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Chronic in vivo imaging in the mouse spinal cord using an implanted chamber

Nature Methods volume 9, pages 297–302 (2012)Cite this article

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Abstract

Understanding and treatment of spinal cord pathology is limited in part by a lack of time-lapse in vivo imaging strategies at the cellular level. We developed a chronically implanted spinal chamber and surgical procedure suitable for time-lapse in vivo multiphoton microscopy of mouse spinal cord without the need for repeat surgical procedures. We routinely imaged mice repeatedly for more than 5 weeks postoperatively with up to ten separate imaging sessions and observed neither motor-function deficit nor neuropathology in the spinal cord as a result of chamber implantation. Using this chamber we quantified microglia and afferent axon dynamics after a laser-induced spinal cord lesion and observed massive microglia infiltration within 1 d along with a heterogeneous dieback of axon stumps. By enabling chronic imaging studies over timescales ranging from minutes to months, our method offers an ideal platform for understanding cellular dynamics in response to injury and therapeutic interventions.

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Figure 1: An imaging chamber for longitudinal optical access to mouse spinal cord without the need for repeated surgeries.
Figure 2: Longitudinal 2PEF imaging of axons and blood vessels over many weeks after surgery.
Figure 3: Histological analysis of reactive microglia and astrocytes, and tissue morphology after chamber implantation.
Figure 4: Imaging and quantification of microglial scar formation at the site of a laser-induced SCI.
Figure 5: 2PEF imaging and quantification of axon dieback after a laser-induced SCI.

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Acknowledgements

We thank the US National Institutes of Health (DP OD006411 to J.R.F. and R01 EB002019 to C.B.S.) and the National Science and Research Council of Canada (to M.J.F.) for financial support, IMRA America, Inc. for the loan of their FCPA μJewel D-400 laser, J. Siebert for critically reading this manuscript, N. Ellis for his assistance in the machine shop and M. Riccio for his assistance with the MicroCT imaging.

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Authors and Affiliations

  1. Department of Physics, Cornell University, Ithaca, New York, USA

    Matthew J Farrar

  2. Department of Biomedical Engineering, Cornell University, Ithaca, New York, USA

    Matthew J Farrar, Ida M Bernstein & Chris B Schaffer

  3. Department of Biomedical Sciences, Cornell University, Ithaca, New York, USA

    Donald H Schlafer

  4. Department of Psychology, Cornell University, Ithaca, New York, USA

    Thomas A Cleland

  5. Department of Neurobiology and Behavior, Cornell University, Ithaca, New York, USA

    Joseph R Fetcho

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  1. Matthew J Farrar
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Contributions

M.J.F., T.A.C., J.R.F. and C.B.S. conceived and designed the experiments. M.J.F. performed surgeries and imaging experiments, I.M.B. performed behavioral assays, and D.H.S. performed histopathology. M.J.F., I.M.B., J.R.F. and C.B.S. analyzed data. J.R.F., T.A.C. and C.B.S. contributed reagents and materials. M.J.F., J.R.F. and C.B.S. wrote the paper.

Corresponding author

Correspondence to Chris B Schaffer.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Notes 1–7, Supplementary Protocol (PDF 1956 kb)

Supplementary Video 1

Rendering of MicroCT image data from a mouse taken 6 d after implantation of the imaging chamber, showing normal spine alignment and no vertebral damage. (MOV 7283 kb)

Supplementary Video 2

Video of a mouse taken 2 weeks after implanting the imaging chamber, showing locomotion, grooming and exploratory behavior. (MOV 7639 kb)

Supplementary Video 3

A series of 2PEF image stacks taken at different times after a laser-induced spinal cord injury, showing GFP-expressing axons (green) and Texas Red-dextran labeled vasculature (red). (MOV 29674 kb)

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Farrar, M., Bernstein, I., Schlafer, D. et al. Chronic in vivo imaging in the mouse spinal cord using an implanted chamber. Nat Methods 9, 297–302 (2012). https://doi.org/10.1038/nmeth.1856

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