Small extracellular vesicles derived from cerebral endothelial cells with elevated microRNA 27a promote ischemic stroke recovery

Axonal remodeling is a critical aspect of ischemic brain repair processes and contributes to spontaneous functional recovery.Our previous in vitro study demonstrated that exosomes/small extracellular vesicles(sEVs)isolated from cerebral endothelial cells(CEC-sEVs)of ischemic brain promote axonal growth of embryonic cortical neurons and that microRNA 27a(miR-27a)is an elevated miRNA in ischemic CEC-sEVs.In the present study,we investigated whether normal CEC-sEVs engineered to enrich their levels of miR-27a(27a-sEVs)further enhance axonal growth and improve neurological outcomes after ischemic stroke when compared with treatment with non-engineered CEC-sEVs.27a-sEVs were isolated from the conditioned medium of healthy mouse CECs transfected with a lentiviral miR-27a expression vector.Small EVs isolated from CECs transfected with a scramble vector(Scra-sEVs)were used as a control.Adult male mice were subjected to permanent middle cerebral artery occlusion and then were randomly treated with 27a-sEVs or Scra-sEVs.An array of behavior assays was used to measure neurological function.Compared with treatment of ischemic stroke with Scra-sEVs,treatment with 27a-sEVs significantly augmented axons and spines in the peri-infarct zone and in the corticospinal tract of the spinal grey matter of the denervated side,and significantly improved neurological outcomes.In vitro studies demonstrated that CEC-sEVs carrying reduced miR-27a abolished 27a-sEV-augmented axonal growth.Ultrastructural analysis revealed that 27a-sEVs systemically administered preferentially localized to the pre-synaptic active zone,while quantitative reverse transcription-polymerase chain reaction and Western Blot analysis showed elevated miR-27a,and reduced axonal inhibitory proteins Semaphorin 6A and Ras Homolog Family Member A in the peri-infarct zone.Blockage of the Clathrin-dependent endocytosis pathway substantially reduced neuronal internalization of 27a-sEVs.Our data provide evidence that 27a-sEVs have a therapeutic effect on stroke recovery by promoting axonal remodeling and improving neurological outcomes.Our findings also suggest that suppression of axonal inhibitory proteins such as Semaphorin 6A may contribute to the beneficial effect of 27a-sEVs on axonal remodeling.

EFFECTS OF DAURISOLINE ON CYTOSOLIC FREE CALCIUMIN FETAL RAT CEREBRAL CELLS

Cytosolic free Ca([Ca]i)was measured in dissociated cerebral cells isolated from fetal rats with the fluorescent indicater fura-2. Increase in[Ca]i occurred rapidly following explsure of the cells to 50 mmol/L KCI,10mol/L Bayk 8644 or 200μmol/L glutamate（Glu）.[Ca]i elevated by

Proliferation and Differentiation of Neural Stem Cells Co-Cultured with Cerebral Microvascular Endothelial Cells after Oxygen-glucose Deprivation

Various stem cells, including neural stem cells （NSCs）, have been extensively studied in stroke models, but how to increase neuronal differentiation rate of NSCs remains unresolved, particu- larly in a damaged environment. The purpose of this study was to investigate the effects of cerebral mi- crovascular endothelial cells （CMECs） on the neurogenesis of NSCs with or without oxygen-glucose deprivation （OGD）. The NSCs acquired from primary culture were immunostained to prove cell purity. Survival and proliferation of NSCs were determined after the co-culture with CMECs for 7 days. After removing the CMECs, NSCs were randomly divided into two groups as follows： OGD and non-OGD groups. Both groups were maintained in differentiation culture for 4 days to evaluate the differentiation rate. Mouse embryo fibroblast （MEF） cells co-cultured with NSCs served as control group. NSCs co-cultured with CMECs had an increase in size （on the 7th day： 89.80±26.12 μm vs. 73.08±15.01μm, P〈0.001） （n=12） and number [on the 7th day： 6.33±5.61/high power objective （HP） vs. 2.23±1.61/HP, P〈0.001] （n=12） as compared with those co-cultured with MEF cells. After further differentiation cul- ture for 4 days, NSCs co-cultured with CMECs had an increase in neuronal differentiation rate in OGD and non-OGD groups, but not in the control group （15.16% and 16.07% vs. 8.81%; both P〈0.001） （n=6） This study provided evidence that OGD could not alter the effects of CMECs in promoting the neuronal differentiation potential of NSCs. These findings may have important implications for the development of new cell therapies for cerebral vascular diseases.

Improvement of learning and memory abilities and motor function in rats with cerebral infarction by intracerebral transplantation of neuron-like cells derived from bone marrow stromal cells

BACKGROUND： Transplantation of fetal cell suspension or blocks of fetal tissue can ameliorate the nerve function after the injury or disease in the central nervous system, and it has been used to treat neurodegenerative disorders induced by Parkinson disease. OBJECTIVE： To observe the effects of the transplantation of neuron-like cells derived from bone marrow stromal cells （rMSCs） into the brain in restoring the dysfunctions of muscle strength and balance as well as learning and memory in rat models of cerebral infarction. DESIGN ： A randomized controlled experiment.SETTING ： Department of Pathophysiology, Zhongshan Medical College of Sun Yat-sen University.MATERIALS ： Twenty-four male SD rats （3-4 weeks of age, weighing 200-220 g） were used in this study （Certification number：2001A027）. METHODS： The experiments were carried out in Zhongshan Medical College of Sun Yat-sen University between December 2003 and December 2004. ① Twenty-four male SD rats randomized into three groups with 8 rats in each： experimental group, control group and sham-operated group. Rats in the experiment al group and control group were induced into models of middle cerebral artery occlusion （MCAO）. After in vitro cultured, purified and identified with digestion, the Fischer344 rMSCs were induced to differentiate by tanshinone IIA, which was locally injected into the striate cortex （18 area） of rats in the experimental group, and the rats in the control group were injected by L-DMEM basic culture media （without serum） of the same volume to the corresponding brain area. In the sham-operated group, only muscle and vessel of neck were separated. ② At 2 and 8 weeks after the transplantation, the rats were given the screen test, prehensile-traction test, balance beam test and Morris water-maze test. ③ The survival and distribution of the induced cells in corresponding brain area were observed with Nissl stained with toluidine blue and hematoxylin and eosin （HE） staining in the groups.MAIN OUTCOME MEASURES ： ① Results of the behavioral tests （time of the Morris water-maze test screen test, prehensile-traction test, balance beam test）; ② Survival and distribution of the induced cells.RESULTS： All the 24 rats were involved in the analysis of results. ① Two weeks after transplantation, rats with neuron-like cells grafts in the experimental group had significant improvement on their general muscle strength than those in the control group [screen test： （9.4±1.7）, （4.7±1.0） s, P 〈 0.01]; forelimb muscle strength [prehensile-traction test： （7.6±1.4）, （5.2±1.2） s, P 〈 0.01], ability to keep balance [balance beam test： （7.9±0.74）, （6.1±0.91） s, P 〈 0.01] and abilities of learning and memory [latency to find the platform： （35.8±5.9）, （117.5±11.6） s, P 〈 0.01; distance： （623.1±43.4）, （1 902.3±98.6） cm, P 〈 0.01] as compared with those in the control group. The functional performances in the experimental group at 8 weeks were better than those at two weeks, which were still obviously different from those in the sham-operated group （P 〈 0.05）. ② The HE and Nissl stained brain tissue section showed that there was nerve cell proliferation at the infarcted cortex in the experiment group, the density was higher than that in the control group, plenty of aggregative or scattered cells could be observed at the site where needle was inserted for transplantation, the cells migrated directively towards the area around them, the cerebral vascular walls were wrapped by plenty of cells; In the control group, most of the cortices were destroyed, karyopyknosis and necrosis of neurons were observed, normal nervous tissue structure disappeared induced by edema, only some nerve fibers and glial cells remained.CONCLUSION： The rMSCs transplantation can obviously enhance the motor function and the abilities of learning and memory in rat models of cerebral infarction.

Effects of L-Tetrahydropalmatine on NOSⅢ Gene Expression in Hypoxia and Cultured Porcine Cerebral Arterial Endothelial Cells during Reoxygenation

To investigate the expression of NOSⅢ mRNA and protein in cultured porcine cerebral arterial endothelial cells (CAEC) during hypoxia and reoxygenation and the effects of L-Tetrahydropalmatine (L-THP) on the gene expression of NOSⅢ in CAEC during hypoxia and reoxygenation. The cultured CAEC were divided into 5 groups: control, hypoxia, hypoxia+reoxygenation, hypoxia+L-THP and reoxygenation+L-THP groups. NOSⅢ mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). Immunocytochemistry was used to detect the level of NOSⅢ protein. The expression of NOSⅢ mRNA and protein were increased when CAEC were exposed to hypoxia for 1 h, and significantly decreased during reoxygenation 2, 6 and 12 h after 1-h of hypoxia. L-THP from 10 -8 mol/L to 10 -3 mol/L could inhibit the up-regulation of NOSⅢ gene expression during hypoxia and down-regulation of NOSⅢ gene expression during reoxygenation.

Stem cells for treatment of cerebral ischemia

Three articles regarding the effects of gene-modified stem cell transplantation and the reinfocing effects of dl-3-butylphthalide on hematopoietic stem cell transplantation and endogenous stem cell mobilization in the treatment of cerebral ischemia were published in Neural Regeneration Research. We hope that our readers find these papers useful to their research.

Transfusion of CXCR4-priming endothelial progenitor cells reduces cerebral ischemic damage and promotes angiogenesis and neurogenesis in db/db diabetic mice

Previous studies suggest that reduction and dysfunction of circulating endothelial progenitor cells(EPCs),and dysregulation in stromal cell derived factor-1/CXC-chemokine receptor 4(SDF-1/ CXCR4) axis in diabetes could be therapeutic targets for diabetic ischemic stroke.This study investigated the efficacy of CXCR4-priming EPCs on cerebral repair following ischemic stroke in db/db diabetic mice.Bone marrow derived EPCs from db/+ control mice were transfected with adenovirus(1×10~7 IU) carrying CXCR4(Ad-CXCR4-EPCs)or null(Ad- null-EPCs).The db/db mice were divided into three groups for EPCs injection(2×10~5 cells/100μl): Ad-CXCR4-EPCs,Ad-null-EPCs or saline(vehicle), via tail vein 2 hrs after middle cerebral artery occlusion (MCAO) surgery.Cerebral blood flow(CBF) was measured with laser Doppler flowmeter.Mice were sacrificed at 2 or 7 days thereafter.Level of circulating EPCs was measured by flow cytometry. Ischemic damage,cerebral microvascular density (MVD),angiogenesis and neurogenesis were determined by histological staining with Fluoro-J,CD31, CD31 +BrdU,NeuN +BrdU,GFAP+BrdU,respectively. Results(table) showed:1) Levels of CXCR4 expression were reduced in the brain and EPCs of db/db mice as measured by real-time RT-PCR and western blot analyses(data not shown);2) The level of circulating EPCs was more in the mice treated with Ad-CXCR4-EPCs;3)EPC transfusion improved CBF,increased MVD,angiogenesis and neurogenesis in peri-infarct area,and decreased ischemic damage.The efficacies were better in Ad-CXCR4 -EPCs group.Data suggest that transfusion of Ad-CXCR4-EPCs could be a therapeutic avenue for ischemia stroke in diabetes.

Changes in the permeability of blood brain barrier and endothelial cell damage after cerebral ischemia

OBJECTIVE： To investigate the effect of endothelial cells on the permeability of blood brain barrier （BBB） after brain injury and its effect mechanism. DATA SOURCES： We searched for the articles of permeability of BBB and endothelial cell injury after brain is- chemia, which were published between January 1982 and December 2005, with the key words of ＂cerebral ischemia damage,blood brain barrier （ BBB）,permeability,effect of endothelial cell （EC） and its variation mechanism＂in English. STUDY SELECTION： The materials were primarily selected. The articles related to the changes in the permeability of BBB and the effect of endothelial cells as well as the change mechanism after cerebral ischemia damage were chosen. Repetitive studies or review articles were excluded. DATA EXTRACTION： Totally 55 related articles were collected, and 35 were excluded due to repetitive or review articles, finally 20 articles were involved. DATA SYNTHESIS： The content or viewpoints of involved literatures were analyzed. Cerebral ischemia had damage for endothelial cells, such as the inflow of a lot of Ca2^＋, the production of nitrogen monoxide and oxygen free radical, and aggravated destruction of BBB. After acceptors of inflammatory mediators on cerebrovascular endothelial cell membrane, such as histamine, bradykinin , 5-hydroxytryptamine and so on are activated, endothelial cells shrink and the permeability of BBB increases. Its mechanism involves in the inflow of extracellular Ca^＋2and the release of intracellular Ca^2＋ in the cells. Glycocalyx molecule on the surface of endothelial cell, having structural polytropy, is the determinative factor of the permeability of BBB. VEGF, intensively increasing the vasopermeability and mainly effecting on postcapillary vein and veinlet, is the strongest known blood vessel permeation reagent. Its chronic overexpression in the brain can lead the destruction of BBB. CONCLUSION： The injury of endothelial cell participants in the pathological mechanism of BBB destruction after cerebral ischemla.

Inhibition of cerebral ischemia/reperfusion injuryinduced apoptosis:nicotiflorin and JAK2/STAT3 pathway

Nicotiflorin is a flavonoid extracted from Carthamus tinctorius.Previous studies have shown its cerebral protective effect,but the mechanism is undefined.In this study,we aimed to determine whether nicotiflorin protects against cerebral ischemia/reperfusion injury-induced apoptosis through the JAK2/STAT3 pathway.The cerebral ischemia/reperfusion injury model was established by middle cerebral artery occlusion/reperfusion.Nicotiflorin（10 mg/kg） was administered by tail vein injection.Cell apoptosis in the ischemic cerebral cortex was examined by hematoxylin-eosin staining and terminal deoxynucleotidyl transferase d UTP nick end labeling assay.Bcl-2 and Bax expression levels in ischemic cerebral cortex were examined by immunohistochemial staining.Additionally,p-JAK2,p-STAT3,Bcl-2,Bax,and caspase-3 levels in ischemic cerebral cortex were examined by western blot assay.Nicotiflorin altered the shape and structure of injured neurons,decreased the number of apoptotic cells,down-regulates expression of p-JAK2,p-STAT3,caspase-3,and Bax,decreased Bax immunoredactivity,and increased Bcl-2 protein expression and immunoreactivity.These results suggest that nicotiflorin protects against cerebral ischemia/reperfusion injury-induced apoptosis via the JAK2/STAT3 pathway.

Effect of ultraviolet blood irradiation and oxygenation on nerve function and function of the red blood cell membrane pump in patients with acute cerebral infarction

BACKGROUND： Ultraviolet blood irradiation and oxygenation （UBIO） has obtained better clinical effect in treating acute cerebral infarction, but the mechanism underlying this effect remains unclear. OBJECTIVE： To observe the effect of UBIO on the nerve function and activities of K^＋-Na^＋-ATPase and Ca2^＋-Mg2^＋-ATPase activities on the red blood cell （RBC） membrane of patients with acute cerebral infarction. DESIGN： A randomized and controlled study.SETTING： Department of Neurology, Xiangfan Central Hospital.PARTICIPANTS： From January 2000 to December 2001, excluding those above 70 years old, 58 cases of 700 patients with acute cerebral infarction admitted in the Department of Neurology, Xiangfan Central Hospital, were recruited and divided into two groups according to the random number table： UBIO treated group （n=28）, including 17 males and 11 females, aged 40-68 years; and control group （n=30）, including 20 males and 10 females, aged 44-69 years. All the patients agreed to participate in the therapeutic program and detected items. The general informations were comparable without obvious differences between the two groups （P 〉 0.05）.METHODS： ① The patients in both groups received routine treatments, besides, those in the UBIO treated group were given UBIO treatment by using the XL-200 type therapeutic apparatus produced in Shijiazhuang, whose ultraviolet wave was set at 253.7 nm with the energy density of 0.568 J/m^2 per second, UBIO treatment started from the second day after admission, once every other day, with a single course consisting of 5-7 treatments. ② In the UBIO treated group, the venous blood was sampled before and after the first, third and the completion of the treatment course respectively, the venous blood was taken at each corresponding time point in the control group. After centrifugation of the blood at 10 000 rounds per minute, the RBC membrane was separated and then the activities of K^＋-Na^＋-ATPase and Ca2^＋-Mg2^＋-ATPase were detected by means of phosphorus determination.③ The nerve function was scored before and after treatment in both groups with European stroke scale, which included 13 items, the total score was 0-100 points, the higher the score, the better the nerve function. MAIN OUTCOME MEASURES ：①Score of European stroke scale before and after treatment in both groups.② Comparison of the activities of K^＋-Na^＋-ATPase and Ca2^＋-Mg2^＋-ATPase on RBC membrane between the two groups before treatment and after the first, third and the completion of the treatment. RESULTS： All the 58 patients with cerebral infarction were involved in the analysis of results.① The score of European stroke scale had no obvious difference between the two groups [（49.31±11.48）, （50.58±12.63）, P 〉 0.05], and it was obviously higher in the UBIO treated group than in the control group after treatment [84.66±13.75）, （77.05±11.17）, P 〈 0.05].②The activity of K^＋-Na^＋-ATPase on RBC membrane in the UBIO treated group was significantly increased after the first and third treatment as compared with before treatment [（31.56±19.25）, （27.64±15.83）, （17.67±13.83）, P 〈 0.01], it was still higher after the completion of the treatment than before treatment without obvious difference [（20.86±14.53）, P 〉 0.05]. After the first and third treatment, it was obviously higher in the UBIO treated group than in the control group [19.31±11.88）, （17.44±10.42）, P 〈 0.01]. ③ In the UBIO treated group, Ca2^＋-Mg2^＋-ATPase activity on RBC membrane significantly increased after the first treatment and remained higher than the pre-treatment level throughout the treatment [（27.49±14.72）, （17.41±4.82）, P 〈 0.01]. The activity of Ca2^＋-Mg2^＋-ATPase on RBC membrane was markedly higher in the UBIO treated group than in the control group after after the first, third and the completion of treatment respectively [（24.83±12.88）, （17.70±5.69）; （28.08±13.44）, （16.32±5.29）; （17.42±6.04）, P〈 0.05-0.01]. CONCLUSION： The effect of UBIO treatment against acute cerebral infarction may be mediated by the increased K^＋-Na^＋ ATPase and Ca2^＋-Mg2^＋-ATPase activities on RBC membrane, which enhances the RBC transformation ability so as to lower RBC aggregation and correct high blood viscosity.

Monitoring the changes in plasm C-reactive protein,fibrinogen and blood white cell in patients with primary hypertension combined with acute cerebral infarction

BACKGROUND： Inflammatory reaction and the increased level of its accompanying active protein play an important role in the occurrence and development of cerebral infarction. C-reactive protein, fibrinogen and white blood cell, as the monitoring index of inflammatory reaction, are very important in the occurrence and development of acute cerebral infarction. OBJECTIVE： To make a comparison between patients with primary hypertension accompanied with acute cerebral infarction and with simple primary hypertension by observing the changes in plasma C-reactive protein and fibrinogen levels as well as white blood cell and differential counts and analyzing their significances. DESIGN ： Controlled observation SETTING ： Ward Building for VIP, Shenzhen Hospital, Peking University. PARTICIPANTS： Totally 133 patients with primary hypertension were selected from Ward Building for VIP, Shenzhen Hospital, Peking University during September 2003 to September 2005, The diagnostic criteria were based on the hypertension diagnosis criteria formulated by the 7^th World Health Organization-International Society of Hypertension Guidelines （WHO-ISH） in 1998. The informed consents were obtained from all the participants. The involved patients were assigned into two groups： primary hypertension group, in which, there were 65 patients with primary hypertension （ degree 2）, including 42 males and 23 females, with mean age of （61 ±14）years and mean blood pressure of （162.7±6.8）/（94.2±8.4） mm Hg （1 mm Hg =0.133 kPa）, and primary hypertension combined with cerebral infarction group, in which, there were 68 patients with primary hypertension combined with cerebral infarction （ meeting the diagnostic criteria formulated in the 4^th National Cerebrovascular Diseases Meeting in 1995 and diagnosed by skull CT or MRI to exclude the patients with lacunar infarction）, including 42 males and 26 females, with mean age of （56±15） years and mean blood pressure of （176.4±9.2）/（96.3±9.7） mm Hg. METHODS： Plasm C-reactive protein and fibrinogen levels, and white blood cell and differential counts of patients in the two groups were examined 24 hours after stroke. The above indexes were re-examined in the primary hypertension combined with cerebral infarction group 72 hours after stroke. White blood cell and differential counts were performed with laser method （East Asia FE-95001 RAM-1, Japan）. The level of C-reactive protein was measured with turbidimetry （BNII Automatic Systems For Analysis, USA）. The level of fibrinogen was measured with algorithm method when prothrombin time was normal and with Clauss method when prothrombin time was abnormal （ACL Automatic Coagulation Analyzer, USA）. MAIN OUTCOME MEASURES： The plasm C-reactive protein and flbrinogen levels, and white blood cell and differential counts 24 hours after stroke in two groups and 72 hours after stroke in primary hypertension combined with cerebral infarction group. RESULTS： All the 133 involved patients participated in the result analysis. The plasm C-reactive protein and fibrinogen levels, and white blood cell and neutrophil counts in patients with primary hypertension were all within the normal range. The plasm C-reactive protein and fibrinogen levels, and white blood cell and neu- trophil counts in patients with primary hypertension combined with cerebral infarction were significantly higher than those in patients with primary hypertension 24 hours after stroke and 72 hours after stroke respectively[24 hours after stroke：（32.12±11.76） mg/L vs. （5.02±3.21 ） mg/L;（4.64±0.75） g/L vs. （3.12±0.49） g/L; （9.32±81）×10^9 L^- 1 vs. （5.78±1.32）×10^9L^- 1 （7.85±2.38）×10^9 L^- 1 vs.（3.49±1.28）×10^9 L^-1,t =7.094, 5.759,4.106,5.491, respectively,all P〈 0.01; 72 hours after stroke： （47.62±18.43） mg/L vs. （32.12±11.76） mg/L; （5.08±0.82） g/L vs. （4.64±0.75） g/L, t =2.864,2.220, respectively, both P 〈 0.05]. CONCLUSION： The increase in fibrinogen level and white blood cell count are the important index in monitoring primary hypertension combined with acute cerebral infarction. The increase in plasm C-reactive protein and fibrinogen levels 72 hours after stroke indicates that plasma C-reactive protein and fibrinogen are very important in the development of disease.

The relationship among amyloid-βdeposition,sphingomyelin level,and the expression and function of P-glycoprotein in Alzheimer’s disease pathological process

In Alzheimer’s disease,the transporter P-glycoprotein is responsible for the clearance of amyloid-βin the brain.Amyloid-βcorrelates with the sphingomyelin metabolism,and sphingomyelin participates in the regulation of P-glycoprotein.The amyloid cascade hypothesis describes amyloid-βas the central cause of Alzheimer’s disease neuropathology.Better understanding of the change of P-glycoprotein and sphingomyelin along with amyloid-βand their potential association in the pathological process of Alzheimer’s disease is critical.Herein,we found that the expression of P-glycoprotein in APP/PS1 mice tended to increase with age and was significantly higher at 9 and 12 months of age than that in wild-type mice at comparable age.The functionality of P-glycoprotein of APP/PS1 mice did not change with age but was significantly lower than that of wild-type mice at 12 months of age.Decreased sphingomyelin levels,increased ceramide levels,and the increased expression and activity of neutral sphingomyelinase 1 were observed in APP/PS1 mice at 9 and 12 months of age compared with the levels in wild-type mice.Similar results were observed in the Alzheimer’s disease mouse model induced by intracerebroventricular injection of amyloid-β1-42 and human cerebral microvascular endothelial cells treated with amyloid-β1-42.In human cerebral microvascular endothelial cells,neutral sphingomyelinase 1 inhibitor interfered with the changes of sphingomyelin metabolism and P-glycoprotein expression and functionality caused by amyloid-β1-42 treatment.Neutral sphingomyelinase 1 regulated the expression and functionality of P-glycoprotein and the levels of sphingomyelin and ceramide.Together,these findings indicate that neutral sphingomyelinase 1 regulates the expression and function of P-glycoprotein via the sphingomyelin/ceramide pathway.These studies may serve as new pursuits for the development of anti-Alzheimer’s disease drugs.

Magnetic resonance imaging and cell-based neurorestorative therapy after brain injury

Restorative cell-based therapies for experimental brain injury, such as stroke and traumatic brain injury,substantially improve functional outcome. We discuss and review state of the art magnetic resonance imaging methodologies and their applications related to cell-based treatment after brain injury. We focus on the potential of magnetic resonance imaging technique and its associated challenges to obtain useful new information related to cell migration, distribution, and quantitation, as well as vascular and neuronal remodeling in response to cell-based therapy after brain injury. The noninvasive nature of imaging might more readily help with translation of cell-based therapy from the laboratory to the clinic.

The Possibility and Molecular Mechanisms of Cell Pyroptosis After Cerebral Ischemia

Introduction Stroke is an important disease that is prevalent worldwide[1–3]. Ischemic stroke accounts for 80%of stroke cases.Currently, evidence-based effective treatments for ischemic stroke are limited, and only intravenous thrombolysis with Alteplase (a commercially available throm-

Roles of cyclooxygenase-2 in microvascular endothelial cell proliferation induced by basic fibroblast growth factor

Background The level of basic fibroblast growth factor （bFGF） increases rapidly after cerebral ischemia. However, the molecular mechanisms for the effects of bFGF on cerebral microvascular endothelial cells （cMVECs） have not yet been fully elucidated. In this study, a murine cMVEC line, bEnd.3, was employed to study the effects of bFGF on cyclooxygenase （COX） expression and its downstream effects in cMVECs. Methods After treatment with bFGF, RT-PCR and Western blotting analyses were carried out to evaluate the changes in COX-2 mRNA and protein expression, respectively. MTT assays were performed to measure cell proliferation. The prostaglandin E2 （PGE2） and vascular endothelial growth factor （VEGF） concentrations in the culture medium were measured by enzyme-linked immunosorbent assay （ELISA）. Results COX-2 mRNA and protein expressions in bEnd.3 cells were induced by bFGF in time- and dose-dependent manners. The bFGF-induced COX-2 upregulation led to enhanced PGE2 production by bEnd.3 cells, and this effect was abolished by the selective COX-2 inhibitor NS-398. bFGF also increased VEGF production by bEnd.3 cells, and this effect was blocked by NS-398 and the EP1/2 （PGE2 receptors） antagonist AH6809. Furthermore, exogenous PGE2 increased VEGF production in bend.3 cells, and AH6809 blocked this effect. Conclusion bFGF increases VEGF production in an autocrine manner by increasing COX-2-generated PGE2 in cMVECs and subsequently stimulates MVEC proliferation and angiogenesis.