History
A 6-month-old sexually intact male Long-Evans rat was evaluated because of acute left hind limb lameness. The rat was enrolled in a research study investigating the role of central chemical signaling pathways in spatial memory and navigation; however, at the time of the evaluation, no experimental treatment had been performed. Prior to the evaluation, the research group noted a mild change in gait but saw no obvious signs of pain or discomfort. Within 24 hours, the rat became unable to effectively move around the cage and was reported for evaluation.
Clinical and Gross Findings
On initial observation, the rat was actively moving around its enclosure with a marked droop in tail carriage and a long-strided, ataxic pelvic limb gait. There were no overt signs of pain elicited during firm palpation of the vertebral column or on manipulation of the pelvic limbs.
Whole body radiography did not reveal any overt structural vertebral abnormalities or evidence of bony or soft tissue disease. A complete neurologic examination revealed ambulatory paraparesis and a long-strided, ataxic pelvic limb gait with the left pelvic limb more severely affected than the right pelvic limb. On the third day following evaluation, the paraparesis had worsened dramatically and motor function in the left pelvic limb was nearly absent. The rat was euthanized by means of CO2 inhalation, and the carcass was perfused with neutral-buffered 10% formalin. The brain was harvested for experimental data collection, and the remaining tissues were provided for examination.
On inspection of the formalin-fixed tissues provided by the research group, pronounced muscular atrophy of both pelvic limbs and the epaxial muscles overlying the sacrum and caudal lumbar vertebrae was evident. The liver was moderately enlarged with smooth rounded edges and an irregular, dimpled capsular surface. The spleen was severely enlarged to approximately 3 times the size expected in a healthy rat and had a firm yet friable texture. On the ventral aspect of the thoracic vertebral column, there was a 0.5 × 1.5-cm irregular, raised, bilobed dark reddish-black mass at the level of T5 through T7 that radiated laterally away from the midline (Figure 1).
Photographs of tissues obtained during the necropsy of a 6-month-old sexually intact male Long-Evans rat that had been evaluated because of acute pelvic limb paraparesis. A—The ventral aspect of the vertebral column (the ribs have been trimmed and removed) has dark red perivertebral discoloration at the level of the caudal thoracic vertebra (T5 through T7). The lesion is raised with a smooth surface and appears to infiltrate adjacent muscle and soft tissue structures. B—The spleen is grossly enlarged with markedly rounded edges and a firm friable texture. Visible fissures on the surface of the spleen are likely a postmortem change that occurred during handling of the tissues.
Citation: Journal of the American Veterinary Medical Association 249, 4; 10.2460/javma.249.4.381
Photographs of tissues obtained during the necropsy of a 6-month-old sexually intact male Long-Evans rat that had been evaluated because of acute pelvic limb paraparesis. A—The ventral aspect of the vertebral column (the ribs have been trimmed and removed) has dark red perivertebral discoloration at the level of the caudal thoracic vertebra (T5 through T7). The lesion is raised with a smooth surface and appears to infiltrate adjacent muscle and soft tissue structures. B—The spleen is grossly enlarged with markedly rounded edges and a firm friable texture. Visible fissures on the surface of the spleen are likely a postmortem change that occurred during handling of the tissues.
Citation: Journal of the American Veterinary Medical Association 249, 4; 10.2460/javma.249.4.381
Photographs of tissues obtained during the necropsy of a 6-month-old sexually intact male Long-Evans rat that had been evaluated because of acute pelvic limb paraparesis. A—The ventral aspect of the vertebral column (the ribs have been trimmed and removed) has dark red perivertebral discoloration at the level of the caudal thoracic vertebra (T5 through T7). The lesion is raised with a smooth surface and appears to infiltrate adjacent muscle and soft tissue structures. B—The spleen is grossly enlarged with markedly rounded edges and a firm friable texture. Visible fissures on the surface of the spleen are likely a postmortem change that occurred during handling of the tissues.
Citation: Journal of the American Veterinary Medical Association 249, 4; 10.2460/javma.249.4.381
Formulate differential diagnoses from the history, clinical findings, and Figure 1—then turn the page→
Histopathologic Findings
All tissues were fixed via whole-body perfusion with neutral-buffered 10% formalin immediately after euthanasia followed by immersion in a similar formalin solution. Tissues were embedded in paraffin, sectioned at a width of 4 to 6 μm, and stained with H&E stain. Histologically, the mass consisted of sheets of a monomorphic population of deeply basophilic round cells (approx 10 to 15 μm in diameter) that contained scant cytoplasm and large, often bizarre nuclei with prominent nucleoli (malignant lymphocytes). Mitotic figures were frequent (approx 15 to 20 mitoses/hpf). Neoplastic cells had infiltrated and expanded the bone marrow of the associated vertebral bodies (T5-T7) and spinal processes as well as the adjacent epaxial muscles and associated soft tissues including peripheral nerves, dorsal root ganglia, and meninges. The periosteum was variably infiltrated and expanded by the invading tumor cells; however, there was no evidence of periosteal reaction. Tumor cells had entered the vertebral canal via a widened vertebral foramen and had expanded the meninges. Rarely, reversal lines could be seen in the associated bone but there was no notable bony lysis. In tumor-affected regions of spinal cord, there was a pattern of multifocal hemorrhage and associated minimal to mild myelomalacia. The liver, spleen, and mesenteric lymph nodes were also infiltrated by similar neoplastic lymphocytes, which displaced parenchymal structures and effaced normal architecture.
Immunohistochemical labeling was performed on sections of affected liver and spleen tissues. Neoplastic lymphocytes did not label with antibody against CD3 antigen (T-cell marker), but there was strong labeling for PAX5, an essential developmental transcription factor and nuclear protein marker found in B lymphocytes at the stages of B cell precursors (pro- and pre-B cells) through to early plasma cells.1,2 The nuclei of neoplastic lymphocytes were also labeled with antibody against terminal deoxynucleotidyl transferase, a specialized DNA polymerase expressed in immature lymphocytes; however, the nuclei were not labeled with antibodies against κ light chain and IgM, which are markers of mature B cells and mature plasmacytes. These results were consistent with a precursor B-cell lymphoma3,4 (Figure 2).
Photomicrographs of sections of the thoracic vertebral column from the rat in Figure 1. The body was perfused with neutral-buffered 10% formalin directly following euthanasia, and subsequently, all tissues were submerged in a similar fixative solution. A—In a subgross cross section, notice the prominence of deeply basophilic cells infiltrating muscle and soft tissue structures ventral to a vertebral body and invading the bone and bone marrow. H&E stain; bar = 8 mm. B—In the spinal cord, there is multifocal hemorrhage in both the white and gray matter. H&E stain; bar = 800 μm. Inset—In a higher-magnification image, there is a prominent region of intraparenchymal hemorrhage. H&E stain; bar = 100 μm. C—In another subgross cross section, neoplastic cells are positive for the B-cell marker PAX5. PAX5-specific immunohistochemical stain; bar = 8 mm. D—In a higher-magnification image, prominent PAX5-positive neoplastic cells can be seen. PAX5-specific immunohistochemical stain; bar = 400 μm.
Citation: Journal of the American Veterinary Medical Association 249, 4; 10.2460/javma.249.4.381
Photomicrographs of sections of the thoracic vertebral column from the rat in Figure 1. The body was perfused with neutral-buffered 10% formalin directly following euthanasia, and subsequently, all tissues were submerged in a similar fixative solution. A—In a subgross cross section, notice the prominence of deeply basophilic cells infiltrating muscle and soft tissue structures ventral to a vertebral body and invading the bone and bone marrow. H&E stain; bar = 8 mm. B—In the spinal cord, there is multifocal hemorrhage in both the white and gray matter. H&E stain; bar = 800 μm. Inset—In a higher-magnification image, there is a prominent region of intraparenchymal hemorrhage. H&E stain; bar = 100 μm. C—In another subgross cross section, neoplastic cells are positive for the B-cell marker PAX5. PAX5-specific immunohistochemical stain; bar = 8 mm. D—In a higher-magnification image, prominent PAX5-positive neoplastic cells can be seen. PAX5-specific immunohistochemical stain; bar = 400 μm.
Citation: Journal of the American Veterinary Medical Association 249, 4; 10.2460/javma.249.4.381
Photomicrographs of sections of the thoracic vertebral column from the rat in Figure 1. The body was perfused with neutral-buffered 10% formalin directly following euthanasia, and subsequently, all tissues were submerged in a similar fixative solution. A—In a subgross cross section, notice the prominence of deeply basophilic cells infiltrating muscle and soft tissue structures ventral to a vertebral body and invading the bone and bone marrow. H&E stain; bar = 8 mm. B—In the spinal cord, there is multifocal hemorrhage in both the white and gray matter. H&E stain; bar = 800 μm. Inset—In a higher-magnification image, there is a prominent region of intraparenchymal hemorrhage. H&E stain; bar = 100 μm. C—In another subgross cross section, neoplastic cells are positive for the B-cell marker PAX5. PAX5-specific immunohistochemical stain; bar = 8 mm. D—In a higher-magnification image, prominent PAX5-positive neoplastic cells can be seen. PAX5-specific immunohistochemical stain; bar = 400 μm.
Citation: Journal of the American Veterinary Medical Association 249, 4; 10.2460/javma.249.4.381
Morphologic Diagnosis and Case Summary
Morphologic diagnosis: B-cell lymphoblastic lymphoma infiltrating the thoracic vertebrae and epaxial muscles and associated soft tissues with secondary spinal cord hemorrhage and degeneration.
Case summary: acute, progressive paraplegia as a result of a B-cell lymphoma in the vertebral column of a Long-Evans rat.
Comments
Spontaneous lymphoma and lymphocytic leukemia are infrequently diagnosed in non-Fischer laboratory rats, although there have been sporadic reports5–7 in inbred and outbred rat strains. Large granular lymphocytic leukemia (LGLL) in Fischer 344 rats has been reported to develop with incidences of as high as 59% (male rats) and 31% (female rats) in specific aged populations (> 2 years).8 However, in contrast to the case described in this report, LGLL is thought to be a disease of natural killer T-cell origin and is found most commonly in the spleens of affected rats. As in Fischer 344 rats with LGLL, most reported cases of spontaneous lymphoma or leukemia in other laboratory rats have involved aged animals.8
Reports of aggressive lymphoma that results in clinical paresis or paralysis in rats are rare, with only 3 published reports9–11 in the previous decade, to our knowledge. Nagamine et al9 described a case of acute paraplegia in a 7.5-month-old sexually intact male Long-Evans (Crl:LE) rat. Postmortem examination of that rat revealed moderate hepatosplenomegaly and a large (3-cm-diameter) friable red mass on the ventral aspect of the vertebral column. Histopathologic examination of the mass identified an infiltrative, aggressive lymphoma, which had invaded the spinal cord, associated vertebral bodies (T5-T7), and adjacent connective tissue. Immunohistochemical analysis revealed a lymphoma of T-cell origin. A similar case involving a 19-week-old sexually intact male Sprague Dawley (Crl:SD) rat was reported.10 This rat had acute pelvic limb paraparesis, which progressed rapidly to a moribund condition; the animal was euthanized. On necropsy, marked hepatosplenomegaly was noted with multiple masses involving the liver and ventral aspect of the vertebral column. Microscopically, an infiltrative lymphoma had invaded the liver, spleen, thoracic vertebrae, bone marrow, and spinal canal resulting in compression of the spinal cord. Immunohistochemical staining was consistent with a CD3-positive T-cell lymphoma. In 2009, Sano et al11 reported acute paraplegia in an aged breeding population of WBN/Kob rats. In that report, 13 rats (mean age, 60 weeks) had abnormal gaits and variable degrees of pelvic limb paresis. The authors identified similar tumors of infiltrative, neoplastic lymphocytes invading the bone marrow, thoracic or lumbar vertebrae, and multiple organs including the spleen, liver, and kidneys. These neoplastic cells were later shown, via immunohistochemical staining, to be B cells.11
B-cell lymphoblastic lymphoma is a neoplasm of immature lymphocytes committed to the B-cell lineage. Precursor lymphoid neoplasms encompass lymphoblastic leukemia and lymphoblastic lymphoma and generally are of either B- or T-cell origin. The distinction between lymphoma and leukemia is arbitrary. If there is major blood and bone marrow involvement, the term lymphoblastic leukemia is used. If the tumor primarily involves extramedullary sites with little or no blood or bone marrow involvement, the term lymphoblastic lymphoma is used. Conventionally, for humans, the presence of < 25% of the total number of neoplastic blasts in the bone marrow as opposed to peripheral tissues has been used as the threshold for differentiating between lymphoblastic lymphoma and lymphoblastic leukemia.12 In rodents, criteria for distinguishing between lymphoblastic lymphoma and lymphoblastic leukemia have not been definitively established. Precursor B-cell tumors in humans are biologically and clinically distinct from precursor T-cell neoplasms. In humans, most neoplasms of precursor B cells are leukemic. This disease is more common in children than in adults. Clinically, patients can have cytopenia, multicentric lymphadenopathy, organomegaly, CNS involvement, and a mediastinal mass.12 Unlike precursor B-cell neoplasms, which are predominantly leukemias, precursor T-cell neoplasms account for 90% of lymphoblastic lymphoma cases in humans.12 A mediastinal mass is more commonly associated with T-cell precursor neoplasms than B-cell precursor neoplasms.
Although cases of spontaneous B-cell lymphoblastic lymphoma in rats are uncommonly reported, a high incidence of spontaneous acute lymphoblastic leukemia has been described in a highly inbred strain of Sprague Dawley rats bred at The Institute of Biology and Medical Genetics at Charles University in Prague. In 2002, Otova et al7 described acute lymphoblastic leukemia–associated disease in up to 87% of male rats and 36% of female rats in a survival study, with death rate peaking between the ages of 6 and 9 months. Clinically affected rats had pronounced enlargement of the spleen and mandibular lymph nodes, with end-stage disease resulting in anemia, cachexia, and spinal cord lesions.7 Cytogenetic analysis of individually isolated malignant lymphocytes taken from these rats revealed what appeared to be a conserved, nonrandom translocation form of trisomy 11, although the importance of this alteration has yet to be determined.
Although rarely diagnosed in rats of an outbred background, spontaneous neoplastic lesions, including lymphoma, should be considered in cases of rats with acute ataxia, particularly in circumstances wherein more commonly diagnosed diseases such as encephalomyelitis, traumatic spinal injury, or thromboembolism have been ruled out. It was likely that spinal cord compression or infiltration by neoplastic cells was responsible for the severe clinical signs observed, although spinal cord compression was not observed on the histologic sections obtained in the rat of the present report. Although the rat was used in a laboratory experiment, it is important to consider that comparable pathological changes could develop in pet rats, which are most often of a similar outbred strain background. Additionally, the lack of a substantial number of reported cases of lymphoma resulting in acute paraparesis in rats may be a reflection of infrequent reporting and incomplete work-up of cases with similar clinical signs.
References
1. Jenson KC, Higgins JPT, Montgomery K, et al. The utility of PAX5 immunohistochemistry in the diagnosis of undifferentiated malignant neoplasms. Mod Pathol 2007; 20: 871–877.
2. Rehg J, Sundberg JP. Utility of antiPax5 in the diagnosis of lymphoproliferative disorders and neoplasia in mice. Comp Med 2008; 58: 246–252.
3. Morse CH, Anver MR, Fredrickson TN, et al. Bethesda proposals for classification of lymphoid neoplasms in mice. Blood 2000; 100: 246–258.
4. Rehg JE, Bush D, Ward JM. The utility of immunohistochemistry for the identification of hematopoietic and lymphoid cells in normal tissues and interpretation of proliferative and inflammatory lesions of mice and rats. Toxicol Pathol 2012; 40: 345–374.
5. Svoboda T, Jiricka Z, Klir P. Spontaneous acute lymphoblastic leukemia in Sprague-Dawley rats. II. Clinicopathologic observations. Neoplasma 1989; 36: 149–154.
6. Gross L, Dreyfuss Y. Spontaneous tumors in Sprague-Dawley and Long-Evans rats and in their F1 hybrids: carcinogenic effect of total-body x-irradiation. Proc Natl Acad Sci U S A 1979; 76: 5910–5913.
7. Otova B, Sladka M, Damoiseaux J, et al. Relevant animal model of human lymphoblastic leukaemia/lymphoma—spontaneous T-cell lymphomas in an inbred Sprague-Dawley rat strain (SD/Cub). Folia Biol (Praha) 2002; 48: 213–226.
8. Thomas J, Haseman JK, Goodman JI, et al. A review of large granular lymphocytic leukemia in Fischer 344 rats as an initial step toward evaluating the implication of the endpoint to human cancer risk assessment. Toxicol Sci 2007; 99: 3–19.
9. Nagamine CM, Jackson CN, Beck A, et al. Acute paraplegia in a young adult Long-Evans rat resulting from T-cell lymphoma. Contemp Top Lab Anim Sci 2005; 44: 53–56.
10. Matsushima K, Yamakawa S, Edamoto H, et al. Spontaneous malignant t-cell lymphoma in a young adult Crl:CD (SD) rat. J Toxicol Pathol 2010; 23: 49–52.
11. Sano T, Osaki K, Kodama Y, et al. Malignant lymphoma with severe infiltrative growth into skeletal muscles in WBN/Kob Rats. J Toxicol Pathol 2009; 22: 173–178.
12. Cortelazzo S, Ponzoni M, Ferreri AJ, et al. Lymphoblastic lymphoma. Crit Rev Oncol Hematol 2011; 79: 330–343.
