Growth factors and corneal epithelium

Regeneration of corneal epithelium following injury is essential for visual rehabilitation. A limited number of approaches are available for treating patients who fail to heal epithelial injuries adequately. The presence of specific receptors for epidermal growth factor (EGF) on epithelial cells suggests that this potent mitogen may play a role in normal epithelial wound healing. Topical application of biosynthetic human EGF significantly accelerated epithelial regeneration in primates following epikeratophakia surgery. Epidermal growth factor alone and with fibronectin accelerated epithelial regeneration of rabbits following mild alkali burns. Since prolonged exposure of cells to EGF is necessary to induce mitosis, the dynamics of EGF in the eye and with various lenses was studied. When applied in methylcellulose-based eye drops, 90% of the EGF was lost from tear film within 10 min, while a small amount (10%) remained associated with conjuctival tissue. Soft contact lenses or epikeratophakia lenticles took up substantial amounts of EGF (50 micrograms) and released 85% of the EGF within 24 h, with a half-life of 4 h in vitro. Epidermal growth factor did not diffuse through corneas or lenticles and did not promote epithelial downgrowth along sutures in primate corneas. These results suggest that biosynthetic growth factors may be useful in the treatment of some epithelial injuries.     

神经生长因子和表皮生长因子对治疗性角膜切削术兔眼角膜上皮和神经纤维的影响

目的比较表皮生长因子(epidermal growth factor,EGF)和神经生长因子(nerve growth factor,NGF)滴眼液对准分子激光治疗性角膜切削术(phototherapeutic keratectomy,PTK)术后角膜上皮和神经纤维再生的影响。方法大耳白兔50只双眼建立角膜浅层瘢痕模型,造模后双眼行PTK,根据术后白天使用的滴眼液不同随机分为5组,每组10只,分别为:空白对照组:双眼滴氯霉素眼液每天4次;羟甲基纤维素组:双眼滴羟甲基纤维素和氯霉素眼液每天各4次;EGF组:双眼滴EGF和氯霉素眼液每天各4次;NGF组:双眼滴NGF和氯霉素眼液每天各4次;EGF+NGF组:双眼滴EGF、NGF和氯霉素眼液每天各4次。分别在PTK术后1d、3d、5d、7d和30d空气栓塞处死各组兔各2只,每组分别取左眼进行免疫组织化学染色,右眼进行角膜神经染色,对角膜上皮和神经纤维进行观察比较。结果术后5～7d各组角膜上皮均已全部愈合,EGF组、NGF组和EGF+NGF组有轻度角膜上皮增生现象,30d各组仍有上皮细胞增生。神经染色术后7d各组之间形态学上未见明显差异。术后30d均可见新生神经纤维出现,从深层未损伤神经和切削区神经干出芽再生。EGF组、NGF组和EGF+NGF组每高倍镜视野中平均新生神经纤维数比其他两组高。透射电镜发现术后30d各组上皮基底细胞仍增大,可见大量扩张的粗面内质网,EGF组和EGF+NGF组基底细胞可见粗面内质网、游离核糖体,NGF组中尚且有较多微丝。结论 EGF和NGF滴眼液对于PTK术后的角膜上皮和神经纤维修复都有明显的促进作用,尤其以EGF+NGF滴眼液的联合应用效果更显著。     

潇来威治疗LASIK术后角膜上皮剥脱500例

目的观察潇来威滴眼液促进LASIK术后角膜上皮剥脱愈合的临床疗效与不良反应。方法LASIK术后角膜上皮剥脱500例(900眼),随机分为A组与B组,A组应用潇来威联合点必舒眼液,B组单纯使用点必舒。疗程10d,术后2、3、7、10d,观察患者症状、体征等指标,裂隙灯显微镜观察角膜上皮剥脱愈合情况。结果A组治愈369眼(82.0％),显效47眼(10.4％),好转34眼(7.6％);B组治愈288眼(64.0％),显效73眼(16.2％),好转81眼(18.0％),无效8眼(1.8％)。两组有效率比较有显著性意义(P<0.05)。结论LASIK术后联合潇来威点眼,能促进角膜上皮细胞的再生修复,提高疗效,缩短疗程。     

Dose-dependent effects of epidermal growth factor on corneal wound healing

The dose-dependent effect of epidermal growth factor (EGF) on the development of wound tensile strength following full-thickness corneal wounds was evaluated in 60 adult rabbits. One eye from each rabbit received a single 7-mm long corneal incision. After injury each rabbit was treated three times daily for 5 or 10 days with either EGF at 0.001 mg/ml (10 eyes), 0.01 mg/ml (10 eyes), 0.1 mg/ml (10 eyes), 1.0 mg/ml (15 eyes), or vehicle (15 eyes). The tensile strength of the wound was evaluated using a 5-mm wide strip of cornea mounted on a tensiometer. We found that EGF at 0.1 mg/ml and at 0.01 mg/ml increased wound strength by 100% at 5 days and by 60% at 10 days (P less than 0.05 and P less than 0.05). However, EGF at 0.001 mg/ml and 1.0 mg/ml appeared to have no effect on wound strength. Histologic examination of full-thickness wounds in a separate series showed an increase in wound fibroblastic response and a diminished fibrin clot at 5 days in rabbits treated with 0.1 mg/ml and 0.01 mg/ml. We conclude that EGF enhances the wound strength of full-thickness corneal wounds in a dose-dependent manner which may be explained in part by an increased fibroblastic response. Concentrations of EGF greater or less than an optimal dose may be less effective in enhancing corneal wound strength.     

Epithel regeneration inhibition in the treatment of Trifluorothymidin (F3TDR) (author's transl)]

After well-defined chemical and mechanical removal of the corneal epithelium from 38 rabbit eyes 19 right eyes were treated with F3TDR (Trifluorothymidine) 6 times daily, whereas the 19 left eyes were with physiologic NaCl-solution 6 times daily. The epithelial regeneration was compared with the contralateral controll-eye. No significant difference was found between both eye-series. The short-timed high dosage of F3TDR seems to have no delaying effect on epithelial regeneration.     

Anti-inflammatory and antiallergic effects of ketorolac tromethamine in the conjunctival provocation model

AIM: To study the effect of the topical anti-inflammatory drug, ketorolac, on (1) the clinical allergic reaction induced by the conjunctival provocation test (CPT); (2) the release of tryptase in tears; and (3) the expression of adhesion molecules on the conjunctival epithelium. METHODS: 10 allergic but non-active patients were challenged in both eyes with increasing doses of specific allergen to obtain a positive bilateral reaction and rechallenged, after 1 week, to confirm the allergic threshold dose response. After 2 weeks, a third CPT was then performed bilaterally 30 minutes after topical application of ketorolac in one eye and placebo in the contralateral eye in a double blind fashion. Clinical symptoms and signs were registered 5, 10, 15, and 20 minutes after challenge. The following objective tests were performed: tear tryptase measurement; tear cytology; and conjunctival impression cytology for immunohistochemical expression of ICAM-1 on epithelial cells. RESULTS: Compared with placebo, ketorolac significantly reduced the total clinical score and the itching score in the 20 minutes after challenge (p<0.0005). Tear levels of tryptase were significantly reduced in the ketorolac pretreated eyes compared with placebo (p<0.03). Eosinophils, neutrophils, and lymphocytes in tear cytology were significantly lower in ketorolac treated eyes compared with placebo. A significant difference in the epithelial expression of ICAM-1 was observed between placebo and ketorolac treated eyes (p<0.05). CONCLUSION: Ketorolac proved to be effective in reducing mast cell degranulation, as indicated by significantly decreased tryptase tear levels, as well as the clinical and cytological allergic reaction.     

Effect of a beta-blocker on altered body position: induced ocular hypertension.

The intraocular pressures (IOP) were measured in both eyes of 25 healthy volunteers in various body positions. One eye was pretreated with levobunolol 0.5% or placebo applied in a masked, randomised fashion, while the other served as control. IOP changes in response to levobunolol and to changes in position were significant (p less than 0.0001). However, pressure rises relative to position were not significantly different in eyes treated with drug vs placebo. Levobunolol did not alter relative changes in IOP from changes in body position. However, the overall lowering effect may offer some protection to patients with glaucoma.     

The influence of epidermal growth factor on cat corneal endothelial wound healing.

Using standardized freeze wounds in cat corneas, we tested the efficacy of human recombinant Epidermal Growth Factor (EGF) to promote endothelial healing when solubilized in either phosphate buffered saline (PBS), 1% methylcellulose (MC), or sodium hyaluronate (NaHA), in final intraocular doses ranging from 2 micrograms to 100 micrograms of EGF. After 6 or 7 days' healing, animals were humanely sacrificed and corneal tissues were fixed and stained for light microscopy and computation of remaining wound areas. EGF in NaHA in final intraocular doses of 2 and 10 micrograms prompted significantly more complete healing of transcorneal freeze wounds to endothelium compared with endothelium of eyes treated with NaHA control solution alone. EGF in PBS or in MC in doses ranging from 2-100 micrograms/eye did not promote more complete wound healing than that seen in eyes treated with their respective vehicle solutions alone. All vehicle solutions were associated with similar degrees of wound healing, implying that they have no intrinsic healing properties.     

Nonsteroidal anti-inflammatory agents after argon laser trabeculoplasty. A trial with flurbiprofen and indomethacin

Seventy glaucomatous eyes received argon laser trabeculoplasty (ALT) to 180 degrees of the trabecular meshwork. Thirty-six eyes were treated with topical .03% sodium flurbiprofen, a nonsteroidal anti-inflammatory agent; 34 eyes received placebo. Eighteen eyes received ALT to 360 degrees of the meshwork; nine of these eyes were treated with topical 1% indomethacin and nine eyes received placebo. Eyes treated with flurbiprofen and indomethacin showed significantly less conjunctival injection following ALT. However, these agents did not significantly alter the anterior chamber reaction after laser therapy compared to placebo. Flurbiprofen-treated eyes showed a significantly smaller percent (32.6%) decrease in intraocular pressure (IOP) one day after ALT as compared to eyes receiving placebo (43.8%). In addition, a higher percentage of placebo treated eyes had a fall in IOP of at least 10 mmHg up to five weeks after ALT. Within the indomethacin protocol, the percentage change in IOP was comparable for both indomethacin and placebo treated eyes. Mild ocular symptoms (itching, burning, foreign-body sensation) developed in 77.8% of flurbiprofen-treated eyes, in 55.6% of indomethacin-treated eyes, and in 20.9% of eyes receiving placebo.     

Screening von myopen LASIK-Patienten mit einer gesteigerten epithelialen Wundheilung Quantitative Bestimmung von EGF mRNA in den kornealen Epithelzellen

BACKGROUND: Wound healing of the cornea is critical for the refractive outcome of myopic laser-assisted in situ keratomileusis (LASIK). As epidermal growth factor (EGF) is important for the origin of epithelial hyperplasia, this study examined preoperative EGF mRNA concentrations in the corneal epithelial cells to detect patients with increased epithelial wound healing response. PATIENTS AND METHODS: The epithelium was biopsied before LASIK in 35 eyes with myopia of -10.0 D. The EGF mRNA concentration in the epithelial cells was quantified by polymerase chain reaction and enzyme-linked oligosorbent assay, and the correlation with postoperative refraction at 6 months was assessed. RESULTS: All eyes were around emmetropia 3 weeks after the surgery. At 6 months postoperatively 27 eyes were within +/- 1.0 D of emmetropia while 8 showed regression of 2.0-4.0 D. Higher EGF mRNA levels were found in eyes with regression than in eyes with postoperative emmetropia. CONCLUSIONS: Preoperative EGF mRNA concentration in the corneal epithelial cells may be an indicator of postoperative refractive outcome of myopic LASIK and offers a new possibility for pharmaceutical manipulation.     

Clinical evaluation of corneal epithelialization after photorefractive keratectomy in patients treated with polydeoxyribonucleotide (PDRN) eye drops: a randomized, double-blind, placebo-controlled trial

PURPOSE: The effect of polydeoxyribonucleotide (PDRN) eye drops vs placebo on corneal epithelial healing after photorefractive keratectomy (PRK) for correction of myopic and myopic-astigmatic defects was evaluated in a randomized, double-blind clinical trial. Primary endpoint for efficacy was the evolution of corneal re-epithelialization. Secondary endpoint was the evaluation of PDRN eye drops tolerability. METHODS: Sixty eyes were enrolled in the study, randomly allocated into standard therapy plus placebo eye drops (30 eyes), or standard therapy plus PDRN eye drops (30 eyes). Checks were carried out preoperatively and at days 1, 2, 3, and 7 of the follow-up. Six eyes dropped out (four in PDRN group, two in placebo group) for reasons unrelated to the study. RESULTS: On day 2, the disepithelialized area was 8.4 mm2+/-9.2 (mean+/-SD) in controls and 6.0 mm2+/-6.8 in PDRN group. On day 3 a complete corneal re-epithelialization was found in 20 out of 26 (77%) eyes of PDRN group and in 17 out of 28 (61%) eyes of placebo group (p<0.05 in percentage terms). On day 7 of follow-up, all eyes appeared to be completely re-epithelialized. The mean score of corneal evaluation on day 3 was 2.9 in PDRN group and 3.75 in control group (p<0.05 between groups). No adverse events occurred during the study. CONCLUSIONS: The data of the study have shown that after PRK, PDRN stimulates corneal epithelium regeneration. PDRN eye drops administration four times a day is well tolerated by patients during the re-epithelialization stage. A much larger clinical study should be performed in order to prove the results obtained in this pilot study.     

Apraclonidine prophylaxis for postcycloplegic intraocular pressure spikes.

A randomized, prospective, double-masked study was undertaken to determine the risk of postcycloplegic intraocular pressure spikes in patients with open-angle glaucoma and to evaluate apraclonidine prophylaxis in minimizing these spikes. Patients were stratified as miotic treated or untreated and each group was randomized to receive either placebo (artificial tears) or apraclonidine in both eyes before instillation of tropicamide. In both the miotic treated and untreated groups that received placebo, there was a high incidence, (37% and 38%, respectively) of clinically significant (greater than or equal to 6 mmHg) intraocular pressure spikes after instillation of tropicamide. In both the miotic treated and untreated groups, there was a statistically significant difference in postcycloplegic intraocular pressure between the subgroup that received placebo and the group that received apraclonidine prophylaxis (P = 0.003 and P = 0.006, respectively). Additionally, four eyes that received placebo had a spike of over 10 mmHg (range, 12 to 27 mmHg), while only one eye had an increase of greater than 10 mmHg (12 mmHg) in the apraclonidine group. Thus, apraclonidine appears to be a useful agent for minimizing precipitous increases in intraocular pressure after cycloplegia in eyes of open-angle glaucoma patients prone to this complication of cycloplegia.     

The effect of epidermal growth factor on epithelial healing after penetrating keratoplasty in human eyes

Two groups of patients were assigned randomly to treatment with epidermal growth factor or placebo after penetrating keratoplasty. In all cases, the epithelium was removed completely from the graft by scrubbing at the close of surgery. One group of patients received topically applied growth factor or placebo in a solution with preservative solution four times a day on the day of surgery and twice a day for two days thereafter. The second group of patients received one-half the concentration of growth factor or placebo in a solution without preservative eight times a day for four days beginning the day before surgery. Healing was assessed twice a day by color photography after instillation of 2% fluorescein; the percent area of the remaining epithelial defect was determined with a planimeter. No therapeutic benefit was demonstrated in the eyes treated with growth factor in either group of patients.     

Contact lenses as a drug delivery device for epidermal growth factor in the treatment of ocular wounds

Background: This work was conducted to investigate the uptake and release of epidermal growth factor (EGF) from hydrogel contact lenses and to determine whether the released protein would be therapeutically active in a rabbit corneal epithelial defect model of ocular trauma, prior to use in humans. Methods: The uptake and release of EGF from hydrogel contact lens materials were determined by high‐pressure liquid chromatography. Contact lenses composed of vasurfilcon A or lotrafilcon A (containing silicone) were incubated in a source solution containing 0.4 ppm EGF for seven hours. To determine the kinetics of drug uptake into the contact lens matrix, drug concentration in the source solution was measured at zero, one, 60, 240 and 420 minutes. To determine the kinetics of release, loaded contact lenses were immersed in a recipient solution of phosphate‐buffered saline. Therapeutic activity in vivo was investigated by placing prepared lenses on the surface of abraded corneas of New Zealand White rabbits, with abraded corneas of contralateral eyes used as controls. Control eyes were treated with contact lenses placed in saline for injection. Wound closure was assessed hourly. Results: Uptake and release of EGF were demonstrated for vasurfilcon A but not lotrafilcon A contact lens materials. The retention time of EGF released from vasurfilcon A contact lenses was similar to control EGF not exposed to contact lens polymers. The greatest adsorption of EGF into the lens material occurred within approximately 120 minutes, with a flattening of the rate of uptake thereafter. Abraded eyes in rabbits showed a significantly higher overall healing rate for EGF‐treated contact lenses compared with control eyes (p < 0.0001). Conclusions: EGF can be delivered from some but not all hydrogel materials. Lens materials composed of silicone may not be useful for delivering EGF to the eye. EGF‐treated contact lenses may be a useful device to facilitate healing of ocular wounds.     

Photodynamic therapy

Photodynamic therapy with Verteporfine (Visudyne Novartis Ophthalmics) achieves a photo occlusion of subfoveal choroidal neovascularization (CNV) resulting from age-related macular degeneration (AMD) in phase 1 and 2 studies. Thereafter, two international randomized clinical trials evaluated the product's efficacy in term of visual acuity. Eyes with neovascular lesions due to AMD of less than 5,400 micro, in size involving the fovea and presenting well-defined CNV on fluorescein angiography could be included when visual acuity was between 5 and 1/10. Six hundred and nine patients were subsequently randomized either to Verteporfine or to placebo (proportion 2:1). A follow-up of 3 months made it possible to consider retreatment when a fluorescein leakage was observed. Functional and angiographic results demonstrated the efficacy of PDT with Verteporfine in comparison with placebo at each examination during follow-up. A visual acuity decrease of less than 15 letters on ETDRS (or 3 lines or approximately 1.5/10) occured less frequently in the treated group with PDT (61% out of 402 treated eyes compared with 46% of 207 placebo eyes). Subgroup analysis of predominantly well-defined CNV (involving 50% or more of the total of the exsudative lesion) showed even more favourable effects (67% of treated eyes versus 39% of placebo eyes). Conversely, when well-defined CNV extended to a smaller area, the results were not significant. The most frequent side effect was Verteporfine extravasation at the injection site. As dynamic phototherapy with Verteporfine limits the decrease in visual acuity always occurring in the natural history of CNV caused by AMD, it is desirable that patients presenting subfoveal predominantly well-defined lesions benefit from this new treatment approach.     

乙酰唑胺治疗黄斑囊样水肿的临床研究

目的：评价乙酰唑胺治疗黄斑囊样水肿(cystoid macular edema，CME)的疗效并探讨其药理机制。 方法：经眼底荧光血管造影(fundus fluorescein anglography，FFA)检查证实的CME患者37例40只眼，随机分为两组，分别给予乙胺唑胺和安慰剂4周，4周后两组交叉给药继续观察4周。在不同阶段进行视力、FFA、阈值化视野和视网膜电图(electro retinogram，ERG)检测；对其中12只眼进行了口服乙酰唑胺前后视网膜中央动脉血流的多普勒超声检测和视网膜血管计算机图像分析。 结果：乙酰唑胺治疗组52.5%的患眼黄斑水肿缓解或消退，75.0%表现为视力提高，总体10°阈值视野视敏度显著提高和ERGb波振幅明显增高，安慰剂组各项指标无明显改善，两组疗效有显著差别(P<0.05)。超声波检测显示CME眼血流缓慢。结论：乙酰唑胺具有明显提高患者视力、改善或缓解黄斑水肿的确切疗效，其机制可能与改善视网膜血流有关。 （中华眼底病杂志，1997，13：89-92）     

重组人BIGH3蛋白滴眼液对兔角膜上皮损伤的修复作用

背景角膜上皮损伤可导致角膜溃疡和基质混浊，甚至出现不可逆的视功能损害，以往采用的药物治疗仅能缓解刺激症状，而促进角膜上皮细胞再生的作用较弱，因此寻求能有效调控角膜上皮细胞生长，从而有效治疗角膜上皮损伤的药物至关重要。目的探讨重组人BIGH3蛋白滴眼液对角膜上皮损伤的修复作用。方法50只清洁级健康成年新西兰大白兔，均取右眼为实验眼，其中2只兔制作角膜上皮损伤模型后即刻行裂隙灯显微镜检查及角膜荧光素染色，眼前节照相后处死；48只兔按照随机数字表法随机分为重组人表皮生长因子（EGF）衍生物组（阳性对照组）、生理盐水组（阴性对照组）、质量分数0．25％重组人BIGH3蛋白滴眼液组及O．5％重组人BIGH3蛋白滴眼液组，每组12只。在直径7mm环钻内滴人体积分数20％乙醇，置于角膜中央区60s，然后用角膜刮匙刮去角膜上皮，制作角膜上皮缺损模型。术后各组兔眼分别点用相应药物，每天4次，分别于术后12、24、36、48、72h行裂隙灯显微镜检查及角膜荧光素染色，于12、24、36、48h采用抽签法各处死2只实验兔，72h时各处死4只实验兔，制备角膜组织标本并行苏木精一伊红染色，观察并比较各组兔眼在不同时间点角膜上皮修复情况，应用计算机图像处理系统测量各组兔眼不同时间点角膜上皮愈合面积。结果各组兔眼用药后均未发现任何眼部刺激症状。组织病理学研究发现，造模后即刻全层角膜上皮缺损，随着时间的延长，实验组及对照组兔角膜上皮缺损区皆逐渐变小，可见周边角膜上皮向中央缺损区移行，细胞层数逐渐增加，细胞排列极向逐渐趋于规则。重组人EGF衍生物组、生理盐水组、0．25％及0．5％重组人BIGH3蛋白滴眼液组角膜上皮愈合速率分别为15．00、13．81、18．05、18．86mm。／d。与生理盐水组比较，术后12、24、36、48h，0．25％、0．5％重组人BIGH3蛋白滴眼液组及重组人EGF衍生物组角膜损伤面积明显缩小，差异均有统计学意义（均P=0．000），但造模12、24、36h，重组人EGF衍生物组与生理盐水组比较差异均无统计学意义（P=0．321、0．057、0．126）。与重组人EGF衍生物组比较，术后各时间点0．5％重组人BIGH3蛋白滴眼液组角膜损伤面积明显缩小，差异均有统计学意义（P=0．042、0．039、0．025、0．008），0．25％重组人BIGH3蛋白滴眼液组角膜损伤面积仅在术后12h、24h明显缩小，差异均有统计学意义（P=0．047、0．042）。术后各时间点0．25％、0．5％重组人BIGH3蛋白滴眼液组间角膜损伤面积的差异均无统计学意义（P：0．358、0．259、0．108、0．062）。术后72h，生理盐水组角膜损伤面积为（0．51-＋0．42）mm2，而其他3个组角膜上皮均完全修复。角膜上皮修复曲线表明，0．25％及0．5％重组人BIGH3蛋白滴眼液组角膜上皮修复的相对面积均优于重组人EGF衍生物组和生理盐水组。结论0．25％及0．5％重组人BIGH3蛋白滴眼液对角膜上皮损伤的愈合有明显的促进作用，其作用优于EGF。     

Surgical occlusion of 2 vortex veins in the treatment of decompensated senile macular degeneration. A longer-term evaluation

Microsurgical occlusion of two vortex veins was carried out on seventeen eyes of 16 patients affected with senile pigment epithelial detachment. Ten eyes had avascular pigment epithelium detachment and 7 eyes had pigment epithelium detachment associated with choroidal new vessels. Regression or stabilisation of the fundus lesions occurred in one of the eyes affected with vascular pigment epithelium detachment. On the other hand 3 months postoperatively, the pigment epithelium detachment had flattened with significant improvement of the visual acuity, in 4 of the 10 eyes affected with avascular pigment epithelium detachment. However the results were not as satisfactory after a longer follow-up. The improved visual acuities noted at 3 months, gradually deteriorated after 6-12 months in most patients and 4 eyes showed subsequent clinical evidence of active disease. The results of the present study lead to conclude that the surgical occlusion of 2 vortex veins does not provide a satisfactory answer to the treatment of senile pigment epithelial detachment.     

Mitotic activity of rabbit corneal epithelium in vitro

It is difficult to maintain corneal epithelial cells for several passages in culture. At a first step to establish a culture system for corneal epithelium, the authors evaluated factors which seemed to be involved in the growth kinetics and mitotic activity of rabbit corneal epithelium in vitro. First of all, to compare the growth kinetics of peripheral and central epithelium, we measured the areas and distances of the epithelium that outgrew from peripheral and central explants in culture. The peripheral corneal epithelium grew 1.2-to 1.6-fold better than the central corneal epithelium. However, there was no significant difference between them. Secondly, we evaluated the effects of epidermal growth factor (EGF), cholera toxin (CTX) and insulin, which were contained in supplemented hormonal epithelial medium (SHEM) developed by Jumblatt et al, on the mitotic activity of rabbit corneal epithelium. Among these drugs, only EGF had the ability to increase the epithelial cell number after 10 days of incubation. By contrast, CTX which elevated the level of cyclic AMP inhibited the proliferation of the epithelium.     

Clinical and histological findings after intravitreal injection of bevacizumab (Avastin®) in a porcine model of choroidal neovascularization

Purpose: To examine the effect of intravitreally injected bevacizumab (Avastin^®) on the histological and angiographic morphology of choroidal neovascularization (CNV) in a masked and placebo‐controlled animal study. Methods: Choroidal neovascularization was induced surgically in 11 porcine eyes by perforating Bruch’s membrane with a retinal perforator. After closure of the ports used for the vitrectomy, which was performed to facilitate the Bruch’s membrane rupture, 0.05 ml of either bevacizumab or Ringer‐Lactat (placebo) was injected into the vitreous cavity. Eyes were enucleated after 14 days. Fundus photographs and fluorescein angiograms (FAs) were obtained immediately prior to enucleation. Sections of formalin‐ and paraffin‐embedded eyes were examined by light microscopy and by immunohistochemical staining. Results: Placebo‐injected eyes exhibited the highest propensity to leak, with five of six eyes leaking on FA, whereas only one of five bevacizumab‐injected eyes exhibited leakage. On histological examination, all 11 eyes contained CNV membranes of similar size, regardless of treatment. The number of vascular endothelial cells was significantly reduced (p = 0.03) in CNV membranes from eyes that had been injected with bevacizumab when compared with CNV membranes from placebo‐injected eyes. There was a trend towards more retinal pigment epithelium cells (p = 0.16) and fewer glial fibres (p = 0.08) in membranes from bevacizumab‐treated eyes compared with placebo‐treated eyes. Bevacizumab was identified immunohistochemically in the inner limiting membrane (ILM) and to a lesser degree in the remaining retina. Strong staining was also detected in both retinal blood vessels and entire CNV membranes with no cellular predisposition. Vascular endothelial growth factor expression was found in the CNV membranes, in the ILM, in the ganglion cell layer, in Müller cells throughout the neuroretina and in retinal blood vessels. Conclusions: Bevacizumab significantly reduced the proliferation of vascular endothelial cells in CNV membranes and showed a strong trend towards a reduction of leakage from these membranes. After a single injection, bevacizumab did not exhibit a size reducing effect on CNV, but it was still present in the membranes 14 days after intravitreal injection.