Three groups of mice were tested. Each mouse in group I (+/+
wild-type) underwent ERG recordings in the following order at three
time points: 1) before sildenafil treatment (n = 11), 2) 1
hour after an intraperitoneal injection of sildenafil at 2 times (2×;
2.9 mg/kg) the equivalent maximum dose by weight recommended for a
70-kg human (n = 11), and 3) 1 hour after an intraperitoneal
injection of sildenafil at 10 times (10×; 14.3 mg/kg) the equivalent
maximum dose by weight recommended for a 70-kg human (n = 7).
Each mouse in group II (Pdeg tm1/+ heterozygotes)
underwent ERG recordings at three time points: 1) before sildenafil
treatment (n = 12), 2) 1 hour after an intraperitoneal
injection of sildenafil at the 2× dose (n = 11), and 3) 1
hour after an intraperitoneal injection of sildenafil at the 10× dose
(n = 12). Group II, Pdeg tm1/+
heterozygous mice (n = 7) were also retested without
sildenafil treatment, after the 10× treatment to evaluate the
potential reversibility of the effect of sildenafil treatment on
the ERGs.
The initial recordings obtained from groups I and II were between P28
and P36. A minimum of 48-hours was maintained between all successive
ERG recordings to ensure drug washout and to allow animal recovery.
Each mouse in group III
(
Pdeg tm1/
Pdeg tm1 homozygotes) underwent ERG recordings at a single time point (P49)
without sildenafil treatment (
n = 5) as a control to verify
the retinal degeneration previously reported at P56.
23
All ERG recordings were performed by the same investigator (DB),
according to a similar method previously reported.
27 Before each recording session, all mice were dark adapted for a minimum
of 12 hours. Under dim illumination, mice receiving treatment were
given an intraperitoneal injection of 2× and of 10× sildenafil 1 hour
before ERG recordings. Just before recording, all mice were
anesthetized with an intramuscular injection of a ketamine (150 mg/kg)
and xylazine (7.5 mg/kg) mixture. The pupils were dilated (1%
cyclopentolate hydrochloride), and a DTL electrode (X-Static
silver-coated conductive nylon yarn; Sauquoit, Scranton, PA) was placed
on the corneal surface, which was kept moist with 0.5%
methylcellulose. A reference electrode was placed in the mouth (E5 disc
electrode; Grass, Quincy, MA), and a ground electrode (E2 subdermal
electrode; Grass) was positioned in the tail, after topical anesthetic
(1% lidocaine jelly) application to minimize any discomfort. Mice were
positioned in front of a Ganzfeld dome on a warmed stage to minimize
body heat loss. Scotopic intensity response functions were generated
with flashes of white light emitted by a visual electrodiagnostic
system (model UTAS E-2000; LKC, Gaithersburg, MD) 20 μsec in
duration, starting with a −4.3 log[
candelas]cd/m
−2 per
second stimulus and progressing to the brightest flash at 0.5 log
cd/m
−2 per second in
0.8-log-unit steps. Each response represented an average of two to five
flashes, depending on the intensity of the stimulus. To avoid the
conditioning effect previously reported to alter dark-adapted ERGs, a
minimum interstimulus interval of 15 seconds was
maintained.
28 29 All responses were amplified, filtered
(low [0.5 Hz] and high [1500 Hz] cutoff), and averaged (LKC
software). For ERG waveform analysis, the a-wave was measured from
baseline to the first negative trough of the ERG response, and the
b-wave amplitude was measured from the a-wave trough to the most
positive peak of the response. At flash intensities that failed to
evoke an a-wave, the b-wave was measured from baseline to the most
positive peak. All implicit times were measure from flash onset to the
corresponding a- and b-wave amplitudes. Statistical significance was
determined using analysis of variance (ANOVA).