To assess retinal functionality via full-field ERG, overnight dark-adapted mice (4, 6, 8, 12, and 20 weeks postdiabetes onset) were stimulated by UTAS Ganzfeld light source (UTAS BigShot E-3000; LKC Technologies, Inc., Gaithersburg, MD, USA) in order to stimulate both eyes simultaneously.
38 Diabetic mice were compared to nondiabetic animals, with 5 to 6 mice per condition. Mice were anesthetized by an IP injection of 75 mg/kg body weight ketamine (Anesketin; Eurovet, Bladel, The Netherlands) combined with 1 mg/kg medetomidine (Domitor; Pfizer, New York, NY, USA) and pupils were dilated with 0.5% tropicamide (Tropicol; Thea Pharma, Wetteren, Belgium) and 15% phenylephrine (Thea Pharma). A ground electrode was placed in the tail base, the reference electrode in the left cheek and contact lenses with gold wire served as signal electrodes. Eyes were kept moist with saline, which was also used as a conductor for the electrical signal. A nine-step stimulation protocol with increasing flash luminance was applied ranging from −3.10 to 1.40 log cd.s/m
2. The results of the lower light intensities (−3.10 to −0.60 log cd.s/m
2) were averaged from three light flashes, interspaced with 10 seconds. For the next two flash stimuli (−0.10 and 0.40 log cd.s/m
2), two flashes with 30 seconds intermediate time were averaged. Step 8 and 9 (highest light intensities, 0.90 and 1.40 log cd.s/m
2) were averaged over two flashes separated with 45 seconds. Oscillatory potentials (OPs) were automatically filtered out by the ERG software. The latencies and amplitudes of OP1-3 were measured separately. Averaged runs were used to measure a-, b-wave and OP amplitudes as well as latency and implicit times. According to the current convention, the a-wave amplitude was determined at a specific time point (8 ms after light flash) preceding the a-wave trough, while b-wave amplitude was measured from the trough of the a-wave to the maximal amplitude following the OPs. Latency was determined from flash onset to the a-wave trough and b-wave implicit time from flash onset to b-wave maximal amplitude, all according to previous literature describing ERG measurements.
39 Final data was retrieved from averaging left and right eye.
40 Electroretinograms (
Figs. 2,
3) were adjusted to start at the zero point, in order to fit the overlays more appropriately. After imaging, an IP injection of atipamezole (1 mg/kg, Antisedan; Pfizer, New York, NY, USA) was administered to reverse anesthesia and eye ointment (Vidisic; Bausch + Lomb, Rochester, NY, USA) was used to prevent dry eyes.