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Will's MS project focused on the evaluation of bladder fatigue in sacral neuromodulation using an injectable electrode.

Longshun's project focused on the development and evaluation of dexamethasone loaded platelet inspired nanoparticles to improve intracortical microelectrode recording performance.

Background and Purpose Spinal cord stimulation (SCS) is approved by the Food and Drug Administration for treating chronic intractable pain in the back, trunk, or limbs through stimulation of the dorsal column. Numerous studies have used swine as an analog of the human spinal cord to better understand SCS and further improve its efficacy. We performed high-resolution imaging of the porcine spinal cord with intact dura mater using micro-computed tomography (μCT) to construct detailed 3-dimensional (3D) visualizations of the spinal cord and characterize the morphology of the dorsal and ventral rootlets. Methods We obtained spinal cords from Yorkshire/Landrace crossbred swine (N = 7), stained samples with osmium tetroxide, and performed μCT imaging of the T12-T15 levels at isotropic voxel resolutions ranging from 3.3 to 50 μm. We measured the anatomical morphology using the 3D volumes and compared our results to measurements previously collected from swine and human spinal cords via microdissection techniques in prior literature. Results While the porcine thoracic-lumbar spinal cord is a popular model for SCS, we highlight multiple notable differences compared to previously published T8-T12 human measurements including rootlet counts (porcine dorsal/ventral: 12.2 ± 2.6, 26.6 ± 3.4; human dorsal/ventral: 5.3 ± 1.3, 4.4 ± 2.4), rootlet angles (porcine ventral-rostral: 161 ± 1°, ventral-caudal: 155 ± 6°, dorsal-rostral: 148 ± 9°, dorsal-caudal: 142 ± 6°; human ventral-rostral: 170 ± 3°, ventral-caudal: 22 ± 10°, dorsal-rostral: 171 ± 3°, dorsal-caudal: 15 ± 7°), and the presence and count of dorsal rootlet bundles. Conclusions Detailed measurements and highlighted differences between human and porcine spinal cords can inform variations in modeling and electrophysiological experiments between the two species. In contrast to other approaches for measuring the spinal cord and rootlet morphology, our method keeps the dura intact, reducing potential artifacts from dissection.

Injectable biomaterials have been engineered to operate within the human body, offering versatile solutions for minimally invasive therapies and meeting several stringent requirements such as biocompatibility, biodegradability, low viscosity for ease of injection, mechanical strength, rapid gelation postinjection, controlled release of therapeutic agents, hydrophobicity/hydrophilicity balance, stability under physiological conditions, and the ability to be sterilized. Their adaptability and performance in diverse clinical settings make them invaluable for modern medical treatments. This article reviews recent advancements in the design, synthesis, and characterization of injectable polymeric biomaterials, providing insights into their emerging applications. We discuss a broad spectrum of these materials, including natural, synthetic, hybrid, and composite types, that are being applied in targeted drug delivery, cell and protein transport, regenerative medicine, tissue adhesives, injectable implants, bioimaging, diagnostics, and 3D bioprinting. Ultimately, the review highlights the critical role of injectable polymeric biomaterials in shaping the future of medical treatments and improving patient outcomes across a wide range of therapeutic and diagnostic applications.

Spinal cord stimulation (SCS) has demonstrated multiple benefits in treating chronic pain and other clinical disorders related to sensorimotor dysfunctions. However, the underlying mechanisms are still not fully understood, including how electrode placement in relation to the spinal cord neuroanatomy influences epidural spinal recordings (ESRs). To characterize this relationship, this study utilized stimulation applied at various anatomical sections of the spinal column, including at levels of the intervertebral disc and regions correlating to the dorsal root entry zone.

SignificanceInformation about the spatial organization of fibers within a nerve is crucial to our understanding of nerve anatomy and its response to neuromodulation therapies. A serial block-face microscopy method [three-dimensional microscopy with ultraviolet surface excitation (3D-MUSE)] has been developed to image nerves over extended depths ex vivo. To routinely visualize and track nerve fibers in these datasets, a dedicated and customizable software tool is required.AimOur objective was to develop custom software that includes image processing and visualization methods to perform microscopic tractography along the length of a peripheral nerve sample.ApproachWe modified common computer vision algorithms (optic flow and structure tensor) to track groups of peripheral nerve fibers along the length of the nerve. Interactive streamline visualization and manual editing tools are provided. Optionally, deep learning segmentation of fascicles (fiber bundles) can be applied to constrain the tracts from inadvertently crossing into the epineurium. As an example, we performed tractography on vagus and tibial nerve datasets and assessed accuracy by comparing the resulting nerve tracts with segmentations of fascicles as they split and merge with each other in the nerve sample stack.ResultsWe found that a normalized Dice overlap (Dicenorm) metric had a mean value above 0.75 across several millimeters along the nerve. We also found that the tractograms were robust to changes in certain image properties (e.g., downsampling in-plane and out-of-plane), which resulted in only a 2% to 9% change to the mean Dicenorm values. In a vagus nerve sample, tractography allowed us to readily identify that subsets of fibers from four distinct fascicles merge into a single fascicle as we move ∼5 mm along the nerve’s length.ConclusionsOverall, we demonstrated the feasibility of performing automated microscopic tractography on 3D-MUSE datasets of peripheral nerves. The software should be applicable to other imaging approaches. The code is available at https://github.com/ckolluru/NerveTracker.

Justin's MS project focused on the development of a microCT methologogy to visualize the porcine spinal cord with intact dura mater.

Anna's MS project focused on the evaluation of bioabsorbable transient neuromodulation electrodes.

Currently there are numerous methods to evaluate peripheral nerve stimulation interfaces in rats, with stimulation-evoked ankle torque being one of the most prominent. Commercial rat ankle torque measurement systems and custom one-off solutions have been published in the literature. However, commercial systems are proprietary and costly and do not allow for customization. One-off lab-built systems have required specialized machining expertise, and building plans have previously not been made easily accessible. Here, detailed building plans are provided for a low-cost, open-source, and basic ankle torque measurement system from which additional customization can be made. A hindlimb stabilization apparatus was developed to secure and stabilize a rat's hindlimb, while allowing for simultaneous ankle-isometric torque and lower limb muscle electromyography (EMG). The design was composed mainly of adjustable 3D-printed components to accommodate anatomical differences between rat hindlimbs. Additionally, construction and calibration procedures of the rat hindlimb stabilization apparatus were demonstrated in this study. In vivo torque measurements were reliably acquired and corresponded to increasing stimulation amplitudes. Furthermore, implanted leads used for intramuscular EMG recordings complemented torque measurements and were used as an additional functional measurement in evaluating the performance of a peripheral nerve stimulation interface. In conclusion, an open-source and noninvasive platform, made primarily with 3D-printed components, was constructed for reliable data acquisition of evoked motor activity in rat models. The purpose of this apparatus is to provide researchers a versatile system with adjustable components that can be tailored to meet user-defined experimental requirements when evaluating motor function of the rat hindlimbs.

Intracortical microelectrodes induce vascular injury upon insertion into the cortex. As blood vessels rupture, blood proteins and blood-derived cells (including platelets) are introduced into the ‘immune privileged’ brain tissues at higher-than-normal levels, passing through the damaged blood-brain barrier. Blood proteins adhere to implant surfaces, increasing the likelihood of cellular recognition leading to activation of immune and inflammatory cells. Persistent neuroinflammation is a major contributing factor to declining microelectrode recording performance. We investigated the spatial and temporal relationship of blood proteins fibrinogen and von Willebrand Factor (vWF), platelets, and type IV collagen, in relation to glial scarring markers for microglia and astrocytes following implantation of non-functional multi-shank silicon microelectrode probes into rats. Together with type IV collagen, fibrinogen and vWF augment platelet recruitment, activation, and aggregation. Our main results indicate blood proteins participating in hemostasis (fibrinogen and vWF) persisted at the microelectrode interface for up to 8-weeks after implantation. Further, type IV collagen and platelets surrounded the probe interface with similar spatial and temporal trends as vWF and fibrinogen. In addition to prolonged blood-brain barrier instability, specific blood and extracellular matrix proteins may play a role in promoting the inflammatory activation of platelets and recruitment to the microelectrode interface.

Introduction:  MicroCT of the three-dimensional fascicular organization of the human vagus nerve provides essential data to inform basic anatomy as well as the development and optimization of neuromodulation therapies. To process the images into usable formats for subsequent analysis and computational modeling, the fascicles must be segmented. Prior segmentations were completed manually due to the complex nature of the images, including variable contrast between tissue types and staining artifacts. Methods: Here, we developed a U-Net convolutional neural network (CNN) to automate segmentation of fascicles in microCT of human vagus nerve.  Results: The U-Net segmentation of ~500 images spanning one cervical vagus nerve was completed in 24 s, versus ~40 h for manual segmentation, i.e., nearly four orders of magnitude faster. The automated segmentations had a Dice coefficient of 0.87, a measure of pixel-wise accuracy, thus suggesting a rapid and accurate segmentation. While Dice coefficients are a commonly used metric to assess segmentation performance, we also adapted a metric to assess fascicle-wise detection accuracy, which showed that our network accurately detects the majority of fascicles, but may under-detect smaller fascicles.  Discussion: This network and the associated performance metrics set a benchmark, using a standard U-Net CNN, for the application of deep-learning algorithms to segment fascicles from microCT images. The process may be further optimized by refining tissue staining methods, modifying network architecture, and expanding the ground-truth training data. The resulting three-dimensional segmentations of the human vagus nerve will provide unprecedented accuracy to define nerve morphology in computational models for the analysis and design of neuromodulation therapies.

Understanding peripheral nerve micro-anatomy can assist in the development of safe and effective neuromodulation devices. However, current approaches for imaging nerve morphology at the fiber level are either cumbersome, require substantial instrumentation, have a limited volume of view, or are limited in resolution/contrast. We present alternative methods based on MUSE (Microscopy with Ultraviolet Surface Excitation) imaging to investigate peripheral nerve morphology, both in 2D and 3D. For 2D imaging, fixed samples are imaged on a conventional MUSE system either label free (via auto-fluorescence) or after staining with fluorescent dyes. This method provides a simple and rapid technique to visualize myelinated nerve fibers at specific locations along the length of the nerve and perform measurements of fiber morphology (e.g., axon diameter and g-ratio). For 3D imaging, a whole-mount staining and MUSE block-face imaging method is developed that can be used to characterize peripheral nerve micro-anatomy and improve the accuracy of computational models in neuromodulation. Images of rat sciatic and human cadaver tibial nerves are presented, illustrating the applicability of the method in different preclinical models. 

Upadhye, A., Chin, J., M. Settell, C. Kolluru, A. Deshmukh, B. Knudsen, M. Laluzerne, O. Buyukcelik, et al. “Imaging the Neural Micro-Anatomy of the Swine Spinal Cord.” Session presented at the NANS Annual Meeting, Las Vegas, January 2023. 

Lam, D., S. Kang, N. Verma, B. Romanauski, Y. Nishiyama, J. Hao, K. Ludwig, H. Park, E. Ross, I. Lavrov, M. Zhang. "Changes in Evoked Responsesl During Epidual Spinal Cord Stimulation Generated by Minimal Movement of Leads." Presented at the NANS 2023, Las Vegas, NV, 2023. 

Mapping the Vagus Nerve, Autonomic Nervous System

“’National Institutes of Health awards $15.75M to research team led by Case Western Reserve University and Duke University to map vagus nerve—body’s ‘super highway’ for controlling major organ functions”

https://thedaily.case.edu/national-institutes-of-health-awards-15-75m-to-research-team-led-by-case-western-reserve-university-and-duke-university-to-map-vagus-nerve-bodys-super-highway-for-c/

https://www.cleveland.com/healthfit/2022/10/cwru-duke-receive-1575-nih-grant-to-map-nerve-develop-treatments-for-heart-intestinal-disease.html

https://www.crainscleveland.com/health-care/nih-awards-1575-million-contract-cwru-duke-led-research-team

https://pratt.duke.edu/about/news/collaborative-team-neural-engineers-receive-sparc-grant-map-bodys-super-highway

https://case.edu/medicine/about/newsroom/national-institutes-health-awards-1575m-research-team-led-case-western-reserve-university-and-duke-university-map-vagus-nerve-bodys-super-highway-controlling-major-organ-functions

Ozge's MS project focused on the

Objective. Vagus nerve stimulation (VNS) is Food and Drug Administration-approved for epilepsy, depression, and obesity, and stroke rehabilitation; however, the morphological anatomy of the vagus nerve targeted by stimulatation is poorly understood. Here, we used microCT to quantify the fascicular structure and neuroanatomy of human cervical vagus nerves (cVNs). Approach. We collected eight mid-cVN specimens from five fixed cadavers (three left nerves, five right nerves). Analysis focused on the 'surgical window': 5 cm of length, centered around the VNS implant location. Tissue was stained with osmium tetroxide, embedded in paraffin, and imaged on a microCT scanner. We visualized and quantified the merging and splitting of fascicles, and report a morphometric analysis of fascicles: count, diameter, and area. Main results. In our sample of human cVNs, a fascicle split or merge event was observed every ∼560 µm (17.8 ± 6.1 events cm−1). Mean morphological outcomes included: fascicle count (6.6 ± 2.8 fascicles; range 1–15), fascicle diameter (514 ± 142 µm; range 147–1360 µm), and total cross-sectional fascicular area (1.32 ± 0.41 mm2; range 0.58–2.27 mm). Significance. The high degree of fascicular splitting and merging, along with wide range in key fascicular morphological parameters across humans may help to explain the clinical heterogeneity in patient responses to VNS. These data will enable modeling and experimental efforts to determine the clinical effect size of such variation. These data will also enable efforts to design improved VNS electrodes.

Electrical stimulation after peripheral nerve injury (PNI) has the potential to promote more rapid and complete recovery of damaged fiber tracts. While permanently implanted devices are commonly used to treat chronic or persistent conditions, they are not ideal solutions for transient medical therapies due to high costs, increased risk of surgical injury, irritation, infection, and persistent inflammation at the site of the implant. Furthermore, removal of temporary leads placed on or around peripheral nerves may have unacceptable risk for nerve injury, which is counterproductive in developing therapies for PNI treatment. Transient devices which provide effective clinical stimulation while being capable of harmless bioabsorption may overcome key challenges in these areas. However, current bioabsorbable devices are limited in their robustness and require complex fabrication strategies and novel materials which may complicate their clinical translation pathway. In this study, we present a simple bioabsorbable / biodegradable electrode fabricated by modifying standard absorbable sutures, and we present data characterizing our prototype's stability in vitro and in vivo.

Buyukcelik, O., and A. Shoffstall. “Deep Learning Based Segmentation of Fascicles in MicroCT Images of the Vagus Nerve.” Presented at the BMES 2022, San Antonio, TX, 2022.

Jitendran, E., D. Menendez, L. Li, and A. Shoffstall. “Evaluating the Route of Systemic Administration for Platelet Inspired Nanoparticles for Targeting Brain-Implanted Microelectrodes.” Presented at the BMES 2022, San Antonio, TX, 2022.

Lam, D., and A. Shoffstall. “Characterizing Feasibility and Performance of Injectable Gold Microwires for Neural Interfacing.” Presented at the BMES 2022, San Antonio, TX, 2022.

Menendez, D., L. Li, and A. Shoffstall. “Platelet Inspired Drug Delivery Vehicle Targets Brain Implanted Microelectrodes.” Presented at the BMES 2022, San Antonio, TX, 2022.

Upadhye, A., and A. Shoffstall. “Imaging Vagus Nerve Micro-Anatomy Using MicroCT.” Presented at the BMES 2022, San Antonio, TX, 2022. 

Sept 2022

Cranial window surgery allows for the imaging of brain tissue in live mice with the use of multiphoton or other intravital imaging techniques. However, when performing any craniotomy by hand, there is often thermal damage to brain tissue, which is inherently variable surgery-to-surgery and may be dependent on individual surgeon technique. Implementing a surgical robot can standardize surgery and lead to a decrease in thermal damage associated with surgery. In this study, three methods of robotic drilling were tested to evaluate thermal damage: horizontal, point-by-point, and pulsed point-by-point. Horizontal drilling utilizes a continuous drilling schematic, while point-by-point drills several holes encompassing the cranial window. Pulsed point-by-point adds a "2 s on, 2 s off" drilling scheme to allow for cooling in between drilling. Fluorescent imaging of Evans Blue (EB) dye injected intravenously measures damage to brain tissue, while a thermocouple placed under the drilling site measures thermal damage. Thermocouple results indicate a significant decrease in temperature change in the pulsed point-by-point (6.90 °C ± 1.35 °C) group compared to the horizontal (16.66 °C ± 2.08 °C) and point-by-point (18.69 °C ± 1.75 °C) groups. Similarly, the pulsed point-by-point group also showed significantly less EB presence after cranial window drilling compared to the horizontal method, indicating less damage to blood vessels in the brain. Thus, a pulsed point-by-point drilling method appears to be the optimal scheme for reducing thermal damage. A robotic drill is a useful tool to help minimize training, variability, and reduce thermal damage. With the expanding use of multiphoton imaging across research labs, it is important to improve the rigor and reproducibility of results. The methods addressed here will help inform others of how to better use these surgical robots to further advance the field. 

Derrick's MS project focused on the development and evaluation of a novel resorbable stimulation electrode.