Effect of bevacizumab on corneal neovascularization in experimental rabbit model

Purpose:  To investigate the effect of bevacizumab in an experimental rabbit model of corneal neovascularization.
Methods:  The right eyes of 24 white New Zealand rabbits were included in a corneal neovascularization model using alkaline burn. They were divided into four groups. Topical bevacizumab was installed three times daily in group 1, 5 mg bevacizumab subconjunctivally every 2 days in group 2, 10 mg bevacizumab subconjunctivally every 2 days in group 3 and 0.2 cc of normal saline in the same way in group 4 (control group). All eyes were treated for 7 days. Then the animals were killed and corneal specimens sent for histopathological analysis. Tear film and aqueous humour samples were obtained to assess vascular endothelial growth factor (VEGF) levels.
Results:  Seven days after topical bevacizumab treatment the neovascular index in group 1 was lower than that in the control group ( P  = 0.028). In groups 2 and 3 the neovascular index was lower 2 days after subconjunctival bevacizumab treatment than that in control group ( P  = 0.009 and P  = 0.009, respectively). In the control group the VEGF level in aqueous humour increased by 66% from day 7 to 14. In groups 1–3 it decreased by 49.80%, 70.20% and 76.44%, respectively ( P  = 0.043). The VEGF level in tear film of the control group increased by 35.23% from day 7 to 14, which was not significant ( P  = 0.893), while in groups 1–3 it decreased by 57.26%, 34.59% and 67.97%, respectively, which was only significant in groups 1 and 3 ( P  = 0.043).
Conclusions:  Subconjunctival 5 mg/mL bevacizumab is effective in reducing corneal neovascularization in animal models and in reducing VEGF levels. Further research is needed to assess the potential side effects and minimal effective dose.     

Comparison of the inhibitory effect of different doses of subconjunctival bevacizumab application in an experimental model of corneal neovascularization

AIM: To evaluate the inhibitory effect of subconjunctival bevacizumab as single- and multiple-dose application, and compare their effects on corneal neovascularization in a rat model. METHODS: Thirty adult Sprague-Dawley rats were used in this experimental study. The central cornea of the rats was cauterized chemically. The rats were randomly enrolled into three groups. All groups received subconjunctival injections. In Group 1 (control group, n=10), 0.05 mL 0.9% NaCl solution was injected on the first day. In Group 2 (single-dose group, n=10), 0.05 mL bevacizumab (1.25 mg) was injected on the first day. In Group 3 (multiple-dose group, n=10), four doses of 0.05 mL bevacizumab (1.25 mg) were injected on the first, third, fifth and seventh day. Slit-lamp examination of all rats was performed at the third and ninth day. Digital images of the corneas were taken and analyzed using image analysis software to calculate corneal neovascularization area. All rats were sacrificed on the tenth day. In corneal sections, the number of blood vessels, state of inflammation and collagen formation was evaluated histopathologically. RESULTS: In Group 3, corneal edema grades were significantly lower than Group 1 and Group 2 (P=0.02, and P=0.035, respectively). The mean percentage of neovascularized corneal area in Group 3 was significantly lower than Group 2 (P=0.005). On histopathological examination, Group 2 and Group 3 showed significantly less number of blood vessels than Group 1 (P=0.005, and P=0.001, respectively). Additionally, Group 3 showed significantly less number of blood vessels compared to Group 2 (P=0.019). Inflammation and edema grades were significantly lower in Group 3 compared to Group 1 (P=0.001). CONCLUSION: Subconjunctival bevacizumab injection is effective in inhibition of newly formed corneal neovascularization. The multiple-dose bevacizumab treatment seems to be more effective compared to single-dose treatment.     

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.     

兔角膜碱烧伤后角膜基质注射脐带间充质干细胞的疗效

目的　观察角膜基质注射脐带间充质干细胞（ｕｍｂｉｌｉｃａｌｃｏｒｄｍｅｓｅｎｃｈｙｍａｌｓｔｅｍｃｅｌｌｓ,ＵＭＳＣｓ）治疗兔角膜碱烧伤的效果。方法　健康新西兰大白兔随机分为３组,每组１２只,每只兔左眼为角膜碱烧伤模型眼。Ａ组兔左眼在碱烧伤２ｄ后角膜基质内注射含有２×１０６个ＵＭＳＣｓ的磷酸盐缓冲液２μＬ,Ｂ组注射相同剂量的磷酸盐缓冲液,Ｃ组为角膜碱烧伤后未进行处理组,在注射后不同时间对角膜混浊程度、新生血管生长情况、角膜上皮缺损情况等进行临床观察并评分,同时进行综合评分。结果　注射后１４ｄＡ组新生血管生长较Ｂ组明显缓慢,Ａ组、Ｂ组注射隧道周围角膜新生血管生长评分分别为０分、２分,差异有统计学意义（Ｐ＜０．０５）。注射后２ｄ和６ｄＡ组角膜混浊程度较Ｂ组明显轻,注射后２ｄＡ组、Ｂ组角膜混浊程度评分分别为２分、４分,注射后６ｄ评分与２ｄ相同,差异均具有统计学意义（均为Ｐ＜０．０５）。注射后２ｄ、７ｄ角膜上皮荧光素染色评分各组之间差异均无统学意义（均为Ｐ＞０．０５）。结论　碱烧伤后角膜基质注射ＵＭＳＣｓ可减少新生血管的形成,为抑制碱烧伤后角膜血管化提供参考,碱烧伤后角膜基质注射ＵＭＳＣｓ可在一定程度上恢复角膜透明度。     

Anti-VEGF monoclonal antibody-induced regression of corneal neovascularization and inflammation in a rabbit model of herpetic stromal keratitis

Background

To determine the efficacy of bevacizumab (Avastin), an anti-VEGF monoclonal antibody, administrated via subconjunctival injection as a corneal anti-angiogenic treatment.

Methods

Right corneas of rabbits were infected with herpes simplex virus type 1, KOS strain. On day 13 post-infection (p.i.), animals were treated subconjunctivally (sc) with a single 10-μl dose (25 μg/μl) of bevacizumab (group A) or with the same volume of an isotype monoclonal antibody, as negative control (group B). All animals were observed clinically on days 2, 5, 7, 14, 21, and 28 p.i., and two corneas each day were obtained for histological assessment and viral titration.

Results

Viral replication was observed no longer than 5 days after infection. By day 7 a dense neutrophil invasion of the cornea was detected, which significantly increased while herpetic stromal keratitis progressed in severity. Positive outcomes observed following the treatment with bevacizumab, compared to control, included: (1) Total involution of neovascularization, (2) reduction in disease severity, (3) improved corneal translucency, (4) absence of scarring, (5) preservation of corneal thickness, (6) no neutrophil infiltration of the cornea.

Conclusions

Subconjunctival administration of bevacizumab induced involution of new vessels, abolished inflammatory response, and resulted in return of corneal function. Furthermore, bevacizumab is a novel approach for the treatment of herpetic stromal keratitis.     

A viral model for corneal scarring and neovascularization following ocular infection of rabbits with a herpes simplex virus type 1 (HSV-1) mutant

PURPOSE: Herpes simplex virus type 1 (HSV-1) remains a major cause of corneal scarring and visual loss. Although efforts have been made, no reproducible animal model is available to examine recurrent corneal disease. Here we propose a rabbit ocular model to study recurrent corneal disease using an HSV-1 mutant that reactivates with high efficiency. METHODS: Rabbits were ocularly infected with 2 x 10 PFU/eye of the parental McKrae, dLAT2903 (a LAT-null virus with a low-reactivation phenotype), or CJLAT (a high-reactivation virus). Acute ocular disease [days 2, 4, 7, and 10 postinfection (pi)], recurrent ocular disease, and neovascularization (days 30 to 58 pi) were monitored. RESULTS: All acute ocular disease symptoms, including conjunctivitis and corneal disease, were similar with all 3 viruses. No corneal scarring was detected in any eyes up to day 30 pi. Between days 35 and 58 pi, corneal scarring was observed in 11/14 (experiment 1) and 18/22 (experiment 2) eyes of CJLAT-infected rabbits. Significantly less corneal scarring was seen in eyes of rabbits infected with McKrae (0/18 and 0/16) or dLAT2903 (0/16 and 3/24) (P < 0.0001). Many of the eyes with corneal scarring developed obvious, measurable neovascularization. CONCLUSIONS: Rabbits infected with CJLAT developed corneal scarring and neovascularization similar to that of clinical ocular HSV-1 recurrent disease. Because this occurred well after the acute infection had resolved, the corneal scarring and neovascularization appeared to be recurrent disease. Thus, CJLAT ocular infection of rabbits may provide a good and reproducible animal model to study factors involved in corneal scarring and neovascularization from recurrent ocular HSV-1.     

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.     

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.     

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.     

人纤溶酶原K5突变体滴眼液对大鼠角膜移植植片存活时间的影响

目的 探讨人纤溶酶原K5突变体(mK5)滴眼液预防大鼠角膜移植排斥反应发生的效果.方法 实验研究.以F344大鼠30只作为供体,Lewis大鼠60只作为受体,建立同种异体角膜移植排斥反应模型.另取15只Lewis大鼠行自体原位角膜移植,即A组为15只F344大鼠自体原位角膜移植.采用完全随机分组设计方案,将60只Lewis大鼠(60只右眼)分为B、C、D及E组.术后术眼4次/d滴眼液,每次1滴,A组和B组滴生理盐水,C组和D组滴mK5滴眼液,浓度分别为5 mg/L和10 mg/L,E组给予0.1%地塞米松滴眼液,连续用药14 d.根据Holland排斥反应评分标准,判断术后植片排斥情况.比较各组角膜植片的平均存活时间,并观察各时间点角膜新生血管生长情况,计算其面积.术后第14天,对各组大鼠角膜植片做组织学检查.采用方差分析和SNK-q检验对以上所得结果进行统计学分析.结果 B、C、D、E组植片平均存活时间分别为(9.3±2.1)、(21.1±7.3)、(23.5±10.8)及(28.2±19.1)d;C、D组分别与B组比较,差异有统计学意义(q=10.24,13.47:P<0.05);E组植片存活情况较C、D组好,但组间比较,差异无统计学意义(q=2.54,1.49;P>0.05).术后A、B、C、D及E组角膜新生血管开始出现的时间分别为(3.1±0.8)、(2.6±0.5)、(6.4±0.5)、(7.8±0.7)及(5.3±1.0)d;C、D、E组与A组比较(q=31.58,51.21,19.98;P<0.05);C、D、E组也较B组明显延长(q=43.87,67.14,24.53;P<0.05);C、D及E组两两组间比较差异有统计学意义(q=11.41,20.37,9.67;P<0.05).术后C、D组角膜新生血管的生长面积明显较B组少(q=30.76,62.14;P<0.05),且与E组比较差异有统计学意义(q=15.20,25.64;P<0.05).病理学检查显示,B组角膜植片可见大量炎性细胞浸润和角膜新生血管形成,而C组与D组植片炎性反应较轻,无明显新生血管形成.结论 mK5滴眼液可抑制同种异体大鼠角膜移植术后新生血管的生成,延缓角膜移植排斥反应的发生.     

Inhibitory effects of topical cyclosporine A 0.05 % on immune-mediated corneal neovascularization in rabbits

Background

We aimed to study the inhibitory effects of topical cyclosporine A (CsA) 0.05 % on immune-mediated corneal neovascularization, and to compare its efficacy with those of dexamethasone 0.1 % and bevacizumab 0.5 %.

Methods

Immune-mediated corneal neovascularization was created in 36 right eyes of 36 rabbits. The rabbits were then randomized into four groups. Group I received CsA 0.05 %, Group II received dexamethasone 0.1 %, Group III received bevacizumab 0.5 %, and Group IV received isotonic saline twice a day for 14 days. The corneal surface covered with neovascular vessels was measured on the photographs. The rabbits were then sacrificed and the corneas excised. Paraffin-embedded sections were stained with hematoxylin-eosin and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay.

Results

The means of percent area of corneal neovascularization in Group I, II, III, and IV were 24.4 %, 5.9 %, 37.1 %, and 44.1 %, respectively. The inhibitory effect of CsA 0.05 % was found to be better than the effect found in the bevacizumab 0.5 % and control groups (p?=?0.03 and p?=?0.02, respectively). CsA 0.05 % was found to have significantly lesser inhibitory effects on corneal neovascularization than dexamethasone 0.1 % (p?<?0.001). Apoptotic cell density was higher in Group III and Group IV than in Group I and Group II. There was no difference between Group I and Group II in terms of apoptotic cell density (p?=?0.7).

Conclusions

Topical CsA 0.05 % was shown to have an inhibitory effect on immune-mediated corneal neovascularization in rabbits.     

雷帕霉素抑制大鼠角膜移植免疫排斥反应的实验研究

目的探讨雷帕霉素(RAPA)对大鼠角膜移植排斥反应的防治效果。方法以SD大鼠为供体,Wistar大鼠为受体建立角膜移植实验模型。采用完全随机分组设计方案,将68只Wistar鼠(68只眼)随机分为4组,A组为Wistar鼠自体原位角膜移植,B、C、D组为SDWistar鼠之间进行同种异体角膜移植。术后灌胃给药,A组和B组给予空白液,C组和D组分别给予RAPA(3mg·kg-1·d-1)和环孢霉素A(CsA)(10mg·kg-1·d-1),连续用药12d。根据Holland排斥反应评分系统,判断术后植片排斥情况。比较各组角膜植片的平均存活时间和植片存活率。采用广义估计方程对角膜的新生血管评分进行统计学分析。术后14d,对各组角膜进行组织学检查。结果RAPA组发生排斥反应的时间明显延迟,同种异体移植对照组植片平均存活时间(MST)为(11.0±1.5)d,RAPA组MST为(36.1±14.9)d,两组比较差异有统计学意义(P<0.05)。RAPA组角膜植片存活情况较CsA组好,但两组植片存活率比较差异无统计学意义(P>0.05)。RAPA组术后角膜新生血管的生长明显低于异体移植对照组(P<0.05)和CsA组(P<0.05)。组织学检查证实,术后14d,RAPA组角膜植片未见明显淋巴细胞浸润。结论口服RAPA能够延缓大鼠角膜移植排斥反应的发生,并能够抑制术后新生血管的生成。     

贝伐单抗抑制角膜新生血管的实验研究

目的评价贝伐单抗（avastin）局部应用对小鼠角膜新生血管（CNV）的抑制作用。方法通过碱烧伤建立CNV模型,将30只Balb／c小鼠随机分成5组,A组贝伐单抗1mg／mL每日点眼2次;B组贝伐单抗3mg／mL每日点眼2次;C组贝伐单抗5mg／mL每日点眼2次;D组0．1％地塞米松每日点眼2次;E组生理盐水每日点眼2次。分别于术后3、7、14d观察CNV情况并拍照。术后第14天,处死全部小鼠,行CNV内皮细胞荧光标记,计算CNV所占全角膜面积的比例。结果各组CNV面积为A组（37．11±3．17）％、B组（29．75±3．56）％、C组（18．76±2．55）％、D组（20．91±2．75）％,E组（41．65±2．11）％。各组小鼠CNV面积依次为c组〈D组〈B组〈A组〈E组,C组同A、B、E组比较差异均有统计学意义（P〈0．01）,c组与D组比较差异无统计学意义（P=0．694）。结论局部应用贝伐单抗对小鼠角膜化学烧伤后的CNV有抑制作用。     

雷帕霉素缓释片防治兔高危角膜移植免疫排斥反应和新生血管增殖的研究

目的探讨雷帕霉素(RAPA)缓释片防治兔高危角膜移植术后免疫排斥反应和角膜新生血管增殖的疗效及其作用机制。方法RAPA缓释片制作:RAPA与载体乙交酯-丙交酯-己内酯三元共聚物(PGLC)制成RAPA缓释片(W/W=50/50),每粒缓释片含RAPA0·5mg。65只健康新西兰大白兔,其中45只兔(45只眼)采用缝线法诱导角膜新生血管生成,选择大于3个象限、新生血管长入角膜超过3mm的40只兔按照随机数字表法分为对照组(A组)、1mgPGLC载体前房植入组(B组)、1%RAPA滴眼液组(C组)及0·5mgRAPA缓释片前房植入组(D组),每组10只兔;余20只兔为供体。对4组兔行右眼同种异体穿透性角膜移植术,术后观察90d,记录排斥指数(RI,为植片水肿、混浊及血管长入评分合计)和新生血管指数(NI,为血管长入植片评分)。定期检测C、D组兔房水中RAPA浓度;术后3周,原位杂交法对4组兔角膜植片中白细胞介素(IL)-2R、单核细胞化学吸引蛋白质(MCP)-1、Fas/FasL进行检测,采用免疫组化法对肿瘤坏死因子(TNF)α和血管内皮细胞生长因子(VEGF)进行检测。术后90d,病理组织学检查视网膜、肝肾结构变化。结果(1)免疫排斥:A、B、C及D组兔发生免疫排斥反应的平均时间分别为(16·5±2·5)、(16·0±2·6)、(47·1±13·2)、(87·6±5·8)d(P=0·000)。术后2周,4组兔植片RI分别为4·9±2·2、3·9±0·9、0·8±0·4、0·3±0·6(P=0·000)。术后12周分别为10·4±0·8、10·0±0·0、7·2±2·2、2·0±3·3(P=0·000)。(2)角膜新生血管:术后2周,4组NI数分别为2·4±0·7、2·1±0·5、0·6±0·5、0·3±0·5(P=0·000)。术后12周分别为3·8±0·5、3·8±0·4、0·8±0·7、0·4±0·8(P=0·000)。(3)房水中RAPA浓度:术后第2、4、8、12周,D组房水中RAPA浓度分别10·7、12·0、9·2、7·0ng/ml,C组房水中RAPA浓度检测不出。(4)细胞因子表达:A、B组植片大量表达IL-2R、MCP-1、TNF-α、VEGF细胞因子,C、D组不表达上述细胞因子,4组植片中Fas/FasL均不表达。(5)组织病理:4组兔视网膜、肝肾组织结构正常。结论局部应用RAPA可以防治兔高危角膜移植术后免疫排斥反应和角膜新生血管增殖,RAPA缓释片疗效优于滴眼液。     

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.     

贝伐单抗对小鼠单纯疱疹病毒性角膜炎角膜新生血管和瘢痕形成的抑制作用

背景 单纯疱疹病毒性角膜炎(HSK)可诱导发生角膜新生血管和炎症反应,传统的治疗药物为阿昔洛韦(ACV),研究已证实贝伐单抗具有抑制新生血管的作用,但其是否对HSK发挥治疗作用值得研究. 目的 研究贝伐单抗对小鼠HSK角膜瘢痕和新生血管的抑制作用.方法 利用体外培养并感染的Vero细胞生产单纯疱疹病毒Ⅰ型(HSV-1),以无血清DMEM培养基于冰上对HSV-1进行10倍梯度稀释后制备成HSV-1液.选用SPF级雄性6～8周龄C57 BL/6小鼠200只,用0.6μl滴度为l×l0 7空斑形成单位(PUF)/ml的HSV-1行小鼠角膜基质注射以制备HSK模型,将模型眼分为单纯ACV注射组、ACV+贝伐单抗注射组和生理盐水注射组,按照分组分别于感染后5、8、11和14d选择角膜混浊评分为1分的模型眼结膜下注射50μg ACV、50 μg ACV+5 μl贝伐单抗和5μl生理盐水.此外采用紫外线照射均有轻度新生血管和瘢痕的6只模型小鼠双眼诱导HSK复发,于复发后0、2、4和6d行5μl贝伐单抗(25 mg/ml)右眼结膜下注射,左眼结膜下注射5 μl生理盐水.于造模后5、7、11、14和17d以及复发的0、2、4和6d行小鼠角膜裂隙灯显微镜检查并用角膜知觉测量仪行中央角膜敏感度检测;制备角膜铺片,采用免疫荧光检测法检测角膜中CD31和βⅢTubulin荧光表达以评估角膜新生血管和角膜神经纤维分布;采用Image J软件测定角膜新生血管面积和瘢痕面积. 结果 HSK成模率达80％以上,造模后7d和复发后2d角膜混浊最重,造模后15 d和复发后2d角膜新生血管面积最大.造模后模型眼中央角膜敏感度逐渐降低,于造模后9d下降到最低.ACV+贝伐单抗注射组角膜病变面积小于单纯ACV注射组,ACV+贝伐单抗注射组小鼠中央角膜敏感度为5.50±0.71,明显高于生理盐水注射组的0.50±1.41,差异有统计学意义(Z=-2.397,P=0.029).ACV+贝伐单抗注射组小鼠角膜病变面积增长率为(167.10±52.53)％,低于生理盐水注射组的(312.30±74.18)％,差异有统计学意义(Z=-1.992,P=0.046).实时荧光定量PCR显示造模后7d,模型眼角膜及同侧三叉神经节(TG)中胸苷激酶(TK)和感染细胞蛋白-27(ICP-27) mRNA相对表达量均较高,造模后45 d明显下降,诱导复发后2d TKmRNA和ICP-27 mRNA均再次升高,复发后7d表达量降至最低.造模后45 d同侧TG中均可见LAT mRNA表达量达峰值,诱导复发后2d相对表达量下降,复发后7d相对表达量再次升高,差异均有统计学意义(均P＜0.01).角膜铺片结果显示,造模后生理盐水注射组小鼠较正常对照小鼠角膜新生血管明显增加,角膜神经纤维明显减少,单纯ACV注射组和ACV+贝伐单抗注射组小鼠角膜新生血管少于生理盐水注射组,ACV+贝伐单抗注射组小鼠角膜神经纤维较生理盐水组注射组和单纯ACV注射组均增加.结论 贝伐单抗结膜下注射可抑制HSK模型小鼠角膜新生血管生成和瘢痕形成,与ACV联合应用时二者有协同作用.     

角膜新生血管对角膜损伤神经再生影响的研究

目的 探讨角膜新生血管对大鼠角膜损伤神经再生的影响。设计 实验研究。研究对象 SD大鼠。方法 采用随机数字表法将18只SD大鼠分为3组,每组6只。A组行缝线铲针角膜基质层间切开及缝线诱导新生血管术,术后0、3、7天给予结膜下注射贝伐单抗;B组行缝线铲针角膜基质层间切开及缝线诱导新生血管术;C组0、3、7天行结膜下注射贝伐单抗操作。分别在术后1天、1周、2周、4周,采用裂隙灯照相法观察记录角膜新生血管面积;角膜共聚焦显微镜记录神经长度。采用Cochet-Bonnet知觉仪测量缝线区的角膜知觉,采用Schirmer试验泪液线测量右眼的泪液分泌量。术后4周角膜全层铺片免疫荧光染色,记录上皮下神经密度。主要指标 角膜新生血管面积比、神经长度、上皮下神经密度、角膜知觉、泪液分泌量。结果 A、B组术后1、2周有角膜新生血管生长,术后4周消退闭锁,C组无角膜新生血管生长。A组术后1、2周新生血管面积比为（10.86±1.57）%和（1.87±0.69）%,分别小于B组的（25.42±2.65）%和（6.48±1.10）%（P均=0.000）。术后1天A、B组神经长度分别为（151.02±4.74）μm、（149.69±4.32）μm（P=0.306）;术后1、2、4周,A组神经长度均长于B组,分别为（193.84±2.25）μm与（155.73±2.98）μm、（217.15±2.08）μm与（166.21±2.41）μm、（220.70±1.41）μm与（203.76±1.74）μm（P均=0.000）。术后A、B组神经长度均有减少并有恢复趋势,C组无明显变化。术后4周A组损伤区上皮下神经密度（22.60%±2.02%）明显高于B组（9.41%±2.01%）（P=0.000）。A、B组上皮下神经短小稀疏、密度低,C组形态正常。A、B组术后1、2、4周时角膜知觉及泪液分泌量均无统计学差异（P均＞0.05）。A、B组均有下降并恢复趋势,C组无明显变化。结论 角膜新生血管可能抑制角膜损伤神经再生,抑制角膜新生血管有利于神经再生。（眼科, 2017, 26： 106-111）     

Inhibition of corneal neovascularization by topical application of nintedanib in rabbit models

AIM: To evaluate the potential efficacy and mechanisms of nintedanib in corneal neovascularization (NV) in rabbit models. METHODS: Corneal NV was induced using 1 mol/L NaOH. Rabbits (n=21) were randomized to 3 groups: Group 1 were treated with 0.9% NaCl, Group 2 with Avastin (5 mg/mL), and Group 3 with nintedanib (1 mg/mL). All treatments started 1d after alkaline burns and were topically performed 3 times a day for 2wk. Photographs were taken on a slit lamp microscope on day 7 and 14. The NV area, the length of the vascularization and angiogenesis index (AI) were used to evaluate the corneal NV. On day 14, the immunohistochemical (IHC) studies of the cornea were examined. Western blot was performed to test the expression levels of vascular endothelial growth factor (VEGF), Akt, p-Akt, P38, p-P38, MMP-2 and MMP-9. RESULTS: The corneal NV area, vessel length and AI in Group 3 were significantly lower than Group 2, with both being lower than Group 1. IHC staining showed that VEGF was significantly overexpressed in the epithelium and stroma of cornea following alkaline burns. In contrast, the level of VEGF was significantly suppressed in both Group 2 and Group 3. Western blot results further confirmed that, compared with Group 1, Group 3 had significantly reduced expressions of VEGF, Akt, p-Akt, p-P38, MMP-2, and MMP-9 in corneal tissues. Trends of lower levels of MMP-2, AKT, and p-AKT in Group 3 than Group 2 were identified. CONCLUSION: Nintedanib and Avastin can effectively inhibit corneal NV, with P38 MAPK and AKT signaling pathways being possibly involved. Nintedanib seems more effective than Avastin and has the potential to be a novel therapy for preventing corneal NV.     

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.     

贝伐单抗联合曲安奈德治疗穿透性角膜移植术后新生血管

目的：观察球结膜下注射贝伐单抗和曲安奈德治疗穿透性角膜移植术后新生血管的临床疗效。
　　方法：对19例20眼因眼外伤行穿透性角膜移植术后3mo,新生血管增生达到Ⅲ度以上患眼随机分为四组。 A组：对照组;B组：贝伐单抗2.5mg(0.1mL);C组：贝伐单抗及曲安奈德各0.1mL;D 组：曲安奈德0.1mL;每月1次,共两次球结膜下注射药物。
　　结果：平均随访3a,B组和C组视力保持稳定或稍有增进,新生血管和免疫反应明显减退(P<0.01),患者眼痛、畏光流泪症状减轻。
　　结论：贝伐单抗联合曲安奈德在治疗穿透性角膜移植术后新生血管的过程中能够有效减退新生血管,并减轻排斥反应,对保持植片的透明性有积极的治疗作用。