Paul Howroyd

Decreased Longevity and Enhancement of Age-Dependent Lesions in Mice Lacking the Nuclear Receptor Peroxisome Proliferator-Activated Receptor α (PPARα)

The nuclear receptor peroxisome proliferator-activated receptor α (PPARα) is activated by peroxisome proliferators (PP), a large class of structurally diverse xenobiotic chemicals, hypolipidemic drugs, and endogenous lipids. PPARα alters the transcriptional programs of genes whose functions include lipid metabolism, inflammation, cell fate, and stress responses in liver, heart, kidney, and skin. Many of these genes are also under control of PPARα in the absence of exogenous peroxisome proliferator exposure. Mice that lack PPAR α (PPARα-null mice) exhibit a number of defects in lipid metabolism and accumulate lipids in the liver. Here, we compared the age-dependent lesions in the liver, kidney, and heart in PPARα-null mice with those observed in wild-type SV129 mice, in the absence of exogenous chemical exposure. Groups of mice were sacrificed, at 6, 12, 18, 21, or 24 months of age, or allowed to age until moribund or found dead. PPARα-null mice had decreased longevity, due to a variety of causes. Statistically significant differences in the occurrence of a number of lesions between strains was observed. Hepatocellular carcinomas and multiple hepatocellular adenomas occurred in PPARα-null mice but not wild type mice. Various nonneoplastic spontaneous aging lesions occurred at higher incidence, shorter latency, or increased severity in PPARα-null mice compared with wild-type mice. In the liver, these included vacuolated hepatocytes and sinusoidal cells and mixed cell inflammation. The kidneys of PPARα-null mice exhibited higher incidences and severities of cortical mineralization. Minimal myocardial mineralization occurred at a higher incidence in PPARα-null mice. Our results imply that PPARα delays the development of some spontaneous lesions associated with aging in the liver, kidney, and heart of SV129 mice.

Histopathological lesions following intramuscular administration of saline in laboratory rodents and rabbits

This review was undertaken to assess the nature and incidence of procedure-related changes in mice, rats and rabbits which received saline solution by intramuscular injection. Data were collected on the injection sites from 7 studies representing 152 animals. The original observations by the different study pathologists from both control and treated animals were evaluated in order to create a glossary of preferred terms to be used in toxicology studies. These standardized terms were then applied to changes observed in the saline-treated animals. The review showed that the most severe of the procedure-related lesions were only of a slight level. Two days post-injection, the local reactions were mainly composed of minimal infiltration by mononuclear cells (lymphocytes and macrophages) with occasional degeneration of myofibres. From 10 to 42 days post-injection, lesions showed regeneration of myofibres and some fibrosis. In rats, the number of injections at each site influenced inflammatory infiltrate and degenerative lesions.

Morphology of the Fetal Rat Testis Preserved in Different Fixatives

Histopathological examination of the testes of exposed fetuses and neonates is important in assessing the developmental effects of environmental toxins, including sex hormone modulators. Modified Davidson’s fluid (mDF) has been suggested as a superior substitute for Bouin’s fluid for fixation of adult animal testes. We compared the morphology of fetal rat testes stained with hematoxylin and eosin (H&E) or immunochemically after fixation in 10% neutral buffered formalin (NBF), Bouin’s fluid, or mDF. Fixation in mDF resulted in more sharply defined nuclear detail and better preservation of cellular cytoplasm on H&E-stained sections of rat testes on gestation day 19. Use of Bouin’s fluid did not allow satisfactory detection of apoptotic cells by fluorescent terminal deoxynucleotide transferase-mediated deoxy-UTP nick labeling. Staining with the immunoperoxidase system and the conventional chromogen diaminobenzidine tetrahydrochloride to visualize 5-bromo-2-deoxyuridine-positive cells demonstrated that the number of positive nuclei and intensity of staining were similar with all 3 fixatives. Immunostaining for cytoskeletal protein vimentin was more intense and provided better details of the Sertoli cell cytoplasm with formalin fixation than with mDF. Our study demonstrates that fixation in mDF provided better morphologic detail in the fetal rat testis compared with 10% NBF and Bouin’s fluid and illustrates the importance of establishing the correct fixation conditions for each immunostaining protocol.

Onset of Puberty and Normal Histological Appearances of the Reproductive Organs in Peripubertal Female Göttingen Minipigs

In preclinical studies, it is important to know whether the animals used are sexually mature or not. Precise data have not yet been published, however, about the histological features of the female reproductive organs during the peripubertal period or about the age of acquisition of sexual maturity in the minipig. The histological characteristics of the genital organs of female control minipigs from toxicology studies were described and, based on the presence of ovarian corpora lutea, used to assess the age at which maturity was reached. Only 50% of females can be considered mature at about 6.5 months old (a body weight of 11.8 kg), and 100% were not mature until about 7.5 months old (13.1 kg), although it is said that females reach sexual maturity at the age of approximately 5 months, by the time the body weight is about 10 to 12 kg. The uterine weights of mature females were higher than 94.4 g, whereas the maximum weight reached in the immature females was 55.2 g. In contrast, the differences between immature and mature ovarian weights were not significant. The histological appearance of the mature vagina in the various stages of the estrous cycle is also described.

Variability in Weight and Histological Appearance of the Prostate of Beagle Dogs Used in Toxicology Studies

This review was performed to assess variations in weight and histologic appearance of the prostate of untreated male beagle dogs between 23 and 108 weeks of age, from two breeding centers. Data from 125 control beagle dogs from twenty-seven regulatory toxicology studies were used. Age, terminal body weight, and prostate weight were analyzed. Prostate sections were examined microscopically, and histological changes—such as development of acini, amount of secretion, and patterns of dilation and inflammation—were recorded and graded when appropriate. The influence of age, terminal body weight, and source on the weight and histological appearance of the prostate, and the degree of interanimal variation were evaluated

Spontaneous testicular tubular hypoplasia/atrophy in the Göttingen minipig: a retrospective study.

The aim of this retrospective study was to assess the incidence and severity of tubular atrophy/hypoplasia in the testes of 104 control Göttingen minipigs aged 4.5 to 15 months. The finding was termed “tubular hypoplasia/atrophy” according to published descriptions for the dog. It included different microscopic changes that were considered part of a continuum, namely seminiferous epithelium vacuolation, presence of multinucleated germ cells in the tubular lumen, and decreased numbers (hypospermatogenesis) or absence of germ cells. This retrospective study demonstrates that tubular hypoplasia/atrophy is present in more than 70% of Göttingen minipigs and can occur at a marked severity in control animals. It correlated with a higher level of cell debris and a decrease in sperm content in the epididymides and with lower absolute and relative testes weights. There was no correlation with the weight of other sexual organs, total bodyweight, or age, which demonstrates that this change was not related to sexual immaturity. The distinction between this background finding and a compound-related effect could be challenging for the pathologist.

Apparent Alveolar Bronchiolar Tumors Arising in the Mediastinum of F344 Rats

Rare tumors were observed in chronic studies in F-344 rats that were purely or largely free in the mediastinal cavity, yet had the histological architecture of alveolar bronchiolar tumors. They had originally been diagnosed as either pulmonary alveolar bronchiolar tumors, mediastinal mesotheliomas, or thymomas. The authors described these tumors, estimated the fraction of thoracic tumors that they represented, and carried out a preliminary immunohistochemical investigation of whether they were of pulmonary or mesothelial origin. Sections of 715 thoracic tumors originally diagnosed as alveolar bronchiolar tumors, mesotheliomas, or thymomas from control or treated F-344 rats in NTP two-year studies were reviewed. Thirty (4%) were found to be purely or largely mediastinal, yet to have an alveolar bronchiolar histological pattern. A subset of these tumors and some typical intrapulmonary alveolar bronchiolar carcinomas and pleural mesotheliomas were immunostained for Clara cell secretory protein (CCSP), β-tubulin IV, and Wilm’s tumor 1 susceptibility gene products (WT1). The tumors with the histological architecture of alveolar bronchiolar tumors immunostained positive for CCSP and negative for WT1, implying they might have been of alveolar bronchiolar origin, despite their predominantly mediastinal location, although more certain identification would require the use of a larger panel of antibodies.

P13: Variation in weight and histological appearance with maturity of the prostate in control beagle dogs used in preclinical studies from two suppliers

Sexual immaturity of dogs can hamper accurate identification of compound-related changes. Knowledge of anatomical variations and development will allow accurate interpretation of observed changes. The aim of this work was to investigate the variation with age of the microscopic appearance of the testis and prostate in control beagle dogs from two sources. Data were extracted from the records of toxicity studies undertaken with control male Beagle dogs obtained from two suppliers, Marshall Farms USA and Harlan France, at MDS between 2001 and 2007. This represented 125 male Beagle dogs, from 23 to 108 weeks of age. HE stained sections of the prostate and testes were examined for all dogs. The degree of acinar development, the absence or presence of secretion in the prostate, and testicular maturity were recorded. We observed that the prostatic secretion increased with the age of dogs, in parallel to the prostatic development and testicular maturity. Moreover a clear difference was observed between suppliers: Acinar development was complete by 31 weeks of age in Harlan dogs against 43 weeks of age in Marshall dogs. This correlated with the testicular maturity. These data were consistent with the literature. Spontaneous variations in the morphology of the prostate may influence the assessment of toxicological data. It is important to be aware of sexual maturity, and to know the normal age-related variations of the prostates histological appearance in the species used.