Yaohui Tang

CXCR4 Antagonist AMD3100 Protects Blood–Brain Barrier Integrity and Reduces Inflammatory Response After Focal Ischemia in Mice

Background and Purpose— Inflammatory response plays a critical role in propagating tissue damage after focal cerebral ischemia. CXCL12 is a key chemokine for leukocyte recruitment. However, the role of CXCL12 and its receptor CXCR4 in ischemia-induced inflammatory response is unclear. Here we use the pharmacological antagonist of CXCR4, AMD3100, to investigate the function of CXCL12/CXCR4 in regulating inflammatory response during acute ischemia. Methods— Adult male CD-1 mice (n=184) underwent permanent suture middle cerebral artery occlusion (MCAO). AMD3100 was injected for 3 days (1 mg/kg/day) after MCAO. Brain water content, infarct volume, neurological score, and myeloperoxidase (MPO) expression and activity were examined at 24, 48, and 72 hours after MCAO. Proinflammatory cytokine RNA and protein levels in brain tissue were measured by RT-PCR and enzyme linked immunosorbent assay. Results— Neurological score was greatly improved in AMD3100-treated mice compared with the control mice 3 days after MCAO (P<0.05). Brain edema–induced change of water content, IgG protein leakage, Evans blue extravasation, occludin, and ZO-1 expression in ipsilateral hemisphere were alleviated by acute treatment of AMD3100. MPO expression and activity revealed that AMD3100 profoundly reduced the number of MPO-positive cells in the ischemic region (P<0.05). It also attenuated proinflammatory cytokines including interleukin 6, tumor necrosis factor &agr;, and interferon &ggr;; their mRNA and protein levels changed accordingly compared with the controls (P<0.05). Conclusions— CXCR4 antagonist AMD3100 significantly suppressed inflammatory response and reduced blood–brain barrier disruption after MCAO. AMD3100 attenuated ischemia-induced acute inflammation by suppressing leukocyte migration and infiltration, in addition to reducing proinflammatory cytokine expression in the ischemic region.

Netrin-1 Hyperexpression in Mouse Brain Promotes Angiogenesis and Long-Term Neurological Recovery After Transient Focal Ischemia

Background and Purpose— Netrin-1 (NT-1) stimulates endothelial cell proliferation and migration in vitro and promotes focal neovascularization in the adult brain in vivo. This in vivo study in mice investigated the effect of NT-1 hyperexpression on focal angiogenesis and long-term functional outcome after transient middle cerebral artery occlusion (tMCAO). Methods— Adeno-associated viral vectors carrying either the NT-1 gene (AAV–NT-1) or GFP (AAV-GFP) were generated and injected into the brains of separate groups of 93 mice. Seven days later, tMCAO followed by 7–28 days of reperfusion were carried out. Histological outcomes and behavioral deficits were quantified 7–28 days after tMCAO. Small cerebral vessel network and angiogenesis were assessed 28 days after tMCAO, using synchrotron radiation microangiography and immunohistochemistry. Results— Western blot and immunohistochemistry showed that on the day of tMCAO, NT-1 hyperexpression had been achieved in both normal and ischemic hemispheres. Immunofluorescence imaging showed that NT-1 expression was primarily in neurons and astrocytes. Ischemia-induced infarction in the NT-1 hyperexpression group was attenuated in comparison to saline or AAV-GFP–treated groups (P<0.01). Similarly, neurological deficits were greatly improved in AAV–NT-1–treated mice compared with mice in saline or AAV-GFP–treated groups (P<0.05). In addition, angiogenesis was increased in AAV–NT-1–treated mice compared with the other 2 groups (P<0.05). In vivo synchrotron radiation microangiography 28 days after tMCAO revealed more branches in AAV–NT-1–treated mice than in other groups. Conclusions— AAV–NT-1 induced NT-1 hyperexpression before tMCAO reduced infarct size, enhanced neovascularization, and improved long-term functional recovery.

Neural stem cell protects aged rat brain from ischemia-reperfusion injury through neurogenesis and angiogenesis

Neural stem cells (NSCs) show therapeutic potential for ischemia in young-adult animals. However, the effect of aging on NSC therapy is largely unknown. In this work, NSCs were transplanted into aged (24-month-old) and young-adult (3-month-old) rats at 1 day after stroke. Infarct volume and neurobehavioral outcomes were examined. The number of differentiated NSCs was compared in aged and young-adult ischemic rats and angiogenesis and neurogenesis were also determined. We found that aged rats developed larger infarcts than young-adult rats after ischemia (P<0.05). The neurobehavioral outcome was also worse for aged rats comparing with young-adult rats. Brain infarction and neurologic deficits were attenuated after NSC transplantation in both aged and young-adult rats. The number of survived NSCs in aged rats was similar to that of the young-adult rats (P>0.05) and most of them were differentiated into glial fibrillary acidic protein+ (GFAP+) cells. More importantly, angiogenesis and neurogenesis were greatly enhanced in both aged and young-adult rats after transplantation compared with phosphate-buffered saline (PBS) control (P<0.05), accompanied by increased expression of vascular endothelial growth factor (VEGF). Our results showed that NSC therapy reduced ischemic brain injury, along with increased angiogenesis and neurogenesis in aged rats, suggesting that aging-related microenvironment does not preclude a beneficial response to NSCs transplantation during cerebral ischemia.

Melatonin pretreatment improves the survival and function of transplanted mesenchymal stem cells after focal cerebral ischemia.

Mesenchymal stem cell (MSC) transplantation has been shown to be beneficial in treating cerebral ischemia. However, such benefit is limited by the low survival of transplanted MSCs in an ischemic microenvironment. Previous studies showed that melatonin pretreatment can increase MSC survival in the ischemic kidney. However, whether it will improve MSC survival in cerebral ischemia is unknown. Our study examined the effect of melatonin pretreatment on MSCs under ischemia-related conditions in vitro and after transplantation into ischemic rat brain. Results showed that melatonin pretreatment greatly increased survival of MSCs in vitro and reduced their apoptosis after transplantation into ischemic brain. Melatonin-treated MSCs (MT-MSCs) further reduced brain infarction and improved neurobehavioral outcomes. Angiogenesis, neurogenesis, and the expression of vascular endothelial growth factor (VEGF) were greatly increased in the MT-MSC-treated rats. Melatonin treatment increased the level of p-ERK1/2 in MSCs, which can be blocked by the melatonin receptor antagonist luzindole. ERK phosphorylation inhibitor U0126 completely reversed the protective effects of melatonin, suggesting that melatonin improves MSC survival and function through activating the ERK1/2 signaling pathway. Thus, stem cells pretreated by melatonin may represent a feasible approach for improving the beneficial effects of stem cell therapy for cerebral ischemia.

Postacute Stromal Cell–Derived Factor-1α Expression Promotes Neurovascular Recovery in Ischemic Mice

Background and Purpose— Acute interventions of stroke are often challenged by a narrow treatment window. In this study, we explore treatments in the postacute phase of stroke with wider windows of opportunity. We investigated the effects of stromal cell–derived factor (SDF-1&agr;) in neurovascular recovery during the postacute phase and downstream signaling pathways, underlying SDF-1&agr;–mediated neurovascular recovery. Methods— Adult male Institute of Cancer Research (ICR) mice underwent middle cerebral artery occlusion. One week after middle cerebral artery occlusion, the animals received stereotactic injection of adenoassociated virus (AAV) carrying SDF-1&agr; gene as treatment or AAV-green fluorescent protein as control and were monitored for 5 weeks. Neurobehavioral outcomes were evaluated, and brain atrophy was measured. Neurogenesis and angiogenesis were examined. The proliferation and migration of neural progenitor cells were evaluated. Downstream pathways of SDF-1&agr; were investigated. Inflammatory response was monitored. Results— Neurobehavioral outcomes were improved, and brain atrophy was greatly reduced for ⩽5 weeks in AAV-SDF-1&agr; groups when compared with the control. SDF-1 receptor CXCR4 was upregulated and colocalized with neural and endothelial progenitor cells. The number of nestin+ and doublecortin+/bromodeoxyuridine+ cells in the subventricular zone, doublecortin+ and neuron+/bromodeoxyuridine+ cells in the perifocal region, and cluster of differentiation (CD)31+ and bromodeoxyuridine+/CD31+ microvessels are also significantly increased in AAV-SDF-1&agr; groups. Administration of CXCR4 antagonist AMD3100 eliminated the beneficial effects of SDF-1&agr;. SDF-1&agr;/CXCR4 interaction activated AKT, extracellular signal-regulated kinases (ERK), and P38 mitogen-activated protein kinase (MAPK) signaling pathways but not the c-Jun N-terminal kinase (JNK) pathway. Conclusions— SDF-1&agr; promoted neurogenesis and angiogenesis during the postacute phase of ischemia without eliciting an inflammatory response. AAV-SDF-1&agr; expression represents a promising avenue for ischemic stroke therapy with a wider treatment window.

High MR sensitive fluorescent magnetite nanocluster for stem cell tracking in ischemic mouse brain

UNLABELLED Stem cells have shown a great potential to treat diseases and injuries, including ischemic brain injury. However, developing agents for the long-term tracking of stem cells with few side effects is still challenging. Our aim is to develop a novel fluorescent-magnetite-nanocluster (FMNC) with high MRI sensitivity and to examine its application in the labeling and tracking of mesenchymal stem cells (MSC). For this purpose, we developed FMNC by embedding individual magnetite nanoparticles (NPs) into a polystyrene scaffold coated with two layers of silica and a sandwiched layer of rhodamine. We examined the efficacy of FMNC in MSC labeling and the feasibility of tracking FMNC-labeled MSCs in the ischemic mouse brain. We found that FMNC has high cell-labeling efficiency with no adverse effects on MSCs. In a mouse middle cerebral artery occlusion model, FMNC-labeled MSCs migrated to and accumulated in the ischemic region after FMNC-labeled MSC transplantation. MRI findings highly correlated to immunohistochemistry results. FROM THE CLINICAL EDITOR In this study, the authors report a novel fluorescent-magnetite-nanocluster with high MRI sensitivity and to labeling and tracking of mesenchymal stem cells, and provide in vivo data utilizing a murine stroke model.

Opportunities and Challenges: Stem Cell‐Based Therapy for the Treatment of Ischemic Stroke

Stem cell‐based therapy for ischemic stroke has been widely explored in animal models and provides strong evidence of benefits. In this review, we summarize the types of stem cells, various delivery routes, and tracking tools for stem cell therapy of ischemic stroke. MSCs, EPCs, and NSCs are the most explored cell types for ischemic stroke treatment. Although the mechanisms of stem cell‐based therapies are not fully understood, the most possible functions of the transplanted cells are releasing growth factors and regulating microenvironment through paracrine mechanism. Clinical application of stem cell‐based therapy is still in its infancy. The next decade of stem cell research in stroke field needs to focus on combining different stem cells and different imaging modalities to fully explore the potential of this therapeutic avenue: from bench to bedside and vice versa.

Optimizing Suture Middle Cerebral Artery Occlusion Model in C57BL/6 Mice Circumvents Posterior Communicating Artery Dysplasia

The suture middle cerebral artery occlusion (MCAO) model is used worldwide in both academia and industry. However, the variable occurrence of dysplasia in posterior communicating arteries (PcomAs) induces high mortality and instability in permanent MCAO models, limiting the models application to transient focal ischemia. In particular, high mortality in intraluminal suture MCAO models is associated with the dysplasia of PcomAs in C57BL/6 mice. Optimization of silicone coating length is critical for reducing mortality and generating stable infarct in this model. The aim of our study is to reduce mortality and improve the reproducibility of the intraluminal suture MCAO model in C57BL/6 mice, which have high variation in PcomA dysplasia. Adult male C57BL/6 mice (n=38) underwent MCAO using sutures with various diameters and silicone coating lengths. The occlusion of cerebral vessels was examined by synchrotron radiation live angiography. The morphology of PcomAs was examined under a microscope after MICROFIL(®) infusion. Neurological outcome, infarct volume, and mortality were examined within 28 days. Optimizing the silicone coating on an 8-0 suture tip, we were able to reduce the model mortality to zero after permanent occlusion in C57BL/6 and produce stable brain infarct volume independent of the patency of PcomAs.

Neurovascular Recovery via Cotransplanted Neural and Vascular Progenitors Leads to Improved Functional Restoration after Ischemic Stroke in Rats

Summary The concept of the “neurovascular unit,” emphasizing the interactions between neural and vascular components in the brain, raised the notion that neural progenitor cell (NPC) transplantation therapy aimed at neural repair may be insufficient for the treatment of ischemic stroke. Here, we demonstrate that enhanced neurovascular recovery via cotransplantation of NPCs and embryonic stem cell-derived vascular progenitor cells (VPCs) in a rat stroke model is correlated with improved functional recovery after stroke. We found that cotransplantation promoted the survival, migration, differentiation, and maturation of neuronal and vascular cells derived from the cotransplanted progenitors. Furthermore, it triggered an increased generation of VEGF-, BDNF-, and IGF1-expressing neural cells derived from the grafted NPCs. Consistently, compared with transplantation of NPCs alone, cotransplantation more effectively improved the neurobehavioral deficits and attenuated the infarct volume. Thus, cotransplantation of NPCs and VPCs represents a more effective therapeutic strategy for the treatment of stroke than transplantation of NPCs alone.

Surgery-Related Thrombosis Critically Affects the Brain Infarct Volume in Mice Following Transient Middle Cerebral Artery Occlusion

Transient middle cerebral artery occlusion (tMCAO) model is widely used to mimic human focal ischemic stroke in order to study ischemia/reperfusion brain injury in rodents. In tMCAO model, intraluminal suture technique is widely used to achieve ischemia and reperfusion. However, variation of infarct volume in this model often requires large sample size, which hinders the progress of preclinical research. Our previous study demonstrated that infarct volume was related to the success of reperfusion although the reason remained unclear. The aim of present study is to explore the relationship between focal thrombus formation and model reproducibility with respect to infarct volume. We hypothesize that suture-induced thrombosis causes infarct volume variability due to insufficient reperfusion after suture withdrawal. Seventy-two adult male CD-1 mice underwent 90 minutes of tMCAO with or without intraperitoneal administration of heparin. Dynamic synchrotron radiation microangiography (SRA) and laser speckle contrast imaging (LSCI) were performed before and after tMCAO to observe the cerebral vascular morphology and to measure the cerebral blood flow in vivo. Infarct volume and neurological score were examined to evaluate severity of ischemic brain injury. We found that the rate of successful reperfusion was much higher in heparin-treated mice compared to that in heparin-free mice according to the result of SRA and LSCI at 1 and 3 hours after suture withdrawal (p<0.05). Pathological features and SRA revealed that thrombus formed in the internal carotid artery, middle cerebral artery or anterior cerebral artery, which blocked reperfusion following tMCAO. LSCI showed that cortical collateral circulation could be disturbed by thrombi. Our results demonstrated that suture-induced thrombosis was a critical element, which affects the success of reperfusion. Appropriate heparin management provides a useful approach for improving reproducibility of reperfusion model in mice.