Abstract
Abstract: :
Purpose: To investigate the role of Sonic hedgehog (Shh), a factor vital for neural development, in regulating the survival of adult retinal cells. The hedgehog genes encode secreted proteins important in many developmental patterning events, and are known to regulate mitogenesis and photoreceptor differentiation in the vertebrate retina. We hypothesized Shh might also decrease photoreceptor death in a transgenic rat model (P23H) carrying a rhodopsin mutation which encodes a dominant negative rhodopsin protein found in a large number of autosomal dominant Retinitis Pigmentosa patients. Methods: VSVg-pseudotyped lentiviral vectors carrying a CMV promoter driving a GFP reporter gene were used to establish cellular sites of expression in the retina. VSVg-lentiviral vectors carrying CMV-Sonic Hedgehog (Shh) were constructed. Subretinal injections of lentiviral vector (2 µl of virus, 1x1010 particles) were performed in the left eye of P23H transgenic and wildtype Sprague-Dawley rats on postnatal days P10 to P20. The right eyes received PBS injections as a control. Animals were sacrificed at 2, 4, 8, weeks, respectively 6 and 12 month after performing in vivo fluorescence imaging and electroretinograms. Results: Using HIV-CMV-GFP, in vivo GFP expression was detectable by 2 days postinjection by fluorescence retinal imaging and GFP expression remained stable for at least 12 months. Cryosections show GFP expression exclusively in RPE cells. Retinas examined by RT-PCR following HIV-CMV-Shh injections performed at P10 to P20 show sonic hedgehog expression, upregulation of the Shh receptor Patched (Ptc) and transcription of the Shh glycoprotein as well as the transmembrane protein Ptc (in RPE and Ganglion cells) by western blot, immunohistochemistry and confocal microscopy. Conclusions: These findings suggest further investigations into application of HIV-Shh as a therapeutic approach for retinal degenerations and to understand more about the Shh signaling pathways in the retina and its influence on photoreceptor cell rescue.
Keywords: gene transfer/gene therapy • retinal degenerations: cell biology • cell death/apoptosis