Tobradex

All animal procedures were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research approved by the Institutional Animal Care and Use Committee of SingHealth Duke-NUS (protocol no. 2019/SHS/1470). Eighteen Sprague-Dawley rats, aged 6 to 8 weeks, were used for the study. Intraperitoneal anesthesia was administered using a combination of xylazine (Troy Laboratories, Glendenning, Australia) and ketamine (Parnell Laboratories, Alexandria, Australia). Corneal wounds were induced by irrPTK as previously described.¹⁶ (link)^,22 (link) In brief, in one randomly selected eye of each rat, the corneal epithelium was removed up to approximately 0.5 mm from the limbus using a #64 surgical blade (BD, Franklin Lakes, NJ). The central 3mm area was then ablated using a Technolas 217z excimer laser (Bausch + Lomb), reaching a depth of 15 µm. A fine metal mesh was placed above the ablation zone after applying 50% of the laser pulse. The contralateral eye, which remained unwounded, served as the control (referred to as the normal group). After laser ablation, the rats received topical tobramycin (Tobrex; Alcon, Geneva, Switzerland) four times a day for 3 days. Seven days after injury, six of the wounded eyes were randomly assigned to the CSK group, which underwent intrastromal injection of quiescent CSKs by an experienced corneal surgeon (J.S.M.). After anesthesia, a stromal tunnel at the edge of the haze region in the anterior stroma was created with a 31G needle. Following that, 4 × 10⁴ cells in 2 µL of 1× PBS were injected through the tunnel using a 30G blunt needle attached to a Hamilton syringe (Hamilton Company, Reno, NV, USA). The chosen dosage was informed by our previous efficacy studies.¹⁸ (link)^,20 Another six random wounded eyes served as sham controls and received intrastromal injection of 2 µL of 1× PBS without CSKs. The other wounded eyes were left untreated (referred to as the injured group). Rats in all four groups received tobramycin and dexamethasone eye drops (Tobradex; Alcon) four times a day for 7 days.

The cDNA encoding H105A was cloned in frame with the signal peptide from Interferon beta^{38 (link)}, to allow secretion of the H105 peptide, in a pAAV2 vector with a CMV promoter resulting in AAV-H105A. The AAV-H105A and AAV-GFP, as control, viruses were generated by InnovaVector (Pozzuoli, NA, Italy)^{39 (link)}. For AAV2 delivery, Rho^P23H/+ pups at P5 were anesthetized for 2 min in ice. After eyelid opening, 0.5 µl of AAV-H105A (1.9 × 10¹² genome copies (GC)/ml) (one eye) or AAV-GFP (in the contralateral eye, 4.5 × 10¹² GC/ml) were intravitreally injected with a 34 GA needle Hamilton syringe. A topical cortisone and antibiotic unguent (TobraDex, 0.3% tobramycin + 0.1% dexamethasone, Alcon) was applied immediately after injection and pups were allowed to recover on a warm pad before reintroduction in the cage.

Diode laser transscleral cyclophotocoagulation was performed with the OcuLight SLx 810 nm diode laser photocoagulator and the handheld fiberoptic G-probe, both from Iris Medical Instruments (Mountain View, CA)
[13 (link),15 (link)]. All the treatments were performed under local anesthesia (2 ml of 2% lidocaine as a retrobulbar injection). The laser was set at an initial power of 1700 mW and a duration of 1 second. The laser power was titrated upwards and downwards during the procedure. The target was to achieve a ‘burst’ sound in roughly half of the laser applications. Laser applications were spaced evenly over the inferior 180 degrees, while sparing the 3- and 9-o’clock regions.
Post-operatively, all preoperative IOP-lowering medications, except systemic medications (such as high-osmotic agents) and topical pilocarpine, were continued. The patients were also given tobramycin and dexamethasone eye drops (TobraDex, S.A. Alcon-Couvreur N.V., Rijksweg, Puurs, Belgium) 6 times per day, 1% atropine gel (Dishan, Xingqi Co., Ltd, Shenyang, China) twice per day, and pranoprofen eye drops (Pranopulin, Senju Pharmaceutical Co., Ltd, Osaka, Japan) 4 times per day.

Male C57BL/6 mice (6–8 weeks old, body weight 19–24 g) were purchased from Charles River Laboratories (Beijing, China) and housed in a pathogen-free facility with free access to sterile acidified water and irradiated food. The retinal IR model was performed by acute elevation of intraocular pressure (IOP) via anterior chamber perfusion, during which a 33-gauge needle penetrated the cornea into the anterior chamber and its attached saline reservoir was elevated to obtain an IOP of 110 mmHg for 90 min. Removal of the needle allowed the release of pressure and natural reperfusion. Then, a topical antibiotic and steroid ointment (TobraDex; Alcon Laboratories, Inc., Texas, USA) was applied to the conjunctival sac. Eyes with cannulation-induced cataracts, iris/retinal bleeding, or anterior chamber leakage (leaks can be easily detected as dripping and wetting) were excluded [39 (link), 40 (link)].
The mice were randomly allocated into six groups: (i) Normal Control group (NC); (ii) IR-Vehicle group (IR-Vehicle) mice were treated by anterior chamber perfusion and intravitreally injected with 2 μl 1% DMSO in normal saline on top of the IR insult; (iii) IR-Fer-1 group (IR-Fer-1); (iv) IR-zVAD group (IR-zVAD); (v) IR-Nec-1 group (IR-Nec-1), mice underwent similar surgical procedures and were injected with 2 μl 100 μM Fer-1 or zVAD or Nec-1 (MCE, New Jersey, USA) dissolved in normal saline with 1% DMSO, respectively; and vi) IR-Fer-1-zVAD-Nec-1 group (IR-FVN), after anterior chamber perfusion, the mice were intravitreally administered with a dose of 100 μM Fer-1, 100 μM zVAD, and 100 μM Nec-1 dissolved in 2 μl normal saline containing 1% DMSO collectively. Each drug dose (100 μM) was optimized by pilot experiments. Fer-1 (formula: C₁₅H₂₂N₂O₂), zVAD (formula: C₂₁H₂₈FN₃O₇), Nec-1 (formula: C₁₃H₁₃N₃OS), RSL-3 (formula: C₂₃H₂₁ClN₂O₅), and Erastin (formula: C₃₀H₃₁ClN₄O₄) were all brought from MedChemExpress LLC, New Jersey, USA. They were originally dissolved in DMSO and then diluted to the target concentration with normal saline (Table S2).
All studies were conducted according to the NIH guidelines (Guide for the Care and Use of Laboratory Animals, 8th Edition, 2011) and approved by the Institutional Animal Care and Use Committee at the Second Affiliated Hospital of Zhejiang University, School of Medicine (No. AIRB-2021-499).

Anesthesia was administered by intraperitoneal injection of ketamine hydrochloride (50–75 mg/kg), xylazine hydrochloride (5 mg/kg), and proparacaine eye drops were topically applied. Corneas were mechanically de-epithelialized spanning a 5 mm diameter. Isotonic 0.22% riboflavin solution eyedrops [10 mg riboflavin in 4.5 ml of 20% dextran T-500 solution (Peschke GmbH, Nürnberg, Germany)] were instilled every 3 min for 30 min. Riboflavin penetration into the anterior chamber was evaluated with slit lamp biomicroscopy using blue light. The limbus and conjunctiva were protected by covering this region with a metal ring affixed to the rat head, designed and produced by the authors, which left only the de-epithelialized cornea exposed to UVA irradiation. The right eye was irradiated at a surface intensity of 9 mW/cm² for varying times listed in Table 1 (UV-360, Medical Photon Corp, Xiamen, China; Fig 1). The indicated riboflavin solution was reapplied topically to the corneal surface every 5 min. Following CXL, Tobramycin eye drops (Tobradex; Alcon Laboratories, Inc) were administered four times daily until the corneal epithelial wound healed.