# En-Face Optical Coherence Tomography Angiography for Longitudinal Monitoring of Retinal Injury
## 3 Results
The lesion that was obscured indicated bleeding and the absorption characteristics of blood severely increasing attenuation of the 488 nm fluorescence; however, IR light as used in OCTA is not attenuated by blood and therefore can still resolve the lesion. Consistent with previous literature, female CNV mice developed larger initial lesions that grew proportionately to the males [34]. For longitudinal analysis 16 sample lesions for each group (CNV and acute injury) with every imaging modality imaged at all timepoints were included for all quantitative analysis. Follow-up subjects used for validation of microvascular vasculature morphometry (Figure 3) and their choroids imaged (Figure A3), and verified for consistency, but were not included in the pooled quantitative longitudinal analysis to keep group size (n = 16) consistent between time points for valid differential analysis. Lesion Dynamic Descriptors: Orthographic views of the lesions were used to landmark and accurately extract the B-scans over time (Figure 1). Acute injury lesions exhibited a cylindrical shape, showing disruption throughout the Outer Nuclear Layer (ONL) and by day 3 began to assume an hourglass shape (Figure 4). Eventually, the hourglass neck would split, and the disruptions would form a pyramidal scar. While there was swelling, the Inner Nuclear Layer (INL) remained relatively undisturbed. It should be noted that although the RPE boundary was distorted, it remained continuous. Early CNV lesions had less definitive borders until day 3, when the swelling was noticeable in all layers. On day 7, there was the formation of edemas with clearly defined layer separation (Figure 4). However, the few lesions that did not present with clear leakage or severe edema still had signs of angiogenesis. By day 14, a reduction in edema and scarring of RPE-ONL was more pronounced. Day 21 was marked by scarring and disruption of RPE. While all the acute injury lesions followed a predictable progression, the CNV lesions size and features were highly variable when evaluated qualitatively. Figure 3: Comparisons of the in-vivo OCTA (color depth map) and retinal flatmounts (binary mask) demonstrate the correlation between findings for the SVP (Superficial Vascular Plexus), IVP (Intermediate Vascular Plexus), DVP (Deep Vascular Plexus), and ONL (Outer Nuclear Layer). At day 14 of the CNV induction, the final OCTA was generated, then perfused with Con A and extracted for flatmount. The left panel is the OCTA images represented as depth maps through the different layers. The right panel is corresponding flatmount images labeled with Con A. The Black arrow denotes a landmark vascular branch in the superficial layer (**a,c**) used to compare lesion locations. The superficial (**a,c**) and IVP (**b,d**) are intact with perfused vasculature at day 14. The DVP (**e.g**) shows a clear disruption of the vasculature where areas of non-perfusion are clear in both OCTA and confirmed in the flatmount. The ONL (**f,h**) should be avascular; however, at this stage of CNV, there is a signal in the OCTA and vascular staining in the flatmounts. Lesion Dynamics: B-Scan Measurements: The female subjects in the CNV group had more severe lesions; however, the growth trends were proportional and consistent with the males and therefore pooled for analysis presented in Figure 5 Panel A. The resulting dynamics traces, plotted in Figure 5 Panel B, were generated from pooled data of treatment groups. The acute injury lesion is confined to the ONL with the major changes seen in the RPE distortions. (Table 1, Figure 5 Panel A) The acute injury lesion expands from \(136\pm 21\)\(\upmu\)m to \(196\pm 44\)\(\upmu\)m (50% change) along the RPE layer at day 3, when the trend reverses. The scar reaches steady state between day 7 and 14 with a 146 \(\pm\) 45 \(\upmu\)m lesion diameter occurring in the RPE. The PR scar is reduced from \(129\pm 10\)\(\upmu\)m to \(78\pm 13\)\(\upmu\)m at day 3 and reduces to a steady state at day 7.