Coherent Anti-Stokes Raman Scattering Microscopy: A Label-Free Method to Compare Spinal Cord Myelin in Different Species

Abstract

Many histological techniques are used to identify and characterize myelin in the mammalian nervous system. Due to the high content of lipids in myelin sheaths, coherent anti-stokes Raman scattering (CARS) microscopy is a label-free method that allows identifying myelin within tissues. CARS excites the CH2 vibrational mode at 2845 cm−1 and CH2 bonds are found in lipids. In this study, we have used CARS for a new biological application in the field of spinal cord analysis. We have indeed compared several parameters of spinal cord myelin sheath in three different species, i.e., mouse, lemur, and human using a label-free method. In all species, we analyzed the dorsal and the lateral funiculi of the adult thoracic spinal cord. We identified g-ratio differences between species. Indeed, in both funiculi, g-ratio was higher in mice than in the two primate species, and the myelin g-ratio in lemurs was higher than in humans. We also detected a difference in g-ratio between the dorsal and the lateral funiculi only in humans. Furthermore, species differences between axon and fiber diameters as well as myelin thickness were observed. These data may reflect species specificities of conduction velocity of myelin fibers. A comparison of data obtained by CARS imaging and fluoromyelin staining, a method that, similar to CARS, does not require resin embedding and dehydration, displays similar results. CARS is, therefore, a label-free alternative to other microscopy techniques to characterize myelin in healthy and neurological disorders affecting the spinal cord.

CSF1R inhibition at chronic stage after spinal cord injury modulates microglia proliferation

“…Acute injury‐induced demyelination is followed by remyelination that commences within the first few weeks after SCI and persists for months (for review, see Pukos et al, 2019). Due to its high content of lipids, myelin integrity can be assessed with CARS microscopy (Poulen, Gerber, et al, 2021). Thus, using CARS, we quantified the density of spinal cord intact myelinated fibers (Figure 3j) at three time points after SCI (14, 42, and 84 dpi; Figure 3) on axial sections at three different levels (epicenter, 3.15 mm rostral and caudal to the lesion site; Figure 3i).…”

“…Coherent anti‐stokes Raman scattering (CARS) images were acquired with LSM 7 MP optical parametric oscillator (OPO) multiphoton microscope (Zeiss, Oberkochen, Germany) with an upright Axio Examiner Z.1 optical microscope associated with a femtosecond Ti: sapphire laser (680–1080 nm, 80 MHz, 140 fs, Chameleon Ultra II, Coherent, France) pumping a tunable OPOs (1000–1500 nm, 80 MHz, 200 fs, Chameleon Compact OPO, Coherent, France) as previously described (Poulen, Aloy, et al, 2021; Poulen, Gerber, et al, 2021). We imaged axial spinal cord sections (14 μm) at three spinal cord levels (lesion epicenter, 3.15 mm rostral and 3.15 mm caudal to the lesion).…”

“…Pictures are a stack of 3 μm (three slices). Density of intact myelin sheaths was manually quantified by an experimenter blind to the conditions as previously described (Poulen, Gerber, et al, 2021). For some acquisitions, eGFP signal was also acquired to observe microglia/macrophages location relative to myelin fibers.…”

Up-Regulation of Astrocytic Fgfr4 Expression in Adult Mice after Spinal Cord Injury

“…Myelin staining: we used fluoromyelin as previously described in [38,39]; in short, sections were rinsed in 0.1 M PBS for 1 min and then incubated in FluoroMyelin™ (1:200, Thermofisher Scientific, Waltham, MA, USA) for 20 min at RT. Sections were washed 3 × 10 mn in 0.1 M PBS, coversliped using fluorescent mounting medium (Dako, Glostrup, Denmark), were kept away from light, and stored at 4 • C. • Neuromuscular junctions: staining on transverse cryosection (16 µm) of the gastrocnemiussoleus-plantaris muscular complex was carried out (Microm HM550, Thermofisher Scientific, Waltham, MA, USA) using enzymatic method [40].…”