The Journal of Toxicological Sciences (J

The Journal of Toxicological Sciences (J. Toxicol. Sci.) 689 Vol.42, No.6, 689-705, 2017

Original Article Background data on NOD/Shi-scid IL-2Rγnull mice (NOG mice)

Kenichiro Kasahara1, Yachiyo Fukunaga1, Saori Igura1, Rie Andoh2, Tsubasa Saito2, Isamu Suzuki2, Hiroyuki Kanemitsu1, Daisuke Suzuki1, Ken Goto2, Daichi Nakamura1, Masahiro Mochizuki2, Masahiko Yasuda3, Ryo Inoue3, Kazutoshi Tamura2 and Mariko Nagatani2

1BoZo Research Center Inc., Tsukuba Research Institute, 8 Okubo, Tsukuba, Ibaraki, 300-2611, Japan 2BoZo Research Center Inc., Gotemba Research Institute, 1284 Kamado, Gotemba, Shizuoka, 412-0039, Japan 3Central Institute for Experimental Animals, 3-25-12, Tonomachi, Kawasaki-ku, Kawasaki, Kanagawa, 210-0821, Japan

(Received June 5, 2017; Accepted August 29, 2017)

ABSTRACT — To obtain background data of NOD/Shi-scid IL-2Rγnull (NOG) mice, severely immune- deﬁcient mice, a total of 120 animals were examined at 7, 26 and 52 weeks-old (20 mice/sex/group). The survival rate at 52 weeks-old was 95% (19/20) in both sexes. Clinically, circling behavior in one direction along the cage wall was observed in males after 8 weeks and females after 47 weeks-old, and hunchback position was found in males after 32 weeks-old. Hematologically, lymphocyte count markedly decreased at all ages, while white blood cell count increased in several mice at 52 weeks-old. Blood chemistry results revealed high values of aspartate aminotransferase, lactate dehydrogenase and creatine phosphokinase in some females at 26 weeks-old, without any related histological change. Histologically, lymphoid hypoplasia characterized by severe lymphocyte depletion with poorly developed tissue architectures was observed. In addition, spongiotic change in the nerve tissue was observed in both sexes at 7 and 26 weeks-old, and intracytoplasmic materials known as tubular aggregates in the skeletal muscles were found in males terminated at 26 and 52 weeks-old and in females at 52 weeks-old. Malignant lymphoma was found in one female euthanized at 20 weeks-old. Further, small intestinal adenoma, hepatocellular adenoma, leukemia, cerebral lipomatous hamartoma, Harderian gland adenoma and uterine polyp were also observed, and their incidences were low except for that of uterine polyp. This study provided detailed background data on NOG mice up to 52 weeks-old and provided information on appropriate use of NOG mice in the various research ﬁelds.

Key words: NOG mice, Background data, Immuno-deﬁcient, Lymphoid hypoplasia, Spongiotic changes, Tubular aggregate

INTRODUCTION larly to the NSG (NOD/SCID/IL2rgKO) mice and SCID/ Beige and DKO (Rag2-γC double-knockout) mice, which NOD/Shi-scid IL-2Rγnull (NOG) mice, formally are characterized by loss of T and B lymphocytes and referred to as NOD.Cg-Prkdcscid I12rgtm1Sug/ShiJic mice, NK cells, NOG mice are able to integrate human cells are severely immuno-deficient animals established at the and tissues into them more efficiently in comparison with Central Laboratories for Experimental Animals by knock- the pre-existing immuno-deficient animals such as nude out introduction of IL-2 receptor γ-chain (IL2Rγc) into mice (Ito et al., 2012; Shibata et al., 1997; Mazurier et the NOD-scid strain (Ito et al., 2002). al., 1999). NOG mice are characterized by the absence of T and Through making good use of their severely immuno- B lymphocytes originated in the scid mutation, decreased deficient properties, NOG mice are attracting great atten- activity of the complements and insufficient function of tion as a possible candidate to re-establish human cells/ macrophages originated in the NOD strain, and absence tissues within them. They are estimated to be a useful of NK cells and malfunction of dendritic cells originat- evaluation system for pharmacological effects of antican- ed in the IL2Rγc knock-out mice (Ito et al., 2002). Simi- cer agents on tumor cells which have been transplanted / Correspondence: Kenichiro Kasahara (E-mail: [email protected])

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K. Kasahara et al. live planted into them and for toxicological and oncologi- Ophthalmologic examinations cal properties of the products for tissue-engineered medi- All survivors were subjected to ophthalmologic exami- cal products. As an important precondition to utilize NOG nations before necropsy. The anterior portion and interme- mice in the above-mentioned evaluation system, it is nec- diate optic media were examined by a slit lamp (SL-15: essary to obtain clinical and especially histological back- Kowa Co., Ltd., Aichi, Japan), and the fundus oculi after ground data on them. Up to the present, there are, how- dilation of the pupil by mydriatic agent (Mydrin P: Santen ever, no reports of the long-term histological background Pharmaceutical Co., Ltd., Osaka, Japan) were exam- data on severely immuno-deficient animals. This study ined by a binocular indirect ophthalmoscope (Omega was carried out to clarify the clinical and histological 500: HEINE Optotechnik GmbH & Co. KG., Bayern, background data on NOG mice up to 52 weeks of age. Germany).

MATERIALS AND METHODS Hematology At necropsy, carried out as scheduled, blood samples Animals were collected via the vena cava inferior into blood col- Sixty male and 60 female NOG mice, a specific path- lection tubes (MicrotainerTM, Nippon Becton Dicking- ogen free, were obtained from the Central Institute for son Co., Ltd., Tokyo, Japan) containing EDTA-2Na using Experimental Animals (Kanagawa, Japan) at 5 weeks of a syringe treated with heparin sodium, and then were age, and they were equally divided into 3 groups. The examined for red blood cell count (RBC), hemoglob- animals were kept untreated until euthanasia under isoflu- in (HGB), hematocrit (HCT), mean corpuscular volume rane anesthesia at 7, 26 or 52 weeks of age. All animals (MCV), mean corpuscular hemoglobin (MCH), mean cor- were housed in polycarbonate flat-bottomed cages (W160 puscular hemoglobin concentration (MCHC), reticulocyte x D370 x H130 mm, Tecniplast Japan Co., Ltd., Tokyo, ratio, platelet count, white blood cell count (WBC) and Japan) installed in a positive pressure rack individual- leukocyte differentiation using ADVIA® 2120i Hematol- ly for males and five animals per cage (W230 x D335 x ogy System (Siemens Healthcare Diagnostics Inc., New H140 mm, Clea Japan Inc., Tokyo, Japan) for females in York, NY, USA). a clean animal room controlled to maintain the tempera- ture at 23 ± 3°C, the relative humidity at 50 ± 20%, the Blood chemistry air ventilation at 10 to 20 times per hour and illumination At the same time as hematological examination, plas- for 12 hr a day, and were allowed free access to pelleted ma samples were obtained by centrifugation of the diet, CE-2 (irradiation sterilized, Clea Japan Inc.) and tap blood collected in tubes containing heparin lithium water. The equipment was disinfected by hypochlorous (Capiject Heparin Lithium: Terumo Co., Ltd., Tokyo, acid or autoclave. Moreover, traffic lines of human and Japan) and then were examined for aspartate aminotrans- equipment for access to animal room were strictly con- ferase (AST), alanine aminotransferase (ALT), lactate trolled. dehydrogenase (LDH), creatine phosphokinase (CPK), To compare with NOG mice, some parameters on alkaline phosphatase (ALP), γ-glutamyl transpeptidase hematology, blood chemistry and organ weights from (γ-GTP), total cholesterol (T-CHO), triglyceride (TG), both sexes of ICR mice that were housed in our facility phospholipid (PL), total bilirubin (T-BIL), glucose (GLU), and sacrificed at same time points as above were used. blood urea nitrogen (BUN), creatinine (CRNN), sodium This experiment was conducted after obtaining (Na), potassium (K), chloride (Cl), calcium (Ca), inor- approval of the Animal Experiment Committee of BoZo ganic phosphorus (P), total protein (TP), albumin (ALB) Research Center Inc. in 2015. It was conducted in accord- using Clinical Laboratory System TBA-120FR (Toshiba ance with the guidelines for the control and welfare of Medical Systems Corporation, Tokyo, Japan), and glob- experimental animals specified by the test facility (Rule ulin, A/G ratio and protein fraction using QuickScan of the Animal Experiment Committee, BoZo Research Densitometer (Helena Laboratories, Inc., Saitama, Japan). Center Inc.). Histology Clinical sings and measurement of body weights All mice were subjected to complete necropsy and Clinical signs were observed once every day and body the brain (cerebrum and cerebellum), spleen, heart, weights were measured once a week until 16 weeks of lungs, liver (including gallbladder), kidneys and testes age and twice a week thereafter. were weighed. Cerebrum, cerebellum, medulla oblongata, spinal cord (cervical, thoracic and lumbar), sciat-

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Background data on NOG mice ic nerves, Harderian glands, pituitary, thyroids, parath- age was 95% in both sexes. yroids, adrenals, thymus, spleen, submandibular lymph node, mesenteric lymph node, heart, thoracic aorta, tra- Clinical signs chea, lung (including the bronchus), tongue, esopha- Circling behavior in one direction along the cage wall gus, stomach, duodenum, jejunum, ileum (including the and hunchback position were observed. The former was Peyer’s patch), cecum, colon, rectum, submandibular found in males and females after 8 weeks and 28 weeks glands, sublingual glands, liver, gall bladder, pancreas, of age, respectively. Although such clinical signs were kidneys, urinary bladder, epididymides, prostate, seminal transient in some cases, hunchback position detected in vesicles, ovary, uterus, vagina, mammary glands (inguinal 8 males at 32 weeks of age continued until and including region, females only), sternum (including the bone mar- 52 weeks of age. row), femurs (including the knee joint and bone marrow), femoral skeletal muscles, skin (inguinal region), nasal Body weights cavity, and any gross lesions were fixed in 10% phos- Except for 3 mice which died or were euthanized, phate-buffered formalin. The eyeballs and optic nerves the body weight was relatively rapidly increased from were fi xed in mixture of 3% glutaraldehyde and 2.5% for- 5 weeks of age (male: 19.7 g, female: 16.4 g) to 30 weeks malin, and the testes were fi xed with Bouin’s fl uid. The of age in male (27.0 g) and 36 weeks of age in female tissues were trimmed, embedded in paraffin, sectioned (24.3 g), and thereafter, declined in gain and reached and stained with hematoxylin and eosin (H&E) for histo- 27.2 g in males and 24.1 g in females at 52 weeks of age logical examination. In addition, the spleen, thymus and (Fig. 1). lymph nodes from some mice and the bone marrow from 2 mice which were suspected to bear hemolymphoretic- Ophthalmology ular tumors were stained with rabbit anti-CD3 mono- Lenticular opacity was observed in a few mice of clonal antibody (Nichirei Biosciences Inc., Tokyo, Japan) both sexes at 7 weeks of age. Thereafter, its incidence as a T cell marker, rabbit anti-PAX5 monoclonal anti- increased with age and reached 100% in both sexes at body (Abcam, Cambridge, UK) as a B cell marker, rabbit 52 weeks of age. Lenticular opacity was a particulate anti-human myeloperoxidase (MPO) polyclonal antibody change in most cases, and the incidence of the particulate (Dako Japan, Tokyo, Japan) as a myeloid cell marker and opacity in the posterior cortex increased with age. How- rabbit anti-Iba1 polyclonal antibody (Wako Pure Chemical ever, they were hardly identifi ed in the histological exam- Industries Ltd., Osaka, Japan) as a macrophage marker. ination because they were sparse and not often captured Moreover, the medulla oblongata and spinal cords were on the slides (Table 1). examined immunohistochemically using rabbit anti-glial fibrillary acidic protein (GFAP) polyclonal antibody (Dako Japan) as an astrocyte marker and Iba1 as a micro- glia marker. Small pieces of the formalin-fixed femoral muscle from 3 mice each per age-group were additionally fi xed in the solution of phosphate buffered 0.5% glutaraldehyde 1.5% paraformaldehyde and followed by post-fi xed in the 1% osmium tetroxide solution, and embedded in epoxy resin. Ultra-thin sections were then prepared, stained with uranyl acetate and lead citrate, and observed under electron microscopy (JEM-1400, JEOL Ltd., Tokyo, Japan).

RESULTS

Mortality and survival rates One male and two females died or were euthanized due to deterioration at 20 (female), 49 (female) and 51 weeks of age (male). One female died of malignant lymphoma, whereas the cause of death of the remaining two mice was unclear. The ﬁ nal survival rate at 52 weeks of Fig. 1. Body weight changes in NOG mice.

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Table 1. Incidences of ophthalmological data in NOG mice. Age (weeks): 7 26 52 Region Sex: Male Female Male Female Male Female -Findings No. of mice: 20 20 20 19 19 19 Cornea -Corneal opacity, focal 1 1 Iris -Posterior synechia 1 Lens Nucleus -Particulate opacity 5 6 5 7 10 1 -Focal opacity 1 1 1 2 Posterior cortex -Particulate opacity 1 2 19 17 -Focal opacity 22 -Adhesion posterior Lens capsule 3 3 1 2 Posterior capsule -Particulate opacity 3 -Focal opacity 1 Vitreous body -Persistent tunica vasculosa lentis 1 -Persistent hyperplastic primary vitreous 1 1 Fundus oculi -Retinal fold, focal 2 -Coloboma of choroid 21

Hematology (Table 4-1, 4-2). One of the characteristic hematological ﬁndings was an extremity low value of lymphocytes (Table 2-2). Fur- Necropsy thermore, there were some mice showing a high value of The most notable macroscopic change was the exist- WBC and a low value of RBC, although the other param- ence of markedly shrunken lymphoid tissues includ- eters were generally not much different from ICR mice ing the thymus, lymph nodes and spleen. In particular, (Table 2-1~2-4). Peyer’s patches in the small intestine were hardly detected macroscopically. On the other hand, splenic enlargement Blood chemistry was observed in some mice of both sexes at 52 weeks of High values of AST, LDH and CPK activities were age and in one leukemia-bearing female. noted in both sexes at 7 weeks of age, high values of AST, In one female, which had malignant lymphoma and LDH and CPK activities in females at 26 weeks of age, was euthanized at 20 weeks of age, an intrathoracic nod- and high values of AST, ALT, LDH and CPK activities in ule probably in the thymic tissue and enlarged spleen females at 52 weeks of age in comparison with ICR mice. were found. ALP activity in both sexes at 7 weeks of age was higher Moreover, discolored and/or elevated foci in the spleen, than those at 26 and 52 weeks of age. Such changes were lungs, jejunum, liver, testes, stomach and spherical bone, similar to ICR mice (Table 3-1, 3-2). and uterine polyps were found in a small number of mice in either or both sexes at 26 and 52 weeks of age (Table 5). Organ weight Corresponding to the macroscopic changes, the spleen Histology weight was obviously low at all points examined. It was Among various histological changes shown in noteworthy that splenic weight was increased in some Table 6 and 7, the most notable changes were observed in mice at 52 weeks-old, while the weight of other organ the hemolymphoreticular, muscular and nervous systems. were roughly similar to ICR mice. In addition, the testis weight was low in some males at 52 weeks of age

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Table 2-1. Hematological data in NOG mice. - Male - Item RBC HGB HCT MCV MCH MCHC Retic PLT WBC Age (No. of mice) Unit x104/μL g/dL % fL pg g/dL % x104/μL x102/μL Mean 858 13.7 46 53.5 16 29.9 2.1 131.5 8.9 7 weeks (19) S.D. 31 0.5 1.4 0.5 0.1 0.3 0.3 7.4 1.9 Mean 835 12.6 42.9 51.3 15.1 29.5 2.4 143.8 8.7 26 weeks (20) S.D. 24 0.5 1.6 1.0 0.3 0.4 0.2 11.3 2.7 Mean 739 11.2 38.2 52.5 15.3 29.2 3.4 155.1 27.8 52 weeks (19) S.D. 130 1.6 4.6 4.8 1.0 0.7 1.3 27.7 41

- Female - Item RBC HGB HCT MCV MCH MCHC Retic PLT WBC Age (No. of mice) Unit x104/μL g/dL % fL pg g/dL % x104/μL x102/μL Mean 886 14.2 47.1 53.2 16.0 30.2 2.3 114.3 14.2 7 weeks (20) S.D. 16 0.2 1.0 0.5 0.2 0.4 0.5 15 6.9 Mean 888 13.8 45.6 51.3 15.5 30.1 2.9 125.2 15.9 26 weeks (19) S.D. 26 0.4 1.5 0.8 0.2 0.4 0.4 12.8 6.0 Mean 812 12.3 40.8 50.9 15.3 30.2 3.4 130.0 27.9 52 weeks (18) S.D. 125 1.5 4.4 4.9 1.0 0.9 1.5 20.6 38.1 RBC: Red blood cell HGB: Hemoglobin HCT: Hematocrit Retic: Reticulocyte PLT: Platelet WBC: White blood cell

Table 2-2. Count and ratio of each WBC in hematological data of NOG mice. - Male - Age Item LYMP NEUT EOS BASO MONO LUC (No. of mice) Unit x102/μL % x102/μL % x102/μL % x102/μL % x102/μL % x102/μL % Mean 1.1 13.3 5.6 62.5 0.6 6.3 0.0 0.6 1.5 17.3 0.0 0.1 7 weeks (19) S.D. 0.3 3.2 1.6 7.1 0.2 1.6 0.1 0.4 0.6 5.8 0.0 0.3 Mean 1.1 12.7 6.3 70.9 0.7 8.9 0.0 0.2 0.6 7.1 0.0 0.2 26weeks (20) S.D. 0.3 3.4 2.3 5.4 0.2 2.9 0.0 0.2 0.3 2.0 0.0 0.2 Mean 5.2 14.3 19.4 71.4 1.0 4.7 0.0 0.1 2.0 9.2 0.1 0.2 52weeks (19) S.D. 10.3 5.5 27.3 5.6 1.1 2.4 0.1 0.1 2.2 3.2 0.5 0.3 - Female - Age Item LYMP NEUT EOS BASO MONO LUC (No. of mice) Unit x102/μL % x102/μL % x102/μL % x102/μL % x102/μL % x102/μL % Mean 1.5 11.4 9.5 64.5 1.0 6.9 0.0 0.5 2.2 16.6 0.0 0.1 7 weeks (20) S.D. 0.4 3.7 6.2 8.7 0.5 1.4 0.1 0.4 0.7 5.9 0.0 0.1 Mean 1.4 9.7 11.9 73.0 0.9 5.4 0.0 0.2 1.7 11.2 0.0 0.4 26weeks (19) S.D. 0.4 2.7 5.5 5.2 0.5 1.6 0.0 0.2 0.5 4.2 0.1 0.3 Mean 3.3 9.3 21.4 76.9 1.3 5.7 0.0 0.2 1.7 7.8 0.1 0.2 52weeks (18) S.D. 6.4 5.0 28.8 4.8 1.1 2.5 0.1 0.2 1.6 3.7 0.4 0.3 LYMP: Lymphocyte NEUT: Neutrophil EOS: Eosinophil BASO: Basophil MONO: Monocyte LUC : Large unstained cell

Hemolymphoreticular system number of lymphoid cells remained in the spleen and The most typical change was systemic lymphoid tis- lymph nodes, and they were immunohistochemically neg- sue hypoplasia characterized by severe depletion of lym- ative for CD3 and PAX5. phocytes with poorly developed tissue structures (Fig. 2a, Malignant lymphoma and leukemia occurred in one 2b and 2c). Especially, lymphoid tissues which were dis- female each at 20 and 52 weeks of age, respectively. tributed through the intestinal, bronchopulmonary and Immunohistochemically, the tumor cells of malignant nasopharyngeal mucosa could not be detected. A small lymphoma were positive for CD3 while those of leuke-

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Table 2-3. Hematological data in ICR mice. - Male - Item RBC HGB HCT MCV MCH MCHC Retic PLT WBC Age (No. of mice) Unit x104/μL g/dL % fL pg g/dL % x104/μL x102/μL Mean 926 14.4 46.6 50.3 15.5 30.8 3.3 108.1 36.3 7 weeks (5) S.D. 18 0.5 2.0 2.3 0.5 0.6 0.2 10.2 13.8 Mean 875 13.0 42.7 48.9 14.9 30.4 3.7 143.1 47.0 26 weeks (10) S.D. 59 0.6 2.0 1.5 0.5 0.5 1.3 24.5 24.5 Mean 877 13.1 41.9 47.8 14.9 31.2 2.7 139.1 42.5 52 weeks (8) S.D. 76 1.0 3.1 1.5 0.5 0.4 0.3 12.0 20.3 - Female - Item RBC HGB HCT MCV MCH MCHC Retic PLT WBC Age (No. of mice) Unit x104/μL g/dL % fL pg g/dL % x104/μL x102/μL Mean 914 14.1 46.1 50.4 15.4 30.7 4.1 90.2 34.6 7 weeks (5) S.D. 33 0.7 1.7 0.9 0.4 0.5 0.6 16.3 14.8 Mean 896 13.3 43.0 48.0 15.0 31.2 3.7 113.6 57.7 26 weeks (10) S.D. 46 0.6 1.9 1.6 0.7 0.7 1.2 11.5 18.4 Mean 900 13.7 43.2 48.0 15.3 31.9 3.1 119.0 22.0 52 weeks (15) S.D. 67 0.9 2.9 1.5 0.7 0.8 0.7 17.0 9.0 RBC: Red blood cell HGB: Hemoglobin HCT: Hematocrit Retic: Reticulocyte PLT: Platelet WBC: White blood cell

Table 2-4. Count and ratio of each WBC in hematological data of ICR mice. - Male - Age Item LYMP NEUT EOS BASO MONO LUC (No. of mice) Unit x102/μL % x102/μL % x102/μL % x102/μL % x102/μL % x102/μL% Mean 30.1 81.8 4.6 13.5 1.0 3.1 0.0 0.2 0.4 1.1 0.1 0.3 7 weeks (5) S.D. 12.4 6.7 1.8 5.2 0.3 1.5 0.1 0.2 0.2 0.2 0.1 0.2 Mean 33.4 71.4 10.4 21.3 1.4 3.2 0.1 0.2 1.3 3.0 0.5 0.8 26weeks (10) S.D. 18.5 7.5 6.6 6.4 0.9 1.1 0.1 0.1 0.6 1.0 0.3 0.4 Mean 19.4 48.5 19.4 43.3 2.1 4.4 0.2 0.4 1.3 2.8 0.2 0.3 52weeks (8) S.D. 11.6 20.8 15.6 19.9 2.9 5.0 0.2 0.4 0.8 0.9 0.1 0.3

- Female - Age Item LYMP NEUT EOS BASO MONO LUC (No. of mice) Unit x102/μL % x102/μL % x102/μL % x102/μL % x102/μL % x102/μL% Mean 21.9 60.9 10.4 32.6 1.6 4.9 0.0 0.1 0.4 1.2 0.1 0.3 7 weeks (5) S.D. 11.2 9.8 3.5 10.0 0.6 1.0 0.0 0.1 0.3 0.4 0.2 0.3 Mean 40.0 69.0 13.1 23.1 2.0 3.7 0.2 0.3 2.1 3.3 0.4 0.7 26weeks (10) S.D. 13.7 8.6 4.7 6.5 0.6 1.5 0.1 0.1 1.7 1.7 0.4 0.7 Mean 12.0 54.1 8.3 37.6 0.9 4.0 0.1 0.4 0.7 3.1 0.1 0.2 52weeks (15) S.D. 6.2 15.0 5.7 15.4 0.5 1.7 0.1 0.5 0.4 1.6 0.1 0.4 LYMP: Lymphocyte NEUT: Neutrophil EOS: Eosinophil BASO: Basophil MONO: Monocyte LUC : Large unstained cell mia were negative for CD3, PAX5, MPO and Iba1. Pro- observed in the femoral muscles of males at 26 weeks liferating cells in the focus of atypical lymphoid hyper- of age and the severity increased at 52 weeks of age plasia which was detected in the thymus of one female (Fig. 2d). Contrary, in females, such change was nev- at 7 weeks of age were positive for CD3 but negative for er detected until 26 weeks of age, but was noted at 50 PAX5. weeks of age. Electron microscopic examination revealed that these materials consisted of dilated and accumulat- Musculoskeletal system ed sarcoplasmic reticula arranging in an orderly manner Intracytoplasmic eosinophilic materials were first (Fig. 2e). In addition, degenerative and/or regenera-

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Table 3-1. Blood Chemical Data in NOG mice. - Male - Item AST ALT LDH CPK ALP γ-GTP T-CHO TG PL T-BIL GLU Age (No. of mice) Unit IU/L IU/L IU/L IU/L IU/L IU/L mg/dL mg/dL mg/dL mg/dL mg/dL Mean 98 38 243 180 355 0 76 77 160 0.1 246 7 weeks (20) S.D. 30 14 111 103 39 0 8 26 17 0.1 42 Mean 38 17 86 31 131 0 88 112 185 0.1 209 26 weeks (20) S.D. 9 3 15 12 17 0 9 42 17 0.0 35 Mean 69 28 208 131 116 0 73 50 158 0.1 212 52 weeks (19) S.D.105 4052130 1026220.124

Item BUN CRNN Na K Cl Ca P TP ALB A/G Age (No. of mice) Unit mg/dL mg/dL mmol/L mmol/L mmol/L mg/dL mg/dL g/dL g/dL Mean 27 0.14 152 4.4 116 8.2 6.7 4.4 3.1 2.5 7 weeks (20) S.D. 2 0.03 1 0.4 2 0.2 0.8 0.2 0.2 0.2 Mean 26 0.18 155 4.3 118 8.1 4.7 4.6 3.3 2.5 26 weeks (20) S.D. 2 0.04 2 0.4 2 0.2 0.6 0.2 0.2 0.2 Mean 25 0.09 152 4.5 117 7.9 4.9 4.5 3.2 2.5 52 weeks (19) S.D. 2 0.02 2 0.4 1 0.3 0.8 0.3 0.2 0.2

- Female - Item AST ALT LDH CPK ALP γ-GTP T-CHO TG PL T-BIL GLU Age (No. of mice) Unit IU/L IU/L IU/L IU/L IU/L IU/L mg/dL mg/dL mg/dL mg/dL mg/dL Mean 109 38 223 139 453 0 63 60 130 0.1 220 7 weeks (20) S.D. 37 10 82 55 42 0 5 25 9 0.0 32 Mean 145 35 582 1778 219 0 55 51 117 0.1 207 26 weeks (19) S.D. 62 7 423 2715 21 0 6 22 12 0.0 23 Mean 149 53 458 427 179 0 49 53 110 0.1 188 52 weeks (18) S.D. 80 77 179 334 30 0 6 25 12 0.1 24

Item BUN CRNN Na K Cl Ca P TP ALB A/G Age (No. of mice) Unit mg/dL mg/dL mmol/L mmol/L mmol/L mg/dL mg/dL g/dL g/dL Mean 25 0.15 152 3.8 118 8.0 6.5 4.3 3.3 3.2 7 weeks (20) S.D. 4 0.03 2 0.3 1 0.1 0.7 0.2 0.2 0.2 Mean 23 0.20 153 3.9 121 7.8 6.4 4.2 3.4 4.0 26 weeks (19) S.D. 2 0.06 2 0.3 2 0.3 1.1 0.2 0.2 0.3 Mean 19 0.11 150 4.3 118 7.6 5.0 4.3 3.4 3.7 52 weeks (18) S.D. 2 0.02 2 0.3 2 0.2 0.4 0.2 0.1 0.5 AST: aspartate aminotransferase ALT: alanine aminotransferase LDH: Lactate dehydrogenase CPK: Creatine phosphokinase ALP: Alkaline Phosphatase γ-GTP: γ-glutamyl transpeptidase T-CHO: Total cholesterol TG: Triglyceride PL: Phospholipid T-BIL: Total bilirubin GLU: Glucose BUN: Blood urea nitrogen CRNN: creatinine Na: Sodium K: Potassium Cl: Chloride Ca: Calcium P: Phosphorus TP: Total protein ALB: Albumin A/G: Aalbumin-globulin ratio tive muscle cells were sporadically seen in both sexes reactive astrocytes and/or microgliosis (Fig. 2f and 2g). at all time points examined, and the incidence in males Such spongy change was ﬁrst observed at 7 weeks of age. increased at 52 weeks of age. Thereafter, except for reactive changes, vacuolation of neuropil gradually became weakened and was almost no Nervous system longer detected in both sexes at 52 weeks of age. Spongy change appeared sporadically in the medulla oblongata and spinal cord. The lesion mainly consist- Other systems ed of vacuolation of neuropil in the white and gray mat- The histological changes other than the above-men- ters, and it was frequently accompanied by hypertrophic tioned ones were as follows. Namely, osseous metapla-

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Table 3-2. Blood Chemical Data in ICR mice. - Male - Item AST ALT LDH CPK ALP γ-GTP T-CHO TG PL T-BIL GLU Age (No. of mice) Unit IU/L IU/L IU/L IU/L IU/L IU/L mg/dL mg/dL mg/dL mg/dL mg/dL Mean 35 24 150 54 313 0 121 77 226 0.2 239 7 weeks (5) S.D. 1 5 27 10 58 0 10 24 22 0.1 18 Mean 44 27 183 30 142 1 136 79 251 0.0 203 26 weeks (10) S.D. 11 11 55 15 76 1 26 25 33 0.1 16 Mean 52 31 178 94 117 0 NE NE NE 0.1 NE 52 weeks (8) S.D. 15 11 94 137 27 0 0.0

Item BUN CRNN Na K Cl Ca P TP ALB A/G Age (No. of mice) Unit mg/dL mg/dL mmol/L mmol/L mmol/L mg/dL mg/dL g/dL g/dL Mean 15 0.11 151 3.9 116 9.5 6.9 4.7 3.2 2.2 7 weeks (5) S.D. 4 0.01 0 0.2 2 0.3 0.5 0.2 0.2 0.1 Mean 28 0.10 147 4.2 114 8.7 4.2 5.1 3.2 1.7 26 weeks (10) S.D. 5 0.01 6 0.4 5 0.4 0.6 0.3 0.2 0.3 Mean 28 0.09 152 4.7 117 NE NE NE 3.1 NE 52 weeks (8) S.D. 7 0.02 1 0.3 2 0.3

- Female - Item AST ALT LDH CPK ALP γ-GTP T-CHO TG PL T-BIL GLU Age (No. of mice) Unit IU/L IU/L IU/L IU/L IU/L IU/L mg/dL mg/dL mg/dL mg/dL mg/dL Mean 52 21 160 73 373 0 109 50 198 0.1 213 7 weeks (5) S.D. 13 8 47 16 98 0 17 21 23 0.0 28 Mean 52 30 134 25 233 0 111 48 189 0.0 191 26 weeks (10) S.D. 21 31 74 5 79 0 19 9 27 0.1 16 Mean 56 23 143 70 211 0 NE NE NE 0.1 NE 52 weeks (15) S.D. 9 4 35 62 85 0 0.0

Item BUN CRNN Na K Cl Ca P TP ALB A/G Age (No. of mice) Unit mg/dL mg/dL mmol/L mmol/L mmol/L mg/dL mg/dL g/dL g/dL Mean 21 0.12 152 3.8 118 9.6 7.3 4.9 3.5 2.5 7 weeks (5) S.D. 3 0.02 2 0.4 2 0.2 1.1 0.2 0.2 0.2 Mean 18 0.13 147 4.1 116 9.3 4.6 5.2 3.5 2.1 26 weeks (10) S.D. 3 0.02 2 0.3 2 0.3 0.6 0.3 0.1 0.3 Mean 24 0.09 152 4.0 119 NE NE NE 3.4 NE 52 weeks (15) S.D. 4 0.02 1 0.4 2 0.2 AST: aspartate aminotransferase ALT: alanine aminotransferase LDH: Lactate dehydrogenase CPK: Creatine phosphokinase ALP: Alkaline Phosphatase γ-GTP: γ-glutamyl transpeptidase T-CHO: Total cholesterol TG: Triglyceride PL: Phospholipid T-BIL: Total bilirubin GLU: Glucose BUN: Blood urea nitrogen CRNN: creatinine Na: Sodium K: Potassium Cl: Chloride Ca: Calcium P: Phosphorus TP: Total protein ALB: Albumin A/G: Aalbumin-globulin ratio NE: Not examined sia was observed in the spleen in both sexes. It was ﬁrst degeneration and/or atrophy of seminiferous tubules in observed at 26 weeks of age, and the incidence increased the testes were observed in some males at 7 weeks of age 52 weeks of age. Osseous metaplasia was sometimes and in almost all males at 26 and 52 weeks of age. More- identiﬁed as a white focus macroscopically. Ectopic exo- over, chondromucinous degeneration was observed in the crine gland tissues were observed in the thymus in both bone in almost all mice of both sexes, and the severity sexes at all points examined, and the incidence increased tended to increase at 52 weeks of age. at 52 weeks of age. Weak basophilic tubules and hyaline casts in the kidneys increased with age in both sexes and

Vol. 42 No. 6 Table 4-1. Absolute and relative organ weight in NOG mice. - Male - Organ F.B.W. * Brain ** Heart Lung Liver Spleen Kidney Testis Age (No. of mice) Unit g mg mg/100g mg mg/100g mg mg/100g g g/100g mg mg/100g mg mg/100g mg mg/100g Mean 20.6 453 2208 103 503 125 610 1.06 5.16 13 64 364 1772 126 616 7 weeks (20) S.D. 1.5 13 144 7 22 9 36 0.13 0.32 2 9 29 83 13 59 Mean 26.9 476 1775 128 478 154 572 1.52 5.64 19 71 447 1664 173 644 26 weeks (20) S.D. 1.7 15 97 11 35 7 24 0.14 0.35 3 9 34 101 34 125 Mean 27.2 477 1754 143 526 161 593 1.4 5.13 43 158 453 1664 133 488 52 weeks (19) S.D. 1.5 26 103 18 55 17 62 0.12 0.36 57 207 40 118 30 112

- Female - Organ F.B.W. * Brain Heart Lung Liver Spleen Kidney Age (No. of mice) Unit g mg mg/100g mg mg/100g mg mg/100g g g/100g mg mg/100g mg mg/100g Mean 17 449 2642 84 493 115 675 0.76 4.48 13 73 222 1304 7 weeks (20) S.D. 0.8 8 125 5 26 7 41 0.05 0.2 4 20 12 60 Mean 22.1 498 2262 108 488 144 651 1.04 4.73 23 104 268 1213 26 weeks (19) S.D. 1.2 20 101 11 39 10 28 0.09 0.24 3 10 19 53 Mean 24.2 508 2101 115 476 156 645 1.11 4.58 48 196 279 1152 Background dataonNOGmice 52 weeks (18) S.D. 1.6 18 110 13 48 12 44 0.13 0.30 72 298 20 47 *: Final body weight **: Left or right data indicates the absolute or relative organ weight, respectively.

Table 4-2. Absolute and relative organ weight in ICR mice. - Male - Organ F.B.W. * Brain ** Heart Lung Liver Spleen Kidney Testis Age (No. of mice) Unit g mg mg/100g mg mg/100g mg mg/100g g g/100g mg mg/100g mg mg/100g mg mg/100g Mean 30.9 488 1579 143 464 184 595 1.60 5.17 79 257 470 1520 215 695 7 weeks (5) S.D. 0.6 6 40 7 21 17 59 0.11 0.33 10 33 49 151 11 35 Mean 43.5 518 1197 198 455 207 477 2.12 4.86 95 217 654 1500 247 573 26 weeks (10) S.D. 3.5 16 93 23 31 16 22 0.26 0.41 20 40 104 170 35 103 Mean 47.6 NE 222 469 221 464 2.31 4.86 93 197 760 1600 NE 52 weeks (8) S.D. 3.5 23 65 18 27 0.19 0.34 25 53 80 186

- Female - Organ F.B.W. * Brain Heart Lung Liver Spleen Kidney Age (No. of mice) Unit g mg mg/100g mg mg/100g mg mg/100g g g/100g mg mg/100g mg mg/100g Mean 23.9 471 1976 122 510 161 675 1.17 4.91 93 389 296 1239 7 weeks (5) S.D. 1.8 11 130 15 60 13 44 0.14 0.49 13 57 21 76 Mean 35.2 513 1465 158 450 213 607 1.65 4.67 140 395 404 1153 26 weeks (10) S.D. 2.3 23 97 13 41 13 27 0.34 0.76 53 124 46 142

Vol. 42No.6 Mean 36.4 NE 147 403 199 548 1.75 4.80 126 352 438 1203 52 weeks (15) S.D. 2.9 14 33 17 37 0.25 0.48 57 193 63 135 *: Final body weight 697 **: Left or right data indicates the absolute or relative organ weight, respectively. NE: Not examined 698

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Table 5. Macroscopic Findings in NOG mice. Sex: Male Female Tissue Age (weeks): 7 26 52 7 26 52 Observation No. of mice: 20 20 20 (1) 20 20 (1) 20 (1) General descriptions -Undernourishment 0 0 0 0 (1) (1) -Discolored skin, pale 0 0 0 0 0 1 -Discoloration, pale, all tissues 0 0 0 0 0 1 -Excess ﬂuid, abdominal 0 0 0 0 0 1 -Focus, dark red, subcutaneous 0 0 0 0 0 1 Thoracic cavity -Excess ﬂuid 0 0 0 0 (1) 0 -Nodule 0 0 0 0 (1) 0 -Thickening, parietal pleura 0 0 0 0 (1) 0 Thymus -Rudimentary 20 20 20 (1) 20 20 (1) 20 (1) Spleen -Large 0 0 2 0 (1) 3 -Small 20 20 18 (1) 20 19 17 (1) -Focus, white 0 1 1 0 0 7 Lymph node, submandibular -Rudimentary 20 20 20 (1) 20 20 (1) 20 (1) Lymph node, mesenteric -Rudimentary 20 20 20 (1) 20 20 (1) 20 (1) Peyer’s patch -Unobservable 20 20 20 (1) 20 20 (1) 20 (1) Lung (bronchus) -Focus, white 0 0 1 0 0 0 -Discoloration, red, lobar 0 0 0 0 (1) 0 Stomach -Focus, raised, glandular stomach 0 0 0 0 0 1 -Dilatation, lumina 0 0 (1) 0 0 0 Intestine, duodenum -Dilatation, lumina 0 0 (1) 0 0 0 Intestine, jejunum -Focus, white 0 0 1 0 0 0 -Dilatation, lumina 0 0 (1) 0 0 0 Intestine, ileum -Diverticulum 1 0 0 0 0 0 -Dilatation, lumina 0 0 (1) 0 0 0 Liver -Discoloration, dark red 0 0 0 0 0 1 -Focus, white 0 0 1 0 0 0 Pancreas -Cyst 0 0 0 0 0 1 Kidney -Cyst 0 0 1 0 0 0 Testis -Small 0 1 0 - - - -Focus, white 0 1 5 (1) - - - Ovary -Large - - - 0 (1) 0 Uterus -Polyp - - - 0 0 2

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Table 5. (Continued). Sex: Male Female Tissue Age (weeks): 7 26 52 7 26 52 Observation No. of mice: 20 20 20 (1) 20 20 (1) 20 (1) -Cyst, endometrial - - - 0 0 1 Clitoral gland -Large - - - 0 1 0 Bone, cranial -Nodule 0 0 0 0 0 1 Tail -Bending 0 0 0 0 0 1 Vertebrae -Stooping 0 0 8 0 0 0 The parenthesis shows the number of dead/moribund sacriﬁced animals.

Table 6. Non-proliferative histological ﬁndings in NOG mice. Sex: Male Female Tissue Age (weeks): 7 26 52 7 26 52 Observation No. of mice: 20 20 20 (1) 20 20 (1) 20 (1) Medulla oblongata -Spongiotic change minimal 7 14 0 5 12 0 mild 1 1 0 0 2 0 -Microgliosis minimal 0 0 0 0 0 1 -Astrocytosis minimal 0 0 0 0 0 1 Spinal cord, cervical -Spongiotic change minimal 5 13 0 4 12 0 mild 0 1 0 0 0 0 -Astrocytosis minimal 0 0 2 0 0 1 Spinal cord, thoracic -Spongiotic change minimal 8 11 0 3 10 0 -Astrocytosis minimal 0 0 1 0 0 1 Spinal cord, lumbar -Spongiotic change minimal 7 12 0 7 8 0 mild 3 4 0 1 0 0 -Astrocytosis minimal 0 0 0 0 0 1 Eye -Degeneration, lens minimal 0 0 2 0 0 0 Bone marrow -Granulopoiesis, increased mild 0 0 1 0 0 1 moderate 0 0 10 (1) 0 0 4 Thymus -Hypocellularity, lymphoid, cortex / medulla severe 16 19 18 (1) 15 19 18 (1) -Hypoplasia, cortex / medulla severe 16 19 18 (1) 15 19 18 (1) -Debris, apoptotic minimal 0 0 0 0 0 1 -Ectopic exocrine gland minimal 3 4 8 (1) 2 1 8 -Ectopic thyroid minimal 0 0 0 1 0 0 -Dermoid cyst present 0 0 0 1 0 0 -Cyst minimal 5 8 1 4 6 3 mild 0 3 16 (1) 0 0 13 (1) Spleen -Hypocellularity, lymphoid, white pulp mild 0 8 2 1 8 3 moderate 17 8 14 (1) 16 10 11

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Table 6. (Continued). Sex: Male Female Tissue Age (weeks): 7 26 52 7 26 52 Observation No. of mice: 20 20 20 (1) 20 20 (1) 20 (1) severe 3 4 4 3 1 5 (1) -Hypoplasia, follicle / marginal zone / PALSa) moderate 17 16 16 (1) 17 18 14 severe 3 4 4 3 1 5 (1) -Metaplasia, osseous mild 0 3 5 0 3 9 -Hematopoiesis, extramedullary minimal 16 5 9 (1) 15 3 7 mild 4 15 8 5 16 7 moderate003002 Lymph node, submandibular -Hypocellularity, lymphoid severe 13 10 16 (1) 20 10 15 -Hypoplasia, cortex / medulla severe 13 10 16 (1) 20 10 15 -Debris, apoptotic minimal 0 2 2 0 0 0 Lymph node, mesenteric -Hypocellularity, lymphoid moderate 6 11 14 7 16 14 severe 8 8 5 53 6 (1) -Hypoplasia, cortex / medulla severe 14 19 19 12 19 20 (1) -Debris, apoptotic minimal 8 9 10 6 10 6 Other lymphoid tissuesb) -Hypoplasia, lymphoid tissue severe 20 20 20 (1) 20 20 (1) 20 (1) Lung (bronchus) -Congestion minimal 0 0 0 0 (1) 0 -Cell inﬁltration, neutrophilic minimal 0 0 0 0 0 (1) Stomach -Erosion, glandular stomach minimal 0 0 0 1 1 0 Liver -Necrosis, focal mild 0 0 0 0 0 1 -Necrosis, single cell, hepatocytic minimal 0 0 0 0 0 (1) -Pigmentation, hepatocytic minimal 0 0 0 0 0 (1) Kidney -Basophilia, tubular minimal 8 14 14 2 8 11 (1) mild 0 0 1 10 0 -Urinary cast, hyaline minimal 0 8 18 (1) 1 10 (1) 14 mild 0 0 1 00 0 -Cystic dilatation, tubular minimal 0 3 1 2 1 0 moderate001 000 Testis -Immature minimal 6 0 0 - - - -Degeneration, seminiferous tubular minimal 7 16 12 - - - mild 0 2 4 (1) -- - moderate 0 0 3 -- - severe 0 0 1 -- - -Atrophy, seminiferous tubular severe 0 1 0 - - - -Spermatocele minimal 1 3 1 - - - Epididymis -Cell debris, luminal minimal 20 0 0 - - - -Reduced sperm, luminal mild 0 2 0 --- moderate 0 0 1 -- - severe 0 1 4 (1) -- - -Sperm granuloma mild 0 1 0 - - - Prostate -Fibrosis minimal 0 0 4 - - -

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Table 6. (Continued). Sex: Male Female Tissue Age (weeks): 7 26 52 7 26 52 Observation No. of mice: 20 20 20 (1) 20 20 (1) 20 (1) Genital organ, female -Immature minimal - - - 12 0 0 Uterus -Adenomyosis mild - - - 0 0 1 Vagina -Increased muciﬁcation minimal - - - 0 3 5 Clitoral gland -Dilatation, ductal mild - - - 0 1 0 Sternum -Degeneration, chondromucinous minimal 18 20 16 (1) 19 19 12 mild 0 0 4 0 0 8 (1) Femur (knee joint) -Degeneration, chondromucinous minimal 3 6 19 2 9 20 (1) Skeletal muscle, femoral -Eosinophilic material, muscular minimal 0 20 6 (1) 0 0 20 (1) mild 0 0 14 0 0 0 -Degeneration, muscular minimal 0 4 14 (1) 0 1 2 -Regeneration, muscular minimal 1 2 14 (1) 0 1 1 Tail -Fracture mild 0 0 0 0 0 1 Numbers in parentheses are the number of dead/moribund sacriﬁced animals. a) Periarterial lymphatic sheath b) Lymphoid tissues in the intestinal, bronchopulmonary and nasopharyngeal mucosa

Proliferative histopathological findings in NOG mice (Kato et al., 2009) and wild-type mice In addition to the above-mentioned malignant lympho- (Pearse, 2006a), and was the only lethal tumor detect- ma, leukemia and atypical lymphoid hyperplasia, focal ed in the present study. Atypical lymphoid hyperplasia, mucosal hyperplasia and adenoma in the small intestine, which is generally said to have a possibility to progress to hepatocellular adenoma and clear cell foci in the liver, malignant lymphoma (Pearse, 2006a), was also detected focal cortical cell hyperplasia in the adrenal glands, ade- only in one female killed as scheduled at 7 weeks of age. noma in the Harderian glands, focal hyperostosis in the The present study showed that NOG mice could live at cranial bone, lipomatous hamartoma in the brain and least up to 52 weeks of age with the survival rate of 95% endometrial stromal polyp in the uterus were observed in in both sexes when they were kept under clean conditions mice of either or both sexes, but their incidences were low set up for immune-deficient animals. except for that of uterine polyp which occurred in approx- In the clinical observation, circling behavior in one imately one-third of females at 52 weeks of age. direction along the cage wall was noticed in males and females after 8 weeks and 47 weeks of age, respectively. DISCUSSION These mice showed no histological changes suggesting a certain relationship with such abnormal behavior, and, Twenty male and 20 female NOG mice were exam- to our knowledge, there were no reports of such abnor- ined at 7, 26 and 52 weeks of age, respectively. Dur- mal behavior in laboratory animals. In addition, hunch- ing the observation period, 1 male and 2 females died or back position was also observed in males after 32 weeks were euthanized due to deterioration. One female died of of age, but its cause was also unclear. malignant lymphoma which probably developed in the Hematologically, the lymphocyte count in NOG mice thymus. However, the cause of death of the remaining 2 was extremely lower than that in ICR mice and oth- mice was unclear. Thymic lymphoma is known to occur er wild-type mice (Serfilippi et al., 2003). The extreme-

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Table 7. Proliferative histopathological findings in NOG mice. Sex: Male Female Tissue Group: 7w 26w 52w 7w 26w 52w Observation Number: 20 20 20 (1) 20 20 (1) 20 (1) Cerebrum -Hamartoma, lipomatous 0 0 0 0 1 (0) 0 Harderian gland -Adenoma 0 0 0 0 0 1 (0) Adrenal -Hyperplasia, cortical, focal 0 0 0 0 1 (0) 1 (0) -Hyperplasia, subcapsular cell 1 8 8 (0) 1 16 (0) 19 (1) Thymus -Hyperplasia, atypical, lymphoid 0 0 0 (0) 1 0 (0) 0 (0) Hemolymphoreticular (all sites) -Lymphoma, malignant 0 0 0 0 1 (1) 0 -Leukemia 0 0 0 0 0 1 (0) Intestine, duodenum -Hyperplasia, mucosal, focal 0 1 1 (0) 0 0 2 (0) Intestine, jejunum -Adenoma 0 0 1 (0) 0 0 0 -Hyperplasia, mucosal, focal 0 0 2 (0) 0 0 1 (0) Liver -Adenoma, hepatocellular 0 0 3 (0) 0 0 0 -Altered cell focus, clear 0 0 1 (0) 0 0 0 Urinary bladder -Hyperplasia, urothelium, diffuse 0 0 1 (0) 0 0 0 Uterus -Polyp, endometrial stromal - - - 0 0 6 (0) -Hyperplasia, glandular, cystic - - - 0 0 10 (1) Bone, cranial -Hyperostosis 0 0 0 0 0 1 Numbers in parentheses are the number of dead/moribund sacrificed animals. ly low lymphocyte count is one of major characters in Histologically, notable changes were observed in the immune-deficient animals. The numerical value of lym- lymphatic tissues, medulla oblongata, spinal cord and phocyte in NOG mice is lower than NOD SCID, highly femoral muscles. The hypoplasia of lymphatic tissues, immune-deficient mice, thus it is considered to indicate the most prominent change in the present study, was char- the severe immunodeficiency state of NOG mice (Charles acterized by severe depletion of lymphocytes with poor- River, 2012). On the other hand, the high values of lym- ly developed tissue structures resulting from loss of T phocyte and neutrophil counts were noted in some NOG and B lymphocytes as previously reported in NOG mice mice, suggesting an existence of inflammatory lesions. (Katano et al., 2011). In this connection, previous inves- However, there were no inflammatory changes detected tigators suggested that hypoplasia of lymphoid tissues in any tissues. might be brought about by abnormalities during the stage In blood chemistry, high values of AST, ALT, LDH, of tissue development (Mebius, 2003; Shores et al., 1991; CPK and ALP activities were noted in both sexes at all van Ewijk et al., 1994; Zuklys et al., 2000; Holländer time points examined. The high value of ALP activity et al., 1995; Rossi et al., 2007). In the present study, a might be related to bone growth, since it began to decline small number of lymphoid cells occasionally remained at 26 weeks of age when the growth curve became more in the hypoplastic lymphoid tissues, but such lymphoid gentle. As to the high values of AST, LDH, CPK and ALP cells were immunohistochemically negative for CD3 nor activities, they might be partially related to the muscular PAX5. Taken together with an appearance of apoptot- degenerative changes detected histologically. ic cell debris in the thymus and lymph nodes, it is rea-

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Fig. 2. Representative histology images of the lymphoid tissues (a, b, c), skeletal muscle (d, e) and spinal cord (f, g). (a) Thymus from male NOG mouse, 7 weeks old. Severe lymphoid depletion with disappearance of the cortico-medullary junction. H&E. (b) Spleen from male NOG mouse, 7 weeks old. Severe lymphoid depletion with loss of normal architectures of the white pulp and marginal zone. H&E. (c) Mesenteric lymph node from male mouse, 7 weeks old. Severe lymphoid depletion with loss of normal cortical and medullary architectures. H&E. (d) Femoral skeletal muscle from male NOG mice, 52 weeks of age. Intracytoplasmic eosinophilic materials in the muscular cells. H&E. (e) Ultrastructure of intracytoplasmic eosinophilic materials in the femoral skeletal muscle. Note tubular aggregates consisted of dilated and accumulated sarcoplasmic reticula with an orderly manner. (f) Spinal cord from male NOG mouse, 7 weeks of age. Spongiotic change characterized by vacuolation of the neuropil in the white and gray matters, with appearance of hypertrophic reactive astrocytes (arrow). H&E. (g) Same area of the photo f. Note appearance of hypertrophic reactive astrocytes (arrow). GFAP immuno- histochemistry.

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K. Kasahara et al. sonable to consider that the hypoplastic lymphoid tissues review of the manuscript. could hardly function, resulting in prevention of survival and/or maturation of lymphocytes. Conflict of interest---- The authors declare that there is In the muscular system of males, intracytoplasmic no conflict of interest. eosinophilic materials sporadically appeared in the muscle cells at 26 weeks of age and increased in incidence REFERENCES and severity at 52 weeks of age. At 52 weeks of age, they were also detected in females. Electron microscop- Agbulut, O., Destombes, J., Thiesson, D. and Butler-Browne, G. ic examination revealed that these inclusions consisted (2000): Age-related appearance of tubular aggregates in the skeletal muscle of almost all male inbred mice. Histochem. Cell of dilated and accumulated sarcoplasmic reticula arrang- Biol., 114, 477-481. ing in an orderly manner. In wild-type mice, they are Boncompagni, S., Protasi, F. and Franzini-Armstrong, C. (2012): known as tubular aggregates in muscle cells and report- Sequential stages in the age-dependent gradual formation and ed to be detected only in male mice (Agbulut et al., 2000; accumulation of tubular aggregates in fast twitch muscle fibers: Boncompagni et al., 2012). Therefore, it was interesting SERCA and calsequestrin involvement. Age (Dordr.), 34, 27-41. Charles River (2012): NOD SCID mouse hematology. North Amer- that in NOG mice, the change occurred not only in males ican colonies, January 2011 - December 2011. http://www.criv- but also in females. er.com/files/pdfs/rms/nod-scid/rm_rm_r_nod_scid_mouse_clini- In the nervous system, spongy change that had not been cal_pathology_data.aspx reported previously developed in neuropil in the medulla Creasy, D., Bube, A., de Rijk, E., Kandori, H. and Kuwahara, M., Masson, R., Nolte, T., Reams, R., Regan, K., Rehm, S., oblongata and spinal cord in both sexes at 7 weeks of age. 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In addition to the above-mentioned histological chang- Pathol., 27, 1S-107S. es, osseous metaplasia in the spleen and ectopic exocrine Hardisty, J.F. and Boorman, G.A (1999): The thyroid and parathy- tissues in the thymus were observed at high incidences. roid glands. In Pathology of the mouse (Maronpot, R.R., ed.), pp. 537-554, Cache River Press, Vienna. Judging from the histological nature and the mode Holländer, G.A., Wang, B., Nichogiannopoulou, A., Platenburg, of incidences, the other histologic changes than the P.P., van Ewijk, W., Burakoff, S.J., Gutierrez-Ramos, J.C. and above-mentioned ones were considered not to be specif- Terhorst, C. (1995): Developmental control point in induction of ic for NOG mice (Kaufmann et al., 2012; Pearse, 2006b; thymic cortex regulated by a subpopulation of prothymocytes. Khosla and Ovalle, 1986; Hardisty and Boorma., 1999: Nature, 373, 350-353. Ito, M., Hiramatsu, H., Kobayashi, K., Suzue, K., Kawahata, M., Leininger et al., 1999; Creasy et al., 2012; Dixon et al., Hioki, K., Ueyama, Y., Koyanagi, Y., Sugamura, K., Tsuji, K., null 2014; Long and Leininger, 1999). Heike, T. and Nakahata, T. (2002): NOD/SCID/γc mouse: an Such hyperplastic and neoplastic changes as malignant excellent recipient mouse model for engraftment of human cells. lymphoma, small intestinal adenoma, hepatocellular ade- Blood. 100, 3175-3182. Ito, R., Takahashi, T., Katano, I. and Ito, M. (2012): Current advanc- noma, leukemia, cerebral lipomatous hamartoma, Harde- es in humanized mouse models. Cell Mol. Immunol., 9, 208- rian gland adenoma and uterine polyp were also detected 214. in the present study on NOG mice, and their incidences Katano, I., Ito, R., Eto, T., Aiso, S. and Ito. M. 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