Yujiro Hayashi

Spatial "omics" technologies provide unprecedented opportunities to study the biology and interactions of individual cells of the gastric neuromuscular apparatus in their native tissue environment. However, the successful application of these approaches and the assessment of structural and molecular differences across the fundus, corpus, and antrum require consistent sampling and tissue orientation. Here, we report a modified "Swiss roll" technique tailored to the mouse stomach, which allows all major anatomical regions to be embedded and visualized in a single cryosection. This approach emphasizes flat fixation and careful positioning to generate sections with reproducible orientation and quality. This study also optimized immunofluorescence staining conditions to ensure high-quality visualization of cellular markers. This protocol enables comprehensive, side-by-side comparisons of gastric regions while minimizing tissue loss and experimental variability. By enhancing tissue preservation and spatial orientation, this method offers a practical and scalable tool for researchers studying gastric biology in both physiological and pathological contexts.

Spatial "omics" technologies provide unprecedented opportunities to study the biology and interactions of individual cells of the gastric neuromuscular apparatus in their native tissue environment. However, the successful application of these approaches and the assessment of structural and molecular differences across the fundus, corpus, and antrum require consistent sampling and tissue orientation. Here, we report a modified "Swiss roll" technique tailored to the mouse stomach, which allows all major anatomical regions to be embedded and visualized in a single cryosection. This approach emphasizes flat fixation and careful positioning to generate sections with reproducible orientation and quality. This study also optimized immunofluorescence staining conditions to ensure high-quality visualization of cellular markers. This protocol enables comprehensive, side-by-side comparisons of gastric regions while minimizing tissue loss and experimental variability. By enhancing tissue preservation and spatial orientation, this method offers a practical and scalable tool for researchers studying gastric biology in both physiological and pathological contexts.

Aging is linked to a gradual decline in the gastric motor function, contributing to reduced food intake, and its association with frailty and sarcopenia. A key cellular change in the gastric neuromuscular apparatus is the loss of interstitial cells of Cajal (ICC), pacemaker cells of the gut. The ICC function as pacemakers that generate electrical slow waves and mediate enteric neurotransmission, playing a critical role in gastric motility. Aging-related ICC depletion leads to impaired gastric compliance and reduced slow wave activity, which contributes to early satiety and reduced food intake. Recent studies have elucidated the molecular and epigenetic mechanisms underlying aging-related ICC decline, highlighting the roles of ICC stem/precursor cells (ICC-SCs), transformation-related protein 53 (TRP53), extracellular signal-regulated kinase (ERK), and insulin-like growth factor 1 (IGF1) pathways, and epigenetic regulation mediated by the histone methyltransferase enhancer of zeste 2 (EZH2). By synthesizing the current findings, this review aims to provide a comprehensive understanding of the mechanisms driving ICC decline and to explore potential therapeutic strategies for preserving gastric motility in aging populations. Future research should aim to translate these discoveries into clinical applications to improve the gastric motor function and overall health in the aging population. Identifying effective interventions targeting ICC maintenance may ultimately help to alleviate age-related gastric motor dysfunction and its associated health burdens, including frailty, malnutrition, and impaired quality of life.

Understanding the molecular interactions within the neuromuscular apparatus in the stomach is crucial for understanding their role in maintaining interstitial cells, such as the interstitial cells of Cajal (ICC), smooth muscle, and enteric neurons, as well as their contribution to gastric motility in both healthy and diseased states. Disruptions of these systems can lead to various gastric motor disorders and diseases, making it essential to explore their functions in detail. We herein present a protocol for gene knockdown using small interfering RNA (siRNAs) in organotypic culture. This ex vivo approach allows the precise manipulation of the gene expression in a tissue environment that closely mimics in vivo conditions, providing valuable insights into the gene function and its effects on gastric physiology. The protocol includes detailed steps for tissue preparation to ensure the preservation of the gastric muscles and the associated neuromuscular apparatus. We then describe the process of siRNA-mediated gene knockdown, offering tips for optimizing transfection efficiency and gene silencing. Additionally, we outline methods for analyzing the effectiveness of knockdown, including both quantitative and qualitative methods for the evaluation of the target gene expression. This protocol is adaptable to various research needs, allowing researchers to focus on specific genes of interest within the neuromuscular system of the stomach. By applying this approach, investigators can deepen their understanding of the molecular mechanisms underlying gastric motility and contribute to the development of new therapeutic strategies for treating gastric motor disorders and diseases.

Macrophages are the originators of inflammatory compounds, phagocytic purifiers in their local environment, and wound healing protectors in oxidative environments. They are molded by the tissue milieu they inhabit, with gastrointestinal (GI) muscularis macrophages (MMs) being a prime example. MMs are located in the muscular layer of the GI tract and contribute to muscle repair and maintenance of GI motility. MMs are often in close proximity to the enteric nervous system, specifically near the enteric neurons and interstitial cells of Cajal (ICCs). Consequently, the anti-inflammatory function of MMs corresponds to the development and maintenance of neural networks in the GI tract. The capacity of MMs to shift from anti-inflammatory to proinflammatory states may contribute to the inflammatory aspects of various GI diseases and disorders such as diabetic gastroparesis or postoperative ileus, functional disorders such as irritable bowel syndrome, and organic diseases such as inflammatory bowel disease. We reviewed the current knowledge of MMs and their influence on neighboring cells due to their important role in the GI tract.

Neurointestinal diseases result from dysregulated interactions between the nervous system and the gastrointestinal (GI) tract, leading to conditions such as Hirschsprung’s disease and irritable bowel syndrome. These disorders affect many people, significantly diminishing their quality of life and overall health. Central to GI motility are the interstitial cells of Cajal (ICC), which play a key role in muscle contractions and neuromuscular transmission. This review highlights the role of ICC in neurointestinal diseases, revealing their association with various GI ailments. Understanding the functions of the ICC could lead to innovative perspectives on the modulation of GI motility and introduce new therapeutic paradigms. These insights have the potential to enhance efforts to combat neurointestinal diseases and may lead to interventions that could alleviate or even reverse these conditions.