Xiaoming Hu

Microglia/Macrophage Polarization Dynamics Reveal Novel Mechanism of Injury Expansion After Focal Cerebral Ischemia

Background and Purpose— Mononuclear phagocytes are highly plastic cells that assume diverse phenotypes in response to microenvironmental signals. The phenotype-specific roles of microglia/macrophages in ischemic brain injury are poorly understood. A comprehensive characterization of microglia/macrophage polarization after ischemia may advance our knowledge of poststroke damage/recovery. Methods— Focal transient cerebral ischemia was induced in mice for 60 minutes; animals were euthanized at 1 to 14 days of reperfusion. Reverse-transcriptase polymerase chain reaction and immunohistochemical staining for M1 and M2 markers were performed to characterize phenotypic changes in brain cells, including microglia and infiltrating macrophages. In vitro experiments using a transwell system, a conditioned medium transfer system, or a coculture system allowing cell-to-cell contacts were used to further elucidate the effect of neuronal ischemia on microglia/macrophage polarization and, conversely, the effect of microglia/macrophage phenotype on the fate of ischemic neurons. Results— Local microglia and newly recruited macrophages assume the M2 phenotype at early stages of ischemic stroke but gradually transformed into the M1 phenotype in peri-infarct regions. In vitro experiments revealed that ischemic neurons prime microglial polarization toward M1 phenotype. M1-polarized microglia or M1-conditioned media exacerbated oxygen glucose deprivation–induced neuronal death. In contrast, maintaining the M2 phenotype of microglia protected neurons against oxygen glucose deprivation. Conclusions— Our results suggest that microglia/macrophages respond dynamically to ischemic injury, experiencing an early “healthy” M2 phenotype, followed by a transition to a “sick” M1 phenotype. These dual and opposing roles of microglia/macrophages suggest that stroke therapies should be shifted from simply suppressing microglia/macrophage toward adjusting the balance between beneficial and detrimental microglia/macrophage responses.

Microglial and macrophage polarization—new prospects for brain repair

The traditional view of the adult brain as a static organ has changed in the past three decades, with the emergence of evidence that it remains plastic and has some regenerative capacity after injury. In the injured brain, microglia and macrophages clear cellular debris and orchestrate neuronal restorative processes. However, activation of these cells can also hinder CNS repair and expand tissue damage. Polarization of macrophage populations toward different phenotypes at different stages of injury might account for this dual role. This Perspectives article highlights the specific roles of polarized microglial and macrophage populations in CNS repair after acute injury, and argues that therapeutic approaches targeting cerebral inflammation should shift from broad suppression of microglia and macrophages towards subtle adjustment of the balance between their phenotypes. Breakthroughs in the identification of regulatory molecules that control these phenotypic shifts could ultimately accelerate research towards curing brain disorders.

Microglia/macrophage polarization dynamics in white matter after traumatic brain injury

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.

Enhanced Oligodendrogenesis and Recovery of Neurological Function by Erythropoietin After Neonatal Hypoxic/Ischemic Brain Injury

Background and Purpose— Neuronal replacement has recently gained attention as a potential therapeutic target under ischemic conditions. However, the oligodendrogenic infrastructure is equally critical for restoration of brain function and is also sensitive to ischemic injury. Erythropoietin (EPO) is a neuroprotective molecule that stimulates neuronal replacement after neonatal hypoxia/ischemia (H/I) when delivered soon after the onset of reperfusion. Because EPO can improve recovery of neurological function in the absence of tissue protection, we hypothesize that EPO may improve neurological function via enhancement of white matter recovery after H/I. Thus, we sought to determine the effects of delayed administration of EPO on white matter injury and recovery of neurological function after neonatal H/I. Methods— EPO (1000 U/kg) was injected intraperitoneally at multiple time points beginning 48 hours after H/I in postnatal day 7 rats. The effects of EPO on oligodendrogenesis, white matter injury, and neurogenesis were evaluated using bromodeoxyuridine incorporation and cell-specific immunohistochemistry. Neurological function was assessed by sensorimotor behavioral tests. Results— Delayed administration of EPO was incapable of reducing brain volume loss but significantly increased oligodendrogenesis and maturation of oligodendrocytes and attenuated white matter injury after H/I. These effects occurred concurrently with enhanced neurogenesis. Delayed EPO treatment improved behavioral neurological outcomes 14 days after H/I injury. Conclusions— Our study demonstrates that delayed administration of EPO promotes oligodendrogenesis and attenuates white matter injury concurrently with increased neurogenesis. These effects likely contribute to the observed improvement in neurological functional outcomes.

Adoptive regulatory T-cell therapy protects against cerebral ischemia

Recent evidence suggests that functional deficiency in regulatory T cells (Tregs), an innate immunomodulator, exacerbates brain damage after cerebral ischemia. We therefore evaluated the effect of Treg transfer in rodent models of ischemic stroke and further investigated the mechanism underlying Treg‐afforded neuroprotection.

HDAC inhibition prevents white matter injury by modulating microglia/macrophage polarization through the GSK3β/PTEN/Akt axis

Significance Moderate or severe traumatic brain injury (TBI) damages white matter, thereby contributing to long-term neurological deficits. Currently, there are no satisfactory therapies to mitigate this white matter injury (WMI). Here we show that inhibition of histone deacetylases (HDACs) exerts robust structural and functional protection of white matter in a murine model of TBI/WMI by polarizing microglia/macrophages toward the beneficial M2 phenotype. HDAC inhibition shifted microglia/macrophage phenotype by up-regulating glycogen synthase kinase 3 beta (GSK3β), which inactivated phosphatase and tensin homologue (PTEN) through phosphorylation, thereby promoting PI3K/Akt signaling. The GSK3β-dependent M2 phenotype exerted potent anti-inflammatory effects that protected myelin-forming oligodendrocytes and diminished WMI. These results reveal a previously unexplored role for GSK3β/PTEN/PI3K signaling in the regulation of microglia/macrophages and demonstrate the promise of HDAC inhibition in the treatment of TBI/WMI. 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 (GSK3β), 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 GSK3β 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 GSK3β may be more subtle than previously thought, in that GSK3β 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.

Molecular dialogs between the ischemic brain and the peripheral immune system: Dualistic roles in injury and repair

Immune and inflammatory responses actively modulate the pathophysiological processes of acute brain injuries such as stroke. Soon after the onset of stroke, signals such as brain-derived antigens, danger-associated molecular patterns (DAMPs), cytokines, and chemokines are released from the injured brain into the systemic circulation. The injured brain also communicates with peripheral organs through the parasympathetic and sympathetic branches of the autonomic nervous system. Many of these diverse signals not only activate resident immune cells in the brain, but also trigger robust immune responses in the periphery. Peripheral immune cells then migrate toward the site of injury and release additional cytokines, chemokines, and other molecules, causing further disruptive or protective effects in the ischemic brain. Bidirectional communication between the injured brain and the peripheral immune system is now known to regulate the progression of stroke pathology as well as tissue repair. In the end, this exquisitely coordinated crosstalk helps determine the fate of animals after stroke. This article reviews the literature on ischemic brain-derived signals through which peripheral immune responses are triggered, and the potential impact of these peripheral responses on brain injury and repair. Pharmacological strategies and cell-based therapies that target the dialog between the brain and peripheral immune system show promise as potential novel treatments for stroke.

Omega-3 Polyunsaturated Fatty Acid Supplementation Confers Long-Term Neuroprotection Against Neonatal Hypoxic–Ischemic Brain Injury Through Anti-Inflammatory Actions

Background and Purpose— Current available therapies for neonatal hypoxia/ischemia (H/I) brain injury are rather limited. Here, we investigated the effect of omega-3 polyunsaturated fatty acids on brain damage and long-term neurological function after H/I in neonates. Methods— Female rats were treated with or without an omega-3 polyunsaturated fatty acids-enriched diet from the second day of pregnancy until 14 days after parturition. Seven-day-old neonates were subjected to H/I and euthanized 5 weeks later for evaluation of tissue loss. Neurological impairment was assessed progressively for 5 weeks after H/I by grid walking, foot fault, and Morris water maze. Activation of microglia and production of inflammatory mediators were examined up to 7 days after H/I. Results— Omega-3 polyunsaturated fatty acid supplementation significantly reduced brain damage and improved long-term neurological outcomes up to 5 weeks after neonatal H/I injury. Omega-3 polyunsaturated fatty acids exerted an anti-inflammatory effect in microglia both in an in vivo model of H/I and in in vitro microglial cultures subjected to inflammatory stimuli by inhibiting NF-&kgr;B activation and subsequent release of inflammatory mediators. Conclusions— Our results suggest that omega-3 polyunsaturated fatty acids confer potent neuroprotection against neonatal H/I brain injury through, at least partially, suppressing a microglial-mediated inflammatory response.

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.

Rapid endothelial cytoskeletal reorganization enables early blood–brain barrier disruption and long-term ischaemic reperfusion brain injury

The mechanism and long-term consequences of early blood–brain barrier (BBB) disruption after cerebral ischaemic/reperfusion (I/R) injury are poorly understood. Here we discover that I/R induces subtle BBB leakage within 30–60 min, likely independent of gelatinase B/MMP-9 activities. The early BBB disruption is caused by the activation of ROCK/MLC signalling, persistent actin polymerization and the disassembly of junctional proteins within microvascular endothelial cells (ECs). Furthermore, the EC alterations facilitate subsequent infiltration of peripheral immune cells, including MMP-9-producing neutrophils/macrophages, resulting in late-onset, irreversible BBB damage. Inactivation of actin depolymerizing factor (ADF) causes sustained actin polymerization in ECs, whereas EC-targeted overexpression of constitutively active mutant ADF reduces actin polymerization and junctional protein disassembly, attenuates both early- and late-onset BBB impairment, and improves long-term histological and neurological outcomes. Thus, we identify a previously unexplored role for early BBB disruption in stroke outcomes, whereby BBB rupture may be a cause rather than a consequence of parenchymal cell injury. Matrix metalloproteinases (MMPs) released from infiltrating immune cells are a major contributor to blood-brain barrier (BBB) breakdown following stroke. Here, the authors identify an early, MMP-independent BBB breakdown mechanism caused by rapid cytoskeletal rearrangements in endothelial cells, which could be inhibited by ADF.