Trimethylamine N-oxide aggravates vascular permeability and endothelial cell dysfunction under diabetic condition: in vitro and in vivo study.

AIM: To provide the direct evidence for the crucial role of trimethylamine N-oxide (TMAO) in vascular permeability and endothelial cell dysfunction under diabetic condition. METHODS: The role of TMAO on the in vitro biological effect of human retinal microvascular endothelial cells (HRMEC) under high glucose conditions was tested by a cell counting kit, wound healing, a transwell and a tube formation assay. The inflammation-related gene expression affected by TMAO was tested by real-time polymerase chain reaction (RT-PCR). The expression of the cell junction was measured by Western blotting (WB) and immunofluorescence staining. In addition, two groups of rat models, diabetic and non-diabetic, were fed with normal or 0.1% TMAO for 16wk, and their plasma levels of TMAO, vascular endothelial growth factor (VEGF), interleukin (IL)-6 and tumor necrosis factor (TNF)-α were tested. The vascular permeability of rat retinas was measured using FITC-Dextran, and the expression of zonula occludens (ZO)-1 and claudin-5 in rat retinas was detected by WB or immunofluorescence staining. RESULTS: TMAO administration significantly increased the cell proliferation, migration, and tube formation of primary HRMEC either in normal or high-glucose conditions. RT-PCR showed elevated inflammation-related gene expression of HRMEC under TMAO stimulation, while WB or immunofluorescence staining indicated decreased cell junction ZO-1 and occludin expression after high-glucose and TMAO treatment. Diabetic rats showed higher plasma levels of TMAO as well as retinal vascular leakage, which were even higher in TMAO-feeding diabetic rats. Furthermore, TMAO administration increased the rat plasma levels of VEGF, IL-6 and TNF-α while decreasing the retinal expression levels of ZO-1 and claudin-5. CONCLUSION: TMAO enhances the proliferation, migration, and tube formation of HRMEC, as well as destroys their vascular integrity and tight connection. It also regulates the expression of VEGF, IL-6, and TNF-α.

Time trends and gender disparities of Chinese cataract burden and their predictions.

AIM: To evaluate the trends and changes in the number and rates of disability-adjusted life years (DALYs) and prevalence of cataract in China between 1990 and 2019, and to predict the trends of cataract burden from 2020 to 2030. METHODS: The Global Burden of Diseases (GBD) database was employed to collect the data on DALYs and the prevalence of cataract in China, which was distinguished by age and sex during the past three decades from 1990 to 2019, and then changes in the number and rates of cataract from 2020 to 2030 were predicted. All data were analyzed by the R program (version 4.2.2) and GraphPad Prism 9.0 statistics software. RESULTS: The number of DALYs of cataract increased from 449 322.84 in 1990 to 1 087 987.61 in 2019, number of cataract cases increased from 5 607 600.94 in 1990 to 18 142 568.96 in 2019. The age-standardized DALY rates (ASDR) generally increased slightly [estimated annual percentage change (EAPC=0.1; 95%CI: -0.24 to 0.45), age-standardized prevalence rates (ASPR) also increased (EAPC=0.88; 95%CI: 0.6 to 1.15). Cataract burden increased with age and female gender. Among the causes of cataract, air pollution was the most important, followed by smoking, high fasting plasma glucose, and high body mass index (BMI). The burden of cataract is predicted to grow persistently from 2020 to 2030, the number of DALYs and prevalence for cataract will rise to 2 336 431 and 43 698 620 respectively by 2030, the ASDR is predicted to be 85/100 000 and ASPR will be 1586/100 000 in 2030, females will still be at greater risk of suffering from cataract than males. CONCLUSION: The burden of cataract in China kept rising from 1990 to 2019. Increasing age and female gender are risk factors for cataract. Air pollution, smoking, high fasting plasma glucose, and high BMI are associated with cataract. The burden of cataract in China will gradually increase from 2020 to 2030, the elderly women in particular need attention. Our results may be of help for providing reference strategies to reduce cataract burden in the near future.

Efficacy and safety of ultrasound cycloplasty for the treatment of glaucoma: a Meta-analysis.

AIM: To evaluate the efficacy and safety of ultrasound cycloplasty (UCP) for glaucoma. METHODS: A comprehensive search of PubMed, Embase, Web of Science, and Google Scholar databases was used to select studies met the inclusion criteria. Meta-analysis was performed by Review Manager and StataCorp LLC. RESULTS: A total of 19 articles met the inclusion criteria. Overall, UCP is effective and safe in the glaucoma treatment, the risk ratio (RR) of the success rate was 2.28 (95%CI, 1.82-2.84). After UCP, patients had a significant reduction in intraocular pressure (IOP; mm Hg), the weighted mean difference (WMD) was 11.39 (95%CI, 9.88-12.90). In addition, UCP brings fewer postoperative complications with RR of 0.30 (95%CI, 0.19-0.49). Most of the complications were short-term and mild. Postoperatively, patients' use of IOP-lowering medications reduced, the standardized mean difference (SMD) was 0.78 (95%CI, 0.40-1.17). However, best corrected visual acuity (BCVA; logMAR) did not have obvious improvement after UCP, the WMD was 0.01 (95%CI, -0.06-0.09). This procedure does provide painfulness relief, with RR of 3.06 (95%CI, 1.95-4.81). CONCLUSION: UCP is effective and safe for suitable glaucoma. It can effectively decrease IOP in glaucoma patients, reduce the patients' dependence on IOP-lowering medications after surgery, relief the painfulness and has fewer long-term or severe postoperative complications, but the BCVA did not improve much.

Subcutaneous pedicled propeller flap technique for microscopic reconstruction of eyelid defects.

AIM: To describe the subcutaneous pedicled propeller flap technique for the microscopic reconstruction of eyelid defects and evaluate its outcomes. METHODS: The clinical data of 23 patients (23 eyes) who underwent microscopic reconstruction of eyelid defects with the subcutaneous pedicled propeller flap technique were retrospectively analyzed. All patients underwent eyelid tumor resection and one-stage microscopic reconstruction with the subcutaneous pedicled propeller flap for anterior- or posterior-layer eyelid defects. The survival rate of the propeller flap, eyelid function and appearance, tumor recurrence rate, and patient satisfaction were evaluated after the surgery. RESULTS: The patients consisted of 12 men and 11 women, aged 31-82y (mean, 58.9y). The longest follow-up time was 5y, and the shortest was 3mo. All the propeller flaps survived well. There was no significant difference in color and luster between the flap and adjacent tissues, and there was no dog ear phenomenon. No obvious scarring was observed. There were no obvious abnormalities of eyelid morphology or function, and no adverse complications such as exposure keratitis, entropion, ectropion, ptosis, and eyelid retraction. No tumor recurrence was found at the time of the last follow-up. All patients were satisfied with the surgical results. CONCLUSION: The subcutaneous pedicled propeller flap technique for the microscopic reconstruction of eyelid defects has satisfactory outcomes in terms of eyelid function and esthetics, and merits clinical application.

Influence of lens position as detected by an anterior segment analysis system on postoperative refraction in cataract surgery.

AIM: To predict postoperative intraocular lens (IOL) position using the Sirius anterior segment analysis system and investigate the effect of lens position and IOL type on postoperative refraction. METHODS: A total of 97 patients (102 eyes) were enrolled in the final analysis. An anterior segment biometry measurement was performed preoperatively with Sirius and Lenstar. The results of predicted lens position (PLP) and IOL power were automatically calculated by the software used by the instruments. Effective lens position (ELP) was measured manually using Sirius 3mo postoperatively. Pearson's correlation analysis and linear regression analysis were used to determine the correlation of lens position to other parameters. RESULTS: PLP and ELP were positively correlated to axial length (AL; r=0.42, P<0.0001 and r=0.49, P<0.0001, respectively). There was a weak correlation between the peLP (ELP-PLP) and the prediction error of spherical refraction (peSR; r=0.34, P<0.0001). The peLP of Softec HD IOL differed statistically from those of both the TECNIS ZCB00 and Sensor AR40E IOLs. Multiple linear regression was used to obtain the prediction formula: ELP=0.66+0.63×[aqueous depth (AQD)+0.6LT] (r=0.61, P<0.0001), and a new variable (AQD+0.6 LT) was found to have the strongest correlation with ELP. CONCLUSION: The Sirius anterior segment analysis system is helpful to predict ELP, which reduces postoperative refraction error.

Proteomic analysis of anti-angiogenic effects by conbercept in the mice with oxygen induced retinopathy.

AIM: To analyze the retinal proteomes with and without conbercept treatments in mice with oxygen-induced retinopathy (OIR) and identify proteins involved in the molecular mechanisms mediated by conbercept. METHODS: OIR was induced in fifty-six C57BL/6J mouse pups and randomly divided into four groups. Group 1: Normal17 (n=7), mice without OIR and treated with normal air. Group 2: OIR12/EXP1 (n=14), mice received 75% oxygen from postnatal day (P) 7 to 12. Group 3: OIR17/Control (n=14), mice received 75% oxygen from P7 to P12 and then normal air to P17. Group 4: Lang17/EXP2 (n=21), mice received 75% oxygen from P7 to P12 with intravitreal injection of 1 µL conbercept at the concentration of 10 mg/mL at P12, and then normal air from P12 to P17. Liquid Chromatography-Mass Spectrometry (LC-MS)/MS data were reviewed to find proteins that were up-regulated after the conbercept treatment. Gene ontology (GO) analysis was performed of conbercept-mediated changes in proteins involved in single-organism processes, biological regulation, cellular processes, immune responses, metabolic processes, locomotion and multiple-organism processes. RESULTS: Conbercept induced a reversal of hypoxia-inducible factor 1 signaling pathway as revealed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and also induced down-regulation of proteins involved in blood coagulation and fibrin clot formation as demonstrated by the Database for Annotation, Visualization and Integrated Discovery (DAVID) and the stimulation of interferon genes studies. These appear to be risk factors of retinal fibrosis. Additional conbercept-specific fibrosis risk factors were also identified and may serve as therapeutic targets for fibrosis. CONCLUSION: Our studies reveal that many novel proteins are differentially regulated by conbercept. The new insights may warrant a valuable resource for conbercept treatment.

Blockade of insulin receptor substrate-1 inhibits biological behavior of choroidal endothelial cells.

AIM: To investigate the effects of blockade of insulin receptor substrate-1 (IRS-1) on the bio-function of tube formation of human choroidal endothelial cells (HCECs). METHODS: Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were performed to determine the expression level of IRS-1 and phospho-IRS-1 in HCECs. Tube formation of HCECs was analyzed using three dimensional in vitro Matrigel assay with or without IRS-1 blockage via IRS-1 inhibitor (GS-101) and vascular endothelial growth factor receptor 2 (VEGFR2) inhibitor. In addition, cell counting kit (CCK)-8 and Transwell migration assay were exerted to analyze the effects of blockade of IRS-1 on the bio-function of proliferation and migration of HCECs, respectively. The apoptosis of HCECs was examined using flow cytometry (FCM). RESULTS: RT-PCR and Western blot revealed that IRS-1 phospho-IRS-1 were expressed in HCECs and the expression level was enhanced by stimulation of VEGF-A. The number of tube formation was decreased significantly in GS-101 treated groups compared to phosphate buffered saline (PBS) treated control groups. Furthermore, both cell proliferation and migration of HCECs were decreased in the presence of GS-101. FCM analysis showed that the apoptosis of HCECs was enhanced when the cells were treated with GS-101. Western blot also showed that the expression level of cleaved-caspase 3 in GS-101 treated group was higher than that in control group. CONCLUSION: Blockade of IRS-1 can inhibit tube formation of HCECs through reducing cell proliferation and migration and promoting cell apoptosis.

An Evaluation System of Fundus Photograph-Based Intelligent Diagnostic Technology for Diabetic Retinopathy and Applicability for Research.

INTRODUCTION: In April 2018, the US Food and Drug Administration (FDA) approved the world's first artificial intelligence (AI) medical device for detecting diabetic retinopathy (DR), the IDx-DR. However, there is a lack of evaluation systems for DR intelligent diagnostic technology. METHODS: Five hundred color fundus photographs of diabetic patients were selected. DR severity varied from grade 0 to 4, with 100 photographs for each grade. Following that, these were diagnosed by both ophthalmologists and the intelligent technology, the results of which were compared by applying the evaluation system. The system includes primary, intermediate, and advanced evaluations, of which the intermediate evaluation incorporated two methods. Main evaluation indicators were sensitivity, specificity, and kappa value. RESULTS: The AI technology diagnosed 93 photographs with no DR, 107 with mild non-proliferative DR (NPDR), 107 with moderate NPDR, 108 with severe NPDR, and 85 with proliferative DR (PDR). The sensitivity, specificity, and kappa value of the AI diagnoses in the primary evaluation were 98.8%, 88.0%, and 0.89, respectively. According to method 1 of the intermediate evaluation, the sensitivity of AI diagnosis was 98.0%, specificity 97.0%, and the kappa value 0.95. In method 2 of the intermediate evaluation, the sensitivity of AI diagnosis was 95.5%, the specificity 99.3%, and kappa value 0.95. In the advanced evaluation, the kappa value of the intelligent diagnosis was 0.86. CONCLUSIONS: This article proposes an evaluation system for color fundus photograph-based intelligent diagnostic technology of DR and demonstrates an application of this system in a clinical setting. The results from this evaluation system serve as the basis for the selection of scenarios in which DR intelligent diagnostic technology can be applied.

High glucose: activating autophagy and affecting the biological behavior of human lens epithelial cells.

AIM: To clarify the effect of autophagy on human lens epithelial cells (HLECs) under high glucose conditions. METHODS: HLECs were cultured with different concentrations of glucose and 3-methyladenine (3-MA); the expression of autophagy-related protein LC3B was detected by Western blotting and immunofluorescence histochemistry. The migration of HLECs was quantified by scratch wound assay and the expression of transforming growth factor-ß (TGF-ß) was measured by real-time polymerase chain reaction. RESULTS: Compared with 5 mmol/L normal glucose treatment, 40 mmol/L glucose treatment can significantly increase the generation of autophagosome in HLECs, which could be inhibited by 0.375 mmol/L 3-MA treatment. The migration of HLECs and the expression of TGF-ß in HLECs induced by high glucose were significantly suppressed by 0.375 mmol/L 3-MA treatment. CONCLUSION: Autophagy promotes HLECs cell migration and increases the expression of TGF-ß after exposed to high glucose, which may relate to the development of diabetic cataract.

Monocyte chemoattractant protein 1 and fractalkine play opposite roles in angiogenesis via recruitment of different macrophage subtypes.

AIM: To explore the interaction between macrophages and chemokines [monocyte chemoattractant protein 1 (MCP-1/CCL2) and fractalkine/CX3CL1] and the effects of their interaction on neovascularization. METHODS: Human peripheral blood mononuclear cells, donated by healthy volunteers, were separated and cultured in RPMI-1640 medium containing 10% fetal bovine serum, then induced into macrophages by stimulation with 30 µg/L granulocyte macrophage-colony stimulating factor (GM-CSF). The expression of CCR2 and/or CX3CR1 in the macrophages was examined using flow cytometry. Macrophages were then stimulated with recombinant human CCL2 (rh-CCL2) or recombinant human CX3CL1 (rh-CX3CL1). The expression of angiogenesis-related genes, including VEGF-A, THBS-1 and ADAMTS-1 were examined using real-time quantitative polymerase chain reaction (PCR). Supernatants from stimulated macrophages were used in an assay of human retinal endothelial cell (HREC) proliferation. Finally, stimulated macrophages were co-cultured with HREC in a migration assay. RESULTS: The expression rate of CCR2 in macrophages stimulated by GM-CSF was 42%±1.9%. The expression rate of CX3CR1 was 71%±3.3%. Compared with vehicle-treated groups, gene expression of VEGF-A in the macrophages was greater in 150 mg/L CCL2-treated groups (P<0.05), while expression of THBS-1 and ADAMTS-1 was significantly lower (P<0.05). By contrast, compared with vehicle-treated groups, expression of VEGF-A in 150 mg/L CX3CL1-treated groups was significantly lower (P<0.05), while expression of THBS-1 and ADAMTS-1 was greater (P<0.05). Supernatants from CCL2 treated macrophages promoted proliferation of HREC (P<0.05), while supernatants from CX3CL1-treated macrophages inhibited the proliferation of HREC (P<0.05). HREC migration increased when co-cultured with CCL2-treated macrophages, but decreased with CX3CL1-treated macrophages (P<0.05). CONCLUSION: CCL2 and CX3CL1 exert different effects in regulation of macrophage in expression of angiogenesis-related factors, including VEGF-A, THBS-1 and ADAMTS-1. Our findings suggest that CCL2 and CX3CL1 may be candidate proteins for further exploration of novel targets for treatment of ocular neovascularization.

Retinal ganglion cell-inner plexiform and nerve fiber layers in neuromyelitis optica.

AIM: To determine the thickness of the retinal ganglion cell-inner plexiform layer (GCIPL) and the retinal nerve fiber layer (RNFL) in patients with neuromyelitis optica (NMO). METHODS: We conducted a cross-sectional study that included 30 NMO patients with a total of 60 eyes. Based on the presence or absence of optic neuritis (ON), subjects were divided into either the NMO-ON group (30 eyes) or the NMO-ON contra group (10 eyes). A detailed ophthalmologic examination was performed for each group; subsequently, the GCIPL and the RNFL were measured using high-definition optical coherence tomography (OCT). RESULTS: In the NMO-ON group, the mean GCIPL thickness was 69.28±21.12 µm, the minimum GCIPL thickness was 66.02±10.02 µm, and the RNFL thickness were 109.33±11.23, 110.47±3.10, 64.92±12.71 and 71.21±50.22 µm in the superior, inferior, temporal and nasal quadrants, respectively. In the NMO-ON contra group, the mean GCIPL thickness was 85.12±17.09 µm, the minimum GCIPL thickness was 25.39±25.1 µm, and the RNFL thicknesses were 148.33±23.22, 126.36±23.45, 82.21±22.30 and 83.36±31.28 µm in the superior, inferior, temporal and nasal quadrants, respectively. In the control group, the mean GCIPL thickness was 86.98±22.37 µm, the minimum GCIPL thickness was 85.28±10.75 µm, and the RNFL thicknesses were 150.22±22.73, 154.79±60.23, 82.33±7.01 and 85.62±13.81 µm in the superior, inferior, temporal and nasal quadrants, respectively. The GCIPL and RNFL were thinner in the NMO-ON contra group than in the control group (P<0.05); additionally, the RNFL was thinner in the inferior quadrant in the NMO-ON group than in the control group (P<0.05). Significant correlations were observed between the GCIPL and RNFL thickness measurements as well as between thickness measurements and the two visual field parameters of mean deviation (MD) and corrected pattern standard deviation (PSD) in the NMO-ON group (P<0.05). CONCLUSION: The thickness of the GCIPL and RNFL, as measured using OCT, may indicate optic nerve damage in patients with NMO.

Optical coherence tomography angiography of optic disc perfusion in non-arteritic anterior ischemic optic neuropathy.

AIM: To compare the optic disc blood flow of non-arteritic ischemic optic neuropathy (NAION) eyes with normal eyes. METHODS: The optic disc blood flow densities of diagnosed non-acute phase NAION eyes (21 eyes, 14 individuals) and normal eyes (19 eyes, 12 individuals) were detected via Optovue optical coherence tomography angiography (OCTA). The optic disc blood flow was measured via Image J software. Correlations between optic disc perfusion and visual function variables were assessed by linear regression analysis. RESULTS: The average percentage of the optic disc non-perfusion areas in the non-acute phase NAION patients (17.84%±6.18%) was increased, when compared to the normal control eyes (8.61%±1.65%), and the difference was statistically significant (P<0.01). Moreover, there was a proportional correlation between the visual field mean defect (MD) and the optic disc non-perfusion area percentage, and the relationship was statistically significant (t=3.65, P<0.01, R2=0.4118). In addition, the critical correlation between the best corrected visual acuity (BCVA) and the optic disc non-perfusion area percentage was statistically significant (t=4.32, P<0.01, R2=0.4957). CONCLUSION: The optic disc non-perfusion area percentages detected via OCTA in NAION eyes were significantly increased when compared with the normal eyes. Both the BCVA and MD were correlated with the optic disc flow detected, revealing that OCTA may be valuable in the diagnosis and estimation of NAION.

Anti-apoptosis effects of vascular endothelial cadherin in experimental corneal neovascularization.

AIM: To explore the effects and mechanism of vascular endothelial cadherin (VE-cadherin) on experimental corneal neovascularization (CRNV). METHODS: Mouse corneas were burned with sodium hydroxide to build a CRNV model. The burned corneas were locally administrated with anti-mouse VE-cadherin neutralizing antibody. Annexin V and cluster of differentiation 31 (CD31) double staining was used to measure vascular endothelial cell apoptosis with the use of flow cytometry (FCM). The protein expression of NADPH oxidase 2 (Nox2), caspase-3, and protein kinase C (PKC) in the burned corneas were examined by Western blot. Human retinal endothelial cell (HREC) proliferation was detected using a Cell Counting Kit 8 (CCK-8) assay in vitro. RESULTS: The amount of CRNV peaked two weeks after the alkali burn. FCM confirmed that VE-cadherin neutralizing antibody treatment increased CD31 positive cell apoptosis. Western blot revealed that the intracorneal protein expression of Nox2 and caspase-3 were up-regulated, while PKC was down-regulated in the VE-cadherin neutralizing antibody administrated group. CCK-8 assay showed that VE-cadherin neutralizing antibody markedly inhibited HREC proliferation. CONCLUSION: VE-cadherin exhibited an anti-apoptosis effect through enhanced PKC signaling and an enhanced cell proliferation pathway.

Corneal collagen crosslinking in keratoconus and other eye disease.

Keratoconus is a condition characterized by biomechanical instability of the cornea, presenting in a progressive, asymmetric and bilateral way. Corneal collagen crosslinking (CXL) with riboflavin and Ultraviolet-A (UVA) is a new technique of corneal tissue strengthening that combines the use of riboflavin as a photo sensitizer and UVA irradiation. Studies showed that CXL was effective in halting the progression of keratoconus over a period of up to four years. The published studies also revealed a reduction of max K readings by more than 2 D, while the postoperative spherical equivalent (SEQ) was reduced by an average of more than 1 D and refractive cylinder decreased by about 1 D. The major indication for the use of CXL is to inhibit the progression of corneal ecstasies, such as keratoconus and pellucid marginal degeneration. CXL may also be effective in the treatment and prophylaxis of iatrogenic keratectasia, resulting from excessively aggressive photo ablation. This treatment has been used to treat infectious corneal ulcers with apparent favorable results. Most recent studies demonstrate the beneficial impact of CXL for iatrogenic ecstasies, pellucid marginal degeneration, infectious keratitis, bullous keratopathy and ulcerative keratitis. Several long-term and short-term complications of CXL have been studied and documented. The possibility of a secondary infection after the procedure exists because the patient is subject to epithelial debridement and the application of a soft contact lens. Formation of temporary corneal haze, permanent scars, endothelial damage, treatment failure, sterile infiltrates, bullous keratopathy and herpes reactivation are the other reported complications of this procedure.

Retinal image graph-cut segmentation algorithm using multiscale Hessian-enhancement-based nonlocal mean filter.

We propose a new method to enhance and extract the retinal vessels. First, we employ a multiscale Hessian-based filter to compute the maximum response of vessel likeness function for each pixel. By this step, blood vessels of different widths are significantly enhanced. Then, we adopt a nonlocal mean filter to suppress the noise of enhanced image and maintain the vessel information at the same time. After that, a radial gradient symmetry transformation is adopted to suppress the nonvessel structures. Finally, an accurate graph-cut segmentation step is performed using the result of previous symmetry transformation as an initial. We test the proposed approach on the publicly available databases: DRIVE. The experimental results show that our method is quite effective.

Inhibitory effect of CCR3 signal on alkali-induced corneal neovascularization.

AIM: To investigate the effect of CC chemokine receptor 3 (CCR3) signal on corneal neovascularization (CRNV) induced by alkali burn and to explore its mechanism. METHODS: Specific pathogen-free male BALB/C mice (aged 6-8 weeks) were randomly divided into CCR3-antagonist treated group (experimental group) and control group. CRNV was induced by alkali burn in mice. The time kinetic CCR3 expression in injured corneas was examined by reverse transcription polymerase chain reaction (RT-PCR). CCR3-antagonist (SB-328437 at different concentration of 125µg/mL, 250µg/mL, and 500µg/mL) was locally administrated after alkali injury. The formation of CRNV was assessed by CD31 corneal whole mount staining at two weeks after injury. Monocyte chemotactic protein 1 (MCP-1), monocyte chemotactic protein 3 (MCP-3) expressions in the early phase after injury were quantified and compared by RT-PCR. Macrophage intracorneal accumulation in the early phase after injury was evaluated and compared by immunohistochemistry. RESULTS: Alkali injury induced the time kinetic intracorneal CCR3 expression. 500µg/mL of CCR3-antagonist treatment in the early phase but not the late phase resulted in significant impaired CRNV as compared to control group (P<0.05). CCR3-antagonist treatment in the early phase significantly reduced the intracorneal MCP-1 and MCP-3 enhancement compare to control group at day 2 and day 4 (P<0.05). Moreover, the number of intracorneal macrophage infiltration in the experimental group was reduced than those in control group at day 4 (P<0.05). CONCLUSION: CCR3 signal is involved in alkali-induced CRNV. CCR3-antagonist can inhibit alkali-induced CRNV by reducing the intracorneal MCP-1 and MCP-3 mRNA expression and the intracorneal macrophage infiltration.

Inhibited experimental corneal neovascularization by neutralizing anti-SDF-1α antibody.

AIM: To explore the effect of SDF-1α on the development of experimental corneal neovascularization (CRNV). METHODS: CRNV was induced by alkali injury in mice. The expression of SDF-1α and CXCR4 in burned corneas was examined by Flow Cytometry. Neutralizing anti-mouse SDF-1α antibody was locally administrated after alkali injury and the formation of CRNV 2 weeks after injury was assessed by Immunohistochemistry. The expression of VEGF and C-Kit in burned corneas was detected by RT-PCR. RESULTS: The number of CRNV peaks at 2 weeks after alkali injury. Compared to control group, SDF-1α neutralizing antibody treatment significantly decreased the number of CRNV. RT-PCR confirmed that SDF-1α neutralizing antibody treatment resulted in decreased intracorneal VEGF and C-Kit expression. CONCLUSION: SDF-1α neutralizing antibody treated mice exhibited impaired experimental CRNV through down regulated VEGF and C-Kit expression.

[Eukaryotic expression and biological activity of recombinant human IL-17B protein].

AIM: To construct pCEP4/hIL-17B recombinant expression vector and express it stably in eukaryotic cells and investigate the biological activity in vitro. METHODS: The CDS region of human IL-17B gene was cloned by RT-PCR. After identification by sequencing, the hIL-17B gene was inserted into expression vector of pCEP4 to construct the recombinant vector pCEP4/hIL-17B, then transfected into 293T cells. The transgenic 293T cell line stably expressing rhIL-17B protein was selected in the presence of Hygromycin B. After FCS-free cultivation and sub-cloning, The IL-17B/mFc gene and protein expression was confirmed by RT-PCR, ELISA and Western blot analysis. To investigate the ability of combination with IL-17B receptor on human leukemic monocytic cell line, THP-1, by Flow cytometrical analysis (FACS) and of stimulation to secret cytokines in vitro. RESULTS: The recombinant pCEP4/hIL-17B and its transgenic 293T cells stably expressing rhIL-17B protein were obtained successfully. FACS analysis showed its high affinity with its receptor and it can stimulated THP-1 cell line to excrete IL-1ß and TNF-α in vitro and consistently caused a dose-dependent influx of neutrophil into the peritoneal cavity by intraperitoneal injection in vivo. CONCLUSION: The obtainment of transgenic 293T cell line stably expressing rhIL-17B protein paved the way for further study on biological functions of hIL-17B.

[Prokaryotic expression and biological activity of recombinant human IL-17F/His protein].

AIM: To Prepare recombinant human IL-17F/His protein and investigate its biological activity in vitro. METHODS: The gene region of human IL-17F was cloned by RT-PCR. After identification by sequencing, the hIL-17F gene encoding function domain was cloned into expression plasmid PQE3.0 and transfected into E.coli M15. By the induction of Isopropyl-ß-D-Thiogalacto-Pyranoside(IPTG), recombinant IL-17F/His protein was effectively expressed in E.coli M15. The recombinant protein was identified by Western blot. RESULTS: After renaturation and purification by HiTrap(TM); affinity column, the recombinant protein can up-regulate macrophages to secret TNF-α, IL-6 and other relative cytokines. It also promoted proliferation of HeLa cells in vitro. CONCLUSION: hIL-17F/His recombinant protein is of high biological activity, which can be used to make further study of its special characteristic.

Potential involvement of nitric oxide synthase but not inducible nitric oxide synthase in the development of experimental corneal neovascularization.

AIM: To investigate the effect of nitric oxide and its synthetase on experimental corneal neovascularization (CRNV). METHODS: CRNV was induced by alkali injury in mice, nitric oxide synthetase (NOS) was inhibited by NG-nitro-L-arginine (L-NAME) and inducible nitric oxide synthetase (iNOS) was inhibited by aminoguanidine hemisulfate salt (AG). The inhibitory effect was detected at day 2 and 4 after corneal alkali injury by reverse transcription polymerase chain reaction (RT-PCR). CRNV was compared between the control and the treated mice by microscopic observation and corneal whole mount CD31 immunostaining. RESULTS: The inhibition of L-NAME to NOS and AG to iNOS after corneal injury was confirmed by RT-PCR (P<0.05). Compared with control mice, L-NAME treated mice exhibited significantly decreased CRNV areas (P<0.05). In contrast, AG treatment failed to attenuate alkali induced CRNV (P>0.05). CONCLUSION: Our findings suggest that NOS but not iNOS plays a critical role in alkali injury induced CRNV.