Subconjunctival Versus Intrascleral Application of Mitomycin C During Trabeculectomy

Abstract

Purpose.: To compare the efficacy and safety between subconjunctival and intrascleral methods of mitomycin C (MMC) application during trabeculectomy.

Methods.: This retrospective study included 165 eyes treated by trabeculectomy and compared clinical outcomes between eyes that received subconjunctival MMC (Group 1, 80 eyes, 48.5%) and eyes that received intrascleral (Group 2, 85 eyes, 51.5%) MMC. Surgical success was defined as an IOP of 6 to 21 mm Hg with or without topical antiglaucoma medication use.

Results.: Mean IOP values were not significantly different between Groups 1 and 2 (P > 0.05) until 2 weeks after surgery, while they differed significantly at 1, 3, 6, 12, and 24 months after surgery (P < 0.05). The mean number of antiglaucoma medications was 3.6 ± 0.6 and 3.5 ± 0.7, respectively, before surgery (P = 0.327), and 0.27 ± 0.32 and 0.10 ± 0.29, respectively, at 24 months after surgery (P = 0.001). The surgical success rate at 24 months was 86.25% and 94.12% in Groups 1 and 2, respectively (P = 0.041, log-rank test). Encysted blebs were observed in 17 and 8 patients in Groups 1 and 2 (P = 0.034), respectively, at 24 months. There was no statistically significant difference in the incidence of hypotony, choroidal detachment, conjunctival leakage, cataract, and the rate of postoperative procedures between Groups 1 and 2 (P > 0.05).

Conclusions.: Compared to subconjunctival MMC application, intrascleral MMC application can increase the long-term success of trabeculectomy without increasing the complication rates.

Introduction

Trabeculectomy is the most common filtration procedure and the gold standard in the surgical treatment of glaucoma. Despite improvements in surgical treatment of glaucoma, the complex pathophysiological mechanisms involved in conjunctival and scleral wound healing have been one of the most important factors limiting surgical success. ¹ However, antiproliferative agents long have been used in conjunction with trabeculectomy to inhibit subconjunctival scarring by decreasing fibroblastic activity and modulating wound healing at the bleb site. ¹ Mitomycin C (MMC) originally was used as a systemic chemotherapeutic agent, and it has been used widely in ophthalmic practice, during and after surgery, for enhancing the success rate of glaucoma filtration surgery. Recent systematic reviews have identified significant improvements in success rates and decreases in postoperative IOP values by the use of MMC during glaucoma filtering surgery. ^2–4 Various toxic effects and complications secondary to the use of MMC are widely known, and several studies have evaluated the different application methods of this drug in terms of exposure time, dose, and surface area of application. ^5–11 Also, MMC is used frequently during trabeculectomy, either under the conjunctival flap (subconjuctival) at the proposed site of trabeculectomy or under a superficial scleral flap (intrascleral), to improve surgical success. However, the effectiveness and safety of application at these two sites have not yet been established. ^8–11  

This study aimed to compare the IOP-lowering effect and complication rate between subconjunctival and intrascleral methods of MMC application in eyes treated by MMC-augmented trabeculectomy. This is the first study in literature, to our knowledge, with a larger number of patients and longer follow-up periods comparing the clinical implications between subconjunctival and intrascleral MMC application. 

Materials and Methods

This retrospective comparative study included 165 eyes in 165 consecutive patients with medically uncontrolled primary open-angle glaucoma (POAG) or primary angle-closure glaucoma (PACG) who underwent trabeculectomy with adjunctive MMC application at The Second People's Hospital of Jinan between November 2007 and March 2010. On the basis of MMC application method as revealed by surgical records, the patients were divided into Group 1, where MMC was applied under the conjunctival flap, and Group 2, where it was applied under a superficial scleral flap. The protocol was approved by the Institutional Ethics Committee of The Second People's Hospital of Jinan and conformed to the tenets of the Declaration of Helsinki. 

Preoperative Assessment

Baseline clinical characteristics and demographic data were collected from the medical records of patients included in this study. Patients followed for less than 24 months, those younger than 25 years, and those with diabetes, secondary glaucoma, a history of ocular surgery, media opacity (keratoleukoma, cataract, etc.), or a history of laser trabeculoplasty were excluded. After applying the exclusion criteria, 226 patients were excluded from the analysis. Biomicroscopic, gonioscopic, and funduscopic examinations were performed in all patients before and after surgery, and IOP levels were measured using a Goldmann applanation tonometer (AT900; Haag-Streit, Koeniz, Switzerland). 

Surgical Technique

All surgeries were performed by the same surgeon using the following technique. After local anesthesia, the bulbus was stabilized with an atraumatic 4-0 silk traction suture suspending the superior rectus muscle. Trabeculectomy was performed by raising a fornix-based conjunctival flap (approximately between the 11 and 1 o'clock positions). A surgical sponge (Medtronic Xomed Surgical Products, Inc., Jacksonville, FL, USA) measuring 6 × 6 mm was soaked in a solution of 0.4 mg/mL MMC (MMC powder was reconstituted in sterile water to achieve the final concentration). In Group 1, this sponge was applied under the conjunctiva for 2 minutes. Then, the MMC application area was irrigated with balanced salt solution and a half-thickness, rectangular, 4 × 5-mm scleral flap was raised. When the entire corneoscleral limbus was exposed, trabeculectomy involving the excision of a trabecular block measuring approximately 1 × 3 mm in size was performed, followed by peripheral iridectomy. In Group 2, after the same scleral flap was raised, the MMC sponge was applied under the scleral flap for 2 minutes. Because the sponge was bigger than the scleral flap, the conjunctival flap outside the scleral flap also was exposed to MMC. Then, the tissue was irrigated with balanced salt solution and trabeculectomy was completed as described above. The scleral flap was sutured with 10/0 nylon sutures (one on each side and two at the apex) and adjusted such that the depth of the anterior chamber was maintained, allowing slow leakage from the edge of the scleral flap. Continuous suturing of the conjunctiva was performed using 10/0 nylon sutures. After surgery, the patients were instructed to stop their preoperative glaucoma medications and were prescribed 0.3% tobramycin four times daily for 1 week and 1% prednisolone acetate eye drops every 2 hours while awake for 2 weeks; this was decreased to 4 times daily for 4 weeks and tapered off over the subsequent 3 to 4 weeks. In addition, 1% atropine twice daily was prescribed if IOP was <6 mm Hg or if the anterior chamber shallowed. Digital massage was recommended for patients in whom appropriate bleb formation was not observed. 

Postoperative Assessment

After surgery, the patients were examined on day 1, and at 1, 2, and 4 weeks, and at 4-week intervals thereafter for 24 months. All patients were followed up for at least 24 months. Evaluated preoperative data included age, sex, IOP, best-corrected visual acuity (BCVA, logMAR Snellen visual acuity was converted to the logMAR scale for comparison, and logMAR values of 1.40, 2.70, and 3.70 were assigned to counting fingers, hand motions, and light perception, respectively), and number of antiglaucoma medications, while postoperative data included IOP values, BCVA, number of antiglaucoma medications, and complications related to surgery. Bleb leakage was confirmed under cobalt blue slit-lamp illumination after a moistened sterile fluorescein strip was applied gently to the bleb surface. A leak was diagnosed if there was any apparent aqueous stream from any leaking point. 

At the 24-month follow-up, photographs of blebs were obtained and graded using the Moorfields Bleb Appearance Grading Scale (MBGS), ¹² which is based on area, height, and vascularization, by a single observer blinded to the respective treatments. An encysted bleb was described as a bleb that was walled off by the Tenon's capsule, and appeared elevated and tense. 

Surgical success was defined as a postoperative IOP value of 6 to 21 mm Hg with or without topical antiglaucoma medication use. Patients with IOP levels of <6 mm Hg were diagnosed with hypotony. Surgical failure was defined as a postoperative IOP value of >21 mm Hg, regardless of medication use. A requirement of bleb needling was not considered a procedural failure. Needling without antimetabolite application was performed in all patients with encysted blebs. 

Statistical Analysis

Data are presented as means ± SD. All statistical analyses were performed using SPSS statistical software (ver. 18.0; SPSS, Inc., Chicago, IL, USA). Independent t-tests were used to evaluate between- and within-group differences. Categorical variables and proportions were analyzed using Pearson χ² tests. Nonparametric data were analyzed using the Mann-Whitney U test. Success rates in both groups were compared using Kaplan-Meier life table analysis and the log-rank tests. Statistical significance was defined as a P value of <0.05. 

Results

Trabeculectomy with adjunctive MMC application was performed in a total of 165 eyes in 165 patients (75 males, 90 females) with POAG (51 eyes, 30.9%) or PACG (114 eyes, 69.1%). Group 1 included 80 eyes (48.5%) and Group 2 included 85 eyes (51.5%). Patients' baseline characteristics are summarized in Table 1. There were no statistically significant differences between the two groups in these baseline characteristics. All patients belonged to a Chinese Han population. 

Table 1

 

Baseline Patient Characteristics

Variable	Group 1, n = 80	Group 2, n = 85	P
Age, y, mean ± SD	52.67 ± 9.86	54.95 ± 7.99	0.104*
Sex, male/female	36/44	39/46	0.909†
BCVA, logMAR, mean ± SD	0.58 ± 0.20	0.57 ± 0.14	0.761*
N of antiglaucoma medications, mean ± SD	3.6 ± 0.6	3.5 ± 0.7	0.327*
Basal IOP, mm Hg, mean ± SD	29.03 ± 10.34	28.46 ± 9.86	0.717*
Type of glaucoma			0.927†
POAG	25	26
PACG	55	59

*  Independent t-test.

†  Pearson χ² test.

There was no statistically significant difference between Groups 1 and 2 in mean IOP values (P > 0.05) until 2 weeks after surgery; however, this difference was significant at 1, 3, 6, 12, and 24 months after surgery (P < 0.05, Table 2). The mean number of antiglaucoma medications was 3.6 ± 0.6 and 3.5 ± 0.7, respectively, before surgery (P = 0.327), and 0.27 ± 0.32 and 0.10 ± 0.29, respectively, at 24 months after surgery (P = 0.001). 

Table 2

 

Pre- and Postoperative Mean IOP (mm Hg)

Time	Group 1, Mean ± SD	Group 2, Mean ± SD	P
Basal IOP	29.03 ± 10.34	28.46 ± 9.86	0.717
Postop IOP
D 1	8.20 ± 1.63	8.19 ± 1.27	0.964
Wk 1	8.35 ± 2.20	8.33 ± 1.91	0.950
Wk 2	8.46 ± 2.95	8.43 ± 2.34	0.942
Mo 1	10.17 ± 2.98	9.24 ± 2.88	0.043
Mo 3	12.15 ± 2.55	11.25 ± 2.18	0.016
Mo 6	12.48 ± 2.56	11.75 ± 1.96	0.041
Mo 12	13.38 ± 2.57	12.45 ± 2.80	0.028
Mo 24	14.15 ± 3.12	12.76 ± 2.55	0.002

The mean BCVA was 0.58 ± 0.20 logMAR in Group 1 and 0.57 ± 0.14 logMAR in Group 2 before surgery (P = 0.761). These values were slightly, but not significantly decreased to 0.64 ± 0.24 logMAR (P = 0.088) and 0.62 ± 0.26 logMAR (P = 0.120), respectively, at the 24-month visit, with no statistically significant difference between groups (P = 0.566). 

Table 3 demonstrates the bleb characteristics at the 24-month visit in both groups. There were no statistically significant differences in MBGS scores for the central bleb area, bleb height, and vascularity between the two groups. The maximal bleb area was significantly different between groups (P = 0.037), with Group 2 exhibiting a more diffuse bleb area. 

Table 3

 

Bleb Morphologic Scores at the 24-Month Visit

	Group 1, n = 88	Group 2, n = 97	P*
Area
1a	2.3 ± 1.1	2.1 ± 0.9	0.475
1b	3.0 ± 1.2	3.4 ± 1.3	0.037
Height	1.5 ± 0.5	1.4 ± 0.5	0.240
Vascularity
3a	2.0 ± 1.1	1.8 ± 0.8	0.199
3b	2.5 ± 1.2	2.7 ± 1.1	0.226
3c	1.5 ± 0.6	1.5 ± 0.6,	0.942

 1a, indicates the central demarcated area of the bleb; 1b, the maximal area; 3a, the vascularity at the central demarcated area of the bleb; 3b, the vascularity at the peripheral part of the bleb; 3c, vascularity at the nonbleb conjunctiva.

*  Mann-Whitney.

Postoperative complications and further surgical interventions are shown in Table 4. Most complications were self-limiting. In 10 eyes of Group 1 and 6 eyes of Group 2, conjunctival leaks were observed in the early postoperative period. Three patients with conjunctival leakage in each group underwent resuturing of the conjunctiva because of persistent leakage (≥2 weeks after surgery). Choroidal detachment almost completely resolved within 2 months of topical prednisolone therapy. Two patients with persistent choroidal detachment in Group 2 required surgical intervention with subretinal fluid drainage. Anterior chamber reformation was undertaken in all patients with prolonged postoperative hypotony, which always was accompanied by a prolonged decrease in anterior chamber depth. An encysted bleb was observed in 17 (21.2%) eyes in Group 1 and 8 (9.4%) eyes in Group 2, with a statistically significant difference between groups (P = 0.034). In all eyes with an encysted bleb, a one-time needling procedure was performed successfully without injecting any antifibrotic agent. The incidence of cataract during the 24 months was not significantly different between the two groups (P = 0.700); two eyes in Group 1 and thee eyes in Group 2 required surgery. 

Table 4

 

Postoperative Complications and Interventions (%)

	Group 1	Group 2	P
Early hypotony, ≤1 mo	10 (12.5)	12 (14.1)	0.760
Prolonged postoperative hypotony, ≥1 mo	2 (2.5)	4 (4.7)	0.449
Choroidal detachment	6 (7.5)	9 (10.6)	0.490
Conjunctival leakage	10 (12.5)	6 (7.1)	0.238
Shallow anterior chamber	10 (12.5)	12 (14.1)	0.760
Encysted blebs	17 (21.2)	8 (9.4)	0.034
Cataract	2 (2.5)	3 (3.5)	0.700
Postsurgical interventions
Conjunctival suture	3 (3.7)	3 (3.5)	0.940
Bleb needling	17 (21.2)	8 (9.4)	0.034
Anterior chamber reformation	2 (2.5)	4 (4.7)	0.449
Subretinal fluid drainage	0	2 (2.1)	0.167

The 24-month life table rates for surgical success were 86.25% (69 eyes) and 94.12% (80 eyes) in Groups 1 and 2, respectively (P = 0.041, log-rank test, Fig.). 

Figure

 

Kaplan-Meier cumulative survival curves for a postoperative IOP value of 6 to 21 mm Hg with or without topical antiglaucoma medication (surgical success). Intercurve analysis using the log-rank test revealed a significant difference between the two groups (P = 0.041).

Discussion

The most common reason for failure of trabeculectomy is fibrosis and extraocular scarring in the subconjunctival–episcleral and bleb areas. ¹ Mitomycin C has become the most commonly used adjuvant agent. ^1,13,14 Different application methods of MMC have been proposed to increase its efficiency while decreasing its toxic effects. In various studies, some risk factors for each patient (such as a history of diabetes, ocular surgery, and laser trabeculoplasty; secondary glaucoma; and young age) have been well described, and scoring systems have been proposed according to these factors to find out the appropriate MMC concentration. ^7,15–17 In the present study, high-risk patients, as described earlier, were excluded from the study, and all patients shared similar preoperative characteristics. Moreover, standard 2-minute MMC was applied in a 0.4-mg/mL dose in all eyes. 

At the 24-months visit, mean IOP values and the number of additional antiglaucoma medications for IOP control were lower in Group 2 than in Group 1 (P < 0.05). The success rate was 86.25% in Group 1 and 94.12% in Group 2 at 24 months (P = 0.041. log-rank test). These findings suggested that intrascleral MMC application seems to be more advantageous than subconjunctival MMC application in the long term. The healing pathway after trabeculectomy can be categorized into four main phases: coagulative, inflammatory, proliferative, and postproliferative remodeling. ¹⁸ Depending on the intensity of the wound-healing response, fibroblasts may be present as early as 24 hours after injury and may persist for several months. ¹⁸ The inhibitory effects of MMC on subconjunctival fibroblast proliferation in vitro have been shown to be dose- and time-dependent. ¹⁹ In the intrascleral MMC application group, a higher MMC dose under the scleral flap can prevent scar formation more effectively in the subconjunctival–episcleral and bleb areas. Corderio et al. ²⁰ reported that the histological examination of surgical eyes demonstrated patency of the tube channel, with obstruction evident mostly at the scleral end of the tube. The hypothesis is that subscleral MMC application works by modulating healing at the edges of the flap. As a result, more aqueous material arrives in the sub-Tenon's area and impairs healing through its antiproliferative action. ^21,22  

Some studies have shown a similar IOP decrease in both groups for a year after surgery. ^8,9 In the present study, the IOP decrease after surgery was similar in both groups until the end of 1 month. The MMC, which cross-links DNA, is an antibiotic derived from streptomycin. It acts at all stages of the cell replication cycle, inhibiting dividing and resting cells. ¹⁶ In animal studies, the effects of MMC was sustained for at least 1 month. ¹⁷ Within 1 month of trabeculectomy, eyes still are in the early wound healing process. Fibroblast proliferation and early scar formation, which heavily influence filtering bleb morphology and function, are ongoing processes at this time. We believe that the inconsistent result of IOP decrease between groups was primarily attributed to the large sample size, which can show the change in IOP more accurately, compared to that in previous studies. ^8–10  

Matlach et al. ¹ have reported a slight decrease in visual acuity after 12 months compared to baseline values. In a similar study, researchers did not note any changes in visual acuity after a 7-month postoperative follow-up. ¹⁹ In our patients, complications affecting visual acuity, such as persistent hypotony and cataract formation, were recorded. A slight decrease in BCVA was observed after 24 months compared to that at baseline in both groups. However, the difference in preoperative and postoperative 24-month BCVA was not statistically significant. This probably is not due to the used adjuvant agents, but it can be explained by the effect of filtering surgery. ²³  

Analysis of bleb morphology at the 24-month follow-up revealed no significant difference in terms of the central bleb area, bleb height, and vascularity. However, we found a statistically significant difference in maximal bleb area between groups, with Group 2 exhibiting more diffuse blebs. Akkan et al. ²³ reported that the mean score of maximal bleb area was 3.04 in the intrascleral MMC application group. Our score for maximal bleb area in Group 2 was similar to that in their study. The incidence of encysted blebs ranges from 2.5% to 29% in the literature. ²⁴ In the present study, we observed more encysted blebs in Group 1 (21.2%) than in Group 2 (9.4%). Corderio et al. ²⁰ proposed that the cellular infiltration was derived from fibroblasts of healthy tissue surrounding the bleb area and that this cellular migration may contribute to the formation of encysted blebs. In Group 2, subscleral administration of MMC worked by modulating healing at the edges of the scleral flap. More aqueous arrives in the sub-Tenon's area and reduces the degree of cellular activity. The degree of cellular activity is reflected in the amount of newly laid scar tissue, which fibroses and contracts as the scarring response develops. We believe that more effectively prevented scar formation resulted in more diffuse blebs and less encysted blebs in Group 2. 

Our study found no statistically significant difference in the incidence of complications, such as hypotony, choroidal detachment, shallow anterior chamber, and conjunctival leakage between the two groups. Earlier studies reported a similar result. Hypotony has been considered to be related to the overfiltration. ^8,9,11 In the present study, early hypotony was observed in 12.5% and 14.1% of patients in Groups 1 and 2, respectively (P > 0.05). Akarsu et al. ²⁵ reported hypotony in 17.6% patients who received 0.4 mg/mL of MMC. In this study, persistent hypotony, which was prolonged for more than 1 month, was observed in 2.5% and 4.7% patients in Groups 1 and 2, respectively (P = 0.449). The incidence of hypotony in our study was not higher than that reported in the literature. ^8,9,11 Conjunctival leakage is a possible risk factor for these complications, and MMC is thought to increase the probability of bleb leakage. ²⁶ In this study, Group 1 encountered a higher incidence of conjunctival leakage, although the differences were not statistically significant. The inhibitory effects of MMC on subconjunctival fibroblast proliferation in vitro have been shown to be dose-dependent. ¹⁹ In the subconjunctival MMC application group, the conjunctiva was directly exposed to a higher MMC dose, which can decelerate conjunctival wound healing. In this study, because of the fornix-based conjunctival flap, slower limbal conjunctival wound healing more easily resulted in leakage. 

Three patients with conjunctival leakage in each group underwent resuturing of the conjunctiva because of persistent leakage 2 weeks after surgery. Choroidal detachment after trabeculectomy was defined as a solid-appearing elevation of the retina and choroid. Because choroidal detachment is a transient complication of trabeculectomy, to our knowledge the risk factors have not been analyzed previously in a large-scale case series. ²⁷ To treat patients suffering from persistent choroidal detachment, suprachoroidal fluid can be drained through a sclerotomy. ²⁸ In the present study, two patients in Group 2 had persistent choroidal detachment, which was resolved with surgical intervention (subretinal fluid drainage). Fortunately, there were no cases of bleb-related endophthalmitis, which has been described previously. ²⁹  

In a study by Tressler et al., ⁹ the intrascleral MMC group required more postoperative procedures for IOP control compared to the subconjunctival MMC group. The results of this study are in agreement with the results of Prata et al., ¹⁰ who reported no significant difference in the rate of postoperative procedures between the two groups. We believe that there are two main reasons for these controversial results. First, the dose of MMC, which has a key role in its effectiveness, was different among studies. The dose was 0.4 mg/mL in our study and 0.5 mg/mL in the study of Prata et al. ¹⁰ and the lowest dose of 0.27 mg/mL was used in the study of Tressler et al. ⁹ Second, the latter study is limited by a small sample size, which can lead to bias. 

Our study has some limitations. First, there were individual factors affecting wound healing, such as immunity level, and this may have influenced the surgical outcomes. Second, the retrospective comparative nature of this study may have caused a bias. A prospective randomized controlled trial should be conducted in the future to clarify these results. 

In conclusion, intrascleral MMC application using a standard dose of 0.4 mg/mL seems to be more effective without increasing the complication rate compared to subconjunctival MMC application, and it can be considered as an alternative approach in filtrating surgery for clinically advanced glaucoma. However, further prospective randomized studies are expected to clarify the benefits of this application method. 

Acknowledgments

The authors alone are responsible for the content and writing of the paper. 

Disclosure: M. Zhang, None; B. Li, None; W. Liu, None; J. Wang, None; X. Wu, None