NMDA and Kainate Induce Internucleosomal DNA Cleavage Associated With Both Apoptotic and Necrotic Cell Death in the Neonatal Rat Brain

Abstract

Injection of N-methyl-D-aspartate (NMDA) or kainate in the striatum of 7-day-old rats induced massive cell loss in the ipsilateral striatum, hippocampus and inner cortical layers. In order to examine whether apoptosis contributes to cell death in this model of excitotoxic injury we examined the progression of internucleosomal DNA fragmentation and changes in cellular ultrastructure. Agarose gel electrophoresis of DNA extracted from the ipsilateral striatum, cerebral cortex and hippocampus clearly showed breakdown of DNA into oligonucleosome-sized fragments, indicative of apoptosis, 12 h post-NMDA injection. In addition, an increase between 12 and 24 h was observed as well as a continuous presence 5 days later. Kainate induced a similar time course of oligonucleosomal DNA fragmentation, but the intensity of the ethidium bromide stained bands was less compared with that observed for NMDA. DNA fragmentation was not detected in animals intrastriatally injected with Tris-HCl or in animals treated with MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohept-5,10-imine hydrogen maleate, 1 mg/kg] 30 min after NMDA injection. MK-801 had no effect on DNA fragmentation induced by kainate. In addition to agarose gel electrophoresis, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labelling (TUNEL) was used for detection of DNA fragmentation in sections. A gradual increase in the density of both apoptotic and non-apoptotic TUNEL nuclei was found in the anterior cingulate (ACC) and retrosplenial (RSC) areas of the cortex, the striatum, and the CA1 area and dentate gyrus of the hippocampus over the first 24 h post-NMDA or kainate injection. In the contralateral hemisphere hardly any TUNEL nuclei were present and their density was comparable with that in animals injected with vehicle only. In the ipsilateral mammillary nucleus (MN), which showed no signs of acute cell swelling after intrastriatal injection with NMDA, internucleosomal DNA fragmentation was found 24 and 48 h after intrastriatal NMDA injection. Here, the density of TUNEL cells with apoptotic morphology was high at 12 and 24 h post-NMDA injection but returned to control levels by 5 days. Electron microscopy showed cells with a clearly apoptotic morphology in the ACC and RSC and in the MN 24 h after NMDA injection. In the CA1 area of the hippocampus a necrotic, rather than an apoptotic, ultrastructure prevailed, indicating that the TUNEL method stained both apoptotic and necrotic cells.(ABSTRACT TRUNCATED AT 400 WORDS)

gp120-Induced Neurotoxicity in Hippocampal Pyramidal Neuron Cultures: Protective Action of TGF-β1

“…Thus, both necrotic and apoptotic cell death may coexist in the same cell population (Bonfoco et al, 1995;van Lookeren Campagne et al, 1995). This seems to be the case for the neuronal damage induced by gp120 under our experimental conditions.…”

“…NMDA-induced neurotoxicity usually has been associated with necrosis (Meldrum and Garthwaite, 1990). However, a growing corpus of data has shown that both apoptosis and necrosis can be involved in excitotoxicity (Bonfoco et al, 1995;van Lookeren et al, 1995). In particular, a study on cortical neurons showed that mild or intense NMDA stimuli can result in predominantly apoptotic or necrotic neuronal damage, respectively (Bonfoco et al, 1995).…”

Slowly triggered excitotoxicity occurs by necrosis in cortical cultures

“…21 Conversely, in necrosis, activation of endonucleases may be a non-specific event occurring in parallel with many other destructive processes, so that DNA laddering appears only evanescently en route to gross cell failure. 9,16,23,51 In summary, the weight of evidence obtained here suggests that even slowly triggered excitotoxic death in neocortical cell cultures occurs primarily by necrosis. This conclusion complements our earlier study indicating that rapidly triggered NMDA receptor-mediated excitotoxic death in the same system also occurs by necrosis.…”

“…1 Some neurons degenerating after intraparenchymal injection of excitotoxins exhibit chromatin condensation and internucleosomal DNA fragmentation. 43,51 Furthermore, evidence has also accumulated that hypoxic-ischemic neuronal death may have an apoptosis component. Internucleosomal DNA fragmentation has been demonstrated in degenerating neurons after ischemic insults, 22,33,40,49 and inhibitors of macromolecule synthesis attenuated neuronal death in certain animal models of ischemia.…”

Glutamate neurotoxicity in mouse cortical neurons: atypical necrosis with DNA ladders and chromatin condensation

“…Kure et al [12] showed that cortical neurons treated with glutamate revealed internucleosomal DNA fragmentation (DNA ladders) and actinomycin D-sensitive neuronal death. NMDA or kainate produced chromatin condensation and DNA ladders in cortical and cerebellar neurons [2,15,17], suggesting that excitotoxicity can induce apoptosis as well as necrosis. In as much as caution that DNA ladders and chromatin condensation may not differentiate apoptosis and necrosis [7], we set out experiments to determine patterns of glutamate neurotoxicity in neuron-rich cortical cell cultures by analyzing ultrastructural change in cytoplasm and nucleus, DNA fragmentation, and chromatin condensation (see abstract, [16]).…”