In-Vitro Assessment of Cortical Repair Induced by Branched-chain Amino Acid Treatment

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2024-03-21

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Purpose: Traumatic Brain Injury (TBI) refers to a constellation of pathologies resulting from mechanical damage to cortical tissue. The neurological sequala of such injuries can be devastating, and definitive treatment does not exist at this time. Branched-chain Amino Acid (BCAA) treatment has demonstrated neuroprotective effects in clinical literature and in various animal models of TBI. However, there is a lack of in-vitro literature referencing the repair capacity of BCAA administration after neuronal injury, particularly in the context of TBI. To fill this gap in knowledge, a scratch assay was repurposed for use in cortical culture, to assess the repair capacity of BCAA treatment.

Methods: Mouse-derived Mixed Cortical Culture (MCC) cells were extracted and seeded in 24 well plates. A scratch assay was performed, where a vertical scratch was drawn across each well in a reproducible manner with a 200 uL pipette tip. This procedure is meant to recapitulate aspects of mechanical damage induced by TBI on cortical tissue. Subsequently, images were taken immediately post-injury (0 hour time point), at 24 hour, and at 48 hour time points post-scratch to quantify the area of scratch unfilled by cells. Various dose concentrations of BCAA were tested in comparison to control (media only) and vehicle control (water). Test conditions included the customary BCAA ratio, which is a 2:1:1 mix of leucine, isoleucine, and valine; additionally, a 1:1:1 ratio of leucine, isoleucine, and valine was also tested.

Results: At 48 hours post-scratch, significant differences were found in open wound area when comparing the media only control to 10 uM (p < 0.01), 30 uM (p < 0.01), 300 uM (p < 0.001), and 1000 uM (p < 0.01) of the 2:1:1 BCAA dose. Significant differences were also found in the wound area when comparing the water vehicle control to 10 uM (p < 0.05), 30 uM (p < 0.01), 300 uM (p < 0.01), and 1000 uM (p < 0.01) of the 2:1:1 BCAA dose. No significant differences were found in the open wound area when comparing the controls and BCAA doses, at the 24 hour time point. Of note, no significant differences were found between control and treatment with the 1:1:1 ratio of leucine, isoleucine, and valine at any time point.

Conclusion: BCAA treatment at the 2:1:1 ratio was seen to accelerate injury recovery at various dose concentrations, as quantified by open wound area after scratch injury was induced. This cell culture model demonstrates the importance of BCAA ratios. While this aligns with animal models and clinical literature, this is the first in-vitro assessment of BCAA repair capacity, in the context of cortical culture. Future studies will be undertaken to further elucidate the constituents of the repair mechanism.

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