Recent evidence has shown that an increase in CD4+CD25+FoxP3+ regulatory T (Treg) cells may contribute to stroke-induced immunosuppression. However, the molecular mechanisms that underlie this increase in Treg cells remain unclear. Here, we used a transient middle cerebral artery occlusion model in mice and specific pathway inhibitors to demonstrate that stroke activates the sympathetic nervous system, which was abolished by 6-OHDA. The consequent activation of β2-adrenergic receptor (AR) signaling increased prostaglandin E2 (PGE2) level in bone marrow. β2-AR antagonist prevented the upregulation of PGE2. PGE2, which acts on prostaglandin E receptor subtype 4 (EP4), upregulated the expression of receptor activator for NF-κB ligand (RANKL) in CD4+ T cells and mediated the increase in Treg cells in bone marrow Treatment of MCAO mice with RANKL antagonist OPG inhibited the increase in percent of bone marrow Treg cells. PGE2 also elevated the expression of indoleamine 2,3 dioxygenase in CD11C+ dendritic cells and promoted the development of functional Treg cells. The effect was neutralized by treatment with indomethacin. Concurrently, stroke reduced production of stromal cell-derived factor-1 (SDF-1) via β3-AR signals in bone marrow but increased the expression of C-X-C chemokine receptor (CXCR) 4 in Treg and other bone marrow cells. Treatment of MCAO mice with β3-AR antagonist SR-59230A reduced the percent of Treg cells in peripheral blood after stroke. The disruption of the CXCR4–SDF-1 axis may facilitate mobilization of Treg cells and other CXCR4+ cells into peripheral blood. This mechanism could account for the increase in Treg cells, hematopoietic stem cells, and progenitor cells in peripheral blood after stroke. We conclude that cerebral ischemia can increase bone marrow CD4+CD25+FoxP3+ regulatory T cells via signals from the sympathetic nervous system.

BackgroundAt low levels, carbon monoxide (CO) has been shown to have beneficial effects on multiple organs and tissues through its potential anti-inflammatory, anti-apoptotic, and anti-proliferative properties. However, the effect of CO-releasing molecule (CORM)-3, a water-soluble CORM, on ischemic stroke and its mechanism of action are still unclear.MethodsWe investigated the role of CORM-3 in the mouse model of transient middle cerebral artery occlusion (tMCAO). CORM-3 or saline was administered to mice by retro-orbital injection at the time of reperfusion after 1-h tMCAO or at 1 h after sham surgery. We assessed infarct volume and brain water content at 24 and 72 h after ischemia, blood-brain barrier permeability at 6 and 72 h after ischemia, and neurologic deficits on days 1, 3, 7, and 14.ResultsAmong mice that underwent tMCAO, those that received CORM-3 had significantly smaller infarct volume and greater expression of neuronal nuclear antigen (NeuN) and microtubule-associated protein 2 than did saline-treated mice. CORM-3-treated mice had significantly fewer activated microglia in the peri-infarction zone than did control mice and exhibited downregulated expression of ionized calcium-binding adapter molecule (Iba)-1, tumor necrosis factor-α, and interleukin 1β. CORM-3-treated mice had significantly lower brain water content and enhanced neurologic outcomes on days 3, 7, and 14 post-tMCAO. Lastly, CORM-3 treatment reduced Evans blue leakage; increased expression of platelet-derived growth factor receptor-β, tight junction protein ZO-1, and matrix protein laminin; and decreased protein level of matrix metalloproteinase-9.ConclusionCORM-3 treatment at the time of reperfusion reduces ischemia-reperfusion-induced brain injury by suppressing neuroinflammation and alleviating blood-brain barrier disruption. Our data suggest that CORM-3 may provide an effective therapy for ischemic stroke.

Transplanted bone marrow-derived mononuclear cells (BMMNCs) can promote arteriogenesis and angiogenesis by incorporating into vascular walls and differentiating into smooth muscle cells (SMCs) and endothelial cells (ECs). Here, we explored whether BMMNCs can enhance arteriogenesis and angiogenesis and promote long-term functional recovery in a rat model of permanent middle cerebral artery occlusion (pMCAO). Sprague-Dawley rats were injected with vehicle or 1×107 BMMNCs labeled with BrdU via femoral vein 24 h after induction of pMCAO. Functional deficits were assessed weekly through day 42 after pMCAO, and infarct volume was assessed on day 7. We visualized the angioarchitecture by latex perfusion on days 14 and 42. BMMNC transplantation significantly reduced infarct volume and neurologic functional deficits compared with untreated or vehicle-treated ischemic groups. In BMMNC-treated rats, BrdU-positive cells were widely distributed in the infarct boundary zone, were incorporated into vessel walls, and enhanced the growth of leptomeningeal anastomoses, the circle of Willis, and basilar arteries. BMMNCs were shown to differentiate into SMCs and ECs from day 14 after stroke and preserved vascular repair function for at least 6 weeks. Our data indicate that BMMNCs can significantly enhance arteriogenesis and angiogenesis, reduce infarct volume, and promote long-term functional recovery after pMCAO in rats.

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Bone marrow mononuclear cells (BMMNCs) are important for angiogenesis after stroke. We investigated the effects of BMMNCs on cognitive function, angiogenesis, and the vascular endothelial growth factor (VEGF)-VEGF receptor 2 (VEGFR2) signaling pathway in a rat model of vascular dementia. We transplanted BMMNCs into rats that had undergone permanent bilateral occlusion of the common carotid arteries (2VO) and observed their migration in vivo. On day 28, we assessed cognitive function with the Morris Water Maze test and examined vascular density and white matter damage within the corpus striatum by staining with fluorescein lycopersicon esculentum (tomato) lectin or Luxol fast blue. We evaluated expression of VEGF, rapidly accelerated fibrosarcoma 1 (Raf1), and extracellular-signal-regulated kinases 1 and 2 (ERK1/2) in the ischemic hemisphere by Western blot analysis on day 7 after cell transplantation. Contribution of the VEGF-VEGFR2 signaling pathway was confirmed by using VEGFR2 inhibitor SU5416. BMMNCs penetrated the blood-brain barrier and reached the ischemic cortex and white matter or incorporated into vascular walls of 2VO rats. BMMNC-treated 2VO rats had better learning and memory, higher vascular density, and less white matter damage than did vehicle-treated rats. The beneficial effects of BMMNCs were abolished by pretreatment of rats with SU5416. Protein expression of VEGF and phosphorylated Raf1 and ERK1/2 was also significantly increased by BMMNC treatment, but this upregulation was reversed by SU5416. BMMNCs can enhance angiogenesis, reduce white matter damage, and promote cognitive recovery in 2VO rats. The angiogenic effect may result from upregulation of the VEGF-VEGFR2 signaling pathway.

In this study we transplanted bone marrow mononuclear cells (BM-MNCs) or microglia into rats that had undergone permanent cerebral ischemia and observed the distribution or morphology of transplanted cells in vivo. In addition, we also compared the effects of BM-MNCs and microglia on infarct volume, brain water content, and functional outcome after permanent cerebral ischemia. BM-MNCs and microglia were obtained from femur and brain, respectively, of newborn rats. Adult rats were injected with vehicle or 3 million BM-MNCs or microglia via the tail vein 24 h after permanent middle cerebral artery occlusion (pMCAO). The distribution or morphologic characteristics of transplanted BM-MNCs (BrdU/double-stained CD34 and CD45) and microglia (BrdU/double-stained Iba-1) were detected with immunofluorescent staining at 3 or 7 and 14 days after pMCAO. Functional deficits were assessed by the modified neurologic severity score at 1, 3, 7 and 14 days after pMCAO. Brain water content was assessed with dry–wet weight method at 3 days, and infarct volume was determined with 2,3,5-triphenyltetrazolium chloride (TTC) staining at 14 days. More BrdU/double-stained CD45- and Iba-1-positive cells were observed than BrdU/double-stained CD34-positive cells around the infarcted area. Some infused microglia show the morphology of innate microglia at 7 days and the increased numbers can be detected at 14 days after pMCAO. BM-MNC-treated rats showed a significant reduction in infarct volume and brain water content compared to vehicle- and microglia-treated rats. In addition, BM-MNC treatment reduced neurologic deficit scores compared to those in the other groups. The results provide evidence that infusion of BM-MNC, but not microglia, is neuroprotective after permanent cerebral ischemia.

Copeptin and NT-proBNP may be useful independent prognostic markers of all-cause or CVD mortality in Chinese patients with AIS.

Cell-based therapy is considered to be a promising therapeutic strategy for stroke treatment. Although unfractionated bone marrow mononuclear cells (BMMNCs) have been tried in both preclinical and clinical trials, the effective subpopulations need to be identified. In this study, we used fluorescence-activated cell sorting to harvest the CXCR4+CD45+ and CXCR4+CD45− BMMNC subpopulations from transgenic mice that express enhanced green fluorescent protein. We then allogeneically grafted unfractionated BMMNCs or a subpopulation into mice subjected to transient middle cerebral artery occlusion (tMCAO) and compared the effects on stroke outcomes. We found that CXCR4+CD45− BMMNCs, but not CXCR4+CD45+ BMMNCs, more effectively reduced infarction volume and neurologic deficits than did unfractionated BMMNCs. Brain tissue from the ischemic hemisphere of mice treated with CXCR4+CD45− BMMNCs had higher levels of vascular endothelial growth factor and lower levels of TNF-α than did tissue from mice treated with unfractionated BMMNCs. In contrast, CXCR4+CD45+ BMMNCs showed an increase in TNF-α. Additionally, CXCR4+CD45+ and CXCR4+CD45− populations exhibited more robust migration into the lesion areas and were better able to express cell-specific markers of different linages than were the unfractionated BMMNCs. Endothelial and astrocyte cell markers did not colocalize with eGFP+ cells in the brains of tMCAO mice that received CXCR4+CD45+ BMMNCs. In vitro, the CXCR4+CD45− BMMNCs expressed significantly more Oct-4 and Nanog mRNA than did the unfractionated BMMNCs. However, we did not detect gene expression of these two pluripotent markers in CXCR4+CD45+ BMMNCs. Taken together, our study shows for the first time that the CXCR4+CD45− BMMNC subpopulation is superior to unfractionated BMMNCs in ameliorating cerebral damage in a mouse model of tMCAO and could represent a new therapeutic approach for stroke treatment.