Consequences of maternal omega-3 polyunsaturated fatty acid supplementation on respiratory function in rat pups

Abstract

Key points: Incomplete development of the neural circuits that control breathing contributes to respiratory disorders in pre-term infants. Manifestations include respiratory instability, prolonged apnoeas and poor ventilatory responses to stimuli. Based on evidence suggesting that omega-3 polyunsaturated fatty acids (n-3 PUFA) improves brain development, we determined whether n-3 PUFA supplementation (via the maternal diet) improves respiratory function in 10-11-day-old rat pups. n-3 PUFA treatment prolonged apnoea duration but augmented the relative pulmonary surface area and the ventilatory response to hypoxia. During hypoxia, the drop in body temperature measured in treated pups was 1 °C less than in controls. n-3 PUFA treatment also reduced microglia cell density in the brainstem. Although heterogeneous, the results obtained in rat pups constitute a proof of concept that n-3 PUFA supplementation can have positive effects on neonatal respiration. This includes a more sustained hypoxic ventilatory response and a decreased respiratory inhibition during laryngeal chemoreflex.

Abstract: Most pre-term infants present respiratory instabilities and apnoeas as a result of incomplete development of the neural circuits that control breathing. Because omega-3 polyunsaturated fatty acids (n-3 PUFA) benefit brain development, we hypothesized that n-3 PUFA supplementation (via the maternal diet) improves respiratory function in rat pups. Pups received n-3 PUFA supplementation from an enriched diet (13 g kg^-1 of n-3 PUFA) administered to the mother from birth until the experiments were performed (postnatal days 10-11). Controls received a standard diet (0.3 g kg^-1 of n-3 PUFA). Breathing was measured in intact pups at rest and during hypoxia (FiO₂ = 0.12; 20 min) using whole body plethysmography. The duration of apnoeas induced by stimulating the laryngeal chemoreflex (LCR) was measured under anaesthesia. Lung morphology was compared between groups. Maternal n-3 PUFA supplementation effectively raised n-3 PUFA levels above control levels both in the blood and brainstem of pups. In intact, resting pups, n-3 PUFA increased the frequency and duration of apnoeas, especially in females. During hypoxia, n-3 PUFA supplemented pups hyperventilated 23% more than controls; their anapyrexic response was 1 °C less than controls. In anaesthetized pups, n-3 PUFA shortened the duration of LCR-induced apnoeas by 32%. The relative pulmonary surface area of n-3 PUFA supplemented pups was 12% higher than controls. Although n-3 PUFA supplementation augments apnoeas, there is no clear evidence of deleterious consequences on these pups. Based on the improved lung architecture and responses to respiratory challenges, this neonatal treatment appears to be beneficial to the offspring. However, further experiments are necessary to establish its overall safety.

Keywords: control of breathing; development; plasticity; prematurity.

Figure 1. Validation of experimental model

A, percentage of n‐3 PUFA (total fatty acids) present in red cell membranes. B, percentage of n‐3 PUFA (total fatty acids) present in brainstem homogenates. C and D, percentage of DHA (C) and EPA (D) in the brainstem. Data are expressed as the mean ± SEM. Post hoc pairwise comparisons were performed only when warranted by ANOVA. Significantly different from control: ^*** P ≤ 0.0001.

Figure 2. n‐3 PUFA supplementation increases body weight

Comparison of body weight (g) in pups (10–11 days old) that received n‐3 PUFA supplementation (black bar) via maternal diet vs. pups raised by mothers receiving control diet (CTRL; white bar). Numbers in brackets indicate the number of pups. Note that data from male and females were pooled because there are no sex‐specific differences for these variables. Data are expressed as the mean ± SEM. Post hoc pairwise comparisons were performed only when warranted by ANOVA. Significantly different from CTRL: ^* P ≤ 0.05.

Figure 3. n‐3 PUFA supplementation augments apnoea frequency in pups

A, plethysmographic recording illustrating one example of a spontaneous (solid arrow) and a post‐sigh apnoea (dashed arrow) in rat pups during baseline conditions. B, proportion of apnoeas in male and female rat pups. C, apnoea index representing the number of total apnoeic events (spontaneous and post‐sigh) per unit of time. D, mean of apnoeas duration (spontaneous and postsigh). Histograms in (C) and (D) represent responses observed in male and female pups and were compared between animals that received n ‐3 PUFA supplementation (black bar) with controls (CTRL) (white bar). Numbers in brackets in (C) indicate the number of pups per group. Data are expressed as the mean ± SEM. Post hoc pairwise comparisons were performed only when warranted by ANOVA. Significantly different from male CTRL: ^* P ≤ 0.05. Significantly different from female CTRL: ^# P ≤ 0.05. Significantly different from male n ‐3 PUFA: ^† P ≤ 0.05.

Figure 4. n‐3 PUFA supplementation delays the breathing frequency response at the onset of hypoxia

Time course of the breathing frequency response to hypoxia (12%). Data are expressed as percentage change from baseline (normoxia) and compared between groups and sexes: males (A) and females (B) that received n ‐3 PUFA supplementation (black circles) and control (CTRL) (white circles). C, gain index of the O₂ chemoreflex calculated as the mean frequency increase observed over the 3rd and 4th minute of hypoxia; this value is reported in arbitrary units reflecting the percentage change from baseline. Numbers in brackets indicate the number of replicates in each group. Data are expressed as the mean ± SEM. Post hoc pairwise comparisons were performed only when warranted by ANOVA. Significantly different from CTRL: ^* P ≤ 0.05.

Figure 5. Sex‐specific effects of n‐3 PUFA supplementation on the correlations between apnoea index and O₂ chemosensitivity gain index

The gain index was obtained A) in males and B) females by calculating the mean increase in the breathing frequency between the 3rd and 4th minute of hypoxia. Data are expressed as absolute values (apnoeas number) and percentage change from baseline (breathing frequency). The treatment effects reported correspond to the ANOVA results.

Figure 6. Sex‐specific effects of n‐3 PUFA supplementation on minute ventilation, tidal volume, breathing frequency responses to hypoxia and O₂ consumption in pups (P10–11)

All variables were measured during normoxia (baseline) and following 20 min of exposure to moderate hypoxia (12% CO₂). Histograms represent responses expressed as a percentage change from baseline and are compared between groups: pups that received n‐3 PUFA supplementation (black bar) and controls (white bar). Data are expressed as the mean ± SEM. The ANOVA results reported are for the entire data set. The number of replicates in each group is reported in Table 2. Post hoc pairwise comparisons were performed only when warranted by ANOVA. Significantly different from male CTRL group: ^* P ≤ 0.05. Significantly different from female CTRL group: ^# P ≤ 0.05. Significantly different from male n‐3 PUFA: ^† P ≤ 0.05.

Figure 7. Effects of n‐3 PUFA supplementation on anapyrexia response to hypoxia

A, comparison of the decrease in T _b° (body temperature) between n ‐3 PUFA supplemented and control (CTRL) pups. B, comparison of the relationship between ΔT _b° and body weight in n ‐3 PUFA supplemented and control pups. Histograms represent responses expressed as the mean ± SEM; numbers in brackets indicate the number of pups per group. Males and females were analysed together because there was no sex‐specific difference for this variable. Significantly different from CTRL group: ^* P ≤ 0.05.

Figure 8. n‐3 PUFA supplementation increases basal respiratory frequency in anaesthetized female pups

Respiratory frequency (beats per minute; bpm) during normoxia in pups that received n‐3 PUFA supplementation (black bars) or control (CTRL; white bars). Data are expressed as the mean ± SEM; numbers in brackets indicate the number of pups in each group. Post hoc pairwise comparisons were performed only when warranted by ANOVA. Significantly different from CTRL: ^* P ≤ 0.05.

Figure 9. n‐3 PUFA supplementation decreases apnoeas duration and has no effect on heart rate in anaesthetized pups during laryngeal chemoreflex (LCR) stimulation

A, original recordings illustrating minute ventilation (red traces) and LCR stimulation in pups that received n ‐3 PUFA supplementation and controls (CTRL). B, mean of apnoeas duration elicited by LCR. C, mean of minimum saturation observed after LCR. D, mean of minimum heart rate observed after LCR. Histograms represent responses observed in pups that received n ‐3 PUFA supplementation (black bar) and CTRL (white bar). Males and females were analysed together because there were no sex‐specific differences for these variables. Data are expressed as the mean ± SEM. Post hoc pairwise comparisons were performed only when warranted by ANOVA. Significantly different from CTRL: ^* P ≤ 0.05.

Figure 10. n‐3 PUFA treatment improves relative alveolar surface area in pups (P10–11)

Typical images of the lung architecture obtained from control (CTRL) rat pup (A) and n‐3 PUFA supplemented pups (B). C, mean linear intercept (L _m) (μm). D, relative alveolar surface area (m² cm^–3). E, lung volume (ml g^–1). Because there was no sex‐specific effect, data from male and female pups were pooled; numbers in brackets indicate the number of replicates in each group. Significantly different from CTRL: ^* P ≤ 0.05.

Figure 11. n‐3 PUFA treatment reduces the number of microglia and promotes an amoeboid phenotype in cNTS

A, schematic representation of cNTS area (Paxinos & Watson, 1998). Photomicrographs illustrating Iba‐1 immunolabelling on tissue from control (CTRL) (B) and n‐3 PUFA supplemented pups (C). D, number of Iba‐1 positive cells in cNTS. E, morphological index of Iba‐1 positive cells in cNTS (illustration of microglia phenotype adapted with permission from Orłowski et al. 2003). Data are expressed as the mean ± SEM. Numbers in brackets indicate the number of replicates in each group. Post hoc pairwise comparisons were performed only when warranted by ANOVA. Significantly different from CTRL: ^* P ≤ 0.05. Significantly different from male CTRL: ^# P ≤ 0.05.