Pathology Report

G1 Mouse ID: S333-1-22

MouseID:	S333-1-22	Sex:	F	Affected:	-	Age(wk):	55	Weight(g):	56.489

Gross Pathology Summary
Animal is large with significant subcutaneous, peritoneal, and mediastinal adipose deposits. External uterine epimetrial nodular lesions. All other tissues and organs examined NSF.
Histopathology Sumamry
Cholangiocarcinoma occurs as an aging lesion in mice and occurs spontaneously in 0.5% of B6C3F1 mice. The lesion can also be induced by 1.2-diethylhydrazine, benzidine dihydrochloride and N-nitrosodiethylamine. In this case, it is difficult to discern if the hepatic lesion is due to age-related changes or from ENU treatment. However, lack of the lesion in G2's leads one to infer that the lesion is associated with aging. Future cases of this carcinoma should be noted and the incidence of it determined if more cases become available. Fibro-osseous lesion (FOL) is a term used to describe a disease entity that has a wide range of histological variability due to the lack of knowledge about it's etiology and pathogenesis. FOL is the most common bone lesion found in B6C3F1 female mice and it's incidence varies from 40-100% of B6C3F1 females. Lesions are often found in long bones and are associated with elevated serum alkaline phosphatase levels, however, no hematological alterations have been associated. The etiology is unknown but age-related, altered estrogen levels and concomitant cystic ovaries or cystic endometrial hyperplasia have been found in female mice with FOL. Given the endometrial lesions, this individual fits the classification of FOL. Cystic endometrial hyperplasia is the most common age related change in B6C3F1 females and the cystic structures in this case appear to fit with the simple cuboidal epithelial cystic form of the disease. The infiltrates in the kidney are age-related, incidental findings. The megakaryocyte reserve in the bone marrow is normal and does not correlate with the low platelet count.
Histopathology Diagnosis
1. Chronic cystic metritis with dystrophic calcification
Histopathology Comments
Liver - There are two well-circumscribed, unencapsulated foci with nests of cells interspersed with schirrous stroma. The cells are small to large in size, polygonal in shape, have moderate amounts of faint basophilic cytoplasm, prominent round to oval nuclei with coarse basophilic chromatin. There are single, large, vacuolated hepatocytes scattered throughout these areas. There is severe anisokaryosis and anisocytosis. There is marked invasion into hepatic tissue. Bone Marrow -The architecture of the proximal tibia is abnormal. There is a large foci that consists of islands of bone spicules interspersed with a prominent fibrovascular stroma.The lesion begins at the metaphyses, extends beyond the cortices and continues towards the diaphysis. The edges of the dense network of bone spicules have the rare osteoclast and evidence of continual new bone formation. The stromal cells are round to elongate in shape, with moderate amounts of pale eosinophilic cytoplasm and have round to elongate nuclei with coarse, basophilic chromatin. There are no histological abnormalties in the hemaotpoeitic cells. Uterus - There are several multifocal, perivascular inflammatory infiltrates. The cells are mononuclear and in one area, extendd into the myometrium from the endometrium. Another foci is located along the serosal surface . There are several endometrial cysts. They each have a thick fibrous capsule and are lined by large, flattened cells that are unidentifiable due to chatter artefact. There are shreds of eosinophilic material within the cystic structures that is consistent with proteinacious secretory fluid. Lung - There are a few large inflammatory aggregates located around large bronchioles and in fat tissue. The cells are mononculear. Spleen - No histological abnormalties. Submaxillary, lingual glands - There are several multifocal aggregates of mononuclear cells throughout the glands. Kidney - There is a moderate amount of multifocal mononuclear aggregates throughout the tissue.2. 2. Special stains: negative for Gram stain; positive for calcium stain. Previous infection may have resolved and calcification can occur after chronic inflammation. Cysts in the uterus is also an age-related lesion. 2. This is an incidental aging-related lesion. 2. This is an incidental aging-related lesion. 2. The infiltrates in the kidney are age-related, incidental findings. 2. Cholangiocarcinoma occurs as an aging lesion in mice and occurs spontaneously in 0.5% of B6C3F1 mice. The lesion can also be induced by 1.2-diethylhydrazine, benzidine dihydrochloride and N-nitrosodiethylamine. In this case, it is difficult to discern if the hepatic lesion is due to age-related changes or from ENU treatment. However, lack of the lesion in G2's leads one to infer that the lesion is associated with aging. Future cases of this carcinoma should be noted and the incidence of it determined if more cases become available. Special stains in this tissue revealed positivity for both calcium and iron.
Histopathology Images
Fibro-osseus lesion. Note replacement of marrow with bone spicules. Cysts and mineralization in uterus. Cholangiocarcinoma Calcium positive staining in cholangiocarcinoma. The pattern of staining is not indicative of fibrosarcoma metastases to liver, another rule out for this tumour.

MouseID:	B4S7238a-3-3	Sex:	M	Affected:	TA	Age(wk):	56	Weight(g):	33.877

Gross Pathology Summary
Spleen small. All other organs and tissues examined NSF.

MouseID:	B4S7238c-1-22	Sex:	F	Affected:	TA	Age(wk):	42	Weight(g):	37.963

Gross Pathology Summary
Animal shows signs of recent pregnancy. Internal uterine mass, bones friable.
Histopathology Sumamry
Liver, Thymus, Spleen, Bone & bone marrow: NSF. Heart: NSF.

MouseID:	I1S7238h-1-21	Sex:	F	Affected:	HA	Age(wk):	15	Weight(g):	20.68

Histopathology Sumamry
Similar histopathological changes are observed in I1S7238h-1-21
Histopathology Diagnosis
1. bone marrow: Morphological Diagnosis: Mild megakaryocyte hyperplasia with predominantly imature megakaryocytes Score:
Histopathology Comments
1. bone marrow: Similar bone marrow histological findings are present in mice I1S7238h-1-21 and I1S7238h-1-22, hence results are discussed together (mouse I1S7238h-1-21 is more severely affected). The overwhelming majority of MK in both mice were considered immature or young (1, 4). Increased nuclear volume and decreased cytoplasmic volume is highly suggestive of ineffective hematopiesis (4) and correlates with thrombocytopenic phenotype observed on hematology. Under normal conditions, lower number of immature than mature forms are expected (1). Indeed, phenotypically normal age and sex matched mouse from the same group (1S7238i-1-24) had nearly 55% mature MK. Moreover additional analysis of other phenotypically/hematologically normal 13 wk old C57BL/6J male (9033-3-2 and 9033-3-1) and female mouse (9033-3-22) showed 45, 55. and 79% mature MK, respectively. The total number of MK/high power field in the affected mice is comparable or even slightly higher than the controls supporting maturation defect rather than a proliferative one. However, we do not rule out the possibility that the increased immature MK may be a result of increased production in response to thrombocytopenia of unknown etiology. Increased production might be confirmed by determing the presence of large ('shift') platelets in the peripheral blood (4). It is also essential to rule out increased platelet consumption as a cause of thrombocytopenia. Generally, thrombocytopenia in animals results from decreased production or increased consumption of platelets (2). Cytological examination of the bone marrow (leukogram) to assess MK maturation (4), assessment of MK ploidy (3), immunophenotyping of MK, and morphological and functional evaluation of platelets will be helpful to support histological observations and to establish pathogeneisis of thrombocytopenia. It should be noted that maturation of MK is a continuum and most of the MK lineage markers such as MPL1, vWF (FVIII-related antigen) or CD61 may not discriminate between immature and mature MK (2). Flow cytometeric immunophenotyping using a panel of late MK markers might be more helpful to generate a maturation profile. MK markers routinely suitable for flow cytometry of on marrow specimen from mice include platelet peroxidase and acetylcholinesterase (reviewed in 2) Reference 1. Bollinger AP (2004). Cytologic Evaluation of Bone Marrow in Rats: Indications, Methods, and Normal Morphology. Veterinary Clinical Pathology. 33:58-67. 2. Car BD, Eng VM (2001).Special considerations in the evaluation of the hematology and hemostasis of mutant mice. Vet Pathol. 2001 38:20-30. 3. Jackson CW et al. (1990). An analysis of megakaryocytopoiesis in the C3H mouse: an animal model whose megakaryocytes have 32N as the modal DNA class. Blood: 76(4):690-6. 4. Valli, VEO.Hematopietic system. In: Pathology of Domestic Animals, ed. Grant M., 5th ed., Vol. 3. p 121, 318 592-593. Saunders-Elsevier, New York, NY, 2007 " There were no significant findings in the following tissues: Calvarium, brain, eyes, ears, tongue, Harderian gland, zymbal gland, salivary glands, nasal sinuses, teeth, trachea, lungs, heart, thymus, thyroid gland, parathyroid gland, liver, gall bladder, exocrine and endocrine pancreas, esophagus, stomach, intestines, urinary organs and tract, adrenal gland, reproductive organs, lymph nodes, spinal cord, bones, skeletal muscles, brown fat, and skin.

MouseID:	I1S7238h-1-22	Sex:	F	Affected:	HA	Age(wk):	15	Weight(g):	20.93

Histopathology Sumamry
Similar lesions are present in I1S7238h-1-22
Histopathology Diagnosis
1. bone marrow: Morphological Diagnosis: Mild megakaryocyte hyperplasia with predominantly immature mekagaryocytes Score:
Histopathology Comments
1. bone marrow: Similar bone marrow histological findings are present in mice I1S7238h-1-21 and I1S7238h-1-22, hence results are discussed together (mouse I1S7238h-1-21 is more severely affected). The overwhelming majority of MK in both mice were considered immature or young (1, 4). Increased nuclear volume and decreased cytoplasmic volume is highly suggestive of ineffective hematopiesis (4) and correlates with thrombocytopenic phenotype observed on hematology. Under normal conditions, lower number of immature than mature forms are expected (1). Indeed, phenotypically normal age and sex matched mouse from the same group (1S7238i-1-24) had nearly 55% mature MK. Moreover additional analysis of other phenotypically/hematologically normal 13 wk old C57BL/6J male (9033-3-2 and 9033-3-1) and female mouse (9033-3-22) showed 45, 55. and 79% mature MK, respectively. The total number of MK/high power field in the affected mice is comparable or even slightly higher than the controls supporting maturation defect rather than a proliferative one. However, we do not rule out the possibility that the increased immature MK may be a result of increased production in response to thrombocytopenia of unknown etiology. Increased production might be confirmed by determing the presence of large ('shift') platelets in the peripheral blood (4). It is also essential to rule out increased platelet consumption as a cause of thrombocytopenia. Generally, thrombocytopenia in animals results from decreased production or increased consumption of platelets (2). Cytological examination of the bone marrow (leukogram) to assess MK maturation (4), assessment of MK ploidy (3), immunophenotyping of MK, and morphological and functional evaluation of platelets will be helpful to support histological observations and to establish pathogeneisis of thrombocytopenia. It should be noted that maturation of MK is a continuum and most of the MK lineage markers such as MPL1, vWF (FVIII-related antigen) or CD61 may not discriminate between immature and mature MK (2). Flow cytometeric immunophenotyping using a panel of late MK markers might be more helpful to generate a maturation profile. MK markers routinely suitable for flow cytometry of on marrow specimen from mice include platelet peroxidase and acetylcholinesterase (reviewed in 2) Reference 1. Bollinger AP (2004). Cytologic Evaluation of Bone Marrow in Rats: Indications, Methods, and Normal Morphology. Veterinary Clinical Pathology. 33:58-67. 2. Car BD, Eng VM (2001).Special considerations in the evaluation of the hematology and hemostasis of mutant mice. Vet Pathol. 2001 38:20-30. 3. Jackson CW et al. (1990). An analysis of megakaryocytopoiesis in the C3H mouse: an animal model whose megakaryocytes have 32N as the modal DNA class. Blood: 76(4):690-6. 4. Valli, VEO.Hematopietic system. In: Pathology of Domestic Animals, ed. Grant M., 5th ed., Vol. 3. p 121, 318 592-593. Saunders-Elsevier, New York, NY, 2007 There were no significant findings in the following tissues: Calvarium, brain, eyes, ears, tongue, Harderian gland, zymbal gland, salivary glands, nasal sinuses, teeth, trachea, lungs, heart, thymus, thyroid gland, parathyroid gland, spleen, liver, gall bladder, exocrine and endocrine pancreas, esophagus, stomach, intestines, urinary organs and tract, adrenal gland, reproductive organs, lymph nodes, spinal cord, bones, skeletal muscles, brown fat, and skin. There were no significant findings in the following tissues: Calvarium, brain, eyes, ears, tongue, Harderian gland, zymbal gland, salivary glands, nasal sinuses, teeth, trachea, lungs, heart, thymus, thyroid gland, parathyroid gland,liver, gall bladder, exocrine and endocrine pancreas, esophagus, stomach, intestines, urinary organs and tract, adrenal gland, reproductive organs, lymph nodes, spinal cord, bones, skeletal muscles, brown fat, and skin.

MouseID:	I1S7238i-1-24	Sex:	F	Affected:	NA	Age(wk):	15	Weight(g):	19.19

Histopathology Sumamry
This mouse is used as a control for I1S7238h-1-21 and I1S7238h-1-22. The bone marrow histology is described below for reference: Marrow cellularity is estimated at 95%. Estimated average myeloid erythroid ratio is 2:1. Granulocyte reserve is within normal limits with synchronous maturation. Megakaryocytes (MK) are abundant. Approximately 54% of the MK have dense hyperchromatic nuclei, low nuclear: cytoplasmic ratio (estimated 1:4), and abundant eosinophilic cytoplasm with distinct cytoplasmic boundaries (interpreted as mature MK). The rest of the MK (estimated 46%) are subjectively smaller in size, have hypolobulated open (hypochromatic) nuclei, high N:C ratio (1:1 or 1:2), scant to moderate amount of lightly basophilic granular cytoplasm (interpreted as immature MK (ref. 1)). Coarse iron is present.
Histopathology Diagnosis
Normal
Histopathology Comments
There were no significant findings in the following tissues: Calvarium, brain, eyes, ears, tongue, Harderian gland, zymbal gland, salivary glands, nasal sinuses, teeth, trachea, lungs, heart, thymus, thyroid gland, parathyroid gland, spleen, liver, gall bladder, exocrine and endocrine pancreas, esophagus, stomach, intestines, urinary organs and tract, adrenal gland, reproductive organs, lymph nodes, spinal cord, bones, bone marrow, skeletal muscles, brown fat, and skin.

MouseID:	I1S7238o-7-2	Sex:	M	Affected:	HA	Age(wk):	36	Weight(g):	26.95

Gross Pathology Summary
Gross dissection showed hard white areas in the muscle of the hind limb and fore limb.
Histopathology Diagnosis
1. bone marrow: Morphological Diagnosis: Megakaryocytic hyperplasia and immaturity MPATH_Diagnosis: hyperplasia Score: moderate 2. prepuce: Morphological Diagnosis: Mild suppurative interstitial preputial gland adenitis Score:
Histopathology Comments
1. bone marrow: The lesions are comparable to other cases with the same phenotype (low platlet count) 2. prepuce: The lesions are considered mild.

MouseID:	I1S7238o-7-3	Sex:	M	Affected:	HA	Age(wk):		Weight(g):	29.62

Gross Pathology Summary
Gross dissection showed hard white areas in the muscle of the hind limb and fore limb.
Histopathology Diagnosis
1. prepuce: Morphological Diagnosis: Mild suppurative interstitial preputial gland adenitis Score:
Histopathology Comments
1. liver: These lesions are mild and are considered incidental. 2. kidney: The renal lesions are considered incidental 3. prepuce: The lesions are considered mild.

MouseID:	I1S7238r-2-1	Sex:	M	Affected:	NA	Age(wk):		Weight(g):	28.44

Gross Pathology Summary
Gross dissection showed hard white areas in the muscle of the hind limb and fore limb.
Histopathology Sumamry
Note: The following summary pertains to all mice/specimens submitted for the grossly observed preputial phenotype by Nicole The overall structure of the preputial glands is within normal limits. There is synchronous maturation of the glandular structures in all groups. The ductular structures have some variation in amount and size. However, these variations are present irrespective of the genetic background, and are likely physiological variations.Mild and moderate inflammatory changes are present in one of the wild type (B10S7238a-2-3), and one of the homozygous mouse (I1S7238o-7-3). The effect of this low degree of inflammation on the grossly observed preputial phenotype is uncertain. The lesions are likely resolving suppurative inflammation of the prepuce (preputial abscess). Preputial abscess is observed in low percentage of usually old mice (Gordon et al., 1996). In summary, we did not observe a histological correlate to the gross observations. Preputial secretions were likely lost during tissue processing. Therefore, we did not rule out possible qualitative or quantitative differences in preputial secretion that might account for the observed phenotype. Reference:Gordon LR. et al. (1996). Spontaneous Non-neoplastic and Neoplastic Lesions and Experimentally Induced Neoplasms of the Testes and Accessroy Sex Glands. In: Pathology of the Aging Mouse. Mohr DL et al. (eds). Vol. 1. pp. 434-435, ILSI Press. Washington, DC, 1996
Histopathology Comments
There were no significant findings in the following tissues: Calvarium, brain, eyes, ears, tongue, Harderian gland, zymbal gland, salivary glands, nasal sinuses, teeth, trachea, lungs, heart, thymus, thyroid gland, parathyroid gland, spleen, liver, gall bladder, exocrine and endocrine pancreas, esophagus, stomach, intestines, urinary organs and tract, adrenal gland, lymph nodes, spinal cord, bones, bone marrow, skeletal muscles, brown fat, and skin.

MouseID:	S7238-5-1	Sex:	M	Affected:	TA	Age(wk):	20	Weight(g):	30.846

Gross Pathology Summary
Testicular abnormality. All other organs and tissues examined NSF.
Histopathology Sumamry
The spermatocele is a common degenerative lesion.
Histopathology Diagnosis
1. Spermatocele
Histopathology Comments
Testes - One testicle has a large cystic structure. The capsule is composed of flat epithelial cells. The centre consists of mature spermatids scattered with large, pale, eosinophilic structures resembling dead, sloughed epithelial cells. Bone Marrow & Spleen - No histological abnormalties

MouseID:	S7238-5-3	Sex:	M	Affected:	TA	Age(wk):	20	Weight(g):	32.113

Gross Pathology Summary
NSF
Histopathology Sumamry
No significant findings.
Histopathology Diagnosis
None
Histopathology Comments
No histological abnormalties

MouseID:	S7238-5-4	Sex:	M	Affected:	TA	Age(wk):	20	Weight(g):	33.185

Gross Pathology Summary
NSF
Histopathology Sumamry
No significant findings
Histopathology Comments
There are no histological abnormalties.

MouseID:	S7238-7-3	Sex:	M	Affected:	TA	Age(wk):	13	Weight(g):	30.218

Gross Pathology Summary
Spleen small, left kidney abnormality. All other organs and tissues examined NSF.
Histopathology Sumamry
No significant findings.
Histopathology Diagnosis
None
Histopathology Comments
There are no histological abnormalties. There were no significant findings in the following tissues: Calvarium, brain, eyes, ears, tongue, Harderian gland, zymbal gland, salivary glands, nasal sinuses, teeth, trachea, lungs, heart, thymus, thyroid gland, parathyroid gland, spleen, liver, gall bladder, exocrine and endocrine pancreas, esophagus, stomach, intestines, urinary organs and tract, adrenal gland, reproductive organs, lymph nodes, spinal cord, bones, bone marrow, skeletal muscles, brown fat, and skin.