Acute and chronic changes in the control of breathing in a rat model of bronchopulmonary dysplasia

Abstract

Infants born very prematurely (<28 wk gestation) have immature lungs and often require supplemental oxygen. However, long-term hyperoxia exposure can arrest lung development, leading to bronchopulmonary dysplasia (BPD), which increases acute and long-term respiratory morbidity and mortality. The neural mechanisms controlling breathing are highly plastic during development. Whether the ventilatory control system adapts to pulmonary disease associated with hyperoxia exposure in infancy remains unclear. Here, we assessed potential age-dependent adaptations in the control of breathing in an established rat model of BPD associated with hyperoxia. Hyperoxia exposure ( ${\text{FI}}_{{\text{O}}_{\text{2}}}$ ; 0.9 from 0 to 10 days of life) led to a BPD-like lung phenotype, including sustained reductions in alveolar surface area and counts, and modest increases in airway resistance. Hyperoxia exposure also led to chronic increases in room air and acute hypoxic minute ventilation (V̇e) and age-dependent changes in breath-to-breath variability. Hyperoxia-exposed rats had normal oxygen saturation ( $\text{S}{\text{p}}_{{\text{O}}_2}$ ) in room air but greater reductions in $\text{S}{\text{p}}_{{\text{O}}_2}$ during acute hypoxia (12% O₂) that were likely due to lung injury. Moreover, acute ventilatory sensitivity was reduced at P12 to P14. Perinatal hyperoxia led to greater glial fibrillary acidic protein expression and an increase in neuron counts within six of eight or one of eight key brainstem regions, respectively, controlling breathing, suggesting astrocytic expansion. In conclusion, perinatal hyperoxia in rats induced a BPD-like phenotype and age-dependent adaptations in V̇e that may be mediated through changes to the neural architecture of the ventilatory control system. Our results suggest chronically altered ventilatory control in BPD.

Keywords: bronchpulmonary dysplasia; chronic hyperxoia; hyperoxia; respiratory control; ventilator control.

Fig. 1.

Perinatal hyperoxia exposure induced a bronchopulmonary dysplasia (BPD)-like phenotype assessed by total lung volume, septal surface area, alveolar density, and alveolar counts. A: representative ×10 hematoxylin and eosin images of lung sections from normoxic and hyperoxic rats at postnatal day (P)10, P21, and P60. B–E: total lung volume was significantly lower only at P10 and not at P21 or P60 (B), whereas septal surface area (C), alveolar density (D), and alveolar counts (E) were significantly lower at P10, P21, and P60 in hyperoxic (n = 7) rats compared with age-matched normoxic (n = 6) rats. *P < 0.05 vs. age-matched normoxic group. Scale bar, 100 µm.

Fig. 2.

Airflow-based plethysmographic metrics indicate a modest increase in airway resistance and no change in respiratory phase timing. A and B: peak inspiratory (A) but not expiratory flow (B) were significantly decreased at postnatal day (P)20–P21 and P60. C–E: the expiratory flow rate at 50% (EF50; C), inspiratory time (D), and expiratory time (E) were unchanged at all ages. F: Rpef (ratio of time to peak expiratory flow divided by expiratory time) was significantly lower in hyperoxic rats at P20–P21 and P60 compared with normoxic rats. P60 values within each group were significantly different from P10 to P11 values for all metrics measured. *P < 0.05 vs. age-matched normoxic group; ^P < 0.05 vs. within-group P10–P11 value. P10–P11: normoxic, n = 15; hyperoxic, n = 7. P20–P21: normoxic, n = 20; hyperoxic, n = 14; P60: normoxic, n = 12; hyperoxic, n = 12.

Fig. 3.

Neonatal hyperoxia exposure caused acute and chronic changes to ventilation measured during room air and acute hypoxia. There were many differences measured between hyperoxic and normoxic rats at each age during [postnatal day (P)10 to P43] and after (P60) development for minute ventilation (V̇e; A and B), breathing frequency (C and D), and tidal volume (V_T; E and F) during room air and acute hypoxia challenges. Notably, there were consistently higher minute ventilations measured acutely at P12–P14 due to a combination of increased breathing frequency and V_T and chronically at P60 due to an increase in V_T in hyperoxic rats compared with normoxic rats during room air and acute hypoxia. Additionally, developmental patterns in breathing were assessed by comparing measured P10 values with subsequent ages within a group, resulting in a different set of significantly different comparisons within normoxic vs. hyperoxic groups, indicating shifts in the developmental pattern of breathing (A–F). *P < 0.05 vs. age-matched normoxic group; ^P < 0.05 vs. within-group P10 value.

Fig. 4.

Blood oxygen saturation and minute ventilation (V̇e)/ oxygen saturation ($\text{S}{\text{p}}_{{\text{O}}_2}$) during development [postnatal day (P)12 to P43] during room air and acute hypoxia. A and B: $\text{S}{\text{p}}_{{\text{O}}_2}$was similar between normoxic and hyperoxic rats during room air breathing (A), but hyperoxic rats had significantly lower $\text{S}{\text{p}}_{{\text{O}}_2}$ from P12 to P17 of age during acute hypoxia (B). C and D: hyperoxic rats had a greater minute ventilation (V̇e) for a given $\text{S}{\text{p}}_{{\text{O}}_2}$ (V̇e/$\text{S}{\text{p}}_{{\text{O}}_2}$) from P12 to P43 during room air breathing (C) and from P12 to P17 during acute hypoxia (D). E: hyperoxic rats had lower hypoxic sensitivity (absolute value of the change in ventilation divided by the change in $\text{S}{\text{p}}_{{\text{O}}_2}$ measured during acute hypoxia and room air) at P12–P14. *P < 0.05 vs. age-matched normoxic group; ^P < 0.05 vs. within group P10 value. Normoxic P12, P14, P17, P21, and P43, n = 7, 9, 14, 12, and 12, respectively. Hyperoxic P12, P14, P17, P21, and P43, n = 10, 6, 11, 10, and 14, respectively.

Fig. 5.

Blood oxygen, blood gas, and minute ventilation (V̇e)/oxygen saturation ($\text{S}{\text{p}}_{{\text{O}}_2}$) and ventilation/$\text{P}{\text{a}}_{{\text{O}}_2}$ at postnatal day (P)60 during room air (RA) and acute hypoxia. A–D: $\text{S}{\text{p}}_{{\text{O}}_2}$, $\text{P}{\text{a}}_{{\text{O}}_2}$, $\text{P}{\text{a}}_{{\text{C}\text{O}}_2}$, and pH were not different between normoxic and hyperoxic rats for either room air or hypoxia breathing. E and F: hyperoxic rats had a greater V̇e for a given $\text{S}{\text{p}}_{{\text{O}}_2}$ (V̇e/$\text{S}{\text{p}}_{{\text{O}}_2}$) during room air and acute hypoxia breathing (E), and V̇e relative to a given $\text{P}{\text{a}}_{{\text{O}}_2}$ (V̇e/$\text{P}{\text{a}}_{{\text{O}}_2}$) was greater during hypoxia breathing and not room air breathing (F). G: no change in hypoxic sensitivity (absolute value of the change in ventilation divided by the change in $\text{S}{\text{p}}_{{\text{O}}_2}$ measured during acute hypoxia and room air) was measured between normoxic and hyperoxic rats at P60. *P < 0.05 vs. age-matched normoxic group; ^P < 0.05 vs. within-group P10 value.

Fig. 6.

Tidal volume (V_T) variability assessed by Poincare analyses was significantly altered acutely and chronically after hyperoxia exposure. A: Poincare plots of every quantified breath per group were plotted for normoxic rats (top plots) and hyperoxic rats (bottom plots) while room air was breathed. B and C: hyperoxia exposure caused significant changes in short-term (SD1) and long-term (SD2) variability during room air (B) and acute hypoxia breathing (C). The developmental pattern of SD1 and SD2 was assessed by comparing all values within a group to respective postnatal day (P)10 values, resulting in significant differences between P10 values and subsequent ages of hyperoxic rats but not normoxic rats. Poincare plots are not shown for acute hypoxia. Points per normoxic plot: P10 = 15,999, P12 = 17,390, P14 = 15,467, P17 = 17,450, P21 = 12,372, and P60 = 4,049. Points per hyperoxic plot: P10 = 21,681, P12 = 32,051, P14 = 29,779, P17 = 21,069, P21 = 12,811, and P60 = 4,720. In the P10 hyperoxic Poincare plot, SD1 and SD2 lines represent examples and not actual values, and the diagonal line represents the line of identification. *P < 0.05 vs. age-matched normoxic group; ^P < 0.05 vs. within group P10 value.

Fig. 7.

Breathing frequency variability assessed by Poincare analyses of the interbreath interval (IBI) was significantly altered acutely and chronically after hyperoxia exposure. A: Poincare plots of every quantified breath per group were plotted for normoxic rats (top plots) and hyperoxic rats (bottom plots) while room air was breathed. B and C: hyperoxia exposure caused significant changes in short-term (SD1) and long-term (SD2) variability during room air (B) and acute hypoxia breathing (C). The developmental pattern of SD1 and SD2 was assessed by comparing all values within a group to respective postnatal day (P)10 values, resulting in different ages being significantly different for normoxic vs. hyperoxic rats (B and C). Poincare plots are not shown for acute hypoxia. Points per normoxic plot: P10 = 16,003, P12 = 16,680, P14 = 15,466, P17 = 16,508, P21 = 12,507, and P60 = 3,781. Points per hyperoxic plot: P10 = 22,637, P12 = 33,734, P14 = 28,669, P17 = 21,124, P21 = 13,737, and P60 = 5,145. *P < 0.05 vs, age-matched normoxic group; ^P < 0.05 vs. within group P10 value.

Fig. 8.

Histological quantification of neuron counts within respiratory control nuclei assessed by counting the number of NeuN (neuron number)-positive cells within a defined region of interest. Neuron counts were normalized to the area of region analyzed. The no. of neurons was elevated in the nucleus of the solitary tract (NTS). However, the no. of neurons in the hypoglossal motor nucleus (HG), dorsal motor nucleus of the vagus (DMV), ventral respiratory column (VRC), retrotrapezoid nucleus (RTN), raphe pallidus (RPa) raphe, raphe magnus (RMg), and raphe obscurus (ROb) was not different between hyperoxic (Hx) and normoxic (Nx) rats. *P < 0.05 vs. age-matched Nx group. Scale bar, 100 µm.

Fig. 9.

Histological quantification of glial fibrillary acid protein (GFAP) expression within respiratory control nuclei assessed by %area fraction of positive signal per ×20 high-powered field following immunofluorescence labeling. GFAP expression was elevated across key respiratory control nuclei at postnatal day (P)60 in hyperoxia-exposed rats. Representative GFAP images are shown for normoxic (Nx; top) and hyperoxic (Hx) rats (bottom), and quantification of GFAP expression within each nucleus is shown under representative histologic images. GFAP expression in the hypoglossal motor nucleus (HG), ventral respiratory column (VRC), retrotrapezoid nucleus (RTN), raphe pallidus (RPa) raphe, raphe magnus (RMg), and raphe obscurus (ROb) was significantly greater in Hx rats compared with Nx rats. No differences in GFAP expression were measured within the nucleus of the solitary tract (NTS) and dorsal motor nucleus of the vagus (DMV). *P < 0.05 vs. age-matched Nx group. Scale bar, 100 µm.