Optimization of Variable Ventilation for Physiology, Immune Response and Surfactant Enhancement in Preterm Lambs

Abstract

Preterm infants often require mechanical ventilation due to lung immaturity including reduced or abnormal surfactant. Since cyclic stretch with cycle-by-cycle variability is known to augment surfactant release by epithelial cells, we hypothesized that such in vivo mechanotransduction improves surfactant maturation and hence lung physiology in preterm subjects. We thus tested whether breath-by-breath variability in tidal volume (VT) in variable ventilation (VV) can be tuned for optimal performance in a preterm lamb model. Preterm lambs were ventilated for 3 h with conventional ventilation (CV) or two variants of VV that used a maximum VT of 1.5 (VV1) or 2.25 (VV2) times the mean VT. VT was adjusted during ventilation to a permissive pCO2 target range. Respiratory mechanics were monitored continuously using the forced oscillation technique, followed by postmortem bronchoalveolar lavage and tissue collection. Both VVs outperformed CV in blood gas parameters (pH, SaO2, cerebral O2 saturation). However, only VV2 lowered PaCO2 and had a higher specific respiratory compliance than CV. VV2 also increased surfactant protein (SP)-B release compared to VV1 and stimulated its production compared to CV. The production and release of proSP-C however, was increased with CV compared to both VVs. There was more SP-A in both VVs than CV in the lung, but VV2 downregulated SP-A in the lavage, whereas SP-D significantly increased in CV in both the lavage and lung. Compared to CV, the cytokines IL-1β, and TNFα decreased with both VVs with less inflammation during VV2. Additionally, VV2 lungs showed the most homogeneous alveolar structure and least inflammatory cell infiltration assessed by histology. CV lungs exhibited over-distension mixed with collapsed and interstitial edematous regions with occasional hemorrhage. Following VV1, some lambs had normal alveolar structure while others were similar to CV. The IgG serum proteins in the lavage, a marker of leakage, were the highest in CV. An overall combined index of performance that included physiological, biochemical and histological markers was the best in VV2 followed by VV1. Thus, VV2 outperformed VV1 by enhancing SP-B metabolism resulting in open alveolar airspaces, less leakage and inflammation and hence better respiratory mechanics.

Keywords: alveolar stability; compliance; inflammation; surfactant protein.

Figures

Figure 1

Comparison of two ventilation patterns applied in preterm lambs. (A) Probability distribution functions of variable ventilations (VV) in which the maximum tidal volume was 1.5 times the mean tidal volume (VT) (VV1, black) or 2.25 times the mean VT (VV2, red). Notice that the tail of the distribution in VV2 is longer than that of VV1 which means that VV2 occasionally includes much larger VT than VV1 even though the mean in both cases was 8 mL/kg. (B,C) Realizations of the time series of VV1 and VV2, respectively, showing 50 consecutive VT's. The solid line represents conventional ventilation (CV).

Figure 2

Mean and standard deviation (SD) of the specific compliance (Cs) and the physiology index (PI) both normalized as a function of time in preterm lambs ventilated with conventional ventilation (CV), variable ventilation with maximum tidal volume of 1.5 times the mean (VV1) and another version of variable ventilation in which maximum tidal volume was 2.25 times the mean tidal volume (VV2) as defined in Figure 1. Data are normalized to unity with the mean of the CV group at the first time point. (A) The difference in the means of the specific compliance among the different levels of ventilation is statistically significant (p = 0.027 independent of time) with values larger during VV2 than CV. (B) The difference in the means of PI among the different levels of ventilation is statistically significant (p < 0.001 independent of time) with values larger during both VV1 and VV2 than CV.

Figure 3

Representative western blots and normalized surfactant protein (SP) levels from lavage fluid and lung tissue as a function of ventilation mode as described in Figure 1. (A) Top: representative western blots for SP-B (~18 kDa) in the bronchoalveolar lavage (BAL), proSP-B (~40 kDa) from lung tissue, the dimer form of SP-B (~18 kDa) from lung tissue and the loading control (LC) β-actin (~42 kDa). Bottom: statistics show that SP-B in BAL increases with VV2 relative to VV1 (p < 0.005). The proSP-B is higher during VV2 than CV (p < 0.005) whereas the levels of the dimer are different for all pairwise comparisons (p < 0.001). (B) Top: representative western blots for proSP-C (~21 kDa) in BAL and lung tissue (~25 kDa) together with the β-actin LC (~42 kDa). Bottom: Compared to CV, proSP-C decreases with VV1 and VV2 both in the lavage (p < 0.001) and the lung (p = 0.006). (C) Top: representative western blots for SP-A (~38 kDa) in BAL and lung tissue (~26 kDa) together with the β-actin LC (~42 kDa). Bottom: Compared to CV, SP-A in the lavage decreases with VV2 (p < 0.001), and in the lung, it increases with VV1 and VV2 (p < 0.001). (D) Top: representative western blots for SP-D (~35 kDa) in BAL and lung tissue (~35 kDa) together with the β-actin LC (~42 kDa). Bottom: Compared to CV, SP-D decreases with VV1 and VV2 both in the lavage and the lung (p < 0.001) and there is also a significant difference between VV1 and VV2. * and # denote statistically significant difference from CV and VV1, respectively.

Figure 4

Representative western blots and the mean and SD of cytokine levels in bronchoalveolar lavage and lung tissue as a function of ventilation mode (see Figure 1). (A) Top: representative western blots in BAL and lung tissue for IL-1β. The IL-1β (~17 kDa) levels significantly decrease with both VV1 and VV2 compared to CV in the lavage (p < 0.001; all three groups are different) as well as in lung tissue (p < 0.001). (B) The TNFα (~17 kDa) (top: blots) levels are lower in the lavage following both VV1 and VV2 than CV (p < 0.001) while the levels from the lung tissue are different between all three groups (p < 0.001). LC: β-actin (~42 kDa). * and # denote statistically significant difference from CV and VV1, respectively.

Figure 5

Mean and SD of the levels of immunoglobulin G light (IgGLC) and heavy (IgGHC) chains from lavage fluid as a function of ventilation mode (see Figure 1). For the light chain (~25 kDa), both VV1 and VV2 produce significantly less IgG than CV (p < 0.01) whereas for the heavy chain (~50 kDa), VV2 had less IgG than both CV and VV1 (p < 0.01). * and # denote statistically significant difference from CV and VV1, respectively.

Figure 6

(A) Representative images of haematoxylin and eosin (H&E) stained pre-term lamb lung sections. A–C represent conventional ventilation (CV), D–F are from VV1 whereas G, H and I are from VV2 (see Figure 1). Large black arrows indicate blood cell infiltration, small black arrows point to weakened, damaged thinner alveolar walls and opened black arrows show dilated or congested capillaries. The walls with retracted tissue are circled in black. A, shows collapsed area near a healthy alveolar region whereas C, shows a totally collapsed region with hemorraghic vascular damage associated with CV. D represents regions associated with excessive alveolar wall thickening and inflammatory cell invasion whereas E, shows vessel damage without collapse with VV1. Alveolar weakening is present in all ventilation regimens with the lowest occurrence in VV2. Scale bar: 100 μm. (B) Representative H,E stained pre-term lamb lung sections with histopathological details. A–C represent CV; D–F are from VV1 whereas G–I are from VV2. A shows alveolar wall weakening and breakage; B shows excessive alveolar wall thickening and inflammatory cell invasion whereas C shows a collapsed region with hemorrhagic vascular damage associated with CV. D represents regions associated with vessel damage (dilated capillaries, inflammatory cell infiltration and small interstitial edema) without alveolar collapse, whereas E shows weakened or broken alveolar regions. In F, inflammatory cell invasion can be seen following VV1. Some alveolar weakening and inflammatory cell infiltration is also present in VV2 but with significantly reduced occurrence shown in G–I. Scale bar: 100 μm.

Figure 7

Representative images of lipid stained preterm lamb lung sections. (A–C) represent conventional ventilation (CV), (D–F) are VV1 whereas (G–I) are VV2 (see Figure 1). (A,D,G) show the phospholipid (green) staining by Pearse's method, where phospholipid positive cells are marked with black arrows. The CV group shows weak staining for phospholipids whereas VV1 and VV2 exhibit stronger staining. The neutral red pinkish counterstain shows the nuclei and lung structure. (B,C,E,F,H,I) show lipid (dark purple and blue) staining using Nile blue. In open lung areas, all conditions exhibit similar number of blue positive cells with dominant acidic lipids indicated by the dark blue staining with slightly larger pools in VV2. VV1 exhibits yellowish blue color indicating a larger amount of neutral lipids compared to the other two conditions, but it also contains acidic lipids indicated by the dark blue staining. However, in the collapsed area (C), CV shows numerous smaller positive staining pattern. The lung tissue is unstained. Insets are magnified regions showing representative positive cells. Scale bar: 25 μm.

Figure 8

Physiology (PI), histology (HI), biochemistry (BI) and combined (CI) indexes as a function of ventilation mode (see Figure 1). * and # denote statistical significance (p < 0.001) compared to CV and VV1, respectively.