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To examine the biological and morphological features of tubular aggregates (TAs) in the skeletal muscles of non-obese diabetic/Cg-PrkdcscidIl2rgtm1Sug /ShiJic (NOG) mice, 73 male and 72 female specific-pathogen-free NOG mice were examined at 7, 18, 22, 26, and 52 weeks of age. TAs were observed as intracytoplasmic eosinophilic materials of the femoral muscles in males at 18, 22, 26, and 52 weeks of age and in females at 52 weeks of age; gender-related differences were noted in the onset time and lesion degree. Intracytoplasmic materials were positive for Gomori's trichrome stain. Electron microscopy revealed that TAs were composed of an accumulation of dilated sarcoplasmic reticulum. In addition, TAs were observed in the femoral and gastrocnemius muscles, but not in the soleus and diaphragm muscles, suggesting that TAs are present in fast muscle fibers. The morphology of TAs and the type of myofibers involved, as well as the gender difference in NOG mice were essentially the same as those of TAs observed in C57BL/6J and MRL+/+ mice.

Biological background data up to 11 weeks of age and tumorigenic susceptibility to xenotransplantation with HeLa cells were compared between severely immuno-deficient NOG and NSG mice. The body weight was lower in NOG mice than in NSG mice. Severe depletion of peripheral blood lymphocytes and lymphoid hypoplasia that are well-known characteristics of these mice were equally observed. No lymphoproliferative lesions developed in any mouse of either strain. The occurrence of ectopic exocrine gland and cyst was a common finding in the thymus of both strains. In addition, minimal spongiotic change was observed in the medulla oblongata and spinal cord in both strains, and its incidence in female NOG mice was a little higher than that in NSG mice. In the adrenal, subcapsular cell hyperplasia that is known as an age-related change in non-genetically modified mice developed earlier and its incidence was higher in NSG mice than in NOG mice. The development of female genital organs of NOG mice was slightly retarded in comparison with that of NSG mice. To evaluate tumorigenic susceptibility to xenotransplantation, female mice were implanted in the dorsal subcutis with 1×103 to 1×106 cells/head of HeLa cells, and were checked up to 16 weeks after implantation. As a result, there was no significant strain difference on tumor formation rate and tumor volume. In conclusion, the present study clearly demonstrated that NOG and NSG mice showed no distinct strain differences in either biological features or biological disadvantages.

To obtain background data of NOD/Shi-scid IL-2Rγnull (NOG) mice, severely immunedeficient 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.

To examine the biological and morphological features of tubular aggregates (TAs) in the skeletal muscles of non-obese diabetic/Cg-PrkdcscidIl2rgtm1Sug/ShiJic (NOG) mice, 73 male and 72 female specific-pathogen-free NOG mice were examined at 7, 18, 22, 26, and 52 weeks of age. TAs were observed as intracytoplasmic eosinophilic materials of the femoral muscles in males at 18, 22, 26, and 52 weeks of age and in females at 52 weeks of age; gender-related differences were noted in the onset time and lesion degree. Intracytoplasmic materials were positive for Gomori’s trichrome stain. Electron microscopy revealed that TAs were composed of an accumulation of dilated sarcoplasmic reticulum. In addition, TAs were observed in the femoral and gastrocnemius muscles, but not in the soleus and diaphragm muscles, suggesting that TAs are present in fast muscle fibers. The morphology of TAs and the type of myofibers involved, as well as the gender difference in NOG mice were essentially the same as those of TAs observed in C57BL/6J and MRL+/+ mice.

In rats, it is sometimes difficult to distinguish malignant reticuloses from astrocytomas in routine histopathological assessment. In the present study, four spontaneous brain neoplasms developing in the cerebrum of one Wistar Hannover rat and three Sprague-Dawley rats were immunohistochemically examined using microglia and macrophage markers. Histopathologically, these neoplasms were localized mainly in the cerebral cortex, hypothalamus or piriform lobe, and the portions showing solid growth did not show characteristic cellular arrangement but had an indistinct boundary with the surrounding brain parenchyma. Neoplastic cells had oval or pleomorphic small nuclei with abundant eosinophilic cytoplasm. Two cases showed neoplastic cell infiltration into the meninges and perivascular spaces. Silver staining showed lack of reticulin fiber production in the stroma of the neoplasms. Immunohistochemically, the neoplastic cells were strongly positive for Iba-1 and sporadically positive for CD68 in all four cases. On the basis of these results, all the neoplasms examined here could be distinguished from astrocytomas and diagnosed as malignant reticuloses. Thus, immunohistochemical demonstration of microglia/macrophage characters, such as using Iba-1, is considered to be helpful for differential diagnosis of malignant reticuloses from astrocytomas among spontaneously occurring primary brain neoplasms in rats.

In order to accurately assess the carcinogenicity of chemicals with regard to rare tumors such as rat CNS tumors, sufficient information about spontaneous tumors are very important. This paper presents the data on the type, incidence and detected age of CNS tumors in F344/DuCrlCrlj (a total of 1363 males and 1363 females) and Crl:CD(SD) rats (a total of 1650 males and 1705 females) collected from in-house background data-collection studies and control groups of carcinogenicity studies at our laboratory, together with those previously reported in F344 and SD rats. The present data on F344/DuCrlCrlj rats (F344 rats) and Crl:CD(SD) rats (SD rats) clarified the following. (1) The incidences of all CNS tumors observed in F344 rats were less than 1%. (2) The incidences of malignant astrocytoma and granular cell tumor were higher in male SD rats than in female SD rats. (3) The incidences of astrocytoma and granular cell tumor were higher in SD rats than in F344 rats. (4) Among astrocytoma, oligodendroglioma and granular cell tumor, oligodendroglioma was detected at the youngest age, followed by astrocytoma, and ultimately, granular cell tumor developed in both strains. The incidences observed in our study were almost consistent with those previously reported in F344 and SD rats.

It is generally said that neoplastic cells are immunohistochemically negative for glial fibrillary acidic protein (GFAP) in rat spontaneous astrocytomas, and there are no reports describing the existence of GFAP-positive neoplastic astrocytes in rat spontaneous oligodendrogliomas and mixed gliomas which contain neoplastic astrocytes. In the present study, to clarify whether GFAP-positive neoplastic astrocytes exist in rat spontaneous oligodendrogliomas and mixed gliomas or not, immunohistochemical examination was performed on spontaneous oligodendrogliomas (26 cases) and mixed gliomas (5 cases) collected from the carcinogenicity studies and short-term toxicity studies. The neoplastic cells that constitute oligodendrogliomas and mixed gliomas were morphologically classified into five types: round A, round B, round C, spindle, and bizarre. The cells of round A, B, and C types were thought to be neoplastic oligodendrocytes because of their positive immunostainability for Olig2. The origin of bizarre cells was obscure because they were negative for Olig2, GFAP, and nestin. The spindle cells were considered to be neoplastic astrocytes, because some of them were positive for GFAP or nestin, and GFAP-positive spindle cells could be morphologically distinguished from reactive astrocytes. In conclusion, the present study clarified for the first time that GFAP-positive neoplastic astrocytes exist in rat spontaneous gliomas.

We investigated a case of spontaneous malignant T-cell lymphoma observed in a 19-week-old male Crl:CD (SD) rat. The rat showed paralysis beginning 1 week before euthanasia. Hematological examination revealed marked lymphocytosis without distinct atypia. Macroscopically, hepatosplenomegaly and partial atrophy of the thoracic spinal cord were observed. Microscopically, neoplastic cells infiltrated into the liver, splenic red pulp, bone marrow and epidural space of the thoracic spinal cord, while no neoplastic cells were observed in the thymus and lymph nodes. Moreover, the spinal cord showed focal degeneration due to compression by marked infiltration of neoplastic cells in the subdural space. The neoplastic cells were generally small-sized round cells that had a round nucleus with/without a single nucleolus and scanty cytoplasm. Immunohistochemically, the neoplastic cells were positive for CD3 and CD8 and negative for CD79α. Judging from these results, the present tumor in this young adult rat was diagnosed as malignant T-cell lymphoma.

In the present survey, the historical data for spontaneous tumors observed in our laboratory was compared in relation to the time-related changes between recent (2000-2004) and past (1990-1999) samples from Fischer 344 rats used in carcinogenicity studies. In the recent samples, there were statistically increased incidences of islet cell adenomas in males and uterine adenocarcinomas in females. On the other hand, there were decreased incidences of pheochromocytomas, prostatic adenomas, pituitary anterior adenomas, large granular lymphocytic (LGL) leukemias and Leydig cell tumors in males and pituitary anterior adenomas in females. Furthermore, there were decreases in the body weights of both sexes at 58 weeks of age in the recent samples, and this may have been related to the decreases in the incidences of pheochromocytomas, Leydig cell tumors, prostatic adenomas and LGL leukemias. Moreover, the decreased incidence of pheochromocytoma in males revealed a positive correlation with a decreased percentage of severe chronic progressive nephropathy (CPN). On the other hand, there were no distinct factors responsible for increased incidences of tumors observed in the recent samples, suggesting a possible genetic drift. In conclusion, the incidences of spontaneous tumors obtained in our laboratory have been changed with time. Smaller body weights in both sexes and reduction in the number of male cases with severe CPN might be related to the reduced incidence of certain types of tumors in recent animals.

The cranial cruciate ligaments (CCL) of 13 dogs with clinical signs of CCL rupture and those of 22 clinically healthy young beagle dogs for laboratory use were examined histopathologically and immunohistopathologically. The most constant changes at an early stage of degenerating ligament tissue in affected dogs were nuclear enlargement and perinuclear halo formation of fibrocytes followed by chondroid metaplasia. These changes were also frequent in apparently healthy young beagles kept under laboratory conditions. PAS and alcian blue positive substance accumulated around activated fibrocytes and within perinuclear halos. S-100 protein was also positive in these cells preceding the morphological change of chondroid metaplasia. Increased mitotic figures and Ki-67 positive cells showed the proliferating nature of these cells at a later stage. Alteration of extracellular matrices from dense collagen fiber type to those of cartilage tissue seemed to predispose dogs to rupture of the CCL along with a degradation in collagen fiber of the primary bundles. Collagen fiber bundles with a parallel fibrillar array never formed in the CCL with degraded primary bundles, whereas activated fibrocytes constantly underwent chondroid metaplasia. The pathogenic mechanism underlying chondroid metaplasia was thought to be nonspecific and attributable to an essential property of activated fibrocytes in the mature tendon tissue.

Historical control data of tumor incidence were collected from the control groups (215 animals of each sex) in four recent carcinogenicity studies that were started between 2005 to 2009 (terminally sacrificed between 2007 and 2011) at BoZo Research Center Inc. (Gotemba, Shizuoka, Japan) using Fischer 344 rats (F344/DuCrlCrlj). These data were compared to the previous historical control data (from 1990 to 2004, previously reported) in the same facility. In the results, the incidence of C-cell adenoma in the thyroid tended to increase in both sexes in recent years (30.8% for males and 24.4% for females in 2005-2009) as compared with the previous data (17.4% and 20.1% for males and 11.5% and 11.8% for females in 1990–1999 and 2000–2004, respectively). In addition, the incidences of pancreatic islet cell adenoma in males and uterine adenocarcinoma tended to increase from around 2000 and remained high in recent years (incidences of islet cell adenoma in males of 10.5%, 17.1% and 20.5% in 1990–1999, 2000–2004 and 2005–2009; incidences of uterine adenocarcinoma of 3.3%, 12.0% and 13.5% in 1990–1999, 2000–2004 and 2005–2009, respectively). There was no apparent difference in the incidence of other tumors.

We investigated a highly metastatic ovarian yolk sac carcinoma in a 52-week-old female Crl:CD(SD) rat. Macroscopically, the present case had severe ascites, bilateral ovarian masses and numerous nodules in the abdominal and thoracic cavities. Histopathologically, these masses and nodules were generally composed of two types of cells mimicking a parietal and visceral yolk sac. The parietal cells were round to polygonal, contained eosinophilic droplets and were arranged in nests and cords in the eosinophilic matrix. Both the intracytoplasmic droplets and the matrix were stained positively with PAS. The visceral cells were cylindriform, and proliferated in papillary and tubular patterns and occasionally formed Shiller-Duval body-like structures. In the dissemination sites, the neoplastic cells proliferated on the surface of the various tissues and often infiltrated into deeper parts of the tissues. Immunohistochemically, both neoplastic cells were positive for α-fetoprotein and keratin, and the eosinophilic matrix was positive for laminin. Ultrastructurally, the parietal cells had dilated rough endoplasmic reticulums, which were filled with electron-lucent laminated structures. The visceral cells had poorly to moderately developed intracytoplasmic organelles and were interconnected with desmosomes. Taken together, the present tumor was diagnosed as yolk sac carcinoma arising from the ovary and was characterized by not only high metastasis but also invasive infiltration with biphasic proliferation of the parietal and visceral cells.

A 9-month-old male beagle suddenly died without any clinical signs. Histopathologically, he showed acute circulatory insufficiency due to diffuse degenerative changes of blood vessels and exudation from arterioles and myocardial contraction band necrosis of the heart. The cardiac lesions were marked in the left ventricle and the vascular lesion consisted of medial degeneration of the arterial system extending from the main branches of the extramural coronary arteries to the arteriolar segments. These lesions showed no inflammatory changes.

Among spontaneous neoplasms of the rat central nervous system, the discrimination between astrocytoma and malignant reticulosis (MR) is sometimes difficult because of their similar cell morphology and infiltration patterns. In the present study, we carried out histological and immunohistochemical analyses on a total of sixty-four cases in Sprague-Dawley and F344 rats. These cases were diagnosed as benign/malignant astrocytoma containing no neoplastic oligodendroglial elements or MR according to the diagnostic criteria of the World Health Organization International Classification of Rodent Tumors ( Mohr et al. 1994 ). Astrocytomas were divided into three types and MR into two types based on the number of lesions, cellularity and infiltration patterns, and so on. Although the neoplastic cells from all types showed various immunoreactivities for RM-4 (anti-rat macrophages and dendritic cells), ED-1, and/or vimentin, there were no distinctive differences among these types, and most cells that were positive for RM-4 were also positive for ED-1. None of the tumor types showed any reactivity for GFAP or S-100 protein. From the results of morphological and immunohistochemical examinations, it was indicated that there are no distinctive differences between spontaneous astrocytomas and MR in rats, and they are probably derived from the same cell lineage, that is, microglia, macrophage, or radial glia.

A highly invasive intracranial malignant schwannoma containing several masses was detected in a 28-week-old male Crl:CD(SD) rat. Macroscopically, 3 masses were noted in the cranial cavity; one was present at the bottom of the cranial cavity and involved the trigeminal nerve, and the other two were in the parietal bone. Histologically, each mass consisted of fusiform cells with interlacing fascicular, wavy and nuclear pseudopalisading arrangements and round cells with cystic lesions. The tumor cells invaded not only the brain but also the parietal bone. In the brain, the tumor cells infiltrated diffusely into the leptomeningeal and perivascular spaces and parenchyma, in which the tumor cell morphology and invasive pattern closely resembled those of malignant astrocytoma and malignant reticulosis. Immunohistochemically, the tumor cells in the masses showed positive reactions for both S-100 protein and GFAP, while those in the cerebral invasion sites were negative for GFAP and less positive for S-100 protein. Electron microscopically, a single basal lamina layer and short intricate cell processes were confirmed in the tumor cells. From these results, the present tumor was diagnosed as a malignant schwannoma arising in the cranial cavity, probably originating from the trigeminal nerve. The present tumor is considered to be a relatively unique malignant schwannoma based on its growth and invasion patterns.

Spontaneous leiomyosarcoma arising from the left ethmoid turbinate was observed microscopically in an 83-week-old male F344 rat. The tumor cells showed smooth-muscle differentiation with prominent nuclear pleomorphism and a small number of mitotic figures. The tumor cells were also immunohistochemically positive for smooth-muscle actin. The tumor protruded slightly into the nasal cavity and invaded the surrounding tissues. The present article is the first case of spontaneous leiomyosarcoma in the rat nasal cavity.

In rat spontaneous neoplasms of the central nervous system, the discrimination between astrocytoma and malignant reticulosis is sometimes difficult because of their similar cell morphologies and growth and infiltration patterns. The derivation of malignant reticulosis is proposed to be lymphocytes, microglias or histiocytes, although their precise origin has not been established yet. In the present retrospective study we examined immunohistochemical analyses from a total of 43 cases in Sprague-Dawley (SD) rats and 21 cases in F344 rats which were diagnosed as benign astrocytoma, malignant astrocytoma or malignant reticulosis according to the diagnostic criteria of WHO International Classification of Rodent Tumours (IARC No. 122). The tumours were classified into five categories as follows: (1) early stage of astrocytoma: 7 cases (SD: 3 cases, F344: 4 cases); they are composed of one lesion of moderate cellularity in the parenchyma. (2) common type of astrocytoma: 41 cases (SD: 29 cases, F344: 12 cases), which includes not only a single lesion with dense cellularity but also multiple extending lesions in the parenchyma, with infiltration into peripheral meninges or perivascular space. (3) glioblastoma type of astrocytoma: 12 cases (SD: 10 cases, F344: 2 cases); necrosis with peripheral palisading is observed. (4) intermediate type of astrocytoma and malignant reticulosis: 2 cases (SD: 1 case, F344: 1 case); neoplastic cells are infiltrated more diffusely in meninges and perivascular space with obvious parenchymal infiltration. (5) malignant reticulosis: 2 cases (both in F344); neoplastic cells are infiltrated diffusely in meninges and perivascular space without obvious parenchymal infiltration. The neoplastic cells from all categories showed various immunoreactivities for vimentin, RM-4 (anti-rat macrophages and interdigitating cells) and ED-1. However, no distinct difference among these categories was observed. The positive cells for RM-4 mainly corresponded to those of ED-1. The reactivity for vimentin was more intense than that of RM-4 and ED-1. A few neoplastic cells from the common and glioblastoma types demonstrated positive reaction for nestin. No reactivity for GFAP or S-100 was observed in any case of any category. Perivascular or intercellular reticulin fibers which were stained with Watanabe's silver impregnation method were observed in some or many cases in each category except for the early stage of astrocytoma. The results of the malignant reticulosis generally coincided with those of astrocytomas. In conclusion, no difference in the cell origin between astrocytoma and malignant reticulosis could be found.

A repeated dose toxicity study of prulifloxacin, a new antibacterial agent, was conducted in Sprague-Dawley rats. Male and female rats were given the test material orally for 4 weeks at doses of 0 (control), 30, 300 and 3000 mg/kg. After discontinuation of the treatment, a 4-week recovery test was also conducted. There was one case of death in the 3000 mg/kg group. Grayish green and soft feces, unkempt fur, transient deep respiration and decreased body weight gain were observed in the 3000 mg/kg group. Decreased food consumption and increased water intake were seen in the 300 and 3000 mg/kg groups. Ophthalmoscopic examination failed to show any abnormalities related to the treatment. In urinalysis, crystalline substance in the urinary sediments, cloudy urine and decreased Na+ excretion were observed in the 300 and 3000 mg/kg groups. Increased urine volume, lowered urine specific gravity and decreased K+ and Cl- excretions were seen in the 3000 mg/kg group. Hematologic examination showed decreased Hb, Ht, MCV and MCH and increased WBC in the 3000 mg/kg group. Blood chemical examination revealed increased BUN and decreased K+ and Cl- in the 3000 mg/kg group, and decreased K+ and gamma-globulin in the 300 mg/kg group. Pathological changes caused by the treatment were as follows. Cecal weight was increased in all dose groups. Cecal distention and swelling of its absorptive cells were seen in the 300 and 3000 mg/kg groups. In kidney, tubular nephrosis with crystalline substance was observed in the 300 and 3000 mg/kg groups, and its organ weight was increased in the 3000 mg/kg group. The above-mentioned changes were reversible except for decreased gamma-globulin, increased BUN and urine volume, and lowered urine specific gravity. Ulcer and small cavities associated with proliferation of fibrous tissue in the femoral articular cartilage were observed in the 3000 mg/kg group at the end of recovery period of 4 weeks. Plasma levels and urinary concentrations of active metabolite of the test material were increased in all dose groups with dose-related manner, whereby no sex difference was observed. No effects caused by the repeated dosing were seen in the plasma concentrations. Increased cecal weight in the 30 mg/kg group was considered to be attributable to the pharmacological effect of the test material. The results show that the NOAEL of prulifloxacin is 30 mg/kg for 4-week repeated dose toxicity in rats.

Stomach tissues from 222 beagle dogs, which were used for toxicological studies, were examined histopathologically. Out of 190 dogs fed the same diet, 185 had gastric lesions, whereas all of 32 dogs fed another diet were free from them. The gastric lesions were noted as an accumulation of eosinophils, infiltration of inflammatory cells including lymphocytes, plasma cells and histiocytes in the gastric lamina propria, formation of foregin body type multinucleated giant cells, focal desquamation and papillary proliferation of mucosal epithelium. The lesions were confined to the superficial layer of the antral and pyloric mucosa, and neither desmoplasia nor clinical signs were seen. Multinucleated giant cells often contained amphophilic foreign bodies of varying appearance with inconsistent reaction for periodic acid-Schiff (PAS), but no definite causal agents were detected.

Hepatic myelolipomas were found in four Goeldi's monkeys (Callimico goeldii) from a small colony originated from 3 animals imported from South America and sustained in Monkey Center of Japan, and the histological features were compared with the hepatic extramedullary foci of hemopoiesis in the fetus or newborn monkeys of this species. The histological features were analogous to those of myelolipomas of the liver, spleen or adrenals in other animals and man. The cause of the relatively high incidence of this rare lesion in these monkeys remains nuclear, but, the close genetic background and identical environmental conditions, including diet are mentioned as possible pathogenic factors.