Adult Long-Evans pigmented rats were used for both acute and 3-week infusion ERG experiments and for immunolabeling of HCN. Other experimental models were wt mice and rd10 mutant mice. All animals were raised in cyclic light (12 hours, 100 lux; 12 hours dark). The experiments were performed in compliance with both the guidelines of the Ethics Committee of the University of Pisa and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
A venous catheter was inserted into the jugular vein with the rats under surgical anesthesia (intraperitoneal injection of urethane 120 mg/100 g) for vehicle and ivabradine acute delivery. Ivabradine (IRIS) dissolved in saline was given at the doses of 3, 6, and 12 mg/kg. The effects of ivabradine were tested on the heart rate (HR), on the ERG in response to flashes and sinusoidal stimuli, and in two cases on the arterial blood pressure (ABP). ERG responses were recurrently tested after the drug injection (sinusoidal response recorded after: 15, 90, 180 minutes, respectively; flash response after 45, 120, 240 minutes). In a separate set of experiments, ivabradine was delivered by means of osmotic pumps (model 2ML4; Alzet Osmotic Pumps, Cupertino, CA); this approach ensured constant delivery rates. The pumps were filled with a solution of ivabradine designed to obtain an average daily amount of 11 mg/kg over 3 weeks. With the rats under surgical anesthesia (2,2,2-tribromethanol 28 mg/100 g; Sigma-Aldrich, Steinheim, Germany). The osmotic pumps were placed subcutaneously between the shoulder blades and inserted into a small pocket, formed by spreading the connective tissue apart. In the first animal group (21-day–treated group), the pumps were maintained for 21 days, in the second (7-day recovery group) on day 21, the pumps were removed, and the animals received no treatment for 7 days. In the third group (control group), the procedure was as for the first group but the pumps were loaded with saline. The amount of drug and vehicle delivered during the infusion time was measured on pump removal by subtraction of the residual from the initial volume.
The repeated treatment of wt and rd10 mice consisted of daily subcutaneous (SC) ivabradine injections (12 mg/kg/d) for a period of 10 days from the postnatal age of 12 days (P12).
Before the ERG recording session, the animals were dark adapted overnight. Anesthesia was induced by a peritoneal injection of urethane 120 mg/100 g. Body temperature was continuously monitored and maintained near 37°C with an electric blanket, and any change greater than 0.1°C was readily compensated for by a feedback circuit. The observed temperature changes were 0.2°C with negligible effects on the ERG.
13 Pupils were dilated with drops of tropicamide 1%. A thin layer of methylcellulose solution (Lacrinorm; Farmigea, Pisa, Italy) protected the cornea. The electrocardiogram (ECG) continuously monitored HR (beats/minute) and general conditions. Corneal transparency and pupil size were regularly checked with an ophthalmoscope.
At the end of the ERG recordings, blood was collected from each anesthetized animal before euthanatization and assayed for ivabradine. After prolonged infusion at a dose of 11 mg/kg, the average ivabradine concentration were 86.3 ± 24 ng/mL (n = 6). These concentrations are sixfold higher than those obtained in patients treated with therapeutic doses of 5 mg.