PhD Defense in Neural Engineering
Aniruddha Upadhye
Ph.D. Candidate
Wickenden 328
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Department of Biomedical Engineering
Cleveland, OH
Thesis Advisors: Andrew Shoffstall, PhD
ABSTRACT
Vagus nerve stimulation (VNS) is a widely used neuromodulation therapy for various disorders, yet its effectiveness is hindered by a limited understanding of fascicular organization and electrode interactions. Many other types of neuromodulation therapies also suffer from similar limitations of side effects and cannot achieve the desired efficacy. In this thesis, we leveraged high-resolution micro-computed tomography (micro-CT) to quantify the complex fascicular architecture of the human cervical vagus nerve and overcame the challenges of acquiring micro-CT scans of long-length samples by developing a specialized workflow. This workflow incorporated 3D-printed molds and image registration techniques to generate high-resolution, long-length scans of the vagus nerve. This was also one of the first studies to quantify fascicular reorganization by analysis as split and merge events. Our findings revealed that fascicles within the vagus nerve undergo frequent splitting and merging events, averaging one reorganization every ~560 μm, with significant inter-individual variability. These findings underscore the challenges in achieving selective stimulation using current electrode designs and highlight the need for personalized approaches to VNS therapy.
In our consecutive study, we evaluated phosphotungstic acid (PTA) as a novel contrast agent for micro-CT imaging of peripheral nerves. Compared to conventional stains like osmium tetroxide and Lugol’s iodine, PTA provided superior visualization of the perineurium, offering clear delineation of fascicular boundaries with minimal tissue artifacts. We optimized micro-CT acquisition parameters to balance image sharpness and noise, ensuring high-fidelity 3D reconstructions of nerve morphology. Our findings demonstrated that PTA staining enhances nerve microstructure visualization, particularly improving contrast at the perineurium-fascicle boundary. Additionally, we assessed PTA’s compatibility with histological techniques, including hematoxylin and eosin staining, Masson’s trichrome, and immunohistochemistry. PTA-stained nerves retained structural integrity, though higher concentrations and prolonged staining altered the optical density of nuclei in special stains. These findings establish PTA as a valuable micro-CT contrast agent that enables high-resolution imaging while preserving compatibility with downstream histology.
By integrating micro-CT with histological validation, this work establishes a robust framework for characterizing nerve morphology and guiding computational models for selective neuromodulation. The insights gained from this research pave the way for designing improved electrode interfaces and refining stimulation paradigms, ultimately advancing the efficacy and precision of VNS therapies. This thesis contributes to the broader field of neural engineering by demonstrating the utility of advanced imaging techniques in understanding peripheral nerve anatomy and optimizing neuromodulatory interventions.