Neoplastic and Nonneoplastic Lesions in Aging Mice of Unique and Common Inbred Strains Contribution to Modeling of Human Neoplastic Diseases

Abstract

The evaluation of spontaneous lesions in classical inbred strains of mice has become increasingly important because genetically engineered mice (GEMs) are created on these backgrounds. Novel inbred strains—genetically diverse from classic strains—are valuable both as a new background for GEM mice and to increase the genetic variation found in laboratory mice. Newly arising spontaneous genetic alterations in commonly used strains may also lead to new and valuable mouse models of disease. This report evaluates gross and histological lesions in relatively new, classic, and rarely explored mouse inbred strains. Pathological lesions of 1273 mice from 12 inbred strains (129S1/SvW, A.CA-H2^f /W, AKR/W, BALB/cW, BN/aW, C57BL/6 W, C57BL/10 W, C3H/W, C3H ^wad /W, CBA/W, DBA/2 W, and WOM/W) are reported. BN/aW, WOM/W, and C3H ^wad /W are novel inbred strains produced and maintained in the Department of Genetics and Laboratory Animal Breeding at the Center of Oncology, Warsaw, Poland. Both neoplastic and nonneoplastic lesions were examined. The prevalence of lung neoplasms was significantly higher in A.CA-H2^f /W (33.3%) and BALB/cW (33.8%) mice (P < .01). The prevalence of liver neoplasms was significantly higher in the CBA/W strain (P < .01). Mammary gland neoplasms arose at a greater frequency in C3H/W mice (P < .01). The occurrence of uterine neoplasms was higher in DBA/W and 129S1/SvW mice. AKR/W and WOM/W mice developed T-cell lymphoblastic lymphoma with high frequency (110/121 [90.9%] and 159/175 [90.9%], respectively) before 1 year of age. The occurrence of nonneoplastic lesions in the kidneys of BN/aW mice was increased (P < .01).

Keywords

mouse inbred strains leukemia lymphoma neoplasms nonneoplastic lesions

The mouse is a useful model for common human diseases such as cancers, type 1 and type 2 diabetes, obesity, and cardiovascular and neurodegenerative disorders. The mouse models are represented by a variety of mice whose phenotypes either arose spontaneously or were specifically engineered via transgenesis.

Because most genetically engineered mice (GEM) models derive from a change in a single gene locus, they do not perfectly mimic the human population, which is heterogeneous. Most mouse models represent only a small number of mouse genetic backgrounds, which limits the mapping of genes involved in carcinogenesis and gene function as well as studies of polygenic networks and gene interactions. A community resource termed the Collaborative Cross was designed to create mice with increased genetic diversity, which will better reflect the complexity of the human population.^14,47,66 In the context of this new approach to mouse modeling, novel inbred strains—genetically diverse from classic inbred strains—appear to be valuable both for their new background for GEM mice and for their increased genetic diversity. Moreover, the possibility of new spontaneously arising genetic alterations in commonly used strains may create new mouse models of human disease.

The documentation of lesions found in inbred strains is increasingly important. The influence of genetic background on genetically engineered mouse phenotypes is apparent, and researchers should be cognizant of the phenotypes of the used strains.⁵ Common lesions of background mouse strains are regarded as “background lesions.” The accurate assessment of the GEM phenotype requires separation of phenotypes due to genetic manipulation from the phenotypes of the background strain.⁷ Therefore, a comprehensive knowledge of the phenotype of background strains is required, especially as inbred strains differ in their susceptibilities to particular neoplastic and nonneoplastic diseases.^{8,25,44,67,73} Furthermore, substrains that arise through prolonged inbreeding within various laboratories can differ significantly from the parent strain.²⁶ There are abundant data on gross and histological lesions of common strains, but this information is widely dispersed. Phenomic databases such as the Mouse Phenome Database at the Jackson Laboratory,³³ Mouse Genome Informatics (MGI),²⁰ International Mouse Strain Resources Database (IMSR),³¹ Japan Mouse/Rat Strain Resources Database (JMSR),³² and others^11,63,77 serve as sources of consolidated mouse strain data, providing links to the references. However, the less commonly used strains are not consistently reported. Much information is available on B6C3F1 (C57BL/6 female × C3H or C3H/He male) as a result of toxicology studies.^35,43,70 Lesions of B6;129 mice, commonly used as a background strain in genetically engineered mice, are also well described.^27,74 Gross and histological lesions of strains commonly used to generate transgenic mice (C57BL/6, 129, FVB, and B6;129) have been recently reviewed.⁹

There is significant background-dependent phenotypic variability in GEM strains. Phenotypes that result from the same genetic alteration can vary on different backgrounds.^29,55 It would be ideal if investigators could choose the background strain that is most appropriate for the studied disease.¹⁷ However, for varying reasons, some strains are favored over others for mouse model production. In particular, 129 embryonic stem (ES) cells and C57BL/6 blastocysts are a favorable combination for transgenesis. The 129 ES cells are easy to modify genetically and have a very high incidence of germline transmission. Furthermore, agouti/black chimerism can be easily identified.^7,18 Interestingly, ES lines have been produced from C57BL strains, as well as F1 or F2 offspring of a cross between inbred C57BL/6 and CBA/2 or FVB/N mice.⁷¹ BALB/c and A mice are used in ENU (N-ethyl-N-nitrosourea) mutagenesis experiments due to their fecundity, tolerance of ENU, and high rates of survival.⁷ The popularity of the C57BL/6 strain results from the fact that it was chosen for the mouse genome-sequencing project and that many early GEMs were created on this background.⁷

This report focuses on spontaneous lesions of aging mice in our unique inbred strains WOM/W and BN/aW, whose genomes vary significantly from the most commonly used strains (unpublished data). Furthermore, we present multiple lesions in traditional inbred strains and in a spontaneously arising variant of the C3H/W strain—C3H ^wad /W.

Materials and Methods

Mice

Strains

In total, 1273 mice from 12 inbred strains (129S1/SvW, A.CA-H2^f /W, AKR/W, BALB/cW, BN/aW, C57BL/6W, C57BL/10W, C3H/W, C3H ^wad /W, CBA/W, DBA/2W, and WOM/W) originated from breeding and research colonies maintained at the Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology in Warsaw, Poland. Inbred mouse strains are included in the register of the Committee on Standardized Nomenclature for Inbred Strains of Mice and the register of the National Academy of Sciences, Washington, DC. In the International Laboratory Code Registry, the code of our breeding center is W.³⁰

The origin of the examined strains and generations of inbreeding are listed in Table 1. BN/aW, WOM/W, and C3H ^wad /W are strains that are unique to our facility and are registered in the Jackson Mouse Database.^15,24,33,64

Table 1.

Origin of the Examined Strains and Generations of Inbreeding.

Strain	Parental Strain	Origin	Date of Origin	Generation (in Conventional Conditions/in SPF Conditions)
129S1/SvW	129/S1/SvJ	The Jackson Laboratory, Bar Harbor, ME	1971	F108 (F66/F42)
A.CA-H2^f /W	A.CA major histocompatibility congenic	Karolinska Institutet, Stockholm, Sweden	1971	F111 (F64/F47)
AKR/W	AKR/A	The Netherlands Cancer Institute, Amsterdam	1984	F120 (F65/F55)
BALB/cW	BALB/c	Memorial Sloan-Kettering Cancer Center (MSKCC), New York, via the Institute of Immunology and Experimental Therapy (IITD), Poland	In the 1970s from MSKCC; 1986 from IITD	F85 (F44/F41)
BN/aW	Origin from unknown mice primarily used for transplantation of vaginal neoplasms	The Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland	1956; registered in 1961 as inbred strain	F167 (F138/F29)
C3H/W	C3H	The National Institute of Oncology, Budapest, Hungary	1955	F173 (F133/F40)
C3H ^wad /W	C3H/W with unknown mutation mapped to the distal region of chromosome 10	Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology	Mutation recorded in 1985; registered in MGI (2010) ID 4818895	F27 SPF
CBA/W	CBA	The Medical Research Council Radiobiological Unit, Harwell, UK	1975	F88 (F62/F26)
C57BL/10W	C57BL/10A	The Netherlands Cancer Institute	1988	F74 (F33/F41)
C57BL/6W	C57BL/10J	The Jackson Laboratory	1999	F30 in SPF
DBA/2W	DBA/2J	The Jackson Laboratory	1999	F40 in SPF
WOM/W	Male from congenic resistance strain B10.BN-H2^bp1 and female of unknown origin (black and tan color)	Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology	Inbreeding since 1985; strain registered in MGI ID: 4820406	F40 in SPF

SPF, specific pathogen free.

Information about the congenic A.CA-H2^f /W strain is not available in the Mouse Phenome Database at the Jackson Laboratory, and currently phenotypic data for this strain are scarce. The other strains are commonly used in biomedical research.

Inbred status

Mice of inbred strains are genetically defined using microsatellite markers. Each strain is characterized by more than 450 microsatellite markers, which are distributed across all autosomes and chromosome X and a smaller portion of chromosome Y. Mice from the studied strains are homozygous at all the examined loci. Based on the obtained microsatellite panel, a set of markers for genetic monitoring has been established. To exclude breeding contamination, every 3 generations, animals from all strains are tested with a set of selected markers that include 17 microsatellite markers and an additional marker on chromosome Y.

Husbandry and housing

The animals were housed in a barrier system under specific pathogen-free (SPF) conditions (see below). Mice were allowed ad libitum access to water and a standard pelleted diet for breeders (LABOFEED H; Feed Factory “MORAWSKI,” Kcynia, Poland). The diet contained crude protein (23%, plant origin), crude fat (4.1%, plant origin), crude fiber (4.6%), crude ash (5.5%), and nitrogen-free extracts (50.8%). The diet was supplemented with dried degreased milk and whey, vitamins, and minerals.

The food, cages with bedding, and filled water bottles were autoclaved. The cages had 400 cm² of floor area. Since 3 to 4 males or 4 to 5 females were maintained in 1 cage, this translates into about 80 cm² of floor area per animal.¹⁶ Mice sharing 1 cage were of the same age and, if possible, siblings. Light cycle was 12 hours dark/12 hours light. The temperature was stabilized at 22 ± 2°C. Mice received humane care and ethical treatment, and all procedures were in accordance with the Polish Animal Protection Act No. 1002/2003 and Polish Animal Experimentation Act No. 289/2005.

Health monitoring

Health monitoring was performed based on the recommendations of the Federation of European Laboratory Animal Science Associations (FELASA).⁵¹ Bacteriology, parasitology, and serology were performed every 3 months (except for some serological tests) on 3 randomly selected animals from each breeding room. Ectoparasites and pinworms were not detected in any examined mouse. Trichomonas muris was found in the cecum and/or colon in wet mounts but did not cause clinical disease or lesions. The trophozoites of Giardia muris were detected in the proximal part of the small intestine, and cysts were found in feces, again without clinical manifestations. Serology was performed every 3 months to identify antibodies to mouse hepatitis virus, epizootic diarrhea of infant mice virus, mouse parvovirus, Sendai virus, pneumonia virus of mice, Theiler’s encephalomyelitis virus, and minute virus of mice (MVM), as well as antibodies to bacteria Clostridium piliforme, cilia-associated respiratory bacillus, and Mycoplasma pulmonis and once yearly for detection of antibodies to ectromelia, Reo-3, mouse cytomegalovirus, adenovirus K-87/FL, and lymphocytic choriomeningitis virus. The results were generally negative, and only ∼4% were equivocal for MVM and C. piliforme (± result for one mouse in 1 control). Mice were not tested for murine norovirus and Helicobacter species.

Observation Protocol

Mice were monitored daily for neoplasm formation and/or signs of chronic progressive diseases. During physical examination, mice were specifically evaluated for inactivity, poor grooming, weight loss, respiratory disorders, pale ears, visible tumors, and palpable internal masses. Mice were maintained until near end of life (EOL) and euthanized when clinical signs suggested death within 24 hours. The criteria for EOL included nonresponsiveness to touch, cold body temperature on touch, slow or labored respiration, hunched body position, matted fur, and failure to drink and eat.⁵⁴ Mice with symptoms of chronic illness observed longer than 3 to 4 weeks were sacrificed for humane reasons. Mice were sacrificed at the age of 1100 days or older if they exhibited age-associated weakness. Very few mice (5) died naturally. The ages of mice with neoplasms and without neoplasms but suffering from nonneoplastic lesions are shown in Table 2. The age of mice on the date of sacrifice was recorded as their neoplasm age. Data were collected from 2003 to 2011.

Table 2.

Age of the Examined Mice in days (Mean, Median, Minimal, and Maximal Age).

		Hematopoietic Neoplasms
		Lymphoid
Strain	T Cell	B Cell	Nonlymphoid	Lung Neoplasms	Liver Neoplasms	Uterine Neoplasms	Ovarian Neoplasms	Mammary Gland Neoplasms	Intestinal Neoplasms	Soft Tissue Neoplasms	Harderian Gland Neoplasms	Neoplasms of Skin	Mice Without Neoplasms
WOM/W
Mean	237	—	—	—	—	—	—	—	292	—	—	—	271
Median	225	—	—	—	—	—	—	292	—	—	—	—	216
Min	91	—	—	—	—	—	—	292	—	—	—	—	89
Max	570	—	—	—	—	—	—	292	—	—	—	—	417
BN/aW
Mean	660	—	—	929	870	869	—	—	—	—	655	—	883
Median	660	—	—	941	864	869	—	—	—	—	655	—	900
Min	660	—	—	655	718	869	—	—	—	—	655	—	393
Max	660	—	—	1065	988	869	—	—	—	—	655	—	1202
C3H ^wad /W
Mean	—	—	—	525	713	—	—	631	—	713	—	—	716
Median	—	—	—	673	709	—	—	631	—	679	—	—	788
Min	—	—	—	143	528	—	—	583	—	638	—	—	150
Max	—	—	—	760	870	—	—	679	—	821	—	—	856
C57BL/6W
Mean	768	779	857	852	812	796	853	—	891	664	851	—	831
Median	852	777	802	851	842	796	853	—	891	808	834	—	864
Min	263	607	779	764	473	796	747	—	891	316	784	—	110
Max	1000	1049	989	967	1061	796	959	—	891	869	935	—	1090
C57BL/10W
Mean	752	866	831	911	888	879	—	—	—	730	856	—	932
Median	867	897	831	867	880	879	—	—	—	730	856	—	1005
Min	372	518	710	799	747	879	—	—	—	611	786	—	586
Max	1017	1165	951	1067	1067	879	—	—	—	848	925	—	1139
129S1/SvW
Mean	567	750	582	765	820	698	460	—	707	596	749	—	663
Median	567	716	582	765	786	797	460	—	743	618	749	—	757
Min	567	646	582	449	616	382	460	—	457	382	741	—	310
Max	567	951	582	1080	869	940	460	—	871	940	757	—	1100
DBA/2W
Mean	462	579	—	—	823	758	773	823	—	767	—	—	702
Median	462	579	—	—	823	718	773	823	—	767	—	—	722
Min	383	579	—	—	823	574	773	823	—	767	—	—	258
Max	540	579	—	—	823	869	773	823	—	767	—	—	1221
C3H/W
Mean	424	—	839	829	812	506	187	421	—	416	—	—	755
Median	424	—	839	823	813	506	187	395	—	416	—	—	806
Min	424	—	703	626	626	506	187	207	—	416	—	—	418
Max	424	—	974	934	934	506	187	732	—	416	—	—	1094
CBA/W
Mean	—	934	—	806	854	1006	1025	822	—	—	—	—	952
Median	—	934	—	776	837	1006	1025	822	—	—	—	—	990
Min	—	863	—	709	642	911	1020	822	—	—	—	—	695
Max	—	1004	—	933	1107	1101	1030	822	—	—	—	—	1082
AKR/W
Mean	304	399	—	—	—	—	241	—	—	—	—	—	452
Median	283	460	—	—	—	—	241	—	—	—	—	—	456
Min	164	202	—	—	—	—	241	—	—	—	—	—	250
Max	583	534	—	—	—	—	241	—	—	—	—	—	459
BALB/cW
Mean	663	764	902	866	888	786	842	532	1111	—	730	222	772
Median	761	767	902	872	929	786	842	499	1111	—	730	222	816
Min	138	680	831	639	746	786	824	400	1111	—	730	222	428
Max	895	968	972	1111	984	786	859	730	1111	—	730	222	1086
A.CA-H2^f /W
Mean	560	—	—	816	873	—	—	227	975	—	982	—	769
Median	542	—	—	834	913	—	—	227	975	—	982	—	766
Min	244	—	—	227	558	—	—	227	975	—	982	—	411
Max	912	—	—	1066	1060	—	—	227	975	—	982	—	1035

Dashes indicate that the given neoplasm did not occur.

Pathological Examination

A complete necropsy was performed on each mouse according to previous recommendations.^19,22 Organ sampling and trimming were based on the Revised Guides for Organ Sampling and Trimming in Rats and Mice.^36,48,58

All tissues (42 samples) and gross lesions from 5 mice per sex, per strain were examined. For all additional animals within a given strain, lungs, trachea with thyroid and parathyroid glands, liver with gallbladder, kidneys with adrenal glands, spleen and testes with epididymides, ovaries, uterus, vagina, pancreas, and visible lesions were sampled. Tissues were fixed in EAFS fixative containing ethanol, acetic acid, formol, and 0.9% NaCl. The intestine was opened, rinsed with 0.9% NaCl, and examined for visible neoplastic foci or enlarged Peyer’s patches or lymphoid follicles. The parts of intestine with visible lesions were recoiled on cotton swabs and fixed. If hematopoietic neoplasms were suspected, thymus, mesenteric lymph nodes, and sternum were fixed for hematoxylin and eosin (HE) and immunohistochemical staining. Blood and smears of bone marrow, obtained from femur, were stained with modified Giemsa stain, and the percentage of neoplastic hematopoietic cells was estimated. Immature granulocytes were identified using Leder stain (naphthol AS-D chloroacetate esterase reaction) on air-dried imprints or paraffin sections.⁴⁶ Congo red stain was used to identify amyloid in suspected cases.

The size of neoplasms detected in each organ was estimated as visible foci (3–4 mm), small (5–7 mm), moderate (8–10 mm), or large (>10 mm). The fixed tissues were routinely processed, embedded in paraffin, and sectioned at 4 μm. Three sections, 100 μm apart, were taken from neoplastic tissues and stained with HE.

Immunophenotyping

Flow cytometry analysis and/or immunohistochemistry were performed when a hematopoietic neoplasm was suspected. Immunophenotypic analysis was performed using flow cytometry analysis on cell suspension from thymus, mesenteric lymph nodes, each enlarged lymph node, and bone marrow. In selected cases of hematopoietic neoplasms, the correct diagnosis required complementary immunohistochemistry.

The single-cell suspension for flow cytometry analysis was obtained by mechanical dissociation of lymphoid tissues in Hank’s balanced salts followed by passage through a fine-gauge needle and filtration of cells through a 40- to 50-μm nylon mesh. The single-cell suspension (1.0 × 10⁶/1 ml) was analyzed by FACSCalibur (Becton-Dickinson, San Jose, CA). The specific 15 monoclonal antibodies (MAbs) for flow cytometry were labeled directly with either FITC (CD90.2/CD90.1, CD3∊, CD8, CD5, CD19, CD45-[B220], CD79b, GAM [goat anti–mouse immunoglobulin], IA^d, IgM) or with PE (CD4, TCRβ, RAM KAPPA [rat anti–mouse κ], IgD). The monoclonal antibody CD3∊ was used to detect both surface (sCD3∊) and intracellular (cCD3∊) antigen. The fixation and permeabilization system (Leucoperm; Serotec Ltd, Oxford, UK) was used for analysis of the intracellular antigen. All antibodies were manufactured by BD Biosciences Worldwide Immunocytometry Systems Pharmingen (San Diego, CA). Working dilution of these antibodies in flow cytometric application was 1 μg to stain 1.0 × 10⁶ cells.

Immunohistochemical staining was performed on paraffin sections using either Avidin-Biotin-Peroxidase Complex (DAKO Denmark A/S, Glostrup, Denmark) or MOM (Mouse on Mouse immunodetection kit; Vector Laboratories, Burlingame, CA). Both methods were used according to the manufacturers’ recommendations. The applied antigen retrieval unmasking method was microwave oven heating for 10 minutes in 0.01 M citrate buffers of pH 6. The specific antibodies for immunohistochemistry were biotin conjugated: anti-IgM, CD45R-(B220), CD5, CD3∊, TCRβ, Gr-1, anti–mouse Igκ light chain, anti–mouse Ig λ₁ and λ₂, or pure (anti-IgD). All antibodies were manufactured by BD Biosciences Worldwide Immunocytometry Systems Pharmingen. The antibodies were titrated, and the determined working dilution was 1:50 for all antibodies except for CD45R-(B220), which was used at a concentration of 1:100.

Classification of Lesions

Neoplasms were classified according to the recommendation of the Mouse Models of Human Cancer Consortium. Pulmonary lesions were classified based on the “Classification of Proliferative Pulmonary Lesions of Mouse: Recommendations of the Mouse Models of Human Cancers Consortium” (Boston, 2001).⁵² The intestinal neoplasms were classified according to “Pathology of Mouse Models of Intestinal Cancer: Consensus Report and Recommendations.”⁴ Classification of mammary gland neoplasms was based on “The Mammary Pathology of Genetically Engineered Mice: The Consensus Report and Recommendations From the Annapolis Meeting” (1999).¹⁰ Lymphoid and nonlymphoid hematopoietic neoplasms were classified according to “Bethesda Proposals for Classification of Lymphoid Neoplasms in Mice”⁴⁹ and “Bethesda Proposals for Classification of Nonlymphoid Hematopoietic Neoplasms in Mice.”³⁸ Lymphoma subclassification was based on a combination of histologic features, cytomorphology, and flow cytometric and immunohistochemical immunophenotyping.^13,60 Neoplasms of the other organs were classified based on classification of rodent neoplasms.^45,69 Nonneoplastic lesions were diagnosed according to the standard criteria used in the description of lesions in mouse pathology and recommended by Frith and Ward.²³

Determination of Cause of Death and Contributed Cause of Death

Cause of death (COD) and contributed COD (CCOD) were determined according to recommendations for toxicological studies.^37,42

Statistical Analysis

Statistical analysis of the occurrence of neoplastic and nonneoplastic lesions was performed using the Kruskal-Wallis test at a level of significance (P = .05), with P < .01 highly significant. The Kruskal-Wallis test was used because the original data set consisted of nominal variables and ranked variables.

Results

The observed strains varied in the prevalence and types of neoplasms; these are presented in Tables 3 and 4. The ages of mice with or without neoplasms are shown in Table 2.

Table 3.

Prevalence of Neoplasms in the Examined Mice.

Strain	No. of Examined Mice	Hematopoietic Neoplasms	Lung Neoplasms	Liver Neoplasms	Uterine Neoplasms	Ovarian Neoplasms	Mammary Gland Neoplasms	Intestinal Neoplasms	Soft Tissue Neoplasms	Harderian Gland Neoplasms	Mice With More Than 1 Type of Neoplasm	Mice Without Neoplasms
WOM/W	175	159 (90.9)	0	0	0	0	1 (1.1)	0	0	0	1 (0.7)	16 (9.1)
Female	91	83 (91.2)	0	0	0	0	1 (1.1)	0	0	0	1 (1.1)	8 (8.8)
Male	84	76 (90.4)	0	0	0	0	0	0	0	0	0	8 (9.5)
BN/aW	101	1 (1.0)	14 (13.9)	6 (5.9)	1 (1.2)	0	0	0	0	1 (1.0)	1 (0.99)	79 (78.2)
Female	86	1 (1.2)	10 (11.6)	4 (4.7)	1 (1.2)	0	0	0	0	0	1 (1.2)	71 (82.6)
Male	15	0	4 (26.7)	2 (13.3)	0	0	0	0	0	1 (6.7)	0	8 (53.3)
C3H ^wad /W	49	0	3 (6.1)	7 (14.2)	0	0	2 (8.0)	0	3 (6.1)	0	1 (2.0)	35 (71.4)
Female	25	0	0	2 (8.0)	0	0	2 (8.0)	0	3 (12.0)	0	0	18 (72.0)
Male	24	0	3 (12.5)	5 (20.8)	0	0	0	0	0	0	1 (4.2)	17 (70.8)
C57BL/6/W	173	40 (23.1)	9 (5.2)	15 (8.6)	1 (1.4)	2 (2.9)	0	1 (0.6)	3 (1.7)	3 (1.7)	4 (2.3)	103 (59.5)
Female	70	19 (24.3)	1 (1.4)	3 (4.3)	1 (1.4)	2 (2.9)	0	1 (1.4)	0	1 (1.4)	2 (2.9)	44 (62.9)
Male	103	21 (20.4)	8 (7.8)	12 (11.7)	0	0	0	0	3 (2.9)	2 (1.9)	2 (1.9)	59 (57.3)
C57BL/10/W	99	32 (32.3)	3 (3.0)	5 (5.0)	1 (2.2)	0	0	0	3 (3.0)	2 (2.0)	3 (3.0)	56 (56.6)
Female	45	13 (28.9)	1 (2.2)	1 (2.2)	1 (2.2)	0	0	0	2 (4.4)	1 (2.2)	2 (4.4)	28 (62.2)
Male	54	19 (35.2)	2 (3.7)	4 (7.4)	0	0	0	0	1 (1.9)	1 (1.9)	1 (1.9)	28 (52.0)
129S1/SvW	111	8 (7.2)	3 (2.7)	4 (3.6)	11 (18.6)	1 (1.7)	0	4 (3.6)	3 (2.7)	2 (1.8)	1 (0.9)	76 (68.0)
Female	59	5 (8.5)	1 (1.9)	1 (1.7)	11 (18.6)	1 (1.7)	0	2 (3.4)	0	1 (1.7)	0	37 (62.7)
Male	52	3 (5.8)	2 (3.4)	3 (5.8)	0	0	0	2 (3.8)	3 (5.8)	1 (1.9)	1 (1.9)	39 (75.0)
DBA/2/W	71	3 (4.2)	0	1 (1.4)	13 (25.4)	1 (2.0)	1 (2.0)	0	1 (1.4)	0	1 (1.4)	52 (74.2)
Female	51	3 (5.9)	0	1 (2.0)	13 (25.4)	1 (2.0)	1 (2.0)	0	0	0	1 (2.0)	33 (64.7)
Male	20	0	0	0	0	0	0	0	1 (5.0)	0	0	19 (95.0)
C3H/W	166	3 (1.8)	4 (2.4)	18 (10.8)	1 (0.9)	1 (0.9)	106 (94.6)	0	1 (0.6)	0	1 (0.6)	33 (19.9)
Female	112	2 (1.8)	1 (0.9)	0	1 (0.9)	1 (1.9)	106 (94.6)	0	0	0	1 (0.9)	2 (1.8)
Male	54	1 (1.9)	3 (5.6)	18 (33.4)	0	0	0	0	1 (1.9)	0	0	31 (57.4)
CBA/W	52	2 (3.8)	6 (11.5)	20 (38.4)	2 (12.5)	4 (25.0)	1 (6.3)	0	0	0	5 (9.6)	22 (42.3)
Female	16	0	2 (12.5)	4 (25.0)	2 (12.5)	4 (25.0)	1 (6.3)	0	0	0	3 (18.8)	6 (38.0)
Male	36	2 (5.6)	4 (11.1)	16 (44.4)	0	0	0	0	0	0	2 (5.6)	16 (44.4)
AKR/W	121	113 (93.3)	0	0	0	1 (1.3)	0	0	0	0	1 (0.8)	8 (6.6)
Female	76	72 (94.7)	0	0	0	1 (1.3)	0	0	0	0	1 (2.2)	4 (5.3)
Male	45	41 (91.1)	0	0	0	0	0	0	0	0	0	4 (8.9)
BALB/cW	71^a	17 (23.9)	24 (33.8)	7 (9.9)	1 (1.4)	2 (2.9)	4 (5.8)	1 (1.4)	0	1 (1.4)	9 (12.7)	22 (31.0)
Female	69^a	17 (23.9)	22 (32.0)	7 (10.1)	1 (1.4)	2 (2.9)	4 (5.8)	1 (1.4)	0	0	8 (11.6)	22 (31.9)
Male	2	0	2	0	0	0	0	0	0	1	1	0
A.CA-H2^f /W	84	4 (4.8)	28 (33.3)	10 (11.9)	0	0	1 (2.1)	1 (1.2)	0	1 (1.2)	0	39 (46.4)
Female	47	1 (2.1)	16 (34.0)	4 (8.5)	0	0	1 (2.1)	0	0	1 (2.1)	0	24 (51.0)
Male	37	3 (8.1)	12 (32.4)	6 (16.2)	0	0	0	1 (2.7)	0	0	0	15 (40.5)

Values are presented as number (%).

^aIn 1 case in BALB/cW females, neoplasm of skin was observed.

Table 4.

Prevalence of Types of Neoplasms in the Examined Mice (Numbers of Affected Animals).

		Hematopoietic Neoplasms			Lung Neoplasms		Liver Neoplasms						Uterine Neoplasms		Ovarian Neoplasms		Mammary Gland Neoplasms					Intestinal Neoplasms	Soft Tissue Neoplasms		Neoplasms of Skin	Harderian Gland Neoplasms
Strain	No. of Examined Mice	Lymphoma		Non lym phoid	Ade noma	Adeno car cinoma	Hepato cellular Adenoma	Hepatocellular Carcinoma			Heman gioma	Heman giosarcoma	Epithelial Neoplasms: Adenoma	Non epithelial Neoplasms	Tubular Adenoma Simple Type and Complex	Granulosa Cell Tumor	Adeno carcinoma A: Adenoma	Adenocarcinoma B		Adeno acanth oma	Fibroma	Adenoma	Liposarcoma	Fibrosarcoma	Squamous Cell Carcinoma	Adenoma
Strain	No. of Examined Mice	T Cell	B Cell	Non lym phoid	Ade noma	Adeno car cinoma	Hepato cellular Adenoma	Well Differen tiated	Moderately Differen tiated	Poorly Differen tiated	Heman gioma	Heman giosarcoma	Epithelial Neoplasms: Adenoma	Non epithelial Neoplasms	Tubular Adenoma Simple Type and Complex	Granulosa Cell Tumor	Adeno carcinoma A: Adenoma	Solid Structure	Mixed Structure	Adeno acanth oma	Fibroma	Adenoma	Liposarcoma	Fibrosarcoma	Squamous Cell Carcinoma	Adenoma
WOM/W	175	159	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1	0	0	0	0	0	0	0	0	0
BN/aW	101	1	0	0	8	6	0	1	1	0	3	1	0	1	0	0	0	0	0	0	0	0	0	0	0	1
C3H ^wad /W	49	0	0	0	2	1	0	1	4	2	0	0	0	0	0	0	0	0	2	0	0	0	1	2	0	0
C57BL/6W	173	7	23	10	7	2	4	6	0	4	0	1	1	0	2	0	0	0	0	0	0	1	0	3	0	3
C57BL/10W	99	3	27	2	3	0	0	1	0	0	1	3	1	0	0	0	0	0	0	0	0	0	0	3	0	2
129S1/SvW	111	1	6	1	2	1	0	2	0	0	1	1	5	6	1	0	0	0	0	0	0	4	0	3	0	2
DBA/2W	71	2	1	0	0	0	0	1	0	0	0	0	7	6	1	0	1	0	0	0	0	0	0	1	0	0
C3H/W	166	1	0	2	4	0	3	3	10	2	0	0	1	0	0	1	37^b	17^b	92^b	0	2	0	0	1	0	0
CBA/W	52	0	2	0	5	1	3	5	9	3	0	0	2	0	3	1	0	0	1	0	0	0	0	0	0	0
AKR/W	121	110	3	0	0	0	0	0	0	0	0	0	0	0	1	0	0	0	0	0	0	0	0	0	0	0
BALB/cW	71	5	9	3	11^a	14^a	0	1	3	2	0	1	1	0	2	0	0	0	1	2	1	1	0	0	1	1
A.CA-H2^f /W	84	4	0	0	11	17	4	3	2	1	0	0	0	0	0	0	0	0	0	0	1	1	0	0	0	1

^aOne case where adenoma coexisted with adenocarcinoma.

^bThirty cases with more than 1 gland affected.

The most frequently observed nonneoplastic lesions are listed in Table 5. The most common nonneoplastic lesions in the examined strains were splenic congestion, reactive lymphoid hyperplasia in lymph nodes and spleen, ovarian cysts, lesions in the accessory male glands (ectasia and cystic degeneration of seminal vesicles, suppurative seminal vesiculitis, cysts and inflammation in preputial glands), otitis media, dental hypertrophy, and rectal prolapse. Nonneoplastic lesions in kidneys were observed mainly in BN/aW mice. Cardiomyopathy was found in C57BL/6W, C57BL/10W, and 129S1/SvW mice. The prevalence of histological types of neoplasms is shown in Table 4. Lymphomas were the most common neoplasia in the examined strains (364 cases/1273 mice, 28.6%). T-cell lymphomas were most commonly identified in AKR/W and WOM/W mice, and B-cell lymphoma was most common in C57BL/6W and C57BL/10W mice. Lymphoma was not observed in 49 C3H ^wad /W mice, and only 1 case was detected each in BN/aW and C3H/W mice. C3H/W mice had a high prevalence of neoplasms of the mammary gland.

Table 5.

Prevalence of Common Nonneoplastic Findings in the Examined Strains (Numbers of Affected Animals).

Organ/System	Lesion	WOM/W	BN/aW	C3H ^wad /W	C57BL/6W	C57BL/10W	129S1/SvW	DBA/2W	C3H/W	CBA/W	AKR/W	BALB/cW	A.CA-H2^f /W
No. of examined mice		175	101	49	173	99	111	71	166	52	121	71	84
Lung	Thickened alveolar walls	0	0	0	3	1	1	0	0	0	0	2	2
	Hemorrhage of obscure etiology	0	2	0	11	4	1	0	0	0	0	1	0
	Acidophilic macrophage pneumonia^a	0	0	0	7	3	0	0	0	0	0	0	1
	Aspiration pneumonia	0	0	0	0	0	1	0	0	1	0	1	0
	Granulocytic infiltration	0	0	0	1	1	0	0	0	0	0	0	0
	Lymphocytic infiltrate	0	0	0	0	0	3	2	0	0	0	0	1
Middle ear	Otitis media, any type	0	1	7	3	1	3	0	1	0	0	1	0
Salivary glands	Lobular hyperplasia (submaxillary and/or parotid)	0	0	0	2	3	0	2	1	0	0	2	0
Skull/brain	Dental hypertrophy	0	10	3	9	5	7	1	0	0	0	3	2
	Fibro-osseous change	0	0	0	1	0	0	0	0	0	0	0	0
	Hydrocephalus	0	0	0	1	0	0	0	0	0	0	0	0
Nasal turbinates	Amyloid-like material in nasal septum	0	0	0	1	1	0	0	1	1	0	0	0
Liver	Necrosis of hepatocytes^b	19	0	0	0	0	0	0	0	5	0	0	0
	Lipidosis	0	0	0	7	0	0	0	0	3	0	0	0
	Karyomegaly and cytomegaly	0	0	0	0	0	0	0	0	4	0	0	0
	Extramedullary hematopoiesis increased	0	0	0	1	2	0	5	1	0	0	0	0
	Mild lymphocytic infiltration in sinusoids	0	0	0	0	0	5	0	1	0	0	0	0
	Intranuclear and intracytoplasmatic inclusions	0	0	0	0	0	0	0	0	6	0	0	0
	Hepatocellular clear cell foci	0	0	0	0	0	0	0	2	4	0	0	0
	Hepatocellular vacuolated cell foci	0	0	0	1	0	0	0	0	4	0	0	0
	Binucleated hepatocytes	0	0	1	0	0	0	0	1	8	0	1	0
	Hepatic fibrosis	0	0	0	2	0	0	1	0	6	0	2	2
	Cysts	0	0	0	0	0	0	4	0	3	0	0	0
Spleen	Congestion	0	0	1	11	5	3	0	5	1	0	0	0
	Lymphoid hyperplasia	0	7	0	24	21	0	9	5	0	0	5	1
	Erythropoietic and granulopoietic activity	0	0	0	2	2	0	4	3	1	0	5	0
	Megakaryocytosis	0	0	0	0	1	0	2	3	1	0	3	0
	Amyloidosis	0	0	0	1	0	4	0	0	0	0	0	0
	Blood cysts	0	0	0	0	1	0	0	0	0	0	2	0
	Atrophy	0	0	1	5	3	2	3	0	0	0	1	2
Lymph nodes	Hyperplasia	0	0	1	28	21	2	9	9	7	0	17	1
Cardiovascular system	Cardiomyopathy, any type/arteriosclerosis	0	3	0	10	8	11	0	0	0	0	0	0
Kidney	Chronic nephritis	0	25	0	0	2	0	0	0	0	0	2	0
	Cysts	0	6	0	0	0	0	0	0	0	0	3	0
	Amyloidosis	0	5	0	0	0	5	0	1	0	0	0	0
	Hyalinization of glomeruli and tubules	0	7	0	0	0	0	0	0	0	0	0	0
	Degeneration of glomeruli	0	17^c	0	0	0	0	0	0	0	0	0	0
	Hydronephrosis	0	1	0	14	5	1	0	0	0	0	0	0
Uterus	Hydrometra	0	0	0	11	1	5	0	0	0	0	0	4
	Pyometra	0	0	0	3	0	1	1	0	0	0	0	3
	Cystic endometrial hyperplasia	0	0	0	11	4	7	2	0	1	0	0	5
	Mummified fetuses	3	0	0	6	5	2	1	0	1	0	2	0
Ovary	Unilateral cysts	0	1	5	8	4	2	3	0	1	0	3	3
	Bilateral cysts	0	0	4	4	2	2	2	0	0	0	0	1
	Hematocysts, unilateral or bilateral	0	0	0	0	0	0	1	0	1	0	1	0
	Atrophy	0	0	1	0	0	1	0	3	0	0	0	0
Clitoral glands	Cysts	0	0	0	5	3	0	5	0	1	0	3	1
Testes	Abscesses	0	1	0	1	2	1	2	2	2	0	0	2
	Atrophy	0	0	0	1	2	1	2	2	1	0	0	0
Male accessory sex glands	Ectasia of seminal vesicles	6	0	1	15	11	8	4	1	1	0	0	3
	Cystic degeneration of seminal vesicles	0	2	1	13	12	3	0	0	1	0	0	2
	Suppurative seminal vesiculitis	0	0	1	2	3	3	1	2	2	0	0	4
	Preputial gland abscesses/inflammation	0	0	1	1	1	7	1	3	4	0	0	0
Urinary system	Mouse urologic syndrome and paraphimosis	0	1	1	7	3	3	1	0	1	0	0	3
Esophagus	Esophageal dilatation	0	0	0	0	0	1	0	0	0	0	0	0
Small/large intestine	Hemorrhagic petechiae	0	0	0	1	0	0	0	0	0	0	1	0
Rectum^c	Prolapse or squamous metaplasia of the rectal mucosa	0	2	1	9	5	3	0	0	2	0	3	6

^aMacrophage accumulations (13 cases) found in alveolar sacs in parenchyma adjacent to lung neoplasms were not taken into account.

^bNecrosis of hepatocytes inside of hepatocellular carcinoma are not included.

^cDegeneration of glomeruli as part of chronic nephritis was observed in 5 cases.

Neoplastic and Nonneoplastic Lesions in the 3 Unique Strains: WOM/W, BN/aW, and C3H ^wad /W

WOM/W mice commonly developed T-cell lymphoblastic lymphoma/leukemias (159/175, 90.9%). Necropsy typically identified an enlarged and nodular thymus with variable splenomegaly and lymphadenomegaly. Histopathologic findings included lymphoblastic infiltration into the liver, lung, and kidneys. In addition, gut-associated lymphoid tissue was involved in 13.8% of lymphoma cases, testes in 20.1%, and pericardium invasion in 11.3%. Neoplastic cells were found in the bone marrow in 137 of 159 cases (86.1%) and in the peripheral blood in 69 of 159 cases (43.4%).

T-cell lymphomas were positive for CD90.2 and cytoplasmic cCD3∊. They were poorly positive for CD5 and negative for CD45-(B220), CD79b, GAM, IA^d, IgD, and IgM. Neoplastic T cells with coexpression of CD4 and CD8 were in the thymus and in peripheral tissues such as lymph nodes and bone marrow in 33 of 159 cases (20.8%). The population of T cells negative for CD4, CD8, and surface sCD3∊ (but cCD3∊⁺) was dominant in the thymus and in peripheral tissues in 78 of 159 cases (49.0%). Abnormal T cells with immunophenotypes CD4⁺CD8^– or CD4^–CD8⁺ in the thymus and peripheral tissues were noted in 11 (6.9%) and 8 (5.0%) cases, respectively. The immunophenotype of neoplastic T cells derived from the thymus and/or lymph nodes was consistent with the immunophenotype of T cells from the affected bone marrow in the same animals.

Other types of neoplasms were not observed, except for 1 case of mammary gland neoplasm (adenocarcinoma type A). That neoplasm was found in a female also diagnosed with T-cell lymphoma. Nonneoplastic lesions were rare in tissues other than the liver in WOM/W mice (see Table 5). Hepatocellular necrosis was interpreted to be the result of infiltrating neoplastic T cells in 19 cases. This strain also had 6 cases of seminal vesicular ectasia (common in aged male mice) without hyperplastic epithelium, and mummified fetuses were detected in 3 found-dead females.

BN/aW mice developed neoplasms in 22 of 101 cases (21.8%). Lung neoplasms were most common (14/101, 13.9%); they were more frequent in males (26.7%) than in females (11.6%). Lung neoplasms were solitary or multiple. The neoplasms were diagnosed as adenomas or adenocarcinomas. The size of adenomas was 3 to 4 mm; adenocarcinomas were greater than 4 mm with irregular nodular growth. Liver neoplasms, identified in 6 of 101 (5.9%), were observed more frequently in males (13.3%) than in females (4.7%). Hepatic vascular neoplasms (hemangiomas and hemangiosarcomas) were more common than hepatocellular carcinomas (see Table 4). Hematopoietic neoplasms, uterine neoplasms, and Harderian gland neoplasms occurred in single cases (see Tables 3, 4).

Nonneoplastic lesions in the kidney were common in BN/aW mice (see Table 5). Sex predilection was not evident (56/101, 55.4%; females 55.9%, males 53.3%). The lesions included chronic interstitial nephritis, glomerular amyloidosis, tubular ectasia, hyaline and proteinaceous casts in tubules, and cortical cysts (see Table 5 and Figs. 1–4).

Figure 1. Kidney; BN/aW mouse, female, age >28 months. Glomerular amyloidosis. The glomerulus is expanded by an amorphous eosinophilic material. Congo red stain. Figure 2. Kidney; BN/aW mouse, male, age >28 months. Ectasia of renal tubules (black arrow), cortical renal cysts, and severe loss of renal tubular epithelium (blue arrow). Hematoxylin and eosin (HE). Figure 3. Kidney; BN/aW mouse, male, age >28 months. Ectasia of renal tubules; glomerulus contains abundant acellular pinkish material. HE. Figure 4. Kidney; BN/aW mouse, male, age >28 months. Chronic inflammation infiltrate (black arrow), interstitial fibrosis (blue arrow), and intralumenal proteinaceous casts. HE.

The lesions of interstitial renal disease were usually limited to the cortex and manifested as foci of mononuclear cells (predominantly lymphocytes) and local tubular dilation. Tubular dilation destroyed tubular architecture and in advanced stages resulted in formation of cortical cysts. Marked dilation of Bowman’s capsules was often observed. Glomeruli varied from normal to sclerotic/nonfunctional depending on the stage of interstitial nephritis. Renal cysts were typically bilateral, although 1 kidney was usually more affected. Nephropathy in BN/aW mice was described as mild or severe. Other nonneoplastic lesions included reactive lymphoid hyperplasia in the spleen and/or lymph nodes, dental hypertrophy, and rectal prolapse (see Table 5).

The C3H ^wad /W strain arose spontaneously from the C3H/W strain in the 1980s. These mice have an unmapped mutation on the distal chromosome 10 (see Table 1).²⁴ C3H ^wad /W mice developed hepatocellular carcinomas with a frequency of 14.2% (7/49). Lung neoplasms occurred in 3 of 49 mice (6.1%), neoplasms of the mammary gland in 2 of 25 females (8%), and soft tissue neoplasms in 3 of 49 mice (6.1%) (see Tables 3, 4). The soft tissue neoplasms consisted of 2 fibrosarcomas and 1 liposarcoma. The subcutaneous fibrosarcomas were medium-size neoplasms arising on the dorsum. The liposarcoma was a large (12 mm diameter) retroperitoneal mass. Common nonneoplastic lesions in C3H ^wad /W mice included ovarian cysts (9/25, 36%), otitis media in males (7/24, 29.1%), lesions in accessory male glands, and mouse urologic syndrome with paraphimosis (see Table 5).

Neoplastic and Nonneoplastic Lesions in Common Inbred Strains

C57BL/6W mice frequently developed hematopoietic neoplasms (40/173, 23.1%), which were typically follicular B-cell lymphomas (FBLs). These lymphomas arose in the spleen and/or mesenteric lymph nodes and infiltrated the liver; less than 10% also involved Peyer’s patches. Neither the bone marrow nor the blood had evidence of neoplastic cells. These FBLs were CD45R-(B220)⁺, CD19⁺, CD79b⁺, IA^d+, GAM⁺, and Igκ light chain expression (RAM KAPPA⁺) by fluorescence-activated cell sorting. T-cell markers CD90.2^–, CD4^–, CD8^–, sCD3∊^–, cCD3∊^–, and TCRβ^– were interpreted as negative, although a small number of T cells were always detected in mesenteric lymph nodes. Immunohistochemistry showed expression of CD45R-(B220), IgM and/or IgD, and Igκ, while λ light chain expression was not noted.

Liver neoplasms (15/173, 8.6%), diagnosed as hepatocellular carcinomas or adenomas, were the second most common neoplasms in C57BL/6W mice; they were more common in males than in females (11.7% and 4.3%, respectively). Pulmonary neoplasms were also relatively common (9/173, 5.2%); most were bronchioloalveolar adenomas (7/9). A male predilection was evident (7.8% males, 1.4% females). Other sporadic neoplasms included soft tissue neoplasms (1.7%), Harderian gland neoplasms (1.7%), ovarian neoplasms (2.9%), uterine neoplasms (1.4%), and intestinal neoplasms (0.6%) (see Tables 3, 4).

The most common nonneoplastic lesion was lymphoid hyperplasia (13.9%) in the spleen and/or lymph nodes. The architecture of the spleen remained normal, but increased numbers of enlarged and coalescing lymphoid follicles were identified. In lymph nodes, follicular hyperplasia was restricted to the cortex; it did not extend into the capsule. Splenic and lymph nodal follicles were cytologically polymorphous. Because lymphoid hyperplasia and lymphoma can be part of a continuum, the clonality of lymphocyte population was assessed. Polyclonal lymphocyte populations were diagnosed as hyperplasia, and monoclonal populations were called lymphoma.

Other nonneoplastic changes included cystic endometrial hyperplasia, ovarian cysts, imperforate vaginas with associated hydrometra or pyometra, hydronephrosis, lesions in male accessory sex glands and cysts in clitoral glands in females, acidophilic macrophage pneumonia, cardiomyopathy, hepatic lipidosis (in males only), dental hypertrophy, and rectal prolapse (see Table 5).

The lesions observed in C57BL/10W mice were similar to lesions in C57BL/6W mice, although hydronephrosis and hydrometra occurred less frequently in C57BL/10W mice, while lipidosis in liver was not found in C57BL/10W mice (see Tables 3–5).

129S1/SvW mice developed uterine neoplasms most frequently (11/59 females, 18.6%). Five were adenomas, 5 were endometrial stromal sarcomas, and 1 was a leiomyosarcoma. Hematopoietic neoplasms (7.2%) were chiefly B-cell lymphomas. Intestinal neoplasms (3.6%) developed in the small intestine and were diagnosed as adenomas. Lung neoplasms occurred in 2.7% of examined mice. Liver neoplasms (3.6%) consisted of both hepatocellular carcinomas and vascular neoplasms. Soft tissue neoplasms developed in 2.7% of examined mice and neoplasms of Harderian glands in 1.8% (2/111) of mice (see Tables 3, 4). Male bias was observed in lung and liver neoplasms, and conversely, female bias was found in lymphoma occurrence. Nonneoplastic lesions that contributed to death included atriomegaly and dilation of the caudal vena cava and renal amyloidosis. Nonneoplastic lesions considered to be incidental included splenic amyloidosis, hydrometra, cystic endometrial hyperplasia, lymphocytic infiltrates within the liver, thyroid and periovarian fat, dental hypertrophy, rectal prolapse, and lesions in male accessory sex glands (see Table 5).

DBA/2W female mice developed uterine neoplasms in 13 of 51 cases (25.4%); 7 were diagnosed as adenomas, and 6 were endometrial stromal sarcomas. Lymphomas (4.2%), liver neoplasms (1.4%), ovarian neoplasms (2.0%), mammary gland neoplasms (2.0%), and neoplasms of soft tissue (1.4%) were diagnosed sporadically (see Tables 3, 4). The observed nonneoplastic changes included hepatic cysts, ovarian cysts, proliferative lesions of the hematopoietic system, clitoral gland cysts, and lesions in testes and male accessory sex glands (see Table 5).

C3H/W mice are carriers of the exogenous milk-transmitted mouse mammary tumor virus (MMTV). Neoplasms developed in mammary glands in 106 of 112 females (94.6%) at age ∼400 days (median, 395 days; min, 207 days; and max, 732 days). The occurrence of neoplasms in all mammary glands was similar.

Mammary gland neoplasms were classified as adenoma (adenocarcinoma A in the Dunn classification) or as adenocarcinoma (adenocarcinoma B in the Dunn classification) (see Table 4). Liver neoplasms (18/166, 10.8%) occurred only in males; 15 were hepatocellular carcinomas, and 3 were adenomas. Lung neoplasms (2.4%) and hematopoietic neoplasms (1.8%) developed rarely, and others were observed only sporadically (see Tables 3, 4). Nonneoplastic lesions were uncommon in C3H/W mice. The observed lesions included splenic congestion, reactive lymphoid hyperplasia, ovarian atrophy, and lesions in male accessory sex glands (see Table 5).

CBA/W mice often developed liver neoplasms (20/52, 38.4%), and hepatocellular carcinomas were more common (17 cases) than hepatocellular adenomas (3 cases). Males were more susceptible than females (44.4% and 25.0%, respectively). Lung neoplasms (6/52, 11.5%) were primarily adenomas; no sex predisposition was observed. Ovarian neoplasms (tubular adenomas) were identified in 4 of 16 females (25%). Hematopoietic, uterine, and mammary gland neoplasms were uncommon (see Tables 3, 4).

Nonneoplastic changes in the liver coexisted with neoplasms or were found separately. They consisted of hepatocyte degeneration, intranuclear and cytoplasmic inclusions, karyomegaly and cytomegaly, the presence of binucleated hepatocytes (more than 20% of hepatocytes), hepatocellular foci of cellular alterations, lipidosis, and hepatic cysts. Several other common nonneoplastic lesions were observed, including cystic endometrial hyperplasia, ovarian cysts, reactive hyperplasia in lymph nodes, and lesions in testes and male accessory sex glands (see Table 5).

AKR/W mice developed hematopoietic neoplasms (113/121, 93.3%), which were typically T-cell lymphoblastic lymphomas (110 cases) and rarely follicular B-cell lymphomas (3 cases). Sex predilection was not observed. T-cell lymphoblastic lymphomas developed in thymus and neoplastic cells infiltrated into the spleen, lymph nodes, liver, and kidneys. Bone marrow was involved in 93 of 110 cases (84.5%) and peripheral blood in 60 of 110 cases (54.5%). Nonneoplastic changes were not identified in this strain.

BALB/cW female mice developed a wide spectrum of neoplasms. Lung neoplasms predominated (24/71, 33.8%) and were diagnosed as adenoma (11) and adenocarcinoma (14); in 1 case, adenoma coexisted with adenocarcinoma. Hematopoietic neoplasms (17/71, 23.9%) were identified as B-cell lymphomas (12.7%), T-cell lymphomas (7.0%), and nonlymphoid hematopoietic neoplasms (4.2%). Liver neoplasms occurred in 9.9% (7/71) of mice and were diagnosed as hepatocellular carcinoma (6) and hemangiosarcoma (1). Mammary gland neoplasms (5.8%) were diagnosed as adenoacanthoma or adenocarcinoma (see Tables 3, 4). The other neoplasms (uterine, ovarian, intestinal, Harderian gland, and skin) occurred sporadically (see Tables 3, 4). The most common nonneoplastic lesions were reactive lymphoid hyperplasia in lymph nodes and spleen, ovarian cysts, renal cysts, dental hypertrophy, and rectal prolapse (see Table 5).

Data from BALB/cW males are scant, since BALB/cW males are highly aggressive and difficult to maintain for a long period during the observation. The data obtained from the observation of BALB/cW males cannot be interpreted. The previous observation (1980–1985) of BALB/cW males showed a prevalence of lung neoplasms (15/140, 36.4%), liver neoplasms (3/140, 2.1%), and hematopoietic neoplasms (2/140, 1.4%).

A.CA-H2^f /W mice frequently developed lung neoplasms (28/84, 33.3%); large adenocarcinomas predominated (17/28) (see Table 4). Liver neoplasms (10/84, 11.9%) developed more frequently in males than in females (16.2% and 8.5%, respectively). Hematopoietic neoplasms (4.8%) were diagnosed as T-cell lymphoblastic lymphomas. Other neoplasms were uncommon (see Tables 3, 4).

Common nonneoplastic lesions included hydrometra, pyometra without a determined cause, cystic endometrial hyperplasia, ovarian cysts, and rectal prolapse. In males, suppurative seminal vesiculitis and mouse urologic syndrome with paraphimosis were observed (see Table 5).

Statistical Results

The prevalence of lung neoplasms was significantly higher in BALB/cW (33.8%) and A.CA -H2 ^f /W (33.3%) mice than in other strains (P < .05). There was no statistical difference between these 2 strains. The prevalence of liver neoplasms was the greatest in CBA/W mice (38.4%, P < .01). C3H/W mice, irrespective of sex, have a lower prevalence of liver neoplasms (10.8%) than CBA/W mice. However, if only males are studied, the prevalence of liver neoplasms (33.3%) is similar to CBA/W mice. The examined strains did not significantly differ in the prevalence of uterine neoplasms, although the highest occurrence was observed in DBA/2W and 129S1/SvW mice. The prevalence of neoplasms of the mammary glands was significantly higher in C3H/W mice (P < .01), which carry exogenous MMTV.

The frequency of T-cell lymphomas was significantly higher in WOM/W (90.9%) and AKR/W mice (90.9%) than in other strains (P < .01). No significant differences were found between these 2 strains. The occurrence of B-cell lymphomas in C57BL/10W mice (27.3%) was significantly higher than in other strains (P < .05). The occurrence of B-cell lymphomas in C57BL/6W mice (13.3%) was higher than in the other strains, but the difference was not statistically significant. No statistically significant differences among strains were found in the occurrence of nonlymphoid hematopoietic neoplasms. There was no significant difference in the frequency of other types of neoplasms between strains. The prevalence of nonneoplastic lesions in kidneys of BN/aW mice (55.4%) was significantly higher than in the other strains (P < .01).

Discussion

The gross and histological evaluation of aged mice provides important information about strain-specific disease predispositions.^56,61

In this article, we have shared pathological data for aged mice of common and new strains to supplement knowledge about strain-specific disease predispositions.

First, we have described neoplastic and nonneoplastic lesions in aged mice from 12 inbred strains. These data are important for differentiating strain-specific lesions in animal models from those due to gene modification. We identified the COD for 1236 mice; in 37 mice, the cause of death was not unequivocally identified. The cause of death was generally attributed to a single disease in the case of neoplasms. Chronic degenerative nonneoplastic lesions were considered the COD if a single disease was observed and the impact of that lesion was evident. Aging mice usually showed a complex set of lesions that contributed to death. The causes of nonneoplastic lesions remain unknown in most cases. However, Helicobacter infection should be taken into consideration in cases of rectal prolapses and lymphocytic infiltration in liver sinusoids, necrosis of hepatocytes, fibrosis, and cytomegaly in liver.^2,76

Second, we have described new strains (WOM/W, BN/aW), which can be considered new mouse models of T-cell lymphoma and age-related nephropathy, respectively. Moreover, we described the type and diversity of mouse diseases that these strains develop as they age.

The first novel strain (WOM/W) is unique in its consistent development of T-cell lymphomas (90.9%) at the young age of ∼230 days (median, 225 days; min, 91 days; max, 570 days). Importantly, the genetic background of WOM/W mice is completely different from that of AKR/W mice (M. Gajewska, unpublished data), a classic strain prone to develop T-cell lymphoblastic lymphomas (60%–90%) at less than 1 year of age.²⁰ AKR mice contain endogenous ecotropic murine leukemia virus (Akv) that is involved in causing a high prevalence of lethal T-cell lymphoblastic lymphomas, primarily developing in the thymus.^28,40 WOM/W mice may be considered a novel strain with a high prevalence of T-cell lymphoblastic lymphomas similar to the AKR, C58, NOD-SCID/SCID, SCID, and HRS/J strains.^8,72

Animal models of lymphoma/leukemia are widely used to study the mechanisms of cancer development and progression as well as to assess the efficacy of new therapies. The Hematopathology Subcommittee of Mouse Models of Human Cancer Consortium (MMHCC) developed criteria to classify mouse lymphoid neoplasms and indicated their relation to diseases in humans.^6,49,62 T-cell lymphoblastic lymphoma/leukemia is recognized as a mouse counterpart of that disease in humans.⁴¹ Moreover, published data on lymphoma in mice indicate that the specific background strain greatly influences the prevalence and type of lymphomas.⁶

Our second new strain (BN/aW) has a high prevalence of renal disease and may be a valuable alternative model to investigate chronic renal disease. Nephropathy has previously been described in C57BL/6, 129, and B6C3F1 hybrid mice.^9,75,78 Spontaneous interstitial nephritis has been reported in CBA/CaH-kdkd mice, which are homozygous at kd on chromosome 10.^50,59 These mice are very susceptible to spontaneous interstitial nephritis and die between 5 and 7 months with end-stage renal failure. However, CBA/CaH-kdkd mice are poor breeders and produce only 1 or 2 litters. The prevalence of nephropathy in C57BL/6 varies by report, from rather low (6%–15%)^3,8,9 to 100% with mild to moderate glomerulonephritis.⁷⁹ Chronic nephropathy has been noted in the 129S4/SvJae strain, but it was not considered the COD.⁸ B6C3F1 hybrid mice are preferred for toxicology studies, but they have a high prevalence of nephropathy. In B6C3F1 mice, nonneoplastic renal lesions include mononuclear cell infiltrates (29.8%), tubular mineralization (11.3%), interstitial nephritis (6.8%), tubular epithelial vacuolization (4.5%), tubular epithelial degeneration/regeneration (2.5%), and cortical cysts (1.3%).¹² A GEM with chronic interstitial nephritis is the [Tg(Cga-LHB/CGB)94Jhn/J] mouse, which expresses a human luteinizing hormone chimera targeted to mouse pituitary gonadotropes. In addition to the expected pathologic ovarian changes, these mice develop chronic interstitial nephritis.⁵⁷ In total, 55.4% of BN/aW mice died after 30 months as a result of nephropathy, which included chronic interstitial nephritis (24.8%), cortical cysts (5.9%), hyalinization of tubules (6.9%), degeneration of glomeruli (16.8%), amyloidosis (5%), and hydronephrosis (1%) (see Table 5). Moreover, BN/aW mice are long lived (median >850 days), develop few neoplasms, and are relatively good breeders (average, 4 litters), increasing their utility as a model of human disease.

Third, we have compared the lesions found in mice from our strains with data obtained from a literature survey. We tabulated the most prominent similarities and differences in morbidity of mice from our substrains and compared them with previously published reports (see Table 6). The traditional inbred strains maintained in our animal facility have been genetically isolated from their parental colonies for more than 20 generations and thus are defined as substrains, although their genetic differences from parental strains have not been evaluated.

Table 6.

Prevalence of Lesions Found in the Observed Strains in Comparison With Data From Literature.

	Prevalence of Lesions in Comparison With Literature Data^a,b,c
Strain	Consistent	Different
C57BL/6 W C57BL/10W	Lymphomas—lower incidence in females Liver neoplasms—male bias Lung neoplasms—male bias Hydrometra, cystic endometrial hyperplasia Hydronephrosis	Ulcerative dermatitis not detected in C57BL/6 W Hydrocephalus not detected in C57BL/6 W Acidophilic macrophage pneumonia—lower incidence in C57BL/6 W
129S1/SvW	Primarily lymphoma Soft tissue sarcomas Lung neoplasms—129S1/SvW lower incidence Liver neoplasms—129S1/SvW lower incidence Ovarian neoplasms—129S1/SvW only observed	Testicular teratomas not detected in 129S1/SvW Acidophilic macrophage pneumonia—rare in 129S1/SvW Uterine neoplasms in 129S1/SvW
DBA/2W	Lymphomas—female bias Liver neoplasms Mammary gland neoplasms low frequency	Lung neoplasms not observed in DBA/W Uterine neoplasms >20% in DBA/W Calcified heart lesions (90% in literature data)—no data from DBA/2 W Glaucoma—no data from DBA/2 W
C3H/W nonfostered	Mammary gland neoplasms Liver neoplasms—male bias Lung neoplasms and lymphomas—observed
CBA/W	Liver neoplasms—male bias Ovarian neoplasms—CBA/W lower incidence	Nonneoplastic lesions in liver in CBA/W
BALB/cW	Lung neoplasms Hematopoietic neoplasms most common in follicular B-cell lymphoma Mammary gland neoplasms—adenoacanthoma, adenocarcinoma Hemangiosarcoma in liver, ovarian cysts	Adrenal subcapsular spindle cell hyperplasia (24% in literature data) not observed in BALB/cW
AKR/W	T-cell lymphomas Nonneoplastic lesions no observed
A.CA-H2^f	No available data in literature

^aFesting.²⁰

^bTumor Frequency Grid on Mouse Tumor Biology Database.⁶⁸

^cBrayton.⁸

Our substrains differ from parental strains in their susceptibilities to neoplasms but generally do not differ with regard to the type of neoplasms they develop. Two strains, DBA/2W and 129S1/SvW, differ most notably from their parental strains in the prevalence of uterine neoplams (increased occurrence in our colony). Another difference is noted in male 129S1/SvW mice, which had no evidence of testicular neoplasms, in contrast to what is seen in the parental strain.

The 129 mice are known for their very high prevalence of testicular teratomas.¹ However, at least 16 recognized substrains of 129 exist.⁵³ According to the revision of 129 strain nomenclature presented by Festing et al,²¹ 129 is followed by letters P, S, T, and X. P stands for derivation from parental stock, S indicates the origin from the line with the Steel allele on the Kitl gene, T means that a line carries or used to carry the Ter (teratoma) gene, and X indicates lines genetically contaminated at The Jackson Laboratory around 1978 by an unknown strain. These lines differ from other 129 substrains at around 25% of single sequence length polymorphic markers and are designated 129cX/Sv.⁶⁵ The prevalence of testicular teratomas is high in the 129/terSv substrain (30%) and low (1%–3%) in 129P parental and many other substrains.³³ More than 15 genes, including Ter (having the strongest effect), Kitl(Sl), Ay, and Trp53, are involved in susceptibility to testicular teratomas.^34,39

The data obtained from this study provide an overview of the types and variety of lesions observed in aging mice of multiple strains. As expected, the examined strains differ in susceptibility to neoplasia as well as prevalence and severity of nonneoplastic lesions. The data presented here add to the growing literature on age- and strain-related lesions in mouse strains commonly used in research and the generation of GEM. This report also shows that substrains are often unique from parental strains and that no assumptions should be made when developing animal models of disease.

Footnotes

Acknowledgements

We thank the medical pathologist Elwira Bakula-Zalewska from the Department of Pathology of the Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology in Warsaw for revision of several diagnoses.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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Neoplastic and Nonneoplastic Lesions in Aging Mice of Unique and Common Inbred Strains Contribution to Modeling of Human Neoplastic Diseases

Abstract

Keywords

Materials and Methods

Mice

Strains

Inbred status

Husbandry and housing

Health monitoring

Observation Protocol

Pathological Examination

Immunophenotyping

Classification of Lesions

Determination of Cause of Death and Contributed Cause of Death

Statistical Analysis

Results

Neoplastic and Nonneoplastic Lesions in the 3 Unique Strains: WOM/W, BN/aW, and C3H wad /W

Neoplastic and Nonneoplastic Lesions in Common Inbred Strains

Statistical Results

Discussion

Footnotes

Acknowledgements

Declaration of Conflicting Interests

Funding

References

Neoplastic and Nonneoplastic Lesions in the 3 Unique Strains: WOM/W, BN/aW, and C3H ^wad /W