Utas e 2000

Visual function was assessed with electroretinography (ERG) performed at pretreatment as baseline, 1 month after AAV1-Rz-SOD2 injection, and 4 months after AAV1-Rz-SOD2 injection as previously described [33 (link)]. For the ERGs, the mice were dark-adapted overnight, and full-field ERGs were recorded with a visual electrodiagnostic system (UTAS-E 2000; LKC Technologies, Gaithersburg, MD) using gold wire loop electrodes placed on each cornea and a reference electrode placed subcutaneously between the eyes. Scotopic rod recordings were performed with stimuli presented at intensities of 0.025, 0.25, and 2.5 log cd-s/m² at 10-, 20-, and 30-s intervals, respectively. Ten responses were recorded and averaged at each light intensity. Photopic cone recordings were performed after the mice were light adapted to a white background light of 100 cds/m² for 5 min. Recordings were performed with four-increasing flash intensities from 0, 5, 10, and 25 log cd-s/m² in the presence of a constant 100 mcds/m² rod suppressing background light. Fifty responses were recorded and averaged at each intensity. The a-waves were measured from the baseline to the peak in the negative direction, and the b-waves were measured from the negative peak to the major positive peak. The ERG data are presented as comparisons between treatment conditions for the mean of the maxima for a- and b-wave responses.

Mice (6-week-old C57BL/6; Koatech) were intravitreally injected with 2 µM BB-Cl-amidine after general anesthesia. Optomotor response measurement (OptoMotry HD; CerebralMechanics) and electroretinography (cat. no. UTAS E-2000; LKC Technologies) were performed 1 week after injection according to previous guidelines (26 (link)). Enucleated eyes were prepared and analyzed using H&E staining and TUNEL assay. Structure and toxicity evaluations were performed according to a previously established protocol (22 (link)). The results of H&E staining were analyzed by measuring the a/b ratio, where ‘a’ is the length from the innermost ganglion cell layer to the outermost inner nuclear layer, while ‘b’ is the length from the innermost ganglion cell layer to the outermost outer nuclear layer. All mice were maintained in a specific-pathogen free facility as previously described, in accordance with the ARVO statement.

Mice were dark adapted for over 16 hours. After deep anesthesia, pupils were dilated with an eye drop containing phenylephrine hydrochloride (5 mg/ml) and tropicamide (5 mg/ml). Full-field electroretinography was performed using the universal testing and electrophysiologic system 2000 (UTAS E-2000, LKC Technologies). The scotopic responses were recorded with a flash of 0 dB at a gain of 2 k using a notch filter at 60 Hz and were bandpass filtered between 0.1 and 1500 Hz. The amplitudes of the a-wave were measured from the baseline to the lowest negative-going voltage, whereas the peak b-wave amplitudes were estimated from the trough of the a-wave to the highest peak of the positive b-wave.

Electroretinogram (ERG) was measured using an electrodiagnostic LKC system (UTAS-E 2000; LKC Technologies, Gaithersburg, MD, USA) to assess the function of the retina. Mice were dark adapted overnight before taking measurements for scotopic a-wave and b-wave responses.¹⁰ (link)^,16 (link) Scotopic rod measurements were recorded following white light flashes of increasing intensities of 0.025, 0.25, and 2.5 log cd·s/m², at time intervals of 10, 20, and 30 seconds. For measurement of photopic cone responses, mice were light adapted to a white background for 10 minutes, followed by flashes of light stimulation at 0, 5, 10, and 25 log cd·s/m² intensities. The trace amplitude between time 0 and the lowest point on the trace was considered the a-wave, and the amplitude between the lowest trace and the highest peak following oscillatory potentials was considered the b-wave.