Specificities of the Skin Morphology in Juvenile Minipigs

Skin morphology documentation in juvenile minipigs from PND1 to week 12 of age was performed on skin samples taken at necropsy from the first litter (i.e., without application of any test item) at Charles River, Les Oncins, France. All samples were fixed in 10% neutral buffered formaldehyde, thereafter sent and processed at the Sophia Antipolis site of Galderma R&D. Twelve samples from one male and one female were obtained per time point from PND1 to 3 months of age (PND1, PND8, PND15, PND29, and W12) as described in Figure 1. The selected anatomical areas allow an extensive documentation of the skin morphology variability and included two samples taken from the lateral region (flank posterior area and flank anterior area), two samples taken on the dorsal line around the spine (rachis posterior area and rachis anterior area), one sample behind the right ear, one sample on the snout, one sample on the chin, one sample taken at the internal face of the forelimb and one on the internal face of the hind limb, two samples taken at the abdomen (anterior and posterior area), and finally one sample taken in the pelvic region (ischium–pelvis). It is to be noted that the term “flank” was used in this study to describe the entire lateral region (region defined using anatomic landmarks: rectangles between the scapulae and hipbones) although this anatomical correct term for the anterior part of this region should be “thoracic region.” This region as defined here corresponds to the area commonly used for dermal application in the dermal toxicology. The other anatomical regions have been selected to document the histological characteristics of areas that could potentially be used for injections or topical administrations.

In the minipig, as in humans, the morphology of the skin layers (stratum corneum, living part of the epidermis, dermis, and hypodermis) differs depending on the anatomical location. The presence and morphology of adnexa, including sweat glands, hair follicles, and sebaceous glands, may also vary. Apocrine glands were seen in all sections; although this type of gland is only seen in specific locations in humans (axillae, areolae, external ear canal, sexual/circumanal, and periumbilical areas), they are widely distributed in the minipig. The apocrine glands are tubular and show a coiled secretory part that is not related to the skin surface but to the hair follicle lumen. From literature data (Makin, Mortensen, and Brock 2012; Swindle 1998; Swindle et al. 2012), eccrine sweat glands may also be seen in minipigs but are thought to be limited to the snout and carpal glands. In the present study, for each animal from PND1 to W12, samples in the snout region were examined; however, we were unable to identify eccrine glands in our samples. Sebaceous glands were seen in association with hair follicles in almost all samples at any given location.

Regardless of the anatomical location, the morphology of the juvenile minipig skin samples taken at PND1 differs dramatically from that of young animals at 12 weeks of age, the main feature being the very high cellularity observed in the dermis. No specific literature data of dermal morphology was found for juvenile minipigs; however, this feature corresponds to what is well known and described for human neonates.

The age range of the animals in this study covered the skin maturation between PND1 and week 12. No specific translational information was found in the literature regarding minipig skin maturation, and therefore, we used the comparative diagram of age categories across species reproduced in the review article from De Schaepdrijver in 2010, although it was based on the central nervous system and the reproductive system development. The age categories used in our study may correspond to the evolution between neonate and child (from birth to approximately 12 years old). More specifically, the period from birth to 2 weeks in minipigs was thought to correspond to the term/neonatal period in humans (i.e., birth to 0.08 years); the period from 2 to 4 weeks in minipigs is considered to correspond to the infant/toddler period in humans (i.e., from 0.08 to 2 years), and the period from 4 to 14 weeks in minipigs was considered as equivalent to the childhood in humans (from 2 to 12 years).

From an embryologic point of view (reviewed in Loomis 2001), epidermis derives exclusively from ectoderm while dermal origins in humans are known to vary depending on the body site: neural crest ectoderm for the dermal mesenchyme of the face and anterior scalp, dermomyotome for the back, and mesoderm for limbs and ventral trunk. During the embryonic period, a presumptive epidermis forms as a single cell layer associated after 6 weeks, but for a short period of time, with a second more superficial layer called periderm. Stratification occurs later during pregnancy, approximately between 11 and 15 weeks for the skin appendages and between 22 and 24 weeks for the interfollicular epidermis. The fetal epidermis consists of a well-differentiated stratified epithelium resembling the adult skin by the middle of the third trimester. The fetal mesenchymal part of the skin is mainly cellular with few fibers and an extracellular gel-like matrix consisting of hydrated proteoglycans. The collagen and elastics fibers assembled in the second half of the pregnancy and the amount of fibers increases over time. In humans, the skin of the neonate shows a thick and well-organized dermis, but when compared to the skin of adults, the cellular component that consists mainly of fibroblasts is still prominent. This corresponds to our observations in neonatal minipigs.

Although several references could be found to support the interest of Göttingen minipig for dermal studies, only scant information was found on the potential impact of the age and/or bodyweight of the minipig on skin thickness. Qvist et al. (2000) documented the thickness of the skin layers according to age ranges in the minipig and compared them to human skin, but these data are based on a limited number of animals (n = 3 for each age category) and were restricted to the dorsal skin. The youngest age evaluated was 1.5 months (epidermis 50.0 ± 5.5 µm and dermis 1.15 ± 0.11 mm), and the authors concluded that the skin at this age was the closest to human skin. In this poster, the authors showed that the epidermal and dermal thickness generally increased with age in the minipig: 64.0 ± 0.0 µm and dermis 1.47 ± 0.19 mm at 3 months and 63.0 ± 5.3 µm and dermis 2.28 ± 0.11 mm at 6 months. In our study, we compared the skin layer thickness at different anatomical locations for juvenile animals from as young as PND1 to 3-month-old minipigs. Qvist et al. did not give an exact location of the examined skin samples in their poster, but we believe what they consider to be “dorsal skin” could correspond to the area around the spine (corresponding to the zones “rachis, anterior” and “rachis, posterior” in our study). Therefore, we could consider that our skin layer measurements were consistent with Qvist et al.’s results for 1-month minipigs (epidermis: 46 to 51 µm; dermis: 954 to 1,152 µm). At 3 months, however, we found no increase in the epidermal thickness (ranging from 35 to 51 µm) and only a limited increase in the dermal thickness (ranging from 1,270 to 1,830 µm). Considering the low number of animals in both studies, the discrepancy may be attributable to interindividual variability or method of measurement, which are not detailed in the poster by Qvist et al.

To our knowledge, this is the first time that an extensive skin anatomical morphology documentation is performed in the minipig, and it demonstrates marked anatomical differences in morphology and skin layer thickness. Some areas on the body, such as the chin and snout, have a very specific morphology, but it is also noted that skin layer thickness may also differ within the anatomical regions that are classically considered as homogeneous. For example, our assessment emphasized the flank region, which was defined using anatomic landmarks, that is, as a rectangle between the scapulae and hipbones, and corresponds to the area commonly used for dermal application in dermal toxicology studies. In this documentation work, two samples were performed taken, and it was noted in all animals that the anterior and posterior samples were almost identical in morphology or thickness except for the subcutis thickness. Indeed, the anterior area consistently showed a thicker subcutis than the posterior area (approximately 2-fold). This example highlights the fact that definition and standardization of skin samples are of major importance in dermal toxicology studies in order to provide adequate controls and reproducible results.

Overall, the neonate skin of minipigs showed the typical 3-layer structure of a mature skin and, as observed in neonate humans, the cellular component of the dermis appeared prominent and dermis and hypodermis thicknesses were smaller when compared to young adult skin. Over time, these unique neonatal characteristics tended to disappear progressively and one month after birth, the skin sections were very similar to those taken at week 12. The utility of the adult Göttingen minipig as an animal model in regulatory toxicology is already recognized for dermal application studies, and our results demonstrate that the minipig may also be the animal of choice for juvenile studies.

We thank Dr. Marie-France Perron Lepage for her very valuable review.