Abstract:We reviews different experimental models of age-related macular degeneration (AMD) used in recent studies. The most widely used one is Laser-induced choroidal neovascularization (CNV), which represents the late severe stage in the exudative form of AMD. Other models are based on several different pathogenesis, like geographic atrophy, drusen formation or multifactorial effects, like age, light, high fat, etc. It is hoped that this article could become a good reference for researchers who need to choose suitable models for AMD study.

Abstract:AIM: To observe the effects of topical 5-fluorouracil (5-FU) application on the rabbits corneal limbus. METHODS: 5-FU was applied topically to the corneoscleral region of rabbits' eyes. The animals were sacrificed immediately or 7, 15, 30 and 60 days later, and tissues were submitted to histological examination. RESULTS: All animals presented corneoscleral deepithelization close to the site of application during the immediate postoperative period, whereas on the 4^th postoperative day ulceration was no longer present. Histological examination showed absence of epithelium and slight edema. After one week(G2), 2 animals presented epithelial defects, thickened epithelium with larger basal cells and loose chromatin, and slight subepithelial edema. The remaining groups showed no alterations. CONCLUSION: The 5-FU topically applied on the corneoscleral limbus postpones the epithelization.

Abstract:AIM: To find new biomarkers in the sera of retinoblastoma (Rb) patients with surface-enhanced laser desorption/ ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and protein chip technique. METHODS: SELDI-TOF-MS, IMAC30 and CM10 protein chips were used to analyze the protein profiles from sera of 18 patients with Rb and 17 age-matched controls. The protein profiling was analyzed statistically by Ciphergen protein chip software 3.0.2. The Student's t -test was applied to compare the protein peak intensity. Fisher's exact test was used to compare the predominance of differential protein peaks appeared in patients. RESULTS: With IMAC30 protein chips, there were 26 proteins which appeared difference in sera of patients with Rb compared to normal children. Among them, 21 proteins, i.e. 7746, 7014, 11713, 3049, 7084, 7299, 5888, 2544, 12575, 5489, 9658, 9575, 9929, 10161, 8955, 1886, 10617, 6209, 2411, 7374, 6614m/z were up-regulated and 5 proteins, i.e. 8382,7923,7972, 8590, 66576m/z, were down-regulated (P < 0.01). Using the 7014m/z protein peak for statistical analysis, we could differentiate the patients with Rb from the healthy children with a sensitivity of 94.4% and a specificity of 82.4%. By CM10 protein chips, 4 proteins, including 3 up-regulated proteins (5888, 6097, 7798m/z) and 1 down-regulated protein (8590m/z), were detected in Rb patients (P <0.01). The sensitivity and specificity were 83.3% and 70.6% respectively when 7798m/z protein peak was selected for statistical analysis. CONCLUSION: There are a few candidates as Rb biomarkers in the sera of Rb patients. SELDI-TOF-MS protein chip technology could be a potential method in the clinical screening test of Rb.

Abstract:AIM: To quantitatively investigate the gene expression of transforming growth factor-β type I receptor (TβR I) and trans- forming growth factor-β type II receptor (TβR II) in rat retina. METHODS: Sprague-Dawley rats were chosen in this research. Gene expression was detected quantitatively by reverse transcription polymerase chain reaction (RT-PCR) analysis. RESULTS: The expression level of TβR I and TβR II were 0.00034±0.00013 and 0.0001±0.00005, respectively. The expression level of TβR I was obviously higher than that of TβR II in the rat retina with statistical significance (P <0.01). The ratio of TβR I /TβR II was 3.9±1.7. CONCLUSION: Real time quantitative RT-PCR is an effective method to detect differential expression genes in retina. The expression change of TβR I and TβR II may play an important role in the pathogenesis of retinopathy, which need further investigation on its significance in the development of proliferation retinopathy.

Abstract:AIM: To explore the inhibitive effects of cervical lympha- denectomy on keratoplasy after alkaline burns. METHODS: The Wistar rats' corneas were transplanted into Sprague-Dawley (SD) rats' eyes which were randomly divided into 4 groups: group A (control group); group B, the cervical lymphadenectomy group; group C, corneal transplantation after the alkali burn injury; group D, cervical lymphaden- ectomy following group C. Out of 6 rats in each group, the cornea of one rat was used for macrophage immuno- histochemistry at day 14 after the transplantation, and the remaining 5 rats were used for studying corneal immune rejection with a slit lamp. The time when allograft rejection occurred was recorded and mean survival times (MST) were compared among the groups. RESULTS: Compared with the MST of group A (10.40±1.14 days),the MST of group B(46.30±9.46 days) was significantly longer (P <0.05). MST of grafts between group C (7.00±1.58 days) and group D (15.00±3.39 days) was also significant (P < 0.05). At 14^th day after the transplantation, there was no CD₆₈immunoreactivity in the graft of group B, and CD₆₈ proteins were expressed to some extent in the grafts of group A and D. However, in the graft of group C, the expression of proteins was dramatically up-regulated. CONCLUSION: Cervical lymphadenectomy therapy has a significant effect in preventing corneal allograft rejection in normal and alkali burned corneal beds.

Abstract:AIM: To investigate the expression of aquaporins-1 (AQP-1) in cultured bovine corneal endothelial cells and to explore the role of AQP-1 in corneal endothelial fluid transport. METHODS: The bovine corneal cells were cultured in DMEM containing 200mL/L neonate bovine serum. AQP-1 expression in the bovine corneal endothelial cells was detected with immunohistochemistry method before and after treatment of the cells with aquaporin inhibitor,p -chloromercuribenzene sulfonate. The osmotic water permeability was determined by monitoring volume changes of cultured bovine corneal endothelial cells. RESULTS: Positive staining was used to reveal the AQP-1 expression in the membrane of cultured bovine (in brown color). The reading of osmotic water permeability of the cultured bovine corneal endothelial cells before treatment with p -chloromercuribenzene sulfonate was 0.044±0.005cm/s, which significantly decreased to 0.017±003cm/s after treatment (n =15). CONCLUSION: AQP-1 expressed in the membrane of cultured bovine corneal endothelial cells may play an important role in fluid transport of corneal endothelial cells. Alteration of the AQP-1 expression may cause abnormal corneal function and corneal edema.

Abstract:AIM: To study the effect of an intravitreal injection of angiostatin on vascular leakage in the retina and iris of oxygen-induced retinopathy of prematurity (ROP). METHODS: Brown Norway rats at postnatal day 7 (P7) were exposed to hyperoxia (750mL/L O₂) for 5 days (P7-12) and then returned to normoxia to induce retinopathy. Angiostatin was reconstituted in sterile Phosphate Buffered Saline (PBS) and diluted to desired different concentrations. Angiostatin solution was injected into the vitreous of the right eye of the ROP rats at P14 and the age-matched normal rats through pars plana using a glass capillary, and the left eye received the same volume of sterile PBS as the control. Vascular permeability was quantified at 1, 2 and 3 days after the injection by measuring albumin leakage from blood vessels into the retina and iris using the Evans blue method and normalized by total protein concentrations. The expression of vascular endothelial growth factor (VEGF) in retina was evaluated using the Western Blot analysis and immunohis- tochemistry 24 hours following the injection. RESULTS: ROP rats showed significant increases of vascular permeability in the retina and iris (P <0.01). Angiostatin reduces vascular permeability in a dose-dependent manner in the retina of ROP rats. The reduction showed a time course trend. Angiostatin injection reduced retinal vascular permeability by approximately 1.5 and 2-fold at P15 (P <0.05) and P16 (P < 0.01), respectively. Angiostatin injection significantly reduced VEGF levels in the retina of ROP rats but did not affect retinal VEGF levels in normal rats. CONCLUSION: Angiostatin significantly decreases patholog- ical vascular permeability in the retina and iris of ROP rats but not in normal rats. Angiostatin down-regulates VEGF expression in retina of ROP rats. These results suggest that angiostatin may have a therapeutic potential in the treatment of ROP and other diseases with vascular leakage.

Abstract:AIM: To investigate the expression and distribution of junction adhesion molecule-1(JAM-1) in human corneal epithelium and compare with those of occludin. METHODS: The expression in RNAs of JAM-1 and occludin was revealed by RT-PCR and the presence of protein was analyzed by the FACS method. Double immunofluorescent staining was used to determine the tissue distribution of JAM-1 and occludin in human corneal epithelium. RESULTS: The expression of JAM-1 and occludin was found in cultured human corneal epithelial cells. The double immunofluorescent study showed positive staining for JAM-1 at cell borders in the entire epithelial layer, while relatively extensive staining was seen in the superficial layer, where it coexisted with the expression of occludin. CONCLUSION: JAM-1 is expressed in entire layer of human corneal epithelium encircling the cells.

Abstract:AIM: To investigate the characteristics of Ets-1 and VEGF expression and distribution in the experimental diabetic rat retina. METHODS: Diabetes was induced by intraperitoneal injection of streptozotocin (STZ). At 4 weeks after STZ-injection, animals were sacrificed. Total proteins were isolated from retinas of experimental eyes and control eyes and assessed by Western blot analysis. Frozen cross sections of eyeballs with 14μm thickness were used to perform double immunofluorescence staining with anti-Ets-1 and anti-VEGF antibodies. RESULTS: Both Ets-1 and VEGF expression were up-regulated in the diabetic retina, the distribution of Ets-1 and VEGF was identical to each other, and the two proteins were almost localized in all retinal layers. CONCLUSION: Ets-1 may contribute to the pathologic progress of the diabetic retina induced by VEGF.

Abstract:AIM: To investigate the expression of hyporia inducing factor-1α (HIF-1α), apoptosis of retinal cells and the role of HIF-1α in apoptosis in rats’ retinal ischemia-reperfusion injury. METHODS: The rat model of experimental retinal ischemia- reperfusion injury was established by increasing the intraocular pressure to 110mmHg (1kPa =7.5mmHg) in rat eyes．At different time points of post ischemia, the expression of HIF-1α of the retina was detected by immunohistochemical staining, and the apoptosis of retinal cell was detected by terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphatebiotin nick end labeling (TUNEL). RESULTS: HIF-1α appeared in the cells of retinal ganglion layer and inner nuclear layer at 2 hours after ischemia .The expression reached to a peak, 12 hours after retinal ischemia-reperfusion, then, the expression was declined. The apoptotic cells were mainly in inner nuclear layer and could be detected at the 12^th, 24^th and 48^th hour after ischemia, the peak value was the group of 24^th hour. CONCLUSION: The expression of HIF-1α in rats’ retina is greatly enhanced after ischemia-reperfusion, which may be involved in the retinal injury; the injury of retinal neurons occurs partly in the form of apoptosis. The expression of HIF-1α may play an important role in cell apoptosis.

Abstract:AIM: To determine the involvement of Ets-1 in the pathological progress of the experimental diabetic retina. METHODS: Diabetes was induced by intraperitoneal injection of STZ. Total RNA and total proteins were isolated from retinas of experimental and control eyes at 4 weeks after STZ-injection and were assessed by Northern blot analysis and Western blot analysis, respectively. RESULTS: Expression of both Ets-1 mRNA and Ets-1 protein was significantly increased in the experimental diabetic rat's retina after STZ-injection compared with the control group (P <0.001). CONCLUSION: Our results indicate that Ets-1 is involved in the pathological progress of experimental diabetic retina. Further studies should be conducted to focus on the relationship between Ets-1 and VEGF in the diabetic retina.

Abstract:AIM: To investigate the expression of vascular endothelial growth factor receptor 2 (VEGFR-2, also known as FLK-1) in laser-induced choroidal neovascularization (CNV) in mouse. METHODS: CNV was induced in C57BL/6 mouse eyes by krypton laser photocoagulation. Choroidal fluorescein angiography and histopathological examination were used to assess the development of experimental CNV. Cryostat sections from lesions on day 10 after laser treatment and normal eyes were prepared for immunohistochemistry for FLK-1. RESULTS: Laser-induced CNV developed in all lesions on day 10. The expression of FLK-1 was detected in endothelial cells, retinal pigmented epithelium (RPE)-like cells and fibroblast-like cells in neovascular lesions. In normal adult mouse retinas, FLK-1 expression was mainly observed in RPE cells, inner nuclear and ganglion cell layers. CONCLUSION: Our findings demonstrate that expression of FLK-1 may play a role in the formation of laser-induced CNV in mice, which suggests that FLK-1 may be a promising potential target for antiangiogenesis therapy for CNV.

Abstract:AIM: To investigate the effect of amniotic membrane transplantation(AMT) on rabbit conjunctival surface recon- struction with severe alkali burns.(2) To evaluate the possibility of AMT treatment for ocular alkali burns during recovering stage. METHODS: Animal models were established on 30 eyes of rabbits by creating severe alkali burns on the conjunctiva from the upper corneal limbus to the upper conjunctival fornix. Preserved human amniotic membrane transplantations and reconstruction of conjunctival fornix were performed at one week after injury (recovering stage). Epithelium growth of burned area after transplantation was observed using light microscope at 1, 2, 3, 4, and 8 weeks. Conjunctival tissue in transplantation area was collected at 1, 4 and 8 weeks. The ultrastructure of the collected tissue was studied by electron microscope. The results were compared with control group, which received only vitamin C subconjunctival injection and antibiotic eye drops as treatment for alkali burn. Exterior eye pictures were also taken at the end of the observation, the width from upper corneal limbus to the edge of upper fornix was measured. Data were analyzed statistically. · RESULTS:(1) In the transplant group, conjunctival epithelium growth was observed in the area of AMT under both light and electron microscope 1 week after surgery. At the 4th week, conjunctival epithelium with goblet cells that resembled normal conjunctival tissues was observed in the whole amniotic membrane area. At the 12th week, the conjunctival epithelium on the amniotic membrane was well formed, and the connective tissue under the epithelium was loose at the fornix. No fibrosis was identified. In contrast, conjunctival epithelium necrosis was observed in the control group at 2 weeks after alkali burns. Reepithelization did not occur through the 12-week observation. Severe fibrosis with inflammatory cells infiltration was observed between 4 to 8 weeks. At the 12th week, fibrosis of the connective tissue at the fornix developed and there were no conjunctival epithelium covering the burned area.(2) In the transplant group, the conjunctiva in transplanted area had no scarring and appeared smooth at the 12th week. Upper fornix was reconstructed. The depth of fornix was 7.9±0.3mm (7.6- 8.2mm), which was approximate to the normal depth 8.2±0.2mm (8.0-8.4mm, P >0.05). While in the control group, the burned area appeared rough with granuloma formation and severe scarring. Upper fornix became shallow. The depth of fornix was 3.1±1.7mm (1.0 - 4.5mm.), and significant difference was found between control and transplant group (P <0.01). CONCLUSION: Human amniotic membrane preserved in glycerin can promote cell adhering, migrating and differenti- ating of normal conjunctival epithelium. Reconstruction of conjunctival surface in early stage of alkali burn can be achieved by AMT. AMT can effectively prevent symblepharon formation.

Abstract:AIM: To study caspase-9 expression on rat retina in the process of chronic elevation of IOP and the changes with the application of amino guanidine (AG), thus to investigate the potential protective function of AG to rat retina with chronic elevation of IOP. METHODS: Immunohistochemistry, RT-PCR and Western blot were used to observe retinal morphology and the expression of caspase-9 at different time points of rat with chronic IOP elevation, both affected and not affected by the application of AG. ·RESULTS: Compared with control group, as time passed retina of experimental group gradually had detectable morphological changes. On 21^st day of chronic IOP elevation, retinas became thinner and the quantity of retinal ganglion cells (RGCs) decreased; caspase-9 expression increased, consistent with the morphological changes. The group using AG presented relatively smaller morphology changes and less expression of caspase-9. CONCLUSION: Apoptosis-related gene caspase-9 plays a part in the process of chronic IOP elevation; AG protects retina by down-regulating expression of caspase-9.

Abstract:AIM: To investigate the effect of the dipeptide Arg-Gln on retinal neovascularization of retinopathy of prematurity (ROP) in the oxygen-induced retinopathy (OIR) animal model. METHODS: Forty-eight 7-day-old C57BL/6J mice were exposed to 750mL/L oxygen for 5 days and then to normal situation to produce the murine model of oxygen-induced retinopathy (OIR). All mice received twice daily intra- peritoneal injections of PBS or the dipeptide Arg-Gln (1.0, 3.0, 5.0g/kg per day), starting on postnatal day 12 and continuing till postnatal day 17. Experimental groups (36 mice, 12 in each group) received Arg-Gln, while the control group (12 mice) received PBS. All mice were executed at postnatal day 17. The changes of retinal vessels of mice were observed by ADPase histochemical technique and HE staining was used to count preretinal neovascular nuclei. RNA was isolated from retinas of 28 mice (7 in each group) selected at random and VEGF mRNA level of each group was measured by real-time RT-PCR. RESULTS: Neovascularization reduced in retinas of the dipeptide Arg-Gln treated group in a dose-dependent manner. Compared with control group, experimental group had diminished non-perfusion area and neovascular tufts in retinal flatmount. The number of the endotheliocyte nuclei of new vessels extending from retina to vitreous was significantly less in the eyes of the experimental group than in control group. Arg-Gln at 5g/kg per day reduced preretinal neovascularization by about 75% (P <0.01). There was a significant reduction in VEGF mRNA at the 17^th day in Arg-Gln treated group compared with control group(P ＜0.01). CONCLUSION: Arg-Gln dramatically inhibits retinal angiogenesis in OIR and this effect is associated with a reduction in retinal VEGF mRNA level. It appears to be a safe way to prevent and treat some neovascular retinal diseases including retinopathy of prematurity.

Abstract:AIM: To investigate the effect of Tetrandrine (Tet) on proliferation of human pterygium fibroblasts (HPF) in vitro and to search for a new method to prevent the recurrence after pterygium surgery. METHODS: With different concentrations (0 to 160μmol/L) of Tet acting on HPF cultured in vitro , the impact was observed at 24, 48, 72 hours respectively after Tet intervention. The MTT method was used to assay the biologic activities of Tet and inhibitive rate of cell growth. The expression of proliferating cell nuclear antigen (PCNA) in each group was detected by immunohistochemistry before and after Tet intervention. RESULTS: With different concentrations of 20, 40, 80 and 160μmol/L and acting for 24 to 72 hours, Tet could inhibit the proliferation of HPF in a dose- and time-dependent manner (P <0.05). After the intervention of Tet, the expression of PCNA protein declined. When the concentration of Tet was in the range of 20 to 160μmol/L, it was able to inhibit the expression of PCNA in a concentration-dependent manner (P <0.05). CONCLUSION: Tet can significantly inhibit the proliferation of pterygium fibroblasts, and the inhibitive action is in a dose- and time-dependent manner within a certain range of concentration. But in high concentration (>160μmol/L), Tet will have cytotoxity.

Abstract:AIM: To investigate the role of retinal pigment epithelium (RPE) in the growth of Müller cells using a co-culture system in vitro. METHODS: Müller cells were co-cultured with RPE cells under both normoxic and hypoxic conditions in Transwell chamber culture system. Müller cell proliferation was eval- uated by MTT assay. The number of cells that migrate through micropores and stay on the outer bottom side of insert systems were observed and counted. RESULTS: The activities of proliferation and migration of Müller cells when co-cultured with RPE cells were significantly higher than those of the Müller cells when cultured alone at all time points under both normoxic and hypoxic conditions. However, for both the co-culture and control groups, there was no significant difference between the measurements at 3 and 6 hours. CONCLUSION: Evidence suggests that RPE, when co- cultured with Müller cells, can stimulate migration and prol- iferation of Müller cells under both hypoxic and normoxic conditions in a time-dependent manner; however, there is no evidence to support the synergetic interaction of RPE and Müller cells co-cultured under hypoxic conditions.

Abstract:AIM: To determine the effect of the tyrphostin AG1295 and AG1296, a selective blocker of platelet-derived growth factor (PDGF) β and α RTK, on proliferative vitreoretinopathy (PVR) development. METHODS: Rabbit conjunctival fibroblasts (RCF) cells were cultured. The effects of AG1295, AG1296, PDGF-AA and PDGF-BB on RCF proliferation were evaluated by MTT assay. Homologous rabbit conjunctival fibroblasts were injected intravitreally to make animal PVR model, followed by injection of 100μmol/L of AG1295 or AG1296 respectively. The presence of tractional retinal detachment (TRD) was assessed to evaluate the effect of AG1295 and AG1296 in vivo. Electroretinography and histologic studies were performed after intravitreal injection of AG1295 into untreated eyes to evaluate toxicity. · RESULTS: Both AG1295 and AG1296 (10μmol/L) significantly inhibited rabbit conjunctival fibroblast cell growth stimulated by PDGF-AA or -BB in vitro . Development of TRD was significantly reduced (P <0.05) with 100μmol/L of AG1295 or AG1296 in vivo , but the effect of AG1295 only present until day 14. Inhibitive effect of AG1296 was longer than that of AG1295. No significant histological or retinal functional damage was found in both drug-treated groups. CONCLUSION: PDGF α and β receptor specific inhibitor AG1296 and AG1295 attenuate PVR without significant side effects in rabbits, and AG1296 is better than AG1295. The much longer and stronger therapeutic effect from PDGF α receptor inhibitor indicates that PDGF α receptor is more important in the development of PVR, and inhibition of this pathway can be a useful treatment alternative to prevent PVR.

Abstract:AIM: To determine the tearing angle and tearing force, and effects of associated pressures in tearing of various materials and human lens capsule in continuous curvilinear capsulorhexis (CCC). METHODS: Tearing was done on different materials such as aluminum-laminated paper, different types of thin transplant plastics and human lens capsule with blunt tip needle. During the procedure, angle and direction of force were measured. Effects of increased underlying pressure on tearing of tearable materials and effect of anterior chamber depth and vitreous pressure on 24 postmortem human eyes with different ages (range from10 to 75 years), was evaluated. RESULTS: Tearing angle in every material was unique for that material Angle and force of tearing was decreased reversely with increasing age (from 85 degree in a 10-years-old to 10 degree in older than 50 years). Increasing vitreous pressure and decrease in AC depth causes higher pressure on point of tearing. Safe methods in controlling CCC were discussed in the context. · CONCLUSION: Understanding the physics and vector of forces during CCC is necessary in good performance and avoidance of radial tears. Change in capsular properties between different ages and different type of cataract causes different tearing angle and tearing force that should be considered during CCC.

Abstract:AIM: To examine a new performance test for detecting eye dominance by testing and re-testing with two different methods of the same subjects for comparing and discussing the reliabilities of these tests. METHODS: A total of 179 university students (mean± SD: 19.37± 1.62 years) were voluntarily participate in this survey consisting of 110 females (61.5%) and 69 males (38.5%). Eye dominances were determined by two different methods which were named McManus and a Gündoğan tests. The reliability of the survey was examined using a test-retest method. RESULTS: Without sex difference, right eyes were found dominant in 128 (71.5 %) participants by McManus test. The same subjects were re-tested by Gündo an method, the right eye dominance were found in 110 (61.5%) subjects. The results of these two methods were related significantly by Fisher exact test(P<0.01), with an agreement score( k=0.256, P<0.001). In females, the right eye dominance was found in 74 (67.3%) and left eye was found in 36 (32.7%) by McManus test. When the same subjects were re-tested by G ndo an method, the right eye dominance was found in 62 (56.4%) and the left eye dominance was found in 48 (43.6%) subjects.McManus and Gündoğan methods results for females were related significantly by Fisher exact test ( P<0.05), with a weak agreement score ( k=0.239, P<0.01). In males, the right/left eye dominances were found respectively 54 (78.3%), 15(21.7%) by McManus test as they were found 48 (69.6 %), 21 (30.4 %) in the same participants when they were re-tested by Gündoğan method.χ ² test and Fisher exact test were used for the analysis of categorical data. The agreement between different methods was analyzed with Kappa statistics. Comparison of proportions was made by two proportions test. value less than 0.05 was considered as significant. CONCLUSION: Without gender difference and also in both females and males marked right eye dominance is observed. The right eye dominance is considering functional laterality may due to the dominance of left hemisphere instead of right hemisphere. It is an important topic for future research in laterality, and it may well become an important model system for future research."

Abstract:Uveitis is an inflammation of any or all parts of the uveal tract including the iris, ciliary body and the choroid. Despite current advances in diagnosis and management, visual loss occurs in 35%-45% of patients with uveitis. The etiopatho- genesis of uveitis remains unknown; it may be associated with environmental and immunogenetic factors. Many studies have demonstrated polymorphisms in major histocompatibility complex (MHC) genes, which may determine involvement in uveitis. Recently polymorphisms in non-MHC genes, including cytokine and chemokine genes, have been reported to play important roles in the pathogenesis of uveitis. We reviewed the advances in the studies on cytokine and chemokine gene polymorphisms associated with uveitis.