Involvement of sex, strain and age factors in high fat diet-induced obesity in C57BL/6J and BALB/cA mice.

Although a number of obesity animal models have been reported, each model possesses different characteristics of obesity, suggesting care should be taken in choosing an animal model suitable for the experimental purpose. In this report, we fed 4-(young) and 52-week-old (middle-aged) C57BL/6J (B6) and young BALB/cA (BALB/c) mice with a high fat diet (HFD) for 9 weeks, and investigated the clinical and histological characteristics of obesity. In BALB/c mice, males gained more body weight and body fat weight and had higher energy intake than females by HFD feeding. Comparing the effect of HFD feeding between the strains of mice, BALB/c male mice accumulated more hepatic lipid than B6 male mice. In addition, middle-aged B6 mice increased the ratio of fat to body weight and hepatic lipid accumulation more than young mice. In conclusion, the characteristics of obesity induced by HFD feeding were influenced by the sex, strain and age of mice. Sex steroid hormones, hepatic lipid metabolism and systemic metabolism might be involved in these factors. The basic data in this study will be useful for the development of animal models of high fat diet-induced obesity.

Repair process of fetal brain after 5-azacytidine-induced damage.

The fetal brain is susceptible to many extrinsic stresses. Some of these stresses induce excessive cell death in the prenatal stage, leading to anomalies in the neonatal brain. However, it is unclear how the developing brain responds to and repairs the prenatal tissue damage. We treated pregnant rats on day 13 of gestation with 5-azacytidine, one of the compounds that induces excessive cell death and inhibits proliferation in neural progenitor cells, to damage the fetal brain, and investigated the repair process up to 60 h after treatment. Histological analysis showed that 5-azacytidine induced strong apoptosis of neural cells. By 60 h, apoptotic cells disappeared and the tissue was repaired, although the telencephalic wall remained thinner than in controls. Flow cytometry analysis showed that the cell cycle distribution also returned to control levels at 60 h, suggesting that the repair process was completed around 60 h. During the repair period, amoeboid microglia infiltrated the brain and ingested the apoptotic cells. These microglial cells were positive for the multiple microglial markers, and mRNAs for the microglia-related cytokines tumor necrosis factor alpha, interleukin 1beta and macrophage colony stimulating factor (M-CSF) were up-regulated. DNA microarray analysis showed the up-regulation of genes relevant to glial cells, inflammation, the extracellular matrix, glycolysis, proliferation and neural development. We show here that the developing brain has the capacity to respond to the damage induced by extrinsic chemical stresses, including changing the expression of numerous genes and the induction of microglia to aid the repair process.

Histopathological characteristics of Ito cells and Kupffer cells in the feline liver.

The histopathological characteristics of Ito cells and Kupffer cells were investigated in the liver of 21 cats (age range: 6 months -18 years) autopsied in our laboratory during 2003. Immunohistochemical examinations were performed using antibodies against lysozyme, desmin and alpha-smooth muscle actin. No Kupffer cells reacted with the antibody against lysozyme. However, macrophages in the lung and spleen showed a positive reaction with the antibody. This finding suggests a possibility that the amount of lysozyme in the Kupffer cells of feline liver is comparatively small. On the other hand, large vacuole-laden cells were observed in the hepatic perisinusoid of some feline cases, and these cells showed a positive reaction with antibodies against desmin and alpha-smooth muscle actin. These cells could be Ito cells with large lipid vacuoles. This conclusion was supported by electron microscopic observation and oil red O staining. However, no such large vacuole-laden perisinusoidal cells were detected in the liver of young cats less than 2 years old. The present study revealed the histopathological features of Kupffer cells and Ito cells in the feline liver.

The critical roles of serum/glucocorticoid-regulated kinase 3 (SGK3) in the hair follicle morphogenesis and homeostasis: the allelic difference provides novel insights into hair follicle biology.

Mutation in the serum/glucocorticoid regulated kinase 3 (Sgk3, also known as Sgkl or Cisk) gene causes both defective hair follicle development and altered hair cycle in mice. We examined Sgk3-mutant YPC mice (YPC-Sgk3(ypc)/Sgk3(ypc)) and found expression of SGK3 protein with altered function. In the hair follicles of YPC mice, the aberrant differentiation and poor proliferation of hair matrix keratinocytes during the period of postnatal hair follicle development resulted in a complete lack of hair medulla and weak hair. Surprisingly, the length of postnatal hair follicle development and anagen term was shown to be dramatically shortened. Also, phosphorylation of GSK3beta at Ser9 and the nuclear accumulation of beta-catenin were reduced in the developing YPC hair follicle, suggesting that phosphorylation of GSK3beta and WNT-beta-catenin pathway takes part in the SGK3-dependent regulation of hair follicle development. Moreover, the above-mentioned features, especially the hair-cycling pattern, differ from those in other Sgk3-null mutant strains, suggesting that the various patterns of dysfunction in the SGK3 protein may result in phenotypic variation. Our results indicate that SGK3 is a very important and characteristic molecule that plays a critical role in both hair follicle morphogenesis and hair cycling.

Encepalomyocarditis virus-induced apoptosis and ultrastructural changes in the lacrimal and parotid glands of mice.

Development of acinar cell apoptosis and ultrastructural changes in the exorbital lacrimal and parotid glands was examined in DBA/2 mice infected with 10(2) PFU/mouse of EMC-D virus. Pyknotic acinar cells, most of which were positive for TUNEL and cleaved caspase-3 and had ultrastructural characteristics of apoptotic cells, developed earlier and were more frequently observed in the parotid gland than in the exorbital lacrimal gland, while the total damage of acinar cells and interstitial infiltration of macrophages were more prominent in the latter than in the former. These findings indicate that EMC-D virus induces acinar cell apoptosis in these glands. In addition, corresponding to the results of the detection of viral RNA signals by in situ hybridization, small aggregates of virus-like particles having typical size and structure of EMC virus were frequently observed in both the cytoplasm and the nucleus of acinar cells in the exorbital lacrimal gland, while they were found only in the cytoplasm of a few acinar cells in the parotid gland. In conclusion, between the exorbital lacrimal and parotid glands, there was a reverse relationship observed between the development of acinar cell apoptosis and that of total damage of acinar cells.

Thymoma in a dog with a part of granular cell proliferation and concurrent lymphoma cells.

A 12-year-old male Shiba dog showed anemia and the swelling of systemic lymph nodes. X-ray and post mortal examinations revealed a anterior mediastinal mass. Histologically, the tumor mass consisted of four different elements; cord-like proliferation of cuboidal epithelial cells, tubular or cystic structures lined with ciliated epithelial cells, proliferation of large round-shaped epithelial cells with PAS-slightly positive granular cytoplasm, and diffuse proliferation of neoplastic lymphocytes. Epithelial cells in cord-like or cystic structures were strongly positive for cytokeratin. Granular or foamy cells were negative for all markers examined and had myelin-like bodies in the cytoplasm by electron microscopy. The neoplastic lymphocytes in the tumor mass were considered being derived from concurrent multicentric lymphoma. Based on these findings, the present case was diagnosed as thymoma with a part of granular cell proliferation and concurrent lymphoma cells.

Ultrastructural changes in the dorsal skin of Wistar-derived hypotrichotic WBN/ILA-Ht rats following UVA-irradiation.

Ultrastructual characteristics of the dorsal skin responses to a single irradiation of UVA (1100 kJ/m2) were examined in Wistar-derived hypotichotic WBN/ILA-Ht rats (HtRs). In the epidermis, mitochondrial swelling of some keratinocytes and dissociation of keratinocytes due to intercellular edema developed at 3 hours (h) after irradiation and continued to 48 h. At 6 h, in addition to these changes, necrosis of keratinocytes accompanied with infiltration of neutrophils was also observed in some portions, and epidermal hyperplasia with many keratinocytes showing nucleolar hypertrophy and some mitotic keratinocytes was observed at 48 h. In the dermis, mitochondrial swelling and/or partial cytoplasmic destruction in capillary endothelial cells and edema with inflammatory cell infiltration were observed at and after 3 h, and extravasation of erythrocytes was found in some capillaries at 48 h. Mitochondrial swelling was also frequently found in pericytes and fibroblasts. Inflammatory cells were mainly composed of neutrophils throughout the experimental period. Mild degranulation of mast cells which also showed mitochondrial swelling was observed at and after 3 h, and a close special relationship between mast cells and fibroblasts or neutrophils was sometimes observed. In conclusion, the most prominent change in the dorsal skin of HtRs exposed to UVA was degeneration of capillary endothelial cells, resulting in edema and inflammatory cell infiltration, and the most characteristic cytopathic effect of UVA was mitochondrial swelling, and it was common to keratinocytes, capillary endothelial cells, pericytes, mast cells, and fibroblasts.

NMDA-induced apoptosis in the developing rat brain.

The N-methyl-D-aspartate receptor (NMDAR), which is one of the glutamate receptors, is considered to have a close relation to synaptic plasticity in the developing brain. In addition, it is also known that the excessive stimulation of NMDARs can trigger neuronal apoptosis. In this study, we examined the expression of neuronal apoptosis in the developing rat brain after the administration of NMDA to pregnant dams or neonates (embryonal days 18 to postnatal days 14). In the NMDA-treated group, the significant increase in nuclei of apoptotic neuronal cells occurred in the dose-dependent manner in the lateral-ventral regions of the fetal cerebral cortex, reaching maximum values at 24 hours after treatment. On the other hand, the induction of apoptosis did not occur in the neonatal brain.

Ultrastructural features of mast cells in picryl chloride (PCL)-induced contact dermatitis in IQI/Jic mice.

Mast cells are one of the major effector cells in the pathogenesis of allergic diseases such as contact dermatitis. In the present study, ultrastructural features of mast cells in contact dermatitis were examined. Namely, the ear of IQI/Jic mice was topically applied with picryl chloride (PCL) at 4 (1st), 11 (2nd), 18 (3rd) and 25 days (4th) after the sensitization with PCL to the abdominal skin. The changes in the ear swelling responses, total serum IgE levels and histology including mast cell numbers were similar to those of previous reports by our research group (Ikeda et al. 2000; Jung et al. 2001). Ultrastructurally, after the 1st application, a close spacial relationship between mast cells and neutrophils and phagocytosis of mast cell granules by neutrophils were observed. Mast cells generally contained non-fused swollen granules filled with altered contents with low electron density and showed an extrusion of membrane-free granules through membrane pores. In addition, interestingly, a few mast cells secreted membrane-bound granules into the dermis without leaving cell membrane damage. After the 4th application when the number of mast cells prominently increased and the total serum IgE level was greatly elevated, in addition to mast cells showing typical anaphylactic degranulation, many mast cells probably in the recovery process from degranulation and several immature mast cells characterized by well-developed Golgi apparatus, many ribosomes and a few electron-dense secretory granules in the peripheral cytoplasm were also observed at the same time. The present results clarified the ultrastructural features of mast cells in the course of PCL-induced contact dermatitis in IQI/Jic mice.

5-Azacytidine (5AzC)-induced histopathological changes in the central nervous system of rat fetuses.

5-Azacytidine (5AzC) is a cytidine analogue which possesses nitrogen atom instead of carbon atom at the position 5 of the pyrimidine ring. In this study, detailed histopathological changes were sequentially examined in the rat fetal brain obtained from dams treated with 5AzC (10 mg/kg) on day 13 of gestation (GD13). At 6 hours after treatment (HAT), a prominent accumulation of neuroepithelial cells showing pleomorphic mitotic figures were observed in the telencephalic wall. The mitosis-index peaked at 6 HAT, and decreased thereafter. Neuroepithelial cells positive for nick end labeling (TUNEL) method, which is widely used for the detection of apoptotic cells, prominently increased at 9 HAT, and the TUNEL-index peaked at 12 HAT. TUNEL-positive cells showed ultrastructural characteristics of apoptosis. At 24 HAT, the formation of rosette-like structures was observed in the fetal brain. From the results of the present study, it was evident that abnormal mitosis and neuronal apoptosis were induced in the rat fetal brain following 5AzC-administration to dams on GD13. In addition, it is suggested that 5AzC-induced apoptosis might occur mainly in the post mitotic phase of cell cycle.

Ultrastructural changes in the dorsal skin of Wistar-derived hypotrichotic WBN/ILA-Ht rats exposed to subchronic ultraviolet B (UVB)-irradiation.

Ultrastructural changes in the dorsal skin were examined in Wistar-derived hypotrichotic WBN/ILA-Ht rats exposed to subchronic UVB-irradiation (10 kJ/m2 per rat per day for up to 3 months). Epidermal hyperplasia developed at I month of UVB-irradiation and progressed thereafter, resulting in epidermal thickening and formation of epidermal ingrowths projecting into the dermis. In some portions of the epidermal ingrowths at 2 and 3 months, keratinocytes were somewhat pleomorphic. In addition, some of the keratinocytes showing cytoplasmic projections migrated into the dermis. The basement membrane and hemidesmosomes at the epidermal-dermal junction became to disappear along with the development of edema spreading from the upper dermis to the epidermis. However, Langerhans cells were still detected in the hyperplastic epidermis even at 3 months. In the dermis, in addition to edema, fibroblast proliferation and mast cell infiltration progressed with time, and degranulation of mast cells was obvious at 2 and 3 months. Only a few basophils as well as eosinophils were also found. In the upper dermis, especially beneath the epidermis, decrease in diameter and disintegration of collagen fibrils were observed. Ultrastructural characteristics of the dorsal skin responses to subchronic UVB-irradiation were clarified in the present study.

Cerebellar myxoid type meningioma in a Shih Tzu dog.

A 6-year and 9 month-old, male, Shih Tzu dog showed ataxia and trembling. By MRI examination, a mass (1 cm) was found in the right cerebellum. As the dog did not respond to radiation therapy, and showed a rise of intracranial pressure, he was euthanized. The cerebellar mass was soft and hemorrhagic. Histologically, the mass contained vimentin-positive spindle- or polyhedral-shaped cells arranged in a cord-like pattern. Mucinous materials were observed in the intercellular spaces. Ultrastructural examination revealed cell processes, microtubule-like structures and desmosomes. The case was diagnosed as myxoid type meningioma.