Colonic oxygen microbubbles augment systemic oxygenation and CO2 removal in a porcine smoke inhalation model of severe hypoxia

Abstract

Inhalation injury can lead to pulmonary complications resulting in the development of respiratory distress and severe hypoxia. Respiratory distress is one of the major causes of death in critically ill patients with a reported mortality rate of up to 45%. The present study focuses on the effect of oxygen microbubble (OMB) infusion via the colon in a porcine model of smoke inhalation-induced lung injury. Juvenile female Duroc pigs (n = 6 colonic OMB, n = 6 no treatment) ranging from 39 to 51 kg in weight were exposed to smoke under general anesthesia for 2 h. Animals developed severe hypoxia 48 h after smoke inhalation as reflected by reduction in SpO₂ to 66.3 ± 13.1% and PaO₂ to 45.3 ± 7.6 mmHg, as well as bilateral diffuse infiltrates demonstrated on chest X-ray. Colonic OMB infusion (75-100 mL/kg dose) resulted in significant improvements in systemic oxygenation as demonstrated by an increase in PaO₂ of 13.2 ± 4.7 mmHg and SpO₂ of 15.2 ± 10.0% out to 2.5 h, compared to no-treatment control animals that experienced a decline in PaO₂ of 8.2 ± 7.9 mmHg and SpO₂ of 12.9 ± 18.7% over the same timeframe. Likewise, colonic OMB decreased PaCO₂ and PmvCO₂ by 19.7 ± 7.6 mmHg and 7.6 ± 6.7 mmHg, respectively, compared to controls that experienced increases in PaCO₂ and PmvCO₂ of 17.9 ± 11.7 mmHg and 18.3 ± 11.2 mmHg. We conclude that colonic delivery of OMB therapy has potential to treat patients experiencing severe hypoxemic respiratory failure.

Fig. 1

Porcine smoke inhalation injury. A, B Before and after (D, E) chest X-ray images confirming the presence of diffuse bilateral infiltrates indicative of ARDS due to smoke inhalation injury. C P_aO₂ (red, p = 0.000147), P_mvO₂ (blue, p < 0.0001), SpO₂ (violet, p < 0.0001), P_aCO₂ (gold, p < 0.0001), P_mvCO₂ (green, p = 0.000123) and ETCO₂ (teal, p < 0.0001) measurements taken both before (t = − 48 h) and after (t = − 0.5 h) smoke inhalation injury. Hematoxylin and eosin (H&E) staining of paraffin-embedded lung tissue sections of baseline (t = − 48 h) (F) and SI + 48 h (t = − 0.5 h) (G) animals (scale bar = 100 µm). H IL-6 marker analysis for baseline and smoke injury (SI) + 2 h (t = − 46 h) for BAL and plasma samples. Comparison of lung injury score (I) and lung wet/dry ratios (J) showing a significant difference between control and SI + 48 h (t = − 0.5 h) samples (p < 0.0001 and p = 0.0188, respectively)

Fig. 2

Colonic OMB administration. A Process flow diagram showing the production process for creating OMB via sonication and differential centrifugation (DSPC = distearoylphosphatidylcholine, PEG40S = polyoxyethylene 40 stearate, QA = quality assurance, OMF = oxygen microfoam). B Particle size by both number percent (black) and volume percent (blue) frequency for the OMB samples. C Microscopy image showing the size of the OMB (scale bar = 10 µm). D Schematic showing the colonic delivery of OMB to a smoke inhalation lung injured pig on minimal mechanical ventilation (FiO₂ = 0.21)

Fig. 3

Colonic OMB systemic oxygenation and CO₂ removal. A Blood oxygen and B CO₂ measurements for NT (top) and colonic OMB (bottom) treatment groups from before smoke inhalation injury (t = − 3000 min) out to t = 150 min post-treatment time. The change in oxygen vitals for NT (hashed) and OMB (solid) treatment groups showing the statistical significance at times t = 15, 30, 45, 60, 90, 120 and 150 min, C P_aO₂ (red), (p = 0.0117, 0.0124, 0.0137, 0.0058, 0.0014, 0.0022 and 0.0005), D P_mvO₂ (blue), (p = 0.0138, 0.0597, 0.0799, 0.1568, 0.1230, 0.0171 and 0.0110), E SpO₂ (violet), (p = 0.1350, 0.1239, 0.0788, 0.0329, 0.0212, 0.0095 and 0.0133), F P_aCO₂ (gold), (p = 0.0025, 0.0004, < 0.0001, < 0.0001, 0.0109, < 0.0001 and 0.0003), G P_mvCO₂ (green), (p = 0.0330, 0.0113, 0.0132, 0.0007, 0.0030, 0.0009 and 0.0018) and H ETCO₂ (teal), (p = 0.0040, 0.0269, 0.0136, 0.0007, 0.0004, 0.0015 and 0.0012)

Fig. 4

Local and systemic injury and inflammation. Hematoxylin and eosin (H&E) staining of paraffin-embedded lung tissue sections of SI + 48 h (t = − 0.5 h) (A) and OMB treatment (t = 3 h) (B) animals (scale bar = 100 µm). Comparison of lung injury score (C) and lung wet/dry ratios (D) between SI + 48 h (t = − 0.5 h) and OMB treatment (t = 3 h) samples. E, F Immunoblot analysis of IL-1β expression levels in fresh frozen lung tissues of baseline (t = − 48 h), NT and OMB samples (t = 3 h). Difference between baseline and NT groups was statistically significant (p = 0.0092). G IL-6 marker analysis for NT and OMB (t = 3 h) for BAL and plasma samples. H–J Proteomic analysis of control (t = − 48 h), SI (NT, t = 3 h) and SI + OMB (t = 3 h) groups for their global protein expression as described in Materials and Methods section. Venn diagram showed 320 proteins with significant differential expression between SI and control groups (H, I). Heat map analysis of 68 proteins that were significantly upregulated or downregulated at 3 h post-OMB treatment (J). A p value of < 0.05 was considered statistically significant

Fig. 5

IL-6 immunoblot and IL-8 and IL-1β ELISA analysis. A, D Immunoblot analysis of IL-6 expression levels in lung tissues of Control (t = − 48 h), SI (t = − 46 h) and SI + OMB (t = 3 h) groups (p < 0.0001). B, C IL-8 expression level in BAL fluid and plasma samples of treated animals at baseline (t = − 48 h), SI + 2 h (t = − 46 h), SI + 48 h (t = − 0.5 h), NT and OMB (t = 3 h) samples. E, F IL-1β expression level in BAL fluid and plasma samples of treated animals at the same timepoints as IL-8 analysis

Fig. 6

Colonic OMB alveolar oxygen pressure. A P_AO₂ measurements for NT (dashed) and colonic OMB (solid) treatment groups from before smoke inhalation injury (t = − 3000 min) out to t = 150 min post-treatment time. The change in P_AO₂ for NT (hashed) and OMB (solid) treatment groups showing the statistical significance at times t = 15, 30, 45, 60, 90, 120 and 150 min (p = 0.0046, 0.0328, 0.0186, 0.0201, 0.0078, 0.0009 and 0.0018)