Effects of inhaled CO administration on acute lung injury in baboons with pneumococcal pneumonia

Abstract

Inhaled carbon monoxide (CO) gas has therapeutic potential for patients with acute respiratory distress syndrome if a safe, evidence-based dosing strategy and a ventilator-compatible CO delivery system can be developed. In this study, we used a clinically relevant baboon model of Streptococcus pneumoniae pneumonia to 1) test a novel, ventilator-compatible CO delivery system; 2) establish a safe and effective CO dosing regimen; and 3) investigate the local and systemic effects of CO therapy on inflammation and acute lung injury (ALI). Animals were inoculated with S. pneumoniae (10(8)-10(9) CFU) (n = 14) or saline vehicle (n = 5); in a subset with pneumonia (n = 5), we administered low-dose, inhaled CO gas (100-300 ppm × 60-90 min) at 0, 6, 24, and/or 48 h postinoculation and serially measured blood carboxyhemoglobin (COHb) levels. We found that CO inhalation at 200 ppm for 60 min is well tolerated and achieves a COHb of 6-8% with ambient CO levels ≤ 1 ppm. The COHb level measured at 20 min predicted the 60-min COHb level by the Coburn-Forster-Kane equation with high accuracy. Animals given inhaled CO + antibiotics displayed significantly less ALI at 8 days postinoculation compared with antibiotics alone. Inhaled CO was associated with activation of mitochondrial biogenesis in the lung and with augmentation of renal antioxidative programs. These data support the feasibility of safely delivering inhaled CO gas during mechanical ventilation and provide preliminary evidence that CO may accelerate the resolution of ALI in a clinically relevant nonhuman primate pneumonia model.

Keywords: Coburn-Forster-Kane equation; Streptococcus pneumoniae; carbon monoxide; drug delivery systems; pneumonia.

Fig. 1.

Carbon monoxide (CO) delivery system. A: front view of carbon monoxide delivery system. B: schematic of experimental setup including ventilator, CO delivery system with injector module, and CO gas cylinder. C: volumetric mixing data using the CO delivery system, a mechanical ventilator, and test lung. Dynamic flow-matching graph demonstrates that the instantaneous flow of CO gas (CO flow) matches the patient's inspiratory flow (Vent Flow) to deliver and maintain a precise and constant concentration of CO (Mixed CO) over time, adjusting for the patient's flow rate, respiratory rate, or tidal volume.

Fig. 2.

Vital signs. A: respiratory rates of both pneumonia groups were significantly elevated above uninfected control animals. B: heart rates of both pneumonia groups were significantly elevated above controls at 24 and 48 h postinoculation. C: temperature of both pneumonia groups was elevated above controls at 24 and 48 h, and the temperature of CO-exposed animals was elevated above control and unexposed pneumonia animals at 48 h. D: mean arterial pressure did not change significantly over the course of the experiment. Gray bars, uninfected, control group mean (n = 5 per time point); white bars, unexposed pneumonia group mean (n = 7–9 per time point); black bars, CO-exposed pneumonia group mean (n = 4–5 per time point); error bars represent standard deviation. *P < 0.05 compared with control, **P < 0.05 compared with control and unexposed pneumonia group. The physiological data for some of these animals have been reported (15, 34, 58).

Fig. 3.

Laboratory data. A: white blood cell (WBC) counts of unexposed pneumonia animals were significantly higher than control and CO-exposed animals at 24 h; however, there was significant within-group variability (increased, decreased, or unchanged from baseline) seen in both pneumonia groups at 24–48 h postinoculation. No other significant differences were noted, and by 168 h, the WBC counts of all groups were near baseline. Circles represent individual animals. B: platelet counts (mean ± SD) were significantly elevated in both pneumonia groups relative to controls at 168 h. C: Pa_O2/Pa_O2 ratios (mean ± SD) were significantly reduced at 48 and 168 h in both pneumonia groups compared with controls. D and E: the experimental strain of S. pneumoniae was isolated from bronchoalveolar lavage (BAL) fluid (BALF) [median (IQR)] at 48 h (D) and from blood [median (IQR)] at 24 and 48 h (E) in both pneumonia groups but not from controls. Antibiotics were administered to all animals once daily × 3 days starting after collection of the 48 h samples. Gray circles, uninfected, control group, n = 5 per time point; white circles, unexposed pneumonia group, n = 7–9 per time point; black circles, CO-exposed pneumonia group, n = 4–5 per time point. *P < 0.05 compared with control. **P < 0.05 compared with control and CO-exposed pneumonia groups. The laboratory data for some of these animals have been reported (15, 34, 58).

Fig. 4.

Delivery of inhaled CO at 200 ppm for 1 h and carboxyhemoglobin (COHb). A and B: adult male baboons (n = 5) were inoculated with S. pneumoniae (10⁸-10⁹ CFU) and given inhaled CO gas (200 ppm) through the ventilator circuit at 24 or 48 h postinoculation for 60–90 min by use of the CO delivery system. COHb levels were measured in arterial blood every 10–15 min using an IL 682 CO-oximeter. A: arterial COHb levels were 1.08 ± 0.18% at baseline and increased linearly to 7.6 ± 1% after 60 min (P < 0.0001) and 10.3% after 90 min of CO administration. B: peak COHb levels decreased following administration of 1.0 Fi_O2, returning to near baseline levels after 82 ± 9.5 min.

Fig. 5.

Correlation between venous and arterial COHb. Animals were given inhaled CO at 200 ppm through the ventilator for 60 min by using the CO delivery system. A and B: in select experiments (n = 2), arterial and venous blood samples were drawn simultaneously for comparison of venous and arterial COHb levels, and comparison of the IL 682 and AVOXimeter 4000 (Avox) CO-oximeters. A: there was excellent correlation between arterial and venous COHb levels obtained by using the IL 682 CO-oximeter (Spearman r, 0.9904; P < 0.0001). Deming Type II linear regression modeling demonstrated a near-perfect diagonal regression line (slope 1.003, y-intercept −0.064; P < 0.0001). B: measurements of COHb levels using the point-of-care AVOXimeter 4000 CO-oximeter correlated poorly with those obtained using the gold standard IL 682 CO-oximeter, especially at lower COHb. The AVOXimeter 4000 CO-oximeter did not perform as accurately when compared with the gold standard IL 682 CO-oximeter.

Fig. 6.

Coburn-Forster-Kane (CFK) equation and COHb prediction at 60 min. Animals (n = 5) were given inhaled CO at 200 ppm for 60–90 min and arterial COHb levels were measured every 10–15 min with an IL 682 CO-oximeter. The CFK equation was used to predict COHb levels by using the measured COHb level at 10 (A) and 20 (B) min. A: correlation between measured COHb and predicted COHb levels by using the 10-min COHb level and CFK equation (Spearman r, 0.9038, P < 0.0001; goodness-of-fit R², 0.735, P < 0.0001). B: correlation between measured COHb and predicted COHb levels by use of the 20-min COHb level and CFK equation (Spearman r, 0.9828, P < 0.0001; goodness-of-fit R², 0.9864, P < 0.0001). C: accuracy of the 10, 20, 30, 40, and 50-min measured COHb levels to predict the 60-min COHb by use of the CFK equation. The 20-min COHb was highly accurate in predicting the 60-min COHb, with a difference between predicted and actual COHb of 0.24 ± 0.33% [95% CI (−0.17–0.66)].

Fig. 7.

Bronchoalveolar fluid studies. Lactate dehydrogenase (LDH) (A), total protein (B), and cell counts (C) in uninfected, control animals (gray circles; n = 3–5 for cell counts and n = 5 for LDH and total protein), unexposed pneumonia animals (white circles; n = 7–9) and CO-exposed pneumonia animals (black circles; n = 4–5) at 0, 48, and 168 h postinoculation. BAL fluid indexes peaked at 48 h in both pneumonia groups and were at or near baseline by 168 h. There were no significant differences between the two pneumonia groups. Bars represent means. *P < 0.05 compared with control values; #P < 0.1 compared with control values. The BAL fluid characteristics for some of these animals are reported (15, 34).

Fig. 8.

Inhaled CO and resolution of pneumonia-induced acute lung injury (ALI). A: representative histopathology at 168 h from a control animal, an unexposed pneumonia animal, and a CO-exposed pneumonia animal. Control photomicrograph demonstrates empty alveoli with delicate septa. Pneumonia photomicrograph demonstrates severe intra-alveolar filling due to edema, leukocytes, erythrocytes, and other debris. Pneumonia + CO photomicrograph demonstrates significantly less alveolar filling with leukocytes and fibrin. Original magnification is ×400. B: ALI scores (reflecting leukocytes, edema, alveolar filling, fibrin, and necrosis) were significantly lower in the CO-exposed pneumonia (PNA + CO) animals (1.9 ± 2.7) than in the unexposed pneumonia (PNA) animals (4.5 ± 2.5) (P < 0.05). C: edema scores were significantly lower in the CO-exposed pneumonia animals (0 ± 0) than in the unexposed animals (0.6 ± 0.7) (P < 0.05). D: wet-to-dry weight ratios of lung tissue taken at 168 h were significantly lower in the CO-exposed pneumonia animals (4.6 ± 0.5) than in the unexposed pneumonia animals (5.3 ± 0.4) (P < 0.05). *P < 0.05; bars represent means. Some of the ALI scores for the uninfected control animals and unexposed pneumonia animals are published (34) and redisplayed in B and C. ALI scores for images shown in A are 0, 6, and 1.3, respectively.

Fig. 9.

Inhaled CO and mitochondrial biogenesis induction in the lung. A: lung tissue was taken from control (gray circles; n = 5), unexposed pneumonia (white circles; n = 5), and CO-exposed pneumonia (black circles; n = 4) animals at necropsy (168 h) and homogenized, and total protein was probed by Western blot for SOD2, HO-1, Tfam, and citrate synthase. Protein expression is relative to Coomassie blue staining. Bars represent means. #P < 0.1 relative to control, *P < 0.05. B: immunofluorescence of nuclear TTF-1 (alveolar type 2 cell marker; green) and ATP synthase (mitochondrial marker; red) in inflation-fixed lung sections from control, unexposed pneumonia, and CO-exposed pneumonia animals. Nuclei are counterstained with DAPI. The distribution of ATP synthase staining is greater in alveolar type 2 cells (arrowheads) and alveolar macrophages (asterisks) in the CO-exposed animals, indicative of increased mitochondrial mass in these cell types. Some of the macrophages in the unexposed pneumonia group appear to stain positively for cytoplasmic TTF-1 (green), which may reflect phagocytosis of alveolar type 2 cell debris. Bars are 100 μM.

Fig. 10.

Inhaled CO and sepsis-induced renal antioxidant defenses. A: kidney tissue was taken from control (gray circles; n = 4), unexposed pneumonia (white circles; n = 5), and CO-exposed pneumonia (black circles; n = 5) animals at necropsy (168 h), homogenized, and total protein was probed by Western blot for Tfam, HO-1, citrate synthase, and SOD2. No discernable pattern was appreciated for Tfam expression. Mean HO-1 expression is similar in control and CO-exposed animals and lower in unexposed pneumonia animals. Both unexposed and CO-exposed pneumonia animals displayed a trend for higher citrate synthase relative to controls, suggestive of sepsis-mediated activation of mitochondrial biogenesis. Sepsis also increased SOD2 expression relative to controls, which was further increased by CO exposure. Protein expression is relative to GAPDH. Bars represent means. #P < 0.1 relative to control, **P < 0.05 relative to control and unexposed pneumonia groups. B: immunofluorescence microscopy of SOD2 (green) and cytochrome c (red) in formalin-fixed kidney sections from control, pneumonia, and CO-exposed pneumonia animals. Compared with controls, SOD2/cytochrome c colocalization (yellow) is decreased in pneumonia animals, indicative of mitochondrial injury and release of cytochrome c into the cytoplasm, whereas colocalization is maintained with inhaled CO, consistent with a mitochondrial protective effect. Original magnification is ×600 for control and PNA and ×400 for PNA + CO.