Seventeen eyes of 17 New Zealand White female rabbits, weighing 3 to 4 kg each, were exposed to high-energy ultrasound by the activation of a phacoemulsification probe in the anterior chamber. Treatment and handling of the rabbits was performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Before surgery, the rabbits were anesthetized by intramuscular injection of 35 mg/kg ketamine hydrochloride and 7 mg/kg xylazine, and the right eye was then examined by specular microscopy (EM-1000 specular microscope; Tomey, Waltham, MA), obtaining three corneal endothelial photographs for analysis, all from within the central 3 mm of cornea. The right eye was cleansed with topical povidone iodine and draped, and a lid speculum was inserted. Next, a clear corneal incision was made in the superotemporal corneal quadrant with a disposable 3.2-mm keratome blade. Using 70% power, and 25 mL/min of irrigation, a 20-g phacoemulsification probe (Series Ten Thousand Phacoemulsification System; Alcon Surgical, Fort Worth, TX) was introduced through the corneal incision into the anterior chamber, taking care to avoid touching all ocular structures including the lens and cornea, and activated in the center of the anterior chamber. Each eye was continuously irrigated, with the power turned alternately on and off every 10 seconds to avoid overheating, until the required 5 minutes of net time (10 minutes in all) was completed.
The rabbits were randomized to receive either balanced salt solution, or BSS with 0.001 M of sterile ascorbic acid introduced into the BSS bottle immediately before phacoemulsification. The two groups were similar in all other respects of surgical and postoperative treatment. Each animal was number encoded before treatment, and surgery, postoperative, and histologic examinations and analysis were all performed with blinding as to the treatment grouping.
On completion of phacoemulsification, the incision was sealed by injection of BSS solution (for both groups) into the incision margins, followed by a subconjunctival injection of betamethasone acetate and betamethasone phosphate (Celestone Chronodase; Schering-Plough, Kenilworth, NJ) and gentamicin. No additional postoperative medications were given.
One week later, the rabbits were again anesthetized as described above, the surgical eye was again examined by specular microscopy, and the rabbits were killed by an overdose of phenobarbital. The right eye of each rabbit was enucleated and preserved in formaldehyde, and corneal sections were stained with hematoxylin and eosin and examined by an experienced ocular pathologist who was blinded to the treatment received by each rabbit, as mentioned previously.
The specular microscopy results were analyzed with the software application for endothelial cell analysis that accompanied the specular microscope (EM-1100 software; Tomey). The results of the three preoperative and three postoperative endothelial cell counts were averaged separately, and the reduction in cell count 1 week after surgery was calculated by subtraction. The nonparametric Mann-Whitney test was used to examine possible differences in preoperative cell counts between the two groups, as well as differences in postoperative cell count reduction. Cell counts are presented as the mean ± SEM.