Modeling oxidative stress on neuronal dendrites and dendritic spines

The role of oxidative stress in chronic pathological conditions of the nervous system is well-established, contributing to neuronal dysfunction and cell death. Numerous studies have sought pharmacologic agents with antioxidant effects for neurons, with controversial results. Our study aims to establish a valid and reproducible in vitro oxidative stress model in neurons, simulating initial stages of neurodegenerative disorders, focusing on morphological alterations in the dendritic field of neurons. Primary hippocampal cells from newborn rat brains were cultured for two weeks, and subsequently, different concentrations of hydrogen peroxide (H₂O₂) were applied for 24 h in cultures. Cell viability assays and flow cytometry were carried out to measure H₂O₂ effects on apoptosis, necrosis, and reactive oxygen species production. Finally, morphometric evaluation of the neuronal dendritic field was conducted, focused on dendritic length, branching, and spine density, with ImageJ software following immunostaining of neurons. Administration of 100 μΜ H₂O₂ for 24 h in primary hippocampal cultures resulted in significant alterations in dendritic morphology (one-way ANOVA, p < 0.05), without significantly affecting cell viability. These results demonstrate the impact of H₂O₂ on neuronal morphology and suggest that dendritic and spinal alterations could serve as sensitive markers of mild oxidative stress on neurons, prior to the activation of apoptotic mechanisms. This can be used as a surrogate model for the initial stages of neurodegenerative disorders when synaptic loss takes place, facilitating study of agent effectiveness with antioxidant therapeutic potential.