Longitudinal Observation of Retinal Response to Optic Nerve Transection in Rats Using Visible Light Optical Coherence Tomography

Abstract

Purpose: To characterize rat retinal responses after optic nerve transection (ONT) by visible-light optical coherence tomography (vis-OCT).

Methods: Unilateral ONT was performed in Brown Norway rats (n = 8). In vivo, vis-OCT retinal imaging was performed on the experimental eyes before ONT (baseline), and two days, one week, two weeks, and four weeks (endpoint) after ONT, as well as on fellow eyes at the endpoint. The system was operated at a 70 kHz A-line sampling rate with both raster scans (512 × 2 × 512 A-lines), and circular scans (2048 × 100 A-lines) acquired around the optic disc. Retinal layers were segmented to calculate layer thicknesses and project en face images for visualization and quantifications. Vessel densities and oxygen saturation were used to evaluate the morphologic and functional impact on the retinal vasculature.

Results: After ONT, retinal nerve fiber bundles demonstrated significant degeneration, starting at two weeks, with a reduction of thicknesses quantified on the nerve fiber layer, ganglion cell complex, and total retina. Along with that, the activation of macrophage-like cells in the vitreoretinal interface was also observed. Vessel densities for all three retinal plexuses were unaffected over the period of observation. However, oxygen saturation in retinal arteries and veins was significantly reduced at four weeks after ONT.

Conclusions: Vis-OCT can provide high-definition, in vivo characterization of retinal responses to ONT in rats. Despite a significant reduction in retinal layer thickness, this was not accompanied by alterations in vascular density. Despite this, oximetry indicates reduced retinal oxygen saturation, suggesting that altered vascular physiology is not reflected in the anatomic appearance of retinal blood vessel density alone.

Figure 1.

Definitions of retinal layer slabs and vascular plexuses in rats as demonstrated in OCT and OCTA B-scans. Vitreous: projected from 40 µm above inner limiting membrane (ILM, blue line) to 3 µm above ILM. NFL, projected from ILM to 25 µm below ILM for en face visualization, or from ILM to the lower boundary (cyan line) of NFL for thickness analysis. Ganglion cell complex (GCC) nerve fiber layer, defined as the slab within the ILM to the lower boundary (green line) of the inner plexiform layer (IPL). Total retina, defined as the slab between the ILM and Bruch's membrane (BM, red line). SVP, defined as the slab within the ILM to the 10 µm above the lower boundary (green line) of IPL. ICP, defined as the slab between the 3 µm above and 10 µm below the lower boundary (green line) of IPL. DCP, defined as the slab between the upper (magenta line) and lower (yellow line) boundaries of the outer plexiform layer (OPL). ONL, outer nuclear layer; EZ, ellipsoid zone. The boundaries are inclusive in the slabs.

Figure 2.

En face structural images projected from the total retina slab and NFL slab, as well as the en face angiographic images projected from SVP, ICP, and DCP plexuses in scans acquired longitudinally at timepoints of before ONT and two days, one week, two weeks, and four weeks after ONT for a representative brown Norway rat. Nerve fiber bundles were appreciated in the en face images before ONT and two days and one week after ONT, whereas they disappeared at two weeks and four weeks after ONT, indicating severe retinal neurodegeneration. However, from visual inspection, the perfusion of the retinal vasculature appears unaffected at all time points up to four weeks after ONT.

Figure 3.

(A) Circular B-scan around optic disc acquired longitudinally at time points of before ONT and two days, one week, two weeks, and four weeks after ONT demonstrating the thinning of retinal layers, especially for the nerve fiber bundles. (B) Statistical results indicate that the thicknesses of the total retina, ganglion cell complex (GCC), and NFL were reduced in the experimental eyes after ONT, especially after two weeks after ONT. (C) Relative reflectance of NFL (defined as the ratio of the averaged OCT reflectance from the NFL slab to that from the total retina slab) for the experimental rat eyes. This was significantly decreased two weeks and four weeks after ONT, compared to baseline. TEMP, temporal; SUP, superior; NAS, nasal; INF, inferior.

Figure 4.

Cells appeared in rat eyes at one week after ONT, as captured by the high-resolution vis-OCT and visualized in the en face projection images of vitreous slabs. In contrast, these cells were not observed before ONT.

Figure 5.

SO₂ in retinal arteries and veins for (A) experimental eyes analyzed using the linear mixed effect model demonstrated a significant decrease in both arteries and veins, (B) fellow eyes at four weeks after ONT compared to baseline and experimental eyes, (C) box plot with the significance (red arrow) observed only between the experimental eyes at four weeks after ONT to baseline, two weeks after ONT, and fellow eyes at four weeks in the pairwise comparison.

Figure 6.

Oxygen saturation in the three retinal vascular plexuses for experimental eyes at baseline and four weeks after ONT surgery. The experimental eye demonstrated lower sO₂ in SVP and DCP after ONT surgery, indicating that reduced vascular sO₂ may be an early indicator of retinal vascular impairment.

Figure 7.

The retinal vascular (orange color) organizations with respect to NFL slab (gray color) shown in OCT/A scan volume (first column), B-scan (second column), and illustrated with schematics (third column) in humans and rats.