Morphological analyses of nephrin expression in progressive glomerulonephropathy of common marmosets.

In this study, we focused on nephrin, one of the key molecules within the slit diaphragm of podocytes, as although there have been reports on its expression in humans and rats, their presence in common marmosets has not been reported. We investigated nephrin expression and changes in glomeruli, depending on the development of spontaneous progressive glomerulonephropathy in common marmosets. Nineteen common marmosets at two to ten years of age were evaluated. The kidney was examined by microscopy with hematoxylin and eosin and immunohistochemical staining for nephrin. The lesions were classified into three grades according to a renal lesion grading system reported previously. The nephrin-positive area was measured by morphometric analysis, and the nephrin-positive ratio was calculated. Nephrin expression was observed along the glomerular capillary loop in a continuous linear pattern in renal lesion grades 0 to 2 and either discontinuous linear or coarse granular pattern in grade 3. Nephrin expression tended to decrease significantly depending on the grade of renal lesions. Alteration in nephrin expression has been suggested to play an important role in the progression of renal lesions.

Relationship between osteoid formation and iron deposition induced by chronic cadmium exposure in ovariectomized rats.

Itai-itai (Japanese, "It hurts! It hurts!") disease (IID), a form of osteomalacia, can be induced in ovariectomized rats by long-term administration of cadmium (Cd). This IID rat model shows severe anemia, severe nephropathy, and osteomalacia accompanied by iron (Fe) deposition at the mineralization front. We characterized the pathogenesis of Cd-induced bone lesions by investigating the relationship between Fe deposition and osteoid tissue formation in ovariectomized rats. The rats were injected with CdCl2 (0.5 mg/kg) for 70 weeks, with or without co-injection of erythropoietin (EPO) for varying lengths of time to elucidate whether EPO prevents and/or cures anemia, and, with the restoration from anemia, lessens the osteoid tissue formation. Necropsies were performed at 25, 50, or 70 weeks. Fe deposition at the mineralization front of bone was found at 50 weeks and increased thereafter. Animals injected with EPO showed decreased Fe deposition, although there was no relation between EPO administration and osteoid formation in the femur. Because the increase in bone lesion severity was independent of the amount of Fe deposition, we suggest that Fe deposition is not involved in the etiology of Cd-induced femoral bone lesions.

A spontaneous middle ear adenocarcinoma characterized by abundant eosinophilic matrix in a young male Crl:CD(SD) rat.

A mass was detected in the right tympanic cavity of a 15-week-old male Crl:CD(SD) rat. Histological examination revealed papillary or tubular proliferations of epithelial cells including ciliated cells that produce mucus and have an abundant eosinophilic matrix. The malignancy of this tumor was revealed by its destructive proliferation, cellular polymorphism, and high proliferative activity. The tumor was diagnosed as a middle ear adenocarcinoma due to its location and histological characteristics. This is the first report of a middle ear adenocarcinoma in rats and the first middle ear tumor accompanied by eosinophilic matrix observed in either humans or animals. The eosinophilic matrix was positive for periodic acid-Schiff and periodic acid-silver methenamine stains and for laminin and collagen Type IV, immunohistochemically, but negative for Congo red, which suggested that this matrix was a basement membrane substance.

Immunohistochemical Characteristics of Thymomas and Hyperplastic Lesions in Wistar Hannover Rats.

Previously, we investigated the higher incidence of hyperplastic lesions and thymomas and histopathological resemblance of cortex-medullary structures between thymomas and normal thymuses in Wistar Hannover (WH) rats. Thymomas had pale-staining cell foci (PA) similar to medulla but without lymphocytes. Here, we focused on the differences in cytokeratin (CK) expression in the thymic epithelia of the cortex and medulla and compared the structures of thymomas and normal thymuses. Thymomas, hyperplastic lesions, and normal thymuses obtained from background studies of WH rats were stained with antibodies against CK14, CK18, and CD20. In normal thymuses, the epithelial cells were positive for CK14 in the medulla and subcapsular area and for CK18 in the cortex, B-cells were positive for CD20 in the medulla. In thymomas, the epithelial cells were positive for CK14 in the medullary differentiation (MD) areas and for CK18 in the cortex-like lymphocyte rich and PA, and B-cells were positive for CD20 in the MD areas.

Supplemental study on 2', 3'-Cyclic Nucleotide 3'-Phosphodiesterase (CNPase) activity in developing rat spinal cord lesions induced by hexachlorophene and cuprizone.

In a previous study, we showed that 2', 3'-Cyclic Nucleotide 3'-Phosphodiesterase (CNPase) expression is induced in different temporal patterns in the cerebrum, cerebellum and medulla oblongata of hexachlorophene (HCP) and cuprizone (CPZ) treated rats. Here, we additionally examined the histopathological changes and CNPase expression in the spinal cord to clarify the reproducibility of different temporal patterns of CNPase expression in the spinal cord showing low degree or lack of spongy changes. Spongy changes were observed in HCP-treated rats, but not in CPZ-treated rats. Immunohistochemistry showed that intense expression of CNPase was not induced following HCP or CPZ treatment. Our data reveal that expression intensity of CNPase may be dependent on the degree of HCP- and CPZ-induced damage of the myelin sheath.

Spontaneous globule leukocyte tumor accompanied by inflammatory cells in a Wistar Hannover rat.

We encountered hematolymphoid neoplastic lesions in the form of many nodules in the spleen and liver in a 110-week-old male Wistar Hannover rat (Crl:WI (Han)). The lesions contained atypical proliferative cells, eosinophils, lymphocytes, and macrophages. The proliferative cells comprised various atypical cell types with or without cytoplasmic eosinophilic granules. The granules were positively stained using periodic acid-Schiff and elastase stains, were bluish purple using phosphotungstic acid and hematoxylin, and showed no metachromasia using toluidine blue. In immunohistochemical staining, the proliferative cells with or without granules were positive for granzyme B, rat mast cell protease II, and Ki67. Electron microscopic examination revealed that single to multiple high-density granules of variable size were covered by a membrane. These findings led to a diagnosis of globule leukocyte tumor. The accompaniment of this tumor by inflammatory cells is likely evoked by mast cell-like active mediators contained in the granules of the globule leukocytes.

Thymomas and Associated Hyperplastic Lesions in Wistar Hannover Rats.

Thymomas occur prevalently in aged Wistar Hannover (WH) rats, along with hyperplastic lesions that cannot be categorized as thymomas. We compared the histological features of hyperplastic lesions and thymomas in WH rats, the incidences of these lesions, and the relationship of these lesions to the degree of thymic involution and also compared these lesions with those of Sprague Dawley (SD) rats in 4-, 13-, 26-, and 104-week studies. There were no morphological differences between hyperplastic cells and benign tumor cells in thymomas. The histological difference between hyperplastic lesions and thymomas was the size of the proliferative areas and the number of medullary differentiation areas. The hyperplastic lesions of the thymus in WH rats might have a potential for progression to thymomas due to the observed multiple hyperplastic lesions or mixed lesions with thymomas. The incidence of these proliferative lesions in the thymus was higher in females than in males. Further, the incidence of these proliferative lesions was higher in WH rats than in SD rats. Thymic involution was more severe in males than in females and more severe in SD rats than in WH rats. The differences in involution progression may have been reflected in the incidence of thymic proliferative lesions in SD and WH rats.

Nonproliferative and Proliferative Lesions of the Rat and Mouse Endocrine System.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) Project (www.toxpath.org/inhand.asp) is a joint initiative among the Societies of Toxicological Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in the endocrine organs (pituitary gland, pineal gland, thyroid gland, parathyroid glands, adrenal glands and pancreatic islets) of laboratory rats and mice, with color photomicrographs illustrating examples of the lesions. The standardized nomenclature presented in this document is also available electronically on the internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous and aging lesions as well as lesions induced by exposure to test materials. A widely accepted and utilized international harmonization of nomenclature for endocrine lesions in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists.

Morphological and immunohistochemical diversity of endometrial stromal sarcoma in rats.

To clarify the histopathological characteristics of rat endometrial stromal sarcoma (ESS), we morphologically reviewed 12 malignant uterine tumors protruding into the lumen in previous rat carcinogenicity studies. The 12 cases were classified into the following 6 types based on their morphological features: spindle cell and collagen rich type, pleomorphic/spindle cell and compact type, decidual alteration type, histiocytic and multinucleated giant cell mixture type, Antoni A-type schwannoma type, and Antoni B-type schwannoma type. Immunohistochemically, tumor cells in all cases exhibited focal or diffuse positive reactions for vimentin, and 11 of the 12 cases were positive for S-100. Interestingly, 9 cases were positive for desmin or αSMA, indicating tumor cells expressing smooth muscle properties. Both Antoni A- and B-type schwannoma types showed low reactions for both muscle markers. Positive results for estrogen receptor α in the 11 cases suggested that they were derived from endometrial stromal cells. On the basis of their immunohistochemical profiles, they were considered to be derived from endometrial stromal cells while they showed morphological variation. The detection of a basement membrane surrounding tumor cells might not be a definitive indicator for differential diagnosis of ESS from malignant schwannoma. In conclusion, ESS could exhibit wide morphological and immunohistochemical variation including features of schwannoma or smooth muscle tumor.

Relationship Between Immunoglobulin Deposition and Early Lesions of Progressive Glomerulonephropathy in Young Common Marmosets.

The authors previously investigated progressive glomerulonephropathy in 2- to 11-year-old common marmosets and characterized age-related changes of the renal glomeruli and development of tubulointerstitial lesions. In this study, immunoglobulin deposition and ultrastructural changes of the glomeruli were investigated in 5 young marmosets from 6 months to 3 years of age with pre-onset or early glomerulonephropathy. In all animals, the foot processes of podocytes were effaced, and IgM was deposited into the glomeruli. In glomeruli without glomerular basement membrane (GBM) alteration, IgM was the only immunoglobulin type deposited in the glomeruli. In cases with more advanced lesions of reticulation and thickening of GBM, IgA and IgG deposits were also observed. Therefore, the authors conclude that IgM may be the primary or earliest immunoglobulin deposited in this nephropathy, whereas IgA and IgG deposition may be connected to the progression of the glomerular lesions. IgM deposition and foot process effacement of podocytes occur early in the life of affected marmosets.

Two types of deposits, hyaline droplets and eosinophilic bodies, associated with α2u-globulin accumulation in the rat kidney.

Alpha2u-globulin is an adult male rat-specific protein that accumulates spontaneously or inductively in the renal proximal tubular epithelium and forms microscopically observable deposits, which are generally referred to as "hyaline droplets," whereas a specific type of deposits is referred to as "eosinophilic bodies" by Japanese toxicologic pathologists. We compared hyaline droplets and eosinophilic bodies using special stains including immunostaining for α2u-globulin and lysosome-associated membrane protein in spontaneously occurring and d-limonene-induced cases. Eosinophilic bodies appeared simultaneously and increased in parallel with the hyaline droplets in the induced case. In both of the spontaneous and induced cases, hyaline droplets and eosinophilic bodies were associated with α2u-globulin and lysosomes, although there were differences in the forms and staining properties that probably reflected the purity or density of α2u-globulin. According to the results, it is not necessary for eosinophilic bodies to be strictly distinguished from hyaline droplets, and it is reasonable to identify eosinophilic bodies as hyaline droplets in α2u-globulin nephropathy in routine toxicity studies, as they have been recognized to be a sequence of changes associated with accumulation of α2u-globulin.

A case report of ventricular septal defect in an adult Sprague Dawley rat.

A 20-week-old male Sprague Dawley rat noted with decreased body weight, dyspnea, and anorexia beginning 2 days before death was necropsied in the recovery period of a sub-chronic toxicity study. The heart was severely enlarged (30 × 20 × 20 mm), 3-4 times larger than normal, with an approximately 6 mm wide defect in the upper, membranous portion of the ventricular septum. Both ventricles measured 4 mm in thickness, and the right ventricle was 4 times thicker than normal. According to a microscopic examination, the myocardial fibers were severely hypertrophic in the right ventricle and mildly hypertrophic in the left ventricle and septum. Myocardial vacuolation, focal hemorrhages with hemosiderin-laden macrophages, myocardial necrosis, focal fibrosis, hyalinized myocardial fibers, and multifocal adhesive pericarditis were also present. This is the first report concerning severe ventricular septal defects in an adult Sprague Dawley rat with a detailed histopathological examination.

Lymphocytic adrenal medullitis and lymphocytic thyroiditis in a laboratory beagle dog.

Lymphocytic adrenal medullitis characterized by inflammation and atrophy in the medulla of the bilateral adrenal glands was observed in an 18-month-old male laboratory beagle dog. It might be that the present lymphocytic adrenal medullitis is an autoimmune-mediated disease as the histological characteristics are consistent with an autoimmune pathogenesis. However, the actual cause remains unclear as the existence of serum autoantibodies against the adrenal medulla could not be confirmed. Although this dog also contracted lymphocytic thyroiditis along with serum thyroglobulin autoantibodies, indicating that the thyroiditis occurred with an autoimmune basis; the relation between the adrenal medullitis and thyroiditis is unknown.

A comparison of the anatomical structure of the pancreas in experimental animals.

As basic knowledge for evaluation of pancreatic toxicity, anatomical structures were compared among experimental animal species, including rats, dogs, monkeys, and minipigs. In terms of gross anatomy, the pancreases of dogs, monkeys, and minipigs are compact and similar to that of humans. The rat pancreas is relatively compact at the splenic segment, but the duodenal segment is dispersed within the mesentery. In terms of histology, the islet of each animal is characterized by a topographic distribution pattern of α- versus ß-cells. ß-cells occupy the large central part of the rat islet, and α-cells are located in the periphery and occasionally exhibit cuffing. In dog islets, ß-cells are distributed in all parts and α-cells are scattered in the center or periphery of the islet (at body and left lobe); whereas ß-cells occupy all parts of the islet and no α-cells are present in the islet (at right lobe). Monkey islets show two distinct patterns, that is, α-cell-rich or ß-cell-rich islets, and the former represent peripheral ß-cells forming an irregular ring. Minipig islets show an irregular outline, and both α- and ß-cells are present in all parts of the islet, intermingling with each other. According to morphometry, the endocrine tissue accounts for <2% of the pancreas roughly in rats and minipigs, and that of monkeys accounts for >7% of the pancreas (at tail). The endocrine tissue proportion tends to increase as the position changes from right to left in the pancreas in each species.

Comparative histopathology of the estrous or menstrual cycle in laboratory animals.

Accurate analysis of female reproductive toxicity requires a thorough understanding the differences in and specifics of estrous or menstrual cycles between laboratory animals. There are some species differences such as the time of sex maturation, the length of the estrous or menstrual cycle, the length of the luteal phase, the number of dominant follicles or corpora lutea, the size of follicles, processes of luteinization, and hormonal changes during the estrous or menstrual cycle. Rodents have a short estrous cycle, and their ovarian cycling features are the same in both ovaries, which contain a large number of follicles and corpora lutea. The dog estrous cycle is much longer than those of other laboratory animals, and it includes a long anestrus phase. The duration of the menstrual cycle of monkeys is roughly 30 days, and their ovarian cycling features are different between the left and right ovaries. In both rodents and dogs, the theca cells invade the early luteum, mixing with granulosa cells during luteinization. However in monkeys, the theca layer dose not mix with the granulosa cells as it invaginates only slightly into the early luteum. In addition, we found that high progesterone levels after ovulation are sustained for a much shorter duration in rodents than in dogs and monkeys due to the comparatively rapid passage of the rodent luteal phase. Based on these species differences, animal species for use in ovarian toxicology studies need to be selected appropriately.

Spontaneous Age-related Lesions of the Kidney Fornices in Sprague-Dawley Rats.

The upper portion of the rat kidney pelvis has specialized anatomic structures referred to as fornices. Fornices have a role in urine concentration. Spontaneous lesions including mineralization, epithelial hyperplasia, and inflammatory cell infiltration may occur in the area of the fornices. However, little information regarding specific historical control data or the spontaneous development of these findings in male and female fornices is known. Understanding spontaneous age-related lesions in the area of the fornices versus other portions of the kidney pelvis may be relevant in the identification of test article-induced changes. A retrospective study was conducted of male and female Sprague-Dawley rat kidney fornices over several time points to determine the incidence and severity of mineralization, epithelial hyperplasia, and inflammatory cell infiltration. Based on this investigation, these lesions appeared to increase over time and, in general, occurred earlier and with a greater incidence in females. Regarding those chemicals that may result in lesions of the kidney pelvis, it may be important for pathologists to separately diagnose lesions of the fornices from other portions of the kidney pelvis to help differentiate between any spontaneous age-related and induced changes.

Histopathological and immunohistochemical characterization of spontaneous pituitary tumors in dwarfs derived from Wistar Hannover GALAS rats.

Histopathological and immunohistochemical characteristics of spontaneous pituitary adenomas found in dwarfs derived from Wistar Hanover GALAS are being described for the first time. The adenomas were seen in 5 males aged 48 weeks or older and in 11 females aged 34 weeks or older. Immunohistochemically, 13 cases without post-mortem changes could be evaluated; 4 Thyroid stimulating hormone (TSH)-containing adenomas, 2 TSH- and prolactin (PRL)-containing adenomas, 1 PRL-containing adenoma and 6 all-negative adenomas that did not react to any of the examined anti-hormone antibodies. The most common type were TSH-containing pituitary adenomas (a total of 6 cases; 46%) which occurred exclusively in females; the tumors consisted mainly of basophilic or amphophilic cells with bizarre nuclei and neoplastic cells and were positive for TSH in varying degrees. The TSH-containing pituitary adenomas, a characteristic of this mutant rat, could be induced by genetically-controlled hypothyroidism in dwarf rats, with higher sensitivity to possible disturbance of the pituitary-thyroid axis in females.

Morphometric analysis of progressive changes in hereditary cerebellar cortical degenerative disease (abiotrophy) in rabbits caused by abnormal synaptogenesis.

We previously investigated rabbit hereditary cerebellar cortical degenerative disease, called cerebellar cortical abiotrophy in the veterinary field, and determined that the pathogenesis of this disease is the result of failed synaptogenesis between parallel fibers and Purkinje cells. In this study, longitudinal changes in the development and atrophy of the cerebellum of rabbits with hereditary abiotrophy after birth were morphometrically examined (postnatal day [PD] 15 and 42) using image analysis. Although development of the cerebellum in rabbits with abiotrophy was observed from PD 15 to PD 42, the growth rate of the cerebellum was less than that in normal rabbits. In rabbits with abiotrophy, the number of granular cells undergoing apoptosis was significantly higher at PD 15 and dramatically decreased at PD 42. The number of granular cells did not increase from PD 15 to 42. The synaptogenesis peak at PD 15 occurred when the largest number of apoptotic granular cells in rabbits with abiotrophy was observed. Although 26% to 36% of parallel fiber terminals formed synaptic junctions with Purkinje cell spines, the remainder did not at PD 15 and 42. The rate of failure of synaptogenesis in the present study might be specific to this case of abiotrophy. Morphometric analysis revealed detailed changes in development and atrophy in animals with postnatal cerebellar disease occurring soon after birth.

Spontaneous and bilateral necrosis of the femoral head in a young experimental beagle dog.

This report describes the pathological characterizations of a rare case of necrosis of the femoral head that was spontaneous, bilateral, avascular and nontraumatic. A 14-month-old beagle dog was presented with pain in the hind limbs. At necropsy, the articular surface in the bilateral femoral head was markedly irregular. There were no gross abnormalities other than in the hip joints. Microscopically, a wide range of trabecular bone necrosis localized in the subchondral area was observed in both femoral heads. In the right femoral head, fibrosis and proliferative vessels were noted in the subchondral area. The articular cartilage was thickened irregularly, but there was no evidence of cartilage necrosis. The bone marrow adjacent to the affected area showed severe depression. In the metaphysis, atrophic bone marrow, but not bone necrosis, was observed. This was a rare case of spontaneous necrosis of the femoral head in an experimental beagle dog.

Histological dermal changes caused by preparation and application procedures in percutaneous dose toxicity studies in dogs, rabbits and rats.

We reevaluated histological slides of dorsal skin in control animals from past percutaneous dose toxicity studies using dogs, rabbits and rats to provide background data concerning histological changes related to preparation and application procedures and vehicles or embrocations of every variety. Acanthosis, dermal or perifollicular inflammatory cell infiltration in dogs; hyperkeratosis, acanthosis, dermal inflammatory cell infiltration or hemorrhage in rabbits; and acanthosis, dermal inflammatory cell infiltration, crust or foreign body granuloma in rats were present as procedure-related underlying histological changes in the control animals. Four mechanical acts, (1) rubbing with gauze to remove an administered substance for reapplication, (2) use of a taut bandage to avoid slipping from the application site, (3) peeling a patch off as a preparation procedure for reapplication, and (4) clipping or shaving, were considered to cause injury to the skin. The degree of influence of the various application procedures was found to be as follows: sham, lotion < cream < ointment and tape in dogs; untreated control, sham < lotion < tape and poultice in rabbits; and sham, sodium carboxymethylcellulose < olive oil and lotion < ointment and tape in rats. The degree of ointment influence on rabbits is equivocal.