Abstract
Purpose::
Glaucoma, characterized by loss of retinal ganglion cells (RGC), is typically accompanied by sensitivity to intraocular pressure (IOP). To assess the contribution of the FasL mediated pathway of apoptosis to RGC death in glaucoma, we examined the comparative expression and localization of FasL and its receptor, CD95, in the C57BL6 reference strain and the DBA/2 mouse model.
Methods::
Retina were obtained from 3 month and 9 month C57BL6 and DBA/2 mice with low or high intraocular pressure as determined by TonopenXL measurement. For gene expression, cDNA was synthesized from harvested RNA and probed by RT-PCR and quantitative PCR. We used immunohistochemistry in whole-mount and cross-sectioned retina to assess the effects of elevated IOP on the levels and localization of FasL and CD95 and fluorescent in situ hybridization (FISH) to identify the cells that respond to elevated pressure and age by upregulation of FasL mRNA.
Results::
RT-PCR and Western blot results demonstrate that CD95 and FasL are present in retinal extracts and that FasL expression quantitatively increased as a function of age and pressure in retina isolated from age-matched animals with low and high IOP. FasL strongly co-localized with glutamine synthetase in Müller cell end-feet, but less so in GFAP-labeled astrocytes. It was heavily concentrated in the extracellular space of RGCs and intravascularly. Membrane bound CD95 was found in neurons of the inner nuclear layer and RGC layer. Interestingly, while CD95 levels decrease within the vasculature as a function of time and pressure, expression levels in RGCs maintained in vitro increase. Moreover, advanced age and increased intraocular pressure resulted in the increased expression of FasL, glutamine synthetase, and GFAP suggesting these stimuli result in a global change in the inflammatory state of the eye.
Conclusions::
CD95 and FasL are expressed and localized to a variety of retinal cells including neurons. CD95 is expressed in RGCs and responds to the effects of time and increased pressure, important glaucoma risk factors. FasL is expressed in the extracellular space around RGCs suggesting that FasL and CD95 may play an apoptotic role in glaucoma. The complex manner by which these binding partners interact suggests a role for FasL-mediated cell signaling in aged RGCs stressed by elevated IOP. Upregulation of CD95 and FasL suggest the death receptor mediated apoptotic pathway is present and functions in RGCs and opens avenues of possible therapeutics in treating glaucoma.
Keywords: ganglion cells • inflammation