Benoxil

SMILE and Femto-LASIK procedure were performed under topical anesthesia (Benoxil, Santen, Inc., Osaka, Japan) and all eyes were performed by the same surgeon (Y. W.).

All the patients were administered levofloxacin eye drops (5 ml: 24.4 mg, Cravit^®, Santen, Osaka, Japan) and sodium hyaluronate eye drops (10 ml: 0.1%, Hylo-comod^®, RSAPHARM Arzneimittel GmbH, Industriestraße, Saarbrücken, Germany) four times a day, 3 days before the surgery. Intraoperatively, oxybuprocaine hydrochloride eye drops (20 ml: 80 mg, Benoxil^®, Santen, Osaka, Japan) were administered twice for topical anesthesia. All SMILE surgeries were conducted using a VisuMax femtosecond laser system (Carl Zeiss Meditec AG, Jena, Germany). The femtosecond laser parameters were set as follows: pulse frequency, 500 kHz; pulse energy, 135 nJ; spot size, 4.3 μm; optical zone diameter, 6.0–6.7 mm; corneal cap diameter, 6.8–7.5 mm; cap thickness, 120 μm; incision length, 2.8 mm; position of incision, 90°; and the edge cutting angle, 90°. After the canning, a micro-separator was used to separate and lift the corneal cap’s edge and separate the lens’s front and back surfaces in turn. Then, micro tweezers were employed to remove the stromal lens from the small incision under the corneal cap, and the wholeness of the stromal lens was carefully checked. Extra water was absorbed by a sterile sponge, and the operation was completed.

Twelve healthy adult New Zealand rabbits (weight: 3.5–4.5 kg, age: 6–7 m) were used in the refractive surgery. Unilateral eyes of these rabbits were randomly selected for the SMILE surgery and divided into two groups; Group Ⅰ: correction of −3.00 diopters (D), and Group Ⅱ: correction of −6.00 diopters (D) (n = 6/group). In the control group, a total of six healthy New Zealand White rabbits were selected and randomly chosen to complete the OCE experiment without any surgical treatment. The SMILE procedures and examinations were performed under anesthesia with an intramuscular injection of ketamine hydrochloride (20 mg/kg; Gutian Pharmaceuticals Co. Ltd., Fuzhou, China) and topical anesthesia with 0.4% Oxybuprocaine (Benoxil; Santen, Osaka, Japan). Subsequently, we euthanized the rabbits with an excess pentobarbital injection. All animal experiments and procedures were approved by the Ethics Committee of Nanchang Ophthalmic Hospital (Zhongshan Ophthalmic Center, Sun Yat-sen University), and the requirements of Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research were fulfilled.

The 2-hydroxypropyl-β-cyclodextrin (HPβCD) was obtained from Nihon Shokuhin Kako Co., Ltd. (Tokyo, Japan). Isoflurane, mannitol, and Ca Test Kits were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Methylcellulose type SM-4 (MC) was supplied by Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan). Lanosterol and the LPO Assay Kit (BIOXYTECH^® LPO-586^TM) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan) and OXIS International, Inc. (Portland, OR, USA), respectively. The Bio-Rad Protein Assay Kit was provided by Bio-Rad Laboratories (Hercules, CA, USA). Benoxil (0.4%) and pivalephrine (0.1%) were obtained from Santen Pharmaceutical Co., Ltd. (Osaka, Japan) and Kanto Chemical Co., Inc. (Tokyo, Japan), respectively. Calpain Activity Fluorometric Assay Kits were provided by BioVision Inc. (San Francisco, CA, USA). All other chemicals were of the highest purity commercially available.

COH modeling was performed as in our previous studies [32 (link)]. Briefly, rats were anesthetized deeply with a mixture of ketamine (25 mg/kg, im) and xylazine (10 mg/kg, im); eyes were locally anesthetized via topical application of 0.4% oxybuprocaine hydrochloride eyedrops (Benoxil, Santen Pharmaceutical Co. Ltd., Osaka, Japan). Micro-magnetic beads (8 μl, BioMag® Superparamagnetic Iron Oxide, Bangs Laboratories, Inc., Fisher, IN, USA) were injected into the anterior chamber of the right eye. Sham injection (0.9% saline) was performed in a conventional manner in the contralateral eye (left eye); this served as the sham-operated group. IOP was measured using a handheld digital tonometer (Tonolab, TioLat, Finland); measurements were performed in the morning to avoid possible circadian differences. The IOPs of both eyes were recorded before surgery (control); they were also recorded at 1 day, 3 days, 1 week, 2 weeks, and 3 weeks after surgery (Fig. 1).Fig. 1

Rat chronic ocular hypertension (COH) model. Mean intraocular pressure (IOP) before (control) and after (≥ 1 day) a single unilateral injection of micro-magnetic beads (8 µl) in rat retinas, showing microbead-induced elevations in IOP (COH). n = 9–47. ***p < 0.001 vs sham-operated treatment at the same timepoint and ^###p < 0.001 vs control

A detailed description of the SMILE procedure was previously published and is only briefly summarized here [13 (link)]. The SMILE surgery was performed using a 500-kHz Visu Max femtosecond laser (Carl Zeiss, Meditec AG, Jena, Germany) with a laser energy of approximately 170 nJ. Following the application of topical anesthesia (oxybuprocaine eye drops, Benoxil, Santen, Inc., Japan), the patient was required to fixate on an internal target light before corneal suction was initiated. The posterior surface of the lenticule was cut first from the periphery to the center, followed by cutting of the anterior surface from the center to the periphery. The diameter of the refractive lenticule measured from 6 to 6.5 mm with a transition zone of 0.1 mm. The incision for lenticule retrieval was made at the 12 o’clock position on the cornea and had a length of 2 to 5 mm with an average of 3.73 mm. The target cap thickness was 110 μm. A manual spatula was inserted through the small incision to dissect the surface plane, and a pair of forceps was used to extract the intrastromal lenticule.