Spatiotemporal evidence of apoptosis-mediated ischemic injury in organotypic hippocampal slice cultures.
Oxygen-glucose deprivation (OGD) induced neuron-specific cell death in organotypic hippocampal slice cultures. Neuronal death was first evident in the CA1 region 24 h after the injury as assessed by propidium iodide (PI) labeling, and continued to extend to the CA3/4 region up to 72 h. At 6 days post-OGD, PI labeling was weak and diffuse with no clear demarcation of pyknotic nuclei. To characterize biochemical changes produced by OGD, cellular efflux of three key amino acid neurotransmitters was evaluated. OGD elicited large increases in the release of GABA and aspartate (55- and 4.5-fold increase over basal, respectively), while there were no detectable changes in extracellular glutamate levels. In order to ascertain the existence of the synaptic pool of glutamate, sister cultures were treated with sodium azide. This evoked a strong increase in glutamate release, suggesting the intactness of the glutamate system. Further studies revealed a time-dependent activation of caspase 3 following OGD, shown by immunoblot analysis as well as by confocal laser scanning microscopy. While we did not observe the activation of caspases 1, 2, or 8 in our model, the activation of caspase 9 was evident, peaking at 12 h post-OGD. Despite no apparent increase in glutamate release by ischemic slices, treatment with a N-methyl-D-aspartate (NMDA) antagonist or an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) antagonist significantly reduced neuronal death. Furthermore, a pan-caspase inhibitor (zVAD-fmk), but not the caspase 3 inhibitor (DEVD-fmk), provided partial neuroprotection. Inhibition of a Ca(2+)-dependent cysteine protease, calpain, by MDL28170 also elicited partial neuroprotective effects.
Cho
Seongeun
S
Neuroscience, Wyeth Research, CN 8000, Princeton, NJ 08543, USA. chos1@wyeth.com
Liu
Danni
D
Fairman
Denise
D
Li
Ping
P
Jenkins
Lorayne
L
McGonigle
Paul
P
Wood
Andrew
A
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D020169:Caspases / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D015687:Cell Hypoxia / Q000187:drug effects / Q000502:physiology; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D018696:Neuroprotective Agents / Q000494:pharmacology; D009924:Organ Culture Techniques; D051381:Rats; D013997:Time Factors
D018696:Neuroprotective Agents; D020169:Caspases