Interfering TRIB3 protects the blood brain barrier through PI3K/Akt pathway to alleviate cerebral ischemia-reperfusion injury in diabetes mellitus mice.

This study aimed to treat diabetic cerebral ischemia-reperfusion injury (CI/RI) by affecting blood brain barrier (BBB) permeability and integrity. The CI/RI model in DM mice and a high glucose (HG) treated oxygen and glucose deprivation/reoxygenation (OGD/R) brain endothelial cell model were established for the study. Evans blue (EB) staining was used to evaluate the permeability of BBB in vivo. TTC staining was used to analyze cerebral infarction. The location and expression of tribbles homolog 3 (TRIB3) in endothelial cells were detected by immunofluorescence. Western blotting was used to detect the protein expressions of TRIB3, tight junction molecules, adhesion molecules, phosphorylated protein kinase B (p-AKT) and AKT. The levels of pro-inflammatory cytokines were detected by qRT-PCR. Trans-epithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)-dextran were used to measure vascular permeability in vitro. TRIB3 ubiquitination and acetylation levels were detected. Acetyltransferase bound to TRIB3 were identified by immunoprecipitation. TRIB3 was localized in cerebral endothelial cells and was highly expressed in diabetic CI/R mice. The BBB permeability in diabetic CI/R mice and HG-treated OGD/R cells was increased, while the junction integrity was decreased. Interference with TRIB3 in vitro reduces BBB permeability and increases junction integrity. In vivo interfering with TRIB3 reduced cerebral infarction volume, BBB permeability and inflammation levels, and upregulated p-AKT levels. The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin reversed the effects of TRIB3-interfering plasmid. In vitro HG treatment induced TRIB3 acetylation through acetyltransferase p300, which in turn reduced ubiquitination and stabilized TRIB3. Interfering TRIB3 protects BBB by activating PI3K/AKT pathway and alleviates brain injury, which provides a new target for diabetic CI/RI.

Analysis of the efficacy and related factors of ventriculoperitoneal shunt for AIDS with cryptococcal meningitis.

Background: Cryptococcus neoformans is an opportunistic pathogen, which is more common in patients with AIDS. Increased intracranial pressure (ICP) is an important complication of cryptococcal meningitis (CM) and affects the therapeutic effect of CM. Objective: To evaluate the effect and treatment for the management of ventriculoperitoneal shunt (VPS) in the treatment of AIDS complicated with CM and to analyze the factors associated with VPS and the indices affecting the outcome of CM patients. Methods: A retrospective case study was conducted on patients with CM treated in the First Affiliated Hospital of Zhejiang University School of Medicine from 2011 to 2019. The Chi-square test was used for categorical variables and the Student's t-test was used for continuous variables. Multivariable analysis of baseline factors related to VPS placement was performed with stepwise logistic regression analysis, factors associated with the outcome of these patients were studied by Cox regression analysis, and Kaplan-Meier survival curves were constructed to assess the outcome of patients. Results: There were 96 patients with AIDS complicated with CM. VPS had a great effect on the patients, especially those with ICP > 350 mmH2O. The outcome, including the mortality rate and modified Rankin scale (MRS) score of these patients, significantly improved after the placement of VPS. The karnofsky performance status (KPS) scores of patients whose ICP > 350 mmH2O improved from 39.3 ± 21.3 at baseline to 88.7 ± 26.9 at 3 months after VPS, better than those without VPS. Multivariable analysis showed that visual impairment (OR, 0.026; 95% CI, 0.001, 0.567; P = 0.021) and ICP > 350 mmH2O (OR, 0.026; 95% CI, 0.002, 0.293; P = 0.003) were related elements with the placement of shunt, and KPS score (HR, 0.968; 95% CI, 0.943, 0.993; P = 0.013) and ICP > 350 mmH2O (HR, 2.801; 95% CI, 1.035, 7.580; P = 0.043) were indices of the outcome of AIDS patients with CM. For patients with ICP > 350 mmHg, Kaplan-Meier analysis showed that the 3-year outcome of patients with VPS was better than that of patients without VPS (P = 0.0067). Conclusion: VPS was associated with better 3-year survival rates, and postshunt placement complications like infections were rare. The identification of factors related to VPS in the initial diagnosis of CM can contribute to more active management and improve the outcome.

Clipping Ophthalmic Segment Artery Aneurysms Using a Modified Subdural Dolenc Approach: Classification and Experience Sharing.

BACKGROUND: Ophthalmic segment artery aneurysms (OSAs) are difficult to clip; therefore, improvement of the surgical method is of great significance to the prevention of complications, and the classification of the aneurysms is essential to formulate a reasonable surgical plan. OBJECTIVE: To explore the strategies and effects of surgery for OSAs using a modified subdural Dolenc approach. METHODS: The clinical data of 38 patients (12 men and 26 women, aged 48-73 years) with OSA were analyzed retrospectively. A total of 44 aneurysms were identified, 40 of which were OSAs. The 40 aneurysms were divided into types Ia1 (n = 2), Ia2 (n = 2), Ib (n = 6), IIa (n = 4), IIb (n = 4), IIIa (n = 0), IIIb (n = 4), IIIc (n = 16), and IV (n = 2) based on preoperative images. Thirty-nine OSAs were operated successfully through pterional craniotomy combined with the modified subdural Dolenc approach, and 1 aneurysm was clipped through the contralateral approach. Clinical outcomes were evaluated using the Glasgow Outcome Scale (GOS). RESULTS: Thirty-nine OSAs were clipped, and one was wrapped. Visual dysfunction, headache, and dizziness improved after the operation in 18 patients. One patient had new visual impairment, and there were no deaths. At discharge, the GOS score was 5 in 36 cases, 4 in 1 case, and 3 in 1 case. Thirty-seven patients had a GOS score of 5, and 1 patient had a score of 3 at 6 months after the operation. CONCLUSION: The modified subdural Dolenc approach (Zheng approach) for clipping OSAs may be associated with less trauma and good postoperative outcomes.

Low-Intensity Focused Ultrasound Technique in Glioblastoma Multiforme Treatment.

Glioblastoma is one of the central nervous system most aggressive and lethal cancers with poor overall survival rate. Systemic treatment of glioblastoma remains the most challenging aspect due to the low permeability of the blood-brain barrier (BBB) and blood-tumor barrier (BTB), limiting therapeutics extravasation mainly in the core tumor as well as in its surrounding invading areas. It is now possible to overcome these barriers by using low-intensity focused ultrasound (LIFU) together with intravenously administered oscillating microbubbles (MBs). LIFU is a non-invasive technique using converging ultrasound waves which can alter the permeability of BBB/BTB to drug delivery in a specific brain/tumor region. This emerging technique has proven to be both safe and repeatable without causing injury to the brain parenchyma including neurons and other structures. Furthermore, LIFU is also approved by the FDA to treat essential tremors and Parkinson's disease. It is currently under clinical trial in patients suffering from glioblastoma as a drug delivery strategy and liquid biopsy for glioblastoma biomarkers. The use of LIFU+MBs is a step-up in the world of drug delivery, where onco-therapeutics of different molecular sizes and weights can be delivered directly into the brain/tumor parenchyma. Initially, several potent drugs targeting glioblastoma were limited to cross the BBB/BTB; however, using LIFU+MBs, diverse therapeutics showed significantly higher uptake, improved tumor control, and overall survival among different species. Here, we highlight the therapeutic approach of LIFU+MBs mediated drug-delivery in the treatment of glioblastoma.

Ventriculoperitoneal shunt is associated with increased cerebrospinal fluid protein level in HIV-infected cryptococcal meningitis patients.

BACKGROUND: The impact of ventriculoperitoneal shunt on cerebrospinal fluid (CSF) biochemical profiles in HIV-associated cryptococcal meningitis (HCM) patients remains unclear. METHODS: Twenty-nine HCM patients who underwent ventriculoperitoneal shunt (the VPS group) and 57 HCM patients who did not undergo ventriculoperitoneal shunt (the non-VPS group) were enrolled in this propensity score matching analysis. Demographic characteristics, symptoms, CSF biochemical profiles, and adverse events were compared between the two groups. The Kaplan-Meier method was used to analyze the survival rate. Univariate and multivariate logistic regression analyses were performed to identify the risk factors for increased CSF protein levels. RESULTS: After 24 weeks of treatment, the intracranial pressure was significantly lower in the VPS group than in the non-VPS group (mmH2O; 155.0 [120.0-190.0] vs. 200.0 [142.5-290.0]; P = 0.025), and the rate of neuroimaging improvement was significantly higher in the VPS group (16/17 [94.1%] vs. 2/10 [20%]; P < 0.001). Furthermore, the 24-week cumulative survival rates were also significantly higher in the VPS group (96.6% vs. 83.5%, P = 0.025). Notably, the CSF protein levels were higher in the VPS group than in the non-VPS group at each examination time, and the CSF glucose was lower in the VPS group than in the non-VPS group even at the 12-week follow-up. In the multivariate analysis, we found that VPS placement was an independent risk factor for increased CSF protein (odds ratio [OR]: 27.8, 95% confidence interval [95% CI] 2.2-348.7; P = 0.010). CONCLUSIONS: VPS decreased the intracranial pressure, improved neuroimaging radiology and reduced the 24-week mortality in HCM patients. However, VPS significantly altered the CSF profiles, which could lead to misdiagnosis of tuberculous meningitis and some of them were diagnosed with immune reconstitution inflammatory syndrome. Physicians should be aware of these changes in the CSF profiles of patients with HCM undergoing VPS.

lncRNA MEG3 restrained the M1 polarization of microglia in acute spinal cord injury through the HuR/A20/NF-κB axis.

The M1 polarization of microglia and neuroinflammation restrict the treatment of acute spinal cord injury (ASCI), and long non-coding ribonucleic acid (lncRNA) maternally expressed gene 3 (MEG3) expression is lessened in ASCI. However, the function and mechanism of lncRNA MEG3 in the M1 polarization of microglia and neuroinflammation in ASCI are unclear. The expressions of lncRNA MEG3 in ASCI mouse spinal cord tissues and lipopolysaccharide (LPS)-treated primary microglia and BV2 cells were quantified through a quantitative real-time polymerase chain reaction. In-vitro assays were conducted to explore the function of lncRNA MEG3 in the M1 polarization of microglia and neuroinflammation in ASCI. RNA degradation, RNA immunoprecipitation, RNA pull-down, cycloheximide-chase, and ubiquitination analyses were carried out to probe into the mechanism of lncRNA MEG3 in the M1 polarization of microglia and neuroinflammation in ASCI. The lncRNA MEG3 expression was lessened in the ASCI mouse spinal cord tissues and LPS-treated primary microglia and BV2 cells, and the overexpression of lncRNA MEG3 restrained the M1 polarization of microglia and the neuroinflammation by regulating the NF-κB signaling pathway. For the investigation of the potential mechanism of such, the overexpression of lncRNA MEG3 restrained the M1 polarization of microglia through the HuR/A20/NF-κB axis and boosted the motor function recovery and neuroinflammation relief in the mice with SCI. The overexpression of lncRNA MEG3 restrained the M1 polarization of microglia through the HuR/A20/NF-κB axis.

Mechanism of ARPP21 antagonistic intron miR-128 on neurological function repair after stroke.

OBJECTIVE: Stroke is a cerebrovascular disorder that often causes neurological function defects. ARPP21 is a conserved host gene of miR-128 controlling neurodevelopmental functions. This study investigated the mechanism of ARPP21 antagonistic intron miR-128 on neurological function repair after stroke. METHODS: Expressions of ARPP21 and miR-128 in stroke patients were detected. The mouse neurons and astrocytes were cultured in vitro and treated with oxygen-glucose deprivation (OGD). The OGD-treated cells were transfected with pc-ARPP21 and miR-128 mimic. The proliferation of astrocytes, and the apoptosis of neurons and astrocytes were detected, and inflammatory factors of astrocytes were measured. The binding relationship between miR-128 and CREB1 was verified. The rat model of middle cerebral artery occlusion (MCAO) was established. ARPP21 expression in model rats was detected. The effects of pc-ARPP21 on neuron injury, brain edema volume, and cerebral infarct in rats were observed. RESULTS: ARPP21 expression was downregulated and miR-128 expression was upregulated in stroke patients. pc-ARPP21 facilitated the proliferation of astrocytes and inhibited apoptosis of neurons and astrocytes, and reduced inflammation of astrocytes. miR-128 mimic could reverse these effects of pc-ARPP21 on neurons and astrocytes. miR-128 targeted CREB1 and reduced BDNF secretion. In vitro experiments confirmed that ARPP21 expression was decreased in MCAO rats, and pc-ARPP21 promoted neurological function repair after stroke. CONCLUSION: ARPP21 upregulated CREB1 and BDNF expressions by antagonizing miR-128, thus inhibiting neuronal apoptosis and promoting neurological function repair after stroke. This study may offer a novel target for the management of stroke.

Inhibition of miR-19a-3p decreases cerebral ischemia/reperfusion injury by targeting IGFBP3 in vivo and in vitro.

BACKGROUND: Inflammation and apoptosis are considered to be two main factors affecting ischemic brain injury and the subsequent reperfusion damage. MiR-19a-3p has been reported to be a possible novel biomarker in ischemic stroke. However, the function and molecular mechanisms of miR-19a-3p remain unclear in cerebral ischemia/reperfusion (I/R) injury. METHODS: The I/R injury model was established in vivo by middle cerebral artery occlusion/reperfusion (MCAO/R) in rats and in vitro by oxygen-glucose deprivation and reperfusion (OGD/R) induced SH-SY5Y cells. The expression of miR-19a-3p was determined by reverse transcription quantitative PCR. The infarction volumes, Neurological deficit scores, apoptosis, cell viability, pro-inflammatory cytokines and apoptosis were evaluated using Longa score, Bederson score, TTC, TUNEL staining, CCK-8, ELISA, flow cytometry assays. Luciferase reporter assay was utilized to validate the target gene of miR-19a-3p. RESULTS: We first found miR-19a-3p was significantly up-regulated in rat I/R brain tissues and OGD/R induced SH-SY5Y cells. Using the in vivo and in vitro I/R injury model, we further demonstrated that miR-19a-3p inhibitor exerted protective role against injury to cerebral I/R, which was reflected by reduced infarct volume, improved neurological outcomes, increased cell viability, inhibited inflammation and apoptosis. Mechanistically, miR-19a-3p binds to 3'UTR region of IGFBP3 mRNA. Inhibition of miR-19a-3p caused the increased expression of IGFBP3 in OGD/R induced SH-SY5Y cells. Furthermore, we showed that IGFBP3 overexpression imitated, while knockdown reversed the protective effects of miR-19a-3p inhibitor against OGD/R-induced injury. CONCLUSIONS: In summary, our findings showed miR-19a-3p regulated I/R-induced inflammation and apoptosis through targeting IGFBP3, which might provide a potential therapeutic target for cerebral I/R injury.

Mechanism of miR-320 in Regulating Biological Characteristics of Ischemic Cerebral Neuron by Mediating Nox2/ROS Pathway.

This study aimed to explore the mechanism of miR-320 in regulating biological characteristics of ischemic cerebral neuron by mediating Nox2/ROS pathway. Primary neurons were cultured and grouped: normal group (normal primary neurons), negative control (NC) group (ischemic primary neurons, transfected with negative control plasmid), model group (ischemic primary neurons), miR-320 mimic group (ischemic primary neurons, transfected with miR-320-overexpressed plasmid), Nox2 vector group (ischemic primary neurons, transfected with Nox2-overexpressed plasmid), and miR-320 mimic + Nox2 vector group (ischemic primary neurons, co-transfected with miR-320- and Nox2-overexpressed plasmid). Dual-luciferase reporter assay showed that there was the target relationship between miR-320 and Nox2. miR-320 expression was significantly decreased, and Nox2 expression was significantly increased in the rest groups compared with normal group (both P < 0.05). There was a co-localization of miR-320 and Nox2 in the cytoplasm. Cell proliferation, contents of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX), and mRNA and protein expressions of Ki67, Bcl-2, and c-myc were significantly declined, and apoptosis rate, contents of malondialdehyde (MDA) and reactive oxygen species (ROS), and caspase-3 mRNA and protein expressions were significantly increased in the rest groups compared with normal group (all P < 0.05). miR-320 promoted cell proliferation; increased contents of SOD, CAT, and GSH-PX; and declined apoptosis and contents of MDA and ROS. Moreover, miR-320 could affect the regulation of Nox2/ROS pathway on ischemic cerebral neuron by negatively regulating Nox2 expression. Overexpressed miR-320 affects the proliferation, apoptosis, and oxidative stress injury of ischemic cerebral neuron by inhibiting Nox2/ROS pathway.

Ginkgolide B promotes the proliferation and differentiation of neural stem cells following cerebral ischemia/reperfusion injury, both in vivo and in vitro.

Neural stem cells have great potential for the development of novel therapies for nervous system diseases. However, the proliferation of endogenous neural stem cells following brain ischemia is insufficient for central nervous system self-repair. Ginkgolide B has a robust neuroprotective effect. In this study, we investigated the cell and molecular mechanisms underlying the neuroprotective effect of ginkgolide B on focal cerebral ischemia/reperfusion injury in vitro and in vivo. Neural stem cells were treated with 20, 40 and 60 mg/L ginkgolide B in vitro. Immunofluorescence staining was used to assess cellular expression of neuron-specific enolase, glial fibrillary acid protein and suppressor of cytokine signaling 2. After treatment with 40 and 60 mg/L ginkgolide B, cells were large, with long processes. Moreover, the proportions of neuron-specific enolase-, glial fibrillary acid protein- and suppressor of cytokine signaling 2-positive cells increased. A rat model of cerebral ischemia/reperfusion injury was established by middle cerebral artery occlusion. Six hours after ischemia, ginkgolide B (20 mg/kg) was intraperitoneally injected, once a day. Zea Longa's method was used to assess neurological function. Immunohistochemistry was performed to evaluate the proportion of nestin-, neuron-specific enolase- and glial fibrillary acid protein-positive cells. Real-time quantitative polymerase chain reaction was used to measure mRNA expression of brain-derived neurotrophic factor and epidermal growth factor. Western blot assay was used to analyze the expression levels of brain-derived neurotrophic factor and suppressor of cytokine signaling 2. Ginkgolide B decreased the neurological deficit score, increased the proportion of nestin-, neuron-specific enolase- and glial fibrillary acid protein-positive cells, increased the mRNA expression of brain-derived neurotrophic factor and epidermal growth factor, and increased the expression levels of brain-derived neurotrophic factor and suppressor of cytokine signaling 2 in the ischemic penumbra. Together, the in vivo and in vitro findings suggest that ginkgolide B improves neurological function by promoting the proliferation and differentiation of neural stem cells in rats with cerebral ischemia/reperfusion injury.

3,4-oxo-isopropylidene-shikimic acid inhibits cerebral ischemia-induced oxidative stress and neuronal apoptosis in rats.

BACKGROUND: To investigate the potential protective effects of 3,4-oxo-isopropylidene-shikimic acid (ISA) on brain ischemic injury in rats. METHODS: Cell Counting Kit-8, flow cytometry, and TUNEL were used to evaluate the cell viability and the apoptosis rate in vitro and in situ. Reactive oxygen species generation was determined by DCFH-DA assay. qPCR and Western blot were used to test the molecular mechanisms related to the anti-apoptosis effects. RESULT: Protective effect of pre-conditioning of ISA on the brain injury caused by ischemia was observed. ISA treatment showed anti-apoptosis effects on isolated primary astrocytes and neurons. ROS generation was also significantly scavenged by treatment of ISA. The treatment with ISA protected astrocytes from hypoxic condition-induced apoptosis and ischemic injury. The underlying mechanisms revealed by qPCR and WB showed that the level of mRNA and protein expression of Bax, Bcl-2, and caspase-3 were significantly down-regulated by ISA treatment (P < 0.05). Pre-conditioning with ISA is beneficial in reducing the neuronal damage caused by brain ischemia. CONCLUSION: Treatment with ISA reduces apoptosis and ROS over-generation caused by ischemic injury. Pre-conditioning with ISA resulted in significantly protective effects on brain under ischemic condition.

Relationship of the angle between the A1 and A2 segments of the anterior cerebral artery with formation and rupture of anterior communicating artery aneurysm.

PURPOSE: To measure the angle between A1 and A2 segments of the anterior cerebral artery and analyze the relationship of this angle with the formation and rupture of anterior communicating artery aneurysm (ACoAA). METHODS: Patients with ACoAA (n=64) and with non-ACoAA (n=187) randomly chosen were included. The A1-A2 segment angles were measured using multislice spiral computed tomography angiography. The angular dimensions and differences were recorded and compared between the ACoAA and non-ACoAA groups and the ruptured (n=23) and unruptured group (n=41). The A1 segment morphology was divided into predominant and balanced type. The ACoAA aneurysm protrusion direction was divided into five types. RESULTS: The incidence of ACoAA was significantly higher in patients with A1 predominance compared to A1 balance (p<0.05). The mean A1-A2 segment angle was significantly smaller in the ACoAA group compared with the non-ACoAA group (p<0.001). There was no significant difference in mean A1-A2 segment angle between ruptured and unruptured groups. There was no significant relationship between aneurysm protrusion rupture and direction. CONCLUSIONS: The formation of ACoAA is more likely when there is A1 segment predominance in the anterior cerebral artery. The A1-A2 angle can help predict the formation of ACoAA but not useful for rupture predicting.

Mechanism of temozolomide-induced antitumour effects on glioma cells.

OBJECTIVE: To investigate the mechanisms of action of the tumoricidal effects of temozolomide against the human glioma cell line U251 in vitro, and to provide preclinical proof-of-concept studies of the effects of temozolomide-containing regimens. METHODS: U251 cells were exposed to 100 µmol/l temozolomide. Morphological alterations were monitored by light microscopy. Cell viability was measured using the 3 -(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle analysis and the rate of apoptosis were determined using flow cytometry and the number of acidic vesicular organelles stained with acridine orange were analysed by fluorescence microscopy. The scratch recovery test was used to measure cell migration. RESULTS: U251 cells that were treated with temozolomide displayed morphological alterations indicative of a rounder shape and impaired cellular adhesion to the cell culture plate compared with control U251 cells. Temozolomide reduced cell viability as measured by the MTT assay, caused cell cycle arrest in the gap 2/mitosis phase, inhibited cell migration and promoted autophagy in U251 cells. CONCLUSION: Temozolomide induced autophagic, but not apoptotic processes, in U251 cells and thus reduced their viability and migration.

Effects of tetrahydrobiopterin on cerebral infarction after transient focal ischemia in rats.

OBJECTIVES: The present study investigated the effects of tetrahydrobiopterin (BH4) on cerebral infarction after transient focal ischemia in rats. METHODS: Focal ischemia (1·5 hours) was created in male Sprague-Dawley rats (250-280 g) by middle cerebral artery occlusion. Some rats were treated with 20 mg/kg tetrahydrobiopterin by intraperitoneal injection 30 minutes before reperfusion. At 2, 6, and 12 hours of reperfusion, the brains were harvested for the nitric oxide synthase (NOS) activity and nitric oxide (NO) level assays. At 12 hours of reperfusion, the brains were harvested for infarct size measurement. RESULTS: NOS activity and NO level were all augmented after reperfusion. BH4 treatment significantly further increased NOS activity and NO level. Cerebral infarct size was significantly bigger in BH4 treatment group compared to that in no treatment group. CONCLUSIONS: The data indicate that BH4 enhances cerebral infarction after transient focal ischemia in rats, through NOS and NO pathway.

Treatment of hypertensive intracerebral hemorrhage through keyhole transsylvian approach.

The present study investigated the therapeutic effects and indications of keyhole transsylvian approach (KTA) in the treatment of hypertensive intracerebral hemorrhage (HICH). Clinical data of 65 cases of HICH were retrospectively analyzed. All the patients were treated by open surgical evacuation either through KTA (KTA group) or through conventional craniotomy approach (CCA group). The operative time, intraoperative bleeding quantity, the length of hospitalization, mortality, and favorable outcome were compared between the 2 groups. Compared with the CCA group, the KTA group had smaller bleeding quantity and shorter length of hospitalization. Favorable outcome at 3 months after admission was higher in the KTA group than that in the CCA group. The present study suggests that treatment of HICH through KTA is a practical and effective surgical procedure.

Phenotypic and functional characteristics of dendritic cells derived from human peripheral blood monocytes.

OBJECTIVE: This study is aimed at developing a simple and easy way to generate dendritic cells (DCs) from human peripheral blood monocytes (PBMCs) in vitro. METHODS: PBMCs were isolated directly from white blood cell rather than whole blood and purified by patching methods (collecting the attached cell and removing the suspension cell). DCs were then generated by culturing PBMCs for six days with 30 ng/ml recombinant human granulocyte-macrophage stimulating factor (rhGM-CSF) and 20 ng/ml recombinant human interleukin-4 (rhIL-4) in vitro. On the sixth day, TNF-alpha (TNFalpha) 30 ng/ml was added into some DC cultures, which were then incubated for two additional days. The morphology was monitored by light microscopy and transmission electronic microscopy, and the phenotypes were determined by flow cytometry. Autologous mixed leukocyte reactions (MLR) were used to characterize DC function after TNFalpha or lipopolysaccharide (LPS) stimulations for 24 h. RESULTS: After six days of culture, the monocytes developed significant dendritic morphology and a portion of cells expressed CD1a, CD80 and CD86, features of DCs. TNFalpha treatment induced DCs maturation and up-regulation of CD80, CD86 and CD83. Autologous MLR demonstrated that these DCs possess potent T-cell stimulatory capacity. CONCLUSION: This study developed a simple and easy way to generate DCs from PBMCs exposed to rhGM-CSF and rhIL-4. The DCs produced by this method acquired morphologic and antigenic characteristics of DCs.

Inhibition of NADPH oxidase attenuates vasospasm after experimental subarachnoid hemorrhage in rats.

BACKGROUND AND PURPOSE: The purpose of this study is to investigate whether apocynin, an NADPH oxidase inhibitor, attenuates vasospasm after experimental subarachnoid hemorrhage (SAH) in rats. METHODS: Rats were subjected to endovascular perforation of the right anterior cerebral artery or sham surgery. Beginning 2 hours after SAH, rats were administered 50 mg/kg apocynin or vehicle by intraperitoneal injection 3 times daily for 2 days. RESULTS: In SAH rats, apocynin treatment enlarged basilar artery diameter (SAH/apocynin=253+/-71 microm, SAH/saline=191+/-60 microm, P<0.01; SAH=190+/-58 microm, sham=276+/-52 microm, P<0.01), reduced neurological deficits (SAH/apocynin=24+/-6.5, SAH/saline=18+/-5.3, P<0.05; SAH=18+/-4.7, sham=27+/-0, P<0.01), decreased NADPH oxidase activity (SAH/apocynin=18.4+/-3.7, SAH/saline=25.7+/-5.2, P<0.05; SAH=27.5+/-5.8, sham=15.4+/-4.5 nmol/min per mg protein, P<0.05), decreased superoxide level (SAH/apocynin=6.5+/-1.8, SAH/saline=9.6+/-2.2, P<0.05; SAH=9.8+/-1.9, sham=4.9+/-0.9 arbitrary units, P<0.05), and lowered membrane translocation of NADPH oxidase subunit p47phox. CONCLUSIONS: Inhibition of NADPH oxidase attenuates delayed cerebral vasospasm after experimental SAH, suggesting that the inhibition of NADPH oxidase may provide a therapeutic strategy for vasospasm after SAH.