Soluble macrophage factors trigger apoptosis in cultured hippocampal neurons.
It is not clear whether macrophages which can phagocytose dead cells, may also contribute to death of potentially viable neurons when they enter brain lesion sites after insult. We have initially examined the effects of macrophage-conditioned medium on the integrity of hippocampal neurons in culture. We assessed qualitative and quantitative changes in neuronal status in terms of nuclear morphology, internucleosomal cleavage, cell membrane integrity and process density. Cell morphology with manual counts to quantitate findings showed that macrophage conditioned medium significantly increased the percentage of neurons with abnormal nuclei. Aurintricarboxylic acid attenuated this effect. Demonstration of laddering of DNA on agarose gels suggested an apoptosis-like event. A commercially available kit used to detect high concentrations of 3'-OH DNA ends showed marked increase in labelled cells. These combined findings confirmed that apoptosis was the main event triggered by conditioned medium. Although the number of cells with incompetent membranes also increased with conditioned medium application the majority of cells with apoptotic nuclei maintained membrane integrity. Conditioned medium also resulted in significant loss of cell processes. Conditioned medium from stimulated microglia showed a similar pattern of injury. The response of stressed neurons to conditioned medium was also tested. Exposure of cultures to mild hypoxia resulted in injury but did not significantly alter their subsequent vulnerability to macrophage-conditioned medium. Early experiments suggest that the documented changes in neuronal status are caused by relatively large and stable secreted macrophage proteins.
Flavin
M P
MP
Department of Pediatrics, Queen's University, Kingston, Ontario, Canada.
Coughlin
K
K
Ho
L T
LT
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001253:Astrocytes / Q000502:physiology; D002478:Cells, Cultured; D018920:Coculture Techniques; D017077:Culture Media, Conditioned; D053938:DNA Fragmentation; D004587:Electrophoresis, Agar Gel; D006624:Hippocampus / Q000166:cytology / Q000502:physiology*; D008264:Macrophages / Q000502:physiology*; D017628:Microglia / Q000502:physiology; D018613:Microscopy, Confocal; D009474:Neurons / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley
D017077:Culture Media, Conditioned