Apoptosis and necrosis occurring in excitotoxic cell death in isolated chick embryo retina

Excitotoxic studies using isolated chick embryo retina indicated that such an in vitro model provides a valid tool to characterize the effect of diffe rent agonists for subtypes of glutamate ionotropic receptors. In retinas ma intained for 24 h in a Krebs medium, after a brief exposure (30 min) to glu tamate agonists, we compared the effects produced by NMDA and non-NMDA-agon ists, such as kainic acid (KA) or alpha -amino-3-hydroxy-5-methyl-4-isoxazo lepropionic acid (AMPA). Delayed retina[ damage was assessed by measuring l actate dehydrogenase (LDH) present in the medium after exposure to the prev iously named agonists. Although at high concentrations, both KA and AMPA pr oduced more relevant release than NMDA, 7-8% of total retinal LDH was relea sed after exposure to a 50 muM concentration of non-NMDA agonists. These va lues were similar to those obtained after 100 muM NMDA. In this regard, ret inal tissue appeared to be less sensitive to excitotoxicity based on the ac tivation of NMDA receptor subtype. All three agents produced histopathologi cal lesions typical for excitotoxic damage. A delayed form of excitotoxicit y observed in retina segments was predominated by necrotic features. Howeve r, the activation of apoptotic machinery early during the incubation period subsequent to brief exposure to NMDA (100 muM) was also present, The activ ation of caspase enzymes was studied by a fluorometric protease activity as say as well as by western blot analysis. Caspase-3-like activity reached th e highest value within 3 h of incubation after exposure to excitotoxin, the n the level, of enzyme activity declined to lower values. As confirmed by a time-related appearance of TUNEL-positive nuclei, apoptotic features appea red to be specific for retina response to NMDA. In contrast, the exposure t o a 50 Lm concentration of KA or AMPA induced necrotic cell damage which wa s evident through the incubation, leading to a delayed mechanism of excitot oxicity. These observations provide evidence that in the retinal model, wit h regard to agonist concentrations and subtype of glutamate receptors, the cascade of events loading to excitotoxicity may result in either apoptotic or necrotic neuronal cell damage.