PARP–1 Inhibition Attenuates the Default of NMDA–Induced Apoptosis to Necrosis in the Rat Retina

Abstract

Abstract: : Purpose: To examine morphological and biochemical changes resulting from the blockade of PARP–1 activity following NMDA–induced toxicity using an in vivo retinal model. Methods: Anaesthetized male Sprague Dawley rats were given an intravitreal injection into the left eye of the PARP–1 specific inhibitor PJ34 two hours prior to a second injection containing PJ34 and NMDA. Right eyes of each rat received either sham treatment followed by NMDA treatment or were given a single injection of NMDA. Retinas were assessed at designated time points for loss of selective membrane permeability properties, TUNEL labeling, polyribosylation of nuclear proteins (PAR) and cell loss in the ganglion cell layer (GCL). Results: NMDA– and PJ34/NMDA–treated retinas showed distinct differences when assessed for ethidium bromide staining (EtBr), TUNEL–labeling and PAR–expression. NMDA alone produced a uniform distribution of EtBr–permeable cells in the GCL. Staining morphologies showed two distinct profiles: cells with staining in condensed nuclei only (early stage apoptosis) and cells that were homogeneously stained (necrotic–like). Retinas that were co–treated with PJ/NMDA showed EtBr–staining of cells in the more advanced stages of apoptosis. These cells were characterized by irregularly–shaped, densely–stained nuclei with some displaying nuclear membrane blebbing. TUNEL labeling confirmed the EtBr observations with NMDA treatment alone resulting in cells typical of early–stage apoptosis, while PJ/NMDA treated retinas contained cells with more advanced apoptotic features. PAR–expression was enhanced with increasing concentrations of NMDA whereas, inclusion of PJ34 dramatically reduced PAR–formation, even under strong excitatory conditions. Cell loss in the GCL was essentially identical between NMDA and PJ34/NMDA–treated retinas. Conclusions: Although blockade of PARP–1 activity attenuates NMDA–induced necrosis in the rat retina, it does not prevent NMDA–induced apoptotic cell death. This indicates that PARP–1 blockade enables cells committed to the apoptotic process to advance without defaulting into necrosis.

Keywords: apoptosis/cell death • ganglion cells • excitatory amino acid receptors