Inflammation and lung maturation from stretch injury in preterm fetal sheep

Abstract

Mechanical ventilation is a risk factor for the development of bronchopulmonary dysplasia in premature infants. Fifteen minutes of high tidal volume (V(T)) ventilation induces inflammatory cytokine expression in small airways and lung parenchyma within 3 h. Our objective was to describe the temporal progression of cytokine and maturation responses to lung injury in fetal sheep exposed to a defined 15-min stretch injury. After maternal anesthesia and hysterotomy, 129-day gestation fetal lambs (n = 7-8/group) had the head and chest exteriorized. Each fetus was intubated, and airway fluid was gently removed. While placental support was maintained, the fetus received ventilation with an escalating V(T) to 15 ml/kg without positive end-expiratory pressure (PEEP) for 15 min using heated, humidified 100% nitrogen. The fetus was then returned to the uterus for 1, 6, or 24 h. Control lambs received a PEEP of 2 cmH(2)O for 15 min. Tissue samples from the lung and systemic organs were evaluated. Stretch injury increased the early response gene Egr-1 and increased expression of pro- and anti-inflammatory cytokines within 1 h. The injury induced granulocyte/macrophage colony-stimulating factor mRNA and matured monocytes to alveolar macrophages by 24 h. The mRNA for the surfactant proteins A, B, and C increased in the lungs by 24 h. The airway epithelium demonstrated dynamic changes in heat shock protein 70 (HSP70) over time. Serum cortisol levels did not increase, and induction of systemic inflammation was minimal. We conclude that a brief period of high V(T) ventilation causes a proinflammatory cascade, a maturation of lung monocytic cells, and an induction of surfactant protein mRNA.

Fig. 1.

Early growth response protein 1 (Egr-1) and cytokine mRNA expression in lung over time. Egr-1 mRNA increased 1 h after 15 min of high-tidal volume (Vt) ventilation and returned to baseline by 6 h. The proinflammatory cytokines IL-1β, IL-6, monocyte chemoattractant protein (MCP)-1, and MCP-2 mRNA all increased by 1 h. The counterregulatory molecule IL-1 receptor antagonist (IL-1ra) also increases at 1 h and remained elevated 24 h after the stretch injury. mRNA level expressed as the degree of change (fold increase) was determined from cDNA using quantitative RT-PCR, with mean values of controls set to 1. *P < 0.05 vs. controls.

Fig. 2.

Lung inflammation, Egr-1, and MCP-1 protein localization. Control lung (A) demonstrated no inflammation and thin airway septations on hematoxylin and eosin (H&E) staining. Airway wall thickenings were apparent by 1 h after the stretch injury (B) with continued congestion and inflammation by 6 h (C, inset). Egr-1 protein was expressed in the nucleus of cells surrounding the small airways at 1 h (E, inset) and had decreased by 6 h (F). MCP-1 protein was in the mesenchyme at 1 h (H, inset) and in inflammatory cells at 6 h (F). Egr-1 and MCP-1 were not increased in control animals (D and G).

Fig. 3.

Maturational signals from initiation of ventilation. A: representative image of large, foamy macrophage-like cells in BALF 24 h after the stretch injury. B: granulocyte/macrophage colony-stimulating factor (GM-CSF) mRNA increased at 1 h in lung after stretch injury and then returned to baseline. Pu.1 protein was minimal in fetal control lungs (C) but increased in the lung parenchyma by 24 h after stretch injury (D).

Fig. 4.

Heat shock protein 70 (HSP70) mRNA expression in the trachea and bronchus. Bronchial epithelium of control lambs expressed HSP70 (A), which was lost 1 h after ventilation (B). HSP70 mRNA was induced in bronchial epithelium at 6 h (C) and returned to baseline by 24 h (D). A similar pattern was seen in the tracheal epithelium (E–H). HSP70 mRNA was induced in the smooth muscle surrounding the larger airways at 1 h after intervention (B) but had resolved by 6 h (C).

Fig. 5.

Smooth muscle actin (SMA) staining in lung parenchyma. A–D: α-SMA protein was increased in the peripheral lungs at 6 h (C) compared with controls (A). E–H: SMAγ mRNA increased in the peripheral lung in animals at 1 h (F) compared with controls (E). Insets: average percentage of tissue staining per high-powered field. *P < 0.05 vs. controls.

Fig. 6.

Regional staining for H&E, Egr-1, and MCP-1 protein did not differ at 1 h. A–D: similar patterns of injury were seen between the right upper lobe (RUL), right middle lobe dependent (RML-D), right middle lobe nondependent (RML-ND), and right lower lobe dependent region (RLL-D) of the lung at 1 h. Egr-1 protein (E–H) and MCP-1 protein (I–L) staining patterns also did not differ between dependent and nondependent regions.