Progressive changes in RCS rat retina during OCT/FAF-based in vivo imaging and their correlation with histological and visual functional analysis

Abstract

Purpose : To determine histological and visual functional correlates of changes observed during fundus autofluorescence (FAF) and optical coherence tomography (OCT) imaging of Royal College of Surgeon (RCS) rats during the progression of retinal degenerative (RD) disease.

Methods : FAF and retinal layer measurements were performed using Heidelberg Spectralis OCT at various postnatal ages (P18 to P180). Visual function was assessed using optokinetic nystagmus (OKN) testing. Retinal sections were stained with H&E, and retinal thickness (RT) and outer nuclear layer (ONL) cell count was analyzed using Aperio ScanScope. Morphological assessments and spectral analysis were used to ascertain correlation between progressive changes in the subretinal debris and the FAF pattern. Immunofluorescent staining for CD68 and GFAP (macrophage and glial cell markers) was performed to assess the source of hyperautofluorescence.

Results : FAF showed homogenous autofluorescence patterns up to P30, followed by homogeneous hyperautofluorescence. OCT showed severe loss of RT in young rats at P37, mostly due to loss of the ONL as demonstrated by histological analysis. Positive correlation between ONL thickness and OKN visual responses was observed at P30 and P37 (Pearson test, r=0.95). No OKN visual responses were recorded after P120. After P60, a hypoautofluorescence developed near the optic nerve. This area gradually spread by P90 and persisted through the entire study. ONL thickness loss and appearance of hypoautofluorescence were positively correlated (p<0.05). Bright hyperautofluorescent spots began appearing in a punctate fashion at P90. Morphological analysis suggested small patches of debris attached to the RPE surface, causing hyperautofluorescence and ruling out the contribution of macrophages and glial cells. Based on spectral analysis, hyperautofluorescent spots had a peak emission wavelength of 538 nm at P60 and P90, with higher wavelengths after P120. CD68 expression was found at P25 in the outer plexiform layer and ONL. By P46, CD68 was expressed predominantly in the outer segments.

Conclusions : Our study demonstrates that changes in FAF pattern can be predictive of morphological anomalies and visual functional loss in RCS rats. FAF is mostly due to subretinal debris as opposed to macrophages and other components within the RPE.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.