Lambs then were stratified into two groups based on the graphical presentations noted in Figure ​Figure11 into those with Sat PC levels < 5 μmol/kg in BAL (n = 18) and those with levels > 5 μmol/kg in BAL (n = 10). Unventilated control lambs had a Sat PC level of 2.0 ± 0.5 μmol/kg. All lambs had less Sat PC than the normal levels of approximately 65 μmol/kg reported for term lambs [25]. There were no differences in birth weight, gestational age, or gender ratio between lambs with Sat PC values greater or less than 5 μmol/kg (Table ​(Table2).2). The differences in Sat PC measured at 2 h did not significantly change the dynamic compliance during the 15 min V_T injury maneuver (Table ​(Table2).2). Although no significant differences in V_T or compliance were apparent during the 15 min stretch injury or at 30 min, lambs with Sat PC levels > 5 μmol/kg had improved ventilation, oxygenation, and compliances when ventilated with lower V_T for the last hour of mechanical ventilation (Figure ​(Figure2).2). Lambs with Sat PC < 5 μmol/kg required a FiO₂ between 0.40 to 0.53 to maintain oxygen saturations, whereas the lambs with higher Sat PC levels required a FiO₂ of only 0.25 to 0.39.

All markers of lung injury were higher in animals with Sat PC levels below 5 μmol/kg in BALF than in the animals with higher Sat PC levels (Table ​(Table2).2). All ventilated animals had airway injury with sloughing of epithelium and inflammatory cells. Injury scoring between groups was higher in low Sat PC animals (Table ​(Table2).2). In situ localization of HSP70 mRNA demonstrated loss of the mRNA from bronchial epithelial cells in the ventilated animals compared with the unventilated controls and increased HSP70 mRNA in the smooth muscle surrounding the airways with ventilation (Figure 3A-C). There were no differences in HSP70 mRNA expression, representing similar airway over-expansion during ventilation. Early growth response protein 1 (Egr-1) surrounded the mesenchyme of the larger airways and was expressed in the medium sized conducting airways (Figure 3D-F). Egr-1 staining tended to be higher (175 vs 128 cells/high power field) in animals with lower Sat PC levels (Figure 3D-F). MCP-1 protein was localized to similar regions, but with more variation within each group (Figure 3G-I). MCP-1 protein levels in the BAL were increased 5 fold in animals with Sat PC less than 5 μmol/kg (Table ​(Table22).

In these studies, we attempted to inhibit important pro-inflammatory mediators of early lung injury response. Since many of the pro-inflammatory cytokines are transcribed after nuclear translocation of NF-κB, we used both IV and intra-tracheal parthenolide to block NF-kB activation in 7 lambs. Parthenolide is a sesquiterpene lactone derived from the plant Feverfew which may block NF-κB activity through inhibition of Iκ-B kinase activity [26]. The NF-kB system is active in preterm sheep and responds to intra-amniotic LPS [27]. Mice also have NF-κB activity by mid-gestation which increases near the end of gestation and in early post-natal life [28]. Mice ventilated with large V_T had NF-κB activation and cytokine production similar to mice exposed to LPS, but the cytokine increases were blocked by pretreatment with dexamthasone [29]. Our previous attempts to block lung inflammation with high dose dexamethasone or hydrocortisone were unsuccessful in the setting of initiation of ventilation in preterm sheep [2]. In a newborn piglet model of RDS, animals receiving a NF-κB inhibitor had no improvement over controls [30]. Inhibition of NF-κB in mice exposed to high V_T ventilation and hyperoxia blocked the additive effect of hyperoxia on volutrauma suggesting differing roles of NF-κB in stretch injury verses oxidative injury [31]. In these three animal models of acute respiratory failure and in these preterm lambs, blockade of NF-κB was unable to prevent inflammation from mechanical ventilation.

We also attempted to block two of the major pro-inflammatory cytokines that are increased with high V_T ventilation. We used recombinant human IL-1ra (Anikinra) to block IL-1 signaling. rhIL-RA is used clinically for treatment of a variety of inflammatory diseases and mutations ofIL-1ra result in severe systemic inflammation in early childhood [32]. We previously demonstrated that rhIL-1ra completely blocked lung inflammation from recombinant sheep IL-1 and decreased, but did not eliminate, the inflammatory response to LPS [16]. When adjusted for the amount of Sat PC in BAL, the IL-1 receptor blocker did not decrease lung injury in these lambs. We also tested nicotinanilide thioglycolate methyl ester (NTME), a CXCR2 inhibitor, to block IL-8 signaling. We previously demonstrated that NTME blocked recombinant ovine IL-8, but did not inhibitLPS induced lung inflammation in preterm sheep [15]. We found no decrease in ventilation mediated lung injury for this IL-8 inhibitor. Although drug levels were not measured in this study, we previously measured bio-availability of Anikinra and NTME in sheep [15,16]. Based on our previous results, the dosage used in the present study should have blocked IL-1 and IL-8 signaling. These studies, in combination with our previous study with postnatal corticosteroid treatment, suggest that blockade of pro-inflammatory responses to the initiation of ventilation of preterm infants will not be accomplished easily.

Previous preterm animal studies demonstrated improved ventilation with increasing endogenous surfactant pool sizes [7,19]. Base on the observation that animals with lower Sat PC levels had exponentially higher indicators of lung injury, we stratified the animals based on Sat PC level. We found animals with Sat PC levels less than < 5 μmol/kg (about 8 mg/kg surfactant based on assumption that Sat PC is 50% of the surfactant lipid pool) had significantly more injury than those with more Sat PC. These findings were similar to our previous findings that lambs with less than 1.9 μmol/kg Sat PC (3 mg/kg surfactant) had high PaCO₂ on CPAP, whereas lambs with more Sat PC had normal PaCO₂ levels [19]. The average surfactant pool of the term newborn sheep is approximately 100 mg/kg in BALF [25]. The average surfactant pool in group of animals with > 5 μmol/kg was about 15% of the levels reported at term. The average Sat PC pool for the lambs with < 5 μmol/kg was 2.5 μmol/kg, about 1.3 fold higher than that for lambs that previously survived on CPAP [19]. This lower effective pool size for the lambs on CPAP may result from the lack of intentional lung injury in those animals. The high V_T and resultant lung injury should cause inhibition of endogenous surfactant pools [7]. In 1970, Adams found surfactant pools less than 5 mg/kg in infants who died with RDS without mechanical ventilation [33]. These lambs were date-mated and the majority of lambs (25/28) were 133 days gestation, suggesting that small variations in lung maturation at the same gestational age can affect lung injury. Small changes in endogenous surfactant levels may have larger effects on lung mechanics than larger surfactant pool size increases with surfactant treatment [7].

Small increases in the endogenous surfactant pool size could decrease the heterogeneity of lung expansion and cause a more even distribution of V_T across the regions of the lung, and thus decrease focal injury [34]. Surfactant decreases surface tension and maintains FRC [35]. Since no PEEP was used during the first 15 min, the lambs with higher Sat PC may have had reduced regions of airway collapse. Newly secreted surfactant following birth is the large aggregate surfactant that has the best functional characteristics. High tidal volume ventilation can convert surfactant from surface-active large-aggregates to less surface-active surfactant forms [36] and these changes in surfactant forms proceed physiologic changes during ventilation [37]. The lambs with less surfactant in BAL may have had less functional surfactant and more surfactant inhibition, although these variables were not measured [38]. The induction of HSP70 in the smooth muscle of the airways was not changed by the amount of Sat PC, demonstrating an airway injury response with ventilation in all groups [1]. Our current analysis of injury based on surfactant pool size stresses the importance of the endogenous surfactant pool size on lung injury induced by the initiation of ventilation at birth.