Modeling Reactive Oxygen Species-Induced Neuronal Death in Mouse Cerebellar Granule Neurons

Take a culture of mouse primary cerebellar granule neurons.

Add media containing hydrogen peroxide, a reactive oxygen species or ROS, and incubate briefly.

Hydrogen peroxide diffuses into the cells and is converted into highly reactive free radicals.

These radicals induce lipid peroxidation, compromising the integrity of the cell membrane.

Additionally, the radicals cause oxidative modifications in cellular proteins, impairing their function.

Furthermore, they induce DNA breaks, leading to genomic instability.

The radicals also cause oxidative damage to intracellular organelles, including mitochondria.

In response, the damaged mitochondria release cytochrome c, which binds to the apoptotic protease activating factor-1, triggering apoptosome formation and the conversion of pro-caspase-9 to active caspase-9.

Caspase 9 activates executioner caspases, further cleaving cellular proteins and leading to apoptotic neuronal death.

Replace the media with fresh, hydrogen peroxide-free media to halt the signaling cascade.

For ROS-induced cell death, treat the neurons with hydrogen peroxide at 75 to 100 micromolar for five minutes. After five minutes, switch it to the conditioned media from parallel cultures.

Due to the instability of hydrogen peroxide, the concentration must be optimized to a level that induces between 50% and 70% cell death after 24 hours. This concentration is usually between 75 and 100 micromolar.