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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.

Remote ischemic postconditioning (RIPostC) has been proved to protect the brain from stroke, but the precise mechanism remains not fully understood. In the present study, we aimed to investigate whether RIPostC attenuates cerebral ischemia-reperfusion injury by abating endoplasmic reticulum (ER) stress response. CHOP, a multifunctional transcription factor in ER stress, regulates the expression of genes related to apoptosis, such as Bim and Bcl-2. Male SD rats were subjected to right middle cerebral artery occlusion (MCAO) for 2 h followed by reperfusion, and RIPostC was induced by three cycles of 10 min ischemia and 10 min reperfusion on bilateral femoral arteries immediately after ischemia. CHOP siRNA (CHOPi) and control siRNA (Coni) were injected into the right lateral ventricle 30 min before the beginning of ischemia. RIPostC, CHOPi, or RIPostC + CHOPi application reduced infarct volume, improved the neurological function, and decreased cell apoptosis. RIPostC increased the protein level of glucose-regulated protein 78 (GRP78) and decreased the protein level of phosphorylated-EIF2α, caspase-12, and CHOP. Furthermore, the expression of CHOP, Bim and cleaved-caspase-3 was decreased, while Bcl-2 expression was increased in response to application of RIPostC, CHOPi, or RIPostC + CHOPi. In sum, RIPostC protects against ischemia-reperfusion brain injury in rats by attenuating ER stress response-induced apoptosis.

Background and Purpose— p53-mediated neuronal death is a central pathway of stroke pathophysiology, but its mechanistic details remain unclear. Here, we identified a novel microRNA mechanism that downregulation of inhibitory member of the apoptosis-stimulating proteins of p53 family (iASPP) by the brain-specific microRNA-124 (miR-124) promotes neuronal death after cerebral ischemia. Methods— In a mouse model of focal permanent cerebral ischemia, the expression of iASPP and miR-124 was quantified by reverse transcription quantitative real-time polymerase chain reaction, immunofluorescence staining, and Western blot. Luciferase reporter assay was used to validate whether miR-124 can directly bind to the 3′-untranslated region of iASPP mRNA. To evaluate the role of miR-124, miR-124 mimic and its inhibitor were transfected into Neuro-2a cells and C57 mice. Results— There was no change in the iASPP mRNA level in cerebral ischemia. However, iASPP protein was remarkably decreased, with a concurrent elevation in miR-124 level. Furthermore, miR-124 can bind to the 3′-untranslated region of iASPP in 293T cells and downregulate its protein levels in Neuro-2a cells. In vivo, infusion of miR-124 decreased brain levels of iASPP, whereas inhibition of miR-124 enhanced iASPP levels and significantly reduced infarction in mouse focal cerebral ischemia. Conclusions— These data demonstrate that p53-mediated neuronal cell death after stroke can be nontranscriptionally regulated by a novel mechanism involving suppression of endogenous cell death inhibitors by miR-124. Further dissection of microRNA regulatory mechanisms may lead to new therapeutic opportunities for preventing neuronal death after stroke.

Mild therapeutic hypothermia, a robust neuroprotectant, reduces neuronal apoptosis, but the precise mechanism is not well understood. Our previous study showed that a novel inhibitor of an apoptosis-stimulating protein of p53 (iASPP) might be involved in neuronal death after stroke. The aim of this study was to confirm the role of iASPP after stroke treated with mild therapeutic hypothermia. To address this, we mimicked ischemia/reperfusion injury in vitro by using oxygen-glucose deprivation/reperfusion (OGD/R) in primary rat neurons. In our in vivo approach, we induced middle cerebral artery occlusion (MCAO) for 60 min in C57/B6 mice. From the beginning of ischemia, focal mild hypothermia was applied for two hours. To evaluate the role of iASPP, small interfering RNA (siRNA) was injected intracerebroventricularly. Our results showed that mild therapeutic hypothermia increased the expression of iASPP and decreased the expression of its targets, Puma and Bax, and an apoptosis marker, cleaved caspase-3, in primary neurons under OGD/R. Increased iASPP expression and decreased ASPP1/2 expression were observed under hypothermia treatment in MCAO mice. iASPP siRNA (iASPPi) or hypothermia plus iASPPi application increased infarct volume, apoptosis and aggravated the neurological deficits in MCAO mice. Furthermore, iASPPi downregulated iASPP expression, and upregulated the expression of proapoptotic effectors, Puma, Bax and cleaved caspase-3, in mice after stroke treated with mild therapeutic hypothermia. In conclusion, mild therapeutic hypothermia protects against ischemia/reperfusion brain injury in mice by upregulating iASPP and thus attenuating apoptosis. iASPP may be a potential target in the therapy of stroke.