Abstract
Abstract: :
Purpose: There is conflict in the current literature regarding the role played by caspases in retinal ganglion cell death. In this study, we have examined the expression patterns of key caspases and related proteins in the rat retina. Methods: Protein expression was initially studied by immunoblot analysis of whole retinas of postnatal day 6 (P6) and postnatal day 60 (P60) Sprague Dawley rats. As the initial step in activation of the intrinsic pathway is the release of cytochrome–C, we looked at several members of the BCL–2 family including Bim, Bax and Bad. Furthermore, we examined caspase–9 and Apaf–1 expression, in addition to the executioners of apoptosis, caspases–7 and –9. Findings were then confirmed by immunohistochemistry, which permitted further localisation of the results to particular layers of the retina. RT PCR was also carried out to determine regulation of these proteins at a transcriptional level. Results: Western blots clearly indicated the presence of pro–apoptotic proteins caspase–3, –7, –9, Apaf–1 and BCL–2 proteins in the P6 retina. Immunohistochemistry further localised these proteins to all layers of the retina, and, in particular, to the retinal ganglion cell layer. In contrast, however, these proteins are downregulated in the P60 retina. Again immunohistochemistry provides further evidence to confirm these findings, indicating that the above proteins are absent in the photoreceptor and ganglion cells in the adult rat retina. Furthermore, RT–PCR confirmed the above findings on a transcriptional level. Of note, caspase–9 is present in both the postnatal and adult photoreceptor and retinal ganglion cells. Conclusions: Pro death proteins are highly expressed in the P6 rat retinal ganglion cell layer. In the young rat, the expression of these proteins is necessary for developmental apoptosis. In contrast, pro–apoptotic proteins are markedly downregulated in the adult rat retina. This confirms a mechanism for the tight control of apoptosis in the mature rat retina. If caspases and related proteins do indeed play a role in rat retinal ganglion cell death in glaucoma, it is clear that they must be re–expressed. Investigation of this upregulation is part of ongoing work.
Keywords: apoptosis/cell death • ganglion cells • retinal development