Distinctive Intrableb Structures of Functioning Blebs following Trabeculectomy according to Amniotic Membrane Transplantation

Abstract

Introduction

Intrableb structures are hallmark features of the filtering bleb. This study aimed to compare the characteristics of functioning blebs using anterior segment optical coherence tomography (AS-OCT) according to amniotic membrane transplantation (AMT).

Methods

Forty eyes from 40 patients diagnosed with primary open-angle glaucoma who underwent trabeculectomy, either with AMT (20 eyes) or without AMT (control group, 20 eyes), were included. Parameters including bleb height, bleb wall thickness, striping layer thickness, striping to bleb wall ratio, bleb wall reflectivity, fluid-filled space score/height/area, and presence of microcysts were assessed using AS-OCT. Surgical success was defined at the time of AS-OCT as an intraocular pressure (IOP) ≤18 mm Hg and IOP reduction ≥30% without medication. In these patients, if the bleb had a clinically diffuse and healthy without any signs of an encapsulated bleb, the bleb was then defined as functioning bleb.

Results

Except for bleb height (p = 0.352) and microcyst formation (p = 0.266), significant differences were observed between the two groups. The functioning blebs of the AMT group exhibited greater fluid-filled space score, area, and height than those of the control group, following adjustment for AS-OCT time (all p < 0.001). Conversely, the functioning bleb of the control group demonstrated thicker bleb wall and striping layer, higher striping to bleb wall ratio, and lower bleb wall reflectivity than those of the AMT group, following adjustment for AS-OCT time (all p ≤ 0.001).

Conclusion

Distinct intrableb structures were identified in functioning blebs according to AMT. The reflectivity and thickness of the bleb wall structures were more pronounced in the functioning bleb after trabeculectomy alone. In contrast, the extent of the fluid-filled space emerged as a more distinctive feature of the intrableb structures in the functioning bleb after trabeculectomy with AMT.

Introduction

Trabeculectomy is the gold standard for glaucoma filtering surgery [1]. The aqueous humor within the functioning bleb traverses the conjunctiva and mingles with the tear film or undergoes absorption by the vascular or perivascular conjunctival tissue and lymphatic vessels proximal to the surgical area [2]. Therefore, both transconjunctival and subconjunctival pathways contribute to the maintenance of optimal intraocular pressure (IOP) control [3, 4]. Nevertheless, relying solely on slit-lamp examination would not be sufficient to evaluate the intrableb structures responsible for the success or failure of trabeculectomy [5, 6]. Numerous prior investigations have analyzed theses pathways of the filtering bleb using anterior segment optical coherence tomography (AS-OCT) [3, 4, 6–9]. Filtering bleb has characteristic intrableb structures which are associated with the surgical outcomes of trabeculectomy [3, 4, 6–9]. Functioning blebs exhibit higher bleb height, thicker bleb wall, greater fluid-filled space, lower bleb wall reflectivity, and more frequent microcyst formation than non-functioning blebs [3, 4, 6–9].

Owing to its anti-scarring and anti-inflammatory properties in fibrotic eye diseases, including glaucoma surgery, amniotic membrane transplantation (AMT) has been integrated with trabeculectomy and proven to be a safe and effective method for reducing IOP [10–15]. Previous studies have identified distinct characteristics of intrableb structures within functioning blebs following trabeculectomy with AMT [3, 4]. In our previous study, bleb wall reflectivity correlated with bleb function, irrespective of AMT, whereas fluid-filled space was associated with favorable IOP control only in the AMT group [3]. Nakamura et al. [4] documented that bleb wall reflectivity in the trabeculectomy alone group and the extent of the subconjunctival fluid-filled space in the AMT-assisted trabeculectomy group were factors associated with IOP control.

A comparison of the intrableb structure of the functioning bleb according to the AMT would shed light on the mechanism of aqueous outflow following trabeculectomy. However, few studies have evaluated intrableb structures after trabeculectomy with AMT using anterior segment imaging devices. Distinctive intrableb structures associated with AMT have been documented [3, 4, 16]. To the best of our knowledge, this is the first study to juxtapose the intrableb structures of functioning blebs according to AMT. In this study, we used AS-OCT to investigate the distinctive features of functioning blebs of eyes with successful IOP control following trabeculectomy with or without AMT in patients with primary open-angle glaucoma (POAG).

Materials and Methods

Ethics Statement

This study was performed in accordance with the tenets of the Declaration of Helsinki and approved by the Institutional Review Board of Pusan National University Hospital (No. 2111-004-108). Written informed consent was obtained from all patients for the surgical procedures as well as for the storage of their information in the hospital database and its use for research purposes. All participants provided written informed consent for the publication of their medical information and accompanying images.

Study Design

This retrospective cohort study analyzed patients diagnosed with POAG who underwent fornix-based trabeculectomy, with or without AMT, between December 2014 and February 2019 at the Department of Ophthalmology, Pusan National University Hospital (Busan, Korea). The patients were followed up for a minimum of 12 months postoperatively. If both eyes underwent a trabeculectomy, only the first operated eye was included. The diagnosis of POAG relies on the presence of glaucomatous optic disc changes and corresponding visual field defects, as confirmed by two reliable visual field tests and an open anterior chamber angle [17]. Since August 2017, we have performed trabeculectomy with AMT on all eligible patients requiring glaucoma surgery who consented to receive AMT. Surgical indications included (1) inadequate IOP control despite maximal tolerated medical therapy, laser trabeculoplasty, or both, and (2) intolerance or allergy to glaucoma medications. The exclusion criteria were secondary glaucoma such as pseudoexfoliation syndrome, pigment dispersion syndrome, uveitis, or any other ocular or systemic disorder affecting the optic nerve, head, macula, or visual field. Patients who had undergone prior ocular surgery, except for those with uncomplicated phacoemulsification, were also excluded.

Preoperatively, all participants underwent a comprehensive ophthalmological examination, including best-corrected visual acuity assessment, slit-lamp examination, IOP measurement with Goldmann applanation tonometry, gonioscopy, dilated fundus examination, stereoscopic optic disc examination, and red-free retinal nerve fiber layer photography (AFC-210; Nidek, Aichi, Japan). Biometry was conducted using the IOLMaster (Carl Zeiss Meditec, Dublin, CA, USA), and standard automated perimetry was performed. Central corneal thickness was performed using ultrasonic pachymetry (Pachmate; DGH Technology, Exton, PA, USA), whereas keratometry was performed using an Auto Kerato-Refractometer (ARK-510A; NIDEK, Hiroshi, Japan).

Surgical Technique

Trabeculectomy using AMT is shown in Figure 1. All surgeries were performed by a single surgeon (J.L.) under local anesthesia. A limbal conjunctival incision of 5–6 mm was made to create a fornix-based conjunctival flap, followed by dissection of the conjunctiva and Tenon’s capsule toward the conjunctival sac. A trapezoidal scleral flap (basal 4.5 mm, apical 2.5 mm, bilateral 2.75 mm) with 2/3 of the scleral thickness was constructed. Surgical sponges (Eye Spear, Huizhou Foryou Medical Devices Co.) soaked in 0.4 mg/mL (0.04%) mitomycin C were placed between the Tenon’s capsule and sclera for 2.5 min, followed by irrigation of the mitomycin C-exposed area with 20 mL of balanced salt solution (BSS) upon sponge removal. Inner sclerostomy and peripheral iridectomy were then performed. The scleral flap was closed using two preplaced 9-0 nylon (Ethicon Inc., Johnson & Johnson, Somerville, NJ, USA) releasable sutures. In the AMT group, a 15 × 15 mm single layer of cryopreserved amniotic membrane (MS Amnion, MS BIO Inc., Seongnam, South Korea) with the stromal side facing up was positioned beneath Tenon’s capsule (Fig. 1a). The amniotic membrane was secured to the lateral aspect of the scleral flap using two interrupted 10-0 nylon sutures (Ethicon Inc., Johnson & Johnson) (Fig. 1b). The Tenon capsule and conjunctiva were pulled anteriorly and closed using interrupted sutures. The anterior chamber was inflated with a BSS, and aqueous outflow through the scleral flap and bleb leakage through the conjunctival sutures were performed. Postoperatively, topical eye drops were initiated, including levofloxacin (Cravit^®, Santen Pharm, Co., Osaka, Japan) four times daily and prednisolone acetate (Predbell^®, CKD Pharm, Co., Seoul, South Korea) six times daily for 1 month, with subsequent tapering over 8–12 weeks based to bleb morphology and IOP. Bleb management involves digital massage or bleb needling if inadequate bleb function is observed.

Fig. 1.

Surgical technique of trabeculectomy with AMT. a Cryopreserved amniotic membrane was peeled from nitrocellulos filter paper, and the stromal side was placed over scleral flap. b Limbal side of amniotic membrane was secured to both sides of scleral flap margin using two micropoint 10-0 nylon vascular needles.

Definition of Surgical Success and Functioning Bleb

Successful IOP control was defined as an IOP ≤18 mm Hg and a reduction in IOP of ≥30% without the need for glaucoma medications at the time of the AS-OCT examination [18, 19]. IOP was assessed at 10:00 a.m. and 5:00 p.m. on the AS-OCT examination date. If the discrepancy between these two measurements exceeded 2 mm Hg, a third IOP measurement was taken, and the mean of the three values was used for analysis [20]. If the IOP criteria for success were met without serious complication such as loss of light perception or necessitating additional glaucoma surgeries (including trabeculectomy or tube shunt surgery), this defines “success of glaucoma surgery.” In these patients, if the bleb had a clinically diffuse and healthy bleb without any signs of an encapsulated bleb, the bled was then defined as a “functioning bleb” [6].

AS-OCT Imaging

All patients underwent AS-OCT for at least 12 months after trabeculectomy. Postoperative blebs were visualized using the Anterior Segment Module (ASM, volume scan vertical-filtering blebs mode, “VolBleb”) of the Spectralis OCT (Heidelberg Engineering, Heidelberg, Germany), utilizing shorter wavelength light sources (870 nm) and higher speed (40,000 A-scans/second) compared to time domain OCT. The high-resolution “VolBleb” mode was used with enhanced depth imaging mode and automatic real time function set at 16 frames. The OCT scan pattern size was 8.3 × 2.8 mm, and the number of B-scans was 21 with a 139 μm-distance between B-scans. Penetration depth was 1.9 mm, with lateral resolution scaling of 10.84 μm/pixel and axial resolution scaling of 3.87 μm/pixel. Only images with quality scores exceeding 25 dB were included in the analysis. In cases where the scleral margin was not clearly delineated in the OCT image, manual adjustment of the contrast setting facilitated identification of the scleral edge. Intrableb structural parameters were measured using the device’s built-in software (Heidelberg Eye Version: 1.10.2.0), whereas bleb wall reflectivity was quantified using ImageJ software (ImageJ 1.50b, http://imagej.nih.gov/ij/; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD, USA) [21].

Horizontal (tangential to the limbus) and vertical (radially perpendicular to the limbus) scans were obtained at the maximum elevation of the bleb for each eye. Quantitative parameters included maximum bleb height, bleb wall thickness, striping layer thickness, striping to bleb wall ratio, fluid-filled space height and area, fluid-filled space score (FFSS), and bleb wall reflectivity (Fig. 2). The average of these measurements from both the horizontal and vertical scans was used for the analysis.

Fig. 2.

Representative AS-OCT images of fluid-filled space score (FFSS) and measurement of functioning intrableb parameters. a Fluid-filled space was diffuse and extented posteriorly beyond the field of image view, classified as FFSS 2. b Fluid-filled space was limited and demarcated with a clear posterior margin, categorized as FFSS 1. c No apparent fluid-filled space was visible, denoted as FFSS 0. White and white dotted two-wayarrows indicate the bleb wall thickness and fluid-filled space height, respectively. Whitedotted line represents the hyporeflective layers with striping phenomenon. Star denotes the fluid-filled space, while the asterisk indicates scleral flap. White dotted arrow points to the microcyst, and the white arrow indicates the visible amniotic membrane beneath the bleb wall.

The bleb height was determined as the maximal vertical distance between the initial reflective signal from the conjunctiva and a straight line perpendicular to the tangent to the sclera (Fig. 2). The bleb wall thickness, consisting of the conjunctiva, Tenon’s capsule, and/or the incorporated amniotic membrane, was measured as the maximum vertical distance between the initial reflective signal of the conjunctiva and the apex of the fluid-filled space (Fig. 2). The striping layer is characterized by multiple parallel and fluid-filled channels within the Tenon’s capsule, resembling a honeycombed structure (Fig. 2) [22, 23].

The fluid-filled space height was assessed as the maximum vertical distance within the signal void or hyporeflective area between the base of the inner bleb wall and the apex of the sclera along a straight line perpendicular to the tangent of the sclera (Fig. 2). The fluid-filled space area was defined as the maximum area of the signal void or hyporeflective area between the base of the inner bleb wall and the apex of the sclera. The FFSS was graded from 0 to 2: (1) a score of 0 indicated an undetectable fluid-filled space, (2) a score of 1 denoted a restricted and well-defined fluid-filled space with a distinct posterior boundary, and (3) a score of 2 represented a diffuse fluid-filled space extending posteriorly beyond the imaging field [4].

Bleb wall reflectivity was assessed using the ImageJ software. Elliptical markers were positioned to outline the background adjacent to the bleb wall, and three equidistant points within the bleb wall (anterior, middle, and posterior) were placed to measure the bleb wall reflectivity. The reflectivity value of the background was subtracted from the values of three points within the bleb wall and the average reflectivity value of the bleb wall was calculated [21]. Microcyst formation, defined as hyporeflective or signal void spaces located directly within or beneath the epithelial layer of the bleb wall [7].

Statistical Analysis

The normality of the data distribution was assessed using the Kolmogorov-Smirnov test. Differences between the two groups were evaluated using either the Mann-Whitney U-test or independent-sample t test for continuous variables and the chi-squared or Fisher’s exact test for categorical variables. The intrableb parameters included bleb height, bleb wall thickness, striping layer thickness, striping to bleb wall ratio, bleb wall reflectivity, fluid-filled space height, fluid-filled space area, FFSS, and presence of microcyst formation. Relationships between variables were examined using Pearson’s correlation and point biserial correlation analysis for parametric tests, and Spearman’s correlation analysis for non-parametric tests. To evaluate the differences over time, a linear regression model was used to examine the interaction effect with adjustments made to the time effect. All statistical analyses were performed using R software (version 4.0.5; R Project for Statistical Computing, Vienna, Austria). A significant level of p < 0.05 was considered statistically significant.

Results

Demographics and Clinical Characteristics in all Patients

We analyzed all eligible patients with POAG who underwent trabeculectomy and postoperative AS-OCT. Overall frequency of successful IOP control (72/85 (84.7%) in the eyes with AMT vs. 23/31 (74.2%) in those without AMT; χ² test, p = 0.274) was similar in the two groups. In the AMT group, 13 eyes were excluded for unsuccessful IOP control. Among these, the bleb morphology in these cases included clinically diffuse blebs in 3 cases, encapsulated blebs in 5 cases, and flat blebs in 5 cases. In the control group, 8 eyes were excluded due to inadequate IOP control. The bleb morphology in these cases included clinically diffuse blebs in 3 cases, encapsulated bleb in 2 cases, and avascular cystic blebs in 3 cases. In this study, all successful IOP control cases had clinically diffuse and healthy bleb without any signs of an encapsulated bleb (IBAGS, online suppl. Table 1; for all online suppl. material, see https://doi.org/10.1159/000542762). Finally, 40 eyes of 40 patients were included in this study, with 20 eyes of 20 patients assigned to the fornix-based trabeculectomy with AMT (AMT group) and 20 eyes of 20 patients to the fornix-based trabeculectomy alone (control group) (online suppl. Fig. 1). The demographic and clinical characteristics of the patients in each group are summarized in Table 1. No significant differences were observed between the two groups (all p ≥ 0.132).

Table 1.

Demographics and clinical characteristics of patients with POAG

	AMT group	Control group	p value
Eyes, n (patients)	20 (20)	20 (20)
Age, years	58.29±12.34	55.36±16.99	0.537
Sex, female	7 (35.0)	6 (30.0)	>0.999
Eye laterality, right	7 (35.0)	9 (45.0)	0.748
Diabetes mellitus	1 (5.0)	5 (25.0)	0.182
Hypertension	4 (20.0)	2 (10.0)	0.661
Preoperative lens status, phakic	7 (35.0)	8 (40.0)	>0.999
Interval between surgery and AS-OCT, year	3.05±0.86 (1.87−4.70, 2.80)	3.41±0.88 (1.37−5.50, 3.37)	0.196
IOP at AS-OCT test, mm Hg	12.40±2.93 (6−17, 12.5)	12.25±2.63 (7−18, 12.5)	0.866
Preoperative IOP, mm Hg	30.85±8.74	29.80±6.68	0.672
Preoperative medications, n	4.15±0.49	3.95±0.60	0.258
Preoperative visual acuity, logMAR	0.26±0.32	0.48±0.58	0.132
Central corneal thickness, µm	541.80±44.80	534.55±41.90	0.600
Axial length, mm	24.63±1.77	25.17±2.33	0.418
Spherical equivalent, D	−1.94±2.66	−2.96±3.09	0.270
Visual field parameter
Visual Field Index, %	52.6±27.18	45.20±32.76	0.442
Mean deviation, dB	−16.98±7.89	−19.44±8.94	0.361
Pattern standard deviation, dB	9.03±3.81	8.30±4.31	0.575

Counting fingers at 30 cm was considered equivalent to the Snellen value of 20/2,000, which corresponds to a logMAR of 2.0. Hand motion acuity was considered equivalent to the Snellen value 20/20,000, which corresponds to a logMAR of 3.0. Values are presented as mean ± standard deviation (range, median) or number (%) unless otherwise indicated. AMT, amniotic membrane transplantation; IOP, intraocular pressure; AS-OCT, anterior segment optical coherence tomography; logMAR, logarithm of the minimum angle of resolution.

Comparison of AS-OCT Images of the Two Successful Groups

The intrableb structures assessed using AS-OCT were compared between the two groups according to the AMT (Table 2; Fig. 3). No significant interaction effects were observed between the AS-OCT examination time and groups for any of the intrableb parameters (Fig. 4, all p ≥ 0.122). As AS-OCT examinations were performed at different times for each patient, adjustments were made for the effect of the interval between surgery and AS-OCT examination (Table 2).

Table 2.

Functioning intrableb parameters assessed with AS-OCT after trabeculectomy according to AMT

Intrableb parameters	Overall (n = 40)	AMT group (n = 20)	Control group (n = 20)	p value	p valuea
Bleb height, μm	1,541.29±218.28	1,501.53±256.96	1,581.05±168.66	0.255	0.352
Bleb wall thickness, μm	1,009.96±406.32	734.58±218.84	1,285.35±362.40	<0.001	<0.001
Striping layer thickness, μm	543.49±422.82	246.32±194.31	840.65±378.54	<0.001	<0.001
Striping/bleb wall ratio	0.47±0.23	0.31±0.18	0.62±0.16	<0.001	<0.001
Bleb wall reflectivity	88.71±20.81	99.90±17.88	77.52±17.49	<0.001	0.001
FFSS	1.29±0.75	1.77±0.34	0.80±0.73	<0.001	<0.001
Fluid-filled space height, μm	531.32±364.53	766.95±245.63	295.70±309.09	<0.001	<0.001
Fluid-filled space area, mm2	2.13±1.64	3.23±1.15	1.04±1.30	<0.0001	<0.0001
Microcyst formation, %	35 (87.5)	19 (95.0)	16 (80.0)	0.182	0.266

Values are presented as mean ± standard deviation unless otherwise indicated. AMT, amniotic membrane transplantation.

^aAdjusted for AS-OCT test interval.

Fig. 3.

Boxplot comparing the functioning bleb parameters assessed with AS-OCT after trabeculectomy according to AMT for the two successful groups. Except for bleb height (a) and microcyst formation (i), the functioning blebs of the control group showed significantly thicker bleb wall (b) and striping layer (c), a higher striping to bleb wall ratio (d), and lower bleb wall reflectivity (e). Functioning blebs of the AMT group had significantly greater FFSS (f), height (g), and area (h) compared to those in the control group.

Fig. 4.

Scatter plot with a simple linear regression line. There were no significant interaction effects between the AS-OCT examination time and groups in all the intrableb parameters (all p ≥ 0.122). In the graph, the red and green lines represent the AMT and control groups, respectively.

The AMT group exhibited a greater FFSS, height, and area than the control group (unadjusted, all p < 0.001; adjusted, all p < 0.001). Conversely, the control group demonstrated greater bleb wall and striping layer thickness, a higher striping to bleb wall ratio, and lower bleb wall reflectivity than the AMT group (unadjusted all p < 0.001; adjusted all p ≤ 0.001). However, no significant differences were observed in bleb height or microcyst formation between the two groups (unadjusted p ≥ 0.182; adjusted all p ≥ 0.266). Furthermore, we compared bleb morphology based on the Indiana Bleb Appearance Grading Scale and found no significant differences between the two groups in bleb height, horizontal extent, vascularity, or Seidel test results (all p ≥ 0.341) (online suppl. Table 2). Avascular cystic blebs (VO and V1 vascularity) or bleb leak (S1 and S2 Seidel tests) were not observed in this study.

Representative AS-OCT Images of Functioning Blebs according to AMT

Figure 5 illustrates the distinct characteristics of the intrableb structures in patients who underwent trabeculectomy with AMT or trabeculectomy alone. In Figure 5a, a 68-year-old man who underwent trabeculectomy with AMT in the right eye exhibited an IOP of 12 mm Hg without the need for glaucoma medications. The functioning bleb displayed a posteriorly extended fluid-filled space, low bleb wall reflectivity, and striped layers. Transplanted amniotic membranes are observed on the inner bleb wall. In Figure 5b, a 68-year-old woman who underwent trabeculectomy alone in the right eye had an IOP of 12 mm Hg without glaucoma medications. In the present case, the functioning bleb exhibited a thick wall with multiple parallel hyporeflective layers and fluid-filled channels within the bleb wall.

Fig. 5.

Representative anterior segment optical coherence tomography (AS-OCT) images of functioning bleb according to AMT. Sixty eight-year-old man (a) and woman (b) received trabeculectomy with and without AMT, respectively. At the time of AS-OCT examination, their IOP was 12 mm Hg without need for glaucoma medications. a AS-OCT image of the functioning bleb in the AMT group showed a diffuse and extended fluid-filled space. b In contrast, the functioning bleb in the control group presented a thick bleb wall with prominent multiple parallel hyporeflective layers with striping phenomenon. The white arrow heads indicate the margin of hyporeflective layers with striping phenomenon. White and white dotted two-way arrows indicate the bleb wall thickness and fluid-filled space height, respectively. White dotted line represents the hyporeflective layers with striping phenomenon, and the star indicates the fluid-filled space.

Discussion

In the present study, we evaluated distinct intrableb structures of functioning blebs according to AMT using AS-OCT in patients who demonstrated successful IOP control following trabeculectomy. Our findings revealed significant differences between the functioning blebs of the AMT and control groups, although there was no significant difference in IOP at AS-OCT examination time between the groups. Specifically, the functioning blebs in the AMT group had significantly greater FFSS, area, and height than those in the control group. In contrast, the functioning blebs of the control group showed a significantly thicker bleb wall and striping layer, a higher striping to bleb wall ratio, and lower bleb wall reflectivity. Bleb height and microcyst formation did not emerge as distinctive features of functioning blebs according to the AMT in this study. These results remained consistent even after adjusting for the effect of AS-OCT examination time.

Two prominent aqueous drainage routes, the transconjunctival and subconjunctival pathways, have been identified [3, 4, 8, 9, 22, 24, 25]. Both pathways are crucial for aqueous drainage and are associated with surgical success, regardless of AMT. However, it is speculated that a more prominent pathway may contribute to the maintenance of good IOP control with or without AMT. In our previous investigations using AS-OCT, both the transconjunctival (characterized by lower bleb wall reflectivity and microcyst formation) and subconjunctival pathways (indicated by a greater FFSS) were significantly associated with good IOP control in multivariate logistic regression analyses [3]. Moon et al. [3] noted that functioning blebs exhibit higher bleb height, thicker bleb wall, lower bleb wall reflectivity, and a higher frequency of microcyst formation than non-functioning blebs. However, the fluid-filled space parameter differed significantly between successful and unsuccessful AMT groups [3]. Therefore, we hypothesized that intrableb structures associated with good IOP control may differ in the functioning bleb according to the AMT.

The findings of this study align with this hypothesis and are consistent with previous research outcomes. The functioning blebs of the AMT group had a wide and elevated fluid-filled space, thick bleb wall, low bleb wall reflectivity with a striping phenomenon, and microcyst formation (Fig. 2a, b; 5a). These fluid field space parameters were significantly greater than those observed in the control group, indicating that the subconjunctival pathway plays a more prominent role in the aqueous drainage pathway following trabeculectomy with AMT (Table 2; Fig. 3). Nakamura et al. [4] reported that a wide subconjunctival fluid-filled space was associated with successful IOL control after trabeculectomy with AMT. Tominaga et al. [8] similarly demonstrated that eyes with posterior episcleral fluid (PEF) beyond the scleral flap exhibited significantly lower IOP than those without PEF. Additionally, Kawana et al. [25] found negative correlations between the IOP and parameters such as the vertical and horizontal lengths, height, and volume of the fluid-filled cavity. These consistent findings across studies provide further support the notion that the subconjunctival pathway plays a pivotal role in achieving successful IOP control after trabeculectomy with AMT.

The functioning bleb of the control group showed a thick bleb wall and striping layer, a high striping to bleb wall ratio, lower bleb wall reflectivity, and a relatively limited or scant fluid-filled space (Fig. 2c; 5b). These thicker or higher bleb wall parameters and lower bleb wall reflectivity were more pronounced than those observed in the AMT group, suggesting that the transconjunctival pathway plays a more prominent role as an aqueous drainage pathway after trabeculectomy alone (Table 2; Fig. 3). Moon et al. [3] reported that a lower bleb wall reflectivity alone was associated with surgical success after trabeculectomy without AMT in a multivariate logistic regression analysis. Nakamura et al. also demonstrated that eyes without AMT exhibited either no or minimal fluid-filled space and that approximately one-third of the blebs with goop IOP had a hyporeflective bleb wall after trabeculectomy alone [4]. Tominaga et al. [8] found that a thicker bleb wall with lower reflectivity was associated with lower IOP levels. Kawana et al. [25] reported that the volume of the hyporeflective area and number of microcysts were negatively correlated with IOP. These findings emphasize the significance of the transconjunctival pathway in achieving successful IOP control after trabeculectomy alone.

As described previously, both subconjunctival and transconjunctival pathways play crucial roles in aqueous drainage following trabeculectomy. However, there were distinctive signs of intrableb structures after trabeculectomy according to AMT. Amniotic membrane possesses anti-scarring and anti-inflammatory properties mediated by downregulation of TGF-β signaling pathway and inhibition of myofibroblast differentiation in fibrotic eye disease, including glaucoma filtering surgeries [10–14, 26]. The implanted amniotic membrane, characterized by its high hydraulic conductivity and semi-permeability to water, may play an essential role as a part of the bleb wall beneath the Tenon’s capsule. In the current study, 12/20 (60.0%) of the functioning blebs in the AMT group had transplanted amniotic membrane at the time of AS-OCT examination (3.05 ± 0.86, year). This can suppress the formation of avascular cystic blebs and potentially promote transconjunctival as well as subconjunctival aqueous absorption for a considerable period of time [3, 10, 16, 27].

In contrast to previous studies that evaluated the relationship between intrableb structure-function in terms of successful versus unsuccessful IOP control, this study focused on comparing the intrableb features of the functioning bleb after trabeculectomy according to AMT. Previous reports have indicated that functioning blebs exhibit higher bleb height, thicker bleb wall, greater fluid-filled space, lower bleb wall reflectivity, and more frequent microcyst formation compared to non-functioning bleb [3, 4, 6–8, 22, 25]. Our results are consistent with these previous findings regarding the intrableb structure-function relationship and reveal unique features of the intrableb structure according to AMT.

Because bleb management procedures are more effective when conducted as soon as possible, earlier detection of non-functioning blebs using AS-OCT could enable glaucoma surgeons to implement appropriate intervention after trabeculectomy [6, 28]. Waibel et al. [6] reported that signs of encapsulation were evident much earlier with AS-OCT, displaying thinner bleb wall thickness and higher bleb cavity height at 1–2 weeks postoperatively compared with functioning blebs. Narita et al. [7] found that taller blebs with thicker blebs and the striping phenomenon at 2 weeks postoperatively appeared to predict good IOP control at 1 year postoperatively. Consistent with previous studies and our own findings, early assessment of intrableb structures using AS-OCT, with a focus on parameters such as fluid-filled space in trabeculectomy with AMT, or the thickness of the bleb wall/striping layer and bleb wall reflectivity in trabeculectomy alone, can be beneficial for managing filtering blebs. Changes in these intrableb structures during the early postoperative period may serve as indicators of long-term surgical success. Further studies are required to test this hypothesis.

This study has some limitations that should be acknowledged. First, despite being a retrospective cohort analysis with no significant differences in demographic and clinical characteristics between the two groups (Table 1), there may still be a selection bias owing to the retrospective nature of the study. Second, the time interval between trabeculectomy and AS-OCT examinations varied in this study, although the difference was not statistically significant (3.41 ± 0.88 years in the control group and 3.05 ± 0.86 years in the AMT group, p = 0.196). Since previous reports have described post-trabeculectomy changes over time in AS-OCT parameters, such as the potential diminishment of the subconjunctival fluid-filled space due to the wound healing process after trabeculectomy alone, this possibility cannot be entirely excluded [4, 7]. As this was a retrospective analysis, there was no uniformity in the timing of AS-OCT evaluations among the patients. To assess the temporal disparities, a linear regression model was used to investigate the interaction effect, which revealed no significant interaction effect between time and group. Our findings remained consistent even after adjusting for the AS-OCT examination time. Furthermore, despite variations in the examination timing for each patient, AS-OCT images were obtained at least 12 months postoperatively. Third, we used a more stringent criterion for success than the TVT study [29]. Although many previous studies (including this study) and recent clinical trials have adopted a cutoff value of IOP ≤18 mm Hg based on glaucoma surgery outcomes, post hoc analyses with stricter IOP criteria may be necessary to generalize the results to patients with glaucoma [4, 9, 18, 19, 30, 31]. Finally, all patients in the study population were of Korean descent; therefore, the influence of AMT on trabeculectomy outcomes may differ in other populations.

Conclusion

Several studies have investigated the relationship between intrableb structures and surgical success. However, research on how this correlation varies according to the use of AMT in patients who achieve successful IOP control is limited. In the current investigation, aimed at evaluating the functioning bleb according to AMT, we observed that the extent of the fluid-filled space was a more discernible characteristic of the intrableb structures after trabeculectomy with AMT. Conversely, the reflectivity and thickness of the bleb wall structures were more prominent in the functioning bleb after trabeculectomy alone. Overall, our study provides valuable insights into the characteristics of functioning blebs in patients who achieved successful IOP control following trabeculectomy according to AMT.

Acknowledgment

We would like to thank Editage (www.editage.co.kr) for English language editing.

Statement of Ethics

This study was performed in accordance with the tenets of the Declaration of Helsinki and approved by the Institutional Review Board of Pusan National University Hospital (No. 2111-004-108). Written informed consent was obtained from all patients for the surgical procedures as well as for the storage of their information in the hospital database and its use for research purposes. All participants provided written informed consent for the publication of their medical information and accompanying images.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

This study was supported by a Biomedical Research Institute Grant (202200300001) from Pusan National University Hospital.

Author Contributions

Jiwoong Lee contributed to the conceptualization and was involved in writing, reviewing, and editing; Jiwoong Lee and Sangwoo Moon contributed to the methodology and data curation; and Sangwoo Moon contributed to the investigation and was involved in writing the original draft preparation. All authors have read and approved the final manuscript and agree to publish the manuscript.

Funding Statement

This study was supported by a Biomedical Research Institute Grant (202200300001) from Pusan National University Hospital.

Data Availability Statement

The data that support the findings of this study are not publicly available because they contain information that could compromise the privacy of research participants, but are available from the corresponding author (J.L.) or Pusan National University Hospital Institutional Ethics Committee IRB (mjkwon@pnuh.co.kr) upon reasonable request.