Skip Navigation

Cardiovascular Research 2003 60(1):156-164; doi:10.1016/S0008-6363(03)00338-9
© 2003 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Rounioja, S.
Right arrow Articles by Hallman, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rounioja, S.
Right arrow Articles by Hallman, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2003, European Society of Cardiology

Intra-amniotic lipopolysaccharide leads to fetal cardiac dysfunction

A mouse model for fetal inflammatory response

Samuli Rouniojaa, Juha Räsänenb, Virpi Glumoffa, Marja Ojaniemia, Kaarin Mäkikalliob and Mikko Hallmana,*

aDepartment of Pediatrics and Biocenter Oulu, University of Oulu, P.O. Box 5000, FIN-90014 Oulu, Finland
bDepartment of Obstetrics and Gynecology, University of Oulu, P.O. Box 5000, FIN-90014 Oulu, Finland

*Corresponding author. Tel.: +358-8-315-5100; fax: +358-8-315-5559. Email address: mikko.hallman{at}oulu.fi

Received 6 November 2002; accepted 7 March 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Intrauterine infection is associated with increased lipopolysaccharide (LPS) and proinflammatory cytokines in amniotic fluid. We hypothesized that intra-amniotic LPS launches a fetal inflammatory response leading to cardiac dysfunction. Methods: A mouse model was established. At 15–16 days of gestation, 52 fetuses of nine dams received LPS and 46 fetuses of nine dams vehicle intra-amniotically. Five dams underwent a sham operation. Echocardiography was performed before and 6 h after the injection to obtain inflow and outflow blood velocity waveforms. Outflow mean velocity (Vmean) and the proportions of isovolumetric relaxation (IRT%) and contraction (ICT%) times of the cardiac cycle were calculated. Pulsatility indices (PI) were calculated from the umbilical and intracranial arteries and the descending aorta. Pulsatility indices for veins (PIV) were obtained from ductus venosus. Toll-like receptor-4 (TLR4) and several other inflammatory mediators were determined using ELISA, immunohistochemistry, or ribonuclease protection assay. Results: In the LPS group, outflow Vmean was significantly lower, and ICT% and IRT% longer than in the other groups. LPS increased PIs, except in the intracranial arteries, which showed a decrease in PIs. In ductus venosus, PIVs were increased after LPS. LPS increased interleukin (IL)-6 in amniotic fluid and induced the expression of proinflammatory cytokines in placenta and fetal membranes, but not in lung. In fetal myocardium, TLR4 was constitutional. LPS induced the expression of IL-1β and tumor necrosis factor (TNF)-{alpha} mRNA in myocardium, whereas inducible nitric oxide synthase (NOS2) protein and nitrotyrosine remained undetectable. Conclusions: As a response to endotoxin in amniotic fluid, fetal myocardium acutely generates cytokines and severe fetal cardiovascular compromise develops. These two may be linked through a mechanism that does not include NO.

KEYWORDS Contractile function; Cytokines; Endotoxins; Hemodynamics; Infection/inflammation


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Cardiac contractile dysfunction is a severe consequence of the systemic inflammatory response syndrome (SIRS) [1]. This sequence has not been studied in the fetus. In intrauterine infection, which is a common perinatal problem, the concentrations of several cytokines increase in amniotic fluid. The proposed fetal inflammatory response syndrome (FIRS) has been associated with spontaneous preterm labor, fetal death, and cardio-respiratory failure, or hypoxic–ischemic brain damage at birth [2–4].

Endotoxins from Gram-negative bacteria (lipopolysaccharide, LPS) have been found in amniotic fluid in intrauterine inflammation [5]. LPS, bound to CD14, activates the Toll-like transmembrane receptor-4 (TLR4) [6]. This leads to the activation of nuclear factor (NF)-{kappa}B, required for transcriptional activation of proinflammatory cytokines and several other inflammatory mediators [7]. Thus intrauterine inflammation may launch a cytokine cascade similar to SIRS.

A number of experiments in vivo and in vitro have been performed to study the mechanism of LPS-induced cardiac depression. According to previous studies, cardiodepression by proinflammatory cytokines is mediated by excessive nitric oxide (NO), generated as a result of the induction of inducible nitric oxide synthase (NOS2) [8–12]. On the other hand, NO-independent cardiodepression by LPS has been documented [13–15]. Some studies have shown a cardiodepressant effect of tumor necrosis factor (TNF)-{alpha} without evidence of activation of NO synthesis [16–18].

To understand the consequences of the intrauterine inflammatory response, we developed a mouse model to study the inflammatory signal transmission and concomitant hemodynamic alterations in the fetus. We hypothesized that intra-amniotic LPS causes a systemic fetal inflammatory response and depresses cardiac function by inducing the cytokine cascade and NO production.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Animal protocols
The protocol was approved by the Animal Research Committee of the University of Oulu. Animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. A total of 23 timed pregnant DBA/2 mice and 123 fetuses (a mean of five per litter) were studied. In preliminary experiments, another 65 dams were studied. The gestational ages of the fetuses (±6 h) were verified by the presence of a vaginal plug (designated as day 0 of pregnancy). LPS (E. coli 055:B5; Sigma) was solubilized in 0.9% sterile saline at a concentration of 1 mg/ml. The solution was diluted into saline and used immediately or frozen once. The final concentration was 10 µg/ml and the final amount per amniotic sac 0.25 µg.

On day 15 or 16 of gestation, the mice were anesthetized with a solution containing 50 µg midazolam, 3.2 µg fentanyl citrate, and 100 µg fluanisone per kg body weight subcutaneously. A heat pad was used to maintain body temperature. The depth of anesthesia was monitored by loss of toe and tailpinch reflexes. Before surgery, the fetuses underwent the first ultrasonographic examination. In surgery, the uterine horns were exposed through a 1.5 cm midline abdominal incision. This allowed identification of the fetuses, placentas, and amniotic sacs through the uterine wall. The dams were randomized to receive either LPS or vehicle. In the LPS group (nine mice, 52 fetuses), 25 µl of saline-containing LPS was injected into the amniotic sac of each fetus using a 30-gauge tuberculin syringe. In the vehicle group (nine mice, 46 fetuses), an identical injection was performed with 25 µl of 0.9% saline. In the control group (five mice, 25 fetuses), a sham operation was performed by exposing the uterine horns and identifying the fetuses in each horn with no intra-amniotic injections. In preliminary experiments, intra-amniotic Trypan blue injections were made. They showed accurate targeting of the amniotic cavity without penetration of the fetal skin or placenta.

After the injections, the abdominal cavity and the abdominal wall were closed in two layers with continuous suture. After surgery, the animals were placed individually in clean cages and transferred into warm (+25 °C) microisolators. The duration of the procedure was approximately 10 min per dam. Six hours after the operation, the animals were anesthetized again and a second ultrasonographic examination identical to the first was performed. Thereafter, the dams were sacrificed by cervical dislocation. The placenta, fetal membranes, fetal heart and lungs were recovered and processed for analysis.

2.2. Doppler echocardiography
After the mouse had been anesthetized, its lower abdomen was shaved and it was placed in a dorsal position on a warm pad. A heat lamp was used to maintain body temperature. The ultrasonographic examination was performed using Acuson Sequoia 512 equipment (Mountain View) with a 13 MHz linear probe.

The fetuses were localized in each uterine horn, starting from the top of each horn. After identifying the fetal heart by color Doppler, the length of the sample volume of pulsed Doppler was adjusted to cover the entire heart. The high-pass filter was set at its minimum. The scanning technique used in this study was similar to that used previously [19,20]. The fetal heart was examined from different views, to minimize the angle between the Doppler beam and the inflow (IF) and outflow (OF) regions of the heart, in order to obtain their maximal velocities. The maximal IF and OF velocities were recorded using a sweep speed of 100 mm/s. From the sagittal view of the fetus, the descending aorta (DAO), the intracranial artery (ICA), and ductus venosus (DV) were located (Fig. 1). Their blood velocity waveforms were obtained by pulsed Doppler. By using the same technique, the umbilical artery (UA) was identified and the blood velocity waveforms were obtained. Immediately after the second ultrasonographic examination, the dam was sacrificed, the abdomen was carefully opened, and the fetuses were identified according to their location at the ultrasonographic examination.


Figure 1
View larger version (152K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Sagittal view of a fetus showing the fetal heart and peripheral vessels with color Doppler ultrasonography at 15 days of gestation. ICA, intracranial artery; DAO, descending aorta; DV, ductus venosus; UA, umbilical artery.

 
The data were videotaped and analyzed afterwards off-line, using the cardiac measurement package of the ultrasound equipment. The fetal heart rate (FHR) and the time–velocity integrals (TVI) of the OF waveforms were measured. The mean velocity (Vmean), which is directly proportional to volume blood flow, was calculated by the formula: Vmean=FHR x TVI. From the IF waveforms, the TVIs of the E- (early passive filling) and A- (active filling during atrial contraction) waves were measured and their ratio was calculated. The proportions of the isovolumetric relaxation (IRT%) and isovolumetric contraction (ICT%) times of the cardiac cycle were calculated. The IRT was measured as the period between the end of ejection and the onset of filling, while the ICT represents the period between the end of ventricular filling and the onset of ejection (Fig. 2). The index of myocardial performance (IMP), which represents the combined systolic and diastolic performance of the heart, was calculated by the formula IMP=(ICT+IRT)/ET, where ET is the ejection time [21]. The pulsatility index value for veins (PIV) was calculated from the DV blood velocity waveforms [PIV=(peak systolic velocity–velocity during atrial contraction)/time-averaged maximum velocity over the cardiac cycle] [22]. Furthermore, the pulsatility index (PI) values were obtained from the UA, DAO and ICA blood velocity waveforms [PI=(peak systolic velocity–end-diastolic velocity)/time-averaged maximum velocity over the cardiac cycle]. The intra-observer variability of the Doppler parameters was analyzed in 15 fetuses from three different litters, by performing the second ultrasonographic examination about 30 min after the first under the same anesthesia.


Figure 2
View larger version (102K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Inflow and outflow blood velocity waveforms of the fetal heart at 15 days of gestation obtained 6 h after an injection of vehicle (A) or lipopolysaccharide (LPS) (B). Note the regular atrial rate (*), the increasing ICT interval (white arrows) and the missing ventricular contraction (open arrow), indicating a second-degree AV block in the fetus with a LPS injection. IRT, isovolumetric relaxation time; ICT, isovolumetric contraction time; ET, ejection time.

 
2.3. Isolation and analysis of RNA
The fetal hearts were homogenized in 0.5 ml of TRIzol reagent (Life Technologies). Isolation was carried out as indicated by the manufacturer. The cytokine mRNA levels were analyzed by Rnase protection assay using a Riboquant multiprobe set (Pharmingen) following the supplier's instructions. The following mRNAs were studied: NOS2, TLR4, interleukin-1 receptor 1 (IL-1R1), TNF-{alpha}, IL-1{alpha}, IL-1β, IL-6, macrophage inflammatory protein (MIP)-2, and IL-10. In addition, the constitutional GAPDH and L32 were analyzed as reference. Total RNA (~10 µg) was hybridized overnight to the 32P-labeled RNA set. Single-stranded RNA and free probe were digested by Rnase A and T1. Protected RNA was phenolized, precipitated, and analyzed on a 5% denaturing polyacrylamide gel. The quantity of protected RNA was determined using a PhosphorImager and associated software (Biorad, Sunnyvale). Cytokine values were expressed as the ratio of the mRNA level in LPS/vehicle fetuses.

For Northern blot analysis, total RNA was isolated and surfactant protein-A (SP-A) mRNA quantified as described previously [23].

2.4. Immunostaining
The organs were fixed in 4% formaldehyde in PBS for 24 h. Thereafter, they were embedded in paraffin and cut into 5-µm sections. After deparaffinization and dehydration, the sections were immunostained for NOS2, TLR4 or nitrotyrosine. Briefly, the sections were incubated for 10 min in boiling 10 mM sodium water (pH 6.0), washed in PBS, and treated with 3% H2O2 in methanol for 15 min at room temperature. After washing with PBS, the sections were blocked with 20% fetal calf serum in PBS.

The polyclonal rabbit antibody specific for mouse NOS2 (N-20, Santa Cruz Biotechnology) was diluted with blocking solution to 4 µg/ml and incubated on sections for 1 h. For TLR4 immunostaining, the sections were blocked with anti-donkey serum and incubated overnight (37 °C) with goat anti-human TLR4 antibody (c-18, Santa Cruz Biotechnology). For nitrotyrosine, polyclonal nitrotyrosine antibody (1:1000; Upstate Biotechnology) was added after dehydration and incubated for 1 h. Coincubation of the antibody with 10 mM nitrotyrosine completely blocked the antibody binding to positive sections, consisting of lungs from LPS-treated adult mice. Detection was carried out with an avidin–biotin peroxidase system using AEC as a substrate. Finally, the sections were counterstained with hematoxylin.

2.5. Amniotic fluid assays
An enzyme-linked immunosorbent assay (ELISA) for mouse IL-6 (M6000, R&D systems) was used for the detection of IL-6. The limit of detection was 3.1 pg/ml. Absorbance values were determined and quantitative concentrations were calculated with a Wallac Victor 1420 multilabel counter with MultiCalc software (Perkin-Elmer Life Sciences, Wallac). The LPS levels in amniotic fluid were measured using a Limulus amebocyte lysate (LAL) kit (Charles River Endosafe).

2.6. Statistical analysis
Statistical analysis of the Doppler ultrasonographic parameters and IL-6 concentrations was performed using one-way ANOVA. If statistical significance was reached, further analysis was performed by the Scheffe F-test. Categorical data were analyzed by the chi-square test. The level of statistical significance was set at P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Dose response of LPS
In preliminary experiments, the effects of different doses of LPS (0.25–2.5 µg per amniotic sac) and different time intervals of LPS challenge (6–24 h) were studied, using fetal survival and premature birth as outcome variables. Regardless of the LPS dosage, the fetal death rate approached 100% at 24 h after the intra-amniotic injection. The fetal death rate ranged between 30 and 70% at 12 h after LPS, and a dose response (0.25–2.5 µg per amniotic sac) was demonstrated. There were no premature births in any of the groups studied. There were no fetal deaths in the vehicle and control groups. The lowest LPS dose (0.25 µg) and the 6 h interval were chosen to determine the primary proinflammatory cytokine response and the pathophysiological effects on fetal cardiovascular function and hemodynamics.

3.2. Amniotic fluid IL-6 and LPS
Six hours after the LPS challenge, endotoxin was still detected in the amniotic fluid. The median concentration of IL-6 was higher (P<0.001) (2373.9 pg/ml, range 69.3–2615.5) in the LPS group than in the vehicle group (391.0 pg/ml, range 28.9–833.0) or in the control group (100.6 pg/ml, range 28.0–515.2). The amniotic fluid IL-6 concentrations were similar in the unoperated animals (data not shown) compared to the vehicle or control groups.

3.3. Ultrasonographic examination
The mean intra-observer variability of the IF and OF TVI calculations ranged from 6.0 to 6.5% (95% confidence interval 4.0–8.4%). In the PI and PIV calculations, the mean intra-observer variability ranged from 3.6 to 6.7% (95% confidence interval 2.3–8.8%). The corresponding variability in the time interval measurements was from 2.5 to 15.9% (95% confidence interval 2.0–21.8%).

The baseline values were similar in the different groups (Table 1). After 6 h, OF Vmean was significantly lower in the LPS group than in the vehicle and control groups (Table 1). ICT% (P<0.05) and IRT% (P<0.005) were increased in the LPS group compared to the other groups. IMP was higher (P<0.005) in the LPS group than in the vehicle and control groups. In the LPS group (15/44) the incidence of arrhythmias was higher (P<0.001) than in the vehicle (1/46) and control groups (0/25) (Fig. 2). The TVI E/A ratio did not differ between the groups.


View this table:
[in this window]
[in a new window]

  		Table 1 Fetal cardiac function in study groups

 
The PI values of UA (P<0.005) and DAO (P<0.005) were higher and ICA lower (P<0.05) in the LPS group compared with the vehicle and control groups. The DV PIV values were higher in the LPS group (P<0.0001) than in the vehicle and control groups (Table 2).


View this table:
[in this window]
[in a new window]

  		Table 2 Fetal arterial and velous circulations in study groups

 
Throughout the experiment, no differences were seen in the measured parameters between the vehicle group and the control group.

3.4. Expression of mRNAs
In fetal myocardium, NOS2, TLR4, and IL-1R1 were constitutive. NOS2, TLR4, IL-1R1 and TNF-{alpha} were constitutive in placenta and TLR4 and IL-1R1 in fetal membranes. There was no detectable induction of the studied mRNAs in the vehicle and control groups.

Following the LPS injection, TNF-{alpha}, IL-1β, and MIP-2 were induced in myocardium (Fig. 3). IL-1β, IL-1{alpha}, and MIP-2 in placenta, and NOS2, TNF-{alpha}, IL-1β, IL-1{alpha}, and IL-6 in fetal membranes were induced after LPS.


Figure 3
View larger version (45K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Expression of inflammatory mediators in fetal myocardium, placenta, fetal membranes, and fetal lung 6 h after the intra-amniotic injection of lipopolysaccharide (LPS) (0.25 µg) or vehicle. Only the mediators induced by LPS are shown. (A) A representative gel electrophoresis of multiple mRNAs after the Rnase protection assay. The undigested anti-sense probe bands are on the left. (B–D) The results of the treatment groups (five to 12 animals per group) on fetal heart, placenta, and fetal membranes. The data are expressed as a fold increase in mRNA levels in the LPS-treated animals relative to the vehicle-treated animals. Results are mean±S.E.M. *P<0.05 vs. vehicle-treated fetuses, {dagger}P<0.005 vs. vehicle-treated fetuses The other mediators analyzed were not increased by LPS. There were no significant differences in the expression of the cytokines between the vehicle-treated and the control animals (data not shown). TNF, tumor necrosis factor; IL, interleukin; MIP, macrophage inflammatory protein.

 
In fetal lung, there was no detectable induction of any of the mRNAs, although IL-1R1 was constitutively expressed. In Northern blot analysis there was no detectable induction in the expression of SP-A.

3.5. Immunohistochemistry of NOS2, TLR4, and nitrotyrosine
Immunohistochemical analysis of the fetal hearts showed positive staining for TLR4. There was no detectable difference in immunostaining following intra-amniotic LPS or vehicle (Fig. 4). NOS2 was not detected in myocardium or in fetal lung in any of the groups studied. There was no detectable immunostaining for nitrotyrosine in fetal heart from LPS- or vehicle-treated fetuses (not shown).


Figure 4
View larger version (122K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Immunostaining of fetal heart for toll-like receptor-4 (TLR4) and inducible nitric oxide synthase (NOS2) 6 h after intra-amniotic lipopolysaccharide (LPS). Positive staining appears in red. (A) Horizontal view of a fetal heart stained with anti-TLR4. (B) Fetal myocardium of the left ventricular wall stained with anti-TLR4. (C) Papillary muscles of the fetal heart stained with anti-TLR4. (D) Control staining without secondary antibody. There was intense cytoplasmic staining with TLR4 in all groups. (E) Fetal myocardium from the LPS group processed with NOS2 antibody. (F) Fetal myocardium from the vehicle group processed with NOS2 antibody. NOS2 was absent in fetal myocardium in all groups. (G) Control staining of adult myocardium 6 h after intraperitoneal LPS with NOS2 antibody. (H) Adult myocardium after intraperitoneal saline. In (A), the scale bar indicates 200 µm, whereas in (B–H) it indicates 50 µm.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Intrauterine infection is associated with premature birth, fetal death, and serious cardio-pulmonary disease in the premature offspring. The possible link between inflammatory mediators and fetal cardiac dysfunction in this setting has not been addressed. In order to study this question, we developed a mouse model of acute intra-amniotic inflammation. Intra-amniotic LPS caused an acute inflammatory reaction, whereas the vehicle-injected fetuses were unaffected. The study period was limited to a few hours after LPS, when the primary cytokine response rather than the secondary inflammatory cascades prevails. Our results demonstrate that intra-amniotic LPS acutely induced cytokines in fetal membranes and placenta and also led to a selective response in fetal organs. In fetal heart the induction of cytokines was associated with impaired myocardial function and altered hemodynamics.

Studies with adult mice with defective TLR4 signaling suggest that TLR4 mediates LPS-induced cardiac dysfunction [24]. Fetal myocardium was found to constitutively express TLR4, the receptor that activates NF-{kappa}B required for the induction of the cytokine cascade [17]. Indeed, intra-amniotic LPS induced TNF-{alpha} and IL-1β expression in myocardium. LPS administration was associated with a decrease in fetal cardiac outflow mean velocity, suggesting decreased cardiac output, and signs of congestive heart failure were found. This is consistent with the in vitro evidence indicating direct depression of the myocardium by TNF-{alpha} and IL-1β [25]. Following intra-amniotic LPS, there was no activation of NOS2 or generation of nitrotyrosine that would indicate NO toxicity in the heart. In contrast, we found induction of NOS2 enzyme following intraperitoneal LPS to adult mice. This is consistent with previous data by Nemoto et al. [24].

The placental and fetal innate immune system is characterized by a predominance of TH2 cell-dependent responses and suppression of TH1 T-cells, leading to downregulation of the cytokines that may cause rejection of the fetus [26]. Thus, feto-placental tissues do not generate TH1 cytokines, including IFN-{gamma}, which augments the induction of NOS2 and potentiates the inhibition of the adrenergic responses by LPS. Although the addition of IL-1β and IFN-{gamma} induce the expression of NOS2 in isolated neonatal cardiac myocytes [27], there was no acute induction of NOS2 in the fetal heart following intra-amniotic LPS. We propose that the lack of NOS2 induction in the fetal heart in vivo is due to a lack of the TH1 response. Nitric oxide-independent cardiodepression has been documented in experiments with neonatal rat cardiomyocytes using TNF-{alpha} at pathophysiologically relevant concentrations [18]. Studies using NOS2 –/– and wild-type littermates revealed that the absence of NOS2 induction in adult mice did not prevent inflammatory myocardial damage or lethal endotoxin shock [28,29]. The present results obtained in fetuses are consistent with the proposal that, in the absence of NOS2, the myocardium may be predisposed to acute inflammatory dysfunction [28].

Intra-amniotic LPS injection prolonged the ICT and IRT periods. ICT represents the time interval from the closure of the atrio-ventricular (AV) valve to the opening of the semilunar valve, i.e. the time needed for the ventricle to increase its pressure from the atrial to the systemic level. On the other hand, the IRT interval represents the period between the closure of the semilunar valve and the opening of the AV valve, and thus the time needed for the ventricle to decrease its pressure from the systemic to the atrial level. Relaxation of the myocardium is an active process that depends on the ability of the myocytes to accelerate calcium transport through Na–Ca channels. The present findings demonstrate impaired contractility and relaxation of the myocardium after LPS. Another possible explanation for the longer IRT and ICT intervals could be the increased pressure gradient between the systemic and atrial levels. This is unlikely, since the endotoxin-induced systemic inflammation at birth is described as a hypotensive state with a decreased pressure gradient between the systemic and atrial level. In addition, global cardiac performance (IMP) was compromised in these fetuses. Our findings are in agreement with the earlier studies showing a negative inotropic effect of TNF-{alpha} and IL-1β on myocytes [30]. The present results further suggest that intra-amniotic LPS induces inflammation in the myocardium which increases the incidence of arrhythmias. Especially second-degree AV blocks were found, suggesting inflammation in the AV nodal tract.

In the peripheral arterial circulation, LPS increased vascular impedances in the placenta and the fetal lower body, as indicated by the increased PI values in the umbilical artery and the descending aorta. On the other hand, vascular impedance was decreased in the intracranial artery. The decrease in the fetal cardiac output and blood pressure triggers compensatory mechanisms, including an increase in catecholamine secretion. Sensitivity to catecholamines varies, and the sensitivity of the cerebral, adrenal and coronary arteries to the catecholamine stimulus is known to be weak, thus leading to redistribution of arterial circulation, called the "brain-sparing" effect. This phenomenon has been described earlier in pregnancies complicated by placental insufficiency [31]. In this way, the fetus tries to maintain an adequate blood and oxygen supply to its brain and myocardium. In severe placental insufficiency associated with redistribution of arterial circulation, the fetus is able to maintain its cardiac output [32]. In LPS-induced myocardial dysfunction, the redistribution of arterial circulation seems to be a secondary mechanism preserving cerebral blood flow in the face of a decrease in cardiac output.

The PIV values of DV were increased in the fetuses after the LPS. This suggests that the systemic venous pressure was elevated, further indicating compromised and failing cardiac function. Previously it has been shown that the pulsatility of blood velocity waveforms in systemic veins correlates positively with the cardiac secretion and production of the N-terminal peptide of proatrial natriuretic peptide, which is released in equivalent amounts compared to atrial natriuretic peptide (ANP). The latter reflects the atrial wall stretch and, thus, the atrial and systemic venous pressures [32].

Because the heart size of the mouse fetus is small, this ultrasonographic method is unable to distinguish between right and left ventricular inflow and outflow areas. However, due to open ductus arteriosus and foramen ovale, ventricles function in parallel and the pressure faced by both ventricles is equal. Based on these facts the pathophysiological effects would be similar in both ventricles. The validity of Doppler measurements has been established both in human fetal and animal studies. The PI and PIV calculations are independent of the angle between the Doppler beam and the vessel, and thus the intra-observer variability of these calculations was less than in calculations which are angle dependent, i.e. TVI for mean velocity. However, the intra-observer variability of the angle-dependent parameters was acceptable, demonstrating the validity of this methodology in fetal mouse studies, in agreement with previous reports [19,20].

According to the current evidence, there is a spectrum of phenotypes associated with the intrauterine inflammatory response syndrome in human pregnancies [2,33,34]. High IL-6 in amniotic fluid or in fetal plasma at premature birth is associated with cardio-respiratory failure, pneumonia, chronic lung disease, intraventricular hemorrhage, and periventricular leukomalacia [2]. Severe placental inflammation (villitis, decidual inflammation) is evident in many cases of sudden fetal death [35]. This is consistent with the present data, suggesting that microbial toxins or cytokines cause progressive cardiac failure.

In the present study, LPS induced several placental inflammatory mediators and IL-6 in amniotic fluid, which is a sensitive index of acute chorioamnionitis [36]. However, in fetal organs the response varied depending upon the expression of the LPS receptor TLR4. The fetal lung showed no detectable expression of TLR4 and there was no cytokine response or induction of SP-A within 6 h after intra-amniotic LPS. Inflammatory activation and the induced maturation of lung surfactant was evident within 2–7 days of the administration of intra-amniotic IL-1{alpha} or LPS to rabbits or sheep [33,37,38]. Fetal myocardium, on the other hand, was found to express the mRNA and protein of TLR4, making the fetal heart a primary target of LPS-induced inflammation, as evidenced by a proinflammatory cytokine response and severe functional disturbances. We propose that the expression levels of TLR4 and other microbial pattern recognition receptors target the primary innate response within the fetal compartment, influencing the fetal outcome.


   Acknowledgements
 
This research was supported by the Academy of Finland, Biocenter Oulu and the Sigrid Juselius Foundation.


   Notes
 
Time for primary review 34 days.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Rangel-Frausto M.S, Pittet D, Costigan M, et al. The natural history of the systemic inflammatory response syndrome (SIRS). A prospective study. J. Am. Med. Assoc. (1995) 273:117–123.[Abstract]
  2. Gomez R, Romero R, Ghezzi F, et al. The fetal inflammatory response syndrome. Am J Obstet Gynecol (1998) 179:194–202.[CrossRef][ISI][Medline]
  3. Hallman M. Cytokines, pulmonary surfactant and consequences of intrauterine infection. Biol Neonate (1999) 76(Suppl_1):2–9.[CrossRef][ISI][Medline]
  4. Romero R, Gomez R, Ghezzi F, et al. A fetal systemic inflammatory response is followed by the spontaneous onset of preterm parturition. Am J Obstet Gynecol (1998) 179:186–193.[CrossRef][ISI][Medline]
  5. Romero R, Roslansky P, Oyarzun E, et al. Labor and infection. II. Bacterial endotoxin in amniotic fluid and its relationship to the onset of preterm labor. Am J Obstet Gynecol (1988) 158:1044–1049.[ISI][Medline]
  6. Beutler B. Endotoxin, toll-like receptor 4, and the afferent limb of innate immunity. Curr Opin Microbiol (2000) 3:23–28.[CrossRef][ISI][Medline]
  7. Tak P.P, Firestein G.S. NF-kappaB: a key role in inflammatory diseases. J Clin Invest (2001) 107:7–11.[CrossRef][ISI][Medline]
  8. Schulz R, Panas D.L, Catena R, et al. The role of nitric oxide in cardiac depression induced by interleukin-1 beta and tumour necrosis factor-alpha. Br J Pharmacol (1995) 114:27–34.[ISI][Medline]
  9. Finkel M.S, Oddis C.V, Jacob T.D, et al. Negative inotropic effects of cytokines on the heart mediated by nitric oxide. Science (1992) 257:387–389.[Abstract/Free Full Text]
  10. Brady A.J, Poole-Wilson P.A, Harding S.E, Warren J.B. Nitric oxide production within cardiac myocytes reduces their contractility in endotoxemia. Am J Physiol (1992) 263:H1963–H1966.[ISI][Medline]
  11. Stein B, Frank P, Schmitz W, Scholz H, Thoenes M. Endotoxin and cytokines induce direct cardiodepressive effects in mammalian cardiomyocytes via induction of nitric oxide synthase. J Mol Cell Cardiol (1996) 28:1631–1639.[CrossRef][ISI][Medline]
  12. Tao S, McKenna T.M. In vitro endotoxin exposure induces contractile dysfunction in adult rat cardiac myocytes. Am J Physiol (1994) 267:H1745–H1752.[ISI][Medline]
  13. Decking U.K, Flesche C.W, Godecke A, Schrader J. Endotoxin-induced contractile dysfunction in guinea pig hearts is not mediated by nitric oxide. Am J Physiol (1995) 268:H2460–H2465.[ISI][Medline]
  14. Klabunde R.E, Coston A.F. Nitric oxide synthase inhibition does not prevent cardiac depression in endotoxic shock. Shock (1995) 3:73–78.[ISI][Medline]
  15. Muller-Werdan U, Engelmann H, Werdan K. Cardiodepression by tumor necrosis factor-alpha. Eur Cytokine Netw (1998) 9:689–691.[ISI][Medline]
  16. Natanson C, Eichenholz P.W, Danner R.L, et al. Endotoxin and tumor necrosis factor challenges in dogs simulate the cardiovascular profile of human septic shock. J Exp Med (1989) 169:823–832.[Abstract/Free Full Text]
  17. Matsumori A. Cytokines in myocarditis and cardiomyopathies. Curr Opin Cardiol (1996) 11:302–309.[CrossRef][ISI][Medline]
  18. Muller-Werdan U, Schumann H, Fuchs R, et al. Tumor necrosis factor alpha (TNF alpha) is cardiodepressant in pathophysiologically relevant concentrations without inducing inducible nitric oxide-(NO)-synthase (iNOS) or triggering serious cytotoxicity. J Mol Cell Cardiol (1997) 29:2915–2923.[CrossRef][ISI][Medline]
  19. Gui Y.H, Linask K.K, Khowsathit P, Huhta J.C. Doppler echocardiography of normal and abnormal embryonic mouse heart. Pediatr Res (1996) 40:633–642.[ISI][Medline]
  20. Mäki JM, Räsänen J, Tikkanen H, et al. Inactivation of lysyl oxidase gene leads to aortic aneurysms, cardiovascular dysfunction and perinatal death in mice. Circulation (in press).
  21. Tsutsumi T, Ishii M, Eto G, Hota M, Kato H. Serial evaluation for myocardial performance in fetuses and neonates using a new Doppler index. Pediatr Int (1999) 41:722–727.[CrossRef][ISI][Medline]
  22. Hecher K, Campbell S, Doyle P, Harrington K, Nicolaides K. Assessment of fetal compromise by Doppler ultrasound investigation of the fetal circulation. Arterial, intracardiac, and venous blood flow velocity studies. Circulation (1995) 91:129–138.[Abstract/Free Full Text]
  23. Glumoff V, Vayrynen O, Kangas T, Hallman M. Degree of lung maturity determines the direction of the interleukin-1-induced effect on the expression of surfactant proteins. Am J Respir Cell Mol Biol (2000) 22:280–288.[Abstract/Free Full Text]
  24. Nemoto S, Vallejo J.G, Knuerfermann P, et al. Escheria coli LPS-induced LV dysfunction: role of toll-like receptor-4 in the adult hearts. Am J Physiol (2002) 282:H2316–H2323.[ISI]
  25. Kumar A, Thota V, Dee L, et al. Tumor necrosis factor alpha and interleukin 1beta are responsible for in vitro myocardial cell depression induced by human septic shock serum. J Exp Med (1996) 183:949–958.[Abstract/Free Full Text]
  26. Prescott S.L, Macaubas C, Holt B.J, et al. Transplacental priming of the human immune system to environmental allergens: universal skewing of initial T cell responses toward the Th2 cytokine profile. J Immunol (1998) 160:4730–4737.[Abstract/Free Full Text]
  27. Arstall M.A, Sawyer D.B, Fukazawa R, Kelly R.A. Cytokine-mediated apoptosis in cardiac myocytes: the role of inducible nitric oxide synthase induction and peroxynitrite generation. Circ Res (1999) 85:829–840.[Abstract/Free Full Text]
  28. Zingarelli B, Hake P.W, Yang Z, et al. Absence of inducible nitric oxide synthase modulates early reperfusion-induced NF-kappaB and AP-1 activation and enhances myocardial damage. FASEB J (2002) 16:327–342.[Abstract/Free Full Text]
  29. Nicholson S.C, Grobmyer S.R, Shiloh M.U, et al. Lethality of endotoxin in mice genetically deficient in the respiratory burst oxidase, inducible nitric oxide synthase, or both. Shock (1999) 11:253–258.[ISI][Medline]
  30. Oral H, Dorn G.W, Mann D.L. Sphingosine mediates the immediate negative inotropic effects of tumor necrosis factor-alpha in the adult mammalian cardiac myocyte. J Biol Chem (1997) 272:4836–4842.[Abstract/Free Full Text]
  31. Wladimiroff J.W, vd Wijngaard J.A, Degani S, et al. Cerebral and umbilical arterial blood flow velocity waveforms in normal and growth-retarded pregnancies. Obstet Gynecol (1987) 69:705–709.[ISI][Medline]
  32. Makikallio K, Vuolteenaho O, Jouppila P, Rasanen J. Ultrasonographic and biochemical markers of human fetal cardiac dysfunction in placental insufficiency. Circulation (2002) 105:2058–2063.[Abstract/Free Full Text]
  33. Jobe A.H, Newnham J.P, Willet K.E, et al. Effects of antenatal endotoxin and glucocorticoids on the lungs of preterm lambs. Am J Obstet Gynecol (2000) 182:401–408.[CrossRef][ISI][Medline]
  34. Watterberg K.L, Demers L.M, Scott S.M, Murphy S. Chorioamnionitis and early lung inflammation in infants in whom bronchopulmonary dysplasia develops. Pediatrics (1996) 97:210–215.[Abstract/Free Full Text]
  35. Tolockiene E, Morsing E, Holst E, et al. Intrauterine infection may be a major cause of stillbirth in Sweden. Acta Obstet Gynecol Scand (2001) 80:511–518.[CrossRef][ISI][Medline]
  36. Yoon B.H, Romero R, Kim C.J, et al. Amniotic fluid interleukin-6: a sensitive test for antenatal diagnosis of acute inflammatory lesions of preterm placenta and prediction of perinatal morbidity. Am J Obstet Gynecol (1995) 172:960–970.[CrossRef][ISI][Medline]
  37. Bry K, Lappalainen U, Hallman M. Intraamniotic interleukin-1 accelerates surfactant protein synthesis in fetal rabbits and improves lung stability after premature birth. J Clin Invest (1997) 99:2992–2999.[ISI][Medline]
  38. Kramer B.W, Moss T.J, Willet K.E, et al. Dose and time response after intraamniotic endotoxin in preterm lambs. Am J Respir Crit Care Med (2001) 164:982–988.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Rounioja, S.
Right arrow Articles by Hallman, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rounioja, S.
Right arrow Articles by Hallman, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?