Pup Weight Research Articles

Maternal periodontitis induces intergenerational injuries on reproductive performance of adult female rat offspring

Existem evidências substanciais de uma relação direta entre periodontite em mulheres grávidas com efeitos adversos reprodutivos. No entanto, nenhum estudo avaliou os efeitos intergeracionais da periodontite sobre o desempenho reprodutivo das gerações subsequentes. O presente estudo investigou se a doença periodontal materna exerce efeitos intergeracionais deletérios sobre o desempenho reprodutivo em ratos fêmeas da geração F1. Assim, filhas de ratas cujas mães foram submetidas a periodontite experimental ou falsamente operadas foram acasaladas com ratos machos sexualmente experientes. O peso corporal e desempenho reprodutivo da geração F1 foram avaliados no dia 21 de gestação, incluindo o peso materno, peso da ninhada, peso da individual dos filhotes, número de filhotes e de reabsorções. A percentagem de fêmeas com reabsorção e o peso da ninhada/número de filhotes também foram calculados. Comparados com o grupo controle, observou-se aumento na porcentagem e número de reabsorções e no peso da ninhada/ número de filhotes, e decréscimo no número de filhotes nascidos no grupo experimental. O peso materno, peso da ninhada e individual dos filhotes não foi diferente entre o controle e experimental. Estes resultados mostram que a periodontite experimental materna prejudica o desempenho reprodutivo da geração F1, mesmo que estes animais não tenham sido expostos diretamente a um processo inflamatório.

Interleukin-11 induces preterm birth and modulates decidual inflammasome gene expression in mice

Decidual inflammation and inflammasome activation contribute to the pathogenesis of preterm birth (PTB). Interleukin-(IL)11 exerts complex regulatory effects on inflammation and IL-11 protein is elevated in PTB decidua. We aimed to determine a functional role for IL-11 in parturition or PTB. IL-11 administration during middle-late gestation pregnancy induced preterm birth in 87.5% of mice versus saline controls. Litters from IL-11-treated mothers had reduced offspring weight and numbers of live pups. IL-11 up-regulated decidual gene expression of key components of the inflammasome including IL-1β, Caspase-1 and ASC. This is the first report of a causal role for IL-11 in PTB in vivo.

Gestational stress, placental norepinephrine transporter and offspring fertility.

Chronic cold stress produces adrenergic overload that can affect fetal development. The placental norepinephrine transporter (NET) clears norepinephrine (NE) from both maternal circulation and the fetus during gestation. If this system fails, NE clearance can be reduced, leading to high fetal exposure to NE. The main aim of this study was to determine the changes in NET expression during gestation and their relationship with the functional capacity of NET to transport NE under stressful conditions. Additionally, this study correlated these findings with the reproductive capacity of 2nd-generation progeny. Pregnant rats were subjected to chronic cold stress at 4°C for 3 h each day throughout their pregnancies. We found that exposure of pregnant rats to sympathetic stress caused the following effects: increased NE and corticosterone levels throughout pregnancy, decreased capacity of the placenta to clear NE from the fetus to the mother's circulation, altered NET protein levels depending on the sex of the fetus and increased placental and body weights of pups. For the first time, we also described the disrupted fertility of progeny as adults. Increased NE plasma levels during pregnancy under sympathetic stress conditions correlated with decreased NET functionality that provoked changes in the development of progeny and their fertility in adulthood.

Combined repeated-dose and reproductive/developmental toxicity screening test of 1-tert-butoxy-4-chlorobenzene in rats

We have carried out animal toxicity tests of chemicals for a chemical safety program implemented by the Ministry of Economy, Trade, and Industry of Japan. Here, we tested 1-tert-butoxy-4-chlorobenzene in a combined repeat-dose and developmental and reproductive toxicity test. The test chemical was administered daily by gavage to 9-week-old Crl:CD (SD) rats at doses of 0, 20, 100, and 500 mg/kg/d. Males were treated for 42 d beginning 14 d before mating. Females were treated from 14 d before mating to day 4 of lactation. Decreased spontaneous locomotion, decreased respiratory rate, and incomplete eyelid opening were observed at 500 mg/kg/d (both sexes), but resolved within 30 min of administration, suggesting central nervous system depression. No notable changes were observed in body weight, food consumption, functional battery tests, or blood test. Increased liver weight with centrilobular or diffuse hepatocyte hypertrophy was observed at 100 and 500 mg/kg/d (both sexes). There were no biochemical or histopathological changes related to hepatotoxicity. Increased kidney weight with basophilic tubules, tubule dilatation, and increased hyaline droplets were observed in males dosed at 100 and 500 mg/kg/d. Immunohistochemical staining indicated α2u-globulin nephropathy, a male rat-specific toxicity. Although kidney weight was also increased in females dosed at 500 mg/kg/d, it was not considered to be an adverse effect because there were no histopathological changes. Pup weights on postnatal day 0 were decreased at 500 mg/kg/d and still decreased on postnatal day 4. Our data indicated the no-observed-adverse-effect-level for repeated-dose and reproductive/developmental toxicity for 1-tert-butoxy-4-chlorobenzene was 100 mg/kg/d.

Reduced uterine perfusion pressure T-helper 17 cells cause pathophysiology associated with preeclampsia during pregnancy.

Preeclampsia is associated with chronic inflammation and an imbalance among T-helper cell subtypes with an increase in T-helper 17 (TH17) cells. The objective of this study was to determine a role for TH17s, from the reduced uterine perfusion pressure (RUPP) rat model of preeclampsia, in the etiology of hypertension and chronic inflammation during pregnancy. CD4+/CD25- T cells were isolated from rat spleens, cultured in TH17 media, and were verified as TH17s via flow cytometry. On day 12 of gestation, 1×106 TH17 cells from RUPP rats were adoptively transferred into NP rats, carotid catheters were inserted on day 18, and on day 19, mean arterial pressure (MAP) was recorded, serum and plasma were collected, and oxidative stress and production of agonistic autoantibodies to the ANG II type I receptor (AT1-AA) were analyzed. MAP increased from 100.3 ± 1.7 mmHg in normal pregnant (NP; n = 17) to 124.8 ± 2.1 mmHg in RUPP (n = 22; P < 0.0001) and to 110.8 ± 2.8 mmHg in NP+RUPP TH17 (n = 11). Pup weights in NP+RUPP TH17s were decreased to 1.92 ± 0.09 g from 2.39 ± 0.14 in NP rats (P < 0.01). AT1-AA significantly increased from 0.1 ± 0.2 beats/min in NP to 15.6 ± 0.7 beats/min in NP+RUPP TH17s. IL-6 was 22.3 ± 5.7 pg/ml in NP and increased to 60.45 ± 13.8 pg/ml in RUPP (P < 0.05) and 75.9 ± 6.8 pg/ml in NP+RUPP TH17 rats (P < 0.01). Placental and renal oxidative stress were 238 ± 27.5 and 411 ± 129.9 relative light units·min-1·mg-1 in NP and 339 ± 104.6 and 833 ± 331.1 relative light units·min-1·mg-1 in NP+RUPP TH17, respectively. In conclusion, RUPP TH17 cells induced intrauterine growth restriction and increased blood pressure, AT1-AA, IL-6, and tissue oxidative stress when transferred to NP rats, indicating a role for autoimmune associated TH17 cells, to cause much of the pathophysiology associated with preeclampsia.

In utero exposure to di-(2-ethylhexyl) phthalate induces metabolic disorder and increases fat accumulation in visceral depots of C57BL/6J mice offspring.

Excessive visceral fat accumulation is associated with metabolic disorders. Di-(2-ethylhexyl) phthalate (DEHP), a candidate environmental obesogen, affects lipid metabolism and adipogenesis. Perinatal exposure to DEHP may be associated with metabolic disorders of dams and offspring. The aim of the present study was to explore the effects of exposure of pregnant dams to DEHP on the metabolism and fat distribution of their offspring, and to determine the mechanisms for these effects. Pregnant C57BL/6J mice were administered DEHP via gavage (0.05 or 500 mg/kg/day) from gestational days 1-19. Pups were sacrificed at nine weeks of age. Serum leptin, insulin, lipid and fasting glucose levels, and the weights of the inguinal (subcutaneous) and gonadal (visceral) fat pads were determined. mRNA expression levels of two developmental genes, T-box 15 (Tbx15) and glypican 4 (Gpc4) were detected in fat tissues. A 100% abortion rate was exhibited in 500 mg/kg DEHP-treated dams, whereas exposure to 0.05 mg/kg DEHP did not affect reproductive outcomes. Pups from the 0.05 mg/kg exposure group were used for subsequent experimentation. Serum leptin, insulin, lipid and fasting glucose concentrations in these pups were significantly higher than those of control pups (P<0.05). Although no significant change in body weight was detected, the visceral fat weights of DEHP-exposed pups were significantly higher than those of control pups (P<0.05). Compared with controls, mRNA expression levels of Tbx15 in subcutaneous fat and Gpc4 in visceral fat were significantly increased among DEHP-exposed pups (P<0.01). The present results suggest that in utero exposure to an environmentally safe dose of DEHP may lead to excessive visceral fat accumulation and metabolic disorders in offspring and that aberrant expression of Tbx15 and Gpc4 may have an important role in these effects.

Effects of prenatal maternal seizure on hippocampal damage and cognitive deficits in offspring rats

Objective: To observe hippocampal damage and cognitive impairment of offspring exposed to prenatal maternal seizure induced by amygdala kindling, and to explore the underlying mechanism by the detection of pathological changes of placenta. Method: Adult female SD rats were randomly divided into three groups: control group(8 rats), kindling group(12 rats) and sham group(8 rats). All the rats were allowed to mate after one week's fully kindling. The pregnant rats in kindling group received electric stimulation every 48 h. Dams were allowed to deliver naturally. Effects of maternal seizure on the number of offspring, the survival rate and body weight of pups were observed. HE staining was used to visualize histopathological changes of placenta. Morris water maze test was used to assess the cognitive function and Nissal's staining to detect hippocampal morphology of the offspring. One-way ANOVA analysis and χ2 test were used. Result: Compared with the sham group (95%(78/82)) and the control group (95%(82/86)), the survival rate of pups in kindling group(81%(66/82))was much lower (χ2=13.817, P=0.001). There were no significant differences in the number of pups per litter and pups birth-weight between kindling group and sham group or control group(F=0.312 and 0.257, P=0.736 and 0.776). HE staining showed that placental tissues from control and sham groups were normal whereas the histologic abnormalities of placentas from kindling group were characterized by thickening of the villus vascular walls, luminal stenosis, trophoblasts hyperplasia, abnormalities of trophoblasts with nuclear pyknosis and karyorrhexis and accumulation of inflammatory lymphocytes in labyrinthine zone. Nissl staining showed that neurons in hippocampus of P0(0 d after birth) and P84(84 d after birth) offspring from control and sham groups were normal, but neuronal damages were obvious in hippocampus of P0 and P84 offspring from kindling groups, and the damages in P0 pups were severe with a marked loss of neuron, shrinkage of cells and nuclear pyknosis and karyorrhexis. In the Morris water maze, compared with the sham group ((29±8), (19±9), (10±4)s) and the control group ((25±6), (17±5), (14±4)s) rats in the kindling group ((36±8), (29±8), (30±11)s) exhibited significantly longer escape latency from the 3rd, 4th, and 5th days (F=6.276, 7.518, 18.422, P=0.030, 0.003, 0.000), significant less time in the target quadrant ((27±8) vs.(58±11)and(68±13)s, F=35.993, P=0.000) and reduced number of crossing the platform ((4.4±1.7) vs. (7.2±1.6) and (8.5±1.3)times, F=18.377, P=0.000). In addition, there was no significant difference between control and sham groups(P all >0.05). Conclusion: The prenatal maternal seizures induced significant pathological damages to hippocampus and cognitive impairment of offspring. Hypoxia-ischemia of placenta might play an important role in this process.

Small Molecule GS-Nitroxide Radiation Mitigator JP4-039/F14 Is Safe and Effective in Pregnant E13.5 Mice

Irradiation during pregnancy can induce fetal death, stunt growth, and/or lead to teratogenic and carcinogenic effects dependent on stage of gestation. A practical radiation mitigator must be safe for pregnant females. We evaluated the mitochondrial targeted GS-nitroxide mitigator JP4-039 for effects on total body irradiated (TBI) pregnant mice. Timed pregnant C57Bl/6NHsd mice at E13.5 were irradiated to 3 Gy, and a subgroup was injected IV 24 hr later (E14.5) with JP4-039. A subgroup of nonirradiated mice received JP-4-039. Mice were followed for number of live births, weight of pups at day 5 after birth, and for number of survivors at weaning. In other studies pups were examined microscopically on E18.5 (day before birth) or on day of birth. Nonirradiated mice showed 97 ± 2% of newborn surviving until weaning. Pups born to 3 Gy irradiated E13.5 pregnant mice had decreased survival (8.3 ± 8.7%) (P < 0.0001). The survival of pups from 3 Gy irradiated E13.5 pregnant mice that received JP4-039/F14 24 hours after irradiation while still significantly decreased (45 ± 16.4% compared to nonirradiated controls (P = 0.0230) was significantly improved compared to irradiated controls (P<0.0001). Newborns surviving 3 Gy in utero had significantly decreased weight at 5 days of 1.64 ± 0.04 g compared to 2.73 ± 0.08 g for nonirradiated controls (P < 0.0001). In contrast, 3 Gy TBI E13.5 irradiated pups from mothers that received JP4-039/F14 showed no significant weight loss compared to control nonirradiated pups (2.44 ± 0.15 and 2.73 ± 0.08, respectively, P = 0.0799) and weight was significantly increased compared to the control 3 Gy irradiated group (2.44 ± 0.15 and 1.64 ± 0.04 g, respectively, P = 0.0433). Pups from nonirradiated mothers that received JP4-039/F14 had significantly increased weight on day 5 compared to nonirradiated mice (3.32 ± 0.10 and 2.73 ± 0.08 g, respectively, P < 0.0001). Microscopic examination of irradiated pups dying on the day of birth, revealed: 1) increased hematopoietic precursors in liver, and decreased glycogen stores in hepatocytes; 2) enlarged adrenal glands with hypertrophied cortical cells; 3) necrosis in brain, with loss of parenchyma within the intermediate zone of white matter and cell debris in the lateral ventricles. These pathologic effects of irradiation were not detected in pups from JP4-039/F14 treated mothers, sacrificed and examined on the day of birth. In addition, while pups in irradiated mothers were alive on E18.5, they had decreased weight and movement compared to E18.5 nonirradiated pups. TBI irradiated E13.5 pregnant mice treated on E14.5 with JP4-039/F14 showed this drug to be a safe and effective radiation mitigator, leading to increased fetal survival, growth and development, and continued survival to weaning at 21 days, with no late deaths.

Effects of Pregnancy on Transplanted Pancreatic Beta Cell Progenitors Derived from Human Embryonic Stem CellsImage 7

Human embryonic stem cells (hESCs) are a potential source of transplantable cells for treating patients with diabetes. However, recent findings from our lab suggest that the cell milieu post-transplant can impact the maturation of hESC-derived pancreatic progenitor cells into functional beta cells. During pregnancy, pancreatic beta cell mass transiently expands to accommodate increased metabolic demands of the mother. Here, we examined the impact of pregnancy on the development of hESC-derived progenitor cells following transplantation into immunodeficient mice. Female transplant recipients and controls (no transplant) were housed with breeder males at 4-weeks post-transplant. A group of male mice also received cell transplants as a positive control. The presence of a cell transplant did not affect fertility, body weight gain during pregnancy, pup weight or litter size in female mice. Between 12 to 15 days of gestation, we observed a significant ~5-fold increase in beta cell proliferation (PCNA+/insulin+ cells) within the endogenous mouse pancreas from pregnant compared to non-pregnant mice, but no difference within the hESC-derived grafts. Exposure to pregnancy also did not impact the secretion of human C-peptide from implanted cells at any stage throughout the study. Interestingly, female transplant recipients displayed glucose-responsive human C-peptide secretion beginning at just 13 weeks post-transplant, whereas grafts from male mice were not glucose responsive until 28 weeks. These data suggest that graft maturation may be faster in female transplant recipients compared to males, and this process is unaffected by pregnancy during the early post-transplant period.

Chronic effects of an anti-angiogenic thrombospondin-1 mimetic peptide, ABT-898, on female mouse reproductive outcomes.

BackgroundAngiogenesis is an essential process in endometriosis disease progression. Earlier, we demonstrated that anti-angiogenic peptide, ABT-898 prevents neoangiogenesis of human endometriotic lesions in a xenograft mouse model. Since angiogenesis is essential for normal ovarian and uterine function, we evaluated effects of ABT-898 on normal female reproductive processes in mice.MethodsCycling female C57BL/6N mice were dosed with ABT-898 (100 mg/kg) or 5 % dextrose control for 21 consecutive days to cover multiple estrous cycles (average estrous cycle 4 to 5 days in mice). Pregnant female mice were dosed with ABT-898 (100 mg/kg) or control on alternate days over the course of gestation, beginning at gestation day 7.5 to 17.5 (gestation length 21 days). Histological analysis along with CD31 and Vimentin immunohistochemistry were performed on ovaries and uteri obtained from treated and control mice. To understand the influence of ABT-898 on systemic angiogenic factors, a Pro Mouse Cytokine 9-plex assay was performed on plasma samples obtained from mice prior to treatment, during the second week of ABAT-898 or control treatment and on the last day of treatment.ResultsABT-898 did not affect the number of estrous cycles over the 21 day treatment compared to control. Histological analysis of ovaries found no difference in the number of primordial, primary, secondary, and antral follicles between ABT-898 treated and control groups. Similarly, no difference was observed in the microvessel density between ABT-898 treated and control uteri, ovarian follicles or corpus luteum when assessed using CD31 or vimentin immunohistochemistry. Electron microscopy revealed similar capillary structure and appearance in both ABT-898 treated and control uteri. Although peripheral blood angiogenic cytokine profiles (IL-15, IL-18, M-CSF, b-FGF, PDGF-bb, MIG, MIP-2, LIF and VEGF) changed over the course of the intervention, there was no significant difference between ABT-898 and control groups at any of the studied time points. Treatment with ABT-898 during pregnancy had no effect on litter size at birth, pup weight at birth or pup weight at weaning.ConclusionOur findings suggest that ABT-898 may not alter angiogenesis dependent reproductive processes in female mice. However, an extensive reproductive toxicology screening is required to substantiate use of ABT-898 in future.Electronic supplementary materialThe online version of this article (doi:10.1186/s12958-016-0192-7) contains supplementary material, which is available to authorized users.

Abstract 032: Soluble fms-like Tyrosine Kinase-1 (sFlt-1)/Placental Growth Factor (PlGF) Imbalance Disrupts Vascular and Uteroplacental Gelatinolytic Matrix Metalloproteinase MMP-2 and -9 and Collagenolytic MMP-1 and -7 Balance in Hypertensive Pregnancy

Preeclampsia is a complication of pregnancy manifested as hypertension-in-pregnancy (HTN-Preg) and fetal growth restriction. Placental ischemia could be an initiating event, but the linking mechanisms and tissue targets are unclear. Placental ischemia is associated with increased sFlt-1/PlGF ratio, and we have shown changes in MMPs in pregnant rat model of reduced uterine perfusion pressure (RUPP). To test the hypothesis that sFlt-1/PlGF imbalance could target vascular and uteroplacental MMPs, we tested if raising sFlt-1/PlGF ratio by infusing sFlt-1 (10 μg/kg/day) in Preg rats would increase BP and alter MMPs, and if correcting sFlt-1/PlGF ratio by infusing PlGF (20 μg/kg/day) in RUPP rats improves BP and reverses the changes in MMPs. On day 19, BP was recorded and the aorta, placenta, uterus and uterine artery were isolated for measuring MMP activity using gelatin and casein zymography and protein levels of MMPs and collagen I and IV using Western blots. BP was in Preg+sFlt 121±3 &gt; Preg 93±7 and in RUPP+PlGF 97±4 &lt; RUPP 118±4 mmHg. Litter size and pup weight were in Preg+sFlt &lt; Preg and in RUPP+PlGF &gt; RUPP. MMP-2 levels were in aorta of Preg+sFlt 3.30±0.05 &lt; Preg 7.75±0.13, and in RUPP+PlGF 6.70±0.19 &gt; RUPP 2.62±0.12. Similarly, MMP-2 in placenta, uterus and uterine artery and MMP-9 in all tissues were in Preg+sFlt &lt; Preg and in RUPP+PlGF &gt; RUPP. In contrast, MMP-1 levels were in aorta of Preg+sFlt 1.73±0.33 &gt; Preg 0.55±0.49, and in RUPP+PlGF 0.53±0.18 &lt; RUPP 4.04±0.30. Also, MMP-1 in placenta, uterus and uterine artery and MMP-7 in all tissues were in Preg+sFlt &gt; Preg and in RUPP+PlGF &lt; RUPP. The MMP-2 and -9 substrate collagen IV was in aorta of Preg+sFlt 0.16±0.00 &gt; Preg 0.06±0.01, and in RUPP+PlGF 0.01±0.00 &lt; RUPP 0.18±0.01. Similarly, collagen IV in placenta, uterus and uterine artery and the MMP-1 and -7 substrate collagen I in all tissues were in Preg+sFlt &gt; Preg and in RUPP+PlGF &lt; RUPP. Thus, sFlt-1/PlGF imbalance is a likely mechanism linking placental ischemia to decreased gelatinases, increased collagenases and improper vascular and uteroplacental remodeling. Correcting sFlt-1/PlGF balance by infusing PlGF could rectify the balance between gelatinolytic and collagenolytic MMPs and thereby restore proper vascular and uteroplacental remodeling in HTN-Preg.

Abstract 133: Depletion of Natural Killer Cell Activation in Response to Placental Ischemia Improves Hypertension and Intrauterine Growth Restriction During Pregnancy

Women with preeclampsia (PE), newly developed hypertension and renal dysfunction during pregnancy, have small-for-gestational-age babies and demonstrate an increase in the cytolytic natural killer (NK) cell activation. The specific role of cytolytic NK cells in the pathophysiology of PE has not been clearly defined. The reduced uterine perfusion pressure (RUPP) model of placental ischemia (PI) exhibits many of the characteristics of PE including hypertension, renal dysfunction, chronic inflammation and intrauterine growth restriction (IUGR). In this study, we tested the hypothesis that PI results in cytolytic activation of NK cells, and examined a role for a reduction in NK cells in RUPP to attenuate PE-like characteristics in response to PI. In this study NK cells were depleted in RUPP rats by intraperitoneal administration of the Anti-asialo GM1 antibody on gestation days 15 and 17. PBMCs and placental lymphocytes were examined via flow cytometry to quantify cytolytic NK cells and to verify NK cell depletion, blood pressure (MAP) and pup weight were measured. While total placental NK cells numbers did not change in response to PI (NP: 14±4.5%; RUPP: 14.3±3.8%), cytolytic activation of placental NK cells significantly increased in response to PI (NP: 3.4±1.1% vs RUPP 10.0±3.4%; p&lt;0.05). Moreover, depletion of NK cells in RUPP (total NK: RUPP: 14.3±3.8% vs RUPP+NKD: 3.5±0.9%) significantly improved blood pressure and intrauterine growth restriction (IUGR): MAP significantly increased in response to PI from 109.5±2.3 mmHg in NP (n=10) to 125.4±2.7 mmHg (n=9) in RUPP rats (p&lt;0.001). Depletion of NK cells with the cell specific depleting antibody significantly lowered blood pressure to 114.4.±1.9 mmHg in RUPP+NKD rats (n=11, p&lt;0.01). Additionally, NK cell depletion in RUPP significantly reduced IUGR in response to PI (1.85±0.06g in RUPP vs. 2.0±0.4g in RUPP+NKD; p&lt;0.05). Depletion of NK cells in RUPP rats was positively associated with lowering blood pressure and blunting IUGR in response to PI. These results suggest a role for cytolytic NK in contributing to hypertension and IUGR in response to PI, potentially identifying previously unknown mechanisms of PE pathophysiology and new therapeutic targets to improve maternal and fetal outcomes of PE.

Abstract P320: Proliferationof Endogenous T-regs Improves the Pathophysiology Associated with Placental Ischemia of Pregnancy

Preeclampsia (PE), new onset hypertension during pregnancy, is associated with pro-inflammatory cytokines and decreased regulatory immune mechanisms such as Tregs, IL-10 and IL-4. We believe this decrease in immune regulatory mechanisms leads to an uncontrolled proinflammatory response which contributes to most of the pathophysiology associated with PE. The Reduced Uterine Perfusion Pressure, RUPP, rat model of induced placental ischemia exhibits similar characteristics as women with PE including high blood pressure, elevated pro-inflammatory cytokines and cells and decreased Tregs, IL-4 and IL-10. Therefore, we hypothesized that stimulating Tregs by administration of a superagonist (SA) would increase the Treg profile in the RUPP rats which could reduce pro-inflammatory cytokines and blood pressure. The RUPP procedure was performed at gestation day 14 (GD14); SA was administered intraperitoneally at GD15, GD18 carotid catheters inserted, and GD19 MAP and pup weight, serum and tissues were collected. MAP in NP rats was 98.6 mmHg ± 4.71, 122.2 mmHg ± 1.84 in RUPPs which was improved to 110.789mmHg ± 1.23 in RUPP+SA. Circulating FoxP3+ Treg cells were 5.987% ± 1.69% of total T-cells population in NP, 0.77% ± 0.49% in RUPP rats but increased to 11.218% ± 2.9% in RUPP+SA; Circulating IL-6 levels were 41.008 ± 4.79 pg/mL in NP, 108.26 ± 25.99 pg/mL in RUPP, and 40.37 ± 4.49 pg/mL in RUPP+SA, Administration of the SA to the NP rats did not affect IL-6 Levels in comparison to NP at 36.79 ± 3.9 pg/mL. Plasma IL-10 Levels were at 58.399 ± 9.527 pg/mL in NP rats, these levels were significantly decreased in the RUPP rats, 26.298 ± 4.33 pg/mL, and treatment of the RUPPs with the SA significantly increased plasma IL-10 levels to 51.5486 ± 3.329 pg/mL. When the SA was given to NP rats, the levels for IL-10 were significantly higher compared to any of the other groups at 85.5207 ± 9.067 pg/mL. Placental Pre-pro Endothelin-1 (PPET-1) was increased 44.42 ± 0.269 fold in RUPP compared to NP 1 ± 0.255, but was decreased to 18.78 ± 0.48 in RUPP+SA. These data suggest an important role for up-regulating Treg cells to enhance the immune regulatory interactions and lower the hypertension without causing further reduction in fetal weight in response to placental ischemia during pregnancy.

Abstract 042: Selective Maternal Hepatic Silencing of Angiotensinogen in Reduced Uterine Placental Perfusion Pressure Rats Improves Blood Pressure and Fetal Weights

Women with Preeclampsia (PE), a form of new onset hypertension during pregnancy, exhibit alterations in the renin angiotension system (RAS). These differences include angiotensin II type 1 receptor agonistic autoantibodies (AT1-AA), ANG II sensitivity, and increased oxidation and levels of angiotensinogen (AGT). While antihypertensive drugs are used in pregnancy, their use is associated with reduced fetal growth, and RAS blockade is specifically contraindicated due to fetotoxicity. AGT is the precursor to ANG II and the initial substrate of the RAS pathway. Genetic mutations that result in an over-expression of AGT are strongly associated with hypertension. We have previously shown that siRNA targeting maternal hepatic AGT in a transgenic model of preeclampsia (hAGT x hREN) resulted in lower blood pressure, a 90% reduction in AT1-AA activity, and normalized IUGR. The purpose of the present study is to examine the effect of silencing maternal hepatic AGT on maternal blood pressure and fetal growth in the reduced uterine placental perfusion pressure (RUPP) model of PE. Methods: Pregnant Sprague Dawley rats were divided into 4 groups: normal pregnant (NP, n=7); RUPP (n= 6); RUPP + siRNA (n =7); NP + siRNA (n=7). On day 12 of gestation (GD), siRNA was subcutaneously injected into rats (10mg/kg), GD14 the RUPP surgery was performed, GD18 carotid catheters inserted, and GD19 conscious blood pressure (MAP) and fetal weight was recorded. Results: MAP was elevated in RUPP vs. NP (134±6 vs. 101±4 mmHg, p&lt; 0.05), but was lower in RUPP + siRNA (117±4 mmHg) and unchanged in the NP+siRNA (92±3 mmHg) group compared to NP. Average fetal weights by group were 2.30±0.09 g (NP), 1.89±0.10 g (RUPP), 2.38±0.09 g (NP+siRNA), and 2.12±0.06 (RUPP+siRNA). A one-way ANOVA indicates a significant reduction in pup weights between NP and RUPP, but not NP+siRNA and RUPP+siRNA, indicating administration of siRNA to RUPP rats prevented the decrease in fetal weight. Conclusion: Administration of AGT siRNA to RUPP rats blunts the increase in blood pressure and prevents a decrease in fetal weights in response to placental ischemia and thus could be a potential therapy for management of preeclampsia and other hypertensive disorders of pregnancy. Research Supported by Alnylam &amp; T32HL105324,

Neuroprotective effects of antenatal magnesium sulfate under inflammatory conditions in a Sprague–Dawley pregnant rat model*

Objective: Antenatal magnesium sulfate (MgSO4) is recommended for fetal neuroprotection. The aim of this animal study was to assess the neuroprotective effect of in utero exposure to MgSO4, under inflammatory conditions.Methods: Timed pregnant Sprague–Dawley (SD) rats (n = 29) received four intra-peritoneal (IP) injections of lipopolysaccharides (LPS; 200 μg/kg), combined with increasing concentrations of MgSO4 (25, 50 or 100 mg/kg, n = 19) or saline solution (SS; n = 10). In the second set of experiments, animals (n = 8) received a single IP injection of i) LPS (500 μg/kg), MgSO4 (50 mg/kg) and SS (n = 4) or ii) LPS (500 μg/kg), MgSO4 (50 mg/kg) and IL-6 (12 μg/kg) (n = 4). Neurodevelopmental outcomes of surviving pups (n = 212) were assessed by the open field and the rotarod tests.Results: Pups’ average weight at postnatal day (P) 25 was 75.77 g and 89.08 g in MgSO4 and control groups, respectively (p = 0.02). Pups in MgSO4 group have traveled a shorter distance and have shown reduced motor balance and coordination (p < 0.01). Average weight of pups receiving (LPS + MgSO4+ IL-6) was 92.26 g at P25, compared to 75.86 g in (LPS + MgSO4+SS) group (p < 0.05).Conclusions: In our model, MgSO4 induces pup’s growth retardation and motor deficits, which may partly be related to a lower IL-6 circulating concentration.

Reproductive toxicity of ZishenYutai pill in rats: Perinatal and postnatal development study

ZishenYutai pill (ZYP) is one of the most commonly used Chinese patent medicines for threatened miscarriage. Although ZYP is widely used, its toxic effects are rarely assessed. We aimed to investigate whether ZYP had reproductive toxicity during perinatal and postnatal period. Pregnant rats (F0) were continuously exposed to 6, 12 and 24 g/kg body weight/d of ZYP by intragastric administration from gestation day15 to post-natal day21. Vehicle and propylthiouracil (PTU, 15 mg/kg) were used as the negative control and positive control, respectively. The mating was done between the treatment (ZYP or PTU) group and negative control group when the F1 pups were born 63–70 days. Body weight, reproductive ability, physical development and neurodevelopment of F0, F1 and F2 pups were observed. The reproductive capacity of F0 and F1 generation decreased significantly after PTU exposure; however, the body weight and reproductive ability of F0, the physical development, weight, feed consumption and reproductive ability of F1, as well as the physical development and body weight of F2 rats were not significantly changed in the ZYP-treated group compared with the negative control group. ZYP exposure had no perinatal toxicity in 3 generations of rats and may be widely used for miscarriage.

Abstract 1660: JP4-039/F14 treatment of E13 pregnant mice 24 hours after total body irradiation (TBI) improves survival, growth and development of fetal mice

Abstract Purpose: A practical radiation mitigator must be safe in pregnant females. Irradiation during pregnancy can induce fetal death, stunt growth, and/or lead to teratogenic and carcinogenic effects dependent on stage of gestation. We evaluated the mitochondrial targeted GS-nitroxide mitigator JP4-039 for effects on total body irradiated (TBI) pregnant mice. Methods: Timed pregnant C57Bl/6NHsd mice at E13 were irradiated to 3 Gy, subgroups were injected IV 24 hr later (E14) with JP4-039 in F14 emulsion (4 ug JP4-039 in 100 ul of F14). Mice were followed for number of pups born, weight of pups at day 5 after birth, and for number of survivors at time of weaning. In other studies of irradiation effects, new- born pups, on the day of delivery, were euthanized, fixed, sectioned and examined microscopically. Results: Nonirradiated mice showed 97 ± 2% of newborn surviving until weaning. Pups born to 3 Gy irradiated E13 pregnant mice had decreased survival (8.3 ± 8.7%) (p &amp;lt; 0.0001). Control nonirradiated pregnant mice receiving JP4-039/F14 or F14 alone showed no effect on pup survival (85 ± 10% at weaning) (p = 0.1453). The survival of pups from 3 Gy irradiated E13 pregnant mice that received JP4-039/F14 24 hr after irradiation was significantly decreased (45 ± 16.4% compared to nonirradiated controls (p = 0.0230). All newborn pups were weighed at 5 days after birth: those surviving 3 Gy in utero had significantly decreased weight of 1.64 ± 0.04 g compared to 2.73 ± 0.08 g for nonirradiated controls (p &amp;lt; 0.0001). In contrast, while the 3 Gy TBI E13 irradiated pups from mothers that received JP4-039/F14 showed no significant weight change compared to control nonirradiated pups (2.44 ± 0.15 and 2.73 ± 0.08, respectively, p = 0.0799). Their weight was significantly increased compared to the 3 Gy irradiated group (2.44 ± 0.15 and 1.64 ± 0.04 g, respectively, p = 0.0433). Pups from nonirradiated mothers that were administered JP4-039/F14 had a significantly increased weight on day 5 compared to nonirradiated mice (3.32 ± 0.10 and 2.73 ± 0.08 g, respectively, p &amp;lt; 0.0001). Microscopic examination of irradiated pups dying at day of birth, revealed: 1) increased number of hematopoietic precursors in liver and decreased glycogen stores in the hepatocytes; 2)adrenal glands were enlarged and contain severely hypertrophied cortical cells; 3) brain, showed necrosis and loss of parenchyma within the intermediate zone of white matter and cell debris in the lateral ventricles. These changes were not observed in pups from JP4-039/F14 treated mothers, sacrificed on the day of birth. Conclusions: Treatment of total body irradiated E13 pregnant mice at E14 with JP4-039/F14 was safe and effective as a radiation mitigator, led to increased numbers of surviving newborns, improved growth and development, and after weaning over 21 days, and increased body weight with no late deaths. Supported by NIAID/NIH U19-AI068021 Citation Format: Michael W. Epperly, Lora Rigatti, Tracy Dixon, Song Li, Joel S. Greenberger. JP4-039/F14 treatment of E13 pregnant mice 24 hours after total body irradiation (TBI) improves survival, growth and development of fetal mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1660.

Abstract 674: WHSC1L1 is a driving oncogene in ERá positive breast cancer

Abstract The 8p11-p12 genomic region is amplified in 15% of breast cancers and is associated with poorer prognosis. This genomic region harbors several oncogenes, including three epigenetic chromatin modifiers: ASH2L, KAT6A, and WHSC1L1. WHSC1L1 is a histone methyltransferase expressed in two isoforms and we have previously demonstrated that this oncogene is involved in overexpression of ERá as well as its activation independent of estrogen signaling in amplicon-bearing SUM44 breast cancer cells. This cell line serves as a model for the subset of breast cancer patients who demonstrate resistance to hormonal therapies in the context of the 8p11 amplicon and WHSC1L1 overexpression. To validate the connection between overexpression of WHSC1L1 and ESR1, we specifically overexpressed the short, non-catalytic isoform of WHSC1L1 in amplicon-bearing cell lines without WHSC1L1 overexpression. Overexpression of WHSC1L1-short resulted in increased expression of ERá in CAMA1 and SUM52 cells, but not in the amplicon null MCF7 cells. To analyze further the oncogenic role of WHSC1L1 in breast cancer, we generated a transgenic mouse model with targeted overexpression of WHSC1L1 in the mammary gland of FVB/N mice. WHSC1L1 transgenic females had normal sized litters, however the pups were profoundly underdeveloped compared to pups born to wild-type females. Pups born to transgenic females displayed delayed hair growth and eye opening and were half the weight of healthy pups nursed by wild-type females. This suggested a lactation deficiency in WHSC1L1-transgenic females. To further investigate this phenotype, mammary glands were harvested for histologic analysis from transgenic and wild-type females at various stages of the reproductive cycle. Mammary glands from virgin WHSC1L1 transgenic females displayed increased branching and terminal bud formation compared to wild-type virgin females. Similarly, alveolar buds of mid-pregnant females were more numerous and densely packed than in wild-type mice. Consistent with a lactation defect, mammary glands from lactating transgenic females showed evidence of decreased alveolar development compared to wild-type females, resulting in mammary glands where half of the gland failed to functionally differentiate. Finally, post-lactation mammary glands from wild-type females underwent involution, whereas glands from transgenic females failed to involute and were hyperplastic. Significantly, we have observed mammary tumor formation in two transgenic female mice thus far. Ongoing studies include further characterization of this novel mouse model, investigation of the effects of WHSC1L1 overexpression on mammary gland development as pertaining to breast cancer tumorigenesis, and the oncogenic role of WHSC1L1 and its effects on ERá. These and other studies are essential for understanding WHSC1L1 and the 8p11 amplicon in ER+ breast cancer, and how this genomic alteration can be exploited to improve patient outcomes. Citation Format: Brittany Turner Ivey. WHSC1L1 is a driving oncogene in ERá positive breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 674.

Tavaborole, a Novel Boron-Containing Small Molecule Pharmaceutical Agent for Topical Treatment of Onychomycosis: II. Prenatal and Postnatal Developmental Toxicity and Maternal Function Study.

Tavaborole is a topical antifungal agent approved by the US Food and Drug Administration for the treatment of toenail onychomycosis. The effects of tavaborole on gestation, parturition (delivery, labor), offspring development, and survival during the perinatal and postnatal periods were assessed in mated female rats. Females (F0 generation) were administered single daily oral (gavage) doses of 15, 60, or 100 mg/kg/d from gestation day 6 through lactation day 20. The females were allowed to deliver naturally and rear their offspring until lactation day 21, at which time the F0 females were euthanized. One male and female from each litter were selected (F1 generation) and retained for assessments, including growth, neurobehavior, fertility, and their ability to produce an F2 generation. Reproductive and offspring parameters were determined for the F1 and F2 generations, as applicable. F1 females and F2 pups were euthanized on postnatal day 7. In the F0 females, decreased activity was observed in the 100 mg/kg/d dose group. Excess salivation was observed in the 60 and 100 mg/kg/d dose groups (slight to moderate), however, this finding was not considered adverse. There were no tavaborole-related effects on the growth, viability, development, neurobehavioral assessments, or reproductive performance of the F1 generation. Survivability and mean body weight of the F2 pups were unaffected. The no observed adverse effect level (NOAEL) for maternal toxicity (F0 generation) was 60 mg/kg/d, based on the decreased activity observed in the 100 mg/kg/d dose group. The NOAEL for the offspring effects was ≥100 mg/kg/d, based on the lack of test article-related changes.

Pregnancy mitigates cardiac pathology in a mouse model of left ventricular pressure overload

In Western countries heart disease is the leading cause of maternal death during pregnancy. The effect of pregnancy on the heart is difficult to study in patients with preexisting heart disease. Since experimental studies are scarce, we investigated the effect of pressure overload, produced by transverse aortic constriction (TAC) in mice, on the ability to conceive, pregnancy outcome, and maternal cardiac structure and function. Four weeks of TAC produced left ventricular (LV) hypertrophy and dysfunction with marked interstitial fibrosis, decreased capillary density, and induced pathological cardiac gene expression. Pregnancy increased relative LV and right ventricular weight without affecting the deterioration of LV function following TAC. Surprisingly, the TAC-induced increase in relative heart and lung weight was mitigated by pregnancy, which was accompanied by a trend towards normalization of capillary density and natriuretic peptide type A expression. Additionally, the combination of pregnancy and TAC increased the cardiac phosphorylation of c-Jun, and STAT1, but reduced phosphoinositide 3-kinase phosphorylation. Finally, TAC did not significantly affect conception rate, pregnancy duration, uterus size, litter size, and pup weight. In conclusion, we found that, rather than exacerbating the changes associated with cardiac pressure overload, pregnancy actually attenuated pathological LV remodeling and mitigated pulmonary congestion, and pathological gene expression produced by TAC, suggesting a positive effect of pregnancy on the pressure-overloaded heart.