Fig 1.
Ten animals were randomly allocated into one of the following groups: Control group (red line, n = 5), which consisted of 6h of standard ventilation; or treatment group (blue line, n = 5), which consisted of 6h of standard ventilation + 160 ppm of iNO + MB 1mg/Kg administered in bolus when metHb was 6% or higher. After the intervention phase, all animals were weaned, extubated and followed clinically for three days. On day 3, animals were sacrificed by exsanguination. Samples collection and assessments were performed during the experiments detailed in the lower part of the Fig 1. Definition of abbreviations: BL = baseline, ppm = parts per million, MB = methylene blue, iNO = inhaled nitric oxide, metHb = methemoglobin, NO2 = nitrogen dioxide and NOx = nitric oxide byproducts, PC = pressure control, PEEP = positive end-expiratory pressure, bpm = beats per minute.
Fig 2.
Schematic representation of the inhaled nitric oxide delivery system, and its components location.
iNO was delivered for 6 hours continuously to reach the target dose of 160 ppm using a medical-grade pressurized cylinder of 2,000 ppm with a custom digital gas regulator device. The gas was administered through a connector attached to the breathing circuit to deliver NO during predetermined ventilation settings using an ICU ventilator. Inspiratory NO and NO2 levels were monitored in real-time using a dedicated gas electrochemical analyzer sampling from a port of the inspiratory limb of the ventilatory circuit, proximal to the endotracheal tube. A multi-absorber scavenger device was used to maintain NO2 below a safety threshold (<5 ppm), and was placed right after the humidification system. Definition of abbreviations: ppm = parts per million, iNO = inhaled nitric oxide, NO2 = nitrogen dioxide, ICU = intensive care unit, h = hours.
Fig 3.
High-dose nitric oxide (NO) delivery and nitrogen dioxide (NO2) formation.
Various parameters of the delivery system were monitored during 6 h of continuous exposure to a pre-set concentration of NO (160 ppm). (a) The NO concentration in the inspiratory line monitored with an electrochemical analyzer (AeroNOx, International Biomedical, TX, United States). The maximum variation observed was 161.6 ± 3.53 ppm, with the highest dose reaching 169 ppm of the pre-set concentration. (b) The buildup of nitrogen dioxide was monitored during the same period of exposure. The NO2 level remained below the safety threshold (5.0 ppm), during the entire experiment, with an average level of 3.24 ± 0.35 ppm. Data are presented as single data points from each individual case: CASE 1 (), CASE 2 (
), CASE 3 (
), CASE 4 (
), and CASE 5 (
). Definition of abbreviations: BL = baseline, ppm = parts per million, NO = nitric oxide, and NO2 = nitrogen dioxide.
Fig 4.
Methemoglobin (metHb) levels over time.
MetHb was measured from an arterial blood sample every 30min during the intervention phase. Inhaled nitric oxide (iNO)-treated animals had significantly higher levels of metHb than controls (a). Variation over time of the metHb percentage and time of methylene blue administration is shown for each treated animal (b-f). The safety threshold for metHb was set at 6%. One dose of methylene blue (MB) during the entire intervention was sufficient to keep metHb levels below the safety threshold (6%). Definition of abbreviation: BL = baseline, MB = methylene blue, metHb = methemoglobin, iNO = inhaled nitric oxide, and ns = non-significant.
Fig 5.
Nitric oxide byproducts formation (NOx) overtime in control versus iNO treated animals.
The uptake of iNO was estimated by monitoring NOx concentrations in plasma, BAL and lung tissue. NOx levels were measured using modified Griess method in combination with Griess reagent system. (a) NOx gradually increased in plasma over time and was significantly higher in the iNO group (blue, n = 5) at 6h when compared to control (red, n = 5) p = 0.032, consistent with iNO delivery. After 72 hours, levels were back to normal and comparable to controls. We did not notice any significant differences in NOx levels in BAL (b) and lung tissue (c). Graphs represented through interleaved scatter with bars with individual values plotted as mean with standard error of mean. * = statistically significant. Definition of abbreviation: BAL = bronchoalveolar lavage, iNO = inhaled nitric oxide, and NOx = nitric oxide byproducts.
Fig 6.
Pulmonary function, inflammation analysis, and histologic examination of the control group (red box/line, n = 5) versus treatment iNO group lungs (blue box/line, n = 5).
The following lung functional parameters were evaluated: (a) lung compliance, (b) tidal volume. There was no significant difference between control and iNO treated animals. Acute lung injury score (c) was similar between the two groups in each evaluated component: Alveolar hemorrhage, capillary congestion, edema and WBC infiltration. Representative images (5X) of H&E stained biopsies of one control (bottom image) and one iNO treated animal (upper image) taken at 72 hours are shown in (d). Additionally, representative bronchoscopic images of one iNO-treated case at baseline (e), after 6 hours of iNO (f), and after three days of survival (g) show no signs of airway injury. Furthermore, after sacrifice, lungs were explanted for visual inspection, and gross appearance is shown in (h-i). No acute lung injuries were noted. Data displayed as mean ± SD for each time-point. Definition of abbreviation: BL = baseline, iNO = inhaled nitric oxide, WBC = white blood cells, H&E = hematoxylin & eosin, and ns = non-significant.
Table 1.
Cytokine levels in plasma, bronchoalveolar lavage and lung tissue at baseline, 3, 6, and 72 hours.
Table 2.
Hemodynamic parameters and arterial blood gas analysis at baseline, 3, 6, and 72 hours.
Fig 7.
Biochemical analyses of control (red line, n = 5) versus iNO treated animals (blue line, n = 5).
Venous blood samples were collected hourly during the intervention phase and daily during the follow-up period in animals of both groups. Kidney function markers, liver enzymes, and coagulation parameters were stable and similar in both groups. Data are displayed as mean ± SD for each time-point. Definition of abbreviation: AST = aspartate aminotransferase, ALT = alanine aminotransferase, LD = lactate dehydrogenase, PT = prothrombin time, INR = international normalized ratio, and ns = non-significant.