Longitudinal In Vivo Imaging of Retinal Ganglion Cells and Retinal Thickness Changes Following Optic Nerve Injury in Mice

Retinal ganglion cells (RGCs) die in sight-threatening eye diseases. Imaging RGCs in humans is not currently possible and proof of principle in experimental models is fundamental for future development. Our objective was to quantify RGC density and retinal thickness following optic nerve transection in transgenic mice expressing cyan fluorescent protein (CFP) under control of the Thy1 promoter, expressed by RGCs and other neurons.A modified confocal scanning laser ophthalmoscopy (CSLO)/spectral-domain optical coherence tomography (SD-OCT) camera was used to image and quantify CFP+ cells in mice from the B6.Cg-Tg(Thy1-CFP)23Jrs/J line. SD-OCT circle (1 B-scan), raster (37 B-scans) and radial (24 B-scans) scans of the retina were also obtained. CSLO was performed at baseline (n = 11) and 3 (n = 11), 5 (n = 4), 7 (n = 10), 10 (n = 6), 14 (n = 7) and 21 (n = 5) days post-transection, while SD-OCT was performed at baseline and 7, 14 and 35 days (n = 9) post-transection. Longitudinal change in CFP+ cell density and retinal thickness were computed. Compared to baseline, the mean (SD) percentage CFP+ cells remaining at 3, 5, 7, 10, 14 and 21 days post-transection was 86 (9)%, 63 (11)%, 45 (11)%, 31 (9)%, 20 (9)% and 8 (4)%, respectively. Compared to baseline, the mean (SD) retinal thickness at 7 days post-transection was 97 (3)%, 98 (2)% and 97 (4)% for the circle, raster and radial scans, respectively. The corresponding figures at 14 and 35 days post-transection were 96 (3)%, 97 (2)% and 95 (3)%; and 93 (3)%, 94 (3)% and 92 (3)%.Longitudinal imaging showed an exponential decline in CFP+ cell density and a small (≤8%) reduction in SD-OCT measured retinal thickness post-transection. SD-OCT is a promising tool for detecting structural changes in experimental optic neuropathy. These results represent an important step towards translation for clinical use

Longitudinal infrared scanning laser ophthalmoscopy images.

<p>Images are from the same animal, and at the same time-points and location (but focused on the retinal nerve fibre layer, RNFL) as the animal shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040352#pone-0040352-g004" target="_blank">Fig. 4</a>. Optic nerve head transection was performed after obtaining the baseline (BL) image. Longitudinal images obtained 3, 7, 10 14 and 21 days after optic nerve transection. The RNFL appears unaltered until there was substantial loss of cyan fluorescent protein positive (CFP+) cells. The numbers at the bottom right of each image after BL show the proportion of CFP+ cells surviving compared to BL in this animal.</p

Proportion of cyan fluorescent protein positive (CFP+) cells surviving after optic nerve transection.

<p>Individual animal (A) and mean data with line showing the exponential function fitted though x = 0 and y = 1 (B). Error bars represent standard deviations.</p

Scanning laser ophthalmoscopy/spectral domain optical coherence tomography set up.

<p>Camera (A) mounted on a customized stereotaxic frame which allowed rotation along the horizontal and vertical axes with two geared systems (controlled by knobs B). The top plate of a rodent stereotaxic frame (C; the ear and bite bar holders were removed for these experiments) was positioned along the horizontal and vertical axes (controlled by knobs D).</p

Variability of retinal thickness measurements with SD-OCT^*.

*<p>values shown are mean (minimum, maximum) standard deviation as a percentage of the mean retinal thickness.</p

Proportional change in retinal thickness after optic nerve transection.

<p>Mean data obtained from the circle, raster and radial scanning patterns. Error bars represent standard deviations.</p

Longitudinal spectral domain optical coherence tomography (SD-OCT) scans.

<p>SD-OCT B-scans at the same line in a raster scan at baseline (BL) and 7, 14 and 35 days after optic nerve head transection (top panel). The segmentation of the retina is also shown (red lines demarcating the inner limiting membrane and retinal pigment epithelium). Retinal thickness values at corresponding time points at corresponding locations (bottom panel).</p

Spectral domain optic coherence tomography (SD-OCT) and corresponding histological section.

<p>SD-OCT B-scan centred on the optic nerve head obtained at baseline (A). Magnified portions of B-scan approximately 1.9 mm from the centre of the optic nerve head (B) and corresponding histological section (C) at the same location. Layers of the retina are clearly visible in the SD-OCT scan, however, at this distance from the optic nerve head, the retinal nerve fibre layer (RNFL) is not consistently visible with either SD-OCT or histology. In the optic nerve head, the SD-OCT signal is obscured by the retinal vessels, At some locations in the SD-OCT scan (A) and in the histological section (D) close to the optic nerve head, the RNFL (arrows) is visible. RGC, retinal ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segments of photoreceptors; OS, outer segments of photoreceptors; RPE, retinal pigment epithelium.</p

Imaging schedule and sample size at each time point.

<p>CFP, cyan fluorescent protein.</p><p>SD-OCT, spectral domain optical coherence tomography.</p><p>BL, baseline (prior to optic nerve transection).</p><p>All observations after BL are post optic nerve transection, except *which represent control (non optic nerve transected) animals for estimating SD-OCT variability.</p

Scanning patterns used for spectral domain optical coherence tomography.

<p>Infrared image of a mouse retina (A) centred on the optic nerve head showing the retinal blood vessels and retinal nerve fibre layer (arrows). Scanning patterns centred on the optic nerve head: circular (B), raster (C) and radial (D).</p