Experimental Corneal Research Articles

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

To explore the effects and mechanism of vascular endothelial cadherin (VE-cadherin) on experimental corneal neovascularization (CRNV). 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. 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. VE-cadherin exhibited an anti-apoptosis effect through enhanced PKC signaling and an enhanced cell proliferation pathway.

A Comparison of Different Corneal Iontophoresis Protocols for Promoting Transepithelial Riboflavin Penetration.

To measure corneal riboflavin penetration using different transepithelial iontophoresis protocols. Freshly enucleated rabbit eyes were divided into nine treatment groups of 4 eyes. One group, in which 0.1% wt/vol riboflavin was applied for 30 minutes without iontophoresis after corneal epithelial debridement, acted as a control. The remaining groups were treated with an intact epithelium using different riboflavin formulations and varying iontophoresis current, soak, and rinse times. After riboflavin application, eyes were snap frozen in liquid nitrogen. Corneal cross sections 35 μm thick were then imaged immediately by two-photon fluorescence microscopy, using image processing software to quantify stromal riboflavin concentration at different corneal depths. In the epithelium-on iontophoresis treatment groups, greater stromal riboflavin penetration was achieved with higher-concentration riboflavin solutions, greater iontophoresis dosage, and longer solution contact times. A protocol utilizing 0.25% wt/vol riboflavin with benzalkonium chloride (BAC) 0.01% and two cycles of applied current and subsequent soaking (1 mA 5 minutes, soak 5 minutes; 0.5 mA 5 minutes, soak 5 minutes) achieved similar stromal riboflavin penetration to epithelium-off controls. The best-performing non-BAC-containing protocol produced stromal riboflavin penetration approximately 60% that of epithelium-off controls. Riboflavin solutions containing saline resulted in minimal stromal penetration. Riboflavin loading within the epithelium was equivalent to or higher than that in the subjacent stroma, despite rinsing the ocular surface with balanced salt solution. Modified iontophoresis protocols can significantly improve transepithelial riboflavin penetration in experimental corneal collagen cross-linking.

Dynamic parameters of blood and aqueous humor endogenous intoxication in case of experimental corneal mechanical nonpenetrative injury and its correction by сeratoxenoimplant

<p>The influence of corneal mechanical non-penetrative injury on indices of endogenous intoxication of blood and<br />aqueous humor in rabbits and its correction by сeratoxenoimplant were studied. It was determined the decrease in<br />the level of endogenous intoxication, as was indicated by decreased content of middle mass molecules blood and<br />aqueous humor as a result of surgical application of сeratoxenoimplant. This demonstrates the feasibility of further<br />clinical testing and possible prospects in wide use of сeratoxenoimplants in ophthalmic practice to reduce metabolic<br />manifestations of the syndrome of endogenous intoxication.</p>

The inhibitory effect of Itraconazole on Corneal neovascularization in Rabbits

PurposeTo evaluate the inhibitory effect of itraconazole on corneal neovascularization in rabbits.MethodsCorneal neovascularization was induced in 36 eyes of 18 rabbits by suture placement in the corneal stroma. Seven days after suture placement, all rabbits were randomly divided into 4 groups and were treated four times daily with balanced salt solution (Group 1, 4 rabbits), topical 0.5% itraconazole (5 mg/mL , Group 2, 4 rabbits), topical 1% itraconazole (10 smg/mL , Group 3, 6 rabbits), and topical 2% itraconazole (20 mg/mL , Group 4, 4 rabbits). After one week, the surface area of corneal neovascularization was assessed on the digital photographs. In the corneal specimens, the concentration of VEGF A (vascular endothelial growth factor), VEGF R2, and PLGF (placental growth factor) mRNAs was measured by RT‐PCR, and the concentration of ERK, p‐ERK, Flk, and p‐Flk was measured by Western Blotting.ResultsThe surface area of induced corneal neovascularization was significantly smaller in Groups 2, 3, and 4 compared to the control group on day 14 (p < 0.05). RT‐PCR analysis showed that the mean concentration of VEGF and PLGF in Groups 2, 3, and 4 was significantly lower than that in the control group after 7 days of treatment. Western Blotting analysis showed that the mean concentration of p‐ERK, Flk, and p‐Flk in Group 3 was significantly lower than that in the control group after 7 days of treatment.ConclusionsTopical itraconazole application was useful for effective inhibition of experimental corneal neovascularization.

The inhibitory effect of Itraconazole on Corneal neovascularization in Rabbits

PurposeTo evaluate the inhibitory effect of itraconazole on corneal neovascularization in rabbits.MethodsCorneal neovascularization was induced in 36 eyes of 18 rabbits by suture placement in the corneal stroma. Seven days after suture placement, all rabbits were randomly divided into 4 groups and were treated four times daily with balanced salt solution (Group 1, 4 rabbits), topical 0.5% itraconazole (5 mg/mL, Group 2, 4 rabbits), topical 1% itraconazole (10 mg/mL, Group 3, 6 rabbits), and topical 2% itraconazole (20 mg/mL, Group 4, 4 rabbits). After one week, the surface area of corneal neovascularization was assessed on the digital photographs. In the corneal specimens, the concentration of VEGF A (vascular endothelial growth factor), VEGF R2, and PLGF (placental growth factor) mRNAs was measured by RT‐PCR, and the concentration of ERK, p‐ERK, Flk, and p‐Flk was measured by Western Blotting.ResultsThe surface area of induced corneal neovascularization was significantly smaller in Groups 2, 3, and 4 compared to the control group on day 14 (p < 0.05). RT‐PCR analysis showed that the mean concentration of VEGF and PLGF in Groups 2, 3, and 4 was significantly lower than that in the control group after 7 days of treatment. Western Blotting analysis showed that the mean concentration of p‐ERK, Flk, and p‐Flk in Group 3 was significantly lower than that in the control group after 7 days of treatment.ConclusionsTopical itraconazole application was useful for effective inhibition of experimental corneal neovascularization.

Dynamic point shifting with null screens using three LCDs as targets for corneal topography.

In this paper, the use of three liquid crystal displays (LCDs) as targets for an experimental corneal topographer is proposed. The main advantage is that the geometrical pattern in the target can be modified without moving any mechanical part to apply the dynamic point shifting (DyPoS) method. Some results on the capabilities of the LCDs, obtained with photo frames, for measuring a 6.37mm radius of curvature calibration sphere, and applying the DyPoS method, are presented. It is shown that the error in measuring the radius of curvature with DyPoS is reduced to 3% of the real value and the RMS in elevation or sagitta differences is around 15μm, 30% or 66% of the values obtained without DyPoS, respectively.

Imidazole-based alkaloid derivative LCB54-0009 suppresses ocular angiogenesis and lymphangiogenesis in models of experimental retinopathy and corneal neovascularization.

Abnormally induced angiogenesis and lymphangiogenesis are associated with human diseases, including neovascular eye disease. Substances that inhibit these processes may have potential as an attractive therapeutic strategy for these diseases. In vitro and in vivo angiogenesis and/or lymphangiogenesis were assessed in VEGF- or hypoxia-stimulated endothelial and retinal cells and in animal models of oxygen-induced retinopathy (OIR), streptozotocin-induced diabetic retinopathy (SIDR), suture-induced inflammatory corneal neovascularization (SICNV) and silver nitrate-induced corneal neovascularization. HUVECs and retinal cells were cultured under hypoxic conditions or incubated with VEGF to identify the molecular mechanisms involved. The imidazole-based alkaloid derivative LCB54-0009 inhibited capillary-like tube formation in VEGF-induced HUVECs without inducing cytotoxic effects. Intravitreal injection of LCB54-0009 into retinas suppressed the formation of the pathological neovascular tufts and increased vascular permeability in both OIR of mice and SIDR of rats. Furthermore, subconjunctival injection of LCB54-0009 into the cornea suppressed corneal inflammation and inflammation-associated angiogenesis and lymphangiogenesis in SICNV of mice and silver nitrate cauterization of rats. These pharmacological activities were associated with effects on HIF-1α protein stability and HIF-1α/NF-κB redox sensitivity through its antioxidant activities. LCB54-0009 also inhibited the hypoxia-induced expression of angiopoietin-2, and VEGF-induced VEGFR-2 activation and downstream signalling, resulting in the down-regulation of the expression of pro-angiogenic factors and pro-inflammatory mediators and an up-regulation of the expression of anti-angiogenic factors. LCB54-0009 is a potential candidate molecule for blocking pathological angiogenesis and lymphangiogenesis mediated by HIF-1α- angiopoietin-2 expression and VEGFR-2 activation.

Topically administered gold nanoparticles inhibit experimental corneal neovascularization in mice.

The aim of this study was to evaluate the in vivo effects and mechanism of topically administered gold nanoparticles (AuNP) in a mouse model of corneal neovascularization. Inflammatory corneal neovascularization was induced by alkali burns, and the corneas were treated with topical AuNPs. After 1 week, the area of corneal neovascularization was measured using image analysis. The levels of vascular endothelial growth factor receptor 2 and extracellular signal-regulated kinase (ERK1/2) were evaluated by Western blotting. The area of corneal neovascularization was significantly reduced by 39.8% in the AuNP group compared with the control group (P = 0.002). Corneal vascular endothelial growth factor receptor 2 level was higher in the control group than in the AuNP-treated group (P = 0.029). AuNP treatment similarly inhibited burn-induced phosphorylation of ERK (P = 0.029). Topical administration of AuNPs significantly reduced development of inflammatory corneal neovascularization by inhibiting the ERK pathway.

Inhibition of FK506 on the expression of vascular endothelial growth factor in retinal Müller cells cultured by high concentration glucose

Background Retinal Muller cells participate in the pathological process of diabetic retinopathy (DR) through expressing vascular endothelial growth factor (VEGF).It is reported that FK506 inhibits the expression of VEGF in solid tumors and experimental corneal neovascularization,but whether FK506 exerts its role on retinal Muller cells or not is still unclear.Objective This study aimed to investigate how FK506 affects the expression vascular endothelial growth factor (VEGF) in rat retinal Muller cells under the condition of high glucose.Methods Immortalized rat retinal Muller cell line was regularly cultivated and logarithmic phase of cells were incubated in 96-well plate with the cell density of 1 × 104/ml.Different concentrations of FK506 (800.00,400.00,200.00,100.00,75.00,50.00,25.00,12.50 and 6.25 pg/ml) (100 μl/well) were added into the culture medium to determine the half maximal inhibitory concentration (IC50) of FK506 by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).The cell lines were cultured with DMEM medium (containing D-glucose of 5.5 mmol/L) or high glucose DMEM (containing D-glucose of 50 mmol/L),and 75 pg/ml FK506 were added into DMEM,respectively,and the cells were divided into the normal control group,FK506 group,high glucose culture group and high glucose + FK506 group.ELISA was employed to assay the content of VEGF protein in the cell supernatant.The expressions of VEGF mRNA and protein in the cells were detected by reverse transcription PCR (RT-PCR) and Western blot,respectively.Results The cells grew well in the normal control group,FK506 group,high glucose culture group and high glucose+FK506 group in 12,24 and 48 hours after culture with the polygon-like shape.The IC50 of FK506 was 75 pg/ml.The contents of FK506 in the cell supernatant were (966.46± 13.59) pg/ml,(1 059.42±67.43) pg/ml,(16 243.11 ±3 926.38) pg/ml and (9 467.25± 1 525.56) pg/ml in the normal control group,FK506 group,high glucose culture group and high glucose+FK506 group,respectively,showing a significant difference among the four groups (F =20.51,P =0.00).The VEGF levels in cell supernatant were significantly higher in the high glucose group than those of the normal group and the high FK506 group (P =0.00,P =0.02),but no significant difference was found in the VEGF level in cell supernatant between the control group and FK506 group (P =0.08).The expressions of VEGF mRNA and protein in the cells were significantly different among the four groups (F=126.06,P=0.00;F=5.44,P=0.01),and the relative expressing values of VEGF mRNA and protein in the cells of the high glucose group were significantly higher than those of the control group and the high+ FK506 group (all at P＜0.01).The relative expressing values of VEGF mRNA and protein were 0.64±0.09 and 0.68±0.18 in the FK506 group,which were lower than those of the normal control group (0.84±0.07 and 0.75± 0.03).However,no significant differences were seen between the two groups (P =0.05,0.07).Conclusions The expression of VEGF in rat retinal Muller cells up-regulates under the high glucose condition.FK506 plays inhibitory effects on VEGF expression to certain extent in vitro. Key words: Antibiotic agent/FK506; Retina; Glial cell/Muller cell; Vascular growth factor; High glucose condition; Cell culture

Critical Role of IP-10 on Reducing Experimental Corneal Neovascularization

Aim and scope: To address the role of interferon-induced protein of 10 kDa (IP-10) in the course of corneal neovascularization (CrNV) in a mouse model of experimental corneal neovascularization. Material and method: BALB/c mice that were 7- to 8-week-old male were included in the study. Corneal injury was induced by NaOH. Mice were randomly divided into 2 groups of IP-10 and vehicle. The alkali-treated eyes received 5 μl of 5 μg/ml IP-10 dissolved in 0.2% sodium hyaluronate for IP-10-treated group, or 5 μl of 0.2% sodium hyaluronate for vehicle-treated group twice a day for 7 days immediately after the alkali injury. 2 weeks after alkali injury, corneas were removed and used for whole mount CD31 staining. The percentages of neovascularization on corneal photographs were examined with digital image analysis. In other experiments, at indicated time intervals, the corneas were removed. Angiogenic factor expression in the early phase after injury was quantified by real-time PCR and western blot. The VEGF expression in macrophages infiltrating into burned corneas was examined by Flow cytometry (FCM) and immunofluorescence. Tube formation and cell proliferation of human retinal endothelial cells (HRECs) were detected after being stimulated with IP-10 in vitro. Results: The mRNA and protein expression of IP-10 and C-X-C motif chemokine receptor 3 (CXCR3) was augmented after the alkali injury (p < 0.05). Compared with vehicle-treated mice, IP-10-treated mice exhibited reduced CrNV 2 weeks after injury, as evidenced by diminished CD31-positive areas (p < 0.05). Concomitantly, the intracorneal mRNA and protein expression enhancement of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) was lower in IP-10-treated mice than in vehicle-treated mice after injury (p < 0.05). Moreover, IP-10 inhibited HREC tube formation and proliferation in vitro. Conclusion: IP-10-treated mice exhibited reduced alkali-induced CrNV through decreasing intracorneal VEGF and bFGF expression, and inhibiting endothelial cell proliferation and tube formation.

Inhibitory effect of tight junction protein on experimental corneal neovascularization

Background Corneal neovascularization (CNV) is one of the causes of corneal blindness.Studies showed that zonula occludens-1 (ZO-1) can inhibit pathological angiogenesis through physical barrier formed by tight junction structure.However,whether ZO-1 plays a role in CNV is unclear.Objective The aim of this study was to explore the effect of ZO-1,a tight junction protein on experimental CNV.Methods The CNV models were established in the left eyes of 24 clear male BALB/c mice aged 7-8 weeks by putting NaOH filter paper in the center of corneas for 15 seconds (15 s group) or 40 seconds (40 s group).CNV was examined and evaluated under the slit lamp microscope,and the expression of ZO-1 mRNA in the corneas were detected and compared by reverse transcription PCR (RT-PCR) between the two groups 2 weeks after modeling.In addition,54 models created by the same method were assigned to 3 groups according to randomized number table,0.2％ hyaluronic acid (HA),antiZO-1 neutralizing antibody (10 mg/L) +0.2％ HA and mouse hypoxia inducible factor-1α (HIF-1α) recombinant protein (5 mg/L)+0.2％ HA were topically administrated in the mice three times a day for 1 week after modeling respectively.The corneas were extracted 2 weeks after application of the drugs.Expression of CD31 in the CNV was assayed to calculate the number and the area of CNV by immunohistochemistry.The expression of VEGF mRNA in the corneas was detected by RT-PCR.The percentages of macrophage-specific F4/80 positive cells and neutrophilsspecific Ly-6G positive cells were calculated to evaluate the infiltrations of inflammatory cells in the corneas by flow cytometry.Results In 2 weeks after alkali burn of corneas,the number of severe CNV was more in the 40 s group than that in the 15 s group (x2 =6.032,P=0.049),and the expression level of ZO-1 mRNA was lower in the 40 s group than that in the 15 s group (1.15±0.08 versus 1.53±0.04) (t=4.157,P=0.014).CD31 positive cell number was more and the staining area was larger in the ZO-1 antibody group and HIF-1α positive control group than those in the 0.2％ HA group (cells:t=-129.590,-226.820,both at P=0.000;area:t =-5.310,-8.840,both at P=0.000).The relative expressions level of vascular endothelial growth factor (VEGF) mRNA was 1.33±0.10 and 1.46±0.11 in the ZO-1 antibody group and HIF-1 α positive control group respectively,which were significantly higher than 0.93±0.06 of the 0.2％ HA group (t =-5.820,-7.284,both at P =0.000).The percentages of positive cells in the ZO-1 antibody group and HIF-1α positive control group were significantly increased in comparison with the 0.2％ HA group for F4/80 (t =-16.750,-17.480,both at P =0.000) and for Ly-6G (t =-21.450,-27.680,both at P=0.000).Conclusions Alkali burn induced CNV downregulates the expression of ZO-1 mRNA.Administration of ZO-1 antibody causes the rise of VEGF mRNA in CNV and the infiltration inflammation cells,which suggests that the influence of ZO-1 on CNV is associated with the expression of VEGF. Key words: Tight junction protein; Neovascularization; Alkali injury; Cornea; Disease model/animal; BALB/c mouse

Corneal viscoelastic properties from finite-element analysis of in vivo air-puff deformation.

Biomechanical properties are an excellent health marker of biological tissues, however they are challenging to be measured in-vivo. Non-invasive approaches to assess tissue biomechanics have been suggested, but there is a clear need for more accurate techniques for diagnosis, surgical guidance and treatment evaluation. Recently air-puff systems have been developed to study the dynamic tissue response, nevertheless the experimental geometrical observations lack from an analysis that addresses specifically the inherent dynamic properties. In this study a viscoelastic finite element model was built that predicts the experimental corneal deformation response to an air-puff for different conditions. A sensitivity analysis reveals significant contributions to corneal deformation of intraocular pressure and corneal thickness, besides corneal biomechanical properties. The results show the capability of dynamic imaging to reveal inherent biomechanical properties in vivo. Estimates of corneal biomechanical parameters will contribute to the basic understanding of corneal structure, shape and integrity and increase the predictability of corneal surgery.

Neovascular growth in an experimental alkali corneal burn model

ObjectiveTo analyze the length and area of corneal surface occupied by vessels, and their location in an experimental model of alkali burn-induced corneal neovascularization. MethodsAn injury to the central cornea of the right eye in 91 Sprague-Dawley rats was induced using a silver nitrate pencil. The rats were divided into seven groups that were sacrificed 2, 4, 6, 8, 10, 12 and 14 days post-injury, and then perfused with a mixture of Chinese ink in PBS (phosphate buffer saline). Corneas were flat-mounted processed and divided into four quadrants. Corneal neovascular growth parameters (length and area) and the location of these vessels were performed blind. The results were statistically analyzed. ResultsNeovascular growth was observed from day 2, reaching its maximum peak in length and area on the 12th day post-injury. A slight reduction in corneal neovascularization was observed after this day. The vessels were initially located in the middle third of the stroma and tended to be observed in the anterior third during the course of the experiment. ConclusionsNeovascularisation was observed on day 2 post-injury in all sectors of corneal surface. Neovascular growth was uniform during the experiment. Neovessels were located in the middle and anterior third of the cornea.

Inhibited experimental mouse corneal neovascularization by CCR3 antagonist

To explore the effect of CCR3 antagonist on the development of experimental corneal neovascularization. Mouse corneas were burned by NaOH to induce corneal neovascularization.Fifty four clean male BABL/c mice aged 7-8 weeks were divided into control group, CCR3 antagonist group and VEGF antibody positive group according to randomized number table. The gene expression of CCR3 and its ligand eotaxin in burned corneas was examined by Real-time PCR. CCR3 antagonist was locally administrated after alkali injury and the formation of corneal neovascular 2 weeks after injury was examined using a digital camera linked to a slit lamp microscope and corneal whole mount staining with CD31. The mRNA and protein expression of chemokines in burned corneas was detected by Real-time PCR and western blot. Compared to control group, CCR3 antagonist treated mice resulted in significantly decreased corneal neovascularization. The related CNV area was 0.51 ± 0.03 in the CCR3 antagonist group, and that in the control group was 0.77 ± 0.15, with significant difference between them (t = 12.91, P = 0.00).Western blot detection did not show significant difference of VEGF protein expression between two groups.Expression level of VEGF in the CCR3 antagonist group was 0.91 ± 0.24, and that in the control group was 1.15 ± 0.30, showing no significant difference (t = 1.08, P = 0.34). Alkali-induced corneal neovascularization was inhibited by CCR3 antagonist. The mechanism that CCR3 pathway plays an important role in corneal neovascularization needs further exploration.

Effects of Topical Janus Kinase Inhibition on Ocular Surface Inflammation and Immunity

To determine the effects of topical Janus kinase inhibition on ocular surface inflammation and immunity. Ophthalmic 0.003% tofacitinib (CP-690,550) was administered topically to inhibit Janus kinase activation at the ocular surface. Male BALB/c mice 6 to 8 weeks of age were subjected to corneal thermocautery and randomized to receive tofacitinib, vehicle, or no treatment. Corneas were subsequently excised for fluorescence-activated cell sorting and quantitative real-time reverse transcription polymerase chain reaction. Female C57BL/6 mice 6 to 8 weeks of age were exposed to desiccating stress to induce experimental dry eye disease and randomized to receive tofacitinib, tofacitinib and vehicle, vehicle, or no treatment. Corneal fluorescein staining was performed to evaluate clinical disease severity. The corneas and conjunctivae were harvested for immunohistochemical staining and quantitative real-time reverse transcription polymerase chain reaction. After corneal thermocautery, it was found that tofacitinib treatment decreased the corneal infiltration of CD45+, Gr-1+, and CD11b+ cells on days 1 and 3. Transcripts encoding interleukin (IL)-1β and IL-6 were significantly decreased by tofacitinib treatment at post-thermocautery day 3. In experimental dry eye disease, tofacitinib treatment twice per day significantly decreased corneal fluorescein staining on days 12 and 15. The corneal infiltration of CD11b+ cells was significantly decreased by tofacitinib treatment twice per day. Tofacitinib treatment twice per day significantly increased the corneal expression of IL-1RA, and significantly decreased the corneal expression of tumor necrosis factor and IL-23. Further, tofacitinib treatment twice per day significantly decreased the conjunctival expression of IL-17A and significantly increased the conjunctival expression of FoxP3. Topical ophthalmic tofacitinib, a Janus kinase inhibitor, suppressed ocular surface inflammation and immunity in experimental corneal thermocautery and dry eye disease.

Induction of corneal collagen cross-linking in experimental corneal alkali burns in rabbits.

Objective: To evaluate the effect of riboflavin-ultraviolet-A-induced cross-linking (CXL) following corneal alkali burns in rabbits. Methods: The right corneas and limbi of ten rabbits were burned using a 1N solution of NaOH and the animals were then divided into two groups: a control group submitted to clinical treatment alone and an experimental group that was treated 1 h after injury with CXL, followed by the same clinical treatment as administered to the controls. Clinical parameters were evaluated post-injury at 1, 7, 15, and 30 days by two independent observers. Following this evaluation, the corneas were excised and examined histologically. Results: There were no statistically significant differences in clinical parameters, such as hyperemia, corneal edema, ciliary injection, limbal ischemia, secretion, corneal neovascularization, symblepharon, or blepharospasm, at any of the time-points evaluated. However, the size of the epithelial defect was significantly smaller in the CXL group (p<0.05) (day 15: p=0.008 and day 30: p=0.008) and the extent of the corneal injury (opacity lesion) was also smaller (day 30: p=0.021). Histopathology showed the presence of collagen bridges linking the collagen fibers in only the CXL group. Conclusions: These results suggest that the use of CXL may improve the prognosis of acute corneal alkali burns.

Local and systemic antioxidant status in experimental purulent corneal ulcer

Aim. To examine the systemic and local (corneal) free radical status in rabbits with experimental purulent corneal ulcers. Methods. The study was performed on the male chinchilla rabbits with an average weight of 3500±200 g 10 intact rabbits (10 eyes) served as controls. Staphylococcal purulent corneal ulcer was modeled on 40 animals (40 eyes) as described by N.A. Adamova (1999). The animals were euthanized at 3rd, 7th, 14th, and 21st day of the experiment. The concentration of malondialdehyde, protein-free thiols, as well as superoxide dismutase, glutathione peroxidase and glutathione-S-transferase activity were determined in cornea homogenate and erythrocyte lysate. The obtained data were processed by the one-factorial dispersive analysis (ANOVA), Newman-Keuls test. Results. In all animals the purulent ulcer occurred 12-24 hours after staphylococcus culture introduction. From day 1 to day 3 of the experiment, the clinical picture of a purulent ulcer corresponded to an infiltration stage. From day 3 to day 7 of the experiment, palpebral edema developed, the purulent discharge lasted out, and in some cases even intensified. From day 7 to day 21 of the experiment, corneal infiltration decreased both by area and intensity. In 11,1% scaring corneal opacity formation began. Experimental purulent corneal ulcer development was accompanied by lipid peroxidation activation both in cornea and in erythrocytes, shown by increased malondialdehyde concentration, decreased protein-free thiols level and antioxidant glutathione peroxidase, glutathione-S-transferase and superoxide dismutase enzymes activity. There was a strong direct relationship between the erythrocyte malondialdehyde level and corneal malondialdehyde concentration, and inverse relationship between erythrocyte malondialdehyde level and erythrocyte superoxide dismutase activity. Conclusion. The development of experimental staphylococcal corneal ulcers is accompanied by the development of local and systemic oxidative stress.

Application of paraffin embedding slice fluorescent staining method in the experimental xenogenic corneal transplantation immune diagnosis

Application of paraffin embedding slice fluorescent staining method in the experimental xenogenic corneal transplantation immune diagnosis

Topically administered bevacizumab had longer standing anti-angiogenic effect than subconjunctivally injected bevacizumab in rat corneal neovacularization.

To compare the effect of topically administered and subconjunctivally injected bevacizumab on experimental corneal neovascularization in rats for two weeks after treatment. Twenty-eight Sprague-Dawley rats were divided into four groups of 7 animals. Each corneal center of right eye was cauterized with silver/potassium nitrate for 8s. After corneal burning, bevacizumab (12.5mg/mL) was topically administered three times per day (TB group) for two weeks or subconjunctivally injected on days 2 and 4 after cauterization (0.02mL; SB group). As negative controls, rats received 0.9% saline topically three times per day (TS group) or subconjunctivally on days 2 and 4 (0.02mL; SS group). Digital photographs of the cornea were taken 1 and 2 weeks after treatment and analyzed to determine the area of cornea covered by neovascularization as the percentage of corneal neovascularization. One week after treatment, the percentage of corneal neovascularization was significantly lower in the TB and SB groups than in the TS and SS groups (all P<0.05). Two weeks after treatment, the percentage of corneal neovascularization was significantly lower in the TB group than in the TS group (P<0.05). In all groups, the percentage of neovascularization was decreasing as time passed (all P<0.05). Topically administered bevacizumab has longer standing anti-angiogenic effect than subconjunctivally injected bevacizumab in corneal neovascularization following chemical injury in rats.

RGTA-based matrix therapy in severe experimental corneal lesions: Safety and efficacy studies

Corneal alteration potentially leading to ulceration remains a major health concern in ocular surface diseases. A treatment that would improve both the quality and speed of healing and control the inflammation would be of great interest. Regenerating agents (RGTAs) have been shown to stimulate wound healing and modulate undesired fibrosis in various in vivo systems. We investigated the effects of RGTA-OTR4120(®) in a rabbit corneal model in order to assess its potential use in ocular surface diseases. First, we assessed its safety for 7 and 28 days using the Draize test criteria in healthy rabbit eyes; then, we investigated the effect of a single dose (50μl, 5μg) in an alkali-burned cornea model. Daily follow-up of clinical signs of healing was scored, and histology was performed at D7. RGTA was well tolerated; no signs of ocular irritation were observed. In the corneal alkali-burn model, non-RGTA-treated eyes showed inflammatory clinical signs, and histology confirmed a loss of superficial corneal layers with epithelial disorganization, neovascularization and infiltration of inflammatory cells. When compared to NaCl control, RGTA treatment appeared effective in reducing clinical signs of inflammation, enhancing re-epithelialization, and improving histological patterns: edema, fibrosis, neovascularization and inflammation. Three to four layers of epithelial cells were already organized, stroma was virtually unvascularized and keratocytes well implanted in parallel collagen fibers with an overall reorganization similar to normal cornea. RGTA appears to be a promising agent for controlling ocular surface inflammation and promoting corneal healing and was well tolerated. This study offers preclinical information and supports the findings of other (compassionate or pilot) studies conducted in patients with various ocular surface diseases.