# En-Face Optical Coherence Tomography Angiography for Longitudinal Monitoring of Retinal Injury
## 2 Materials and Methods  
Animal Use: All animal handling and care protocols were approved by the University of Texas Medical Branch IACUC board, and procedures were performed per the standards set forth in the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research. For retinal imaging, wild-type C57 BL/6 mice (The Jackson Laboratory) were used. Both male and female mice were randomly assigned to acute injury and L-CNV groups. Since CNV is a degenerative disorder affecting elderly patients, aged mice (80 weeks) were used in the initial experimental group as they exhibit a realistically variable response, including an increase in RPE autofluorescence, and the aged female BL6 mice develop a severe response to L-CNV [34; 38; 39]. With aging, the ability for wound healing or regulation of inflammatory response may be impaired [40]. For each of the experimental repetitions, adult mice (18 weeks) were used to verify the findings of the longitudinal group. The experimental groups were chosen as litters of 4-female and 4-male mice. In total, 24 animals were used for in-vivo imaging and confocal imaging of tissue. From the data, complete sets of longitudinal data (16 lesions per condition) were measured at all seven time-points. The aged matched group subjects were screened for any apparent retinal disruptions or defects. Mice were anesthetized with a ketamine dexmedetomidine mixture, and their eyes were dilated with phenylephrine and tropicamide (Alcon). Corneal hydration was maintained with lubricating drops (Alcon). Laser lesions were induced with an image-guided laser system (Micron III Phoenix Research Laboratories, Pleasanton, CA), to guide a photocoagulation laser (Meridian Merilas) to the precise location on the retina. The focused 532 nm laser beam onto the retina reliably induced 4 lesions in the right eye of each subject, typically at the 3,6,9, and 12 o'clock positions corresponding to the retinal quadrants, as previously described by [PERSON] et. al. [34]. Prior to this experiment, a dosing study was used to determine threshold parameters for reliably inducing acute injury lesions and L-CNV, using the Micron III system in place of a slit lamp ophthalmoscope [35; 41]. The output power was verified using a thermopile power meter (Field Max II, Coherent). The L-CNV model [38; 42; 43] was induced with 180 mW for 70 ms (0.012 joules), and acute injury with 46 mW of power for 200 ms (0.009 joules), with a fixed spot size of 50 \(\upmu\)m. Following the laser treatment, we imaged the lesions at the timepoints of 1h, 1 day, 2 days, 1 week, 2 weeks, and 3 weeks. In-Vivo Lesion Verification: Each lesion was verified as acute injury or CNV using traditional in-vivo ophthalmology modalities using a Spectralis HRA +OCT imaging system. White light, Infrared (IR), and FA fundus imaging modes were all used to image the retina (Figure 11). In conjunction with the fundus imaging, volumetric scans were acquired with the Bioptigen OCT. Prescreening of each subject was used to establish baseline health, specifically scanning for preexisting retinal degeneration or physiological abnormalities. Any signs of bleeding or redness were noted, as they could be symptomatic of choroidal bleeding or retinal detachment. As an adjunct to color fundus imaging, IR SLO imaging was used to confirm lesions. For FA imaging, 100 mg/kg of 10% Sodium Fluorescein (AK-FLUOR, Akorn, Decatur, IL) was diluted 1:20 in normal saline for intraperitoneal injection prior to fluorescence fundus imaging with the Spectralis HRA. Multiple FA images were captured through the late stage of perfusion. FA imaging was used to validate vascular leakage from day 3 to 21. Reference IR images were acquired concurrently for comparisons to ensure the focus and accuracy of the FA images. Images were calibrated, and pertinent measurements are in \(\upmu\)m or \(\upmu\)m\({}^{2}\). Acute injury lesions were verified as transient hyperreflectivity and the low autofluorescence signal on fundus imaging.