Ketamine

OCT was used to analyze the structure of the retina in vivo and the measurement of the ganglion cell layer, including the retinal never fiber layer (RNFL) and the inner plexiform layer (GCL/IPL) complex; thickness was measured as described previously [20 (link)]. Briefly, borders of the RNFL and the IPL were determined manually, and thickness was measured at a distance of 400 µm away from the center of the optic nerve head in the superior, inferior, nasal, and temporal quadrants. At week 8, all remaining mice of both cohorts were anesthetized with intraperitoneal injection of ketamine (30 mg/kg, Mylan) with xylazine (5 mg/kg, Akorn Inc.). Pupils were dilated using 1% tropicamide (Alcon). OCT images were obtained using an SD-OCT instrument (Bioptigen, Morrisville, NC, USA) and the average regional GCL/IPL complex thickness was calculated as an indicator of RGC degeneration. Data of mouse eyes from 46 naïve animals, 49 EAE mice, and 28 EAE mice that underwent GA treatment were used for statistics. OCT data were excluded either because of poor images or questionable detection of borders between retinal layers.

Forty-two days after EAE induction, all animals underwent pattern VEP recordings on a Celeris platform (Diagnosys, Lowell, MA, USA). Briefly, after dark adaption overnight, the animals were anesthetized with intraperitoneal injection of ketamine (30 mg/kg, Mylan, Canonsburg, PA, USA), xylazine (5 mg/kg, Akorn Inc., Lake Forest, IL, USA), and acepromazine (2.3 mg/kg, Rattlesnake Drugs, Scottsdale, AZ, USA). Pupils were dilated using 1% tropicamide (Alcon Laboratories, Geneva, Switzerland), and GenTeal (Alcon Laboratories, Geneva, Switzerland) gel was used to keep the corneas moistened. The animals were kept on heating pads to maintain a constant body temperature of 37°C. Eyes were randomly selected, and 600 pattern VEP traces were recorded using a 2 Hz reversal vertical stripe pattern stimulus at a luminescence of 50 candela/m² and 100% contrast. Amplitudes were measured in microvolts (µV) from the zero line to the N1 trough (N1 amplitude) and from the N1 trough to the P2 peak (N1 to P2 amplitude). Latencies in milliseconds (ms) were determined as the time from presentation of the stimulus to the N1 trough and the P2 peak. Data from eyes with flat or atypical pattern VEP waveforms or non-unique peak identification were excluded from the statistical analysis.

Mice were anesthetized by intraperitoneal injection of ketamine (30 mg/kg, Mylan, Canonsburg, PA, USA), xylazine (5 mg/kg, Akorn Inc., Lake Forest, IL, USA) and acepromazine (2.3 mg/kg, Rattlesnake Drugs, Scottsdale, AZ, USA). After using 1% tropicamide to dilate the pupil, a drop of GenTeal gel (Alcon Laboratories, Fort Worth, TX, USA) was placed on the corneal surface to maintain corneal integrity. PERG was recorded using a pattern stimulator (Diagnosys Celeris System, Diagnosys LLC, Lowell, MA, USA) at 1 Hz (2 inversions per second) and 50 cd/m² alternating, reverse, black and white vertical stimulation response. Six hundred traces were recorded from each eye, the average waveform was calculated, and the amplitude (µV) from the peak of P1 to the valley of N2 was measured. All recordings were carried out while maintaining the animals’ body temperature between 37 °C and 38 °C using the system’s thermal pad.