Preservation of the Tenon’s capsule and Sub-Tenon’s space after the autologous limbal-conjunctival transplantation in pterygium surgery

Background No previous studies have evaluated the scarring of the conjunctiva and Tenon’s capsule (TC) separately when only the conjunctiva is removed, and TC is preserved in the upper part of the bulb: the site designated for trabeculectomy. Thus, this cohort interventional study aims to investigate the scarring of the donor area after autologous limbal-conjunctival transplantation (ALCT) using anterior segment optical coherence tomography (AS-OCT). Methods Twenty-three eyes with pterygium were submitted to resection and local reconstruction with ALCT. AS-OCT images obtained from each eye preoperatively served as postoperative control. ALCT was removed superiorly with preservation of TC. The cure of this donor area occurred by secondary intention. AS-OCT was performed preoperatively and 30 and 180 days postoperatively in the donor area, measuring the thickness of the conjunctiva (epithelium and stroma) and TC. The sub-Tenon’s space (STS) was clinically assessed. Results The mean thickness of the conjunctival epithelium was 48.04 ± 11.37µm in the preoperative period and 51.87±15.04µm 180 days after surgery, without statistically signicant difference (P=0.282). A statistically signicant increase (P=0.005) in the mean thickness of the conjunctival stroma, from 85.35±23.10µm in the preoperative period to 101.61±20.19µm 180 days after surgery was found. TC had no signicant changes, slightly increasing from 117.13±24.26µm preoperatively to 118.09±19.24µm (P=0.808) 180 days after surgery. STS was found in 19 (82%) eyes 180 days after surgery. Conclusions Following ALCT with TC preservation, scarring took place in the conjunctiva, epithelium, and stroma. The TC and the STS were preserved not showing any signicant changes.


Background
Pterygium is one of the most prevalent eye diseases in tropical regions [1]. It is mainly treated with surgery, and its recurrence is reduced via an ALCT [2][3][4]. The donor area for ALCT is the superior bulbar conjunctiva, region often used in trabeculectomy (TRAB). TRAB creates a new pathway for aqueous humor drainage. The aqueous humor is diverted into the STS via a trabecular-scleral stula [5,6]. Closure of this stula by brotic tissue is the most common cause of TRAB failure [7]. Several studies have focused on the scarring of the conjunctiva and TC, characteristically a mobile connective tissue situated below the conjunctiva [5,[8][9][10][11][12]. TC has a dense amorphous histological structure and contains broblasts [13][14][15][16][17]. Being one connective tissue, TC heals completely by brosis, [7,14,19,20] compromising its mobility and providing poor TRAB outcomes. Furthermore, the TC adheres only weakly to the episclera, and is separated from it by the STS [20].
Regarding the increased risk of TRAB failure in eyes with a history of surgery, this cohort interventional study aims to assess, using AS-OCT, scar evolution in the donor superior bulbar conjunctiva, TC, and STS, of which the latter two are not excised during ALCT [12,21,22]. From the best of our knowledge, this work is the rst to assess the preservation of TC and STS at the tissue donor site.

Methods
All patients with pterygium examined in our o ce between May 2017 and May 2018 were invited to participate. Surgical procedures were performed by the same surgeon (FSV) during the same period. The inclusion criteria were age over 25 years and primary or recurrent pterygium in eyes with an intact superior limbal-conjunctival donor area. No restrictions were placed in the length of the pterygium.
Exclusion criteria were recurrent pterygium in which the superior limbal-conjunctival region had been used as a donor in a previous surgery; eyes with diseases other than pterygium; very small or asymptomatic pterygium; patients with diffuse or sectoral limbic failure involving the superior limbal area; chronic in ammation from other etiologies not related to the pterygium itself; patients who failed to attend all postoperative assessments. Clinical evaluation in slit lamp and digital photography Patients were clinically evaluated pre-and postoperatively by the same surgeon (FSV) for the biomicroscopic variables in the superior part of their eyeballs. In the pre-and postoperative visits (1, 7, 30, and 180 days after surgery), photographs of donor area were taken without ash at 10-x and 16-x magni cation using a camera (Nikon ® ; Nikon Imaging Japan Inc.) attached to a slit-lamp (HR Asapt; ® São Paulo, Brazil). We assessed: (1) conjunctival hyperemia (present or absent); (2) vessel path alteration in the donor area (congestion, path distortion, and variation in the number of vessels; their presence or absence, only considered ≥7 days after surgery.

Assessment of conjunctival mobility
It was assessed by the surgeon (FSV) using a cotton swab after instilling eye drops of 0.5% proxymetacaine (Anestalcon ® ; Alcon ® , São Paulo-SP, Brazil). Conjunctiva was slightly pressed and displaced in the vertical and horizontal directions. Mobility was present when tissue displacement was seen. Data was recorded in a speci c research protocol.

Assessment by external examiners
Six months after surgery, another clinical slit-lamp assessment of patients was performed by two ophthalmologists, glaucoma specialists, with over ten years of experience in TRAB. They were unaware of the preoperative presentation of patients and the scarring evolution up to examination. They answered questions about (1) observation of blood vessels from the ocular surface towards the limbus (presence or absence of changes such as congestion, path distortion, and variation in the number of vessels), (2) tissue mobility in the donor and recipient areas (presence or absence, assessed by touching with a cotton swab, and (3) the viability of surgical reintervention at the site, including TRAB.

AS-OCT Evaluation
AS-OCT exams were performed one day before the pterygium excision and, 30 and 180 days afterwards, using the Optovue-Avanti ® device (Optovue Inc., Fremont, California, USA) with the lens as the anterior segment. This device acquires images at a speed of 70,000 A scans/second and an axial resolution of 5µm [24]. Conjunctival thickness and TC measurements were assessed based on the study by Howlett et al. who evaluated the conjunctiva-Tenon's capsule complex in the superior bulbar conjunctiva [25].
The following pre-and postoperative measurements were compared ( To determine the site where TRAB trans-scleral stula normally drains the aqueous humor, usually 3 mm from the limbus, the beginning of the epithelial limbus was marked, coinciding with the end of Bowman's layer [26]. This point of reference is accurate, unlike the transition from the epithelium limbus to the conjunctival epithelium, which is not a speci c anatomical marker reproducible in all eyes. Measurements were taken 4 mm from the beginning of the limbus, instead of 3 mm from the end of the limbus (Fig. 2*).
These measurements were performed using the imaging software built into the device. Each structure of interest was determined, and the thickness was recorded. The inbuilt software also obtained comparative AS-OCT pre-and postoperative images (Fig. 3).

Sample calculation
Sample calculation was determined using the conjunctival epithelium and conjunctival/TC stromal thickness from our pilot study, which used data from the rst 10 patients and had a normal distribution.
Standard deviation values of the conjunctival epithelium and conjunctival/TC stromal thickness were 7.35µm and 32.61µm, respectively. These values were similar to those reported by Zhang et al. [27,28] involving AS-OCT of the bulbar conjunctiva. Thus, the standard deviation values considered for the sample calculation were those obtained by Zhang et al. of a conjunctival epithelial thickness of 7.4µm and a conjunctival/TC stromal set of 32.5µm. With a sample of at least 23 eyes, a statistical power of 90% would be obtained in detecting the differences when comparing the pre-and postoperative mean values of the conjunctival epithelial thickness, as well as of the stromal/TC complex.

Statistical Analysis
A descriptive analysis was performed by evaluating the mean and the standard deviation of variables with normal distributions. The Shapiro-Wilk test was used to verify the hypothesis of normality for continuous variables in micrometers (µm). Paired Student's t-test was used to compare the mean pre-and postoperative measurements of conjunctival epithelium thickness and the conjunctiva/TC stromal complex thickness.
Analyses were performed using the SPSS ® Software (IBM, Chicago, IL) version 18, with a level of statistical signi cance of P<0.05.

Results
Thirty-two patients agreed to participate, but only 22 had all exams and postoperative assessments, yielding a total of 23 eyes (one patient had both eyes operated). Ten patients were excluded because they did not complete the follow-up. Among patients, 65% were female and 12% had recurrent pterygium. Patients' age ranged from 31 to 67 years, mean age of 49.0±11.8.

Vascular changes
It was analyzed seven days after surgery. Vascular changes were compared between pre-and postoperative photographic images from the donor area. Vascular changes were seen in 22 (95.6%) eyes seven days after surgery (Fig. 4C). Thirty days after surgery, they were seen in 23 (100%) eyes (Fig. 4D).
After 180 days, they were seen in six (26%) eyes, while the remaining 17 (74%) had a vascular path similar to their respective preoperative one ( Figure 4E).

AS-OCT Evaluation
We made a statistical comparison between the mean thicknesses of the conjunctival epithelium, conjunctival stroma, and TC preoperatively with those 180 days after surgery (Chart 1). The same comparison was made using charts between the mean thickness in the preoperative and postoperative periods over 6 months (Charts 1.a, 1.b, and 1.c). Chart 1.a shows a small variation in the means, with an overlapping con dence interval. There was no statistically signi cant difference (P=0.282) between the mean conjunctival epithelium measurements in the preoperative (48.04±11.37µm) and postoperative periods (51.87±15.04µm). Chart 1.b shows a signi cant difference between the means of the pre-and postoperative measures of the conjunctival stroma (P=0.005). An increase is seen in the mean from 85.35±23.10µm in the preoperative period to 101.61±20.19µm after six months. Chart 1.c shows a difference in the mean TC thickness in the preoperative period (117.13±24.26µm) and that measured 180 days after surgery (118.09±19.24µm). This increase in the postoperative period did not have a statistically signi cant difference (P=0.808). These anatomical structures were assessed for changes in the presence or absence of the STS. In the postoperative period, the STS was preserved in 19 (82.6%) eyes.

Discussion
In the present study, clinical assessments were performed to identify signs of brosis or scar distortion in the region of ALCT. Hyperemia in the donor area and vascular changes, which were seen during the scarring from seven to 30 days after surgery in all 23 eyes, did not persist after 180 days after surgery. This suggests that its occurrence was due to the scarring in ammatory phase in the donor conjunctiva, without residual brosis after the end of scarring. The mobility of the conjunctiva-TC complex was normal in all eyes in the pre-and postoperative period in the donor area. This preservation of tissue mobility indirectly indicated that TC and STS showed no scarring adhesions to episclera and sclera. Therefore, it is important to highlight that the healing of the donor area occurs by secondary intention, without approaching its edges.

The external examiners
When evaluating the donor area six months after ACLT, external examiners identi ed no signs of brosis, except for one patient who had the largest pterygia. The donor area was considered feasible for surgical reintervention, including TRAB, in all eyes, except for one eye.

AS-OCT
The images, measured pre-and postoperatively by AS-OCT, showed preserved TC and STS in most patients. Changes in thickness and re ectivity only occurred in the conjunctival stroma in most eyes. Consequently, this suggests that the in ammatory scarring reaction occurred in the conjunctival stromal level without compromising the deep levels. Comparing clinical ndings with those obtained by AS-OCT, it is observed that there exists correlation of the preserved conjunctiva-Tenon's mobility. By preserving TC, the brous reaction of the deep layers is prevented, and therefore, its preservation acts as a shield for the STS. Thus, the three tissue levels above the STS, as seen in AS-OCT, showed distinct behaviors after conjunctival donation. The conjunctival epithelium regenerated through its stem cells, resuming its preoperative appearance and measurements. The conjunctival stroma showed changes resembling brosis, such as the increase of its re ectivity and its thickness. The TC remained unchanged in most eyes. Preservation of the TC in the perioperative period probably prevented the in ammatory scar response in this tissue as well as brosis and adhesions to episclera, and also preserving the STS. Given that the superior bulbar region must scar well to allow for surgical treatment of glaucoma, it is not surprising that several studies have addressed this topic [5,7,8,10,12,29,30]. However, most of these studies do not clearly distinguish between the conjunctiva and the TC. The TC was often identi ed as subepithelial tissue or subconjunctival space [5,10]. Nevertheless, the trans-scleral stula produced by TRAB releases aqueous humor into the sub-Tenon's region, not into the subconjunctival region [6]. In other words, the aqueous humor is released between the TC and episclera and not between the conjunctiva and TC. In studies analyzing the association between TRAB failure and the previous surgery, surgeries involved the incision of the conjunctiva, the TC, and the episclera, leading to brotic adhesions in the TC and episclera and there is no STS in the area where the surgery was performed [8,10,31]. Broadway et al. concluded that TRAB failure was more common in patients undergoing procedures after previous conjunctival management [10]. However, surgeries considered by these authors in 1998, such as cataract and retinopexy, were trans-scleral procedures. They did not distinguish between structures and considered conjunctiva and CT, collectively, as the conjunctiva. Gozawa et al. [31] used AS-OCT in the superior bulbar region in patients submitted to trans-scleral cataract removal, using the access to the lens through the superior bulbar region in half the patients and temporal access in the other half. They compared the procedure performed in rabbit eyes, which were later submitted to histological sectioning of the superior bulbar region in the pre-and postoperative periods. Structural changes were seen in the conjunctiva and TC after the superior trans-scleral incision in the AS-OCT, as well as in ammatory ndings in the histological evaluation. In the eyes with temporal incisions, the histological sections of the superior bulbar region, which was not operated upon, showed no in ammatory signs. AS-OCT has shown that untouched tissues had been preserved. Thus, Gozawa et al. concluded that the TRAB altered the connective tissue and TC and that these ndings were not seen with temporal incisions, preserving the superior bulbar region. Nevertheless, the surgical technique for a trans-scleral phakectomy was performed within the conjunctiva, TC, STS, and sclera [31]. Furthermore, the AS-OCT images were collected using Casia SS-1000 ® (Tomey Corporation, Nagoya-Aichi-Japan), which has an axial resolution of 10µm-half of the AS-OCT resolution used in the present study [24]. Thus, in the study of Gozawa et al., images had a lower resolution, making it di cult to differentiate the structures. In contrast to the ndings of Gozawa et al., our results showed that by preserving the TC and STS during surgery it was possible to maintain their re ectivity and thickness during the postoperative period. Previous studies involving restrictive syndrome were also considered. This syndrome is seen during the postoperative period in patients with strabismus or retinal detachment treated with scleral xation. During the scleral xation, TC was excessively manipulated with extensive muscle dissection [18, 32, 33], with brotic restriction of TC and its adherence to episclera [32]. As a preventive measure for this surgical complication, these studies recommended minimal TC manipulation during such procedures [34]. Comparing analysis of this surgical complication with our ndings, we must highlight the importance of TC preservation for maintaining the conjunctiva-TC complex mobility. Studies examining TC broblasts have provided a better understanding of the development of the brosis responsible for TRAB failure [7,16,34,35]. In an animal model of scarring after a stulizing surgery, broblasts were seen to arise from episclera and the subconjunctival connective tissue in a study conducted by Joseph et al. [11]. In other words, they come from the structures that delimit the STS, the TC and episclera, as well as from sites neighboring muscle emergence [11], demonstrating the importance of these structures in the brosis of the STS. Therefore, our ndings corroborate the literature regarding the importance of preserving the TC in order to avoid postoperative brosis. In studies that investigated the scarring of the conjunctiva, TC, and STS separately [21,31], surgical procedures caused injuries in all of these structures, without any attempts of preservation, since procedures involved trans-scleral incisions. The conjunctival excision with TC preservation allows for full scarring similar to that of the preoperative state, both with the slit lamp photography and with AS-OCT. In addition, the procedure preserves the STS, maintaining tissue mobility without adhesions to the episclera. These ndings are important for patients undergoing surgical treatment of the ocular surface. This is particularly important for those who need multiple procedures in the same region, such as those with pterygium and glaucoma. Further studies evaluating surgical reintervention in the donor area and the following scarring process should be performed to assess the effectiveness of TRAB after donating conjunctival tissue with the preservation of TC.

Conclusions
After ALCT with TC preservation, scarring occurs in the conjunctiva, epithelium, and stroma. The underlying TC and the STS structures remain preserved for eventual surgery.

Declarations
Funding: No funds, grants, or other support was received.
Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Con icts of interest/Competing interests: The authors declare that they have no competing interests Authors' contributions: Fernanda S. Vidal and Sebastião Cronemberger conceived and designed the protocol. All authors were involved in the analysis and interpretation of the data. FSV, SC and JAM wrote the rst draft of the manuscript, revised the manuscript and produced the nal version. All authors read and approved the nal manuscript.
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