O252: Studying the neuronal regenerative response to in-vitro laser axotomy

Abstract Aims Outcomes of peripheral nerve injury (PNI) remain suboptimal, and the intrinsic mechanisms directing regeneration of injured peripheral neurons are not completely understood. To study the cellular response to injury, it is critical to deploy a model that facilitates a precise and controlled transection. We aim to recreate clinical outcomes at a micrometer level and analyse the impact of injury parameters, such as the proximity of injury to the cell body, on neuronal survival and regeneration. Methods We cultured adult rat dorsal root ganglion (DRG) neurons and transected individual neurites with a high-precision laser at 25um and 125um from the cell body. We monitored for 24 hours using time-lapse microscopy and analysed cell survival and neurite regeneration, comparing between two distance groups. Results Following laser transection at 25um from the cell body, 56% (n=25) of neurons survived for 24 hours and 33.3% (n=12) regenerated. When laser-transected at 125um, 75% (n=40) of neurons survived and 73.1% (n=26) regenerated. The mean regenerative distance was 19.9um (n=4) when injured at 25um and 173.9um (n=19) at 125um. Discussion Our laser axotomy method facilitated efficient and precise transection of individual neurites. Distal injuries generated significantly higher survival rates and regenerative length and rates compared to proximal injuries. These findings mimic clinical outcomes where distal injuries have greater recovery than proximal injuries, thereby demonstrating the efficacy of this model for investigating cellular mechanisms directing peripheral neuron regeneration. We envisage that this model will facilitate future studies aiming to accelerate peripheral neuron regeneration in a clinical context.