Spectrochemical Analysis of Peripheral Nerve Myelin for use in Living Biological Systems
Joey Grochmal, MD; Wulin Teo; Hardeep Ghambir, PhD; Ranjan Kumar; Peter K. Stys, MD; Rajiv Midha, MD, MSc, FRCS(C) Neuroscience, University of Calgary, Calgary, AB, Canada
Introduction: Current experimental techniques for the chemical analysis of peripheral nerve myelin are not well applied in living biological systems. We introduce the technique of myelin analysis using spectral confocal fluorescence microscopy of the solvatochromic myelin-incorporated dye Nile Red, and then apply our method to the study of myelination by Schwann cells and Schwann cell-like stem cells (Skin-derived precursor Schwann cells) both in vitro and in-vivo.
Hypothesis: Our hypothesis is that myelin conferred by SKP-SCs and SCs will reliably move through states of progressive chemical maturity as the tissue gradually approaches an adult phenotype, and that this progression can be characterized by Nile Red emission spectra in living systems.
Methods: In vitro analysis- We co-cultured BFP transduced SCs and SKP-SCs with isolated Thy-1 GFP DRG explants. Cell derived myelin was stained with Nile Red and imaged at sequential time points with spectral confocal microscopy, then analyzed using ImageTrak spectral analysis software (Peter Stys).
In-vivo analysis- GFP positive SCs and SKP-SCs were injected into the sciatic nerves of Lewis rats 9 days post doxorubicin injury (focal demyelination injury). Rats were sacrificed at 12,15,18,21, and 24 days post injection and sciatic nerves were fixed, frozen, and sectioned (4nerves/cell group/time point). SKP-SC and SC Nile Red stained myelin was identified on cross-sections with circumferential GFP co-localization, and imaged and analyzed as above.
Live rodent analysis- The above experimental paradigm was repeated using BFP transduced SCs and SKP-SCs, and live sciatic nerve imaging (SD Thy-1 GFP rat) was performed sequentially following topical exposure to 50uM Nile Red at 15,21, and 27 days post cell-injection. Cell-derived myelin figures were identified and analyzed as above.
Results: All three experimental paradigms demonstrate that myelin polarity changes in a reliable fashion with progressive maturity, irrespective of paradigm or cell type used. Preliminary in-vitro data suggests that these changes may in part reflect interaction of immature myelin with cytoplasmic lipid-rich vacuoles. Preliminary results also suggest that SKP-SC conferred myelin may progress to chemical maturity faster than SC myelin.
Conclusions: PNS myelin analysis by Nile Red emission spectra can yield spectro-chemical information regarding myelination in living systems, while Nile Red can function as a non-toxic probe for live cell and animal imaging. Sequential and reliable changes in NR emission patterns with progressive myelin maturity can be used to measure myelination "velocity" by SC grafts, while single cell imaging yields novel clues as to the cellular machinery of myelin formation.
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