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Soluble macrophage factors trigger apoptosis in cultured hippocampal neurons
M P Flavin1, K Coughlin, L T Ho
1Department of Pediatrics, Queen's University, Kingston, Ontario, Canada.
Abstract:
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.
Insights
Macrophages in brain lesions may harm viable neurons. Macrophage-conditioned medium triggers apoptosis and process loss in cultured neurons, suggesting secreted proteins contribute to neuronal injury after brain insult.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Macrophages are immune cells involved in clearing cellular debris.
- Their role in neuronal death within brain lesions is not fully understood.
- Investigating macrophage-conditioned medium's effect on neurons is crucial for understanding neuroinflammation.
Purpose of the Study:
- To determine if macrophage-conditioned medium induces death in potentially viable neurons.
- To characterize the mechanisms of neuronal injury induced by macrophage factors.
- To assess the impact on neuronal morphology and integrity.
Main Methods:
- Primary hippocampal neurons cultured in vitro.
- Treatment with macrophage-conditioned medium.
- Assessment of nuclear morphology, DNA fragmentation (apoptosis), membrane integrity, and neurite density.
- Use of aurintricarboxylic acid as a potential inhibitor.
Main Results:
- Macrophage-conditioned medium significantly increased abnormal nuclear morphology and DNA fragmentation, indicating apoptosis.
- Apoptotic neurons largely maintained membrane integrity, but process density significantly decreased.
- Aurintricarboxylic acid partially attenuated the observed effects.
- Conditioned medium from stimulated microglia induced similar neuronal injury.
Conclusions:
- Secreted proteins in macrophage-conditioned medium trigger apoptosis and neurite damage in cultured neurons.
- These findings suggest macrophages may contribute to neuronal loss in brain lesions.
- Further research is needed to identify specific secreted proteins responsible for neurotoxicity.