Spontaneous Age-Related Histopathological Changes in Microminipigs.

Microminipigs have become an attractive animal model for toxicology and pharmacology studies and for human disease models, owing to their manageable size. Although there are numerous reports of spontaneous age-related lesions in mice, rats, dogs, and monkeys, those in minipigs are scarce. In the present study, spontaneous age-related histopathological changes were investigated using 37 microminipigs (20 males and 17 females) that were 6 months to 10 years of age. Abnormal deposits of materials were evident in several animals from 6 years of age, and these deposits included amyloid in the renal medulla, thyroid gland, and adrenal gland, hyaline droplets in glomeruli, and fibrillar inclusions in neurons. Arterial sclerosing changes (intimal thickening, intimal proliferation, and medial mineralization) and proliferative lesions (hyperplasia of hepatocytes, follicular cells, Leydig cells, and uterine endometrial glands) were present at 4 years of age and beyond. Renal adenoma, uterine leiomyoma, and Leydig cell tumor were observed in several microminipigs. Moreover, glomerulosclerosis, renal interstitial fibrosis, thymic involution, and adrenocortical cell vacuolation were common in aging microminipigs. Since knowledge of age-related changes is helpful for pathologists, the basic information obtained in this study will be a useful reference for all future toxicity evaluations in microminipigs.

Histomorphometric evaluation of the Göttingen minipig eye.

OBJECTIVE: The Göttingen minipig is becoming a popular nonrodent animal model in ocular research; however, there is a paucity of literature regarding normative ocular reference data in this breed. We, therefore, investigated the characteristics of the cornea, retina, and sclera in order to establish baseline histomorphometric data in male and female Göttingen minipigs. PROCEDURES: This study utilized paraffin-embedded, Davidson's-fixed, control Göttingen minipig eyes (six males and eight females). Hematoxylin and eosin stained slides of the eyes were scanned via an Aperio slide scanner and analyzed using Aperio ImageScope™. Linear measurements were made of the cornea, retina, and sclera. RESULTS: There were no statistically significant differences between males and females in corneal or scleral measurements or total retinal thickness. There were, however, statistically significant differences between the sexes in the thickness of the ventral peripheral ganglion cell layer [13.15 µm (±3.65) in males; 10.68 µm (±3.37) in females; P = 0.03], ventral peripheral inner plexiform layer [23.47 µm (±2.85) in males; 21.16 µm (±3.62) in females; P = 0.03], ventral central outer plexiform layer [7.97 µm (±2.43) in males; 6.63 µm (±1.73) in females; P = 0.02], and ventral peripheral outer plexiform layer [8.79 µm (±1.82) in males; 11.23 µm (±3.11) in females; P = 0.01]. CONCLUSIONS: This study provides normative histomorphometric reference data for the Göttingen minipig eye. These data will aid researchers in study design and interpretation of findings in Göttingen minipig ocular studies.

Expression patterns of tight junction proteins in porcine major salivary glands: a comparison study with human and murine glands.

Tight junction (TJ) proteins play a dynamic role in paracellular fluid transport in salivary gland epithelia. Most TJ studies are carried out in mice and rats. However, the morphology of rodent salivary glands differs from that of human glands. This study aimed to compare the histological features and the expression pattern of TJ proteins in porcine salivary glands with those of human and mouse. The results showed that porcine parotid glands were pure serous glands. Submandibular glands (SMGs) were serous acinar cell-predominated mixed glands, whereas sublingual glands were mucous acinar cell-predominated. Human SMGs were mixed glands containing fewer mucous cells than porcine SMGs, whereas the acinar cells of murine SMGs are seromucous. The histological features of the duct system in the porcine and human SMGs were similar and included intercalated, striated and excretory ducts, but the murine SMG contained a specific structure, the granular convoluted tubule. TJ proteins, including claudin-1 to claudin-12, occludin and zonula occludin-1 (ZO-1), were detected in the porcine major salivary glands and human SMGs by RT-PCR; however, claudin-6, claudin-9 and claudin-11 were not detected in the murine SMG. As shown by immunofluorescence, claudin-1, claudin-3, claudin-4, occludin and ZO-1 were distributed in both acinar and ductal cells in the porcine and human SMGs, whereas claudin-1 and claudin-3 were mainly present in acinar cells, and claudin-4 was mainly distributed in ductal cells in the murine SMG. In addition, 3D images showed that the TJ proteins arranged in a honeycomb-like structure on the luminal surface of the ducts, whereas their arrangements in acini were irregular in porcine SMGs. In summary, the expression pattern of TJ proteins in salivary glands is similar between human and miniature pig, which may be a candidate animal for studies on salivary gland TJ function.

A reproducible swine model of proximal descending thoracic aortic aneurysm created with intra-adventitial application of elastase.

OBJECTIVE: Animal models are required to explore the mechanisms of and therapy for proximal descending thoracic aortic aneurysm (TAA). This study aimed to establish a reproducible swine model of proximal descending TAA that can further explain the occurrence and progression of proximal descending TAA. METHODS: Eighteen Chinese Wuzhishan miniature pigs (30.32 ± 1.34 kg) were randomized into the elastase group (n = 12) and the control group (n = 6). The elastase group received intra-adventitial injections of elastase (5 mL, 20 mg/mL), and the control group received injections of physiologic saline solution. A 4-cm descending thoracic aortic segment proximal to the left subclavian artery was isolated. The distance between the left subclavian artery and the injection starting point of the descending thoracic aorta was 0.5 cm. Elastic protease was circumferentially injected intra-adventitially into the isolated segment of the aortic wall in the elastase group by a handmade bent syringe. The length of the elastic protease injection was 2 cm. An average of 12 injection points were distributed in this 2-cm aortic segment. Each injection point used about 0.4 mL of elastic protease. The distance between two injection points was about 1.5 cm. All animals underwent digital subtraction angiography preoperatively and 3 weeks after operation. Three weeks after TAA induction, aortas were harvested for biochemical and histologic measurements. RESULTS: All animals in the elastase group developed TAAs. No aneurysms were observed in the control group. The distance between the left subclavian artery and the TAA was 8.00 ± 4.19 mm. Preoperative and postoperative aortic diameters of the elastase group were 15.42 ± 0.43 mm and 24.53 ± 1.41 mm, respectively (P < .0001). Preoperative and postoperative aortic diameters of the control group were 15.31 ± 0.33 mm and 15.57 ± 0.40 mm, respectively (P = .5211). The changes of aortic structure and composition included reduction of smooth muscle cells and degradation of elastic fibers. Levels of matrix metalloproteinases 2 and 9 were increased in TAA tissue. CONCLUSIONS: This study established a reproducible large animal model of proximal descending TAA. This model has the same biochemical characteristics as human aneurysms in the aspects of aortic expansion, aortic middle-level degeneration, and changes in the levels of matrix metalloproteinases and provides a platform for further study.

Normative Retinal Thicknesses in Common Animal Models of Eye Disease Using Spectral Domain Optical Coherence Tomography.

Purpose This study demonstrates a standardized approach to measuring retinal thickness (RT) using spectral domain optical coherence tomography (SD-OCT) in commonly used animal models of disease and reports a normative data set for future use. Materials and Methods Twenty normal eyes of 4 adult animal models (5 rats, 5 rabbits, 5 canines, and 5 mini-pigs) were used. Manual measurements were made on the commercially available Heidelberg Spectralis™ SD-OCT to determine the total, inner, and outer retinal thickness (RT) at fixed distances from the optic nerve head (ONH) (1, 2, 3, 4, 5, and 6 mm away) in order to control for normal variation in retinal thickness. Analysis of variance (ANOVA) with P value <0.05 indicated statistical significance. Results Total RT significantly decreased with increasing distance from the ONH for the canine, mini-pig, and rabbit vascular models. Inner RT significantly decreased for the canine, mini-pig, rabbit vascular, and rabbit avascular models; and outer RT significantly decreased for only the canine model. Among the animal models, RT at similar distances from the ONH were significantly different for total, inner, and outer RT. Conclusion There are significant differences in the total, inner, and outer RT of normal canine, mini-pig, rabbit, and rat retinas with SD-OCT using a standardized approach. These measurements provide a normative reference for future studies and illustrate a standardized method of assessing RT.

Normal Developmental and Estrous Cycle-dependent Histological Features of the Female Reproductive Organs in Microminipigs.

The microminipig has become an increasingly attractive animal model for various experimental practices because of its manageable size; however, studies of the histological features of the female reproductive organs in microminipigs are limited. The present study investigates the sexual development of the reproductive organs and the cyclical changes during the estrous cycle in female microminipigs. The ovaries, oviducts, uteri, and vaginal tissues from 33 animals aged 0 to 26 months were utilized in this study. By evaluating the large tertiary follicles, corpora lutea, and the regressing corpora lutea, we estimated that female microminipigs reached puberty at approximately 5 months of age and sexual maturity at 8 months of age. The appearance of the follicles and corpora lutea in the ovaries, as well as the epithelium in other reproductive organs, was synchronized with each phase of the estrous cycle and was identical to that in common domestic pigs. In addition, several spontaneous findings were observed, including mesonephric duct remnants adjacent to oviducts and mineralization in ovaries. Understanding the normal histology of the reproductive organs in microminipigs is crucial for advancing pathological evaluations for future toxicological studies.

The importance of minipigs in dermal safety assessment: an overview.

The use of miniature swine as a non-rodent species in safety assessment has continued to expand for over a decade and their use has become routine, particularly in pharmacology as a model for human integumentary diseases. Translational preclinical swine study data are now favorably compared and contrasted to human data, and miniature swine models provide important information in dermal safety assessment and skin pharmacology. For example, the miniature swine model has been well-accepted for cutaneous absorption and toxicity studies due to swine integument being morphologically and functionally similar to human skin. Subsequently, this model is important to dermal drug development programs, and it is the animal model of choice for assessment of dermal absorption, local tolerance and systemic toxicity following dermal exposures. In conclusion, the miniature swine model has an important role to play in the safety assessment of pharmaceutical products and in multiple aspects of human dermal drug development.

Miniature Swine Breeds in Toxicology and Drug Safety Assessments: What to Expect during Clinical and Pathology Evaluations.

The use of miniature swine as a nonrodent species in safety assessment has continued to expand for over a decade, and they are becoming routinely used in toxicology and in pharmacology as well as a model for human diseases. Miniature swine models are regularly used for regulatory toxicity studies designed to assess safety of new therapeutic compounds given through different routes of exposure and are used as an alternative model to the canine or the nonhuman primate. Translational preclinical swine study data presented support the current finding that miniature swine are the animal model of choice for assessment of drug absorption, tolerance, and systemic toxicity following systemic exposures. Because research investigators need to be familiar with important anatomic and histopathologic features of the miniature swine in order to place toxicopathologic findings in their proper perspective, clinical and anatomic pathology data from a large number of Sinclair, Hanford, Yucatan, and Göttingen breeds from control groups from a wide variety of studies performed between 2004 and 2014 will be presented, compared, and partially illustrated.

Sexual Maturation in the Female Göttingen Minipig.

In the literature, experimental data on sexual maturation of female Göttingen minipigs are lacking. This may impede a reliable evaluation of reproductive functioning, particularly in the young (immature) sow used in toxicity studies. To find suitable method(s) to detect ovulation during in-life, a pilot study was performed with 3 adult sows (approximately 10-11 months), followed by a study with 14 immature females (approximately 3-4 months). From the tested parameters, progesterone analysis was the most reliable predictor. First progesterone peaks were observed in 13 sows at 3.7-4.2 or 5.5-6.5 months with a cycle length of 17-22 days. One sow did not show progesterone release until necropsy at 7 months of age. Histopathology of the reproductive organs confirmed sexual maturity for all sows, except the one without progesterone peak. In conclusion, the age range of sexual maturity of female Göttingen minipigs (3.7-6.5 months) is much wider than previously thought, and in-life progesterone analysis is a useful tool to determine sexual maturity of individual animals.

Histological Development of Male Reproductive Organs in Microminipigs.

Microminipigs are becoming increasingly attractive alternatives for various experimental applications, such as general toxicology studies, owing to their manageable size. However, there are limited studies on the male reproductive organs of microminipigs, particularly on the histological aspects of sexual maturity. To clarify the development of male reproductive organs, 35 male microminipigs, aged 0 to 12 months, were used in this study. Histological and histomorphological evaluation was performed based on spermatogenic development, measurement of tubular structure in testes and epididymides, and histological progress of accessory glands. In addition, spontaneous testicular changes were quantitatively assessed. Histologically, male microminipigs sexually matured around 4.5 months of age, when spermatogenesis in testes and structural development in genital organs were completed. Spontaneous testicular changes occurred in all the animals investigated. Multinucleated giant cell was most commonly observed, followed by hypospermatogenesis and tubular atrophy/hypoplasia. However, the number of affected tubules was less than 1% in testes after 4.5 months of age, suggesting that the influence of these changes on evaluation of toxicity studies may be minimal. It is preferable to use sexually mature animals in toxicology studies; therefore, the information obtained by the present study will be helpful for future toxicity evaluations in microminipigs.

Method for perfusion decellularization of porcine whole liver and kidney for use as a scaffold for clinical-scale bioengineering engrafts.

BACKGROUND: Whole-organ engineering provides a new alternative source of donor organs for xenotransplantation. Utilization of decellularized whole-organ scaffolds, which can be created by detergent perfusion, is a strategy for tissue engineering. In this article, our aim is to scale up the decellularization process to human-sized liver and kidney to generate a decellularized matrix with optimal and stable characteristics on a clinically relevant scale. METHODS: Whole porcine liver and kidney were decellularized by perfusion using different detergents (1% SDS, 1% Triton X-100, 1% peracetic acid (PAA), and 1% NaDOC) via the portal vein and renal artery of the liver and kidney, respectively. After rinsing with PBS to remove the detergents, the obtained liver and kidney extracellular matrix (ECM) were processed for histology, residual cellular content analysis, and ECM components evaluation to investigate decellularization efficiency, xenoantigens removal, and ECM preservation. RESULTS: The resulting liver and kidney scaffolds in the SDS-treated group showed the most efficient clearance of cellular components and xenoantigens, including DNA and protein, and preservation of the extracellular matrix composition. In comparison, cell debris was observed in the other decellularized groups that were generated using Triton X-100, PAA, and NaDOC. Special staining and immunochemistry of the porcine liver and kidney ECMs further confirmed the disrupted three-dimension ultrastructure of the ECM in the Triton X-100 and NaDOC groups. Additionally, Triton X-100 effectively eliminated the residual SDS in the SDS-treated group, which ensured the scaffolds were not cytotoxic to cells. Thus, we have developed an optimal method that can be scaled up for use with other solid whole organs. CONCLUSIONS: Our SDS-perfusion protocol can be used for porcine liver and kidney decellularization to obtain organ scaffolds cleared of cellular material, xenoimmunogens, and preserved vital ECM components.

Three-dimensional structure of minipig fibrolamellar bone: adaptation to axial loading.

Fibrolamellar bone is transiently produced by large, fast growing mammals. The fibrolamellar bone unit is initially formed by elaboration of a network of blood vessels. This is followed by the deposition of a thin, porous and hypercalcified layer, then by the infilling of the vascular cavities by the sequential deposition of a relatively thick rapidly forming bone on both sides of the hypercalcified layer, and finally by lamellar bone. We investigated the 3D structure of the collagenous network of fibrolamellar bone from the femora of a young minipig using mainly the FIB-SEM dual beam microscope and the Serial Surface View method. This enabled us to identify the fibril orientation, the canalicular network organization and other structural motifs within each element of the fibrolamellar unit. The first formed primary hypercalcified layer (PHL) is composed of fibril arrays and multiple small pores, and appears to have an isotropic structure. The major bone component is deposited on both sides of the PHL, and is composed of collagen fibrils with a preferred orientation, mainly aligned parallel to the bone long axis. This bone component is therefore parallel-fibered bone and not woven bone. We also observed that the collagen fibers are organized into bundles. The lamellar bone has most of its collagen fibrils aligned with the bone long axis. This study therefore shows that the large majority of collagen fibrils in fibrolamellar bone are aligned with the bone long axis. This anisotropic structure therefore appears to be adapted to loading along the bone long axis.

Estrous cycle-dependent morphology in the reproductive organs of the female Göttingen minipig.

The present study describes the normal histology of female reproductive organs during the estrous cycle in the Göttingen minipig. For this purpose, sexually mature females were sacrificed at different phases of the cycle (follicular/proliferation, ovulation, and early-, mid-, and late-luteal/secretory phase). Ovaries, uterus, cervix, vagina, and mammary gland tissues were processed for microscopic evaluation. Sexual maturity was assured by selecting females in which at least 1 progesterone peak was measured. Stage-distinguishing features in ovaries were the Graafian follicles (disrupted vs. nondisrupted) and corpora lutea of recent and preceding cycles (size, cell morphology, and structural composition). In the uterus, stage-specific markers were epithelial morphology, secretory activity (using periodic acid-Schiff/hematoxylin staining), and epithelial mitosis and/or apoptosis. The other reproductive organs were not suitable to discriminate between the different phases of the cycle due to a high morphologic variability (mammary gland, and vagina) or absence of clear morphologic differences between the phases (cervix). The increased use of young minipigs (frequently immature/peripubertal) in preclinical testing requires more knowledge on the histologic cyclic changes. With the present morphologic description of the morphologic characteristics of the reproductive tract in recently ovulating minipigs, a guidance for staging the estrous cycle and determination of sexual immaturity is provided.

Onset of puberty and normal histological appearances of the reproductive organs in peripubertal female Göttingen minipigs.

In preclinical studies, it is important to know whether the animals used are sexually mature or not. Precise data have not yet been published, however, about the histological features of the female reproductive organs during the peripubertal period or about the age of acquisition of sexual maturity in the minipig. The histological characteristics of the genital organs of female control minipigs from toxicology studies were described and, based on the presence of ovarian corpora lutea, used to assess the age at which maturity was reached. Only 50% of females can be considered mature at about 6.5 months old (a body weight of 11.8 kg), and 100% were not mature until about 7.5 months old (13.1 kg), although it is said that females reach sexual maturity at the age of approximately 5 months, by the time the body weight is about 10 to 12 kg. The uterine weights of mature females were higher than 94.4 g, whereas the maximum weight reached in the immature females was 55.2 g. In contrast, the differences between immature and mature ovarian weights were not significant. The histological appearance of the mature vagina in the various stages of the estrous cycle is also described.

Dynamic MR imaging of a minipig's knee using a high-density multi-channel receive array and a movement device.

OBJECT: To construct an optimised, high-density receive array and a movement device to achieve dynamic imaging of the knee in orthopedic large animal models (e.g., minipigs) at 1.5 T. MATERIALS AND METHODS: A 13-channel RF receive array was constructed, and the crucial choice of the array element size (based on considerations like region of interest, geometry of the minipig's knee, achievable signal-to-noise ratio, applicability of parallel imaging, etc.) was determined using the Q factors of loops with different sizes. A special movement device was constructed to guide and produce a reproducible motion of the minipig's knee during acquisition. RESULTS: The constructed array was electrically characterised and the reproducibility of the cyclic motion was validated. Snapshots of dynamic in vivo images taken at a temporal resolution (308 ms) are presented. Some of the fine internal structures within the minipig's knee, like cruciate ligaments, are traced in the snapshots. CONCLUSION: This study is a step towards making dynamic imaging which can give additional information about joint injuries when static MRI is not able to give sufficient information, a routine clinical application. There, the combination of a high-density receive array and a movement device will be highly helpful in the diagnosis and therapy monitoring of knee injuries in the future.

Nasal passages of Göttingen minipigs from the neonatal period to young adult.

Histopathological examination of the nasal passages requires a standardized approach for recording lesion distribution patterns. Nasal diagrams provide guidance to map the lesions. Information on lesions exists for rodents, dogs, and monkeys, which all have been used in inhalation studies. Recently, minipigs have garnered interest as an inhalation model because minipigs resemble humans in many features of anatomy, physiology, and biochemistry and may be a good alternative to monkeys and dogs. The present work explored the microanatomy and histology of the nasal passages of Göttingen minipigs from postnatal day 1 until 6 months of age. Six nasal levels were selected, which allow examination of the squamous, transitional (nonciliated) and ciliated respiratory, and olfactory epithelia; the nasopharynx; and relevant structures such as the vomeronasal organ, olfactory bulb, and nasal/nasopharynx-associated lymphoid tissue.

Swine as models in biomedical research and toxicology testing.

Swine are considered to be one of the major animal species used in translational research, surgical models, and procedural training and are increasingly being used as an alternative to the dog or monkey as the choice of nonrodent species in preclinical toxicologic testing of pharmaceuticals. There are unique advantages to the use of swine in this setting given that they share with humans similar anatomic and physiologic characteristics involving the cardiovascular, urinary, integumentary, and digestive systems. However, the investigator needs to be familiar with important anatomic, histopathologic, and clinicopathologic features of the laboratory pig and minipig in order to put background lesions or xenobiotically induced toxicologic changes in their proper perspective and also needs to consider specific anatomic differences when using the pig as a surgical model. Ethical considerations, as well as the existence of significant amounts of background data, from a regulatory perspective, provide further support for the use of this species in experimental or pharmaceutical research studies. It is likely that pigs and minipigs will become an increasingly important animal model for research and pharmaceutical development applications.

Contribution of large pig for renal ischemia-reperfusion and transplantation studies: the preclinical model.

Animal experimentation is necessary to characterize human diseases and design adequate therapeutic interventions. In renal transplantation research, the limited number of in vitro models involves a crucial role for in vivo models and particularly for the porcine model. Pig and human kidneys are anatomically similar (characterized by multilobular structure in contrast to rodent and dog kidneys unilobular). The human proximity of porcine physiology and immune systems provides a basic knowledge of graft recovery and inflammatory physiopathology through in vivo studies. In addition, pig large body size allows surgical procedures similar to humans, repeated collections of peripheral blood or renal biopsies making pigs ideal for medical training and for the assessment of preclinical technologies. However, its size is also its main drawback implying expensive housing. Nevertheless, pig models are relevant alternatives to primate models, offering promising perspectives with developments of transgenic modulation and marginal donor models facilitating data extrapolation to human conditions.

Uterine lesions in 32 female miniature pet pigs.

Thirty-two 4-month-old to 19-year-old female miniature pet pigs were spayed. Uterine lesions were present in all except 8 pigs. The 24 remaining pigs had diffuse cystic endometrial hyperplasia, of which 14 had smooth muscle tumors, including leiomyomas and leiomyosarcomas, in the uterus or broad ligament. Nodular endometrial lesions-including adenocarcinomas, adenomas, and/or adenomyosis-were present in 10 pigs, 3 of which had concurrent smooth muscle tumors. Pyometra was present in 3 pigs. In uterine sections with cystic endometrial hyperplasia, adenomyosis, or adenomas, approximately 70% of epithelial nuclei expressed estrogen receptor and progesterone receptor immunohistochemically; in adenocarcinomas, expression was 20%. Regardless of malignancy, more than 50% of nuclei in smooth muscle tumors expressed estrogen receptor and progesterone receptor. Aging was associated with the development of uterine lesions in miniature pet pigs.