Xiaojie Fu

Cerebral ischemia increases bone marrow CD4+CD25+FoxP3+ regulatory T cells in mice via signals from sympathetic nervous system

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.

Bone marrow mononuclear cell transplantation promotes therapeutic angiogenesis via upregulation of the VEGF–VEGFR2 signaling pathway in a rat model of vascular dementia

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.

CXCR4+CD45− BMMNC subpopulation is superior to unfractionated BMMNCs for protection after ischemic stroke in mice

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.

Erythropoietin improves hypoxic-ischemic encephalopathy in Neonatal Rats after Short-term Anoxia by Enhancing Angiogenesis.

Erythropoietin (EPO) is important for angiogenesis after hypoxia/ischemia. In this study, we investigated whether recombinant human erythropoietin (rhEPO) can enhance angiogenesis, and promote cognitive function through vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) signaling pathway in a rat model of hypoxic-ischemic encephalopathy (HIE). RhEPO, selective VEGFR2 inhibitor (SU5416) or vehicle was administrated by intraperitoneal injection. The assessment for cognitive function begins on day 60 after anoxia. Vascular density in hippocampus and white matter damage within corpus callosum were examined on day 28 after anoxia. The expression of erythropoietin receptor (EPOR), VEGF, rapidly accelerated fibrosarcoma 1 (Raf1), and extracellular-signal-regulated kinases 1 and 2 (ERK1/2) in hippocampus were evaluated on day 7 after anoxia. RhEPO-treated anoxia rats had better cognitive recovery, higher vascular density, and less white matter damage than in the vehicle anoxia rats. These protective effects associated with increased expression of EPOR, VEGF; and increased phosphorylation of Raf1 and ERK1/2. While this up-regulation, and changes in the histopathologic and functional outcomes were abolished by SU5416. Our data indicate that rhEPO can enhance angiogenesis, reduce white matter damage, and promote cognitive recovery through VEGF/VEGFR2 signaling pathway in anoxia rats.

A modified bilateral carotid artery stenosis procedure to develop a chronic cerebral hypoperfusion rat model with an increased survival rate.

BACKGROUND Bilateral carotid artery occlusion (2-vessel occlusion, 2VO) in rats is a classic and frequently used approach to develop an animal model of chronic cerebral hypoperfusion. However, this method results in substantial mortality in rats. NEW METHOD We investigated whether a modified 2VO procedure, which induces bilateral carotid artery stenosis via ligation of each bilateral common carotid artery (CCA) with a 32 gauge (G) needle followed by needle removal, could produce a chronic cerebral hypoperfusion rat model with an increased survival rate. Sprague-Dawley (SD) rats were treated with the standard or modified 2VO procedure, and changes in cerebral blood flow (CBF) and survival rates were determined. On day 28, cognitive function was assessed with the Morris Water Maze (MWM) test, and neuronal survival and degeneration within the hippocampal CA1 area were measured. Damage to the white matter (WM) within the corpus striatum was assessed via Luxol fast blue (LFB) staining and analyses analyzing the levels of the myelin basic protein (MBP) protein levels. RESULTS The modified 2VO procedure induced similar cognitive impairments, hippocampal lesions and WM damage compared with the standard 2VO procedure in rats; however, it had an increased survival rate. Comparison with existing methods This novel method can be used to quickly and effectively establish a chronic cerebral hypoperfusion rat model with common materials and an improved survival rate. CONCLUSION Bilateral carotid artery stenosis using a 32 G needle is a useful and reliable method to develop a rat model of chronic cerebral hypoperfusion with increased survival.

Neuroprotective Effects of Cistanches Herba Therapy on Patients with Moderate Alzheimer’s Disease

Cistanches Herba (CH) is thought to be a “Yang-invigorating” material in traditional Chinese medicine. We evaluated neuroprotective effects of Cistanches Herba on Alzheimers disease (AD) patients. Moderate AD participants were divided into 3 groups: Cistanches Herba capsule (CH, n = 10), Donepezil tablet (DON, n = 8), and control group without treatment (n = 6). We assessed efficacy by MMSE and ADAS-cog, and investigated the volume changes of hippocampus by 1.5 T MRI scans. Protein, mRNA levels, and secretions of total-tau (T-tau), tumor necrosis factor-α (TNF-α), and interleukin- (IL) 1β (IL-1β) in cerebrospinal fluid (CSF) were detected by Western blot, RT-PCR, and ELISA. The scores showed statistical difference after 48 weeks of treatment compared to control group. Meanwhile, volume changes of hippocampus were slight in drug treatment groups but distinct in control group; the levels of T-tau, TNF-α, and IL-1β were decreased compared to those in control group. Cistanches Herba could improve cognitive and independent living ability of moderate AD patients, slow down volume changes of hippocampus, and reduce the levels of T-tau, TNF-α, and IL-1β. It suggested that Cistanches Herba had potential neuroprotective effects for moderate AD.

ChAT-positive neurons participate in subventricular zone neurogenesis after middle cerebral artery occlusion in mice.

HighlightsSVZ choline acetyltransferase (ChAT)+ neurons promote neurogenesis in adult mice.SVZ neurogenesis was elevated after middle cerebral artery occlusion (MCAO) in mice.The cholinergic system in the SVZ was activated after MACO.Post‐stroke neurogenesis was enhanced by donepezil and abolished by atropine.SVZ ChAT+ neurons may promote neurogenesis via the FGFR signaling pathway. ABSTRACT The mechanisms of post‐stroke neurogenesis in the subventricular zone (SVZ) are unclear. However, neural stem cell‐intrinsic and neurogenic niche mechanisms, as well as neurotransmitters, have been shown to play important roles in SVZ neurogenesis. Recently, a previously unknown population of choline acetyltransferase (ChAT)+ neurons residing in rodent SVZ were identified to have direct control over neural stem cell proliferation by indirectly activating fibroblast growth factor receptor (FGFR). This finding revealed possible neuronal control over SVZ neurogenesis. In this study, we assessed whether these ChAT+ neurons also participate in stroke‐induced neurogenesis. We used a permanent middle cerebral artery occlusion (MCAO) model produced by transcranial electrocoagulation in mice, atropine (muscarinic cholinergic receptor [mAchR] antagonist), and donepezil (acetylcholinesterase inhibitor) to investigate the role of ChAT+ neurons in stroke‐induced neurogenesis. We found that mAchRs, phosphorylated protein kinase C (p‐PKC), and p‐38 levels in the SVZ were upregulated in mice on day 7 after MCAO. MCAO also significantly increased the number of BrdU/doublecortin‐positive cells and protein levels of phosphorylated–neural cell adhesion molecule and mammalian achaete scute homolog‐1. FGFR was activated in the SVZ, and doublecortin‐positive cells increased in the peri‐infarction region. These post‐stroke neurogenic effects were enhanced by donepezil and partially decreased by atropine. Neither atropine nor donepezil affected peri‐infarct microglial activation or serum concentrations of TNF‐&agr;, IFN‐&ggr;, or TGF‐&bgr; on day 7 after MCAO. We conclude that ChAT+ neurons in the SVZ may participate in stroke‐induced neurogenesis, suggesting a new mechanism for neurogenesis after stroke.

Effects of an amyloid-beta 1-42 oligomers antibody screened from a phage display library in APP/PS1 transgenic mice

We screened anti-Aβ1-42 antibodies from a human Alzheimers disease (AD) specific single chain variable fragment (scFv) phage display library and assessed their effects in APP/PS1 transgenic mice. Reverse transcription-PCR was used to construct the scFv phage display library, and screening identified 11A5 as an anti-Aβ1-42 antibody. We mixed 11A5 and the monoclonal antibody 6E10 with Aβ1-42 and administered the mixture to Sprague-Dawley rats via intracerebroventricular injection. After 30 days, rats injected with the antibody/Aβ1-42 mixture and those injected with Aβ1-42 alone were tested on the Morris water maze. We also injected 11A5 and 6E10 into APP/PS1 transgenic mice and assessed the concentrations of Aβ in brain and peripheral blood by ELISA at 1-month intervals for 3 months. Finally we evaluated behavior changes in the Morris water maze. Rats injected with Aβ1-42 and mixed antibodies showed better performance in the Morris water maze than did rats injected with Aβ1-42 alone. In APP/PS1 transgenic mice, Aβ concentration was lower in the brains of the antibody-treated group than in the control group, but higher in the peripheral blood. The antibody-treated mice also exhibited improved behavioral performance in the Morris water maze. In conclusion, anti-Aβ1-42 antibodies (11A5) screened from the human scFv antibody phage display library promoted the efflux or clearance of Aβ1-42 and effectively decreased the cerebral Aβ burden in an AD mouse model.

Alpha-7 Nicotinic Receptor Signaling Pathway Participates in the Neurogenesis Induced by ChAT-Positive Neurons in the Subventricular Zone

Choline acetyltransferase-positive (ChAT+) neurons within the subventricular zone (SVZ) have been shown to promote neurogenesis after stroke in mice by secreting acetylcholine (ACh); however, the mechanisms remain unclear. Receptors known to bind ACh include the nicotinic ACh receptors (nAChRs), which are present in the SVZ and have been shown to be important for cell proliferation, differentiation, and survival. In this study, we investigated the neurogenic role of the alpha-7 nAChR (α7 nAChR) in a mouse model of middle cerebral artery occlusion (MCAO) by using α7 nAChR inhibitor methyllycaconitine. Mice subjected to MCAO exhibited elevated expression of cytomembrane and nuclear fibroblast growth factor receptor 1 (FGFR1), as well as increased expression of PI3K, pAkt, doublecortin (DCX), polysialylated - neuronal cell adhesion molecule (PSA-NCAM), and mammalian achaete-scute homolog 1 (Mash1). MCAO mice also had more glial fibrillary acidic protein (GFAP)/5-bromo-2′-deoxyuridine (BrdU)-positive cells and DCX-positive cells in the SVZ than did the sham-operated group. Methyllycaconitine treatment increased cytomembrane FGFR1 expression and GFAP/BrdU-positive cells, upregulated the levels of phosphoinositide 3-kinase (PI3K) and phospho-Akt (pAkt), decreased nuclear FGFR1 expression, decreased the number of DCX-positive cells, and reduced the levels of DCX, PSA-NCAM, and Mash1 in the SVZ of MCAO mice compared with levels in vehicle-treated MCAO mice. MCAO mice treated with α7 nAChR agonist PNU-282987 exhibited the opposite effects. Our data show that α7 nAChR may decrease the proliferation of neural stem cells and promote differentiation of existing neural stem cells after stroke. These results identify a new mechanism of SVZ ChAT+ neuron-induced neurogenesis.

Effects of crenolanib, a nonselective inhibitor of PDGFR, in a mouse model of transient middle cerebral artery occlusion

Neurogenesis in the subventricular zone (SVZ) plays a vital role in neurologic recovery after stroke. However, only a small fraction of newly generated neuroblasts from the SVZ will survive long-term. Successful migration and survival of neuroblasts requires angiogenesis, lesion-derived chemo-attractants, and appropriate local microenvironments, which are partly regulated by the platelet-derived growth factor receptor (PDGFR) signaling pathway. In this study, we investigated the effects of PDGFR inhibition in a mouse model of transient middle cerebral artery occlusion (MCAO). We blocked the pathway using a nonselective PDGFR inhibitor, crenolanib, during the acute post-MCAO phase (days 1-3) or during the sub-acute phase (days 7-9). Downregulating the PDGFR signaling pathway with crenolanib from day 1 to day 3 after MCAO significantly decreased the migration of neuroblasts from the SVZ to the peri-infarct region, decreased angiogenesis, and lowered expression of vascular endothelial growth factor, stromal cell-derived factor-1, and monocyte chemotactic protein-1. Downregulation of the PDGFR signaling pathway on days 7-9 with crenolanib significantly increased apoptosis of the neuroblasts that had migrated to the peri-infarct region, increased the number of activated microglia, and decreased the expression of brain-derived neurotrophic factor, neurotrophin-3, and interleukin-10. Crenolanib treatment increased the apoptosis of pericytes and decreased the pericyte/vascular coverage, but had no effects on apoptosis of astrocytes. We conclude that the PDGFR signaling pathway plays a vital role in the SVZ neurogenesis after stroke. It can also affect angiogenesis, lesion-derived chemo-attractants, and the local microenvironment, which are all important to stroke-induced neurogenesis.