Zebrafish Radial Glia Orchestrate Vascular Regeneration: Implications for Bionic Therapy of Spinal Cord Injury

Abstract

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

1. Introduction

Neurologic function in mammals is often irreversibly compromised following spinal cord injury (SCI) [1]. To overcome this limited regenerative capacity, we introduce the concept of spine bionic treatment. Inspired by organisms with remarkable self-repair abilities, this approach aims to enhance the efficiency of the human body's own repair processes after SCI by mimicking natural regeneration process or morphological structures found in nature. In this study, we utilized larval zebrafish, which at this stage exhibit exceptional regenerative capacity and optimal optical transparency suitable for high-resolution longitudinal live imaging. To precisely quantify the vascular response to SCI, we focused on the perineurial vascular plexus (PNVP). This foundational vascular network forms reproducibly at a defined developmental stage (around 18 dpf, Fig. 1a), providing a clear and standardized baseline for injury experiments. In contrast, the intra-neural vascular plexus (INVP) remains underdeveloped and more variable at these stages, and its visualization in later stages can be confounded by the increasing complexity of surrounding tissues and vessels. Thus, the larval PNVP model offers a unique and powerful system for dissecting the cellular and molecular dynamics of spinal vascular regeneration [2].

The regeneration of the vasculature after SCI is crucial prerequisite for of neurological function recovery [3]. The disruption of blood supply post-SCI exacerbates ischemia and promotes neuronal apoptosis of spinal cord tissue; conversely, early functional vascular remodeling is beneficial to mitigate ischemic damage, supply essential nutrients, and clear inflammatory debris [4]. New blood vessels sprout from the remaining blood vessels and proliferate in the injured tissue, which is an essential part of spinal cord regeneration. Zebrafish showed superior regenerative capacity after SCI compared to mammals; their larvae spinal cord can heal within 3 days post-injury, and adult zebrafish can also achieve spinal cord remodeling 30 days after injury [5]. Although revascularization is integral to this remarkable regeneration process, the strategies to actively promote vascular repair after spinal cord injury have been insufficiently emphasized, despite the fact that this is an important basis for providing the microenvironment support necessary for regeneration [6].