1556 - Effect of Traumatic Brain Injury on Drosophila Dopaminergic Mitochondrial Hydrogen Peroxide Production

Objectives: Increased mitochondrial reactive oxygen species (ROS) levels occur in response to inflammatory mediators released during brain injury and may lead to neurodegeneration. We investigated the effect of mitochondrial production of hydrogen peroxide (H₂O₂) as a marker of neuroinflammation in Drosophila melanogaster and hypothesized that high impact trauma (HIT) flies will show elevated H₂O₂ within dopaminergic (DA) neuron mitochondria. To address this hypothesis, a redox-sensitive fluorescent protein was genetically driven into tyrosine hydroxylase (TH)-containing DA neurons (mito-roGFP-ORP1). The maximal excitation wavelength of this redox-sensitive biosensor shifts when oxidized; thus, its fusion with oxidant receptor peroxidase 1 (Orp1) allows it to report relative H₂O₂ levels.

Design: 10-day-old, non-anesthetized flies expressing mito-roGFP-ORP1 were administered 1 strike in a 60° angle HIT device, while sham flies were placed in the HIT device without the pendulum drop. 4 hours post-injury, brains were incubated with 1% rabbit anti-TH antibodies followed by 0.5% goat anti-rabbit IgG to identify DA neurons. Confocal microscope captures of brains at 630x were excited at 405 nm (oxidized) and 488 nm (non-oxidized). To determine relative levels of H₂O₂, the sum of the total volume of oxidized fluorophore emission per region was divided by that of the non-oxidized fluorophore from Image Pro Premier 3D data. Unpaired t-tests determined the effects of injury.

Results: Compared to sham, female HIT files showed a statistically significant decrease in H₂O₂ production (p = 0.0036; male HIT decreased, p = 0.3389).

Conclusions: Although contrasting to the hypothesis, this data may represent neuroprotective mechanisms within Drosophila DA neurons prior ROS elevation. Previous Rodent studies have shown increased formation of ROS and nitrogen intermediates are affected by injury magnitude and post-injury intervals, and secondary injury mechanisms likely vary across TBI models. Studies investigating alternate post-injury time points of ROS quantification post-TBI are warranted.