Effect of subconjunctival bevacizumab (Avastin) on experimental corneal neovascularization in guinea pigs

PURPOSE: To evaluate the effect of subconjunctival bevacizumab (Avastin) on experimental corneal neovascularization in guinea pigs. METHODS: Forty eyes of 40 guinea pigs were chemically cauterized with 75% silver nitrate and 25% potassium nitrate sticks. Fifteen eyes (group 1) received 2 subconjunctival injections of bevacizumab (0.1 mL, 1.25 mg) simultaneously with cauterization and 3 days later. Fifteen eyes (group 2) received 2 subconjunctival injections of bevacizumab (0.1 mL, 1.25 mg) 3 and 5 days after cauterization. Ten eyes (group 3, control group) received 2 subconjunctival injections of 0.1 mL of balanced salt solution 3 and 5 days after cauterization. After we determined the burn and neovascularization scores for all groups, the animals were killed on the 10th day. The percentages of neovascularization on the surface of the cornea were measured in terms of pixels on digital photographs. The average number of vessels at maximally vascularized areas was determined for each specimen. RESULTS: Neovascularization score was 1.1 +/- 0.3 in group 1, 2.46 +/- 1.3 in group 2, and 3.5 +/- 0.5 in the control group. The difference was statistically significant (P < 0.001). The area of neovascularization at the cornea surface was 15.6% +/- 10.1% in group 1, 19.74% +/- 11.2% in group 2, and 23.5% +/- 7.4% in the control group (P = 0.194). The average number of neovascular vessels at group 1 was significantly reduced in comparison with group 2 and the control group (P < 0.001). CONCLUSIONS: Subconjunctival injection of bevacizumab decreases the extent of chemically induced corneal neovascularization in guinea pigs. The antineovascular effect of bevacizumab is higher if the injection is performed simultaneously with the chemical cauterization.     

Subconjunctival bevacizumab injection for corneal neovascularization

PURPOSE: To report on the clinical use of subconjunctival bevacizumab in patients with corneal neovascularization. METHODS: The charts of 10 consecutive patients with corneal neovascularization who received subconjunctival injections of bevacizumab (2.5 mg/0.1 mL) were reviewed. Digital photographs of the cornea were graded by 2 masked observers for density, extent, and centricity of corneal vascularization. Image analysis was used to determine the area of cornea covered by neovascularization as a percentage of the total corneal area. RESULTS: No significant ocular or systemic adverse events were observed during 3.5 +/- 1.1 months of follow-up. Seven patients showed partial regression of vessels. The extent decreased from 6.0 +/- 1.2 (SD) clock hours before the injection to 4.6 +/- 1.0 clock hours after bevacizumab injection (P = 0.008). Density decreased from 2.7 +/- 0.2 to 1.9 +/- 0.3, respectively. (P = 0.007). No change was noticed in the centricity of corneal vessels. Corneal neovascularization covered, on average, 14.8% +/- 2.5% (SD) of the corneal surface before the injections, compared with 10.5% +/- 2.8% (P = 0.36, t test) after bevacizumab injection. Therefore, bevacizumab decreased corneal neovascularization by 29%. CONCLUSIONS: Short-term results suggest that subconjunctival bevacizumab is well tolerated and associated with a partial regression of corneal neovascularization.     

Inhibition of experimental corneal neovascularization by using subconjunctival injection of bevacizumab (Avastin)

PURPOSE: To evaluate the effect of subconjunctival bevacizumab (Avastin) administration on corneal neovascularization (NV) in rabbits. METHODS: NV was induced by placing a suture at the corneal periphery of the right eye of 20 rabbits. Immediately after suturing and again 1 week later, rabbits were divided into 2 groups and administered a subconjunctival injection of normal saline (control) or bevacizumab (Avastin; 5 mg/0.2 mL), respectively. On day 14, digital photographs of the cornea were taken and analyzed to determine the area of the cornea covered by NV. In addition, immunohistochemical analysis was used to determine CD31 and vascular endothelial growth factor (VEGF) expression in corneal tissue. RESULTS: Analysis of digital photographs showed that there was less corneal NV in bevacizumab-treated eyes than in controls (P < 0.001, Mann-Whitney U test). In addition, there was less staining for VEGF and CD31 in corneas from bevacizumab-treated eyes than in control eyes. Subconjunctival bevacizumab injections were not associated with any complications during observation. CONCLUSIONS: Subconjunctival bevacizumab administration decreased suture-induced corneal neovascularization in rabbits.     

Increased expression of inflammatory cytokines and matrix metalloproteinases in pseudophakic corneal edema

PURPOSE: To evaluate the expression of inflammatory cytokines and matrix metalloproteinases in the corneal epithelium in pseudophakic corneal edema (PCE). METHODS: Tissue sections were prepared from formalin-fixed, paraffin-embedded blocks of corneal buttons removed from 20 patients with PCE during penetrating keratoplasty (PKP) and from 11 age-matched control eyes enucleated because of uveal melanoma. Expression of interleukin (IL)-1beta, -6, and -8; vascular endothelial growth factor (VEGF); and matrix metalloproteinase (MMP)-1, -3, and -9 proteins in the corneal epithelium was evaluated by immunohistochemistry. Digital image analysis was performed to quantify the expression of the various cytokines and MMPs. A mean intensity stain index (ISI), based on the staining density and the area stained, was calculated from digital images captured from sequential areas of the corneal epithelium. RESULTS: The expression of most of the inflammatory cytokines and MMPs was significantly higher in the corneal epithelium of PCE corneal buttons than in the control specimens. MMP-9 had the highest expression when compared with the control (ISI = 55.08 +/- 23.71 in PCE compared with 0.169 +/- 0.156 in the control; P < 0.0001). Significantly higher ISIs were also recorded for MMP-1 (16.14 +/- 8.49 vs. 1.13 +/- 1.79; P < 0.0001), IL-1beta (62.62 +/- 27.23.97 vs. 1.61 +/- 1.27; P < 0.0001), IL-8 (37.91 +/- 21.18 vs. 4.24 +/- 3.60; P < 0.0001), and VEGF (81.67 +/- 26.22 vs. 19.40 +/- 16.85; P = 0.0001). The expression of MMP-3, IL-6, and TNF-alpha in PCE was not different from control expression. Significant positive correlations were found between the expression of IL-1beta and MMP-9 (r(2) = 0.37; P = 0.015), between VEGF and IL-8 (r(2) = 0.22; P = 0.042), and a significant correlation was found between the expression of MMP-3 and TNF-alpha (r(2) = 0.5197; P = 0.0007). The expression of TNF-alpha correlated significantly with the patient's age (r(2) = 0.28; P = 0.0195). CONCLUSIONS: The corneal epithelium in PCE expresses high levels of cytokines and matrix-degrading enzymes, which are associated with inflammation, wound healing, angiogenesis, and tissue degradation. The expression of these mediators may partially explain the pathologic features associated with this disease, such as bulla formation, recurrent epithelial desquamation, and corneal neovascularization.     

结膜下注射Avastin抑制角膜新生血管的实验研究

目的:观察结膜下注射Avastin对实验性兔眼角膜新生血管(neovascularization,NV)的抑制作用,初步探讨作用机制。方法:应用5mm直径的加样器(末端附有棉片)吸入1mol/LNaOH接触新西兰兔右眼(20眼)中央角膜区烧灼30s,制作碱烧伤兔眼角膜NV模型。将实验兔随机分成2组,10眼(A组)碱烧伤后立即结膜下注射Avastin 2.5mg;其余10眼为对照组(B组),结膜下注射等量生理盐水。烧灼后次日每天裂隙灯观察角膜NV、角膜水肿情况,分别于3,7,14,21,28d裂隙灯照相并计算NV面积及NV抑制率。伤后7,28d各组随即处死5只实验兔,取角膜组织做石蜡切片行组织病理学检查及VEGF免疫组织化学检测。结果:两组兔眼伤后第1d角膜缘血管网明显扩张充血,3d时血管开始侵入角膜,7～14d时NV达到高峰,14～21d后NV稳定并逐渐回退。两组角膜NV长度、NV面积及角膜水肿程度存在差异(P<0.05);A组各时间点角膜NV抑制率为44.2%～55%。A组角膜上皮及实质层水肿较轻,NV较少,后弹力层基本完整,VEGF表达明显弱于B组。结论:结膜下注射Avastin对碱烧伤诱导的兔眼角膜NV形成及生长具有明显的抑制作用,可能通过下调VEGF表达发挥作用。     

Flt-1 intraceptors inhibit hypoxia-induced VEGF expression in vitro and corneal neovascularization in vivo

PURPOSE: To determine whether subunits of VEGF receptor-1 coupled with an endoplasmic reticulum retention signal can block hypoxia-induced upregulation of VEGF secretion in corneal epithelial cells and block murine corneal angiogenesis induced by corneal injury. METHODS: Human corneal epithelial cells, transfected with plasmids encoding Flt23K or Flt24K (the VEGF-binding domains of the Flt-1 receptor coupled with the endoplasmic reticulum retention peptide KDEL), were subjected 2 days after transfection to 5% hypoxia for 24 hours. Supernatant was sampled at 24 hours and assayed for VEGF by ELISA. For in vivo models, mouse corneas underwent intrastromal injections of plasmids encoding Flt23K or Flt24K, and 2 days later, sustained injury induced by topical NaOH and mechanical scraping. Corneas were assessed 2 days later for VEGF ELISA and leukocyte counting or 1 week later for quantification of neovascularization. RESULTS: Hypoxia induced VEGF by human corneal epithelial cells was sequestered by both Flt23K and Flt24K; Flt-1 23K suppressed VEGF secretion as well. Intrastromal delivery of plasmid Flt23K suppressed VEGF by 40.4% (P = 0.009), leukocytes by 49.4% (P < 0.001), and neovascularization by 66.8% (P = 0.001). Flt24K suppressed VEGF expression by 30.8% (P = 0.042), leukocytes by 25.8% (P < 0.001), and neovascularization by 49.5% (P = 0.015). CONCLUSIONS: Flt-1 intraceptors, which are endoplasmic reticulum retention signal-coupled VEGF receptors, significantly suppress hypoxia-induced VEGF secretion by corneal epithelial cells in vitro. In vivo, delivery of naked plasmids expressing these intraceptors inhibits injury-induced upregulation of VEGF, leukocyte infiltration, and corneal neovascularization.     

反义寡核苷酸对人视网膜色素上皮细胞EGFR表达的影响

目的　研究反义寡核苷酸 (ODN)对人视网膜色素上皮 (RPE)细胞表皮生长因子受体 (EGFR)表达的影响。方法　采用人工合成反义ODN经阳性脂质体包裹后转染人RPE细胞。采用半定量RT PCR法与ELISA法检测EGFRmRNA及蛋白的表达水平。结果　显示转染反义ODN 2 4h后 ,EGFRmRNA的表达抑制率为 35 .2 % ,EGFR蛋白抑制率为 6 6 .4 5 % .4 8h后 ,反义EGFRODNs对EGFR的表达抑制作用消失。结论　EGFR反义ODN可抑制人视网膜色素上皮细胞的EGFRmRNA及其蛋白的表达。     

The effect of different doses of subconjunctival bevacizumab injection on corneal neovascularization

To evaluate the effects of various doses of subconjunctival bevacizumab injections in the treatment of patients with corneal neovascularization. During the 6-month-follow-up, no significant ocular or systemic adverse events were observed related to the subconjunctival bevacizumab injection. In Group 1, the total area of corneal neovascularization before injection was 14.8 ± 3.2 % of the corneal surface and 10.2 ± 2.8 % 6 months after injection (p < 0.01). The mean decrease in Group 1 was 32.0 ± 3.0 %. In Group 2, the total area of corneal neovascularization before and 6 months after the injection was 14.2 ± 2.5 and 9.8 ± 2.3 %, respectively (p < 0.01). The mean decrease in Group 2 was 31.0 ± 2.3 %. The difference between the two groups was not statistically significant (p > 0.05). Twenty-four eyes of 24 patients with corneal neovascularization who were treated with a subconjunctival injection of bevacizumab were included in this retrospective study. Fourteen eyes were treated with 2.5 mg/0.1 ml (Group 1), and 10 eyes were treated with 5.0 mg/0.2 ml (Group 2) of subconjunctival bevacizumab. Digital photographs of the cornea were used to determine the area of corneal neovascularization before injection and at 1 month, 3 months, and 6 months after treatment. Subconjunctival injection of bevacizumab is well tolerated and associated with a partial regression of corneal neovascularization. The efficacy of this treatment is not correlated to the injection dose.     

The inflammatory milieu associated with conjunctivalized cornea and its alteration with IL-1 RA gene therapy

PURPOSE: This study was designed to gain an insight into the inflammatory milieu into which a donor limbal graft is routinely introduced. The objective of this study was to modulate this environment by gene therapy with the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1 RA). METHODS: In a mouse model, the ocular surface cytokine environment associated with a conjunctivalized cornea was assessed 4 weeks after injury. Total corneal epithelial and limbal debridement was performed with a combination of alkali and scrape injury. The cytokines and adhesion molecules measured included IL-1alpha, IL-1beta, IL-6, VEGF, intercellular adhesion molecule (ICAM)-1, and vascular adhesion molecule (VCAM)-1, by real-time PCR or ELISA. Injured corneas were transfected with IL-1 RA by injection of naked plasmid vector pIRES-EGFP-IL-1 RA immediately after injury. Corneas transfected with pIRES-EGFP served as the control. Expression of corneal IL-1 RA after transfection with pIRES-EGFP-IL1-RA was assessed over a 2-week period by real-time PCR and Western blot analysis. In addition, limbal stem cell grafts transfected with IL-1 RA were assessed for leukocyte influx. RESULTS: Conjunctivalized corneas showed increased expression of IL-1alpha, IL-1beta, IL-1 RA, IL-6, VEGF, ICAM-1, and VCAM-1, compared with normal cornea. Transfection-efficiency experiments indicated that corneal expression of IL-1 RA peaked between 12 and 24 hours and lasted up to 2 weeks after the initial transfection. IL-1 RA corneal gene therapy resulted in a downregulation of IL-1beta and VCAM-1 expression at 4 weeks after injury, whereas downregulation of IL-6 was evident only at 1 week after injury. Corneal neovascularization was also reduced. In addition, corneal limbal stem cell grafts transfected with IL-1 RA showed a decreased leukocyte influx compared with control grafts. CONCLUSIONS: Transfection of a cornea with IL-1 RA immediately after epithelial injury selectively altered the cytokine profile of the resultant conjunctivalized cornea and suppressed corneal neovascularization. Transfection of corneal limbal donor tissue with IL-1 RA before engraftment can reduce leukocyte influx into the graft. The findings demonstrate the feasibility of using transient cytokine gene expression, either in donor or recipient corneal tissue, to alter the ocular surface environment beneficially.     

VEGF-dependent conjunctivalization of the corneal surface

PURPOSE: To investigate the mechanisms governing corneal neovascularization and the appearance of goblet cells in a murine model of limbal insufficiency. METHODS: The spatial and time-dependent relationship between corneal neovascularization and goblet cell density was analyzed in corneal flatmounts. Immunohistochemical detection of the vascular endothelial growth factor (VEGF) receptor Flt-1 (VEGFR1) was performed in paraffin-embedded sections. A transgenic mouse that expresses the reporter gene lacZ targeted to the Flt-1 locus through homologous recombination was used to analyze corneal expression of Flt-1. The presence of soluble and membranous goblet cell Flt-1 mRNA and protein content was assessed with Northern and Western blot analyses, respectively. Finally, systemic adenoviral expression of a soluble Flt-1/Fc construct was used to study the effect of inhibition of VEGF bioactivity on the appearance of goblet cells and neovascularization. RESULTS: Corneal neovascularization preceded the appearance of goblet cells, although both processes overlapped temporally. Flt-1 was abundant in the conjunctiva-like epithelium covering the cornea, as well as in the goblet cells, invading leukocytes, and vasculature. A similar expression pattern was observed in the transgenic mice expressing the lacZ gene downstream from the Flt-1 promoter. Isolated human and rat goblet cells in culture expressed Flt-1 mRNA and protein, as did freshly isolated human conjunctiva. The systemic inhibition of VEGF bioactivity potently suppressed both corneal neovascularization (8.3% +/- 8.1% vs. 41.1% +/- 15.3% corneal area; P < 0.001) and corneal goblet cell density (1.6% +/- 2.5% vs. 12.2% +/- 2.4% corneal area; P < 0.001). CONCLUSIONS: Two important features of corneal conjunctivalization, the appearance of goblet cells and neovascularization, are regulated by VEGF. Both processes are probably mediated, in part, through the Flt-1 receptor. Taken together, these data indicate that an anti-VEGF therapeutic approach may limit the visual loss associated with conjunctivalization of the corneal surface.     

Subconjunctival bevacizumab injection for corneal neovascularization in recurrent pterygium

PURPOSE: We report on the use of subconjunctival bevacizumab on corneal vessel density in recurrent pterygia. METHODS: The charts of 5 patients with recurrent pterygium, who received subconjunctival injections of bevacizumab (2.5 mg/0.1 ml) were retrospectively reviewed. Ophthalmic evaluation included Snellen visual acuity (VA), tonometry and complete examination before the injection and at 1 week and 1 and 3 months thereafter. Digital photographs of the eyes were analyzed by image analysis software to determine the area of cornea covered by new vessels as a percentage of the total corneal area. RESULTS: No ocular or systemic adverse events were observed. No change in visual acuity was noted in any patient following the injection. The mean change in corneal vascularization after one bevacizumab injection was 0.03%+/-0.45, while after two injections the change was 0.025%+/-0.19 (both not statistically different than zero, t-test). CONCLUSIONS: Short-term results suggest that subconjunctival bevacizumab is well tolerated but does not cause regression of corneal vessels in recurrent pterygium.     

The Effect of Bevacizumab on Corneal Neovascularization in Rabbits

Purpose

To determine the efficacy of topical application and subconjunctival injection of bevacizumab in the treatment of corneal neovascularization.

Methods

Corneal neovascularization was induced with a silk suture of the corneal stroma in 12 rabbits (24 eyes). One week after suturing, four rabbits were treated with topical bevacizumab at 5 mg/mL (group A) and another four rabbits were treated with topical bevacizumab 10 mg/mL (group B) in the right eyes twice a day for two weeks. A subconjunctival injection of bevacizumab 1.25 mg/mL was done in the right eyes of four rabbits (group C). All of the left eyes (12 eyes) were used as controls. The area of corneal neovascularization was measured after one and two weeks, and the concentration of vascular endothelial growth factor (VEGF) in corneal tissue was measured after two weeks.

Results

The neovascularized area was smaller in all treated groups than in the control group (p<0.001). Upon analysis of the neovascularized area, there was no significant difference between groups A and B. However, the mean neovascularized area of group B was significantly smaller than that of group C after two weeks of treatment (p=0.043). The histologic examination revealed fewer new corneal vessels in all treated groups than the control group. The concentration of VEGF was significantly lower in all treated groups compared to the control group (p<0.01), but no difference was shown between treated groups.

Conclusions

Topical and subconjunctival bevacizumab application may be useful in the treatment of corneal neovascularization and further study is necessary.     

反义寡核苷酸对人视网膜色素上皮细胞EGFR表达和增生抑制作用的研究

目的 研究反义寡核苷酸(ODN)对人视网膜色素上皮(RPE)细胞，表皮生长因子受体(EGFR)表达和增生的影响。方法 采用人工合成反义ODN经阳性脂质体包裹后转染人RPE细胞。用MTT和细胞周期分析检测转染ODN后对细胞的生长抑制作用，采用半定量RT-PCR法与ELISA法检测EGFR mRNA及蛋白的表达水平。结果 显示转染反义ODN 24 h后，EGFR mRNA的表达抑制率为35．2％，EGFR蛋白抑制率为66．45％。48 h后．反义EGFR ODNS对EGFR蛋白的抑制作用消失。EGFR反义ODN对RPE细胞生长增生的抑制率约为40％。细胞周期分析EGFR反义ODN作用于细胞后，细胞G0／G1期细胞数明显增多，S期细胞数相应减少，而G2 M期细胞数亦明显减少。结论 EGFR反义SON可抑制人RPE细胞的生长和EGFR mRNA及其蛋白的表达。     

白细胞介素-1受体拮抗剂防治角膜移植免疫排斥反应的实验研究

目的观察白细胞介素-1受体拮抗剂(IL-1ra)抑制大鼠正位穿透性角膜移植术后免疫排斥反应、促进植片存活的作用.方法将主要组织相容性系统完全不同的近交系F344大鼠的角膜植片移植到另一近交系LOU大鼠的角膜上,将手术成功大鼠分为实验Ⅰ、Ⅱ、Ⅲ组及对照Ⅳ组,均于术后第1d起结膜下分别注射50、100、200μg的IL-1ra,对照组(Ⅳ组)结膜下注射等量生理盐水,连续2周.对角膜植片存活情况进行评分,观察其临床效果.结果各实验组角膜植片平均存活天数(meansurvivaltime,MST)分别为(11.13±1.46)d、(12.00±1.60)d及(13.44±1.13)d,明显高于对照组(8.00±1.25)d,差异有显著性(t=0.00,P＜0.01).IL-1ra200μg组与50μg组间比较,差异有显著性(t=0.00,P＜0.01).结论结膜下注射IL-1ra可抑制角膜移植术后免疫排斥反应,减少新生血管的生成,减轻免疫性炎性反应,延长角膜植片的存活时间.IL-1ra的剂量越大,效果越好.     

Intraoperative mitomycin versus postoperative 5-fluorouracil in high-risk glaucoma filtering surgery.

In a randomized clinical trial, the authors compared the use of postoperative subconjunctival injections of 5-fluorouracil (5-FU) in 19 eyes with a single intraoperative application of subconjunctival mitomycin (MMC) at the filtering site in 20 eyes at high risk for failure of glaucoma filtering surgery. Six months after surgery, intraocular pressures averaged 10.9 +/- 5.3 mmHg (mean +/- standard deviation) in the MMC-treated eyes versus 14.2 +/- 5.5 mmHg in the 5-FU-treated eyes (P = 0.08) and were less than or equal to 12 mmHg in 60.0% of MMC-treated eyes and 21.1% of 5-FU-treated eyes (P = 0.03). Mitomycin-treated eyes were receiving an average of 0.3 +/- 0.5 medications for intraocular pressure control, and 5-FU-treated eyes were receiving an average of 1.1 +/- 1.1 medications (P = 0.01). Drug-induced corneal epithelial defects were seen in nine 5-FU-treated eyes and in no MMC-treated eyes (P = 0.0004). These results suggest that intraoperative MMC may be a viable alternative to postoperative 5-FU, with lower overall intraocular pressures, decreased dependence on postoperative ocular antihypertensive medications, and decreased corneal toxicity.     

Unique homologous siRNA blocks hypoxia-induced VEGF upregulation in human corneal cells and inhibits and regresses murine corneal neovascularization

PURPOSE: To determine whether RNA interference (RNAi) could block hypoxia-induced upregulation of vascular endothelial growth factor (VEGF) in human corneal epithelial cells in vitro and inhibit and regress injury-induced murine corneal neovascularization in vivo. METHODS: siRNA selected on the basis of target sequence homology between mouse and human VEGF was placed into expression cassettes and transfected into human corneal epithelial cells. Hypoxia-induced VEGF synthesis was assayed. Also, the effect of a plasmid capable of directing the expression of an siRNA against VEGF when injected into mouse corneas 8 hours before alkali-mechanical trauma was studied. Leukocyte count, VEGF protein levels, and degree of neovascularization in corneas were compared with that of a control siRNA plasmid. Plasmids were injected 1 week after injury to assess the ability of RNAi to regress corneal neovascularization. RESULTS: Hypoxia-induced VEGF mRNA synthesis and protein secretion by human corneal epithelial cells was efficiently suppressed by an siRNA targeted against a sequence uniquely identical for the mouse and human VEGF genes. Intrastromal delivery of a plasmid expressing this siRNA before murine corneal injury suppressed corneal VEGF by 55.7% versus control (P = 0.014), leukocyte infiltration by 69.5% (P < 0.001), and neovascularization 1 week after injury by 72.3% (P = 0.001). At the regression time point, treated corneas had 72.8% less neovascularization (P < 0.001). CONCLUSIONS: RNAi significantly suppresses expression of VEGF induced by hypoxia in human corneal epithelial cells in vitro. In vivo, intrastromal delivery of a plasmid expressing siRNA against VEGF suppresses injury-induced VEGF expression, leukocyte infiltration, and angiogenesis and was able to regress corneal neovascularization.     

Experimental inhibition of corneal neovascularization by photodynamic therapy with verteporfin

PURPOSE: To investigate the anti-angiogenic effects of photodynamic therapy with verteporfin in a rabbit model of corneal neovascularization. METHODS: One week after suturing, the localization of verteporfin in the neovascularized cornea was examined through fluorescent microscopy 1 hr after administration. Rabbits were treated with one or two times of photodynamic therapy with verteporfin at 1-week intervals. Analysis of corneal neovascularization was performed by biomicroscopic and histological examinations. RESULTS: Fluorescent microscopy showed green fluorescence in the vascular walls and interstitial tissue of the corneal stroma. The mean percentages of neovascularized corneal area at 3 days, 1 week, and 2 weeks after one time of photodynamic therapy were 90.3% +/- 3.5%, 71.6% +/- 6.2%, and 43.6% +/- 15.1% in treated eyes and 96.4% +/- 1.9% (p = 0.10), 88.6% +/- 4.6% (p = 0.01), and 76.8% +/- 4.4% (p < 0.01) in control eyes, respectively. The mean percentages 3 days, 1 week, and 2 weeks after two times of photodynamic therapy were also significantly lower in treated eyes compared with control eyes. In quantitative histological examination at 1 and 2 weeks after therapy, treated eyes showed significantly less neovascular area and number of vessels than control eyes. CONCLUSIONS: Photodynamic therapy with verteporfin is a safe and useful procedure to reduce experimental corneal neovascularization and can be used to inhibit angiogenesis in the cornea.     

Local suppression of IL-1 by receptor antagonist in the rat model of corneal alkali injury

Proinflammatory cytokines, including interleukin-1 (IL-1) have been implicated in the inflammation that follows corneal alkali injury. The purpose of this series of experiments was to test whether topically applied interleukin-1 receptor antagonist (IL-1ra) could suppress corneal inflammation and promote transparency after alkali injury. Alkali injury was induced on day 0 by application of 1N NaOH to both eyes of Wistar rats (n = 28). Immediately thereafter, eyes received either topical IL-1ra (20 mg ml(-1)) in 0.2% sodium hyaluronate or vehicle alone three times daily during days 0-14. Biomicroscopic features including corneal opacity and neovascularization were assessed using a standard grading scheme. Inflammation was quantified histologically. Corneas excised at day 3 and 7 (randomly selected six eyes in each group per time point studied) were homogenized, and levels of IL-1alpha, IL-1beta, IL-6, IL-8, IL-10, and RANTES were quantified by enzyme-linked immunosorbent assay. Epithelial wound healing was examined by computed analysis of fluorescein stained corneal photographs taken daily until day 14. According to these evaluations, eyes treated with IL-1ra maintained corneal transparency with minimal neovascular invasion. Additionally, corneal damage and cell infiltration were reduced on day 7 (infiltration cells were almost 40% decreased). All cytokine/chemokine levels in IL-1ra treated eyes were significantly lower at day 3, and IL-6 and IL-10 remained significantly lower at day 7 compared to vehicle-treated eyes. IL-1ra treatment retarded epithelial wound healing in the early stage (day 1-4); however, subsequently IL-1ra treated eyes had enhanced healing with full epithelial closure at nearly the same time point as vehicle-treated eyes (day 10). We conclude that local antagonism of IL-1 after alkali injury can significantly decrease corneal inflammation and lead to enhanced corneal transparency.     

血管内皮细胞生长因子反义寡聚脱氧核苷酸 在缺血性视网膜病变中的作用

目的研究血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)反义寡聚脱氧核苷酸在缺血性视网膜病变中的作用。方法以高浓度氧诱导C57BL/6J小鼠建立视网膜缺血性病变动物模型,应用血管内皮细胞生长因子反义寡聚脱氧核苷酸不同浓度进行球后注射,计数并比较正常和实验条件下以及不同浓度药物注射后视网膜新生血管芽细胞核数。结果高浓度氧诱导的小鼠视网膜发生较多的新生血管芽,而球后注射VEGF反义寡聚脱氧核 苷酸后视网膜新生血管芽的细胞核数明显减少,特别是大剂量注射后减少达59.3%(P<0.01)。结论VEGF反义寡聚脱 氧核苷酸能有效抑制缺血性视网膜病变新生血管的增生。(中华眼底病杂志,2001,17:141-143)     

Expression of matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) in inflammation-associated corneal neovascularization

Matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) are all implicated in the development of neovascularization. To investigate the possible role of these factors in corneal neovascularization we have analysed the expression of MMP-2, MMP-9 and VEGF in a rat model of inflammation-associated corneal neovascularization. In this model, corneal neovascularization was induced in Long-Evans rats by krypton laser photocoagulation whereafter eyes were enucleated at 1, 4, 7, 10 and 20 days. Slit-lamp biomicroscopy and histologic analysis revealed a gradual development of corneal neovascularization that peaked 7-10 days after treatment when newly formed vessels could be seen throughout the corneal surface reaching deep into the stroma. Antisense and sense riboprobes were generated using DNA complementary to MMP-2, MMP-9 and VEGF, and mRNA expression was analysed using in situ hybridization. The expression of MMP-2 and MMP-9 in untreated corneas was low or absent whereas VEGF was weakly expressed in the corneal epithelium. MMP-2 expression was increased during corneal neovascularization and was mainly localized to the cells infiltrating areas of new vessel formation. Many of these cells appeared to be inflammatory cells. VEGF expression had a similar overall distribution to MMP-2 during neovascularization with the exception that its expression in the corneal epithelium remained and even increased slightly. MMP-9 was prominently expressed at the border of regenerating corneal epithelium in areas with epithelial wounding but was not detected in the vascularized stroma. Together, the results of the present study support a role for MMP-2 and VEGF in inflammation-associated corneal neovascularization whereas MMP-9 instead appears to be involved in corneal epithelial wound-healing.