1. Failed glaucoma surgery
2. Previous surgery with conjunctival incision like retinal surgery
3. Inflammatory glaucoma
4. Neovascular glaucoma
5. Irido corneal endothelial (ICE) syndrome
6. Pseudoexfoliation
7. Pseudophakic and aphakic eyes with glaucoma
8. Anterior segment dysgenesis
9. Congenital and juvenile glaucoma with failure of one conventional surgery without adjunct antimetabolites
10. Ocular pemphigoid, Stevens Johnson syndrome
11. Chronic use of multiple topical medications
12. Low target IOP as in progressive Normal Tension Glaucoma
13. Post Keratoplasty associated with Glaucoma

5 FU the first antimetabolite used by glaucoma surgeons is a fluorinated pyramidine analogue which interferes with s phase and G 2 phase of cell cycle, that corresponds to DNA and RNA synthesis, respectively. Hence it is more toxic to proliferative than nonproliferative cells. In a pilot study the dose administered was 3 mg daily as a subconjunctival injection on postoperative days 1 to 14 (1). This dosage was selected based on experimental studies (2) and the clinical observation that a single subconjunctival dose of 10 mg of 5 FU is welltolerated as an adjunctive in the treatment of complicated retinal detachment associated with proliferative vitreoretinopathy (3).

However subsequently it was increased to 5 mg injections twice a day for 1 week followed by 5 mg daily injection for 1 week, based on clinical observation of rapid scarring noted in patients undergoing filtration surgery for neovascular glaucoma. The total dose administered based on this protocol was 125 mg of 5 FU. This dose is adjusted depending on the nature of wound closure, presence of any leak and on the presence of postoperative shallow anterior chamber. By adjusting the dose of 5 FU based on the clinical response, certain complications associated with 5 FU can be reduced (4).

While subconjunctival injection of 5 FU has been shown to be effective in minimizing the scar formation and enhance the success of filtration surgery, complications due to repeated subconjunctival injection including corneal epithelial defects, conjunctival wound leaks, risk of globe perforation and need for repeated injections cause patient discomfort, which limit its use (5, 6). Intraoperative application of 5 FU avoids the risk of epithelial defects and ocular perforation; it decreases discomfort to the patient associated with repeated injections and requires less frequent visits.

A single application of 5 FU has been shown to inhibit fibroblast proliferation up to 36 days in-vitro (7). In vitro studies have shown highest levels of 5 FU in the scleral flap and scleral bed when a 5 FU soaked sponge was placed on the scleral bed for 5-minute period (8). Intraoperative 5-FU has been shown to be effective in patients with high risk for failure, without corneal complications noted with subconjunctival injection of 5 FU (9-12). The variable success rates noted can be explained by differences in the study population, study design, dosage of antimetabolite, and success criteriaemployed. A single intraoperative application of 5 FU at a concentration of 50 mg/ml for a period of 5 minutes appears effective in low-risk eyes without risk of complications associated with repeated subconjunctival injections. The technique of application is to use a sponge or filter papers soaked in commercially available 5 FU at a concentration of 50 mg/ml and apply it on the scleral bed before raising the scleral flap. The sponge or filter papers are then removed and the area rinsed thoroughly with balanced salt solution. One can proceed with trabeculectomy taking care to meticulously close the scleral flap and the conjunctiva and secure the anterior chamber formation, at the conclusion of the surgery. Postoperative 5 FU is given as a subconjunctival injection. 0.1 ml of commercially available 5 FU in a concentration of 50 mg /ml (i.e. 5 mg) is loaded in a tuberculin or insulin syringe. Using a fine caliber needle, preferably 30 G needle, the injection is done away from the site of bleb. This is necessary to prevent accidental entry of 5 FU into the anterior chamber. 5 FU has a pH of 9 and would irreversibly damage corneal endothelium. The pain can be minimized by applying a cotton bud soaked in anesthetic at the proposed site of injection for a minute after topical anaesthesia.

The use of 5 FU either intraoperatively or postoperatively can cause complications. These include corneal epitheliopathy, filamentary keratitis, epithelial erosions, corneal ulcer, perforation and corneal opacity. Also the thin and pale bleb seen postoperatively can leak with resultant hypotony and choroidal detachment.

These thin blebs are also prone for blebitis and endophthalmitis.

Mitomycin is an antitumor antibiotic, which is isolated from Streptomyces caespitosus. It inhibits e DNA replication causing cell death. Mitomycin inhibits fibroblasts like 5 FU but it is 100 times more potent on cultured rabbit fibroblasts (13). This difference is due to the difference in the administered dose and not due to their mechanisms in inhibiting fibroblast proliferation. Based on the in vitro study, the potency to inhibit fibroblast of 0.2 mg of mitomycin would be equivalent to 20 mg of 5 FU (13). But this high dose of 5 FU cannot be given as subconjunctival injection, since it can produce severe corneal toxicity and complications. In rabbit model it has been shown that, a 5minute application of 5 FU has a temporary delaying effect on the proliferation of fibroblasts; similar application of mitomycin showed a lasting effect that may be appropriate in patients with a high risk for filtration failure (14). In vitro sensitivity of human Tenon's capsule fibroblasts to Mitomycin C and its correlation with outcome of glaucoma filtration surgery, showed positive correlation (15).

Clinical application was first reported by Chen in 1986 (16) and subsequently by Palmer (17). Randomized study comparing the efficacy of 5 FU with mitomycin in patients undergoing trabeculectomy for glaucoma with poor surgical prognosis; complete success i.e. IOP controlled without supplementary antiglaucoma medication was achieved 88.2% in mitomycin group compared to 40% in 5 FU group (18). Based on this study, mitomycin is definitely more favorable to create a good filtration bleb compared to 5 FU. The probability of successful IOP control without postoperative antiglaucoma medication is higher with the use of mitomycin compared to 5 FU. In patients with previously failed glaucoma filtration surgery who were undergoing glaucoma triple procedure i.e. Phacoemulsification with IOL combined with trabeculectomy, adjunctive use of mitomycin increased the success compared to control group of patients where mitomycin was not used (19). The technique of intraoperative application of mitomycin is similar to that of 5 FU. We use a sponge or filter papers soaked in commercially available mitomycin at a concentration of 0.2 to 0.4 mg/ml and apply it on the scleral bed before raising the scleral flap for period of 1 to 3 minutes depending the risk factors in the patient, status of conjunctiva, extent of scarring, preoperative IOP and the target IOP aimed for in the patient. Contact with the cut edge of conjunctival flap to the sponge soaked in mitomycin should be avoided. The sponge or filter papers are then removed and the area rinsed thoroughly with balanced salt solution.

One can proceed with trabeculectomy taking care to meticulously close the scleral flap and the conjunctiva and ensure the anterior chamber formation, at the conclusion of the surgery.

Figure 2 shows a typical ischemic bleb 3 months after trabeculectomy with mitomycin .

Figure 2: Trab with Mitomycin post-operative ischemic bleb

Postoperatively mitomycin can be given as a subconjunctival injection of 0.02 mg with a fine needle, preferably 30 G adjacent to the bleb. Avoid entry of mitomycin into the anterior chamber, as it would result in irreversible endothelial damage.

Recently, topical application of mitomycin has been reported in the postoperative period, where a sponge soaked in mitomycin is placed in the bleb area for 3 days following trabeculectomy (20).

The suggested dosage of antimetabolites is given in tables 1 and 2.

Table-1

Table-2

Hypotony that is persistent and causing decrease in vision is the most dreaded complication. Late onset bleb leaks and blebitis and bleb-related endophthalmitis can occur. Patients who have thin blebs should be warned about the early symptoms of infection and must be advised to seek medical help early, to prevent devastating complications that follow even a mild conjunctival infection. Mitomycin induced scleritis has been reported in a few cases where inferior trabeculectomy was done; this has been seen first reported when mitomycin was used as an adjunct in the form of eye drops following pterygium excision surgery. The risk of hypotony and choroidal detachment is higher with longer the duration of application of mitomycin (21).

Hypotonous maculopathy is due to thin avascular bleb caused by extensive cellular effects, focal oozing or leaking of bleb as well as toxic effects on the ciliary body. Myopic individuals, young patients (< 40 yrs) and usage of higher concentration and longer duration of application can be associated with this condition.

Retino choroidal folds, optic disc oedema, characterize hypotonous maculopathy. Young and myopic patients are more prone to develop hypotonous maculopathy due to longer axial length and, thus reduction of scleral rigidity (22). Hypotonous maculopathy, which is persistent for more than 4 to 6 months following surgery, would require surgical correction, if non incision methods like autologous blood injection into the bleb or compression sutures do not resolve the problem. Bleb excision with conjunctival-Tenon's autograft from a healthy adjacent area would reverse the hypotony and improve the visual acuity. If the sclera is extensively thin and fragmented due to toxic effects of mitomycin, a scleral auto graft or preserved donor sclera would be required. Mild bleb leaks can be managed by conjunctival advancement or amniotic membrane grafts.

Antitransforming Growth Factor β2 monoclonal antibody:

Transforming growth factor-β is one of the potent stimulants of bleb scarring following surgery. Out of the three isoforms found in humans i.e. β-1, β-2 and β-3, β-2 is predominantly ocular isoform (23, 24). The neutralization of the activity of transforming growth factor β-2 makes it a target for modulating the scarring response following trabeculectomy. It has also been noted that a high concentration of transforming growth factor β-2 in the aqueous of glaucomatous eyes (25).

Human monoclonal antibody specific to human TGFβ-2, CAT-152 (Cambridge Antibody Technology) has been developed. Evaluation of this antibody following filtration surgery in the rabbits has shown that it significantly improves the outcome compared to placebo (26).

In a landmark, randomized placebo controlled trial in patients undergoing primary trabeculectomy, human monoclonal antibody (CAT-152) was evaluated and found to be safe and well tolerated. Human monoclonal antibody (CAT-152) was administered as 4, 100 μl (100 μg in 100 ml) subconjunctival injections. The injection was given immediately before and after surgery and at day 1 and a week postoperatively. At 12 months follow-up blebs with CAT-152-antibody treatment, were more diffuse, avascular and noncystic much unlike thin blebs seen with the use of 5 FU and mitomycin (27). The synthetically engineered TGF-β antibody is not yet commercially available. More trials are underway and it is likely that we will see new advances in wound modulation following trabeculectomy.

Suramin:

This was initially developed as an antiparasite drug, which inhibits reverse transcriptase and is being in clinical trials of AIDS, for selected malignancies. Suramin is a heparin analogue and hence binds to heparin binding proteins. Growth factors inhibited by suramin include TGF β 1. 2, 3; as well as PDGF A, PDGF b, EGF, bFGF, IGF-1 and IGF-II. Suramin affects cytokines that play an important role in fibroblast proliferation. Suramin inhibits production of collagen type I and type III.

In a clinical study Suramin was applied during surgery at a concentration of 200 mg/ml for a period of 5 minutes. On day 1, 2 and 3 following surgery a subconjunctival injection of 0.1 ml (200 mg/ml) was administered close to the bleb. A comparison with a historical group of mitomycin-treated eyes showed (0.2 mg/ml) showed no difference in the IOP before and after surgery (28).

Photodynamic Therapy:

Photodynamic therapy with carboxyfluorescein ester followed by irradiation with blue light which selectively destroys the fibroblasts, has been shown to inhibit proliferation of human Tenon's fibroblasts in vitro and in experimental animal eyes (29,30). The first human study of photodynamic modulation of wound healing following glaucoma surgery has been reported recently (31). In this study of 42 eyes, complete success was achieved in 59.5%; additional 16.7% had success with supplementary medical treatment and 23.8% failed. Further studies are awaited using this technique for wound modulation.

Beta radiation:

Recent report of 1000 rad of b radiation as a single dose during trabeculectomy with good results has renewed the interest in this modality for fibroblast inhibition. Though it is a long-term retrospective study (7 yrs), there is lack of control of group (32). The long-term effect on the crystalline lens as well as the risk of radiation retinopathy should be kept in mind before using this modality.

Nanomolecules:

A new class of nanomolecules, dendrimers coupled with glucosamine has been shown to profoundly reduce the scarring after experimental glaucoma filtration surgery (33). This gives us a unique opportunity to reduce the failure of filtration surgery.

Genetherapy:

The insertion of antiproliferative genes which are associated with control of cell cycle like p21(WAF-1/Cip-1) using an adenovirus system has been promising in experimental eyes without bleb thinning seen with antimetabolites like mitomycin (34).

Bleb revision of failed trabeculectomy can be done in many patients by needling, supplemented by subconjunctival injection of 5 FU or mitomycin. It is best done under topical anaesthesia, with a fine 29G or 30G needle which is inserted subconjunctivally 7 mm away from the bleb and thick bleb wall is opened with multiple punctures and needle withdrawn; this is followed by a subconjunctival injection of 5 FU 5mg or 0.1 ml of 0.1mg or 0.2 mg/ml mitomycin(35,36). Although the long-term results of this procedure are not known, this technique is definitely worth a try, compared to an extensive repeat filtration procedure.

Antimetabolites have been used to supplement glaucoma drainage devices with some success though all of them are small short-term studies without controls. Partial Tenon's capsule resection with adjunctive mitomycin did not result in avascular blebs over Ahmed Glaucoma valves compared to Ahmed valve without Tenon's capsule resection in patients with neovascular glaucoma. The beneficial effects of mitomycin cannot be ascertained in this study since all the patients received mitomycin (37). In a randomized placebo controlled, masked study, mitomycin 0.4 mg/ml for 2 minutes was compared with balanced salt solution in 25 patients undergoing double plate Molteno implant. Mitomycin was no better than balanced salt solution with respect to IOP or percentage change from baseline IOP (38). The addition of mitomycin was reported to increase the likelihood of a 2-3 year period of medication-free IOP control compared to placebo or 5-FU in patients undergoing Molteno implantation (39).

Precautions & recommendations:

There is no doubt that antimetabolites have improved the surgical success of glaucoma filtration procedure through its' effects on wound modulation. It is important to understand that these agents are toxic and can cause severe ocular morbidity including vision loss. In order to maximize the safety and decrease the risk of ocular complications, a thorough knowledge and complete understanding of all these agents are essential. Careful patient selection and thorough preoperative evaluation is essential to choose the appropriate antimetabolites, their dose and duration of application. This will not only enhance the success rate but also limit the risk of untoward ocular complications. It is imperative to adopt impeccable surgical techniques to prevent wound related complications. Use of atraumatic needle to close the conjunctiva and adjustment of filtration with releasable suture or with laser suturolysis will help in avoiding the immediate postoperative hypotony, shallow anterior chamber and enhance the success of filtration. It is possible that the more desirable diffuse blebs can be achieved by increasing the surface area of application of mitomycin (40). The features common to cystic blebs are anterior aqueous drainage at the limbus and a “ring of steel” scar tissue (Figure 3).

Figure 3: Trabeculectomy with mitomycin anterior bleb

Studies conducted by Khaw and Co-workers have shown that the diffuse blebs can be obtained by increasing the area of application of mitomycin, there by dramatically reducing the risk of bleb-related complications over a three- year period from 20% to 0% (41).

Postoperatively the appearance of the bleb is monitored and surgical intervention like needling of the bleb with injection of 5FU would be needed to suppress the fibroblastic activity. This will enhance the bleb survival for prolonged periods. Those who end up in thin avascular blebs should be warned about symptoms of bleb leaks and bleb-related infection and advised prompt medical attention.

1. Heuer DK, Parrish RK II, Gressel MG et al. 5-fluorouracil and glaucoma filtrationSurgery: II. A Pilot study. Ophthalmology 1984;91:384-94
2. Gressel MG, Parrish RK II, Folberg R. 5 fluorouracil and glaucoma filtration surgery.An animal model. Ophthalmology 1984;91:378-383
3. Blumenkranz M, Hernandez E, Ophir A, Norton EWD. 5-fluoruracil new applicationsIn compli-cated retinal detachment for an established antimetabolite. Ophthalmology1984;91:122-30.
4. Weinreb RN. Adjusting the dose of 5-fluorouracil after filtration surgery to minimizeside effects, Ophthalmology 1987;94:564-570
5. Knapp A, Heuer DK, Stern GA, Drebe WT Jr. Serious corneal complications ofglaucoma filtering surgery with postoperative 5-fluorouracil. Am J Ophthalmol1987;103:183-7
6. Lee DA, Hersh P, Kersten D, Melamed S. Complications of subconjunctival 5-fluorouracil following glaucoma filtering surgery, Ophthalmic Surg 1987;18:187-90.
7. Khaw PT, Sherwood MB, MacKay SLD, Rossi MJ, Schultz G. Five-minutetreatments with fluorouracil, floxuridine, and mitomycin have long-term effects onhuman Tenon's capsule Fibroblasts. Arch Ophthalmol 1992;110:1150-4.
8. Kitazawa Y, Taniguchi T, Nakano Y et al. 5-Fluorouracil for trabeculectomy inglaucoma. Grefes Arch Clin Exp Ophthalmol 1992;225:403-5.
9. Feldman RM. Dietze PJ, Gross RL, Oram O. Intraoperative 5-Flurorouraciladministration in Trabeculectomy J of Glaucoma 1994;3:302-307.
10. Egbert PR, Williams AS, Singh K et al. A prospective trial of intraoperativefluorouracil during trabeculectomy in a black population. Am J Ophthalmol 1993;116:612-616.
11. Lanigan L, Sturmer J, Baez KA, Hitching RA, Khaw PT. Single intraoperative application of of 5-fluorouracil during filtration surgery: early results. Br J Ophthalmol 1994;78:33-37.
12. Shelat B, Sridhar Rao B, Vijaya L et al. Results of Intraoperative 5-Fluorouracil inPatients undergoing Trabeculectomy - Pilot trial, Ind Journal Ophthalmol.1996;44 91-94.
13. Yamamoto T, Varani J, Soong KH, Lichter PR. Effects of 5-flurouracil and mitomycin C on cultured rabbit fibroblasts. Ophthalmol 1990;97:1204-1210.
14. Khaw PT, Doyle JW, Sherwood MB, Grierson I, Schultz G, McGorray S. Prolonged localized tissue effects from 5-minute exposures to fluorouracil and mitomycin C. Arch Ophthalmol 1993;111:263-267.
15. Madhavan H N, Sridhar Rao B, Vijaya L, Neelakantan A. In Vitro Sensitivity of Human Tenon's capsule fibroblasts to Mitomycin C and its correlation with outcome of Glaucoma Filtration Surgery, Ophthalmic Surgery, 1995;26:61-65.
16. Chen CW, Huang HT, Shen MM. Enhancement of IOP control effect of Trabeculectomy by local application of anticancer drug. In Acta XXV Concilinium Ophthalmologicum (Rome) Amsterdam the Netherlands: Kugler & Ghedini 1986;2:1481-1491.
17. Palmer SS. Mitomycin as adjunct chemotherapy trabeculectomy. Ophthalmolgy 1991;98:317-321
18. Kitazawa Y, Kawase K, Matsushita H, Minobe M. Trabeculectomy with Mitomycin, A comparative study with fluorouracil. Arch Ophthalmol 1991;109:1693-1698.
19. Shin D, Kim YY, Sheth N et al. The role of adjunctive mitomycin C in secondary glaucoma triple procedure as compared to primary glaucoma triple procedure. Ophthalmology 1998;105:740-745.
20. Mietz H, Jacobi P C, Krieglestein G K. Intraoperative episcleral versus postoperative topical application of mitomycin- C for trabeculectomies. Ophthalmol 2002;109:1343-1349.
21. Neelakantan A, Rao BS, Vijaya L et al. Effect of the Concentration and Duration of Application of Mitomycin C in Trabeculectomy, Ophthalmic Surgery, 1994; 25:612-615,
22. Stamper RL, McMenemy MG, Lieberman MF. Hypotonous maculopathy after trabeculectomy subconjunctival 5-fluorouracil. Am J Ophthalmol 1992;114:544-553.
23. Jampel HD, Roche N, Stark WJ, Roberts AB. Transforming growth factor-beta in human aqueous humor. Curr Eye Res 1990;9:963-969.
24. Pasquale LR, Dorman-Pease ME, Lutty GA, et al. Immunolocalization of TGF-beta 1, TGF-beta 2, And TGF beta-3 in the anterior segment of the human eye. Invest Ophthalmol Vis Sci 1993;34:23-30.
25. Tripathi RC, Li J, Chan WF, Tripathi BJ Aqueous humor in glaucomatous eyes contain an increased level of TGF-beta 2. Exp Eye Res 1994;59:723-727.
26. Cordeiro MF, Gay JA, Khaw PT. Human anti-transforming growth factor antibody: a new glaucoma anti-scarring agent. Invest Ophthalmol Vis Sci 199;40:1975-1982.
27. Siriwardena D, Khaw PT, King AJ, Donalson ML et al. Human antitransforming growth factor b 2 monoclonal antibody a new modulator of wound healing in Trabeculectomy: A randomized placebo controlled clinical study. Ophthalmol 2002; 109:427-431.
28. Mietz H, Krieglestein G K. Suramin to enhance glaucoma filtration procedure: a clinical comparison with mitomycin. Ophthalmic Surg Lasers 2001;32:358-369.
29. Grisanti S,Gralla A, MauereP, et al: Cellular photoablation to control postoperative fibrosis in filtration surgery: in vitro studies. Exp Eye Res 2000;70:145-152.
30. Grisanti S, Diestelhorst M, Heinmann K et al: Cellular photoablation to control postoperative fibrosis in a rabbit model of filtration surgery. Br J Ophthamol 2000;83:1353-1359.
31. Jordon JF, Diestelhorst M, Grisanti S, et al: Photodynamic modulation of wound healing in glaucoma filtration surgery. Br J Ophthamol 2003;87:870-875.
32. Lai JSM, Poon ASY, Tham CCY et al: Trabeculectomy with b radiation. Long-term follow-up. Ophthalmology 2003;110:1822-1826.
33. Shaunak S, Thomas S, Gianasi A et al. Control of excessive scar formation in the eye with dendrimer based constructs of glucosamine. Nat Biotechnol. 2004;22:1-11.
34. Perkins TW, Faha B, Ni M et al. Adenovirus-mediated gene therapy using human p21WAF-1Cip-1 to Prevent wound healing in a rabbit model of glaucoma filtration surgery. Arch Ophthalmol 2002;120:941-949.
35. Shin DH, Juzych MS,, Khatana A et al: Needling revision of failed filtering blebs with adjunctive 5-fluorouracil . Ophthalmology 1993;24:242-248.
36. Greenfield DS, Milller MP, Suner U et al: Slit-lamp needle revision of failed filtering blebs using mitomycin C. Ophthalmology 1996;122:195-204.
37. Susanna R Jr and The Latin American Glaucoma Society (SLAG)investigators. Partial Tenon's capsule resection with adjunctive mitomycin C in Ahmed glaucoma valve implant surgery. Br J Ophthalmol 2003;87:994-998.
38. Cantor L, Burgoyne J, Sanders S, et al. The effect of mitomycin C on Molteno implant surgery: a 1-year randomized masked, prospective study. J Glaucoma 1998;7:240-246.
39. Perkins TW, Gangnon R, Ladd W, et al. Molteno implant with mitomycin C: intermediate-term results. J Glaucoma 1998;7:86-92.
40. Khaw PT, Wellls AP, Lim KS. Surgery for glaucoma in the 21st century. Br J Ophthalmol 2005; 86:710-711.
41. Wells AP, Cordeiro MF, Bunce C, Khaw PT. Cystic bleb formation and related complicationsin limbus versus fornix based conjunctival flaps in paediatric and young adult trabeculectomy with mitomycin C. Ophthalmology 2003;110:2192-2197.