A method for quantitative spatial analysis of immunolabeled fibers at regenerative electrode interfaces

• Published in: Journal of neuroscience methods 2024-12, Vol.412, p.110295, Article 110295
• Main Authors: Rosario, Michael, Zhang, Jingyuan, Kaleem, Muhammad Irfan, Chandra, Nikhil, Yan, Ying, Moran, Daniel, Wood, Matthew, Ray, Wilson Z., MacEwan, Matthew
• Format: Article
• Language: English
• Subjects: Animals; Confocal Microscopy; Electrodes, Implanted; Histology; Immunofluorescence; Immunohistochemistry; Male; Microscopy, Confocal - methods; Motor Neurons - physiology; Nerve Fibers - physiology; Nerve Regeneration - physiology; Neuroprosthetics; Peripheral nerve interface; Rats; Rats, Sprague-Dawley; Regenerative electrode; Sciatic Nerve - physiology; Spatial Analysis
• ISSN: 0165-0270; 1872-678X; 1872-678X
• DOI: 10.1016/j.jneumeth.2024.110295

Regenerative electrodes are being explored as robust peripheral nerve interfaces for neuro-prosthetic control and sensory feedback. Current designs differ in electrode number, spatial arrangement, and porosity which impacts the regeneration, activation, and spatial distribution of fibers at the device interface. Knowledge of sensory and motor fiber distributions are important in optimizing selective fiber activation and recording. We use confocal microscopy and immunofluorescence methods to conduct spatial analysis of immunolabeled fibers across whole nerve cross sections. This protocol was implemented to characterize motor fiber distribution within 3 macro-sieve electrode regenerated (MSE), 3 silicone-conduit regenerated, and 3 unmanipulated control rodent sciatic nerves. Total motor fiber counts were 1485 [SD: +/- 50.11], 1899 [SD: +/- 359], and 5732 [SD: +/- 1410] for control, MSE, and conduit nerves respectively. MSE motor fiber distributions exhibited evidence of deviation from complete spatial randomness and evidence of dispersion and clustering tendencies at varying scales. Notably, MSE motor fibers exhibited clustering within the central portion of the cross section, whereas conduit regenerated motor fibers exhibited clustering along the periphery. Prior exploration of fiber distributions at regenerative interfaces was limited to either quadrant-based density analysis of randomly sampled subregions or qualitative description. This method extends existing sample preparation and microscopy techniques to quantitatively assess immunolabeled fiber distributions within whole nerve cross-sections. This approach is an effective way to examine the spatial organization of fiber subsets at regenerative electrode interfaces, enabling robust assessment of fiber distributions relative to electrode arrangement. •We implement a method for spatial analysis of immunolabeled fibers at regenerative electrode interfaces.•Immunofluorescence enables robust multiplexing for visualization of distinct fiber types in the same section.•Confocal imaging enables axon tracking over stacked sections, facilitating fiber identity confirmation during segmentation.•Spatial analysis is performed on free, well documented open-source software.