From static to dynamic:live observation of the support system after ischemic stroke by two photon-excited fluorescence laser-scanning microscopy

Ischemic stroke is one of the most common causes of mortality and disability worldwide.However,treatment efficacy and the progress of research remain unsatisfactory.As the critical support system and essential components in neurovascular units,glial cells and blood vessels(including the bloodbrain barrier)together maintain an optimal microenvironment for neuronal function.They provide nutrients,regulate neuronal excitability,and prevent harmful substances from entering brain tissue.The highly dynamic networks of this support system play an essential role in ischemic stroke through processes including brain homeostasis,supporting neuronal function,and reacting to injuries.However,most studies have focused on postmortem animals,which inevitably lack critical information about the dynamic changes that occur after ischemic stroke.Therefore,a high-precision technique for research in living animals is urgently needed.Two-photon fluorescence laser-scanning microscopy is a powerful imaging technique that can facilitate live imaging at high spatiotemporal resolutions.Twophoton fluorescence laser-scanning microscopy can provide images of the whole-cortex vascular 3D structure,information on multicellular component interactions,and provide images of structure and function in the cranial window.This technique shifts the existing research paradigm from static to dynamic,from flat to stereoscopic,and from single-cell function to multicellular intercommunication,thus providing direct and reliable evidence to identify the pathophysiological mechanisms following ischemic stroke in an intact brain.In this review,we discuss exciting findings from research on the support system after ischemic stroke using two-photon fluorescence laser-scanning microscopy,highlighting the importance of dynamic observations of cellular behavior and interactions in the networks of the brain’s support systems.We show the excellent application prospects and advantages of two-photon fluorescence laser-scanning microscopy and predict future research developments and directions in the study of ischemic stroke.

P2X7 receptor activation aggravates NADPH oxidase 2-induced oxidative stress after intracerebral hemorrhage

Oxidative stress is a crucial pathological process that contributes to secondary injury following intracerebral hemorrhage. P2X7 receptor(P2X7R), which is activated by the abnormal accumulation of extracellular ATP, plays an important role in the regulation of oxidative stress in the central nervous system, although the effects of activated P2X7R-associated oxidative stress after intracerebral hemorrhage remain unclear. Mouse models of intracerebral hemorrhage were established through the stereotactic injection of 0.075 U VII collagenase into the right basal ganglia. The results revealed that P2X7R expression peaked 24 hours after intracerebral hemorrhage, and P2X7R expressed primarily in neurons. The inhibition of P2X7R, using A438079(100 mg/kg, intraperitoneal), reduced nicotinamide adenine dinucleotide phosphate oxidase 2(NOX2) expression and malondialdehyde generation, increased superoxide dismutase and glutathione/oxidized glutathione levels, and alleviated neurological damage, brain edema, and apoptosis after intracellular hemorrhage. The P2X7R inhibitor A438079(100 mg/kg, intraperitoneal injection) inhibited the activation of extracellular signal-regulated kinase 1/2(ERK1/2) and nuclear factor kappa-B(NF-κB) after intracerebral hemorrhage. Blocking ERK1/2 activation, using the ERK1/2 inhibitor U0126(2 μg, intraventricular injection), reduced the level of NOX2-mediated oxidative stress induced by P2X7R activation after intracellular hemorrhage. Similarly, the inhibition of NF-κB, using the NF-κB inhibitor JSH-23(3.5 μg, intraventricular), reduced the level of NOX2-mediated oxidative stress induced by P2X7R activation. Finally, GSK2795039(100 mg/kg, intraperitoneal), a NOX2 antagonist, attenuated P2X7R-mediated oxidative stress, neurological damage, and brain edema after intracerebral hemorrhage. The results indicated that P2X7R activation aggravated NOX2-induced oxidative stress through the activation of the ERK1/2 and NF-κB pathways following intracerebral hemorrhage in mice. The present study was approved by the Ethics Committee of Huazhong University of Science and Technology, China(approval No. TJ-A20160805) on August 26, 2016.

Experimental Study of Cell Migration and Functional Differentiation of Transplanted Neural Stem Cells Co-labeled with Superparamagnetic Iron Oxide and Brdu in an Ischemic Rat Model

Objective To explore the migration of transplanted neural stem cells co-labeled with superparamagnetic iron oxide （SPIO） and bromodeoxyuridine （Brdu） using the 4.7T MR system and to study the cell differentiation with immuno-histochemical method in ischemic rats. Methods Rat neural stem cells （NSCs） co-labelled with SPIO mediated by poly-L-lysine and bromodeoxyuridine （BrdU） were transplanted into the unaffected side of rat brain with middle cerebral artery occlusion （MCAO）. At weeks 1, 2, 3, 4, 5, and 6 after MCAO, migration of the labelled cells was monitored by MRI. At week 6 the rats were killed and their brain tissue was cut according to the migration site of transplanted ceils indicated by MRI and subjected to Prussian blue staining and immunohistochemical staining to observe the migration and differentiation of the transplanted NSCs. Results Three weeks after transplantation, the linear hypointensity area derived from the migration of labelled NSCs was observed by MRI in the corpus callosum adjacent to the injection site. Six weeks after the transplantation, the linear hypointensity area was moved toward the midline along the corpus callosum. MRI findings were confirmed by Prussian blue staining and immunohistochemical staining of the specimen at week 6 after the transplantation. Flourescence co-labelled immunohistochemical methods demonstrated that the transplanted NSCs could differentiate into astrocytes and neurons. Conclusion MRI can monitor the migration of SPIO-labelled NSCs after transplantation in a dynamical and non-invasive manner. NSCs transplanted into ischemic rats can differentiate into astrocytes and neurons during the process of migration.

Apolipoprotein E Gene Polymorphisms Are Risk Factors for Spontaneous Intracerebral Hemorrhage:A Systematic Review and Meta-analysis

Intracerebral hemorrhage(ICH)is a serious clinical disease with high morbidity,whose pathogenesis might be related to apolipoprotein E(APOE)gene polymorphisms.To comprehensively evaluate the risk factors for ICH occurrence,we performed a meta-analysis.We searched online databases to identify eligible studies based on the relationship between APOE genetic polymorphisms and ICH occurrence risk.Specific and pooled odds ratios(ORs)were calculated and by assessing small study bias,we drew the relationship between APOE polymorphisms and ICH risk.We included 15 eligible studies in our study containing a total of 1642 ICH samples and 5545 normal controls.The comparison of e4 andε3 APOE genotypes revealed that specific and pooled ORs showed a significantly increased odds ratio in ICH patients with theε4 genotype,indicating thatε4 gene is a risk factor for ICH occurrence,and the heterogeneity is acceptable.Similarly,it was found that theε2 genotype also contributed to the incidence rate of ICH.However,after the subgroup analysis by ethnicity,this APOE genetic polymorphism acted as a harmful factor only in white populations,but did not show an effect in Asian populations.It was suggested that both 82 andε4 APOE alleles were risk factors for ICH in general.They were risk factors in white populations only,neither had a detectable effect in Asian populations after subgroup analysing by ethnicity.

MRI Features and Site-specific Factors of Ischemic Changes in White Matter： A Retrospective Study

Brain magnetic resonance imaging （MRI） of the elderly often reveals white matter changes （WMCs） with substantial variability across individuals. Our study was designed to explore MRI features and site-specific factors of ischemic WMCs. Clinical data of consecutive patients diagnosed with ischemic cerebral vascular disease who had undergone brain MRI were collected and analyzed. Multi-logistic regression analysis comparing patients with mild versus severe WMCs was performed to detect independent associations. Analyses of variance （ANOVAs） were used to detect regionally specific differences in lesions. We found that lesion distribution differed significantly across five cerebral areas, with lesions being predominant in the frontal lobe and parieto-occipital area. To explore WMCs risk factors, after adjusting for gender, diabetes mellitus, and hypertension, only age （P〈0.01）, creatinine （P=0.01）, alkaline phosphatase （ALP） （P=0.01） and low-density lipoprotein cholesterol （LDL-C） （P=0.03） were found to be independently associated with severe WMCs. Age （P〈0.001） was strongly associated with WMCs in the frontal lobe while hypertension was independently related to lesions in the basal ganglia （P=0.048） or infratentorial area （P=0.016）. In conclusion, MRI of WMCs showed that ischemic WMCs occurred mostly in the frontal lobe and parieto-occipital area. The infratentorial area was least affected by WMCs. Typically, age-related WMCs were observed in the frontal lobes, while hypertension-related WMCs tended to occur in the basal ganglia and infratentorial area.

Insights into Initial Demyelinating Episodes of Central Nervous System during Puerperium

Background：Inflammatory demyelinating disease of central nervous system （CNS） is an inflammatory disease characterized by a high childbearing female predominance.Labor-related alterations for postpartum demyelinating attacks are not entirely clear.This study aimed to summarize clinical features of female patients of reproductive age with initial CNS inflammatory demyelinating attacks during puerperium.Methods：Fourteen female patients with initial demyelinating events during puerperium between January 2013 and December 2016 were retrospectively studied.Records of clinical features,neuroimaging,serum antibodies,cerebrospinal fluid （CSF） findings,annualized relapse rate （ARR）,and treatment were analyzed.Results：Among 14 patients,5 patients were diagnosed with multiple sclerosis （MS）,four as neuromyelitis optica （NMO）,two as longitudinal extensive transverse myelitis,two as clinical isolated syndrome （CIS）,and one as acute brainstem syndrome.All the 14 puerperal female patients presented with more than one manifestation of hemiplegia,paraplegia,uroschesis,visual loss or dysarthria,and with mild to moderate abnormalities of CSF.Attacks occurred during the first trimester postpartum and cesarean section was the main delivery way （n =10）.Median Expanded Disability Status Scale （EDSS） scores were 5.0 （range：2.0-9.0） at the onset and 2.5 （range：0-7.0） at the end of follow-ups.Patients with MS and CIS had a significantly lower EDSS scores than patients with NMO spectrum disorders （P 〈 0.05）.Median ARR was 0.46 （range：0-1.16）;all patients had a low ARR （0.49 ± 0.34,95% confidence interval：0.29-0.69） with standardized treatments.Conclusion：Labor-related alterations in the mother＇s immune system might result in newly-onset demyelinating diseases of central nervous system.

Effects of Prior Antiplatelet Therapy on the Prognosis of Primary Intracerebral Hemorrhage： A Meta-analysis

Background：Antiplatelet therapy （APT） was prevalently being used in the prevention of vascular disease,but the influence of prior APT on the prognosis of patients with intracerebral hemorrhage （ICH） remains controversial.This meta-analysis was to explore the effects of prior APT on the prognosis of patients with primary ICH.Methods：PubMed and Embase were searched to identify the eligible studies.The studies comparing the mortality of ICH patients with or without prior APT were included.The quality of these studies was evaluated by the Newcastle-Ottawa quality assessment scale.The adjusted or unadjusted odds ratio （OR） for mortality between ICH patients with and without prior APT were pooled with 95% confidence interval （95% CI） as the effect of this meta-analysis.Results：Twenty-two studies fulfilled the inclusion criteria and exhibited high qualities.The pooled OR was 1.37 （95% CI：1.13-1.66,P =0.001） for univariate analysis and 1.41 （95% CI：1.05-1.90,P =0.024） for multivariate analysis.The meta-regression indicated that for each 1-day increase in the time of assessment,the adjusted OR for the mortality of APT patients decreased by 0.0049 （95% CI：0.0006-0.0091,P =0.026） as compared to non-APT patients.Conclusion：Prior APT was associated with high mortality in patients with ICH that might be attributed primarily to its strong effect on early time.