Effects Of SMILE-Derived Decellularized Lenticule As An Adhesion Barrier In A Rabbit Model of Glaucoma Filtration Surgery

Houfa Yin Zhejiang University School of Medicine Xinyi Chen Zhejiang University School of Medicine Xiaogang Hong People's Hospital of Kaihua Jian Ma Zhejiang University School of Medicine Fang Wu Zhejiang University School of Medicine Ting Wan Zhejiang University School of Medicine Yiwen Sang Zhejiang University School of Medicine Qiuli Fu Zhejiang University School of Medicine Zhenwei Qin Zhejiang University School of Medicine Danni Lyu Zhejiang University School of Medicine Wei Wu Zhejiang University School of Medicine Jinfu Yin Zhejiang University School of Medicine Yabo Yang (  yangyabo@zju.edu.cn ) Zhejiang University School of Medicine


Background
Glaucoma is one of the leading causes of irreversible blindness worldwide.
Lowering of intraocular pressure (IOP) remains the only proven treatment to slow the progression of glaucoma [1]. Trabeculectomy, in which a drainage bypass is created to allow excess aqueous humour to drain into a conjunctival ltering bleb, is one of the most effective glaucoma ltration surgeries in reducing IOP [2]. However, ltration bleb dysfunction often occurs due to excessive scar tissue formation at the surgical site [3]. To reduce excessive scar formation, antimetabolic agents such as mitomycin C (MMC) and 5-uorouracil (5-FU) are often used during trabeculectomy surgery and have been shown to improve the surgical outcome [4,5]. However, these antimetabolic agents may lead to serious postoperative complications, such as persistent postoperative hypotony, corneal toxicity, ltering bleb leakage, blebitis, and endophthalmitis [4,5]. Thus, a more physiological approach to suppressing subconjunctival brosis is needed.
Several investigations have been conducted of the prevention of bleb adhesion and brosis using physical barriers that are placed in the subconjunctival space or underneath the scleral ap. These include hyaluronate hydrogel, biodegradable polymers, or and nonbiodegradable polymers [5][6][7]. Recently, small incision lenticule extraction (SMILE) has proved to be a safe, e cient, and predictable corneal refractive surgery [8,9]. With increasing patients undergoing SMILE, the extracted lenticules could be used for various purposes in the ophthalmic eld, such as hyperopia correction, keratoconus treatment, and management of corneal perforation [10][11][12]. Decellularized lenticules are thin and transparent and also exhibit good biocompatibility in vivo [13]. Therefore, we suggest that decellularized lenticule will act as a physical adhesion barrier during trabeculectomy surgery.
The aim of this study was to evaluate the e cacy of SMILE-derived decellularized lenticule in reducing adhesions between the conjunctiva and sclera, and keeping ltering bleb active after trabeculectomy in rabbit eyes.

Materials And Methods
The use of SMILE-derived lenticules was approved by the Ethics Committee of the Second A liated Hospital, Zhejiang University School of Medicine, and the procedures used conformed to the tenets of the Declaration of Helsinki. Male New Zealand white rabbits (weighing 2-2.5 kg, aging 3-4 months) were supplied by the Academy of Medical Sciences of Zhejiang province. All animal experiments were approved by the Animal Ethics Committee of the Second A liated Hospital, School of Medicine, Zhejiang University and were in accordance with the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in ophthalmic and vision research. Study was carried out in compliance with the ARRIVE guidelines.
Decellularization of SMILE-derived lenticule SMILE-derived lenticule were collected during refractive surgery using the VisuMax femtosecond laser system (Carl Zeiss Meditec AG, Jena, Germany) as our previous study [13]. Lenticules with a diameter of 6.6mm and a central thickness of 50 μm were selected for the following procedure. The fresh lenticules were decellularized using sodium chloride (NaCl) and nucleases as our previous study (Fig. 1A) [13].

Surgical Procedure
As different rabbits had signi cantly different baseline IOP and wound-healing reactions, surgery was performed on both eyes of the rabbits. After creating the scleral ap, the eye was randomly assigned to the decellularized lenticule group or control group. Twelve eyes of 6 rabbits were used in this study. The rabbits were anesthetized with an auricular vein injection of sodium pentobarbital (30 mg/kg), and topical anaesthesia using 0.4% oxybuprocaine hydrochloride eye drops was administered before surgery. Trabeculectomy was then performed with previously reported methods by an experienced glaucoma specialist (J.F.Y) with few modi cations [14]. Brie y, a fornix-based ap of conjunctiva was carefully dissected and a 3 × 3 mm partial thickness scleral ap was separated. After a 1 × 2 mm trabecular tissue was removed, peripheral iridectomy was performed. The scleral ap was not sutured, but the conjunctiva was closed with a 10-0 nylon suture. In the decellularized lenticule group, the decellularized lenticule was loosely secured by suturing on the sclera with 10-0 nylon (Fig. 1B). Only trabeculectomy was conducted on the control group, and no decellularized lenticule was placed.

Clinical evaluation
After topical anaesthesia, IOP was measured by Tono-pen (Reichert, Depew, NY, USA) at baseline and 3, 7, 14, 21, and 28 days after surgery. An average of three measurements taken from each eye was recorded. Bleb appearance was examined via a slit lamp and was graded as previously described at 3, 7, 14, 21, and 28 days after surgery.

Histological analysis and immunohistochemistry
Rabbits were euthanized 28 days after surgery by an overdose intravenous injection of sodium pentobarbital. Eyeballs were enucleated and xed in 4% paraformaldehyde solution overnight. Then the eyeballs were dissected at the equator and embedded in para n. Four-micrometre-thick serial sections were cut through the centre of the operation site, and stained with hematoxylin and eosin (H&E) for general histologic examination. Masson trichrome staining was performed to evaluate scar tissue formation. To examine the myo broblast adjacent to the surgical site, we immunohistochemically measured the expressions of α-smooth muscle actin (α-SMA).

Statistical analysis
Each measurement was expressed as the mean ± standard deviation (SD). The Mann-Whitney U test and an unpaired t test were used to compare Bleb score and IOP between the 2 groups. A P value less than 0.05 was considered statistically signi cant. All analyses were performed using Statistical Package for the Social Sciences software (version 22.0, International Business Machines Corp.)

Slit-lamp examination and bleb appearance
Slit-lamp examination revealed no severe postoperative in ammation in the anterior chamber, and no bleb leakage, blebitis, or endophthalmitis was observed during the postoperative period in both groups.
Bleb morphology was scored based on its appearance and size as previously described at 3, 7, 14, 21, and 28 days after surgery [15]. Filtering blebs were maintained over the scleral ap in the decellularized lenticule group for at least 28 days in the decellularized lenticule group, whereas the ltering bleb collapsed within 14 days after surgery in the control group. Bleb scores were signi cantly higher in the decellularized lenticule group than those in the control group on day 3 and from day 14 to day 28 (P<0.05, Fig. 2).

Postoperative IOP changes
There was no statistically signi cant difference of the initial IOP between the decellularized lenticule group and the control group (Fig. 3). The IOP was reduced 3 days after surgery in both groups, and it did not differ signi cantly between two groups within 7 days after surgery (Fig. 3). However, IOP began to increase again in the control group 7 days after surgery, and the IOP of the decellularized lenticule group was signi cantly lower than that of the control group from day 14 to day 28 (P<0.05, Fig. 3).

Histopathologic features
Histopathologic examination was performed 28 days after surgery to evaluate the effects of decellularized lenticule on bleb scarring. H&E staining revealed that ltering space between the conjunctiva and lenticule remained prominent in the decellularized lenticule group while no ltering space was observed in the control group; however, massive scarring was observed in the control group ( Fig. 4A and 4B). No evidence of obvious in ammatory change or tissue damage was observed in either group (Fig. 5B). To assess the degree of subconjunctival brotic response, we performed immunohistochemical staining for α-SMA (a marker of myo broblasts). Many cells with intensive α-SMA expression were observed in the subconjunctival area in the control group, which indicated severe brosis (Fig. 5A). However, the bleb brosis was signi cantly attenuated in the decellularized lenticule group (Fig. 5B).
Consistent with α-SMA expression, Masson's trichrome staining demonstrated signi cant collagen deposition in the subconjunctival region of the control group (Fig. 6A). In contrast, there was less collagen deposition in the decellularized lenticule group (Fig. 6B).

Discussion
The present study demonstrates for the rst time that the use of decellularized lenticule for trabeculectomy in rabbits keeps the ltering bleb active and maintains IOP reduction by inhibiting the formation of subconjunctival brosis.
Antimetabolites such as MMC and 5-FU are commonly used during trabeculectomy to inhibit subconjunctival brosis [4,5]. However, the usage of these antimetabolites has been associated with higher risk for wound healing disorders and severe infections due to their non-selectivity [4,5]. In recent years, adhesion barriers between the conjunctiva and sclera have been investigated as alternative methods for preventing bleb adhesion and brosis, including PDMAA polymer, expanded polytetra uoroethylene (Gore-Tex) membrane, sepra lm, biodegradable collagen, and honeycombpatterned lm [5][6][7]14]. Although some of these adhesion barriers have proven effective in reducing bleb adhesion and brosis in animal models, clinical trials showed inconsistent results concerning the surgical outcome [5].
SMILE has become clinically available as an alternative to laser in situ keratomileusis since 2011 [16,17]. The extracted lenticule is the immediate by-product of this procedure and is typically discarded after surgery. The increasing popularity of this surgery has made it easier to obtain SMILE-derived lenticules.
The decellularized lenticule is a thin stromal layer with low immunogenicity and good biocompatibility, which makes it an excellent candidate for corneal stromal regeneration [18,19]. We have previously shown that decellularized lenticule could safely and effectively repair damage to the anterior cornea in rabbits [13]. Recently, Gu et al also reported that subretinally transplanted decellularized lenticule exhibited excellent biocompatibility without obvious adverse reactions and brosis [20]. Therefore, decellularized lenticule might be a useful biomaterial in various types of ophthalmic surgery.
The present study reveals that decellularized lenticule promotes IOP reduction and prolongs bleb survival in trabeculectomy in rabbits with no complications. A strategy to reduce scar formation following glaucoma ltration surgery is to reduce the adhesion of the tenon broblasts to the underlying sclera at the surgical site [21]. Our in vivo studies suggest that decellularized lenticule has a space-keeping effect that prevents adhesion between the tenon broblasts and sclera (Fig. 1C). To x the decellularized lenticule precisely in the desired area, it was loosely sutured onto the sclera. However, we speculate that there is a passage between the sclera and the decellularized lenticule to divert aqueous humour from the anterior chamber to the subconjunctival space, given that the IOP reduction and bleb formation observed in the postoperative period (Fig. 1C).
Myo broblast accumulation and excessive collagen deposition in the subconjunctiva are major causes of bleb failure [22]. Histopathologic examination showed that myo broblasts in ltrated the subconjunctival area with compact collagen deposition in the control group; however, there were fewer myo broblasts and less collagen deposition in the subconjunctiva in the decellularized lenticule. These ndings indicate that decellularized lenticule may effectively inhibit excessive scar formation in glaucoma ltering surgery and improve the surgical outcome.

Conclusions
Although further studies with larger sample sizes and longer follow-ups are warranted to clarify the safety and e cacy of decellularized lenticule in glaucoma ltering surgery, our study provides a novel separating agent to prevent subconjunctival brosis after trabeculectomy and increase the success rate of glaucoma ltering surgery.