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Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue
Published on: August 15, 2012
Apoptosis of primary-culture rat microglial cells induced by pathogenic Acanthamoeba spp
1Department of Microbiology, Seoul 121-752, Korea. hjshin@madang.ajou.ac.kr
Abstract:
To determine whether trophozoites and lysates of pathogenic Acanthamoeba spp. induce apoptosis in primary-culture microglial cells, transmission electron microscopic (TEM) examinations, assessment of DNA fragmentation by agarose gel electrophoresis, and the TdT-mediated dUTP nick-end labeling assay were performed. When a trophozoite of pathogenic Acanthamoeba culbertsoni came in contact with a microglial cell, the digipodium was observed by TEM. Nuclear chromatin condensation was observed in 10% of microglial cells, while it was not revealed when they were cocultured with weakly pathogenic Acanthamoeba royreba trophozoites. DNA fragmentation in microglial cells cocultured with the A. culbertsoni lysate was detected by electrophoresis, showing DNA ladder formation, whereas it was hardly observed in microglial cells cocultured with A. royreba. DNA fragmentation of microglial cells was also confirmed by flow cytometry analysis. The fluorescence of TdT-stained apoptotic bodies became intensely visible with microglial cells cocultured with the A. culbertsoni lysate. In contrast, with microglial cells cocultured with the A. royreba lysate, only a background level of fluorescence of TdT-stained apoptotic bodies was detected. These results suggest that some rat microglial cells cocultured with pathogenic A. culbertsoni undergo cytopathic changes which show the characteristics of the apoptotic process, such as nuclear condensation and DNA fragmentation.
Insights
Pathogenic Acanthamoeba culbertsoni trophozoites and their lysates induce apoptosis in microglial cells, characterized by nuclear condensation and DNA fragmentation. Weakly pathogenic Acanthamoeba royreba did not elicit these apoptotic changes.
Area of Science:
- Neuroscience
- Immunology
- Microbiology
Background:
- Microglial cells are the primary immune cells of the central nervous system.
- Acanthamoeba species are opportunistic pathogens that can cause serious infections.
- The interaction between Acanthamoeba and microglial cells is not fully understood.
Purpose of the Study:
- To investigate whether pathogenic Acanthamoeba species induce apoptosis in primary microglial cells.
- To compare the effects of pathogenic Acanthamoeba culbertsoni and weakly pathogenic Acanthamoeba royreba on microglial cells.
Main Methods:
- Transmission electron microscopy (TEM) for cellular morphology.
- Agarose gel electrophoresis to assess DNA fragmentation.
- TdT-mediated dUTP nick-end labeling (TUNEL) assay and flow cytometry for apoptosis detection.
Main Results:
- Acanthamoeba culbertsoni trophozoites induced nuclear chromatin condensation in 10% of microglial cells.
- DNA fragmentation, indicative of apoptosis, was observed in microglial cells exposed to A. culbertsoni lysate.
- A. royreba showed minimal induction of apoptotic markers in microglial cells.
Conclusions:
- Pathogenic Acanthamoeba culbertsoni can induce apoptosis in rat microglial cells.
- Apoptotic changes include nuclear condensation and DNA fragmentation.
- These findings highlight the cytopathic potential of Acanthamoeba in the CNS immune environment.
