Abstract
Purpose: :
We have previously demonstrated that TNFR1 and TRADD are up-regulated in optic nerve injured retinas and expressed by axotomized retinal ganglion cells (RGCs) (Agudo et al, Mol Vision 2008, 14:1050-63). Here we further analyse the temporal regulation of FADD and Caspase 8, adaptor and effectors downstream the TNFR1 death signalling pathway, in retinas undergoing intraorbital optic nerve transection (IONT) or crush (IONC)
Methods: :
In control adult albino rats and in ON injured retinas analyzed at 12h, 48h or 7d after IONT or IONC (n=4, per treatment and time point), retinas were either freshly dissected for protein extraction and western blotting, or perfused, frozen and cut on the cryostat on 15µm thick radial sections. RGCs were identified with Fluorogold (FG) applied to both superior colliculi (SCi) one week before ON injury. Sections and westerns were incubated with primary antibodies against FADD and Caspase 8, and revealed with secondary antibodies conjugated to alexa-568 and HRP, respectively.
Results: :
Western blotting assays revealed that Caspase 8 precursor and active forms, and FADD were up-regulated as soon as 12h post-IONT and IONC This up-regulation is maintained up to 7dpl. This up-regulation is maintained up to 7dpl. Immunohistofluorescence to radial sections of control and injured retinas showed that both proteins were expressed by RGCs as seen by their co-localization with FG, among other cells of the ganglion cell later. Western blotting assays revealed that Caspase 8 precursor and active forms, and FADD were up-regulated as soon as 12h post-IONT and IONC.
Conclusions: :
The up-regulation of FADD and Caspase 8 after optic nerve injury further implies an active role for the extrinsic apoptotic pathway triggered by TNFR1 in signalling axotomy induced RGC death.
Keywords: retina • ganglion cells • neuroprotection