角膜碱烧伤后VEGF的表达与新生血管的关系

目的研究碱烧伤后角膜血管内皮生长因子（vascular endothelial growthfactor，VEGF）的表达与角膜新生血管化的关系。方法30只Sprague-Dawleg（SD）大鼠碱烧伤，诱导角膜新生血管模型。碱烧伤后不同时间形态学分析来评价角膜新生血管的情况，免疫组化及Westem blot评价角膜VEGF的表达。结果角膜碱烧伤后24hVEGF的表达开始增高，伤后4d达高峰，伤后7d VEGF的表达下降，14d后显著下降。碱烧伤后，角膜新生血管与VEGF的表达呈明显的平行关系。结论碱烧伤后大鼠角膜VEGF的表达水平与新生血管的形成有相关性，并在其中发挥重要作用。     

大鼠角膜碱烧伤后CD105在新生血管化角膜中的表达

目的：研究大鼠角膜碱烧伤后CD105在新生血管化角膜组织中的表达。方法：采用角膜碱烧伤法对60只Wistar大鼠制作角膜新生血管模型。裂隙灯显微镜下观察大鼠角膜碱烧伤后角膜新生血管生长情况,免疫组化法检测碱烧伤后不同时间角膜组织中CD105的表达。结果：大鼠角膜碱烧伤后2d角膜新生血管开始形成,3～7d生长旺盛,新生血管面积达到最大,10～14d角膜新生血管生长停滞。在新生血管化角膜组织中,CD105于3～7d表达最明显,14d后表达微弱。结论：大鼠角膜碱烧伤后CD105在新生血管化角膜组织中有明显表达。     

The effect of oxygen on corneal neovascularization.

Since tissue oxygen levels are believed to play a pivotal role in new vessel growth in several situations, we studied the effect of several oxygen concentrations (0, 10, 21, 50, 75, or 100%) on corneal vascularization induced in the rat by chemical cautery. We achieved this by perfusing known concentrations of oxygen through goggles fitted over both eyes of the rat after corneal cauterization. Neovascularization was measured in flat corneal preparations with India ink-filled vessels 4 days postcautery using computerized image analysis. The angiogenic response of rats whose eyes were continuously exposed to 0-75% oxygen were not significantly different from each other. The mean response in corneas exposed to 100% oxygen was 10-21% lower than all of the other groups, and this difference was statistically significant when compared to oxygen concentrations of 0, 21 and 75%. The reason for the inhibitory effect of 100% oxygen remains to be determined, but it may represent a toxic effect of oxygen free radicals on the vascular endothelium.     

Comparison of the effects of bevacizumab and ranibizumab injection on corneal angiogenesis in an alkali burn induced model

AIM: To investigate the effects of bevacizumab and ranibizumab on corneal neovascularization in an alkali burn-induced model of corneal angiogenesis. METHODS: Fifteen Wistar albino rats were divided randomly into 3 groups after chemical cauterization of the cornea. The first group received a single dose of 0.1mL saline solution as a control group whereas second and third groups received a single dose of 2.5mg bevacizumab or 1mg ranibizumab by subconjunctival injection, respectively. After three weeks, the rat corneas were evaluated by biomicroscopy and corneal photographs were taken. The percentage of neovascularization area, length of the longest new vessel, corneal edema and corneal opacity scores were assessed. RESULTS: The analysis of digital photographs showed that the percentage of neovascularization area to the total corneal area, the length of the longest new vessel, corneal edema and opacity scores were significantly lower in both study groups compared to the control group (P<0.05). Additionally, the percentage of corneal neovascularization area, the length of the longest new vessel and corneal opacity score were less with bevacizumab than ranibizumab. CONCLUSION: Subconjunctival bevacizumab and ranibizumab treatments may be effective methods in reducing corneal neovascularization. Furthermore, bevacizumab is more effective than ranibizumab in the inhibition of corneal neovascularization.     

大鼠角膜碱烧伤后CD105在新生血管化角膜中的表达

目的:研究大鼠角膜碱烧伤后CD105在新生血管化角膜组织中的表达。方法:采用角膜碱烧伤法对60只Wistar大鼠制作角膜新生血管模型。裂隙灯显微镜下观察大鼠角膜碱烧伤后角膜新生血管生长情况,免疫组化法检测碱烧伤后不同时间角膜组织中CD105的表达。结果:大鼠角膜碱烧伤后2d角膜新生血管开始形成,3～7d生长旺盛,新生血管面积达到最大,10～14d角膜新生血管生长停滞。在新生血管化角膜组织中,CD105于3～7d表达最明显,14d后表达微弱。结论:大鼠角膜碱烧伤后CD105在新生血管化角膜组织中有明显表达。     

Retrobulbar anesthesia and eyelid closure--effect on corneal angiogenesis.

We evaluated the effect of corneal anesthesia produced by retrobulbar injections of bupivicaine and epinephrine on corneal neovascularization in the rat. The eyelids were sutured closed to prevent dessication and ulceration of the insensitive corneas. The amount of corneal neovascularization induced in animals receiving a retrobulbar anesthetic did not differ from those receiving control solutions. However, rats receiving retrobulbar injections exhibited greater corneal neovascularization than those that did not. Rats that had their eyelids sutured or patched closed also had more neovascularization than animals that did not. Likewise, rats that had sutures placed in the upper and lower eyelids, without palpebral immobilization, manifested more neovascularization than rats without sutures. These studies suggest that corneal anesthesia does not affect neovascularization induced by silver/potassium nitrate cauterization and that retrobulbar injections and eyelid closure enhance corneal neovascularization.     

Role of protein tyrosine phosphorylation in rat corneal neovascularization

Background: Recent studies have suggested that tyrosine kinase pathways that are activated by angiogenic growth factors may play a role in corneal neovascularization. Methods: Corneal neovascularization was induced in rat corneas by chemical cauterization. At 6, 24, 48, 96, and 168 h after chemical cauterization the rat corneas without the corneal epithelium were prepared for gel electrophoresis. Total protein profiles of the corneal samples were examined by staining gels with Coomassie brilliant blue. Tyrosine-phosphorylated proteins, three angiogenic growth factors (basic fibroblast growth factor, vascular endothelial growth factor, and platelet-derived growth factor-B chain), and three intracellular signal proteins in the tyrosine kinase pathways (phospholipase C, SHC, and mitogen-activated protein kinase) in the corneal samples were examined by western blot analysis. A topical treatment of genistein eye drop (5 mg/ml) was used for inhibition of corneal neovascularization after chemical cauterization in rats. Results: In total protein profiles, three bands in the corneal samples were increased after cauterization. Overall tyrosine-phosphorylated proteins and all three angiogenic growth factors increased with progression of corneal neovascularization. The tyrosine-phosphorylated forms of three intracellular signal proteins were also increased after cauterization. Treatment with topical genistein was effective in inhibiting corneal neovascularization in rats. Conclusion: Protein tyrosine phosphorylation was involved in inflammation-induced corneal neovascularization. Tyrosine kinase inhibitors may have utility as inhibitors of corneal neovascularization.     

Suppression of experimental corneal angiogenesis by focal X-ray irradiation.

PURPOSE: To investigate the effect of focal X-ray irradiation on experimental corneal angiogenesis in the rabbit. METHODS: A gelatin hydrogel sheet impregnated with basic fibroblast growth factor was implanted into the corneal stroma of rabbits; this induced corneal angiogenesis. After the first sign of corneal angiogenesis was noted, the corneal region was irradiated with a dose of 10 Gy or 20 Gy. The control rabbits received no irradiation. The eyes were examined by slitlamp biomicroscopy and photographed, over a period of 28 days. The maximum length and total surface area of corneal angiogenesis were quantified by computerized image analysis. RESULTS: Corneal angiogenesis was noted on day 3 following implantation of the hydrogel sheet. In the rabbits irradiated with 10 Gy, the maximum length and total surface area of corneal angiogenesis were both significantly lower on day 4 and 7 following irradiation, compared to the respective measurement in the controls. In the rabbits irradiated with 20 Gy, the maximum length and total surface area of corneal angiogenesis were significantly lower between days 4 and 21, and between days 4 and 14, respectively, compared to the respective measurement in the controls. CONCLUSIONS: Focal X-ray irradiation to the corneal region suppressed corneal angiogenesis in a dose-dependent manner. Focal X-ray irradiation may be beneficial in treating ocular angiogenesis.     

Inhibitory effects of topical thymoquinone on corneal neovascularization

PURPOSE: Thymoquinone, one of the biologically active components of black seed oil, has anti-inflammatory and antioxidant properties. We aimed to study the effect of thymoquinone on corneal neovascularization in rats and to compare its efficacy with that of triamcinolone acetonide. METHODS: Chemical cauterization of the cornea was performed with silver nitrate/potassium nitrate sticks in 40 eyes in 40 rats. An examiner blinded to the experiments scored the intensity of the cauterization. Topical instillation of thymoquinone 0.1%, thymoquinone 0.4%, and triamcinolone acetonide was continued for 7 days. The inhibitory effects of the drugs on corneal neovascularization were tested and compared with each other and with controls with a computer program that evaluates percent areas of cornea covered by neovascularization. RESULTS: The means of percent area of corneal neovascularization in the thymoquinone 0.1%, thymoquinone 0.4%, triamcinolone acetonide, and control groups were 60.1%, 45%, 46%, and 72%, respectively. The inhibitory effect of thymoquinone 0.4% was found to be equal to that of triamcinolone acetonide (P = 0.87). The thymoquinone 0.4% and triamcinolone groups were different from the thymoquinone 0.1% and control groups (P < 0.05). There was also a significant difference between the percent area of corneal neovascularization in the thymoquinone 0.1% group and that of the controls (P < 0.05). The mean burn stimulus intensities were not different among the groups (P = 0.54). CONCLUSIONS: Thymoquinone was shown to have an inhibitory effect, comparable with that of triamcinolone, on corneal neovascularization in this rat model. However, thymoquinone decreased corneal neovascularization in a dose-dependent manner.     

Inhibition of corneal neovascularization after alkali burn: comparison of different doses of bevacizumab in monotherapy or associated with dexamethasone

Background: To compare the effects of different doses of bevacizumab with both saline and dexamethasone on inflammatory angiogenesis in the rat cornea induced by small chemical lesions. Methods: Corneal chemical cauterization was performed on 24 rats. Animals were divided randomly into six groups and received a daily subconjunctival injection for 7 days of: balanced salt solution 0.1 mL or dexamethasone phosphate 4 mg/day or bevacizumab 2.5 mg/day, 3.75 mg/day, 5.0 mg/day or bevacizumab 5.0 mg/day + dexamethasone phosphate 4 mg/day. Clinical examination under slit lamp was performed daily for 7 days to evaluate corneal opacity and vessel size evolution. Computer‐assisted quantitative image analysis was used to measure the total corneal area covered by neovascularization. Results: At final examination, the dexamethasone, bevacizumab 5.0 mg/day and dexamethasone + bevacizumab groups showed a significant lowering in corneal opacity score as compared with control (P = 0.024, P = 0.006 and P = 0.013, respectively). Also, a significant reduction on new vessels size score was observed. Surface of corneal neovascularization was significantly reduced in dexamethasone, bevacizumab 5.0 mg/day and dexamethasone + bevacizumab groups compared with control (P = 0.045, P = 0.047 and P = 0.044, respectively). Conclusion: Our study demonstrates the ability of a 5.0 mg/day bevacizumab subconjunctival injection, in monotherapy or associated with dexamethasone, to cause a short‐term involution of corneal neovascularization after corneal alkali burn. Combination of both of these treatments may have advantages to monotherapy approaches.     

Antineovascular effect of α-interferon on corneal neovascularization in rats

Clinical and histologic effects of topical recombinant α-interferon (IFN), systemic IFN, topical dexamethasone, and dexamethasone plus topical IFN on corneal neovascularization in response to silver nitrate cauterization were investigated in 110 eyes of 60 Wistar rats. The eyes were evaluated according to the burn stimulus, neovascularization, and histologic inflammation grades 5 days after cauterization. Antineovascular effects of topical and systemic IFN were significantly greater than a placebo but less than dexamethasone alone or in combination with IFN. This research was presented in part at the 27th Turkish National Ophthalmology Congress, Marmaris, Turkey, October 1993.     

Effect of Octreotide on experimental corneal neovascularization.

PURPOSE: To examine the ability of subcutaneously administered Octreotide ( a long acting somatostatin analoque) to serve as an inhibitory agent for corneal neovascularization in eyes of Wistar Albino rats. METHODS: Neovascular growth into the corneas of all the animals was induced by silver nitrate cauterization. Half of the animals which were randomly selected for the Octreotide group received 30 micrograms systemic Octreotide for 7 days. The treatment was initiated on the same day as chemical cauterization. The rest of the animals (control group) received no treatment. Slit lamp and histopathologic examination of the corneas of both groups were performed at the end of the study period. RESULTS: It was observed that the corneal neovascularization and histopatologic scores of the Octreotide group were significantly lower than those of the control group (p < 0.001, p < 0.01). CONCLUSION: Systemic administration of Octreotide inhibits the corneal neovascular response in a rat model.     

Expression of matrix metalloproteinases 2 and 9 and tissue inhibitors of metalloproteinase 1 and 2 in inflammation-induced corneal neovascularization.

PURPOSE: Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) have been linked to the angiogenic process in general. In order to understand the potential roles of MMP-2, MMP-9 and TIMPs in the corneal neovascularization process, we examined the expression and activities of MMP-2, MMP-9 and TIMPs during the course of cauterization-induced corneal neovascularization in a rat model. METHODS: Neovascularization of rat corneas was induced by silver nitrate cauterization. The expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 was examined by immunohistochemistry and RT-PCR. The protein activities of MMPs and TIMPs were compared in pre- and postcauterization corneas by gelatin zymography and reverse zymography, respectively. RESULTS: MMP-2, TIMP-1 and TIMP-2 immunoreactivities were expressed in normal corneas, predominantly in the corneal epithelium. After injury, immunoreactivities of both MMPs and TIMPs were increased, notably in the healing corneal epithelium, infiltrating inflammatory cells, stromal fibroblasts and ingrowing vascular endothelial cells. The increase in gross MMP-2 enzymatic activity paralleled the maximal vascular ingrowth on day 4, while the gross MMP-9 enzymatic activity rose immediately on day 1, then decreased steadily, which paralleled the magnitude of inflammatory cell infiltration. The immunoreactivity of MMPs/TIMPs decreased significantly 2 weeks after cauterization. On day 35, MMP-2, TIMP-1 and TIMP-2 staining was seen only in corneal epithelium and vascular endothelial cells. Both the RT-PCR and reverse zymography results revealed a more constant expression of TIMP-2, while the TIMP-1 expression appeared to be more inducible. CONCLUSION: MMPs as well as TIMPs were upregulated in cauterization-induced corneal neovascularization, suggesting that both may participate in extracellular matrix remodeling in the corneal wound healing, inflammation and neovascularization processes.     

Measurement of central corneal thickness and pre-corneal tear film thickness of rabbits using the Scheimpflug system

AIM:To measure central corneal thickness (CCT) and pre-corneal tear film thickness using the Galilei dual-Scheimpflug analyzer (GSA) in New Zealand white rabbits.METHODS:Ten normal New Zealand white rabbits (20 eyes) were included in this study. With the assistance of 0.1% fluorescein, the pre-corneal tear film can be well visualized. Both eyes of each rabbit were scanned once with the GSA pre- and post-instillation of 1μL 0.1% fluorescein. The difference between the two measurements of CCT (4-mm diameter) was recorded as the pachymetric values of the central tear film.RESULTS:The CCT of pre- and post-instillation was 388.8±9.5μm and 407.0±10.5μm, respectively. After a paired t-test analysis, the central pre-corneal tear film thickness of 4mm diameter was 18.2±5.31μm with a 95% confidence interval of (15.7, 20.6)μm (P<0.001).CONCLUSION:GSA can be used to measure CCT and analyze central tear film thickness of rabbits with the help of fluorescein.     

鼠角膜碱烧伤后血管内皮细胞生长因子在其角膜中的表达及意义

目的：探讨角膜碱烧伤后血管内皮细胞生长因子（vascular endothelial growth factor,VEGF)在鼠角膜中的表达及意义。方法：采用1mol/L氢氧化钠溶液烧伤Sprayue-Dawley(SD)大鼠角膜,建立炎症性角膜新生血管动物模型;用免疫印迹分析SD大鼠角膜烧伤后不同时间段VEGF蛋白在角膜中的表达量;免疫组织化学染色方法检测SD大鼠角膜烧伤后VEGF蛋白在角膜各层中的分布。结果：正常SD大鼠角膜中未检出VEGF蛋白;伤后6h,鼠角膜可检测出VEGF蛋白;伤后96h时,角膜中VEGF蛋白达高峰;伤后168hVEGF蛋白表达量下降,伤后SD大鼠角膜基质层,尤其是毗邻于烧灼区的浅基质层中有大量炎性细胞浸润,浸润的炎性细胞和新生血管内皮细胞均有VEGF蛋白表达。结论;炎性细胞分泌的VEGF蛋白参与了炎症性角膜新生血管增殖的调控,对新生血管的生长有诱导和维持作用。     

The inhibitory effect of different concentrations of topical bevacizumab on corneal neovascularization

Acta Ophthalmol. 2010: 88: 862–867

Abstract.

Purpose: This study aimed to evaluate the effects of different concentrations of topically administered bevacizumab (Avastin) on experimental corneal neovascularization (NV) in rats. Methods: Corneal NV was induced by chemical cauterization with silver nitrate sticks applied to the centre of the corneas of 37 Wistar rats. The rats were then randomized to four topical treatment groups: group 1 (n = 10) received 4 mg/ml bevacizumab; group 2 (n = 9) received 2 mg/ml bevacizumab; group 3 (n = 10) received 1 mg/ml bevacizumab, and group 4 (n = 8) represented a control group and received saline. All drops were initiated immediately after cauterization and applied twice per day for 7 days. Corneal NV was assessed 8 days after cauterization in a masked fashion, both qualitatively by clinical evaluation and quantitatively by blood vessel count in photographs of histological sections. Results: On clinical evaluation, groups 1 and 2 showed significantly less NV compared with the saline‐treated control group (p = 0.006 and p = 0.024, respectively). Histopathological evaluation showed that only group 1 differed significantly from controls (5% significance level) and normal corneal epithelium was seen in all groups. Conclusions: Topically administered bevacizumab at a concentration of 4 mg/ml significantly reduces corneal NV according to both clinical and histopathological evaluations; lower concentrations were less effective on both parameters. No corneal epitheliopathy was found using these concentrations.     

^99Tc-MDP对碱烧伤性大鼠角膜新生血管生成的抑制作用

目的：探讨^99Tc-MDP对碱烧伤性大鼠角膜新生血管生成的抑制作用。方法：采用碱烧伤法制备角膜新生血管模型,24只健康SD大鼠随机分成两组,实验组予以腹腔注射^99Tc-MDP,每天1次,至模型建立后14d。对照组以等体积的生理盐水腹腔注射。观察新生血管形成时间、第21d时角膜新生血管长度和新生血管面积。结果：实验组新生血管长入的时间为6．25±1．93d,对照组长入时间为5．83±1．86d,差异无统计学意义（P=0．30）。新生血管长度实验组为1．7±0．33mm,对照组为2．82±0．25mm;新生血管的面积实验组为17．3±3．26mm^2,对照组为24．8±5．10mm^2,两者之间差异均有统计学意义（P〈0．01）。结论：使用^99Tc-MDP可以有效抑制碱烧伤引起的角膜新生血管的生成。