Longitudinal Imaging of Vitreous Immune Cell Recruitment After Retinal Injury Using Visible-Light OCT

Abstract

Purpose : Immune response helps maintain intraocular homeostasis after injury; thus, visualizing and quantifying immune cell concentration in the retina and vitreous improves the understanding of eye injuries. We aim to apply visible-light OCT (vis-OCT) to image and localize vitreous immune cell recruitment in living mice after partial optic nerve crush (pONC) longitudinally.

Methods : We performed pONC in wild type C57BL/6 mice to induce optic nerve damage. We then used vis-OCT to image the retina and vitreous before pONC and 12 hrs, 24hrs, and three days after pONC. Vis-OCT data was acquired following a temporal speckle averaging protocol, where each acquisition consisted of 512 A-lines × 512 B-scans, with each B-scan repeated twice and each volume repeated five times. At an A-line rate of 75 kHz, each acquisition took ~35 s. The field of view was 1×1 mm² with the optic nerve head placed at the corner of the volume. After reconstruction, the five repeated volumes were registered and averaged to reduce speckle noise.
The inner limiting membrane (ILM) was segmented using the second derivative of image intensity, and the whole retina image was flattened with respect to the segmented boundary. The volume 10-100 μm above the ILM was extracted for analyzing immune cells in vitreous. A maximum intensity project (MIP) and a 3D cell localization algorithm were used to visualize the cell distribution inside the vitreous.

Results : The transparent nature of the vitreous allows the visualization of hyperreflective spherical cells by vis-OCT. Compared to eyes imaged before ONC, we observed a higher immune cell concentration at 12 hrs and 24 hrs after pONC (Fig 1a). Three days after pONC, the immune cell concentration decreased. Computational analysis extracts the spherical cells against the background, enabling the localization of vitreous immune cells within the 3D volume.

Conclusions : These results demonstrate the feasibility of visualizing and localizing vitreous immune cell response in vivo after pONC injury with vis-OCT for the first time. This work has the potential to improve our understanding of various blinding diseases, including glaucoma.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

 

Fig 1. (a) MIP of vitreal cells (green) overlap with the retinal fiber bundle images (red). (b) Immune cells localization in 3D vitreous volume from 12 hrs after pONC. (c) Vis-OCT B-scan with the retina flattened at ILM. The cells are visible in the vitreous and retina.