Modeling gut neuro-epithelial connections in a novel microfluidic device.

The intestinal lumen is filled with diverse chemical and physical stimuli. Intestinal epithelial cells sense these stimuli and signal to enteric neurons which coordinate a range of physiologic processes required for normal digestive tract function. Yet, the neuro-epithelial connections remain poorly resolved, in part because the tools for orchestrating interactions between these cellular compartments are lacking. We describe the development of a two-compartment microfluidic device for co-culturing enteric neurons with intestinal epithelial cells. The device contains epithelial and neuronal compartments connected by microgrooves. The epithelial compartment was designed for cell seeding via injection and confinement of intestinal epithelial cells derived from human intestinal organoids. We demonstrated that organoids planarized effectively and retained epithelial phenotype for over a week. In the second chamber we dissociated and cultured intestinal myenteric neurons including intrinsic primary afferent neurons (IPANs) from transgenic mice that expressed the fluorescent protein tdTomato. IPANs extended projections into microgrooves, surrounded and frequently made contacts with epithelial cells. The density and directionality of neuronal projections were enhanced by the presence of epithelial cells in the adjacent compartment. Our microfluidic device represents a platform that may, in the future, be used to dissect structure and function of neuro-epithelial connections in the gut and other organs (skin, lung, bladder, and others) in health and disease.

Modeling Gut Neuro-Epithelial Connections in a Novel Micro uidic Device.

Organs that face external environments, such as skin and gut, are lined by epithelia, which have two functions - to provide a semi-permeable barrier and to sense stimuli. The intestinal lumen is filled with diverse chemical and physical stimuli. Intestinal epithelial cells sense these stimuli and signal to enteric neurons which coordinate a range of physiologic processes required for normal digestive tract function. Yet, the neuro-epithelial connections between intestinal epithelial cells and enteric neurons remain poorly resolved, which leaves us with limited mechanistic understanding of their function. We describe the development of a two-compartment microfluidic device for modeling neuro-epithelial interactions, and apply it to form the gut's neuro-epithelial connections. The device contains epithelial and neuronal compartments connected by microgrooves. The epithelial compartment was designed for cell seeding via injection and confinement of intestinal epithelial cells derived from human intestinal organoids. We demonstrated that organoids planarized effectively and retained epithelial phenotype for over a week. In the second chamber we dissociated and cultured intestinal myenteric neurons including intrinsic primary afferent neurons (IPANs) from transgenic mice that expressed the fluorescent protein tdTomato. IPANs extended projections into microgrooves, surrounded and frequently made contacts with epithelial cells. The density and directionality of neuronal projections were enhanced by the presence of epithelial cells in the adjacent compartment. Our microfluidic device represents a platform for dissecting structure and function of neuro-epithelial connections in the gut and other organs (skin, lung, bladder, and others) in health and disease.

Intestinal enteroendocrine cells rely on ryanodine and IP3 calcium store receptors for mechanotransduction.

Enteroendocrine cells (EECs) are specialized sensors of luminal forces and chemicals in the gastrointestinal (GI) epithelium that respond to stimulation with a release of signalling molecules such as serotonin (5-HT). For mechanosensitive EECs, force activates Piezo2 channels, which generate a very rapidly activating and inactivating (∼10 ms) cationic (Na+ , K+ , Ca2+ ) receptor current. Piezo2 receptor currents lead to a large and persistent increase in intracellular calcium (Ca2+ ) that lasts many seconds to sometimes minutes, suggesting signal amplification. However, intracellular calcium dynamics in EEC mechanotransduction remain poorly understood. The aim of this study was to determine the role of Ca2+ stores in EEC mechanotransduction. Mechanical stimulation of a human EEC cell model (QGP-1) resulted in a rapid increase in cytoplasmic Ca2+ and a slower decrease in ER stores Ca2+ , suggesting the involvement of intracellular Ca2+ stores. Comparing murine primary colonic EECs with colonocytes showed expression of intercellular Ca2+ store receptors, a similar expression of IP3 receptors, but a >30-fold enriched expression of Ryr3 in EECs. In mechanically stimulated primary EECs, Ca2+ responses decreased dramatically by emptying stores and pharmacologically blocking IP3 and RyR1/3 receptors. RyR3 genetic knockdown by siRNA led to a significant decrease in mechanosensitive Ca2+ responses and 5-HT release. In tissue, pressure-induced increase in the Ussing short circuit current was significantly decreased by ryanodine receptor blockade. Our data show that mechanosensitive EECs use intracellular Ca2+ stores to amplify mechanically induced Ca2+ entry, with RyR3 receptors selectively expressed in EECs and involved in Ca2+ signalling, 5-HT release and epithelial secretion. KEY POINTS: A population of enteroendocrine cells (EECs) are specialized mechanosensors of the gastrointestinal (GI) epithelium that respond to mechanical stimulation with the release of important signalling molecules such as serotonin. Mechanical activation of these EECs leads to an increase in intracellular calcium (Ca2+ ) with a longer duration than the stimulus, suggesting intracellular Ca2+ signal amplification. In this study, we profiled the expression of intracellular Ca2+ store receptors and found an enriched expression of the intracellular Ca2+ receptor Ryr3, which contributed to the mechanically evoked increases in intracellular calcium, 5-HT release and epithelial secretion. Our data suggest that mechanosensitive EECs rely on intracellular Ca2+ stores and are selective in their use of Ryr3 for amplification of intracellular Ca2+ . This work advances our understanding of EEC mechanotransduction and may provide novel diagnostic and therapeutic targets for GI motility disorders.

Mechano-Sensing Channel PIEZO2 Enhances Invasive Phenotype in Triple-Negative Breast Cancer.

BACKGROUND: Mechanically gated PIEZO channels lead to an influx of cations, activation of additional Ca2+ channels, and cell depolarization. This study aimed to investigate PIEZO2's role in breast cancer. METHODS: The clinical relevance of PIEZO2 expression in breast cancer patient was analyzed in a publicly available dataset. Utilizing PIEZO2 overexpressed breast cancer cells, and in vitro and in vivo experiments were conducted. RESULTS: High expression of PIEZO2 was correlated with a worse survival in triple-negative breast cancer (TNBC) but not in other subtypes. Increased PEIZO2 channel function was confirmed in PIEZO2 overexpressed cells after mechanical stimulation. PIEZO2 overexpressed cells showed increased motility and invasive phenotypes as well as higher expression of SNAIL and Vimentin and lower expression of E-cadherin in TNBC cells. Correspondingly, high expression of PIEZO2 was correlated with the increased expression of epithelial-mesenchymal transition (EMT)-related genes in a TNBC patient. Activated Akt signaling was observed in PIEZO2 overexpressed TNBC cells. PIEZO2 overexpressed MDA-MB-231 cells formed a significantly higher number of lung metastases after orthotopic implantation. CONCLUSION: PIEZO2 activation led to enhanced SNAIL stabilization through Akt activation. It enhanced Vimentin and repressed E-cadherin transcription, resulting in increased metastatic potential and poor clinical outcomes in TNBC patients.

Quantitative Mobility Analysis of the Face and its Relevance for Surgical and Non-surgical Aesthetic Facial Procedures.

BACKGROUND: Understanding the degree of facial mobility upon postural changes is of great clinical relevance especially if facial assessment, facial measurements and/or facial markings are done in an upright position, but facial procedures are performed in a supine position. OBJECTIVE: The objective of this study is to investigate regional facial skin displacement and facial volume changes in individuals between upright and supine positions. METHODS: This multi-center study analyzed a total of 175 study participants with a mean age of 35.0 (10.2) years and a mean body mass index of 24.71 (3.5) kg/m2. 3D surface scanning technology with automated registration and alignment was utilized, and multivariate analyses were performed with adjustment for age, gender, body mass index, facial skin sagging and laxity. RESULTS: The medial face displaced less than the lateral face in both cranial (0.88 mm) and in lateral (0.76 mm) directions, and the lower face displaced more than the middle face in both cranial (1.17 mm) and lateral directions (1.37 mm). Additionally, the medial face lost, on average, 3.00cc whereas the lateral face increased by 5.86cc in volume; the middle face increased by 2.95cc, whereas the lower face decreased by 0.98cc in volume. All p < 0.001. CONCLUSION: Practitioners should be mindful that there is a statistically significant change in facial soft tissues between the upright and supine positions and that the magnitude of the change does not necessarily reflect on the aging process alone but is a multi-factorial process which should be individualized for each patient's needs. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Ultrasound Anatomy of the Dorsal Nasal Artery as it Relates to Liquid Rhinoplasty Procedures.

Nonsurgical rhinoplasty procedures using soft tissue fillers have gained popularity. With the increasing frequency of such procedures, the incidence of intra-arterial injection of soft tissue filler material and subsequent ischemia has also risen. This article analyzes the topographic anatomy of the dorsal nasal artery in the nasal soft tissue to potentially enhance patient safety in nonsurgical rhinoplasty procedures. The dorsal nasal artery shows a variable topographic course, especially in relationship to the procerus muscle. By understanding the topographic courses of the dorsal nasal artery, aesthetic practitioners may be able to perform nonsurgical rhinoplasty procedures with increased safety and efficacy.

Studying Murine Small Bowel Mechanosensing of Luminal Particulates.

Gastrointestinal (GI) motility is critical for normal digestion and absorption. In the small bowel, which absorbs nutrients, motility optimizes digestion and absorption. For this reason, some of the motility patterns in the small bowel include segmentation for mixing of luminal contents and peristalsis for their propulsion. Physical properties of luminal contents modulate the patterns of small bowel motility. The mechanical stimulation of GI mechanosensory circuits by transiting luminal contents and underlying gut motility initiate and modulate complex GI motor patterns. Yet, the mechanosensory mechanisms that drive this process remain poorly understood. This is primarily due to a lack of tools to dissect how the small bowel handles materials of different physical properties. To study how the small bowel handles particulates of varying sizes, we have modified an established in vivo method to determine small bowel transit. We gavage live mice with fluorescent liquid or tiny fluorescent beads. After 30 minutes, we dissect out the bowels to image the distribution of fluorescent contents across the entirety of the GI tract. In addition to high-resolution measurements of the geometric center, we use variable size binning and spectral analysis to determine how different materials affect small bowel transit. We have explored how a recently discovered "gut touch" mechanism affects small bowel motility using this approach.

The subjectively perceived injectability as an early indicator for adverse events?

BACKGROUND: With an increasing demand of aesthetic soft-tissue filler treatments, the occurrence of adverse events rises likewise. An optimized injection algorithm adapted to product characteristics (eg, rheology) of the soft-tissue filler is crucial in order to ensure satisfying clinical outcomes and high patient safety. OBJECTIVE: To identify a subjective feedback mechanism for the avoidance of adverse events after soft-tissue filler injection procedures. METHODS: A retrospective data analysis of n = 387 aesthetic treatments performed on n = 291 patients (4 males, 287 females) with different soft-tissue fillers with regard to loss of volume (filling effect), injected layer, injectability ("ease of injection"), injected volume, and injection technique was conducted. RESULTS: The subjectively perceived injectability during the injection process was statistically significantly related to G-Prime value with rs = 0.101 with p = 0.048, indicating an increased difficulty while injecting products with higher G-Prime. The occurrence of adverse events was also statistically significantly related to the injectability: injections with increased subjectively perceived difficulty showed increasing odds of developing adverse events by OR 0.157 with p = 0.002. CONCLUSION: Injections that were subjectively more difficult to perform are more likely to develop adverse events. Respecting the layered arrangement of the face, the recommended and approved depth and facial region for each specific treatment enable practitioners to achieve satisfying outcomes while keeping the rate of adverse events low.

Gut feelings: mechanosensing in the gastrointestinal tract.

The primary function of the gut is to procure nutrients. Synchronized mechanical activities underlie nearly all its endeavours. Coordination of mechanical activities depends on sensing of the mechanical forces, in a process called mechanosensation. The gut has a range of mechanosensory cells. They function either as specialized mechanoreceptors, which convert mechanical stimuli into coordinated physiological responses at the organ level, or as non-specialized mechanosensory cells that adjust their function based on the mechanical state of their environment. All major cell types in the gastrointestinal tract contain subpopulations that act as specialized mechanoreceptors: epithelia, smooth muscle, neurons, immune cells, and others. These cells are tuned to the physical properties of the surrounding tissue, so they can discriminate mechanical stimuli from the baseline mechanical state. The importance of gastrointestinal mechanosensation has long been recognized, but the latest discoveries of molecular identities of mechanosensors and technical advances that resolve the relevant circuitry have poised the field to make important intellectual leaps. This Review describes the mechanical factors relevant for normal function, as well as the molecules, cells and circuits involved in gastrointestinal mechanosensing. It concludes by outlining important unanswered questions in gastrointestinal mechanosensing.

Specialized Mechanosensory Epithelial Cells in Mouse Gut Intrinsic Tactile Sensitivity.

BACKGROUND AND AIMS: The gastrointestinal (GI) tract extracts nutrients from ingested meals while protecting the organism from infectious agents frequently present in meals. Consequently, most animals conduct the entire digestive process within the GI tract while keeping the luminal contents entirely outside the body, separated by the tightly sealed GI epithelium. Therefore, like the skin and oral cavity, the GI tract must sense the chemical and physical properties of the its external interface to optimize its function. Specialized sensory enteroendocrine cells (EECs) in GI epithelium interact intimately with luminal contents. A subpopulation of EECs express the mechanically gated ion channel Piezo2 and are developmentally and functionally like the skin's touch sensor- the Merkel cell. We hypothesized that Piezo2+ EECs endow the gut with intrinsic tactile sensitivity. METHODS: We generated transgenic mouse models with optogenetic activators in EECs and Piezo2 conditional knockouts. We used a range of reference standard and novel techniques from single cells to living animals, including single-cell RNA sequencing and opto-electrophysiology, opto-organ baths with luminal shear forces, and in vivo studies that assayed GI transit while manipulating the physical properties of luminal contents. RESULTS: Piezo2+ EECs have transcriptomic features of synaptically connected, mechanosensory epithelial cells. EEC activation by optogenetics and forces led to Piezo2-dependent alterations in colonic propagating contractions driven by intrinsic circuitry, with Piezo2+ EECs detecting the small luminal forces and physical properties of the luminal contents to regulate transit times in the small and large bowel. CONCLUSIONS: The GI tract has intrinsic tactile sensitivity that depends on Piezo2+ EECs and allows it to detect luminal forces and physical properties of luminal contents to modulate physiology.

Safety and efficacy of microneedling technology in the treatment of acne scars.

BACKGROUND: Current options for the reduction of acne scarring (eg, ablative laser resurfacing) are associated with considerable side effects and limitations in terms of patient population. Percutaneous collagen induction via microneedling poses an alternative treatment method due to its low rates of reported adverse events and side effects. OBJECTIVE: To assess the safety and effectiveness of microneedling treatments in reducing acne scars. METHODS: A total of 22 patients (18 females and 4 males) with a mean age of 38 ± 7.6 years were assessed regarding the appearance of facial acne scarring. Acne scars were assessed via the Acne Scar Assessment Scale (ASAS) and the Goodman and Baron acne scar grading scale before and after two/three treatments. Additionally, the post-interventional development of side reactions, adverse events, and patient-reported outcomes (eg, pain/discomfort, skin redness) was reported. RESULTS: Compared to baseline, the mean ASAS value was improved statistically significantly on average by 1.41 and 1.46 after the second treatment as assessed by the independent raters and the patients, respectively. In patients who received a total of three treatments, a statistically significant mean improvement in ASAS value of 1.35 and 1.66 compared to baseline was assessed by the independent raters and patients, respectively. No unexpected adverse events were reported. The severity and rate of side reactions decreased over the course of this study. CONCLUSION: Microneedling treatments can pose a safe and effective option in the reduction of acne scarring. In this study, microneedling helped achieving a significant reduction of acne scars while showing high patient safety.

Understanding Facial Muscle Aging: A Surface Electromyography Study.

BACKGROUND: Facial aging is a multifactorial process that involves all tissues of the face, including skin, muscles, fat, ligaments, and bone. Whereas robust evidence is available for age-related changes of bone and facial fat, the influence of age on facial muscle activity is poorly understood. OBJECTIVES: The objective of this study was to investigate the motor unit action potential of facial muscles by utilizing surface-derived, noninvasive electromyography in young and old healthy volunteers. METHODS: The study investigated a total of 32 healthy volunteers with a mean [standard deviation] age of 42.6 [19.6] years (range, 21-82 years) and a mean BMI of 23.9 [2.7] kg/m2 (range, 18.5-29.7 kg/m2) by performing surface-derived, noninvasive facial electromyography. Nine facial muscles were investigated bilaterally, resulting in a total of 1632 measurements of the signal, baseline noise, and signal-to-noise ratio of these muscles. RESULTS: The results of the study revealed that age does not significantly influence the signal (P = 0.234), the baseline noise (P = 0.225), or the signal-to-noise ratio (P = 0.432) of younger individuals (<30 years) vs older individuals (>50 years) in a gender- and BMI-matched statistical model. Exceptions were the zygomaticus major muscle (reduced activity), procerus muscle (increased activity), and corrugator supercilii muscle (increased activity). CONCLUSIONS: The results of this facial electromyography study may help to increase the understanding of facial aging. Future studies need to reproduce the results presented herein to further increase our understanding of facial aging.

SCN5A mutation G615E results in NaV1.5 voltage-gated sodium channels with normal voltage-dependent function yet loss of mechanosensitivity.

SCN5A is expressed in cardiomyocytes and gastrointestinal (GI) smooth muscle cells (SMCs) as the voltage-gated mechanosensitive sodium channel NaV1.5. The influx of Na+ through NaV1.5 produces a fast depolarization in membrane potential, indispensable for electrical excitability in cardiomyocytes and important for electrical slow waves in GI smooth muscle. As such, abnormal NaV1.5 voltage gating or mechanosensitivity may result in channelopathies. SCN5A mutation G615E - found separately in cases of acquired long-QT syndrome, sudden cardiac death, and irritable bowel syndrome - has a relatively minor effect on NaV1.5 voltage gating. The aim of this study was to test whether G615E impacts mechanosensitivity. Mechanosensitivity of wild-type (WT) or G615E-NaV1.5 in HEK-293 cells was examined by shear stress on voltage- or current-clamped whole cells or pressure on macroscopic patches. Unlike WT, voltage-clamped G615E-NaV1.5 showed a loss in shear- and pressure-sensitivity of peak current yet a normal leftward shift in the voltage-dependence of activation. In current-clamp, shear stress led to a significant increase in firing spike frequency with a decrease in firing threshold for WT but not G615E-NaV1.5. Our results show that the G615E mutation leads to functionally abnormal NaV1.5 channels, which cause disruptions in mechanosensitivity and mechano-electrical feedback and suggest a potential contribution to smooth muscle pathophysiology.