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Balwantray C. Chauhan, Kelly T. Steven, Julie M. Levesque, Glen P. Sharpe, Neil O'Leary, Michele L. Archibald, Xu Wang; In vivo Longitudinal Quantification of Retinal Ganglion Cell Density and Retinal Thickness Changes with Spectral-Domain OCT Following Optic Nerve Injury in Mice. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1646.
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© ARVO (1962-2015); The Authors (2016-present)
To quantify retinal ganglion cell (RGC) density and retinal thickness following optic nerve transection in transgenic mice that express cyan fluorescent protein (CFP) under Thy1 promotion.
21 mice from the B6.Cg-Tg(Thy1-CFP)23Jrs/J line were used. A modified Spectralis spectral-domain (SD) OCT was used to image CFP+ cells. SD-OCT circle, volume (37 B-scans) and radial (24 B-scans) scans were also obtained. CFP imaging was done at baseline (n=9) and 3 (n=6), 5 (n=6), 7 (n=5), 10 (n=3), 14 (n=4) and 21 (n=2) days post-transection, and SD-OCT at baseline and 7 (n=4) and 14 (n=4) days post-transection. Isofluorane anaesthesia was used and imaging took 15-20 mins. Follow-up images were acquired in the same locations with the registration software. Longitudinal change in CFP+ cell density and retinal thickness were computed. Whole-mounted retinas at baseline, 7, 14 and 21 days post-transection were stained with the RGC marker Brn3a. CFP+ and Brn3a+ cells were counted. Potential effects of isofluorane on Thy1 expression was examined with Thy1 mRNA levels in mice exposed to 0 (n=2) and 30 (n=2) mins of isofluorane.
The mean percentage CFP+ cells remaining post-transection was 83%, 63%, 47%, 32%, 18% and 11% compared to baseline at 3, 5, 7, 10, 14 and 21 days respectively. The decline in individual mice was similar with an inter-animal difference of ≤10% at any time point. The mouse retinal nerve fibre layer is thin and not resolvable for segmention. Compared to baseline, the mean (range) retinal thickness was 97 (90-100)%, 96 (91-99)% and 98 (95-99)% for the circle, volume and radial scans at 7 days and 97 (94-100)%, 97 (89-100)% and 95 (88-92)% at 14 days post-transection. The mean density of CFP+ cells from whole-mounted retinas was 1801, 384, 128 and 55 cells/mm2 at baseline, 7, 14 and 21 days post-transection. Brn3a+ cell density was 2667, 520, 197 and 106 cells/mm2. Thy1 mRNA increased by 5.9% after 30 mins of anaesthesia.
(1) Longitudinal imaging showed an exponential decline in CFP+ cell density and a small (≤5%) reduction in SD-OCT measured retinal thickness post-transection (2) Brn3a+ cell density was higher than CFP+ cell density at all time points. (3) CFP imaging is not significantly impacted by isofluorane anaesthesia. (4) SD-OCT is a promising tool for structural changes in experimental studies.
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