Abstract
AbstractStimulation of retinal neurons using optogenetics via use of chanelrhodopsin-2 (ChR2) has opened up a new direction for restoration of vision for treatment of retinitis pigmentosa (RP). Here, we report non-viral in-vivo electroporation of degenerated retina of adult RP-mice with ChR2-plasmids and subsequent in-vivo imaging of retina to confirm expression. Further, we demonstrate that in addition to efficient non-viral delivery of ChR2 to a specific retinal layer, threshold level of stimulation light needs to be delivered onto the retina for achieving successful behavioral outcome. Measurement of intensity of light reaching the retina of RP-mouse models along with geometrical optics simulation of light propagation in the eye is reported in order to determine the stimulating source position for optimal light delivery to the retina. The light-guided navigation of mice with ChR2 expressing retinal ganglion cells was found to be significantly improved over a long distance in correlation with stimulation intensity.
Highlights
Vision has a significant impact on physical, emotional as well as the psychological state of human health
Since very low intensity (~mW/ mm2) light is necessary for optogenetic stimulation[11,12], high-resolution activation of the degenerated retina is feasible using light from external μLED arrays projected to various retinal neuronal layers after photo-sensitization using ChR2
Subretinal injection of plasmid DNA combined with electroporation resulted in efficient transgene expression
Summary
Stimulation of retinal neurons using optogenetics via use of chanelrhodopsin-2 (ChR2) has opened up a new direction for restoration of vision for treatment of retinitis pigmentosa (RP). Though the total power reaching the retina decreases at larger distance of the source fiber, the peak irradiance increases (Fig. 2b) This can be attributed to the fact that though some light is lost at a larger source distance due to the finite size of the collecting eye-optics and the pupil size. The mouse was placed at various distances along the beam path in order to find out the threshold Both non-specific transfection of the retina as well as promoter-specific targeting of bipolar and retinal ganglion cells of adult mice and rats using viral methods have been established[17,18,19,20,21,22,23,24]. Our success in guiding the optogenetically treated rd1/rd[1] mouse over a distance of 100cm was attributed to efficient transfection of retinal ganglion cells, optimal light (intensity) delivery and careful training
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