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Live imaging of experimental animals is now possible thanks to recent technological advances that overcome the limitations of conventional histological analysis. In contrast to conventional histological microscopy techniques, this intravital approach can reveal previously unknown morphogenetic and functional processes in live tissues. In addition, this approach can capture real-time information on these processes, compared with conventional histological microscopy and other techniques that only provide snapshots in time. We used multi-photon laser-scanning microscopy (MPLSM) for in vivo real-time imaging of intra-abdominal organs, and investigated the intravital microscopic changes in various disease-model mice, referred to as ‘dynamic pathology’. For example, we used this technology to examine bacterial translocation in dextran sodium sulfate (DSS)-induced colitis, thrombus formation in laser-induced endothelial injury, neutrophil extracellular traps, the dynamics of circulating free DNA in a model of DSS-induced colitis, and to obtain a comprehensive understanding of the development and blood flow dynamics of the small intestinal microcirculation in a mouse model of necrotizing enterocolitis. This mini review summarizes the in vivo observation methods that we have developed to observe the dynamic pathology in various disease-model mice using MPLSM.

Abstract Background Tobacco ingestion is widely known to cause nicotine toxicity, which may result in severe symptoms. Two heated tobacco sticks, called TEREA™ and SENTIA™, were launched in 2021 by Philip Morris International (New York, NY, USA), and their ingestion is associated with a risk of bowel injury because they contain a partially pointed metallic susceptor. However, this risk is not well known to the general public or healthcare providers. To increase awareness of this risk, we herein report a case involving extraction of a metallic susceptor after ingestion of the heated tobacco stick TEREA™. Case presentation A 7-month-old girl presented to the emergency department of a nearby hospital because she was suspected to have accidentally swallowed heated tobacco. Although she presented with no symptoms related to nicotine poisoning, abdominal X-ray examination revealed a metal object in her stomach. According to a statement released by the Japan Poison Information Center, the TEREA™ heated tobacco stick contains a metallic susceptor with a rectangular shape and sharp corners. The patient was transferred to our department because of the risk of bowel injury, and upper gastrointestinal endoscopy was performed. No cigarettes were found by endoscopic observation; however, a metallic susceptor was located in the second part of the duodenum. We grasped it with biopsy forceps and carefully removed it using an endoscope with a cap attached to the tip. The post-endoscopic course was uneventful. Conclusions Some patients who ingest heated tobacco sticks might be exposed not only to the effects of nicotine but also to physical damage caused by a metallic susceptor. Infants and toddlers especially could swallow these sticks, therefore tobacco companies need to make the problem more public. Clinicians also should alert the problem, and pay attention to this risk in the clinical setting.

Background and Objectives: Anti-vascular endothelial growth factor (anti-VEGF) therapy has become the first-line treatment for diabetic macular edema. However, it is still not clear whether anti-VEGF agents act on systemic blood vessels. The aim of this study is to determine whether a direct topical application or intravitreal injection of anti-VEGF will change the intestinal blood vessels of mice. Materials and Methods: C57BL/6 mice were laparotomied under deep anesthesia, and the blood vessels on the surface of the intestines were exposed, examined, and photographed through a dissecting microscope. Vascular changes were evaluated before and at 1, 5, and 15 min after the topical application of 50 µL of the different anti-VEGF agents onto the surface of the intestine (group S) or after the intravitreal injection (group V). The vascular density (VD) was determined for five mice in each group before and after 40 μg/μL of aflibercept (Af), or 25 μg/μL of bevacizumab (Be), or 10 μg/μL of ranibizumab (Ra) were applied. Endothelin-1 (ET1), a potent vasoconstrictor, was used as a positive control, and phosphate-buffered saline (PBS) was used as a control. Results: For group S, no significant changes were observed after PBS (baseline, 1, 5, and 15 min: 46.3, 44.5, 44.8, and 43.2%), Be (46.1, 46.7, 46.7, and 46.3%), Ra (44.7, 45.0, 44.7, and 45.6%), and Af (46.5, 46.2, 45.9, and 46.1%, repeated ANOVA) were applied topically. Significant decreases in the VD were observed after ET1 (46.7, 28.1, 32.1, and 34.0%, p < 0.05) was topically applied. For group V, no significant differences were observed for all anti-VEGF agents. Conclusions: The topical application or intravitreal injections of anti-VEGF agents do not cause a change in the VD of the intestinal vessels, which may be related to its safety.

Necrotizing enterocolitis (NEC) is one of the most lethal gastrointestinal emergencies in neonates needing precision treatment. Here the authors show that remote ischemic conditioning is a non-invasive therapeutic method that enhances blood flow in the intestine, reduces damage, and improves NEC outcome.

Abstract Background Sarcopenia frequently occurs in metastatic cancer patients. Emerging evidence has revealed that various secretory products from metastatic tumours can influence host organs and promote sarcopenia in patients with malignancies. Furthermore, the biological functions of microRNAs in cell‐to‐cell communication by incorporating into neighbouring or distal cells, which have been gradually elucidated in various diseases, including sarcopenia, have been elucidated. Methods We evaluated psoas muscle mass index (PMI) and intramuscular adipose tissue content (IMAC) using pre‐operative computed tomography imaging in 183 colorectal cancer (CRC) patients. miR‐203 expression levels in CRC tissues and pre‐operative serum were evaluated using quantitative polymerase chain reaction. Functional analysis of miR‐203 overexpression was investigated in human skeletal muscle cells (SkMCs), and cells were analysed for proliferation and apoptosis. Expressions of several putative miR‐203 target genes (CASP3, CASP10, BIRC5, BMI1, BIRC2, and BIRC3) in SKMCs were validated. Results A total of 183 patients (108 men and 75 women) were included. The median age of enrolled patients at diagnosis was 68.0 years (range 35–89 years). High IMAC status significantly correlated with female gender (P = 0.004) and older age (P = 0.0003); however, no other clinicopathological factors correlated with IMAC status in CRC patients. In contrast, decreased PMI significantly correlated with female gender (P = 0.006) and all well‐established disease development factors, including advanced T stage (P = 0.035), presence of venous invasion (P = 0.034), lymphovascular invasion (P = 0.012), lymph node (P = 0.001), distant metastasis (P = 0.002), and advanced Union for International Cancer Control tumour–node–metastasis stage classification (P = 0.0004). Although both high IMAC status and low PMI status significantly correlated with poor overall survival (IMAC: P = 0.0002; PMI: P

Background: Treatment modalities for neonates with congenital diaphragmatic hernia (CDH) have greatly improved in recent years, with a concomitant increase in survival. However, long-term outcomes restrict the identification of optimal care pathways for CDH survivors in adolescence and adulthood. Therefore, we evaluated the long-term outcomes within the Japanese CDH Study Group (JCDHSG). Methods: Participants were born with CDH between 2006 and 2018 according to the JCDHSG. Participants were enrolled in the database at 1.5, 3, 6, and 12 years old. Follow-up items included long-term complications, operations for long-term complication, and home medical care. Results: A total of 747 patients were included in this study, with 626 survivors (83.8%) and 121 non-survivors (16.2%). At 1.5, 3, 6, and 12 years old, 45.4%, 36.5%, 34.8%, and 43.6% developed complications, and 20.1%, 14.7%, 11.5%, and 5.1% of participants required home care, respectively. Recurrence, pneumonia, pneumothorax, gastroesophageal reflux disease, and intestinal obstruction decreased with age, and thoracic deformity increased with age. Conclusions: As CDH survival rates improve, there is a need for continued research and fine-tuning of long-term care to optimize appropriate surveillance and long-term follow-up.

This protocol describes a novel technique to investigate the microcirculation dynamics underlying the pathology in the small intestine of neonatal mice using two-photon laser-scanning microscopy (TPLSM). Recent technological advances in multi-photon microscopy allow intravital analysis of different organs such as the liver, brain and intestine. Despite these advances, live visualization and analysis of the small intestine in neonatal rodents remain technically challenging. We herein provide a detailed description of a novel method to capture high resolution and stable images of the small intestine in neonatal mice as early as postnatal day 0. This imaging technique allows a comprehensive understanding of the development and blood flow dynamics in small intestine microcirculation.

Severe neonatal gastrointestinal diseases such as necrotizing enterocolitis or spontaneous intestinal perforation are potentially lethal conditions which predominantly occur in preterm infants. Cytomegalovirus (CMV), which is known to cause congenital and acquired infections in the newborns, has also been implicated in such severe gastrointestinal diseases in premature infants. However, the pathogenic role of CMV and effect of antiviral therapy in severe gastrointestinal disease in premature neonates is currently unclear. We present an infant, born at 26-weeks' gestation, presented with progressive dyspepsia and abdominal distention after the closure of the symptomatic patent ductus arteriosus at the day of life (DOL) 4, requiring the emergent surgery for ileal perforation at the DOL8. After the surgery, abdominal symptoms persisted and the second emergent surgery was performed for the recurrent ileal perforation at DOL17. Even then the abdominal symptoms prolonged and pathological examination in the affected intestine at the second surgery showed CMV inclusion body. Immunoreactivity for CMV antigen was detected in the specimen at the first surgery on DOL8. Blood and urinary CMV-DNA were detected at DOL28. CMV-DNA was also detected in the dried umbilical cord which was obtained within a week from birth. A 6-week course of intravenous ganciclovir (12 mg/kg/day) was started at DOL34 and then symptoms resolved along with decreasing blood CMV-DNA. Pathological findings characteristic of CMV were not detected in the resection specimen at the ileostomy closure at DOL94. These observations indicate that anti-CMV therapy may be beneficial for some premature infants with severe CMV-associated gastrointestinal diseases and warrants further studies focusing on pathogenic role, diagnosis, treatment and prevention of this underrecognized etiology of severe gastrointestinal diseases particularly in premature neonates.

Neutrophil extracellular traps (NETs) are known to promote metastasis in mouse models. Here the authors show plasma redox imbalance caused by albumin oxidation to induce inflammation-independent NETosis and lung metastasis, and albumin oxidation and reduced plasma free thiol to be associated with lung metastasis in a cohort of head and neck cancer patients.

Major risk factors for necrotizing enterocolitis (NEC) are formula feeding and prematurity; however, their pathogenic mechanisms are unknown. Here, we found that insufficient arginine/nitric oxide synthesis limits blood flow in the intestinal microvasculature, leading to hypoxia, mucosal damage and NEC in the premature intestine after formula feeding. Formula feeding led to increased intestinal hypoxia in pups at postnatal day (P)1 and P5, but not in more mature pups at P9. Accordingly, blood flow in the intestinal microvasculature increased after formula feeding in P9 pups only. mRNA profiling revealed that regulators of arginine/nitric oxide synthesis are at higher levels in endothelial cells of the intestine in P9 than in P1 pups. Importantly, arginine supplementation increased intestinal microvasculature blood flow and prevented NEC, whereas an arginine antagonist exacerbated NEC. Our results suggest that balancing intestinal oxygen demand and supply in the premature intestine by modulating arginine/nitric oxide could be used to prevent NEC. This article has an associated First Person interview with the first author of the paper.

Abstract Background Prebiotics are non-digestible food ingredients that enhance the growth of certain microbes within the gut microbiota. Prebiotic consumption generates immune-modulatory effects that are traditionally thought to reflect microbial interactions within the gut. However, recent evidence suggests they may also impart direct microbe-independent effects on the host, though the mechanisms of which are currently unclear. Methods Kinome arrays were used to profile the host intestinal signaling responses to prebiotic exposures in the absence of microbes. Identified pathways were functionally validated in Caco-2Bbe1 intestinal cell line and in vivo model of murine endotoxemia. Results We found that prebiotics directly regulate host mucosal signaling to alter response to bacterial infection. Intestinal epithelial cells (IECs) exposed to prebiotics are hyporesponsive to pathogen-induced mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) activations, and have a kinome profile distinct from non-treated cells pertaining to multiple innate immune signaling pathways. Consistent with this finding, mice orally gavaged with prebiotics showed dampened inflammatory response to lipopolysaccharide (LPS) without alterations in the gut microbiota. Conclusions These findings provide molecular mechanisms of direct host-prebiotic interactions to support prebiotics as potent modulators of host inflammation.

Necrotizing enterocolitis (NEC) is characterized by intestinal injury and impaired mucin synthesis. We recently showed that breast milk exosomes from rodents promote intestinal cell viability, epithelial proliferation, and stem cell activity, but whether they also affect mucus production is unknown. Therefore, the aim of this study was to investigate the effects of bovine milk-derived exosomes on goblet cell expression in experimental NEC and delineate potential underlying mechanisms of action. Exosomes were isolated from bovine milk by ultracentrifugation and confirmed by Nanoparticle Tracking Analysis and through the detection of exosome membrane markers. To study the effect on mucin production, human colonic LS174T cells were cultured and exposed to exosomes. Compared to control, exosomes promoted goblet cell expression, as demonstrated by increased mucin production and relative expression levels of goblet cell expression markers trefoil factor 3 (TFF3) and mucin 2 (MUC2). In addition, exosome treatment enhanced the expression of glucose-regulated protein 94 (GRP94), the most abundant intraluminal endoplasmic reticulum (ER) chaperone protein that aids in protein synthesis. Furthermore, experimental NEC was induced in mouse pups by hyperosmolar formula feeding, lipopolysaccharide administration and hypoxia exposure on postnatal days 5-9. Milk exosomes were given with each gavage feed. NEC was associated with ileal morphological injury and reduction in MUC2+ goblet cells and GRP94+ cells per villus. Exosome administration to NEC pups prevented these changes. This research highlights the potential novel application of milk-derived exosomes in preventing the development of NEC in high-risk infants when breast milk is not available.

We report the case of an 8-year-old boy with a 30-mm solid mass in the right lower quadrant of the abdominal wall. Computed tomography revealed that the tumor was on the lateral border of the rectus abdominis, and surgical resection was performed. Despite difficulty in differentiating this mass from nodular fasciitis, pathologic analysis and immunohistochemical staining led to the diagnosis of leiomyosarcoma.

Background/Aims. The aim of this study is to clarify the differences of CCL20 and CCR6 expression, chemokine correlated to intestinal homeostasis, between pediatric and adult ulcerative colitis (UC) patients. Methods. Onehundred forty-one patients who underwent proctocolectomy were divided to two groups including childhood-onset UC (CUC,

Neutrophil extracellular traps (NETs) represent extracellular microbial trapping and killing. Recently, it has been implicated in thrombogenesis, autoimmune disease, and cancer progression. The aim of this study was to characterize NETs in various organs of a murine sepsis model in vivo and to investigate their associations with platelets, leukocytes, or vascular endothelium. NETs were classified as two distinct forms; cell-free NETs that were released away from neutrophils and anchored NETs that were anchored to neutrophils. Circulating cell-free NETs were characterized as fragmented or cotton-like structures, while anchored NETs were characterized as linear, reticular, membranous, or spot-like structures. In septic mice, both anchored and cell-free NETs were significantly increased in postcapillary venules of the cecum and hepatic sinusoids with increased leukocyte-endothelial interactions. NETs were also observed in both alveolar space and pulmonary capillaries of the lung. The interactions of NETs with platelet aggregates, leukocyte-platelet aggregates or vascular endothelium of arterioles and venules were observed in the microcirculation of septic mice. Microvessel occlusions which may be caused by platelet aggregates or leukocyte-platelet aggregates and heterogeneously decreased blood flow were also observed in septic mice. NETs appeared to be associated with the formation of platelet aggregates or leukocyte-platelet aggregates. These observational findings may suggest the adverse effect of intravascular NETs on the host during a sepsis.