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Label-free in vivo imaging of myelinated axons in health and disease with spectral confocal reflectance microscopy

Abstract

We report a newly developed technique for high-resolution in vivo imaging of myelinated axons in the brain, spinal cord and peripheral nerve that requires no fluorescent labeling. This method, based on spectral confocal reflectance microscopy (SCoRe), uses a conventional laser-scanning confocal system to generate images by merging the simultaneously reflected signals from multiple lasers of different wavelengths. Striking color patterns unique to individual myelinated fibers are generated that facilitate their tracing in dense axonal areas. These patterns highlight nodes of Ranvier and Schmidt-Lanterman incisures and can be used to detect various myelin pathologies. Using SCoRe we carried out chronic brain imaging up to 400 μm deep, capturing de novo myelination of mouse cortical axons in vivo. We also established the feasibility of imaging myelinated axons in the human cerebral cortex. SCoRe adds a powerful component to the evolving toolbox for imaging myelination in living animals and potentially in humans.

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Figure 1: In vivo imaging of mouse cortex using spectral confocal reflectance microscopy (SCoRe).
Figure 2: SCoRe signal is dependent on myelination.
Figure 3: Transcranial time-lapse imaging of the mouse cortex reveals progressive age-dependent myelination.
Figure 4: Multicolor reflection spectrum reveals distinct myelin structures in the spinal cord and sciatic nerve in vivo.
Figure 5: Myelin pathology and human myelinated axons imaged with SCoRe.

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References

  1. Nave, K.-A. Myelination and support of axonal integrity by glia. Nature 468, 244–252 (2010).

    Article  CAS  Google Scholar 

  2. Zatorre, R.J., Fields, R.D. & Johansen-Berg, H. Plasticity in gray and white: neuroimaging changes in brain structure during learning. Nat. Neurosci. 15, 528–536 (2012).

    Article  CAS  Google Scholar 

  3. Liu, J. et al. Impaired adult myelination in the prefrontal cortex of socially isolated mice. Nat. Neurosci. 15, 1621–1623 (2012).

    Article  CAS  Google Scholar 

  4. Franklin, R.J.M. & Ffrench-Constant, C. Remyelination in the CNS: from biology to therapy. Nat. Rev. Neurosci. 9, 839–855 (2008).

    Article  CAS  Google Scholar 

  5. Fancy, S.P.J., Chan, J.R., Baranzini, S.E., Franklin, R.J.M. & Rowitch, D.H. Myelin regeneration: a recapitulation of development? Annu. Rev. Neurosci. 34, 21–43 (2011).

    Article  CAS  Google Scholar 

  6. Bartzokis, G. et al. Multimodal magnetic resonance imaging assessment of white matter aging trajectories over the lifespan of healthy individuals. Biol. Psychiatry 72, 1026–1034 (2012).

    Article  Google Scholar 

  7. Kirby, B.B. et al. In vivo time-lapse imaging shows dynamic oligodendrocyte progenitor behavior during zebrafish development. Nat. Neurosci. 9, 1506–1511 (2006).

    Article  CAS  Google Scholar 

  8. Kaya, F. et al. Live imaging of targeted cell ablation in Xenopus: a new model to study demyelination and repair. J. Neurosci. 32, 12885–12895 (2012).

    Article  CAS  Google Scholar 

  9. Wang, H., Fu, Y., Zickmund, P., Shi, R. & Cheng, J.-X. Coherent anti-stokes Raman scattering imaging of axonal myelin in live spinal tissues. Biophys. J. 89, 581–591 (2005).

    Article  CAS  Google Scholar 

  10. Fu, Y., Huff, T.B., Wang, H.-W., Wang, H. & Cheng, J.-X. Ex vivo and in vivo imaging of myelin fibers in mouse brain by coherent anti-Stokes Raman scattering microscopy. Opt. Express 16, 19396–19409 (2008).

    Article  CAS  Google Scholar 

  11. Imitola, J. et al. Multimodal coherent anti-Stokes Raman scattering microscopy reveals microglia-associated myelin and axonal dysfunction in multiple sclerosis-like lesions in mice. J. Biomed. Opt. 16, 021109 (2011).

    Article  Google Scholar 

  12. Ben Arous, J. et al. Single myelin fiber imaging in living rodents without labeling by deep optical coherence microscopy. J. Biomed. Opt. 16, 116012 (2011).

    Article  Google Scholar 

  13. Witte, S. et al. Label-free live brain imaging and targeted patching with third-harmonic generation microscopy. Proc. Natl. Acad. Sci. USA 108, 5970–5975 (2011).

    Article  CAS  Google Scholar 

  14. Farrar, M.J., Wise, F.W., Fetcho, J.R. & Schaffer, C.B. In vivo imaging of myelin in the vertebrate central nervous system using third harmonic generation microscopy. Biophys. J. 100, 1362–1371 (2011).

    Article  CAS  Google Scholar 

  15. Filler, T.J. & Peuker, E.T. Reflection contrast microscopy (RCM): a forgotten technique? J. Pathol. 190, 635–638 (2000).

    Article  CAS  Google Scholar 

  16. Xiao, J., Levitt, J.B. & Buffenstein, R. The use of a novel and simple method of revealing neural fibers to show the regression of the lateral geniculate nucleus in the naked mole-rat (Heterocephalus glaber). Brain Res. 1077, 81–89 (2006).

    Article  CAS  Google Scholar 

  17. Grutzendler, J., Kasthuri, N. & Gan, W.-B. Long-term dendritic spine stability in the adult cortex. Nature 420, 812–816 (2002).

    Article  CAS  Google Scholar 

  18. Waxman, S.G. Ultrastructural observations on branching patterns of central axons. Neurosci. Lett. 1, 251–256 (1975).

    Article  CAS  Google Scholar 

  19. Ha, H. Axonal bifurcation in the dorsal root ganglion of the cat: a light and electron microscopic study. J. Comp. Neurol. 140, 227–240 (1970).

    Article  CAS  Google Scholar 

  20. Hirrlinger, P.G. et al. Expression of reef coral fluorescent proteins in the central nervous system of transgenic mice. Mol. Cell. Neurosci. 30, 291–303 (2005).

    Article  CAS  Google Scholar 

  21. Jacobson, S. Sequence of myelination in the brain of the albino rat. A. Cerebral cortex, thalamus and related structures. J. Comp. Neurol. 121, 5–29 (1963).

    Article  CAS  Google Scholar 

  22. Chong, S.Y.C. et al. Neurite outgrowth inhibitor Nogo-A establishes spatial segregation and extent of oligodendrocyte myelination. Proc. Natl. Acad. Sci. USA 109, 1299–1304 (2012).

    Article  CAS  Google Scholar 

  23. Binding, J. et al. Brain refractive index measured in vivo with high-NA defocus-corrected full-field OCT and consequences for two-photon microscopy. Opt. Express 19, 4833–4847 (2011).

    Article  CAS  Google Scholar 

  24. Macleod, H.A. Thin-Film Optical Filters (Institute of Physics Publishing, 2001).

  25. Balice-Gordon, R.J., Bone, L.J. & Scherer, S.S. Functional gap junctions in the schwann cell myelin sheath. J. Cell Biol. 142, 1095–1104 (1998).

    Article  CAS  Google Scholar 

  26. Gould, R.M., Byrd, A.L. & Barbarese, E. The number of Schmidt-Lanterman incisures is more than doubled in shiverer PNS myelin sheaths. J. Neurocytol. 24, 85–98 (1995).

    Article  CAS  Google Scholar 

  27. Lam, C.K., Yoo, T., Hiner, B., Liu, Z. & Grutzendler, J. Embolus extravasation is an alternative mechanism for cerebral microvascular recanalization. Nature 465, 478–482 (2010).

    Article  CAS  Google Scholar 

  28. Davalos, D. et al. ATP mediates rapid microglial response to local brain injury in vivo. Nat. Neurosci. 8, 752–758 (2005).

    Article  CAS  Google Scholar 

  29. Harb, R., Whiteus, C., Freitas, C. & Grutzendler, J. In vivo imaging of cerebral microvascular plasticity from birth to death. J. Cereb. Blood Flow Metab. 33, 146–156 (2013).

    Article  CAS  Google Scholar 

  30. Livet, J. et al. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450, 56–62 (2007).

    Article  CAS  Google Scholar 

  31. Gan, W.B., Grutzendler, J., Wong, W.T., Wong, R.O. & Lichtman, J.W. Multicolor “DiOlistic” labeling of the nervous system using lipophilic dye combinations. Neuron 27, 219–225 (2000).

    Article  CAS  Google Scholar 

  32. Hong, G. et al. Multifunctional in vivo vascular imaging using near-infrared II fluorescence. Nat. Med. 18, 1841–1846 (2012).

    Article  CAS  Google Scholar 

  33. Gupta, N. et al. Neural stem cell engraftment and myelination in the human brain. Sci. Transl. Med. 4, 155ra137 (2012).

    Article  Google Scholar 

  34. Windrem, M.S. et al. Neonatal chimerization with human glial progenitor cells can both remyelinate and rescue the otherwise lethally hypomyelinated shiverer mouse. Cell Stem Cell 2, 553–565 (2008).

    Article  CAS  Google Scholar 

  35. Bø, L., Vedeler, C.A., Nyland, H.I., Trapp, B.D. & Mørk, S.J. Subpial demyelination in the cerebral cortex of multiple sclerosis patients. J. Neuropathol. Exp. Neurol. 62, 723–732 (2003).

    Article  Google Scholar 

  36. Lucchinetti, C.F. et al. Inflammatory cortical demyelination in early multiple sclerosis. N. Engl. J. Med. 365, 2188–2197 (2011).

    Article  CAS  Google Scholar 

  37. Feng, G. et al. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 41–51 (2000).

    Article  CAS  Google Scholar 

  38. Jung, S. et al. Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion. Mol. Cell. Biol. 20, 4106–4114 (2000).

    Article  CAS  Google Scholar 

  39. Muzumdar, M.D., Tasic, B., Miyamichi, K., Li, L. & Luo, L. A global double-fluorescent Cre reporter mouse. Genesis 45, 593–605 (2007).

    Article  CAS  Google Scholar 

  40. Nimmerjahn, A., Kirchhoff, F., Kerr, J.N.D. & Helmchen, F. Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo. Nat. Methods 1, 31–37 (2004).

    Article  CAS  Google Scholar 

  41. Fenrich, K.K. et al. Long-term in vivo imaging of normal and pathological mouse spinal cord with subcellular resolution using implanted glass windows. J. Physiol. (Lond.) 590, 3665–3675 (2012).

    Article  CAS  Google Scholar 

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Acknowledgements

This study was supported by the following grants: R01AG027855 and R01HL106815 (J.G.). We would like to thank J. Bewersdorf and D. Toomre for helpful discussions and P. Yuan for critical reading of the manuscript. We thank A. Nishiyama (University of Connecticut) and F. Kirchhoff (University of Saarland) for providing PLPDsRed mice. Postmortem human specimen was obtained from the brain bank in the Cognitive Neurology and Alzheimer's disease Center (CNADC) at Northwestern University (grant AG13854).

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Authors

Contributions

A.J.S. and J.G. conceived and designed the initial project. A.J.S. and R.A.H. carried out the experiments. All authors contributed to experimental design, data analysis and manuscript. J.G. supervised the project.

Corresponding author

Correspondence to Jaime Grutzendler.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 15505 kb)

Three dimensional rendering of a SCoRe image captured in vivo in a Thy1-YFP transgenic mouse.

A single YFP-labeled (green) reflective (magenta) axon (arrow) is shown among many non myelinated YFP-labeled axons and dendrites. (AVI 8154 kb)

Representative 450 μm z stack of the mouse cortex acquired in vivo with SCoRe.

Video demonstrates the depth capabilities of SCoRe which maintains a high signal to noise ratio even at ~400 μm. The decrease in the number of reflected fibers with depth indicates the drop-off in the number of myelinated fibers just below cortical layer 1 projection axons in addition to the change in the orientation of some of the myelinated axons as SCoRe is not able to efficiently detect myelinated axons running orthogonally. Step size 1 μm, depth indicated in upper left corner and video displayed at 10 frames per second. (AVI 23477 kb)

Three dimensional rendering of a SCoRe image from the sciatic nerve.

Video shows the unique reflected spectrum from individual axons after removing the layers of the highly reflective and disordered signal from the sciatic epineurium. (AVI 6094 kb)

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Schain, A., Hill, R. & Grutzendler, J. Label-free in vivo imaging of myelinated axons in health and disease with spectral confocal reflectance microscopy. Nat Med 20, 443–449 (2014). https://doi.org/10.1038/nm.3495

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