A Predictive Test of Outflow Enhancement by Ab Interno Trabeculectomy

Purpose: To investigate trabeculopuncture (TP) for predicting the outcome of ab interno trabeculectomy (AIT). AIT is an effective, low-risk procedure for open angle glaucoma. Despite widespread utilization, it fails in patients with an unidentified distal outflow resistance. Methods: We bisected 81 enucleated porcine eyes and perfused them for 72 hours. They were assigned to two groups: trial (n=42) and control (n=39). Intraocular pressure (IOP) was measured continuously. At 24 hours, four YAG-laser trabeculopunctures on the nasal trabecular meshwork were performed, followed by a 180° AIT at the same site at 48 hours. Eyes were divided into TP and AIT responders and non-responders; the proportion of TP responders between both AIT groups was compared. Results: Both post-TP and post-AIT IOPs were lower than baseline IOP (p=0.015 and p<0.01, respectively). The success rates of TP and AIT were 69% and 85.7%, respectively. The proportion of TP responders among AIT responders was greater than that of AIT non-responders (p<0.01). Sensitivity and specificity values of TP as predictive test for AIT success were 77.7% and 83.3%, respectively. The positive and negative predictive values were 96.6% and 38.5%, respectively. Conclusion: A 10% reduction in IOP after TP can be used as predictor for the success (>20% IOP decrease) of 180° AIT in porcine eyes.


Introduction
Intraocular pressure (IOP) reduction is the only treatment for glaucoma 1 demonstrated to be effective with high-quality level I evidence. [2][3][4] Selective laser trabeculoplasty, now a recommended first line of treatment for most open angle glaucomas, 5 and medications may achieve the desired IOP levels in many patients, but about 50% still require surgery eventually. 6 Traditional glaucoma filtering surgeries 7 lower the IOP by bypassing the trabecular meshwork (TM) and draining the aqueous humor into a newly created epibulbar space 8 , but they are associated with a high rate of complications and require intensive postoperative care. 9,10 Canal-based minimally invasive glaucoma surgeries produce far fewer complications and allow to intervene earlier 11,12 because they lower the IOP by bypassing or removing the TM to enhance the physiological aqueous humor outflow route. 11,13,14 Leading modalities are trabecular bypass stents (TBS) [15][16][17] and ab interno trabeculectomy (AIT) in which the TM is either ablated, 18 incised, 19 or excised. 20 However, approximately 30% of patients experience an insufficient IOP reduction. 21 One would expect the IOP to be close to the level of episcleral venous pressure, approximately 8 mmHg but this pressure can rarely be achieved 21 due to an increased post-trabecular resistance. 22,23 So far, there is no presurgical test that could assess the post-trabecular resistance predict the outcome of AIT.
A noninvasive procedure that allows aqueous to bypass the proximal outflow resistance at least temporarily is ND:YAG laser-assisted trabeculopuncture (TP). Not unlike trabecular bypass stents, TP creates a focal opening through the trabecular meshwork (TM) and inner wall of Schlemm's canal (SC). 24 In 1985, Epstein et al. investigated TP as a treatment for glaucoma but the subsequent IOP reduction was short-lived. 24 Here, we hypothesized that TP could be used to assess the distal outflow tract function before AIT or TBS are considered. We deployed our porcine anterior chamber ex vivo perfusion model 25,26 to develop a predictive test and address this unmet need.

Study Design
In total, 81 hemisected, perfused porcine eyes were assigned to one of two groups: trial (T) (n = 42) and control (C) (n = 39). Eyes in the trial group underwent trabeculopuncture using a Nd:YAG laser 24 hours after incubation, followed by ab interno trabeculectomy a day later. The IOP was measured continuously for 72 hours, with baseline values being recorded 24 hours (IOPBL) after the start of the experiment. Post-trabeculopuncture IOP (IOPTP) was measured 48 hours and post-ab interno trabeculectomy IOP (IOPAIT) at 72 hours.
Eyes in the control group did not undergo any procedures, but were incubated and monitored similarly for 72 hours.

Preparation and Incubation
Freshly enucleated porcine eyes were obtained from a local abattoir (Landschlachterei Issing, Retzbach, Bavaria, Germany) and processed within three hours postmortem. Institutional Animal Care and Use Committee review was waived because animals were not being sacrificed for the purpose of

Nd:YAG-Laser TP and AIT
After perfusion for 24 hours, the anterior segments were removed from the dish and four evenly spaced trabeculopunctures were placed along the nasal 180° of trabecular meshwork using a Qswitched Nd:YAG laser (VISUALS YAGIII, Zeiss, Oberkochen, Germany). Fifteen shots with an energy of 7-10 mJ were applied for each puncture. A 180-degree ab interno trabeculectomy was performed 24 hours later along the same nasal180° of trabecular meshwork. [26][27][28]

Histology
We obtained sagittal sections before TP as well as after TP and AIT and fixed them with 4% paraformaldehyde in PBS for 24 hours. After rinsing them three times in PBS, they were embedded in paraffin, sectioned at 6-micron thickness, and stained with hematoxylin and eosin.

Statistical Analysis
Our sample size calculation indicated a minimum requirement of 35 eyes per group to achieve a testing power of 0.9. We analyzed data with SPSS Statistics (Version 26, IBM, New York, USA). Means and standard deviations were reported for all parameters. We tested for normal distribution with the Kolmogorov-Smirnov test and used a paired t-test or a Wilcoxon Signed Rank test to compare dependent means; an unpaired t-test or Mann-Whitney U test was used for independent means. Oneway repeated measures multivariate analysis of variances (MANOVA) was used to compare more than two means. TP and AIT success were defined as a decrease of 5% and 10% from baseline IOP, respectively. We used the Fisher's exact test to compare the number of eyes that responded to TP and AIT to the number of eyes that responded to TP but failed AIT. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated. A receiver operating characteristic curve (ROC curve) was plotted for IOP reduction after TP and AIT success. For all tests, a p-value of 0.05 or less was considered statistically significant.

Results
Pilot experiments with whole eyes showed that a transcorneal TP using a Ritch trabeculoplasty lens could not be accomplished reliably. Therefore, anterior segments were inverted and the TM to be lasered directly. The TM could be readily identified (Figure 1, BL1). The procedure resulted in small, well circumscribed pits of approximately 500 µm in length and 250 µm in depth (Figure 1, TP1). No obvious damage to adjacent tissue was detected upon inspection with a microscope. AIT removed the TM extensively, leading to a narrow continuous groove along the nasal quadrants (Figure 1, AIT1).    (Figure 3).
A subanalysis showed a mean IOPBL of 17.1±4.4 mmHg and 11.9±2.7 mmHg for AIT responders and AIT non-responders, respectively. These values differed significantly (p = 0.008). There was no difference in IOPTP and IOPAIT in both subgroups (p = 0.48 and p = 0.45, Table 3). Out of all 13 TP nonresponders, 76.9% (n = 10) showed an IOP increase after TP of at least 10%.

Discussion
In this paper, we used a porcine anterior segment model to assess the utility of TP in predicting the success of AIT. The lack of good predictive tests combined with the relatively high rate (30%) of canalbased surgeries and implants led us to explore simple options to avoid unnecessary surgeries. Porcine eyes have been used as a model to study glaucoma extensively and were used here as well for that reason. 29,30 We expected TP to cause an IOP drop in our porcine anterior segment perfusion model, similar to that reported in human eyes. TP is not unlike trabecular bypass stents, which also cause a focal opening in the TM, and increase outflow in our model. 17 The amount of total energy used was higher than what is normally required in humans due to an at least three times thicker TM compared to human eyes. 30 After AIT, a further decrease in IOP was seen because of the comprehensive excision of the nasal TM. Compared to a study by Dang et al., which tested outflow enhancements of three different AIT devices on porcine eyes, we found similar baseline IOP values (16.35±4.52 mmHg vs 15.93±2.08 mmHg) 26 but observed a post-AIT IOP decrease by only 31%, in contrast to 48% as reported in that study. 26 The control group in our cohort experienced a small IOP increase by approximately 13% over 72 hours. This is in line with Dang et al. who observed a 10% IOP increase in control eyes during 72 hours of incubation. 31 To adjust for this, we chose 5% and 10% to be a satisfactory post-TP and post-AIT IOP reduction, respectively. These values correspond to a 10% and 20% IOP reduction after each of these procedures. Clinically, a 20% post-AIT IOP reduction is often regarded as sufficient for mild to moderate glaucoma. 32 Being able to predict this outcome will help avoid unnecessary procedures and decrease the burden on the healthcare system.
Interestingly, our subanalysis revealed that AIT responders had a higher baseline IOP compared to non-responders. This is perhaps to be expected, as AIT generally caused a greater decrease in eyes with higher baseline IOPs in clinical studies. 33 AIT non-responders also had a higher mean IOP after TP, which did not reach significance with the number examined here. It is possible that the collapse of laser-induced trabeculopunctures temporarily decreases outflow facility as described before, 34,35 which could similarly affect our non-TP responders if their TM was compromised in the area of TP and AIT. Additionally, there was no difference between baseline and post-AIT IOP levels in non-responders, which is indicative of a post-trabecular meshwork resistance in these eyes. These were not glaucomatous eyes, however. Ocular hypertension can be induced experimentally in pig eyes 36 but does not occur in pigs naturally. We suspect that inadvertent compression of key elements of the distal outflow tract in the nasal quadrants by the compression ring of the perfusion mount is responsible for this. This would not necessarily lead to an increased IOP because at least 3/4th of the outflow tract has to be compromised 37 , but it can explain the failure to respond to TP and AIT. One has to be careful interpreting the results of this subanalysis, however, as there were only six AIT non-responders in our study.
A simple and noninvasive predictive test for canal-based surgeries that ablate, excise, disrupt or bypass the TM is urgently needed because of the rapidly increasing demand for these procedures.
The implementation of Nd:YAG laser-TP for such a test is straightforward as this device is ubiquitously available in ophthalmology practices and clinics and most ophthalmologists are familiar with its use. 38 Although the effect of TP is too short-lived to be useful for glaucoma treatment, 39,40 it is precisely this benign nature that may afford a low-risk test of distal outflow resistance.
The positive predictive value (PPV) of 96.6% for TP as a test for AIT success is promising.
Moreover, our sensitivity and specificity values of 77% and 83%, respectively, are sufficient for a clinical test. Our data did not show a high negative predictive value (NPV, 38.5%) in porcine eyes, however, because these are nonglaucomatous eyes. The NPV in human eyes should be higher, matching the AIT failure rate caused by a presumed higher rate of posttrabecular resistance than in pigs.
One limitation of our study is the ex-vivo setting. Hence, wound healing of the TM and its effect on IOP cannot be observed. Another limitation is the anatomical difference between porcine eyes and human eyes. In porcine eyes, the outflow tract consists of an angular aqueous plexus, whereas humans have a Schlemm's canal often with a single lumen. 41,42 We used four evenly spaced TPs over the nasal angle not only to cover the extent of an AIT but also because of the decreased circumferential flow compared to a human Schlemm's canal. Clinically, reflux of blood from SC can normally be seen after a TP, a useful indicator of completion that is absent in an ex vivo model. We had to use the IOP decrease and an increased outflow of fluorescent beads as an indicator instead.
In conclusion, a 10% IOP reduction after trabeculopuncture can be used to predict a successful ab interno trabeculectomy in porcine eyes.    Fig. 1 Visualization of the effects of trabeculopuncture (TP) and ab interno trabeculectomy (AIT)