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New functional vessels form after spinal cord injury in zebrafish

Ana Ribeiro, Mariana Rebocho da Costa, Carmen de Sena-Tomás, Elsa Charas Rodrigues, Raquel Quitéria, Tiago Maçarico, Susana Constantino Rosa Santos, Leonor Saúde

Preprint posted on 12 June 2022 https://www.biorxiv.org/content/10.1101/2022.06.09.495446v1

Back to (the) back: regenerated blood vessels repopulate the injured spinal cord in zebrafish!

Selected by Arsila Ashraf

Why I chose this preprint

This study introduces the vascular system as a new component of the regenerative program in the zebrafish spinal cord and provides an in-depth analysis of vasculature during spinal cord repair. The findings of this study are impactful and could help design revascularization strategies that could be applied to the mammalian spinal cord. Furthermore, I was fascinated to see how newly formed blood vessels appear to be associated with regrowing axons at the injury site. This preprint highlights the importance of understanding the mechanisms that govern vascular repair and their possible role in neuronal regeneration, which could inform the development of new treatment strategies for spinal cord injury.

Background

Spinal cord injury (SCI) breaks axonal fibers and blood vessels, leading to severe and long-lasting sensory-motor deficits (Ahuja et al., 2017). In mammals, the vascular system is repaired inefficiently after SCI, which results in altered vessel distribution, as well as a compromised blood-spinal cord barrier (BSCB) (Oudega, 2012; Yao et al., 2021). Unlike in mammals, spinal cord repair and functional recovery after injury are remarkably efficient in zebrafish (Becker et al., 2004; Hui et al., 2010). However, how zebrafish blood vessels respond to SCI has not been investigated yet.

In this study, Ribeiro and colleagues addressed the distribution of the spinal cord vasculature in the adult zebrafish spinal cord before and after injury. Following injury, the zebrafish spinal cord re-establishes a stable and functional vascular network driven by endothelial proliferation, pericyte recruitment, and BSCB recovery. Overall, the authors present a detailed spatiotemporal description of the vascular repair response to SCI and demonstrate that zebrafish can successfully revascularize the injured spinal tissue.

Key findings

The zebrafish spinal cord was injured with the contusion method; that is spinal cord was squeezed dorsoventrally with forceps (Hui et al., 2010). The response of blood vessels (labeled with endothelial marker) was examined in cleared wholemount or sections of the spinal cord on different days post-injury (dpi) with light sheet or confocal microscopy. Spinal cords from zebrafish subjected to sham injury were included as controls.

Blood vessels revascularize the injured spinal cord

After the injury, multiple blood vessels revascularized the lesion site by 7 dpi, and most of the newly formed blood vessels persisted at the wound for as long as 90 dpi. Closer morphological analysis in the early post-injury phase revealed large and tortuous vessels that agglomerated in the central region of the spinal cord. However, towards the later time points – between 60 and 90 dpi – the authors observed a remodeling and reduction in vascular complexity, suggesting the removal of dysmorphic and dysfunctional vessels.

Interestingly, the regrowth of blood vessels at the injury site preceded the glial bridge formation and the regeneration of neurons. Moreover, some axons growing into the injured tissue were detected close to the new blood vessels, raising the possibility that blood vessels act as pioneers for regenerating axons.

Angiogenesis contributes to the formation of new vessels

Next, using an endothelial nuclear label, the authors showed that vascular repair in response to injury involved an initial phase (from 1 to 7 dpi) of rapid endothelial growth. The authors hypothesized that the proliferation of endothelial cells contributes to the new vessel formation. Endothelial proliferation and its dynamics were evaluated by tracking EdU incorporation after spinal cord/sham injury. The results indicated that an early and transient endothelial proliferative response promotes the repair and regrowth of blood vessels into the injured tissue. Furthermore, gene expression analysis in contused spinal cords revealed an upregulation of the expression of angiogenesis-related genes, like those belonging to the VEGF signaling pathway.

Barrier properties are re-established in the repaired vasculature

A vessel perfusion/permeability assay was employed to determine whether the repaired vasculature was functional. In this assay, a fluorescent tracer size restricted by the BSCB was injected into the heart and allowed to perfuse for either 1 minute (to measure vessel perfusion) or 30 minutes (to analyze vessel leakiness). Vessels at the lesion site were filled with the tracer by 7 dpi, indicating that the growing blood vessels had successfully fused, and blood was flowing across the injury site. However, barrier properties of the repaired vasculature were re-established slowly, between 28 and 60 dpi.

Pericytes cover vessels during vessel stabilization

Finally, the authors examined whether revascularization involves the recruitment of pericytes. Pericytes are critically involved in vascular development and maintenance of barrier integrity (Gaengel et al., 2009; Armulik et al., 2010). A significant increase in the number of pericytes that surrounded repairing blood vessels occurred from 7 to 30 dpi. In addition, the rise in pericyte number was consistent with the activation of the PDGF pathway (from 7 dpi) as indicated by gene expression analysis. The peak of pericyte coverage around the vessels was observed at 30 and 60 dpi, corresponding to the period of BSCB restoration. Within 90 dpi, pericyte coverage returned to homeostatic levels after vasculature remodeling and barrier recovery.

In conclusion, this study reveals vascular repair to be an integral part of zebrafish spinal cord regeneration. As depicted in the figure above, the repair of injured vasculature is a process characterized by an early burst of angiogenesis (A), followed by intermediate/late phases of pericyte recruitment (B) and vessel remodeling (C).

Questions for the authors

  1. In your opinion, are the findings of your study relevant to other forms of central nervous system traumatic injuries, for example, stroke?
  2. Do you think the interactions between different cell types in the spinal niche orchestrate the repair and recovery of the spinal cord?
  3. This study demonstrates the involvement of pericytes during the vascular repair. Have you considered examining the vascular smooth muscle cells (another mural cell type) in the context of SCI in zebrafish?

References

  1. Ahuja C S, et al. (2017). Traumatic spinal cord injury. Nature Reviews Disease Primers.
  2. Armulik A, et al. (2010). Pericytes regulate the blood-brain barrier. Nature.
  3. Becker C G, et al. (2004). L1.1 is involved in spinal cord regeneration in adult zebrafish. Journal of Neuroscience.
  4. Gaengel K, et al. (2009). Endothelial-mural cell signaling in vascular development and angiogenesis. Arteriosclerosis, Thrombosis and Vascular Biology.
  5. Hui S P, et al. (2010). Cellular response after crush injury in adult zebrafish spinal cord. Developmental Dynamics.
  6. Oudega M. (2012). Molecular and cellular mechanisms underlying the role of blood vessels in spinal cord injury and repair. Cell and Tissue Research.
  7. Yao C, et al. (2021). Revascularization after traumatic spinal cord injury. Frontiers in Physiology.

Tags: injury, regenaration, repair, vascularization, zebrafish

Posted on: 31 October 2022

doi: https://doi.org/10.1242/prelights.32899

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Author's response

Ana Ribeiro shared

1. In your opinion, are the findings of your study relevant to other forms of central nervous system traumatic injuries, for example, stroke?

Our study reveals the dynamics of blood vessel regrowth and maturation in the injured spinal cord. These findings may serve as a template for traumatic injury studies in the adult zebrafish brain, where brain re-vascularisation has only been addressed in larvae (Chen et al, 2019; PMID: 31006646). The knowledge of how the injured nervous tissue can be efficiently re-vascularised can also be useful to identify key differences (and potential therapeutic targets) in the vascular response after CNS traumatic injuries in mammals. The identification of therapeutic targets would be further aided by the uncovering of the pathways involved in the spinal cord re-vascularisation in zebafish, which we plan to address in future studies.

2. Do you think the interactions between different cell types in the spinal niche orchestrate the repair and recovery of the spinal cord?

The cooperation between different cell types during zebrafish spinal cord regeneration has been shown before, for example between glial bridge cells and neurons with regrowing axons (Mokalled et al, 2016; PMID: 27811277) and between macrophages and ependymal-radial glial progenitor cells (Cavone et al, 2021; PMID: 34033756). In this study we also find an interaction between blood vessels and regrowing axons, mimicking what is observed during development (Wälchli et al, 2015; PMID: 26182414) and raising the possibility that endothelial cells influence or are influenced by axonogenesis. However, we have not been able to test this interaction, since we were unable to inhibit vascular repair.

3. This study demonstrates the involvement of pericytes during the vascular repair. Have you considered examining the vascular smooth muscle cells (another mural cell type) in the context of SCI in zebrafish? 

In this study we focused on pericytes, since this mural cell population was shown to have deleterious or permissive roles in spinal cord injury in mice and zebrafish, respectively. In mice pericytes detach from blood vessels and contribute to the fibrotic scar that forms at the injury core (Goritz et al, 2011; PMID: 21737741), whereas pericytes in zebrafish larvae secrete ECM components that are conducive for axonal regrowth (Tsata et al, 2021; PMID: 33412105). In our study we only detected pericytes (labelled with a pdgfrb reporter transgene) in association with blood vessels, suggesting that in the adult spinal cord pericytes have a vascular-restricted role (i.e. regulation of the blood-spinal cord barrier or vascular constriction). However, other perivascular populations may be present in the adult zebrafish spinal cord that are not labelled by our pdgfrb reporter transgene and could have similar roles to the larval pericytes. We are currently testing the expression of other mural cell markers in our adult model, to determine if other perivascular cells are present and participate in the regenerative process.

 

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