Animals: care, husbandry, and general experimental procedure
The experiment was based on the recommendations of the Association for Research in Vision and Ophthalmology (ARVO) and Animal Research: Reporting of in Vivo Experiment (ARRIVE) guidelines. The study was also submitted under Proposal No. 027/12, and was approved by the Ethics Committee on Animal Use of the Federal University of Goiás (UFG).
The study involved 14 healthy adult male New Zealand white rabbits weighing on average 2.5 kg. The sample size was based on the number required to obtain reliable statistical results. The animals were acquired from a local supplier (MH Cunicultura Coelho Forte), located at Rodovia Bela Vista-Hidrolândia Km 9, Bela Vista de Goiás, Goiás – Brazil. The animals’ eyes and overall health were considered normal after a clinical examination done by an experienced veterinarian, as recommended by TALIERI et al. . The animals were kept in a bioterium, under veterinary supervision, and transported to the laboratory when needed for the experiments. In the bioterium, the rabbits were housed in individual cages with free access to food and water, and the local temperature was kept at 24 ± 1 °C.
The rabbits were divided into two groups of seven animals each (n = 7), a control group (C) and a treatment group (S). Group (C) was given 1.5 mL of saline orally, while group (S) was given 10 mg of sildenafil citrate (Viagra®, Pfizer, Guarulhos, SP) orally. Both groups were treated at 24-h intervals for 30 consecutive days.
The experimental protocol followed the sequence: (1) oral administration of the vasodilator sildenafil, (2) measurement of intraocular pressure (IOP), (3) measurement of mean arterial pressure (MAP), and (4) color Doppler imaging of the external ophthalmic artery. Because sildenafil citrate was administered orally, a 45 to 60 min wait time was allowed between ingestion of the drug and the subsequent steps of the protocol.
The evaluations were performed weekly, as follows: Day one - moment 1 (M1), Day seven - moment 2 (M2), Day fourteen - moment 3 (M3), Day twenty-one - moment 4 (M4), and day thirty - moment 5 (M5).
To measure the IOP and MAP and for the Doppler study, the unsedated rabbits were wrapped in a towel, leaving only the head exposed for the investigator to take the measurements. Care was taken not to apply excessive force in restraining the animals during these evaluations.
All the evaluations were performed in triplicate by the same investigator, in a blind study. To reduce the period of restraint, only the right eye of each animal was examined, thus avoiding the effects of stress on the retrobulbar circulation and optimizing the action time of the drug.
Measurement of IOP
The intraocular pressure of rabbits was measured with a Tono-Pen AVIA VET® applanation tonometer (Reichert®, New York, USA). The procedure consisted in gently lifting the eyelid, applying a drop of 0.5 % proparacaine (Anestalcon®, Alcon, São Paulo, SP) on the eye, and taking a reading with the tonometer five minutes later.
Measurement of MAP
After the trichotomy and antisepsis of the dorsal surface of the rabbits’ ears, the central artery was cannulated with a 24G catheter (BD AngiocathTM, Becton Dickinson Indústrias Cirúrgicas Ltda, Juiz de Fora, MG, Brazil). The catheter was then plugged to a semi-rigid silicone tubing system. In this system, two silicone tubes were connected to a three-way stopcock. The free end of one the tube was attached a catheter and the other end to a BD sphygmomanometer (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). The system was then filled with 1 mL/1000 mL of 0.9 % heparinized saline (Heparin, Cristália Produtos Químicos Farmaceuticos Ltda, Itapira, SP, Brazil). The air/liquid interface was positioned at the height of the right atrium and eye, and the mean arterial pressure was read with the sphygmomanometer .
Calculation of OPP
The ocular perfusion pressure (OPP) was determined by subtracting the mean arterial pressure (MAP) from the intraocular pressure (IOP), as described by KIEL & HEUVEN .
Color Doppler imaging
The color Doppler evaluation of the external ophthalmic artery was performed using a MyLab™ 30 VET ultrasound system (The Esaote Group, Genoa, Italy) coupled to a 13–18 MHz linear transducer.
Before the examination, the cornea was anesthetized by applying one drop of 0.5 % proparacaine hydrochloride topical ophthalmic anesthetic (Anestalcon®, Alcon, São Paulo, SP, Brazil). A layer of sterile aqueous gel was then applied to the corneal surface, and the transducer was gently placed in a longitudinal position, with the position indicator facing the upper eyelid.
The sagittal image of the eyeball and optic nerve were recorded in two-dimensional mode. The external ophthalmic artery was identified near the entrance of the optic nerve by color Doppler imaging. The cursor of the pulsed wave Doppler was then immediately positioned over the ophthalmic artery, within the vessel lumen, using the uniform insonation method, to record its blood flow curve, after which the peak systolic velocity (PSV) and end diastolic velocity (EDV) were evaluated based on this curve. The angle was not corrected and measurements greater than 60° were not included. The ophthalmic artery resistance index (RI) was calculated, automatically by the software Mylab desk, witch in based on the Pourcelot equation (RI = PSV − EVS/ PSV), and the curves were analyzed by the same operator (APAC), whom marked the PSV and the EDV of three consecutive curves .
A randomized 2 x 5 split-plot experimental design was used, with the treatments corresponding to the plots (with and without sildenafil citrate), the evaluation periods to the subplots (M1 - M2 - M3 - M4 - M5), and each animal to an experimental unit.
Using R® statistical software, the data were tested for normality by the Shapiro-Wilk test and subjected to an analysis of variance. The means were compared by the Tukey test, adopting a 5 % level of significance.