Topical Bevacizumab as Adjunctive Therapy for Bleb Survival after Trabeculectomy in the Rabbit Model
Jaime Rafeal Hubilla Tripon, MD and Ma. Imelda Yap-Veloso, MD
Glaucoma is the second leading cause of blindness worldwide.1 At present, intraocular pressure (IOP) is the only modifiable risk factor in the treatment of glaucoma. It has been found that patients with glaucoma require normal to low IOPs to prevent progression of the disease.2 Current treatment modalities to lower IOP include medical therapy, laser treatment, and glaucoma filtering surgery (trabeculectomy). Trabeculectomy is the procedure of choice when medical and laser therapy fail to lower IOP to optimum levels. This procedure lowers IOP by creating a fistula in which aqueous flows from the posterior chamber, through a peripheral iridectomy into the anterior chamber, then through a sclerostomy and out into the subconjunctival space, forming a bleb. Survival of this bleb, and thus its IOP-lowering function, is dependent on the wound healing and the degree of postoperative scarring3.
Ocular wound healing involves overlapping phases of hemostasis, inflammation, angiogenesis, fibroblast migration, and tissue remodeling4. Surgical damage to the ocular tissues induces the activation of the clotting and complement systems, as well as stimulating the release of growth factors such as transforming growth factor-β (TGF-β) and vascular endothelial growth factor (VEGF). These in turn stimulate the migration and proliferation of inflammatory cells. The inflammatory cells then activate and stimulate the migration and proliferation of fibroblasts. This is followed by blood vessel endothelial migration and proliferation as modulated by the continued release of VEGF. After resolution of healing, what is left is a fibrous conjunctival scar5. Modulating these phases will reduce scarring and fibrosis and prolong bleb survival.
Jose Rizal became a marked man when he published Noli Me Tangere (Berlin, 1887), a satire on the excesses of the friars in the Philippines. In 1892, he was banished to Dapitan in Zamboanga where he continued to practice ophthalmology. In addition, he taught the locals agriculture and sound public-health practices. It was in Dapitan where he met his future wife, Josephine Bracken, who brought her stepfather for eye treatment. His interest in local flora and fauna even allowed him to send species of animals and plants abroad, which his European scientist friends later named after him.
Anti-metabolites, such as mitomycin-C, have been found to reduce postoperative fibrosis and improve the success rate of trabeculectomy by targeting the DNA replication of fibroblasts, inhibiting their proliferation, and thereby limiting scarring.6,7,8 However, the use of mitomycin-C has been shown to increase susceptibility to long term complications, such as bleb leak, blebitis, and endophthalmitis.9 It is because of these long term complications that other methods of wound modulation are being developed.
An expanding amount of evidence points to the secreted protein, vascular endothelial growth factor (VEGF), as an important mediator in the OPHTHALMOLOGYprocess of angiogenesis. VEGF has been found to be a potent stimulator of endothelial cell growth in vitro and neovascularization in vivo.10,11 Inhibition of VEGF has been shown to prevent the formation, halt progression, and even cause the regression of ocular neovascularization12.
VEGF is a homodimeric glycoprotein specific for endothelial cells. It has been shown to be a critical regulator of vasculogenesis, angiogenesis, and vascular permeability13. An increase in VEGF is associated with proliferation and migration of endothelial cells, and breakdown of basement membrane via activation of metalloproteinase production, which facilitates the invasion of new blood vessels into the surrounding tissue stroma. VEGF also serves as a chemotactic agent for monocytes, with VEGF receptors on all inflammatory cells12.
Li and associates showed that VEGF levels increased in the aqueous humor of glaucoma patients undergoing trabeculectomy14. They found that VEGF could stimulate the growth of human Tenon’s capsule fibroblasts and hypothesized that inhibition of VEGF could improve the success rate of filtering surgery.
Bevacizumab (Avastin, Genentech) is a full-length, humanized, murine monoclonal antibody directed against all the biologically active forms of VEGF-A.15 Bevacizumab has been approved by the FDA for the treatment of colorectal cancer. It is currently being used off-label, for the treatment of neovascular age-related macular degeneration (AMD). The success of bevacizumab in the treatment of retinal and choroidal neovascularization prompted investigators to study its possible applications in anterior segment neovascularization. Topical administration of bevacizumab has been shown to improve corneal neovascularization in several animal studies15, 16. Qin et al found that bevacizumab, at concentrations of 5 mg/mL and above, induced a dose-dependent reduction of viable Tenon’s fibroblast cells in vitro17. Bevacizumab reduced Tenon’s fibroblast cell numbers by inducing apoptosis. Rate of reduction was found to be comparable with 5-FU in vitro. The safety of bevacizumab for ocular use has been the subject of several studies. Yoeruek and colleagues investigated the toxicity of bevacizumab on cultures of human corneal cells and concluded that it was nontoxic to the cornea in vitro18. It was thus deemed safe for both intravitreal and topical use in humans.
revision of encapsulated filtering bleb with bevacizumab. After needling and injection of 1 mg, the bleb was noted to be more diffuse with a decrease in vascularity19. Other studies compared the outcomes of bevacizumab-augmented trabeculectomy with 5-FU-augmented trabeculectomy and found comparable mean IOPs20. Intracameral bevacizumab during trabeculectomy in patients at high risk of bleb failure showed a mean IOP drop of 11.5 mmHg at 3 months postoperatively21. These studies showed the beneficial use of bevacizumab for the treatment not only of retinal and choroidal neovascularization but also of anterior segment neovascularization, specifically wound modulation for a successful trabeculectomy.
This study determined the effect on bleb survival of topically administered bevacizumab, alone or as an adjunct to mitomycin-C, after trabeculectomy in rabbit eyes.
An experimental interventional comparative animal study was conducted using 16 albino rabbits weighing between 1-1.5 kg. Rabbits were then randomly allocated to 4 groups. Group 1 underwent plain trabeculectomy and received topical balanced salt solution (BSS) and served as the negative control. Group 2 underwent plain trabeculectomy and received topical bevacizumab. Groups 3 and 4 underwent mitomycin-C-enhanced trabeculectomy and received topical BSS and bevacizumab, respectively. Group 3 served as the positive control. All rabbits were handled in accordance with the Association for Research in Vision and Ophthalmology resolution on the use of animals in research. Baseline IOPs of both eyes of each rabbit were measured with a Medtronic Tonopen (currently Avia Tonopen, Reichert Corp., CA, USA). General anesthesia was induced with an intramuscular injection of ketamine at a dose of 50 mg/kg. Topical 5% betadine and proparacaine were instilled. An 8-0 Vicryl corneal traction suture was placed and a limbal-based conjunctival flap was made. A limbal groove was created with blade #15 and extended to clear cornea, after which a paracentesis was made with a 15-degree stab knife. For eyes undergoing mitomycin-C-enhanced trabeculectomy, a cotton tip saturated with 0.4 mg mitomycin-C was placed underneath the
conjunctival flap for 2 minutes and washed with 20 cc BSS. The anterior chamber was entered through the limbal groove with a 15-degree stab knife. A sclerostomy was created using a 1.5 mm scleral punch. Peripheral iridectomy was done, and the chamber reformed with BSS. The conjunctival incision was repaired with running 8-0 Vicryl sutures. BSS was injected through the paracentesis to inflate the bleb. Eight right eyes underwent plain trabeculectomy and 8 left eyes underwent mitomycin-C-enhanced trabeculectomy. Contralateral eyes served as controls for IOP.
Rabbits were randomized to receive either topical BSS or topical 12.5 mg/mL bevacizumab, given four times daily, receiving a total of 3.33 mg daily. Postoperatively, atropine once daily and gatifloxacin 4 times daily were instilled. Topical steroids were not included in the regimen so as not to interfere with the wound modulation effects of the test drug. Observers were masked with regards to treatment assignments.
IOPs of both postoperative and control eyes were measured with a tonopen and recorded. Bleb height, width, and length were measured with a caliper. Digital pictures were also taken. Bleb vascularity was graded as 0 = avascular, 1 = normal (based on vascularity of non-operated contralateral eye), 2 = hyperemic, 3 = very hyperemic (Figure 1). Observations were done on days 1,3,5,7, then every 3 days thereafter until bleb failure was detected or after 21 days postoperatively. To minimize variance between animals due to baseline differences, IOP was recorded as a ratio of the IOP of the experimental postoperative eye divided by the IOP of the contralateral control eye (IOPratio = IOPpostop /
IOPcontrol) and bleb morphology was recorded as a percentage of the maximum estimated bleb volume (% bleb = estimated bleb volume / maximum bleb volume). Bleb failure was said to have occurred if IOPratio ≥0.8, or if % bleb = 0. One randomly selected rabbit from each group was sacrificed at day 21. The eyes were enucleated, preserving the superior conjunctiva and fixed in 10% buffered formaline solution. Sections containing the bleb were processed in paraffin and stained with hematoxylin-eosin. Fibroblast cell counts were done at sites within the bleb areas. Mean IOP, mean IOPratio, mean estimated bleb volume, and mean % bleb were plotted as a function of time. Mann-Whitney U test was used to compare
survival times were recorded. One-way analysis of variance (ANOVA) was done to compare rates of bleb failure between treatment groups. Correlation analysis was performed using Pearson’s Product Moment.
One rabbit died 1 week postoperatively and was dropped from the analyses. A total of 15 eyes out of 16 were analyzed. Baseline mean IOP ratios and % bleb volume achieved better homogeneity when compared with the mean IOP and bleb volume.
The mean IOP ratio as a function of time (Figure 2) and the linear regression plots (Figure 3) showed an increase in IOP ratio after baseline. One-way ANOVA of the slopes of the linear regression plots showed no statistical significant differences (ρ=0.64) among the groups.
In eyes that underwent plain trabeculectomy, mean bleb survival in terms of IOP ratio (Table 2) were 6.3 (±1.2) and 9.2 (±1.5) days in the BSS and topical bevacizumab groups (ρ=0.25) respectively. In eyes that underwent mitomycin-C-enhanced trabeculectomy, mean bleb survival were 16 and 18.2 days (ρ=0.40) respectively. Significant differences were found between the following groups: BSS and mitomycin + BSS (ρ=0.00), BSS and mitomycin + bevacizumab (ρ=0.00), bevacizumab and mitomycin + BSS (ρ=0.002), bevacizumab and mitomycin + bevacizumab (ρ=0.00).
Table 2. Bleb survival time in terms of IOP.
The mean maximum extended bleb volume was plotted as a function of time (Figure 4) and the slopes of the linear regression plots showed no statistically significant differences (ρ=0.74).
In eyes that underwent plain trabeculectomy, mean bleb survival in terms of bleb morphology (Table 3) were 8 (±1.7) and 12.2 (±2.9) days for the BSS and bevacizumab groups (ρ=0.08) respectively. In eyes that underwent mitomycin-C-enhanced trabeculectomy, mean bleb survival were 19.5 (±1.0) and 20 (±1.1) days (ρ=0.99) respectively. Significant differences were shown between the following groups: BSS and mitomycin + BSS (ρ=0.00), BSS and mitomycin + bevacizumab (ρ=0.00), bevacizumab and mitomycin + BSS (ρ=0.002), bevacizumab and mitomycin + bevacizumab (ρ=0.001).
There were no significant differences in mean bleb vascularity among the groups (Table 4). However, there was a trend favoring less vascularization for the bevacizumab groups.
The relationships between mean IOP ratio slope, % bleb slope, bleb vascularity, and bleb survival time are shown in Table 5. The only significant correlation was found between bleb survival time based on IOP and survival time based on % bleb ( ρ < 0.01).
Wound healing occurs in several overlapping phases.4 After tissue trauma, the wound healing process begins via increased vascular permeability, which gives blood elements such as fibroblasts, inflammatory cells, cytokines, and macrophages, access to the wound. This is followed by fibroblastic proliferation. These fibroblasts then produce collagen which serves as a scaffold for angiogenesis. New vessel formation, as facilitated by VEGF, starts as capillary buds which extend into the collagen matrix. These new vessels provide nutrients for the fibroblasts, which produce more collagen. The end result is scarring.
Targeting more than one phase in the wound healing process would theoretically maximize efficacy of therapy. Mitomycin-C inhibits the DNA replication of the fibroblast cells, targeting fibroblast migration and scar formation. Bevacizumab targets the angiogenesis and chemotactic properties of VEGF during wound healing. This two-prong approach is designed to attack the inflammatory process at two separate but co-dependent sites: fibroblast proliferation and angiogenesis.
Our results showed a trend towards lower IOPs, larger blebs, less vascularity, and lower fibroblast counts in eyes treated with topical bevacizumab as compared with those treated with BSS. Mean bleb survival time was also longer in the bevacizumab groups. However, when compared with the respective negative (plain trabeculectomy + BSS) and positive controls (Mitomycin-C-enhanced trabeculectomy + BSS), the differences were not statistically significant. Statistical significance was noted between eyes that underwent enhanced trabectulectomy and those that underwent plain trabeculectomy, consistent with results of other studies on mitomycin-C. From this, we can infer that better bleb survival rates could be obtained using topical bevacizumab as an adjunct to mitomycin-C-enhanced trabeculectomy as opposed to plain trabeculectomy alone.
Correlational analyses in this study suggested that increased vascularity would result in lower bleb survival rates, consistent with other studies. Although not statistically significant, there was a trend toward positive correlation between vascularity and rate of postoperative rise in IOP, as well as a negative correlation with postoperative decrease in bleb size.
In summary, topical application of bevacizumab has been shown to decrease bleb vascularization in rabbit eyes. This trend implied that topical bevacizumab may have a contributory role in wound modulation after trabeculectomy. Larger sample sizes, higher dose or more frequent application, or a different route of administration such as subconjunctival or intracameral injection, may yield better statistical results.
1. Broman AT, Quigley HA. The number of people with glaucoma worldwide in 2010 and 2020. Br J Ophthalmol
2. AGIS investigators. The Advanced Glaucoma Intervention Study (AGIS): The relationship between control of intraocular pressure and visual field deterioration. Am J Ophthalmol 2002;130:429-440.
3. Hitchings RA, Grierson I. Clinico-pathological correlation in eyes with failed fistulizing surgery. Trans Ophthalmol Soc UK 1983;103:84-88.
4. Seetner A, Motin JD. Healing of trabeculectomies in rabbits. Can J Ophthalmol 1979;14:536-547.
5. Khaw PT, Jones E, Mireskandari K, et al. Modulating wound healing after glaucoma surgery. Glaucoma Today. 2004. July/ Aug:12-19.
6. Lama PJ, Fechtner RD. Antifibrotics and wound healing in glaucoma surgery. Surv Ophthalmol 2003;48:314-346.
7. Skuta GL. Antifibrotic agents in glaucoma filtering surgery. Int Ophthalmol Clin 1993;33:165-182.
8. Akarsu C, Önol M, Hasanreisoglu B. Postoperative 5- fluorouracil versus intraoperative mitomycin-C in high-risk glaucoma filtering: extended follow-up. Clin Exp Ophthalmol 2003;31:199-205.
9. Debry P, Perkins T, Heatley G, et al. Incidence of late-onset bleb-related complications following trabeculectomy with mitomycin. Arch Ophthalmol 2002;120:297-300.
10. Adamis A, Shima D. The role of vascular endothelial growth factor in ocular health and disease. Retina 2005; 25:111-118.
11. Leung DW, Cachianes G, Kuang WJ, et al. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 1989; 246:1306-1309.
12. Adamis AP, Shima DT, Tolentino MJ, et al. Inhibition of vascular endothelial growth factor prevents retinal ischemiaassociated iris neovascularization in a non-human primate. Arch Ophthalmol 1996;114:66-71.
13 Bhisitkul RB. Vascular endothelial growth factor biology: clinical implications for ocular treatments. Br J Ophthalmol 2006;90:1542-1547.
14. Li Z, Van Bergen T, de Viere SV, et al. Role of vascular endothelial growth factor and placental growth factor
in glaucoma and scar formation after glaucoma filtration surgery. Invest Ophthalmol Vis Sci 2009;50:5217-5225.
15. Rosenfeld P. An update on bevacizumab. Rev Ophthalmol 2005;12:12.
16. Manzano R, Peyman G, Khan P, et al. Inhibition of experimental corneal neovascularization by bevacizumab
(Avastin). Br J Ophthalmol 2007;91:804-807.
17. O’Neil EO, Qin Q, Van Bergen NJ, et al. Antifibrotic activity of bevacizumab on human Tenon’s fibroblasts. Invest Ophthalmol Vis Sci 2010;51:6524-6532.
18. Yoeruek E. Safety profile of bevacizumab on cultured human corneal cells. Cornea 2007;26:977-982.
19. Kahook MY, Schuman JS, Noecker RJ, et al. Needle bleb revision of encapsulated filtering bleb with bevacizumab. Ophthalmic Surg Lasers Imaging 2006;37:148-150.
20. Jurkowska-Dudzinska J, Kosior-Jarecka E, Zarnowski T. Comparison of the use of 5-fluorouracil and bevacizumab in primary trabeculectomy: results at 1 year. Clin Exp Ophthalmol 2012;40:135-42.
21. Surinder P. Intracameral bevacizumab during trabeculectomy in patients at high risk of bleb failure: initial results. Clin Exp Ophthalmol 2007;35(Suppl):A65-A91.
22. Gruezo K, Khu P. Efficacy of intraoperative triamcinolone acetonide as antifibrotic agent in filtration surgery. Philipp J Ophthalmol 2007;32:60-65.