Figure 5
From: Novel 199 base pair NEFH promoter drives expression in retinal ganglion cells
EGFP expression from NEFH promoter constructs in retinal wholemounts and optic nerves. 1 × 109 vg of NEFH-P or hA-P AAV were delivered by IV injection to adult wild type mice, and EGFP positive cells analysed 4 weeks post-injection. Eyes were enucleated and fixed in 4% pfa; retinas wholemounted and optic nerves (ONs) cryosectioned longitudinally. Retinas were immunostained for BRN3A (Cy3 label, red) and EGFP (Alexa-Fluor-488 label, green). ONs were immunostained for EGFP (Alexa-Fluor-488 label, green), and counterstained with DAPI. (A,B) Overview of wholemount retinas with EGFP signal and BRN3A label (retinal ganglion cell, RGC) overlaid on the right half of the images. (C,D) Magnified view of wholemount retinas with EGFP signal and BRN3A label overlaid on the right half of the images. Some BRN3A-positive cells (RGCs) were co-labelled with EGFP; closed arrowheads indicate some double positive cells. Open arrowheads delineate an example of an EGFP-positive RGC axon. (E,F) EGFP-positive RGC axons detected in ONs. Scale bars: 500 μm (B), 50 μm (D), 100 μm (F). EGFP-positive RGCs were counted in six representative areas imaged at ×20 magnification (such as C and D) in each wholemount transduced with either NEFH-P or hA-P AAV (n = 6) using automated quantification in cellSens. The number of RGCs in the analysed areas (G) and the percent of EGFP-positive RGCs in the transduced retinas (F) are given in bar charts; bars represent mean + SD. Statistical significance between NEFH-P and hA-P AAV-transduced retinas was carried out using a t-test.
