Assessment of P-glycoprotein function using canine intestinal organoid-derived epithelial interfaces.

P-glycoprotein (P-gp), a multidrug efflux pump encoded by the ABCB1 (formerly MDR1) gene, plays a crucial role in limiting drug absorption and eliminating toxic compounds in both humans and dogs. However, species-specific differences in P-gp substrates necessitate the development of canine-specific evaluation systems. Canine intestinal organoids derived monolayers offer a promising platform for studying drug transport, yet P-gp-mediated transport in these models remains unexplored.We generated canine colonoid-derived 2D monolayers to investigate ABCB1 gene expression and P-gp function. We employed widely recognised P-gp substrates, Rhodamine 123 and Doxorubicin, in conjunction with the P-gp inhibitor PSC833 at Days 5 and 10 of culture.A significant increase in gene expression of P-gp encoded by the ABCB1 was noted on Day 10 compared to Day 5 of culture. Despite this disparity in gene expression, the transport activity of P-gp, as assessed by the efflux of Rhodamine 123 and Doxorubicin with PSC833 inhibition, did not exhibit significant differences between these two time points. However, the inhibition of P-gp function by PSC833 confirms the presence of functional P-gp in our model.Canine intestinal organoid-derived monolayers provide a valuable tool for investigating P-gp-mediated drug transport. These findings highlight the potential for predicting drug bioavailability and adverse reactions in veterinary medicine, aligning with principles of ethical and sustainable research.

Assessment of cytochrome P450 induction in canine intestinal organoid models.

Understanding cytochrome P450 (CYP) enzymes in the canine intestine is vital for predicting drug metabolism and developing safer oral medications. This study evaluates canine colonoids as a model to assess the expression and induction of essential intestinal CYP enzymes.Canine colonoids were cultured in expansion medium (EM) with Wnt-3A and in differentiation medium (DM) without Wnt-3A. We assessed the mRNA expression of CYP2B11, CYP2C21, CYP3A12, and CYP3A98 using qPCR and examined the effects of rifampicin and phenobarbital as inducers.Our findings show that DM significantly increased the mRNA expression of CYP3A98 and CYP2B11, but not CYP3A12, compared to EM. CYP2C21, not typically expressed in the intestine, remained unexpressed in colonoids. Rifampicin induced CYP3A98, aligning with pregnane x receptor (PXR) regulation, while phenobarbital did not, suggesting no constitutive androstane receptor (CAR) involvement. CYP2B11 did not respond to either inducer, suggesting alternative regulatory pathways in canine colonoids.This study is a pioneering effort to establish conditions for studying P450 expression in canine colonoids, confirming significant CYP3A98 expression in the canine intestine. It demonstrated colonoids can induce CYP activity post drug treatments. Further research is needed to enhance species-specific drug metabolism understanding and validate this model for broader applications.

High-fat diet enhances cell proliferation and compromises intestinal permeability in a translational canine intestinal organoid model.

BACKGROUND: Emerging evidence underscores the responsiveness of the mammalian intestine to dietary cues, notably through the involvement of LGR5 + intestinal stem cells in orchestrating responses to diet-driven signals. However, the effects of high-fat diet (HFD) on these cellular dynamics and their impact on gut integrity remain insufficiently understood. Our study aims to assess the multifaceted interactions between palmitic acid (PA), cell proliferation, and the intestinal epithelial barrier using a canine colonoid model. Canine models, due to their relevance in simulating human intestinal diseases, offer a unique platform to explore the molecular mechanisms underlying HFD derived intestinal dysfunction. RESULTS: Canine colonoids were subjected to PA exposure, a surrogate for the effects of HFD. This intervention revealed a remarkable augmentation of cell proliferative activity. Furthermore, we observed a parallel reduction in transepithelial electrical resistance (TEER), indicating altered epithelium barrier integrity. While E-cadherin exhibited consistency, ZO-1 displayed a noteworthy reduction in fluorescence intensity within the PA-exposed group. CONCLUSIONS: By employing canine intestinal organoid systems, we provide compelling insights into the impact of PA on intestinal physiology. These findings underscore the importance of considering both cell proliferative activity and epithelial integrity in comprehending the repercussions of HFDs on intestinal health. Our study contributes to a deeper understanding of the consequences of HFD on intestinal homeostasis, utilizing valuable translational in vitro models derived from dogs.

Three-Dimensional Morphogenesis in Canine Gut-on-a-Chip Using Intestinal Organoids Derived from Inflammatory Bowel Disease Patients.

Canine intestines possess similarities in anatomy, microbiology, and physiology to those of humans, and dogs naturally develop spontaneous intestinal disorders similar to humans. Overcoming the inherent limitation of three-dimensional (3D) organoids in accessing the apical surface of the intestinal epithelium has led to the generation of two-dimensional (2D) monolayer cultures, which expose the accessible luminal surface using cells derived from the organoids. The integration of these organoids and organoid-derived monolayer cultures into a microfluidic Gut-on-a-Chip system has further evolved the technology, allowing for the development of more physiologically relevant dynamic in vitro intestinal models. In this study, we present a protocol for generating 3D morphogenesis of canine intestinal epithelium using primary intestinal tissue samples obtained from dogs affected by inflammatory bowel disease (IBD). We also outline a protocol for generating and maintaining 2D monolayer cultures and intestine-on-a-chip systems using cells derived from the 3D intestinal organoids. The protocols presented in this study serve as a foundational framework for establishing a microfluidic Gut-on-a-Chip system specifically designed for canines. By laying the groundwork for this innovative approach, we aim to expand the application of these techniques in biomedical and translational research, aligning with the principles of the One Health Initiative. By utilizing this approach, we can develop more physiologically relevant dynamic in vitro models for studying intestinal physiology in both dogs and humans. This has significant implications for biomedical and pharmaceutical applications, as it can aid in the development of more effective treatments for intestinal diseases in both species.

Proinflammatory cytokines suppress stemness-related properties and expression of tight junction in canine intestinal organoids.

Recent advancements in canine intestinal organoid research have paved the way for the development of enhanced in vitro models, crucial for exploring intestinal physiology and diseases. Despite these strides, there is a notable gap in creating specific in vitro models that focus on intestinal inflammation. Our study aims to bridge this gap by investigating the impact of proinflammatory cytokines on canine intestinal epithelial cells (IECs) within the context of organoid models. Canine intestinal organoids were treated with proinflammatory cytokines TNF-α, IFN-γ, and IL-1ß. The expression of stem cell markers Lgr5, Sox9, Hopx, and Olfm4 was evaluated through RT-qPCR, while membrane integrity was assessed using immunofluorescence staining for tight junction proteins and transport assays for permeability. IFN-γ significantly decreased Lgr5 expression, a key intestinal stem cell marker, at both 24 and 48 h post-treatment (p=0.030 and p=0.002, respectively). Conversely, TNF-α increased Olfm4 expression during the same intervals (p=0.018 and p=0.011, respectively). A reduction in EdU-positive cells, indicative of decreased cell proliferation, was observed following IFN-γ treatment. Additionally, a decrease in tight junction proteins E-cadherin and ZO-1 (p<0.001 and p=0.003, respectively) and increased permeability in IECs (p=0.012) were noted, particularly following treatment with IFN-γ. The study highlights the profound impact of proinflammatory cytokines on canine IECs, influencing both stem cell dynamics and membrane integrity. These insights shed light on the intricate cellular processes underlying inflammation in the gut and open avenues for more in-depth research into the long-term effects of inflammation on intestinal health.

Ion channel function in translational bovine gallbladder cholangiocyte organoids: establishment and characterization of a novel model system.

The study of biliary physiology and pathophysiology has long been hindered by the lack of in vitro models that accurately reflect the complex functions of the biliary system. Recent advancements in 3D organoid technology may offer a promising solution to this issue. Bovine gallbladder models have recently gained attention in the investigation of human diseases due to their remarkable similarities in physiology and pathophysiology with the human gallbladder. In this study, we have successfully established and characterized bovine gallbladder cholangiocyte organoids (GCOs) that retain key characteristics of the gallbladder in vivo, including stem cell properties and proliferative capacity. Notably, our findings demonstrate that these organoids exhibit specific and functional CFTR activity. We believe that these bovine GCOs represent a valuable tool for studying the physiology and pathophysiology of the gallbladder with human significance.

MUC5AC and MUC5B expression in canine gallbladder mucocele epithelial cells.

Gallbladder mucocele (GBM) is one of the most common gallbladder diseases in dogs. Its pathogenesis has not yet been clarified, but excessive accumulation of a secretory gel-forming mucin, MUC5AC in the gallbladder has been reported. This study aimed to ascertain if MUC5AC overproduction resulted in mucus accumulation in the gallbladder during GBM development. Eleven dogs undergoing cholecystectomy who were pathologically diagnosed with GBM were included, and the expression level of mucins, particularly MUC5AC and MUC5B, in their gallbladder epithelial cells was compared with those in normal gallbladder epithelial cells. On reverse transcription-quantitative polymerase chain reaction screening, there was a significant difference (P<0.05) in the mRNA expression level of MUC1, but not of other mucins including MUC5AC and MUC5B, between mucocele and normal gallbladder epithelial cells. Protein expression levels were also evaluated for MUC5AC and MUC5B using immunohistochemistry. There was little immunoreactivity for MUC5AC, whereas MUC5B showed definitive staining in gallbladder epithelial cells. There was no difference in MUC5AC and MUC5B protein expression levels between mucocele and normal gallbladder epithelial cells. These data suggest that excessive production of mucin, especially MUC5AC and MUC5B, does not occur in canine GBM, and that abnormal mucus excretion, rather than excessive mucus production, may be the cause of GBM development.

Polyoxazoline-conjugated porcine serum albumin as an artificial plasma expander for dogs.

Veterinary medicine has made tremendous progress for domestic dogs, which are irreplaceable family members enriching human life. Nevertheless, no adequate supply system exists for their blood products. This study examined the synthesis, structure, safety, and efficacy of poly(2-ethyl-2-oxazoline)-conjugated porcine serum albumin (POx-PSA) as an artificial plasma expander for dogs. The aqueous POx-PSA solution showed moderately high colloid osmotic pressure and good blood cell compatibility. Actually, lyophilized powder stored for 1 year can regenerate into a homogeneous solution. The circulation half-life of POx-PSA in rats was 2.1-fold longer than that of naked PSA. Rats produced neither anti-PSA IgG antibody nor anti-POx IgG antibody, which suggests excellent immunological stealth properties of POx-PSA. Complete resuscitation of hemorrhagic shock in rats was achieved soon after injection of POx-PSA solution. Serum biochemistry tests and histopathological observations indicated no abnormality in the related organs. When POx-PSA was administered to dogs intravenously, (i) no serum biochemical or hematological alteration was observed, also (ii) no overt deterioration of animal health was observed. These results indicate that POx-PSA has potential as an artificial plasma expander for dogs.

Farm and Companion Animal Organoid Models in Translational Research: A Powerful Tool to Bridge the Gap Between Mice and Humans.

Animal organoid models derived from farm and companion animals have great potential to contribute to human health as a One Health initiative, which recognize a close inter-relationship among humans, animals and their shared environment and adopt multi-and trans-disciplinary approaches to optimize health outcomes. With recent advances in organoid technology, studies on farm and companion animal organoids have gained more attention in various fields including veterinary medicine, translational medicine and biomedical research. Not only is this because three-dimensional organoids possess unique characteristics from traditional two-dimensional cell cultures including their self-organizing and self-renewing properties and high structural and functional similarities to the originating tissue, but also because relative to conventional genetically modified or artificially induced murine models, companion animal organoids can provide an excellent model for spontaneously occurring diseases which resemble human diseases. These features of companion animal organoids offer a paradigm-shifting approach in biomedical research and improve translatability of in vitro studies to subsequent in vivo studies with spontaneously diseased animals while reducing the use of conventional animal models prior to human clinical trials. Farm animal organoids also could play an important role in investigations of the pathophysiology of zoonotic and reproductive diseases by contributing to public health and improving agricultural production. Here, we discuss a brief history of organoids and the most recent updates on farm and companion animal organoids, followed by discussion on their potential in public health, food security, and comparative medicine as One Health initiatives. We highlight recent evolution in the culturing of organoids and their integration with organ-on-a-chip systems to overcome current limitations in in vitro studies. We envision multidisciplinary work integrating organoid culture and organ-on-a-chip technology can contribute to improving both human and animal health.

Case Report: Tricuspid Annuloplasty for Right-Sided Congestive Heart Failure Secondary to Pulmonary Hypertension in a Dog.

An 11-year-old, 12.3-kg, female Miniature Dachshund was presented to our institution with ascites of unknown etiology. The dog had been administered moxidectin for 3 years to treat a heartworm infection. Thoracic radiographs showed enlargement of the right heart. Echocardiography revealed right atrial and ventricular dilatation as well as flattening of the interventricular septum. Heartworm was identified in the main pulmonary artery, which was dilated. Tricuspid regurgitation (TR) was observed using color Doppler ultrasonography, and 2.5 L of ascites were removed. The dog was diagnosed with pulmonary hypertension, severe TR, and right-sided congestive heart failure. Except at the initial site, heartworm was not detected using echocardiography, and the antigen test was negative. However, pharmacological treatment did not improve the right-sided congestive heart failure. Instead, De Vega tricuspid annuloplasty (TAP) was performed on the beating heart under cardiopulmonary bypass with the owner's consent. Sutures terminated between the two commissures in the middle of the annulus and were secured using another pledget. Annular reduction was performed by tying down the plication suture while the cylindrical sizer was inserted into the tricuspid valve orifice. The size of the cylindrical sizer was 16 mm, which was set based on the height and width of the septal leaflet. A 6-month follow-up showed a reduction of TR and right-sided volume overload with no evidence of ascites retention/recurrence or any other complication. Our findings indicate that TAP may be a valid treatment option for dogs with right-sided congestive heart failure caused by secondary TR.

Evaluation of the degree and distribution of lymphangiectasia in full-thickness canine small intestinal specimens diagnosed with lymphoplasmacytic enteritis and granulomatous lymphangitis.

Intestinal lymphangiectasia (IL) is often observed in dogs with chronic small intestinal diseases. Hypoplasia of the lymphatic vessel due to decreased lymphangiogenesis, which has been suggested in human idiopathic IL, may contribute to the pathogenesis of canine IL. This study aimed to evaluate the diameter and number of lymphatic vessels in full-thickness small intestinal specimens of dogs with IL. Immunohistochemical labeling of lymphatic endothelial cell markers was performed on retrospectively retrieved full-thickness small intestinal specimens. Sixteen dogs with histologically confirmed IL were included, of which 10 had lymphoplasmacytic enteritis (LPE), and six had granulomatous lymphangitis (GL). Nine dogs that died from non-gastrointestinal disorders and with little or no abnormalities in the small intestine were used as controls. Lymphatic vessel diameters in dogs with IL were significantly increased in all layers of the small intestine, including the villus lacteal, lamina propria, submucosa, muscularis, and mesentery, compared with controls (all P<0.01). There was no significant difference in the lymphatic vessel diameters between dogs with LPE and GL (all P>0.05). There was no significant difference in the number of lymphatic vessels between dogs with IL and the controls in all layers of the small intestine (all P>0.05). This study demonstrated that IL was observed in all layers of the small intestine, including the submucosa, muscularis, and mesentery, independent of the underlying disease. Factors other than reduced lymphatic vessels would contribute to the pathogenesis of IL in dogs.

Association between intestinal lymphangiectasia and expression of inducible nitric oxide synthase in dogs with lymphoplasmacytic enteritis.

Intestinal lymphangiectasia (IL) is a common complication in dogs. Since nitric oxide (NO) is known to relax the lymphatic vessel, we evaluated inducible NO synthase (iNOS) expression using immunohistochemistry in 13 dogs with lymphoplasmacytic enteritis (LPE) with or without IL. The duodenal iNOS expressing cells were significantly increased in dogs with IL-negative or IL-positive LPE dogs (P=0.025, P=0.007) compared with control dogs. However, there was no significant difference in iNOS expression between IL-positive and IL-negative tissues. Based on these results, there is no clear evidence for the NO overproduction in the pathogenesis of IL in dogs with LPE. Factors other than NO could, thus, contribute to IL in dogs with LPE.

Effects of proportions of carbohydrates and fats in diets on mucin concentration and bile composition in gallbladder of dogs.

The associations of diet compositions with mucin secretion in gallbladder have not been investigated in dogs. This study aimed to examine the effects of a low-carbohydrate diet (LC) and a low-fat diet (LF) on bile mucin concentration and composition of gallbladder bile in six clinically healthy beagle dogs. After feeding of both diets, the bile mucin concentration was significantly decreased. In addition, there were significant decreases in the concentrations of taurochenodeoxycholic acid in bile, which is considered to promote mucin secretion, after feeding of both diets. The present study suggested that the proportions of carbohydrate and fat in diet affect the composition of gallbladder bile in dogs.

Changes in the coagulation parameters in dogs with protein-losing enteropathy between before and after treatment.

Protein-losing enteropathy (PLE) is known to induce hypercoagulability and resultant thromboembolism in dogs. We hypothesized that hypercoagulability would improve if remission was obtained in dogs with PLE after treatment. This study aimed to evaluate the changes in the coagulation parameters after treatment in dogs diagnosed with PLE. As coagulation parameters, prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, thrombin-antithrombin complex (TAT), D-dimer, and antithrombin (AT) were measured. In addition to these parameters, rotational thromboelastometry (ROTEM), which evaluates the comprehensive coagulation and fibrinolysis reactions of whole blood, was conducted and the data of clotting time (CT), clot formation time (CFT), α angle (α), maximum clot firmness (MCF) and lysis index at 60 min (LI60) were obtained. Eleven of the 14 dogs diagnosed with PLE were classified as responders to the treatment based on the changes in their plasma albumin (ALB) concentration after treatment. Significant increase in CFT and decrease of α and MCF indicating the resolution of hypercoagulability were found after treatment in responder dogs; however, there was no significant change in the coagulation and fibrinolysis parameters other than those measured by ROTEM. This study demonstrated that the hypercoagulability detected by ROTEM was significantly improved after treatment in dogs with PLE.

Evaluation of visceral fat mass in dogs by computed tomography.

In human medicine, computed tomography (CT) is the gold standard for visceral fat measurement. Research shows that the visceral fat area (VFA) of the umbilical slice is significantly correlated with the visceral fat volume (VFV). In veterinary medicine, however, few studies have evaluated visceral fat using CT. This study aimed to evaluate the visceral fat in dogs using CT images, and determine if the slice significantly correlated with VFV to simplify visceral fat measurements. This retrospective study includes data on 90 dogs that underwent whole-body CT scans for diagnostic purposes. VFV was calculated as the product of VFA and thickness in each CT slice; the correlation between VFV and VFA was analyzed at the level of each lumbar vertebra. Visceral fat percentage (VF%) was calculated as the ratio of the product of VFV and fat density to the body weight. Visceral fat area percentage (VFA%) was calculated as the ratio of VFA to the body area, and its correlation with the VF% and the body condition score (BCS) was analyzed. VFA was highly correlated with VFV at the level of each lumbar vertebra, with the highest correlation (r=0.964) at the L3 level. VFA% was significantly correlated with VF% (r=0.930) and weakly correlated with BCS (r=0.523). This study demonstrates that it is sufficient to use only the L3 slice for visceral fat evaluation and that the evaluation can be based on VFA% of the L3 level.