Increased Prostaglandin E2 in Brainstem Respiratory Centers Is Associated With Inhibition of Breathing Movements in Fetal Sheep Exposed to Progressive Systemic Inflammation

Abstract

Background: Preterm newborns commonly experience apnoeas after birth and require respiratory stimulants and support. Antenatal inflammation is a common antecedent of preterm birth and inflammatory mediators, particularly prostaglandin E2 (PGE₂), are associated with inhibition of vital brainstem respiratory centers. In this study, we tested the hypothesis that exposure to antenatal inflammation inhibits fetal breathing movements (FBMs) and increases inflammation and PGE₂ levels in brainstem respiratory centers, cerebrospinal fluid (CSF) and blood plasma.

Methods: Chronically instrumented late preterm fetal sheep at 0.85 of gestation were randomly assigned to receive repeated intravenous saline (n = 8) or lipopolysaccharide (LPS) infusions (experimental day 1 = 300 ng, day 2 = 600 ng, day 3 = 1200 ng, n = 8). Fetal breathing movements were recorded throughout the experimental period. Sheep were euthanized 4 days after starting infusions for assessment of brainstem respiratory center histology.

Results: LPS infusions increased circulating and cerebrospinal fluid PGE₂ levels, decreased arterial oxygen saturation, increased the partial pressure of carbon dioxide and lactate concentration, and decreased pH (p < 0.05 for all) compared to controls. LPS infusions caused transient reductions in the % of time fetuses spent breathing and the proportion of vigorous fetal breathing movements (P < 0.05 vs. control). LPS-exposure increased PGE₂ expression in the RTN/pFRG (P < 0.05 vs. control) but not the pBÖTC (P < 0.07 vs. control) of the brainstem. No significant changes in gene expression were observed for PGE₂ enzymes or caspase 3. LPS-exposure reduced the numbers of GFAP-immunoreactive astrocytes in the RTN/pFRG, NTS and XII of the brainstem (P < 0.05 vs. control for all) and increased microglial activation in the RTN/pFRG, preBÖTC, NTS, and XII brainstem respiratory centers (P < 0.05 vs. control for all).

Conclusion: Chronic LPS-exposure in late preterm fetal sheep increased PGE₂ levels within the brainstem, CSF and plasma, and was associated with inhibition of FBMs, astrocyte loss and microglial activation within the brainstem respiratory centers. Further studies are needed to determine whether the inflammation-induced increase in PGE₂ levels plays a key role in depressing respiratory drive in the perinatal period.

Keywords: PGE2; brainstem; fetal breathing movements; inflammation; respiratory centers.

FIGURE 1

Fetal arterial blood gases measured across 3 days at baseline (0 h), +2 h, and +6 h following saline [SAL (control)]/LPS infusions. Partial pressure of oxygen (PaO₂, A), oxygen saturation (SaO₂, B), partial pressure of carbon dioxide (PaCO₂, C), lactate (D), and pH (E) in SAL (control = black) and LPS-exposed (blue) fetuses. Gray bars indicate SAL or LPS infusion on each day after baseline sampling. Data are means ± SEM, *P < 0.05 vs. control, **P < 0.01 vs. control, ***P < 0.001 vs. control, ****P < 0.0001 vs. control.

FIGURE 2

Fetal breathing movements (FBMs) measured across 3 days at baseline (1 h before SAL/LPS infusions), 0 h (the hour of SAL/LPS infusion), +1, 2, 3, 4, 5, 6, and 12 h epochs following saline (SAL) and LPS infusions. Percentage of time fetuses spent breathing (A), total duration of FBMs (B), average amplitude of FBMs (average depth of breaths during each 1-h epoch, C), frequency of FBMs (average number of breaths/second during an episode of FBMs over each 1 h epoch, D), and percentage of vigorous fetal breathing movements ≥ 5 mmHg (E) in SAL (control = black) and LPS-exposed (blue) fetuses. Gray bars indicate SAL or LPS infusion on each day after baseline sampling. Data are means ± SEM, *P < 0.05 vs. control.

FIGURE 3

Plasma PGE₂ concentrations measured across the 3 days of SAL/LPS infusions at baseline (before infusions), +2 and +6 h after infusions (A). Gray bars indicate SAL or LPS infusion on each day after baseline sampling. Data are means ± SEM. Cerebrospinal fluid (CSF) PGE₂ concentration in SAL (control = black, n = 6) and LPS-exposed (blue, n = 6) fetuses measured on experimental day 5 (4 days after starting infusions, B). Data are means ± SD, *P < 0.05 vs. control.

FIGURE 4

PGE₂ expression in brainstem respiratory centers. Schematic diagram of brainstem respiratory centers analyzed: RTN/pFRG (A), preBÖTC, NTS, XII, and raphe nucleus (B). Representative photomicrographs of PGE₂ (green) staining in the RTN/pFRG in SAL (C,C’) and LPS exposed fetuses (D,D’). Area fraction of PGE₂ staining in brainstem respiratory centers in SAL (control = black, n = 8) and LPS-exposed fetuses (blue, E, n = 8). Data are means ± SD, **P < 0.01 vs. control. Representative photomicrographs of GFAP (red) merged with PGE₂ (green) showing co-localization of PGE₂ to GFAP + astrocytes within the preBÖTC. White arrowheads indicate PGE₂ positive neurons (F,F’). Scale = 20 μm.

FIGURE 5

mRNA levels of PGES (A), PGHS-2 (B), and CASP3 (C) in the medulla of SAL (black, n = 7) and LPS-exposed fetuses (blue, n = 7). Data are means ± SD and expressed as fold change from the SAL (control) group.

FIGURE 6

GFAP⁺ astrocytes within brainstem respiratory centers. Representative photomicrographs of GFAP + labeling (red) and the nuclear stain HOECHST (blue) in the RTN/pFRG of a control (A) and LPS exposed fetus (A’). Scale = 20 μm. Numbers of GFAP + astrocytes in the brainstem respiratory centers in SAL (control = black, n = 8) and LPS-exposed fetuses (blue, B, n = 8). Data are mean ± SD, *P < 0.05 vs. control, **P < 0.01 vs. control.

FIGURE 7

IBA-1⁺ microglia within brainstem respiratory centers. Representative images of microglial phenotypes assessed (A). Total numbers of IBA-1 + microglia and numbers of ramified, hyper ramified, reactive, and ameboid IBA-1 + microglia in the RTN/pFRG (B), preBÖTC (C), XII (D), NTS (E), and raphe nucleus (F) in SAL (control = black, n = 8) and LPS-exposed fetuses (blue, n = 8). Data are means ± SD, *P < 0.05 vs. control.