10.1 Background and development
The integumentary system in the mouse is similar to that of other mammals, consisting of the skin and its associated specialized structures (mammary tissue, hair and nails). Components of the skin are derived from multiple embryonic layers with the epidermis deriving from ectoderm, connective tissues of the dermis and subcutis from mesoderm and nerve endings and melanocytes from the neural crest. The epidermis develops from a single layer in the embryo to become a multilayered structure which is thickest at birth and then thins to two to three layers in the first two weeks postnatal. The first hair follicle cycle of development also begins in the embryo but does not complete until after birth. Different types of hair follicle develop at different rates with the vibrissae being most advanced at birth (Sundberg and King 2000).
In the female mouse the mammary gland arises from ectoderm around embryonic day 11, proliferating to invade the mammary fat pad around day 17 and then forming a simple branching structure with a single duct leading to the nipple prior to birth. In the male embryo development of the mammary epithelium is curtailed at day 13 leaving only remnant tubular structures within the mammary fat. The majority of mammary development occurs postnatally in the female with the onset of ovarian hormone production from week 3 (Richert et al. 2000).
The origins of adipocytes have been under intense investigation in recent years and, while most adipose tissue is still thought to arise from mesoderm, there is evidence of neurectodermal origin for some fat deposits and postnatal development from bone-marrow stem cells. White adipocytes and brown adipocytes also appear to arise from different lineages with brown adipocytes expressing genes suggestive of a myogenic cell origin (Majka et al. 2011).
10.2 Sampling technique
For routine sampling neutral buffered formalin is sufficient for fixation of skin, mammary and adipose tissue for morphological analysis, although acid alcohol fixatives and freezing may be needed for immunohistochemistry (Paus et al. 1999; Sundberg and King 2000). The site of sampling may reflect the study requirements. A single section from the inguinal region will enable analysis of the skin, mammary and white adipose tissue (including nipple in female mice) (Ruehl-Fehlert et al. 2003) in one sample. Mammary tissue is extensive in the female mouse (Chapter 1, Figure 1.14) and so may also be seen incidentally in skin samples from multiple sites.
For more detailed analysis of hair-follicle development, a sample of dorsal skin is preferred. Ideally the samples should be taken from a consistent site, for example in the dorsal midline at the thoracolumbar junction (Paus et al. 1999). To aid orientation after fixation, samples of skin can be laid flat onto cardboard at the time of sampling to prevent distortion (Paus et al. 1999; Sundberg and King 2000). Labelling the cranial and caudal edges of the sample will also help ensure that samples are sectioned to produce longitudinal sections through the hair follicles which are easier to analyse than cross or tangential sections. Although H&E staining is adequate for basic analysis of skin and hair follicles, other histochemical and immunohistochemical stains may be useful to delineate accurately all the substages of hair follicle development (Muller-Rover et al. 2001).
A single skin sample taken from mice at 10–12 weeks of age may be suitable for a routine screen where no skin or hair phenotype is suspected. Where a phenotype is suspected then it is important to sample animals at different stages of the hair developmental cycle (Sundberg et al. 2005) and also to analyse plucked hair samples. In depth approaches to phenotyping have been described in detail elsewhere and will not be covered in this chapter (Sundberg and King 2000).
Additional samples of skin may be needed to examine skin of different types (e.g. ear, eyelid, muzzle, tail) and nails. Muzzle or facial skin samples may be taken for analysis of vibrissae. Tail skin is most easily examined by fixation of the whole tail and decalcification prior to sectioning. Nails can be sectioned longitudinally after dissection, fixation and decalcification of individual digits. Sampling of pinna and eyelids are discussed in Chapter 12.
Analysis of mammary tissue from models of mammary tumours may require more extensive tissue sampling of specific glands including representative thoracic and inguinal mammary glands in addition to any tumour masses (Cardiff et al. 2000). Trimming of excess fat from the mammary tissue will aid fixation and processing. At necropsy, lesions can be noted using a map of mammary tissue (Rasmussen et al. 2000). For detailed analysis of the branching structure of the mammary glands, whole mount sections may be useful (Rasmussen et al. 2000).
Until recently, adipose tissue was routinely divided into two main subtypes—brown adipose tissue (BAT) and white adipose tissue (WAT), based on their morphological appearance and the ability of BAT to express UCP1. It is now accepted that a third subtype known as ‘brite’ adipose tissue consists of adipose tissue that is WAT in morphological appearance but can also express UCP1 when stimulated, for example by exposure to cold. In routine studies, adipose tissues may not be sampled specifically but their presence may be noted in association with other tissue samples. For example BAT is often found surrounding the aorta in mice (Chapter 4) and WAT is often seen subcutaneously in skin or surrounding mammary tissue. Where specific investigation of adipose tissue is required, for example in the study of metabolic and obese phenotypes, appropriate deposits can be sampled (Casteilla et al. 2008) to take into account BAT, WAT and brite phenotypes (Walden et al. 2012). In the mouse the interscapular, cervical, axillary and mediastinal fat deposits all express UCP-1. The bi-lobed interscapular deposit is the most well developed and is a convenient site to sample in all ages of mice. Cardiac, inguinal and retoperitoneal fat deposits can express UCP-1 under certain conditions and are classified as ‘brite’ adipose tissue. Brite tissues are rarely sampled but it is important to be aware of this mixed phenotype. Mesenteric and epididymal do not normally express UCP-1 and can be sampled as examples of WAT (Casteilla et al. 2008). Tissues from specific fat deposits should be embedded in a consistent way. It is sometimes useful to take a cross-section through the interscapular fat at the widest point as this allows analysis of the size and consistency of the BAT and the sample also usually includes some peripheral WAT for ease of comparison.
10.3 Anatomy and histology
The histology of mouse skin is generally similar to that of other mammals, consisting of an outer epithelial epidermis covering the connective tissue dermis and an underlying hypodermis largely composed of fat (Figure 10.1). A thin layer of skeletal muscle, the panniculus carnosus, separates the hypodermal fat from the adventitia, a loose connective tissue layer attached to the muscle of the underlying body wall.
The epidermis may appear very thin (1–2 layers) in adult mice but still consists of the four epithelial-derived cell layers seen in other species—stratum basale, stratum spinosum and stratum granulosum covered by the keratinized stratum corneum (Figure 10.2), which can be distinguished by different patterns of keratin expression using IHC. The stratum basale contains the proliferating cells and so is the only layer where mitoses may be seen in normal skin. Darkly basophilic granules may be seen in the stratum granulosum cells. The epidermis is thicker in neonatal mice (Figure 10.3) and the hair follicles are still in an embryonic hair cycle. The first mature hair cycle starts 2 to 3 weeks after birth. In pigmented strains of mice, melanin pigment is found in the cells of the stratum basale.
Figure 10.2 High-power image of hyperplastic epidermis. The epidermis in this section is slightly thicker than normal but allows all the layers to be seen, including the stratum granulosum, which can be hard to visualize in normal skin sections.
The dermis consists of loose connective tissue with blood vessels and normally a sparse resident population of inflammatory cells. Hair follicles and associated adnexal glands and muscle are also seen in the dermis and extending into the hypodermis. The sebaceous glands are made up of large vacuolated polygonal cells with a central nucleus. The cytoplasm of these secretory cells can be stained for neutral lipid, for example with oil-red-O. The secretory cells are surrounded by a population of small, cuboidal, darkly staining cells known as reserve cells which differentiate into secretory cells (Figure 10.4). The hypodermis is composed predominantly of adipose tissue the thickness of which changes with the stage of the hair follicle, being thicker in anagen and at its thinnest in telogen.
Figure 10.4 Sebaceous glands composed of vacuolated secretory cells surrounded by small reserve cells.
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