Corneal endothelial cell loss after EX-PRESS surgery depends on site of insertion, cornea or trabecular meshwork

Previously, we reported that the Ex-press® shunt (EXP) was associated with more rapid reduction in corneal endothelial cells when inserted into the cornea rather than the trabecular meshwork (TM). We compared the reduction rate of corneal endothelial cells between the corneal insertion group and TM insertion group. This was a retrospective study. We included patients who had undergone EXP surgery and were followed for > 5 years. We analyzed the corneal endothelial cell density (ECD) before and after EXP implantation. We included 25 patients in the corneal insertion group and 53 patients in the TM insertion group. One patient in the corneal insertion group developed bullous keratopathy. The ECD decreased significantly more rapidly in the corneal insertion group (p < 0.0001), in whom the mean ECD decreased from 2227 ± 443 to 1415 ± 573 cells/mm2 at 5 years with a mean 5-year survival rate of 64.9 ± 21.9%. By contrast, in the TM insertion group, the mean ECD decreased from 2356 ± 364 to 2124 ± 579 cells/mm2 at 5 years, and the mean 5-year survival rate was 89.3 ± 18.0%. The decrease rate of ECD was calculated as 8.3%/year in the corneal insertion group and 2.2%/year in the TM insertion group. Insertion into cornea is a risk factor for rapid ECD loss. The EXP should be inserted into the TM to preserve the corneal endothelial cells.


Introduction
Filtration surgery using an Ex-Press® shunt (EXP; Alcon Laboratories, Fort Worth, TX, USA) is performed worldwide and was approved in Japan in December 2011.This surgery is performed to reduce intraocular pressure (IOP) for glaucoma patients.EXP is a stainless-steel filtration device designed to shunt the aqueous humor from the anterior chamber to the subconjunctival space.This surgery is performed to reduce intraocular pressure (IOP) for glaucoma patients.EXP surgery is expected to be a minimally invasive surgery to replace trabeculectomy (Trab), and many complication studies of surgical outcomes between EXP and Trab have been reported.Across several studies, the surgical outcomes of EXP were similar to those of Trab [1][2][3][4][5].As a feature of EXP surgery, it is not necessary to remove the trabecular meshwork or resect the peripheral iris.For these reasons, the risk of vitreous hemorrhage and vitreous prolapse is low.There are several reports showing that postoperative complications are fewer with EXP than with Trab [1,4].In addition, it has also been reported that postoperative astigmatism is less prevalent and visual acuity recovers more quickly with EXP [6,7].There are also reports that EXP results in lower corneal endothelial cell loss from inflammation [8].However, there have been reports that EXP surgery led to a rapid decrease in corneal endothelial cells [9,10].In fact, we experienced a case who developed bullous keratopathy after EXP [11].
Bullous keratopathy due to corneal endothelial cell reduction is one of the serious complications of filtration surgery.The corneal endothelial cells work to maintain corneal transparency [12].Some previous studies have reported that Trab reduced corneal endothelial cell density (ECD) from 2.2 to 9.3% after two years [10,13].There are several reports of corneal endothelial cell loss after EXP surgery [8-10, 14, 15], but these previous reports have shown large differences in the reduction ratio of ECD.The factors that reduce ECD are not known in detail.We previously investigated the factors affecting reduction in corneal endothelial cells after EXP [15] and found that insertion of EXP into the cornea causes a faster decrease than insertion into the trabecular meshwork (TM).
We followed patients who underwent EXP surgery for more than 5 years and measured the density of their corneal endothelial cells.There has been no previous report with long-term data of ECD loss after EXP.We compared the reduction rates of ECD for the corneal insertion group and TM insertion group.

Patients
This was a retrospective single-facility study.We included consecutive glaucoma patients who underwent EXP surgery for the first time at Toyama University Hospital and were followed for > 5 years.EXP is contraindicated for use in patients with uveitis or primary angle closure glaucoma because these diseases pose a risk of EXP obstruction due to the transformation of peripheral anterior synechia.As a result, we performed EXP surgery for patients with primary open-angle glaucoma (POAG) or pseudo-exfoliation glaucoma (PEXG).In patients who underwent binocular surgery, we used unilateral data from the eye that was operated on first.We excluded patients who had a history of conventional trabeculectomy, laser iridotomy, peripheral iridotomy, long tube shunt surgery or keratoplasty.We excluded who had corneal disease as corneal dystrophy.We included patients with a history of cataract surgery, trabeculotomy (TLO) (including the Trabectome® procedure, a canaloplasty) or vitrectomy.Finally, we recruited 78 eyes to the study.
All subjects were recruited during the period from September 2012 to March 2017.All patients underwent a comprehensive ophthalmic examination including refraction (KR-800PA; Topcom, Tokyo), Goldmann gonioscopy (AU-910-4(O4GFA); Ocular; Washington, USA), Goldmann applanation tonometry (GAT) (AT900; HAGG; Bern, Switzerland), a fundus examination, automated perimetry (Humphrey Field Analyzer; Carl Zeiss Meditec, Dublin, CA), measurement with optical coherent tomography (OCT) RS-3000 (Nidek; Aichi, Japan) and measurement of the central corneal thickness (CCT) with AS-OCT (CASIA SS-1000; Tomey, Nagoya, Japan).Orthoptists helped these examinations.We confirmed the diagnosis of glaucoma from these test results.The IOP was measured using GAT.The patients were already taking glaucoma medications, if tolerated, but required further treatment to lower their IOP due to the progression of their visual field disorder.This study is retrospective and did not use specific surgical criteria for glaucoma and cataracts.Surgical indications were decided by the judgment of one glaucoma specialist (N.T).The preoperative (baseline) IOP was defined as the mean of two measurements with Goldmann applanation tonometry (GAT) at the patient's visits immediately before surgery.
The research protocol was approved by the Institutional Review Board of the University of Toyama, and the procedures used conformed to the tenets of the Declaration of Helsinki.After the nature and possible consequences of the study were explained to the patients, written informed consent was obtained from all participants in the study.

Surgical techniques
All operations were performed by one surgeon (N.T.).The surgeon in this study has abundant experience of EXP surgery.Retrobulbar anesthesia was administered.A standard fornix-based conjunctival incision was made to gain access to the scleral bed adjacent to the limbus.A single 3.5 × 3.5 mm square scleral flap was created at superior nasal or superior temporal.Mitomycin C (MMC) solution (0.04 mg/ ml) was applied below the conjunctiva and below the scleral flap for 4 min.At this point, the eye was a completely enclosed space, and thus the MMC solution could not flow into the anterior chamber.The treated area was then irrigated with approx.100 ml of balanced salt solution.If the patient required simultaneous cataract surgery, the cataract surgery was performed at this time.Phacoemulsification was performed with a WhiteStar Signature system (Abbott Medical Optics, Santa Ana, CA), and the intraocular lens (IOL) was implanted from a clear temporal cornea.Since this was a retrospective study, no clear criteria for surgery were established such as VA or Emery grades or age; rather, cataract surgery was performed according to the judgment of the operator.
The scleral flap was lifted, and a 25-ga.needle was horizontally inserted into the anterior chamber at the surgical limbus from the sclera-cornea transition zone parallel with the iris to create a path for the EXP (model P50).The EXP shunt was then inserted into the anterior chamber.We did not use gonioscopy intraoperatively.The scleral flap was sutured using 10-0 nylon, and the tension on the sutures was adjusted to maintain the anterior chamber depth with a slow flow of aqueous humor around the margins of the scleral flap.Most of the cases received 2 sutures.The conjunctiva was meticulously closed with 10-0 nylon sutures.We confirmed that there was no leakage from the blebs.

Postoperative treatment
The postoperative treatment protocol was the same in both groups.The postoperative treatments consisted of topical steroids, antibiotics, and non-steroidal antiinflammatory drugs (NSAIDs).The antibiotics were applied for 4-6 weeks.The steroid and NSAID doses were reduced over the 12-week period after the intervention.After surgery, glaucoma medications were stopped in all cases.Glaucoma medications were later added back at the discretion of the physicians.We counted a compounded agent as two medications.We performed laser suture-lysis or needling when the operator determined that these treatments were necessary.

Grouping
We characterized the insertion position of EXP as corneal insertion or TM insertion, and divided subjects into two groups accordingly.We defined a case as TM insertion if any part of the EXP touched the TM (Fig. 1); and defined the remainder as corneal insertion.We confirmed the insertion position of the EXP with a Gonio angle mirror at 3 months after surgery.

Evaluation of reduction rate of ECD
We examined the ECD with an EM-4000 specular microscope (Tomey Corp., Nagoya, Japan) that could calculate the value of the density automatically.We measured ECD every 6 months after surgery.The values were measured just once before surgery and once after, and do not represent an average of multiple measurements.We measured only the central ECD.We defined the reduction rate of ECD as the ratio of post-operative ECD from preoperative ECD.We excluded the data of patients who underwent additional surgery after EXP.

Statistical analysis
We used a paired t-test and Fisher's exact test for comparisons between two groups.We used a Wilcoxon signed-rank test to compare preoperative and post-operative IOP, glaucoma medication and ECD.We used JMP Pro 14 software (SAS, Cary, NC) for all statistical analyses.Significance was defined as a p-value < 0.05.

Ophthalmic data
The surgeries were conducted without intraoperative complications such as expulsive hemorrhage.There were two cases who could not be inserted EXP.These two cases underwent conventional trabeculectomy.These cases were excluded from this study.Finally, we analyzed 78 eyes.Table 1 shows the analyzed ophthalmic data: patient age, central corneal thickness (CCT), insertion position (cornea insertion/TM insertion), history of TLO (Yes/ No), type of glaucoma (POAG/PEXG) and surgical method (EXP surgery alone/simultaneous cataract surgery).There was no case in which the EXP was inserted into the ciliary body.There were no factors significantly different.Post-operative IOPs are summarized in Table 2. Post-operative IOP was  3.

Evaluation of ECD
One case of the corneal insertion group developed bullous keratopathy.After 1 year, the ECD in that eye could not be measured.The results of the mean ECD values are shown in Table 4 and Fig. 2. The mean ECD gradually decreased and showed a significantly greater decrease in the corneal insertion group than the TM group at all points of the followup period (p < 0.0001).The mean survival ratios of the ECD values are shown in Table 5.The mean survival ratio was significantly lower in the corneal insertion group at all points of the follow-up period (p < 0.0001).The decrease rate of ECD was calculated as 8.3%/year in the corneal insertion group and 2.2%/year in the TM insertion group.

Discussion
In this study, we excluded patients who underwent additional glaucoma surgery.In other words, the IOP data only pertained to cases with good postoperative IOP that did not require additional glaucoma surgery.On average, EXP decreased the IOP from 23.9 to 11.4 mmHg.The cornea insertion group showed a faster ECD loss than the TM insertion group.In our study, EXP surgery with corneal insertion showed an ECD decrease of 35.3% after 5 years, while that with TM insertion showed a 10.7% decrease.[13].PEXG has been shown to be a factor of rapid ECD loss [16].We included PEXG patients in our study.The mean ECD survival ratios of POAG and PEXG after 5 years were 84.5 ± 27.3 and 79.1 ± 22.4%; the difference was not significant (p = 0.292).In TM insertion, our result after 2 years was 4.2%.This result seemed comparable to Trab.Li et al. reported that minimally invasive surgery causes less inflammation and had less effect in ECD [17].EXP surgery is a minimally invasive glaucoma surgery and might have less effect on ECD loss than Trab.
Soro-Martínez et al. reported that Trab simultaneous cataract surgery causes more ECD loss than Trab alone [18].In this study, the mean ECD survival ratio of EXP alone and EXP and simultaneous cataract surgery after 5 years were 80.1 ± 21.6 and 84.1 ± 23.7%, respectively, with no significant difference (p = 0.460).Kasahara et al. reported that Trabec-tome® surgery decreased ECD by 2.4% after one year [19].The mean 5-year ECD survival ratios for groups with and without a history of TLO were 79.8 ± 20.5 and 80.1 ± 22.9%, respectively, with no significant difference (p = 0.292).
The reason that corneal insertion of EXP resulted in faster ECD loss was not clear.Dahlan et al. reported that the presence of a foreign body in the cornea caused rapid ECD loss [20].Alfawaz et al. reported that anterior segment inflammation adversely affects the ECD [21].As a foreign body, EXP might cause chronic inflammation and rapid ECD loss [22].The mechanisms of ECD reduction after EXP surgery might be multifactorial.Other possible mechanisms include abnormal outflow [23].
Our study has some limitations.This is a retrospective single-facility study.The technical impact of the surgery cannot be ignored.We included patients who had undergone cataract surgery and TLO, surgeries associated with ECD loss [19,24].We included patients who underwent Ex-press surgery with simultaneous cataract surgery.Cataract surgery could decrease the ECD [25].In this study, there was not significantly different ECD reduction ratio between Ex-press surgery alone and Ex-press surgery with simultaneous cataract extraction in both groups.(TM insertion group; p = 0.976, Corneal extraction group; p = 0.860) The measurement site of the ECD at the central cornea was not identical in all cases, and the value of the ECD might be different for each measurement.We measured the ECD only once at the central cornea.It would have been better to take several measurements and use the average value of ECD.We did not analyze the anterior chamber configuration.We did not measure the distance from the limbs or the insertion angle of the EXP.These factors might affect the ECD loss.There are many factors for effect to ECD, as the effect of glaucoma medications (carbonic anhydrase inhibitor), peripheral anterior synechia, depth of anterior chamber and phaco-power.We did not consider these factors.Finally, the number of patients was limited.
In conclusion, insertion of EXP into the cornea was a risk factor for rapid corneal endothelial cell loss.If the EXP is inserted into the cornea, it is difficult to reinsert it into the TM.Thus, surgeons need to be very careful regarding the position of the insertion.

Fig. 1
Fig. 1 Example of the insertion position of the EXP device A The EXP is inserted into the trabecular meshwork (TM).B The EXP is inserted between the cornea and the TM.We included cases in which the EXP has even slight contact with the TM in the TM insertion group.C The EXP is inserted into the cornea

Table 4
The mean postoperative ECD ECD: endothelial cells density, TM: trabecular meshwork

Table 5
[8]]mean survival rate of ECDArimura et al.reported that the mean ECD decrease at 24 months was 18.0%[10].On the other hand, Ishida et al. reported a mean ECD decrease at 24 months of 4.0%[9].Omatsu et al. reported that Ex-Press® surgery decreased the mean ECD from 2377 to 2317 after 2 years[8], only 2.5%.In short, the results of previous studies have been very different.One reason for the large variations among these studies might be the differences in the insertion position of the EXP.In our study, the decrease was 15.1% in the corneal insertion group and 4.2% in the TM insertion group.Aoyamaet al. reported that EXP decreased ECD by 1.8%/year after 3 years [14].Our 5-year result was 2.2%/year in the TM insertion group.This study does not compare ECD loss between EXP and Trab.Arimura et al. reported that Trab decreased the ECD by 2.2% [10].Higashide et al. reported that Trab decreased ECD by 4.8% in case of POAG and 18.2% in the case of PEXG