Timed-pregnant Rats Research Articles

Dysregulation of CITED2 in abnormal lung development in the nitrofen rat model.

CITED2 both modulates lung, heart and diaphragm development. The role of CITED2 in the pathogenesis of congenital diaphragmatic hernia (CDH) is unknown. We aimed to study CITED2 during abnormal lung development in the nitrofen model. Timed-pregnant rats were given nitrofen on embryonic day (E) 9 to induce CDH. Fetal lungs were harvested on E15, 18 and 21. We performed RT-qPCR, RNAscope™ in situ hybridization and immunofluorescence staining for CITED2. We observed no difference in RT-qPCR (control: 1.09 ± 0.22 and nitrofen: 0.95 ± 0.18, p = 0.64) and in situ hybridization (1.03 ± 0.03; 1.04 ± 0.03, p = 0.97) for CITED2 expression in E15 nitrofen and control pups. At E18, CITED2 expression was reduced in in situ hybridization of nitrofen lungs (1.47 ± 0.05; 1.14 ± 0.07, p = 0.0006), but not altered in RT-qPCR (1.04 ± 0.16; 0.81 ± 0.13, p = 0.33). In E21 nitrofen lungs, CITED2 RNA expression was increased in RT-qPCR (1.04 ± 0.11; 1.52 ± 0.17, p = 0.03) and in situ hybridization (1.08 ± 0.07, 1.29 ± 0.04, p = 0.02). CITED2 protein abundance was higher in immunofluorescence staining of E21 nitrofen lungs (2.96 × 109 ± 0.13 × 109; 4.82 × 109 ± 0.25 × 109, p < 0.0001). Our data suggest that dysregulation of CITED2 contributes to abnormal lung development of CDH, as demonstrated by the distinct spatial-temporal distribution in nitrofen-induced lungs.

Abstract P426: Novel Peptide B7-33 Demonstrates Efficacy In Rat Models Of Preeclampsia

Background: Preeclampsia (PreE) is a hypertensive pregnancy disorder, which occurs in approximately 10% of all gestations. The literature suggests potential therapeutic role of H2 relaxin in PreE. Due to the complex insulin-like structure of relaxin (A- and B- chains, 53 amino acids, 3 disulfide bonds), a novel H2 relaxin B-chain-only peptide variant B7-33 (27 amino acids without any disulfide bonds) has recently been developed. Objective: This single-chain peptide displayed equivalent efficacy to the natural H2 relaxin in several functional assays both in vitro and in vivo . The Aim of this study is to evaluate whether B7-33 attenuates preE syndrome in rat models of PreE. Methods: Rat Model 1. The efficacy of B7-33 was evaluated in the Reduction of Uterine Perfusion Pressure (RUPP) model as described previously. RUPP rats are randomly assigned to 4 groups (N=8/group): 1) vehicle, 2) B733; 3) B733-Fc; and 4) B733-HSA. Rats are dosed twice weekly (i.v.) from GD10 to GD 20. Rat Model 2. Timed-pregnant rats are used, and the model is conducted as previously described. DOCA and saline administration begin at approximately GD2. Three groups of animals are studied (n=8 per group). 1) normal pregnant rats; 2) pregnant animals injected (i.p.) initially with 12.5 mg of DOCA, followed by a weekly injection of 6.5 mg, and whose drinking water was replaced with 0.9% saline; 3) rats administered DOCA and saline as for Group 2, and given B733 35 ug i.p. biweekly from GD10-20. BP, proteinuria and inflammatory markers were evaluated. Statistical comparisons were performed using analysis of variance with Duncan’s post hoc test. Results: RUPP rats have increased MAP, plasma TNF-α, and plasma sFlt-1 along with decreased NO index compared to normal pregnancy. Treatment with B733 and B7-33 fusion proteins lowers MAP, TNF-α, and sFlt-1 back to that of normal pregnancy. B7-33 data are consistent with earlier data with serelaxin in the RUPP model. All fusions tested ameliorate hypertension in the RUPP animals. B7-33 normalizes BP and proteinuria in the DOCA rat model of preE. Conclusion: Both B7-33 and B7-33 fusion proteins attenuate preE syndrome in RUPP and DOCA rat models of PreE. We conclude that B7-33 is an ideal candidate for development as a novel therapeutic in preE.

Simulated gastric hydrolysis and developmental toxicity of dimethyltin bis(2-ethylhexylthioglycolate) in rats.

Dimethyltin dichloride is used as the putative toxophore for dimethyltin bis-alkylthio esters in a read-across approach. Recent chemical and toxicological investigations challenges this read across as data on dioctyltin bis(2-ethylhexyl thioglycolate) and dibutyltin bis(2-ethylhexyl thioglycolate) showed the dialkyltin thioglycolates do not generate dialkyltin dichloride. Results obtained by 119Sn-NMR spectroscopy demonstrated that dimethyltin bis(2-ethylhexyl thioglycolate), the smallest commercially manufactured dialkyltin thioester molecule of this kind, hydrolyzed to dimethyltin chloro-(2-ethylhexyl) thioglycolate under simulated gastric conditions. These studies did not detect dimethyltin dichloride. Dimethyltin bis(2-ethylhexyl thioglycolate) was administered orally to timed-pregnant Wistar-Han rats in an Arachis oil vehicle at 5, 10, and 25mg/kg/day [Gestation Day 6 (GD6) through GD20] with no maternal deaths observed. At 25mg/kg/day treatment statistically significant reductions occurred in feed consumption (-9%), maternal body weight (-2.4%) and adjusted maternal weight gain (-68%). There were no adverse gestational findings. Maternal thymus weight was significantly reduced in rats at 25mg/kg in the absence of changes in hormone levels of T3, T4 or TSH. There were no effects on fetal growth, no dose-dependent pattern of external, visceral, or skeletal malformations and no toxicologically relevant increase in anatomical variations at any dose group. Based on the obtained experimental data it is concluded that dimethyltin bis(2-ethylhexyl thioglycolate) forms dimethyltin chloro-(2-ethylhexyl thioglycolate), not dimethyltin dichloride, in the stomach environment at pH 1.2, and dimethyltin bis(2-ethylhexyl thioglycolate) was not teratogenic nor fetotoxic in rats. The maternal NOAEL was 10mg/kg/day, and the developmental NOAEL was 25mg/kg/day, the high dose. The maternal LOAEL was 25mg/kg/day based on decreased food consumption, lower adjusted mean body weight gain and reduced maternal thymus weight.

Sexually concordant and dimorphic transcriptional responses to maternal trichloroethylene and/or N-acetyl cysteine exposure in Wistar rat placental tissue

Numerous Superfund sites are contaminated with the volatile organic chemical trichloroethylene (TCE). In women, exposure to TCE in pregnancy is associated with reduced birth weight. Our previous study reported that TCE exposure in pregnant rats decreased fetal weight and elevated oxidative stress biomarkers in placentae, suggesting placental injury as a potential mechanism of TCE-induced adverse birth outcomes. In this study, we investigated if co-exposure with the antioxidant N-acetylcysteine (NAC) attenuates TCE exposure effects on RNA expression. Timed-pregnant Wistar rats were exposed orally to 480 mg TCE/kg/day on gestation days 6–16. Exposure of 200 mg NAC/kg/day alone or as a pre/co-exposure with TCE occurred on gestation days 5–16 to stimulate antioxidant genes prior to TCE exposure. Tissue was collected on gestation day 16. In male and female placentae, we evaluated TCE- and/or NAC-induced changes to gene expression and pathway enrichment analyses using false discovery rate (FDR) and fold-change criteria. In female placentae, exposure to TCE caused significant differential expression 129 genes while the TCE+NAC altered 125 genes, compared with controls (FDR< 0.05 + fold-change >1). In contrast, in male placentae TCE exposure differentially expressed 9 genes and TCE+NAC differentially expressed 35 genes, compared with controls (FDR< 0.05 + fold-change >1). NAC alone did not significantly alter gene expression in either sex. Differentially expressed genes observed with TCE exposure were enriched in mitochondrial biogenesis and oxidative phosphorylation pathways in females whereas immune system pathways and endoplasmic reticulum stress pathways were differentially expressed in both sexes (FDR<0.05). TCE treatment was differentially enriched for genes regulated by the transcription factors ATF6 (both sexes) and ATF4 (males only), indicating a cellular condition triggered by misfolded proteins during endoplasmic reticulum stress. This study demonstrates novel genes and pathways involved in TCE-induced placental injury and showed antioxidant co-treatment largely did not attenuate TCE exposure effects.

Impact of in Utero Rat Exposure to 17Alpha-Ethinylestradiol or Genistein on Testicular Development and Germ Cell Gene Expression.

Although the decline in male fertility is believed to partially result from environmental exposures to xenoestrogens during critical developmental windows, the underlying mechanisms are still poorly understood. Experimental in utero exposures in rodents have demonstrated the negative impact of xenoestrogens on reproductive development, long-term adult reproductive function and offspring health. In addition, transcriptomic studies have demonstrated immediate effects on gene expression in fetal reproductive tissues, However, the immediate molecular effects on the developing germ cells have been poorly investigated. Here, we took advantage of a transgenic rat expressing the green fluorescent protein specifically in germ cells allowing purification of perinatal GFP-positive germ cells. Timed-pregnant rats were exposed to ethinylestradiol (EE2, 2 μg/kg/d), genistein (GE, 10 mg/kg/d) or vehicle by gavage, from gestational days (GD) 13–19; testes were sampled at GD20 or post-natal (PND) 5 for histological analysis and sorting of GFP-positive cells. While EE2-exposed females gained less weight during treatment compared to controls, neither treatment affected the number of pups per litter, sex ratio, anogenital distance, or body and gonadal weights of the offspring. Although GE significantly decreased circulating testosterone at GD20, no change was observed in either testicular histology or germ cell and sertoli cell densities. Gene expression was assessed in GFP-positive cells using Affymetrix Rat Gene 2.0 ST microarrays. Analysis of differentially expressed genes (DEGs) (p < 0.05; fold change 1.5) identified expression changes of 149 and 128 transcripts by EE2 and GE respectively at GD20, and 287 and 207 transcripts at PND5, revealing an increased effect after the end of treatment. Only about 1% of DEGs were common to both stages for each treatment. Functional analysis of coding DEG revealed an overrepresentation of olfactory transduction in all groups. In parallel, many non-coding RNAs were affected by both treatments, the most represented being small nucleolar and small nuclear RNAs. Our data suggest that despite no immediate toxic effects, fetal exposure to xenoestrogens can induce subtle immediate changes in germ cell gene expression. Moreover, the increased number of DEGs between GD20 and PND5 suggests an effect of early exposures with latent impact on later germ cell differentiation.

Effects of prenatal opioid exposure and early life adversity on opioid‐induced antinociception

Diagnoses of maternal opioid use disorder (MOUD) at delivery increased more than 500% between 1999 and 2017 in the United States. Today, cases of in utero opioid exposure due to MOUD exceed cases of the most common birth defects, including cleft lip, cleft palate, clubfoot, and Down syndrome, combined. Children exposed to opioids in utero are at increased risk of adverse childhood experiences (ACEs) due to parental care being compromised by SUDs. These ACEs include abuse, neglect, loss of a parent from death or incarceration, parental divorce, and household domestic violence. Altered sensitivity to opioid analgesia later in life is one potential outcome shared by in utero opioid exposure and ACEs that may contribute to poorly controlled pain and increased risk of opioid addiction. Studies using rat models of prenatal opioid exposure (POE) or early life adversity (ELA) have shown that each decreases opioid‐induced antinociception long after exposure has ceased. However, the contributions of POE and ELA, alone and in combination, to opioid analgesic response in humans remains unknown. In this study, we used a rodent model of combined POE and ELA to disambiguate their long‐term effects more quickly and with greater experimental control than human studies. We hypothesized that combined POE and ELA will decrease morphine‐induced antinociception in adolescence relative to POE or ELA alone. We used a two‐by‐two between‐subjects factorial design in which timed‐pregnant Long‐Evans rats were exposed to morphine (15 mg/kg/day; “POE” group) or saline (“vehicle” group) via subcutaneous osmotic minipump from gestation day 9 until delivery. Litters were fostered to untreated dams then randomly assigned to normal housing or ELA conditions using a limited bedding and nesting procedure from postnatal days 3‐11. Morphine‐induced antinociception was measured between postnatal days 30 and 54 using a warm‐water tail withdrawal (WWTW) assay. Cumulative dose‐response of morphine‐induced antinociception was determined on test day 0 (1.0‐30 mg/kg morphine, s.c.) and test day 13 (3.0‐100 mg/kg, s.c.). On test days 1‐12, rats were dosed with 18 mg/kg, s.c., every 12 hours, and WWTW was conducted on odd test days. Interesting, preliminary results of this ongoing study indicate that morphine‐induced antinociception was enhanced on test day 0 in POE + normal‐housed males (n = 2). These rats exhibited 100% maximum possible effect (MPE) and significantly higher potency (mean ED50=5.96 mg/kg; 95% CI = ‐12.12, 24.05), while mean MPE for the other groups (n=2‐3) was less than 70% and mean ED50&gt;17 mg/kg. Males exhibited partial tolerance throughout the 12‐day chronic morphine treatment period. In the POE + normal‐housed males, MPE decreased from 100% on test day 1 to 58% on test day 11. For the remaining groups, MPE decreased from 44‐57% on test day 1 to 12‐23% on test day 11. Morphine was more potent in the POE + normal‐housed males on test day 13 (mean ED50=66.21 mg/kg; 95% CI = ‐281.6, 414) than in other males (mean ED50&gt;104.4 mg/kg). No clear group effect was observed in females. These unexpected results suggest a complex sex‐dependent interaction between POE and ELA on opioid antinociception.

Trichloroethylene modifies energy metabolites in the amniotic fluid of Wistar rats

Exposure to trichloroethylene (TCE), an industrial solvent, is associated with several adverse pregnancy outcomes in humans and decreased fetal weight in rats. However, effects of TCE on energy metabolites in amniotic fluid, which have associations with pregnancy outcomes, has not been published previously. In the current exploratory study, timed-pregnant Wistar rats were exposed to 480 mg TCE/kg/day via vanilla wafer or to vehicle (wafer) alone from gestational day (GD) 6–16. Amniotic fluid collected on GD 16 was analyzed for metabolites important in energy metabolism using short chain fatty acid and tricarboxylic acid plus platforms (N = 4 samples/sex/treatment). TCE decreased concentrations of the following metabolites in amniotic fluid for both fetal sexes: 6-phosphogluconate, guanosine diphosphate, adenosine diphosphate, adenosine triphosphate, and flavin adenine dinucleotide. TCE decreased fructose 1,6-bisphosphate and guanosine triphosphate concentrations in amniotic fluid of male but not female fetuses. Moreover, TCE decreased uridine diphosphate-D-glucuronate concentrations, and increased arginine and phosphocreatine concentrations, in amniotic fluid of female fetuses only. No metabolites were increased in amniotic fluid of male fetuses. Pathway analysis suggested that TCE altered folate biosynthesis and pentose phosphate pathway in both sexes. Using metabolite ratios to investigate changes within specific pathways, some ratio alterations, including those in arginine metabolism and phenylalanine metabolism, were detected in females only. Ratio analysis also suggested enzymes, including gluconokinase, as potential TCE targets. Together, results from this exploratory study suggest that TCE differentially modified energy metabolites in amniotic fluid based on sex. These findings may inform future studies of TCE reproductive toxicity.

Evidence of Anxiety, Depression and Learning Impairments following Prenatal Hypertension.

Background: Hypertensive disorders of pregnancy, such as Preeclampsia (PreE) and HELLP (hemolysis, elevated liver enzyme, low platelet) syndrome, affects approximately 5–10% of pregnancies and increases the risk of women developing disorders, such as anxiety or depression, in the postpartum period. Using preclinical rodent models, we set out to determine whether rats with a history of PreE or HELLP had evidence of anxiety, depression or cognitive impairment and whether immune suppression during pregnancy prevented these changes in mood and/or cognition. Methods: Timed-pregnant rats were infused with sFlt-1 and/or sEng to induce PreE or HELLP beginning on gestational day 12. After delivery, a battery of validated behavioral assays was used to assess post-partum depression, anxiety and learning. Results: There was no negative effect on maternal pup interaction due to PreE or HELLP; however, hypertensive dams spent more time immobile in the forced swim test (p < 0.0001). Hypertensive dams also spent less time in the open area of the open field (p = 0.001). There were no significant changes in recognition memory (p = 0.08); however, spatial learning was impaired in hypertensive dams (p = 0.003). Immobility time in the forced swim test was positively correlated with increased circulating S100B (p = 0.04), while increased time spent in the outer zones of the open field was negatively correlated with BDNF levels (p < 0.0001). Conclusion: The results from this study suggest that hypertensive pregnancy disorders are associated with depression, anxiety and learning impairments in the post-partum period.

Autism-Like Behavior in the Offspring of CYP11A1-Overexpressing Pregnant Rats.

Autism spectrum disorders (ASD) represent a complex group of neurodevelopmental disorders that are characterized by impaired social behavior and communication as well as repetitive behavior and restricted interests. Prenatal exposure to high levels of testosterone and preeclampsia are thought to be risk factors of ASD. We had previously reported that overexpression of the mitochondrial cholesterol side-chain cleavage enzyme (CYP11A1) could lead to both preeclampsia-like symptoms and increased testosterone levels in pregnant rats. In this study, we investigated the association between high CYP11A1 levels in pregnant rats and autism-like behavior in their offspring. Timed-pregnant Sprague-Dawley (SD) rats were injected with CYP11A1 gene-carrying adenoviruses on gestational day 8.5, and their offspring were then compared with those from timed-pregnant control SD rats. Compared with their control counterparts, the offspring of the CYP11A1-ovexpressing dams displayed more symptoms of anxiety and spent less time in social interactions and more time in self-grooming and rearing, all indicators of autism-like behavior. Sequencing of the transcriptome in primary microglia from the offspring of CYP11A1-overexpressing dams revealed that immune pathways were highly activated, and the gamma-aminobutyric acid type A (GABAA) receptor genes were among the top differentially expressed genes. Using primary microglia cultures generated from neonatal rats, tumor necrosis factor-alpha expression was found to be elevated in the cells transfected with CYP11A1-carrying adenoviruses. Additionally, the offspring of CYP11A1-overexpressing dams displayed dysregulated GABAA receptor expression. Taken together, these results suggest that abnormal CYP11A1 gene expression in pregnant rats could lead to microglial immune activation and dysregulated GABAA receptor expression in their offspring and thereby anxiety and autism-related behavior. Our study suggests that the pathways regulated by CYP11A1 could be promising preventative and therapeutic targets for ASD.

The role of perinatal testosterone on exploratory behavior, sexual motivation, and spatial working memory in adult rats

Aim: Sex differentiation occurs during the perinatal period (four days before and after birth) in the rat brain. The brain remains a female brain unless exposed to testosterone, which gets metabolized to 17β-estradiol and dihydrotestosterone. While aromatase converts testosterone to 17β-estradiol, 5α-reductase converts testosterone to dihydrotestosterone. 17β-estradiol exerts its effects by binding to an estrogen receptor, whereas dihydrotestosterone via an androgen receptor. The role of dihydrotestosterone and 17β-estradiol during the organizational phase of brain differentiation was tested using adult rat behaviors. The study sought to examine the effects of perinatal manipulations on adult rat behaviors. Materials and methods: Timed-pregnant rats received either the vehicle (5% ethanol and 95% sesame oil), exemestane (4 mg/kg/ml; aromatase inhibitor), or flutamide (20 mg/kg/ml; androgen receptor blocker) subcutaneously during the last four days of pregnancy. Pups from each group continued to receive their treatments during the first four days after birth. From postnatal day 65, these animals were subjected to open field, sexual motivation, and spatial working memory tests. Results: Male rats receiving flutamide exhibited exploratory behavior significantly more compared to exemestane and control groups. Male rats receiving exemestane had substantially higher plasma testosterone and displayed an increased interest in the estrus rat than the control and flutamide group. Conclusion: Androgen receptor blockade resulted in increased exploratory behavior in male rats where 17β-estradiol was testosterone's primary metabolite. The blocking of aromatase using exemestane resulted in higher levels of plasma testosterone and enhanced sexual motivation in male rats. Thus, 17β-estradiol and dihydrotestosterone's presence during the organizational phase is essential for normal socio-sexual and exploratory behaviors.

Linseed ameliorates renal apoptosis in rat fetuses induced by single or combined exposure to diesel nanoparticles or fenitrothion by inhibiting transcriptional activation of p21/p53 and caspase‐3/9 through pro‐oxidant stimulus

Gestational exposure to environmental pollutants can induce oxidative injury and apoptosis since the fetal organs are sensitively vulnerable to these chemicals. In this work, we have investigated the renal anti-apoptotic efficiency of linseed (LS) against the oxidative stress-mediated upregulation of the fetal apoptosis-related genes following the prenatal intoxication with diesel nanoparticles (DNPs) and/or fenitrothion (FNT). A fifty-six timed-pregnant rats were equally divided to eight groups; control, LS (20% in diet), DNPs (0.5 mg/kg by intratracheal inoculation), FNT (3.76 mg/kg by gavage), DNPs+FNT, LS + DNPs, LS + FNT, and LS + DNPs+FNT. The transmission electron microscope analysis revealed the spherical shape of diesel particles with a homogeneous nanosized range (20-92.3 nm) and the crystallinity was confirmed by electron diffraction microscopy. Administration of DNPs and/or FNT significantly increased fetal renal malondialdehyde, nitric oxide, and glutathione reductase as compared with the control group. However, they declined the level of glutathione together with the activities of glutathione peroxidase, glutathione-S-transferase, superoxide dismutase, and catalase. Furthermore, DNPs and/or FNT elicited many histopathological changes in fetal renal cells, markedly up-regulated apoptosis-related gene expressions (p53, p21 caspase-3, and caspase-9), and evoked DNA breaks as detected by comet assay. Interestingly, LS supplementation significantly ameliorated the disturbances in oxidant/antioxidant biomarkers, downregulated the apoptosis gene expressions, and alleviated DNA damage alongside renal cell architecture. These findings reveal that the antioxidant and anti-apoptotic characteristics of LS are acceptable defender pointers for the renal injury especially during gestational exposure to DNPs and/or FNT.

Unfractionated heparin displaces sFlt-1 from the placental extracellular matrix

Soluble vascular endothelial growth factor receptor-1 (sFlt-1) is an anti-angiogenic protein which is secreted by numerous cell types and acts as a decoy receptor for the angiogenic protein vascular endothelial growth factor (VEGF). Despite its physiologic importance in maintaining angiogenic balance, excess sFlt-1 levels are associated with the pathogenesis of many diseases, especially those with angiogenic imbalance, endothelial dysfunction, and hypertension. Although sFlt-1 is a soluble protein, it contains a binding site for the extracellular matrix component heparan sulfate. This allows cells to retain and localize sFlt-1 in order to prevent excessive VEGF signaling. During pregnancy, placental syncytiotrophoblasts develop a large extracellular matrix which contains significant amounts of heparan sulfate. Consequently, the placenta becomes a potential storage site for large amounts of sFlt-1 bound to extracellular heparan sulfate. Additionally, it should be noted that sFlt-1 can bind to the anticoagulant unfractionated heparin due to its molecular mimicry to heparan sulfate. However, it remains unknown whether unfractionated heparin can compete with heparan sulfate for binding of localized sFlt-1. In this study, we hypothesized that administration of unfractionated heparin would displace and solubilize placental extracellular matrix(ECM)-bound sFlt-1. If unfractionated heparin can displace this large reservoir of sFlt-1 in the placenta and mobilized it into the maternal circulation, we should be able to observe its effects on maternal angiogenic balance and blood pressure. To test this hypothesis, we utilized in vitro, ex vivo, and in vivo methods. Using the BeWo placental trophoblast cell line, we observed increased sFlt-1 in the media of cells treated with unfractionated heparin compared to controls. The increase in media sFlt-1 was found in conjunction with decreased localized cellular Flt (sFlt-1 and Flt-1) as measured by total cell fluorescence. Similar results were observed using ex vivo placental villous explants treated with unfractionated heparin. Real-time quantitative PCR of the explants showed no change in sFlt-1 or heparanase-1 mRNA expression, eliminating increased production and enzymatic cleavage of heparan sulfate as causes for sFlt-1 media increase. Timed-pregnant rats given a continuous infusion of unfractionated heparin exhibited an increased mean arterial pressure as well as decreased bioavailable VEGF compared to vehicle-treated animals. These data demonstrate that chronic unfractionated heparin treatment is able to displace matrix-bound sFlt-1 into the maternal circulation to such a degree that mean arterial pressure is significantly affected. Here we have shown that the placental ECM is a storage site for large quantities of sFlt-1, and that it should be carefully considered in future studies concerning angiogenic balance in pregnancy.

Effects of Perinatal Inhibition of Gonadal Steroid Hormones on Behavioral Changes in Rats

Sex differentiation of a rat brain occurs during the perinatal period (four days prior to and four days after birth). The undifferentiated brain becomes a female brain unless it is exposed to testosterone (T) which gets metabolized to 17β‐estradiol (E2) and dihydrotestosterone (DHT). While aromatase converts T to E2, 5α‐reductase converts T to DHT. 17β‐estradiol exerts its effects by binding to an estrogen receptor (ER) whereas DHT via an androgen receptor (AR). We tested the significance of DHT and E2 during the organizational phase in the differentiation of the rat brain. During the last four days of pregnancy, seven timed‐pregnant rats received either the vehicle (5% ethanol and 95% sesame oil), exemestane (Aromasin; 4mg/kg/ml), or flutamide (20 mg/kg/ml) subcutaneously. Pups from each group continued to receive their specific treatments during the first four days after birth. From postnatal day 65, these animals were subjected to open field (OF), spatial working memory, and sexual motivation (SM) tests. Male rats receiving flutamide exhibited a significant increase in exploratory behavior. Additionally, male rats receiving exemestane exhibited significantly greater interest in the estrus rat compared to the control group. In conclusion, under AR blockade, where E2 becomes the main gonadal hormone, improvements in exploratory behavior in male rats were observed. Moreover, when exemestane was used to block E2 conversion, and DHT becomes the main gonadal hormone, increased SM in male rats was observed. Thus, the presence of both E2 and DHT during organizational phase appears to be essential for normal socio‐sexual and exploratory behaviors.

Prenatal exposures to bisphenol A and di (2-ethylhexyl) phthalate disrupted seminiferous tubular development in growing male rats

Endocrine-disrupting compounds (EDCs) are found in the environment due to their use in industrial and manufacturing activities. Exposure of the population to bisphenol A (BPA) and di (2-ethylhexyl) phthalate (DEHP) is significant because they are present in many consumer products. EDCs target the reproductive tract because they express high levels of steroid hormone receptors, which act as transcriptional factors to regulate reproductive development. In the present study, timed-pregnant Long-Evans female rats (n = 8–10) were administered BPA and DEHP by oral gavage at 2.5 or 25 μg/kg body weight and 5 or 50 μg/kg body weight, respectively. Exposures to chemicals were limited to the period between gestational days 12 and 21 followed by assessment of testicular development in male offspring in the postnatal period. Leydig cells and Sertoli cells are the two major somatic cells present in the testis. The 17β-hydroxysteroid dehydrogenase (17β-HSD) steroidogenic enzyme is a marker for Leydig cell maturation, whereas transferrin is a marker for Sertoli cell differentiation. At day 10 post-partum, testes were obtained from cohorts of control and chemical-exposed male rats and processed to measure 17β-HSD and transferrin expression levels in western blots. Compared to control, 17βHSD enzyme protein was increased in BPA-treated rats but levels were decreased in animals exposed to DEHP (P < 0.05). Transferrin protein was decreased in male rats exposed to both BPA and DEHP compared to control animals (P < 0.05). To assess qualitative cellular changes within the spermatogenic epithelium, testes were obtained from separate cohorts of male rats at 35 days of age and processed for histopathological analysis. Results showed that prenatal exposures of male rats to BPA and DEHP caused disruption of the spermatogenic epithelium evident as disorganization and atrophy of seminiferous tubules as well as desquamation of germ cells into the tubular lumen. Together, results from the present study support the view that developmental exposures to environmentally relevant levels of BPA and DEHP are associated with disruptions of testicular cell development, which have implications for endocrine and exocrine functions of testis.

Di (2-ethylhexyl) phthalate-induced hypospadias in SD rats is related with Mafb expression: a transcriptome profiling-based study

To investigate the transcriptome profile of genital tubercles (GTs) in male SD rats and explore the mechanism of hypospadias induced by Di (2-ethylhexyl) phthalate (DEHP). Forty time-pregnant SD rats were randomly divided into 4 equal groups, namely GD16 group and GD19 group (in which the male GTs were collected on gestation day[GD]16 and GD19 for RNA-seq, respectively), control group and DEHP exposure group (with administration of oil and 750 mg/kg DEHP by gavage from GD12 to GD19, respectively).In the control and DEHP exposure groups, the GTs were collected from the male fetuses on GD19.5, and scanning electron microscopy and HE staining were used to observe the morphological changes.The differentially expressed genes (DEGs) in the GTs were screened using lllumina HiSeq 2000 followed by GO and KEGG enrichment analyses to characterize the transcriptome profile.Immunofluorescence assay was performed to verify the DEGs (Mafb) identified by RNA-seq results.Immunofluorescence assay and Western blotting were used to examine the expression levels of Mafb in the penile tissue. A total of 1360 DEGs were detected in the GTs between GD16 group and GD19 group by RNA-seq.Among these genes, 797 were up-regulated and 563 were down-regulated.These DEGs were mainly enriched in the cell adhesion plaque signaling pathway, axon guidance signaling pathway, and extracellular matrix receptor signaling pathway.Compared with that in GD16 group, Mafb was significantly up-regulated in GD19 group, which was consistent with the sequencing results.Mafb and β-catenin were significantly down-regulated in DEHP-exposed group compared with the control group (P &lt; 0.01). Mafb expression increases progressively with the development of GTs in male SD rats.DEHP exposure causes significant down-regulation of Mafb and β-catenin, suggesting that β-catenin signaling pathway that affects Mafb is related to DEHP-induced hypospadias in SD rats.

The Effect of Perinatal Blockade of Androgen Receptors on Adult Rat Behaviors and the Expression of Estrogen and Androgen Receptors in specific brain regions

The brain, by default, differentiates into a female brain unless testosterone (T) is present during a critical window of time. In rats, there is a surge of T around the time of birth, more specifically four days prior to and four days after birth, referred to as the perinatal period. The purpose of this study was to examine the effects of blockade of androgen receptors (AR) during the perinatal period using flutamide (F) on sexual motivation (SM) and an open field (OF) test as well as the expression of AR and estrogen receptors (ER)‐alpha and ER‐beta in the hypothalamus, hippocampus, and amygdala. Four timed‐pregnant rats were divided into two groups: one group (N=2) received F during perinatal period whereas the other group (N=2) served as the control group receiving vehicle (95% Olive oil‐5% ethanol). Once the pups reached adulthood, the male rats underwent SM testing where they were exposed to either an estrus or an ovariectomized rat. Both F and control groups of male rats spent more time with the estrus rat compared to the ovariectomized rat suggesting that SM was not affected by their AR blockade during perinatal period. Open field testing recorded animal movements between the two zones, the center and the periphery, within an open arena. Male rats moved between the two zones more than the female rats did. At the end of the behavioral study, animals were sacrificed and the brains were harvested to isolate the hippocampus, hypothalamus, and amygdala. Real time PCR testing was carried out to quantify the levels of AR, ER alpha, and ER beta using B‐Actin as an internal control. Perinatal blockade of AR had no effect on the expression of any of the receptors in the three regions tested in the adult rats. Thus, the increased movement by the male rats in the OF testing is not associated with changes in the expression of AR, ER‐alpha, or ER‐beta.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Does Shipping Late‐Gestation Rats Alter Respiratory Control in Their Offspring?

Ordering timed‐pregnant rats is convenient for many developmental studies, but the shipping process may be stressful for the animals. Given the sensitivity of the developing respiratory control system to perinatal stress (e.g., Joseph et al., Respir Physiol Neurobiol 185:75–86, 2013), we investigated whether prenatal “shipping stress” alters the respiratory control of neonatal rats. In the first set of experiments, timed‐pregnant rats were shipped to arrive at our animal facility at embryonic age 17 (E17) and their pups were compared to those of rats bred in‐house (IH) at 8–9 days of age (P8–9). Normoxic ventilation (measured by head‐body plethysmography) was 30% greater in E17 pups than in IH pups (138±6 vs. 106±4 ml min−1 100g−1; mean±SEM, P&lt;0.001), and their hypoxic ventilatory responses (HVR) to 12% O2 were lower (45±6 vs. 67±4 % increase from baseline; P=0.02). We then repeated the experiment with timed‐pregnant rats shipped to arrive at E13 or E19. At P4–5, ventilation was approximately 10% greater in E19 pups than in E13 or IH pups (treatment, P=0.02), but the magnitude and time course of the HVR was similar among groups (treatment × time, P=0.53). At P11–12, no differences in normoxic or hypoxic ventilation were detected among the treatment groups (treatment, P=0.24; treatment × time, P=0.08). Similarly, in vitro carotid body responses to hypoxia did not differ among E13, E19, and IH groups at either age (both P&gt;0.05). The different outcomes of these studies could reflect the different gestational ages at which the rats were shipped or different origins/shipping distances of the pregnant rats (Portage, MI vs. Kingston, NY).Support or Funding InformationSupported by NIH grant P20 GM‐103423 (Maine INBRE).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Placental Ischemia Leads to Regional Astrocyte Activation: Role of TNFα

Preeclampsia, a hypertensive disorder of pregnancy, can quickly progress to eclampsia when seizure develops in the peri-partum period. We have shown that the preclinical rat model of preeclampsia, induced by surgical reduction of uterine perfusion pressure (RUPP), has reduced latency to drug induced seizures and cerebral edema. The increased seizure sensitivity was associated with increased cerebrospinal fluid (CSF) cytokines, an indicator of tissue inflammation and potential activation of neuroglial cells. Moreover, other labs have shown that treatment of brain slices with serum from pregnant women induced increased neuronal activity and neuro-inflammation through TNFα. Thus, we tested the hypothesis that placental ischemia induces regional astrocyte activation that can be reversed by pharmacological blockade of TNFα. On GD14, time pregnant rats were randomly assigned to normal pregnant (NP, n=6) or RUPP (n=6) groups and further assigned to no treatment or etanercept injection (0.8 mg/kg, sc) on GD18. RUPP rats underwent surgery on GD14 to induce placental ischemia by inserting silver clips around the abdominal aorta and each of the uterine artery branches. Normal pregnant rats did not undergo surgery. Brains were harvested on GD19 and processed for immunofluorescence staining. Anterior brain was cut into 20 μm thick sections and stained for glial fibrillary acidic protein (GFAP), an astrocyte marker. Z-stacks (0.5 μm steps) were obtained and percent area of GFAP positive astrocytes was analyzed using Image J in the anterior commissure, corpus callosum, cortex, and caudate putamen. Two-Way Analysis of Variance revealed a significant increase in the percent area of astrocytes in the caudate putamen in RUPP rats [RUPP: 5.05 ± 1.09%; NP: 1.32 ± 0.28% (P = 0.01)] which was reduced by etanercept (2.11 ± 0.85%). There were no significant changes in % area in the anterior commissure, corpus callosum, or cortex (P> 0.05). Increased astrocyte area in the caudate putamen was associated with higher number of astrocytes [NP: 7 ± 1; RUPP: 13 ± 1 (p<0.01)] which was unchanged by etanercept (12 ± 3 cells). Our results are consistent with the hypothesis that placental ischemia induces cytotoxic edema (cell swelling) in the anterior cerebrum through regional astrocyte activation (caudate putamen). Moreover, increased circulating TNFα in placental ischemic rats may contribute to astrocyte activation, and in turn induce the release of TNFα and other pro-inflammatory cytokines, increasing seizure susceptibility and cytotoxic edema. Additional analyses of astrocytic morphology are ongoing and the direct role of pro-inflammatory cytokines on seizure susceptibility during pregnancy are areas of future study. Support or Funding Information NIH K99/R00HL129192, COBRE Pilot Grant, P20GM104357, P01HL051971 Representative images of GFAP+ astrocytes in the Caudate Putamen (CP) taken at 60X. Graph shows % area covered by astrocytes for each group. Individual points represent average data per section (1–3 images - 2 sections per rat). NP-normal pregnant, RUPP - reduced uterine perfusion pressure. *p<0.05, **p<0.01. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Effect of Flutamide on Spatial Working Memory and Serum Estrogen Levels in Female Rats

In the absence of testosterone, the brain, by default, differentiates into a female brain. Sex differentiation of the rat brain occurs during the perinatal period (four days prior to and four days after birth) during which time both estrogen and androgen receptors are expressed in the female brain. However, the role of androgen receptors in the differentiation of the female brain during the perinatal period is unknown. Our purpose of this study was to examine the effects of blockade of androgen receptors on circulating estrogen levels and spatial working memory. Four timed‐pregnant Long Evans rats were divided into two groups: one group (N=2) received flutamide during perinatal period whereas the other group (N=2) served as control receiving vehicle (95% Olive oil‐5% ethanol). Female pups from the flutamide group (N=8) continued to receive flutamide whereas the pups from the control group received vehicle for the additional four days (N=15). Prior to performing Radial Arm Maze test to assess their spatial working memory, adult animals were placed on a 60 % food restriction to bring their body weights close to 85% of their original weight. spatial working memory testing involved a three‐day habituation, three‐day training, and one testing day. During the habituation period, all eight arms of the Radial Arm Maze contained food. During the training and the testing periods, however, four of the eight arms contained food and the other four did not. Any‐maze software linked to a video‐tracking device tracked the amount of time each animal spent in the arms with or without food. Flutamide‐treated animals spent more time in the arms with food compared to the control group suggesting greater choice accuracy or modestly improved spatial working memory (p= 0.06). At the end of the behavioral study, animals were sacrificed and trunk blood was collected to measure serum estrogen levels using ELISA. Flutamide‐treated animals showed an increase in serum estrogen levels (p&lt;0.05) implying a relationship between improved spatial working memory and increased levels of estrogen. Thus, this study suggests that the blockade of androgen receptors during perinatal period increases serum estrogen levels and marginally improves spatial working memory in female rats.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of Nitric Oxide Synthase on Blood Pressure Regulation and Vascular Function in Pregnant Rats on a High-Fat Diet.

While obesity is a leading risk factor for preeclampsia, the mechanisms whereby obese women are more susceptible to pregnancy-induced hypertension are unclear. As high-fat diet (HFD) is an important contributor to the development of obesity, we tested the hypothesis that pregnant rats on HFD have hypertension and endothelial dysfunction due to reduced nitric oxide synthase (NOS). Twelve-week-old Sprague-Dawley female rats were fed normal diet (ND, 13% fat kcal) or HFD (40% fat kcal) for 9 weeks. Timed-pregnant rats were then generated and the effect of HFD on mean arterial blood pressure (MAP) and vascular function was assessed on gestational day (GD) 19. MAP was not different between HFD and ND pregnant rats. Intriguingly, sensitivity to acetylcholine-induced endothelium-dependent vasorelaxation was enhanced in small mesenteric arteries of HFD dams compared to ND controls (logEC50 -7.9 ± 0.3 vs. -6.7 ± 0.3 M; P < 0.05). Additionally, HFD dams exhibited higher mesenteric artery expression of NOS3 and plasma levels of NO metabolites than ND controls (1738.0 ± 316.4 vs. 1094.0 ± 82.5 pg/mg and 72.5 ± 8.7 vs. 39.7 ± 4.5 µM, respectively; both P < 0.05). Further, to determine the role of NOS in modulating blood pressure in HFD pregnant rats, animals were treated with the nonselective inhibitor Nω-Nitro-l-arginine methyl ester hydrochloride (100 mg/l, drinking water) from GD 14 to 19. It was found that NOS inhibition increased MAP equally in HFD and ND groups. Contrary to our initial hypothesis, HFD dams were normotensive and presented increased endothelial function and NO/NOS3 levels. This enhanced NOS-mediated vascular function does not appear to have a major impact on blood pressure regulation of HFD-fed pregnant rats.