NMDA AND KAINATE INDUCE INTERNUCLEOSOMAL DNA CLEAVAGE ASSOCIATED WITHBOTH APOPTOTIC AND NECROTIC CELL-DEATH IN THE NEONATAL RAT-BRAIN

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 ap optosis 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 hippocam pus clearly showed breakdown of DNA into oligonucleosomesized fragment s, indicative of apoptosis, 12 h post-NMDA injection. In addition, an increase between 12 and 24 h was observed as well as a continuous pres ence 5 days later. Kainate induced a similar time course of oligonucle osomal DNA fragmentation, but the intensity of the ethidium bromide st ained bands was less compared with that observed for NMDA, DNA fragmen tation was not detected in animals intrastriatally injected with Tris- HCl or in animals treated with MK-801 -10,11-dihydro-5H-dibenzo[a,d]cy clohept-5,10-imine hydrogen maleate, 1 mg/kg] 30 min after NMDA inject ion. MK-801 had no effect on DNA fragmentation induced by kainate. In addition to agarose gel electrophoresis, terminal deoxynucleotidyltran sferase-mediated dUTP-biotin nick end labelling (TUNEL) was used for d etection of DNA fragmentation in sections. A gradual increase in the d ensity of both apoptotic and non-apoptotic TUNEL nuclei was found in t he anterior cingulate (ACC) and retrosplenial (RSC) areas of the corte x, the striatum, and the CA1 area and dentate gyrus of the hippocampus over the first 24 h post-NMDA or kainate injection. In the contralate ral 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 c ell swelling after intrastriatal injection with NMDA, internucleosomal DNA fragmentation was found 24 and 48 h after intrastriatal NMDA inje ction. 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 apoptot ic, ultrastructure prevailed, indicating that the TUNEL method stained both apoptotic and necrotic cells. Based on biochemical and morpholog ical criteria this study provides strong evidence that both apoptosis and necrosis are involved in NMDA- or kainate-induced excitotoxic cell death in the neonatal rat brain.