Comprehensive Evaluation of Peripheral Nerve Regeneration in the Acute Healing Phase Using Tissue Clearing and Optical Microscopy in a Rodent Model

Abstract

Peripheral nerve injury (PNI), a common injury in both the civilian and military arenas, is usually associated with high healthcare costs and with patients enduring slow recovery times, diminished quality of life, and potential long-term disability. Patients with PNI typically undergo complex interventions but the factors that govern optimal response are not fully characterized. A fundamental understanding of the cellular and tissue-level events in the immediate postoperative period is essential for improving treatment and optimizing repair. Here, we demonstrate a comprehensive imaging approach to evaluate peripheral nerve axonal regeneration in a rodent PNI model using a tissue clearing method to improve depth penetration while preserving neural architecture. Sciatic nerve transaction and end-to-end repair were performed in both wild type and thy-1 GFP rats. The nerves were harvested at time points after repair before undergoing whole mount immunofluorescence staining and tissue clearing. By increasing the optic depth penetration, tissue clearing allowed the visualization and evaluation of Wallerian degeneration and nerve regrowth throughout entire sciatic nerves with subcellular resolution. The tissue clearing protocol did not affect immunofluorescence labeling and no observable decrease in the fluorescence signal was observed. Large-area, high-resolution tissue volumes could be quantified to provide structural and connectivity information not available from current gold-standard approaches for evaluating axonal regeneration following PNI. The results are suggestive of observed behavioral recovery in vivo after neurorrhaphy, providing a method of evaluating axonal regeneration following repair that can serve as an adjunct to current standard outcomes measurements. This study demonstrates that tissue clearing following whole mount immunofluorescence staining enables the complete visualization and quantitative evaluation of axons throughout nerves in a PNI model. The methods developed in this study could advance PNI research allowing both researchers and clinicians to further understand the individual events of axonal degeneration and regeneration on a multifaceted level.

Monitoring peripheral nerve degeneration in ALS by label-free stimulated Raman scattering imaging

“…1, Supplementary Fig. 1)111920212223242526. Collection of pump light, after the lasers' SRS interaction with the sample in focus, was achieved by the use of a high numerical aperture condenser and band-pass filter before detection with a photodiode (Methods section and Supplementary Fig.…”

“…We therefore next tested the utility of SRS imaging in the context of a mouse model of axonal injury by sciatic nerve crush22232642. The sciatic nerve of one hind limb was crushed in WT and SOD1 G93A animals then imaged distally of the injury at both 1 week and 3 weeks after initial injury (Fig.…”

“…SRS and its precursor coherent anti-Stokes Raman scattering have been demonstrated as a powerful tool for myelin imaging181920212223242526. Although there is growing interest in the importance of demyelination in ALS, systematic studies of peripheral nerve degeneration have not yet been carried out with SRS27.…”

Coherent Raman Scattering Microscopy in Biology and Medicine

“…An important application is the visualization of the myelin sheath in the central nervous system (26–28, 96, 97) and the peripheral nervous system (98, 99), which provides an effective way to study spinal cord injury and myelin disease ex vivo (Figure 2 c , left ) and in vivo (Figure 2 c , middle and right ). In Figure 2 c , the myelin sheath, myelinated axons, and the node of Ranvier are highlighted (from left to right) with good contrast in CARS images.…”

In Situ and In Vivo Molecular Analysis by Coherent Raman Scattering Microscopy

“…The quantitative measurement of lipid storage has also been applied to identify 9 out of 272 genes whose inactivation increased fat content by more than 25% (98). Studies of myelin disease and spinal cord injury used CRS microscopy at the tissue level to visualize the myelin sheath in the central (99–103) and peripheral (104, 105) nervous systems. For medical imaging, Freudiger et al (89) and Ji et al (106) showed that, by the linear combination of SRS images at peaks of 2,850 and 2,940 cm −1 for CH 2 and CH 3 vibration modes, a stain-free histopathological image comparable to a hematoxylin and eosin (H&E) stained image can be achieved (Figure 3 a ).…”