Changes of statistical structural fluctuations unveils an early compacted degraded stage of PNS myelin

Abstract

Degradation of the myelin sheath is a common pathology underlying demyelinating neurological diseases from Multiple Sclerosis to Leukodistrophies. Although large malformations of myelin ultrastructure in the advanced stages of Wallerian degradation is known, its subtle structural variations at early stages of demyelination remains poorly characterized. This is partly due to the lack of suitable and non-invasive experimental probes possessing sufficient resolution to detect the degradation. Here we report the feasibility of the application of an innovative non-invasive local structure experimental approach for imaging the changes of statistical structural fluctuations in the first stage of myelin degeneration. Scanning micro X-ray diffraction, using advances in synchrotron x-ray beam focusing, fast data collection, paired with spatial statistical analysis, has been used to unveil temporal changes in the myelin structure of dissected nerves following extraction of the Xenopus laevis sciatic nerve. The early myelin degeneration is a specific ordered compacted phase preceding the swollen myelin phase of Wallerian degradation. Our demonstration of the feasibility of the statistical analysis of SµXRD measurements using biological tissue paves the way for further structural investigations of degradation and death of neurons and other cells and tissues in diverse pathological states where nanoscale structural changes may be uncovered.

Figure 1

(A) The experimental apparatus is located at the ID13 beamline of the European Synchrotron Radiation Facility (ESRF) and features an electron undulator providing 12.4 keV X-rays to crystal optics delivering a 1 µm beam spot on the sample. The sample holder houses a 1 mm-diameter quartz capillary containing the nerve and the culture medium. The x-y translator allows the sample to move in both horizontal and vertical directions with a sampling step of 5 µm within a selected area. The X-rays scattered by the sample are recorded by a FReLoN detector. (B) Intensity of the x-ray diffraction reflections after background subtraction, with respect to the impinging beam of the sciatic nerve bathed in normal Ringer's solution at pH 7.3 for the native nerve. The intensity (in arbitrary units) is plotted against the reciprocal distance (Å⁻¹). (C) Colormap of the integrated intensity of the myelin XRD diffraction pattern measured point by point of a full native nerve. The intensity is greater in the centre and decreased at the edges of the nerve due to the differing quantities of myelinated axons traversed by the beam in these different locations. This is caused by the generally cylindrical shape of the nerve. The bar corresponds to 100 μm.

Figure 2

Integrated intensity of X ray diffraction patterns measured pixel by pixel in a typical selected (40 × 120 µm) central area, of (A) native and (B) early degraded nerve. The bar corresponds to 10 µm. (C) Probability density function of Integrated intensity measured in the areas (A) and (B) for (full circles) native and (empty squares) early degraded nerve. Both distributions are fit by Lognormal lineshape (solid lines). The average intensity increases by a factor 2 in the early degraded nerve.

Figure 3

Period measured pixel by pixel in a central 40 × 120 µm area, of the (A) native and (B) early degraded nerve. The bar corresponds to 10 µm. (C) Probability density function of period measured in areas (A) and (B) for (full circles) native and (empty squares) early degraded nerve. The periodicity presents intrinsic fluctuations of about 2 Angströms. We observe a distribution shift of 0.8 ± 0.1 Angströms, indicated by the arrow, towards lower values for the early degraded nerve.

Figure 4. Scatter plot of myelin period as a function of diffraction intensity for (blue dots) native and (red dots) early degraded nerve.

Figure 5. Two-point spatial correlation function G(r) for (upper panel) the integrated intensity and (lower panel) myelin period.

In each panel the exponential behavior of (full circles) native and (empty squares) early degraded nerve, is reported. The noise level, representing the spatial correlation of the randomized matrices, is reported by triangles.