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Rationale: Glial scars present a major obstacle for neuronal regeneration after stroke. Thus, approaches to promote their degradation and inhibit their formation are beneficial for stroke recovery. The interaction of microglia and astrocytes is known to be involved in glial scar formation after stroke; however, how microglia affect glial scar formation remains unclear. Methods: Mice were treated daily with M2 microglial small extracellular vesicles through tail intravenous injections from day 1 to day 7 after middle cerebral artery occlusion. Glial scar, infarct volume, neurological score were detected after ischemia. microRNA and related protein were examined in peri-infarct areas of the brain following ischemia. Results: M2 microglial small extracellular vesicles reduced glial scar formation and promoted recovery after stroke and were enriched in miR-124. Furthermore, M2 microglial small extracellular vesicle treatment decreased the expression of the astrocyte proliferation gene signal transducer and activator of transcription 3, one of the targets of miR-124, and glial fibrillary acidic protein and inhibited astrocyte proliferation both in vitro and in vivo. It also decreased Notch 1 expression and increased Sox2 expression in astrocytes, which suggested that astrocytes had transformed into neuronal progenitor cells. Finally, miR-124 knockdown in M2 microglial small extracellular vesicles blocked their effects on glial scars and stroke recovery. Conclusions: Our results showed, for the first time, that microglia regulate glial scar formation via small extracellular vesicles, indicating that M2 microglial small extracellular vesicles could represent a new therapeutic approach for stroke.

Blood-brain barrier disruption is one of the hallmarks of multiple sclerosis. Mesenchymal stem cells showed great potential for the multiple sclerosis therapy. However, the effect of mesenchymal stem cells on blood-brain barrier in multiple sclerosis remains unclear. Here, we investigated whether mesenchymal stem cells transplantation protected blood-brain barrier integrity and further explored possible underlying mechanisms. Adult female C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein peptide33-55 (MOG33-55) to induce experimental autoimmune encephalomyelitis (EAE). Mesenchymal stem cells (5 × 105) were transplanted via tail vein at disease onset. In the cell culture, we examined lipopolysaccharide-induced AQP4 upregulation in astrocytes. Results indicated that mesenchymal stem cells therapy improved neurobehavioral outcomes in EAE mice, reduced inflammatory cell infiltration, IgG protein leakage, and demyelination in spinal cord. Mesenchymal stem cells therapy also increased tight junction protein expression. In addition, mesenchymal stem cells downregulated AQP4 and A2B adenosine receptor (A2BAR) expression in EAE mice in spinal cord. We found that MSCs-conditioned medium (MCM) reduced the expression of inflammatory cytokines, AQP4 and A2BAR in lipopolysaccharide-activated astrocytes. BAY-60-6583 (a selective A2BAR agonist) reversed the MCM-induced AQP4 downregulation and increased p38 MAPK phosphorylation. Furthermore, the upregulation effects of A2BAR agonist were eliminated when treated with p38 MAPK inhibitor SB203580. Thus, we concluded that mesenchymal stem cells alleviated blood-brain barrier disruption by downregulating AQP4 in multiple sclerosis, possibly through inhibiting the A2BAR/p38 MAPK signaling pathway. Our work suggests that mesenchymal stem cells exert beneficial effect through maintaining blood-brain barrier integrity in EAE mice.

Background Intracellular acidosis in the ischemic penumbra can contribute to further cell death, effectively enlarging the infarct core. Restoring the acid-base balance may enhance tissue survivability after cerebral ischemia. Objective This study investigated whether translocating protons out of penumbral neurons could mitigate tissue acidification and induce neuroprotection in a rodent model of acute cerebral ischemia. Methods We modulated the penumbral neurons via a light-driven pump to translocate protons out (i.e., archaerhodopsin/ArchT group) or into (i.e., channelrhodopsin-2/ChR2 group) neurons after focal cerebral ischemia in rats. Intracellular pH values were imaged via neutral red (NR) fluorescence and cerebral blood flow (CBF) was monitored through laser speckle contrast imaging (LSCI). Global CBF responses to electrical stimulation of the hindlimbs were obtained 24 h and 48 h after ischemia to assess neurological function. Behavioral and histological outcomes were evaluated 48 h after ischemia. A control group without gene modification was included. Results The reduction of relative pH (RpH), the amplitude of negative peak of hypoemic response (RNP) and the hemispheric lateralization index (LI) in ArchT group were significantly less than those of the ChR2 or control group. Moreover, RpH was strongly correlated with RNP (r = 0.60) and LI (r24h = 0.80, r48h = 0.59). In addition, behavioral and histological results supported a neuroprotective effect of countering neuronal acidosis in penumbra through optogenetic stimulation. Conclusion(s) These results indicate that countering intracellular acidosis by optogenetically translocating protons out of penumbral neurons during the acute ischemic stage could induce protection after ischemic brain injury.

Microglial activation participates in white matter injury after cerebral hypoperfusion. However, the underlying mechanism is unclear. Here, we explore whether activated microglia aggravate white matter injury via complement C3-C3aR pathway after chronic cerebral hypoperfusion. Methods: Adult male Sprague-Dawley rats (n = 80) underwent bilateral common carotid artery occlusion for 7, 14, and 28 days. Cerebral vessel density and blood flow were examined by synchrotron radiation angiography and three-dimensional arterial spin labeling. Neurobehavioral assessments, CLARITY imaging, and immunohistochemistry were performed to evaluate activation of microglia and C3-C3aR pathway. Furthermore, C3aR knockout mice were used to establish the causal relationship of C3-C3aR signaling on microglia activation and white matter injury after hypoperfusion. Results: Cerebral vessel density and blood flow were reduced after hypoperfusion (p

Background Adipose-derived stem cells (ADSCs) and their extracellular vesicles (EVs) have therapeutic potential in ischemic brain injury, but the underlying mechanism is poorly understood. The current study aimed to explore the contribution of miRNAs in ADSC-EVs to the treatment of cerebral ischemia. Methods After the intravenous injection of ADSC-EVs, therapeutic efficacy was evaluated by neurobehavioral tests and brain atrophy volume. The polarization of microglia was assessed by immunostaining and qPCR. We further performed miRNA sequencing of ADSC-EVs and analyzed the relationship between the upregulated miRNAs in ADSC-EVs and microglial polarization-related proteins using Ingenuity Pathway Analysis (IPA). Results The results showed that ADSC-EVs reduced brain atrophy volume, improved neuromotor and cognitive functions after mouse ischemic stroke. The loss of oligodendrocytes was attenuated after ADSC-EVs injection. The number of blood vessels, as well as newly proliferated endothelial cells in the peri-ischemia area were higher in the ADSC-EVs treated group than that in the PBS group. In addition, ADSC-EVs regulated the polarization of microglia, resulting in increased repair-promoting M2 phenotype and decreased pro-inflammatory M1 phenotype. Finally, STAT1 and PTEN were highlighted as two downstream targets of up-regulated miRNAs in ADSC-EVs among 85 microglia/macrophage polarization related proteins by IPA. The inhibition of STAT1 and PTEN by ADSC-EVs were confirmed in cultured microglia. Conclusions In summary, ADSC-EVs reduced ischemic brain injury, which was associated with the regulation of microglial polarization. miRNAs in ADSC-EVs partly contributed to their function in regulating microglial polarization by targeting PTEN and STAT1.

White matter injury is a critical pathological characteristic during ischemic stroke. Oligodendrocyte precursor cells participate in white matter repairing and remodeling during ischemic brain injury. Since oligodendrocyte precursor cells could promote Wnt-dependent angiogenesis and migrate along vasculature for the myelination during the development in the central nervous system, we explore whether exogenous oligodendrocyte precursor cell transplantation promotes angiogenesis and remyelination after middle cerebral artery occlusion in mice. Here, oligodendrocyte precursor cell transplantation improved motor and cognitive function, and alleviated brain atrophy. Furthermore, oligodendrocyte precursor cell transplantation promoted functional angiogenesis, and increased myelin basic protein expression after ischemic stroke. The further study suggested that white matter repairing after oligodendrocyte precursor cell transplantation depended on angiogenesis induced by Wnt/β-catenin signal pathway. Our results demonstrated a novel pathway that Wnt7a from oligodendrocyte precursor cells acting on endothelial β-catenin promoted angiogenesis and improved neurobehavioral outcomes, which facilitated white matter repair and remodeling during ischemic stroke.

Endothelial progenitor cell transplantation is a potential therapeutic approach in brain ischemia. However, whether the therapeutic effect of endothelial progenitor cells is via affecting complement activation is unknown. We established a mouse focal ischemia model ( n = 111) and transplanted endothelial progenitor cells into the peri-infarct region immediately after brain ischemia. Neurological outcomes and brain infarct/atrophy volume were examined after ischemia. Expression of C3, C3aR and pro-inflammatory factors were further examined to explore the role of endothelial progenitor cells in ischemic brain. We found that endothelial progenitor cells improved neurological outcomes and reduced brain infarct/atrophy volume after 1 to 14 days of ischemia compared to the control ( p < 0.05). C3 and C3aR expression in the brain was up-regulated at 1 day up to 14 days ( p < 0.05). Endothelial progenitor cells reduced astrocyte-derived C3 ( p < 0.05) and C3aR expression ( p < 0.05) after ischemia. Endothelial progenitor cells also reduced inflammatory response after ischemia ( p < 0.05). Endothelial progenitor cell transplantation reduced astrocyte-derived C3 expression in the brain after ischemic stroke, together with decreased C3aR and inflammatory response contributing to neurological function recovery. Our results indicate that modulating complement C3/C3aR pathway is a novel therapeutic target for the ischemic stroke.

Objective: Directly modulating targeted cortical function, brain stimulation provides promising techniques for stroke intervention. However, the cellular level mechanisms underlying preserved neurovascular function remains unclear. Optogenetics provides a cell-specific approach to modulate the neuronal activity. This study aims to investigate whether the exclusive excitation of sensorimotor neurons using optogenetics in an acute stroke can protect neurovascular function and reduce infarct size. Methods: Sensorimotor neurons were transfected with channelrhodopsin-2 and excited by a 473-nm laser. The photothrombotic stroke was induced in the ipsilateral parietal cortex and the targeted area for modulation remained intact. Optogenetic stimulation was carried out within 2 h after stroke in the modulation group. Using a laser speckle contrast imaging technique, we measured the cerebral blood flow at baseline, 0, 2, and 24 h after stroke, and analyzed the hemodynamic changes in both modulation ( n = 12) and control ( n = 9) groups. Also, the neurovascular response was measured 24 h after stroke. Results: We found that neuronal-specific excitation of an ipsilesional sensorimotor cortex at an acute stage could reduce the expansion of an ischemic area and promote the neurovascular response at 24 h after stroke. The histological and behavioral results consolidate the protective effects of optogenetic-guided neuronal modulation in acute stroke. Conclusion: Excitatory stimulation of ipsilesional sensorimotor neurons in an acute stroke could protect neurovascular function and reduces the expansion of ischemic area. Significance: For the first time, this work demonstrates that specific neuronal modulation in the acute stroke is neuroprotective and reduces the infarct size.

Summary Introduction Dl‐3‐N‐butylphthalide (NBP), a small molecule drug used clinically in the acute phase of ischemic stroke, has been shown to improve functional recovery and promote angiogenesis and collateral vessel circulation after experimental cerebral ischemia. However, the underlying molecular mechanism is unknown. Aims To explore the potential molecular mechanism of angiogenesis induced by NBP after cerebral ischemia. Results NBP treatment attenuated body weight loss, reduced brain infarct volume, and improved neurobehavioral outcomes during focal ischemia compared to the control rats (P

Rationale: Microglia play a critical role in modulating cell death and neurobehavioral recovery in response to brain injury either by direct cell-cell interaction or indirect secretion of trophic factors. Exosomes secreted from cells are well documented to deliver bioactive molecules to recipient cells to modulate cell function. Here, we aimed to identify whether M2 microglia exert neuroprotection after ischemic attack through an exosome-mediated cell-cell interaction. Methods: M2 microglia-derived exosomes were intravenously injected into the mouse brain immediately after middle cerebral artery occlusion. Infarct volume, neurological score, and neuronal apoptosis were examined 3 days after ischemic attack. Exosome RNA and target protein expression levels in neurons and brain tissue were determined for the mechanistic study. Results: Our results showed that the M2 microglia-derived exosomes were taken up by neurons in vitro and in vivo. M2 microglia-derived exosome treatment attenuated neuronal apoptosis after oxygen-glucose deprivation (p

Rationale: Brain collaterals contribute to improving ischemic stroke outcomes. However, dynamic and timely investigations of collateral blood flow and collateral restoration in whole brains of living animals have rarely been reported. Methods: Using multiple modalities of imaging, including synchrotron radiation angiography, laser speckle imaging, and micro-CT imaging, we dynamically explored collateral circulation throughout the whole brain in the rodent middle cerebral artery occlusion model. Results: We demonstrated that compared to control animals, 4 neocollaterals gradually formed between the intra- and extra-arteries in the skull base of model animals after occlusion (p

The pathological role of reactive gliosis in CNS repair remains controversial. In this study, using murine ischemic and hemorrhagic stroke models, we demonstrated that microglia/macrophages and astrocytes are differentially involved in engulfing synapses in the reactive gliosis region. By specifically deleting MEGF10 and MERTK phagocytic receptors, we determined that inhibiting phagocytosis of microglia/macrophages or astrocytes in ischemic stroke improved neurobehavioral outcomes and attenuated brain damage. In hemorrhagic stroke, inhibiting phagocytosis of microglia/macrophages but not astrocytes improved neurobehavioral outcomes. Single-cell RNA sequencing revealed that phagocytosis related biological processes and pathways were downregulated in astrocytes of the hemorrhagic brain compared to the ischemic brain. Together, these findings suggest that reactive microgliosis and astrogliosis play individual roles in mediating synapse engulfment in pathologically distinct murine stroke models and preventing this process could rescue synapse loss., Microglia and astrocytes clear neuronal debris after stroke, whether glia remain phagocytic and cause synapse loss at the subacute stage remains unknown. Here, the authors show in a murine model of ischemic stroke that inhibition of phagocytosis by MEGF10 and MERTK deletion in microglia and astrocytes attenuated damage and improved neurological outcomes by preventing synapse loss.

Background Farnesoid X receptor (FXR) is a nuclear receptor that plays a critical role in controlling cell apoptosis in diverse diseases. Previous studies have shown that knocking out FXR improved cardiac function by reducing cardiomyocyte apoptosis in myocardial ischemic mice. However, the role of FXR after cerebral ischemia remains unknown. In this study, we explored the effects and mechanisms of FXR knockout (KO) on the functional recovery of mice post cerebral ischemia-reperfusion. Methods Adult male C57BL/6 wild type and FXR KO mice were subjected to 90-min transient middle cerebral artery occlusion (tMCAO). The mice were divided into five groups: sham, wild-type tMCAO, FXR KO tMCAO, wild-type tMCAO treated with calcium agonist Bayk8644, and FXR KO tMCAO treated with Bayk8644. FXR expression was examined using immunohistochemistry and Western blot. Brain infarct and brain atrophy volume were examined at 3 and 14 days after stroke respectively. Neurobehavioral tests were conducted up to 14 days after stroke. The protein levels of apoptotic factors (Bcl-2, Bax, and Cleaved caspase-3) and mRNA levels of pro-inflammatory factors (TNF-α, IL-6, IL-1β, IL-17, and IL-18) were examined using Western blot and RT-PCR. TUNEL staining and calcium imaging were obtained using confocal and two-photon microscopy. Results The expression of FXR was upregulated after ischemic stroke, which is located in the nucleus of the neurons. FXR KO was found to reduce infarct volume and promote neurobehavioral recovery following tMCAO compared to the vehicle. The expression of apoptotic and pro-inflammatory factors decreased in FXR KO mice compared to the control. The number of NeuN+/TUNEL+ cells declined in the peri-infarct area of FXR KO mice compared to the vehicle. We further demonstrated that inhibition of FXR reduced calcium overload and addition of ionomycin could reverse this neuroprotective effect in vitro. What is more, in vivo results showed that enhancement of intracellular calcium concentrations could aggravate ischemic injury and reverse the neuroprotective effect of FXR KO in mice. Conclusions FXR KO can promote neurobehavioral recovery and attenuate ischemic brain injury, inflammatory release, and neuronal apoptosis via reducing calcium influx, suggesting its role as a therapeutic target for stroke treatments.

Background Netrin-1 functions largely via combined receptors and downstream effectors. Evidence has shown that astrocytes express netrin-1 receptors, including DCC and UNC5H2. However, whether netrin-1 influences the function of astrocytes was previously unknown. Methods Lipopolysaccharide was used to stimulate the primary cultured astrocytes; interleukin release was used to track astrocyte activation. In vivo, shRNA and netrin-1 protein were injected in the mouse brain. Infarct volume, astrocyte activation, and interleukin release were used to observe the function of netrin-1 in neuroinflammation and brain injury after middle cerebral artery occlusion. Results Our results demonstrated that netrin-1 reduced lipopolysaccharide-induced interleukin-1β and interleukin-12β release in cultured astrocytes, and blockade of the UNC5H2 receptor with an antibody reversed this effect. Additionally, netrin-1 increased p-AKT and PPAR-γ expression in primary cultured astrocytes. In vivo studies showed that knockdown of netrin-1 increased astrocyte activation in the mouse brain after middle cerebral artery occlusion (p

Background Ischemic stroke causes a multitude of brain damage. Neurovascular injury and myelin sheath degradation are two manifestations of ischemic brain damage. Therapeutic strategies aiming only at repairing the neural components or the vessels cannot efficiently restore neurological function. Endothelial progenitor cells (EPCs) have the advantages of both promoting angiogenesis and secreting trophic factors that would promote neurogenesis. Chemokine cxcl12 gene therapy has also been shown to promote angiogenesis, neurogenesis, and remyelination, attracting EPCs, neural progenitor cells, and oligodendrocyte progenitor cells (OPCs) to the injured sites of the brain. In this work, we tested whether these two therapeutics can be combined by genetically engineering the EPCs with cxcl12 to harness the synergistic effects of these two interventions. Methods We used lentivirus (LV) to deliver cxcl12 gene into human umbilical cord blood EPCs to generate the engineered CXCL12-EPCs, which were then delivered into the perifocal region at 1 week after permanent middle cerebral artery occlusion to investigate the effects of CXCL12-EPCs on the functional recovery and angiogenesis, neurogenesis, and remyelination in ischemic stroke mice. Green fluorescent protein (gfp) gene-modified EPCs and LV-CXCL12 gene therapy were used as controls. Results CXCL12-EPC treatment significantly reduced brain atrophy and improved neurobehavioral function at 5 weeks after brain ischemia. The treatment resulted in increased blood vessel density and myelin sheath integrity, and promoted neurogenesis, angiogenesis, and the proliferation and migration of OPCs. In-vitro data showed that CXCL12-EPCs performed better in proliferation and tube formation assays and expressed a higher level of vascular endothelial growth factor compared to GFP-EPCs. Conclusions The synergistic treatment of CXCL12-EPCs outperformed the single therapies of GFP-EPCs or LV-CXCL12 gene therapy in various aspects related to post-ischemic brain repair. cxcl12-engineered EPCs hold great potential in the treatment of ischemic stroke.

Microglia are brain resident macrophages originated from primitive progenitor cells in the yolk sac. Microglia can be activated within hours and recruited to the lesion site. Traditionally, microglia activation is considered to play a deleterious role in ischemic stroke, as inhibition of microglia activation attenuates ischemia induced brain injury. However, increasing evidence show that microglia activation is critical for attenuating neuronal apoptosis, enhancing neurogenesis, and promoting functional recovery after cerebral ischemia. Differential polarization of microglia could likely explain the biphasic role of microglia in ischemia. We comprehensively reviewed the mechanisms involved in regulating microglia activation and polarization. The latest discoveries of microRNAs in modulating microglia function are discussed. In addition, the interaction between microglia and other cells including neurons, astrocytes, oligodendrocytes, and stem cells were also reviewed. Future therapies targeting microglia may not exclusively aim at suppressing microglia activation, but also at modulating microglia polarization at different stages of ischemic stroke. More work is needed to elucidate the cellular and molecular mechanisms of microglia polarization under ischemic environment. The roles of microRNAs and transplanted stem cells in mediating microglia activation and polarization during brain ischemia also need to be further studied.

Matrix metalloproteinase 9 (MMP-9) plays a beneficial role in the delayed phase of middle cerebral artery occlusion (MCAO). However, the mechanism is obscure. Here, we constructed hypoxia response element (HRE)-regulated MMP-9 to explore its effect on glial scars and neurogenesis in delayed ischemic stroke. Adult male Institute of Cancer Research (ICR) mice underwent MCAO and received a stereotactic injection of lentivirus carrying HRE-MMP-9 or normal saline (NS)/lentivirus-GFP 7 days after ischemia. We found that HRE-MMP-9 improved neurological outcomes, reduced ischemia-induced brain atrophy, and degraded glial scars (p < 0.05). Furthermore, HRE-MMP-9 increased the number of microvessels in the peri-infarct area (p < 0.001), which may have been due to the accumulation of endogenous endothelial progenitor cells (EPCs) in the peri-infarct area after glial scar degradation. Finally, HRE-MMP-9 increased the number of bromodeoxyuridine-positive (BrdU+)/NeuN+ cells and the expression of PSD-95 in the peri-infarct area (p < 0.01). These changes could be blocked by vascular endothelial growth factor receptor 2 (VEGFR2) inhibitor SU5416 and MMP-9 inhibitor 2-[[(4-phenoxyphenyl)sulfonyl]methyl]-thiirane (SB-3CT). Our results provided a novel mechanism by which glial scar degradation and vascular endothelial growth factor (VEGF)/VEGFR2-dependent angiogenesis may be key procedures for neurological recovery in delayed ischemic stroke after HRE-MMP-9 treatment. Therefore, HRE-MMP-9 overexpression in the delayed ischemic brain is a promising approach for neurological recovery.

Very limited experimental data is available regarding the safe dosages related to synchrotron radiation (SR) procedures. We used young rats and macaques to address bone marrow and skin tolerance to various doses of synchrotron radiation.Rats were subjected to 0, 0.5, 2.5, 5, 25 or 100 Gy local SR X-ray irradiation at left hind limb. Rat blood samples were analyzed at 2-90 days after irradiation. The SR X-ray irradiated skin and tibia were sectioned for morphological examination. For non-human primate study, three male macaques were subjected to 0.5 or 2.5 Gy SR X-ray on crus. Skin responses of macaques were observed.All rats that received SR X-ray irradiation doses greater than 2.5 Gy experienced hair loss and bone-growth inhibition, which were accompanied by decreased number of follicles, thickened epidermal layer, and decreased density of bone marrow cells (p 0.05). Macaque skin could tolerate 0.5 Gy SR X-ray but showed significant hair loss when the dose was raised above 2.5 Gy.The safety threshold doses of SR X-ray for rat skin, bone marrow and macaque skin are between 0.5 and 2.5 Gy. Our study provided essential information regarding the biosafety of SR X-ray irradiation.

Introduction L‐glutamine is an antioxidant that plays a role in a variety of biochemical processes. Given that oxidative stress is a key component of stroke pathology, the potential of L‐glutamine in the treatment of ischemic stroke is worth exploring. Aims In this study, we investigated the effect and mechanisms of action of L‐glutamine after cerebral ischemic injury. Results L‐glutamine reduced brain infarct volume and promoted neurobehavioral recovery in mice. L‐glutamine administration increased the expression of heat‐shock protein 70 (HSP70) in astrocytes and endothelial cells. Such effects were abolished by the coadministration of Apoptozole, an inhibitor of the ATPase activity of HSP70. L‐glutamine also reduced oxidative stress and neuronal apoptosis, and increased the level of superoxide dismutase, glutathione, and brain‐derived neurotrophic factor. Cotreatment with Apoptozole abolished these effects. Cell culture study further revealed that the conditioned medium from astrocytes cultured with L‐glutamine reduced the apoptosis of neurons after oxygen‐glucose deprivation. Conclusion L‐glutamine attenuated ischemic brain injury and promoted functional recovery via HSP70, suggesting its potential in ischemic stroke therapy.

Objective Priapism refers to a condition with persistent abnormal erection of the penis, which is usually caused by disease or injury in the brain or spinal cord, or obstruction to the outflow of blood through the dorsal vein at the root of the penis, without sexual desires. The effect of cerebral ischaemia on sexual function is unknown. The aim of this study is to explore whether priapism occurs in adult mice. Furthermore, we examined the relationship between priapism and the region of infarct in the brain. Design Adult male CD-1 mice who underwent permanent middle cerebral artery occlusion (pMCAO) were closely examined from 2 hours to 14 days postoperation. Results We found that priapism occurs in ∼80% of the mice with pMCAO, which could persist up to 14 days. Further study has demonstrated that the occurrence of priapism is related to the infarct region: priapism is found only in mice with ischaemic injury extending to the hypothalamus and the hippocampus regions. Conclusion Our result suggested priapism may be used as a deep brain injury marker for evaluating brain injury in mice after pMCAO.

A rodent model of embolic middle cerebral artery occlusion is used to mimic cerebral embolism in clinical patients. Thrombolytic therapy is the effective treatment for this ischemic injury. However, it is difficult to detect thrombolysis dynamically in living animals. Synchrotron radiation angiography may provide a novel approach to directly monitor the thrombolytic process and assess collateral circulation after embolic stroke. Thirty-six adult Sprague-Dawley rats underwent the embolic stroke model procedure and were then treated with tissue plasminogen activator. The angiographic images were obtained in vivo by synchrotron radiation angiography. Synchrotron radiation angiography confirmed the successful establishment of occlusion and detected the thrombolysis process after the thrombolytic treatment. The time of thrombolytic recanalization was unstable during embolic stroke. The infarct volume increased as the recanalization time was delayed from 2 to 6 h (p

Dl-3-N-butylphthalide (NBP) is approved in China for the treatment of ischemic stroke. Previous studies have shown that NBP promotes recovery after stroke via multiple mechanisms. However, the effect of NBP on vascular function and thrombosis remains unclear. Here, we aim to study the effect of NBP on vascular function using a rat model of transient middle cerebral artery occlusion (MCAO) and a state-of-the-art high-resolution synchrotron radiation angiography. Eighty SD rats underwent MCAO surgery. NBP (90 mg/kg) was administrated daily by gavage. Synchrotron radiation angiography was used to evaluate the cerebral vascular perfusion, vasoconstriction, and vasodilation in real-time. Neurological scores, brain infarction and atrophy were evaluated. Real-time PCR was used to assess the expression levels of thrombosis and vasoconstriction-related genes. Results revealed that NBP attenuated thrombosis after MCAO and reduced brain infarct and atrophy volume. NBP administrated at 1 and 4 h after MCAO prevented the vasoconstriction of the artery and maintained its diameter at normal level. Administrated at one week after surgery, NBP functioned as a vasodilator in rats after MCAO while displayed no vasodilating effect in sham group. Our results suggested that NBP attenuates brain injury via increasing the regional blood flow by reducing thrombosis and vasoconstriction.

MicroRNA-29b (miR-29b) is involved in regulating ischemia process, but the molecular mechanism is unclear. In this work, we explored the function of miR-29b in cerebral ischemia. The level of miR-29b in white blood cells was evaluated in patients and mice after ischemic stroke. Brain infarct volume and National Institute of Health stroke scale (NIHSS) scores were analyzed to determine the relationship between miR-29b expression and the severity of stroke. The relationship of miR-29b and aquaporin-4 (AQP4) was further studied in mice. We found that miR-29b was significantly downregulated in stroke patients ( P < 0.05). MiR-29b level negatively associated with NIHSS scores ( r = −0.349, P < 0.01) and brain infarct volume ( r = −0.321, P < 0.05). In ischemic mice, miR-29b in the brain and blood were both downregulated ( r =0.723, P < 0.05). MiR-29b overexpression reduced infarct volume (49.50 ±6.55 versus 35.48 ±2.28 mm3, P < 0.05), edema (164±4% versus 108±4%, P < 0.05), and blood-brain barrier (BBB) disruption compared with controls (15 ±9% versus 7 ±3%, P < 0.05). Aquaporin-4 expression greatly decreased after miR-29b overexpression (28±7% versus 11 ±3%, P < 0.05). Dual-luciferase reporter system showed that AQP-4 was the direct target of miR-29b ( P < 0.05). We concluded that miR-29b could potentially predict stroke outcomes as a novel circulating biomarker, and miR-29b overexpression reduced BBB disruption after ischemic stroke via downregulating AQP-4.

Matrix metalloproteinase-9 (MMP-9) plays a beneficial role in the sub-acute phase after ischemic stroke. However, unrestrained MMP-9 may disrupt the blood-brain barrier (BBB), which has limited its use for the treatment of brain ischemia. In the present study, we constructed lentivirus mediated hypoxia-controlled MMP-9 expression and explored its role after stroke. Hypoxia response element (HRE) was used to confine MMP-9 expression only to the hypoxic region of mouse brain after 120-min transient middle cerebral artery occlusion. Lentiviruses were injected into the peri-infarct area on day 7 after transient ischemia. We found hyperexpression of exogenous HRE-MMP-9 under the control of hypoxia, and its expression was mainly located in neurons and astrocytes without aggravation of BBB damage compared to the CMV group. Furthermore, mice in the HRE-MMP-9 group showed the best behavioral recovery compared with the normal saline, GFP, and SB-3CT groups. Therefore, hypoxia-controlled MMP-9 hyperexpression during the sub-acute phase of ischemia may provide a novel promising approach of gene therapy for stroke.

The lenticulostriate artery plays a vital role in the onset and development of cerebral ischemia. However, current imaging techniques cannot assess the in vivo functioning of small arteries such as the lenticulostriate artery in the brain of rats. Here, we report a novel method to achieve a high resolution multi-functional imaging of the cerebrovascular system using synchrotron radiation angiography, which is based on spatio-temporal analysis of contrast density in the arterial cross section. This method provides a unique tool for studying the sub-cortical vascular elasticity after cerebral ischemia in rats. Using this technique, we demonstrated that the vascular elasticity of the lenticulostriate artery decreased from day 1 to day 7 after transient middle cerebral artery occlusion in rats and recovered from day 7 to day 28 compared to the controls (p < 0.001), which paralleled with brain edema formation and inversely correlated with blood flow velocity (p < 0.05). Our results demonstrated that the change of vascular elasticity was related to the levels of brain edema and the velocity of focal blood flow, suggesting that reducing brain edema is important for the improvement of the function of the lenticulostriate artery in the ischemic brain.

Background and Purpose— Striatal GABAergic neuron is known as a key regulator in adult neurogenesis. However, the specific role of striatal GABAergic neuronal activity in the promotion of neurological recovery after ischemic stroke remains unknown. Here, we used optogenetic approach to investigate these effects and mechanism. Methods— Laser stimulation was delivered via an implanted optical fiber to inhibit or activate the striatal GABAergic neurons in Gad2-Arch-GFP or Gad2-ChR2-tdTomato mice (n=80) 1 week after 60-minute transient middle cerebral artery occlusion. Neurological severity score, brain atrophy volume, microvessel density, and cell morphological changes were examined using immunohistochemistry. Gene expression and protein levels of related growth factors were further examined using real-time polymerase chain reaction and Western blotting. Results— Inhibiting striatal GABAergic neuronal activity improved functional recovery, reduced brain atrophy volume, and prohibited cell death compared with the control ( P P P Conclusions— After ischemic stroke, optogenetic inhibition of GABAergic neurons upregulated bFGF expression by endothelial cells and promoted neurobehavioral recovery, possibly orchestrated by astrocytes. Optogenetically inhibiting neuronal activity provides a novel approach to promote neurological recovery.

Rationale: Cerebral ischemia upregulates aquaporin-4 expression, increases blood-brain barrier (BBB) permeability, and induces brain edema. Mesenchymal stem cells (MSCs) can repress inflammatory cytokines and show great potential for ischemic stroke therapy. However, the effect of MSCs regarding the protection of ischemia-induced BBB break down is unknown. Objective: We test whether MSCs therapy protects BBB integrity and explore the molecular mechanisms of aquaporin-4 on BBB integrity. Methods and Results: Two hundred and twenty-eight adult CD1 male mice underwent 90 minutes transient middle cerebral artery occlusion and received 2 × 105 MSCs intracranial transplantation. The neurological severity score was improved and both ischemia-induced brain edema and BBB leakage were reduced in MSC-treated mice. MSCs therapy reduced astrocyte apoptosis and inhibited ischemia-induced aquaporin-4 upregulation. In addition, small-interfering RNA knockdown of aquaporin-4 after cerebral ischemia effectively reduced aquaporin-4 expression, brain edema, BBB leakage, and astrocyte apoptosis. Conditional medium from lipopolysaccharide (LPS)-activated microglia enhanced aquaporin-4 expression, p38 and JNK phosphorylation, and apoptosis of cultured astrocytes. MSC treatment reduced the expression of inflammatory cytokines in LPS-activated microglia, and subsequently reduced aquaporin-4 expression and apoptosis of astrocytes. Knockdown of aquaporin-4 in cultured astrocytes also reduced apoptosis. Treatment with p38 and JNK inhibitors showed that p38, but not the JNK signaling pathway, was responsible for the aquaporin-4 upregulation. Conclusion: MSCs protected BBB integrity by reducing the apoptosis of astrocytes after ischemic attack, which was due to the attenuation of inflammatory response and downregulation of aquaporin-4 expression via p38 signaling pathway. Stem Cells 2014;32:3150–3162

Current studies demonstrated that metformin is not only a hypoglycemic drug, but also a neuro-protective agent. However, the effect of metformin during ischemic brain injury is unclear. The aim of the present study is to explore the effect of metformin during ischemic brain injury. Adult male CD1 mice underwent 90min transient middle cerebral artery occlusion. Metformin (200mg/kg) was given at the time of reperfusion daily until sacrifice. Results showed that metformin treatment significantly reduced ischemia-induced brain atrophy volume compared to the control (p

Objective:To study the interactive influence of implanted nano platinum black electrodes (as compared with pure platinum electrodes) on rabbit orbicularis oculi muscle morphology and function.Methods:The influence of the two types of electrode on the orbicularis oculi muscle was monitored in a rabbit model of facial paralysis. Biological electric current and exciting current were administered to biological tissue, and morphological and functional changes were identified. Changes in orbicularis oculi muscle contraction, electrode configuration and performance associated with long-term electrical stimulation were observed over 28 days of implantation.Results:The nano platinum black electrode was superior to the pure platinum electrode in the following aspects: morphology and functionality, electrical excitation function of the orbicularis oculi muscle (as assessed by electromyography), muscle contraction function and biological tissue changes. Furthermore, the nano platinum black electrode features had good stability.Conclusion:Microelectrode surface modification with nano platinum black can effectively increase the microelectrode surface area and improve electrode performance, and is associated with good tissue compatibility.

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-1α) in neurovascular recovery during the postacute phase and downstream signaling pathways, underlying SDF-1α–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-1α 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-1α were investigated. Inflammatory response was monitored. Results— Neurobehavioral outcomes were improved, and brain atrophy was greatly reduced for ≤5 weeks in AAV-SDF-1α 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-1α groups. Administration of CXCR4 antagonist AMD3100 eliminated the beneficial effects of SDF-1α. SDF-1α/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-1α promoted neurogenesis and angiogenesis during the postacute phase of ischemia without eliciting an inflammatory response. AAV-SDF-1α expression represents a promising avenue for ischemic stroke therapy with a wider treatment window.

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 ( P0.05) 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

Intravenous transplantation of endothelial progenitor cells (EPCs) reduced ischemic brain injury. However, less cell homing to damaged sites limited its functions. In present study, we labeled EPCs with silica-coated superparamagnetic iron oxide nanoparticles (SiO4@SPIONs) and applied exterior magnetic field to guide SiO4@SPIONs-labeled EPCs (SiO4@SPIONs-EPCs) to the ischemic hemisphere of the brain. We optimized SiO4@SPIONs labeling dose, which did not affect proliferation, migration and tube formation of EPCs in vitro. SiO4@SPIONs-EPCs homing was greatly increased in ischemic hemisphere with magnetic field treatment in mice underwent transient middle cerebral artery occlusion (tMCAO). Injection of SiO4@SPIONs-EPCs and followed by magnetic field treatment showed improved neurobehavioral outcomes, reduced brain atrophic volume, increased microvessel density and VEGF expression in the ischemic perifocal region compared to groups without magnetic field treatment (p < 0.05). Our results demonstrated that exterior magnetic field could guide SiO4@SPIONs-EPCs to ischemic region and enhance therapeutic effect, suggesting that magnetic-guided SiO4@SPIONs-EPCs delivery is a promising approach in cerebral ischemic therapy.

Necroptosis was reported as one backup way of programmed cell death when apoptosis was blocked, and the receptor interacting protein 1 was considered as the key necroptosis regulator protein. Here, we report the neuroprotective effects of curcumin which attenuates necroptosis. Primary cortical neurons were cultured and were injured by ferrous chloride, z.vad.fmk was applied to block apoptosis, curcumin was administrated to protect neurons, necrostatin-1 was applied to inhibit necroptosis if needed. Cell viability was measured by detecting lactate dehydrogenase activity in lysates of surviving cells, and assessed by cell counting kit-8. The expression of receptor interacting protein 1 was detected by immunoblot and immunofluorescence. Results showed that necroptosis mainly occurred in the concentrations of ferrous chloride ranging from 100 to 200μM, curcumin attenuated necroptosis in a dose-dependent manner. Furthermore, curcumin decreased expression of receptor interacting protein 1 in a dose- and time-dependent manner. Taken together, these findings suggest that curcumin protects against iron induced neurotoxicity in primary cortical neurons by attenuating necroptosis.

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 P P 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.

Detection of microvascular changes in experimental stroke models is limited by current technologies. Using state-of-the-art synchrotron radiation (SR), we explored the feasibility of detecting the normal morphological variations of lenticulostriate arteries (LSAs) and the changes to LSAs following middle cerebral artery occlusion (MCAO). Cerebral microvessels of ICR mice were imaged with synchrotron radiation microangiography using nonionic iodine and barium sulfate as contrast agents. Using SR we reproducibly observed the detailed cerebral microvasculature of LSAs arising from the origin of middle cerebral artery (MCA) with a resolution of approximately 5 micrometers, at least a 20-fold greater resolution compared to CT or MRI imaging. Notably, SR microangiography was able to reveal ischemia/reperfusion induced leakage in the lenticulostriate artery territory. To our knowledge this is the first time that the three-dimensional morphology of LSAs and real time visualization of LSA hemorrhage have been characterized in live mice. This work demonstrates that SR microangiography can provide a unique tool for furthering experimental stroke research to examine the efficacy of neuroprotective therapies on parameters such as angiogenesis and vascular integrity.

SummaryAims MicroRNAs play an important role in the pathogenesis of ischemic brain injury and in the repair process during postischemic condition. However, the key miRNAs and their function in these processes remain unclear. Methods Circulating blood MicroRNAs profiles were examined in the ischemic stroke patients. The predicted network of difference was analyzed by ingenuity pathway analysis. The key MicroRNAs were selected, and the function was further studied in a mouse ischemia model. The predicted downstream target was confirmed. Results We found that 24 MicroRNAs were differently expressed in stroke patients compared to the control (P < 0.05). Bioinformatic analysis showed a MicroRNAs regulated network with the highest score in the stroke cascade, which was consisted of 10 MicroRNAs including key hypoxia-related miR-210 and its predicted downstream target brain derived neurotrophic factor (BDNF). Lentivirus-mediated miR-210 overexpression enhanced the microvessel density and the number of neural progenitor cells in the ischemic mouse brain (P < 0.05) and improved neurobehavioral outcomes in the ischemic mouse (P < 0.05). MiR-210 upregulation increased mBDNF/proBDNF protein expression in the normal and ischemic mouse brain. The dual-luciferase reporter assay identified that BDNF was the direct target of miR-210. Conclusion MiR-210 is a crucial ischemic stroke-associated MicroRNAs and a potential target for the stroke therapy.

Striatal neurons regulate the activity of neural progenitor cells in the subventricular zone, but the effect of striatal neuronal activity on neurogenesis after ischemic stroke is unclear. In this study, we used optogenetic tools to investigate the impact of striatal neuronal activity on the neurogenesis and functional recovery after cerebral ischemia. We transfected striatal neurons with channelrhodopsin-2 or halorhodopsin from Natronomonas so that they can be excited by 473 nm laser or inhibited by 594 nm laser, respectively. Neural inhibition but not excitation at 4–7 days after middle cerebral artery occlusion resulted in reduced atrophy volume (6.8 ± 0.7 vs 8.5 ± 1.2 mm3, p +, BrdU+/doublecortin+ and BrdU+/NeuN+ cells ( p

Summary Objectives Dynamically observe cerebral vascular changes in hyperglycemic rats in vivo and explore the effect of diabetes on endothelial function after ischemic stroke. Background Diabetes affects both large and small vessels in the brain, but the dynamic process and mechanism are unclear. Methods We investigated the structural and functional changes of brain vasculature in living hyperglycemic rats and their impact on stroke outcomes via a novel technique: synchrotron radiation angiography. We also examined the effect of prolonged fasudil treatment on arterial reactivity and hemorrhagic transformation. Adult Sprague Dawley rats were treated by streptozotocin to induce type 1 diabetes. These hyperglycemic rats received fasudil pretreatment and then underwent transient middle cerebral artery occlusion. Results We found that diabetes caused arteries narrowing in the circus Willis as early as 2 weeks after streptozotocin injection (P

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 model's 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.

Chemokine CXC ligand 12 (CXCL12), originally named stromal cell-derived factor-1 (SDF-1), is a member of the CXC chemokine subfamily. CXCL12 is found to be expressed by all cell types that are presented in the central nervous system (CNS). It works in conjunction with the G-protein coupled receptor CXCR4, which is found at the surface of a variety of cells including neurons, astrocytes, microglia, bone marrow-derived cells, as well as other progenitor cells. Recent studies revealed that CXCL12 could also bind and signal through receptor CXCR7. CXCL12 and CXCR4 are constitutively expressed in the brain but are up-regulated in the ischemic penumbra regions following ischemic stroke. CXCL12/CXCR4 play important roles in multiple processes after ischemic stroke, which include inflammatory response, focal angiogenesis, and the recruitment of bone marrow-derived cells (BMCs) and neural progenitor cell (NPC) to injury. In addition to its roles in stroke pathology, CXCL12 is also thought to be a key regulator in stroke repairing. This review will focus on the function of CXCL12/CXCR4 in post-stroke inflammation and neurovascular repairing. The potential application of CXCL12 modulation in clinical stroke treatment is also discussed.

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.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.

Previous studies revealed that curcumin is neuroprotective in diseases of the central nervous system such as cerebral ischemia and traumatic brain injury. However, the effect of curcumin on intracerebral hemorrhage remains unclear. We, therefore, investigated the pre-clinical effect of curcumin treatment on neurological outcomes following intracerebral hemorrhage, using a mouse model. Intracerebral hemorrhage was induced by autologous blood injection into the right basal ganglia. Curcumin (150 mg/kg) was administered 15 min after intracerebral hemorrhage. Grid walk and neurological scores were evaluated at 1, 3, 7, and 14 days post-injury. Mice were killed at 24 h or 28 days following injury, for histological examination. Evans Blue and water content in the ipsilateral and contralateral hemispheres were measured to evaluate the extent of blood–brain barrier disruption and brain edema. Zonula occludens-1 was detected by immunostaining. In situ zymography was used to measure the localization and focal enzymatic activity of matrix metalloproteinase. Our results demonstrated that curcumin reduced brain edema, measured by alleviated water content and Evans Blue leakage at 24 h (p

The purpose of the study was to evaluate the feasibility of microbubbles as phase contrast imaging (PCI) agents for angiography applications. The hypothesis was that the introduction of microbubbles into tissue produces a significant change in the refractive index and highlights the lumen of the vessel in PCI. The absorption and phase contrast images of commercially available microbubbles were obtained and compared in vitro. A further increase in contrast was observed in PCI. Microbubbles highlighted the lumen of the renal microvessels, acting as a positive contrast medium in ex vivo imaging. In addition, home-made microbubbles with larger diameters were introduced for image contrast enhancement in living tumor-bearing mice, demonstrating the feasibility of microbubble-based x-ray phase-contrast imaging for tumor vasculature in vivo.

Netrin-1 (NT-1) is one of the axon-guiding molecules that are critical for neuronal development. Because of its structural homology to the endothelial mitogens, NT-1 may have similar effects on vascular network formation. NT-1 was shown to be able to stimulate the proliferation and migration of human cerebral endothelial cells in vitro and also promote focal neovascularization in adult brain in vivo. In the present study, we reported the delivery of NT-1 using an adeno-associated virus (AAV) vector (AAV-NT-1) into mouse brain followed by transient middle cerebral artery occlusion (tMCAO). We found that AAV vectors did not elicit a detectable inflammatory response, cell loss or neuronal damage after brain transduction. The level of NT-1 was increased in the AAV-NT-1-transduced tMCAO mice compared with the control mice. Furthermore, the neurobehavioral outcomes were significantly improved in AAV-NT-1-transduced mice compared with the control animals (P

Remyelination is an important repair process after ischemic stroke-induced white matter injury. It often fails because of the insufficient recruitment of oligodendrocyte progenitor cells (OPCs) to the demyelinated site or the inefficient differentiation of OPCs to oligodendrocytes. We investigated whether CXCL12 gene therapy promoted remyelination after middle cerebral artery occlusion in adult mice. The results showed that CXCL12 gene therapy at 1 week after ischemia could protect myelin sheath integrity in the perifocal region, increase the number of platelet-derived growth factor receptor-α (PDGFRα)-positive and PDGFRα/bromodeoxyuridine-double positive OPCs in the subventricular zone, and further enhance their migration to the ischemic lesion area. Coadministration of AMD3100, the antagonist for CXCL12 receptor CXCR4, eliminated the beneficial effect of CXCL12 on myelin sheath integrity and negatively influenced OPC proliferation and migration. At 5 weeks after ischemia, CXCR4 was found on the PDGFRα- and/or neuron/glia type 2 (NG2)-positive OPCs but not on the myelin basic protein-positive mature myelin sheaths, and CXCR7 was only expressed on the mature myelin sheath in the ischemic mouse brain. Our data indicated that CXCL12 gene therapy effectively protected white matter and promoted its repair after ischemic injury. The treatment at 1 week after ischemia is effective, suggesting that this strategy has a longer therapeutic time window than the treatments currently available. Significance This study has demonstrated for the first time that CXCL12 gene therapy significantly ameliorates brain ischemia-induced white matter injury and promotes oligodendrocyte progenitor cell proliferation in the subventricular zone and migration to the perifocal area in the ischemic mouse brain. Additional data showed that CXCR4 receptor plays an important role during the proliferation and migration of oligodendrocyte progenitor cells, and CXCR7 might play a role during maturation. In contrast to many experimental studies that provide treatment before ischemic insult, CXCL12 gene therapy was performed 1 week after brain ischemia, which significantly prolonged the therapeutic time window of brain ischemia.

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.

The rat suture middle cerebral artery occlusion (MCAO) is a frequently used animal model for investigating the mechanisms of ischemic brain injury. During suture MCAO, transection of the external carotid artery (ECA) potentially restrains blood flow and impairs masticatory muscle and other ECA-supported territories, consequently influencing post-operation animal survival. This study was aimed at investigating the effect of ECA transection on the hemodynamic alterations using a novel synchrotron radiation (SR) angiography technique and magnetic resonance imaging in live animals. Fifteen male adult Sprague-Dawley rats were used in this study. Animals underwent MCAO, in which the ECA was transected. SR angiography was performed before and after MCAO. Rats then underwent magnetic resonance imaging (MRI) to detect the tissue lesion both intra- and extra-cranially. Animals with SR angiography without other manipulations were used as control. High-resolution cerebrovascular morphology was analyzed using a novel technique of SR angiography. The masticatory muscle lesion was further examined by hematoxylin and eosin staining. MRI and histological results showed that there was no masticatory muscle lesion at 1, 7 and 28 days following MCAO with ECA transection. In normal condition, the ECA and its branch external maxillary artery were clearly detected. Following ECA transection, the external maxillary artery was still observed and the blood supply appeared from the anastomotic branch from the pterygopalatine artery. SR angiography further revealed the inter-relationship of hemisphere extra- and intra-cranial vasculature in the rat following MCAO. Transection of the ECA did not impair masticatory muscles in rat suture MCAO. Interrupted blood flow could be compensated by the collateral circulation from the pterygopalatine artery.

Angiogenesis, an important process for long term neurological recovery, could be induced by ischemic brain injury. In this chapter, we describe a system to deliver adeno-associated viral (AAV) vector-mediated gene therapy for ischemic stroke. This includes the methods to construct, produce, and purify an AAV vector expressing target gene and an approach to quantify the number of microvessels and capillary density with synchrotron radiation angiography (SRA) imaging.

Background The relationship between circulating microRNA-223 and pathogenesis of acute ischemic stroke is unknown. Here we investigated the roles and possible targets of circulating microRNA-223 in human ischemic stroke within the first 72 hours. Methods Blood samples were collected from patients within 72 hours after cerebral ischemia (n = 79) and compared with healthy control samples (n = 75). The level of possible downstream factors of microRNA-223 including insulin-like growth factor-1, insulin-like growth factor-1 receptor and interleukin-6 was examined by ELISA assay. The relationship between the microRNA-223 level and NIHSS scores, TOAST subtypes, and infarct volume was analyzed respectively. In addition, twelve adult male CD-1 mice underwent middle cerebral artery occlusion using the suture technique. Circulating blood and brain tissue in the ischemic ipsilateral hemisphere were collected at 24 hours after middle cerebral artery occlusion. microRNA-223 was detected by real-time polymerase chain reactions. Results microRNA-223 levels in the circulating blood of acute ischemic stroke patients were greatly increased compared to the control (p