Fresh human corneoscleral tissue, preserved in storage medium (Optisol; Chiron Vision, Irvine, CA) and not suitable for transplantation was obtained from the Mississippi Lions Eyebank (Flowood, MS). The tissue was obtained and managed according to the Declaration of Helsinki. The corneoscleral tissue was mounted on a Barron artificial anterior chamber (Katena Products Inc., Denville, NJ). In a control group (
n = 5), air was injected into the deep stroma, although most likely there was some variation in the depth at which it was injected in each eye. Air injection was performed with a 27-gauge needle with the beveled edge facing the Descemet's membrane as described in the big bubble technique.
12 A small air bubble was left in the artificial chamber that helped to visualize Descemet's folds. A second group (
n = 5) received a stromal injection of 0.3 mL of 2.5 mg/mL collagenase type 2 (Worthington, Lakewood, NJ) in commercial balanced salt solution (BSS; Alcon, Forth Worth, TX) also using a 27-gauge, beveled needle. The solution of collagenase was left in the stroma for 1 hour and 15 minutes at room temperature. Finally, a third group (
n = 18) received an injection with 0.3 mL of 2.5 mg/mL collagenase (
Figs. 1A–C) that was left in the stroma for 1 hour and 15 minutes, at which point an injection of 0.7 mL of air into the deep stroma with a 27-gauge, beveled needle was performed (
Fig. 1D). Air was easily dispersed into the cornea stroma (see
Supplementary Video S1). After the separation of Descemet's was attempted, corneal tissue was fixed in formaldehyde for paraffin sectioning or in OCT (optimal cutting temperature compound; Tissue-Tek; Sakura Finetek, Torrance, CA) for cryosections.