Subretinal Electrical Stimulation Induces FGF2 Protein Accumulation in RCS Rat Retina

Abstract

Purpose: : We previously showed that subretinal electrical stimulation (SES) from a microphotodiode diode array (MPA) transiently preserves photoreceptors adjacent to the implant and dark- and light-adapted ERG b-wave amplitudes, while inducing Fgf2 mRNA expression in pigmented, dystrophic RCS rats Ciavatta et al, IOVS, 2009). The current purpose was to test the hypothesis that SES of RCS rats would cause FGF2 protein accumulation in close proximity to the MPA.

Methods: : At postnatal day 21 (P21), RCS rats were subretinally implanted in one eye with an MPA that was either active (A; ~1600 µA/cm²), n=11, or nominally active (NA; ~6 µA/cm²), n=5. Weekly for 3 weeks, rats were dark-adapted and received light stimulation using electroretinography (ERG) xenon flashes for MPA stimulation. Retinal function was evaluated at P49 by scotopic (3.9x10^-4 to 768 cd s/m²) and photopic (0.151 to 75 cd s/m²) ERGs. Rats were sacrificed on P51. Both eyes were enucleated, fixed, and embedded for cryosectioning at 10 µm thickness (A, n=4; NA, n=3). FGF2 and GFAP were assessed by immunofluorescence.

Results: : Similar to previous ERG results, rats in the A group tended to have higher dark adapted b-wave amplitude ratios (treated:opposite eyes) than rats in the NA- or naïve control groups. In A-treated treated eyes, FGF2 labeling was dense and punctate in the inner nuclear layer (INL), outer plexiform layer (OPL), and along the RPE side of the MPA, but diffuse in the outer retina. GFAP labeling was elevated near the MPA region along the ganglion cell layer (GCL) with strong labeling penetrating to the INL. Strong GFAP labeling colocalized with FGF2 labeling along the RPE side of the MPA. FGF2 and GFAP labeling decreased with distance from the implant region. FGF2 and GFAP labeling in NA-treated eyes was similar to A treated eyes, but less intense overall: punctate FGF2 in the INL and OPL; GFAP in the GCL. However, FGF2 was stronger along the outer retina surface of the NA implant, and FGF2 and GFAP labeling were both absent at the RPE side of the implant. Labeling in peripheral retina, distal to the implant region, in A and NA groups was indistinguishable from contralateral eyes.

Conclusions: : SES from MPA induces not only upregulation of Fgf2 mRNA, but also elevated synthesis of FGF2 protein adjacent to the device.

Keywords: neuroprotection • growth factors/growth factor receptors • electroretinography: non-clinical