9.1 Introduction

The lymphoid and haematopoietic system is composed of multiple organs and tissues distributed throughout the body and is responsible for the development of the immune response, and the production of the blood’s cellular components.

The primary lymphoid organs are the bone marrow and thymus, responsible for production and maturation of the B- and T- lymphocytes respectively. The secondary lymphoid organs, the lymph nodes, spleen and mucosa-associated lymphoid tissues maintain populations of mature lymphocytes and are the sites of antigenic stimulation and clonal expansion. The lymphoid organs are composed of two tissue components, reticular connective tissue and lymphatic tissue, composed of lymphocytes, macrophages and antigen presenting cells.

Haematopoietic stem cells are active in the mouse liver from embryonic day 10, and in the spleen from embryonic day 13, but the bone marrow becomes the primary site of haematopoiesis from embryonic day 18 onwards (Holsapple et al. 2003). The predominant site of haematopoiesis in the adult mouse is the bone marrow of the long bones, but the spleen retains haematopoietic activity throughout adult life and haematopoietic activity in the liver can be seen in response to disease (Haley 2003; Taylor 2011; Linden et al. 2012).

The anatomy and histology of the structural components of each of the different lymphoid tissues are presented below but there are several sources of information (some of which are available online) that provide details of the basic anatomy of the mouse (Cook 1965, 1983; Hummel et al. 1966), and provide guidance on general necropsy and histology practices, which include descriptions of procedures to follow for the dissection and trimming of these tissues. Reviews of the normal histopathology of the lymphoid system of the mouse, and changes associated with exposure to xenobiotics in preclinical toxicity tests are also available (Ward et al. 1999; Maronpot 2006).

There are complex interactions between the different organs of the lymphoid system, which is a dynamic system reacting to changes in antigenic stimulation throughout life. These reactions can be manifested as morphological changes in the different components of the system. There can also be pronounced strain-, genetic-, age-, and sex-dependent variations in the function (Sellers et al. 2012) and normal appearance of lymphoid organs, which need to be taken into account when performing histopathological evaluation of these tissues (Elmore 2012). Careful comparison of findings seen in lymphoid tissues with the range of changes observed in concurrent control animals is required to ensure that normal background variations are taken into account when interpreting these findings (Figure 9.1). It can also be difficult to differentiate between severe hyperplastic reactions in lymphoid tissues and lymphoma or leukaemia. Knowledge of the range of reactive changes in the species being examined, and of species-, sex- and strain-related differences in anatomical and histological appearance of the lymphoid tissues, as well as an understanding of the biological behaviour of the neoplastic changes being diagnosed is important in reaching correct diagnoses (Ward et al. 2012).

Figure 9.1 Comparison of morphology of axillary lymph nodes from control mice at 20 weeks of age, illustrating the type of variation that can be seen in the size of the nodes and the proportions of the different components: (a) relatively small axillary lymph node with few primary follicles; (b) relatively large axillary lymph node with prominent paracortical areas, secondary follicles, sinus erythrocytosis and pigmented macrophages.

9.2 Lymph nodes

As mentioned above, inappropriate trimming or sectioning of small lymph nodes can introduce artefactual variation in apparent size of the different compartments of the lymph node (Figure 9.2). The presence of erythrocytes within the sinusoids can be indicative of haemorrhage in the tissues drained by the lymph node, but this change can also be agonal or artefactual as a result of damage during necropsy procedures (Figure 9.3). The presence of haemosiderin laden macrophages and erythrophagocytosis is indicative of the ante-mortem nature of the change (Elmore 2006a; Taylor 2011). Crush artefacts of lymph nodes are a result of inappropriate handling at necropsy or histology (Figure 9.4).

Figure 9.2 Variation in morphology of lymph node associated with off-centre (a) or central (b) sectioning of the node.

Figure 9.3 The presence of blood within the sinuses of an axillary lymph node associated with agonal haemorrhage or damage during necropsy procedures.

Figure 9.4 Crush artefact of lymph node due to inappropriate handling of fresh or fixed tissue. Smearing of the lymphocytes in affected region (arrows).

9.2.4 Anatomy and histology

Correct identification and nomenclature of the murine lymph nodes is important to ensure reproducibility of findings in scientific studies (Van den Broeck et al. 2006) and a clear understanding of lymph node structure, and regional variations in structure, is important in correctly identifying changes in the nodes (Willard-Mack 2006). A generalized illustration of the structure of a ‘typical’ lymph node is presented in Figure 9.5.

Figure 9.5 Representation of the structure of a lymph node, illustrating on the left the vascular structure, in the centre, the reticular meshwork superimposed on the vasculature, and on the right, a lobule as it appears histopathologically. (Reprinted by permission of SAGE Publications from Willard-Mack, C.L. (2006) Normal structure, function, and histology of lymph nodes. Toxicologic Pathology, 34, 409–424, and by permission of the illustrator, David A Sabio.)

There are relatively few lymph nodes in the mouse compared to other species (Haley 2003). Van den Broeck et al. (2006) identified 22 different nodes in the mouse. These are bean-shaped structures connected to lymph vessels and distributed throughout the body. They are small and can be difficult to identify within fat and connective tissues. The peripheral lymph nodes are bilateral and include the mandibular, axillary and popliteal lymph nodes. Central lymph nodes in the thoracic or abdominal cavities are usually not bilateral (including the mediastinal, pancreatic and mesenteric) (Van den Broeck et al. 2006; Linden et al. 2012). The distribution and nomenclature of the lymph nodes in the mouse has been well described by Van den Broeck et al. (2006) (Figure 1.9 and Table 9.1).

Table 9.1 Lymph nodes.

A reticular meshwork supports the lymphatic components and the lymph node is composed of lymphoid lobules, separated by lymph filled sinuses and surrounded by a fibrous capsule (Linden et al. 2012). Afferent lymph vessels enter the lymph node at the convex edge, connecting to subcapsular sinuses and draining through the cortex and paracortex into the medullary sinuses, and draining from the lymph node via efferent lymphatics at the hilus. The hilus is also the point of entry of arterioles and nerves, and of exit of veins.

The basic anatomical and functional unit of the lymph node is the nodule, present in varying numbers dependent on the size and location of the lymph node. The differentiation of structures and lymphoid cell populations within the nodules gives rise to the structural differences noted histopathologically, namely the cortex, paracortex and medulla. The peripheral cortex, below the subcapsular sinus, is composed of follicular structures consisting mainly of B lymphocytes. The size and appearance of the follicles is dependent on antigenic stimulation. Primary follicles are unstimulated and appear as dense collections of small lymphocytes surrounding a small follicular centre containing a small number of pale lymphoid cells. Stimulated follicles are classed as secondary follicles and are larger, containing proliferating B cells forming germinal centres containing large lymphoblasts, macrophages and tingible body macrophages (Figure 9.6). The paracortex represents the area of the lymph nodes containing predominantly T lymphocytes, and is situated between the follicles and the medullary sinuses. Antigenic stimulation of T lymphocytes leads to proliferation of these cells in the paracortex but without the formation of follicular structures or germinal centres. High endothelial venules (HEV) are the site of entry of vascular lymphocytes into the stroma of the lymph nodes (Figure 9.7). They are located throughout the interfollicular cortex and paracortex but appear more obvious at the periphery of the paracortex. As these vessels transition into the medulla, the high endothelium is lost and they become lined by squamous endothelium typical of the medullary venules. The medulla is composed of cords and sinuses, with variable numbers of lymphocytes, plasma cells and macrophages. Plasma cell precursors from the cortex migrate to the medulla following B-cell stimulation, where they mature and they release antibodies into the lymph. Following antigenic stimulation, the cords can be packed with plasma cells and small lymphocytes (Figure 9.8). A more detailed description of the anatomy of the lymph node and the nodular structure in relation to immune function is presented in the review by Willard-Mack (2006).

Figure 9.6 Comparison of primary and secondary follicles in lymph nodes. (a) Collection of small lymphocytes surrounding a small follicular centre containing a small number of pale lymphoid cells; (b) Large secondary follicle with increased number of pale staining cells and tingible body macrophages.

Figure 9.7 High endothelial venules.

Figure 9.8 Plasmacytosis. Medullary sinuses packed with plasma cells in an enlarged mandibular lymph node.

A variety of spontaneous changes are seen in the lymph nodes of mice, some of which represent changes related to antigenic stimulation, often of an unknown aetiology. Proliferative changes may be secondary to the presence of tumours or degenerative changes in the animal, particularly the presence of skin lesions which can result in hyperplasia of cellular elements in draining lymph nodes. The incidence of lymphocyte hyperplasia (Figure 9.9) increases with age and tends to be more common in females than males (Frith et al. 2001; Elmore 2006a). Plasma cell hyperplasia and mast cell hyperplasia (Figure 9.10) are also commonly seen but there are strain differences in the background incidence of mast cells in lymph nodes (Ward et al. 1999; Frith et al. 2001; Elmore 2006a).

Figure 9.9 Lymphocytic hyperplasia of the lymph node with an overall increase in lymphocytic-rich compartments and increased germinal centres (arrows).

Figure 9.10 Increased heavily granulated mast cells (arrows) in the medullary sinuses of a lymph node.

Atrophy is often seen in the lymph nodes of ageing mice (Ward et al. 1999) and can be associated with fatty infiltration of the lymph node (Figure 9.11), or sinus ectasia (Elmore 2006a; Taylor 2011) (Figure 9.12). Angiectasis is most often seen in the mesenteric lymph nodes and can be distinguished from sinus ectasia by the presence of endothelial lining cells (Figure 9.13). Sinus erythrocytosis can be related to the lymph node draining a site of haemorrhage but can also be artefactual. The levels of erythrophagocytosis and haemosiderin pigment seen are indicative of the chronicity of the lesion (Elmore 2006a; Taylor 2011) (Figure 9.14). Sinus histiocytosis is characterized by the presence of large macrophages with eosinophilic cytoplasm in the medullary and subcapsular sinuses of the lymph nodes, often associated with phagocytosis of pigment (commonly haemosiderin or lipofuschin) or other materials (Figure 9.15). Hyperplasia of dendritic reticular cells is occasionally seen in young mice and is considered to be secondary to viral infections (Taylor 2011) (Figure 9.16). Extramedullary haematopoiesis may occasionally be seen in lymph nodes, secondary to haemorrhage or severe inflammation (Frith et al. 2001; Elmore 2006a; Taylor 2011). The lymph nodes are described as a common site for deposition of amyloid in different strains of mice, particularly the CD-1 mouse (Elmore 2006a; Percy and Barthold 2007). However, the occurrence of amyloidosis in CD-1 mice has shown time-related changes and can vary considerably between animal suppliers (Maita et al. 1988; Engelhardt et al. 1993; Taylor 2011).

Figure 9.11 Atrophy and fatty infiltration of a lymph node in an aged mouse.

Figure 9.12 Cystic dilatation of the sinuses of a mandibular lymph node.

Figure 9.13 Angiectasis in the mesenteric lymph node of an aged CD-1 mouse.

Figure 9.14 Sinus erythrocytosis with erythrophagocytosis (black arrow) of the axillary lymph node. Presence of haemosiderin pigment in macrophages (white arrow).

Figure 9.15 Sinus histiocytosis of a mesenteric lymph node. The sinuses contain increased numbers of large, pale eosinophilic histiocytes (arrow).

Figure 9.16 Hyperplasia of dendritic reticular cells. (a) Low power view of axillary lymph node showing increased numbers of eosinophilic cells within sinusoids. (b) High power view illustrating eosinophilic cells with elongated nuclei.

Neoplastic changes of the lymph nodes, other than secondary involvement of haematopoietic neoplasms are rarely seen in mice and almost never in mice less than a year old. Haemangiomas are occasionally seen in lymph nodes in chronic toxicity studies (Faccini et al. 1990).

9.3 Spleen

The spleen is composed of two compartments, the red pulp and the white pulp. The red pulp is composed of a vascular network and sinuses involved in filtration of the blood, and macrophages which phagocytose foreign material and cellular debris. The spleen is often enlarged at necropsy and there are a number of physiological and pathological explanations for this so this change should not be automatically mistaken for lymphoma (Table 9.2). Extramedullary haematopoiesis is common in the red pulp of the mouse spleen and reactive haematopoiesis involving myeloid, erythroid and megakaryocyte hyperplasia is seen in response to increased demand, as a result of anaemia (due to chronic haemorrhage for example from uterine and ovarian lesions in ageing female mice), neoplasia or inflammation (Figure 9.17). In severe cases of extramedullary haematopoiesis the spleen can be very large and the distinction between haematopoiesis and granulocytic leukaemia needs to be made based on the presence of an inciting lesion and the lack of involvement of other organs. Also, in leukaemia, a single stage of the developing granulocyte is typically present whereas in reactive granulopoiesis a complete series of developing cells are usually present (Frith et al. 1983, 2007; Ward 1990). The white pulp is composed of three compartments: the PALS, the marginal zone, and follicles. The white pulp of the mouse tends to be more prominent than that of the rat, while the marginal zones are less prominent and more variable in the mouse. The PALS are divided into inner PALS and outer PALS, the inner being a T-cell dependant region stains more intensely then the outer PALS. The marginal zone is composed mainly of B-cells and macrophages, and a smaller population of T-cells. B-cell containing follicles are associated with the PALS, and show similar changes in morphology to those described in the lymph nodes (Cesta 2006a; Suttie 2006; Linden et al. 2012).

Table 9.2 Differential diagnosis of enlarged spleen.

Source: Frith et al. (1996, 2001; Ward et al. (1999).

Figure 9.17 Extramedullary haematopoiesis in red pulp of the spleen characterized by the presence of megakaryocytes (black arrow) and cluster of small darkly basophilic staining erythrocyte precursors (white arrow).

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