Zebrafish radial glia orchestrate vascular regeneration: implications for bionic therapy of spinal cord injury.

BACKGROUND

Bionic treatment is a strategy designed to facilitate functional recovery after clinical spinal cord injury (SCI) by emulating the natural morphological structure and regeneration process. We used zebrafish model, an animal with remarkable regenerative capabilities to investigate the regulatory pattern of spinal vascular regeneration following SCI, with the hope of providing inspirations for the development of bionic SCI treatment.

METHODS

The experimental zebrafish were monitored and evaluated via live imaging. We first determined the formation time of the spinal perineural vessel plexus (PNVP) and used this as the timepoint to initiate SCI. Subsequently, a SCI model was established to observe the pattern of vascular repair without intervention; Furthermore, radial glial (RGs) of Tg(gfap: NTR-mCherry) report line fish were chemically ablated using metronidazole (Mtz) or nitrofuropyrinol (Nfp). We assessed the patterns of vascular repair, the vascular coverage of the injured area, and the number of vascular endothelial cells (ECs). Concomitantly, by analyzing the expression profile of vascular endothelial growth factor aa (Vegfaa) in the injured region following RGs ablation, and leveraging a public available single-cell sequencing dataset, we postulated the potential downstream pathways involved. The functional relevance of these pathways was finally evaluated by applying specific inhibitors.

RESULTS

The zebrafish PNVP forms at approximately 18 dpf; therefore, SCI modeling was explicitly timed at 19 dpf in this study to coincide with this development milestone. In the Tg(gfap: NTR-mCherry) report line, RGs were successfully ablated using either Mtz or Nfp. Following ablation, both vascular coverage in the injured area and the number of ECs were significantly reduced in the Mtz/Nfp + SCI group compared to the DMSO + SCI group. Moreover, The vegfaa reporter line revealed a notable decline in vegfaa signal within the injured region post-ablation, suggesting its involvement in the repair process. This implication was further supported by inhibitor experiments, where intervention against the Notch and PI3K/Akt-mTOR pathways significantly altered the extend of vascular repair, indicating a potential correlation between these pathways and RGs-regulated vascular repair.

CONCLUSION

Our findings demonstrate that RGs are a pivotal regulators of spinal vasculature regeneration in zebrafish SCI model. The underlying mechanisms may involve the Vegfa-PI3K/Akt-mTOR and Notch signaling pathways. Therefore, it can be postulated that pro-vascular repair therapy in mammals following SCI could potentially be achieved by therapeutically mimicking pro-regenerative functions of RGs.