in the third wave around ED 10.5 from the hemogenic endothelium of the dorsal aorta of the aorta‐gonad‐mesonephros (AGM) tissue. The generation of HSCs is dependent on proinflammatory cytokines secreted by macrophages in the arterial wall and by catecholamines supplied by the sympathetic nervous system in the developing embryo [2, 3]. The HSCs migrate from the AGM to the fetal liver where they undergo marked proliferation between ED 11 and ED 16. Additional HSCs are supplied to the fetal liver by the vitelline and umbilical arteries. From the fetal liver, HSCs move to the fetal spleen from around ED15.5 until a few weeks after birth. Hemopoietic stem cells in the spleen of adult mice are mostly committed to erythropoiesis. In addition, HSCs begin to populate the bone marrow starting at ED17.5. They undergo a rapid expansion in the bone marrow during the first three weeks after birth after which they become quiescent [1].
We describe here the pathology of the hemopoietic and lymphoid tissues with an emphasis on changes in mice with spontaneous and genetically engineered mutations. A detailed review of the pathology of these tissues in mice and rats was recently published as part of the INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) [4].
Bone Marrow
The bone marrow occupies the cavities of the trabecular meshwork of the axial and long bones. It is highly vascularized and supplied by nerves, but it lacks lymphatic drainage. The bone marrow forms a network of hemopoietic and nonhemopoietic cells, including endothelial cells, pericytes, fibroblasts, and osteoblasts. Together, these cells form a niche, a microenvironment for HSCs that controls their self‐renewal and differentiation [5]. The HSCs generate progenitor cells that differentiate into erythrocytes, leukocytes, including immature B lymphocytes, and platelets. In addition to its major role in generating blood cells, the bone marrow is also the site where populations of plasma cells and memory T cells reside following their induction in secondary lymphoid organs.
Examination of bone marrow: Routine assessment of the bone marrow is performed on sections of bones following formalin fixation and decalcification. Decalcification is usually achieved with organic acids such as formic acid or with chelating agents such as ethylenediaminetetraacetic acid (EDTA) solution. The latter procedure takes longer but is preferred for immunohistochemistry because of enhanced antigen preservation. The knee joint with the distal femur and proximal tibia and sternum are good choices. Light microscopy of H&E‐stained sections, preferably less than 4 μm thick, can assess overall cellularity, ratio of erythroid to myeloid precursors, and number of megakaryocytes. Examination of smears or cytospin preparations of bone marrow cells can provide a more detailed analysis of the cellular composition of the bone marrow. Cell suspensions can be obtained by flushing the long bones with a 25G needle and syringe after removing the ends (epi‐ and metaphyses). In depth analysis of the cellular composition of bone marrow and the different maturation stages of blood cell lineages requires flow cytometry of cells labeled with fluorochrome‐conjugated antibodies.
Hypoplasia and aplasia: Mutations or chemical treatments that induce DNA damage or interfere with cell division will induce bone marrow aplasia, a general loss of all hematopoietic lineages. Hematopoietic cells are absent, and bone marrow space is filled with venous sinuses and a few adipocytes (Figure 7.1). Such changes are often associated with a reduction of the red pulp of the spleen. Loss of specific lineages may be seen with genetic deletion of growth factors. A selective decrease of erythropoiesis is associated with chronic inflammation. There is increased myelopoiesis and an increase of hemosiderin.
Hyperplasia: Increased numbers of hematopoietic cells may involve all or selected cell lineages. The hyperplasia is usually the result of increased demand induced by increased secretion of growth factors or cytokines, and is typically associated with increased extramedullary hematopoiesis (EMH) in the spleen and liver. An increase of erythropoiesis is caused by anemia and may be associated with megakaryocyte hyperplasia if there is loss of platelets. Increased myelopoiesis is often seen with chronic inflammation and can result in a dramatic shift of the myeloid:erythroid ratio.Figure 7.1 Bone marrow. Bone marrow hypoplasia four days after deletion of Cul4A in Cul4a/Pcid2tm2Ktc mice (b) compared with C57BL/6J mice (a). (c) Fibro‐osseous lesions in 821‐day old female 129S1/SvlmJ and 427‐day old female KK/H1J (d) mice. Higher magnification of fibro‐osseous lesion (e).
Aging‐associated changes: The overall cellularity of the bone marrow in old mice remains high at >90% in contrast to humans in which the hematopoietic cells are gradually replaced by adipocytes, although there are strain and regional differences in the distribution and accumulation of adipocytes. Aging is associated with an increased number of HSCs, increased myelopoiesis, and increased accumulation of plasma cells. The plasma cells promote myelopoiesis through the local secretion of inflammatory cytokines [6]. These changes may be difficult to assess in routine H&E sections of the bone marrow. Fibro‐osseous lesions are proliferative alterations comprised of fibrovascular tissue intermixed with fine bone trabeculae in the bone marrow of aged mice (Figure 7.1). Although they replace hemopoietic tissue, the lesions are not sufficiently expansive to compromise the production of hemopoietic cells. Fibro‐osseous lesions occur predominantly in female mice with a high incidence in B6C3F1 mice and in certain inbred strains including 129S1/SvImJ, KK/H1J, and NZW/LacJ [7–9].
Lymphoid Tissues
Lymphoid tissue can be divided into primary, secondary, and tertiary lymphoid tissues. The primary lymphoid organs are the sites of development and maturation of B and T lymphocytes. The developing B and T lymphocytes undergo somatic recombination of the immunoglobulin and T cell receptor gene segments resulting in a vast repertoire of antigen‐specific receptors. This process is largely antigen‐independent. The primary lymphoid organs in the mouse are the bone marrow for B cell development and the thymus for T cell development. The initiation of the adaptive immune response mediated by antibodies and T cells occurs in the secondary lymphoid organs which include the lymph nodes, white pulp of the spleen, and mucosa‐associated lymphoid tissues. Mature, naïve B and T lymphocytes circulate through the secondary lymphoid organs which are connected via the lymphatic and blood circulation. Naïve lymphocytes enter the lymph nodes and mucosa‐associated lymphoid tissues via high endothelial venules and leave via the efferent lymphatics. The spleen has open‐ended arterioles that deliver lymphocytes to the marginal zone and red pulp. Lymphocytes leave the spleen primarily via the splenic vein. The restricted circulation pattern of naïve lymphocytes maximizes the chances that the few lymphocytes with receptors for a specific antigen will encounter that antigen which is delivered to the lymphoid tissues. Lymph nodes are strategically positioned throughout the body to capture antigens delivered via the afferent lymphatics from the tributary areas. Blood‐borne antigens are captured by macrophages, B cells and dendritic cells in the marginal zone of the spleen and transported into the white pulp. The epithelium that overlies the mucosa‐associated lymphoid tissues has specialized epithelial cells (M cells) that facilitate the uptake of antigens from the mucosal surface and transport them into the underlying lymphoid tissue.
Tertiary lymphoid organs develop at sites of chronic inflammation associated with infections, autoimmune disease, and cancer. The organization of tertiary lymphoid organs mimics that of secondary lymphoid organs with varying degrees of differentiation.
Primary Lymphoid Organs
The primary lymphoid organs in the mouse are the bone marrow for the antigen‐independent generation of B cells and the thymus for T cells. The general assessment of bone marrow is described above. Microscopy of routinely stained sections of bone marrow does not provide specific information about the development of B cells. This requires isolation of bone marrow cells followed by labeling with appropriate reagents and analysis by flow cytometry.
