Superficial punctate keratitis (SPK) scoring of the cornea

After 14 days of RS9 topical administration to scopolamine-treated rats, they were anesthetized by inhaled induction of 3% isoflurane. SPK in the corneal surface was stained by applying a 0.1% fluorescein solution. After 30 seconds, extra fluorescein solution was washed out by saline solution. Corneas of anaesthetized animals were observed by slit lamp examination. The severity of punctate staining was evaluated using a modification of the method of Murakami et al [23]. Briefly, the corneal score of fluorescein infiltration was determined at each 1/3 area of the cornea (upper, intermediate, and lower). The score was categorized from 0 to 3 with 0.5 grade steps depending on the observation: 0 = no fluorescence, 1 = fluorescence resembling sparse dots, 2 = dense dot-like pattern, and 3 = very dense dot-like fluorescence. After SPK scoring, cornea were subjected to immunostaining, histological examination and RT-PCR respectively.