Nonpregnant Female Mice Research Articles

Gestational influenza A virus infection elicits nonresolving vascular dysfunction and T-cell accumulation in the aorta of mice.

T-cell accumulation within the aorta promotes endothelial dysfunction and the genesis of cardiovascular disease, including hypertension and atherosclerosis. Viral infection during pregnancy is also known to mediate marked acute endothelial dysfunction, but it is not clear whether T cells are recruited to the aorta and whether the dysfunction persists postpartum. Here, we demonstrate that influenza A virus (IAV) infection during pregnancy in a murine model resulted in endothelial dysfunction of the aorta, which persisted for up to 60 days postinfection and was associated with higher levels of IFN-γ mRNA expression within the tissue. In the absence of infection, low numbers of naïve CD4+ and CD8+ T cells, central memory T cells, and effector memory T cells were observed in the aorta. However, with IAV infection, these T-cell subsets were significantly increased with a notable accumulation of IAV-specific CD8+ effector memory T cells. Critically, this increase was maintained out to at least 60 days. In contrast, IAV infection in nonpregnant female mice resulted in modest endothelial dysfunction with no accumulation of T cells within the aorta. These data, therefore, demonstrate that the aorta is a site of T-cell recruitment and retention after IAV infection during pregnancy. Although IAV-specific memory T cells could theoretically confer protection against future influenza infection, nonspecific memory T-cell activation and IFN-γ production in the aorta could also contribute to future endothelial dysfunction and cardiovascular disease.NEW & NOTEWORTHY Pregnancy is a risk factor for cardiovascular complications to influenza A virus (IAV) infection. We demonstrate that gestational IAV infection caused endothelial dysfunction of the maternal aorta, which persisted for 60 days postinfection in mice. Various T cells accumulated within the aorta at 60 days because of the infection, and this was associated with elevated levels of the proinflammatory cytokine, IFN-γ. Our study demonstrates a novel "long influenza" cardiovascular phenotype in female mice.

Cardiac automaticity is modulated by IKACh in sinoatrial node during pregnancy.

Pregnant women have a significantly elevated resting heart rate (HR), which makes cardiac arrhythmias more likely to occur. Although electrical remodeling of the sinoatrial node (SAN) has been documented, the underlying mechanism is not fully understood. The acetylcholine-activated potassium current (IKACh), one of the major repolarizing currents in the SAN, plays a critical role in HR control by hyperpolarizing the maximal diastolic potential (MDP) of the SAN action potential (AP), thereby reducing SAN automaticity and HR. Thus, considering its essential role in cardiac automaticity, this study aims to determine whether changes in IKACh are potentially involved in the increased HR associated with pregnancy. Experiments were conducted on non-pregnant (NP, 2-3 months old) and pregnant (P, 17-18 gestation days) female CD-1 mice. IKACh was recorded on spontaneously beating SAN cells using the muscarinic agonist carbachol (CCh). Voltage-clamp data showed a reduction in IKACh density during pregnancy, which returned to control values shortly after delivery. The reduction in IKACh was explained by a decrease in protein expression of Kir3.1 channel subunit and the muscarinic type 2 receptor. In agreement with these findings, current-clamp data shows that the MDP of SAN cells from P mice were less hyperpolarized following CCh administration. Surface electrocardiograms (ECGs) recorded on anesthetized mice revealed that the cholinergic antagonist atropine and the selective KACh channel blocker tertiapin-Q increased HR in NP mice and had only a minimal effect on P mice. AP and ECG data also showed that pregnancy is associated with a decrease in beating and heart rate variability, respectively. IKACh function and expression are decreased in the mouse SAN during pregnancy, strongly suggesting that, in addition to other electrical remodeling of the SAN, reduced IKACh also plays an important role in the pregnancy-induced increased HR.

Correction to: “Impact of Ghrelin on Islet Size in Nonpregnant and Pregnant Female Mice”

Correction to: “Impact of Ghrelin on Islet Size in Nonpregnant and Pregnant Female Mice”

Impact of Ghrelin on Islet Size in Nonpregnant and Pregnant Female Mice.

Reducing ghrelin by ghrelin gene knockout (GKO), ghrelin-cell ablation, or high-fat diet feeding increases islet size and β-cell mass in male mice. Here we determined if reducing ghrelin also enlarges islets in females and if pregnancy-associated changes in islet size are related to reduced ghrelin. Islet size and β-cell mass were larger (P = .057 for β-cell mass) in female GKO mice. Pregnancy was associated with reduced ghrelin and increased liver-expressed antimicrobial peptide-2 (LEAP2; a ghrelin receptor antagonist) in wild-type mice. Ghrelin deletion and pregnancy each increased islet size (by ∼19.9-30.2% and ∼34.9-46.4%, respectively), percentage of large islets (>25 µm2×103, by ∼21.8-42% and ∼21.2-41.2%, respectively), and β-cell mass (by ∼15.7-23.8% and ∼65.2-76.8%, respectively). Neither islet cross-sectional area, β-cell cross-sectional area, nor β-cell mass correlated with plasma ghrelin, although all positively correlated with LEAP2 (P = .081 for islet cross-sectional area). In ad lib-fed mice, there was an effect of pregnancy, but not ghrelin deletion, to change (raise) plasma insulin without impacting blood glucose. Similarly, there was an effect of pregnancy, but not ghrelin deletion, to change (lower) blood glucose area under the curve during a glucose tolerance test. Thus, genetic deletion of ghrelin increases islet size and β-cell cross-sectional area in female mice, similar to males. Yet, despite pregnancy-associated reductions in ghrelin, other factors appear to govern islet enlargement and changes to insulin sensitivity and glucose tolerance in the setting of pregnancy. In the case of islet size and β-cell mass, one of those factors may be the pregnancy-associated increase in LEAP2.

Fetal Maternal Hemorrhage As a Trigger for Fetal/Neonatal Alloimmune Thrombocytopenia: Evidence from a Murine Model

Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a severe bleeding disorder caused by maternal alloantibodies targeting fetal platelet alloantigens. Among the 37 human platelet alloantigens (HPAs) identified, the HPA-1a/HPA-1b polymorphism, involving a Leu33Pro amino acid substitution in the integrin β3 subunit, is the most common cause of severe FNAIT, particularly in Caucasians. Leu33 serves as a key target for alloantibody binding, leading to fetal and neonatal thrombocytopenia and, in severe cases, spontaneous or trauma-induced intracranial hemorrhage. FNAIT shares a pathophysiologic basis with Hemolytic Disease of the Fetus and Newborn (HDFN), which stem from maternal immunization to alloantigens on fetal platelets and red blood cells (RBCs), respectively. While alloimmunization against RBC alloantigens typically occurs in response to fetal maternal hemorrhage (FMH), a clear link between FMH and maternal alloimmunization against HPA-1a on circulating fetal platelets has remained elusive. To assess whether FMH can induce platelet alloimmunization and lead to the development of FNAIT during pregnancy, we utilized recently developed transgenic mice expressing the HPA-1a allogeneic epitope on a murine GPIIIa backbone. To simulate varying degrees of FMH, we transfused different doses of HPA-1a platelets into wild-type (WT), non-pregnant female mice. We found that transfusing as few as 1x10 7 HPA-1a-positive platelets (equivalent to a FMH of approximately 30 ml in humans) resulted in production of high-titer HPA-1a-specific alloantibodies. Since >25% of the cases of FNAIT in humans are thought to occur in what are experienced as first pregnancies, we further investigated whether exposure to HPA-1a in pregnant female mice could still induce FNAIT. We found that when WT female mice pregnant with HPA-1a-positive fetuses were transfused with HPA-1a-positive platelets, they consistently developed HPA-1a-specific antibodies with similar titers as observed in non-pregnant females and delivered thrombocytopenic pups with FNAIT. Taken together, these findings demonstrate that exposure to HPA-1a-positive platelets in the maternal circulation during pregnancy is sufficient to trigger FNAIT, and that maternal tolerance, normally increased during pregnancy, does not play a significantly immunosuppressive role to prevent alloimmunization to the HPA-1a epitope. These findings support the notion that FMH may be a possible cause of alloimmunization in humans, even during a first pregnancy. Finally, the result of this study further validates the usefulness of this HPA-1a alloantigen-specific humanized transgenic mouse model to explore the etiology and pathophysiology of FNAIT. Further studies should significantly advance preventative and therapeutic strategies for this life-threatening immunologic disorder.

THU309 Ghrelin Deficiency Does Not Improve Glucose Tolerance And Insulin Secretion In Female Mice

Abstract Disclosure: D. Gupta: None. K. Shankar: None. S. Varshney: None. O. Singh: None. S. Paul: None. S.B. Ogden: None. S. Osborne-Lawrence: None. A.W. Burstein: None. N.P. Metzger: None. C.P. Richard: None. J.M. Zigman: None. Background: Ghrelin is a peptide hormone secreted primarily from specialized enteroendocrine cells of the stomach. Ghrelin’s ability to regulate blood glucose has emerged as one of its well-recognized actions. For instance, administered ghrelin increases blood glucose in rodents and humans, while ghrelin-knockout (GKO) mice and ghrelin receptor-knockout (GHSR-KO) mice exhibit reduced blood glucose upon fasting, improved glucose tolerance when on regular chow or when on obesogenic diets, and frank hypoglycemia when chronically food restricted. Upon obesogenic diet or glucose challenge, plasma insulin is higher in GKO and GHSR-KO mice, suggesting that ghrelin affects blood glucose at least in part by inhibiting insulin secretion. Notably, ghrelin’s glucoregulatory actions, including its effects to alter insulin secretion, have almost exclusively been studied in males. Because sex significantly impacts the pathogenesis of metabolic disorders and type 2 diabetes, here we studied ghrelin’s effects on glucose tolerance and insulin secretion in female mice and in the setting of pregnancy. Methods: Separate cohorts of 12-13 week-old female GKO and wildtype (WT) littermates with 11.5 gestational days induced by timed pregnancy, and aged matched GKO and WT non-pregnant female mice were fasted for 6 h after which oral glucose tolerance test (OGTT) was performed. Briefly, mice were administered D-glucose (2 g/kg BW) by oral gavage at t = 0 min. Blood glucose was measured from nicked tails at t = 0, 15, 30, 60, 90 and 120 min and insulin was assayed in blood samples collected at t = 0, 15 and 30 min of glucose administration. Results: In OGTT, blood glucose increased significantly and similarly 15 min after oral glucose gavage in GKO and WT mice with or without pregnancy. Blood glucose AUC was 74.2% and 72.8% decreased with pregnancy in WT and GKO mice, respectively. No difference in plasma insulin was observed 15 min after glucose gavage in GKO mice vs WT mice with or without pregnancy. AUC of plasma insulin was significantly increased with pregnancy by 112.6% and by 123.8% in WT mice and GKO mice, respectively. Conclusions: With pregnancy, plasma insulin elevates (presumably as a means to maintain or improve glucose tolerance). Ghrelin deficiency during pregnancy is dispensable for this elevation in insulin and improvement in glucose tolerance. Ghrelin’s response to glucose tolerance and insulin secretion was blunted in female mice suggesting ghrelin’s actions on glucose tolerance and insulin secretion exhibit sexual diversity. Disclosure: The authors have no competing interest. Funding. This work was supported through research grants from the NIH (R01 DK103884 and R01 DK119341 to J.M.Z) Presentation: Thursday, June 15, 2023

Gestational exposure to ambient fine particulate matter disrupts maternal hepatic lipid metabolism

Pregnancy is a unique physiological stage for females as well as a vulnerable period for pollutant exposure. The effect of gestational ambient fine particulate matter (PM2.5) exposure on maternal lipid metabolism during pregnancy is rarely observed, and the mechanism is unknown. In the current study, pregnant C57BL/6 mice were randomly assigned to either ambient PM2.5 or filtered air exposure chambers since gestational day (GD) 0. Meanwhile, non-pregnant female mice were housed as controls in each exposure chamber. PM2.5 exposure exerted no significant effect on body weight gain or the body composition during pregnancy. Pregnant mice exposed to PM2.5 demonstrated improved glucose tolerance, whereas non-pregnant mice showed an increased fasting blood glucose level after PM2.5 exposure with no alterations in glucose tolerance. PM2.5 exposure exerted no significant effect on total lipid content in serum during pregnancy, while an increased serum total lipid level was found in non-pregnant mice exposed to PM2.5. PM2.5 exposure had no effect on total liver lipid levels, it increased several triacylglycerol (TAG) species and total cholesterol esters (CEs) in pregnant mice but lowered a considerable amount in non-pregnant mice’ livers. Furthermore, gestational exposure to PM2.5 enhanced the expression of key enzymes in fatty acid uptake, de novo lipid synthesis, and β oxidation, and inhibited molecules for lipid export in mice liver. Conversely, PM2.5 exposure upregulated proteins involved in hepatic lipolysis and lipid export in non-pregnant mice. These results suggest that the interference of PM2.5 exposure during pregnancy on the lipid metabolism, particularly the hepatic lipid metabolism, differs from that during non-pregnancy. This study provides toxicological evidence that PM2.5 exposure during pregnancy disrupts the lipid metabolism of the liver and provides a basis for protecting vulnerable populations.

Intestinal permeability and peripheral immune cell composition are altered by pregnancy and adiposity at mid- and late-gestation in the mouse.

It is clear that the gastrointestinal tract influences metabolism and immune function. Most studies to date have used male test subjects, with a focus on effects of obesity and dietary challenges. Despite significant physiological maternal adaptations that occur across gestation, relatively few studies have examined pregnancy-related gut function. Moreover, it remains unknown how pregnancy and diet can interact to alter intestinal barrier function. In this study, we investigated the impacts of pregnancy and adiposity on maternal intestinal epithelium morphology, in vivo intestinal permeability, and peripheral blood immunophenotype, using control (CTL) and high-fat (HF) fed non-pregnant female mice and pregnant mice at mid- (embryonic day (E)14.5) and late (E18.5) gestation. We found that small intestine length increased between non-pregnant mice and dams at late-gestation, but ileum villus length, and ileum and colon crypt depths and goblet cell numbers remained similar. Compared to CTL-fed mice, HF-fed mice had reduced small intestine length, ileum crypt depth and villus length. Goblet cell numbers were only consistently reduced in HF-fed non-pregnant mice. Pregnancy increased in vivo gut permeability, with a greater effect at mid- versus late-gestation. Non-pregnant HF-fed mice had greater gut permeability, and permeability was also increased in HF-fed pregnant dams at mid but not late-gestation. The impaired maternal gut barrier in HF-fed dams at mid-gestation coincided with changes in maternal blood and bone marrow immune cell composition, including an expansion of circulating inflammatory Ly6Chigh monocytes. In summary, pregnancy has temporal effects on maternal intestinal structure and barrier function, and on peripheral immunophenotype, which are further modified by HF diet-induced maternal adiposity. Maternal adaptations in pregnancy are thus vulnerable to excess maternal adiposity, which may both affect maternal and child health.

Fruit Extract of Oil Palm (<i>Elaeis Guineensis</i> (Jacq.)) Inhibits Uterine Contractility in <i>Ex-Vivo</i> Mouse Models

Background: Different parts of Elaeis guineensis (EG) plants are used ethno-medicinally for treating numerous illnesses including abdominal contraction.
 Objectives: Thus, this study evaluated the inhibitory potential of the fruit-extract of EG on the uterine contractility in ex-vivo mouse models.
 Material and Methods: The effect of EG extract on spontaneous uterine contractions, oxytocin (OT)-induced uterine contractions, high potassium chloride induced uterine contractions and oxytocin-induced calcium free contraction were evaluated in adult non-pregnant female albino mice.
 Results: EG in a concentration dependent mode diminished spontaneous uterine contractions, while amplitude and frequency of contractions were inhibited significantly, but the frequency of contraction was at the highest concentration of EG. EG also produced a significant inhibition of the frequency of OT-induced contraction in calciumfree media.
 Conclusion: The aqueous fruit extracts of EG inhibited uterine contraction in the uterus through inhibition of intracellular calcium stores.

Midgestation Leptin Infusion Induces Characteristics of Clinical Preeclampsia in Mice, Which Is Ablated by Endothelial Mineralocorticoid Receptor Deletion.

Patients with preeclampsia demonstrate increases in placental leptin production in midgestation, and an associated increase in late gestation plasma leptin levels. The consequences of mid-late gestation increases in leptin production in pregnancy is unknown. Our previous work indicates that leptin infusion induces endothelial dysfunction in nonpregnant female mice via leptin-mediated aldosterone production and endothelial mineralocorticoid receptor (ECMR) activation, which is ablated by ECMR deletion. Therefore, we hypothesized that leptin infusion in mid-gestation of pregnancy induces endothelial dysfunction and hypertension, hallmarks of clinical preeclampsia, which are prevented by ECMR deletion. Leptin was infused via miniosmotic pump (0.9 mg/kg per day) into timed-pregnant ECMR-intact (WT) and littermate-mice with ECMR deletion (KO) on gestation day (GD)11-18. Leptin infusion decreased fetal weight and placental efficiency in WT mice compared with WT+vehicle. Radiotelemetry recording demonstrated that blood pressure increased in leptin-infused WT mice during infusion. Leptin infusion reduced endothelial-dependent relaxation responses to acetylcholine (ACh) in both resistance (second-order mesenteric) and conduit (aorta) vessels in WT pregnant mice. Leptin infusion increased placental ET-1 (endothelin-1) production evidenced by increased PPET-1 (preproendothelin-1) and ECE-1 (endothelin-converting enzyme-1) expressions in WT mice. Adrenal aldosterone synthase (CYP11B2) and angiotensin II type 1 receptor b (AT1Rb) expression increased with leptin infusion in pregnant WT mice. KO pregnant mice demonstrated protection from leptin-induced reductions in pup weight, placental efficiency, increased BP, and endothelial dysfunction. Collectively, these data indicate that leptin infusion in midgestation induces endothelial dysfunction, hypertension, and fetal growth restriction in pregnant mice, which is ablated by ECMR deletion.

The investigation of hippo signaling pathway in mouse uterus during peri-implantation period.

Events in the uterus during the peri-implantation period include embryo development, acquisition of uterine receptivity, implantation and decidualization. Hippo signaling pathway regulates cell proliferation, apoptosis and differentiation. We aimed to determine localization and expressions of pYAP (Phospho Yes-associated protein), YAP (Yes-associated protein), TEAD1 (TEA domain family member 1) and CTGF (Connective tissue growth factor), members of the Hippo signaling pathway, in the mouse uterus during the peri-implantation period. Pregnant mice were randomly separated into 5 groups: 1st, 4th, 5th, 6th, and 8th days of pregnancy groups. Non-pregnant female mice in estrous phase were included in the estrous group. Uteri and implantation sites were collected. Also, inter-implantation sites were collected from the 5th day of pregnancy group. pYAP, YAP, TEAD-1 and CTGF were detected by immunohistochemistry and Western blotting. We observed that the expressions of YAP, TEAD-1 and CTGF were increased in the luminal and glandular epithelium on the 1st and 4th days of pregnancy when epithelial proliferation occurred. pYAP expression was high, and YAP and CTGF expressions were low in the luminal epithelium of the implantation sites on the 5th day of pregnancy, when epithelial differentiation occurred. pYAP expression was low, YAP and CTGF expressions were high at implantation sites on the 6th and 8th days of pregnancy, where decidua was formed. Our findings suggest that the Hippo signaling pathway might be involved in implantation and decidualization. Our findings will guide further studies and may help to elucidate underlying causes of implantation failure and pregnancy loss.

The Establishment of a Mouse Model of Recurrent Primary Dysmenorrhea.

Primary dysmenorrhea is one of the most common reasons for gynecologic visits, but due to the lack of suitable animal models, the pathologic mechanisms and related drug development are limited. Herein, we establish a new mouse model which can mimic the periodic occurrence of primary dysmenorrhea to solve this problem. Non-pregnant female mice were pretreated with estradiol benzoate for 3 consecutive days. After that, mice were injected with oxytocin to simulate menstrual pain on the 4th, 8th, 12th, and 16th days (four estrus cycles). Assessment of the cumulative writhing score, uterine tissue morphology, and uterine artery blood flow and biochemical analysis were performed at each time point. Oxytocin injection induced an equally severe writhing reaction and increased PGF2α accompanied with upregulated expression of COX-2 on the 4th and 8th days. In addition, decreased uterine artery blood flow but increased resistive index (RI) and pulsatility index (PI) were also observed. Furthermore, the metabolomics analysis results indicated that arachidonic acid metabolism; linoleic acid metabolism; glycerophospholipid metabolism; valine, leucine, and isoleucine biosynthesis; alpha-linolenic acid metabolism; and biosynthesis of unsaturated fatty acids might play important roles in the recurrence of primary dysmenorrhea. This new mouse model is able to mimic the clinical characteristics of primary dysmenorrhea for up to two estrous cycles.

EVALUATION OF ACUTE ORAL TOXICITY OF A HERBAL UTERINE

Maintaining optimum reproductive health of animal is crucial for profitability in livestock enterprises and boosting the economy of farmers. However, the reproductive efficiency of animals is commonly reduced by problems such as puerperal metritis, clinical endometritis, pyometra, subclinical endometritis, early embryonic mortality, repeat breeding, retention of foetal membranes, and post-partum anestrus. ExaparTM Liquid (M/s Ayurvet Limited, India) is a polyherbal uterine cleanser and restorative that helps to achieve and restore better post-partum reproductive efficiency. This study aimed to evaluate the potential of Exapar TM Liquid to induce acute oral toxicity in mice as per OECD 423 guidelines. Nine healthy and adult nulliparous, non-pregnant Swiss albino female mice, weighing 24-28 g, were used for the study. The animals were observed for the manifestation of toxic effects and mortality after the oral administration of the test substance. Toxicity was evaluated on the basis of changes in body weight, signs of toxicity, gross and histological appearances of vital organs, and blood biochemistry. Exapar TM Liquid was found safe for oral use as no toxic effects or mortalities were observed up to the completion of experiment.

Evaluation of acute oral toxicity of a herbal semen quality enhancer

The objective of this study was to assess the acute oral toxicity of AV/SGB/27 (M/s Ayurvet Limited) in accordance with OECD-423 guideline. AV/SGB/27 is a herbal formulation for enhancing semen quality of livestock. Nine healthy and adult nulliparous nonpregnant Swiss albino female mice, weighing 24-28 g, were used in this study. After being given test material orally, the mice were examined for toxic effects and mortality. Changes in body weights, symptoms of toxicity, histological appearances of liver, kidney, and lungs, and biochemical parameters were used to assess the toxicity. No toxic effects or mortalities were noticed until the trial was completed, indicating that AV/SGB/27 is safe for oral consumption.

The Acute Toxicity of Rutin in Mice

Acute toxicity is a step to evaluate the toxicity of a substance. Rutin is one of the flavonoid compounds with a variety of pharmacological effects. The aim of the study is to calculate the lethal dose that affect fifty percent of the mice used in the experiment (LD50). Thirty Swiss albino male and 30 non-pregnant female mice have been divided equally and randomly into 5 treated groups and one control group (n=5) Rutin has been administered with concentrations 5, 2.5.1.25,0.625 and 0.312 g/kg administered as a single dose intraperitoneally (IP) while the control group received 1% DMSO (IP). Animals were observed for any morbidity and mortality for 14 days. After 14 days the animal blood collected for biochemical and hematological analysis then all animals are euthanized for histopathological evaluation. The results showed the LD50 was 1.51 g/kg for male mice while for female mice was1.49 g/kg. No significant changes were observed at dose of 1.25glkg (female) and 0.625, 0.312 glkg (both sexes) in body weight measurements and in biochemical or hematological assays. Moreover no significant histopathological changes were reported compared to control.. It can be concluded that Rutin is practically a non-toxic substance.

A reduction in voluntary physical activity in early pregnancy in mice is mediated by prolactin.

As part of the maternal adaptations to pregnancy, mice show a rapid, profound reduction in voluntary running wheel activity (RWA) as soon as pregnancy is achieved. Here, we evaluate the hypothesis that prolactin, one of the first hormones to change secretion pattern following mating, is involved in driving this suppression of physical activity levels during pregnancy. We show that prolactin can acutely suppress RWA in non-pregnant female mice, and that conditional deletion of prolactin receptors (Prlr) from either most forebrain neurons or from GABA neurons prevented the early pregnancy-induced suppression of RWA. Deletion of Prlr specifically from the medial preoptic area, a brain region associated with multiple homeostatic and behavioral roles including parental behavior, completely abolished the early pregnancy-induced suppression of RWA. As pregnancy progresses, prolactin action continues to contribute to the further suppression of RWA, although it is not the only factor involved. Our data demonstrate a key role for prolactin in suppressing voluntary physical activity during early pregnancy, highlighting a novel biological basis for reduced physical activity in pregnancy.

Acute and sub-acute toxicity studies of the chloroform extract of Crinum asiaticum bulbs in mice.

Crinum asiaticum bulbs are widely distributed in parts of Africa and Asia, most evidence suggest this plant is an Asian in origin with some occurrence in the Indian Ocean islands. In Ghana, it is mostly found in the southern part and used ethno-medicinally to treat upper respiratory tract infection, severe cough, skin infection and healing of wounds. In spite of the usage among the indigenous people, there is very little published research on toxicity pertaining to the short or long term use of the bulbs of Crinum asiaticum. The aim of this study is to assess the acute and sub-acute toxicity profile of the chloroform extract of Crinum asiaticum bulbs (CCAE) in ICR mice. In the acute toxicity test, healthy male and non-pregnant female ICR mice (15-20 g) were used. Mice were given single low dose of CCAE orally (2000 mg/kg) in one group and 5000 mg/kg in another group as the highest dose and the control group recieved 1 ml/kg of normal saline (p.o). Gross observation from the acute toxicity study was made for seven days. In the sub-acute toxicity study, doses of 500 and 1500 mg/kg of CCAE were administered orally for 14 days consecutively and observations made throughout the period. In both studies, the animals were closely observed for clinical behavioral changes such as fixed posture, vasodilatation, irritabilities, change in body weight, loss of appetite, hair loss and mortalities. The toxic effect of the extract on liver, kidneys, haematological, and biochemical parameters were assessed including histopathological examination to assess the integrity of the cells of kidney and liver. In conclusion the toxicity study of CCAE showed no significant toxic effect on the liver, kidney, body weight, neurological functions, haematological and biochemical parameters in oral acute toxicity test followed by sub-acute oral toxicity test. Histopathological studies on the cells of kidney and liver appeared normal. The LD50 was determined to be greater than 5000 mg/kg. Hence the chloroform extract of Crinum asiaticum bulbs are not toxic when the dosing is below 5000 mg/kg.

ACE2 CONTRIBUTES TO THE NORMAL REGULATION OF ARTERIAL PRESSURE AND IMMUNITY IN FEMALES OF REPRODUCTIVE AGE

Objective: Hypertension and cardiovascular disease are age and sex dependent. These differences may, in part, be mediated by the depressor/pressor balance of the renin angiotensin system. Here we determined the role of angiotensin converting enzyme 2 (ACE2) in the regulation of arterial pressure in females of reproductive age and investigated whether targeting deficits in ACE2-generated angiotensin (Ang)-(1–7) restores the normal regulation of arterial pressure during pregnancy. Design and method: Mean arterial pressure (MAP) was measured via telemetry in 14 week old wild-type (WT) and ACE2 knockout (ACE2-KO) male mice and WT and ACE2-KO female mice receiving vehicle or the MasR agonist, AVE-0991 (24 ug/kg/min s.c) prior to and during pregnancy. FACS analysis was used to determine circulating immune cell activation and infiltration into kidneys (baseline and Gd18) and placentae (Gd18). Results: Basal MAP was lower in WT females than ACE2-KO females, WT males and ACE2-KO males (91 ± 2 vs 100 ± 1, 98 ± 1 and 102 ± 2 mmHg, respectively; all P < 0.05 vs WT female). In ACE2-KO females, AVE-0991 lowered basal MAP by 5 ± 1 mmHg (P = 0.03). In WT females, MAP decreased during pregnancy reaching a nadir at Gd9 before returning to pre-conception levels during late gestation. In contrast, in ACE2-KO mice, MAP increased significantly during late gestation (P < 0.0001 vs WT) and this effect was prevented by AVE-0991 (P < 0.05 vs vehicle). This effect of AVE-0991 on MAP was due to changes in diastolic rather than systolic arterial pressure. ACE2-KO mice had smaller litters but greater birth/pup weight than WT mice. AVE-0991 normalised litter size and birth/pup weight in ACE2-KO mice to that observed in WT mice. Circulating and renal T-regulatory cells were lower in non-pregnant and pregnant female and male ACE2-KO mice than their WT counterparts. Treatment with AVE-0991 did not alter the proportion of T-regulatory cells. Conclusions: These data indicate that ACE2 plays an important role in the regulation of arterial pressure and immunity in females of reproductive age. A corollary of this is that deficits in ACE2-generated Ang-(1–7) may contribute to an increased risk of hypertension in non-pregnant and pregnant premenopausal females and therefore may be a novel therapeutic target.

Changes in the Distribution of Intrauterine Microbiota May Attribute to Immune Imbalance in the CBA/J×DBA/2 Abortion-Prone Mice Model.

Background: Female Genital Tract (FGT) is an important micro-ecological area of human body. Microbiota in the lower reproductive tract may subsequently invade the uterine cavity during embryo implantation and produce immune responses. CBA/J×DBA/2 mating combination has been widely used as an abortion-prone mice model but whether microbiota existed in their uterine cavity remains unclear. In this context, the role of the microbial communities in immune response deserves attention.Objective: To investigate the relationship between the distribution of microbiota in the uterine cavity of CBA/J×DBA/2 abortion-prone mouse model and the immune imbalance of the maternal-fetal interface.Methods: In this study, female CBA/J mice were paired with male DBA/2 mice to develop an abortion-prone model (BA group), and with male BALB/c mice to build a standard pregnancy model (BC group). The non-pregnant female mice were served as the control group (C group). Uterine flushing fluid and sera were collected on day 13.5 of pregnancy. 16S rRNA sequencing technology was used to analyze the distribution of intrauterine microbiota. Phylogenetic Investigation of Communities were conducted to predict the microbiota functions by Reconstruction of Unobserved States (PICRUST) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The serum IL 10, INF-γ, and TNF-α levels were examined using Enzyme-linked immunosorbent assay (ELISA) method.Results: All samples were detected with microbial communities. The α diversity (p = 0.00077) had significant differences among three groups. Proteobacteria was the most dominant phylum in C group (mean = 83.21%) and BA group (mean = 43.23%). Firmicutes was dominant in BC group (mean = 46.4%), as well as the second dominant one in C group (mean = 12.63%) and BA group (mean = 40.55%). Microbiota functions were associated with metabolism and immune response through the NOD-like receptor signaling pathway. The serum IL 10 level in BA group were significantly lower than that in BC group (10.14 ± 1.90 pg/ml, n = 8; vs. 19.03 ± 1.82 pg/ml, n = 10; p = 0.004). The serum TNF-α and INF-γ level in BA group were also significantly higher than that in BC group (523.1 ± 58.14 pg/ml, n = 8 vs. 310.3 ± 28.51 pg/ml, n = 10, p = 0.0029; 69.22 ± 5.38 pg/ml, n = 8 vs. 50.85 ± 2.45 pg/ml, n = 10, p = 0.0042).Conclusion: Microbial communities were colonized in uterine cavity of CBA/J mice both at non-pregnant stage and pregnant stage when mated with both BALB/c and DBA/2 male mice. The differentially abundant microbiome may be attributed to the immune tolerance through binding to the NOD-like receptor.

Pregnancy-related plasticity of gastric vagal afferent signals in mice.

Gastric vagal afferents (GVAs) sense food-related mechanical stimuli and signal to the central nervous system, to integrate control of meal termination. Pregnancy is characterized by increased maternal food intake, which is essential for normal fetal growth and to maximize progeny survival and health. However, it is unknown whether GVA function is altered during pregnancy to promote food intake. This study aimed to determine the mechanosensitivity of GVAs and food intake during early, mid-, and late stages of pregnancy in mice. Pregnant mice consumed more food compared with nonpregnant mice, notably in the light phase during mid- and late pregnancy. The increased food intake was predominantly due to light-phase increases in meal size across all stages of pregnancy. The sensitivity of GVA tension receptors to gastric distension was significantly attenuated in mid- and late pregnancy, whereas the sensitivity of GVA mucosal receptors to mucosal stroking was unchanged during pregnancy. To determine whether pregnancy-associated hormonal changes drive these adaptations, the effects of estradiol, progesterone, prolactin, and growth hormone on GVA tension receptor mechanosensitivity were determined in nonpregnant female mice. The sensitivity of GVA tension receptors to gastric distension was augmented by estradiol, attenuated by growth hormone, and unaffected by progesterone or prolactin. Together, the data indicate that the sensitivity of GVA tension receptors to tension is reduced during pregnancy, which may attenuate the perception of gastric fullness and explain increased food intake. Further, these adaptations may be driven by increases in maternal circulating growth hormone levels during pregnancy.NEW & NOTEWORTHY This study provides first evidence that gastric vagal afferent signaling is attenuated during pregnancy and inversely associated with meal size. Growth hormone attenuated mechanosensitivity of gastric vagal afferents, adding support that increases in maternal growth hormone may mediate adaptations in gastric vagal afferent signaling during pregnancy. These findings have important implications for the peripheral control of food intake during pregnancy.