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Neurobasal®/B27 is a gold standard culture media used to study primary neurons in vitro. An alternative media (BrainPhys®/SM1) was recently developed which robustly enhances neuronal activity vs. Neurobasal® or DMEM. To the best of our knowledge BrainPhys® has not been explored in the setting of neuronal injury. Here we characterized the utility of BrainPhys® in a model of in vitro mechanical-stretch injury.Primary rat cortical neurons were maintained in classic Neurobasal®, or sequentially maintained in Neurocult® followed by BrainPhys® (hereafter simply referred to as “BrainPhys® maintained neurons”). The levels of axonal markers and proteins involved in neurotransmission were compared on day in vitro 10 (DIV10). BrainPhys® maintained neurons had higher levels of GluN2B, GluR1, Neurofilament light/heavy chain (NF-L & NF-H), and protein phosphatase 2 A (PP2A) vs. neurons in Neurobasal®. Mechanical stretch-injury (50 ms/54% biaxial stretch) to BrainPhys® maintained neurons modestly (albeit significantly) increased 24 h lactate dehydrogenase (LDH) levels but markedly decreased axonal NF-L levels post-injury vs. uninjured controls or neurons given a milder 38% stretch-injury. Furthermore, two 54% stretch-injuries (in tandem) exacerbated 24 h LDH release, increased α-spectrin breakdown products (SBDPs), and decreased Tau levels. Also, BrainPhys® maintained cultures had decreased markers of cell damage 24 h after a single 54% stretch-injury vs. neurons in Neurobasal®. Finally, we tested the hypothesis that lentivirus mediated overexpression of the pro-death protein RBM5 exacerbates neuronal and/or axonal injury in primary CNS cultures. RBM5 overexpression vs. empty-vector controls increased 24 h LDH release, and SBDP levels, after a single 54% stretch-injury but did not affect NF-L levels or Tau.BrainPhys® is a promising new reagent which facilities the investigation of molecular targets involved in axonal and/or neuronal injury in vitro.BrainPhys®/SM1 increases the expression of axonal proteins.BrainPhys®/SM1 increases the expression of proteins involved in neurotransmission.Stretch-injury decreases neurofilament light chain and Tau levels.Neurobasal®/B27 exacerbates stretch-injury.Lentivirus mediated overexpression of pro-death RBM5 exacerbates stretch-injury.