T. J. M. Moss and G. R. Polglase are joint senior authors.
Intrauterine inflammation alters cardiopulmonary and cerebral haemodynamics at birth in preterm lambs
Article first published online: 20 MAR 2013
© 2013 The Authors. The Journal of Physiology © 2013 The Physiological Society
The Journal of Physiology
Volume 591, Issue 8, pages 2127–2137, April 2013
How to Cite
Galinsky, R., Hooper, S. B., Wallace, M. J., Westover, A. J., Black, M. J., Moss, T. J. M. and Polglase, G. R. (2013), Intrauterine inflammation alters cardiopulmonary and cerebral haemodynamics at birth in preterm lambs. The Journal of Physiology, 591: 2127–2137. doi: 10.1113/jphysiol.2012.249680
- Issue published online: 15 APR 2013
- Article first published online: 20 MAR 2013
- Accepted manuscript online: 20 FEB 2013 04:04AM EST
- (Received 10 December 2012; accepted after revision 13 February 2013; first published online 18 February 2013)
- • Intrauterine inflammation is associated with preterm birth and poor long-term cardiopulmonary and neurological outcomes. The effect of intrauterine inflammation on the cardiopulmonary and cerebral haemodynamic transition after preterm birth is poorly understood.
- • We demonstrated that intrauterine inflammation increased pulmonary vascular resistance, reduced pulmonary blood flow and left ventricular output and increased cerebral blood flow and cerebral oxygen delivery after preterm birth.
- • Increasing positive end-expiratory pressure, which causes a haemodynamic challenge, reduced pulmonary blood flow and left ventricular output in controls but not in lipopolysaccharide-exposed lambs. A transient reduction in brachiocephalic arterial pressure was observed in lipopolysaccharide-exposed lambs.
- • Intrauterine inflammation altered the cardiopulmonary and cerebral haemodynamic transition at birth and reduced the responsiveness of the pulmonary circulation to an increase in positive end-expiratory pressure.
Abstract Intrauterine inflammation is associated with preterm birth and poor long-term cardiopulmonary outcomes. We aimed to determine the effect of intrauterine inflammation on the cardiopulmonary and cerebral haemodynamic transition at birth, and the response to subsequent haemodynamic challenge. Fetal instrumentation was performed at ∼112 days gestation (term is 147 days) for measurement of cardiopulmonary and cerebral haemodynamics. At 118 days, inflammation was induced by intra-amniotic administration of lipopolysaccharide (LPS; n= 7); controls (n= 5) received intra-amniotic saline. At 125 days lambs were delivered and mechanically ventilated. Arterial blood gases, pulmonary and systemic arterial blood pressures and flows were measured during the perinatal period. At 10 min a haemodynamic challenge was administered by increasing positive end-expiratory pressure. During the first 10 min after birth, LPS-exposed lambs had higher pulmonary vascular resistance and lower pulmonary blood flow and left ventricular output than controls. Carotid arterial blood flow was higher in LPS-exposed lambs than controls between 3 and 7 min after delivery, and cerebral oxygen delivery was higher at 5 min. During the haemodynamic challenge, pulmonary blood flow and left ventricular output were reduced in controls but not in LPS-exposed lambs; a transient reduction in brachiocephalic arterial pressure occurred in LPS-exposed lambs but not in controls. Intrauterine inflammation altered the cardiopulmonary and cerebral haemodynamic transition at birth and reduced the cardiopulmonary response to a haemodynamic challenge after birth. The transient reduction in brachiocephalic arterial pressure suggests intrauterine inflammation may alter cerebrovascular control following an increase in positive end-expiratory pressure.
alveolar arterial difference in oxygen
carotid arterial pulsatility index
carotid blood flow
- F IO2
fraction of inspired oxygen
- P MPA
main pulmonary arterial pressure
- D O2
peak inspiratory pressure
positive end-expiratory pressure
pulmonary blood flow
persistent pulmonary hypertension of the newborn
pulmonary vascular resistance
- C dyn,spec
specific dynamic lung compliance
ventilation efficiency index
During fetal life, the lungs are liquid-filled and play no role in gas exchange (Rudolph, 1979; Abman, 2007). As a result, fetal pulmonary vascular resistance (PVR) is high and 90% of right ventricular output bypasses the lungs and passes through the ductus arteriosus into the systemic circulation. At birth, the initiation of ventilation aerates the lungs, which reduces PVR and transforms the pulmonary circulation into a low-pressure circuit, which receives 100% of right ventricular output (Polglase et al. 2005). The increase in pulmonary blood flow (PBF) after birth is essential for providing venous return to the left ventricle (preload), which mostly comes from umbilical venous return in the fetus (Rudolph, 1979). Thus, the increase in PBF at birth supports both pulmonary gas exchange and left ventricular output (LVO), thereby maintaining cardiovascular stability (Crossley et al. 2009).
Failure to reduce PVR at birth contributes to the pathogenesis of persistent pulmonary hypertension of the newborn (PPHN), which impairs the cardiopulmonary haemodynamic transition after birth (Woldesenbet & Perlman, 2005; Abman, 2007; Polglase et al. 2010). PPHN is associated with low PBF and a persistent right-to-left shunt across the ductus arteriosus (Kinsella & Abman, 1995), which may impair LVO and cerebral perfusion (Polglase et al. 2010). Exposure to intrauterine inflammation can induce PPHN (Woldesenbet & Perlman, 2005; Woldesenbet et al. 2008), which is probably mediated by inflammation-induced changes in pulmonary vascular development (Woldesenbet et al. 2008). Intrauterine inflammation, induced by intra-amniotic administration of lipopolysaccharide (LPS), attenuates vascular growth and remodels the resistance arterioles in the lung of preterm lambs (Kallapur et al. 2004). These structural alterations translate into an increase in PVR and a reduction in PBF in preterm lambs (Polglase et al. 2010).
Intrauterine inflammation increases the risk of diffuse white matter injury in the brains of preterm neonates, probably due to activation of a systemic inflammatory cascade and induction of cerebral blood flow instability (Grether & Nelson, 1997; Wu & Colford, 2000; Kaukola et al. 2006; Hansen-Pupp et al. 2008). With respect to the latter, the initial fetal-to-neonatal transition may be a particularly vulnerable time for brain injury in preterm infants because they commonly have poor pulmonary and systemic perfusion during this time (Noori et al. 2009).
Inappropriate positive pressure ventilation procedures in preterm infants can also compromise the fetal-to-neonatal haemodynamic transition. For instance, although a positive end-expiratory pressure (PEEP) improves lung inflation and development of a functional residual capacity (Siew et al. 2009; te Pas et al. 2009), high PEEPs increase PVR and reduce PBF; thus reducing cardiac venous return and cardiac output (Polglase et al. 2009). In preterm neonates with compromised pulmonary vascular development and altered cerebral haemodynamic control, an elevation in airway pressure may further compromise the circulatory transition at birth.
We aimed to determine the effect of intrauterine inflammation on cardiopulmonary and cerebral haemodynamics during the fetal-to-neonatal transition in preterm lambs. We hypothesized that intrauterine inflammation would impair the cardiopulmonary and cerebral haemodynamic transition at birth in preterm lambs. Further, we hypothesized that the adverse haemodynamic consequences of an increase in airway pressure would be exacerbated in LPS-exposed preterm lambs.
All procedures were approved by the relevant Monash University Animal Ethics Committee. At 112 ± 1 (mean ± standard deviation) days of gestation (term ∼147 days), pregnant ewes bearing singleton or twin fetuses underwent aseptic surgery under general anaesthesia (2% Isoflurane in oxygen; Bomac Animal Health, NSW, Australia) for instrumentation as described previously (Polglase et al. 2005). Polyvinyl catheters containing heparinized saline were inserted into the fetal brachiocephalic artery (via the right brachial artery), main pulmonary artery (MPA) and the amniotic cavity. Vascular flow probes (Transonic Systems, Ithaca, NY, USA) were placed around the left main pulmonary artery (size: 4 mm) and left carotid artery (size: 3 mm) for monitoring PBF and carotid arterial blood flows (CaBF), respectively. After surgery, ewes received analgesia for 72 h using a transdermal fentanyl patch (75 μg h−1; Janssen Cilag, North Ryde, NSW, Australia). At 118 days, ewes were randomly allocated to receive a bolus of LPS (Escherichia coli 055:B5, 20 mg; Sigma Aldrich, NSW, Australia: n= 7) or saline (controls; 4 ml; n= 5) into the amniotic sac. Fetal well-being was regularly monitored by blood–gas measurements (ABL30; Radiometer, Copenhagen, Denmark).
Preterm delivery and mechanical ventilation
At 125 days, 7 days after intra-amniotic LPS or saline administration, lambs were delivered by caesarean section under general anaesthesia (2% Isoflurane in oxygen; Bomac Animal Health). Following caesarean section, ewes were humanely killed by pentobarbital sodium overdose. Lambs were intubated and lung liquid was passively drained before the umbilical cord was clamped and cut. After delivery, lambs were weighed, fitted with a pulse oximeter for pre-ductal measurement of transcutaneous oxyhaemoglobin saturation levels (Masimo, Irvine, CA, USA) and connected to a mechanical ventilator (Babylog 8000+; Drager, Lubec, Germany). Ventilation commenced in volume guarantee mode with a target tidal volume of 7.5 ml kg−1, a PEEP of 4 cmH2O, and a peak inspiratory pressure of 40 cmH2O. The inspired oxygen fraction (FIO2) began at 21% but was adjusted to maintain SpO2 between 85 and 95%. The lower and upper limits of FIO2 were 21% and 40%, respectively. Throughout ventilation, lambs were sedated by continuous infusion of Alfaxane (3 mg kg−1 min−1) through an umbilical vein catheter implanted immediately after delivery. Left pulmonary arterial blood flow, CaBF and pulmonary and systemic arterial pressures were measured and digitally recorded in real time. Lamb well-being was assessed by regular arterial blood gas measurements (ABL30; Radiometer, Copenhagen, Denmark) of samples collected from the brachiocephalic arterial catheter.
After 10 min of mechanical ventilation, PEEP was increased from 4 to 8 cmH2O for 10 min, to induce a haemodynamic challenge. At the end of the 10 min period, PEEP was returned to 4 cmH2O. We have previously demonstrated that increasing PEEP from 4 to 8 cmH2O increases PVR and decreases PBF in preterm lambs (Polglase et al. 2005).
At the end of the experiment, lambs were humanely killed by pentobarbital sodium overdose (100 mg kg−1 i.v.; Valabarb, Jurox, Rutherford, NSW, Australia). Lungs were removed and tissue from the right lower lobe was frozen in liquid nitrogen for subsequent measurement of lung injury and inflammation using RT-PCR.
Specific dynamic lung compliance (Cdyn,spec) was calculated as VT (ml kg−1 birthweight)/ΔP (cmH2O), where ΔP= (PIP-PEEP). Ventilation efficiency index (VEI) was calculated as 3800/ΔP×f×PaCO2, where 3800 = CO2 production constant and f= breathing frequency (Ikegami et al. 2004). The alveolar arterial difference in oxygen (aADO2) was calculated as P[(barometric) −P(H2O)]×FIO2−(PaCO2/0.93)−PaO2, where P(barometric) is barometric pressure and P(H2O) is water vapour pressure at 39°C, as described previously (Polglase et al. 2005). Oxygenation index was calculated as (FIO2× mean airway pressure × 100/PaO2). Estimated cerebral oxygen delivery (DO2) was calculated as arterial blood oxygen content × CaBF, where arterial oxygen content = ([Hb]×SaO2× 1.36)+(PaO2× 0.003).
Left ventricular output, end-diastolic and minimum postsystolic pulmonary blood flow and carotid arterial pulsatility index
LVO was calculated from estimates of total PBF, based on measured left pulmonary artery flow and weight differences between the left and right lung as previously described (Crossley et al. 2009). Total PBF was integrated over 10 consecutive cardiac cycles and the mean integral was multiplied by the corresponding heart rate to derive LVO. End-diastolic PBF and minimum postsystolic PBF was calculated as described previously (Polglase et al. 2005, 2006, 2010), from five consecutive cardiac cycles recorded at 1 min intervals throughout the ventilation period. Carotid arterial pulsatility index (CaPI) was calculated as (peak systolic CaBF – minimum diastolic carotid blood flow)/mean CaBF. All calculations were performed using Labchart Pro (version 7.3.1, ADInstruments, Castle Hill, NSW, Australia).
The rate of change in pulmonary blood flow (PBF), postsystolic minimum PBF, end-diastolic PBF and left ventricular output during the fetal-to-neonatal haemodynamic transition
The rates of increase in PBF, postsystolic minimum PBF, end-diastolic PBF and LVO immediately after birth were derived by calculating the slope (y) within each group between measurements made before delivery and at 1 and 2 min time points after delivery for each of the variables outlined, where y= difference in flow/time. Use of data from the period before birth to the second minute after birth ensured data collection early enough to reflect the immediate haemodynamic transition at birth.
Assessment of lung injury and inflammation
We measured mRNA levels of genes: connective tissue growth factor (CTGF), cysteine-rich-61 (CYR-61) and early growth response-1 (EGR-1), previously identified as early markers of ventilator-induced lung injury (Wallace et al. 2009) and inflammatory cytokines: interleukin (IL)-1β, IL-8 and IL-6, known to be upregulated after ventilator-induced lung injury (Polglase et al. 2012b).
Total RNA was extracted from lung tissue and reverse transcribed to produce cDNA (Qiagen, Melbourne, Victoria, Australia). The mRNA levels for CTGF, CYR-61 EGR-1 and IL-1β, IL-8 and IL-6 were determined using quantitative real-time PCR as described previously (Wallace et al. 2009). CTGF, CYR-6, EGR-1 and IL mRNA levels in the lung tissue of ventilated LPS-exposed lambs were compared to ventilated controls.
Data are presented as means ±s.e.m. Statistical analyses of physiological data were undertaken using Graphpad Prism software (v5.0; Graphpad software, San Diego, CA, USA). Serial data were compared by two-way repeated measures ANOVA with treatment (control vs. LPS) and time as factors. Post hoc comparisons were performed using the Holm–Sidak method. The rate of change between the fetal time-point and minutes 1 and 2 after delivery were calculated by statistically comparing the slope between groups. The mRNA levels of CTGF, CYR-61, EGR-1, IL-1β, IL-8 and IL-6 were compared using an unpaired t test. Statistical significance was defined as P < 0.05.
Birth weights of preterm lambs were not different between groups (LPS, 2.99 ± 0.27 kg; controls, 2.84 ± 0.14 kg: P= 0.7). The ratios of males-to-females and singletons-to-twins are presented in Table 1. Fetal arterial PO2, SO2, CO2, pH and lactate levels at birth did not differ between groups (Table 1).
|Body weight (kg)||2.84 ± 0.14||2.99 ± 0.27|
|Fetal PaO2 (mmHg)||21.8 ± 1.2||21.0 ± 1.0|
|Fetal SaO2 (%)||65.2 ± 2.1||61.8 ± 4.0|
|Fetal CO2 (mmHg)||47.0 ± 1.0||53.9 ± 1.9|
|Fetal pH||7.37 ± 0.03||7.32 ± 0.03|
|Fetal lactate (mmol l−1)||1.3 ± 0.1||1.4 ± 0.2|
Cardiopulmonary and cerebral haemodynamics during the first 10 min after birth
PBF was lower in LPS-exposed lambs compared to controls during the first 10 min after delivery (P= 0.04; Fig. 1A). Postsystolic minimum PBF, end-diastolic PBF and LVO were reduced during the first 10 min after delivery in LPS-exposed lambs (P= 0.01, 0.01 and 0.02, respectively; Fig. 1B, C and D). There was retrograde postsystolic minimum PBF in three of seven LPS-exposed lambs, but no retrograde flow was observed in controls (data not shown). PMPA was not different between groups during the initial 10 min ventilation period (Fig. 1E).
Carotid blood flow was increased between 3 and 7 min after delivery in LPS-exposed lambs compared to controls (P < 0.05; Fig. 2A). CaPI was reduced compared to the fetal value during the first 10 min after delivery, but was not different between groups. CaPI was 20% lower in LPS-exposed lambs compared to controls during the first 8 min after delivery; however, this did not reach statistical significance (Fig. 2B). Brachicephalic arterial pressure was not different between groups during the first 10 min after delivery (Fig. 2C). Cerebral DO2 was higher in LPS-exposed lambs compared to controls at 5 min after delivery (P= 0.04; Fig. 3).
The rate of change in pulmonary blood flow (PBF), postsystolic minimum PBF, end-diastolic PBF and left ventricular output during the fetal-to-neonatal transition after birth
The rate of change in PBF from prenatal levels until 2 min after birth was lower in LPS-exposed lambs than controls (P < 0.05; Fig. 4A). Postsystolic minimum PBF was lower in LPS-exposed lambs between fetal–2 min and 1–2 min time-points compared to controls (P= 0.01; Fig. 4B). The rate of increase in end-diastolic PBF, indicative of the rate of reduction in PVR was lower in LPS-exposed lambs compared to controls (P= 0.02; Fig. 4C). The rate of increase in LVO was lower in LPS-exposed lambs compared to controls between 1 and 2 min after delivery (P= 0.01; Fig. 4D).
Ventilation, arterial blood gases, oxygenation and acid–base status
During the neonatal period, no differences were observed in PIP, Cdyn.spec or VEI. Similarly, PaO2, SaO2, PaCO2, FIO2 and oxygenation index did not differ between groups. The aADO2 was lower in LPS-exposed lambs compared to controls 5 min after delivery (P < 0.05; Table 2). Arterial lactate concentration tended to be higher in LPS-exposed lambs compared to controls 5 min after delivery (P= 0.08; Table 2). Arterial pH did not differ between groups (Table 2).
|Minutes after delivery|
|PIP (cmH2O)||35.0 ± 3.0||36 ± 1.3||34.2 ± 3.3||35.6 ± 1.5||36.2 ± 2.8||35.0 ± 2.5||29.0 ± 3.8||25.6 ± 1.5||28.6 ± 3.4||26.3 ± 1.2|
|Cdyn,spec||0.22 ± 0.04||0.22 ± 0.01||0.25 ± 0.04||0.22 ± 0.02||0.25 ± 0.04||0.23 ± 0.02||0.32 ± 0.06||0.33 ± 0.04||0.32 ± 0.04||0.31 ± 0.03|
|aADO2||89.7 ± 34.6||30.8 ± 8.9*||62.8 ± 20.8||59.6 ± 14.4||70.2 ± 29.5||101.9 ± 56.3||95.7 ± 26.2||82.4 ± 20.7||108.9 ± 29.1||98.6 ± 29.1|
|F IO2||29 ± 5||21 ± 0||25 ± 4||21 ± 0||25 ± 4||26 ± 5||25 ± 4||23 ± 2||25 ± 4||24 ± 3|
|OI||9.0 ± 3.5||8.1 ± 0.9||5.7 ± 0.8||7.9 ± 0.7||9.1 ± 2.5||12.8 ± 3.2||6.0 ± 0.6||8.3 ± 1.1||9.8 ± 1.7||10.1 ± 1.4|
|VEI||0.06 ± 0.02||0.05 ± 0.01||0.07 ± 0.01||0.08 ± 0.01||0.09 ± 0.03||0.08 ± 0.01||0.14 ± 0.02||0.11 ± 0.02||0.15 ± 0.03||0.10 ± 0.02|
|pH||7.20 ± 0.04||7.23 ± 0.03||7.26 ± 0.03||7.28 ± 0.04||7.28 ± 0.05||7.29 ± 0.05||7.36 ± 0.04||7.35 ± 0.05||7.35 ± 0.05||7.34 ± 0.06|
|P aCO2 (mmHg)||56.3 ± 9.8||56.7 ± 3.9||47.7 ± 5.8||38.3 ± 4.8||45.6 ± 9.2||44.8 ± 5.5||31.1 ± 4.3||41.5 ± 5.2||31.4 ± 5.7||42.7 ± 4.8|
|Lactate (mmol l−1)||2.2 ± 0.3||3.2 ± 0.5§||2.2 ± 0.2||2.5 ± 0.5||2.1 ± 0.3||2.5 ± 0.4||1.9 ± 0.3||2.3 ± 0.5||1.8 ± 0.4||2.1 ± 0.5|
|P aO2 (mmHg)||40.7 ± 6.7||40.7 ± 7.2||49.4 ± 8.3||42.2 ± 7.3||46.0 ± 10.1||36.7 ± 4.2||38.7 ± 4.9||32.1 ± 3.7||25.1 ± 2.3||29.2 ± 3.6|
|S aO2 (%)||84.6 ± 8.9||84.3 ± 3.8||91.3 ± 5.3||89.2 ± 4.0||85.4 ± 9.9||86.2 ± 4.8||81.4 ± 9.2||84.8 ± 4.1||77.1 ± 9.4||77.3 ± 7.7|
Cardiopulmonary and cerebral haemodynamic response to increased positive end-expiratory pressure
The increase in PEEP caused an increase in heart rate in both groups (Fig. 5A). A reduction in PBF was observed in controls after increasing PEEP but no change was detected in LPS-exposed lambs (Fig. 5B). Increasing PEEP caused a slower reduction in end-diastolic PBF in LPS-exposed lambs compared to controls (Fig. 5C). A reduction in LVO was observed in controls after increasing PEEP but no change was detected in LPS-exposed lambs (Fig. 5D). In LPS-exposed lambs, increasing PEEP from 4 to 8 cmH2O reduced brachiocephalic arterial pressure by 9 mmHg at 2 min (P= 0.04), but no change was detected in controls (Fig. 5E). Increasing PEEP did not alter SaO2, PMPA, CaBF or CaPI in either group (data not shown).
Indices of lung injury and inflammation
After ventilation, mRNA levels of CTGF, CYR61 and EGR-1 were not different between LPS-exposed lambs and controls (P= 0.15, P= 0.28 and P= 0.87, respectively; Fig. 6A, B and C).
Similarly, mRNA levels of IL-1β, IL-8 and IL-6 were not different between controls and LPS-exposed lambs (P= 0.50, P= 0.44 and P= 0.54, respectively; Fig. 6D, E and F).
A stable circulatory transition at birth is critical for ensuring neonatal well-being and survival, especially in extremely preterm infants. Given the high prevalence of chorioamnionitis in preterm infants (Lahra & Jeffery, 2004), we aimed to determine the effect of intrauterine inflammation on cardiopulmonary and cerebral haemodynamics during the fetal-to-neonatal transition. Our major findings were that intra-amniotic LPS increased PVR and reduced PBF and LVO. Further, cerebral blood flow and oxygen delivery were increased during the initial transition at birth in LPS-exposed lambs. PBF and LVO were reduced in controls but unaffected in LPS-exposed lambs following an increase in PEEP. Brachiocephalic arterial pressure decreased transiently in LPS-exposed lambs but not controls after increasing PEEP. These data suggest cardiopulmonary and cerebral haemodynamics are altered by intrauterine inflammation during the initial circulatory transition at birth and during an increase in PEEP.
Subclinical/histological chorioamnionitis was modelled in this study using a single injection of LPS into the amniotic cavity of pregnant sheep (Newnham et al. 2002; Nitsos et al. 2002). This causes a fetal inflammatory response that is tolerated without fetal demise or any clinical symptoms in the ewe (Kramer et al. 2001; Newnham et al. 2002; Nitsos et al. 2002). Intra-amniotic LPS injection in sheep causes alterations in fetal lung and brain development (Willet et al. 2000; Moss et al. 2002a; Nitsos et al. 2006; Gavilanes et al. 2009; Polglase et al. 2012b). These data are consistent with altered risk of respiratory disease and brain injury observed in preterm infants exposed to histological chorioamnionitis (Watterberg et al. 1996; Wu & Colford, 2000; Wu, 2002; Rocha et al. 2007; Hansen-Pupp et al. 2008; Lahra et al. 2009).
A single intra-amniotic injection of LPS in sheep elicits pulmonary and systemic inflammation in the fetus as early as 5 h after injection (Kramer et al. 2001). Inflammatory cytokine mRNA levels are increased in the chorioamnion and amniotic fluid by 5 and 24 h, respectively, and remain elevated for 7 days (Kramer et al. 2001). Elevations in inflammatory cell number in the fetal lungs and amniotic fluid persist for up to 45 days after intra-amniotic LPS exposure (Moss et al. 2002a). This fetal inflammatory response alters development of the fetal lungs and brain (Moss et al. 2002b; Nitsos et al. 2006; Wolfs et al. 2009). In our present study, fetal sheep were exposed to LPS at 118 days, which marks the late canalicular/early alveolar stage of lung development. At this stage the pulmonary vascular bed is immature (Hooper & Harding, 2001) and susceptible to remodelling and abnormal development caused by inflammation. Induction of intrauterine inflammation at 118 days reduces the expression of microvascular markers and endothelial vasodilators (Kallapur et al. 2003, 2004). The developmental consequences of these inflammation-induced alterations to vascular development include hypertrophy of the smooth muscle layer and deposition of collagen in the adventitial layer of pulmonary arterioles (Willet et al. 2000; Kallapur et al. 2004; Polglase et al. 2010). These inflammation-induced changes to pulmonary vascular development and structure probably contribute to an increased PVR and thus impair the circulatory transition after preterm birth.
During the first 10 min after birth, PVR was increased in LPS-exposed lambs, as reflected by a reduction in end-diastolic PBF, which is an indicator of downstream resistance to blood flow (Polglase et al. 2005). Consequently, PBF and LVO were reduced during the first 10 min after birth in LPS-exposed preterm lambs. Intrauterine inflammation caused a reduction in postsystolic minimum PBF; indicating that vessel wall resistance in the left pulmonary artery is increased in LPS-exposed lambs compared to controls and increases the likelihood that a right-to-left shunt is maintained across the ductus arteriosus immediately after the systolic phase of the cardiac cycle (Grant et al. 1999). This is confirmed by the persistence of retrograde flow (right-to-left) during postsystolic minimum PBF in three of seven LPS-exposed lambs; no retrograde flow was observed in controls. This represents abnormal pulmonary vascular development and/or function because a persistent right-to-left shunt is associated with both PPHN and a requirement for greater respiratory support (Kinsella & Abman, 1995; Woldesenbet & Perlman, 2005; Polglase et al. 2010). During the immediate neonatal period the majority of shunting across the ductus arteriosus is normally left-to-right (Evans, 1993; Crossley et al. 2009; Noori et al. 2009; de Waal & Kluckow, 2010), because of the rapid decrease in PVR and the reversal in the pressure gradient across the ductus arteriosus; after removal of the placental circulation, pressure is normally 3–5 mmHg lower in the pulmonary artery than the aorta.
The rate of increase in mean, end-systolic and end-diastolic PBF and LVO was slower immediately after birth in LPS-exposed lambs. We have shown that the rate of reduction in PVR is greatest during the first minute after birth (Polglase et al. 2005). This demonstrates that the cardiopulmonary haemodynamic transition is impaired in LPS-exposed lambs immediately after delivery. These observations confirm previous studies from our laboratory (Polglase et al. 2010), and suggest the normal reduction in PVR after birth, which is critical to accommodating the entire output of the right ventricle along with reverse flow from the ductus arteriosus (left-to-right shunting), is attenuated after exposure to intrauterine inflammation.
We speculate that the increased cerebral perfusion and oxygen delivery in LPS-exposed lambs is due to an inflammation-induced increase in cerebral metabolic demand. There is an increase in cerebral oxygen delivery in preterm fetal sheep 2 and 4 days after intra-amniotic LPS, indicating an increased cerebral metabolic demand before birth (Andersen et al. 2011). Our data suggest that increased cerebral oxygen delivery persists until 7 days after LPS exposure.
The inflammation-induced alteration in cerebral perfusion may be associated with upregulation in systemic proinflammatory cytokines, which can compromise the integrity of the blood–brain barrier and cerebrovasculature (Polglase et al. 2012b). In preterm neonatal lambs, intrauterine inflammation causes vascular damage in the periventricular white matter and increases inflammatory cytokine mRNA expression in both the periventricular and subcortical white matter, 2 and 4 days after LPS exposure (Polglase et al. 2012b). We did not observe a difference in lung mRNA levels of inflammatory cytokines between groups, probably due to the presence of ventilator-induced lung injury in the control and LPS-exposed lambs, as shown previously (Polglase et al. 2012b), thus masking antenatal inflammation caused by LPS exposure (Kramer et al. 2001).
We have demonstrated that increasing PEEP causes a reduction in PBF and LVO due to an increase in PVR and a re-establishment of right-to-left shunting through the ductus arteriosus (Polglase et al. 2009). In LPS-exposed lambs, increasing PEEP 30 min after birth, when the transition of the circulation has stabilized, caused a greater increase in PVR and reduction to PBF compared to controls (Polglase et al. 2010). In this study, the increase in PEEP caused an increase in PVR in both groups (but it was delayed in LPS-exposed lambs) and a reduction in PBF and LVO in controls, which was not evident in the LPS-exposed lambs. The discrepancy between our current and previous experiments probably relates to the timing of the increase in PEEP. Residual liquid may still be present in distal airways 10 min after delivery, which, combined with inflammation-induced remodelling of the pulmonary vascular bed (Kallapur et al. 2004; Polglase et al. 2010), may have reduced compliance of the pulmonary arterioles and therefore restricted the circulatory effects of an increase in airway pressure. It is not known whether exposure to inflammation in utero alters lung liquid production but limited data suggest it does not (Moss et al. 2002b). To our knowledge, the effects of intrauterine inflammation on lung liquid clearance after birth are unknown.
We are unsure of the exact mechanism(s) responsible for the transient reduction in brachiocephalic arterial pressure that occurred in LPS-exposed lambs soon after increasing PEEP. However, our data suggest that cerebral vascular control is altered following an increase in PEEP in LPS-exposed lambs. We cannot discount that the transient reduction in brachiocephalic arterial pressure was associated with a reduction in right ventricular output or preload caused by an increase in intrathoracic pressure. However, this is unlikely given main pulmonary arterial pressure, PBF and end-diastolic PBF were unaffected by increasing PEEP in LPS-exposed lambs.
Ventilator-induced lung injury is known to impair cardiopulmonary and cerebral haemodynamics (Wallace et al. 2009; Polglase et al. 2012a). Therefore, we measured mRNA levels of CTGF, CYR-61 and EGR-1, which have previously been identified as early markers of ventilator-induced lung injury (Wallace et al. 2009). Exposure to intrauterine inflammation did not alter mRNA levels of CTGF, CYR-61 and EGR-1, suggesting that the degree of ventilator-induced lung injury was equivalent between LPS and control groups. This is further confirmed by the lack of difference in lung mRNA levels of IL-1β, IL-6 and IL-8 between groups. It remains possible that a longer duration of ventilation, or ventilation at a different point in time after intra-amniotic injection of LPS could result in an altered lung injury response.
In conclusion, we found that intrauterine inflammation attenuated the cardiopulmonary haemodynamic transition at birth and increased cerebral blood flow and oxygen delivery during the immediate neonatal period. Our study provides novel insight into the pathophysiology that may underlie the increased incidence of abnormal cardiopulmonary, systemic and cerebral haemodynamics observed in preterm neonates exposed to chorioamnionitis. Further studies are required to elucidate the mechanisms responsible for alterations in cardiopulmonary–cerebral perfusion in preterm neonates following exposure to intrauterine inflammation.
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R.G., S.B.H., M.J.W., M.J.B., G.R.P. and T.J.M.M. conceived and designed the experiments. R.G., S.B.H., M.J.W., A.J.W., M.J.B., G.R.P. and T.J.M.M. were involved in the collection, analysis and interpretation of data. R.G., S.B.H., M.J.W., A.J.W., M.J.B., G.R.P. and T.J.M.M. drafted the article or revised it critically for important intellectual content. All authors approved the final version of the manuscript.
The authors gratefully acknowledge the technical assistance of Alex Satragno, Alison Moxham, Karyn Rodgers and Valerie Zahra. This study was supported by NHMRC Program grants (384100 and 606789), an Australian Postgraduate Award (R. Galinsky), a National Heart Foundation of Australia (NHFA) grant in aid (G.R.P.), National Health and Medical Research Council of Australia (NHMRC) Career Development Fellowships (T.J.M.M.; 303261: G.R.P.: 1026890), NHMRC Fellowship (S.B.H.; 545921), a Rebecca L. Cooper Medical Research Foundation Fellowship (G.R.P.) and the Victorian Government's Operational Infrastructure Support Program. The authors report no conflict of interest in accordance with journal policy.