Pathology of Genetically Engineered and Other Mutant Mice. Группа авторов

EBS and MBS contain large numbers of definitive RBCs. Stain: H&E.

      Source: Bolon and Ward [39] with permission of CRC Press.

The maternal immune response against the embryo is restrained by the presence of a transient immune‐modulating structure in the decidua of mice and rats designated variously as the decidualized mesometrial triangle, mesometrial lymphoid aggregates of pregnancy (MLAp), or metrial gland (Figure 5.6) [49–52]. This metrial “gland” isn't really a gland but an area of the placenta composed of arteriolar‐like blood vessels and abundant uterine natural killer (uNK) cells (also called granulated metrial gland [GMG] cells). These uNK cells are maternal bone marrow‐derived, perforin‐positive leukocytes that express NK cell biomarkers [52, 53]. The uNK cell population and the entire metrial gland begin to regress by mid‐gestation.

Placental size differs among mouse strains. Outbred ICR (Institute of Cancer Research) mice have placentas that are about 25% larger than those of age‐matched inbred C57BL/6J (B6) mice. This discrepancy is correlated with embryonic growth as ICR embryos are approximately 25% bigger than age‐matched B6 embryos [54]. A similar placental size increase of 20% has been reported in hybrid mice, where embryos are larger relative to those generated by the inbred C57BL/Mcl or C3H/BiMcl parental strains [55], and even larger increases have been described in cloned mice [56]. Strain differences in placental biology are not limited to structural effects, as metabolic capacities and pathways in placental tissue also vary among mouse strains [57].

Figure 5.6 The nascent metrial gland (also designated the mesometrial lymphoid aggregate of pregnancy), shown here at GD8.5 (Panel (a)), appears as a dense collection of granulated metrial gland (GMG) cells (Panel (b); also called uterine natural killer [uNK] cells, Panel (c)) that surrounds large maternal blood vessels in the mesometrial decidua. Lectin histochemistry for the uNK cell marker Dolichos biflorus agglutinin (DBA) lectin (Panels (a) and (c)). Stain (panel (b)): H&E.

      Sources: Dr. Bruno Zavan, University of Alfenas, Brazil, and from Bolon [96] with permission of Elsevier.

Technical Considerations for Pathologic Evaluation of Developing Mice

      The plan for pathologic evaluation for any phenotyping project in developing mice will utilize techniques comparable to those applied when examining adult mice. However, the choice of methods will depend on several factors unique to prenatal and early postnatal animals, including the developmental age of the subjects, the presumed timing of the lethal event(s), and in many instances the expertise and interest of the investigators and cost. In general, the initial pathologic evaluation of a novel phenotype will center on known anatomic, clinical, and molecular pathology procedures [5, 58]. For prenatal phenotypes, both the embryo and placenta must be examined since developmental and lethal events may occur in either or both of these specimens.

      An important consideration for success in mouse developmental pathology is a sufficient degree of prior knowledge and experience by the morphologist. Developmental pathology is an endeavor where DIY (“do it yourself”) assessments [59] are rarely efficient or effective, and often are incorrect or misleading. Substantial money, time, and animals will likely be wasted if individuals with limited or no foundation in mouse developmental biology and pathology attempt to lead a developmental pathology project.

      Noninvasive Imaging of Developing Mice

The traditional basis for characterizing structural changes in embryonic lethal and neonatal lethal phenotypes in mice has concentrated on qualitative macroscopic and light microscopic analysis, supplemented as needed by quantitative measurements of organ dimensions [60, 61], volumes [62], or cell numbers [48]. Historically, these strategies have been undertaken at one or more specific time points and have been complicated by some degree of sample destruction (e.g. via continuous or interval [“step”] sectioning to make multiple μm‐thick, two‐dimensional [2D] tissue slices) and subsequent three‐dimensional [3D] tissue reconstruction (e.g. by mental or physical realignment [“stacking”] many 2D tissue slices). In recent decades, the advent of noninvasive imaging methods for rodents has been adapted to allow one‐time or serial evaluation of adult [63] and developing [22, 64, 65] mice. These technologies facilitate the digitized, relatively high‐resolution, 2D and 3D macroscopic and microscopic analysis of anatomic features and/or functional attributes without destroying or greatly distorting the specimen (Figure 5.7). Key modalities include computer‐assisted tomography (CAT or μCT), confocal microscopy, echocardiography, magnetic resonance microscopy (MRM), optical coherence tomography (OCT), and ultrasound biomicroscopy (UBM), among others [66–69]. These noninvasive imaging techniques will not replace standard microscopy since both light and electron microscopy afford higher resolution of cell and tissue architecture. Instead, the use of imaging techniques provides the best data when employed in tandem with conventional pathology techniques.

      Anatomic Pathology Evaluation ofDeveloping Mice

For prenatal phenotypes, the first task is to measure maternal total body weight and then count the numbers of total and viable conceptuses (i.e. embryo/placental units) as well as abnormal and resorbed implantation sites. For isolated embryos and neonates, relevant individual animal characteristics are recorded including sex (based on anogenital distance; Figure 5.8), body weight, and/or crown/rump length as well as the kind and severity of external gross defects. The embryos and neonates then are relegated to different subgroups for conventional necropsy to harvest tissues (Figure 5.9), free‐hand sectioning of the torso (Figure 5.10) and head (Figure 5.11) in multiple planes to view organs in situ to find gross internal defects, double staining to highlight skeletal (bone and cartilage) differentiation (Figure 5.12), or fixation to permit histopathological evaluation [70]. Placentas should be fixed together with their embryos.

Histological processing is a key step in preparing the tissues of developing mice for microscopic evaluation. The choice or processing techniques depend on the age of the animal and the types of tissues that need to be preserved. Commonly, fixation is done by immersion, which is simple and rapid, but perfusion fixation may be needed to preserve deep, dense tissues that degenerate quickly, such as bone marrow and brain [58, 71]. For early embryos, tissues generally are fixed by immersion in neutral buffered 10% formalin (NBF), Bouin's solution, or modified Davidson's solution. All three of these solutions penetrate well, and both Bouin's and modified Davidson's solutions harden soft embryonic tissues so that they may be handled for macroscopic evaluation. For late‐stage embryos (GD14.0 and later) and neonates, the same three fixatives still may be used, but for NBF the thick skin will needed to be opened (usually by a long slit in the ventral thoracic and abdominal regions) to permit penetration. Tissue dehydration and clearing, embedding media, and routine staining protocols for use with embryos and neonates generally are equivalent to those used for adult tissues [72]. The two biggest differences between embryos and neonates vs. adults are that gentle decalcification is required for all samples of developing mice beginning at GD15.5 and later due to progressive skeletal mineralization, and that all embryos and neonates should