Abstract

Peripheral nerve injuries in animal models require imaging approaches that can reveal the dynamic, multiscale process of regeneration over time without disrupting repair mechanisms. Traditional methods lack the ability to visualize cellular behavior and matrix remodeling continuously. We present an imaging technique based on an implantable nerve window and fluorescent labeling, enabling high-resolution, longitudinal cellular-scale observation of peripheral nerve regeneration in living mice. We first evaluated near-infrared fluorophore-conjugated fibrin as a transient marker for early regenerative tissue within nerve guidance conduits. Imaging performed upon surgical re-exposure of the nerve revealed the incorporation of fibrin into early-stage regenerative cables, with gradual signal degradation over two weeks. This approach demonstrated the feasibility of using extracellular matrix-based tracers for dynamic visualization of regenerative tissue and motivated the development of longer-term imaging strategies. To enable stable, chronic optical access to the sciatic nerve in a mouse model, we developed a surgical method to expose the nerve and implant a permanent, polydimethylsiloxane (PDMS) skin-embedded window, which outperformed rigid, 3D-printed designs. This technique allowed imaging of the nerve for more than 90 days post-implantation, with no detectable structural or functional impact, minimal inflammation, and limited fibrotic response, permitting continuous observation of regeneration within the same animal. Using this platform, we achieved multiplexed, in vivo imaging of the peripheral nerve environment by combining a transgenic fluorescent reporter (Thy1-YFP for axons, S100-GFP for Schwann cells, Tie2-GFP for vascular endothelial cells) with optional additional labeling (second-harmonic generation for collagen, Nile Red for myelin/lipids, and/or fluorophore-conjugated tomato lectin for vasculature). We applied the nerve window across multiple injury models, including compression, crush, partial transection, and gap transection followed by conduit repair, to longitudinally observe distinct regenerative responses, such as immune infiltration, axonal degeneration, Schwann cell migration, axon–collagen interactions, and vascular changes. We further refined the nerve window approach in the conduit repair model by testing conduit technologies and tracking regeneration in different mouse strains. FVB/N mice exhibited less fibrosis but also reduced regenerative capacity compared to C57BL/6 and B6D2. Statistical evaluation showed that early-stage visual observations could predict later regenerative progress. Customized PDMS and poly(glycerol sebacate) conduits were successfully imaged and supported gap regeneration. Finally, we tested fluorescent fibrin as a passive reporter scaffold within the nerve repair conduit lumen and showed that it exhibited gradual degradation in vivo. Together, this work introduces a system combining in vivo optical imaging, multimodal fluorescent labeling, and adaptable injury models to enable noninvasive, high-resolution tracking of peripheral nerve regeneration in live mice. These techniques open the door to mechanistic investigation of regenerative processes at a cellular scale and accelerated preclinical evaluation of therapies targeting peripheral nerve repair.

Committee Chair

Mikhail Berezin

Committee Members

David Brogan; Guy Genin; Matthew MacEwan; Matthew Wood

Degree

Doctor of Philosophy (PhD)

Author's Department

Interdisciplinary Programs

Author's School

McKelvey School of Engineering

Document Type

Dissertation

Date of Award

8-18-2025

Language

English (en)

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