Novel nerve regeneration assessment method using adult zebrafish with crush spinal cord injury.

Abstract

Zebrafish (Danio rerio), an alternative to rodents, are widely used in neurological, genetic, and toxicology research. The zebrafish larval spinal cord injury model has been used in neural mechanistic analyses owing to its high regenerative capacity and throughput; however, it also had several limitations in imitating rodents. Therefore, we investigated the use of adult zebrafish as an alternative model to rodents for evaluating nerve regeneration. Here, we established a novel spinal cord regeneration evaluation method, which was based on the maximum swimming speed of adult zebrafish in a custom-built hydrodynamic-based aquarium. The spinal cords of adult male zebrafish were crushed using forceps, and maximum swimming speed and histological spinal cord regeneration were evaluated. Spinal cord-injured zebrafish showed a significant decline in motor function, followed by recovery at 3 weeks postoperatively, accompanied by histological regeneration. Spinal cord regeneration can be indirectly assessed by monitoring maximum swimming speed. They were also fed a diet containing fig extract, which can promote peripheral nerve regeneration; they were fed daily starting 1 week before the operation. Maximum swimming speed was measured time-dependently until 3 weeks postoperatively. Fig-consuming fish showed improved recovery of maximum swimming speed compared to the controls, which was consistent with the histological analysis. In summary, we established a spinal cord regeneration assessment system using adult zebrafish in a customized aquarium, which enables researchers to evaluate spinal cord regeneration in adult zebrafish similar to that of rodent experiments, contributing to faster and easier screening of neuroregenerative technology.