Abstract

Traumatic brain injury (TBI) initiates a cascade of cellular damage, with mechanical stress leading to immediate disruption of neuronal homeostasis, particularly concerning ion regulation and mitochondrial function. This study investigates the protective role of insulin in mitigating the cellular pathology induced by mechanical damage in a primary culture of brain neurons, serving as an *in vitro* model for TBI. Using fluorescent indicators, we monitored changes in intracellular calcium ([Ca²⁺]ᵢ) and sodium ([Na⁺]ᵢ) concentrations, alongside mitochondrial membrane potential (ΔΨm), following a standardized mechanical scratch injury. The injury caused an abrupt increase in [Ca²⁺]ᵢ and [Na⁺]ᵢ and a sharp drop in ΔΨm. Crucially, a subset of neurons exhibited delayed calcium deregulation (DCD) and a sustained high [Na⁺]ᵢ plateau, indicative of irreversible damage. Pre-treatment with 100 nM insulin significantly reduced the proportion of neurons progressing to DCD and sustained [Na⁺]ᵢ elevation. These findings demonstrate that insulin contributes to the normalization of ionic homeostasis and preserves mitochondrial integrity following mechanical trauma. The results highlight a potential neuroprotective mechanism of insulin, suggesting its therapeutic relevance in managing the acute cellular consequences of TBI by stabilizing critical membrane and mitochondrial functions.