# En-Face Optical Coherence Tomography Angiography for Longitudinal Monitoring of Retinal Injury
## 3 Results
(**c**) 3 days, the vessels are disorganized or non-perfused as lesion height increases. (**d**) 7 days, vascular perfusion is low. (**e**) 14 days, perfusion can be visualized; however, the capillary organization has changed. (**f**) 21 days, a full field of capillaries returned with remodeling of the lesion. Figure 9: OCTA images of the Choroidal layer for acute injury lesions and CNV. While the choroid below the acute injury lesions (**a–c**, L1 and L2) appears to be intact, the CNV lesions (**d–f**, L2 and L3) clearly penetrate [PERSON]’s membrane appearing as a void in OCTA images. Assessment is immediate, and the separation differentiates acute insults that do not form CNV. As evidenced by OCT assessment of the lesion, the vaporization bubble began to swell and displace the inner retinal layers immediately following the lesion induction (Figure 4). The RPE disruption was apparent at the 1h time point in the en-face OCT, corresponding with the discontinuous layers seen in the B-scan, and is an early sign of successful CNV induction (Figure 11). In non-pathogenic conditions, the acute phase response ends, and the lesion entered the resolving phase between day 3 and 7; however, in that period, the CNV development continued forming sub-retinal edemas (fully apparent at day 7) indicative of the transition into the chronic inflammatory response. The ONL view identified the early sign of inflammation, edema formation at day 7, and eventual scarring (Figure 7). The optical slab containing the IPL/INL was most affected by the retinal swelling and edema. The region above and around the lesion was displaced, and moreover, the IVP and DVP showed regions of capillary non-perfusion. The non-perfusion was obvious on days 3 and 7; the reperfusion was not apparent until day 14 (Figures 6, 7 and 10). This capillary non-perfusion or loss of flow was not directly observable through FA, as the inner retina presents as either void or obscured by the leakage from the choroid vasculature. Immunofluorescent Staining: A final group of age-matched mice was utilized for producing the retinal and choroidal flatmounts. To assess the OCTA findings in the inner retinal layers, the SVP, IVP, DVP, and ONL were mapped with OCTA projections and ConA Lectin stained flatmounts at day 14 (Figure 3). The vascular features disrupting the IVP and DVP were confirmed in the ConA Staining. Additionally, the CNV vascular intrusion into the ONL was validated in the flatmount. Neither the OCTA nor Lectin staining in the acute injury and control conditions exhibited abnormal vasculature. Isolectin staining was used to validate the en-face OCT assessment of the integrity of the RPE layer with choroidal flatmounts (Figure 11). The flatmounts were co-stained with isolectin and DAPI to visualize the choroid. At one week, the choroid of the acute injury lesions was comparable to the naive control eyes, with neither showing signs of disruptions or abnormal vasculature. The CNV choroids showed clear neovascular scaring. While the swelling in CNV is consistent with the previously published histopathological studies showing cellular infiltration and wound healing in the chronic phase [49], the early impact on the  
Figure 11: Alternative processing of the OCTA data to show the changes in the Retinal Pigment Epithelium/Choroid over time. (**a**) pretreatment shows no vasculature. (**b**) 1 hour, the initial lesion is a dark void. (**c**) 3 days, the distorted vasculature and fluid can be seen in the center of the lesion. (**d**) 7 days, A ring of neovascularization begins to form. (**e**) 14 days, The lesion forms scar and ring patterns typical of flatmounts of angiogenesis. (**f**) 21 days, the border ring is no longer visualized but infiltrating vasculature can be seen. inner retina has not been explored. Our data shows that the early swelling and capillary non-perfusion precede edema and neovascularization. We found that the en-face OCTA provides new information on the early inner retina capillary non-perfusion in CNV (Figure 8).