Espion system

The ERG was measured to evaluate the RP function of rats before intraperitoneal injection and three days after retinal light damages separately and was recorded by an Espion Diagnosys System (Diagnosys, Littleton, MA, United States). As described in Miyai et al. (2019) (link), after 24 h of dark adaptation, the rats were given intraperitoneal anesthesia, and their pupils were dilated with phenylephrine hydrochloride and tropicamide (0.5%). Two wire loop electrodes were placed on the corneal surface of the eyes and served as the ERG signal-recording electrodes. In addition, two subdermal needle electrodes were inserted into the base of the tail and nasal part and separately served as the ground electrode and the common reference electrode. Retinal responses were recorded for 30 min.
Light stimulation was performed using a white LED following the protocol described in Miyai et al. (2019) (link). Dark and light adaptation was performed in four steps, and the light intensity was switched from weak to strong. Electroretinographic waveforms were recorded and sampled, and the data were analyzed by using a Diagnosys digital acquisition system. The waveforms of ERG were measured from trough to peak (Lazarou et al., 2015 (link)), and the values of ERG amplitudes were compared among the four groups.

Electroretinography (ERG) was performed on both treated and untreated eyes at 5 weeks following microbead injection using an Espion Diagnosys system (Diagnosys LLC, Littleton, MA, USA). Tropicamide (0.5%, Akorn, Lake Forest, IL, USA) and 0.5% proparacaine hydrochloride (Falcon Pharmaceuticals, Fort Worth, TX, USA) were used for pupil dilation. Body temperature was maintained at 37 °C with a homeothermic controller and unit. Electrical signals were recorded with two 3 mm gold wire loop electrodes (Diagnosys) contacting the corneal surface of eyes precoated with a 2.5% hydroxypropyl-methylcellulose solution (Gonak, Akorn, Lake Forest, IL, USA). A subdermal needle electrode (Viasys Healthcare, Chicago, IL, USA) between the ears served as common reference while the other subdermal needle electrode inserted at the base of the tail acted as ground. Retinal responses were recorded simultaneously from both eyes. Light stimuli were delivered via a ColorDome unit. Three different light intensities, 1, 5 and 7 cd.s/m², were used in a 3-step examination. The stimuli were blue flashes (6.28 cd/m²) and green (560 nm) background (10 cd/m²). Each recording was an average of 10 sweeps with an inter-stimulus interval of 0.4 s. Signals were band-pass filtered at 1 kHz. The PhNR amplitude was measured from the baseline to the trough following the b-wave.

Full-field ERG was performed 3 weeks after EVC using an Espion Diagnosys System (Diagnosys LLC, Littleton, MA, USA). Electrical signals were recorded with two 3-mm platinum wire loop electrodes placed on the corneal surfaces of eyes that had been pre-coated with 2.5% hydroxypropyl-methylcellulose solution (Gonak; Akorn, Lake Forest, IL, USA). One subdermal needle electrode inserted into the base of the right leg served as the ground, while the other subdermal needle electrode placed over the nasal bone served as the common reference. Light stimuli were delivered using a ColorDome unit at four different stimulus strengths (11.38 cd.s/m²–0.33 Hz, 11.38 cd.s/m²–1 Hz, 22.76 cd.s/m²–0.33 Hz, and 22.76 cd.s/m²–0.33 Hz) in a 4-step test. In each step, the stimulus frequency was 2 Hz, and a green light with an intensity of 10 cd/m² was presented for 4 ms against a green background.

The photopic negative response (PhNR) procedure was conducted by using an Espion Diagnosys system (Diagnosys, Littleton, MA, USA) as previously described [26 (link)]. Briefly, the rats were intraperitoneally injected with 1% sodium pentobarbital. Their pupils were dilated with tropicamide (0.5%). Then, the PhNR procedure was performed. Data were collected and analyzed by using GraphPad Prism 9.

Eight weeks after the model was established, full-field ERG was performed, and the phNR was evaluated using an Espion Diagnosys System (Diagnosys LLC, Littleton, MA, USA). According to the procedures described (Zhou et al., 2017a (link)), rats were dark-adapted for at least 6 h and prepared under dim red illumination. After the rats were anesthetized, the pupils were dilated, and the corneas were precoated with 2.5% hydroxypropyl-methylcellulose solution (Gonak; Akorn, Lake Forest, IL, USA). ERG was performed with a pair of gold wire loop electrodes that contacted the surface of the cornea. The responses were amplified and bandpass-filtered between 0.3 and 300 Hz, and the luminance range of the light stimuli was 0.003 to 10.0 (P) cd.s/m2. The amplitudes of the a- and b-waves and OPs were measured. After full-field ERG was performed and OPs were recorded, light stimuli were delivered at four different stimulus strengths (11.38 cd.s/m2–0.33 Hz, 11.38 cd.s/m2–1 Hz, 22.76 cd.s/m2–0.33 Hz, and 22.76 cd.s/m2–0.33 Hz) in a four-step test using a ColorDome unit to measure the phNR.

Mice were dark-adapted for a minimum of 12 hours. All ERG recordings were performed using an Espion system (Diagnosys LLC, Lowell, MA, USA) in a dark room under dim red light illumination. Mice were anesthetized, and the eyes were dilated as described above. Body temperature was maintained at 37 °C using pumped-water heat pad fixed on the ERG stage. Coupling gel was applied to each eye, before placing a monopolar electrode on the cornea. A gold-cup electrode was placed in the mouth to serve as the reference electrode, and a silver-silver chloride electrode was inserted into the tail as the ground electrode. The mouse head was positioned in front of a Ganzfeld stimulus. Scotopic ERG responses were recorded across increasing light intensities from −3.3 to 1.5 log cd.s.m⁻² in 0.3-log-unit increments. Full-field ERG was recorded, and each response was the average of 3–5 trials. Signals were acquired at 2 kHz with high- and low-pass filtering at 0.1 and 100 Hz respectively.