Deep-learning segmentation of fascicles from microCT of the human vagus nerve.

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.

Fascicles split or merge every ∼560 microns within the human cervical vagus nerve.

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 ∼560µm (17.8 ± 6.1 events cm-1). Mean morphological outcomes included: fascicle count (6.6 ± 2.8 fascicles; range 1-15), fascicle diameter (514 ± 142µm; range 147-1360µm), 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.

Imaging peripheral nerve micro-anatomy with MUSE, 2D and 3D approaches.

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.

In vivo Visualization of Pig Vagus Nerve "Vagotopy" Using Ultrasound.

Background: Placement of the clinical vagus nerve stimulating cuff is a standard surgical procedure based on anatomical landmarks, with limited patient specificity in terms of fascicular organization or vagal anatomy. As such, the therapeutic effects are generally limited by unwanted side effects of neck muscle contractions, demonstrated by previous studies to result from stimulation of (1) motor fibers near the cuff in the superior laryngeal and (2) motor fibers within the cuff projecting to the recurrent laryngeal. Objective: Conventional non-invasive ultrasound, where the transducer is placed on the surface of the skin, has been previously used to visualize the vagus with respect to other landmarks such as the carotid and internal jugular vein. However, it lacks sufficient resolution to provide details about the vagus fascicular organization, or detail about smaller neural structures such as the recurrent and superior laryngeal branch responsible for therapy limiting side effects. Here, we characterize the use of ultrasound with the transducer placed in the surgical pocket to improve resolution without adding significant additional risk to the surgical procedure in the pig model. Methods: Ultrasound images were obtained from a point of known functional organization at the nodose ganglia to the point of placement of stimulating electrodes within the surgical window. Naïve volunteers with minimal training were then asked to use these ultrasound videos to trace afferent groupings of fascicles from the nodose to their location within the surgical window where a stimulating cuff would normally be placed. Volunteers were asked to select a location for epineural electrode placement away from the fascicles containing efferent motor nerves responsible for therapy limiting side effects. 2-D and 3-D reconstructions of the ultrasound were directly compared to post-mortem histology in the same animals. Results: High-resolution ultrasound from the surgical pocket enabled 2-D and 3-D reconstruction of the cervical vagus and surrounding structures that accurately depicted the functional vagotopy of the pig vagus nerve as confirmed via histology. Although resolution was not sufficient to match specific fascicles between ultrasound and histology 1 to 1, it was sufficient to trace fascicle groupings from a point of known functional organization at the nodose ganglia to their locations within the surgical window at stimulating electrode placement. Naïve volunteers were able place an electrode proximal to the sensory afferent grouping of fascicles and away from the motor nerve efferent grouping of fascicles in each subject (n = 3). Conclusion: The surgical pocket itself provides a unique opportunity to obtain higher resolution ultrasound images of neural targets responsible for intended therapeutic effect and limiting off-target effects. We demonstrate the increase in resolution is sufficient to aid patient-specific electrode placement to optimize outcomes. This simple technique could be easily adopted for multiple neuromodulation targets to better understand how patient specific anatomy impacts functional outcomes.

3D imaging of the vagus nerve fascicular anatomy with cryo-imaging and UV excitation.

Vagus nerve stimulation (VNS) is a method to treat drug-resistant epilepsy and depression, but therapeutic outcomes are often not ideal. Newer electrode designs such as intra-fascicular electrodes offer potential improvements in reducing off-target effects but require a detailed understanding of the fascicular anatomy of the vagus nerve. We have adapted a section-and-image technique, cryo-imaging, with UV excitation to visualize fascicles along the length of the vagus nerve. In addition to offering optical sectioning at the surface via reduced penetration depth, UV illumination also produces sufficient contrast between fascicular structures and connective tissue. Here we demonstrate the utility of this approach in pilot experiments. We imaged fixed, cadaver vagus nerve samples, segmented fascicles, and demonstrated 3D tracking of fascicles. Such data can serve as input for computer models of vagus nerve stimulation.

Plasmon-enhanced photoelectrochemical water splitting with size-controllable gold nanodot arrays.

Size-controllable Au nanodot arrays (50, 63, and 83 nm dot size) with a narrow size distribution (± 5%) were prepared by a direct contact printing method on an indium tin oxide (ITO) substrate. Titania was added to the Au nanodots using TiO(2) sols of 2-3 nm in size. This created a precisely controlled Au nanodot with 110 nm of TiO(2) overcoats. Using these precisely controlled nanodot arrays, the effects of Au nanodot size and TiO(2) overcoats were investigated for photoelectrochemical water splitting using a three-electrode system with a fiber-optic visible light source. From UV-vis measurement, the localized surface plasmon resonance (LSPR) peak energy (ELSPR) increased and the LSPR line width (Γ) decreased with decreasing Au nanodot size. The generated plasmonic enhancement for the photoelectrochemical water splitting reaction increased with decreasing Au particle size. The measured plasmonic enhancement for light on/off experiments was 25 times for the 50 nm Au size and 10 times for the 83 nm Au nanodot size. The activity of each catalyst increased by a factor of 6 when TiO2 was added to the Au nanodots for all the samples. The activity of the catalyst was proportional to the quality factor (defined as Q = E(LSPR)/Γ) of the plasmonic metal nanostructure. The enhanced water splitting performance with the decreased Au nanodot size is probably due to more generated charge carriers (electron/hole pair) by local field enhancement as the quality factor increases.