KEYWORDS: Nerve, Animal model studies, Windows, Animals, Visualization, Visual process modeling, Process modeling, Muscles, In vivo imaging, Temporal resolution
In vivo imaging of a nerve injury and longitudinal monitoring of peripheral nerve processes in preclinical models can shed light on many biological process within the nerve. Optical imaging with its high spatial and temporal resolution is highly promising, however high scattering from the skin and other adjacent organs such as muscle limit the application to only in vitro or ex vivo studies. To address these limiting issues, we developed a minimally invasive in vivo model that enables continuous imaging of a peripheral nerve with a high, single axon resolution. Our approach uses a flexible skin-imbedded transparent optical window with the nerve surgically repositioned above the muscle layer. This modality allows longitudinal single-axonal resolution microscopy of the nerve with virtually any optical reporter that can be used in living animals.
Significance: Exogenous extracellular matrix (ECM) proteins, such as fibrinogen and the thrombin-polymerized scaffold fibrin, are used in surgical repair of severe nerve injuries to supplement ECM produced via the injury response. Monitoring the dynamic changes of fibrin during nerve regeneration may shed light on the frequent failure of grafts in the repair of long nerve gaps.Aim: We explored whether monitoring of fibrin dynamics can be carried out using nerve guidance conduits (NGCs) containing fibrin tagged with covalently bound fluorophores.Approach: Fibrinogen was conjugated to a near-infrared (NIR) fluorescent dye. NGCs consisting of silicone tubes filled with the fluorescent fibrin were used to repair a 5-mm gap injury in rat sciatic nerve (n = 6).Results: Axonal regeneration in fluorescent fibrin-filled NGCs was confirmed at 14 days after implantation. Intraoperative fluorescence imaging after implantation showed that the exogenous fibrin was embedded in the early stage regenerative tissue. The fluorescent signal temporarily highlighted a cable-like structure within the conduit and gradually degraded over two weeks.Conclusions: This study, for the first time, visualized in vivo intraneural fibrin degradation, potentially a useful prospective indicator of regeneration success, and showed that fluorescent ECM, in this case fibrin, can facilitate imaging of regeneration in peripheral nerve conduits without significantly affecting the regeneration process.
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