Lipid Order Degradation in Autoimmune Demyelination Probed by Polarized Coherent Raman Microscopy

Abstract

Myelin around axons is currently widely studied by structural analyses and large-scale imaging techniques, with the goal to decipher its critical role in neuronal protection. Although there is strong evidence that in myelin, lipid composition, and lipid membrane morphology are affected during the progression of neurodegenerative diseases, there is no quantitative method yet to report its ultrastructure in tissues at both molecular and macroscopic levels, in conditions potentially compatible with in vivo observations. In this work, we study and quantify the molecular order of lipids in myelin at subdiffraction scales, using label-free polarization-resolved coherent anti-Stokes Raman, which exploits coherent anti-Stokes Raman sensitivity to coupling between light polarization and oriented molecular vibrational bonds. Importantly, the method does not use any a priori parameters in the sample such as lipid type, orientational organization, and composition. We show that lipid molecular order of myelin in the mouse spinal cord is significantly reduced throughout the progression of experimental autoimmune encephalomyelitis, a model for multiple sclerosis, even in myelin regions that appear morphologically unaffected. This technique permits us to unravel molecular-scale perturbations of lipid layers at an early stage of the demyelination progression, whereas the membrane architecture at the mesoscopic scale (here ∼100 nm) seems much less affected. Such information cannot be brought by pure morphological observation and, to our knowledge, brings a new perspective to molecular-scale understanding of neurodegenerative diseases.

Figure 1

(A) Schematic view of the PR-CARS microscope. The vector E represents the direction of linear polarization of ω_s and ω_p (α-angle), rotated by a half-wave plate (λ/2). DM, dichroic mirror; x/y, galvanometric mirrors; P, polarizer; PMT, photomultiplier tube; OBJ: objective. (B) Given here is an example of PR-CARS data (left panel) recorded in one pixel (right panel) of the myelin sheath region (control sample), averaged over the diffraction limit size (schematically represented by a circle). Scale bars represent 5 μm. (C) (Top) Given here is a schematic of the multilamellar lipid membrane in the focal spot (represented as a circle) and zoom on a lipid showing the dominant nonlinear bond dipoles (arrows) probed by PR-CARS. (Bottom) Distribution function p(φ) is read-out from PR-CARS (see text), as a sum of orders of symmetry. (D) Given here is a large-scale CARS image (total intensity summed over 18 input polarization angles) of a spinal cord tissue (score 1.5). Scale bars represent 50 μm. To see this figure in color, go online.

Figure 2

(A) Given here is a PR-CARS (centered at 2845 cm^–1) total intensity image (sum over 18 input polarization angles) of the myelin sheath from a spinal tissue (control sample). Scale bars represent 5 μm. (B) Given here is a corresponding S₄^(s) map. (C) Given here is a S₄^(a) map. (D) Given here is a S₂ map. For those maps (not for data analysis), the PR-CARS intensity is averaged over 3×3 pixels. A region of the S₂ map is zoomed-in to make more visible the complete orientational information, represented by a stick oriented with the φ₂ angle with respect to the horizontal axis, and which color is S₂. (E) Given here are experimental values of S₂, S₄^(s) taken at intensities above the threshold 1300 analog signal value in the myelin sheath (## in the intensity image (A)) and at lower intensity signals from a surrounding background (# in the intensity image (A)). For the myelin sheath region, the inset image is color-coded in intensity. (Solid lines) Given here are theoretical (S₂, S₄^(s)) values for a Gaussian distribution of increasing width σ, and Gaussian superimposed with an isotropic distribution of proportions 25%. (F) Given here is a S₄^(a),S₄^(s) scatter plot following a similar color-coded intensity scale to that in (E). To see this figure in color, go online.

Figure 3

PR-CARS analysis of the different characteristic morphological features visible in the demyelination process. (A) Total intensity images were taken from clinical score 1 (left) and 2 (right). Symbols (^∗, #, ^∗#) refer to different membrane locations. White square ROIs are used for statistical analysis in (B). Scale bars represent 5 μm. (B) (Left) Given here are S₂ maps (superimposed on the intensity image) of characteristic features of myelin degradation in white ROIs of (A): “healthy” myelin, “swollen”, and “blebbed”; and products of degradation: “debris not filled (NF)” and “debris filled (F)”. (Middle) Given here are corresponding histograms of S₂ values, taken within the dashed square ROIs in (B). (Right) Given here are combined images of S₂ and φ₂, showing S₂ as a color scale and φ₂ as the orientation of sticks for each measured pixel. (C) Given here is a schematic morphological interpretation of the myelin multilayer (enlarged) in positions in (A) corresponding to (^∗) “healthy”, (^∗#) “swollen”, and (#) “blebbed” myelin. The circular region represents the focal spot (not to scale). To see this figure in color, go online.

Figure 4

(A) Given here are general normalized histograms (over all measured pixels) of characteristic features of myelin degradation plotted for all measurements, regardless of the clinical score of the EAE disease (∼1000 pixels per regions). Number of regions measured: normal myelin (N = 136), swollen (N = 91), blebbed (N = 82), debris filled (F) (N = 30), debris not-filled (NF) (N = 20). ^∗∗∗, p ≤ 0.001. (B) Given here are corresponding SD ${\sigma }_{S_2}$ values within each region, taken per category. Error bars are SD values over all regions measured. To see this figure in color, go online.

Figure 5

(A) (Top) Given here are mean g-ratio values versus the EAE score (see Fig. S5). (Bottom) Given here are S₂ values versus different scores of EAE, for the myelin regions of normal morphology (see text). Statistical analysis is shown relative to the control sample except when notified with a bar (^∗, p ≤ 0.05, ^∗∗, p ≤ 0.01, ^∗∗∗, p ≤ 0.001). Given is the number of regions considered in the analysis: control (N = 26), CFA (N = 30), score 0 (N = 28), score 1 (N = 17), score 1.5 (N = 9), score 2 (N = 22), score 2.5 (N = 19), score 4 (N = 11). Error bars are SD values. (B) Shown here are (S₂, S₄^(s)) values for myelin at different EAE scores (straight myelin sheaths are considered, and only the most statistically relevant scores are depicted for clarity) and MLVs from different lipid/cholesterol compositions: pure DPPC/Chol or DOPC/Chol at 0, 5, 10, 20% (v/v), or DPPC/DOPC/Chol 30% (v/v/v) ternary mixtures with DPPC/DOPC ratios 1:1; 1:2; 2:1. Error bars are SD values (for MLV samples N = 4). To see this figure in color, go online.