Thirty-six male Wistar rats weighing 275–295 g were obtained from Envigo RMS Laboratories, Israel. The rats were maintained and handled according to the recommendations of the Association for Research in Vision and Ophthalmology Statement for Use of Animals in Ophthalmic and Visual Research. The experimental protocol was approved by our institutional Animal Care and Use Committee of Rabin Medical Center (approval #02720).

All rats were weighed, and basal blood glucose level was quantified with a glucometer (Performa, Accu-Chek) from venous blood drawn from the tail. Nineteen rats were selected at random to undergo induction of diabetes type 1 by intravenous injection of 60 mg/kg of streptozocin (Merck KGaA, Darmstadt, Germany) in ice-cold citrate buffer pH 4.4 [27]. The procedure was performed under inhalational anesthesia with isoflurane using a glass dome.

Before the corneal wound was induced, blood glucose levels were monitored every two days for the first week and once again after six weeks. A glucose level of over 200 mg/dL was considered diabetes mellitus (Gheibi et al. 2017) [28].

The remaining (non-diabetic) rats served as the control group.

Two rats died during the induction of diabetes, leaving 17 rats in each group.

Six weeks after induction of diabetes, all rats were placed under anesthesia with isoflurane gas and topical oxybuprocaine hydrochloride 0.4% drops (Localin, Fischer Pharmaceuticals, Bnei Brak, Israel) applied in the right eye. With the aid of a surgical microscope, a corneal wound was created by placing an incision in the epithelium using a disposable biopsy punch 4 mm in diameter (Uni-Punch®, Premier, Plymouth Meeting, PA, USA) and then softly pilling the epithelium with a no.11 surgical scalpel blade (Swann-Morton, Sheffield, England).

IDEI drops were concocted with ADT21 peptide (GenScript, Inc. and the Blavatnik Centre for Drug Discovery, Tel Aviv University, Tel Aviv, Israel) [11]. The peptide was dissolved in dimethyl sulfoxide (DMSO) 0.02% and diluted in sodium chloride (NaCl) 0.9% to a concentration of 2 mmol/L and kept at −20 °C. Control drops were prepared from NaCl 0.9% mixed with DMSO 0.02%. [The DMSO was mixed with NaCl in light of reports that at high concentrations, DMSO may benefit corneal epithelial healing [29].] In each group of 17 rats, nine were treated with IDEI drops, and eight were treated with NaCl (sham) drops. Drops were administered four times daily, at 09:00, 13:00, 17:00, and 21:00. Eyes were examined with a slit-lamp twice daily until complete healing. The epithelial defect was stained with topical fluorescein (Fluoro Touch, Fluorescein Sodium Ophthalmic Strips, Madhu Instruments, New Delhi. India) and photographed under cobalt blue light. The eyes were examined and photographed with a slit lamp twice a day until complete healing. The area of corneal erosion was calculated and analyzed using ImageJ (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, MD, USA; https://imagej.nih.gov/ij/, 1997–2018).

Groups were compared for time to complete healing using a t-test for independent samples and for a percentage of rats that achieved complete wound closure at the different time points by Fisher’s exact test. The log-rank (Mantel-Cox) test was used to compare rates of complete closure over time. The data was tested for normal distribution. Statistical significance was defined as p < 0.05.