Xylazine

Twelve week old male Noble (NBL/Crl) rats were obtained from an in-house breeding colony. Prostate cancer was induced as described by Özten et al. (30 ). While under ketamine-xylazine anesthesia (100 mg/kg ketamine [Henry Schein Animal Health, Dublin, OH] and 5 mg/kg xylazine [Lloyd Laboratories, Shenandoah, IA]), each rat surgically received two Silastic tubing implants (Dow Corning, ID 0.078 inch; OD 0.125 inch) containing crystalline testosterone tightly packed over 2 cm length and one implant containing crystalline 17β-estradiol tightly packed over 1 cm length. Rats were randomly divided into three groups of 30 animals. One week after hormone implantation, rats were switched from a standard chow obtained from Harlan Teklad (currently Envigo, Madison, WI) to AIN-93M diet (control) or isoenergetic AIN-93M diets containing 5% or 10% lyophilized BRB powder at the expense of the starch component of the AIN-93M diet. The AIN-93M diet was also obtained from Harlan Teklad and was stored at 4°C; the berry powder was mixed into the diet in-house using a Patterson-Kelly mixer and was stored at −20°C until fed freshly three times per week. Rats were euthanized when moribund or surviving for 48 weeks.

Immediately prior to 25h post-infection, a slit lamp biomicroscope (Topcon SL D, Kogaku Kikai K.K., Tokyo, Japan) was used to examine the infected but untreated and infected but iCAP treated eyes. Slit lamp examination (SLE) scores for the clinical parameters related to each anatomical site were assigned. Digital photographs of the eyes were captured. At 25h post-infection all rabbits were anesthetized with a subcutaneous injection of a mixture of 35–50 mg/kg ketamine and 2–10 mg/kg xylazine (Henry Schein Animal Health, Dublin, Ohio, USA) and then euthanized with an overdose (>390 mg per rabbit) of sodium pentobarbital (Fatal-Plus solution, Henry Schein Animal Health, Dublin, Ohio, USA) followed by bilateral pneumothorax induction. Each infected cornea, whether untreated or treated with iCAP, was extracted, coarsely chopped, placed in 3mL sterile phosphate buffered saline (PBS) and homogenized with an electric tissue homogenizer. The tissue homogenates were serially diluted, plated on TSA in triplicate and incubated overnight at 37°C until colonies appeared (12 – 16h). The bacterial loads in each infected cornea, whether untreated or treated with iCAP, were then enumerated based on these colony counts.

A total of thirty female New Zealand White rabbits weighing 1.8 – 2.3kg and of average age 7 – 9 weeks were included in the study. The rabbits were anesthetized with a subcutaneous injection of a mixture of 35 – 50mg/kg ketamine and 2 – 10mg/kg xylazine (Henry Schein Animal Health, Dublin, Ohio, USA). One eye of each rabbit received 1 – 2 topical drops of proparacaine (0.5%; Bausch & Lomb, Bridgewater, New Jersey, USA). This was followed by intrastromal injection of 10μL of the diluted subculture of P. aeruginosa using a 30.5-gauge sterile needle. This procedure ensures that the actual amount of P. aeruginosa injected in the stroma is between 10² – 10³ CFU.