Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
DOI: 10.5301/ejo.5000391 Eur J Ophthalmol 2014; 24 ( 3 ): 299-308 Original Article free on line The effect of subconjunctival ranibizumab on corneal and anterior segment neovascularization: study on an animal model Vasilios S. Liarakos1-3, Dimitrios Papaconstantinou4, Ioannis Vergados2, Maria Douvali2, Panagiotis G. Theodossiadis2 Naval Hospital, Athens - Greece 2nd Department of Ophthalmology, “Attikon” Hospital, University of Athens - Greece 3 Netherlands Institute for Innovative Ocular Surgery, Rotterdam - the Netherlands 4 st 1 Department of Ophthalmology, University of Athens - Greece 1 2 Purpose: To evaluate the effect of subconjunctival anti–vascular endothelial growth factor (VEGF) ranibizumab on corneal and anterior segment neovascularization. Methods: In this experimental study and laboratory investigation, chemical cauterization was utilized to induce corneal neovascularization in 16 rabbits randomly divided in 2 equal groups. Cauterized eyes were either treated with 0.1 mL (1 mg) of subconjunctival ranibizumab or administered a sham injection. A third group of 4 rabbits served as control for side effects after ranibizumab administration. All animals were monitored daily for 14 days and the extent of corneal scarring and neovascularization was measured on days 1, 7, and 14. After enucleation, ocular tissues were separated under a surgical microscope and VEGF levels were measured with ELISA. Statistical analysis was performed to compare the extent of corneal neovascularization and VEGF levels between treated and untreated eyes. Results: Subconjunctival ranibizumab inhibited corneal neovascularization significantly both in the first and the second week compared to untreated controls (p = 0.006 and p = 0.001, respectively). The VEGF levels were significantly lower in all anterior segment tissues like the cornea, iris, aqueous humor, and conjunctiva of the treated eyes (p<0.01). The reduction of VEGF levels ranged from 19% to 73% in different ocular tissues. Corneal scarring was not significantly affected by anti-VEGF treatment (p = 0.7). No side effects were noticed. Conclusions: Early subconjunctival administration of ranibizumab may successfully inhibit alkali-induced corneal neovascularization in an animal model. Subconjunctival ranibizumab reduces VEGF levels significantly not only in the cornea and the bulbar conjunctiva but also in the aqueous humor and the iris. Keywords: Cornea, Iris, Neovascularization, Ranibizumab, Vascular endothelial growth factor Accepted: October 22, 2013 INTRODUCTION Corneal clarity and avascularity are important for the proper optical performance of the cornea. Corneal neovascularization is a sight-threatening condition usually associated with inflammatory or infectious disorders of the ocular surface. There is a balance between angiogenic factors and anti-angiogenic molecules in the cornea, which tilts towards angiogenesis in case of various inflammatory, infectious, degenerative, or traumatic disorders (1). Corneal neovascularization is a common clinical problem with serious consequences in vision; it can compromise © 2013 Wichtig Editore - ISSN 1120-6721 299 Subconjunctival ranibizumab for anterior segment neovascularization corneal transparency and plays a major role in corneal graft rejection (2) and the prognosis of penetrating keratoplasty (3-5) by breaching corneal immune privilege (6). Diseases associated with corneal neovascularization include inflammatory disorders, corneal graft rejection, infectious keratitis, contact lens–related hypoxia, alkali burns, stromal ulceration, aniridia, and limbal stem cell deficiency. In these conditions, the balance between angiogenic and antiangiogenic factors may be tilted in favor of neovascularization due to the upregulation of angiogenic factors and/or the downregulation of antiangiogenic factors (7). Twenty percent of corneal specimens obtained during corneal transplantation show histopathologic evidence of neovascularization (4, 5, 8). Subsequently, topical antiangiogenic therapy could reduce the need for corneal transplantation and retransplantation by improving long-term graft survival. Vascular endothelial growth factor (VEGF)–A is upregulated in inflamed and vascularized corneas in both human and animal models (9-11). Enhanced VEGF production has been shown in hypoxia and inflammatory response. The importance of VEGF in corneal angiogenesis has been demonstrated, among others, also by the inhibition of neovascularization with the administration of anti-VEGF agents in rabbit cornea models (12, 13). Additional VEGF members include VEGF-B, VEGF-C, and VEGF-D, which bind differently to VEGF receptors and regulate angiogenesis and lymphangiogenesis (14). Ranibizumab is an anti-VEGF agent already approved for age-related macular degeneration (15), diabetic retinopathy (16), and retinal vein occlusions (17, 18). The primary purpose of our study was to evaluate the effect of subconjunctival ranibizumab on corneal neovascularization. Additionally, we aimed to evaluate the potential effect of subconjunctival ranibizumab on VEGF levels in various ocular tissues. MATERIALS AND METHODS New Zealand albino rabbits were used in this experimental study. Twenty male 4-month-old animals weighing 2.83.5 kg were included in the protocol. The animals were housed under a 12 hour:12 hour light-dark cycle with free access to standard chow and water ad libitum. All rabbits were examined prior to the experimental study and confirmed to have no apparent corneal abnormality. All 300 procedures were performed with animals under general and topical anesthesia. General anesthesia was induced by intramuscular injection of 5 mg/kg ketamine hydrochloride (Ketalar®; Pfizer, Hellas) and 2 mg/kg xylazine hydrochloride (Rompun®; Bayer Schering Pharma AG, Leverkusen, Germany), as previously described (13). The eyes were additionally anesthetized topically with 0.5% proparacaine hydrochloride (Alcaine®; Alcon Hellas) before manipulation. Every procedure was conducted according to the ARVO statement for the use of animals in ophthalmic and vision research. Every procedure was conducted in accordance with the Declaration of Helsinki, to Council Directive 86/609/EEC of 24-11-1986 of the European Union, as well as Greek laws and regulations (Presidential Decree 160/1991, Act No 2015/2001) regarding the protection of animals used for experimental and other scientific purposes. The experimental protocol was approved by the Ethics Committee of Attikon University Hospital. No human material or human data were used in this study. Induction of corneal neovascularization The rabbit model of corneal neovascularization subsequent to corneal alkali injury has been utilized effectively in the past (13, 19). Therefore, an alkali cauterization model was generated for this study and checked for reproducibility. This is a model of corneal angiogenesis characterized by epithelial defect and stromal scarring. An alkali burn injury was induced in only 1 eye of 16 rabbits. Whatman filter papers (5 x 5mm) were soaked in 5% NaOH solution for 10 seconds and then applied on the surface of the upper half of the cornea for 60 seconds. The eyelids were held open for another 30 seconds after application and were then rinsed thoroughly with 20 mL of balanced salt solution (BSS). To increase the reproducibility of the alkali burn model, the whole process was carried out on all animals in the same manner by the same investigator. Only the one eye was cauterized so that the animals were not blinded. Group formation and follow-up Immediately after alkali cauterization, rabbits were randomly and equally divided in 2 groups. One hour after the chemical cauterization, 0.1 mL (1 mg) of ranibizumab (Lucentis®; Novartis, Hellas, Greece) was administered subconjunctivally in the cauterized eye of the one group (treated group) with a 30-G needle, 2 mm from the superior © 2013 Wichtig Editore - ISSN 1120-6721 Liarakos et al limbus. A sham injection of 0.1 mL of BSS was administered also with a 30-G needle, 2 mm from the superior limbus, in the cauterized eyes of the second group (untreated eyes). A third group of 4 rabbits was only injected subconjunctivally with 0.1 mL (1 mg) of ranibizumab in the one eye in order to evaluate possible side effects. A topical antibiotic (Tobramycin; Tobrex®, Alcon, Hellas) was administered every 4 hours for the first 24 hours to avoid bacterial infection. In case of infection or corneal perforation during follow-up, eyes had to be excluded from the study. All 20 animals were monitored daily. To minimize observer bias, all observations were performed by an investigator who was masked to the allocation of the animals in each group. Digital pictures (Sony 8.1 megapixels digital camera, Carl Zeiss lens, Oberkochen, Germany) were taken on days 1, 7, and 14. Image analysis was performed on corneal digital photographs using an image processing and analysis software program (IMAGEnet 2000® v2.5x for Windows). The area of corneal scarring and neovascularization was measured in pixels and expressed as the percentage of corneal surface, as described previously (13). In order to achieve a more detailed evaluation of the neovascularization, a qualitative evaluation of the density and diameter of the vessels was performed. The precise measurement of the diameter of the vessels has been suggested for human patients in the literature (20); however, it was difficult to apply in laboratory conditions. Laboratory investigation All animals were euthanased on day 14 and eyes were enucleated. First, the aqueous humor was aspirated immediately after enucleation with a 27-G needle inserted from the limbus parallel to the iris plane. Then, the vitreous humor was aspirated with a 16-G needle inserted 2 mm behind the limbus. Ocular tissues were surgically separated under a surgical microscope. The cornea was excised at the corneal limbus (including the limbus) with a curved cornea scissors. The iris was separated from the ciliary body manually with a forceps and a blade. A 7 x 7-mm piece of the superior bulbar conjunctiva was also cut with a Westcott tenotomy scissors and excised. All samples were placed in preweighed laboratory microcentrifuge tubes. Then, they were weighed with an electronic precision laboratory balance (Kertus, Hellas, Greece) and stored in a −80oC freezer. Cornea, iris, and conjunctiva samples were homogenized after adding phosphate buffered saline (PBS), and then centrifuged at 1000 g, at 4oC for 10 minutes. The supernatant was used to measure the concentration of VEGF. Aqueous and vitreous samples were also diluted with PBS. VEGF levels were determined by a commercially available ELISA kit (R&D Systems; Minneapolis, Minnesota, USA), as described before (21-23). Previous studies have shown that the antibody against human VEGF crossreacts with rabbit VEGF (24) and comparison of the nucleotide sequences of rabbit and human VEGF revealed a homology of 91%. The assay was performed according to the manufacturer’s instructions. The tissue samples’ VEGF concentrations were calculated from the standard curve, corrected for total protein, and expressed in picograms of protein per gram of tissue (pg/g). Statistical analysis Statistical analysis was performed using SPSS® v15.0 for Windows (SPSS Inc., Chicago, Illinois, USA). Nonparametric Mann-Whitney U test was used to compare corneal neovascularization, scarring, and VEGF levels between treated and untreated eyes. One-way analysis of variance (ANOVA) was used to evaluate the differences in corneal neovascularization and scarring over time as well as the differences in the VEGF levels of different tissues in the same eyes. Statistical significance was defined as a p<0.05. Values were expressed as mean ± SD. The standard error of the mean (SEM) regarding VEGF levels is mentioned to indicate 95% confidence interval (95% CI). RESULTS The effect of subconjunctival ranibizumab on corneal scarring and neovascularization Profound corneal scarring was evident in all cauterized eyes within 24 hours after the alkali cauterization. Corneal opacity extended slightly beyond the primary alkali injury and was similar between the untreated and the treated eyes (36.75 ± 4.98% and 34.88 ± 3.00%, respectively; p = 0.385, MannWhitney U test). Adjacent bulbar conjunctiva appeared hyperemic in both groups during the first 5 days following cauterization; however, these signs regressed during followup. During the 14-day follow-up, no significant change was observed in the area of corneal scarring in both groups © 2013 Wichtig Editore - ISSN 1120-6721 301 Subconjunctival ranibizumab for anterior segment neovascularization TABLE I - C ORNEAL SCAR AND NEOVASCULARIZATION IN UNTREATED EYES AND EYES TREATED WITH SUBCONJUNCTIVAL RANIBIZUMAB Corneal scar, % Corneal neovascularization, % Untreated, mean ± SD Treated, mean ± SD Mann-Whitney U, p value Untreated, mean ± SD Treated, mean ± SD Mann-Whitney U, p value Day 1 37.63 ± 5.24 34.88 ± 3.00 0.197 0 0 NA Day 7 42.63 ± 8.55 40.75 ± 7.34 0.632 9.25 ± 3.20 3.63 ± 3.02 0.006a Day 14 37.75 ± 6.14 36.38 ± 4.66 0.709 22.63 ± 4.03 6.13 ± 4.22 0.001a ANOVA p = 0.266 p = 0.095 p<0.001a p = 0.002a ANOVA = analysis of variance; NA = not applicable. The area of corneal neovascularization changed significantly over time in both groups (ANOVA) but it was significantly smaller in the treated eyes compared to the untreated eyes during the 14-day follow-up (Mann-Whitney U). The area with corneal scar did not change significantly over time (ANOVA) and was similar between the 2 groups (Mann-Whitney U). a Statistically significant difference. Fig. 1 - Evolution of corneal neovascularization and scar in eyes treated with subconjunctival ranibizumab vs untreated eyes. Corneal scar and neovascularization (NV) are expressed as a percentage of the corneal surface in eyes treated with subconjunctival ranibizumab (dark gray) and untreated eyes (light gray). Median values and 95% confidence intervals are shown. *The difference is statistically significant at the 0.01 level (Mann-Whitney U test). #The difference is not statistically significant (p>0.05; Mann-Whitney U test). (p = 0.404 and p = 0.095, respectively, one-way ANOVA), as shown in Table I and Figure 1. Mild peripheral neovascularization consisting of multiple, short (<2 mm long), fine and dense vessels emerging from the limbus appeared during the first week in all cauterized corneas in the untreated group (Fig. 2). Fourteen days after cauterization, there was clear evidence of neovascularization covering almost one quarter of the cornea, with major centripetal vessels and multiple smaller branch vessels (Fig. 2). Neovascular tissue progressed significantly from 8.38% ± 3.29% of the corneal surface on day 7 to 21.88% ± 5.03% on day 14 (p<0.001, one-way ANOVA), as shown in Table I. Very restricted scattered areas of faint neovascularization were observed in eyes treated with ranibizumab 302 7 days after cauterization (Fig. 2). Although neovascularization progressed during the second week (p = 0.002; one-way ANOVA), neovascular area did not exceed 10% of the cornea in most of the treated eyes (Fig. 2). The area of neovascularization was significantly smaller in the corneas treated with subconjunctival ranibizumab, compared to untreated corneas, on both days 7 and 14 (p = 0.01 and p<0.001, respectively; Mann-Whitney U test), as shown in Table I and Figure 1. No recurrence or significant progression of corneal neovascularization was recorded after day 10. No significant inflammation, corneal melt, or other major complication that would be a reason for exclusion were noted during follow-up. © 2013 Wichtig Editore - ISSN 1120-6721 Liarakos et al Control corneas in the third group presented neither scarring nor neovascularization. Moreover, no apparent clinical ophthalmic or systemic side effects were noted. The effect of subconjunctival ranibizumab on VEGF levels in different ocular tissues Fig. 2 - Pictures of corneal neovascularization in untreated eyes and after subconjunctival ranibizumab. Corneal scar and neovascularization in untreated control eyes 7 days (A) and 14 days (B) after alkali burn injury. Mild peripheral neovascularization appeared by the end of the first week (A) and progressed significantly during the second week, covering almost one-quarter of the cornea with major centripetal and multiple smaller branch vessels (B). Corneal scar and neovascularization in eyes treated with subconjunctival ranibizumab 7 days (C) and 14 days (D) after alkali burn injury followed by a single injection of 1 mg of ranibizumab. Only restricted scattered areas of faint neovascularization were observed 7 days after cauterization (C). Neovascular area did not exceed 10% of the cornea in most of the treated eyes 14 days after chemical injury (D). All tissue samples were excised from the enucleated globes as described above and were weighed before preparing them for the ELISA. Differences between treated and untreated eyes regarding the average weight of the tissue samples were not statistically significant (p>0.1, Mann-Whitney U test), as shown in Table II. The VEGF levels (per unit of tissue mass) were found to be lower in the vitreous humor and, by ascending order of concentration, were higher in the aqueous humor, even higher in the cornea, and much higher in the iris and the conjunctiva (Tab. II). Corneal VEGF levels were found to be significantly lower in treated eyes compared to untreated ones by more than 45% (p = 0.009; Mann-Whitney U test). The VEGF levels were measured to be lower in the conjunctiva, the iris, and the aqueous humor of treated eyes compared to untreated ones by 44%-71% (p = 0.001; Mann-Whitney U test), as shown in Table II and Figure 3. Although vitreous VEGF levels were also lower in the treated group, the difference was not statistically significant (p = 0.093). TABLE II - VEGF LEVELS AND TISSUE WEIGHT IN UNTREATED EYES AND EYES TREATED WITH SUBCONJUNCTIVAL RANIBIZUMAB Tissue weight VEGF Untreated, mg Treated, mg MannWhitney U, p value Untreated, pg/g, mean ± SEM Treated, pg/g, mean ± SEM Difference, % MannWhitney U, p value Cornea 80.92 ± 3.16 71.36 ± 12.41 0.150 2365.46 ± 213.32 1166.88 ± 150.09 -50.7 0.005a Iris 46.90 ± 9.08 34.29 ± 7.24 0.078 23186.80 ± 2064.31 6343.35 ± 251.33 -72.6 0.001a Conjunctiva 107.05 ± 76.48 126.38 ± 54.23 1.000 28411.14 ± 1517.47 16432.25 ± 929.24 -42.2 0.001a Aqueous humor 217.52 ± 33.74 195.54 ± 44.28 0.873 424.73 ± 31.83 211.02 ± 25.46 -50.2 0.001a 1264.86 ± 92.95 1542.27 ± 382.58 0.140 112.75 ± 4.69 91.89 ± 8.19 -18.8 0.059 p<0.001a p<0.001a Vitreous ANOVA ANOVA = analysis of variance; VEGF = vascular endothelial growth factor. The VEGF levels were found to be significantly lower in the eyes treated with subconjunctival ranibizumab in almost all ocular tissues. No significant differences were noted between the 2 groups regarding the weight of the tissue samples. a Statistically significant difference. © 2013 Wichtig Editore - ISSN 1120-6721 303 Subconjunctival ranibizumab for anterior segment neovascularization are not aware of any other experimental or clinical study addressing the effect of subconjunctival ranibizumab on iris neovascularization. Advantages of early subconjunctival administration Fig. 3 - Significant decrease of vascular endothelial growth factor (VEGF) levels in all anterior segment tissues after subconjunctival administration of ranibizumab. The VEGF levels in different tissue samples of eyes treated with subconjunctival ranibizumab (dark gray) and untreated eyes (light gray). Mean values and 95% confidence intervals are shown. *The difference is statistically significant at the 0.01 level (Mann-Whitney U test). #The difference is not statistically significant (p>0.05; Mann-Whitney U test). DISCUSSION Ranibizumab is an anti-VEGF agent already approved for ophthalmic use. Our initial results regarding subconjunctival administration of ranibizumab in the early posttraumatic period were encouraging (Liarakos VS, et al. IOVS 2008; 49: ARVO E-Abstract 3743) and triggered further experimental research. Recently, the effectiveness of subconjunctival ranibizumab for corneal neovascularization was well-documented by an experimental study on rats (25). In our study, we used a dose twice as high as in the study of Sener et al (25) and extended the follow-up to 15 days instead of 8, because, according to Dastjerdi et al (26), the concentration of the anti-VEGF agent in the stroma begins to fade after that time. During this period, ranibizumab proved to be well-tolerated when administered subconjunctivally even at a dose twice as high as the intravitreal dose and twice as high as used by Sener et al (25). Moreover, we tried to provide additional evidence based on the effect of ranibizumab on the actual levels of VEGF in the cornea as well as the iris and other ocular tissues. We 304 We chose to administer subconjunctival ranibizumab in the early posttraumatic period, soon after cauterization, because we have already documented the favorable effect of early administration of anti-VEGF treatment in a corneal neovascularization model (13). Early administration of antiVEGF treatment achieves a desirable concentration in the early phases of corneal wound healing, as described by Azar (7). This is a period when keratocytes proliferate and transform to fibroblasts and then migrate to populate the wound. This procedure is accompanied by the production of high levels of epithelial and stromal growth factors (tumor growth factor [TGF]–α, TGF-β, basic fibroblast growth factor, platelet-derived growth factor) as well as cytokines (tumor necrosis factor–α, interleukin [IL]–1) (3) and apparently also VEGF (27). We chose subconjunctival administration instead of eyedrops because it has been shown that subconjunctival bevacizumab may achieve favorable results when compared to eyedrops (28). Although bevacizumab eyedrops are known to penetrate the cornea and enter the anterior chamber (29), prolonged topical application may cause epitheliopathy and even corneal thinning (30). In a recent experimental study, it has been shown that subconjunctival administration of bevacizumab is superior to topical application in the form of drops with regard to stromal penetration, especially in intact corneas (26). Targeting VEGF to treat corneal neovascularization Topical steroids remain a first-choice treatment for corneal neovascularization. However, steroids are not always ideally indicated, as they can either delay corneal wound healing, both in humans (31) and in experimental animal models (32), or trigger the onset or recurrence of herpetic or fungal inflammation (33). It is also known that chronic treatment may lead to glaucoma and cataract formation. In terms of the pathophysiology of corneal neovascularization, inflammation is not necessarily the only reason. Corneal hypoxia, limbal stem cell deficiency, persistent or recurrent epithelial defects, and keratocyte activation should also be considered (7, 34). Moreover, polymorphonuclear chemoattractants © 2013 Wichtig Editore - ISSN 1120-6721 Liarakos et al IL-8 and cytokines like IL-6 and IL-1β are upregulated by closed-eye conditions or contact lens wear (35). The VEGF expression is localized to corneal epithelial cells, corneal endothelial cells, vascular endothelial cells of limbal vessels and of newly formed stromal vessels, and weakly by keratocytes (36). In addition, VEGF expression has markedly increased in epithelial cells of inflamed corneas, vascular endothelial cells, macrophage infiltrates, and fibroblasts in corneal scar tissue (7). The VEGF concentrations are significantly higher in vascularized corneas than in normal control corneas (27, 36). Additionally, VEGF promotes several steps of angiogenesis, including proteolytic activities (dissolution of the membrane of the original vessel), vascular endothelial cell proliferation, migration, and capillary tube formation. Subsequently, adjuvant or alternative treatments for corneal neovascularization also targeting VEGF should be evaluated. Pharmacokinetics of ranibizumab Although both ranibizumab and bevacizumab neutralize all isoforms of VEGF-A, thus having a similar clinical effect at least on choroidal neovascularization (Comparison of AMD Treatments Trials study [37]), their small structural differences do not ensure identical behavior in all ocular tissues. In vitro studies showed that the binding affinity of ranibizumab is much more specific than bevacizumab, but bevacizumab has a longer half-life than ranibizumab (38). When administered intravitreally, ranibizumab’s concentration is described to decline in a monoexponential fashion with a similar half-life in both the vitreous and the aqueous humor (2.88 and 2.84 days, respectively) (38). Subsequently, pharmacokinetics may differ not only in the retina (39) but also in other tissues. In our study, VEGF levels were significantly lower in the treated eyes 14 days after a single subconjunctival ranibizumab injection. A time-dependent diffusion of subconjunctival bevacizumab in the rabbit cornea has been described (19). Although diffusion extends during the first days after administration, it declines significantly after the first week. However, its clinical effect may last longer (13). Possible effect of subconjunctival ranibizumab on iris neovascularization The VEGF levels in the cornea of untreated cauterized eyes were almost 5 times higher than VEGF levels in the aqueous humor and almost 20 times higher than VEGF levels in the vitreous of the same eyes. On the contrary, VEGF levels in the cornea of the same eyes were 10-14 times lower than VEGF levels in highly vasculated tissues, like the iris and the conjunctiva. This shows that, in general, VEGF levels are related to the vasculature of the ocular tissues, either normally preexisting or newly established. Based on the significant reduction of VEGF levels in the various ocular tissue samples of the treated cauterized eyes, it seems that ranibizumab not only diffuses into the conjunctiva and the adjacent cornea. It probably penetrates the ocular surface and enters into the anterior chamber, where it reduces VEGF levels in the iris and the aqueous humor significantly. Ranibizumab could have been distributed into the ocular tissues via the systemic circulation and reached the anterior chamber through the ciliary vessels or through direct diffusion into the tissues. A similar pattern of systemic distribution has been described for subconjunctival bevacizumab (40). However, this has not been confirmed for ranibizumab. The VEGF levels were reduced in the iris of treated eyes by more than 70%. Based on the reduction of VEGF levels, the effect on the iris was larger than the effect on the conjunctiva, which happened to be the primary site of ranibizumab injection. This indicates that subconjunctival ranibizumab may also be a therapeutic alternative for secondary iris neovascularization. So far, mostly intravitreal injections of bevacizumab have been suggested for treating iris neovascularization secondary to diabetic retinopathy (41) or other ischemic retinal disorders (42). Intravitreal ranibizumab may also inhibit iris neovascularization secondary to central retinal vein occlusion (43). However, subconjunctival administration is safer, more convenient for the patient, and easier for the doctor to perform. Although the minimum therapeutic concentration of ranibizumab within the cornea is not known, our study demonstrated that ranibizumab was clinically effective and reduced VEGF levels significantly in almost all ocular tissues, at least in the first 14 days after administration. Its potency in animal models should be further evaluated with regard to the dosage regimen before interpretation of the experimental results. Clinical application We have already depicted the effect of subconjunctival bevacizumab on experimental corneal neovascularization © 2013 Wichtig Editore - ISSN 1120-6721 305 Subconjunctival ranibizumab for anterior segment neovascularization and we have documented the supremacy of early administration (13). Other experimental studies agree with these results (12, 19, 21). Experimental results encouraged clinical application with several reports of subconjunctival bevacizumab for corneal neovascularization associated with various corneal or ocular surface pathologies (4446). It is shown that a single subconjunctival injection of bevacizumab in its usual concentration of 25 mg/mL may achieve better results than topical instillation in the form of eyedrops at least in the first week of treatment (28). In a clinical study, it is shown that subconjunctival bevacizumab repeated every 4-7 days is superior to intense topical administration of bevacizumab eyedrops (47). There are no such reports with ranibizumab. The frequency of administration also could be related to the severity of the corneal neovascularization as well as its etiology. Further clinical studies are needed to determine this. In an ongoing clinical study regarding administration of a single subconjunctival injection of 0.5 mg ranibizumab in primary pterygium preoperatively or intraoperatively, ranibizumab is well-tolerated and does not affect the survival of the conjunctival autograft (48). However, the possible effect of ranibizumab on VEGF levels or the vessels themselves was not evaluated. Prompt regression of conjunctival microvessels was also reported in a single patient with pterygium (49). Subconjunctival ranibizumab was well-tolerated by 5 patients who were injected 3 days to 2 months prior to pterygium excision (50). CONCLUSIONS REFERENCES 6. 1. 2. 3. 4. 5. 306 Chang JH, Gabison EE, Kato T, Azar DT. Corneal neovascularization. Curr Opin Ophthalmol 2001;12:242-9. Garcia DD, Shtein RM, Musch DC, Elner VM. Herpes simplex virus keratitis: histopathologic neovascularization and corneal allograft failure. Cornea 2009;28:963-5. Cursiefen C, Cao J, Chen L, et al. Inhibition of hemangiogenesis and lymphangiogenesis after normal-risk corneal transplantation by neutralizing VEGF promotes graft survival. Invest Ophthalmol Vis Sci 2004;45:2666-73. Cursiefen C, Küchle M, Naumann GO. Angiogenesis in corneal diseases: histopathologic evaluation of 254 human corneal buttons with neovascularization. Cornea 1998;17:611-3. Dana MR, Schaumberg DA, Kowal VO, et al. Corneal neovascularization after penetrating keratoplasty. Cornea 1995;14:604-9. This experimental and laboratory study indicates that early subconjunctival administration of ranibizumab may successfully inhibit alkali-induced corneal neovascularization in an animal model. Subconjunctival ranibizumab reduces VEGF levels significantly not only in the cornea and the bulbar conjunctiva but also in the aqueous humor and the iris, suggesting a possible treatment for secondary neovascularization of the anterior segment of the eye. ACKNOWLEDGEMENT Dr. Liarakos received the 2011 Annual Scholarship from the Hellenic Society of Intraocular Implants and Refractive Surgery. Financial Support: No financial support was received for this submission. Conflict of Interest Statement: None of the authors has conflict of interest with this submission. Meeting Presentation: Primary results were presented at the 2008 ARVO meeting (Liarakos VS, et al. IOVS 2008; 49: ARVO E-Abstract 3743). Address for correspondence: Vasilios S. Liarakos Adrianoupoleos 5 Papagou 15669 Athens Greece [email protected] Cursiefen C, Colin J, Dana R, et al. Consensus statement on indications for anti-angiogenic therapy in the management of corneal diseases associated with neovascularisation: outcome of an expert roundtable. Br J Ophthalmol 2012;96:3-9. 7. Azar DT. Corneal angiogenic privilege: angiogenic and antiangiogenic factors in corneal avascularity, vasculogenesis, and wound healing (an American Ophthalmological Society thesis). Trans Am Ophthalmol Soc 2006;104:264-302. 8. Lee P, Wang CC, Adamis AP. Ocular neovascularization: an epidemiologic review. Surv Ophthalmol 1998;43:245-69. 9. Amano S, Rohan R, Kuroki M, Tolentino M, Adamis AP. Requirement for vascular endothelial growth factor in woundand inflammation-related corneal neovascularization. Invest Ophthalmol Vis Sci 1998;39:18-22. 10. Kvanta A, Sarman S, Fagerholm P, Seregard S, Steen B. Expression of matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) in inflammation-associated corneal neovascularization. Exp Eye Res 2000;70:419-28. © 2013 Wichtig Editore - ISSN 1120-6721 Liarakos et al 11. Mastyugin V, Mosaed S, Bonazzi A, Dunn MW, Schwartzman ML. Corneal epithelial VEGF and cytochrome P450 4B1 expression in a rabbit model of closed eye contact lens wear. Curr Eye Res 2001;23:1-10. 12. Kim TI, Kim SW, Kim S, Kim T, Kim EK. Inhibition of experimental corneal neovascularization by using subconjunctival injection of bevacizumab (Avastin). Cornea 2008;27: 349-52. 13. Papathanassiou M, Theodossiadis PG, Liarakos VS, Rouvas A, Giamarellos-Bourboulis EJ, Vergados IA. Inhibition of corneal neovascularization by subconjunctival bevacizumab in an animal model. Am J Ophthalmol 2008;145:424-31. 14. Li X, Eriksson U. Novel VEGF family members: VEGF-B, VEGF-C and VEGF-D. Int J Biochem Cell Biol 2001;33:421-6. 15. Rosenfeld PJ, Brown DM, Heier JS, et al; MARINA Study Group. Ranibizumab for neovascular age-related macular degeneration. N Engl J Med 2006;355:1419-31. 16. Mitchell P, Bandello F, Schmidt-Erfurth U, et al; RESTORE study group. The RESTORE study: ranibizumab monotherapy or combined with laser versus laser monotherapy for diabetic macular edema. Ophthalmology 2011;118:615-25. 17. Campochiaro PA, Brown DM, Awh CC, et al. Sustained benefits from ranibizumab for macular edema following central retinal vein occlusion: twelve-month outcomes of a phase III study. Ophthalmology 2011;118:2041-9. 18. Brown DM, Campochiaro PA, Bhisitkul RB, et al. Sustained benefits from ranibizumab for macular edema following branch retinal vein occlusion: 12-month outcomes of a phase III study. Ophthalmology 2011;118:1594-602. 19. Chen WL, Lin CT, Lin NT, et al. Subconjunctival injection of bevacizumab (Avastin) on corneal neovascularization in different rabbit models of corneal angiogenesis. Invest Ophthalmol Vis Sci 2009;50:1659-65. 20. Dastjerdi MH, Al-Arfaj KM, Nallasamy N, et al. Topical bevacizumab in the treatment of corneal neovascularization: results of a prospective, open-label, noncomparative study. Arch Ophthalmol 2009;127:381-9. 21. Kim WJ, Jeong HO, Chung SK. The effect of bevacizumab on corneal neovascularization in rabbits. Korean J Ophthalmol 2010;24:230-6. 22. Li Z, Van Bergen T, Van de Veire S, et al. Inhibition of vascular endothelial growth factor reduces scar formation after glaucoma filtration surgery. Invest Ophthalmol Vis Sci 2009;50:5217-25. 23. Ambati BK, Anand A, Joussen AM, Kuziel WA, Adamis AP, Ambati J. Sustained inhibition of corneal neovascularization by genetic ablation of CCR5. Invest Ophthalmol Vis Sci 2003;44:590-3. 24. Kinnunen K, Korpisalo P, Rissanen TT, et al. Overexpression of VEGF-A induces neovascularization and increased vascular leakage in rabbit eye after intravitreal adenoviral gene transfer. Acta Physiol 2006;187:447-57. 25. Sener E, Yuksel N, Yildiz DK, et al. The impact of subconjuctivally injected EGF and VEGF inhibitors on experimental 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. corneal neovascularization in rat model. Curr Eye Res 2011;36:1005-13. Dastjerdi MH, Sadrai Z, Saban DR, Zhang Q, Dana R. Corneal penetration of topical and subconjunctival bevacizumab. Invest Ophthalmol Vis Sci 2011;52:8718-23. Zheng M, Deshpande S, Lee S, Ferrara N, Rouse BT. Contribution of vascular endothelial growth factor in the neovascularization process during the pathogenesis of herpetic stromal keratitis. J Virol 2001;75:9828-35. Hashemian MN, Z-Mehrjardi H, Moghimi S, Tahvildari M, Mojazi-Amiri H. Prevention of corneal neovascularization: comparison of different doses of subconjunctival bevacizumab with its topical form in experimental rats. Ophthalmic Res 2011;46:50-4. Yoeruek E, Ziemssen F, Henke-Fahle S, et al. Safety, penetration and efficacy of topically applied bevacizumab: evaluation of eyedrops in corneal neovascularization after chemical burn. Acta Ophthalmol 2008;86:322-8. Kim SW, Ha BJ, Kim EK, Tchah H, Kim TI. The effect of topical bevacizumab on corneal neovascularization. Ophthalmology 2008;115:e33-8. Borderie VM, Touzeau O, Bourcier T, Allouch C, Laroche L. Graft reepithelialization after penetrating keratoplasty using organ-cultured donor tissue. Ophthalmology 2006;113: 2181-6. Sarchahi AA, Maimandi A, Tafti AK, Amani M. Effects of acetylcysteine and dexamethasone on experimental corneal wounds in rabbits. Ophthalmic Res 2008;40:41-8. Wilhelmus KR, Dawson CR, Barron BA, et al; Herpetic Eye Disease Study Group. Risk factors for herpes simplex virus epithelial keratitis recurring during treatment of stromal keratitis or iridocyclitis. Br J Ophthalmol 1996;80:969-72. Ellenberg D, Azar DT, Hallak JA, et al. Novel aspects of corneal angiogenic and lymphangiogenic privilege. Prog Retin Eye Res 2010;29:208-48. Thakur A, Willcox MD. Contact lens wear alters the production of certain inflammatory mediators in tears. Exp Eye Res 2000;70:255-9. Philipp W, Speicher L, Humpel C. Expression of vascular endothelial growth factor and its receptors in inflamed and vascularized human corneas. Invest Ophthalmol Vis Sci 2000;41:2514-22. Martin DF, Maguire MG, Ying GS, Grunwald JE, Fine SL, Jaffe GJ; CATT Research Group. Ranibizumab and bevacizumab for neovascular age-related macular degeneration. N Engl J Med 2011;364:1897-908. Bakri SJ, Snyder MR, Reid JM, Pulido JS, Ezzat MK, Singh RJ. Pharmacokinetics of intravitreal ranibizumab (Lucentis). Ophthalmology 2007;114:2179-82. Veurink M, Stella C, Tabatabay C, Pournaras CJ, Gurny R. Association of ranibizumab (Lucentis®) or bevacizumab (Avastin®) with dexamethasone and triamcinolone acetonide: an in vitro stability assessment. Eur J Pharm Biopharm 2011;78:271-7. © 2013 Wichtig Editore - ISSN 1120-6721 307 Subconjunctival ranibizumab for anterior segment neovascularization 40. Nomoto H, Shiraga F, Kuno N, et al. Pharmacokinetics of bevacizumab after topical, subconjunctival, and intravitreal administration in rabbits. Invest Ophthalmol Vis Sci 2009;50:4807-13. 41. Oshima Y, Sakaguchi H, Gomi F, Tano Y. Regression of iris neovascularization after intravitreal injection of bevacizumab in patients with proliferative diabetic retinopathy. Am J Ophthalmol 2006;142:155-8. 42. Wakabayashi T, Oshima Y, Sakaguchi H et al. Intravitreal bevacizumab to treat iris neovascularization and neovascular glaucoma secondary to ischemic retinal diseases in 41 consecutive cases. Ophthalmology 2008;115:1571-80. 43. Risard SM, Pieramici DJ, Rabena MD, et al. Intravitreal ranibizumab for macular edema secondary to central retinal vein occlusion. Retina 2011;31:1060-7. 44. Bahar I, Kaiserman I, McAllum P, Rootman D, Slomovic A. Subconjunctival bevacizumab injection for corneal neovascularization. Cornea 2008;27:142-7. 45. Georgakopoulos CD, Vasilakis P, Makri OE, Beredima E, Pharmakakis N. Subconjunctival bevacizumab for corneal 308 46. 47. 48. 49. 50. neovascularization secondary to topical anesthetic abuse. Cutan Ocul Toxicol 2011;30:320-2. Yeung SN, Lichtinger A, Kim P, Amiran MD, Slomovic AR. Combined use of subconjunctival and intracorneal bevacizumab injection for corneal neovascularization. Cornea 2011;30:1110-4. Dastjerdi MH, Saban DR, Okanobo A, et al. Effects of topical and subconjunctival bevacizumab in high-risk corneal transplant survival. Invest Ophthalmol Vis Sci 2010;51: 2411-7. Galor A, Yoo SH, Piccoli FV, Schmitt AJ, Chang V, Perez VL. Phase I study of subconjunctival ranibizumab in patients with primary pterygium undergoing pterygium surgery. Am J Ophthalmol 2010;149:926-31. Mansour AM. Treatment of inflamed pterygia or residual pterygial bed. Br J Ophthalmol 2009;93:864-5. Mandalos A, Tsakpinis D, Karayannopoulou G, et al. The effect of subconjunctival ranibizumab on primary pterygium: a pilot study. Cornea 2010;29:1373-9. © 2013 Wichtig Editore - ISSN 1120-6721