Purpose. To evaluate the role of SPARC in the antiproliferation effect of MMC on human Tenon’s fibroblasts (HTF). Method. Sixteen PACG patients aged 59 ± 10 years (31–72 years), including 6 males and 10 females, were recruited. Tenon tissue was harvested during filtering surgery. Cell density was evaluated after MMC application with different concentrations and application times, by which the optimized MMC application modality was determined. MMC, si-SPARC, or SPARC protein was used when needed to evaluate the cell densities under different conditions, by which the role of SPARC in MMC-mediated antifibrotic process was identified. Results. Considering that the cell densities, as well as SPARC expression on mRNA and protein levels, are relatively stable when the MMC concentration is higher than 0.02% and exposure time longer than 90 s, we chose the MMC application pattern with 0.02% and 90 s as an optimized pattern for the downstream work. Compared to control, the si-SPARC and MMC downregulated the SPARC protein by 91% (P<0.01) and 65% (P<0.01) and mRNA by 96% (P<0.01) and 64% (P<0.01), respectively. MMC decreases the cell densities by 53.50% compared to control. si-SPARC + MMC dramatically deceased the cell density no matter compared to the control group (P<0.01) or MMC group (P<0.01); correspondingly, the relative collagen gel area in the MMC + si-SPARC group was higher than that in the MMC group or si-SPARC group (P<0.05). The reactive oxygen species expression in the MMC + si-SPARC group is higher than that in the MMC group (P<0.05). Conclusion. This study demonstrates that in HTF, (1) MMC downregulates the expression of SPARC in protein and mRNA levels; (2) SPARC depletion has synergistic effect on the antifibrotic effect of MMC; and (3) reactive oxygen species are the possible mediator in the antifibrotic effect of MMC and si-SPARC.

Proliferative vitreoretinopathy (PVR) is a blinding eye disease and there is no effective pharmacological measure to prevent PVR development. The difficulty comes from lack of potent antiproliferative agent and lack of sustained delivery to cover high-risk time window for PVR to develop. Lipid prodrug of PMEG, hexadecyloxypropyl 9-[(2-phosphonomethoxy)ethyl]guanine (HDP-PMEG), was prepared and was evaluated as a pharmacological adjuvant to surgical management of PVR. A dose-escalation study determined that the highest nontoxic dose for intravitreal use in pigmented rabbits was 3 µg per eye. The genotoxicity of HDP-PMEG was harnessed as a perioperative preventative measure against PVR in a rabbit eye model while the sustained intravitreal pharmacological effect was evaluated on a laser-induced fibrovascular model in rat eye. After intravitreal 3 µg, HDP-PMEG particles in the rabbit vitreous was visible for at least 6 weeks. A single 50-min intravitreal infusion of HDP-PMEG demonstrated significant inhibition of PVR formation when compared with the eyes infused with only BSS (BSS vs. HDP-PMEG: estimate = 1.14, OR = 3.1, p = .027). A single intravitreal 104 ng (equivalent to 3 µg for rabbit eye) of HDP-PMEG significantly inhibit laser-induced fibrovascular proliferation in rat eye by 55% (least square mean pixel, BSS = 4763569.5 vs. HDP-PMEG = 2148129.7, p < .0001, generalized estimating equation [GEE]). Retinal fluorescein angiography showed the odds for BSS intervened eyes to have higher-rated FA leaking grades were 38.5 times compared with HDP-PMEG treated eyes (p < .0001, GEE). Our study results indicate that single intravitreal HDP-PMEG may be a promising ocular drug delivery as a perioperative intervention to prevent PVR reoccurrence following primary surgical management.

Background: Fetuin-A, a liver-derived glycoprotein, is correlated with diabetes. The aim of the present investigation was to evaluate serum and vitreous concentrations of fetuin-A in patients with diabetic retinopathy (DR).Material/Methods: We randomly selected 224 diabetic patients and 68 control subjects for this study.Results: There were markedly higher serum and vitreous fetuin-A concentrations in proliferative diabetic retinopathy (PDR) patients than in the other three groups. NPDR patients exhibited elevated vitreous fetuin-A concentrations compared with patients without DR. However, no significant differences in serum fetuin-A concentrations were observed between NPDR patients and patients without DR. In addition, there were significantly lower concentrations of serum and vitreous fetuin-A in control subjects compared with the other three groups.Conclusions: The occurrence and severity of DR is correlated with serum and vitreous fetuin-A concentrations.

Background Dimethyl sulfoxide(DMSO) is a commonly used adjuvant to promote testing drug solubility to prepare multi-levels testing drug concentrations.DMSO is cell type-dependent cytotoxic and its toxicity can interfere the testing drug evaluation.Determining its safe concentration on commonly used cell types is important for ocular drug development.Objective This study was to determine the minimal toxic concentration of DMSO for in vitro ocular cell lines in a simulated drug screening setting.Methods Retinal pigment epithelial (RPE) cells were isolated from one pigmented rabbit and primarily cultured.Human RPE cell strain (ARPE19),scleral fibroblasts line (S75-Fron),human Müller cell line (MIO-M1),human lens epithelial cell line (HLEC),human choroidal melanoma cell line (OCM-1),human umbilical endothelial cell (HUVEC) and human HeLa cell line (HELA) were cultured.Different concentrations of DMSO (1.6％,1.0％,0.8％,0.4％,0.2％ and 0.1％) were prepared with 160 μl DMSO solution and 9.84 ml RPMI1640 (or DMEM/F12 or DMEM) containing 2％ fetal bovine serum.Different concentrations of DMSO were added in medium for 96 hours,and the and viability (absorbance) of the cells was detected using MTS to evaluate the cytotoxicity of DMSO.Results Rabbit primary RPE cells showed the yellow-green fluorescence for cytokeratin(CK) and HMB45 red fluorescence for S100.The viability of the cells was gradually declined as the increase of DMSO dose,showing significant differences in ARPE19,S75-Fron,HLEC,OCM-1,HUVEC and primary RPE cells (all at P＜0.05),and when DMSO concentrations were ≥ 0.8％,the cell viabilities were significantly lower.But no significant difference was found in MIO-M1 cells among different doses of DMSO (F=0.830,P=0.547).The minimal toxic concentration of DMSO for ARPE19,HUVEC,HELA,HLEC,MIO-M1,OCM-1,primary RPE cells and S75-Fron was 0.8％,0.1％,0.8％,＞1.6％,＞1.6％,0.2％,0.2％,0.2％,respectively,and HUVEC was more sensitive to the cytotoxicity of DMSO (P=0.02),and MIO-M1 was the least sensitive to DMSO (P =0.39).The viability of HUVEC and primary RPE cells went down with the increase of DMSO dose,and S75-Fron viability started to decline in 0.1％ DMSO and then stabilize with the higher concentrations until 1.6％ DMSO at which the viability showed further decline.Conclusions The tolerability of ocular cells in vitro to DMSO varies depending on the cell types.The minimal toxic concentration ranged from 0.1％ to 1.6％.The result suggests that a concurrent DMSO control should be set up along with the testing compound.

OBJECTIVE To characterize the safety profile of triamcinolone acetonide made in China (Transton) and triamcinolone acetonide acetate (Tongyong) for their ocular application. METHODS Experimental study. In vitro cell viability assay was performed on 3 types of human ocular cells to evaluate the cytotoxicity of the simulated vitreal concentrations (from a 1:15 dilution as if injected into 1.5 ml of rabbit vitreous to 1:50 dilution as if injected into 5 ml of human vitreous) of Transton and Tongyong using MTT method.In vivo 28 guinea pigs, randomly divided into four groups, were used for evaluating either intravitreal 6 µl of the two types of triamcinolone suspension or 18 µl of their supernatant. Following the injections, the eyes were monitored by biomicroscopy, ophthalmoscopy, tonometry, electroretinography, and histology. The Dunnett's test was used to analyze in vitro cell viability. Paired sample t-test and Generalized Estimating Equations (GEE) were respectively used to compare electroretinography data and intraocular pressure between experimental eyes and the control eyes. RESULTS The undiluted supernatant of Transton and Tongyong was toxic to human scleral fibroblasts when compared with their control groups (MTT values = 0.046 ± 0.036 and 0.044 ± 0.05 versus 0.367 ± 0.106 and 0.413 ± 0.128) (P < 0.01) or with the BSS group (0.368 ± 0.106 and 0.441 ± 0.137) (P < 0.01). 1:15 or greater dilution of supernatant of Transton and Tongyong did not show cytotoxicity on cultured human retinal pigment epithelium cells or Müller cell (P > 0.05), but 1:15 dilution of Tongyong supernatant showed cytotoxicity on the Müller cells (MTT value = 0.366 ± 0.062 versus 0.417 ± 0.042 for BSS) (P = 0.03). In vivo, neither intravitreal 6 µl (0.25 mg, equivalent to 4 mg in 0.1 ml for human eyes) of the suspension nor 18 µl (equivalent resultant preservative concentration to intravitreal 4 mg in 0.1 ml for rabbit eyes) of the supernatant of Transton and Tongyong showed ocular toxicity. CONCLUSION The equivalent dose (6 µl) to 4 mg in 0.1 ml intravitreal suspension for human eye or equivalent resultant preservative concentration to 0.1 ml intravitreal suspension for rabbit eye is found safe in guinea pig eyes.

PURPOSEWe sought to better characterize the intravitreal profile of different triamcinolone formulations.METHODSThe study was performed in vitro and in vivo. Kenalog-40, Triesence, and Transton were characterized for ocular pharmacokinetics, particle size, crystallinity, and dissolving kinetics in vitreous following an intravitreal injection into 12 rabbit eyes. The relationship of free drug levels in the aqueous and vitreous was investigated through a dual-probe microdialysis and liquid chromatography tandem mass spectrometry.RESULTSTriesence had the most uniform particle size distribution (mean 11.51 μm) and Kenalog-40 had the largest particle sizes (mean 18.86 μm). Triesence and Kenalog-40 had 100% crystallinity, while Transton had 89% crystallinity. Triesence had a slower dissolution in vitreous than that of Kenalog-40, and Transton had the fastest dissolution, though their solubility was very similar. Following a 1.2 mg intravitreal injection in the rabbit eye, Triesence had a significantly lower ocular free drug level than Kenolog-40 (P = 0.025) and Transton (P = 0.007). Quantitative dual-probe microdialysis revealed that the aqueous free triamcinolone (Kenolog-40) was less than 1% of the vitreous free triamcinolone during the first few hours, and this percentage increased to 26.8% at 2 weeks and was 11.7% at 3 weeks following an intravitreal injection.CONCLUSIONSTriesence demonstrated a significantly slower dissolution profile and lower free drug level in the vitreous than the other preserved triamcinolone, which may translate into a longer therapeutic duration and lower rate of drug-associated complications.

Purpose: To characterize the safety profile of triamcinolone acetonide (TA) for intraocular application.Methods: In vitro cell viability assay was performed on 2 types of human ocular cells to evaluate the cytotoxicity of the simulated different vitreal concentrations (from a 1: 15 dilution as if injected into 1.5 mL of rabbit vitreous to 1: 50 dilution as if injected into 5 mL of human vitreous) of preservative and excipient (supernatant) from Kenalog-40. In vivo 35 guinea pigs were used for evaluating either a dose of intravitreal triamcinolone acetonide (Kenalog-40 and Triesence) or the supernatant of Kenalog-40. The animal eyes were monitored by biomicroscopy, ophthalmoscopy, tonometry, electroretinography, and histology.Results: A >= 1:15 dilution of triamcinolone acetonide supernatant from Kenalog-40 did not show cytotoxicity on cultured human pigment epithelial (retinal pigment epithelium) cells or Muller cells. In vivo, neither intravitreal 6 mu L (0.248 mg, equivalent to 4 mg in 0.1 mL for human eyes) nor 18 mu L (0.744 mg, equivalent to 4 mg in 0.1 mL for rabbit eyes and equivalent to 12 mg in 0.1 mL for human eyes) of triamcinolone acetonide suspension showed ocular toxicity. No significant difference was noted between Kenalog-40 and Triesence clinically and histopathologically.Conclusion: The equivalent triamcinolone acetonide doses to 0.1 mL (4 or 12 mg) intravitreal injection for human eye were found safe in guinea pig eyes. No significant difference was noted for 0.1 mL intravitreal injection between Kenalog-40 and Triesence. RETINA 32:364-374, 2012

As graphene becomes one of the most exciting candidates for multifunctional biomedical applications, contact between eyes and graphene-based materials is inevitable. On the other hand, eyes, as a special organ in the human body, have unique advantages to be used for testing new biomedical research and development, such as drug delivery. Intraocular biocompatible studies on graphene-related materials are thus essential. Here, we report our recent studies on intraocular biocompatibility and cytotoxicity of graphene oxide (GO) both in vitro and in vivo. The successful preparation of GO nanosheets was confirmed using atomic force microscopy, contact angle analyzer, Fourier transform infrared spectroscopy, and Raman spectroscopy. The influence of GO on human retinal pigment epithelium (RPE) cells in terms of the cell morphology, viability, membrane integrity, and apoptosis was investigated using various techniques, including optical micrography, cell counting kit-8 (CCK-8) assay, lactate dehydrogenase (LDH) assay, and apoptosis assay. The addition of GO had little influence on cell morphology, but the change was visible after long-time culturing. RPE cells showed higher than 60% cell viability by CCK-8 assay in GO solutions and less than 8% LDH release, although a small amount of apoptosis (1.5%) was observed. In vitro results suggested good biocompatibility of GO to RPE cells with slight adverse influence, on the cell viability and morphology in long-time periods, along with aggregation of GO. Thus, some further studies are needed to clarify the cytotoxicity mechanism of GO. GO intravitreally injected eyes showed few changes in eyeball appearance, intraocular pressure (IOP), eyesight, and histological photos. Our results suggested that GO did not cause any significant toxicity to the cell growth and proliferation. Intravitreal injection of GO into rabbits' eyes did not lead to much change in the eyeball appearance, IOP, electroretinogram, and histological examination.

Leber congenital amaurosis (LCA) is one of the most severe forms of inherited retinal degeneration and can be caused by mutations in at least 15 different genes. To clarify the proteomic differences in LCA eyes, a cohort of retinal degeneration 12 (rd12) mice, an LCA2 model caused by a mutation in the RPE65 gene, were injected subretinally with an AAV vector (scAAV5-smCBA-hRPE65) in one eye, while the contralateral eye served as a control. Proteomics were compared between untreated rd12 and normal control retinas on P14 and P21, and among treated and untreated rd12 retinas and control retinas on P42. Gene therapy in rd12 mice restored retinal function in treated eyes, which was demonstrated by electroretinography (ERG). Proteomic analysis successfully identified 39 proteins expressed differently among the 3 groups. The expression of 3 proteins involved in regulation of apoptosis and neuroptotection (alpha A crystallin, heat shock protein 70 and peroxiredoxin 6) were investigated further. Immunofluorescence, Western blot and real-time PCR confirmed the quantitative changes in their expression. Furthermore, cell culture studies suggested that peroxiredoxin 6 could act in an antioxidant role in rd12 mice. Our findings support the feasibility of gene therapy in LCA2 patients and support a role for alpha A crystallin, heat shock protein 70 and peroxiredoxin 6 in the pathogenetic mechanisms involved in LCA2 disease process.

OBJECTIVE:To investigate the differential expression of complement C4b and transthyretin in proliferative vitreoretinopathy (PVR).METHODS:It was a controlled experimental study. Human vitreous samples of 5 patients with PVR were analyzed by using two-dimensional gel electrophoresis and mass spectrometry, and the results were compared with those from normal control vitreous obtained from donor eyes. An in vivo model of PVR was created by intravitreous injection of cultured rabbit retinal pigment epithelial (RPE) cells. The vitreous of PVR models were analyzed by enzyme linked immunosorbent assay (ELISA) to confirm the proteomic results from the PVR patients.RESULTS:Seventy nine various proteins were expressed differently between PVR and normal vitreous, among which nine up-regulated proteins including complement C4b, transthyretin (TTR), and 7 albumins were identified by mass spectrometry. The up-regulation of complement C4b and TTR in PVR patients was also confirmed by ELISA. The concentration of complement C4b and TTR in normal vitreous were (20.18 ± 1.97) mg/L and (88.58 ± 8.84) mg/L respectively, in PVR patients were (38.1 ± 5.79) mg/L and (112.57 ± 6.89) mg/L respectively, difference significantly between these two groups (C4b: t = 11.54, TTR:t = 9.24; P < 0.05).CONCLUSIONS:Differences of complement C4b and TTR expression were observed between PVR and normal vitreous. These results have lead to the assumption that there is a connection between elevated concentrations of both complement C4b and TTR and the pathogenesis of PVR and further studies on the functions of these proteins are required.

PURPOSE:To investigate the safety and inhibitory effects of hexadecyloxypropyl 9-[2-(phosphonomethoxy) ethyl] guanine (HDP-PMEG) on ocular cell proliferation and collagen matrix contraction.METHODS:For the antiproliferation studies, various ocular cell monolayers were exposed to HDP-PMEG, PMEG, 5-fluorouracil (5-FU), and daunorubicin (DNB). For the collagen contraction studies, retinal pigment epithelium (RPE) cells seeded onto type I collagen lattices were exposed for a single 5- or 50-min period to various concentrations of HDP-PMEG or 5-FU. For the cytotoxicity study, trypan blue exclusion tests were performed using a human Müller cell line. Cytotoxicity was determined up to 4 days after treatment.RESULTS:The proliferation of RPE cells, scleral fibroblasts, vessel endothelial cells, and ocular melanoma cells can all be significantly inhibited by HDP-PMEG. Its inhibitory effects on those cells were uniformly stronger than that of 5-FU. Contraction of the collagen matrix containing RPE cells was significantly inhibited by HDP-PMEG and by 5-FU at concentrations of 20 µM and 2,000 µM, respectively, as compared with controls (p<0.05). The safety profile of HDP-PMEG was significantly better than 5-FU and daunorubicin. The ocular therapeutic index is 1,100 for HDP-PMEG, 17.2 for 5-FU, and 1.25 for daunorubicin.CONCLUSIONS:HDP-PMEG possesses a significant inhibitory effect on the proliferation of RPE, retinal glial cells, scleral fibroblasts, and ocular melanoma cells. HDP-PMEG is also genotoxic and may be used as a single short application for the modulation of unwanted ocular proliferation.

Background: Corneal intrastromalcorneal implantation is the ideal model for biocompatibility assessment of the implanted materials, but different implanting positions may influence the material degradation and corneal metabolism, finally impacting on the biocompatibility evaluation of intracorneal implantation. OBJECTIVE: To investigate the histocompatibility of chitosan-collagen membrane (30% collagen) implanted into different positions of rabbit cornea. METHODS: Chitosan-collagen composite membrane at 20-μm thick was prepared. New Zealand rabbits were randomized divided into two groups: peripheral corneal intra-stroma implantation group of chitosan-collagen membrane; central corneal intra-stroma implantation group of the membrane. Slitlamp microscopy examinations were performed to observe the reaction of ocular surface and anterior chamber after the implantation of the membrane, the interaction between cornea and implanted membrane, the transparency of cornea were also observed. Cornea slices were obtained 6 weeks later to observe the histological changes of cornea and implanted materials at implantation site. RESULTS AND CONCLUSION: In peripheral corneal intra-stroma implantation group, the complex membrane gradually degraded and finally completed degraded, and the corneal structure was not markedly influenced, indicating good histocompatibility of corneal tissues. In central corneal intra-stroma implantation group, the ocular surface was stable at early stage of the implantation, also suggesting good biocompatibility. Two weeks later, the material rim became untransparent with the materials gradually degraded. At days 34 and 42, each one cornea melted accompanying inflammatory cells infiltration. Results demonstrated that thin chitosan-collagen composite membrane has good transparency and matched mechanical properties, materials implanted into the peripheral cornea may completely degrade within a short time. It may be used as a corneal restorative material, especially as the carrier of corneal limbal stem cells to repair corneal damage.

目的：通过动物实验观察羧基单壁碳纳米管的眼内生物安全性。方法：选择健康成年雄性SD大鼠，随机分为3组，每组5只，羧基单壁碳纳米管剂量分别为 0.05 、0.1、 0.2 mg/ml，各5μl注入实验眼，各组均以右眼为实验眼，左眼注入等量的磷酸盐缓冲液（PBS）为对照，共观察1个月。采用裂隙灯和间接眼底镜等方法进行临床观察，玻璃体腔注入羧基单壁碳纳米管后观察眼压变化，视网膜电图评价注入前及注入后各时间点视网膜功能状态，光镜观察视网膜各层结构改变。结果：玻璃体腔注入羧基单壁碳纳米管前后眼压没有明显的变化，视网膜电图未见明显的改变，光镜观察视网膜各层结构未见明显改变。结论：实验剂量的羧基单壁碳纳米管注入SD大鼠玻璃体腔未见明显毒性。