Minipig Research Articles

The Inhibition of Cell Proliferation of Bile Duct Scar Fibroblasts in Miniature Pigs by 5-fluorouracil and its Mechanism

The Inhibition of Cell Proliferation of Bile Duct Scar Fibroblasts in Miniature Pigs by 5-fluorouracil and its Mechanism

Species Difference of Asarinin Metabolism in vitro and its Effect on the Activity of Cytochrome P450 Enzymes

Aim: To investigate the metabolic behaviors of asarinin in different species and found animal models with metabolic pathways similar to those in humans and to study the effect (inhibition or induction) between asarinin and cytochrome P450 enzymes (CYP450s). Materials and Methods: The interspecies difference in asarinin metabolism was studied using liver microsomes (LMs) from humans and six mammals including rabbits, mini pigs, dogs, monkeys, rats, and mice. The potential drug–drug interactions (DDIs) between asarinin and cytochrome P450 enzymes was studied by incubating with six CYP enzymes in human liver microsomes (HLMs). Results: The metabolic profiles revealed substantially different metabolism of asarinin among the seven species. Moreover, the potential DDIs between asarinin and cytochrome P450 enzymes were studied in vitro by incubating with six CYP enzymes in HLMs, it was found that asarinin inhibited CYP2C9, CYP3A4, and CYP2E1 activity. Conclusion: The interspecies comparison can help find other species that have similar metabolic pathways to humans. There were potential DDIs between asarinin and some drugs metabolized by CYP2C9, CYP2E1, and CYP3A4.

Effects of Cryofrequency in the Different Swine Skin Tissue Layers: <i>In Vitro</i> and <i>in Vivo</i> Study

Introduction: Cryofrequency is a novel therapy which was developed with the combination of cold (cryotherapy) and heat (radiofrequency) application, causing a thermal shock effect on skin tissue. The thermal shock is believed to alter local metabolism. Objective: To investigate the effect of cryofrequency on the skin tissue of a mini pig in an in vitro and in vivo experimental model. Methodology: A small swine of the S. domesticus species, a male mini-pig (30 kg weight) was elected for the in vivo procedure subject. The in vitro step was performed on the extracted abdominal part of another subject of the same species and breed. Then, after the application of cryofrequency in the monopolar and bipolar form in vivo, a histological analysis was performed. Results: A higher temperature variation was observed comparing the pilot study in vitro with respect to the experimental one in vivo. Microscopically, an increase of collagen fibers and blood supply and decreased adipose tissue was observed after cryofrequency application. Conclusion: With the bipolar cryofrequency, no reduction of adipose tissue was observed, but regarding the connective tissue, extensive areas of collagen deposition were observed, as well as a great amount of new-formed collagen fibers.

Two dimensional automatic active shape model of degenerative disc repaired by low-intensity laser.

Intervertebral disc degeneration is the main factor causing low back pain, and the related long-term treatment can improve the situation of degeneration. This study aimed to investigate the effect of low-intensity laser irradiation on the repair of degenerative intervertebral disc by two dimensional automatic active shape model (2D-AASM). Nine Bama miniature pigs were randomly divided into three groups: control group (Con), model group (Mod) and laser treatment group (Las). After one month, the discs were treated with low-energy laser for another month. MRI was performed for one month, and the statistical shape model and 2D-AASM of intervertebral disc were established based on the minimum description length method. The model established by the proposed method is more accurate and the segmentation result is more accurate. From the segmented T2-weighted image, the signal intensity of the Mod group decreased significantly, and the signal intensity in the Las group was moderate and high compared with the Mod group. The HE staining display the structure of Con group was damaged, and the construction of Las group was restored compared with Mod group. The 2D-AASM method effectively improves the accuracy of intervertebral disc segmentation. The low-intensity laser has a protective effect on the repair of the degenerative intervertebral disc.

Impairment of CRH in the intestinal mucosal epithelial barrier of pregnant Bama miniature pig induced by restraint stress.

Female, especially for pregnant female, are vulnerable to psychological stress. The morphology and metabolism of the maternal intestine are both obviously changed during pregnancy, thus making intestinal health status more fragile under psychological stress. The aim of the present study was to investigate the role of CRH and CRHR1 in the pregnant maternal intestine under psychological stress, thus exploring the mechanism of psychological stress in the pregnant maternal intestine. Bama miniature pigs were divided into the control and restraint stress groups from the first day of pregnancy. After restraint stress treatment for 18 consecutive days (D18), the plasma, duodenum, jejunum, ileum and colon were collected for study. Pregnant Bama miniature pigs subjected to restraint stress had significantly elevated CRH, adrenocorticotropic hormone (ACTH) and cortisol (COR) levels in plasma. Consistent with the increase in CRH levels, we observed enhanced oxidative stress levels in the intestine, which resulted in intestinal mucosal injury, including impaired intestinal morphology, a reduced number of goblet cells and proliferating cell nuclear antigen-positive cells, decreased expression of MUC2 and tight junctions, and elevated expression of CRHR1 and caspase-3. Moreover, exogenous CRH could directly promote IPEC-J2 cell apoptosis and influence its cell cycle (S and G2 phase) through CRHR1, and antalarmin could alleviate this phenomenon. Therefore, our results illustrated that the intestinal dysfunction of pregnant Bama miniature pigs was caused by restraint stress, and these changes were associated with the enhanced expression of CRH and CRHR1 in the intestine.

Pathology of coronary artery after third-generation drug-eluting stent implantation in low-density lipoprotein receptor knockout mini pigs and human autopsy cases

Background and Aims: Third-generation drug-eluting stent (3rd DES) is widely used in percutaneous coronary intervention. Clinical superiority of 3rd DES has been demonstrated, however, pathological response after 3rd DES remains unclear. We aimed to examine the histology after 3rd DESs in low-density lipoprotein receptor knockout (LDLR -/-) mini pigs and human autopsy cases.

A Bama miniature pig model of monoallelic TSC1 mutation for human tuberous sclerosis complex

Tuberous sclerosis complex (TSC) is a dominant genetic neurocutaneous syndrome characterized by multiple organ hamartomas. Although rodent models bearing a germline mutation in either TSC1 or TSC2 gene have been generated, they do not develop pathogenic lesions matching those seen in patients with TSC because of the significant differences between mice and humans, highlighting the need for an improved large animal model of TSC. Here, we successfully generate monoallelic TSC1-modified Bama miniature pigs using the CRISPR/Cas9 system along with somatic cell nuclear transfer (SCNT) technology. The expression of phosphorylated target ribosomal protein S6 is significantly enhanced in the piglets, indicating that disruption of a TSC1 allele activate the mechanistic target of rapamycin (mTOR) signaling pathway. Notably, differing from the mouse TSC models reported previously, the TSC1+/− Bama miniature pig developed cardiac rhabdomyoma and subependymal nodules, resembling the major clinical features that occur in patients with TSC. These TSC1+/− Bama miniature pigs could serve as valuable large animal models for further elucidation of the pathogenesis of TSC and the development of therapeutic strategies for TSC disease.

Passage of spermatozoa through the epididymis of the boar (Sus scrofa domesticus)

Previous studies of epididymal transit in the pig have had insufficient animal numbers to provide a comprehensive picture of a continuous process. The present study attempts to addresses this issue. Radioactively labeled thymidine was infused into the testicular arteries of 48 sexually rested young adult boars of the Goettingen Miniature Pig breed. Hemicastrations were performed in random order in 2 animals daily on Days 21–24 following thymidine injection and in 4 animals daily on Days 25–41 and 43, 45, 47 and 49. Sperm obtained from 12 epididymal sites between the proximal caput and distal cauda were autoradiographically examined to record the percentage of labeled sperm and labeling intensity at different times after thymidine infusion. An initial surge of labeled spermatozoa emerged in the proximal caput 28 days after thymidine injection. After 2 d labeled sperm had arrived at the distal caput and, after another 2, the bulk of labeled sperm was found in the corpus. From there the sperm advanced to the transition of corpus and cauda, where progress was arrested until Day11. On Day 12, transit was resumed and by Day 13 sperm had passed through the cauda and vacated the epididymis via the ductus deferens.

Pathology of coronary artery after drug-eluting stent implantation in low-density lipoprotein receptor knockout mini pigs; comparison with wild type pigs

Pathology of coronary artery after drug-eluting stent implantation in low-density lipoprotein receptor knockout mini pigs; comparison with wild type pigs

LC-MS/MS quantification of progesterone in ovariectomized miniature swine and its application for preliminary pharmacokinetic study of a novel vaginal sustained release system

Objective We here established a simple, fast, robust and sensitive LC-MS/MS method and validated as well for the quantitative analysis of progesterone (PGT) in ovariectomized (OVX) miniature swine plasma. The method was successfully applied to characterize the pharmacokinetics of a progesterone vaginal drug delivery system. Methods Megestrol acetate was utilized as an internal standard (IS). The separation and detection of PGT from endogenous interference was performed successfully by liquid chromatography with gradient elution and mass spectrometry equipped with positive ESI source using MRM mode. The EVA intravaginal rings (IVRs) were manufactured by hot-melt extrusion (HME), afterward were administrated vaginally to OVX minipigs to evaluate PK study. Results The calibration curve for swine plasma samples across the concentration ranged between 0.25 ng/mL and 100 ng/mL. The intra- and inter-assay accuracy and precision were lower than ±5% and 5.88%, respectively. Recoveries of PGT and IS were ranging from 114–119% and 96.5–112%, respectively. In vitro study showed that the EVA IVRs release 18 mg/day of PGT continuously over 7 days, and corresponding mean PGT plasma concentration in OVX minipigs (CAVG) was 4.892 ng/mL. Conclusion All the study produced reliable results for the measurement of PGT concentration in miniature swine plasma and the method was successfully applied to a PK study for PGT vaginal ring in miniature pigs, which may lay the foundation for further research on the progesterone preparations intended for in assisted reproductive technology.

Testosterone deficiency accelerates early stage atherosclerosis in miniature pigs fed a high-fat and high-cholesterol diet: urine 1H NMR metabolomics targeted analysis.

To gain insights into the role of testosterone in the development of atherosclerosis and its related metabolic pathways, we applied a proton nuclear magnetic resonance (1H NMR)-based metabolomics approach to investigate urine metabolic profiles in miniature pigs fed a high-fat and high-cholesterol (HFC) diet among intact male pigs (IM), castrated male pigs (CM) and castrated male pigs with testosterone replacement (CMT). Our results showed that testosterone deficiency significantly increased atherosclerotic lesion areas, intima-media thickness, as well as serum lipid levels in the CM pigs. Moreover, seventeen significantly changed metabolites were identified in both IM vs. CM and CMT vs. CM groups. Among these, seven were shared between the two comparative groups and were all significantly reduced in the urine of the CM group but rescued in the CMT group. In addition, the correlation analysis demonstrated that several metabolites, including niacinamide, myo-inositol, choline and 3-hydroxyisovalerate, were negatively correlated with atherosclerotic lesion areas. Our study demonstrated that testosterone deficiency accelerated early AS formation in HFC diet-fed pigs, which involved several metabolites predominantly related to lipid metabolism, inflammation, oxidative stress and endothelial disorders. Our results reveal potential pathways in the pathogenesis of atherosclerosis caused by testosterone deficiency and HFC diet.

Establishment and Characterization of Immortalized Miniature Pig Pancreatic Cell Lines Expressing Oncogenic K-RasG12D.

In recent decades, many studies on the treatment and prevention of pancreatic cancer have been conducted. However, pancreatic cancer remains incurable, with a high mortality rate. Although mouse models have been widely used for preclinical pancreatic cancer research, these models have many differences from humans. Therefore, large animals may be more useful for the investigation of pancreatic cancer. Pigs have recently emerged as a new model of pancreatic cancer due to their similarities to humans, but no pig pancreatic cancer cell lines have been established for use in drug screening or analysis of tumor biology. Here, we established and characterized an immortalized miniature pig pancreatic cell line derived from primary pancreatic cells and pancreatic cancer-like cells expressing K-rasG12D regulated by the human PTF1A promoter. Using this immortalized cell line, we analyzed the gene expression and phenotypes associated with cancer cell characteristics. Notably, we found that acinar-to-ductal transition was caused by K-rasG12D in the cell line constructed from acinar cells. This may constitute a good research model for the analysis of acinar-to-ductal metaplasia in human pancreatic cancer.

Porcine skin damage threshold from mid-infrared optical parametric oscillator radiation at 3.743 µm.

There is increasing use of mid-infrared optical parametric oscillator radiation operating in the wavelength range of 3-5 µm. To expand existing damage data for skin exposure to lasers in this wavelength region, the in-vivo damage threshold at the wavelength of 3.743 µm was determined in a Guizhou miniature pig model for an exposure duration of 1.0 s. The irradiance of the laser spot was nearly Gaussian-distributed and the 1/e2 beam diameter on the animal skin surface was fixed at 0.94 and 0.88 cm along horizontal and vertical directions. Damage lesion determinations were performed at 1- and 24-hour post-exposure. The probit analysis was employed to establish the ED50 values. The ED50 expressed in peak radiant exposure for the Gaussian spot was 4.04 J/cm2 at the 24-hour post-exposure. Sufficient margin existed between the damage threshold and MPE from the current laser safety standard. The obtained data may contribute to the knowledge base for refinement of laser safety standard in the wavelength range of 2.6-1000 µm.

A novel cryo-embedding method for in-depth analysis of craniofacial mini pig bone specimens

The disconnect between preclinical and clinical results underscores the imperative for establishing good animal models, then gleaning all available data on efficacy, safety, and potential toxicities associated with a device or drug. Mini pigs are a commonly used animal model for testing orthopedic and dental devices because their skeletons are large enough to accommodate human-sized implants. The challenge comes with the analyses of their hard tissues: current methods are time-consuming, destructive, and largely limited to histological observations made from the analysis of very few tissue sections. We developed and employed cryo-based methods that preserved the microarchitecture and the cellular/molecular integrity of mini pig hard tissues, then demonstrated that the results of these histological, histochemical, immunohistochemical, and dynamic histomorphometric analyses e.g., mineral apposition rates were comparable with similar data from preclinical rodent models. Thus, the ability to assess static and dynamic bone states increases the translational value of mini pig and other large animal model studies. In sum, this method represents logical means to minimize the number of animals in a study while simultaneously maximizing the amount of information collected from each specimen.

Lentilactobacillus kribbianus sp. nov., isolated from the small intestine of a mini pig.

A Gram-stain-positive, facultative anaerobic, rod-shaped bacteria isolated from the small intestine of a mini pig was designated as strain YH-lac9T. 16S rRNA gene sequence analysis revealed that the strain belongs to the genus Lentilactobacillus and is closely related to Lentilactobacillus senioris JCM 17472T, Lentilactobacillus rapi JCM 15042T and Lentilactobacillus diolivorans JCM 13927T, with 97.6, 96.2 and 95.7 % sequence similarity, respectively. Analysis of housekeeping gene sequences (pheS and recA) revealed that the strain formed a sub-cluster with L. senioris, supporting the results of 16S rRNA gene sequences analysis. The average nucleotide identity value for YH-lac9T and the most closely related strain is 74.1 %. The main fatty acids are C18 : 1ω9c, summed feature 7, C16 : 0 and summed feature 8. The G+C content of the genomic DNA is 37.8 mol%. In view of its chemotaxonomic, phenotypic and phylogenetic properties, YH-lac9T (=KCTC 25005=JCM 33997) represents a novel taxon. The name Lentilactobacillus kribbianus sp. nov. is proposed.

Novel imaging-based approaches for predicting the hepatic venous pressure gradient in a porcine model of liver cirrhosis and portal hypertension

AimsHepatic venous pressure gradient (HVPG) is critical for staging and prognosis prediction of portal hypertension (PH). However, HVPG measurement has limitations (e.g., invasiveness). This study examined the value of non-invasive, imaging-based approaches including magnetic resonance elastography (MRE) and intravoxel incoherent motion (IVIM) diffusion-weighted imaging (DWI) for the prediction of HVPG in a porcine model of liver cirrhosis and PH. Main methodsMale Bama miniature pigs were used to establish a porcine model of liver cirrhosis and PH induced by embolization. They were randomly assigned to an experimental group (n = 12) and control group (n = 3). HVPG was examined before and after transjugular intrahepatic portosystemic shunt (TIPS). MRE and IVIM-DWI were performed to obtain quantitative parameters including liver stiffness (LS) in MRE, tissue diffusivity (D), pseudo-diffusion coefficient (D*), and perfusion fraction (f) in IVIM-DWI. The correlation between HVPG and the parameters was assessed. Key findingsLS values were significantly greater in the experimental group, while f values were significantly decreased at 4, 8, and 12 weeks after embolization compared to the control group. Furthermore, HVPG was significantly lower immediately after versus before TIPS. In parallel, LS and f values showed significant alterations after TIPS, and these changes were consistent with a reduction in HVPG. Spearman analysis revealed a significant correlation between the parameters (LS and f) and HVPG. The equation was eventually generated for prediction of HVPG. SignificanceThe findings show a good correlation between HVPG and the quantitative parameters; thus, imaging-based techniques have potential as non-invasive methods for predicting HVPG.

Bone tissue engineering in the greater omentum with computer-aided design/computer-aided manufacturing scaffolds is enhanced by a periosteum transplant.

Aim: This study aimed to evaluate two different vascularized bone flap scaffolds and the impact of two barrier membranes for the reconstruction of critical-size bone defects. Materials & methods: 3D-printed scaffolds of biodegradable calcium phosphate and bioinert titanium were loaded with rhBMP-2bone marrow aspirate, wrapped by a collagen membrane or a periosteum transplant and implanted into the greater omentum of miniature pigs. Results: Histological evaluation demonstrated significant bone formation within the first 8weeks in both scaffolds. The periosteum transplant led to enhanced bone formation and a homogenous distribution in the scaffolds. The omentum tissue grew out a robust vascular supply. Conclusion: Endocultivation using 3D-printed scaffolds in the greater omentum is a very promising approach in defect-specific bone tissue regeneration.

Burn wound assessment system using near-infrared hyperspectral imaging and deep transfer features

The instant and accurate burn assessment is very important for burn wound treatment. But due to the limited burn wound samples, the accurate classification of burn wounds is difficult. In this work, a full-field burn wound classification system is proposed using the near-infrared hyperspectral imaging (NIHSI) with the deep transfer features. The convolutional neural network (CNN) is applied to establish a nonlinear relationship between the hyperspectral image data and the burn severity. And the transfer learning strategy is used to boost the high classification accuracy. The burn wounds are created on bilateral sides of the abdomen of a bama miniature pig. The burn depth model (BdasNet) is pre-trained based on side A, and then fine-tuned with data of side B. The proposed model BdasNet can get the classification accuracy more than 96% for burn depth, which demonstrated the proposed method can perform an accurate classification of burn wounds and provide more practical references for clinicians.

Pancreatic GLP-1r binding potential is reduced in insulin-resistant pigs

IntroductionThe insulinotropic capacity of exogenous glucagon like peptide-1 (GLP-1) is reduced in type 2 diabetes and the insulin-resistant obese. We have tested the hypothesis that this response is the consequence...

P.695 The involvement of GPR39 (zinc receptor) in epileptogenesis – studies in knockout mice

The instant and accurate burn assessment is very important for burn wound treatment. But due to the limited burn wound samples, the accurate classification of burn wounds is difficult. In this work, a full-field burn wound classification system is proposed using the near-infrared hyperspectral imaging (NIHSI) with the deep transfer features. The convolutional neural network (CNN) is applied to establish a nonlinear relationship between the hyperspectral image data and the burn severity. And the transfer learning strategy is used to boost the high classification accuracy. The burn wounds are created on bilateral sides of the abdomen of a bama miniature pig. The burn depth model (BdasNet) is pre-trained based on side A, and then fine-tuned with data of side B. The proposed model BdasNet can get the classification accuracy more than 96% for burn depth, which demonstrated the proposed method can perform an accurate classification of burn wounds and provide more practical references for clinicians.