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Single Administration of Ultra-Low-Dose Lipopolysaccharide in Rat Early Pregnancy Induces TLR4 Activation in the Placenta Contributing to Preeclampsia
research-article
Author(s):
Pingping Xue
1 ,
Mingming Zheng
2 ,
Ping Gong
3 ,
Caimei Lin
1 ,
Jianjun Zhou
2 ,
Yujing Li
4 ,
Li Shen
1 ,
Zhenyu Diao
2 ,
Guijun Yan
2 ,
Haixiang Sun
2
,
* ,
Yali Hu
1
,
*
Publication date (Electronic):
8 April 2015
Journal:
PLoS ONE
Publisher:
Public Library of Science
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Abstract
Balanced immune responses are essential for the maintenance of successful pregnancy. Aberrant responses of immune system during pregnancy increase the risk of preeclampsia. Toll-like receptor 4 (TLR4) plays a crucial role in the activation of immune system at the maternal-fetal interface. This study aimed to generate a rat model of preeclampsia by lipopolysaccharide (LPS, a TLR4 agonist) administration on gestational day (GD) 5 as rats are subjected to placentation immediately after implantation between GDs 4 and 5, and to assess the contribution of TLR4 signaling to the development of preeclampsia. Single administration of 0.5 μg/kg LPS significantly increased blood pressure of pregnant rats since GD 6 (systolic blood pressure, 124.89 ± 1.79 mmHg versus 119.02 ± 1.80 mmHg, P < 0.05) and urinary protein level since GD 9 (2.02 ± 0.29 mg versus 1.11 ± 0.18 mg, P < 0.01), but barely affected blood pressure or proteinuria of virgin rats compared with those of saline-treated pregnant rats. This was accompanied with adverse pregnancy outcomes including fetal growth restriction. The expression of TLR4 and NF-κB p65 were both increased in the placenta but not the kidney from LPS-treated pregnant rats, with deficient trophoblast invasion and spiral artery remodeling. Furthermore, the levels of inflammatory cytokines were elevated systemically and locally in the placenta from pregnant rats treated with LPS. TLR4 signaling in the placenta was activated, to which that in the placenta of humans with preeclampsia changed similarly. In conclusion, LPS administration to pregnant rats in early pregnancy could elicit TLR4-mediated immune response at the maternal-fetal interface contributing to poor early placentation that may culminate in the preeclampsia-like syndrome.
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Most cited references32
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Spiral artery remodeling and trophoblast invasion in preeclampsia and fetal growth restriction: relationship to clinical outcome.
Fiona Lyall, Stephen C Robson, Barret Bulmer (2013)
Failure to transform uteroplacental spiral arteries is thought to underpin disorders of pregnancy, including preeclampsia and fetal growth restriction (FGR). In this study, spiral artery remodeling and extravillous-cytotrophoblast were examined in placental bed biopsies from normal pregnancy (n = 25), preeclampsia (n = 22), and severe FGR (n = 10) and then compared with clinical parameters. Biopsies were immunostained to determine vessel wall integrity, extravillous-cytotrophoblast location/density, periarterial fibrinoid, and endothelium. Muscle disruption was reduced in myometrial spiral arteries in preeclampsia (P = 0.0001) and FGR (P = 0.0001) compared with controls. Myometrial vessels from cases with birth weight 5th percentile. Fewer extravillous-cytotrophoblast surrounded both decidual and myometrial vessels in the normal group and preeclampsia group compared with the FGR group (P = 0.001). For myometrial vessels, the normal group contained more intramural extravillous-cytotrophoblast than in preeclampsia (P = 0.015). Decidual vessels in the FGR group had less fibrinoid deposition compared with controls (P = 0.013). For myometrial vessels, less fibrinoid was deposited in both the preeclampsia group (P = 0.0001) and the FGR group (P = 0.01) when compared with controls, and less fibrinoid was deposited in the preeclampsia group when compared with FGR group (P 5th percentile (P<0.02). A major defect in myometrial spiral artery remodeling occurs in preeclampsia and FGR that is linked to clinical parameters. Interstitial extravillous-cytotrophoblast is not reduced in preeclampsia but is increased in FGR.
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Essential role of microRNA-155 in regulating endothelium-dependent vasorelaxation by targeting endothelial nitric oxide synthase.
Yong Tang, Yuan-Ping Pang, Shiuh-Lin Huang … (2012)
Nitric oxide generated by endothelial nitric oxide synthase (eNOS) plays an important role in maintaining cardiovascular homeostasis. Under various pathological conditions, abnormal expression of eNOS contributes to endothelial dysfunction and the development of cardiovascular diseases. A variety of pathological stimuli has been reported to decrease eNOS expression mainly through decreasing eNOS mRNA stability by regulating the binding of several cytosolic proteins to the cis-acting sequences within eNOS mRNA 3' untranslated regions. However, the detailed mechanisms remain elusive. Because microRNAs inhibit gene expression through binding to the 3' untranslated regions of their target mRNAs, microRNAs may be the important posttranscriptional modulators of eNOS expression. Here, we provided evidence that eNOS is a direct target of miR-155. Overexpression of miR-155 decreased, whereas inhibition of miR-155 increased, eNOS expression and NO production in human umbilical vein endothelial cells and acetylcholine-induced endothelium-dependent vasorelaxation in human internal mammary arteries. Inflammatory cytokines including tumor necrosis factor-α increased miR-155 expression. Inhibition of miR-155 reversed tumor necrosis factor-α-induced downregulation of eNOS expression and impairment of endothelium-dependent vasorelaxation. Moreover, we observed that simvastatin attenuated tumor necrosis factor-α-induced upregulation of miR-155 and ameliorated the effects of tumor necrosis factor-α on eNOS expression and endothelium-dependent vasodilation. Simvastatin decreased miR-155 expression through interfering mevalonate-geranylgeranyl-pyrophosphate-RhoA signaling pathway. These findings indicated that miR-155 is an essential regulator of eNOS expression and endothelium-dependent vasorelaxation. Inhibition of miR-155 may be a new therapeutic approach to improve endothelial dysfunction during the development of cardiovascular diseases.
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Inflammation in rat pregnancy inhibits spiral artery remodeling leading to fetal growth restriction and features of preeclampsia
Tiziana Cotechini, Maria Komisarenko, Arissa Sperou … (2014)
Preeclampsia (PE) is a serious condition that affects 3–10% of all pregnancies (Lyall and Belfort, 2007), and is characterized by the development of maternal hypertension (>140/90 mm Hg) and proteinuria (≥300 mg/24 h; Redman and Jefferies, 1988). PE usually develops after 20 wk of gestation, and is difficult to treat except by early delivery, which can result in neonatal complications. In addition to increasing the maternal and fetal risk of morbidity and mortality, the pathological processes associated with PE can restrict fetal growth and impair development. Fetal growth restriction (FGR) occurs when the fetus fails to achieve its genetically predetermined growth potential (Gardosi et al., 1992). It is the second leading cause of fetal death and is often associated with PE (Peleg et al., 1998; Vatten and Skjaerven, 2004). Although the precise mechanisms leading to the development of FGR/PE remain unknown, there is evidence that these complications are associated with an aberrant maternal inflammatory response. Women afflicted by FGR/PE exhibit a heightened inflammatory state; proinflammatory cytokines and chemokines, such as TNF, IL-6, and MCP-1, are elevated systemically and locally in the placenta (Redman et al., 1999; Borzychowski et al., 2006; Jain et al., 2007; LaMarca et al., 2007; Szarka et al., 2010). This abnormal inflammatory response leads to oxidative and nitrosative stress associated with decreased nitric oxide (NO) bioavailability (Roggensack et al., 1999; Lowe, 2000; Borzychowski et al., 2006). It is widely recognized that the placenta plays an important role in the pathophysiology of FGR/PE (Page, 1939; Koga et al., 2010). In the two-stage model of PE initially proposed by Redman (1991), deficient placental perfusion (stage one) leads to the release of vasoactive factors that precipitate the onset of the maternal syndrome (stage two) (Roberts and Gammill, 2005). Placental perfusion is dependent on adequate remodeling of the uterine spiral arteries, a process whereby endovascular trophoblast cells invade and replace the endothelium and vascular smooth muscle of these vessels (Boyd and Hamilton, 1970). Hence, a deficiency in this remodeling process is thought to account for the poor placental perfusion associated with FGR/PE. Despite the strong association between abnormal maternal inflammation and FGR/PE, it is not known whether inflammation is causally linked to the deficient spiral artery (SA) remodeling that characterizes these pregnancy complications. Reister et al. (1999) examined vessels from preeclamptic pregnancies and found that an increase in the distribution of macrophages around the spiral arteries was associated with impaired trophoblast invasion. Moreover, our in vitro studies revealed that activated macrophages inhibit trophoblast invasion by secreting TNF at subapoptotic levels (Renaud et al., 2005). Other studies have additionally described an inhibitory role of TNF in the regulation of trophoblast motility and migration (Todt et al., 1996; Renaud et al., 2007; Venegas-Pont et al., 2010). Using a rat model, we describe a novel mechanism by which abnormal maternal inflammation results in deficient SA remodeling and altered uteroplacental perfusion. Our study also reveals that this inflammation-induced deficiency in vascular adaptation is associated with FGR and features of PE. RESULTS LPS administration induces a systemic and local inflammatory response and results in TNF-mediated FGR To assess whether LPS administration affects in utero growth, we defined FGR in our rats as a fetal weight falling below the 10th percentile for gestational age. To determine the threshold of FGR, we evaluated the distribution of all fetal weights from the saline-treated control cohort (n = 22 dams; n = 305 fetuses; mean fetal weight = 0.9244 ± 0.007 g); fetuses with weights 200 preeclamptic women revealed that at the time they were discharged from hospital, 78% were still hypertensive. Moreover, hypertension was found in 54% of women at 6 wk postpartum and in 39% of women by 3 mo postpartum (Berks et al., 2009). The reduced uteroplacental perfusion (RUPP) model of PE is characterized by elevated blood pressure, and studies using this model revealed a physiological role for TNF as a key molecule mediating the elevation in blood pressure (LaMarca et al., 2005). In our study, administration of LPS resulted in a significant elevation in MAP that was mediated by TNF. High levels of TNF are known to play an inhibitory role in endothelium-dependent and NO-mediated vasodilation (Zhang et al., 2009). Moreover, there is evidence that TNF induces the release of the potent vasoconstrictor endothelin-1 (ET-1) from bovine aortic endothelial cells in a time- and concentration-dependent manner (Marsden and Brenner, 1992). Thus, we propose that Eta prevents the increase in MAP observed in LPS-treated pregnant rats, at least partly, by inhibiting TNF-mediated release of ET-1 by the maternal endothelium. Furthermore, our data revealed that transdermal administration of GTN attenuated the LPS-induced increase in MAP. Studies indicate that the blood pressure lowering effects of NO donors are at least partly due to inhibition of ET-1–mediated vasoconstriction (Bourque et al., 2011; Bourque et al., 2012). Moreover, it has been reported that endothelium-derived NO inhibits the release of ET-1 from the porcine aorta (Boulanger and Lüscher, 1990) and that exogenous administration of NO inhibits ET-1 gene transcription (Kourembanas et al., 1993). Therefore, it is likely that GTN prevents the increases in MAP observed in LPS-treated pregnant rats by interfering with a mechanism of vasoconstriction involving TNF-induced ET-1 activity. Through this mechanism GTN may also ensure adequate placental perfusion, thereby preventing the development of an exaggerated inflammatory response to LPS and the downstream sequelae. In addition to these indirect antiinflammatory actions of GTN, it is also possible that GTN has direct antiinflammatory properties. We have preliminary results indicating that GTN can inhibit in vitro TNF release by LPS-activated THP-1 cells, a monocytic cell line (unpublished data). Nevertheless, to our knowledge, the present study provides the first evidence that GTN can inhibit acute proinflammatory effects of LPS. The critical role of the kidneys in the regulation and maintenance of blood pressure suggests that the maternal renal abnormalities, characteristic of PE, are closely linked to blood pressure alterations. Our rat model revealed that treatment with LPS resulted in renal alterations similar to those present in women with severe PE. These alterations included mesangial hypercellularity, occlusion of the capillary loops and urinary space, and thickening of the GBM. Renal abnormalities were not observed in dams treated with Eta + LPS or GTN + LPS. Our data agree with a recent study that mechanistically linked high levels of TNF and subsequent oxidative stress to hypertension and renal abnormalities in a mouse model of the chronic inflammatory autoimmune disorder systemic lupus erythematosus (Venegas-Pont et al., 2010). Our conceptual model proposes that during pregnancy, an otherwise mild inflammatory stimulus causes severe restrictions in uteroplacental perfusion leading to FGR and an exaggerated maternal inflammatory response characterized by increased circulating TNF levels, increased MAP, and renal structural alterations. Whereas administration of LPS to nonpregnant rats led to a small increase in MAP, LPS administration to nonpregnant animals did not cause renal alterations and did not result in substantial increases in circulating WBCs and TNF levels. Although we focused on investigating the role of abnormal inflammation in SA remodeling, we cannot rule out the possibility that other inflammation-related factors contribute to the pathogenesis of pregnancy complications. For example, abnormal inflammation may be linked to FGR/PE via disruption of hemostasis (Pijnenborg et al., 2006; Cotechini et al., 2012; Falcón et al., 2012), release of other proinflammatory molecules (Borzychowski et al., 2006), local placental damage (Burton et al., 2009), and/or syncytiotrophoblast microparticles (Knight et al., 1998) that may induce systemic endothelial dysfunction. In addition to the nonpathological, low-grade inflammatory state of healthy pregnancy, normal pregnant women are more susceptible to infections compared with their nonpregnant counterparts (Mor and Cardenas, 2010). Indeed, our study and other studies found the effects of LPS to be pregnancy specific (Faas et al., 1994, 1995). Our study indicates that TNF levels in nonpregnant rats, measured 2 h after LPS administration, were not significantly different from TNF levels measured in saline-treated rats during pregnancy. The excessive inflammation characteristic of women afflicted by PE may therefore be an exacerbation of an underlying inflammatory pathology. Obesity, urinary tract infections, periodontal disease, and viral infections are associated with the development of PE (Conde-Agudelo et al., 2008; Rustveld et al., 2008; Chaparro et al., 2013). Based on our conceptual model, we propose that deficient placental perfusion is a critical aspect of the pathophysiology of FGR and PE associated with abnormal inflammation. The use of LPS to trigger inflammation is well described and has been used to study the pathophysiology of various disease states including Parkinson’s disease (Liu and Bing, 2011), acute lung injury (Rittirsch et al., 2008), and pathological pregnancies (Faas et al., 1994). Recently, Lin et al. (2012) reported that the endogenous antiinflammatory molecule Lipoxin A(4) could alleviate LPS-induced PE-like symptoms in pregnant Sprague–Dawley rats. The authors of that study did not report FGR, possibly because a single, lower dose of LPS was used. Although LPS is often used to induce sepsis, the doses used in our model were much lower than those required to induce septic shock in rodents (McDonald et al., 2000; Nemzek et al., 2008). In other studies, LPS has been used to induce preterm labor in mice (Salminen et al., 2008; Shynlova et al., 2013); however, with the low doses of LPS used in our study, we did not observe preterm labor. Modulation of inflammation may have therapeutic benefit in the prevention and management of FGR/PE. Despite low transplacental transfer, Eta is currently classified as a category B drug by the United States Federal Drug Administration and is not routinely prescribed to pregnant women due to a lack of well-controlled prospective studies (Berthelsen et al., 2010). Two recent retrospective studies examining the safety of Eta use during pregnancy were conflicting. One study linked Eta to the development of congenital malformations (Carter et al., 2009), whereas the most recent report did not find increased incidence of malformations when compared with acceptable rates in Western countries (Viktil et al., 2012). The results of our study also highlight the potential use of NO-mimetics as antiinflammatory agents in the prevention of FGR/PE. The safety and use of GTN in obstetrics as a tocolytic agent are well characterized (Smith et al., 1999; de Pace et al., 2007). In summary, our study demonstrates a novel mechanism by which abnormal maternal inflammation decreases SA remodeling associated with deficient uteroplacental perfusion and the development of FGR/PE. Given the similarities of hemochorial placentation between humans and rats, in particular the pattern of deep intrauterine trophoblast invasion and SA remodeling (Soares et al., 2012), we believe that our rat model of LPS-induced FGR/PE is useful for the study of inflammation-associated pregnancy complications. The results of our study indicate that judicious management of inflammation, either systemically or by blocking the release of placental proinflammatory molecules, is a potential strategy for the prevention and/or treatment of FGR/PE. MATERIALS AND METHODS Animals. Studies were conducted in accordance with the guidelines of the Canadian Council on Animal Care and protocols were approved by the Queen’s University Animal Care Committee. Virgin female Wistar rats (3–6 mo old; Charles River Laboratories) were housed in a light- and humidity-controlled environment and fed ad libitum with free access to tap water. Vaginal smears were performed the morning after females were housed overnight with a fertile male at a 2:1 ratio. The presence of spermatozoa in the vaginal lavage confirmed pregnancy and was designated GD 0.5. Experimental protocol. Pregnant rats received i.p. injections of saline or LPS (Escherichia coli serotype 0111:B4; Sigma-Aldrich) on GD 13.5, 14.5, 15.5, and 16.5 of a 22-d gestation. To induce an inflammatory response, dams received a single low dose (10 µg/kg) of LPS on GD 13.5, followed by higher doses (40 µg/kg) daily until GD 16.5 (n = 28). Control animals (n = 22) received saline (1 ml/kg) injections on the corresponding GDs. Rats received 5 ml of lactated ringers subcutaneously with each injection. Dams were sacrificed 24 h after the last LPS/saline injection on GD 17.5. Animals used in the radiotelemetry experiments (see below) were sacrificed on PD 7.5. After euthanasia, fetal weights were measured and recorded in conjunction with the pup’s location along the length of the uterine horn. To evaluate the role of TNF on the development of FGR, dams additionally received i.p. injections of the TNF inhibitor Eta (10 mg/kg; Enbrel; Amgen and Wyeth Pharmaceuticals) on GD 13.5 and 15.5, 6 h before administration of LPS (n = 16). To determine whether the effects of LPS are pregnancy specific, nonpregnant rats were exposed to the same doses of LPS as pregnant animals over 4 d (10 µg/kg followed by 40 µg/kg) and sacrificed on the fifth day, 24 h after the last LPS injection. To examine the acute effects of LPS, additional experiments were performed on rats 2 h after the initial LPS (10 µg/kg) injection. For these studies, maternal blood was collected at euthanasia from rats across all treatment groups on GD 13.5, 2 h after LPS administration (n = 5–7). Corresponding experiments were performed on nonpregnant rats 2 h after saline or LPS administration (n = 4). Administration of GTN. To assess a potential role of deficient NO signaling in the pathogenesis of inflammation-induced FGR/PE, pregnant dams (n = 16) receiving LPS were additionally treated with the NO-mimetic GTN. This was accomplished via daily application and replacement of Minitran transdermal patches (Graceway Pharmaceuticals) starting on GD 12.5 and ending on GD 16.5. Patches (cut to a surface area of 1.7 cm2, providing a GTN steady-state delivery rate of 25 µg/h) were placed on the nape of the neck and were covered with a thin layer of New Skin Liquid Bandage (Prestige Brands Inc.). The last patch was removed at euthanasia on GD 17.5. Tissue preparation. Rats were sacrificed on GD 17.5 via an i.p. injection (40–50 mg/kg) of sodium pentobarbital (Ceva Santé Animale). Following euthanasia, the wet weight of each fetus was measured and recorded in combination with its location along the length of the respective uterine horn. Implantation sites (uterus + placenta) were placed in 4% paraformaldehyde for at least 24 h. Before tissue processing, implantation sites were carefully bisected (through the MT and placenta) to ensure that the mid-portion of each unit was used for further analysis. Tissues were subsequently transferred to 70% ethanol and were processed and embedded in paraffin according to standard procedures. Serial sections (5 µm) were cut from each uteroplacental unit for immunohistochemical analysis. Identification of trophoblast and smooth muscle cells. Trophoblast cells were identified by immunohistochemistry for cytokeratin using mouse monoclonal anticytokeratin antibody (DAKO) and the method described by Vercruysse et al. (2006). Negative controls consisted of sections in which the primary antibody was substituted with an equal concentration of mouse IgG (DAKO). Immunohistochemistry for α-actin was used to identify vascular smooth muscle cells. In brief, tissues were rehydrated using a graded ethanol series and sections were subsequently blocked using 10% normal horse serum (NHS) + 0.1% Tween-80 (Thermo Fisher Scientific) for 20 min followed by serum-free DAKO block for 7 min. Sections were then incubated for 30 min in the presence of antibody against smooth muscle α-actin (Sigma-Aldrich; 1:400 in 2% NHS); this was followed by incubation with biotinylated anti–mouse IgG antibody (1:200 in 1% NHS + 3% normal rat serum; 30 min; Vector Laboratories). A Vectastain ABC Elite kit (Vector Laboratories) and diaminobenzidine (DAKO) were used to detect antigenic sites. Tissues were counterstained with Gill’s hematoxylin (Thermo Fisher Scientific), dehydrated, and mounted. Negative controls consisted of sections incubated with an equal concentration of mouse IgG (DAKO) in place of the primary α-actin antibody. Identification of tissue macrophages using CD68 immunohistochemistry. After rehydration, proteinase K antigen retrieval in sections of implantation sites was performed, and endogenous peroxidase was quenched using 3% H2O2 + 0.3% Triton X-100 (Bio-Rad Laboratories) in PBS. Nonspecific sites in sections were subsequently blocked using 10% NHS for 20 min, followed by serum-free DAKO block for 7 min (DAKO). Primary antibody (AbD Serotec; 1:250 in 2% NHS) was added overnight at 4°C. Secondary antibody (1:200 in 1% NHS; Vector Laboratories) was added for 30 min and detected using the Vectastain ABC Elite kit (Vector Laboratories). Sections were developed using diaminobenzidine (DAKO), counterstained with Gill’s hematoxylin (Thermo Fisher Scientific), dehydrated, and mounted. Negative controls consisted of sections in which primary antibody was substituted with equal concentrations of mouse IgG (DAKO). Image analysis. Assessment of activated macrophage localization, interstitial trophoblast invasion, and SA remodeling was performed on slides scanned at 20× using an Aperio ImageScope system and software (Aperio). A positive pixel count was used to quantify labeled cells in the MT to assess the presence of activated macrophages. Interstitial trophoblast invasion was calculated as the total area occupied by cytokeratin-positive interstitial trophoblast cells within the MT. Only sections exhibiting the “break through” of interstitial trophoblast cells through the giant cell layer were used to quantify interstitial invasion for consistency (Bridgman, 1948). The cross-sectional area of spiral arteries exhibiting endovascular trophoblast cells was measured by a blinded observer and confirmed by a second investigator. Specifically, the fibrinoid layer around spiral arteries and/or the basal surface of cytokeratin-positive or endothelial cells were used as the boundary with which to manually delineate the luminal contour of each vessel. Doppler ultrasonography. Doppler ultrasonography was used to evaluate maternal and fetal hemodynamic alterations on GD 17.5 using a previously described method (Renaud et al., 2011) adapted from Mu and Adamson (2006). In brief, abdominal hair was removed from anaesthetized (∼3% isoflurane in oxygen by nose cone) dams using a mechanical shaver followed, by application of a chemical depilatory. Maternal heart rate and breathing were monitored constantly to ensure surgical plane for the duration of the measurements. The high frame rate 707B 30 mHz scanhead was used in Doppler mode to obtain pulse-wave recordings for offline analysis. Data acquisition was performed on 3–4 viable implantation sites per dam; measurements were recorded from an implantation site located adjacent to the bladder and a site in close proximity to the ovary within each horn. Doppler waveforms were recorded for 2–3 spiral arteries and the corresponding fetal umbilical artery for each implantation site. After acquisition, a blinded observer performed analysis. PSV and end diastolic flow velocity (EDV) of each SA was measured to determine the mean SA RI of the implantation site based on the following equation (RI = [PSV – EDV]/PSV). Umbilical PSV of the corresponding implantation site was also measured. Complete blood cell counts (CBCs). Whole blood was collected by cardiac puncture into tubes containing ethylenediaminetetraacetic acid (EDTA) for CBC analysis. Blood samples (12 µl) were analyzed using the ABC Vet Animal Blood Counter (Scil Animal Care Company) according to the manufacturer’s instructions. Analysis of maternal plasma TNF. To confirm that LPS administration induced maternal inflammation, plasma levels of TNF were quantified by ELISA (R&D Systems) according to the manufacturer’s instructions. Levels falling below the minimum detectable dose of the kit (5 pg/ml) were considered undetectable. At euthanasia, maternal blood was collected via cardiac puncture into EDTA-containing syringes to prevent clotting. Blood samples were obtained from dams at the following time-points: 2 h after saline or LPS ± Eta/GTN administration at GD 13.5; 2 h after LPS administration at GD 15.5; 2 h after LPS administration to nonpregnant animals. Implantation of radiotelemetry transducers. Continuous assessment of MAP was achieved through femoral implantation of PA-C40 radiotransmitters (Data Sciences International). In brief, virgin female rats were anaesthetized using ∼3.0% isoflurane in oxygen by nose cone. The left thigh was shaved, and a 2-cm incision was made to expose the femoral vessels and nerve. Using a dissection microscope, the femoral artery was carefully retracted, and a permanent occlusion suture was placed around the vessel. Proximal to the occlusion ligature, a 26-gauge needle was used to puncture the vessel to facilitate the insertion of the tip of the transmitter catheter. Cannulation forceps were used to advance the catheter tip into the abdominal aorta, whereupon sutures were used to hold the device in place. Transmitters were secured subcutaneously over the right flank and the incision was closed with staples. Postoperative care consisted of administration of meloxicam (1 mg/kg), dexamethasone (0.05 mg/kg), and tramadol (20 mg/kg) in 10 ml of subcutaneous lactated ringers administered daily for 4–7 d as needed. Dams were allowed to recover for at least 14 d before overnight mating. Pregnant animals were randomized into the four treatment groups (n = 5/group): saline, LPS, Eta + LPS, and GTN + LPS. Dams and pups were euthanized on PD 7.5. Radiotelemetry data acquisition and representation. Upon recovery, continuous 24-h data were collected for 30 s every 8 min using Dataquest A.R.T. Acquisition System (Data Sciences International, version 4.1) until the study endpoint at PD 7.5. All values from a 24-h period (midnight–midnight) were used to calculate a daily 24-h mean data point for each GD. Although absolute MAP values exhibited a normal range of variability among groups before treatments started, importantly, MAP profiles did not differ significantly between animals before treatment initiation (GD 0–11). To evaluate the effect of treatment on changes in blood pressure, MAP measured on GD 11 was set as baseline for each animal, and the delta value was calculated for subsequent GDs/PDs. To determine whether the effects of LPS on MAP are pregnancy-specific, we performed an additional set of experiments on nonpregnant animals. Animals in the nonpregnant study group (n = 4) were instrumented with radiotelemetry transducers, as previously described. After recovery, all animals were subjected to the following procedures: 2 d of baseline recording; four saline injections (once/day); 1 d of rest; 2 d of baseline recording; four LPS injections (10–40 µg/kg/day, as per the doses used in the pregnancy studies). For each experimental animal, MAP recorded over the 2 d preceding the onset of each treatment was set as baseline, and the delta value was determined for subsequent experimental days. Baseline MAP before the onset of saline treatment was not significantly different than baseline MAP before LPS administration. Urine analysis. Urine was collected from animals instrumented with radiotransmitters on GD 10.5–17.5, GD 19.5, and PD 7.5 using a modified metabolic cage technique. For all days of urine collection that corresponded with injection days, urine was collected in the morning, before animals received any treatment. In brief, dams were placed in cages lined with 96-well plates for 3–4 h on the morning of collection. Urine was collected from the plates and centrifuged (300 g, for 5 min) before storing at −80°C for future analysis. Total protein concentration was determined using a protein assay (Bio-Rad Laboratories) and was normalized against total creatinine concentration (Cayman Chemical Company) to assess proteinuria. Because each animal had different protein/creatinine ratios before the onset of any treatment, a baseline protein/creatinine measurement for each rat was determined by taking the mean protein/creatinine values from urine collected each day from GD 10.5 to 12.5. This baseline protein/creatinine ratio was used to calculate the change in proteinuria from urine collected on subsequent days after initiation of treatments. To determine whether proteinuria correlates with changes in MAP, changes in protein/creatinine ratios were matched against corresponding changes in MAP for all animals in each treatment group. Assessment of postnatal pup weight. Pups from all treatment groups were weighed on PD 1.5 and 7.5. It has recently been suggested that the standardization of litter sizes is beneficial to account for litter size–induced effects (i.e., physical space restraints and access to maternal milk supplies), which would otherwise dilute the ability to detect treatment-related outcomes on fetal growth and postnatal development. Given that pup weight assessment was performed several days after treatment was administered and may have affected litter sizes, we applied a correction factor to standardize pup weights based on litter size as described by Chahoud and Paumgartten (2009). Histopathological analysis of kidneys. Kidneys harvested from dams on GD 17.5 were fixed, processed, sectioned (3 µm), and stained with hematoxylin and eosin and periodic acid-Schiff for histopathological assessment of renal alterations. The degree of glomerular pathology was assessed by a blinded observer based on criteria and scoring methods adapted from other studies (Strevens et al., 2003; Li et al., 2007), and after consultation with an experienced renal pathologist at Kingston General Hospital. For each kidney, 20 glomeruli were individually scored and the severity of glomerular pathology was determined from the mean score for each kidney. Confirmatory scoring was performed on each section by an additional blinded observer to ensure that mean scores for each section were consistent. Glomeruli from nonpregnant animals exposed to the same doses of LPS (n = 4) were additionally assessed as previously described. Electron microscopic analysis of renal ultrastructure. Glutaraldehyde-fixed 1-mm3 cortical samples from kidneys were processed, embedded in epon, and cut into ultra-thin (90-nm) sections. Images captured by a blinded observer were later used to evaluate ultrastructural alterations of the GBM. Quantitative assessment of GBM thickness (GBM area/GBM length) was performed using Image-Pro Plus version 6.0 software. Quantitative analysis of placental nitrotyrosine. Placental nitrotyrosine levels were quantified by ELISA using a commercially available kit (Millipore). As previously described (Roberts et al., 2009), placentas randomly chosen from all treatment groups were homogenized in lysis buffer on ice using a Polytron tissue homogenizer (Biospec Products Inc.). Homogenates were centrifuged (20,000 g, for 5 min) and the supernatant was transferred to separate tubes. A modified Lowry assay (DC Protein Assay; Bio-Rad Laboratories) was used to measure and subsequently adjust protein concentrations between samples. Samples (saline, n = 9 from seven mothers; LPS, n = 21 from nine mothers; LPS + GTN, n = 9 from seven mothers) were assayed in duplicate. Statistics. Data were analyzed using GraphPad Prism 6.0 Software (GraphPad). Parametric data are presented as mean ± SEM and were analyzed using two-tailed Student’s t test (two groups), one-way ANOVA (three or more groups), or two-way ANOVA (two-variables). Significant differences between individual treatment groups were determined using the Bonferroni post-hoc test when comparing fewer than five groups, or Tukey’s multiple comparison post-hoc test when comparing more than five groups. Kruskal-Wallis followed by Dunn’s multiple comparison post-hoc test was used to assess alterations in maternal plasma TNF levels, as these did not follow a normal distribution. χ2 was used to analyze data obtained from nitrotyrosine determination experiments. For the radiotelemetry studies in pregnant animals, changes in MAP relative to baseline (GD 11) were analyzed using repeated measures two-way ANOVA, followed by Bonferroni post-hoc test to assess for statistical differences between groups. Within each treatment group, differences in raw MAP from baseline (GD 11) were determined by repeated measures two-way ANOVA, followed by Dunnett’s multiple comparisons test. Expectation maximization imputation was performed for data points missing at random (a total of six data points) from the radiotelemetry experiments using SPSS Statistics software. This method has been previously used and validated for imputation of missing data in rat studies using small sample sizes (Rubin et al., 2007). Changes in MAP relative to baseline in nonpregnant animals were analyzed using repeated measures two-way ANOVA, followed by Bonferroni post-hoc test to assess for statistical differences between groups. To compare overall differences in proteinuria, the mean change of the protein/creatinine ratio was calculated for each day of urine collection and analyzed using one-way ANOVA, followed by Bonferroni post-hoc test. Nonlinear, nonparametric data were assessed by Spearman correlation, whereas linear parametric data were assessed by Pearson correlation. For all statistical tests, P < 0.05 was considered significant, whereas P < 0.1 was considered a trend toward significance.
Author and article information
Contributors
Haibin Wang: Role: Academic Editor
Journal
Journal ID (nlm-ta): PLoS One
Journal ID (iso-abbrev): PLoS ONE
Journal ID (publisher-id): plos
Journal ID (pmc): plosone
Title:
PLoS ONE
Publisher:
Public Library of Science
(San Francisco, CA USA
)
ISSN
(Electronic):
1932-6203
Publication date
(Electronic):
8
April
2015
Publication date Collection: 2015
Volume: 10
Issue: 4
Electronic Location Identifier: e0124001
Affiliations
[2
]Department of Obstetrics and Gynecology, Nanjing Drum Tower Hospital, Nanjing University
Medical School, Nanjing, China
Author notes
Conceived and designed the experiments: MZ GY HS YH. Performed the experiments: PX PG CL JZ YL LS. Analyzed the data: PX MZ GY HS YH. Contributed reagents/materials/analysis tools: PX MZ ZD GY HS YH. Wrote the paper: PX MZ HS YH.
* E-mail:
stevensunz@
123456163.com
(HS);
dtylhu@
123456126.com
(YH)
Article
Publisher ID: PONE-D-14-53075DOI: 10.1371/journal.pone.0124001
PMC ID: 4390151
PubMed ID: 25853857
SO-VID: 69774046-7907-49ce-a80d-514527e4ec63
Copyright statement:
Copyright @
2015
License:
This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
History
Date
received
: 8
December
2014
Date
accepted
: 25
February
2015
Page count
Figures: 8,
Tables: 2,
Pages: 16
Funding
This study was supported by the following grants: Chinese National Natural Science
Foundation (81370724, 81070508); Maternal and Fetus Medicine Key Lab of Jiangsu Province,
China (XK 201102); Innovative Research Program for Postgraduate in Higher Education
Institutions of Jiangsu Province for the year 2013 (CXLX13_568). The funders had no
role in study design, data collection and analysis, decision to publish, or preparation
of the manuscript.
Categories
Subject:
Research Article
Custom metadata
Data Availability All relevant data are within the paper and its Supporting Information files.
ScienceOpen disciplines: Uncategorized
Data availability:
ScienceOpen disciplines: Uncategorized