A novel statistical methodology for quantifying the spatial arrangements of axons in peripheral nerves.

A thorough understanding of the neuroanatomy of peripheral nerves is required for a better insight into their function and the development of neuromodulation tools and strategies. In biophysical modeling, it is commonly assumed that the complex spatial arrangement of myelinated and unmyelinated axons in peripheral nerves is random, however, in reality the axonal organization is inhomogeneous and anisotropic. Present quantitative neuroanatomy methods analyze peripheral nerves in terms of the number of axons and the morphometric characteristics of the axons, such as area and diameter. In this study, we employed spatial statistics and point process models to describe the spatial arrangement of axons and Sinkhorn distances to compute the similarities between these arrangements (in terms of first- and second-order statistics) in various vagus and pelvic nerve cross-sections. We utilized high-resolution transmission electron microscopy (TEM) images that have been segmented using a custom-built high-throughput deep learning system based on a highly modified U-Net architecture. Our findings show a novel and innovative approach to quantifying similarities between spatial point patterns using metrics derived from the solution to the optimal transport problem. We also present a generalizable pipeline for quantitative analysis of peripheral nerve architecture. Our data demonstrate differences between male- and female-originating samples and similarities between the pelvic and abdominal vagus nerves.

High-throughput segmentation of unmyelinated axons by deep learning.

Axonal characterizations of connectomes in healthy and disease phenotypes are surprisingly incomplete and biased because unmyelinated axons, the most prevalent type of fibers in the nervous system, have largely been ignored as their quantitative assessment quickly becomes unmanageable as the number of axons increases. Herein, we introduce the first prototype of a high-throughput processing pipeline for automated segmentation of unmyelinated fibers. Our team has used transmission electron microscopy images of vagus and pelvic nerves in rats. All unmyelinated axons in these images are individually annotated and used as labeled data to train and validate a deep instance segmentation network. We investigate the effect of different training strategies on the overall segmentation accuracy of the network. We extensively validate the segmentation algorithm as a stand-alone segmentation tool as well as in an expert-in-the-loop hybrid segmentation setting with preliminary, albeit remarkably encouraging results. Our algorithm achieves an instance-level [Formula: see text] score of between 0.7 and 0.9 on various test images in the stand-alone mode and reduces expert annotation labor by 80% in the hybrid setting. We hope that this new high-throughput segmentation pipeline will enable quick and accurate characterization of unmyelinated fibers at scale and become instrumental in significantly advancing our understanding of connectomes in both the peripheral and the central nervous systems.

Automatic assessment of human gastric motility and emptying from dynamic 3D magnetic resonance imaging.

BACKGROUND: Time-sequenced magnetic resonance imaging (MRI) of the stomach is an emerging technique for non-invasive assessment of gastric emptying and motility. However, an automated and systematic image processing pipeline for analyzing dynamic 3D (ie, 4D) gastric MRI data has not been established. This study uses an MRI protocol for imaging the stomach with high spatiotemporal resolution and provides a pipeline for assessing gastric emptying and motility. METHODS: Diet contrast-enhanced MRI images were acquired from seventeen healthy humans after they consumed a naturalistic contrast meal. An automated image processing pipeline was developed to correct for respiratory motion, to segment and compartmentalize the lumen-enhanced stomach, to quantify total gastric and compartmental emptying, and to compute and visualize gastric motility on the luminal surface of the stomach. KEY RESULTS: The gastric segmentation reached an accuracy of 91.10 ± 0.43% with the Type-I error and Type-II error being 0.11 ± 0.01% and 0.22 ± 0.01%, respectively. Gastric volume decreased 34.64 ± 2.8% over 1 h where the emptying followed a linear-exponential pattern. The gastric motility showed peristaltic patterns with a median = 4 wave fronts (range 3-6) and a mean frequency of 3.09 ± 0.07 cycles per minute. Further, the contractile amplitude was stronger in the antrum than in the corpus (antrum vs. corpus: 5.18 ± 0.24 vs. 3.30 ± 0.16 mm; p < 0.001). CONCLUSIONS & INFERENCES: Our analysis pipeline can process dynamic 3D MRI images and produce personalized profiles of gastric motility and emptying. It will facilitate the application of MRI for monitoring gastric dynamics in research and clinical settings.

Human organ donor-derived vagus nerve biopsies allow for well-preserved ultrastructure and high-resolution mapping of myelinated and unmyelinated fibers.

The vagus nerve provides motor, sensory, and autonomic innervation of multiple organs, and electrical vagus nerve stimulation (VNS) provides an adjunctive treatment option for e.g. medication-refractory epilepsy and treatment-resistant depression. The mechanisms of action for VNS are not known, and high-resolution anatomical mapping of the human vagus nerve is needed to better understand its functional organization. Electron microscopy (EM) is required for the detection of both myelinated and unmyelinated axons, but access to well-preserved human vagus nerves for ultrastructural studies is sparse. Intact human vagus nerve samples were procured intra-operatively from deceased organ donors, and tissues were immediately immersion fixed and processed for EM. Ultrastructural studies of cervical and sub-diaphragmatic vagus nerve segments showed excellent preservation of the lamellated wall of myelin sheaths, and the axolemma of myelinated and unmyelinated fibers were intact. Microtubules, neurofilaments, and mitochondria were readily identified in the axoplasm, and the ultrastructural integrity of Schwann cell nuclei, Remak bundles, and basal lamina was also well preserved. Digital segmentation of myelinated and unmyelinated axons allowed for determination of fiber size and myelination. We propose a novel source of human vagus nerve tissues for detailed ultrastructural studies and mapping to support efforts to refine neuromodulation strategies, including VNS.

Stomach serosal arteries distinguish gastric regions of the rat.

Because the stomach in situ has few distinctive surface features and changes shape dramatically with food intake, we have used micro-CT imaging combined with two distinct contrast agents to (1) characterize the pattern of arteries, potential landmarks, on the stomach wall and (2) evaluate how meal-related shape changes affect the size of the different regions. Images generated with a contrast agent injected directly into the heart during perfusion enabled a thorough look at the organizational features of the stomach angioarchitecture. The stomach receives its blood supply primarily from two pairs of vessels, the gastric and gastroepiploic arteries. Each of the three regions of the stomach is delineated by a distinctive combination of arterial fields: the corpus, consistent with its dynamic secretory activity and extensive mucosa, is supplied by extensive arterial trees formed by the left and right gastric arteries, travelling, respectively, on the ventral and dorsal stomach surfaces. These major arteries course circularly from the lesser towards the greater curvature, distally along both left (or ventral) and right (or dorsal) walls of the corpus, and branch rostrally to supply the region. The muscular antrum is characterized by smaller arterial branches arising primarily from the right gastroepiploic artery that follows the distal greater curvature and secondarily from small, distally directed arteries supplied by the large vessels of the left and right gastric arteries. The forestomach, essentially devoid of mucosal tissue and separated from the corpus by the limiting ridge, is vascularized predominantly by a network of small arteries issued from the left gastroepiploic artery coursing around the proximal greater curvature, as well as from higher order and smaller branches issued by the gastric and celiac arteries. These distinctive arterial fields appear to distinguish the major gastric regions, irrespective of the degree of fill of the stomach. Volume assessments of stomach compartments were made from images of iodine-stained stomachs. By varying the delay time between eating and perfusion, we were able to probe the emptying behavior of the stomach and demonstrate that the regions of the stomach empty at different rates, thus changing the relative dimensions of the organ regions. Notably, and despite these shape changes, the gastric arteries appear to form a regular, particularly recognizable, and lateralized pattern corresponding to the corpus that should be of use in guiding surgical and experimental interventions.

Stomach region stimulated determines effects on duodenal motility in rats.

Gastric electrical stimulation (GES) is used clinically to promote proximal GI emptying and motility. In acute experiments, we measured duodenal motor responses elicited by GES applied at 141 randomly chosen electrode sites on the stomach serosal surface. Overnight-fasted (H2O available) anesthetized male rats (n = 81) received intermittent biphasic GES for 5 min (20-s-on/40-s-off cycles; I = 0.3 mA; pw = 0.2 ms; 10 Hz). A strain gauge on the serosal surface of the proximal duodenum of each animal was used to evaluate baseline motor activity and the effect of GES. Using ratios of time blocks compared with a 15-min prestimulation baseline, we evaluated the effects of the 5-min stimulation on concurrent activity, on the 10 min immediately after the stimulation, and on the 15-min period beginning with the onset of stimulation. We mapped the magnitude of the duodenal response (three different motility indices) elicited from the 141 stomach sites. Post hoc electrode site maps associated with duodenal responses suggested three zones similar to the classic regions of forestomach, corpus, and antrum. Maximal excitatory duodenal motor responses were elicited from forestomach sites, whereas inhibitory responses occurred with stimulation of the corpus. Moderate excitatory duodenal responses occurred with stimulation of the antrum. Complex, weak inhibitory/excitatory responses were produced by stimulation at boundaries between stomach regions. Patterns of GES efficacies coincided with distributions of previously mapped vagal afferents, suggesting that excitation of the duodenum is strongest when GES electrodes are situated over stomach concentrations of vagal intramuscular arrays, putative stretch receptors in the muscle wall.

The identification of neuronal control pathways supplying effector tissues in the stomach.

The stomach acts as a buffer between the ingestion of food and its processing in the small intestine. It signals to the brain to modulate food intake and it in turn regulates the passage of a nutrient-rich fluid, containing partly digested food, into the duodenum. These processes need to be finely controlled, for example to restrict reflux into the esophagus and to transfer digesta to the duodenum at an appropriate rate. Thus, the efferent pathways that control gastric volume, gastric peristalsis and digestive juice production are critically important. We review these pathways with an emphasis on the identities of the final motor neurons and comparisons between species. The major types of motor neurons arising from gastric enteric ganglia are as follows: immunohistochemically distinguishable excitatory and inhibitory muscle motor neurons; four neuron types innervating mucosal effectors (parietal cells, chief cells, gastrin cells and somatostatin cells); and vasodilator neurons. Sympathetic efferent neurons innervate intramural arteries, myenteric ganglia and gastric muscle. Vagal efferent neurons with cell bodies in the brain stem do not directly innervate gastric effector tissues; they are pre-enteric neurons that innervate each type of gastric enteric motor neuron. The principal transmitters and co-transmitters of gastric motor neurons, as well as key immunohistochemical markers, are the same in rat, pig, human and other species.

Vagal innervation of the stomach reassessed: brain-gut connectome uses smart terminals.

Brain-gut neural communications have long been considered limited because of conspicuous numerical mismatches. The vagus, the parasympathetic nerve connecting brain and gut, contains thousands of axons, whereas the gastrointestinal (GI) tract contains millions of intrinsic neurons in local plexuses. The numerical paradox was initially recognized in terms of efferent projections, but the number of afferents, which comprise the majority (≈ 80%) of neurites in the vagus, is also relatively small. The present survey of recent morphological observations suggests that vagal terminals, and more generally autonomic and visceral afferent arbors in the stomach as well as throughout the gut, elaborate arbors that are extensive, regionally specialized, polymorphic, polytopic, and polymodal, commonly with multiplicities of receptors and binding sites-smart terminals. The morphological specializations and dynamic tuning of one-to-many efferent projections and many-to-one convergences of contacts onto afferents create a complex architecture capable of extensive peripheral integration in the brain-gut connectome and offset many of the disparities between axon and target numbers. Appreciating this complex architecture can help in the design of therapies for GI disorders.

Gastric stimulation drives fast BOLD responses of neural origin.

Functional magnetic resonance imaging (fMRI) is commonly thought to be too slow to capture any neural dynamics faster than 0.1 Hz. However, recent findings demonstrate the feasibility of detecting fMRI activity at higher frequencies beyond 0.2 Hz. The origin, reliability, and generalizability of fast fMRI responses are still under debate and await confirmation through animal experiments with fMRI and invasive electrophysiology. Here, we acquired single-echo and multi-echo fMRI, as well as local field potentials, from anesthetized rat brains given gastric electrical stimulation modulated at 0.2, 0.4 and 0.8 Hz. Such gastric stimuli could drive widespread fMRI responses at corresponding frequencies from the somatosensory and cingulate cortices. Such fast fMRI responses were linearly dependent on echo times and thus indicative of blood oxygenation level dependent nature (BOLD). Local field potentials recorded during the same gastric stimuli revealed transient and phase-locked broadband neural responses, preceding the fMRI responses by as short as 0.5 s. Taken together, these results suggest that gastric stimulation can drive widespread and rapid fMRI responses of BOLD and neural origin, lending support to the feasibility of using fMRI to detect rapid changes in neural activity up to 0.8 Hz under visceral stimulation.