Label‐free non‐linear microscopy to measure myelin outcome in a rodent model of Charcot‐Marie‐Tooth diseases

Abstract

Myelin sheath produced by Schwann cells covers and nurtures axons to speed up nerve conduction in peripheral nerves. Demyelinating peripheral neuropathies result from the loss of this myelin sheath and so far, no treatment exists to prevent Schwann cell demyelination. One major hurdle to design a therapy for demyelination is the lack of reliable measures to evaluate the outcome of the treatment on peripheral myelin in patients but also in living animal models. Non-linear microscopy techniques which include second harmonic generation (SHG), third harmonic generation (THG) and coherent anti-stokes Raman scattering (CARS) were used to image myelin ex vivo and in vivo in the sciatic nerve of healthy and demyelinating mice and rats. SHG did not label myelin and THG required too much light power to be compatible with live imaging. CARS is the most reliable of these techniques for in vivo imaging and it allows for the analysis and quantification of myelin defects in a rat model of CMT1A disease. This microscopic technique therefore constitutes a promising, reliable and robust readout tool in the development of new treatments for demyelinating peripheral neuropathies.

Long term AAV2/9-mediated silencing of PMP22 prevents CMT1A disease in rats and validates skin biomarkers as treatment outcome measure

“…First, we imaged adult rat sciatic nerves 12 months after injection in newborn pups using Coherent Antistokes Raman Scattering (CARS) non-linear microscopy technique. This allows myelin sheath imaging without any labeling [21]. Using this technique on an intact nerve in a longitudinal view, we observed that the majority of transduced cells were myelinated cells ( Fig.…”

“…Second, we focused on the specific myelin defects observed in the CMT1A rat model using CARS imaging on freshly fixed nerves. Indeed, in a previous study, we had characterized CMT1A rat myelin sheath defects using this imaging technique [21]. The CMT1A myelin sheath showed a high heterogeneity compared to WT rat littermates: thin myelin sheath, demyelination ( Fig.…”

“…All CARS images were obtained as described previously [21] with a two-photon microscope LSM 7 MP coupled to an OPO (Zeiss, France) and complemented by a delay line [42]. A ×20 water immersion lens (W Plan Apochromat DIC VIS-IR) was used for image acquisition.…”

Schwann cell demyelination is triggered by a transient mitochondrial calcium release through Voltage Dependent Anion Channel 1

“…Finally, we tested the anti-demyelinating effect of TRO19622 and the pro-demyelinating effect of MJ in vivo using CARS imaging of myelin as read-out for demyelination. Coherent Anti-stokes Raman Scattering (CARS) is a non-linear microscopy approach that allows imaging lipids, hence myelin, in vivo or ex vivo (37,38). First, two months old mice were treated with 3mg/kg of TRO19622 or vehicle subcutaneously for 5 consecutive days.…”

“…Four days after MJ injection or nerve crush, animal were sacrificed and their sciatic nerves were collected, washed by PBS and fixed for 1h in 4% PFA solution at room temperature. All CARS images were obtained as described previously (37) with a two-photon microscope LSM 7 MP coupled to an OPO (Zeiss, France) and complemented by a delay line (53). A ×20 water immersion lens (W Plan Apochromat DIC VIS-IR) was used for image acquisition.…”