Development of osteoporosis animal model using micropigs / 한국실험동물학회지

Osteoporosis is a known major health problem and a serious disease of the bone, there has been a great need to develop more and newer animal models for this disease. Among animal models used for testing drug efficacy, the minipig model has become useful and effective due to its close similarity with humans (validity), particularly with the pharmacokinetics of compounds via subcutaneous administration, the structure and function of the organs, the morphology of bone and the overall metabolic nature. Based on these advantages, we sought to develop a new animal model of osteoporosis using micropig, which differs from other miniature pigs in the genetic background. Female micropigs were used for the induction of a moderate osteoporosis model by bilateral ovariectomy (OVX) and compared with shamoperated animals. For osteoporosis evaluation, clinical biomarkers such as blood osteocalcin (OSC) and parathyroid hormone (PTH) levels were measured, as well as bone mineral density (BMD) using micro-computed tomography (micro-CT). Compared to sham, OVX animals have decreased blood OSC level, while the blood PTH level increased in blood sera. In addition, we observed the significantly decreased BMDs of tibia region in OVX animals. Based on these results, we report that the micropig model developed in this study can be used to develop a new and effective medical method for diagnosis and treatment of osteoporosis.

Human leukocytes regulate ganglioside expression in cultured micro-pig aortic endothelial cells / 한국실험동물학회지

Gangliosides are ubiquitous components of the membranes of mammalian cells that are thought to play important roles in various cell functions such as cell-cell interaction, cell adhesion, cell differentiation, growth control, and signaling. However, the role that gangliosides play in the immune rejection response after xenotransplantation is not yet clearly understood. In this study, the regulatory effects of human leukocytes on ganglioside expression in primary cultured micro-pig aortic endothelial cells (PAECs) were investigated. To determine the impact of human leukocytes on the expression of gangliosides in PAECs, we performed high-performance thin layer chromatography (HPTLC) in PAECs incubated with FBS, FBS containing human leukocytes, human serum containing human leukocytes, and FBS containing TNF-alpha. Both HPTLC and immunohistochemistry analyses revealed that PAECs incubated with FBS predominantly express the gangliosides GM3, GM1, and GD3. However, the expression of GM1 significantly decreased in PAECs incubated for 5 h with TNF-alpha (10 ng/mL), 10% human serum containing human leukocytes, and 10% FBS containing human leukocytes. Taken together, these results suggest that human leukocytes induced changes in the expression profile of ganglioside GM1 similar to those seen upon treatment of PAECs with TNF-alpha. This finding may be relevant for designing future therapeutic strategies intended to prolong xenograft survival.

Contrast-enhanced ultrasound analysis of renal perfusion in normal micropigs

Contrast-enhanced ultrasound is one of method for evaluating renal perfusion. The purpose of this project was to assess perfusion patterns and dynamics in normal micropig kidney using ultrasonographic contrast media. Eight young healthy micropigs were included in this study. Micropigs were anesthetized with propofol and received an intravenous bolus of microbubble contrast media through an ear vein. Time/mean pixel value (MPV) curves were generated for selected regions in the right renal cortex and medulla. The parenchyma was enhanced in two phases. The cortex was first enhanced followed by a more gradual enhancement of the medulla. A significant difference in perfusion was detected between the cortex and medulla. Following the bolus injection, the average upslope was 0.68 +/- 0.27 MPV/sec, downslope was -0.27 +/- 0.13 MPV/sec, baseline was 73.9 +/- 16.5 MPV, peak was 84.6 +/- 17.2 MPV, and time-to-peak (from injection) was 17.5 +/- 6.6 sec for the cortex. For the medulla, the average upslope was 0.50 +/- 0.24 MPV/sec, downslope was -0.12 +/- 0.06 MPV/sec, baseline was 52.7 +/- 7.0 MPV, peak was 65.2 +/- 9.3 MPV, and time-to-peak (from injection) was 27.5 +/- 5.0 sec. These data can be used as normal reference values for studying young micropigs.

Cerebellar maturation ratio of forebrain and brainstem at magnetic resonance imaging in the micropig / 대한수의학회지

The study of pigs as a human disease model has been conducted in neuroscience. But the morphological development of pig brain by using MRI is rare. The purpose of this study is to determine whether cerebellum maintains consistent proportion to other brain regions in aging. Clinically healthy sixteen micropigs, 1, 2, 4, and 8 months were studied. The micropigs were anesthetized with isoflorane. MRI was acquired using a 0.3T system. To figure out development of ratio that allowed identification of normal cerebellum size, we measured the area of the cerebellum, brainstem, and forebrain from the mid-sagittal brain images on T1W. Mid-sagittal cross-sectional area (CSA) of total brain, forebrain, brainstem, and cerebellum were expressed as absolute values and also as percentages which were compared between the four age groups of micropigs for the purpose to define the effect of age on brain morphometry. It was found that there was not a significant difference in the percentage of the brain occupied by an individual region between groups although the absolute CSA differed significantly among age groups. There was no effect of age on the ratio between the cerebellum and total brain in 4 age groups. The normal size of cerebellum changes during brain development maintained a consistent ratio to other brain regions in normal micropigs. The ratio of CSA quantified on the mid-sagittal MR images offers a suitable method to detect presence of cerebellar anomalies in micropigs.

Development of a type II diabetic mellitus animal model using Micropig(R) / 한국실험동물학회지

Diabetes, which has shown an explosive increase in terms of its incidence, is regarded as a serious disease that must be overcome for the sake of human life. Among animal models used for testing of drug efficacy, the mini-pig model has shown a rapid upload due to its many similarities with human, particularly concerning the pharmacokinetics of compounds after subcutaneous administration, the structure and function of the gastrointestinal tract, the morphology of the pancreas, and overall metabolic status. Based on these various advantages, we sought to develop an animal model of type II diabetic mellitus using the Micro-pig, which differs from other miniature pigs. We used six male Micro-pigs for induction of a moderate insulin deficient model with nicotinamide (NIA)/streptozotocin (STZ) treatment and three animals for control. For evaluation of incidence of type II diabetes, we measured blood glucose level, and performed oral glucose tolerance test and immunohistochemistry on pancreatic tissue using insulin antibody. Compared to control animals, all animals treated with NIA/STZ showed high levels of glucose and low levels of insulin. In addition, we observed the partially destroyed beta cell population from tissue of the pancreas in treated animals. Based on these results, we report that the Micro-pig model developed in this study can be used for testing of the efficacy of therapeutic agents for treatment of Type 2 diabetic mellitus.

Characteristics of Wound Contraction according to the Shape and Antomical Regions of the Wound in Porcine Model

PURPOSE: The shape and location, the amount of the wound and the characteristics of the remaining tissues are known to influence wound contraction. The previous studies using small animals have not been an appropriate model because the wound healing mechanisms and skin structures are different from those of the human. The purpose of this study is to evaluate wound contraction according to the shape and location of the wound using a Micropig(R). METHODS: Four Micropigs(R)(Medikinetics, Pyeongtaek, Korea) that were 10 months of age weighed 25kg were used. Full thickness skin defects were made by clearing all the tissues above the fascial layer in the shape of square, a regular triangle and a circle of 9cm2 each on the back around the spine. Eight wounds were created on the back of each pig, 50mm apart from each other. The randomly chosen wound shapes included 11 squares, 11 regular triangles, and 10 circles. Wound dressing was done every other day with polyurethane foam. The wound size was measured using a Visitrak Digital(R)(Smith & Nephew, Hull, UK) on every other day after surgery from day 2 to day 28. A biopsy was performed on day 3, and 1, 2, 3 and 4 weeks to investigate the degree of acute and chronic inflammation, the number of microvesssel and myofibroblast density using H & E stain and immunohistochemistry. The wound contraction rate was calculated to figure out the differences among each of the shapes and the locations. RESULTS: The ultimate shape of the circle wound was oval, and that of the regular triangle and square were stellate. The maximum contraction rate was obtained on 8 to 10 days for all the shapes, which corresponds with the immunohistochemical finding that myofibroblast increases in the earlier 2 weeks whereas it decreases in the later 2 weeks. Epithelialization was seen in the wound margin on day 7 and afterwards. The final wound contraction rates were highest for the regular triangle shapes; however, there were no statistically significant differences. The wound contraction rates by locations showed statistically significant differences. The wound in the cephalic area presented more contractions than that of the wounds in the caudal area. CONCLUSION: The location of a wound is more important factor than the wound shape in wound contraction.

Multidetector computed tomographic angiography evaluation of micropig major systemic vessels for xenotransplantation

Due primarily to the increasing shortage of allogeneic donor organs, xenotransplantation has become the focus of a growing field of research. Currently, micropigs are the most suitable donor animal for humans. However, no standard method has been developed to evaluate the systemic vascular anatomy of micropigs and standard reference values to aid in the selection of normal healthy animals as potential organ donors are lacking. Using 64-channel multidetector row computed tomographic angiography (MDCTA), we evaluated morphological features of the major systemic vessels in micropigs and compared our results to published human data. The main vasculature of the animals was similar to that of humans, except for the iliac arterial system. However, diameters of the major systemic vessels were significantly different between micropigs and humans. Specifically, the diameter of the aortic arch, abdominal aorta, external iliac artery, and femoral artery, were measured as 1.50 +/- 0.07 cm, 0.85 +/- 0.06 cm, 0.52 +/- 0.05 cm, and 0.48 +/- 0.05 cm, respectively, in the micropigs. This MDCTA data for micropig major systemic vessels can be used as standard reference values for xenotransplantation studies. The use of 64-channel MDCTA enables accurate evaluation of the major systemic vasculature in micropigs.

64-Channel multi-detector row CT angiographic evaluation of the micropigs for potential living donor lung transplantation

Micropigs are the most likely source animals for xenotransplantation. However, an appropriate method for evaluating the lung of micropigs had not been established. Therefore, this study was performed to evaluate the feasibility of 64-channel multi-detector row computed tomography (MDCT) to measure the diameter of the pulmonary arteries and the lung volume in micropigs. The mean diameters of the trachea, and left and right bronchi were 1.6 +/- 0.17, 1.18 +/- 0.14, and 1.1 +/- 0.11 cm, respectively. The mean diameters of the main, right, and left pulmonary arteries were 1.38 +/- 0.09, 1.07 +/- 0.26, and 0.98 +/- 0.13 cm and the diameters of right, left, and common inferior pulmonary veins were 0.97 +/- 0.20, 0.76 +/- 0.20, and 1.99 +/- 0.26 cm, respectively. The mean lung volume was 820.3 +/- 77.11 mL. The data presented in this study suggest that the MDCT may be a noninvasive, rapid, and accurate investigational method for pulmonary anatomy in living lung donors.

Multidetector row computed tomography evaluation of the micropig kidney as a potential renal donor

Multidetector row computed tomography (MDCT) provides anatomical information about the kidney and other internal organs. Presently, the suitability of 64-channel MDCT to assess the kidney of healthy micropigs was evaluated. Morphological evaluations of the kidney and the major renal vessels of six healthy micropigs were carried out using MDCT, recording kidney volume and the diameter and length of renal arteries and veins. The mean diameters and lengths of the renal artery were 0.44 +/- 0.05 and 4.51 +/- 0.55 cm on the right side and 0.46 +/- 0.06 and 3.36 +/- 0.27 cm on the left side, respectively. The mean diameters and lengths of the renal vein were 1.44 +/- 0.52 and 4.22 +/- 1.29 cm on the right side and 1.38 +/- 0.17 and 5.15 +/- 0.87 cm on the left side, respectively. The mean volume of the right kidney was 79.3 +/- 14.5 mL and of the left kidney was 78.0 +/- 13.9 mL. The data presented in this study suggest that the MDCT offers a noninvasive, rapid, and accurate method for the evaluation of the renal anatomy in living kidney donors. It also provides sufficient information about extra-renal anatomy important for donor surgery and determination of organ suitability.

Imaging evaluation of the liver using multi-detector row computed tomography in micropigs as potential living liver donors

The shortage of organ donors has stimulated interest in the possibility of using animal organs for transplantation into humans. In addition, pigs are now considered to be the most likely source animals for human xenotransplantation because of their advantages over non-human primates. However, the appropriate standard values for estimations of the liver of micropigs have not been established. The determination of standard values for the micropig liver using multi-detector row computed tomography (MDCT) would help to select a suitable donor for an individual patient, determine the condition of the liver of the micropigs and help predict patient prognosis. Therefore, we determined the standard values for the livers of micropigs using MDCT. The liver parenchyma showed homogenous enhancement and had no space-occupying lesions. The total and right lobe volumes of the liver were 698.57 +/- 47.81 ml and 420.14 +/- 26.70 ml, which are 51.74% and 49.35% of the human liver volume, respectively. In micropigs, the percentage of liver volume to body weight was approximately 2.05%. The diameters of the common hepatic artery and proper hepatic artery were 6.24 +/- 0.20 mm and 4.68 +/- 0.13 mm, respectively. The hepatic vascular system of the micropigs was similar to that of humans, except for the variation in the length of the proper hepatic artery. In addition, the diameter of the portal vein was 11.27 +/- 0.38 mm. In conclusion, imaging evaluation using the MDCT was a reliable method for liver evaluation and its vascular anatomy for xenotransplantation using micropigs.

Comparison of cardiac function and coronary angiography between conventional pigs and micropigs as measured by multidetector row computed tomography

Pigs are the most likely source animals for cardiac xenotransplantation. However, an appropriate method for estimating the cardiac function of micropigs had not been established. Computed tomography (CT) analysis aimed at estimating cardiac function and assessing the coronary arteries has not been carried out in micropigs. This study determined the feasibility of evaluating cardiac function in a micropig model using multidetector row computed tomography (MDCT) and compared the cardiac function values with those of conventional pigs. The mean age of the conventional pigs and micropigs was approximately 80 days and approximately 360 days, respectively. The mean body weight in the conventional pigs and micropigs was 29.70 +/- 0.73 and 34.10 +/- 0.98 kg, respectively. Cardiac MDCT detected ejection fractions of 52.93 +/- 3.10% and 59.00 +/- 5.56% and cardiac outputs of 1.46 +/- 0.64 l/min and 1.21 +/- 0.24 l/min in conventional pigs and micropigs, respectively. There were no significant differences in cardiac function between conventional pigs and micropigs in the reconstructed CT images. There were also no differences in the coronary angiographic images obtained by MDCT. It is expected that the results of this study will help improve understanding of cardiac function in micropigs. The data presented in this study suggest that MDCT is a feasible method for evaluating cardiac function in micropigs.

Effectiveness of 99mTc-tetrofosmin for assessment of heart functions in micropigs

This study examined the suitability of a nuclear imagingtechnique using 99mTc-tetrofosmin as an agent to assess theheart functions of healthy micropigs. The mean age of thepigs was 360 days (male), and the mean body weight was35.3kg ranging from 34.5-36kg. There were no significantperfusion defects in any of the reconstructed images.Gated single-photon emission computed tomographyimaging can be used to calculate the ventricular volumeand ejection fraction (EF). In this case, an EF of 79% wascalculated from the ventricular volume of the end-systolicimage (10 ml) subtracted from that of the end-diastolicvolume (49 ml). A perfusion defect (particularly the apex,lateral wall) is unlikely because of the presence of apreserved wall motion in a segment with a defect. It isconcluded that quantitative cardiac scintigraphy, using99mTc-tetrofosmin is an adequate technique for estimatingthe heart functions of healthy micropigs.

Comparative analysis of heart functions in micropigs and conventional pigs using echocardiography and radiography

The production of miniature animals has been suggested for use in organ transplantation. At present, many of the studies about application of animal organs to human have been focused on pigs because of the number of advantages involved and due to their similarities with human. However, a physiological analysis of the organs to be transplanted has not yet been carried out. Therefore, this study analyzed whether or not there were physiological and morphological differences in the hearts of conventionallyreared pigs and micropigs. In this study, the morphological and physiological functions of the heart were examined using radiographic and echocardiographic equipment. In the lateral radiographic view, the heart of the micropig has a larger cardiac long axis : short axis ratio than does the conventional pig, but the difference in the vertebral heart score was not significant. In addition, there were no morphological differences on the X-ray fluoroscopic view. There were no differences in echocardiographic values, except for several values in the left ventricle traces. Overall, it is expected that the values measured in this study will contribute to understanding of the physiological characteristics of micropigs.