Leilei Mao

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.

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.

Essential Role of Program Death 1-Ligand 1 in Regulatory T-Cell–Afforded Protection Against Blood–Brain Barrier Damage After Stroke

Background and Purpose— Our recent research revealed that adoptively transferred regulatory T cells (Tregs) reduced acute ischemic brain injury by inhibiting neutrophil-derived matrix metalloproteinase-9 (MMP-9) and protecting against blood–brain barrier damage. The mechanisms underlying Treg interactions with neutrophils remain elusive. This study evaluates the contribution of program death 1-ligand 1 (PD-L1) to Treg-mediated neutrophil inhibition and neuroprotection after cerebral ischemia. Methods— In vitro experiments were performed using a transwell system or a coculture system allowing cell-to-cell contact. Focal cerebral ischemia was induced in mice for 60 minutes. Tregs (2×106) isolated from donor animals (wild-type or PD-L1–/–) were intravenously injected into ischemic recipients 2 hours after middle cerebral artery occlusion (MCAO). MMP-9 production, blood–brain barrier permeability, and brain infarct were assessed at 1 or 3 days after MCAO. Results— In vitro experiments reveal that Treg-mediated inhibition of neutrophil MMP-9 required direct cell-to-cell contact. The suppression of MMP-9 was abolished when Tregs were pretreated with PD-L1 neutralizing antibodies or when neutrophils were pretreated with PD-1 antibodies. In vivo studies confirmed that intravenous administration of Tregs pretreated with PD-L1 antibodies or Tregs isolated from PD-L1–deficient mice failed to inhibit MMP-9 production by blood neutrophils 1 day after 60 minutes MCAO. Furthermore, the blood–brain barrier damage after MCAO was greatly ameliorated in PD-L1–competent Treg-treated mice but not in PD-L1–compromised Treg-treated mice. Consequently, PD-L1 dysfunction abolished Treg-mediated brain protection and neurological improvements 3 days after MCAO. Conclusions— PD-L1 plays an essential role in the neuroprotection afforded by Tregs against cerebral ischemia by mediating the suppressive effect of Tregs on neutrophil-derived MMP-9.

Adoptive Regulatory T-Cell Therapy Preserves Systemic Immune Homeostasis After Cerebral Ischemia

Background and Purpose— Cerebral ischemia has been shown to result in peripheral inflammatory responses followed by long-lasting immunosuppression. Our recent study demonstrated that intravenous delivery of regulatory T cells (Tregs) markedly protected against transient cerebral ischemia by suppressing neutrophil-derived matrix metallopeptidase 9 production in the periphery. However, the effect of Tregs on systemic inflammatory responses and immune status has not been fully characterized. Methods— Cerebral ischemia was induced by middle cerebral artery occlusion for 60 minutes in mice or 120 minutes in rats. Tregs were isolated from donor animals by CD4 and CD25 double selection and transferred intravenously to ischemic recipients at 2 hours after middle cerebral artery occlusion. Animals were euthanized on different days after reperfusion. The effects of Tregs on systemic inflammation and immune status were evaluated using flow cytometry, ELISAs, and immunohistochemistry. Results— Systemic administration of purified Tregs raises functional Tregs in the blood and peripheral organs, including spleen and lymph nodes. These exogenous Tregs remain in the blood and peripheral organs for ≥12 days. Functionally, Treg adoptive transfer markedly inhibits middle cerebral artery occlusion–induced elevation of inflammatory cytokines (interleukin-6 and tumor necrosis factor &agr;) in the blood. Furthermore, Treg treatment corrects long-term lymphopenia and improves cellular immune functions after ischemic brain injury. As a result, Treg-treated animals exhibit decreased bacterial loads in the blood during recovery from cerebral ischemic attack. Conclusions— Treg treatment did not exacerbate poststroke immunosuppression. On the contrary, Treg-treated animals displayed improved immune status after focal cerebral ischemia.

Rosiglitazone Promotes White Matter Integrity and Long-Term Functional Recovery After Focal Cerebral Ischemia

Background and Purpose— Oligodendrogenesis is essential for white matter repair after stroke. Although agonists of peroxisome proliferator–activated receptors &ggr; confer neuroprotection in models of cerebral ischemia, it is not known whether this effect extends to white matter protection. This study tested the hypothesis that the peroxisome proliferator–activated receptors &ggr; agonist rosiglitazone enhances oligodendrogenesis and improves long-term white matter integrity after ischemia/reperfusion. Methods— Male adult C57/BL6 mice (25–30 g) were subjected to 60-minute middle cerebral artery occlusion and reperfusion. Rosiglitazone (3 mg/kg) was injected intraperitoneally once daily for 14 days beginning 2 hours after reperfusion. Sensorimotor and cognitive functions were evaluated ⩽21 days after middle cerebral artery occlusion. Immunostaining was used to assess infarct volume, myelin loss, and microglial activation. Bromodeoxyuridine (BrdU) was injected for measurements of proliferating NG2+ oligodendrocyte precursor cells (OPCs) and newly generated adenomatous polyposis coli+ oligodendrocytes. Mixed glial cultures were used to confirm the effect of rosiglitazone on oligodendrocyte differentiation and microglial polarization. Results— Rosiglitazone significantly reduced brain tissue loss, ameliorated white matter injury, and improved sensorimotor and cognitive functions for at least 21 days after middle cerebral artery occlusion. Rosiglitazone enhanced OPC proliferation and increased the numbers of newly generated mature oligodendrocytes after middle cerebral artery occlusion. Rosiglitazone treatment also reduced the numbers of Iba1+/CD16+ M1 microglia and increased the numbers of Iba1+/CD206+ M2 microglia after stroke. Glial culture experiments confirmed that rosiglitazone promoted oligodendrocyte differentiation, perhaps by promoting microglial M2 polarization. Conclusions— Rosiglitazone treatment improves long-term white matter integrity after cerebral ischemia, at least, in part, by promoting oligodendrogenesis and facilitating microglial polarization toward the beneficial M2 phenotype.

ST2/IL-33-Dependent Microglial Response Limits Acute Ischemic Brain Injury

ST2, a member of the interleukin (IL) 1 receptor family, and its ligand IL-33 play critical roles in immune regulation and inflammatory responses. This study explores the roles of endogenous IL-33/ST2 signaling in ischemic brain injury and elucidates the underlying mechanisms of action. The expression of IL-33 rapidly increased in oligodendrocytes and astrocytes after 60 min transient middle cerebral artery occlusion (tMCAO). ST2 receptor deficiency exacerbated brain infarction 3 d after tMCAO as well as distal permanent MCAO. ST2 deficiency also aggravated neurological deficits up to 7 d after tMCAO. Conversely, intracerebroventricular infusions of IL-33 after tMCAO attenuated brain infarction. Flow cytometry analyses demonstrated high levels of ST2 expression on microglia, and this expression was dramatically enhanced after tMCAO. The absence of ST2 enhanced the expression of M1 polarization markers on microglia/macrophages, and impaired the expression of M2 polarization markers after tMCAO. In vitro studies on various types of cultures and coculture systems confirmed that IL-33/ST2 signaling potentiated expression of IL-10 and other M2 genes in primary microglia. The activation of ST2 on microglia led to a protective phenotype that enhanced neuronal survival against oxygen glucose deprivation. Further in vitro studies revealed that IL-33-activated microglia released IL-10, and that this was critical for their neuroprotective effects. Similarly, intracerebroventricular infusions of IL-33 into IL-10 knock-out mice failed to provide neuroprotection against tMCAO in vivo. These results shed new light on the IL-33/ST2 axis as an immune regulatory mechanism that serves as a natural brake on the progression of ischemic brain injury. SIGNIFICANCE STATEMENT This is the first study to identify the function of interleukin (IL) 33/ST2 signaling in poststroke microglial responses and neuroprotection against ischemia. Using two models of ischemic stroke, we demonstrate here that ST2 deficiency shifted microglia/macrophages toward a M1-like phenotype, thereby expanding brain infarcts and exacerbating long-term behavioral deficits after stroke. Using stroke models and various in vitro culture and coculture systems, we further characterized a previously undefined mechanism whereby IL-33/ST2 engagement stimulates the production of IL-10 from microglia, which, in turn, enhances neuronal survival upon ischemic challenge. These results shed light on endogenous IL-33/ST2 signaling as a potential immune regulatory mechanism that serves to promote beneficial microglial responses and mitigate ischemic brain injury after stroke.

Strategy to Suppress Oxidative Damage-Induced Neurotoxicity in PC12 Cells by Curcumin: the Role of ROS-Mediated DNA Damage and the MAPK and AKT Pathways

Oxidative damage plays a key role in causation and progression of neurodegenerative diseases. Inhibition of oxidative stress represents one of the most effective ways in treating human neurologic diseases. Herein, we evaluated the protective effect of curcumin on PC12 cells against H2O2-induced neurotoxicity and investigated its underlying mechanism. The results indicated that curcumin pre-treatment significantly suppressed H2O2-induced cytotoxicity, inhibited the loss of mitochondrial membrane potential (Δψm) through regulation of Bcl-2 family expression, and ultimately reversed H2O2-induced apoptotic cell death in PC12 cells. Attenuation of caspase activation, poly(ADP-ribose) polymerase (PARP) cleavage, DNA damage, and accumulation of reactive oxygen species (ROS) all confirmed its protective effects. Moreover, curcumin markedly alleviated the dysregulation of the MAPK and AKT pathways induced by H2O2. Taken together, our findings suggest that the strategy of using curcumin could be a highly effective way in combating oxidative damage-mediated human neurodegenerative diseases.

Enhanced Therapeutic Potential of Nano-Curcumin Against Subarachnoid Hemorrhage-Induced Blood-Brain Barrier Disruption Through Inhibition of Inflammatory Response and Oxidative Stress

Curcumin and nano-curcumin both exhibit neuroprotective effects in early brain injury (EBI) after experimental subarachnoid hemorrhage (SAH). However, the mechanism that whether curcumin and its nanoparticles affect the blood–brain barrier (BBB) following SAH remains unclear. This study investigated the effect of curcumin and the poly(lactide-co-glycolide) (PLGA)-encapsulated curcumin nanoparticles (Cur-NPs) on BBB disruption and evaluated the possible mechanism underlying BBB dysfunction in EBI using the endovascular perforation rat SAH model. The results indicated that Cur-NPs showed enhanced therapeutic effects than that of curcumin in improving neurological function, reducing brain water content, and Evans blue dye extravasation after SAH. Mechanically, Cur-NPs attenuated BBB dysfunction after SAH by preventing the disruption of tight junction protein (ZO-1, occludin, and claudin-5). Cur-NPs also up-regulated glutamate transporter-1 and attenuated glutamate concentration of cerebrospinal fluid following SAH. Moreover, inhibition of inflammatory response and microglia activation both contributed to Cur-NPs’ protective effects. Additionally, Cur-NPs markedly suppressed SAH-mediated oxidative stress and eventually reversed SAH-induced cell apoptosis in rats. Our findings revealed that the strategy of using Cur-NPs could be a promising way in improving neurological function in EBI after experimental rat SAH.

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.