation and OL survival and differentiation in a murine TBI model induced by controlled cortical impact. Similar TBI-induced contusion volumes were detected in the Cx3cr1-Cre ERT2 control (Ctrl) mice and selective microglial Nhe1 knockout (Cx3cr1-Cre ERT2 ;Nhe1 flox/flox , Nhe1 cKO) mice. Compared to the Ctrl mice, the Nhe1 cKO mice displayed increased resistance to initial TBI-induced white matter damage and accelerated chronic phase of OL regeneration at 30 days post-TBI. The cKO brains presented increased anti-inflammatory phenotypes of microglia and infiltrated myeloid cells, with reduced proinflammatory transcriptome profiles. Moreover, the cKO mice exhibited accelerated post-TBI sensorimotor and cognitive functional recovery than the Ctrl mice. These phenotypic outcomes in cKO mice were recapitulated in C57BL6J wild-type TBI mice receiving treatment of a potent NHE1 inhibitor HOE642 for 1-7 days post-TBI. Taken together, these findings collectively demonstrated that blocking NHE1 protein stimulates restorative microglial activation in oligodendrogenesis and neuroprotection, which contributes to accelerated brain repair and neurological function recovery after TBI., (© 2022. The Author(s).)

Background: Chronic cerebral hypoperfusion (CCH) causes white matter damage and cognitive impairment, in which astrogliosis is the major pathology. However, underlying cellular mechanisms are not well defined. Activation of Na + /H + exchanger-1 (NHE1) in reactive astrocytes causes astrocytic hypertrophy and swelling. In this study, we examined the role of NHE1 protein in astrogliosis, white matter demyelination, and cognitive function in a murine CCH model with bilateral carotid artery stenosis (BCAS)., Methods: Sham, BCAS, or BCAS mice receiving vehicle or a selective NHE1 inhibitor HOE642 were monitored for changes of the regional cerebral blood flow and behavioral performance for 28 days. Ex vivo MRI-DTI was subsequently conducted to detect brain injury and demyelination. Astrogliosis and demyelination were further examined by immunofluorescence staining. Astrocytic transcriptional profiles were analyzed with bulk RNA-sequencing and RT-qPCR., Results: Chronic cerebral blood flow reduction and spatial working memory deficits were detected in the BCAS mice, along with significantly reduced mean fractional anisotropy (FA) values in the corpus callosum, external capsule, and hippocampus in MRI DTI analysis. Compared with the sham control mice, the BCAS mice displayed demyelination and axonal damage and increased GFAP + astrocytes and Iba1 + microglia. Pharmacological inhibition of NHE1 protein with its inhibitor HOE642 prevented the BCAS-induced gliosis, damage of white matter tracts and hippocampus, and significantly improved cognitive performance. Transcriptome and immunostaining analysis further revealed that NHE1 inhibition specifically attenuated pro-inflammatory pathways and NADPH oxidase activation., Conclusion: Our study demonstrates that NHE1 protein is involved in astrogliosis with pro-inflammatory transformation induced by CCH, and its blockade has potentials for reducing astrogliosis, demyelination, and cognitive impairment., (© 2021. The Author(s).)

The precise mechanisms underlying the beneficial effects of regulatory T (Treg) cells on long-term tissue repair remain elusive. Here, using single-cell RNA sequencing and flow cytometry, we found that Treg cells infiltrated the brain 1 to 5 weeks after experimental stroke in mice. Selective depletion of Treg cells diminished oligodendrogenesis, white matter repair, and functional recovery after stroke. Transcriptomic analyses revealed potent immunomodulatory effects of brain-infiltrating Treg cells on other immune cells, including monocyte-lineage cells. Microglia depletion, but not T cell lymphopenia, mitigated the beneficial effects of transferred Treg cells on white matter regeneration. Mechanistically, Treg cell-derived osteopontin acted through integrin receptors on microglia to enhance microglial reparative activity, consequently promoting oligodendrogenesis and white matter repair. Increasing Treg cell numbers by delivering IL-2:IL-2 antibody complexes after stroke improved white matter integrity and rescued neurological functions over the long term. These findings reveal Treg cells as a neurorestorative target for stroke recovery., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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.

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.

Persistent endoplasmic reticulum (ER) stress in neurons is associated with activation of inflammatory cells and subsequent neuroinflammation following traumatic brain injury (TBI); however, the underlying mechanism remains elusive. We found that induction of neuronal-ER stress, which was mostly characterized by an increase in phosphorylation of a protein kinase R-like ER kinase (PERK) leads to release of excess interferon (IFN)β due to atypical activation of the neuronal-STING signaling pathway. IFNβ enforced activation and polarization of the primary microglial cells to inflammatory M1 phenotype with the secretion of a proinflammatory chemokine CXCL10 due to activation of STAT1 signaling. The secreted CXCL10, in turn, stimulated the T-cell infiltration by serving as the ligand and chemoattractant for CXCR3 + T-helper 1 (Th1) cells. The activation of microglial cells and infiltration of Th1 cells resulted in white matter injury, characterized by impaired myelin basic protein and neurofilament NF200, the reduced thickness of corpus callosum and external capsule, and decline of mature oligodendrocytes and oligodendrocyte precursor cells. Intranasal delivery of CXCL10 siRNA blocked Th1 infiltration but did not fully rescue microglial activation and white matter injury after TBI. However, impeding PERK-phosphorylation through the administration of GSK2656157 abrogated neuronal induction of IFNβ, switched microglial polarization to M2 phenotype, prevented Th1 infiltration, and increased Th2 and Treg levels. These events ultimately attenuated the white matter injury and improved anxiety and depressive-like behavior following TBI. SIGNIFICANCE STATEMENT A recent clinical study showed that human brain trauma patients had enhanced expression of type-1 IFN; suggests that type-1 IFN signaling may potentially influence clinical outcome in TBI patients. However, it was not understood how TBI leads to an increase in IFNβ and whether induction of IFNβ has any influence on neuroinflammation, which is the primary reason for morbidity and mortality in TBI. Our study suggests that induction of PERK phosphorylation, a characteristic feature of ER stress is responsible for an increase in neuronal IFNβ, which, in turn, activates microglial cells and subsequently manifests the infiltration of T cells to induce neuroinflammation and subsequently white matter injury. Blocking PERK phosphorylation using GSK2656157 (or PERK knockdown) the whole cascade of neuroinflammation was attenuated and improved cognitive function after TBI., (Copyright © 2020 the authors.)

Na + /H + exchanger (NHE1) activation is required for multiple microglial functions. We investigated effects of selective deletion of microglial Nhe1 in Cx3cr1-Cre ER ;Nhe1 f/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 CD11b + /CD45 low-med microglia in the ischemic hemisphere at day 3 post-stroke, but stimulated expression of Ym1, CD68, TGF-β, IL-10, decreased expression of CD86 and IL-1β, and reduced GFAP + 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-Cre +/- ;Nhe1 f/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 CD11b + /CD45 low-med microglia or CD11b + /CD45 hi 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., (© 2018 Wiley Periodicals, Inc.)

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., Competing Interests: The authors declare no conflict of interest.

OBJECT Craniosynostosis is a condition in which one or more of the calvarial sutures fuses prematurely. In addition to the cosmetic ramifications attributable to premature suture fusion, aberrations in neurophysiological parameters are seen, which may result in more significant damage. This work examines the microstructural integrity of white matter, using diffusion tensor imaging (DTI) in a homogeneous strain of rabbits with simple, familial coronal suture synostosis before and after surgical correction. METHODS After diagnosis, rabbits were assigned to different groups: wild-type (WT), rabbits with early-onset complete fusion of the coronal suture (BC), and rabbits that had undergone surgical correction with suturectomy (BC-SU) at 10 days of age. Fixed rabbit heads were imaged at 12, 25, or 42 days of life using a 4.7-T, 40-cm bore Avance scanner with a 7.2-cm radiofrequency coil. For DTI, a 3D spin echo sequence was used with a diffusion gradient (b = 2000 sec/mm(2)) applied in 6 directions. RESULTS As age increased from 12 to 42 days, the DTI differences between WT and BC groups became more pronounced (p < 0.05, 1-way ANOVA), especially in the corpus callosum, cingulum, and fimbriae. Suturectomy resulted in rabbits with no significant differences compared with WT animals, as assessed by DTI of white matter tracts. Also, it was possible to predict to which group an animal belonged (WT, BC, and BC-SU) with high accuracy based on imaging data alone using a linear support vector machine classifier. The ability to predict to which group the animal belonged improved as the age of the animal increased (71% accurate at 12 days and 100% accurate at 42 days). CONCLUSIONS Craniosynostosis results in characteristic changes of major white matter tracts, with differences becoming more apparent as the age of the rabbits increases. Early suturectomy (at 10 days of life) appears to mitigate these differences.

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]