DADOS Spectroscopy of Organotypic Brain Slices to Determine Hydrogen Peroxide-Induced Oxidative Stress

Organotypic brain slices are state-of-the-art ex vivo preparations that allow the study of all types of brain cells for several weeks to months. The brain slices are cultured on semi-permeable membranes, and as the slices flatten and become transparent, remain viable for several weeks. However, without cellular staining, assessing slice health remains challenging. Thus, this study aimed to determine whether assessing spectroscopy of the living brain slices can detect oxidative stress.

Brain slices (300 µm) were prepared from postnatal day 8-10 mouse brains and cultured as quarter-brain slices at the hippocampal level. Spectrography was performed using an adapted DADOS spectrograph. Fresh, living slices were tested with exposure to three laser wavelengths: red-green-blue, ultraviolet (UV), and infrared (IR) light, and a neon and mercury lamp. To induce oxidative stress, some slices were treated with 9.7 mM hydrogen peroxide for 2 days, and others were then stained with the red fluorescent nuclear cell death dye propidium iodide.

Our data showed that the DADOS spectrograph can detect spectra in living brain slices using all seven light sources. PI treatment alone decreased the detected wavelength signal when exposed to mercury, but increased it when exposed to neon. Hydrogen peroxide reduced the wavelength after exposure to IR and the red laser, while the area (intensity) was enhanced after exposure to UV, mercury, neon, and IR.

Our data show that oxidative stress-mediated processes can be measured with a DADOS spectrograph in living organotypic brain slices. The slices showed a differential spectral pattern when stimulated with seven different light sources. This study pioneers a novel, low-cost, easy-to-use DADOS spectroscopy tool for living brain slices.