this study was approved by the institutional animal care and use committee of korea , prior to experiments , and all the in vivo experiment procedures were performed according to regulation of association for research in vision and ophthalmology for the ophthalmic field and studies on visual function . twelve new zealand white rabbits ( samtako , osan , korea ) , weighing between 2.0 kg and 2.5 kg were used regardless of their sex ; all rabbits were examined prior to surgery and confirmed to have normal cornea . systemic anesthesia was induced by the intramuscular injection of the mixture of tilemine and zolazepam , zoletil ( vibrac , carros , france ) , at a 0.2 mg / kg dose , and topical anesthesia was induced by proparacaine eye drop ( alcaine ; alcon , fort worth , tx , usa ) . in 12 house rabbits ( 24 eyes ) , a corneal suture 3 mm in length passing through the corneal stroma area was performed using 7 - 0 black silk ( sofsilk ; syneture , quebec , canada ) , at the 12 o'clock direction distanced from the corneal limbus by 1 mm . after suturing , to prevent infection , ofloxacin eye drops ( ocuflox ; samil , seoul , korea ) were administered four times per day for seven days . one week later , the suture was removed after confirming the sufficient formation of corneal neovascularization . to prevent error caused by the result of systemic absorption , saline was administered to the left eye of all 12 animals without special treatments and used as the control group ( 12 eyes ) . among 12 right eyes , in four eyes cases , a 5 mg / ml bevacizumab eye drop was administered twice a day for two weeks , and in the other four eyes cases , a 10 mg / ml bevacizumab eye drop was administered twice a day for two weeks . the remaining four eyes were treated with the subconjunctival injection of 1.25 mg ( 0.01 ml ) bevacizumab once , and afterward , no other treatments were administered . the picture of the cornea of each experiment group was taken one week and two weeks after treatment with a camera ( contax d-7 , stutgart , germany ) attached to a microscope ( s21 ; carl zeiss , jena , germany ) at 25 times magnification , and the neovascularization area was measured using axiovision ac software ( carl zeiss ) . considering the area prior to treatment as one , the relative reduction level was calculated and analyzed . two weeks after treatment , both eyes of 12 animals were extracted and the neovascularization area was cut into halves . the area with neovascularization was prepared as sections , and a histological test was performed . of corneal sections obtained from each eye , one half was fixed in 10% neutral formalin , and after a dehydration process , embedded in paraffin . sections were then prepared , stained with hematoxylin & eosin , and examined under a biomicroscope ( bx-50 ; olympus , tokyo , japan ) . from the remaining corneal sections , the area with neovasculatures was measured accurately , and then immediately stored in a -80 freezer . for these tissues , 1 mm phenylmethylsulfonylfloride was added to phosphate buffered saline , and then homogenized as 200 l / g volume . afterward , the samples were centrifuged at 1,000 g , at 4 for ten minutes , and only the supernatant was used . the concentration of vegf in tissues was measured by luminometer using the human vegf immunoassay kit ( r&d system , minneapolis , mn , usa ) . the statistical analysis on the change of vascularization area and vegf concentration was performed by mann - whitney u - test and a p - value less than 0.05 was considered to be significant . this study was approved by the institutional animal care and use committee of korea , prior to experiments , and all the in vivo experiment procedures were performed according to regulation of association for research in vision and ophthalmology for the ophthalmic field and studies on visual function . twelve new zealand white rabbits ( samtako , osan , korea ) , weighing between 2.0 kg and 2.5 kg were used regardless of their sex ; all rabbits were examined prior to surgery and confirmed to have normal cornea . systemic anesthesia was induced by the intramuscular injection of the mixture of tilemine and zolazepam , zoletil ( vibrac , carros , france ) , at a 0.2 mg / kg dose , and topical anesthesia was induced by proparacaine eye drop ( alcaine ; alcon , fort worth , tx , usa ) . in 12 house rabbits ( 24 eyes ) , a corneal suture 3 mm in length passing through the corneal stroma area was performed using 7 - 0 black silk ( sofsilk ; syneture , quebec , canada ) , at the 12 o'clock direction distanced from the corneal limbus by 1 mm . after suturing , to prevent infection , ofloxacin eye drops ( ocuflox ; samil , seoul , korea ) were administered four times per day for seven days . one week later , the suture was removed after confirming the sufficient formation of corneal neovascularization . to prevent error caused by the result of systemic absorption , saline was administered to the left eye of all 12 animals without special treatments and used as the control group ( 12 eyes ) . among 12 right eyes , in four eyes cases , a 5 mg / ml bevacizumab eye drop was administered twice a day for two weeks , and in the other four eyes cases , a 10 mg / ml bevacizumab eye drop was administered twice a day for two weeks . the remaining four eyes were treated with the subconjunctival injection of 1.25 mg ( 0.01 ml ) bevacizumab once , and afterward , no other treatments were administered . the picture of the cornea of each experiment group was taken one week and two weeks after treatment with a camera ( contax d-7 , stutgart , germany ) attached to a microscope ( s21 ; carl zeiss , jena , germany ) at 25 times magnification , and the neovascularization area was measured using axiovision ac software ( carl zeiss ) . considering the area prior to treatment as one , the relative reduction level was calculated and analyzed . two weeks after treatment , both eyes of 12 animals were extracted and the neovascularization area was cut into halves . the area with neovascularization was prepared as sections , and a histological test was performed . of corneal sections obtained from each eye , one half was fixed in 10% neutral formalin , and after a dehydration process , embedded in paraffin . sections were then prepared , stained with hematoxylin & eosin , and examined under a biomicroscope ( bx-50 ; olympus , tokyo , japan ) . from the remaining corneal sections , the area with neovasculatures was measured accurately , and then immediately stored in a -80 freezer . for these tissues , 1 mm phenylmethylsulfonylfloride was added to phosphate buffered saline , and then homogenized as 200 l / g volume . afterward , the samples were centrifuged at 1,000 g , at 4 for ten minutes , and only the supernatant was used . the concentration of vegf in tissues was measured by luminometer using the human vegf immunoassay kit ( r&d system , minneapolis , mn , usa ) . the statistical analysis on the change of vascularization area and vegf concentration was performed by mann - whitney u - test and a p - value less than 0.05 was considered to be significant . in all rabbits , sufficient neovasculature was formed on day seven after corneal suture , while infection , as well as other specific findings , were not detected . images of the corneal neovascularization area enlarged 25 times were taken using a microscope , and the images prior to surgery , the first week , and the second week were compared and analyzed ( fig . 1 ) . the area of neovascularization of each group was analyzed and compared , and the result revealed that the groups administered 5 mg / ml bevacizumab , 10 mg / ml bevacizumab , and subconjunctival injection of 1.25 mg bevacizumab showed greater reduction of neovascularization area than the control group ( fig . 2 ) . comparing the difference between the groups administered 5 mg / ml bevacizumab eye drop and 10 mg / ml eye drop , the p - value ( mann - whitney u - test ) of week 1 was 0.248 , of week 2 was 0.083 ; a significant difference was not shown . comparing the groups administered bevacizumab eye drop and treated by subconjunctival injection , a significant difference was not detected at the first week . however , the group treated with 10 mg / ml bevacizumab eye drop showed a more significant reduction of neovascularization area than the group treated by subconjunctival injection at the second week ( fig . 2 ) . treatment revealed that a greater neovascularization reduction within the corneal stroma than the control group was shown in the three treated groups . however , among the treated groups , a great difference in the number and area of neovasculatures was difficult to detect . over the study period , significant injury in the corneal epithelium , stroma , and endothelium , that is , superficial punctate erosion , change of corneal thickness , conjunctival injection or any other type of corneal complication were not observed in all eyes ( fig . the concentration of vegf in each corneal section was compared , and the result showed that the control group contained 1,284.33223.01 pg / ml , while the groups treated with 5 mg / ml bevacizumab eye drop , 10 mg / ml eye drop , and subconjunctival injection were shown to be 998.33130.93 pg / ml , 942.0032.86 pg / ml , and 968.2483.34 pg / ml , respectively . the treated group showed a reduction of significant concentrations in comparison with the control group ( p<0.05 ) , but a significant difference of concentrations was not detected among the three treated groups ( p>0.05 ) ( fig . in all rabbits , sufficient neovasculature was formed on day seven after corneal suture , while infection , as well as other specific findings , were not detected . images of the corneal neovascularization area enlarged 25 times were taken using a microscope , and the images prior to surgery , the first week , and the second week were compared and analyzed ( fig . 1 ) . the area of neovascularization of each group was analyzed and compared , and the result revealed that the groups administered 5 mg / ml bevacizumab , 10 mg / ml bevacizumab , and subconjunctival injection of 1.25 mg bevacizumab showed greater reduction of neovascularization area than the control group ( fig . 2 ) . comparing the difference between the groups administered 5 mg / ml bevacizumab eye drop and 10 mg / ml eye drop , the p - value ( mann - whitney u - test ) of week 1 was 0.248 , of week 2 was 0.083 ; a significant difference was not shown . comparing the groups administered bevacizumab eye drop and treated by subconjunctival injection , a significant difference was not detected at the first week . however , the group treated with 10 mg / ml bevacizumab eye drop showed a more significant reduction of neovascularization area than the group treated by subconjunctival injection at the second week ( fig . treatment revealed that a greater neovascularization reduction within the corneal stroma than the control group was shown in the three treated groups . however , among the treated groups , a great difference in the number and area of neovasculatures was difficult to detect . over the study period , significant injury in the corneal epithelium , stroma , and endothelium , that is , superficial punctate erosion , change of corneal thickness , conjunctival injection or any other type of corneal complication were not observed in all eyes ( fig . the concentration of vegf in each corneal section was compared , and the result showed that the control group contained 1,284.33223.01 pg / ml , while the groups treated with 5 mg / ml bevacizumab eye drop , 10 mg / ml eye drop , and subconjunctival injection were shown to be 998.33130.93 pg / ml , 942.0032.86 pg / ml , and 968.2483.34 pg / ml , respectively . the treated group showed a reduction of significant concentrations in comparison with the control group ( p<0.05 ) , but a significant difference of concentrations was not detected among the three treated groups ( p>0.05 ) ( fig . to maintain the transparency of the cornea , it is very important to maintain avascularity , and for this , the appropriate homeostasis of vascular inhibitor factors and vascular growth factors should be maintained . when the balance of angiogenic factors such as fibroblast growth factor ( fgf ) and vegf , and angiogenic suppressors such as angiostatin , endostatin , and pigment epithelium derived factor ( pedf ) are disrupted by diseases , neovascularization develops . for the treatment of corneal neovasculature , various drug therapies , laser photocoagulation , and surgical therapy previously , drug therapies including , steroid , non - steroid anti - inflammatory agents , cyclosporin a , thalidomide , prolactine , methotrexate , and angiostatin , have been reported . although steroid eye drop therapy has been used most widely for the treatment and prevention of corneal neovascularization presently , it may cause glaucoma , cataract , infection , and other complications . hence , its long term use can pose complications . studies attempted to treat corneal neovasculature by the application of angiogenesis suppressor factors such as angiostatin or pedf , have been conducted , however , they eventually failed . corneal laser photocoagulation using an argon laser may accompany recurrence of neovascularization , heat injury of adjacent tissues , and the consequent increase of inflammation . as surgical therapy , limbal transplantation   or amniotic membrane transplantation   has been attempted , however , positive effects were not reported . recently , photodynamic treatment using verteporfin also has been used for the treatment of corneal neovascularization . its short term effect has been reported to be very positive , but its long term effect has not been proven . in addition , it has to be injected intravenously and repeated treatments are required , therefore its cost may become extremely high . because of such diverse problems , verteporfin has been rarely used in clinics to date . vegf has been reported to play a very important role in numerous ophthalmic diseases accompanying neovascularization . vegf stimulates and accelerates the various processes of neovascularization ( protein degradation , proliferation , migration of endothelial cells , and formation of capillary blood vessels ) . in addition , in recent studies , vegf - a has shown involvement not only in neovascularization , but also lymphangiogenesis . vegf is not only involved in the regulation of neovascularization , it also has been proven in animal studies that if vegf were suppressed at the level of mrna or protein , corneal neovascularization was also decreased . bevacizumab is a recombinant monoclonal antibody and it inhibits the binding of vegf - a to its receptor by binding to vegf - a . recently , in the treatment of choroidal neovascularization associated with age - related macular degeneration , positive results of the injection treatment of bevacizumab into the vitreous body have been reported [ 16 - 18 ] . moreover , the injection of bevacizumab within the vitreous body has been reported to be very effective on the degeneration of the neovasculature in the iris or the iridial corneal margin area . the safety of the drug has been reported to be excellent , and its systemic use has been reported to increase hypertension and thrombosis at a mild level in the treatment of colorectal cancer . however , its probability is very low , and it is thought that in cases of eye drop or subconjunctival injection , the incidence would be substantially decreased . also in cases where bevacizumab is injected within the vitreous body , special systemic side effects have not yet been reported . during the injection of bevacizumab within the vitreous body , 1 - 2 mg is injected , and considering the vitreous body volume ( 5.2 ml ) , the concentration corresponding to 0.2 - 0.4 mg / ml [ 16 - 18 ] . considering that the vitreous body is a closed space , it is thought that a concentration higher than this is required for the eye drop to be effective on corneal neovasculature . therefore , in this study , neovascularization was induced in the cornea of house rabbits , 5 mg / ml or 10 mg / ml bevacizumab eye drop was administered to the eye , and 1.25 mg was used for the injection within the vitreous body . our experiments showed that the results of the groups treated with eye drop and subconjunctival injection were significant ( p<0.05 ) , but corneal neovasculature could not be completely removed . considering the several causes of neovasculature formation , administration of bevacizumab twice a day alone via eye drop may not be effective . it is possible that the administered drug is removed by tears , and thus it could not react with all vegf receptors . in fact , in our experiment , upon analysis of the vegf concentration of corneal sections , the treatment group showed significantly lower values than the control group . nevertheless , the vegf concentration of the control group was still maintained at a constant level ( fig . 4 ) . in other words , even after treatment , a large number of vegf still remained in tissues . however , even in cases of subconjunctival injection , the result was not significantly different from eye drop treatments , and considering that the half - life of bevacizumab is less than 30 minutes , it appears that removal of the drug via tears may not be a great factor . in addition , the difference of the treatment according to eye drop concentrations was shown to be not significant ( fig . moreover , our findings may be due to the presence of other cytokines ( transforming growth factor a and b2 ; tgf a and b1 , and fgf involved in corneal neovascularization in addition to vegf ) . in the future , actually , the eye drop treatment was terminated in two weeks , and the subconjunctival injection was terminated after only one treatment . nonetheless , at two weeks after treatment , the neovasculature area was definitely smaller than after one week , and at the second week , the outcome of eye drop treatment was significantly better than the subconjunctival treatment ( p=0.043 ) . to improve the treatment effectiveness , prolonging the duration of eye drop treatment or repeated subconjunctival treatments may be one option . presently , the focus of bevacizumab application to the treatment of corneal neovascularization was the administration route and dose . regarding the subjects , have proven the fact that in experimental rat models , bevacizumab suppressed not only corneal neovascularization , but also neo - lymphangiogenesis , and both systemic administration and eye drop administration were reported to be effective . in addition , during the experiment processes , toxicity on the cornea was not detected . have reported that for the treatment of the neovascularization in the rat cornea , a 4 mg / ml bevacizumab eye drop was administered , and satisfactory results were obtained . erdurmus and totan   have reported that in human eyes , 2.5 mg bevacizumab ( 0.1 ml ) was injected subconjunctivally to two eyes with the neovasculature , which proved effective within one week , and special systemic complications were not detected . only in one eye case , the degeneration of large blood vessels was not noticeable , which shows the necessity of diverse clinical studies on the dose of bevacizumab , with or without repeated injections , or in combination with other therapies . in our study , regarding the effectiveness differences according to the eye drop concentration , the effectiveness was shown to not vary by the analysis of the neovasculature area and vegf concentration , which shows that both routes are effective . however , if a difference between the eye drop treatment and the subconjunctival injection treatment was detected at the second week of treatment , the repeated treatment of the subconjunctival injection should be considered . in addition to bevacizumab , ranibizumab ( lucentis ; genentech , san francisco , ca , usa ) , and pegaptanib sodium ( macugen ; eyetech pharmaceuticals , new york , ny , usa ) , are antibodies to vegf developed recently . while these two drugs were already approved for the ophthalmic application , their shortcoming is excessively high cost . in the future , if the cost problem were solved , the use of these drugs for the treatment of corneal neovascularization should be also considered . because bevacizumab suppresses the proliferation of blood vessel and lymphoid tissues , it is hypothesized that it may elevate the survival rate of grafts after corneal transplantation , and it may be used for diverse ophthalmic diseases such as the treatment of herpes keratitis with neovascularization and the prevention of recurrence after the pterygium surgery . based on our study , it is thought that bevacizumab would be used as a definite supplement therapy for the treatment of corneal neovascularization in the future , and clinical studies on the dose , administration route , administration duration , and frequency are required .