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Corey A Smith, Balwantray C Chauhan; Longitudinal In Vivo Imaging of Retinal Ganglion Cells Labeled with Cholera Toxin Subunit B. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2176.
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To assess retinal ganglion cell (RGC) labeling with cholera toxin subunit B (CTB) via intravitreal injection for in vivo non-invasive and real-time imaging in wild type mice. It was hypothesized that uptake of CTB by RGCs act as a detectable tracer for longitudinal in vivo imaging of viable RGCs.
1 μl of CTB, a non-toxic neuronal tracer, conjugated to Alexa Fluor® 488 (CTB-488) was injected into the vitreous of C57BL/6 mice. The retina was imaged non-invasively by fluorescence imaging (488 nm excitation) with a modified Spectralis (Heidelberg Engineering) device at multiple time points in the same animal (n = 16) post-injection. Proof-of-principle work was completed to determine if CTB-488 is specific to RGCs in mice with immunohistochemistry of retinal flat-mount and section preparations for RGC (RBPMS) and amacrine (ChAT and GABA) cell labeling, respectively (n = 8).
Individual cells were detected by in vivo imaging after 10-15 days and to date have been successfully imaged consecutively up to 100-days post-injection (Figure). Fifteen days post-injection, the median (interquartile range, IQR) density for CTB-488 labeled cells was 3110 (2980, 3240) cells/mm2 and RBPMS labeled cells was 2560 (2500, 2740) cells/mm2. The percent difference between CTB-488 and RBPMS cell counts in the same animals was 19.4% with the number of CTB-488 expressing cells significantly higher than those labeled with RBPMS (p < 0.05). This difference was explained by amacrine cell labeling that co-localized with approximately 18.6% of CTB-488 labeled cells in the ganglion cell layer (GCL). This indicates some of the CTB-488 positive cells are amacrine cells and aligns with cell count differences between CTB-488 and RBPMS.
While it was found that CTB-488 uptake was not restricted to RGCs, the majority of RGCs were labeled. These findings have shown that intravitreal injection of CTB-488 is a reliable and effective label for RGCs in mice, by providing a clear, long-lasting and strong labeling of cells in the GCL in vivo. This work is developing techniques for non-invasive in vivo single cell resolution retinal imaging in wild type animals. This will allow for more robust and longitudinal studies to be performed when studying degenerative diseases of the retina in animal models.
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