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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

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.

Diabetes is usually associated with cerebrovascular disease, especially stroke. In practice, fasudil is widely accepted to be applied for the treatment of vascular disease. This article demonstrates the study concentrating on the effects of fasudil pretreatment on the prognosis of diabetic stroke. 250–300 g Sprague-Dawley rats were randomly divided into three groups, non-diabetic stroke group, diabetic stroke group, and fasudil pretreatment group. The rats of diabetes group were treated with intraperitoneal injection of streptozotocin (60 mg/kg), in the meantime the same dose of citrate buffer was injected into those of the control group. The rats of the fasudil group received daily fasudil intraperitoneal injection at 10 mg/kg for three consecutive weeks. After four weeks, all the rats of the experimental group were treated with middle cerebral artery occlusion for 90 min. After sacrifice, the fresh brain samples were collected for following experiments, including infarct volume, edema volume, blood-brain barrier (BBB), which were detected by immunohistochemistry. Inflammatory factors were examined by real-time polymerase chain reaction (RT-PCR) using tissue Ribonucleic Acid (RNA). The concentration of blood glucose is 15 mmol/L or more, which proved that the diabetes model was a success. Fasudil pretreatment decreases the percentage of stroke mortality of diabetes from 43.75% to 31.25%, while the infarction volume decreases from 52.95% ± 12.7% to 45.97% ± 6.7%. Gap formation of tight junction and Immunoglobulin G (IgG) leakage were reduced (P < 0.05), and the expression of inflammatory factors decreases (P < 0.05) in fasudil pretreatment after diabetic stroke. Diabetes aggravates the mortality of cerebral ischemic rats. Prolonged fasudil pretreatment can reduce mortality of diabetic stroke, decrease cerebral infarction volume and undermine inflammatory factors expression, and protect the BBB.

Studies demonstrate that microRNA-126 plays a critical role in promoting angiogenesis. However, its effects on angiogenesis following ischemic stroke are unclear. Here, we explored the effect of microRNA-126-3p and microRNA-126-5p on angiogenesis and neurogenesis after brain ischemia. We demonstrated that both microRNA (miRNA)-126-3p and microRNA-126-5p increased the proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) compared with the scrambled miRNA control (p < 0.05). Transferring microRNA-126 into a mouse middle cerebral artery occlusion model via lentivirus, we found that microRNA-126 overexpression increased the number of CD31+/BrdU+ (5-bromo-2′-deoxyuridine-positive) proliferating endothelial cells and DCX+/BrdU+ neuroblasts in the ischemic mouse brain, improved neurobehavioral outcomes (p < 0.05), and reduced brain atrophy volume (p < 0.05) compared with control mice. Western blot results showed that AKT and ERK signaling pathways were activated in the lentiviral-microRNA-126-treated group (p < 0.05). Both PCR and western blot results demonstrated that tyrosine-protein phosphatase non-receptor type 9 (PTPN9) was decreased in the lentiviral-microRNA-126-treated group (p < 0.05). Dual-luciferase gene reporter assay also showed that PTPN9 was the direct target of microRNA-126-3p and microRNA-126-5p in the ischemic brain. We demonstrated that microRNA-126-3p and microRNA-126-5p promoted angiogenesis and neurogenesis in ischemic mouse brain, and further improved neurobehavioral outcomes. Our mechanistic study further showed that microRNA-126 mediated angiogenesis through directly inhibiting its target PTPN9 and activating AKT and ERK signaling pathways. Keywords: angiogenesis, ischemic stroke, microRNA, neurogenesis, PTPN9

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

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 contributed to their function in regulating microglial polarization by targeting PTEN and STAT1.

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.

Objective: DL-3n-butylphthalide (NBP) has beneficial effects in different stages of ischemic stroke. Our previous studies have demonstrated that NBP promoted angiogenesis in the perifocal region of the ischemic brain. However, the molecular mechanism of NBP for blood–brain barrier protection in acute ischemic stroke was unclear. Here, we explored the neuroprotective effects of NBP on blood–brain barrier integrity in the acute phase of ischemic stroke in a rat model.Methods: Adult male Sprague–Dawley rats (n = 82) underwent 2 h of transient middle cerebral artery occlusion and received 90 mg/kg of NBP for 3 days. Brain edema, infarct volume, surface blood flow, and neurological severity score were evaluated. Blood–brain barrier integrity was evaluated by Evans blue leakage and changes in tight junction proteins. We further examined AQP4 and eNOS expression, MMP-9 enzyme activity, and possible signaling pathways for the role of NBP after ischemic stroke.Results: NBP treatment significantly increased eNOS expression and surface blood flow in the brain, reduced brain edema and infarct volume, and improved neurological severity score compared to the control group (p < 0.05). Furthermore, NBP attenuated Evans blue and IgG leakage and increased tight junction protein expression compared to the control after 1 and 3 days of ischemic stroke (p < 0.05). Finally, NBP decreased AQP4 expression, MMP-9 enzyme activity, and increased MAPK expression during acute ischemic stroke.Conclusion: NBP protected blood–brain barrier integrity and attenuated brain injury in the acute phase of ischemic stroke by decreasing AQP4 expression and MMP-9 enzyme activity. The MAPK signaling pathway may be associated in this process.

Multisensory stimulation plays an important role in the recovery of ischemic stroke. However, little is known about the interactions between neuronal activities with multi-afferent stimulations and their effects on hemodynamic responses. Optogenetics has been a useful tool in neuroscience research to unravel the mechanisms of neurovascular coupling at cell-specific level. In this study, we applied laser speckle contrast imaging (LSCI) to map the cortical hemodynamic response with high spatiotemporal resolution. The results showed that optogenetic inhibition of pyramidal neurons in sensorimotor cortex induced both local and distant increases of cerebral blood flow (CBF) with dual peaks, and the full width at half maximum (FWHM) was significantly larger than that of the CBF response to optogenetic excitation. Furthermore, optogenetic excitation of pyramidal neurons could significantly increase the local CBF response to sensory stimulation, whereas optogenetic inhibition of pyramidal neurons decreased the local CBF response at the early stage after sensory stimulation and increased the distant CBF response during the recovery period. Our work provided useful insights into the mechanisms of brain stimulation, which might help in clinical neurological applications.

Background and Purpose: Diabetes mellitus increases stroke incidence and mortality and hampers functional recovery after stroke. Fingolimod has been shown to improve neurofunctional recovery and reduce brain infarction after ischemic injury in mice without comorbidities. In this work, we investigated the effects of fingolimod in diabetic mice after transient middle cerebral artery occlusion (tMCAO). Methods: Hyperglycemia was induced by a single bolus streptozotocin injection. Adult male ICR mice (n = 86) underwent 1-h tMCAO surgery and received intraperitoneal injection of fingolimod (1 mg/kg) or vehicle immediately after reperfusion. Clark neurological score, brain infarction and edema, blood–brain barrier (BBB) integrity, apoptosis, and inflammation were evaluated at 24 h after tMCAO. Results: Fingolimod treatment reduced the number of infiltrated inflammatory cells and lowered the mRNA level of Tnfα. It also increased the ratio of Bcl-2/Bax. However, fingolimod significantly aggravated brain edema and reduced the expression levels of tight junction proteins ZO-1 and Occludin. The negative impacts of fingolimod on BBB integrity outweighed its beneficial effects in anti-inflammation, which resulted in the lack of improvement in endpoint outcomes at 24 h after tMCAO. Conclusion: Caution should be taken in considering the acute treatment using fingolimod for ischemic stroke with diabetes comorbidity.

The coupling between neuronal activity and cerebral blood flow (CBF), known as neurovascular coupling, has been reported to be impaired after stroke. This study aims to investigate the neurovascular coupling impairment at the acute stage after ischemic stroke. Laser speckle contrast imaging (LSCI) was applied to measure the hemodynamic response to optogenetic excitation of sensorimotor neurons in healthy and ischemic brain. The results showed that the hemodynamic response to optogenetic stimulation decreased and the regional CBF response was correlated with the distance from the ischemic core at the acute stage, regardless of the change in resting CBF. Our results also demonstrated that excitatory neuronal stimulation of intact area could promote the recovery of neurovascular coupling, whereas peri-infarct neuronal excitation failed to restore neurovascular function 24 hrs after ischemia. These results suggested the intact periphery of penumbra as the target for excitatory stimulation in aspect of restoring the perfusion after ischemic stroke.

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.

Objectives: Mesenchymal stem cell therapy has shown therapeutic potential for ischemic stroke. However, low cell viability and lack of multimodal imaging to track stem cell distribution limit its clinical translation. Here we developed a multifunctional probe to track stem cell viability and migration in a mouse model of focal brain ischemia. Methods: A theragnostic probe was developed by cobalt protoporphyrin IX (CoPP), with photoacoustic imaging detectability into 125 Iodine modified mesoporous silica nanoparticles ( 125 I-CoPP@MSN). The effect on cell viability and differentiation were examined in vitro . Adult male ICR mice (n=40) were subjected to 90 min transient middle cerebral artery occlusion. 125 I-CoPP@MSN labeled stem cells were transplanted into the peri-infarct region after 1 day of brain ischemia. Grafted cells was monitored by SPECT and photoacoustic imaging. The cell survival was evaluated by bioluminescence imaging. Results: 125 I-CoPP@MSNs have high efficiency for labeling cells without affecting their viability and differentiation. 125 I-CoPP@MSNs increased the viability of stem cells subjected to H 2 O 2 -induced oxidant stress in vitro , compared with controls ( p 125 I-CoPP@MSN labeling increased graft cell viability, reduced brain atrophy volume, and improved behavioral outcomes, compared to the controls ( p + and DCX + cells were increased in CoPP@MSN labeled group than that in controls (p Conclusion: We conclude that 125 I-CoPP@MSNs is a novel probe for the real-time tracking and enhancing its therapeutic efficacy in ischemic stroke therapy.

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.

Elucidating the normal structure and distribution of cerebral vascular system is fundamental for understanding its function. However, studies on visualization and whole-brain quantification of vasculature with cellular resolution are limited. Here, we explored the structure of vasculature at the whole-brain level using the newly developed CLARITY technique. Adult male C57BL/6J mice undergoing transient middle cerebral artery occlusion and Tie2-RFP transgenic mice were used. Whole mouse brains were extracted for CLARITY processing. Immunostaining was performed to label vessels. Customized MATLAB code was used for image processing and quantification. Three-dimensional images were visualized using the Vaa3D software. Our results showed that whole mouse brain became transparent using the CLARITY method. Three-dimensional imaging and visualization of vasculature were achieved at the whole-brain level with a 1-μm voxel resolution. The quantitative results showed that the fractional vascular volume was 0.018 ± 0.004 mm3 per mm3, the normalized vascular length was 0.44 ± 0.04 m per mm3, and the mean diameter of the microvessels was 4.25 ± 0.08 μm. Furthermore, a decrease in the fractional vascular volume and a decrease in the normalized vascular length were found in the penumbra of ischemic mice compared to controls (p < 0.05). In conclusion, CLARITY provides a novel approach for mapping vasculature in the whole mouse brain at cellular resolution. CLARITY-optimized algorithms facilitate the assessment of structural change in vasculature after brain injury.

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.

Neural stem cell (NSC) transplantation is a promising treatment to improve the recovery after brain ischemia. However, how the survival, proliferation, migration, and differentiation of implanted NSC are influenced by endogenous neuronal activity remains unclear. In this work, we used optogenetic techniques to control the activity of striatal neurons and investigated how their activity affected the survival and migration of transplanted NSCs and overall neurological outcome after ischemic stroke. NSCs cultured from transgenic mice expressing fluorescent protein were transplanted into the peri-infarct region of the striatum after transient middle cerebral artery occlusion (tMCAO) surgery. The striatal neurons were excited or inhibited for 15 minutes daily via implanted optical fiber after tMCAO. The results revealed that mice which received NSC transplantation and optogenetic inhibition had smaller brain infarct volume and increased NSC migration compared to the NSC alone or PBS group (p<0.05). In contrast, mice which received NSC transplantation and optogenetic excitation showed no difference in infarct volume and neurological behavior improvement compared to the PBS control group. In vitro experiments further revealed that the conditioned media from excited GABAergic neurons reduced NSC viability through paracrine mechanisms.Conclusion. Optogenetic inhibition of striatal neuronal activity further improved neurological recovery after NSC transplantation at the subacute phase after brain ischemia.

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.

Introduction: Oxidative stress plays an important role in ischemic stroke pathogenesis. L-glutamine (L-GLN) was shown to have antioxidant activity and was approved by the FDA in July 2017 for the treatment of sickle cell disease. In this study, we aim to investigate the effect of L-GLN in attenuating ischemic brain injury using a mouse middle cerebral artery occlusion (tMCAO) model. Methods: Ninety-minute tMCAO was used to induce a focal cerebral ischemic injury. Animals (n=135) were divided into four groups: (1) Sham, (2) Saline, (3) L-GLN, and (4) L-GLN plus HSP70 ATPase activity inhibitor Apoptozole (Az). Treatments were delivered intraperitoneally once daily for the first three days after ischemic surgery. Neurobehavioral tests were performed before tMCAO and at 1, 3, 7, and 14 days after tMCAO using the modified neurological severity score (mNSS), elevated body swing test (EBST), and rotarod test. Infarction volume was assessed by cresyl violet staining of brain sections. The expression levels of heat shock proteins and its signaling pathway related proteins were evaluated by Western Blot and real time-PCR. The level of oxidative stress was evaluated by malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) assays. Astrocyte and neuron cell cultures and oxygen glucose deprivation (OGD) model was used to further examine the molecular pathways involved in the protective effects by L-GLN. Results: L-GLN reduced brain infarct volume and promoted neurobehavioral recovery in mice suffering from ischemic injury. Such beneficial effect was abolished by the coadministration of AZ. L-GLN induced the up-regulation of HSP70, reduced oxidative stress level, and reduced the expression of IL-1beta and IL-6 in ischemic mice brain. In L-GLN group, SOD and GSH-PX activities are significantly increased compared to the Saline group. Nrf2 was upregulated in the L-GLN group while co-treatment with AZ abolished this upregulation. Cell culture experiments revealed that the conditioned media from astrocytes cultured with the presents of L-GLN attenuated the apoptosis of neurons after OGD. Conclusion: L-GLN attenuates ischemic brain injury in part through HSP70 related pathways and holds potential in post-stroke treatment.

State estimation suffers some new challenging problems when wireless sensor networks (WSNs) are used in mobile target tracking systems. An important problem among them is low observability, which makes it necessary to further study performance of tracking estimators from an observable degree point of view. This paper studies observable degree analysis (ODA) to formulate the estimator performance for a kind of wireless tracking filter. For a kind of time-varying Kalman estimator, an improved ODA method is proposed by using weighted least square, Cauchy Schwarz inequality and normalization processing. The main contributions are that the relation is firstly analytically established between the observable degree and the estimation performance, and finally the observable degree is normalized in [0,1]. Meanwhile, observable degrees are both given for local state components and the global state. Thereby, we have the conclusion that higher observable degree corresponds to better estimation performance of the Kalman filter. Accordingly, it is potentially hopeful to achieve an effective function to study estimation performance of tracking estimators by directly using the observable degree. Simulation is provided to verify the results on real-time and generality, completeness and consistency/matching of the proposed observable degree analysis.

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.

For multi-sensor tracking system, track initialization is a problem due to crossed data association between different frames and different sources. In this paper, a RANSAC-based track initialization algorithm for multi-sensor system is proposed. Considering the fact that target motion is contiguous in time-space domain, measurements of the target are homologous to the target motion model. Therefore, the proposed algorithm uses homology to associate all measurements regardless of frames and sources. RANSAC technology is used to recursively learn the target motion model, associating continuous measurements from all sensors. It is better to avoid missing measurement effects in some frames or in some sensors through a homology association mechanism. Compared with the traditional track initialization algorithm, the proposed algorithm maintains good robustness in noise and clutter environment, which is confirmed by simulation results in this paper.

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

Brain edema primarily occurs as a consequence of various cerebral injuries including ischemic stroke. Excessive accumulation of brain water content causes a gradual expansion of brain parenchyma, decreased blood flow and increased intracranial pressure and, ultimately, cerebral herniation and death. Current clinical treatment for ischemic edema is very limited, therefore, it is urgent to develop novel treatment strategies. Mounting evidence has demonstrated that AQP4, a water channel protein, is closely correlated with brain edema and could be an optimal therapeutic target for the reduction of ischemic brain edema. AQP4 is prevalently distributed in the central nervous system, and mainly regulates water flux in brain cells under normal and pathological conditions. This review focuses on the underlying mechanisms of AQP4 related to its dual role in edema formation and elimination.

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.

Patients with diabetes suffer the higher risk of dementia and the underlying pathological mechanism of cognitive dysfunction in diabetes is not fully understood. In this study, we explore whether the cognitive impairment in the diabetic rat is associated with increased blood brain barrier (BBB) permeability and the change of the inflammatory cytokine. Experimental diabetic rats were induced by single intraperitoneal injection of streptozotocin (STZ). Cognitive function was evaluated by Morris water maze in the normal and the diabetic rats, respectively. The spatial acquisition trials were conducted over five consecutive days and the probe test was performed on day 6, followed by working memory test on the next 4 days. Escape latency was recorded in the acquisition trials and working memory test; time spent in the target quadrant and the number of crossing the former platform were recorded in the probe test. BBB permeability was assessed by measuring the extravasation of IgG. The image of occludin and claudin-5 staining by a confocal microscope were acquired to measure the gap in the tight junction. Cytokines TNF-α, IL-1β and IL-6 mRNA expression were further examined by Real-time PCR. The time spent in the target quadrant within 30 s decreased in the 8-week STZ rats compared to that of the normal rats (p < 0.05), while no difference was seen in the performance of working memory between the diabetic and normal rats. IgG leakage significantly increased in the brain parenchyma of the 8-week STZ rats compared to the normal rats (p < 0.05). The immunostaining of occludin and claudin-5 suggested the gap in the tight junction increased in the 8-week STZ rats compared to the normal rats (p < 0.05). Moreover, TNF-α and IL-6 mRNA also increased in the brain of 8-week STZ rats compared to the normal rats (p < 0.05). These results suggested that loss of BBB integrity might contribute to progressive impairment of cognitive in the diabetic rats. The increase of TNF-α and IL-6 expression might trigger the disruption of BBB in the brain, which eventually caused cognitive impairment in the 8-week STZ rats.

The main idea of the recent proposed clustering by fast search and find of density peaks (CFSFDP) clustering algorithm depicts the cluster center with the local density and distance and it achieves significant effects in typical applications. But it can't select the cluster centers adaptively. Therefore, an improved CFSFDP algorithm is proposed in this paper, which determines the cluster centers by the Max-min algorithm. First, the Max-min algorithm is introduced to obtain the number of categories. Then the local density and distance information is used to determine the cluster centers as do in CFSFDP algorithm. It can not only adaptively obtain categories number of the data, but also obtain the corresponding clustering centers. The simulation results show that the proposed algorithm can find the number of categories and find the cluster centers. Meanwhile, it can find the cluster centers which are hard to be obtained through decision diagram.

Introduction: Diabetes is a risk factor for stroke. Fingolimod is a small molecule drug approved by FDA in 2010 for the treatment of multiple sclerosis. It has been shown that fingolimod reduced inflammatory infiltration and brain edema, as well as exhibited neuroprotective effects after ischemic stroke. In this work, we investigated the effects of fingolimod on diabetic stroke mice in the acute and post-acute stages. Methods: Diabetes was induced by streptozotocin (150mg/kg) injection. For acute phase studies, adult male ICR mice underwent 1-hour transient middle cerebral artery occlusion (tMCAO) (n=55), and received intraperitoneal injection of fingolimod (1mg/kg) immediately after reperfusion. Death rate, neurological score, brain infarction, edema, and blood brain barrier permeability were evaluated at 24 hours after MCAO. The number of CD3+ T cells in peripheral blood was analyzed by flow cytometry. Cellular apoptosis, tight junction changes and inflammatory cell infiltration were measured by immunofluorescence staining. To investigate the long-term function of fingolimod, mice (n=25) were treated with fingolimod (1mg/kg) immediately after reperfusion after 1-hour tMCAO. Neurobehavioral outcomes, brain atrophy, neurogenesis, and angiogenesis were evaluated at 14 days after tMCAO. Results: At 24 hours after tMCAO, mice mortality was reduced by fingolimod treatment. Fingolimod reduced the number of TUNEL positive cells and inflammatory cell infiltration, as well as lowered the mRNA level of inflammatory cytokines including IL-6 and TNF-α. However, it caused aggravated edema which is accompanied by reduced ZO-1 and occludin level, and raised AQP4 expression. In the post-acute stage, fingolimod improved the functional recovery and reduced brain atrophy. The numbers of nestin and DCX/BrdU positive neural progenitor cells in the subventricular zone and CD31/BrdU positive angiogenic microvessels were increased in fingolimod treated group. Conclusion: Fingolimod reduced the mortality of diabetic stroke mice in the acute phase through neuroprotection and anti-inflammation, and promoted the functional recovery in post-acute phase by promoting neurogenesis and angiogenesis.

Phase-contrast imaging (PCI) on synchrotron radiation (SR) light source has become a paradigm in X-ray biomedical imaging. In vitro and in vivo biomedical research has attracted high attention thanks to its capability of providing high diagnostic significant images in a wide range of organs or viscera, including the breast, joints, cartilage, lung, eyes, vasculature, brain and central nervous system. In this chapter, the basic principle, main phase-contrast imaging methods and their bio-medical applications are introduced. Various imaging methods are described and evaluated. The prospect and limitations of synchrotron-based medical imaging are briefly discussed as well.

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.

Hypertension has a profound influence on the structure and function of blood vessels. Cerebral vessels undergo both structural and functional changes in hypertensive animals. However, dynamic changes of cerebrovasculature and the factors involved in this process are largely unknown. In this study, we explored the dynamic changes of vascular structure in hypertensive rats using novel synchrotron radiation angiography. Twenty-four spontaneously hypertensive rats (SHR) and 24 Sprague–Dawley (SD) rats underwent synchrotron radiation (SR) angiography. Each group had 8 animals. We studied the cerebral vascular changes in SHR over a time period of 3–12-month and performed quantitative analysis. No vascular morphology differences between SHR and SD rats were observed in the early stage of hypertension. The number of twisted blood vessels in the front brain significantly increased at the 9- and 12-month observation time-points in the SHR compared to the SD rats (p < 0.01). The vessel density of the cortex and the striatum in SHR was consistently higher than that in SD rats at time points of 3-, 9-, and 12-month (p < 0.001). Vascular elasticity decreased both in SHR and SD rats with aging. There were statistically significant differences in the relative vascular elasticity of extracranial/intracranial internal carotid artery, middle cerebral artery, posterior cerebral artery and anterior cerebral artery between SHR and SD rats at 12-month (p < 0.01). We concluded that the dynamic vascular alterations detected by SR angiography provided novel imaging data for the study of hypertension in vivo. The longer the course of hypertension was, the more obvious the vascular differences between the SHR and the SD rats became.

In order to assist the military operator to make accurate analysis of the situation in the modern war, which has the characteristics of large amount of information. Based on ArcGIS Engine, in this paper, a modular two-dimensional battlefield situation display system is designed and implemented. The function of each module in the system is discussed and analyzed, then some key technologies of the system are introduced. The demonstration results show the validation of the situation display system.

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.

Brain arteriovenous malformation (BAVM) is frequently described as vascular malformation. Although computer tomography (CT), magnetic resonance imaging (MRI) and angiography can clearly detect lesions, there are no diagnostic biological markers of BAVM available. Current study demonstrated that microRNA (miRNA) showed a feasible marker for vascular disease. To find key correlations between these miRNAs and the onset of BAVM, we carried out chip analysis of serum miRNAs by identifying 18 potential markers of BAVM. We then constructed a principle component analysis and logistic regression (PCA-LR) model to analyze the 18 miRNAs collected from 77 patients. Another 9 independent samples were used to test the resulting model. The results showed that miRNAs hsa-mir-126-3p and hsa-mir-140 are important protective factors, while hsa-mir-338 is a dominating risk factor, all of which have stronger correlation with BAVM than others. We also compared the testing results using PCA-LR model with those using LR model. The comparison revealed that PCA-LR model is better in predicting the disease.

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.

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.

Endogenous Netrin-1 (NT-1) protein was significantly increased after cerebral ischemia, which may participate in the repair after transient cerebral ischemic injury. In this work, we explored whether NT-1 can be steadily overexpressed by adeno-associated virus (AAV) and the exogenous NT-1 can promote neural stem cells migration from the subventricular zone (SVZ) region after cerebral ischemia. Adult CD-1 mice were injected stereotacticly with AAV carrying NT-1 gene (AAV-NT-1). Mice underwent 60 min of middle cerebral artery (MCA) occlusion 1 week after injection. We found that NT-1 mainly expressed in neuron and astrocyte, and the expression level of NT-1 significantly increased 1 week after AAV-NT-1 gene transfer and lasted for 28 days, even after transient middle cerebral artery occlusion (tMCAO) as well (p < 0.05). Immunohistochemistry results showed that the number of neural stem cells was greatly increased in the SVZ region of AAV-NT-1-transduced mice compared with control mice. Our study showed that overexpressed NT-1 promoted neural stem cells migration from SVZ. This result suggested that NT-1 is a promising factor for repairing and remodeling after focal cerebral ischemia.