Histopathological and immunohistochemical evaluation for detecting changes in blood hormone levels caused by endocrine disruptors in a 28-day repeated-dose study in rats.

Although measurements of blood hormone levels in rodent toxicological studies can provide important information on the mechanisms of toxicity and extrapolation to humans, there are several difficulties such as large individual differences and limited sample volume. To develop a more simplified method that does not depend solely on blood samples, we examined the possible application of immunohistochemistry for detecting endocrine disruptors in short-term studies. Aminotriazole (AMT), propylthiouracil (PTU), phenobarbital, aminoglutethimide (AGT), estradiol, and vitamin D3 were administered orally to 6-week-old male and female SD rats (five/group) for 28 days. Measurements of serum hormone levels revealed decreases in triiodothyronine (T3) and thyroxine (T4) in the AMT and PTU groups, an increase in thyroid stimulating hormone (TSH) in the AMT, PTU, and AGT groups, and an increase in adrenocorticotrophic hormone in the AGT group. Increased thyroid, pituitary, and adrenal gland weights; histopathological lesions, including follicular hypertrophy/hyperplasia, hypertrophy/vacuolation of anterior pituitary cells, and increased adrenocortical vacuolation were observed in association with the hormone level changes. Immunohistochemical analysis revealed a decreased T4 level in the thyroid gland of the AMT and PTU groups and an increased area of TSH positive immunostaining in the pituitary gland of the AMT, PTU, and AGT groups, consistent with the changes in serum T4 and TSH levels, respectively. These results suggest that histopathological analysis and immunohistochemistry for T4 and TSH might be useful and sensitive methods of detecting thyroid dysfunction, and that combining organ weight measurements is a reliable parameter of detecting endocrine disruptors.

Spontaneous adenolipoma of the mammary gland in the male F344 rat.

A 110-week-old male F344 rat from the high-dose group of a 104-week carcinogenicity study, exhibited a spontaneously occurring subcutaneous mass in the left axilla extending to the chest. Histologically, the mass was well-demarcated from the adjacent mammary tissue and slightly encapsulated without evidence of infiltration into the surrounding tissues. The mass contained both epithelial and adipose components. The epithelial component consisted of ductal structures of various sizes lined by a single layer of flattened to cuboidal epithelial cells with relatively clear or vacuolated cytoplasm. These ductal structures were well-intermingled with an adipose component that consisted of a uniform monomorphic cell population of mature adipocytes. Both cell types were well-differentiated and did not exhibit cellular atypia. Within the mass, fibrous connective tissue was found in the stroma with infiltration of numerous mast cells. Based on these findings, the mass was diagnosed as an adenolipoma of the mammary gland.

Neuromuscular and vascular hamartoma of the small intestine in an F344 rat.

An intestinal mass was found in the border area of the jejunum and ileum of a 110-week-old male F344 rat. Histopathologically, the mass protruded into the lumen and was covered with intestinal epithelium, exhibiting a normal architecture. The lesion was located in the submucosa and consisted of loose connective tissue, smooth muscle, scattered ganglion cells, and blood vessels of various sizes. Although these components showed an irregular and disordered structure, no cellular atypia, increased proliferation activity, or invasive growth to adjacent tissues were detected. Immunohistochemical analyses revealed that smooth muscle, ganglion, and endothelial cells were positive for α-smooth muscle actin and vimentin, S-100, and CD34 and von Willebrand factor, respectively, indicating maturation of these cells. Thus, the mass was diagnosed as a neuromuscular and vascular hamartoma of the small intestine. To the best of our knowledge, this is the first report of this type of lesion in rodents.

A 90-day repeated oral dose toxicity study of four stereoisomers of 2,4-dimethyl-4-phenyltetrahydrofuran, a synthetic flavoring substance, in F344 rats.

2,4-Dimethyl-4-phenyltetrahydrofuran (CAS no. 82461-14-1) is a food additive used as a synthetic flavoring substance. To investigate the toxicological properties and determine the no-observed-adverse-effect level (NOAEL), a 90-day repeated oral dose toxicity study of 2,4-dimethyl-4-phenyltetrahydrofuran containing four stereoisomers was conducted in F344 rats at doses of 0, 6, 24, and 96 mg/kg body weight (BW)/day. No mortality or abnormal clinical signs related to treatment in any group was observed. At a dose of 96 mg/kg BW, fluctuated serum total protein and total cholesterol and increased absolute and relative liver weights and relative kidney weights were observed in both sexes. Increased serum albumin in males and decreased Na and Cl in females were also observed. On histopathological assessment, at a dose of 96 mg/kg BW, diffuse hepatocellular hypertrophy in the liver in both sexes and tubular regeneration with scattered proximal tubular degeneration and/or necrosis throughout the cortex in the kidney in males were detected. Based on these findings, the NOAEL for 2,4-dimethyl-4-phenyltetrahydrofuran used in the current study was found to be 24 mg/kg BW/day for both sexes.

Accumulation of brown pigment-laden macrophages associated with vascular lesions in the lungs of cynomolgus monkeys(Macaca fascicularis).

Accumulation of macrophages containing brown pigments in the lungs is a well-known spontaneous lesion found in cynomolgus monkey. However, its pathogenesis has not been clearly described. In our survey, brown pigment-laden macrophages were found in the lungs of 4 out of 43 cases. Brown pigments were mostly found in the macrophages of the perivascular interstitium, which proved to be hemosiderin. Some small- to medium-sized vessels that exhibited prominent accumulation of brown pigment-laden macrophages showed degeneration and necrosis of the smooth muscle cells of tunica media. Furthermore, ruptures of the internal and external elastic laminae were seen in some of the vessels. These findings suggested that partial fragmentation of the vascular elastic lamina followed by degeneration and necrosis of the tunica media caused blood leakage leading to the accumulation of hemosiderin-laden macrophages in the perivascular interstitium of the lungs.

Neuroblastoma at the trigeminal nerve in a cynomolgus monkey (Macaca fascicularis).

A male cynomolgus monkey (Macaca fascicularis) of 5 years and 11 months of age from the vehicle control group of a 4-week repeated oral dose toxicity study had a spontaneously occurring mass lesion directly attached to the proximal part of the left trigeminal nerve. Histologically, the mass was characterized by a multifocal nodular appearance. Nodular zones showed low to moderate cellularity and were composed of small round cells exhibiting nuclear uniformity. On the other hand, inter-nodular zones were composed of nerve fiber containing septa and closely aggregated highly pleomorphic cells. Immunohistochemically, the small round cells were strongly immunopositive for synaptophysin, neuN, and class III beta-tubulin, while the highly pleomorphic cells were weakly immunopositive for neuN and occasionally immunopositive for class III beta-tubulin and doublecortin, suggesting that the tumor had originated from a neuronal lineage cell. Based on these findings, the mass was diagnosed as a neuroblastoma at the trigeminal nerve.

Fetal critical aortic stenosis with natural improvement of hydrops fetalis due to spontaneous relief of severe restrictive atrial communication.

We describe a rare case of fetal critical aortic stenosis with spontaneous relief of severe restrictive atrial communication, resulting in complete resolution of hydrops fetalis in utero. Fetal ultrasonography showed hydrops fetalis caused by critical aortic stenosis with a severely restrictive foramen ovale and severe mitral regurgitation at 23 weeks of gestation. Hydrops fetalis, however, spontaneously resolved, showing an obvious increase of flow through the foramen ovale and pulmonary vein at 26 weeks of gestation. The neonate required balloon dilation of the aortic valve and balloon atrioseptostomy immediately after birth and also received bilateral pulmonary artery banding and arterial duct stenting 1 week later. The patient was in good condition after conversion to biventricular circulation via Ross procedure at 8 months old. The present case suggests that atrioseptostomy as a fetal intervention may improve outcome in even a hydropic condition.

Histiocytic sarcoma in the brain of a cat.

A mass lesion in the subependymal region of the lateral ventricle in a 13-year-old neutered male mongrel cat with a complaint of somnolence, right circling movement and posture abnormality was examined. The magnetic resonance image examination revealed a relatively large T1-hypointense and T2-hyperintense mass lesion in the left interventricular foramen region, and there were no abnormalities in the chest and abdominal x-ray radiographic, funduscopic, and electric retinogram findings. The cat was died 43 days after the initial referral, and the post-mortem examinations revealed a poorly demarcated subependymal mass. Histologically, the brain lesion consisted of complex proliferation of highly pleomorphic cells resembling histiocytes with atypia and abundant mitotic figures. Moderate infiltrates of small reactive lymphocytes were admixed with the pleomorphic cell population. Gemistcytic astrocytes were also intermingled with the periphery of neoplastic foci. Immunohistochemically, most of the pleomorphic cells were positive for HLA-DR alpha-chain and ionized calcium binding adaptor molecule 1, and few were positive for lysozyme and alpha-1 antichymotrypsin. The atypical pleomorphic cells were negative for CD3, IgG (H and L), glial fibrillary acidic protein and neurofilament, suggesting monocytic/histiocytic-origin of the cells. The number of Ki-67-positive cell nuclei was extremely large, reflecting the high growth activity of these cells. Based on the findings, the lesion was considered as histiocytic sarcoma.

Eosinophilia and eosinophilic infiltration into splenic B-cell high-grade lymphoma in a dog.

A 13-year-old mixed-breed dog showing ascites, anorexia and anemia was found to have leukocytosis with marked eosinophilia, splenomegaly and hepatomegaly. The dog died 4 days after initial presentation and was diagnosed with splenic high-grade B-cell lymphoma at necropsy. Remarkable infiltrations of eosinophils were observed in spleen and liver tissues. The eosinophilia and infiltration of eosinophils into the lesions could have been associated with B-cell lymphoma because causes other than lymphoma were excluded. This is the first report of eosinophilia and eosinophilic infiltrations into neoplastic lesions in a dog with high-grade B-cell lymphoma.

Hamartoma in the medulla oblongata with marked mineral deposits in a dog.

A mass lesion in the medulla oblongata in a 9-year-old female Golden Retriever was examined pathologically. The medullary mass was 1 to 2 cm in diameter and poorly demarcated. The cut surface was discolored with numerous sand-like materials. Histologically, the mass lesion consisted of complex proliferation of irregularly arranged vessels as well as astrocytes with numerous mineral deposits. The astrocytic components exhibited no apparent anaplastic morphology. The proliferating vessels were either veins with muscle layer and capillaries. A few axons and myelinated fibers were also found in the lesion. The number of ki-67-positive cell nuclei was extremely small, suggesting the poor growth activity of these cells. Based on the findings, the lesion was considered to be a non-neoplastic, hamartomatous change.

Prediction of postnatal arrhythmia in fetuses with cardiac rhabdomyoma.

OBJECTIVES: Fetal cardiac rhabdomyomas are rare but well-known to be associated with arrhythmia or conduction abnormalities. However, since in utero electrophysiological information is quite limited, it remains unclear which type of rhabdomyoma will develop arrhythmia after birth. The aim of this study is to identify factors that predict postnatal arrhythmia requiring therapy in fetuses with cardiac rhabdomyoma. STUDY DESIGN: A retrospective review of infants prenatally diagnosed with cardiac rhabdomyoma was performed at our tertiary pediatric cardiac center between 1990 and 2016. Fetal arrhythmia was diagnosed using fetal echocardiography and magnetocardiography. We compared the characteristics of cases with and without antiarrhythmic therapy after birth. Cases without antiarrhythmic therapy after birth consisted of those who had postnatal arrhythmia but did not require antiarrhythmic therapy and those who had no postnatal arrhythmia. RESULTS: A total of 20 fetuses with cardiac rhabdomyoma were included in this study. Ten cases (50%) were confirmed as having tuberous sclerosis after birth. The mean gestational week at diagnosis and delivery were 32.1 ± 2.7 and 37.6 ± 2.8 weeks, respectively. Mean cardiac tumor size in utero was 21 ± 11 mm (range, 7-54 mm) in diameter. Fetal arrhythmia was found in six cases; three resolved in utero with transplacental antiarrhythmic therapy. Postnatal arrhythmia or conduction abnormalities were found in 12 cases; 7 required antiarrhythmic therapy. Cases with antiarrhythmic therapy after birth had larger cardiac tumor in utero than those without therapy (29.6 ± 12.8 mm versus 16.3 ± 5.8 mm, p < .01). Cardiac tumor size >30 mm in diameter predicted postnatal arrhythmia requiring therapy with sensitivity of 57.1% and specificity of 100%. Location and number of cardiac tumor and presence of arrhythmia or conduction abnormalities in utero were similar between the two groups. CONCLUSIONS: Cardiac rhabdomyomas >30 mm in diameter are associated with postnatal arrhythmia requiring therapy regardless of number and location.

Fetal echocardiographic findings in right pulmonary artery to left atrium communication: a case report and review of the literature.

Right pulmonary artery to left atrium communication (RPALA com) is a fatal congenital heart disease with right-to-left shunting. We present a rare case of RPALA com that was prenatally diagnosed and confirmed by autopsy. We focus on some unique echocardiographic findings for prenatal diagnosis of RPALA com by reviewing our case and the literature. Asymmetric cardiomegaly with a pear sign and dilatation of the pulmonary artery might be useful for screening tests. A larger stalk with the pear sign, high-velocity of shunt flow, and absence of a dilated pulmonary vein might support the prenatal diagnosis of RPALA com.

MicroRNA-21 is associated with fibrosis in a rat model of nonalcoholic steatohepatitis and serves as a plasma biomarker for fibrotic liver disease.

Evidence indicates that hepatic fibrosis is the initial lesion of cirrhosis or hepatocellular carcinoma in diseases such as nonalcoholic steatohepatitis (NASH). To induce NASH, we fed rats a choline-deficient and iron-supplemented L-amino acid-defined (CDAA) diet. Histopathological examination revealed that fibrosis appeared from week 4 and progressed to bridging fibrosis from week 12. Using qRT-PCR assays, we detected increased expression of miR-21, Mmp-9, and Timp-1 in liver that peaked during week 4, when fibrosis was first detected. The expression pattern of miR-21 in plasma paralleled that in liver. Fibrosis tended to be resolved within 12 weeks of a recovery period after 12 weeks of feeding, and the expression of miR-21, Timp-1, and Mmp-9 decreased in liver. Comprehensive analyses of miRNA and mRNA expression in the liver using samples acquired at week 4 detected 16 miRNAs and 11 mRNAs that are mutually-interacting fibrosis-related factors. We therefore conclude that miR-21 was closely associated with fibrosis in a rat model of NASH and has potential as a plasma biomarker for hepatic fibrosis.

Pathological features of polyneuropathy in three dogs.

Canine polyneuropathy is a neurological disorder characterized by a dysfunction of multiple peripheral nerves. The etiology of the disease is diverse; it may occur in cases of infectious, immune-mediated, or hereditary conditions or in association with endocrinopathy, neoplasm, or chemical intoxication. It is often difficult to determine the etiology through clinical symptoms. The aim of this study is to investigate pathological differences among three canine polyneuropathy cases with each presumably having a different etiology. Cases included a 13-month-old female border collie (Dog No.1), a 21-month-old male chihuahua (Dog No.2) and an 11-year-old male beagle (Dog No.3). Clinical examinations revealed hindlimb ataxia and sensory loss in Dog No.1, forelimb paralysis and vertebral pain in Dog No.2, and paddling-gait and hypothyroidism in Dog No.3. Histopathologically, axonal swelling and pale myelin were observed in Dog No.1. Giant axons mimicking giant axonal neuropathy were obvious in Dog No.2. Dog No.3 showed atrophic axons and severe interstitial edema. Distributions of peripheral nerve lesions coincided with respective clinical symptoms. According to their clinical and pathological features, Dogs No.1 and No.2 were suspected of hereditary polyneuropathy, while Dog No.3 seemed to have hypothyroidism-associated polyneuropathy. As each case demonstrated unique pathological features, different pathogeneses of peripheral nerve dysfunction were suggested.

Pathological and immunohistochemical features of subdural histiocytic sarcomas in 15 dogs.

Subdural histiocytic sarcomas from 15 dogs (mean age 7.8 years) were histopathologically examined. Among the 15 dogs, there was a marked breed predominance (toward Pembroke Welsh Corgi dogs, 47%), but no gender predilection. Focal solitary subdural masses were detected in the cerebrum (12 cases) and spinal cord (1 case), whereas diffuse infiltrative lesions were observed in the cerebral leptomeninges in 2 cases. All neoplastic lesions had common histological features characterized by the proliferation of pleomorphic histiocytic cells combined with various inflammatory reactions. Multinucleated giant cells, phagocytosis, and atypical mitotic figures in the neoplastic cells were commonly observed. Most of the pleomorphic neoplastic cells in the present cases were immunopositive for monocytic, histiocytic, or both markers, such as human leukocyte antigen (HLA)-DR, ionized calcium-binding adaptor molecule 1 (Iba1), cluster of differentiation (CD)163, and CD204, except for the neoplastic cells in 2 focal and 2 diffuse histiocytic sarcomas. The findings suggest that differences in cell origin, molecular expression, or both patterns are responsible for the distribution patterns of canine subdural histiocytic sarcomas.

Establishment of a novel feline leukemia virus (FeLV)-negative B-cell cell line from a cat with B-cell lymphoma.

We established a novel feline B-cell line, MS4, from the neoplastic pleural effusion of a cat with cutaneous B-cell lymphoma. Immunophenotype staining of the MS4 cells was positive for CD20, CD79α, and IgA and negative for CD3, CD4, CD5, CD8α, CD18, CD21, CD22, IgM, IgG, Ig light chain, and MHC class II. PCR analysis for immunoglobulin heavy chain gene rearrangements revealed a monoclonal rearrangement, whereas no clonal rearrangement of the T-cell receptor γ gene was detected. Southern blotting with an exogenous feline leukemia virus (FeLV) U3 probe revealed no integration of exogenous FeLV provirus. The MS4 cell line is the first FeLV-negative feline B-cell lymphoma cell line, and may be used to investigate the pathogenesis of spontaneously occurring feline lymphoma and the development of new therapies.