Espion e2

BN rats underwent electroretinography assessments at baseline and 12-months post-injection. Rats were dark adapted overnight, and all experimental manipulations were conducted under dim red illumination. An Espion E2 system attached to a Color Dome (Diagnosys LLC, Cambridge, UK) with Bessel filters installed for 0, 1000, and 60 Hz positioned in a Faraday cage to reduce electrical noise was utilized for recording. After anesthetization with isoflurane, rats were placed on a heated stage to maintain body temperature while Refresh lubricating eye drops were applied to the corneas to maintain hydration and provide electrical conductivity. Solid core subdermal electrodes were inserted into the scalp and haunch serving as reference and ground electrodes, respectively, while gold wire loop electrodes were placed on the cornea of both eyes. Responses were recorded after brief (4 ms) single (1 Hz) white flash stimuli over a 6-log luminance series (-4 through 0.48 log cd.s/m²). A- and B-wave amplitudes were measured using Espion E2 software (Diagnosys LLC, Cambridge, UK).

The effect of chronic implantation on retinal structure was investigated using colour fundus photography (TRC-50Dx, Topcon Medical Systems, NJ, USA) and Fourier domain OCT (Spectralis, Heidelberg Engineering GmbH, Heidelberg, Germany). Retinal function was assessed using full-field ERG (Espion E2, Diagnosys LLC, Lowell, MA, USA). Clinical assessment tests have been used previously in preclinical studies evaluating the safety of retinal prostheses [10] (link), [15] (link), [16] (link), [26] (link), and are also common clinical ophthalmological tests.

Retinal function was analyzed in wild type and TLR5−/− mice by electroretinography (ERG) as previously described [30] (link), [37] (link), [38] (link). ERGs were performed at 8 and 12 hours postinfection (Espion E2, Diagnosys LLC, Lowell MA). After dark adaptation for at least 6 hours, eyes were exposed to a transient flash of light. Bright flashes resulted in a response which consisted of an A wave initial negative amplitude followed by a B wave positive deflection. A-wave provides a direct measure of photoreceptor activity, while B-wave represents the action of Muller cells, bipolar cells, and second order neurons. A- and B-wave amplitudes were recorded for each infected eye and compared with the uninfected eye. The percentage of retinal function retained was then calculated using the formula 100 – {[1 – (experimental A-wave amplitude/control A-wave amplitude)] ×100} or 100 – {[1 – (experimental B-wave amplitude/control B-wave amplitude)] ×100} [38] (link). Values represent the mean ±SD for N≥4 eyes per time point.

Electroretinography was performed 3 days before the injection (day -3) and repeated at days 15, 30 and 70 post injection. We used the day of injection as reference day 0. Animals were dark adapted overnight for at least 12 hours before being anesthetized with intramuscular injection of ketamine (80 mg/kg) and xylazine (7.5 mg/kg). Topical anesthesia of tropicamide 0.5% and phenylephrine 2.5% were used to dilate the pupils. Body temperature was kept at 36°C with a warm blanket.
ERGs were recorded with a gold wire loop placed on cornea and good contact between cornea and electrode was assured with a drop of saline. The reference gold-cup electrode was placed in the mouth and the ground needle electrode was inserted to the tail. Preparation for ERG recordings were conducted under dim red light.
All recordings were performed using an Espion system (Espion e2, Diagnosys LLC; USA). An average of at least three consistent recordings was taken for each stimulus intensity. The stimulus intensity ranged from -4.0 to 1.0 log cd.s/m². In addition, a double flash recording protocol presented at 1.0 cd.s/m² with the inter-stimulus interval of 0.7s was performed to obtain an isolated cone response.

Dark and light-adapted ERGs were performed under dim red light using an Espion E² (Diagnosys, Lowell, MA, USA). ERGs were performed on 1-, 3-, 6-, 9-, and 12-month-old Crb1^KOCrb2^LowMGC and Crb1^KO mice. The ERG values of the right and left eye were averaged for the analysis in Figures 1 and S2. One eye was used for analysis for other experiments (treatment versus control eye). Mice were anesthetized using 100 mg/kg ketamine and 10 mg/kg xylazine intraperitoneally, and the pupils were dilated using atropine drops (5 mg/mL). ERGs were recorded as previously described.¹⁶ (link) Scotopic recordings were obtained at −4, −3, −2, −1, 0, 1, 1.5, and 1.9 log cd⋅s/m² light intensity. Flicker recordings were obtained at 0.5 log⋅cd s/m² fixed light intensity at the frequencies 0.5, 1, 2, 3, 5, 7, 10, 12, 15, 18, 20, and 30 Hz. Photopic recordings were obtained at 30 cd/m² background light at −2, −1, 0, 1, 1.5, and 1.9 log⋅cd s/m² light intensity. The ERG tests were performed consecutively as follows: (1) scotopic, (2) flicker, (3) 10-min light exposure (30 cd⋅s/m² light intensity), and (4) photopic.