4.9. In Vivo Wound Healing of the Corneal Epithelium of the Rats

The abrasion of the corneal epithelium in the normal and STZ rats was performed as described previously [41,55,70,71,72,73]. The rats were anesthetized with sodium pentobarbital (30 mg/kg, i.p.), and a 3.0 mm diameter circle was outlined in the center of the cornea with a disposable dermatological skin punch (BIOPSY PUNCH, Kai Industries Co., Ltd., Gifu, Japan). The encircled corneal epithelia were removed with a BD Micro-SharpTM (blade 3.5 mm, 30°, Becton Dickinson, Fukushima, Japan). The corneal area, from which the epithelium was removed, was dyed by instilling a solution containing 1% fluorescein (Alcon, Tokyo, Japan) and 0.4% Benoxil (Santen Pharmaceutical Co., Ltd., Osaka, Japan) [74]. Changes in the corneal wound area were monitored using a TRC-50X (Topcon, Tokyo, Japan) equipped with a digital camera (EOS Kiss Digital N, Canon Inc., Tokyo, Japan) [41] 6 h and 25 h after the corneal epithelial abrasion; the wound area was analyzed with image analyzing software Image J. Corneal wound healing (%) was calculated using the following equation.