Overexpression of CDKN1B inhibits fibroblast proliferation in a rabbit model of experimental glaucoma filtration surgery.

PURPOSE: To investigate the potential antiproliferative effect of cyclin-dependent kinase inhibitor 1B (CDKN1B) overexpression in a rabbit model of glaucoma filtration surgery (GFS). METHODS: The recombinant adenovector expressing exogenous CDKN1B was delivered to Tenon's capsule by subconjunctival injection during unilateral filtration surgery. The time course of CDKN1B expression was monitored by immunohistochemistry and Western blot analysis. Evaluation of proliferating activity was performed by proliferating cell nuclear antigen (PCNA), argyrophilic nucleolar organizing region (AgNOR) staining, and fibroblast-specific protein 1 (FSP-1). Cyclin-dependent kinase 2 (Cdk2) and Cdk4 expression were detected with immunohistochemical analysis. RESULTS: The overexpression of CDKN1B in Tenon's capsule was monitored throughout the experimental period. Immunoreactivity to CDKN1B was mainly observed in the nucleus of fibroblasts. The increased expression of CDKN1B in sclera was detected up to 21 days after viral infection, whereas the level of CDKN1B protein in corneal stroma was not significantly increased. The overexpression of CDKN1B induced a significant decrease in AgNOR number/nucleus and area/nucleus, PCNA staining, FSP-1 positive cells, and the decreased expressions of Cdk2 and Cdk4, as evidenced by nuclear and cytoplasmic immunoreactivity to Cdk2 and Cdk4 antibodies in positive fibroblasts. CONCLUSIONS: The persistent overexpression of CDKN1B mediated by the recombinant adenovector expressing exogenous CDKN1B in Tenon's fibroblasts after GFS may lead to the inhibition of fibroblast proliferation and the downregulation of Cdk2 and Cdk4 activity, thereby reducing the severity of scar formation and the surgical outcome.

Interference of Y-27632 on the signal transduction of transforming growth factor beta type 1 in ocular Tenon capsule fibroblasts.

AIM: To investigate the interfering effect of Y-27632, a ROCK-I selective inhibitor, on the signal transduction pathway of transforming growth factor-ß1 (TGF-ß1) in ocular Tenon capsule fibroblasts (OTFS) in vitro. METHODS: After OTFS from passages 4 to 6 in vitro were induced by TGF-ß1 and then treated by Y-27632, the changes of the OTFS cell cycles were analyzed via flow cytometry, and the proteins expression of the α-smooth muscular actin (α-SMA), connective tissue growth factor (CTGF), collagen I were calculated by Western blot. After OTFS treated by the different concentrations of Y-27632, the expression levels of the α-SMA, CTGF and collagen I mRNA were assayed by RT-PCR. RESULTS: Y-27632 had no markedly effect on the OTFS cell cycles. After treated by TGF-ß1, OTFS in G1 period significantly increased. The cell cycles distribution by both TGF-ß1 and Y-27632 had no remarkable difference from that in control group. Y-27632 significantly inhibited the proteins expressions of both α-SMA and CTGF, while to some extent inhibited that of collagen I. TGF-ß1 significantly promoted the proteins expressions of α-SMA, CTGF and collagen I. After OTFS treated by both TGF-ß1 and Y-27632, of α-SMA, the protein expression was similar with that in control group (P=0.066>0.05), but the protein expression of CTGF or collagen I, respectively, was significantly different from that in control group (P=0.000<0.01). The differences of expressions of the α-SMA, CTGF and collagen I mRNA in 30, 150, 750µmol/L Y-27632 group were statistically significant, compared with those in control group, respectively (α-SMA, P=0.002, 0.000, 0.000; CTGF, P=0.014, 0.002, 0.001; collagen I, P=0.003, 0.002, 0.000). CONCLUSION: Blocking the Rho/ROCK signaling pathway by using of Y-27632 could inhibit the cellular proliferation and the expression of both CTGF and α-SMA whatever OTFS induced by TGF-ß1 or not. Y-27632 suppressed the expression of collagen I mRNA without induction.

[Effects of oxidative stress on barrier function of human retina pigment epithelium and its molecular mechanisms].

OBJECTIVE: To investigate the effects of hydrogen peroxide (H2O2) on the barrier function and expression of tight junction protein in human retinal pigment epithelium (RPE) cells. METHODS: Experimental study. The human RPE cell line (D407) were cultured and treated with (H2O2 treated group) or without H2O2 (normal control group). The effect of H2O2 on cell viability of RPE cells was determined by MTT test. After treated with low concentration of H2O2 for 24 h to 72 h, transepithelial electrical resistance (TER) of confluent RPE cells was measured by epithelial voltmeter. The permeability of RPE cells to sodium fluorescein was measured. The expressions of the occludin and claudin-1 to -4 were determined by real-time polymerase chain reaction and Western blot analysis.t-text and one-way ANOVA were used to assess statistical significance between H2O2 treated and normal control groups. RESULTS: H2O2 at 0.2 mmol/L showed no decrease of cell viability of D407 cells, and this concentration was selected for the present study. The TER of D407 cells gradually increased, peaking at day 8 and then remained stable for 1 week. As compared to the control group, a reduction in the TER was first evident after 3 hours of treatment. Continuous culturing of cells for longer periods further reduced the TER, with a maximum effect after 24 hours of treatment and was maintained to 72 hours (24 h: 11.86 ± 1.19 vs. 24.13 ± 1.26, t = 12.260, P = 0.000; 72 h: 11.56 ± 1.47 vs. 24.33 ± 1.52, t = 10.460, P = 0.000). At any time point after adding sodium fluorescein, the permeability values of cells after treated with H2O2 for 24 hours were significantly higher than those of cells without H2O2 treatment (20 min: 25% ± 3% vs. 12% ± 4%, t = -4.50, P = 0.011; 40 min: 36% ± 4% vs. 16% ± 5%, t = -5.41, P = 0.006; 60 min: 51% ± 5% vs. 29% ± 6%, t = -4.88, P = 0.008). The expression of mRNA and protein in claudin-1, -3, and -4 were all downregulated in D407 cells treated with H2O2, whereas the expression of claudin-2 was upregulated (claudin-1 mRNA: 0.98 ± 0.18 vs. 0.28 ± 0.12, t = 5.60, P = 0.005, claudin-1 protein, 48 ± 10 vs. 100 ± 12, t = 5.77, P = 0.004; claudin-3 mRNA: 0.37 ± 0.12 vs.1.03 ± 0.15, t = 5.95, P = 0.004; claudin-3 protein: 63 ± 13 vs. 100 ± 15, t = 3.23, P = 0.032; claudin-4 mRNA: 0.38 ± 0.11 vs.0.99 ± 0.17, t = 5.22, P = 0.002, claudin-4 protein, 57 ± 12 vs. 100 ± 13, t = 4.21, P = 0.014). However, the expression of these occluding did not differ between cells treated with and without H2O2 (mRNA:1.30 ± 0.21 vs. 1.02 ± 0.16, t = -1.84, P = 0.140; protein: 109 ± 15 vs. 100 ± 14, t = -0.76, P = 0.490). CONCLUSION: Oxidative stress causes increase in the paracellular permeability of RPE cells in vitro, which may depends on the changes in expression of certain transmembrane proteins associated with the tight junction.

17-Alpha-estradiol ameliorating oxygen-induced retinopathy in a murine model.

PURPOSE: Our aim was to explore the effects and mechanism of 17-alpha-estradiol (17α-E2) on oxygen-induced retinopathy (OIR) in a murine model. METHODS: Newborn mice exposed to hyperoxia underwent subcutaneous injections of different doses of 17α-E2 from postnatal days (PND) 7 to 17. The retinal flat mounts were scored for avascular/total retinal area on PND 17. Vascular endothelial growth factor (VEGF), malondialdehyde (MDA) concentrations, and intensity, activity, and quality of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the retina were determined on PND 9, 13 (14), and 17. RESULTS: The avascular area, which is found in retinas of hyperoxia-exposed pups but not in retinas of normoxia-exposed ones, was significantly smaller in retinas of 17α-E2-treated pups. MDA and VEGF concentrations and intensity, activity, and quality of NADPH oxidase were stable in retinas of normoxia pups on PND 9, 13 (14), and 17, whereas in retinas of hyperoxia-exposed and 17α-E2-treated pups, they fluctuated markedly. VEGF concentrations were lower in retinas of hyperoxia-exposed pups than in those of normoxia ones on PND 9. Elevated VEGF concentrations were found in retinas of 17α-E2-treated pups on PND 9 and in hyperoxia-exposed pups on PND 14 and 17. Low VEGF concentrations were found in retinas of 17α-E2-treated pups on PND 14 and 17. MDA concentrations and NADPH oxidase concentration and activity, which were higher in retinas of hyperoxia-exposed pups, were lower in retinas of 17α-E2-treated pups on PND 9, 13, and 17. The most effective outcome in retinas of 1.0 µg 17α-E2-treated pups was markedly reversed by ICI182780. CONCLUSIONS: We found that 17α-E2 mitigates oxidative stress reactions and ameliorates OIR severity by decreasing NADPH oxidase expression and activity via the receptor and other pathways.

Comparative efficacy and safety of the fixed versus unfixed combination of latanoprost and timolol in Chinese patients with open-angle glaucoma or ocular hypertension.

BACKGROUND: A noninferiority trial was conducted to evaluate the efficacy of a single evening dose of fixed-combination latanoprost 50 µg/mL and timolol 0.5 mg/mL (Xalacom®; LTFC), in Chinese patients with primary open-angle glaucoma (POAG) or ocular hypertension (OH) who were insufficiently controlled on ß-blocker monotherapy or ß-blocker-based dual therapy. METHODS: This 8-week, randomized, open-label, parallel-group, noninferiority study compared once-daily evening dosing of LTFC with the unfixed combination of latanoprost, one drop in the evening, and timolol, one drop in the morning (LTuFC). The primary efficacy endpoint was the mean change from baseline to week 8 in diurnal intraocular pressure (IOP; mean of 8 AM, 10 AM, 2 PM, 4 PM IOPs). LTFC was considered noninferior to LTuFC if the upper limit of the 95% confidence interval (CI) of the difference was < 1.5 mmHg (analysis of covariance). RESULTS: Baseline characteristics were similar for LTFC (N = 125; POAG, 70%; mean IOP, 25.8 mmHg) and LTuFC (N = 125; POAG, 69%; mean IOP, 26.0 mmHg). Mean diurnal IOP changes from baseline to week 8 were -8.6 mmHg with LTFC and -8.9 mmHg with LTuFC (between-treatment difference: 0.3 mmHg; 95%-CI, -0.3 to 1.0). Both treatments were well tolerated. CONCLUSIONS: A single evening dose of LTFC was at least as effective as the unfixed combination of latanoprost in the PM and timolol in the AM in reducing IOP in Chinese subjects with POAG or OH whose IOP was insufficiently reduced with ß-blocker monotherapy or ß-blocker-based dual therapy. LTFC is an effective and well tolerated once-daily treatment for POAG and OH.

Filtering bleb area and intraocular pressure following subconjunctival injection of CTGF antibody after glaucoma filtration surgery in rabbits.

AIM: To study the role of connective tissue growth factor (CTGF) antibody in inhibiting bleb scarring after glaucoma filtration surgery (GFS) in rabbit model. METHODS: GFS was performed on both eyes in five rabbits. One eye of each rabbit was chosen randomly as antibody group and received subconjunctival injection of 0.1mL CTGF antibody (50mg/L) immediately after GFS applied and on the 5 th day after GFS. The other eye of each rabbit as control group was received subconjunctival injection of 0.1mL PBS at the same time as antibody group. On postoperative days 1, 3, 5, 7, 10, and 14, the appearance of filtrating blebs was observed under slit lamp, the area and the intraocular pressure (IOP) were measured with micrometer and applanation tonometer, respectively. RESULTS: On postoperative days 1, 3, 5, 7, 10, and 14, areas of filtrating blebs in antibody group were all larger comparing with the control group (P<0.05) and IOPs of antibody group were lower than the control group (P<0.05). CONCLUSION: Subconjunctival injection of CTGF antibody can maintain larger bleb area and lower IOP after GFS in rabbit.

[Effect of recombinant human erythropoietin on bcl-2 protein expression in the retina in a rabbit model of acute high intraocular pressure].

OBJECTIVE: To investigate the effect of recombinant human erythropoietin (rhEPO) on the expression of bcl-2 protein in the retina of rabbits with acute high intraocular pressure and explore the mechanism underlying the protective effect of rhEPO on the retina against ischemia-reperfusion injury. METHODS: rhEPO was injected subcutaneously in the ear of a rabbit model of acute high intraocular pressure induced by physiological saline perfusion into the anterior chamber. Bcl-2 protein expression in the retina of the rabbits was observed by immunohistochemical staining on days 1, 3, 7, and 14 after retinal ischemia-reperfusion and compared with that in normal rabbits and untreated rabbit models. RESULTS: bcl-2-positive cells were observed in the retina of normal rabbits with a mean positive cell number of 10.5-/+1.2 in each high-power visual field. Compared with that in the normal control group, the number of the positive cells decreased significantly in both the model group and EPO group (P<0.05, P<0.01), but the latter group showed a significantly greater number than the former (P<0.05 at day 7 and P<0.01 at day 14). CONCLUSION: Systemic administration of rhEPO can up-regulate the expression of bcl-2 protein in the retina of rabbits with acute high intraocular pressure, which is probably one of the mechanisms for the protective effect of rhEPO on the retina against ischemia-reperfusion injury.

[Effects of rAAV-mediated rhBDNF gene transfection on BDNF gene expression in the retina of a rabbit model of acute high intraocular pressure].

OBJECTIVE: To observe the changes in the expression of brain derived neurotrophic factor (BDNF) gene in the retina of rabbits with acute high intraocular pressure (IOP) after injection of recombinant adeno-associated virus (rAAV) vector containing human BDNF gene (rAAV-hBDNF), and investigate the neuroprotective mechanism of rAAV-hBDNF. METHODS: The unilateral eyes of 24 white rabbits were randomly chosen as the model group with high IOP induced by saline perfusion into the anterior chamber, and the contralateral eyes served as the control group without treatment. In another 24 white rabbits, 10 microl rAAV-BDNF was injected into the vitreous body of one of the eyes 3 days before induction of high IOP. On days 1, 3, 7, and 14 after perfusion, the bilateral eyes of 6 rabbits were excised for immunohistochemistry for the expression of endogenous BDNF gene in the retina. RESULTS: The number of BDNF-positive cells in the retina decreased after induction of high IOP, and injection of rAAV-hBDNF resulted in a significant increase in BDNF-positive cells as compared with the positive cell number in the high IOP model and control groups (P<0.05, P<0.01). CONCLUSION: rAAV-mediated BDNF gene transfection can increase endogenous BDNF expression in the retina of rabbits with acute high IOP. Intravitreous injection is an effective pathway for rAAV-hBDNF gene transfection into the retina.

Downregulation of lentivirus-mediated ILK RNAi on tractional force generation in human retinal Müller cells.

AIM: To investigate the effect of lentivirus-mediated integrin-linked kinase (ILK) RNA interference (RNAi) on human retinal Müller cells transdifferentiation into contractile myofibroblasts. METHODS: A lentiviral vector expressing ILK-specific shRNA was constructed and introduced into cultured retinal Müller cells. Silencing of the ILK gene was identified by real time RT-PCR and Western blot. The Müller cell phenotype change was confirmed by immunodetection of alpha-smooth muscle actin (alpha-SMA) stress fiber formation. The generation of tractional force was assessed using a tissue culture assay with cells incubated in three-dimensional collagen gels; cell migration was determined by the Boyden chamber method, using 10% FBS as a chemotactic factor. RESULTS: Significant decreases in ILK mRNA and protein expression were detected in Müller cells carrying lentiviral ILK-shRNA vector. Cells treated with anti-ILK siRNA showed less alpha-SMA stress fiber formation under hypoxic conditions or cell subcultivation. Lentiviral ILK-shRNA vector transfection also significantly reduced cell migration and cell-mediated gel contraction. CONCLUSION: Lentivirus-mediated ILK RNAi decreased cell migration and contractile force generation by inhibiting alpha-SMA stress fiber formation in human retinal Müller cells. This tool might be useful to treat ocular fibroproliferative diseases associated with transdifferentiated Müller cells.

[Neuroprotective effect of rAAV-mediated rhBDNF gene transfection on rabbit retina against acute high intraocular pressure].

OBJECTIVE: To investigate the neuroprotective effect of human brain-derived neurotrophic factor gene transfection into rabbit retina against acute high intraocular pressure (HIOP). METHODS: Acute HIPO was induced in one eye of 24 white rabbits via saline perfusion into the anterior chamber (model group), and the contralateral eye without treatment served as the control group. In another 24 rabbits, 10 microl recombinant adeno-associated virus (rAAV) vector containing human BDNF gene (rAAV-BDNF) was injected into the vitreous body of one of the eyes 3 days before the operation for HIPO (BDNF group). At 1, 3, 7, and 14 days after HIOP model establishment, 6 eyes in each group were excised to observe the number of retinal ganglion cells (RGCs) and the thickness of the inner retina layer. For the eyes dissected on day 14, electroretinogram b (ERG-b) wave was detected 30 min before (baseline) and on days 1, 3, 7 and 14 after HIOP. Another 5 rabbits were used for ultrastructural observation of the RGCs using transmission electron microscopy, including 1 without treatment, 2 with unilateral HIOP and 2 with rAAV-BDNF transfection before HIOP. RESULTS: The amplitude of ERG-b wave showed no significant difference between the 3 groups before HIOP (P>0.05). In HIOP model group and BDNF group, the amplitude decreased to the lowest at 1 day after HIOP and failed to recover the baseline level at 14 days (P<0.01); at the end of the observation, the amplitude was significantly higher in BDNF group than in the model group (P<0.01). Decreased number of RGCs and thickness of inner retina layer occurred in the model group, but these changes were milder in BDNF group (P<0.05, P<0.01). Electron microscopy revealed ultrastructural changes in the RGCs following acute HIOP, and transfection with rAAV-BDNF ameliorated these changes. CONCLUSION: rAAV-BDNF transfection protects the retinal structure and improves the amplitude of ERG-b wave after acute high IOP suggesting its neuroprotective effects.

Adenovirus-mediated delivery of p27(KIP1) to prevent wound healing after experimental glaucoma filtration surgery.

AIM: The aim of the study was to evaluate the outcome of adenovirus-mediated p27(KIP1) (Ad-p27) expression on wound healing after filtration surgery and to investigate the inhibition of cell proliferation induced by Ad-p27. METHODS: We constructed the adenovirus recombinant vector Ad-p27 and administered it to a rabbit model of glaucoma filtration surgery by subconjunctival injection; phosphate-buffered saline (PBS) and mitomycin C (MMC) were used as controls. Intraocular pressure (IOP), bleb scores, and anterior chamber depths were observed during a 28-d period. Histological examinations, fluorescence observations and Western blot analyses were evaluated. RESULTS: Ad-p27 enhanced the surgical outcome and inhibited cell proliferation when compared with PBS. Bleb scores in the Ad-p27-treated eyes were higher than those in the PBS-treated eyes on d 7 (P<0.01), 14 (P<0.01) and 21 (P<0.05). On d 28, IOP remained significantly decreased in the Ad-p27 group compared with the PBS group (P<0.05). However, no differences in bleb scores or IOPs were observed between the Ad-p27 and MMC groups. Histological analysis showed that total cell numbers were markedly reduced, and less scar tissue was observed at the surgical site in eyes treated with Ad-p27. The number of fibroblasts was decreased in Tenon's capsule in Ad-p27-treated eyes; however, a marked and diffuse signal from the green fluorescent protein (GFP) was observed in fibroblasts. Western blot analysis revealed a high level of p27(KIP1) expression in conjunctival epithelium (P<0.01), relatively high expression in superficial scleral stroma (P<0.01), and low expression in corneal epithelium in the Ad-p27 group. CONCLUSIONS: Ad-p27 administration significantly improves the outcome of filtration surgery and inhibits postoperative proliferation in rabbit eyes. These findings suggest that p27(KIP1) is a potential adjunctive agent for inhibition of wound healing after filtration surgery.

[Effects of recombinant human erythropoietin on hypoxia inducible factor-1alpha expression in the retina of rabbits with acute high intraocular pressure].

OBJECTIVE: To observe the effect of recombinant human erythropoietin (rhEPO) on the expression of hypoxia inducible factor-1alpha (HIF-1alpha) in the retina of rabbits with acute high intraocular pressure and investigate the mechanism of rhEPO in protecting the retina from ischemia-reperfusion injury. METHODS: Acute high intraocular pressure was induced in the rabbits by perfusion of normal saline into the anterior chamber, and rhEPO was injected subcutaneously. The changes in HIF-1alpha protein expression in the retina was observed by immunohistochemistry on days 1, 3, 7, and 14 after retinal ischemia- reperfusion. RESULTS: HIF-1alpha expression was not observed in the retina of the normal control rats, but intense HIF-1alpha expression was found in the model group (P<0.01). In rabbits with rhEPO injection and those in the model group, the patterns of HIF-1alpha expression alterations were similar, but the HIF-1alpha-positive cells in the retina were significantly fewer in rhEPO group (P<0.05). CONCLUSION: rhEPO can down-regulate HIF-1alpha expression in the retina of rabbits with acute high intraocular pressure, which may be one of the mechanisms that rhEPO protects the retina from ischemia-reperfusion injury.

Subconjunctival sustained release 5-fluorouracil for glaucoma filtration surgery.

AIM: To determine the release characteristics of a 5-fluorouracil-loaded poly (lactic acid) disc (5-FU-PLA-DS) and the effect of sustained drug delivery on the success of glaucoma filtration surgery in rabbit eyes. METHODS: A method of microspheres accumulated by excessive carriers was used in the preparation of the 5-FU-PLA-DS. The disc was characterized for drug loading, entrapment efficiency, in vitro release, and external morphology. It was then implanted subconjunctivally into rabbit eyes with trabeculectomy. Intraocular pressure, ocular inflammatory reaction, filtration bleb appearance, and persistence were evaluated up to postoperative d 90. A quantitative analysis of 5-fluorouracil (5-FU) was performed in the aqueous humor. Ultrasound biomicroscopy was used to assess the appearance of the filtering fistula. RESULTS: The 5-FU-PLA-DS was produced with the drug-loading of 3.07+/-0.08 mg (mean+/-SD). 5-FU was released for 91 d with suppressive concentrations. The decrease in intraocular pressure from baseline was significantly more marked in the 5-FU-PLA-DSimplanted eyes during postoperative d 3-90, and the persistence of bleb and filtration fistula was longer than the control eyes (P<0.05). Corneal toxicity and hyperemia triggered by 5-FU was lower in the 5-FU-PLA-DS-implanted eyes than those exposed to 5-FU intraoperatively. The 5-FU concentration in the aqueous humor was insufficient for corneal endothelial damage. No evidence of toxic reaction was found in the conjunctival biopsy. CONCLUSION: 5-FU-PLADS displaying sustained intraocular release of 5-FU, reduced intraocular pressure, and prolonged bleb persistence, while significantly reducing 5-FU toxicity.

[Effect of neuropeptide NAP on cultured neuroepithelial cells from rabbit retina].

OBJECTIVE: With the rabbit iris pigment epithelial cells (IPECs), which were containing the NT4-NAP fusion gene, taken as the substituting secreting cells producing the neuropeptide NAP, the effect of neuropeptide NAP on the growth status of retinal neuroepithelial cells of rabbit was examined and explored. METHODS: The iris pigment epithelial cells and retinal neuroepithelial cells of rabbit were cultured; rAAV-GFP and rAAV-NAP (containing NT4-NAP fusion gene) were constructed; the rabbit IPECs were infected with rAAV-GFP and rAAV-NAP; the infections of viruses were detected by GFP fluorescence expression; the supernatant of culture from rabbit IPECs with NAP was collected and added into the culture medium of rabbit retinal neuroepithelial cells, and the growth state of retinal neuroepithelial cell was observed. RESULTS: Compared to control cells, the rabbit IPECs could express the GFP fluorescence. The rabbit retinal neuroepithelial cells with NAP supernatant could survive more, and the survival cells showed longer and stronger axons. On 14's day of cell culture, the mean axon length of NAP group was (14. 6+/-1. 1) microm, while that of control group was only (3. 1+/-0. 6) mircom. Obviously, a significant difference existed between two groups (P<0. 05). CONCLUSION: The rabbit IPECs with NT4-NAP fusion gene can secrete NAP, and the NAP can promise the growth of rabbit retinal

[The effect of NT4-NAP fusion gene transfection on photoprotection of rabbit iris pigment epithelium cells].

OBJECTIVE: To investigate the effect of infection with adeno-associated virus (AAV) vector containing NT4-NAP fusion gene on photoprotection of rabbit iris pigment epithelium cells (IPECs). METHODS: rAAV-GFP and rAAV-NAP (containing NT4-NAP fusion gene) were constructed; rabbit IPECs were cultured and infected with rAAV-GFP and rAAV-NAP; transfection of viruses was detected by GFP fluorescence expression; NAP protect rabbit iris pigment epithelium from light stimulation was evaluated by MTT and flow cytometry. RESULTS: Rabbit IPECs expressed the GFP fluorescence; comparing with control cells, IPECs transfected with rAAV-NAP remained normal proliferation and showed lower apoptosis percentage after ultra-violet stimulation. CONCLUSION: rAAV-NAP constructed in the study can infect rabbit IPECs, and NAP may protect rabbit IPECs from ultra-violet damage.

[The role of specific ROCK inhibitor in the prevention of experimental PVR in rabbits].

OBJECTIVE: To determine the effect of Y27632, a specific inhibitor of p160 Rho-associated coiled-coil forming protein kinase (ROCK), on experimental rabbit PVR. METHODS: Cultured rabbit retinal pigment epithelial cells were used in the experiments. The effects of Y27632 on RPE alpha-SMA (smooth muscle actin) stress fiber formation were studied by immuno-fluorescent staining. An in vitro type I collagen gel contraction assay and MTT assay were used to detect the effect of Y27632 on RPE cell contractile force and proliferation. Cultured 6 th passage rabbit RPE cells were injected intravitreally to induce the PVR model and then followed injection of 50 micromol/L of Y27632. The presence of tractional retinal detachment (TRD) was assessed to evaluate the effect of this agent in vivo. Electroretinography and histological studies were performed after intravitreal injection of Y27632 into untreated eyes to evaluate toxicity. RESULTS: The results showed that Y27632 disrupted SMA stress fiber formation in the cultured RPE cells and impaired contractile force generated by RPE cells (t = 16.212, P < 0.01). Development of TRD was significantly reduced (P < 0.01) with 50 micromol/L of Y27632. No obvious histological or retinal functional damage was found in the Y27632-treated group. CONCLUSION: p160 ROCK specific inhibitor Y27632 decreased contractile force generated by RPE cells and attenuated PVR without significant side effect in rabbit. This reagent could be potential therapeutically method in the treatment of PVR.