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.
Highlights
Improving treatment outcomes after peripheral nerve injury (PNI) remains an important goal for researchers and clinicians worldwide
Tissue clearing enables imaging of individual axons throughout the sciatic nerve As previously mentioned, light scattering caused by the numerous heterogeneities in nerve optical refractive index leads to poor depth penetration for many optical microscopy tools
As the thickness of rat sciatic nerves is approximately 1 mm in diameter, confocal imaging following tissue clearing allowed for complete fluorescence imaging throughout entire nerve bundles
Summary
Improving treatment outcomes after peripheral nerve injury (PNI) remains an important goal for researchers and clinicians worldwide. With over 300,000 annual upper extremity injuries, approximately 100,000 surgical repairs in Europe alone [1,2], and an increasing incidence of blast injuries sustained during warfare [3,4], there is a recognized and compelling need to accelerate peripheral nerve regeneration techniques and technologies; this has resulted in the development of peripheral nerve microsurgery in the 1960s and the subsequent refinement of a range of surgical techniques and treatments [5,6] Despite these advances, motor outcomes for patients remain delayed, unpredictable and usually incomplete [1,2,3]. Measurements of compound nerve action potentials (CNAP) in small animals can be difficult due to the inherent short distances between probe sites [11]; often, there exist several different neural pathways to carry the same evoked electrical current to a distal target other than the pathway of interest
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