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High-resolution 3D imaging and analysis of axon regeneration in unsectioned spinal cord with or without tissue clearing

Abstract

Here we present a protocol for analyses of axon regeneration and density in unsectioned adult mouse spinal cord. This includes methods for injury and tracing of dorsal column sensory and corticospinal axons; clearing and staining of unsectioned spinal cord; visualization of axon degeneration and regeneration in cleared and uncleared specimens using two-photon microscopy; and either manual or semi-automatic analysis of axon density and regeneration in 3D space using Imaris and ImageJ software. This protocol can be used to elucidate the molecular and cellular mechanisms underlying nervous system degeneration and regeneration and to establish the therapeutic efficacy of candidate neuroregenerative treatments. Because tissue sectioning is not required, this protocol enables unambiguous evaluation of regeneration and greatly accelerates the speed at which analyses can be conducted. Surgical procedures take <30 min per mouse, with a wait period of 2 weeks between axonal injury and tracing and 2–8 weeks between tracing and tissue processing. Clearing and immunolabeling take ~1–2 weeks, depending on the size of the sample. Imaging and analysis can be performed in 1 d. All these procedures can be accomplished by a competent graduate student or experienced technician.

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Fig. 1: Incomplete lateral hemisection spinal cord injury.
Fig. 2: Tracing dorsal column sensory axons.
Fig. 3: Tracing corticospinal axons.
Fig. 4: Tissue mounting and imaging.
Fig. 5: Identifying the lesion site.
Fig. 6: Schematic representation of the steps required for manual or semi-automated analysis of axon regeneration in 3D.
Fig. 7: Workflow for the manual analysis of axon regeneration.
Fig. 8: Workflow for semi-automated analysis of axon regeneration.
Fig. 9: 3D anatomy and injury of the corticospinal tract.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank S. Stern, B. Schaffran, S. Cerqueira, G. Duncan, A. Husch, and T. Van Rossum for helpful feedback on the protocol. We also thank the DZNE Light Microscope Facility (LMF), the Image and Data Analysis Facility (IDAF), and the Animal Research Facility (ARF) for technical support. We particularly thank K. Keppler (DZNE LMF) and C. Moehl (DZNE IDAF) for their help. We are grateful to J. Liu and W. Tetzlaff (UBC) for helpful discussions of mouse models of spinal cord injury. B.J.H. is supported by a Wings for Life (WfL) Aguayo-Tator Mentoring Fellowship and a non-stipendiary European Molecular Biology Organization (EMBO) Long-Term Fellowship (ALTF 28-2017). A.T. is supported by the Craig H. Neilsen Foundation, the Marina Romoli Onlus Association and Discovery Themes on Chronic Brain Injury. F.B. is supported by the Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), the International Foundation for Research in Paraplegia, WfL, the Deutsche Forschungsgemeinschaft, ERANET AXON REPAIR, and ERANET RATER SCI. F.B. is a member of the excellence cluster ImmunoSensation2 (EXC2151–390873048) and the SFB 1089 and is a recipient of the Roger de Spoelberch Prize.

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Contributions

B.J.H., O.B., A.T., and F.B. conceived and designed the protocol. B.J.H., O.B., and A.T. performed the experiments. B.J.H. and O.B. wrote the manuscript. F.B. supervised the project and edited the manuscript.

Corresponding authors

Correspondence to Brett J. Hilton, Andrea Tedeschi or Frank Bradke.

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The authors declare no competing interests.

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Journal peer review information: Nature Protocols thanks Kai Liu and other anonymous reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Tedeschi, A. et al. Neuron 92, 419−434 (2016): https://doi.org/10.1016/j.neuron.2016.09.026

Ertürk, A. et al. Nat. Med. 18, 166−171 (2012): https://doi.org/10.1038/nm.2600

Integrated supplementary information

Supplementary Figure 1 Comparison of analysis in unsectioned vs. sectioned spinal cord.

(a) One hour after thoracic incomplete lateral hemisection spinal cord injury, AAV1-CMV-GFP was injected into the left sciatic nerve and a conditioning lesion (n = 4) or sham procedure (n = 3) was performed. Four weeks after injury, animals were perfused and unsectioned spinal cords were cleared and imaged. Unsectioned spinal cords were then cut into 60 µm sagittal sections and stained for GFAP. Images were taken of 2–3 consecutive sections containing the highest density of GFP+ sensory axons using a Leica 8 Confocal microscope and axon regeneration was analyzed on these sections. Images are representative of the extent of axon regeneration for each group and are of the same samples with and without sectioning. Scale bars, 200 µm. (b) Inset of a. Arrowheads point to regenerating axons rostral to the lesion site after a conditioning lesion. Scale bar, 100 µm. (c) Quantification of regenerating axons and comparison of sectioned vs. unsectioned analysis. Sham n = 3 and PNL n = 4.

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1

Reporting Summary

Supplementary Video 1

Volume rendering of conditioned sensory axons regenerating in unsectioned and uncleared spinal cord.

Supplementary Video 2

Rotation and ortho-slicing of the sample.

Supplementary Video 3

Volume rendering of corticospinal axons in a cleared sample with a spinal cord injury.

Supplementary Data 1

Raw data in Imaris file format of GFP+ sensory axons regenerating past a spinal cord injury site.

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Hilton, B.J., Blanquie, O., Tedeschi, A. et al. High-resolution 3D imaging and analysis of axon regeneration in unsectioned spinal cord with or without tissue clearing. Nat Protoc 14, 1235–1260 (2019). https://doi.org/10.1038/s41596-019-0140-z

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