Use of Both Fluoro-Jade B and Hematoxylin and Eosin to Detect Cell Death in the Juvenile Rat Brain Exposed to NMDA-Receptor Antagonists or GABA-Receptor Agonists in Safety Assessment.

Administration of pediatric anesthetics with N-methyl D-aspartate (NMDA)-receptor antagonist and/or γ-aminobutyric acid (GABA) agonist activities may result in neuronal degeneration and/or neuronal cell death in neonatal rats. Evaluating pediatric drug candidates for this potential neurotoxicity is often part of overall preclinical new drug development strategy. This specialized assessment may require dosing neonatal rats at postnatal day 7 at the peak of the brain growth spurt and evaluating brain tissue 24 to 48 hours following dosing. The need to identify methods to aid in the accurate and reproducible detection of lesions associated with this type of neurotoxic profile is paramount for meeting the changing needs of neuropathology assessment and addressing emerging challenges in the neuroscience field. We document the use of Fluoro-Jade B (FJB) staining, to be used in conjunction with standard hematoxylin and eosin staining, to detect acute neurodegeneration and neuronal cell death that can be caused by some NMDA-receptor antagonists and/or GABA agonists in the neonatal rat brain. The FJB staining is simple, specific, and sensitive and can be performed on brain specimens from the same cohort of animals utilized for standard neurotoxicity assessment, thus satisfying animal welfare recommendations with no effect on achievement of scientific and regulatory goals.

Stereology of the Peripheral Nervous System.

Qualitative histopathology has been the gold standard for evaluation of morphological tissue changes in all organ systems, including the peripheral nervous system. However, the human eye is not sensitive enough to detect small changes in quantity or size. Peripheral nervous system toxicity can manifest as subtle changes in neuron size, neuron number, axon size, number of myelinated or unmyelinated axons, or number of nerve fibers. Detection of these changes may be beyond the sensitivity of the human eye alone, necessitating quantitative approaches in some cases. Although 2-dimensional (2D) histomorphometry can provide additional information and is more sensitive than qualitative evaluation alone, the results are not always representative of the entire tissue and assumptions about the tissue can lead to bias, or inaccuracies, in the data. Design-based stereology provides 3D estimates of number, volume, surface area, or length, and stereological principles can be applied to peripheral nervous system tissues to obtain accurate and precise estimates, such as neuron number and size, axon number, and total intraepidermal nerve fiber length. This review describes practical stereological approaches to 3 compartments of the peripheral nervous system: ganglia, peripheral nerves, and intraepidermal nerve fibers.

Histologic Features of Postnatal Development of Immune System Organs in the Sprague-Dawley Rat.

The immune system of the rat undergoes substantial functional and morphological development during the postnatal period. Some aspects of this development are genetically predetermined, while other aspects depend on environmental influences. Detailed information on postnatal development is important in the interpretation of histopathologic findings in juvenile toxicology and pubertal assay studies, as well as other studies conducted in juvenile rats. Studies were conducted to provide detailed characterization of histologic features of the major functional compartments of immune system organs in male and female Sprague-Dawley rats at weekly intervals from the day of birth through postnatal day (PND) 42. Maturation of the individual immune system organs occurred across a range of ages, with histologic maturation of T-cell-related compartments typically occurring prior to maturation of B-cell-related compartments. The sequence of histologic maturation was bone marrow and thymus on PND 14, mesenteric lymph node on PND 21, Peyer's patches and bronchus-associated lymphoid tissue on PND 28, mandibular lymph node, nasopharynx-associated lymphoid tissue, and diffuse mucosal mononuclear cell population of small intestine on PND 35, and spleen on PND 42. An estimation of functional maturation can be made based on the morphological indications of maturity of each compartment of immune system organs, but histologic indications of maturity do not confirm functional immunocompetence.

The metrial gland in the rat and its similarities to granular cell tumors.

Metrial glands are normal structures located in the mesometrial triangle of the pregnant rat uterus from gestational day (GD) 8 through termination of pregnancy. Metrial glands are composed of a dynamic mixed cell population of granulated metrial gland (GMG) cells, endometrial stromal cells, trophoblasts, blood vessels, and fibroblasts. Collections of similar cells may be seen in association with pseudopregnancy and other hormonal disturbances. Granulated metrial gland cells are the hallmark cell of the metrial gland. They are bone-marrow-derived, perforin-positive, natural killer cells that proliferate in the pregnant uterus. Understanding the normal histogenesis of the metrial gland and recognizing the possible existence of GMG cells and a reactive metrial gland in the nonpregnant state are important when examining any uterine lesion that contains granulated cells. This report demonstrates that the cellular composition, morphology, and immunohistochemical staining profile of normal metrial glands are similar to reported granular cell neoplasms in rats and mice. The possibility of a non-neoplastic lesion involving the metrial gland should be considered when proliferative lesions involving granulated cells are observed in the uterus of mice and rats from nonclinical toxicity studies. Positive immunohistochemical staining for perforin and S100 would assist in the classification of such lesions as a reactive metrial gland or decidual reaction.

Dose-dependent effects on cell proliferation, seminiferous tubules, and male germ cells in the fetal rat testis following exposure to di(n-butyl) phthalate.

UNLABELLED: Adult male rats gestationally exposed to di(n-butyl)phthalate (DBP) have dysgenetic testes characterized by seminiferous epithelial degeneration, clustering of Leydig cells, and decreased spermatogenesis. Cell proliferation and apoptosis are key processes regulating development of the testis, and alterations in these processes may underlie testicular dysgenesis. OBJECTIVE: To determine whether gestational exposure to DBP affects cell proliferation and apoptosis in the developing rat testis. DESIGN: Pregnant dams were exposed to different dose levels of DBP in mid-gestation and cellular outcomes in fetal and early postnatal testes were assayed by histological and morphometric approaches. RESULTS: Gestational exposure to high dose DBP inhibited proliferation of fetal testicular somatic cells but did not affect apoptosis. Exposed fetal testes had a smaller volume and decreased cell numbers, with decreases in both the tubular and interstitial cell populations. A reduction was observed in the testis volume and altered seminiferous tubule morphometry at > or =50 mg/kg/d, and a decreased testicular cell number at > or =30 mg/kg/d DBP. The number of multinucleated gonocytes in DBP-exposed fetal testes increased after exposure to > or =100 mg/kg/d. The number of proliferating cells in the DBP-exposed testis rapidly rose after birth (when exposure stopped), and the testis volume and the total cell number was comparable to control by postnatal day 2. CONCLUSION: DBP reversibly inhibits proliferation of somatic cells in the fetal rat testis. Decreased proliferation, rather than increased apoptosis, is the underlying mechanism of altered fetal development of DBP-exposed seminiferous tubules contributing to testicular dysgenesis.

Ovarian follicle counts using proliferating cell nuclear antigen (PCNA) and semi-automated image analysis in rats.

Ovarian follicle counting is a method to assess ovarian toxicity in reproductive toxicity studies in rats. Although ovarian follicle counting has been traditionally performed manually on hematoxylin and eosin (H&E)-stained sections, the use of immunohistochemical methods, including human cytochrome P450 1B1 (CYP1B1) and proliferating cell nuclear antigen (PCNA), have been used to enhance the visibility of the primordial and primary follicles to facilitate manual counting. In this study, serial sections from both ovaries from ten 3-month-old female Sprague Dawley rats were stained using routine H&E and immunohistochemistry for PCNA. Counting of primordial and primary follicles was performed manually using these two stains and by semi-automated image analysis of PCNA-stained slides. Although manual counting of PCNA-stained slides is preferable to manual counting of H&E-stained slides, manual counting involves variability between individual counters. Semi-automated image analysis of PCNA-stained slides yields an accurate and consistent count of these primordial/primary follicles and eliminates variability between individual counters.

Activation of peroxisome proliferator-activated receptor alpha enhances apoptosis in the mouse liver.

Chronic exposure to peroxisome proliferators (PPs) leads to increased incidence of liver tumors in rodents. Liver tumor induction is thought to require increased hepatocyte proliferation and suppression of apoptosis. Transcript profiling showed increased expression of proapoptotic genes and decreased expression of antiapoptotic genes in the livers of mice exposed to the PP WY-14,643 (WY). We tested the hypothesis that prior exposure to WY would increase susceptibility to apoptosis inducers such as Jo2, an antibody which activates the Fas (Apo-1/CD95) death pathway. When compared to their untreated counterparts, wild-type mice pretreated with WY exhibited increased caspase-3 activation and hepatocyte apoptosis following challenge with Jo2. Livers from WY-treated peroxisome proliferator-activated receptor alpha (PPARalpha)-null mice were resistant to the effects of Jo2. In the absence of Jo2 and detectable apoptosis, wild-type mice treated with WY exhibited increases in the activated form of caspase-9. As caspase-9 is a component of the apoptosome, we examined the expression of upstream effectors of apoptosome activity including members of the Bcl-2 family. The levels of the antiapoptotic Mcl-1 transcript and protein were significantly decreased by PPs. PPARalpha-null mice were also resistant to another treatment (concanavalin A) that induces hepatocyte apoptosis. These results (1) indicate that PPARalpha activation increases sensitivity of the liver to apoptosis and (2) identify a mechanism by which PPARalpha could serve as a pharmacological target in diseases where apoptosis is a contributing feature.

Exposure in utero to di(n-butyl) phthalate alters the vimentin cytoskeleton of fetal rat Sertoli cells and disrupts Sertoli cell-gonocyte contact.

Di(n-butyl) phthalate (DBP) is commonly used in personal care products and as a plasticizer to soften consumer plastic products. Male rats exposed to DBP in utero have malformations of the male reproductive tract and testicular atrophy characterized by degeneration of seminiferous epithelium and decreased sperm production. In the fetal testis, in utero exposure to DBP reportedly resulted in reduced testosterone levels, Leydig cell aggregates, and multinucleated gonocytes (MNG). We investigated whether exposure in utero to DBP affects rat fetal Sertoli cells and compromises interactions between Sertoli and germ cells in the developing testis. Histological examination showed that MNG occurred at low frequency in the normal fetal rat testis. Exposure in utero at the dose level of DBP above estimated environmental or occupational human exposure levels significantly increased the number of these abnormal germ cells. Postnatally, MNG exhibited aberrant mitoses and were detected at the basal lamina. MNG were not apoptotic in the fetal and postnatal rat testes, as indicated by TUNEL. Sertoli cells in DBP-exposed fetal testis had retracted apical processes, altered organization of the vimentin cytoskeleton, and abnormal cell-cell contacts with gonocytes. The effect of DBP on Sertoli cell morphology at the level of light microscopy was reversed after birth and cessation of exposure. Our data indicate that fetal Sertoli cells are targeted by exposure in utero to DBP and suggest that abnormal interactions between Sertoli and germ cells during fetal life play a role in the development of MNG.

Constitutive expression of peroxisome proliferator-activated receptor alpha-regulated genes in dwarf mice.

Defects in growth hormone secretion or signaling in mice are associated with decreased body weights (dwarfism), increased longevity, increased resistance to stress, and decreases in factors that contribute to cardiovascular disease and cancer. Peroxisome proliferators (PP) alter a subset of these changes in wild-type mice through activation of the nuclear receptor family member PP-activated receptor alpha (PPARalpha). We tested the hypothesis that an overlap in the transcriptional programs between untreated dwarf mice and PP-treated wild-type mice underlies these similarities. Using transcript profiling, we observed a statistically significant overlap in the expression of genes differentially regulated in control Snell dwarf mice (Pit-1dw) compared with phenotypically normal heterozygote (+/dw) control mice and those altered by the PP 4-chloro-6-(2,3-xylidino)-2-pyrimidinyl)thioacetic acid (WY-14,643) in +/dw mice. The genes included those involved in beta- and omega-oxidation of fatty acids (Acox1, Cyp4a10, Cyp4a14) and those involved in stress responses (the chaperonin, T-complex protein1epsilon) and cardiovascular disease (fibrinogen). The levels of some of these gene products were also altered in other dwarf mouse models, including Ames, Little, and growth hormone receptor-null mice. The constitutive increases in PPARalpha-regulated genes may be partly caused by increased expression of PPARalpha mRNA and protein as observed in the livers of control Snell dwarf mice. These results indicate that some of the beneficial effects associated with the dwarf phenotype may be caused by constitutive activation of PPARalpha and regulated genes.

Overlapping transcriptional programs regulated by the nuclear receptors peroxisome proliferator-activated receptor alpha, retinoid X receptor, and liver X receptor in mouse liver.

Lipid homeostasis is controlled in part by the nuclear receptors peroxisome proliferator (PP)-activated receptor alpha (PPARalpha) and liver X receptor (LXR) through regulation of genes involved in fatty acid and cholesterol metabolism. Exposure to agonists of retinoid X receptor (RXR), the obligate heterodimer partner of PPARalpha, and LXR results in responses that partially overlap with those of PP. To better understand the gene networks regulated by these nuclear receptors, transcript profiles were generated from the livers of wild-type and PPARalpha-null mice exposed to the RXR pan-agonist 3,7-dimethyl-6S,7S-methano, 7-[1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphth-7-yl]-2E,4E-heptadienoic acid (AGN194,204) or the PPAR pan-agonist WY-14,643 (WY; pirinixic acid) and compared with the profiles from the livers of wild-type and LXRalpha/LXRbeta-null mice after exposure to the LXR agonist N-(2,2,2-trifluoroethyl)-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethylethyl)phenyl] sulfonamide (T0901317). All 218 WY-regulated genes altered in wild-type mice required PPARalpha. Remarkably, approximately 80% of genes regulated by AGN194,204 required PPARalpha including cell-cycle genes, consistent with AGN-induced hepatocyte proliferation having both PPARalpha-dependent and -independent components. Overlaps of approximately 31 to 62% in the transcript profiles of WY, AGN194,204, and T0901317 required PPARalpha and LXRalpha/LXRbeta for statistical significance. Ofthe 50 overlapping genes regulated by T0901317 and WY, all but one were regulated in a similar direction. These results 1) identify new transcriptional targets of PPARalpha and RXR important in regulating lipid metabolism and liver homeostasis, 2) illustrate the importance of PPARalpha in regulation of gene expression by a prototypical PP and by an RXR agonist, and 3) provide support for an axis of PPARalpha-RXR-LXR in which agonists for each nuclear receptor regulate an overlapping set of genes in the mouse liver.

Role of the peroxisome proliferator-activated receptor alpha (PPARalpha) in responses to trichloroethylene and metabolites, trichloroacetate and dichloroacetate in mouse liver.

Trichloroethylene (TCE) is an industrial solvent and a widespread environmental contaminant. Induction of liver cancer in mice by TCE is thought to be mediated by two carcinogenic metabolites, dichloroacetate (DCA) and trichloroacetate (TCA). TCE is considered to be a relatively weak peroxisome proliferator (PP), a group of rodent hepatocarcinogens that cause adaptive responses in liver through the PP-activated receptor alpha (PPARalpha). The objectives of this study were to determine whether effects of TCE, TCA and DCA in the liver associated with carcinogenesis are mediated by PPARalpha. Male wild-type and PPARalpha-null mice were given TCE by gavage for 3 days or 3 weeks; TCA or DCA were given in the drinking water for 1 week. Increases in relative liver and kidney weights by TCE were dependent on PPARalpha whereas liver weight increases by DCA were PPARalpha-independent. Dose-dependent increases in hepatocyte proliferation observed in wild-type mice after TCE exposure as determined by BrdU-labeling of hepatocytes were PPARalpha-dependent. Transcript profiling using macroarrays containing approximately 1200 genes showed that 93% (40 out of 43) of all expression changes observed in wild-type mice upon TCE exposure were dependent on PPARalpha and included known targets of PP (Cyp4a12, epidermal growth factor receptor) and additional genes involved in cell growth. Increases in enzymes that catalyze beta- and omega-oxidation of fatty acids were dependent on PPARalpha after exposure to TCE, TCA or DCA. TCE altered a unique set of genes in the livers of PPARalpha-null mice compared to wild-type mice including those that respond to different forms of stress. These data support the hypothesis that PPARalpha plays a dominant role in mediating the effects associated with hepatocarcinogenesis upon TCE exposure.

The transcriptional response to a peroxisome proliferator-activated receptor alpha agonist includes increased expression of proteome maintenance genes.

The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha), in addition to regulating lipid homeostasis, controls the level of tissue damage after chemical or physical stress. To determine the role of PPARalpha in oxidative stress responses, we examined damage after exposure to chemicals that increase oxidative stress in wild-type or PPARalpha-null mice. Primary hepatocytes from wild-type but not PPARalpha-null mice pretreated with the PPAR pan-agonist WY-14,643 (WY) were protected from damage to cadmium and paraquat. The livers from intact wild-type but not PPARalpha-null mice were more resistant to damage after carbon tetrachloride treatment. To determine the molecular basis of the protection by PPARalpha, we identified by transcript profiling genes whose expression was altered by a 7-day exposure to WY in wild-type and PPARalpha-null mice. Of the 815 genes regulated by WY in wild-type mice (p < or = 0.001; > or =1.5-fold or < or =-1.5-fold), only two genes were regulated similarly by WY in PPARalpha-null mice. WY increased expression of stress modifier genes that maintain the health of the proteome, including those that prevent protein aggregation (heat stress-inducible chaperones) and eliminate damaged proteins (proteasome components). Although the induction of proteasomal genes significantly overlapped with those regulated by 1,2-dithiole-3-thione, an activator of oxidant-inducible Nrf2, WY increased expression of proteasomal genes independently of Nrf2. Thus, PPARalpha controls the vast majority of gene expression changes after exposure to WY in the mouse liver and protects the liver from oxidant-induced damage, possibly through regulation of a distinct set of proteome maintenance genes.

Mimetics of caloric restriction include agonists of lipid-activated nuclear receptors.

The obesity epidemic in industrialized countries is associated with increases in cardiovascular disease (CVD) and certain types of cancer. In animal models, caloric restriction (CR) suppresses these diseases as well as chemical-induced tissue damage. These beneficial effects of CR overlap with those altered by agonists of nuclear receptors (NR) under control of the fasting-responsive transcriptional co-activator, peroxisome proliferator-activated co-activator 1alpha (PGC-1alpha). In a screen for compounds that mimic CR effects in the liver, we found statistically significant overlaps between the CR transcript profile in wild-type mice and the profiles altered by agonists of lipid-activated NR, including peroxisome proliferator-activated receptor alpha (PPARalpha), liver X receptor, and their obligate heterodimer partner, retinoid X receptor. The overlapping genes included those involved in CVD (lipid metabolism and inflammation) and cancer (cell fate). Based on this overlap, we hypothesized that some effects of CR are mediated by PPARalpha. As determined by transcript profiling, 19% of all gene expression changes in wild-type mice were dependent on PPARalpha, including Cyp4a10 and Cyp4a14, involved in fatty acid omega-oxidation, acute phase response genes, and epidermal growth factor receptor but not increases in PGC-1alpha. CR protected the livers of wild-type mice from damage induced by thioacetamide, a liver toxicant and hepatocarcinogen. CR protection was lost in PPARalpha-null mice due to inadequate tissue repair. These results demonstrate that PPARalpha mediates some of the effects of CR and indicate that a pharmacological approach to mimicking many of the beneficial effects of CR may be possible.

Role of the peroxisome proliferator-activated receptor alpha in responses to diisononyl phthalate.

Diisononyl phthalate (DINP) is a compound widely used as a plasticizer in the production of polyvinyl chloride products. Chronic exposure to DINP leads to liver cancer in rats and mice. Many phthalates are considered to be relatively weak peroxisome proliferators (PP), a group of rodent hepatocarcinogens that cause a variety of adaptive responses in liver through the PP-activated receptor alpha (PPARalpha). The objectives of this study were to determine whether DINP-induced effects in the liver associated with carcinogenesis are mediated by PPARalpha and to identify novel gene targets of DINP. Male and female SV129 wild-type, SV129 PPARalpha-null, and B6C3F1 mice were administered DINP by gavage or in the feed. Transcript profile technology and reverse transcriptase (RT)-polymerase chain reaction (PCR) were used to identify gene targets. Dose-dependent increases in relative liver weights were dependent on PPARalpha in 10- or 12-week-old male and female mice and 30-week-old male mice. Female 30-week-old mice exhibited PPARalpha-independent increases in relative liver weights. Increases in hepatocyte proliferation, palmitoyl-CoA oxidase (PCO) activity, and levels of enzymes involved in beta- and omega-oxidation of fatty acids were shown to be dependent on PPARalpha. Five novel genes were shown to be altered in the livers of female wild-type mice after a 3-week exposure, but not in PPARalpha-null, mice. These genes included those involved in DNA repair and recombination (ATP-dependent helicase and Endonuclease III homolog), drug metabolism (Cyp2a4) and protein trafficking (FKBP-1, FKBP-13). An additional gene (Cyp2d9) was shown to be down-regulated in wild-type mice but up-regulated in PPARalpha-null mice indicating more complex regulation by PPARalpha and additional factors. These data support the hypothesis that PPARalpha plays a dominant role in mediating the effects associated with hepatocarcinogenesis after DINP exposure.