Hyper-reflective dots in optical coherence tomography imaging and inflammation markers in diabetic retinopathy

Abstract

The aim of this study is to correlate small dot hyper-reflective foci (HRF) observed in spectral domain optical coherence tomography (SD-OCT) scans of an animal model of hyperglycaemia with focal electroretinography (fERG) response and immunolabelling of retinal markers. The eyes of an animal model of hyperglycaemia showing signs of diabetic retinopathy (DR) were imaged using SD-OCT. Areas showing dot HRF were further evaluated using fERG. Retinal areas enclosing the HRF were dissected and serially sectioned, stained and labelled for glial fibrillary acidic protein (GFAP) and a microglial marker (Iba-1). Small dot HRF were frequently seen in OCT scans in all retinal quadrants in the inner nuclear layer or outer nuclear layer in the DR rat model. Retinal function in the HRF and adjacent areas was reduced compared with normal control rats. Microglial activation was detected by Iba-1 labelling and retinal stress identified by GFAP expression in Müller cells observed in discrete areas around small dot HRF. Small dot HRF seen in OCT images of the retina are associated with a local microglial response. This study provides the first evidence of dot HRF correlating with microglial activation, which may allow clinicians to better evaluate the microglia-mediated inflammatory component of progressive diseases showing HRF.

Keywords: HRF; hyper-reflective spot; microaneurysm; microglia; retina.

FIGURE 1

Hyperglycaemic rat model showing dot hyper‐reflective foci in OCT scans. (a, b) Fundus images do not reveal obvious vascular changes in the albino rat. The white circles in (a, b) indicate the location of the HRF observed in the OCT images. (c) OCT image corresponding to the green line in (a) shows a hyper‐reflective dot in the outer retina (red arrow). See inset (d). OCT image corresponding to the green line in (b) shows a large blood vessel (white arrow); note the shadowing and elevation of the surface of the retina over it and small hyper‐reflective dots above it (red arrows). Scale bar 100 μm. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigmented epithelium.

FIGURE 2

Big and small hyper‐reflective foci observed in the OCT images. (a) Fundus imaging does not reveal obvious changes. (b) OCT image corresponding to the green line in (a) shows a 100 μm thick lesion in inner retina. (c) Section of the HRF area in B and staining with DAPI defines an enlarged capillary lumen area (asterisk) distorting the INL. (d) Immunohistochemical labelling with anti‐glial fibrillary acidic protein (GFAP) shows stress areas (white encircled area) around the vascular lesion. (e) Shows increased labelling of inflammatory cells (Iba‐1, white encircled areas) around the location of the HRF lesion and in the RPE‐choriocapillaris interface. The asterisks in (c)–(e) show the histological appearance of a large HRF in (b), corresponding to an enlarged blood vessel. (f) Quantification of GFAP labelling. Scale bar 100 μm.

FIGURE 3

Histological examination of the small dot HRF in the hyperglycaemic rat model. (a) Retinal area without HRF does not show microglial activation in the corresponding retinal section (a′). (b) OCT image showing two dot HRF in outer retina (arrows). (b′) Retinal section of the area of HRF in (b) shows an increased number of branched microglia (Iba‐1) in the inner retina. (c) Quantification of Iba‐1 labelling. Scale bar 30 μm. INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; RPE, retinal pigmented epithelium.

FIGURE 4

Retinal function on the dot HRF areas in the hyperglycaemic rat model. (a) OCT scan showing several dot HRF in the ONL and INL (red arrows), two of them visible surrounding a blood vessel in the GCL. (b) fERG results (blue line, average value) of areas adjacent to the HRF. Black double arrow indicates differences in a‐wave and in the b‐wave values compared with normal retina. (c) fERG results on HRF areas (blue line is average result) showing reduced a‐wave and b‐wave (indicated by double arrow) compared with normal retina. (d) a‐wave of the ERG is unaffected in low light levels compared with normal retinal areas but is significantly reduced in dot HRF areas when high light stimulus is used. (e) b‐wave of the ERG is significantly reduced in both HRF and HRF‐adjacent retinal areas compared with normal areas. The asterisk (*) denotes that a post hoc analysis revealed significant differences in fERG function between normal area and the HRF area, with a p value <0.05. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.