A short guide on in vivo transfection of flag-HDAC4 in dentate gyrus for study of adult neurogenesis in repetitive mild traumatic brain injury.

HDAC4, a class II epigenetic regulator, plays a critical role in adult hippocampal neurogenesis and determines the fate of newly proliferating and/or differentiating neural stem cells (NSCs). However, repetitive mild traumatic brain injury (rmTBI) impairs neurogenesis, contributing to the pathophysiology of cognitive deficits observed in neurodegenerative and neuropsychiatric disorders. Here, we present a protocol: (1) to induce rmTBI in mice using a closed head injury (CHI) device and to validate the model by assessing cognitive/behavioral performances and (2) to perform intracranial injection of FLAG-HDAC4 into the dentate gyrus of adult mice using a stereotaxic apparatus, in order to investigate the role of HDAC4 in impaired adult neurogenesis. This is achieved by analyzing BrdU⁺ and Nestin⁺ cells (markers of NSC proliferation), DCX⁺ and ND1⁺ cells (markers of neuronal differentiation), along with immunohistochemistry, fluorescence microscopy, and image analysis.