Background: The use of resuscitative endovascular balloon occlusion of the aorta (REBOA) for hemorrhagic shock in the torso has become increasingly common as a bridge to definitive hemostasis. Hydrogen molecules, distributed throughout the bloodstream, alleviate ischemic injury but cannot reach ischemic organs during REBOA use. This study investigates whether intra-aortic irrigation with hydrogen-dissolved saline under REBOA use delivers hydrogen to the intestine in a swine hemorrhagic shock model. Methods: We induced volume-regulated hemorrhagic shock in a 40 kg female swine. Following this, hydrogen-dissolved saline irrigation was initiated through an intra-aortic catheter positioned distal to the REBOA balloon. Hydrogen concentration in the portal vein was determined in four models: controlled hemorrhagic shock with full REBOA inflation during the standard occlusion time, uncontrolled hemorrhagic shock with liver injury and full REBOA inflation during the extended occlusion time, uncontrolled hemorrhagic shock with liver injury and partial REBOA inflation during the extended occlusion time, and as the control model, controlled hemorrhagic shock with full REBOA inflation during the standard occlusion time with normal saline irrigation without hydrogen. Results: Hydrogen concentration in the portal vein was found to be 0.224 mg/L (13.998%) in the controlled hemorrhagic shock model with full REBOA inflation, 0.049 mg/L (3.063%) in the uncontrolled hemorrhagic shock model with liver injury and full REBOA inflation, 0.018 mg/L (1.125%) in the uncontrolled hemorrhagic shock model with liver injury and partial REBOA inflation, and 0.002 mg/L (0.015%) in the control model. These results demonstrate the presence of hydrogen in the portal vein under different REBOA applications. Conclusions: Increased hydrogen concentration in the portal vein indicated that hydrogen was delivered to the intestine. These findings suggest an approach for drug administration during REBOA use. However, further investigations are required to establish its application in clinical settings.

Organoid technologies have emerged as powerful tools in regenerative medicine; however, current organoid technologies are predominantly based on pluripotent stem cell-derived systems, which poses challenges related to complexity, maturation, and clinical translation. In this commentary, we highlight a recent study by Huang et al. describing a tissue-level strategy for direct generation of functional multilineage organoids from human adult adipose tissue using reaggregated micro-fat (RMF). By preserving native tissue architecture and cellular heterogeneity, this approach enables the formation of mesoderm-, endoderm-, and ectoderm-derived organoids without genetic reprogramming or prolonged expansion culture. RMF-derived organoids demonstrate functional properties in vivo, including support of hematopoiesis and restoration of glycemic control in disease models. While further optimization and standardization will be required for clinical translation, this work provides a compelling alternative to conventional pluripotent stem cell-based paradigms and underscores the importance of tissue context in regenerative strategies. The study reframes adipose tissue as an integrated regenerative substrate and broadens the conceptual landscape of organoid generation toward more clinically accessible approaches.

ABSTRACT Objectives This study aimed to investigate the neuroprotective effects of sweet potato petioles and leaves (PL), which are rich in polyphenols with antioxidant and anti‐inflammatory properties, using a gerbil model of transient cerebral ischaemia. We also evaluated the preventive potential of continuous PL intake against ischaemic brain injury. Methods Custom diets, formulated by adding 1% or 10% PL powder to MF standard diet, were provided ad libitum to gerbils after a 5‐min bilateral common carotid artery occlusion for 7 days (post‐ischaemia PL providing group). Diets were also provided from 7 days before to 7 days after the ischaemic episode (pre‐ischaemia PL providing group, total 14 days). The number of viable neurons in the hippocampal CA1 region was assessed on postoperative Day 7. Results The number of viable neurons was significantly higher in both post‐ and pre‐10% PL groups compared with that in the MF group (43.90 ± 3.28 in MF, 66.55 ± 6.60 in post 10% PL and 69.56 ± 3.30 in pre 10% PL; p < 0.01), although no significant differences were observed in the post‐ and pre‐1% PL groups (55.49 ± 3.19 in post 1% PL 47.27 ± 2.61 in pre 1% PL). Moreover, comparison between the pre‐ and post‐10% PL groups indicated that pre‐ischaemic administration resulted in slightly higher number of viable neurons. Conclusion Continuous intake of sweet potato PL may support the prevention and mitigation of neuronal damage in ischaemic events.

ABSTRACT Purpose: To detail a technique to implant a double-lumen catheter to remove anti-pig antibodies. We transplanted fetal pig kidneys into cynomolgus monkeys using a double filtration plasma exchange (DFPP) protocol. Methods: Two approaches for double-lumen catheter insertion in monkeys (3–9.2 kg) were developed. DFPP was performed using hydroxyethyl starch (6%) as a replacement fluid. We transplanted fetal porcine kidneys, administered immunosuppressive agents, and evaluated the grafts. Results: The catheter insertion site was large, with postoperative hemostasis similar to blind subcutaneous puncture. Monkeys tolerated DFPP well, maintaining stable blood pressure. The technique reduced anti-pig antibodies by 67%, though acute rejection was not fully suppressed. Conclusion: A safe technique for double-lumen catheter placement in cynomolgus monkeys was developed, along with a DFPP protocol for reducing anti-pig antibodies.

Background: Hydrogen gas (H2) has been shown to be effective in the treatment of various clinical conditions, from acute illnesses to chronic illnesses. However, its clinical indications and the corresponding appropriate hydrogen delivery methods have yet to be determined. This is due to the fact that the pharmacokinetics and pharmacodynamics of hydrogen in each delivery method have not been experimentally proven. Here, we verified the pharmacokinetics of hydrogen after the infusion of hydrogen-saturated saline. Methods: Hydrogen-saturated saline was prepared and checked for sterility and component specifications. Hydrogen-saturated saline was administered intravenously (125 mL/h) through the left internal jugular vein of pigs, and the blood hydrogen concentration was measured over time. Results: It was confirmed that hydrogen can be safely mixed under pressure into intravenous solutions (pharmaceutical products) without the contamination of foreign substances by using a needle-less vial access cannula. No change in the PH or composition of the solution was observed due to hydrogen filling. The hydrogen concentrations of blood samples collected from the left internal jugular vein 3 cm to the heart from the tip of the infusion line were 6.4 (30 min), 4.7 (60 min), 4.9 (90 min), and 5.3 (120 min) ppb w/w, respectively. The hydrogen concentrations of blood samples collected from the right atrium were 0.7 (30 min), 0.5 (60 min), 0.7 (90 min), and 0.7 (120 min) ppb, respectively. The hydrogen concentration of blood samples collected from the right internal carotid artery were 0.1 (pre), 0.2 (30 min), 0.3 (60 min), 0.0 (90 min), and 0.0 (120 min) ppb w/w, respectively. Conclusions: We confirmed that hydrogen could be safely pressurized and filled into intravenous (pharmaceutical) solution without contamination by foreign objects using a needle-free vial access cannula. When saturated hydrogen saline was dripped intravenously, almost all of the hydrogen was expelled during its passage through the lungs and could not be supplied to the arterial side.

Background/Objectives: Mandarin orange peel (MOP) is recognized for its traditional medicinal properties due to its high flavonoid content. This study aimed to analyze MOP harvested in Japan for specific bioactive compounds and to explore its health applications in dogs, including effects on gut microbiota and cognitive symptoms. Methods: Flavonoid content (particularly hesperidin and nobiletin) of MOP was measured. High-flavonoid MOP was then incorporated into gelatin cubes. In the time–course blood concentration study, experimental beagle dogs received a single oral dose (4 g). For intestinal microbiota analysis and blood biochemical tests, beagle dogs were administered 1 g twice daily. For the Demonstration Test, older dogs (a 14-year-old female Shiba Inu, a 14-year-old female Miniature Dachshund, and a 19-year-old male Miniature Dachshund) were administered 1 g twice daily. Analysis included microbiota profiling via 16S rDNA sequencing and observational assessment of cognitive indicators in a pilot study involving senior dogs with Alzheimer’s disease. Results: The MOP powder contained 9.3% hesperidin in early-ripening varieties (October harvest) and 6.9% in ripe varieties (December harvest). Nobiletin content was 41 mg/100 g (0.041%) and 35 mg/100 g (0.035%) for the early and late harvests, respectively. Administration of MOP-enriched gelatin cubes reduced Fusobacteriaceae and increased Eggerthellaceae. Cognitive symptoms like howling and counterclockwise turning showed improvement in senior dogs (n = 3). Conclusions: This study provides preliminary support for the potential health benefits of MOP in canine dietary applications, particularly for gut health and cognitive function. Improvement in cognitive symptoms may be due to the anxiolytic effects of mandarin peel.

Background This study describes the development of a long-term short bowel syndrome (SBS) model that does not require parenteral nutrition (PN) using Microminipigs (MMP), the smallest experimental pigs, with an adult weight of ≤ 30 kg. Methods Two MMPs were used to establish an SBS model. A 70 % small bowel resection was performed at 6 months of age (body weight, 5 kg), followed by an 85 % resection 4 months later. Untreated MMPs served as controls. Postoperative clinical conditions, weight, blood, and stool samples were monitored longitudinally. Nine months after the initial surgery, the animals were euthanized for organ collection and histological assessments. Results Both treated MMPs remained in good general condition and continued to gain weight postoperatively. Plasma citrulline levels decreased after the second surgery, reflecting a reduced intestinal epithelial mass. At 7 months postoperatively (13 months of age), fecal fat was graded as 2 + in both treated MMPs, whereas the 38-month-old control showed a grade of 0. Histologically, jejunal villi were shorter, and goblet cells were smaller and fewer in the treated MMPs. Colonic crypts were taller in the treated MMPs, with no signs of inflammation or fibrosis in any tissue. Histological examination of the liver revealed no changes in fatty acid levels or fibrosis. Conclusions The MMP SBS model demonstrated prolonged survival and maintained good health for nearly 9 months without PN, which is a significantly longer period than that reported in previous models. This model offers a practical and physiologically relevant platform for future translational and reverse translational studies on intestinal adaptation.

Potter sequence consists of various symptoms associated with renal dysplasia. For bilateral renal agenesis, there is no hope of survival. As a novel therapeutic approach for Potter sequence, we develop a unique approach of "transplantation of fetal kidneys from a different species during the fetal stage." In this study, we first validate the approach using allogeneic transplantation. Fetal kidneys with bladders from green fluorescent protein-expressing rats (embryonic day 14.0-16.5) are subcutaneously transplanted into allogeneic rat fetuses in utero (embryonic day 18.0-18.5). After birth, the transplanted fetal kidneys are confirmed to have urine production capability. Furthermore, long-term (up to 150 days) urine production is sustained. Next, we perform xenotransplantation. The transplantation of mouse fetal kidneys into rat fetuses in utero leads to the maturation of renal tissue structures. We demonstrate organ transplantation into in utero fetuses using fetal kidneys as donor organs for fetal therapy.

Background/Objectives: Although many healthy foods are used for elderly humans, there are no suitable animal models to test them. We reared microminipigs (MMPs) for over 10 years, establishing aged MMP models. Using this aged preclinical model, we evaluated the effects of a polyphenol-rich diet without sacrificing the animals. Methods: A polyphenol-rich diet containing sweet potato petioles and leaves was administered to the aged MMPs daily for one month. Changes in fecal microbiota and aging-related cells in the peripheral blood before and after administration were assessed. Results: Administration of a diet containing sweet potato petiole and leaf resulted in increased abundance of the genera Muribaculaceae, Oscillibacter, and Desulfovibrio and a decreased abundance of the genus UCG-002 within the family Oscillospiraceae. Prediction of metabolic enzyme activity from microbiota composition identified 77 enzymes significantly altered after administration. KEGG Mapper analysis of these enzymes revealed their involvement in 27 pathways. Flow cytometry analysis of peripheral blood revealed no significant differences in the proportion of ß-galactosidase-positive cells in either group. In contrast, a significant increase in the number of Ki-67-positive cells was observed in some individuals in the treatment group. However, no significant differences in Ki-67 expression were detected after stimulation with anti-CD3/CD28 antibodies. Conclusions: We established an aged MMP model to evaluate the efficacy and safety of drugs and health foods in elderly humans. Using this model, sweet potato petioles and leaves were shown to have potential as candidate materials for future health food research.

Bioluminescence imaging (BLI) in rodent models has revolutionized preclinical research in recent decades, enabling precise and noninvasive observation of cellular and molecular processes in vivo. Among various bioluminescent systems, the firefly luciferase-luciferin system is one of the most widely employed for in vivo cell tracking. This comprehensive review focuses on using luciferase-transgenic (Luc-Tg) rat models, known as firefly rats, in conjunction with BLI to investigate tissue regeneration and stem cell dynamics. Compared with other imaging modalities, BLI offers enhanced tissue penetration, reduced background noise, and the capacity to perform longitudinal studies with fewer animals, aligning with ethical research principles. Applications of Luc-Tg rats in fat grafting, soft tissue expansion, hair growth cycle analysis, and other skin studies are discussed, demonstrating the versatility and precision of BLI in tracking complex biological processes. Integrating advanced analytical and genome-editing techniques with BLI promises to enhance data interpretation’s accuracy and efficiency. These advancements have deepened our understanding of the cellular fate and mechanisms underlying tissue regeneration, presenting promising avenues for optimizing therapeutic strategies in reconstructive surgery and regenerative medicine. Combining luciferase reporter genes and BLI is crucial to unraveling complex biological phenomena, advancing soft tissue regeneration research, and developing innovative therapeutic strategies for various medical conditions.

Purpose: Amid rising health awareness, natural products which has milder effects than medical drugs are becoming popular. However, only few systems can quantitatively assess their impact on living organisms. Therefore, we developed a deep-learning system to automate the counting of cells in a gerbil model, aiming to assess a natural product's effectiveness against ischemia. Methods: The image acquired from paraffin blocks containing gerbil brains was analyzed by a deep-learning model (fine-tuned Detectron2). Results: The counting system achieved a 79%-positive predictive value and 85%-sensitivity when visual judgment by an expert was used as ground truth. Conclusion: Our system evaluated hydrogen water's potential against ischemia and found it potentially useful, which is consistent with expert assessment. Due to natural product's milder effects, large data sets are needed for evaluation, making manual measurement labor-intensive. Hence, our system offers a promising new approach for evaluating natural products.

Imaging technologies are used to observe the morphology and function of various organs in the body and have become indispensable in a multitude of fields, ranging from basic research to clinical medicine. The luminescence technology based on the luciferin–luciferase reaction has been used in many research fields as an imaging technique, enabling quantitative analysis and detection at high sensitivity. Specifically in gene therapy and cell therapy, it has been developed as an in vivo bioimaging technique mainly for small animal models because of its non-invasive and time-sequential analysis. Currently, translational research using this luminescence imaging technology in pigs for clinical applications is ongoing. In this review, we discuss the progress of these technologies and issues for their clinical application, focusing on pigs, by comparing conventional imaging techniques, including fluorescent probes, with luminescence imaging techniques.

Purpose: To develop a new 4/6 infarct nephrectomy (INx) model rat mimicking moderate chronic kidney disease (CKD) and to evaluate its application. Methods: We modified the conventional 5/6 INx rat model to create the 4/6 INx model by ligating the renal artery branch to induce infarction of one-third of the left kidney after right kidney removal and compared biochemically and histologically both models. To demonstrate the application of the 4/6 INx model, the effects of a supplementary compound containing calcium carbonate, chitosan, palm shell activated charcoal etc., that is effective for both CKD and its complications, were compared between both models. Results: Impairment of renal function in the 4/6 INx group was significantly more moderate than in the 5/6 INx group (P < 0.05). The 4/6 INx group showed less histological damage in kidney than in the 5/6 INx group. The supplementary compound did not improve CKD in the 5/6 INx group, but ameliorated elevation of blood urea nitrogen in the 4/6 INx group. Conclusion: We developed the 4/6 INx model, which is more moderate than the conventional 5/6 INx model. This model could potentially demonstrate the effectiveness of drugs and supplements intended to prevent CKD and its progression.

Porcine organs and human induced pluripotent stem cell (iPSC)-derived organoids as alternative organs for human transplantation have garnered attention, but both face technical challenges. Interspecies chimeric organ production using human iPSCs shows promise in overcoming these challenges. Our group successfully generated chimeric renal organoids using human iPSC-derived nephron progenitor cells (NPCs) and fetal mouse kidneys. However, the current technology is limited to rodents. Therefore, this study focused on producing human-pig chimeric renal organoids, as pigs are the most promising species for xenotransplantation. Modification of existing culture systems enables continuous renal development in both species, resulting in the successful creation of human-pig chimeric renal organoids. Moreover, this method can be applied to generate humanized xenogeneic kidneys for future clinical applications. This study provides evidence that optimizing culture conditions enables the early-stage kidney development beyond species barriers, thus laying the foundation for accelerating research on humanized xenogeneic kidney fabrication for clinical purposes. Creation of chimeric renal organoids using human iPSC technology with fetal pig kidneys demonstrates inter-species coexistence and codevelopment in early kidney development, paving the way for humanized xenogeneic kidney creation for clinical use.

Background: During skin expansion, subcutaneous adipose tissue undergoes the greatest change. The adipose layer appears to gradually thin or even disappear in long-term expansion. The response and contribution of adipose tissue to skin expansion remain to be elucidated. Methods: The authors established a novel expansion model by transplanting luciferase-transgenic adipose tissue into the rat dorsum, followed by integrated expansion, to trace the dynamic changes in subcutaneous adipose tissue during expansion and the migration of adipose tissue–derived cells. In vivo luminescent imaging was performed to continuously track the adipose tissue changes. Histologic analysis and immunohistochemical staining evaluated the regeneration and vascularization of the expanded skin. Growth factor expression in expanded skin with or without adipose tissue was determined to evaluate the paracrine effect of adipose tissue. Adipose tissue–derived cells were traced in vitro by anti-luciferase staining, and their fate was determined by costaining for PDGFRα, DLK1, and CD31. Results: In vivo bioimaging showed that cells in adipose tissue were alive during expansion. After expansion, the adipose tissue exhibited fibrotic-like structures, with more DLK1 + preadipocytes. Skin expanded with adipose tissue was significantly thicker than that without adipose tissue, with more blood vessels and cell proliferation. Vascular endothelial growth factor, epidermal growth factor, and basic fibroblast growth factor expression was higher in adipose tissue than in skin, indicating paracrine support from adipose tissue. Luciferase-positive adipose tissue-derived cells were observed in expanded skin, indicating direct participation in skin regeneration. Conclusion: Adipose tissue transplantation can effectively promote long-term skin expansion by contributing to vascularization and cell proliferation by means of various mechanisms. Clinical Relevance Statement: The authors’ findings suggest that it would be better if the expander pocket is dissected over the superficial fascia to preserve a layer of adipose tissue with skin. In addition, their findings support the treatment of fat grafting when expanded skin presents with thinning.

Fetal organs and organoids are important tools for studying organ development. Recently, porcine organs have garnered attention as potential organs for xenotransplantation because of their high degree of similarity to human organs. However, to meet the prompt demand for porcine fetal organs by patients and researchers, effective methods for producing, retrieving, and cryopreserving pig fetuses are indispensable. Therefore, in this study, to collect fetuses for kidney extraction, we employed cesarean sections to preserve the survival and fertility of the mother pig and a method for storing fetal kidneys by long-term cryopreservation. Subsequently, we evaluated the utility of these two methods. We confirmed that the kidneys of pig fetuses retrieved by cesarean section that were cryopreserved for an extended period could resume renal growth when grafted into mice and were capable of forming renal organoids. These results demonstrate the usefulness of long-term cryopreserved fetal pig organs and strongly suggest the effectiveness of our comprehensive system of pig fetus retrieval and fetal organ preservation, thereby highlighting its potential as an accelerator of xenotransplantation research and clinical innovation.