Diffusion tensor imaging to assess axonal regeneration in peripheral nerves

Abstract

Development of outcome measures to assess ongoing nerve regeneration in the living animal that can be translated to human can provide extremely useful tools for monitoring the effects of therapeutic interventions to promote nerve regeneration. Diffusion tensor imaging (DTI), a magnetic resonance based technique, provides image contrast for nerve tracts and can be applied serially on the same subject with potential to monitor nerve fiber content. In this study, we examined the use of ex vivo high-resolution DTI for imaging intact and regenerating peripheral nerves in mice and correlated the MRI findings with electrophysiology and histology. DTI was done on sciatic nerves with crush, without crush, and after complete transection in different mouse strains. DTI measures, including fractional anisotropy (FA), parallel diffusivity, and perpendicular diffusivity were acquired and compared in segments of uninjured and crushed/transected nerves and correlated with morphometry. A comparison of axon regeneration after sciatic nerve crush showed a comparable pattern of regeneration in different mice strains. FA values were significantly lower in completely denervated nerve segments compared to uninjured sciatic nerve and this signal was restored toward normal in regenerating nerve segments (crushed nerves). Histology data indicate that the FA values and the parallel diffusivity showed a positive correlation with the total number of regenerating axons. These studies suggest that DTI is a sensitive measure of axon regeneration in mouse models and provide basis for further development of imaging technology for application to living animals and humans.

Figure 1. Axonal regeneration in four different mouse inbred strains

Compound muscle action potential (CMAP) amplitudes recorded in the hindpaw on day 9,15 and 17 after nerve crush were similar in PWK/PHj (■), WSB/EiJ (▲), DBA/2J (◆) and C57BL/6 (●) mice.

Figure 2. Changes in fractional anisotropy (FA) after transection and 17 days after nerve crush

Representative light micrographs of an uninjured sciatic nerve (A), 10 days after nerve transection (B), and 17 days after nerve crush (C) (bar = 75 μm). (D) Average FA values of sciatic nerves after transection and 17 days after crush injury. In comparison to control (black bars) nerves, FA is reduced in transected and in regenerated sciatic nerve segments after crush injury.

Figure 3. Correlation between morphology and diffusion tensor imaging (DTI) parameters

Correlations between DTI parameters and numbers of myelinated axons in PWK/PHj (■), WSB/EiJ (▲), DBA/2J (◆) and C57BL/6 (●) mice. (A) The fractional anisotropy (FA) and numbers of regenerating axons are significantly correlated. (B) The parallel diffusivity (λ_‖) correlates with axon counts. (C) The perpendicular diffusivity (λ_┴) is not correlated with the number of regenerating axons.

Figure 4. Diffusion tensor imaging with 3D reconstruction and tractography of ex vivo mouse peripheral nerves

Fiber tracking can visualize axon degeneration and regeneration in the mouse sciatic nerve injury. Fiber tracking with a FA threshold of 0.5 was performed on diffusion tensor data collected from uninjured nerves, transected nerves and in regenerating nerves 17 days after crush injury. Seed points were selected at the proximal end of the sciatic nerves. For each case, the entire nerve tissue specimen (left, gray, reconstructed from T2-weighted images) and fiber tracking result (right, red) are displayed. Arrows mark the transection (yellow) and crush site (white), respectively.

Figure 5. Comparison of conventional T2 (T2W) and DTI-based contrast (anisotropy map [FA] and direction encoded color maps [DEC])

Axial and horizontal slices with different angles were extracted from 3D images of a fixed mouse hind limb. White arrows indicate location of sciatic nerves. It is difficult to distinguish nerve in T2-weighted images. Anisotropy and color maps provide a much better contrast to identify peripheral nerves. The color map visualizes the orientation of axonal fibers with RGB color. This further distinguishes nerve from surrounding tissues due to unique and coherent color along its trajectory that is dictated by the orientation of axons/nerve fibers (bar = 1mm).

Figure 6. 3D reconstruction of ex vivo mouse peripheral nerves

In the left panel, muscle is indicated by transparent gray structures, which is removed in the middle panel for better visualization of the nerves. The light gray structure is the bone. The 3D trajectories of the sciatic nerves are reconstructed from 3D DTI data. The three major branches of the sciatic nerves are displayed with different colors: yellow = tibial, blue= superficial peroneal, red = deep peroneal nerves, green = saphenous nerve. The branching can be clearly appreciated in the right panel.