Microglia and macrophages in the neuro-glia-vascular unit: From identity to functions.

Although both are myeloid cells located surrounding cerebral vasculature, vessel-associated microglia (VAM) and perivascular macrophages (PVMs) can be distinguished by their distinct morphologies, signatures and microscopic location. As key component of neuro-glia-vascular unit (NGVU), they play prominent roles in neurovasculature development and pathological process of various central nervous system (CNS) diseases, including phagocytosis, angiogenesis, vessel damage/protection and blood flow regulation, therefore serving as potential targets for therapeutics of a broad array of CNS diseases. Herein, we will provide a comprehensive overview of heterogeneity of VAM/PVMs, highlight limitations of current understanding in this field, and discuss possible directions of future investigations.

TGF-ß1 ameliorates BBB injury and improves long-term outcomes in mice after ICH.

Intracerebral hemorrhage (ICH) is a devastating subtype of stroke characterized by high mortality and morbidity rates with no effective treatment. TGF-ß/ALK-5 signaling is reported to participated in the regulation of blood-brain barrier (BBB) integrity in the inflammation pain model, the effects of transforming growth factor (TGF)-ß1 and the potential mechanisms on BBB after ICH have not been fully elucidated. Herein, we have demonstrated that peripheral administration of TGF-ß1 reduces brain edema and ameliorated BBB injury after ICH. Consistent with previous results, TGF-ß1 is shown to promote activation of anti-inflammatory microglia and reduce the inflammatory response after ICH. Furthermore, TGF-ß1 administration improves long-term outcomes after ICH. Our data suggest that TGF-ß1 may be a promising therapeutic agent for ICH.

ASK1-K716R reduces neuroinflammation and white matter injury via preserving blood-brain barrier integrity after traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) is a significant worldwide public health concern that necessitates attention. Apoptosis signal-regulating kinase 1 (ASK1), a key player in various central nervous system (CNS) diseases, has garnered interest for its potential neuroprotective effects against ischemic stroke and epilepsy when deleted. Nonetheless, the specific impact of ASK1 on TBI and its underlying mechanisms remain elusive. Notably, mutation of ATP-binding sites, such as lysine residues, can lead to catalytic inactivation of ASK1. To address these knowledge gaps, we generated transgenic mice harboring a site-specific mutant ASK1 Map3k5-e (K716R), enabling us to assess its effects and elucidate potential underlying mechanisms following TBI. METHODS: We employed the CRIPR/Cas9 system to generate a transgenic mouse model carrying the ASK1-K716R mutation, aming to investigate the functional implications of this specific mutant. The controlled cortical impact method was utilized to induce TBI. Expression and distribution of ASK1 were detected through Western blotting and immunofluorescence staining, respectively. The ASK1 kinase activity after TBI was detected by a specific ASK1 kinase activity kit. Cerebral microvessels were isolated by gradient centrifugation using dextran. Immunofluorescence staining was performed to evaluate blood-brain barrier (BBB) damage. BBB ultrastructure was visualized using transmission electron microscopy, while the expression levels of endothelial tight junction proteins and ASK1 signaling pathway proteins was detected by Western blotting. To investigate TBI-induced neuroinflammation, we conducted immunofluorescence staining, quantitative real-time polymerase chain reaction (qRT-PCR) and flow cytometry analyses. Additionally, immunofluorescence staining and electrophysiological compound action potentials were conducted to evaluate gray and white matter injury. Finally, sensorimotor function and cognitive function were assessed by a battery of behavioral tests. RESULTS: The activity of ASK1-K716R was significantly decreased following TBI. Western blotting confirmed that ASK1-K716R effectively inhibited the phosphorylation of ASK1, JNKs, and p38 in response to TBI. Additionally, ASK1-K716R demonstrated a protective function in maintaining BBB integrity by suppressing ASK1/JNKs activity in endothelial cells, thereby reducing the degradation of tight junction proteins following TBI. Besides, ASK1-K716R effectively suppressed the infiltration of peripheral immune cells into the brain parenchyma, decreased the number of proinflammatory-like microglia/macrophages, increased the number of anti-inflammatory-like microglia/macrophages, and downregulated expression of several proinflammatory factors. Furthermore, ASK1-K716R attenuated white matter injury and improved the nerve conduction function of both myelinated and unmyelinated fibers after TBI. Finally, our findings demonstrated that ASK1-K716R exhibited favorable long-term functional and histological outcomes in the aftermath of TBI. CONCLUSION: ASK1-K716R preserves BBB integrity by inhibiting ASK1/JNKs pathway in endothelial cells, consequently reducing the degradation of tight junction proteins. Additionally, it alleviates early neuroinflammation by inhibiting the infiltration of peripheral immune cells into the brain parenchyma and modulating the polarization of microglia/macrophages. These beneficial effects of ASK1-K716R subsequently result in a reduction in white matter injury and promote the long-term recovery of neurological function following TBI.

The role of microglial/macrophagic salt-inducible kinase 3 on normal and excessive phagocytosis after transient focal cerebral ischemia.

Previous studies suggested that anti-inflammatory microglia/macrophages (Mi/MΦ) play a role in "normal phagocytosis," which promoted the rapid clearance of necrotic substances and apoptotic cells. More recently, a few studies have found that Mi/MΦ also play a role in "pathological phagocytosis" in the form of excessive or reduced phagocytosis, thereby worsening damage induced by CNS diseases. However, the underlying mechanisms and the Mi/MΦ subtypes related to this pathological phagocytosis are still unknown. Salt-inducible kinase 3 (SIK3), a member of the 5' adenosine monophosphate-activated protein kinase (AMPK) family, has been shown to regulate inflammation in several peripheral diseases. Whether SIK3 also regulates the inflammatory response in CNS diseases is currently unknown. Therefore, in this study, we created a transgenic tamoxifen-induced Mi/MΦ-specific SIK3 conditional knockout (SIK3-cKO) mouse to examine SIK3's role in phagocytotic function induced by transient focal cerebral ischemia (tFCI). By single-cell RNA-seq, we found the pro-inflammatory Mi/MΦ phenotype performed an excessive phagocytotic function, but the anti-inflammatory Mi/MΦ phenotype performed a normal phagocytotic function. We found that SIK3-cKO caused Mi/MΦ heterogenization from the transitional phenotype to the anti-inflammatory phenotype after tFCI. This phenotypic shift corresponded with enhanced phagocytosis of both apoptotic and live neurons. Interestingly, SIK3-cKO enhanced normal phagocytosis of myelin debris but attenuating excessive phagocytosis of non-damaged myelin sheath, thereby protecting white matter integrity after tFCI. CD16, a pro-inflammation marker, was decreased significantly by SIK3-cKO and correlated with "excessive phagocytosis." SIK3-cKO promoted long-term recovery of white matter function and neurological function as assessed with electrophysiological compound action potential (CAPs) and behavioral analysis. This study is the first to show a role of SIK3 in Mi/MΦ phagocytosis in CNS diseases, and reveals that promoting Mi/MΦ anti-inflammatory heterogenization inhibits "excessive phagocytosis" of live cells and facilitates "normal phagocytosis" of apoptotic cells. Therefore, inhibition of SIK3 in Mi/MΦ may be a potential therapeutic target in stroke and other CNS diseases with accompanying white matter destruction. In the acute stage of tFCI, Mi/MΦ polarized into different phenotypes. The pro-inflammatory Mi/MΦ phenotype performed an excessive phagocytotic function. In contrast, the anti-inflammatory Mi/MΦ phenotype performed a normal phagocytotic function. After tFCI, SIK3-cKO promoted anti-inflammatory phenotypic heterogenization of Mi/MΦ. SIK3-cKO promoted Mi/MΦ phagocytosis of apoptotic (normal phagocytosis) and living neuronal cell bodies (excessive phagocytosis) in gray matter. Interestingly, SIK3-cKO specifically increased normal phagocytosis of myelin debris concurrent with an attenuation of excessive phagocytosis of myelin sheath in white matter. These changes induced by SIK3-cKO were associated with protection of white matter integrity and long-term neurofunctional recovery after tFCI.

Interleukin 13 promotes long-term recovery after ischemic stroke by inhibiting the activation of STAT3.

BACKGROUND: Microglia/macrophages are activated after cerebral ischemic stroke and can contribute to either brain injury or recovery by polarizing microglia/macrophage into distinctive functional phenotypes with pro- or anti-inflammatory properties. Interleukin-13 (IL-13) is an anti-inflammatory cytokine that regulates microglia/macrophage polarization toward an anti-inflammatory phenotype. However, it is not clear whether IL-13 is beneficial after ischemic stroke long-term and the underlying molecular mechanism(s) remain unknown. Thus, we examined the effect of IL-13 on long-term recovery and microglia/macrophage polarization in mice with transient middle cerebral artery occlusion model (tMCAO). METHODS: tMCAO was induced in adult male C57BL/6J mice. IL-13 (60 µg/kg) was administered intranasally starting 2 h after stroke and continued for seven consecutive days. Sensorimotor function, spatial learning and memory function, as well as brain infarct volume were assessed up to 35 days after stroke. White matter integrity was evaluated by electrophysiology, immunofluorescence staining, and transmission electron microscopy. Microglia/macrophage activation was assessed using immunofluorescence staining and quantitative real-time polymerase chain reaction. Changes in immune cells in the brain and the periphery, and expression of IL-13 receptors in different brain cells were detected by flow cytometry. Primary neuron/microglia co-cultures and a STAT3 inhibitor were used for mechanistic studies. RESULTS: Post-treatment with IL-13 improved long-term neurofunctional recovery and decreased brain tissue atrophy after stroke. Intranasal delivery of IL-13 enhanced the structural and functional integrity of white matter after stroke. Furthermore, the neuroprotection afforded by IL-13 administration was not due to a direct effect on neurons, but by indirectly regulating the anti-inflammatory phenotype of microglia/macrophages. IL-13 treatment also had no effect on peripheral immune cells. Mechanistically, IL-13 improved the long-term outcome after ischemic stroke by promoting the polarization of microglia/macrophages toward the anti-inflammatory phenotype at least partially by inhibiting the phosphorylation of STAT3. CONCLUSIONS: IL-13 promotes white matter repair and improves neurofunctional outcomes after ischemic stroke by modulating microglia/macrophages via inhibition of STAT3 phosphorylation.

Irisin ameliorates neuroinflammation and neuronal apoptosis through integrin αVß5/AMPK signaling pathway after intracerebral hemorrhage in mice.

BACKGROUND: Neuroinflammation is a crucial factor in the development of secondary brain injury after intracerebral hemorrhage (ICH). Irisin is a newly identified myokine that confers strong neuroprotective effects in experimental ischemic stroke. However, whether this myokine can exert neuroprotection effects after ICH remains unknown. This study aimed to investigate the impact of irisin treatment on neuroinflammation and neuronal apoptosis and the underlying mechanism involving integrin αVß5/AMPK pathway after ICH. METHODS: Two hundred and eighty-five adult (8-week-old) male C57BL/6 mice were randomly assigned to sham and ICH surgery groups. ICH was induced via intrastriatal injection of autologous blood. Irisin was administered intranasally at 30 min after ICH. To elucidate the underlying mechanism, cilengitide (a selective integrin αVß5 inhibitor) and dorsomorphin (a selective phosphorylated AMPK inhibitor) were administered before irisin treatment. The short- and long-term neurobehavior tests, brain edema, quantitative-PCR, western blotting, Fluoro-Jade C, TUNEL, and immunofluorescence staining were performed to assess the neurofunctional outcome at the level of molecular, cell, histology, and function. RESULTS: Endogenous irisin and its receptor, integrin αVß5, were increased, peaked at 24 h after ICH. irisin post-treatment improved both short- and long-term neurological functions, reduced brain edema after ICH. Interestingly, integrin αVß5 was mainly located in the microglia after ICH, and irisin post-treatment inhibited microglia/macrophage pro-inflammatory polarization and promoted anti-inflammatory polarization. Moreover, irisin treatment inhibited neutrophil infiltration and suppressed neuronal apoptotic cell death in perihematomal areas after ICH. Mechanistically, irisin post-treatment significantly increased the expression of integrin αVß5, p-AMPK and Bcl-2, and decreased the expression of IL-1ß, TNF-α, MPO, and Bax following ICH. The neuroprotective effects of irisin were abolished by both integrin αVß5 inhibitor cilengitide and AMPK inhibitor dorsomorphin. CONCLUSIONS: This study demonstrated that irisin post-treatment ameliorated neurological deficits, reduced brain edema, and ameliorated neuroinflammation and neuronal apoptosis, at least in part, through the integrin αVß5/AMPK signaling pathway after ICH. Thus, irisin post-treatment may provide a promising therapeutic approach for the early management of ICH.

Conditional knockout of ASK1 in microglia/macrophages attenuates epileptic seizures and long-term neurobehavioural comorbidities by modulating the inflammatory responses of microglia/macrophages.

BACKGROUND: Apoptosis signal-regulating kinase 1 (ASK1) not only causes neuronal programmed cell death via the mitochondrial pathway but also is an essential component of the signalling cascade during microglial activation. We hypothesize that ASK1 selective deletion modulates inflammatory responses in microglia/macrophages(Mi/Mϕ) and attenuates seizure severity and long-term cognitive impairments in an epileptic mouse model. METHODS: Mi/Mϕ-specific ASK1 conditional knockout (ASK1 cKO) mice were obtained for experiments by mating ASK1flox/flox mice with CX3CR1creER mice with tamoxifen induction. Epileptic seizures were induced by intrahippocampal injection of kainic acid (KA). ASK1 expression and distribution were detected by western blotting and immunofluorescence staining. Seizures were monitored for 24 h per day with video recordings. Cognition, social and stress related activities were assessed with the Y maze test and the three-chamber social novelty preference test. The heterogeneous Mi/Mϕ status and inflammatory profiles were assessed with immunofluorescence staining and real-time polymerase chain reaction (q-PCR). Immunofluorescence staining was used to detect the proportion of Mi/Mϕ in contact with apoptotic neurons, as well as neuronal damage. RESULTS: ASK1 was highly expressed in Mi/Mϕ during the acute phase of epilepsy. Conditional knockout of ASK1 in Mi/Mϕ markedly reduced the frequency of seizures in the acute phase and the frequency of spontaneous recurrent seizures (SRSs) in the chronic phase. In addition, ASK1 conditional knockout mice displayed long-term neurobehavioral improvements during the Y maze test and the three-chamber social novelty preference test. ASK1 selective knockout mitigated neuroinflammation, as evidenced by lower levels of Iba1+/CD16+ proinflammatory Mi/Mϕ. Conditional knockout of ASK1 increased Mi/Mϕ proportion in contact with apoptotic neurons. Neuronal loss was partially restored by ASK1 selective knockout. CONCLUSION: Conditional knockout of ASK1 in Mi/Mϕ reduced seizure severity, neurobehavioral impairments, and histological damage, at least via inhibiting proinflammatory microglia/macrophages responses. ASK1 in microglia/macrophages is a potential therapeutic target for inflammatory responses in epilepsy.

The interleukin-4/PPARγ signaling axis promotes oligodendrocyte differentiation and remyelination after brain injury.

The repair of white matter damage is of paramount importance for functional recovery after brain injuries. Here, we report that interleukin-4 (IL-4) promotes oligodendrocyte regeneration and remyelination. IL-4 receptor expression was detected in a variety of glial cells after ischemic brain injury, including oligodendrocyte lineage cells. IL-4 deficiency in knockout mice resulted in greater deterioration of white matter over 14 d after stroke. Consistent with these findings, intranasal delivery of IL-4 nanoparticles after stroke improved white matter integrity and attenuated long-term sensorimotor and cognitive deficits in wild-type mice, as revealed by histological immunostaining, electron microscopy, diffusion tensor imaging, and electrophysiology. The selective effect of IL-4 on remyelination was verified in an ex vivo organotypic model of demyelination. By leveraging primary oligodendrocyte progenitor cells (OPCs), microglia-depleted mice, and conditional OPC-specific peroxisome proliferator-activated receptor gamma (PPARγ) knockout mice, we discovered a direct salutary effect of IL-4 on oligodendrocyte differentiation that was mediated by the PPARγ axis. Our findings reveal a new regenerative role of IL-4 in the central nervous system (CNS), which lies beyond its known immunoregulatory functions on microglia/macrophages or peripheral lymphocytes. Therefore, intranasal IL-4 delivery may represent a novel therapeutic strategy to improve white matter integrity in stroke and other brain injuries.

IL-13 Ameliorates Neuroinflammation and Promotes Functional Recovery after Traumatic Brain Injury.

Microglia play essential roles in neuroinflammatory responses after traumatic brain injury (TBI). Our previous studies showed that phenotypes of microglia, as well as infiltrating macrophages, altered at different stages after CNS injury, which was correlated to functional outcomes. IL-13 is an anti-inflammatory cytokine that has been reported to protect against demyelination and spinal cord injury through immunomodulation. The effects of IL-13 in microglia/macrophage-mediated immune responses after TBI remain unknown. In this study, we showed that intranasal administration of IL-13 in male C57BL/6J mice accelerated functional recovery in the controlled cortical impact model of TBI. IL-13 treatment increased the time to fall off in the Rotarod test, reduced the number of foot faults in the foot fault test, and improved the score in the wire hang test up to 28 d after TBI. Consistent with functional improvement, IL-13 reduced neuronal tissue loss and preserved white matter integrity 6 d after TBI. Furthermore, IL-13 ameliorated the elevation of proinflammatory factors and reduced the number of proinflammatory microglia/macrophages 6 d after TBI. Additionally, IL-13 enhanced microglia/macrophage phagocytosis of damaged neurons in the peri-lesion areas. In vitro studies confirmed that IL-13 treatment inhibited the production of proinflammatory cytokines in rat primary microglia in response to LPS or dead neuron stimulation and increased the ability of microglia to engulf fluorophore-labeled latex beads or dead neurons. Collectively, we demonstrated that IL-13 treatment improved neurologic outcomes after TBI through adjusting microglia/macrophage phenotypes and inhibiting inflammatory responses. IL-13 may represent a potential immunotherapy to promote long-term recovery from TBI.

Cordycepin confers long-term neuroprotection via inhibiting neutrophil infiltration and neuroinflammation after traumatic brain injury.

BACKGROUND: The secondary injury caused by traumatic brain injury (TBI), especially white matter injury (WMI), is highly sensitive to neuroinflammation, which further leads to unfavored long-term outcomes. Although the cross-talk between the three active events, immune cell infiltration, BBB breakdown, and proinflammatory microglial/macrophage polarization, plays a role in the vicious cycle, its mechanisms are not fully understood. It has been reported that cordycepin, an extract from Cordyceps militaris, can inhibit TBI-induced neuroinflammation although the long-term effects of cordycepin remain unknown. Here, we report our investigation of cordycepin's long-term neuroprotective function and its underlying immunological mechanism. METHODS: TBI mice model was established with a controlled cortical impact (CCI) method. Cordycepin was intraperitoneally administered twice daily for a week. Neurological outcomes were assessed by behavioral tests, including grid walking test, cylinder test, wire hang test, and rotarod test. Immunofluorescence staining, transmission electron microscopy, and electrophysiology recording were employed to assess histological and functional lesions. Quantitative-PCR and flow cytometry were used to detect neuroinflammation. The tracers of Sulfo-NHS-biotin and Evans blue were assessed for the blood-brain barrier (BBB) leakage. Western blot and gelatin zymography were used to analyze protein activity or expression. Neutrophil depletion in vivo was performed via using Ly6G antibody intraperitoneal injection. RESULTS: Cordycepin administration ameliorated long-term neurological deficits and reduced neuronal tissue loss in TBI mice. Meanwhile, the long-term integrity of white matter was also preserved, which was revealed in multiple dimensions, such as morphology, histology, ultrastructure, and electrical conductivity. Cordycepin administration inhibited microglia/macrophage pro-inflammatory polarization and promoted anti-inflammatory polarization after TBI. BBB breach was attenuated by cordycepin administration at 3 days after TBI. Cordycepin suppressed the activities of MMP-2 and MMP-9 and the neutrophil infiltration at 3 days after TBI. Moreover, neutrophil depletion provided a cordycepin-like effect, and cordycepin administration united with neutrophil depletion did not show a benefit of superposition. CONCLUSIONS: The long-term neuroprotective function of cordycepin via suppressing neutrophil infiltration after TBI, thereby preserving BBB integrity and changing microglia/macrophage polarization. These findings provide significant clinical potentials to improve the quality of life for TBI patients.

Endothelium-targeted overexpression of heat shock protein 27 ameliorates blood-brain barrier disruption after ischemic brain injury.

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.

The effect of age-related risk factors and comorbidities on white matter injury and repair after ischemic 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.

APE1/Ref-1 facilitates recovery of gray and white matter and neurological function after mild stroke injury.

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.

Regulatory T cells ameliorate tissue plasminogen activator-induced brain haemorrhage after stroke.

Delayed thrombolytic treatment with recombinant tissue plasminogen activator (tPA) may exacerbate blood-brain barrier breakdown after ischaemic stroke and lead to lethal haemorrhagic transformation. The immune system is a dynamic modulator of stroke response, and excessive immune cell accumulation in the cerebral vasculature is associated with compromised integrity of the blood-brain barrier. We previously reported that regulatory T cells, which function to suppress excessive immune responses, ameliorated blood-brain barrier damage after cerebral ischaemia. This study assessed the impact of regulatory T cells in the context of tPA-induced brain haemorrhage and investigated the underlying mechanisms of action. The number of circulating regulatory T cells in stroke patients was dramatically reduced soon after stroke onset (84 acute ischaemic stroke patients with or without intravenous tPA treatment, compared to 115 age and gender-matched healthy controls). Although stroke patients without tPA treatment gradually repopulated the numbers of circulating regulatory T cells within the first 7 days after stroke, post-ischaemic tPA treatment led to sustained suppression of regulatory T cells in the blood. We then used the murine suture and embolic middle cerebral artery occlusion models of stroke to investigate the therapeutic potential of adoptive regulatory T cell transfer against tPA-induced haemorrhagic transformation. Delayed administration of tPA (10 mg/kg) resulted in haemorrhagic transformation in the ischaemic territory 1 day after ischaemia. When regulatory T cells (2 × 106/mouse) were intravenously administered immediately after delayed tPA treatment in ischaemic mice, haemorrhagic transformation was significantly decreased, and this was associated with improved sensorimotor functions. Blood-brain barrier disruption and tight junction damages were observed in the presence of delayed tPA after stroke, but were mitigated by regulatory T cell transfer. Mechanistic studies demonstrated that regulatory T cells completely abolished the tPA-induced elevation of MMP9 and CCL2 after stroke. Using MMP9 and CCL2 knockout mice, we discovered that both molecules partially contributed to the protective actions of regulatory T cells. In an in vitro endothelial cell-based model of the blood-brain barrier, we confirmed that regulatory T cells inhibited tPA-induced endothelial expression of CCL2 and preserved blood-brain barrier integrity after an ischaemic challenge. Lentivirus-mediated CCL2 knockdown in endothelial cells completely abolished the blood-brain barrier protective effect of regulatory T cells in vitro. Altogether, our studies suggest that regulatory T cell adoptive transfer may alleviate thrombolytic treatment-induced haemorrhage in stroke victims. Furthermore, regulatory T cell-afforded protection in the tPA-treated stroke model is mediated by two inhibitory mechanisms involving CCL2 and MMP9. Thus, regulatory T cell adoptive transfer may be useful as a cell-based therapy to improve the efficacy and safety of thrombolytic treatment for ischaemic stroke.

HDAC inhibition prevents white matter injury by modulating microglia/macrophage polarization through the GSK3ß/PTEN/Akt axis.

Severe traumatic brain injury (TBI) elicits destruction of both gray and white matter, which is exacerbated by secondary proinflammatory responses. Although white matter injury (WMI) is strongly correlated with poor neurological status, the maintenance of white matter integrity is poorly understood, and no current therapies protect both gray and white matter. One candidate approach that may fulfill this role is inhibition of class I/II histone deacetylases (HDACs). Here we demonstrate that the HDAC inhibitor Scriptaid protects white matter up to 35 d after TBI, as shown by reductions in abnormally dephosphorylated neurofilament protein, increases in myelin basic protein, anatomic preservation of myelinated axons, and improved nerve conduction. Furthermore, Scriptaid shifted microglia/macrophage polarization toward the protective M2 phenotype and mitigated inflammation. In primary cocultures of microglia and oligodendrocytes, Scriptaid increased expression of microglial glycogen synthase kinase 3 beta (GSK3ß), which phosphorylated and inactivated phosphatase and tensin homologue (PTEN), thereby enhancing phosphatidylinositide 3-kinases (PI3K)/Akt signaling and polarizing microglia toward M2. The increase in GSK3ß in microglia and their phenotypic switch to M2 was associated with increased preservation of neighboring oligodendrocytes. These findings are consistent with recent findings that microglial phenotypic switching modulates white matter repair and axonal remyelination and highlight a previously unexplored role for HDAC activity in this process. Furthermore, the functions of GSK3ß may be more subtle than previously thought, in that GSK3ß can modulate microglial functions via the PTEN/PI3K/Akt signaling pathway and preserve white matter homeostasis. Thus, inhibition of HDACs in microglia is a potential future therapy in TBI and other neurological conditions with white matter destruction.

Interleukin-4 Is Essential for Microglia/Macrophage M2 Polarization and Long-Term Recovery After Cerebral Ischemia.

BACKGROUND AND PURPOSE: Interleukin-4 (IL-4) is a unique cytokine that may contribute to brain repair by regulating microglia/macrophage functions. Thus, we examined the effect of IL-4 on long-term recovery and microglia/macrophage polarization in 2 well-established stroke models. METHODS: Transient middle cerebral artery occlusion or permanent distal middle cerebral artery occlusion was induced in wild-type and IL-4 knockout C57/BL6 mice. In a separate cohort of wild-type animals, IL-4 (60 ng/d for 7 days) or vehicle was infused into the cerebroventricle after transient middle cerebral artery occlusion. Behavioral outcomes were assessed by the Rotarod, corner, foot fault, and Morris water maze tests. Neuronal tissue loss was verified by 2 independent neuron markers. Markers of classically activated (M1) and alternatively activated (M2) microglia were assessed by real-time polymerase chain reaction, immunofluorescence, and flow cytometry. RESULTS: Loss of IL-4 exacerbated sensorimotor deficits and impaired cognitive functions ≤21 days post injury. In contrast to the delayed deterioration of neurological functions, IL-4 deficiency increased neuronal tissue loss only in the acute phase (5 days) after stroke and had no impact on neuronal tissue loss 14 or 21 days post injury. Loss of IL-4 promoted expression of M1 microglia/macrophage markers and impaired expression of M2 markers at 5 and 14 days post injury. Administration of IL-4 into the ischemic brain also enhanced long-term functional recovery. CONCLUSIONS: The cytokine IL-4 improves long-term neurological outcomes after stroke, perhaps through M2 phenotype induction in microglia/macrophages. These results are the first to suggest that immunomodulation with IL-4 is a promising approach to promote long-term functional recovery after stroke.

Omega-3 polyunsaturated fatty acids mitigate blood-brain barrier disruption after hypoxic-ischemic brain injury.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) have been shown to protect the neonatal brain against hypoxic/ischemic (H/I) injury. However, the mechanism of n-3 PUFA-afforded neuroprotection is not well understood. One major determinant of H/I vulnerability is the permeability of the blood-brain barrier (BBB). Therefore, we examined the effects of n-3 PUFAs on BBB integrity after neonatal H/I. Female rats were fed a diet with or without n-3 PUFA enrichment from day 2 of pregnancy to 14days after parturition. H/I was introduced in 7day-old offspring. We observed relatively rapid BBB penetration of the small molecule cadaverine (640Da) at 4h post-H/I and a delayed penetration of larger dextrans (3kD-40kD) 24-48h after injury. Surprisingly, the neonatal BBB was impermeable to Evans Blue or 70kD dextran leakage for up to 48h post-H/I, despite evidence of IgG extravasation at this time. As expected, n-3 PUFAs ameliorated H/I-induced BBB damage, as shown by reductions in tracer efflux and IgG extravasation, preservation of BBB ultrastructure, and enhanced tight junction protein expression. Furthermore, n-3 PUFAs prevented the elevation in matrix metalloproteinase (MMP) activity in the brain and blood after H/I. Thus, n-3 PUFAs may protect neonates against BBB damage by blunting MMPs activation after H/I.

Sevoflurane Preconditioning Confers Neuroprotection via Anti-apoptosis Effects.

Neuroprotection against cerebral ischemia afforded by volatile anesthetic preconditioning (APC) has been demonstrated both in vivo and in vitro, yet the underlying mechanism is poorly understood. We previously reported that repeated sevoflurane APC reduced infarct size in rats after focal ischemia. In this study, we investigated whether inhibition of apoptotic signaling cascades contributes to sevoflurane APC-induced neuroprotection. Male Sprague-Dawley rats were exposed to ambient air or 2.4 % sevoflurane for 30 min per day for 4 consecutive days and then subjected to occlusion of the middle cerebral artery (MCAO) for 60 min at 24 h after the last sevoflurane intervention. APC with sevoflurane markedly decreased apoptotic cell death in rat brains, which was accompanied by decreased caspase-3 cleavage and cytochrome c release. The apoptotic suppression was associated with increased ratios of anti-apoptotic Bcl-2 family proteins over pro-apoptotic proteins and with decreased activation of JNK and p53 pathways. Thus, our data suggest that suppression of apoptotic cell death contributes to the neuroprotection against ischemic brain injury conferred by sevoflurane preconditioning.

Omega-3 fatty acids protect the brain against ischemic injury by activating Nrf2 and upregulating heme oxygenase 1.

Ischemic stroke is a debilitating clinical disorder that affects millions of people, yet lacks effective neuroprotective treatments. Fish oil is known to exert beneficial effects against cerebral ischemia. However, the underlying protective mechanisms are not fully understood. The present study tests the hypothesis that omega-3 polyunsaturated fatty acids (n-3 PUFAs) attenuate ischemic neuronal injury by activating nuclear factor E2-related factor 2 (Nrf2) and upregulating heme oxygenase-1 (HO-1) in both in vitro and in vivo models. We observed that pretreatment of rat primary neurons with docosahexaenoic acid (DHA) significantly reduced neuronal death following oxygen-glucose deprivation. This protection was associated with increased Nrf2 activation and HO-1 upregulation. Inhibition of HO-1 activity with tin protoporphyrin IX attenuated the protective effects of DHA. Further studies showed that 4-hydroxy-2E-hexenal (4-HHE), an end-product of peroxidation of n-3 PUFAs, was a more potent Nrf2 inducer than 4-hydroxy-2E-nonenal derived from n-6 PUFAs. In an in vivo setting, transgenic mice overexpressing fatty acid metabolism-1, an enzyme that converts n-6 PUFAs to n-3 PUFAs, were remarkably resistant to focal cerebral ischemia compared with their wild-type littermates. Regular mice fed with a fish oil-enhanced diet also demonstrated significant resistance to ischemia compared with mice fed with a regular diet. As expected, the protection was associated with HO-1 upregulation, Nrf2 activation, and 4-HHE generation. Together, our data demonstrate that n-3 PUFAs are highly effective in protecting the brain, and that the protective mechanisms involve Nrf2 activation and HO-1 upregulation by 4-HHE. Further investigation of n-3 PUFA neuroprotective mechanisms may accelerate the development of stroke therapies.