Abstract
Abstract: :
Purpose: To investigate the developmental regulation of apoptosis in Retinal Ganglion Cells (RGCs) from both neonatal and adult mice following axotomy and excitotoxicity using in vitro retinal explants. Methods: TUNEL staining was used to identify cells undergoing apoptosis on whole eye sections (P1–7) and sections of retinal explant cultures at various time points and conditions. The expression of caspases, in particular 3 and 9, was determined by immunohistochemistry (IHC). IHC was also used to examine the expression of other proteins known to regulate apoptosis such as Apaf–1 in both neonatal and adult mice. RGCs were isolated by immunopanning, RNA extracted and RT–PCR carried out to detect expression of proteins of interest. Results: Developmental apoptosis in RGCs between P1–7 involves the activation of caspases 3 and 9. During degeneration following Optic Nerve (ON) axotomy, death is seen as early as 6h in the Ganglion Cell Layer (GCL) of P6 and was dependent on caspases 3 and 9. In contrast, adult RGCs were completely resistant at equivalent time points. Likewise, following Glutamate treatment [10 mM], adult retinal explants were more resistant than P6 explants to apoptosis at equivalent time points in culture. We also found a significant down regulation of Apaf–1 and caspase–3 in adults compared to neonatal mice at both RNA and protein level. Conclusions: Retinal Ganglion Cell developmental apoptosis is caspase–dependent. RGCs from adult animals are more resistant to apoptosis than neonatal RGCs following ON axotomy and excitotoxic insult. The expression of caspase 3 and Apaf–1 present in neonatal mice is absent in adult mice. Thus, age–dependent differential susceptibility to apoptosis is likely to be due to this down regulation.
Keywords: apoptosis/cell death • retinal development • ganglion cells