Use of Miniature Pig for Biomedical Research, with Reference to Toxicologic Studies

J Toxicol Pathol 2007; 20: 125–132

Review Use of Miniature Pig for Biomedical Research, with Reference to Toxicologic Studies

Tetsuo Nunoya1, Kazumoto Shibuya1, Toshiki Saitoh1, Hajime Yazawa1, Keigo Nakamura1, Yasuko Baba1, and Takuya Hirai1

1Nippon Institute for Biological Science, 9–2221–1 Shinmachi, Ome, Tokyo 198–0024, Japan

Abstract: The pigs have been a well-recognized experimental animal in biomedical research for many years. Minipigs particularly have gained in massive importance in biomedical research over the last few years. Pigs are increasingly being used as an alternative non-rodent animal species to the dog or monkey in toxicology because of the morphological and physiological similarities between porcine and human organs, especially the skin, cardiovascular system, gastrointestinal tract, and the urinary system. Accumulating data indicate that the minipig can be used for all routes of administration and is preferable to the dog or monkey in many cases. The advantages of the minipig compared to the domestic pig are its smaller size, even at full maturity, slower growth during studies, ease of handling, and controlled genotype as well as microbiologically obvious characteristics. Minipigs also have an advantage over traditional non- rodent animals because of increasing ethical concerns about the use of them in experiments. Reservoir of information from studies using minipigs is the keystone for the future diffusion of them as a good alternative to the non-rodent animals traditionally used in toxicology. (J Toxicol Pathol 2007; 20: 125–132) Key words: miniature pig, animal model, biomedical research, toxicology

Introduction and regulatory studies are liable to be avoided as far as possible from the perspective of animal protection and their A crucial prerequisite for the development of safe rising costs. Along with the increased use of pigs in preclinical protocols in biomedical research is to ensure the research, technical advancements in surgery, anesthesia, suitability of animal models that would allow human-related husbandry and handling of the animal as well as improving validation of the valuable research information gathered the humane care have accelerated making it easier to use pigs from experimentation with them. Among animal models, in research institutions. Compared with the domestic pig, the pig has played a significant role in the scientific the advantages of the minipig are its ease of handling community as an alternative non-rodent species to the dog or because of its inherently smaller size, even at full maturity, monkey. The miniature pigs (minipigs) in particular have which is particularly suitable for long-term studies, and its gained increasing importance in the biomedical research well characterized and controlled genotype as well as areas of radiology, traumatology, dermatology, pathology, phenotype. Accordingly minipigs such as Yucatan, Hanford embryology, gastroenterology, cardiology, nephrology, and Göttingen have been widely used for chronic studies, in surgery, pediatrics, and odontology over the last few years. which the significantly rapid growth of domestic breeds More recently, minipigs have been used for such fields of would be an issue. Minipigs enable collection of larger investigation as the generation of organs with human volumes of multiple samples of body fluids and biopsies transgene expression for xenotransplantation1, and compared to rodents, thus making it possible to conduct neurotransplantation using porcine neural stem cells grafted studies that approximate to those performed in humans. into inbred miniature pigs as an alternative model to non- Minipigs are mild and good in nature, and the commercially human primates xenografted with human cells2. Behind the available ones tend to be more tractable than the domestic research trend is the fact that the anatomy of pig’s organ breeds raised in an agricultural setting, allowing successful systems, as well as physiological and pathophysiological performance of experiments under unstressed conditions. responses, has been found similar to those of the human3 and Minipigs are bred and raised under strictly controlled that monkeys and dogs widely used for drug development conditions including a defined environment, restricted access to food, and microbiological monitoring.

Received: 1 May 2007, Accepted: 14 May 2007 General Considerations Mailing address: Tetsuo Nunoya, Nippon Institute for Biological Science, 9–2221–1 Shinmachi, Ome, Tokyo 198–0024, Japan The pig (Sus scrofa domestica) has been widely used in TEL: 81-428-33-1059 FAX: 81-428-31-6166 E-mail: [email protected] biomedical research for decades because of its 126 Miniature Pig in Toxicology morphological and physiological similarities to the human, Types of Minipigs e.g. with respect to the cardiovascular, integumentary, digestive, and urinary systems. Most of the pigs used as There are several breeds of minipigs available in the experimental animals have been small domestic breeds, but US, Europe and Japan. The well known breeds are the the domestic pig was found unsuitable for specific purposes Yucatan16, Hanford14, and Sinclair minipigs17 in the US, such as chronic studies because of the disadvantage of its Göttingen in Europe3, and Ohmini18, CLAWN19 and NIBS20 rapid growth during the studies. The development of minipigs in Japan. minipigs has resulted in strains of more manageable size The Yucatan minipig is a naturally occurring breed of with genetically and microbiologically obvious miniature swine and the only miniature breed in North characteristics. The main difference between the farm pig America derived from one primary gene pool without the and the minipig is the size at birth and sexual maturity. The introduction of genes from other breeds21. The Yucatan former typically increases from an average weight of 1 kg at minipig was developed at Colorado State University from birth to 100 kg at 4 months of age and the latter usually foundation animals imported from the Yucatan peninsula in grows from a birth weight of 0.5 kg to 12–45 kg at 4 months 196022. The Yucatan minipig is slate-gray, essentially of age4,5. Minipigs reach puberty at an earlier age than larger hairless, and has a docile temperament. The tractable nature domestic pigs. Gilts reach puberty at 3.5–6 months of age of this pig minimizes risks for animal handlers as well as and boars have been known to successfully serve and sire 4– researchers. The absence of a hair coat facilitates a number 8 litters at 3–6 months of age with breed variation6. of laboratory procedures and reduced the heavy odor found The cardiovascular system of the pig is anatomically in most breeds of swine16. With regular human contact, close to that of human, excepting the presence of the left these pigs become amenable to handling and tolerate azygous vein which enters the coronary sinus7. The growth restraint well. Their mean birth weight is 0.74 kg, and their of the heart and great vessels in young pigs is comparable to mean weaning weight at 60 days is 6.47 kg. The mean that of growing children, and the heart of a 40–50 kg weight of mature non-obese boars is 83 kg while that of miniature pig is approximately equivalent to that of an adult mature sows is 70 kg16. The weight at sexual maturity has human. Moreover, the heart size to body weight ratio in pigs been lowered through selective breeding to, currently, commonly used in the laboratory (25–30 kg) is identical to between 30 and 50 kg, with an ultimate goal of 20–25 kg22. that of humans8. Though the hemodynamics of the pig have The Hanford miniature swine was developed at the been shown to be similar in cardiac function to humans, Hanford Laboratory (Battelle Northwest Research variations among breeds and age of the animal need to be Laboratories) in Richard, Washington in 1958. The Hanford taken into consideration9. Experimental atherosclerosis has minipig was produced by crossing Falouse gilts with a been induced by feeding minipigs an atherosclerotic diet and Pittman-Moore boar which had been bred at the University the induced lesions appeared to be similar to those observed of Minnesota and derived from swamp hogs from the in humans10,11. southern US. The Hanford minipig has a white haircoat and The skin of the pig is relatively hairless and tightly skin with less subcutaneous fat than other breeds and weighs attached to the subcutaneous tissues like that of humans. 75 kg at maturity24, making them excellent for dermal With the exception of the absence of apocrine sweat glands, studies. The heart size of the 40–50 kg Hanford minipig is the similarities of pig skin to that of humans have long been similar in size to that of humans, with few collaterals, recognized in overall morphology including epidermal making the pig excellent cardiovascular model4. thickness, cellular composition and cutaneous blood supply The Sinclair miniature swine was developed at the characteristics12,13. Because of these anatomical as well as University of Missouri Comparative Medicine Research physiological similarities, more than any other animal Farm, i.e. the Sinclair Farm23. The Sinclair minipig has a species, pigs have been used as a standard model for surgical significant incidence of cutaneous melanoma with a and wound healing studies of the skin and as a model of histopathology similar to that of humans17 and has been dermal and transdermal toxicology4. extensively studied as an appropriate animal model for the The digestive and urinary systems of pigs function like study of melanoma17,23. those of humans more than most other species of animal. The Göttingen minipig is the most widely used Many of gastrointestinal models have been developed for experimental breed of recent years. The Göttingen minipig nutritional studies including digestion and intestinal enzyme is one of the smallest purpose-bred pigs among micropigs or development14, and urinary models in decades for minipigs available for research. The Göttingen minipig was transplantation biology including xenograft studies4. The developed in the early 1960s at the Institute of Animal brain development of pigs and the similar topical, histologic Breeding and Genetics of the University of Göttingen, and vascular anatomy make them useful as general Germany3. The minipig was obtained by crossbreeding the mammalian models. Neurotransplantation using porcine Minnesota minipig, first with the Vietnamese Pot-Bellied neural stem cells grafted into inbred minipigs has been pig, and then with the German Landrace to obtain a pale skin, studied as an alternative model to non-human primates which resulted in a minipig of white color and small size xenografted with human cells4,15. with stable genetics and phenotype3. The Göttingen minipig is a white non-pigmented and small-sized one with an adult Nunoya, Shibuya, Saitoh et al. 127 body weight of 30–40 kg, if raised on restricted diet25. systems and the glucuronidation of propafenone’s principal The CLAWN miniature swine has been developed by metabolite (5-hydroxypropafenone) was more efficient in mating F1 progeny of the Göttingen minipig with Ohmini the minipig28. miniature swine. Their white color, downsizing, and The deposition of olestra, a fat substitute made from reproductive performance were improved by the fatty acids esterified to sucrose, was examined by following introduction of genetic materials from the Landrace and a single oral gavage dose in weanling Hanford minipigs. Large Yorkshire pigs26. The ingested olestra was essentially not absorbed by the The Ohmini is a minipig which has been developed animals with a young developing gastrointestinal tract which from the foundation stock of the Chinese breed by Hiroshi is similar to that of young children (2–5 yr). The study Omi at the Japan Domestic Animals Institute Co. Ltd. in suggested that the weaning minipig is considered a potential 1971. The Ohmini minipig is black and has a long-haired model for studies on absorption and metabolism of body with drooping ears and belly. Its mean body weight is compounds in children32. 35 kg at one-year-old and its litter size approximates ten18. The pharmacokinetics and metabolism of diclofenac The NIBS minipig has been produced through mating sodium (Voltaren), a nonsteroidal anti-inflammatory drug, of three kinds of breeds, Pitman-Moore, Chinese native were studied in Yucatan minipigs after intravenous or oral (Short-ear pig of Taiwan), and Göttingen in 199327. The administration in a solution, and the results were compared established NIBS strain is a white, non-pigmented and with historical data from humans. The total plasma small-sized pig, and weighs 20–25 kg at 6 months of age if clearance in minipigs was fivefold slower than in humans raised on a specified low calorie feed. The NIBS minipig and the plasma levels of the metabolites 4’-hydroxy, 5- attains sexual maturity at approximate 5 months of age in hydroxy, 3’-hydroxy, 4’,5-dihydroxy, and 3’-hydroxy-4’- females and 7 months of age in males, and has a mean litter methoxy diclofenac were considerably lower in minipigs size of 4.2 piglets. Analyses of the major histocompatibility than in humans after both i.v. and oral administration. While complex (MHC) class II DRB1, DQB, and DQB loci in the these results suggested slower metabolism and/or NIBS minipig have revealed that they are fixed homogenously enterohepatic recirculation of the parent drug in minipigs, to d/d, d/d, and S09/S09 or S10/S10, respectively27. the trends were concluded similar between minipigs and humans33. Drug Metabolism and Pharmacokinetics In contrast to the usefulness of minipigs as an alternative species to the dog or monkey for evaluating In drug pharmacokinetics the generally used rodent potential risks to humans, there has been a report that they models such as rats have been shown not to correspond well are not always sensitive to the specific poisoning, such as to the metabolic properties of human monooxygenases methanol-induced neuro-ocular toxicosis34. When 4-month- (cytochromes P450, CYP)28. Since there are interspecies old female Yucatan minipigs were given a single oral dose of differences and variations in the CYP subfamily, the methanol at 1.0, 2.5, or 5.0 g/kg body weight, comparable to selection of an appropriate experimental model relevant to or greater than the minimal lethal dose in humans, by the human metabolism is crucial. Examples of appropriate gavage, methanol-dosed pigs did not develop optic nerve models are dogs for CYP2D, monkeys for CYP2C, and pigs lesions, toxicologically significant formate accumulation, or or minipigs for CYP3A29. As opposed to other routinely metabolic acidosis which were characteristics in the human used animals, the minipigs possess the main human liver methanol toxicity. Based on these results, it was concluded enzyme of drug biotransformation, CYP3A, in comparable that female minipigs do not appear to be overtly sensitive to amounts and activities30, and microsomal enzymes, CYP2A, methanol and thus may not be a suitable animal model for 2C, 3A with very similar N-terminal sequences to the human the acute methanol-induced neuro-ocular toxicosis34. enzymes31. To prove the suitability of minipigs as an experimental animal for drug metabolism and Dermatotoxicology pharmacokinetics in humans, several kinds of chemicals were examined for their metabolism in vitro at the microsomal The skin of such laboratory animals as mice, rats, level as well as in the minipig. The study described that guinea pigs, rabbits, dogs and monkeys is distinctively minipig-derived systems at the subcellular (microsomal) or different from that of humans, whereas the pig skin has molecular level mimic those of human origin28. similarities in view of general morphologies including The antiarrhythmic drug, propaferone, is a suitable drug epidermal thickness, cellular composition and turnover, for evaluation of interspecies differences in metabolism immunological reactivity, and steroid-induced response25. because of considerable involvement of CYP2D6 and Pigs have a relatively hairless skin that enables the clinical CYP3A4 together with CYP1A2 for its conversion in evaluation of surface alterations and facilitates sequential humans. When the in vitro metabolism of the drug was biopsies. Unlike rodents, minipigs possess sufficient body compared, the microsomal activities of minipig and human weight to tolerate prolonged topical administration of potent origins did not significantly differ. The pharmacokinetics in drugs free from the complications of systemic absorption35. minipigs was, however, more complex reflecting differences While the skin of pigs is thicker and less vascular than that of in substrate specificities of many enzymatic and transport humans, the blood supply characteristics such as size, 128 Miniature Pig in Toxicology distribution and communications of the blood vessels are external skin changes. These advantages made the Yucatan closely similar to those described for the human skin46. The minipig a desirable alternative to other species of animals for skin of pigs has no apocrine sweat gland and a substantial use in skin flap research39. In addition, the skin of minipig amount of subcutaneous fat is deposited as the animal has been preferably used for the evaluation of dermal grows4. In view of closer morphological and physiological irritation by topical drugs40,41, the effect of collagenase in similarities between the human and porcine skin compared promoting injury and wound healing in vivo42, and with other laboratory animals, the pig has been ascribed as a estimating the antifungal activity of drugs in vitro43. preferred animal to evaluate the safety aspect of dermally On the other hands, there is a report that the domestic applied drugs. Chemical compounds, depending on their pig is a better animal model than the Yucatan minipig for physicochemical properties and the area of dermal preclinical studies. The permeation kinetics of tacrine application, generally show a high percutaneous penetration (THA), a centrally acting acetylcholine-esterase inhibitor for through the skin of densely haired animals, whereas the the treatment of Alzheimer’s disease, was compared using permeability and metabolism of the compounds in the the dorsal skins and the absorptive mucosae of both animals porcine skin appear to be more comparable to those in as routes for their systemic delivery of THA44. The results humans, in contrast to the absence of significant correlation indicated that the domestic pig has a significantly higher skin between the human skin and the hairless dog or nude mouse permeability than the Yucatan minipig, with more than a 2- skin37. Dermal studies conducted in minipigs allow the fold difference in intrinsic permeability. The diffusivity for evaluation of both local as well as systemic toxicity similarly the domestic pig skin was very similar to that for the human in other non-rodent species. skin, and it was concluded that the domestic pig is a better As an optimal skin type for in vitro permeability animal model than the Yucatan minipig for preclinical studies, the potential use of minipig skin was evaluated by studies on the delivery of tacrine44. studying three model drug substances, nicotine, salicylic The usefulness of pig skin was evaluated in acid and testosterone, which have different physicochemical photodermatology and sunburn cell (SBC) formation in the properties corresponding to substances with high, epidermis of minipigs and the erythema production was intermediate and low permeation rates, respectively38. When studied45. SBCs were readily formed in the epidermis of the evaluating Göttingen minipigs and domestic pigs, it was animals by ultraviolet radiation. Suberythemal doses of the found that the skin of both animals possessed transdermal broad-band UVB radiation were effective in producing permeabilities, which correlated with the human skin and SBCs, and there was a dose-response relationship between exhibited a lower intra- and inter-variation. Furthermore, it SBC formation and ultraviolet radiation. When the SBC was shown that the permeability and variation of fluxes formation was related to the ultraviolet radiation in terms of through the skin of Göttingen minipigs were dependent on biological dose (i.e. minimal erythema dose), neither the the age of minipigs and the drug substance. The study spectral characteristics of the sources nor the degree of concluded that the Göttingen minipig, like the domestic pig, pigmentation in the skin affected the response. When SBC is a good skin model for in vitro permeation through the formation was related to physical dose (J/m2), the protection human skin38. Since the degree of resemblance in value of tanning was evident. From these results, when permeation of various compounds through the pig skin and coupled with the morphological and physiological human skin varies with compounds with different chemical similarities between the human and porcine skin, the characteristics, it is desirable in every new study to check the usefulness of minipigs was demonstrated as an animal model degree of resemblance in the literature or, if there is no data, in photodermatology too45. to establish the similarities of the particular compound under Damage mechanisms involved in near-infrared laser studies36. injuries are believed to partly result from the energy Porcine models have been used extensively for the skin absorption by the skin pigmentation (melanin). A highly flap research because of the established similarity between pigmented animal model, therefore, may be a suitable the cutaneous blood supply of pigs and humans. The subject for laser exposure studies. On the basis of epidermal Yucatan minipig provided an excellent model for skin thickness measurements and melanin assessment, the researching the properties of random cutaneous flaps, flank and dorsal neck of the Yucatan minipigs were closely offering several advantages over other breeds of pigs39. A comparable to those of the human epidermis from the face, total of 67 random cutaneous dorsal flank flaps measuring 4 neck and arms, and better suited to laser injury studies than × 14 cm were raised on nine Yucatan minipigs. The mean the Yorkshire pig models of the human skin because of their survival length of the non-delayed flaps was greater than higher pigmentation levels44,46. In a later study, however, no others reported in the literature. The well-defined plane statistical difference was found between the 24 h effective between the subcutaneous tissue and the panniculus dose of laser pulses in two breeds, Yorkshire pigs and carnosus facilitated flap elevation consistently above the Yucatan minipigs, nor were there any differences in level of the panniculus carnosus, thereby ensuring the histologic lesions represented by desiccation of the creation of a true random cutaneous flap. Furthermore, the superficial layer of epidermis to desiccation with hairless skin, particularly beneficial in studying chemical inflammatory centers, and finally replacement of peels, permits visual observation and easy monitoring of any inflammatory centers with areas of epidermal ulceration47. Nunoya, Shibuya, Saitoh et al. 129

Cardiovascular Research humans. Eight minipigs were injected with either 20 mg per kg of azoxymethane weekly or 10 mg per kg of the substance For cardiovascular research dogs have been frequently every other week or a combination of both treatments for up used as a large species, but there is an increasing concern to 20 weeks. Chronic toxic effects were limited to the liver over animal welfare regarding the use of dogs. Additionally, and no colon tumors were induced. Hepatic lesions included there are some anatomic and physiologic differences, which proliferation of bile ducts with fibrosis, cholestasis, and make the extrapolation of data to human difficult, in dogs. widespread hepatocellular atypia including hepatocellular Pigs, on the other hand, have many similarities to humans in anisocytosis, megalocytosis and binucleated hepatocytes50. the cardiovascular system, including anatomy and Although the adequacy of experimental schedule for tumor electrophysiology, that can expedite the transition from induction must be verified, these findings may be relevant to research to clinical application8. investigators considering the minipigs as an alternative large Pigs have been utilized in the evaluation of therapeutic animal model for human colon carcinogenesis. compounds focused on altering the detrimental Minipigs may present an alternative animal model for consequences of myocardial infarction. Myocardial teratogenicity and reproductive studies where there is a lack infarction studies in pigs have been complicated by the use of suitability for traditional animals such as mice, rats or of antiarrhythmic drugs or the high incidence of ventricular rabbits25. A study of reproductive and developmental fibrillation. As a new model of experimental myocardial toxicology, including teratology, was described in relation to infarction, studies were successfully performed in Yucatan the effects of exposure of Hanford minipigs to electric fields. minipigs by thiamylal-anesthetization without the use of When female minipigs were bred after 18 months of uniform intravenous antiarrhythmic drugs48. Myocardial infarction exposure to a vertical, 60-Hz, 30-kV/m electric field for 20 can be achieved in anesthetized minipigs without the aid of h/day, 7 days/week and killed at 100 days of gestation, the intravenous antiarrhythmic drugs and reduced cardioversion. malformation incidence in litters (75%) of the exposed Therefore, Yucatan minipigs were concluded as being useful females was significantly greater than in litters (29%) of the for the evaluation of therapeutic compounds focused on sham-exposed minipigs. When F1 gilts were bred again at altering the detrimental consequences of myocardial 18 months of age, defective offsprings were found more in infarction. litters (71%) of the F1 gilts born from the exposed females The effects of different substances on blood pressure, than litters of the F1 gilts born from the sham-exposed heart rate, and electrocardiograms were examined with a females, suggesting an association between chronic telemetry system in conscious NIBS minipigs aged 6–9 exposure to a strong electric field and developmental effects months (weighing 17.0–17.5 kg)20. The plasma in minipigs51. Another teratogenicity study was reported for concentration of the dopamine D2 antagonist, haloperidol, Göttingen minipigs which were given mashed feed mixed which is known to cause QT interval prolongation and with pyrimethamine (PY), an antiparasitic compound. associated arrhythmias in humans, was almost equal to that Major malformations such as cleft palate, clubfoot and in humans. Although the adrenergic β receptor antagonist, micrognathia were observed in 11 out of 16 newborns from dl-propranolol, shortened QTcF interval, the receptor 3 pregnant sows which received 3.6 mg/kg/day, whereas no antagonist, dl-sotalol, prolonged QTcF interval. These malformations were found in 22 newborns from sows which results reveal that NIBS minipigs can be used for prediction were given low doses (0.9 and 1.8 mg/kg/day) of PY52. of the effects of drugs on cardiovascular parameters such as prolongation of QT interval in humans in the safety Naturally Occurring and Induced Diseases pharmacology studies too. Pigs, like other experimental animals, are vulnerable to Miscellaneous infectious microorganisms and easily affected with viral and bacterial diseases. Several examples of naturally occurring Studies of inhalation exposure of Göttingen minipigs to and experimentally induced non-infectious diseases are aerosol and gaseous drugs have been reported. The minipigs discussed here. were exposed via mask inhalation to the test substance using Salt poisoning sometimes occurs in minipigs after a computer-assisted exposure system that allowed prolonged consumption of sodium chloride, and toxicity also simultaneous exposure of groups of four animals in parallel occurs with water deprivation even when a diet containing at different controlled dose levels. Inhalation treatment of normal levels of salt is fed. Therefore, all sources of water verapamil, a cardiovascular drug, showed a good absorption should be checked daily, especially for young minipigs first and favorable pharmacokinetics when compared with i.v. trying to drink from nipple drinkers in new animal cages, and drug application. These results have highlighted the during the transportation of minipigs for a long time. The usefulness of minipigs for inhalation studies49. early clinical signs are thirst, constipation, skin irritation and Minipigs can offer a number of investigational inappetence. Then nervous signs and sometimes deaths advantages over rodents, and thus the possibility of inducing follow, especially when unrestricted water is suddenly colon cancer was studied in Hanford minipigs which have a available after a period with no water. The signs include similar system of absorption and metabolism to that seen in aimless wandering, bumping into objects, walking backward 130 Miniature Pig in Toxicology or in circles, and convulsive seizures. The confirmatory cholesterol distribution into the LDL fraction (81%) versus diagnosis is usually made by recognition of the control (57%) pigs. Diabetic pigs showed several early signs pathognomonic changes, eosinophilic encephalomyelitis in of the excess vascular disease and 75% of the coronary artery the brain of pigs, and brain sodium levels6,53. segments showed contractile oscillations in response to Sinclair minipigs display cutaneous melanoma lesions prostaglandin F2α. Sudan IV staining of the carotid artery for and develop a generalized depigmentation subsequent to fatty streaks was significantly increased only in diabetic tumor regression. The melanoma is an inherited malignancy pigs. These minipig models may provide insights into the with many of the histopathological characteristics of human etiology of human diabetic dyslipidemia and atherosclerosis melanoma54. The incidence of melanoma is about 75% at and facilitate research on the etiology and treatment of the birth with no sex or site preference. The lesions are peripheral vascular and coronary artery diseases in diabetic histopathologically similar to human junctional and patients. compound nevi and malignant cutaneous melanoma55. A To evaluate and study the link between obesity and significant number of swines exhibit metastasis to the lipoprotein metabolism, animal models have been made in regional lymph nodes, liver, lungs, and approximately 35% minipigs with overfeeding unbalanced diets or feeding a of all primary lesions regress following puberty, leaving the high-fat high-energy diet. Yucatan minipigs aged 4 months the appearance of a halo nervus and a generalized were studied over a 12-month period from childhood to vitiligo54,55. The information obtained in studies of Sinclair adulthood. Differences between groups were observed only minipigs should facilitate the understanding of the after 10 months old, the end of sexual maturity59. The mechanisms by which the melanoma and vitiligo develop overfed minipigs had a higher LDL-cholesterol versus HDL- and provides possible therapeutic treatments for human cholesterol ratio compared to normally fed pigs and VLDL- counterparts54. triacylglycerol evolution in the overfed minipigs was Because of the similarity of skin anatomy, physiology, associated with an increase in LDL-triacylglycerol plasma and immunology, many investigative studies in dermatology concentrations after sexual maturation. This phenomenon have been performed prefereably in pigs. One of frequently has not been observed in similar studies when obesity was used models, allergic contact dermatitis (ACD), was induced in adult minipigs and may represent a specific experimentally induced in Göttingen minipigs using 2,4- hallmark of an obesity induced during sexual maturity. dinitrofluorobenzene as a hapten56. The major epidermal Obesity can also be induced in female Göttingen minipigs60. changes were acanthosis, spongiosis, intracellular edema, To establish a model of human metabolic syndrome, exocytosis, and abscesses. In addition to these findings, CD Göttingen minipigs received a hight-fat high-energy diet antigen expression patterns of the dermal infiltrates closely with 55% of energy contents derived from fat. After resembled findings in the human ACD. treatment, the tryglyceride concentration of treated minipigs Studies of the pathogenic components of the diabetic with higher body weight was significantly increased and the milieu and the mechanisms of accelerated atherosclerosis insulin area under the curve during intravenous glucose have been limited by the lack of suitable animal models. A tolerance testing was significantly high in the obese animals potentially appropriate animal model for human diabetic although the plasma glucose and insulin concentrations were dyslipidemia is the pig, because it carries a large fraction of not affected. These findings share some of the metabolic total cholesterol in low-density lipoprotein (LDL), similar to impairments seen in obese humans and the disease humans. Feeding minipigs (Chinese Guizhou minipigs) with conditions may serve as a model of metabolic syndrome60. a high-fat/high-sucrose diet (HFSD) could induce insulin resistance, mild diabetes and atherosclerotic lesions57. When Conclusions plasma total cholesterol (TC), high-density lipoprotein cholesterol, triglyceride (TG), insulin and glucose were In preclinical research laboratory animals serve for the quantified at monthly intervals, a mild high plasma TC and assessment of pharmacodynamic effects or the identification TG were induced during the feeding period. In addition, of unanticipated adverse effects of drug candidates. In hypertrophic adipocytes, fat deposit in the liver, loss of particular the results obtained from animal toxicity studies pancreatic beta-cells, and aortic fatty streak lesions were should be reliably predictive for the condition in humans. induced in HFSD-fed minipigs at the end of 6 months of Therefore, the use of animal species that mimic as closely as feeding. Meanwhile Sinclair minipigs were made diabetic possible the human condition is desired for studies. by destroying the insulin-producing cells of the pancreas Minipigs were introduced in Europe for scientific with alloxan, followed by feeding with a high fat and high researches in the early 1970s and for toxicologic studies as cholesterol diet58. Diabetic pigs exhibited hyperglycemia, an alternative species to the rodents in the mid-1980s61. but plasma urea nitrogen, creatinine, and transaminase levels Since then minipigs have been increasingly used for were in the normal range, indicating no adverse effects on biomedical researches because they are appropriately small the kidney and liver functions. The lipoprotein profile in to be easily handled in standard laboratory animal facilities, diabetic pigs was similar to that found in human diabetic but large enough for sampling body fluids and biopsies over patients and was characterized by hypertriglyceridemia (2.8- extended periods, and unlike the domestic pigs genetically fold increase versus control) and a profound shift of and phenotypically controlled. Minipigs provide advantages Nunoya, Shibuya, Saitoh et al. 131 on long term studies in terms of slow and limited growth too. Atherosclerosis. 43: 119–132, 1982. Commercially available minipigs such as NIBS and 11. 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