Pig Model Research Articles

Wuzhishan miniature pig-derived intestinal 2D monolayer organoids to investigate the enteric coronavirus infection.

Intestinal organoids are valuable tools for investigating intestinal physiology and pathology ex vivo. In previous studies, intestinal organoids of commercial pigs have been developed. Here, we established intestinal organoids derived from Wuzhishan miniature pigs (WZS pigs), a unique kind of pig in the Hainan province of China. Three-dimensional (3D) intestinal organoids and organoid monolayers were developed and assessed. Furthermore, the susceptibility of organoid monolayers of WZS pigs to transmissible gastroenteritis virus (TGEV) was demonstrated. An RNA-seq analysis revealed that the TGEV infection stimulated antiviral and inflammatory immune responses in organoid monolayer models. The study implied the transmission risk of swine enteric coronavirus on WZS pigs and provided useful tools for further research on WZS pigs as laboratory miniature pig models.

Placental Nanoparticle-mediated IGF1 Gene Therapy Corrects Fetal Growth Restriction in a Guinea Pig Model.

Fetal growth restriction (FGR) caused by placental insufficiency is a major contributor to neonatal morbidity and mortality. There is currently no in utero treatment for placental insufficiency or FGR. The placenta serves as the vital communication, supply, exchange, and defense organ for the developing fetus and offers an excellent opportunity for therapeutic interventions. Here we show efficacy of repeated treatments of trophoblast-specific human insulin-like 1 growth factor (IGF1) gene therapy delivered in a non-viral, polymer nanoparticle to the placenta for the treatment of FGR. Using a guinea pig maternal nutrient restriction model (70% food intake) of FGR, nanoparticle-mediated IGF1 treatment was delivered to the placenta via ultrasound guidance across the second half of pregnancy, after establishment of FGR. This treatment resulted in correction of fetal weight in MNR + IGF1 animals compared to sham treated controls on an ad libitum diet, increased fetal blood glucose and decreased fetal blood cortisol levels compared to sham treated MNR, and showed no negative maternal side-effects. Overall, we show a therapy capable of positively impacting the entire pregnancy environment: maternal, placental, and fetal. This combined with our previous studies using this therapy at mid pregnancy in the guinea pig and in two different mouse model and three different human in vitro/ex vivo models, demonstrate the plausibility of this therapy for future human translation. Our overall goal is to improve health outcomes of neonates and decrease numerous morbidities associated with the developmental origins of disease.

Novel systematic processing of cardiac magnetic resonance imaging identifies target regions associated with infarct-related ventricular tachycardia.

There is lack of agreement on late gadolinium enhancement cardiac magnetic resonance (LGE-CMR) imaging processing for guiding ventricular tachycardia (VT) ablation. We aim at developing and validating a systematic processing approach on LGE-CMR images to identify VT corridors that contain critical VT isthmus sites. This is a translational study including 18 pigs with established myocardial infarction and inducible VT undergoing in vivo characterization of the anatomical and functional myocardial substrate associated with VT maintenance. Clinical validation was conducted in a multicentre series of 33 patients with ischaemic cardiomyopathy undergoing VT ablation. Three-dimensional LGE-CMR images were processed using systematic scanning of 15 signal intensity (SI) cut-off ranges to obtain surface visualization of all potential VT corridors. Analysis and comparisons of imaging and electrophysiological data were performed in individuals with full electrophysiological characterization of the isthmus sites of at least one VT morphology. In both the experimental pig model and patients undergoing VT ablation, all the electrophysiologically defined isthmus sites (n = 11 and n = 19, respectively) showed overlapping regions with CMR-based potential VT corridors. Such imaging-based VT corridors were less specific than electrophysiologically guided ablation lesions at critical isthmus sites. However, an optimized strategy using the 7 most relevant SI cut-off ranges among patients showed an increase in specificity compared to using 15 SI cut-off ranges (70 vs. 62%, respectively), without diminishing the capability to detect VT isthmus sites (sensitivity 100%). Systematic imaging processing of LGE-CMR sequences using several SI cut-off ranges may improve and standardize procedure planning to identify VT isthmus sites.

Cardioprotective microRNAs (protectomiRs) in a pig model of acute myocardial infarction and cardioprotection by ischaemic conditioning: MiR-450a.

Cardioprotective miRNAs (protectomiRs) are promising therapeutic tools. Here, we aimed to identify protectomiRs in a translational porcine model of acute myocardial infarction (AMI) and to validate their cardiocytoprotective effect. ProtectomiR candidates were selected after systematic analysis of miRNA expression changes in cardiac tissue samples from a closed-chest AMI model in pigs subjected to sham operation, AMI and ischaemic preconditioning, postconditioning or remote preconditioning, respectively. Cross-species orthologue protectomiR candidates were validated in simulated ischaemia-reperfusion injury (sI/R) model of isolated rat ocardiomyocytes and in human AC16 cells as well. For miR-450a, we performed target prediction and analysed the potential mechanisms of action by GO enrichment and KEGG pathway analysis. Out of the 220 detected miRNAs, four were up-regulated and 10 were down-regulated due to all three conditionings versus AMI. MiR-450a and miR-451 mimics at 25 nM were protective in rat cardiomyocytes, and miR-450a showed protection in human cardiomyocytes as well. MiR-450a has 3987 predicted mRNA targets in pigs, 4279 in rats and 8328 in humans. Of these, 607 genes are expressed in all three species. A total of 421 common enriched GO terms were identified in all three species, whereas KEGG pathway analysis revealed 13 common pathways. This is the first demonstration that miR-450a is associated with cardioprotection by ischaemic conditioning in a clinically relevant porcine model and shows cardiocytoprotective effect in human cardiomyocytes, making it a promising drug candidate. The mechanism of action of miR-450a involves multiple cardioprotective pathways.

Emodin repairs interstitial cells of Cajal damaged by cholelithiasis in the gallbladder.

Hypercholesterolemia induces cholelithiasis and dysfunction of gallbladder motility. Interstitial cells of Cajal (ICCs) contribute to gallbladder motility. Emodin modulates the contractility of the gallbladder muscle; however, the underlying mechanism is unknown. This study aimed to explore the effects of emodin on gallbladder ICCs with cholelithiasis in a guinea pig model. Animals were randomly divided into a healthy control group and three study groups. All study groups received a high-cholesterol diet (HCD) for 8 weeks. Subsequently, they were randomly assigned to either the HCD group or one of the emodin treatment groups lasting 4 or 8weeks. Total cholesterol (TC) and triglycerides (TG) were measured to determine changes in serum lipid levels. Immunohistochemistry was performed to detect the morphology and number of ICCs. TUNEL assays were performed to detect ICC apoptosis. Transmission electron microscopy was employed to observe ICC structure. Western blotting and real-time polymerase chain reaction were used to detect changes in stem cell factor (SCF)/c-kit pathway expression. Serum TC and TG were higher in all study groups. In cases of cholelithiasis, the SCF/c-kit pathway was downregulated, the number of gallbladder ICCs decreased, apoptosis increased, and the ICC network structure was damaged. After emodin treatment, the SCF/c-kit pathway was upregulated, the number of gallbladder ICCs increased, apoptosis decreased, and the ICC network structure recovered. Cholelithiasis downregulates the SCF/c-kit pathway and damages gallbladder ICCs. Emodin upregulates the SCF/c-kit pathway and increases gallbladder ICCs, contributing to recovery from gallbladder motility disorders.\.

Gentamicin and clindamycin antibiotic-eluting depot technology eradicates S. aureus in an implant-associated osteomyelitis pig model without systemic antibiotics.

The therapeutic challenges of orthopedic device-related infections and emerging antimicrobial resistance have attracted attention to drug delivery technologies. This study evaluates the preclinical efficacy of local single- and dual-antibiotic therapy against implant-associated osteomyelitis (IAO) using a drug-eluting depot technology, CarboCell, that provides sustained release of high-dose antibiotics and allows for strategic in situ placement in relation to infectious lesions. Clindamycin and gentamicin were formulated in CarboCell compositions. One-stage-revision of tibial Staphylococcus aureus IAO was conducted in 19 pigs. Pigs were treated locally with CarboCell containing either gentamicin alone for 1 week or a co-formulation of gentamicin and clindamycin for 1 or 3 weeks. Bone, soft tissue, and antibiotic depots were collected for microbiology, histology, and HPLC analyses. Supporting in vivo release studies of CarboCell formulations were performed on mice. Both single- and dual-antibiotic CarboCell formulations were developed and capable of eradicating the infectious bacteria in bone and preventing colonization of implants inserted at revision. Eradication in soft tissue was observed in all pigs after 3 weeks and in 6/9 pigs after 1 week of treatment. Neutrophil counts in bone tissue were below the infection cut-off in all pigs receiving the dual-antibiotic therapies, but above in all pigs receiving the single-antibiotic therapy. Histological signs of active bone reorganization and healing were observed at 3 weeks. In conclusion, all CarboCell formulations demonstrated strong therapeutic activity against IAO, eradicating S. aureus in bone tissue and preventing colonization of implants even without the addition of systemic antibiotic therapy.

Gastrointestinal microbiota and metabolites responses to dietary cereal grains in an adult pig model.

Corn (C), wheat (W), and paddy rice (PR) are important energy sources and are commonly used in feed production for swine. This study mainly focuses on the variation and regularities of microbiota and metabolites in the gastrointestinal tract (GIT) of pigs in response to C, W, and PR. A total of 18 pigs were allotted into three dietary groups with six replicated pigs and received diets containing C, W, or PR as the sole energy source, respectively. The results showed that digestive parts significantly affected the diversity of microbial communities. Cereal grain sources significantly influenced the β-diversity of microbial communities in the colon and rectum. Campylobacterota and Proteobacteria are mainly distributed in the duodenum, Lactobacillus in the jejunum, and Bacteroidota in the colon and rectum. The W diet increased the Bacteroidota, Spirochaetota, and Prevotellaceae_NK3B31_group abundances and showed the highest concentrations of all short-chain fatty acids (SCFAs) in the hindgut. Fibrobacterota, Bacteroidota, Spirochaetota, Prevotellaceae_NK3B31_group, Prevotella, and Treponema in the colon or rectum were positively correlated with acetate, propionate, butyrate, and total SCFAs. These findings suggested that aerobic bacteria and facultative anaerobes in the foregut will gradually be replaced by anaerobes in the hindgut. The W diet had the best fermentability and was beneficial to the colonization of microbial communities that mainly used carbohydrates. The hindgut flora of the PR diet group may be more balanced with fewer potential pathogenic bacteria. Many microbial communities have been identified to contribute positively to the SCFA production of the hindgut. Collectively, our study revealed the spatial variation regularities of GIT microbial communities in an adult pig model and provided new insights into GIT microbiota and responses of metabolites to cereal grain diets.

Biodegradable Piezoelectric-Conductive Integrated Hydrogel Scaffold for Repair of Osteochondral Defects.

Osteochondral injury is a prevalent condition for which no specific treatment is currently available. This study presents a piezoelectric-conductive scaffold composed of a piezoelectric cartilage-decellularized extracellular matrix (dECM) and piezoelectric-conductive modified gelatin (Gel-PC). The piezoelectricity of the scaffold is achieved through the modification of diphenylalanine (FF) assembly on the pore surface, while the conductive properties of scaffold are achieved by the incorporating poly(3,4-ethylenedioxythiophene). In vitro experiments demonstrate that bone marrow mesenchymal stem cells (BMSCs) undergo biphasic division during differentiation. In vivo studies using a Parma pig model of osteochondral defects demonstrate that the piezoelectric-conductive scaffold exhibits superior reparative efficacy. Notably, the generation of electrical stimulation is linked to joint movement. During joint activity, mechanical forces compress the scaffold, leading to deformation and the subsequent generation of an electric potential difference. The positive charges accumulated on the upper layer of the scaffold attract BMSCs, promoting their migration to the upper layer and chondrogenic differentiation. Meanwhile, the negative charges in the lower layer induce the osteogenic differentiation of BMSCs. Overall, this piezoelectric-conducive scaffold provides a promising platform for the effective repair of osteochondral defects.

Impaired Skeletal Development by Disruption of Presenilin-1 in Pigs and Generation of Novel Pig Models for Alzheimer's Disease.

Presenilin 1 (PSEN1) is one of the genes linked to the prevalence of early onset Alzheimer's disease. In mice, inactivation of Psen1 leads to developmental defects, including vertebral malformation and neural development. However, little is known about the role of PSEN1 during the development in other species. To investigate the role of PSEN1 in vertebral development and the pathogenic mechanism of neurodegeneration using a pig model. CRISPR/Cas9 system was used to generate pigs with different mutations flanking exon 9 of PSEN1, including those with a deleted exon 9 (Δexon9). Vertebral malformations in PSEN1 mutant pigs were examined by X-ray, micro-CT and micro-MRI. Neuronal cells from the brains of PSEN1 mutant pigs were analyzed by immunoflourescence, followed by image analysis including morphometric evaluation via image J and 3D reconstruction. Pigs with a PSEN1 null mutation (Δexon9-12) died shortly after birth and had significant axial skeletal defects, whereas pigs carrying at least one Δexon9 allele developed normally and remained healthy. Effects of the null mutation on abnormal skeletal development were also observed in fetuses at day 40 of gestation. Abnormal distribution of astrocytes and microglia in the brain was detected in two PSEN1 mutant pigs examined compared to age-matched control pigs. The founder pigs were bred to establish and age PSEN1ΔE9/+ pigs to study their relevance to clinical Alzheimer's diseases. PSEN1 has a critical role for normal vertebral development and PSEN1 mutant pigs serves as novel resources to study Alzheimer's disease.

Evaluating the ability of non-tuberculous mycobacteria to induce non-specific reactions in bovine tuberculosis diagnostic tests in guinea pigs and cattle

Limitations in diagnostic test performance are one of the major challenges hampering the eradication of bovine tuberculosis (bTB). Non-tuberculous mycobacteria (NTM) are considered one of the main causes of non-specific reactions in the intradermal tuberculin test, the most widely used bTB diagnostic test. To determine the role of NTMs in bTB misdiagnosis in Spain, an experimental study including the NTM species most commonly found in bTB-positive animals from bTB-free farms in the country (M. avium subsp. avium (Maa), “Mycobacterium avium subsp. hominissuis” (Mah), M. bourgelatii, M. intermedium, M. kansasii and M. nonchromogenicum) was carried out on guinea pigs and cattle. First, guinea pigs were sensitized with the selected NTMs, and six weeks post-sensitization four antigen mixtures (bovine-PPD, avian-PPD, P22 and ESAT6-CFP10) were inoculated intradermally and their effect was measured 24- and 48-h post-inoculation. Larger erythematous reactions were observed in guinea pigs sensitized with Mah, M. kansasii, and Maa, with significant differences in the reactions measured at the bovine-PPD inoculation site for the two first bacteria compared with other NTMs. The sensitization process was repeated in cattle, and five months post-sensitization the same antigen mixtures were inoculated in the cervical region and responses were measured at 48- and 72-h post-inoculation. A significantly higher increase in the skinfold thickness measured at the bovine-PPD inoculation site was observed in calves sensitized with Mah, Maa, M. intermedium and M. kansasii. These results demonstrate that certain NTM species may play a more significant role in bTB diagnostic interferences and show that results obtained in guinea pig and bovine models do not always coincide.

Sulforaphane improves post-resuscitation myocardial dysfunction by inhibiting cardiomyocytes ferroptosis via the Nrf2/IRF1/GPX4 pathway

BackgroundFerroptosis is an important type of cell death contributing to myocardial dysfunction induced by whole body ischemia reperfusion following cardiac arrest (CA) and resuscitation. Sulforaphane (SFN), known as the activator of the nuclear factor E2-related factor 2 (Nrf2), has been proven to effectively alleviate regional myocardial ischemia reperfusion injury. The present study was designed to investigate whether SFN could improve post-resuscitation myocardial dysfunction by inhibiting cardiomyocytes ferroptosis and its potential regulatory mechanism. Methods and resultsAn in vivo pig model of CA and resuscitation was established. Hypoxia/reoxygenation (H/R)-stimulated AC16 cardiomyocytes was constructed as an in vitro model to simulate the process of CA and resuscitation. In vitro experiment, SFN reduced ferroptosis-related ferrous iron, lipid reactive oxygen species, and malondialdehyde, increased glutathione, and further promoted cell survival after H/R stimulation in AC16 cardiomyocytes. Mechanistically, the activation of Nrf2 with the SFN decreased interferon regulatory factor 1 (IRF1) expression, then reduced its binding to the promoter of glutathione peroxidase 4 (GPX4), and finally recovered the latter’s transcription after H/R stimulation in AC16 cardiomyocytes. In vivo experiment, SFN reversed abnormal expression of IRF1 and GPX4, inhibited cardiac ferroptosis, and improved myocardial dysfunction after CA and resuscitation in pigs. ConclusionsSFN could effectively improve myocardial dysfunction after CA and resuscitation, in which the mechanism was potentially related to the inhibition of cardiomyocytes ferroptosis through the regulation of Nrf2/IRF1/GPX4 pathway.

Virus Shedding and Diarrhea: A Review of Human Norovirus Genogroup II Infection in Gnotobiotic Pigs.

For nearly twenty years, gnotobiotic (Gn) pigs have been used as a model of human norovirus (HuNoV) infection and disease. Unique in their ability to develop diarrhea and shed virus post oral challenge, Gn pigs have since been used to evaluate the infectivity of several genogroup II HuNoV strains. Nearly all major pandemic GII.4 variants have been tested in Gn pigs, with varying rates of infectivity. Some induce an asymptomatic state despite being shed in large quantities in stool, and others induce high incidence of both diarrhea and virus shedding. Non-GII.4 strains, including GII.12 and GII.6, have also been evaluated in Gn pigs. Again, rates of diarrhea and virus shedding tend to vary between studies. Several factors may influence these findings, including age, dosage, biological host factors, or bacterial presence. The impact of these factors is nuanced and requires further evaluation to elucidate the exact mechanisms behind increases or decreases in infection rates. Regardless, the value of Gn pig models in HuNoV research cannot be understated, and the model will surely continue to contribute to the field in years to come.

Neuropathological mRNA Expression Changes after Single Mild Traumatic Brain Injury in Pigs.

Traumatic brain injury (TBI) is a public health concern, with an estimated 42 million cases globally every year. The majority of TBIs are mild TBIs, also known as concussion, and result from the application of mechanical forces on the head. Most patients make a complete recovery and mortality is rare; therefore, studies investigating cellular changes after mild TBI in a clinical setting are limited. To address this constraint, our group utilized a pig model of closed-head rotational acceleration-induced TBI, which recreated the biomechanical loading parameters associated with concussion on a large gyrencephalic brain similar to humans. While our previous research has focused on immunohistochemical characterization of neuropathology, the current study utilized transcriptomic assays to evaluate an array of TBI-induced neurodegenerative analytes. Pigs subjected to mild TBI were survived for 3 days post-injury (DPI) (n = 3), 30 DPI (n = 3), or 1 year post-injury (YPI) (n = 3) and compared to animals undergoing a sham procedure (n = 8). RNA was isolated from whole coronal sections of fixed tissue and multiplexed on a Nanostring neuropathology panel. Differential expression analysis revealed 11 differentially expressed genes at 3 DPI versus sham, including downregulation of the synaptotagmin calcium sensor gene (SYT1), upregulation of the neurofibromin gene (NF1), and upregulation of the Alzheimer's disease-associated receptor gene (SORL1). There were no differentially expressed genes at 30 DPI or 1 YPI compared to shams. Additionally, high-magnitude undirected global significance scores (GSS) were detected at 3 DPI for chromatin modification and autophagy gene sets, and at 30 DPI for cytokine gene sets, while many dysregulated gene sets were highlighted by directed GSSs out to 1 YPI. This study adds to a growing body of literature on transcriptomic changes in a clinically relevant large animal model of closed-head TBI, which highlights potential therapeutic targets following mild TBI.

Unraveling the impact of hyperleptinemia on female reproduction: insights from transgenic pig model

BackgroundInfertility is a growing global health concern affecting millions of couples worldwide. Among several factors, an extreme body weight adversely affects reproductive functions. Leptin is a well-known adipokine that serves as an endocrine signal between adiposity and fertility. However, the exact mechanisms underlying the effects of high leptin level on female reproduction remain unclear.MethodsTransgenic pigs overexpressing leptin (♀) were produced by backcrossing and screened for leptin overexpression. The growth curve, fat deposition, reproductive performance, apoptosis, serum hormones and cholesterol production, RNA sequencing, and single-nucleus RNA sequencing (snRNA-seq) of the leptin-overexpressing pigs and wild-type group were evaluated.ResultsTransgenic pigs overexpressing leptin (♀) were obtained, which exhibited significantly reduced body weight, body size, and back fat thickness. These pigs manifested a late onset of puberty (330 ± 54.3 vs. 155 ± 14.7 days), irregular estrous behavior characterized by increased inter-estrous interval (29.2 ± 0 vs. 21.3 ± 0.7 days), and more number of matings until pregnancy (at least 3 times). This reproductive impairment in leptin pigs was related to hormonal imbalances characterized by increased levels of FSH, LH, prolactin, E2, P4, and TSH, altered steroidogenesis such as increased levels of serum cholesterol esters along with steroidogenic markers (StAR, CYP19A), and ovarian dysfunctions manifested by neutrophilic infiltration and low expression of caspase-3 positive cells in the ovaries. Moreover, bulk RNA sequencing of the ovaries also revealed neutrophilic infiltration followed by upregulation of inflammation-related genes. Furthermore, snRNA-seq reflected that leptin overexpression triggered immune response, suppressed follicle development and luteinization, resulting in metabolic dysfunction and hormone imbalance in the ovary.ConclusionsLow body weight in leptin overexpressing pigs adversely affects the reproductive performance, causing delayed puberty, irregular estrous cycles, and reduced breeding efficiency. This is linked to metabolic imbalances, an increased immune response, and altered ovarian functions. This study provides a theoretical basis for the complex mechanisms underlying leptin, and infertility by employing leptin-overexpressing female pigs.

A Decrease in Autophagy Increases the Level of Collagen Type I Expression in Scleral Fibroblasts.

Autophagy dysregulation triggers extracellular matrix remodeling via changes in cellular collagen levels and protease secretion. However, the effect of autophagy on scleral extracellular matrix remodeling in the context of myopia is not fully understood. In this study, we measured the level of autophagy in sclera of form deprivation myopic guinea pigs; we also sought a correlation between the level of autophagy in human scleral fibroblasts and the extent of COL1A1 synthesis. We measured the level of COL1A1 expression and the levels of autophagic protein markers in scleral tissues in vivo using a form deprivation myopic guinea pig model. Rapamycin and chloroquine were respectively used to activate and inhibit autophagy in cultured human scleral fibroblasts. COL1A1 gene and protein expression levels were analyzed via quantitative real-time polymerase chain reaction, Western blotting, and immunofluorescence. Levels of autophagy-related proteins were assessed via Western blotting. The sclera of form deprivation myopic guinea pig eyes exhibited decreased expression of COL1A1 and increased expression level of autophagy. After chloroquine exposure, human scleral fibroblasts exhibited decreased autophagy and increased COL1A1 expression. Inhibition of scleral fibroblast autophagy increased COL1A1 expression at the gene and protein levels, thus explaining the effect of autophagy on collagen synthesis by scleral fibroblasts.

An orally administered glucose-responsive polymeric complex for high-efficiency and safe delivery of insulin in mice and pigs.

Contrary to current insulin formulations, endogenous insulin has direct access to the portal vein, regulating glucose metabolism in the liver with minimal hypoglycaemia. Here we report the synthesis of an amphiphilic diblock copolymer comprising a glucose-responsive positively charged segment and polycarboxybetaine. The mixing of this polymer with insulin facilitates the formation of worm-like micelles, achieving highly efficient absorption by the gastrointestinal tract and the creation of a glucose-responsive reservoir in the liver. Under hyperglycaemic conditions, the polymer triggers a rapid release of insulin, establishing a portal-to-peripheral insulin gradient-similarly to endogenous insulin-for the safe regulation of blood glucose. This insulin formulation exhibits a dose-dependent blood-glucose-regulating effect in a streptozotocin-induced mouse model of type 1 diabetes and controls the blood glucose at normoglycaemia for one day in non-obese diabetic mice. In addition, the formulation demonstrates a blood-glucose-lowering effect for one day in a pig model of type 1 diabetes without observable hypoglycaemia, showing promise for the safe and effective management of type 1 diabetes.

Circulating factors, in both donor and ex-vivo heart perfusion, correlate with heart recovery in a pig model of DCD

BackgroundHeart transplantation with donation after circulatory death and ex-situ heart perfusion offers excellent outcomes and increased transplantation rates. However, improved graft evaluation techniques are required to ensure effective utilization of grafts. Therefore, we investigated circulating factors, both in-situ and ex-situ, as potential biomarkers for cardiac graft quality. MethodsCirculatory death was simulated in anesthetized male pigs with warm ischemic durations of 0, 10, 20, or 30 min. Hearts were explanted and underwent ex-situ perfusion for 3h in an unloaded mode, followed by left ventricular loading for 1h, to evaluate cardiac recovery (outcomes). Multiple donor blood and ex-situ perfusate samples were used for biomarker evaluation with either standard biochemical techniques or nuclear magnetic resonance spectroscopy. ResultsCirculating adrenaline, both in the donor and at 10 min ex-situ heart perfusion, negatively correlated with cardiac recovery (p <0.05 for all). We identified several new potential biomarkers for cardiac graft quality that can be measured rapidly and simultaneously with nuclear magnetic resonance spectroscopy. At multiple timepoints during unloaded ex-situ heart perfusion, perfusate levels of acetone, betaine, creatine, creatinine, fumarate, hypoxanthine, lactate, pyruvate and succinate (p <0.05 for all) significantly correlated with outcomes; the optimal timepoint being 60 min. ConclusionsIn heart donation after circulatory death, circulating adrenaline levels are valuable for cardiac graft evaluation. Nuclear magnetic resonance spectroscopy is of particular interest, as it measures multiple metabolites in a short timeframe. Improved biomarkers may allow more precision and therefore better support clinical decisions about transplantation suitability.

Myosin Vb Traffics P-glycoprotein to the Apical Membrane of Intestinal Epithelial Cells

The xenobiotic efflux pump P-glycoprotein is highly expressed on the apical membrane of the gastrointestinal tract, where it regulates the levels of intracellular substrates. P-glycoprotein is altered in disease, but the mechanisms that regulate the levels of P-glycoprotein are still being explored. The molecular motor myosin Vb (Myo5b) traffics diverse cargo to the apical membrane of intestinal epithelial cells. We hypothesized that Myo5b was responsible for the delivery of P-glycoprotein to the apical membrane of enterocytes. We used multiple murine models that lack functional Myo5b or the myosin binding partner Rab11a to analyze P-glycoprotein localization. Pig and human tissue were analyzed to determine P-glycoprotein localization in the setting of MYO5B mutations. Intestinal organoids were used to examine P-glycoprotein trafficking and to assay P-glycoprotein function when MYO5 is inhibited. In mice lacking Myo5b or the binding partner Rab11a, P-glycoprotein was improperly trafficked and had decreased presence in the brush border of enterocytes. Immunostaining of a pig model lacking functional Myo5b and human biopsies from a patient with an inactivating mutation in Myo5b also showed altered localization of intestinal P-glycoprotein. Human intestinal organoids expressing the motorless MYO5B tail domain had colocalization with P-glycoprotein, confirming that P-glycoprotein was trafficked by MYO5B in human enterocytes. Inhibition of MYO5 in human intestinal cell lines and organoids resulted in decreased P-glycoprotein capacity. Additionally, inhibition of MYO5 in human colon cancer cells diminished P-glycoprotein activity and increased cell death in response to a chemotherapeutic drug. Collectively, these data demonstrate that Myo5b is necessary for the apical delivery of P-glycoprotein.

Airways Relaxant and Antiasthmatic Activity of Aconitum heterophyllum Wall ex Royle. Roots: A Mechanistic Insight.

Aconitum heterophyllum Wall ex Royle. (Ranunculaceae) is a traditional medicinal herb that has shown extensive pharmacological potential to treat cough, diarrhea, and infectious diseases but no scientific evidence is available to validate its antiasthmatic potential. In this study, we have investigated the tracheal relaxation and antiasthmatic activity of the selected bioactive fraction of A. heterophyllum. Chemical profiling of the most effective fraction obtained via bioassay-guided fractionation was done using LC-MS (Liquid chromatography-mass spectrometry, a technique utilized in the identification, separation, and quantification of known and unknown compounds). Molecular docking analysis of characterized constituents was performed to recognize the binding receptors, followed by an evaluation of the tracheal relaxation ability of active fraction. An acute oral toxicity study of the most effective fraction was done using OECD guidelines 423. Further, the therapeutic efficacy of the fraction was validated in asthma using a guinea pig model of ovalbumin (OVA) induced allergic asthma. The bio-guided activity revealed that hydro-methanolic extract of A. heterophyllum roots (F-1) was the most active fraction. LC-MS analysis of F-1 showed the presence of six major bioactive compounds in F-1. Molecular docking studies revealed strong binding affinities of identified constituents with histaminic receptor (H1) and muscarinic receptor (M3). The ex vivo study demonstrated smooth muscle relaxant activity of F-1 via dysregulating diverse signal transduction pathways viz. histaminic and muscarinic receptors antagonism (non-competitive), stimulation of β2-adrenergic receptor pathway, and soluble guanylyl cyclase activation. The findings of acute oral toxicity studies revealed that F-1 had no toxicity up to the dose of 2000 mg/Kg. The anti-asthmatic therapeutic efficacy of F-1 was further confirmed by the amelioration of respiratory hyperresponsiveness in asthmatic guinea pigs. This is the first evidence-based study showing the antiasthmatic therapeutic potential of the traditionally used herb A. heterophyllum through, computational and animal studies.

Stimulation of the internal superior laryngeal nerve as a potential therapy for obstructive sleep apnea in a porcine model.

Impaired pharyngeal sensing of negative pressure (NP) can lead to a blunted response of the upper airway dilator muscles and contribute to the development of obstructive sleep apnea (OSA). This response is modulated by the nerve fibers in the internal branch of the superior laryngeal nerve (iSLN), mediating negative pressure sensation. Artificial excitation of these fibers could be a potential treatment target for OSA. To evaluate this, electrostimulation of the iSLN was performed in a porcine-isolated upper airway model. Artificial obstructions were induced by varying the levels of negative pressure, and the ability of the animal to resolve these obstructions was evaluated. The pressure at which the animal was still able to resolve the obstruction was quantified as "Resolvable Pressure." Thereby, the effects on pharyngeal patency (n = 35) and the duration of the therapeutic effect outlasting the stimulation (n = 6) were quantified. Electrostimulation before the introduction of an artificial obstruction improved the median resolvable pressure from -28.3 cmH2O [IQR: -45.9; -26.1] to -92.6 cmH2O [IQR: -105.1; -78.6]. The median therapeutic effect was found to outlast the last stimulation burst applied by 163 s when five stimulation bursts were applied in short succession [IQR: 58; 231], 58 s when two were applied [IQR: 7; 65], and 6 s when one was applied [IQR: 0; 51]. Stimulation of the iSLN increased electromyography (EMG) in the genioglossus (GG). The proposed treatment concept can improve pharyngeal patency in the model. Transfer of the results to clinical application could enable the development of a new neuromodulation therapy for OSA.NEW & NOTEWORTHY Electrostimulation before the introduction of an artificial obstruction to induce artificial sleep apnea in the pig model improves the response of the upper airway to negative pressure (NP). The electrostimulation creates a sustained therapeutic effect that outlasts the initial electrostimulation. The use of this therapy in clinical practice has the potential to treat obstructive sleep apnea (OSA).