New Zealand white rabbits (female, 3.0–3.5 kg, 6 months old) were used in this study. Use, care and treatment of all animals followed the regulation of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All experimental procedures were approved by the Committee for Animal Research of the National Taiwan University Hospital. All surgical procedures and in vivo confocal microscopic observations were performed with animals under general anesthesia induced by intramuscular injection (Zoletil 20 mg/kg; Virbac Animal Health, Carros, France) and 4 mg/kg of xylazine (Rompun; Bayer Animal Health, Leverkusen, Germany). The eyes were topically anesthetized with 0.5% proparacaine hydrochloride (Alcaine; Alcon, Fort Worth, TX, USA) before manipulation. The right eye of each animal was used for the experiments and the left eye was not treated. Fifteen recipient eyes (six for DSAEK and nine for nDSAEK) were included. The corneas were inspected under a surgical microscope, and observations were recorded every 2 weeks after surgery for 3 to 6 months (transmission electron microscopy). For donor preparation, six rabbits (12 eyes) were euthanized with intravenous injection of 240 mg/kg thiamylal sodium (Shinlin Singseng Pharmaceutical, Taoyuan, Taiwan). The same agents were also used for euthanizing rabbits at the end of the experiments.
The preparation of donor grafts was performed immediately before the DSAEK or nDSAEK procedures. First, all donor eyeballs received corneal pachymetric measurement (Ocuscan; Alcon) to determine the corneal thickness. A lamellar corneal cut was performed using a laser system (IntraLase; Abbot Medical Optics, Santa Ana, CA, USA) with the posterior donor discs aimed at 120 μm. Anterior segment optical coherence tomography (Visante; Carl Zeiss Meditec AG, Oberkochen, Germany) was performed to confirm graft thickness of 120 ± 20 μm. The corneas were then trephined to obtain donor discs at 8-mm diameter. These graft tissues were then implanted unilaterally into the right eyes of the recipient rabbits using either DSAEK or nDSAEK techniques.
In the recipient eyes immediately before the DSAEK and nDSAEK surgeries, lensectomies were performed. At surgery, pupils were dilated with topical phenylephrine and tropicamide (Mydrin P, Santen Pharmaceutical Co., Ltd., Osaka, Japan). For Descemet's stripping automated endothelial keratoplasty, a 4-mm limbal-corneal incision was made and the endothelium/Descemet's membrane was mechanically scraped using a modified Price-Sinskey hook (Asico, Westmont, IL, USA) with continuous injection of air into the anterior chamber through an anterior chamber maintainer. The scraped area measured at least 5 mm in diameter, and the denuded area was checked by indocyanine green staining during the operation in the DSAEK group. Descemet's membrane scraping was not performed in the nDSAEK group. An endothelial insertion system (Tan's Endoglide; Angiotech Pharmaceuticals, Inc., Vancouver, Canada) was loaded with the donor tissue, oriented so that its endothelial side faced the anterior chamber. An intraocular viscoelastic injection (Viscoat; Alcon) was applied to protect the donor corneal endothelial cells from mechanical damage during insertion. The graft was pulled into the endothelial insertion system (Angiotech Pharmaceuticals, Inc.) and entered the opposite side of the anterior chamber through the corneal incision. The inserted graft was then attached to the recipient cornea by air injection, and the incision was closed with 10-0 nylon sutures. After surgery, all animals received topical medications including 0.5% levofloxacin (Cravit; Santen Pharmaceutical Co., Ltd., Osaka, Japan); 0.1% betamethasone, (Rinderon; Shionogi & Co., Ltd., Osaka, Japan); tropicamide (Mydrin M, Santen Pharmaceutical Co., Ltd.); and dexamethasone, neomycin and polymyxin B ointment (Maxitrol; Alcon) and were maintained with the right side of the face facing upward for 30 to 60 minutes.