Assistant Professor University of Miami Miami, Florida, United States
Introduction: Facial paralysis is a debilitating condition resulting from nerve damage, leading to the immobility of facial muscles, and significantly impacting patients' quality of life. Despite the limited success of current treatment options, interventions such as facial reanimation surgeries often yield variable outcomes in reconnecting lost nerves. Biomaterials have shown promise in previous studies to promote regeneration and regrowth in damaged nerve tissues. Hydrogel tubes, leveraging their mechanical and structural properties, provide a biomimetic environment similar to the peripheral nervous system (PNS). These tubes play a critical role in guiding nerve regrowth and facilitating cell infiltration by serving as scaffolds. Given the crucial role of the neuromuscular junction (NMJ) in converting electrical impulses from the motor neuron into muscle activity, it is essential to ensure its stability and proper reinnervation following injury. The present study evaluates NMJ stability and reinnervation following facial nerve injury and biomaterial implantation.
Materials and
Methods: Anesthetized Sprague Dawley female rats sustained a 5-millimeter nerve gap defect in the left buccal and marginal mandibular branch of the facial nerve. The animal conditions were assigned to the following groups: sham, injury, autograft facial nerve, and PEG tube implantation. The PEG hydrogel tube implants were made of degradable micro annealed microgels and were fabricated as previously described (Ciciriello et al., 2022). At 8 weeks post-injury, electrophysiology data acquisition was performed, and the whisker pad muscles were harvested, sectioned, and stained. Electrophysiological data acquisition involved stimulating the buccal and marginal mandibular nerves with a tungsten electrode and recording the resulting compound muscle action potentials (CMAPs) from the whisker pad using a 16-channel microwire electrode array and Synapse software. Following histological analysis, synaptic vesicle 2 (SV2) was utilized to identify the presynaptic terminal, neurofilament 200 (NF200) to identify the reinnervating axon, and alpha-bungarotoxin (α-BTX) to identify the post-synaptic terminal. The NMJ’s after PEG implantation were categorized as healthy, stable, disassembled, and/or disorganized (Ross et al., 2016). The synapses were further analyzed with the ImageJ software and NMJ-morph plug-in previously described (Burns et al., 2007) for the following post-synaptic variables: AChR perimeter (μm), AChR area (μm2), end-plate diameter (μm), endplate perimeter (μm), compactness (%), end- plate area (μm2), and fragmentation. The NMJ-morph tool operates on a repeatable workflow which is specifically tailored for use on confocal z-stack projections of individual NMJs. The standard built-in ImageJ functions were leveraged to record the ‘core variables and ‘derived’ morphologic data.
Results, Conclusions, and Discussions: The following study focused on a comprehensive analysis of NMJ morphology following biomaterial intervention in a facial nerve injury rodent model. The implementation of an ImageJ-based platform, NMJ-morph, provided a workflow for statistical comparison of the experimental conditions ex vivo. Histological analysis evaluation of NMJ morphology and integrity were detected across all conditions. (Figure 1A). Preliminary results of the electrophysiological recordings demonstrate significant improvements in muscle activation following biomaterial intervention. Moving forward, we will continue to evaluate NMJ morphology and integrity, with a hypothesis of improved NMJ morphological outcomes following biomaterial intervention due to the increased electrical activity demonstrated via electrophysiological recordings. The evaluation of NMJ integrity is essential to further understand effective nerve regeneration and functional recovery. The experimental outcome of the present study aims to provide valuable insights into advancements in future therapeutic strategies for nerve repair, emphasizing the importance of NMJ stability in successful nerve regeneration.
