Clinical research of EX-PRESS drainage device and modified trabeculectomy combined with intravitreal conbercept treatment for neovascular glaucoma.

AIM: To evaluate the efficacy and safety of modified trabeculectomy (experimental group) and implantation of EX-PRESS drainage device (control group), combined with intravitreal conbercept injection for neovascular glaucoma (NVG). METHODS: Totally 30 patients with NVG were selected from June 2014 to June 2017, and randomly divided into experimental group and control group. All patients were underwent intravitreal conbercept (0.5 mg/0.05 mL) treatment before surgery. Modified trabeculectomy was performed in MT group, while EX-PRESS drainage device implantation was performed in EX group. The success rates, best corrected visual acuity (BCVA), intraocular pressure (IOP), filtering bleb and complications were observed and compared. RESULTS: The differences of success rate, BCVA and filtering bleb were not statistically significant 12mo after the surgery (P>0.05), however, the difference of IOP at 1d, 1wk, 1, 3, and 6mo after surgery was statistically significant (F time=390.64, P time<0.0001) between two groups. The interactions between two groups in the given time showed no significant difference (F intergroup×time=0.181, P intergroup×time=0.57), and also there was no significant difference in IOP between the two groups (F=3.16, P=0.09). The results of pairwise comparison at each time point showed no significant difference in IOP between 1d and 1wk, 3 and 6, 3mo and 12mo after surgery (P>0.05), while the results at other time point indicate statistical differences (P<0.05). CONCLUSION: The modified trabeculectomy and the implantation of EX-PRESS drainage device have clinical application value in reducing IOP and postoperative complications of refractory NVG.

LncRNA AK148321 alleviates neuroinflammation in LPS-stimulated BV2 microglial cell through regulating microRNA-1199-5p/HSPA5 axis.

AIMS: Dysregulated long non-coding RNA (lncRNA) expression is closely related to neuroinflammation, leading to multiple neurodegenerative diseases. In this study, we investigated the function and regulation of lncRNA AK148321 in neuroinflammation using an in vitro lipopolysaccharide (LPS)-stimulated BV2 microglial cell system. METHODS: Expression of AK148321 was analyzed by qPCR. Inflammatory cytokine expression levels were determined by ELISA assay. The interaction between AK148321, microRNA (miRNA), and its target gene was validated by luciferase reporter assay and RNA immunoprecipitation (RIP). Cell apoptosis was analyzed by Annexin V/PI staining. RESULTS: LPS treatment suppressed AK148321 expression in BV2 cells. Overexpression of AK148321 inhibited LPS-induced BV2 microglial cell activation and decreased the expression of inflammatory cytokine TNF-α and IL-1ß. AK148321 function as a competing endogenous RNA (ceRNA) by sponging microRNA-1199-5p (MiR-1199-5p). In LPS-stimulated BV2 cells, AK148321 exerted its inhibitory function via negatively modulating miR-1199-5p expression. Moreover, we identified that Heat Shock Protein Family A Member 5 (HSPA5) was a direct target of miR-1199-5p. RIP assay using the anti-Ago2 antibody further validated the relationship among AK148321, miR-1199-5p and HSPA5. The AK148321/miR-1199-5p/HSPA5 axis regulated the neuroinflammation in LPS-induced BV2 microglial cells. Microglial cell culture supernatant from LPS-stimulated, AK148321-overexpressing BV2 cells suppressed the cell apoptosis of mouse hippocampal neuronal cell HT22, while HSPA5 knockdown abrogated the suppression effect. CONCLUSION: Our findings suggest that AK148321 alleviates neuroinflammation in LPS-stimulated BV2 microglial cells through miR-1199-5p/HSPA5 axis.

Compound anisodine affects the proliferation and calcium overload of hypoxia-induced rat retinal progenitor cells and brain neural stem cells via the p-ERK1/2/HIF-1α/VEGF pathway.

As a Traditional Chinese Medicine, compound anisodine (CA) has previously been shown to regulate the vegetative nervous system, improve microcirculation and scavenge reactive oxygen species, and has been commonly utilized as a neuroprotective agent to treat ischemic optic neuropathy and choroidoretinopathy. The present study aimed to investigate the neuroprotective effects of CA on the proliferation and calcium overload of hypoxia-induced rat retinal progenitor cells (RPCs) and brain neural stem cells (BNSCs) harvested from neonatal Sprague-Dawley rats. Cells were treated with CA at 0.126, 0.252, 0.505 or 1.010 g/l for four hours prior to or after hypoxia (<1% oxygen) for four h, followed by re-oxygenation for four hours; a normal control group and a CA-untreated hypoxia model group were also included. An MTT assay demonstrated that the cell viability was markedly improved following treatment with 0.126-1.010 g/l CA, compared with that in the hypoxia model group (P<0.05). Bromodeoxyuridine (BrdU) immunocytochemical staining and flow cytometry indicated that after culture in hypoxia for 4 h, the number of BrdU+ RPCs and BNSCs was significant decreased, as well as the cell population in S+G2 phase of the cell cycle, which was significantly attenuated by treatment with 1.010 g/l CA for 4 h prior to hypoxia (P<0.05). Furthermore, laser scanning confocal microscopy showed that the intracellular calcium concentration in hypoxia-cultured RPCs and BNSCs was markedly increased, which was attenuated by 0.126-1.010 g/l CA in a concentration-dependent manner (P<0.05). Furthermore, western blot analysis demonstrated that after hypoxia, the protein levels of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) were upregulated in RPCs and BNSCs, whereas phosphorylated extracellular signal-regulated kinase (phospho-ERK 1/2Thr202/Tyr204) and Cyclin D1 were downregulated; of note, treatment with 1.010 g/l CA significantly attenuated these changes (P<0.05). The results of the present study suggested that CA may improve the proliferation and inhibit calcium overload in hypoxia-induced RPCs and BNSCs by altering the protein levels of Cyclin D1 as well as signaling through the p-ERK1/2/HIF-1α/VEGF pathway.

Anti-aging effect of simvastatin on retinas of rats and its potential mechanisms / 国际眼科杂志(Guoji Yanke Zazhi)

·AIM: To investigate the anti - aging effect and its potential mechanisms of simvastatin on retinas of physiological aging rats.·METHODS:Totally 40 three-month old healthy SD rats which had no eye diseases, were randomly assigned into two groups: simvastatin group ( n = 20 ) and control group ( n=20 ) . All rats were cultivated under the same conditions until they were nine - month old when interventions started to be given. Simvastatin group was given intragastric administration of 5mg/kg simvastatin every day until 17-month old. Control group was given intragastric administration of same amount of saline gavage. Retinal thickness was measured by HE staining, while Cu - Zn - SOD, NOX2, Bcl - 2 and Bax were determined by immunohistochemistry ( IHC) .·RESULTS:HE staining showed that the retinal structure was clearer; the morphology of cell was more homogeneous;the number of cells was more stable and the structure of retinal pigment epithelium was more compact when compared with control group. Thickness of retinal neuroepithelium layer and retinal pigment epithelium increased significantly in the simvastatin group. Immunohistochemistry analysis showed that the expressions of Cu - Zn - SOD and Bax statistically increased while the NOX2, Bcl-2 as well as Bcl-2/Bax decreased in simvastatin group when compared with control group (P<0. 05).·CONCLUSION: Simvastatin plays a protective role in retinal aging by decreasing oxidative stress reaction and promoting cell apoptosis.

Effect of dihydrotestosterone on the expression of mucin 1 and the activity of Wnt signaling in mouse corneal epithelial cells.

AIM: To explore the effects of the androgen dihydrotestosterone on the expression of mucin 1 (MUC1) and the activity of Wnt signaling in mouse corneal epithelial cells. METHODS: Primary mouse corneal epithelial cells were isolated from the corneas of BALB/c mice. Quantitative real-time polymerase chain reaction, immunofluorescence and Western blot analysis were used to quantify the differential expression of selected genes. The androgen receptor was silenced by transfecting cells with androgen receptor shRNAs. TOP-Flash and FOP-flash reporter plasmids were used to measure ß-catenin-driven transcription. RESULTS: Dihydrotestosterone treatment increased MUC1 expression and activated the Wnt signaling pathway and led to the translocation of ß-catenin and upregulation of the Wnt downstream target gene TATA box binding protein and urokinase plasminogen activator. These effects were prevented by downregulating the androgen receptor. CONCLUSION: Androgens may protect against dry eye by regulating the expression of MUC1 which is stimulated by the activation of Wnt signaling via the androgen receptor. An understanding of the mechanisms associated with androgen-mediated protection against dry eye is an important step in developing new therapies for this disease.

Effects of Low-dose Triamcinolone Acetonide on Rat Retinal Progenitor Cells under Hypoxia Condition.

BACKGROUND: Retinal degenerative diseases are the leading causes of blindness in developed world. Retinal progenitor cells (RPCs) play a key role in retina restoration. Triamcinolone acetonide (TA) is widely used for the treatment of retinal degenerative diseases. In this study, we investigated the role of TA on RPCs in hypoxia condition. METHODS: RPCs were primary cultured and identified by immunofluorescence staining. Cells were cultured under normoxia, hypoxia 6 h, and hypoxia 6 h with TA treatment conditions. For the TA treatment groups, after being cultured under hypoxia condition for 6 h, RPCs were treated with different concentrations of TA for 48-72 h. Cell viability was measured by cell counting kit-8 (CCK-8) assay. Cell cycle was detected by flow cytometry. Western blotting was employed to examine the expression of cyclin D1, Akt, p-Akt, nuclear factor (NF)-κB p65, and caspase-3. RESULTS: CCK-8 assays indicated that the viability of RPCs treated with 0.01 mg/ml TA in hypoxia group was improved after 48 h, comparing with control group (P < 0.05). After 72 h, the cell viability was enhanced in both 0.01 mg/ml and 0.02 mg/ml TA groups compared with control group (all P < 0.05). Flow cytometry revealed that there were more cells in S-phase in hypoxia 6 h group than in normoxia control group (P < 0.05). RPCs in S and G2/M phases decreased in groups given TA, comparing with other groups (all P < 0.05). There was no significant difference in the total Akt protein expression among different groups, whereas upregulation of p-Akt and NF-κB p65 protein expression and downregulation of caspase-3 and cyclin D1 protein expression were observed in 0.01 mg/ml TA group, comparing with hypoxia 6 h group and control group (all P < 0.05). CONCLUSION: Low-dose TA has anti-apoptosis effect on RPCs while it has no stimulatory effect on cell proliferation.

Optical coherence tomography characteristics of responses to intravitreal bevacizumab in idiopathic choroidal neovascularization.

AIM: To investigate factors associated with responses to intravitreal bevacizumab (IVB) in naive idiopathic choroidal neovascularization (iCNV) by high domain optical coherence tomography (OCT). METHODS: We retrospectively reviewed clinical data of 40 eyes of iCNV patients who received a single or multiple IVB on an as-needed basis (1.25 mg/0.05 mL). One month after the first injection, subretinal fluid (SRF) volume was evaluated and the eyes were divided into 3 groups based on responses to IVB. Good, moderate, and poor responses were defined as 61%-99%, 30%-60%, and <30% resolution of SRF on OCT after IVB in iCNV, respectively. OCT findings were analyzed to find factors associated with difference in response levels. Comparisons were made using Wilcoxon's matched-pairs signed-rank test, the Mann-Whitney U test for means with continuous data and Fisher's exact test for categorical data. RESULTS: The mean number of IVB was 1.28±1.50 and mean follow up time was 3.60±1.20mo. At postoperative 1mo, there were 8 (20%) eyes in good response, 20 (50%) in moderate response and 12 (30%) eyes in poor response group and at last visit there were 28 good responders (70%), 8 (20%) moderate responders and 4 (10%) poor responders. Statistically significant difference was detected between good responders and non good responders in choroidal neovessels thickness (P=0.029), SRF height (P=0.049) and SRF volume (P=0.031) at post treatment 1mo. CONCLUSION: OCT is a valuable diagnostic tool. Decrease in choroidal neovessels thickness, SRF height and volume predicts favorable response of iCNV to IVB therapy.

In Vivo Retinal Vein Bypass Surgery in a Porcine Model.

PURPOSE: To evaluate the feasibility of retinal vein bypass surgery for induced branch retinal-vein occlusion (BRVO) in the living porcine eye. METHODS: Fifteen minipigs were used in the study. Seven days before vascular surgery, hyaluronidase and plasmin were intravitreally injected for induction of posterior vitreous detachment. Aspirin and warfarin were oral administered daily starting 5 d prior to vascular surgery for anti-coagulation. The minipigs were anethetized with an intraperitoneal injection of 300 mg/kg chloral hydrate for intravitreal injection procedure and vascular surgery. Temporary keratoprosthesis vitrectomy was performed, and intraoperative video fluorescein angiography (VFA) was possible. The central and posterior vitreous was removed together with the posterior hyaloid membrane to facilitate vascular maneuvers. BRVO was induced by bipolar diathermy on the vein at the main vein's first branching. Polyimide tubes (50.8-µm internal diameter and 7.6-µm wall thickness) were used as artificial vessels. Vascular manipulation was performed in a bimanual manner. Both end of a prepared tubing was inserted into venous lumen by puncturing and catheterization, and the vein bypass bridging the occlusion was created. Then, the patency of the bypass graft was assessed by intraoperative VFA. RESULTS: The retinal vein bypass surgery was surgically accomplished in 33% (5/15) of the eyes, and the immediate graft patency was confirmed by intraoperative VFA only in one eye. We observed and recorded fluorescein flow from the branch vein to the main vein through the bypass graft which bridging the occlusive vein segment. CONCLUSIONS: We demonstrated the feasibility of retinal vein bypass for induced BRVO in the living porcine eye, and the immediate graft patency was successfully evaluated by intraoperative VFA. Despite the potential, there are still some significant hurdles in vivo retinal vein bypass surgery, and modification of both surgical instruments and maneuvers is needed for further study.

MICROSTRUCTURAL EFFECTS OF INTRAVITREAL BEVACIZUMAB IN IDIOPATHIC CHOROIDAL NEOVASCULARISATION.

BACKGROUND: Idiopathic choroidal neovascularization (ICNV) is a unilateral ocular disease which occurs in patients younger than 50 years and accounts for approximately 17% of patients with CNV. We evaluated microstructural effects of intravitreal bevacizumab in eyes with treatment-naïve idiopathic choroidal neovascularisation. METHODS: In this case series study we reviewed the treatment and follow up records of 40 symptomatic eyes having ICNV, who received an intravitreal injection of bevacizumab (1.25 mg/0.05 mL) followed by additional doses based on optical coherence tomography findings, including intraretinal fluid, subretinal fluid, or pigment epithelial detachment. We analysed the results of best-corrected visual acuity, central retinal thickness, neovessels size (thickness and diameter), and disrupted photoreceptor length at baseline and at final visit with paired t-test. Difference in best corrected visual acuity was correlated with difference in optical coherence tomography parameters by Pearson's correlation. RESULTS: Mean logarithm of the minimum angle of resolution best-corrected visual acuity improved from 0.60 initially to 0.24 after treatment (p=0.01). Difference in mean central retinal thickness (82.65 +/- 44.1) pm, choroidal neovessels thickness (149.58 +/- 71.1) microm, choroidal neovessels diameter (1250.8 +/- 145.1) pm, photoreceptor disruption length (2141.20 +/- 318.8) microm were all statistically significant (p=0.01). Difference in best corrected visual acuity was correlated with optical coherence tomography parameters found no statistically significant difference. CONCLUSION: Intravitreal bevacizumab therapy is safe and well tolerated in ICNV eyes. Restoration of photoreceptor disruption length, decrease in central retinal thickness and choroidal neovessels size has association with visual improvement in idiopathic choroidal neovascularisation.

Whole-exome sequencing identifies USH2A mutations in a pseudo-dominant Usher syndrome family.

Usher syndrome (USH) is an autosomal recessive (AR) multi-sensory degenerative disorder leading to deaf-blindness. USH is clinically subdivided into three subclasses, and 10 genes have been identified thus far. Clinical and genetic heterogeneities in USH make a precise diagnosis difficult. A dominant­like USH family in successive generations was identified, and the present study aimed to determine the genetic predisposition of this family. Whole­exome sequencing was performed in two affected patients and an unaffected relative. Systematic data were analyzed by bioinformatic analysis to remove the candidate mutations via step­wise filtering. Direct Sanger sequencing and co­segregation analysis were performed in the pedigree. One novel and two known mutations in the USH2A gene were identified, and were further confirmed by direct sequencing and co­segregation analysis. The affected mother carried compound mutations in the USH2A gene, while the unaffected father carried a heterozygous mutation. The present study demonstrates that whole­exome sequencing is a robust approach for the molecular diagnosis of disorders with high levels of genetic heterogeneity.

OPTICAL COHERENCE TOMOGRAPHIC CHARACTERISTICS OF IDIOPATHIC CHOROIDAL NEOVASCULARISATION.

BACKGROUND: Idiopathic choroidal neovascularisation (ICNV) is the development of choroidal neovascularisation (CNV) in young adults without any apparent manifestations of primary ocular or systemic diseases We aim to assess characteristics and pathological changes at various stages in ICNV by optical coherence tomography (OCT). METHODS: We reviewed clinical charts of 40 ICNV eyes and classified them into three stages. Active stage < 1 month, intermediate 1-3 months and cicatricial > 3 months period after initiation of treatment in naïve ICNV eyes. OCT characteristics of these morphological changes were determined. Parameters such as mean volume (MV), central macular thickness (CMT) and neovessels size (thickness and diameter) were analyzed and compared using one -way ANOVA. RESULTS: We have 12 males and 28 females with a mean age of 30.1 ± 7.80 years. In active stage, heterogenous activity of CNV was observed, along with disrupted RPE layer, surrounded by subretinal fluids and loss of foveal depression. In intermediate stage, CNV reflection appears homogenous with smooth peripheral Retinal pigment epithelium (RPE) lesion and reduction in retinal thickness. In cicatricial stage, OCT presents dome shaped elevation, strong homogenous reflection, absence of subretinal fluids and reformation of foveal depression. We have found that difference in mean volume and choroidal neovessels thickness was statistically significant in the three stages. CONCLUSIONS: In our study we have concluded that OCT is useful tool for following the clinical course of ICNV and understanding the pathological changes in CNV regression.

[Effects of transforming growth factor-ß2 on transdifferentiation, extracellular matrix synthesis and connective tissue growth factor expression of human lens epithelium cells].

OBJECTIVE: To explore the effects of transforming growth factor-ß2 (TGF-ß2) on the transdifferentiation, extracellular matrix synthesis and connective tissue growth factor (CTGF) expression in human lens epithelium cells (HLEC) in vitro. METHODS: HLEC were incubated with different concentrations of TGF-ß2 (0.0, 0.1, 1.0 and 10.0 µg/L) for 24 h in vitro. The morphological changes of HLEC were observed under inverted phase-contrast microscope. The expression of CTGF and α-smooth muscle actin (α-SMA, as a landmark protein of epithelial mesenchymal transition) in HLEC was measured by immunofluorescence method. Real-time PCR and Western blot were used to evaluate the expression of CTGF, α-SMA, fibronectin (Fn) and COL-I (as the major components of extracellular matrix) after stimulating by TGF-ß2. RESULTS: Normal HLEC presented polygonal shape and were anchorage-dependent. After incubated with different concentrations of TGF-ß2 for 24 h, the morphology of polygonal HLEC was changed into fibroblast-like shape and changed from monolayer and to multilayer cells, and the intercellular space became bigger. CTGF and α-SMA were expressed in the cytoplasm after induction of TGF-ß2. Expression of CTGF in HLEC was increased with increasing concentrations of TGF-ß2 (CTGF protein expression: 0.53 ± 0.03, 0.73 ± 0.01, 0.65 ± 0.03 in cells cultured with 0.1, 1.0 and 10 µg/L TGF-ß2, respectively; CTGF gene induction: 1.00 ± 0.00, 7.18 ± 0.41, 12.88 ± 0.45, 32.84 ± 1.61 in cells cultured with 0.0, 0.1, 1.0 and 10.0 µg/L TGF-ß2, respectively) (F = 77.55, P < 0.05; F = 379.0, P < 0.05). TGF-ß2 could induce HLEC transdifferentiation and accelerate. α-SMA expression was increased by TGF-ß2 dose-dependently (protein expression: 0.48 ± 0.01,0.78 ± 0.04, 0.69 ± 0.04; gene induction: 1.00 ± 0.00, 2.30 ± 0.22, 3.1 ± 0.21, 3.86 ± 0.10) (F = 62.73, P < 0.05; F = 80.22, P < 0.05). TGF-ß2 also promoted expression of Fn and COL-I in a dose-dependent manner (COL-I gene induction: 1.00 ± 0.00, 5.52 ± 0.96, 18.31 ± 1.2, 82.51 ± 1.45;COL-I protein expression: 0.78 ± 0.05, 1.15 ± 0.11, 2.16 ± 0.14; Fn gene induction: 1.00 ± 0.00, 2.36 ± 0.25, 3.27 ± 0.24, 4.25 ± 0.24; Fn protein expression: 0.64 ± 0.01,0.95 ± 0.02, 1.23 ± 0.14) (F = 1881.52, 105.30, P < 0.05; F = 64.44, 51.81, P < 0.05). CONCLUSION: TGF-ß2 induces the expression of CTGF by HLEC, promotes transdifferentiation of and extracellular matrix synthesis by HLEC.

Septicemia caused by Leifsonia aquatica in a healthy patient after retinal reattachment surgery.

Leifsonia aquatica is an aquatic bacterium that is typically found in environmental water habitats. Infections due to L. aquatica are rare and commonly catheter associated in immunocompromised patients. We report the first case of an acute septicemia caused by L. aquatica in a healthy immunocompetent host after cryopexy in the absence of a catheter.

Effects of transforming growth factor ß2 and connective tissue growth factor on induction of epithelial mesenchymal transition and extracellular matrix synthesis in human lens epithelial cells.

AIM: To investigate the effects of transforming growth factor ß2 (TGF-ß2) and connective tissue growth factor (CTGF) on transdifferentiation of human lens epithelial cells (HLECs) cultured in vitro and synthesis of extracellular matrix (ECM). METHODS: HLECs were treated with TGF-ß2 (0, 0.5, 1.0, 5, 10µg/L) and CTGF (0, 15, 30, 60, 100µg/L) for different times (0, 24, 48, 72h) in vitro and the expression of α-smooth muscle actin (α-SMA), the main component of the extracellular matrix type I collagen (Col-1) and fibronectin (Fn) were measured by using real-time polymerase chain reaction (PCR) and western-blot. RESULTS: TGF-ß2 and CTGF significantly increased expression of α-SMA mRNA and protein (P<0.05, P<0.001), Fn mRNA and protein (P<0.001), Col-1 mRNA and protein (P<0.001). TGF-ß2 could induce HLECs expression of CTGF mRNA and protein in dose-dependent manner (P<0.05, P<0.001). TGF-ß2 and CTGF could induce HLECs to express α-SMA, Fn and Col-1 in time-dependent manner. Each time of TGF-ß2 and CTGF induced HELCs expression of α-SMA, Fn, Col-1 mRNA and protein was significant increase compared with control (P<0.05, P<0.001). CONCLUSION: TGF-ß2 and CTGF could induce HLECs epithelial mesenchymal transition and ECM synthesis.

[A simple method for assessment of RNA integrity in laser capture microdissection samples].

OBJECTIVE: To develop a simple method for assessment of RNA integrity in laser capture microdissection (LCM) samples. METHODS: The total RNA were isolated from the LCM samples and the sections before and after microdissection and examined by agarose gel electrophoresis. Real-time PCR was employed to assess the RNA from LCM samples, and the quantity of RNA was theoretically estimated according to the average total RNA product in mammalian cells (10 ng/1000 cells). RESULTS: When the total RNA from the sections before and after microdissection was intact, the RNA from LCM samples also had good quality, and the 28S and 18S rRNAs were visualized by ethidium bromide staining. Real-time PCR also showed good RNA quality in the LCM samples. CONCLUSION: A simple method for quantitative and qualitative assessment of the RNA from LCM samples is established, which can also be applied to assessment of DNA or proteins in LCM samples.

[Construction and identification of a plasmid with EGFP reporter gene for enhancer function analysis].

OBJECTIVE: To construct a plasmid vector with EGFP reporter gene for functional analysis of enhancers. METHODS: EGFP DNA was amplified by PCR from plasmid pEGFP-N1 DNA and subcloned into plasmid PGL3-promoter backbone without luc(+) gene to construct the enhancer-identifying vector pEGFP-enhancer. Different copies of hypoxia response element (HRE) sequence were synthetized and subcloned into the multiple cloning site of the plasmid pEGFP-enhancer. Using Lipofectamine 2000, the recombined pEGFP-HRE and pEGFP-5HRE plasmids were transfected into the Hela cells respectively. After hypoxic or normoxic cell culture, EGFP expression in the cells was detected by flow cytometry and fluorescence microscopy. RESULTS: After hypoxic exposure, the fluorescence intensity of EGFP in the Hela cells transfected with the plasmid increased with the enhancer HRE copies, while the fluorescence intensity underwent no significant changes after normoxic cell culture. CONCLUSION: we have successfully constructed the enhancer expression vector plasmid pEGFP-enhancer, which can identify the activity of the enhancers through EGFP expression.

[In vivo labeling of cells in the ependyma/subventricular zone of adult rats].

OBJECTIVE: To identify the labeled cells in the ependyma/subventricular zone (SVZ) of normal adult rats by DiI injected into the lateral ventricle. METHODS: Fifty male Sprague-Dawley rats were divided randomly into five groups (10 per group). All of the rats were injected with 10 microL of 2 g/L fluorescence dye DiI into the right lateral ventricle. The five groups of rats were sacrificed at 6 h, 12 h, 24 h, 36 h or 48 h after injection respectively. Hoechst 33258 staining was used to identify nuclei and laser confocal microscopy was used to detect the DiI-labeled cells and to measure the thickness of the tissue with DiI fluorescence in the wall of the left lateral ventricle. RESULTS: After injection of the DiI into the right lateral ventricle, DiI- Hoechst 33258 double positive cells were found in the ependymal layer of the left lateral ventricular wall at 24 h and in the SVZ at 48 h as well. The thickness of the tissue with DiI fluorescence in the left ependyma/septal subventricular zone (SVZspt) and ependyma/postnatal equivalent of the ganglionic eminences (SVZge) remained unchanged at 12 h and 24 h after DiI injection. The thickness of the tissue with DiI fluorescence in the left ependyma/SVZge was significantly greater than that in the ependyma/SVZspt at all of the time points (P<0.05). CONCLUSION: The ependyma/SVZ cells can be labeled by Dil 24-48 h after injection (10 microL of 2 g/L) into the lateral ventricle.

[Effect of histamine on intracortical blood vessels of rats].

OBJECTIVE: To investigate histamine-induced changes of the intracortical vessels in the cortical slice of rat brain. METHODS: Immunohistochemistry was employed to detect the expression of H1 and H2 receptors in the intracortical blood vessels of rats. Histamine-induced constriction of the intracortical blood vessels of the brain slices was observed with differential interference contrast microscope. Measurements of the luminal diameter were made on-line during the course of the experiment and confirmed off-line from the stored images. In order to observe whether histamine H1 and H2 receptors affected histamine-induced constriction, the intracortical blood vessels in the brain slices were pre-treated with H1 receptor antagonist diphenhydramine and H2 receptor antagonist cimetidine. RESULTS: Expression of H1 and H2 receptors was detected in the intracortical blood vessels of the rat brain. Histamine (1-100 micromol/L) induced a concentration-dependent constriction from (1.48-/+0.67)% to (32.91-/+7.91)%. The reactions to each histamine concentration were significantly (P<0.01) different from each other, with the exception of the highest histamine concentrations (30 and 100 micromol/L) when maximal constriction due to histamine were observed (P>0.05). With pre-treatment of the slice with 10 micromol/L diphenhydramine, application of histamine did not elicit constriction. Pre-treatment of the slice with 10 micromol/L cimetidine did not completely inhibit but somehow significantly weakened vascular constriction in response to histamine treatment at 10 and 30 micromol/L (P<0.05). CONCLUSION: Histamine can induce constriction of the intracortical blood vessels, which is mediated by H1 receptor.

[Effect of citicoline on spatial learning and memory of rats after focal cerebral ischemia].

OBJECTIVE: To investigate the effects of citicoline on spatial learning and memory of rats after focal cerebral ischemia. METHODS: The rats were randomly divided into sham-operation group, ischemia control group and citicoline group. In the later two groups, focal cerebral ischemia model was established by introducing an intraluminal filament into the left middle cerebral artery, and citicoline (500 mg/kg) or 0.9% NaCl was administered intraperitoneally once a day for 2 weeks after the operation. The rats in the sham-operation group were not subjected to middle cerebral artery occlusion (MCAO) with intraluminal filament. The spatial learning and memory functions of the rats were evaluated by Morris water maze test 15 days after MCAO for 5 days. RESULTS: The rats in ischemia control group exhibited serious spatial learning and memory deficits in both place navigation test and spatial probe test. In the former test, the mean escape latency of citicoline-treated rats were significantly shorter than that of ischemia control rats (P<0.01), and in the latter test significant diffidence was noted between citicoline and ischemia control groups in the percentage time spent in the former platform quadrant and frequency of crossing the former platform (P<0.05). CONCLUSION: Citicoline can improve the spatial learning and memory function of rats after focal cerebral ischemia.

[A comparison study for differentiation between human fetal retinal progenitor cells and brain neural stem cells in vitro].

OBJECTIVE: Investigating the potential of differentiation of human fetal retinal progenitor cells (hRPCs) and brain neural stem cells (hBNSCs) in vitro. METHODS: hRPCs and hBNSCs were isolated from human fetuses (8-12 weeks of gestation) and cultured in serum-free DMEM/F12 culture medium with N2 supplement, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) or culture medium with 10% fetal bovine serum (FBS) but without EGF and bFGF. Immunocytochemical and immunofluorescence studies were conducted for identification of neural stem cells, retinal progenitors or the subtypes of neurons, astrocytes, retinal ganglion cells and rod photoreceptors with the specific antibodies for Nestin, Pax6, Map2, GFAP, Thy-1 and Rhodopsin, respectively. RESULTS: Both hRPCs and hBNSCs could proliferate and differentiate in DMEM/F12 + N2 with or without 10% FBS and expressed specific markers of immature neuroepithelial cells, retinal progenitors, mature neurons, astrocytes, retinal ganglion cells and rod photoreceptors. hBNSCs easily attached, spread out longer neurites and to form a network when cultured with serum contained medium. hRPCs were more difficult to attach and had only short dendrites. CONCLUSIONS: Both hRPCs and hBNSCs can differentiate into retinal specific cell types in vitro. The adherent, migration and differential capacity of hRPCs and hBNSCs are different when these cells are induced by the serum-contained culture medium.