Pediatric Pulmonology 49:898–904 (2014)
Natural surfactant combined with superoxide dismutase and catalase decreases oxidative lung injury in the preterm lamb Carlo Dani, MD,1* Iuri Corsini, MD,1 Mariangela Longini, DVM,2 Silvia Burchielli, Giulia Dichiara, DVM,3 Carlo Cantile, MD,4 and Giuseppe Buonocore, MD2
3 DVM,
Summary. We performed a randomized study in preterm lambs to assess the hypothesis that treatment with a natural surfactant combined with superoxide dismutase (SOD) and catalase (CAT) might decrease pulmonary oxidative stress in an animal model of respiratory distress syndrome (RDS). Animals received 200 mg/kg of porcine natural surfactant or 200 mg/kg of natural surfactant combined with 2 mg/ml of SOD and 3,000 U/ml of CAT. Lung tissue oxidation was studied by measuring total hydroperoxide (TH), advanced oxidation protein products (AOPP), and non-protein bound iron (NPBI) in bronchial aspirate samples. In addition, the animal’s lung mechanics were evaluated. TH, AOPP, and NPBI were lower in the groups treated with surfactant plus SOD and CAT than in the surfactant group, while lung mechanics did not vary. We concluded that natural surfactant combined with SOD and CAT is effective in reducing the oxidative lung stress in an animal model of RDS. Pediatr Pulmonol. 2014; 49:898–904. ß 2013 Wiley Periodicals, Inc.
Key words: oxidative stress; superoxide dismutase; catalase; surfactant; respiratory distress syndrome; preterm lamb. Funding source: Chiesi Farmaceutici Srl
INTRODUCTION
Preterm infants are frequently affected by lung disorders such as respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD). Several investigators have assessed the role of reactive oxygen species (ROS) in the development of these disorders and the importance of antioxidant enzymes (AOEs) in preventing them.1,2 It is interesting that surfactant, a cornerstones of RDS treatment, has been found to be effective in decreasing patients’ mortality, but without consistently reducing the incidence of BPD.3 Actually, an important aspect of increased oxidant stress to the lung is injury to pulmonary surfactant, with surfactant replacement mitigating the pathophysiology of oxygen toxicity.4 Since surfactant metabolism and function is damaged by hyperoxia, it seems reasonable to supplement commercial surfactant preparations with antioxidant enzymes, which are often deficient in the premature lung.5,6 Moreover, because surfactant and associated proteins are continuously taken up by type II cells, it may serve as effective vehicle for the delivery of antioxidant enzymes to the lung epithelium. Matalon et al.7 demonstrated that natural calf lung surfactant contains significant amounts of superoxide dismutase (SOD) and catalase (CAT), and we recently ß 2013 Wiley Periodicals, Inc.
observed that commercial natural surfactants show measurable SOD and CAT activity, which differs between surfactants likely due their different origins and preparation procedures.8 These commercial surfactants exhibit scavenger activity against H2O2 and addition of SOD and CAT induces a remarkable increase in their antioxidant effects in vitro.8 It has also been demonstrated that intratracheal instillation of natural surfactant plus SOD
1 Department of Neuroscience, Psychology, Drug Research and Child Health, Careggi University Hospital of Florence, Florence, Italy. 2 Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena, Siena, Italy. 3 Institute of Clinical Physiology, Consiglio Nazionale delle Ricerche, Pisa, Italy. 4
Department of Animal Pathology, University of Pisa, Pisa, Italy.
Correspondence to: Carlo Dani, MD, Division of Neonatology, Careggi University Hospital, University of Florence School of Medicine, Viale Morgagni, 85 Firenze, Italy. E-mail: [email protected] Received 13 May 2013; Revised 1 August 2013; Accepted 13 August 2013. DOI 10.1002/ppul.22955 Published online 13 December 2013 in Wiley Online Library (wileyonlinelibrary.com).
Surfactant Plus SOD and CAT for RDS
and CAT results in a significant increase in antioxidant enzymes in the lung of RDS animal models.9,10 On the basis of these considerations, we hypothesized that the treatment with natural surfactant combined with SOD and CAT might decrease the lung tissue oxidative stress in preterm animals with RDS in comparison with treatment with surfactant alone. To assess this hypothesis, we evaluated the effect of the intratracheal administration of a porcine surfactant combined with SOD and CAT on pulmonary oxidative stress in preterm lambs with RDS and compared the results with a group of preterm lambs administered only surfactant. MATERIALS AND METHODS
The experimental protocol was designed in compliance with the recommendations of the European Economic Community (86/609/CEE) for the care and use of laboratory animals and was approved by the animal care committee of the University of Florence. Animals and Instrumentation
All animals were delivered by caesarean section at 125 1.3 days of gestational age from Massa ewes (term 145 days of gestational age). After exposure of the fetal head and neck, a carotid artery catheter was inserted for continuous blood pressure monitoring and blood sampling; the catheter was connected to pressure transducers, zero-referenced to mid-chest level. A venous catheter was inserted in the right internal jugular vein for continuous infusion of fluids (dextrose 10%, 100 ml/kg/day) and medications and an endotracheal tube was tied into the trachea. The fetal lung fluid that could be easily aspirated by syringe was recovered, and the lambs were delivered and weighed (2,430 320 g). A standard limb lead electrocardiogram (ECG) was used. Rectal temperature was kept within a normal range using electric warming pads. Before the first breath the lambs received randomly 200 mg/kg of natural surfactant (Curosurf1, Chiesi Spa, Parma, Italy), followed by 10 ml air given into the airways by syringe, or 200 mg/kg of natural surfactant combined with 2 mg/ml of SOD (Superoxide Dismutase, Sigma Chemical Co., St. Louis, MO), and 3,000 U/ml of CAT (Sigma Chemical Co.). The amount of SOD and CAT was decided on the basis of a previous study in which we
ABBREVIATIONS: AOEs antioxidant enzymes BPD bronchopulmonary dysplasia CAT catalase RDS respiratory distress syndrome ROS reactive oxygen species SOD superoxide dismutase
899
demonstrated in vitro that they were effective in increasing significantly the surfactant’s scavenger activity against H2O2.8 Randomization was performed using sealed opaque envelopes before the delivery. All animals were ventilated for 6 h with time-cycled and pressure-limited infant ventilators (Newport Breeze Ventilator, Soma Technology, Inc., Bloomfield, CT) using similar ventilation strategies. The initial ventilator settings included positive endexpiratory pressure (PEEP) of 4 cm H2O and a peak inspiratory pressure (PIP) sufficient to obtain a target tidal volume (VT) of about 10 ml/kg. The inspired oxygen fraction was adjusted to keep a target pO2 (100– 150 mm Hg). These ventilation parameters can injure the lung, thus evidencing the therapeutic effects of surfactant with or without SOD and CAT. To exclude the possibility that changes in ventilator setting might affect the results of the study, researchers responsible for ventilator adjustments were masked to the treatment assignment. Blood gases, pH, and base excess (BE) were analyzed by a blood gas, electrolyte, and metabolite system (Radiometer Copenhagen USA, West Lake, OH) at least every 30 min or when ventilatory status changed as indicated by changes in chest movement and tidal volumes. During the whole experiment the plasma expander polygeline (Emagel1, Novaselect, Potenza, Italy) and/or dopamine, 10–20 mg/kg/min were administered as needed to maintain a mean systemic arterial pressure above 30 mm Hg. Metabolic acidosis (pH < 7.25 and BE < 8 mmol/L) was corrected with sodium bicarbonate or THAM infusion (in case of hypercapnia: PaCO2 > 45 mm Hg). The pH, pO2, and pCO2, of each animal were recorded as soon as possible after delivery (baseline), and 1 (T1), 2 (T2), 4 (T4), and 6 (T6) hr after surfactant administration. Lung Mechanics Monitoring
Mean airway pressure (MAP), dynamic lung compliance (Cdyn), exhaled tidal volume (TV), and expiratory resistance (Raw) were measured with a neonatal respiratory monitor (Florian Neonatal Respiration MonitorTM, Acutronic, Hirzel, Switzerland) and their values at baseline, T1, T2, T4, and T6 were recorded. Experimental Procedures
Bronchial aspirate samples from animals were obtained with the following technique: 1 ml/kg sterile 0.9% saline was instilled using a 10 ml syringe through a 8F gauge feeding catheter that had been placed in the endotracheal tube so that the tip extended 1 cm beyond the distal end of the tube. The saline was instilled and immediately aspirated back into the syringe. The mean volume of saline returned was 0.8 (0.2) ml/kg. Pediatric Pulmonology
900
Dani et al.
Lung Oxidative Stress Assays
Statistical Analysis
All samples were clarified by centrifugation (1,000 rpm 5 min) and the supernatant was immediately frozen at 708C and stored for subsequent analysis. Bronchial aspirate samples were collected from each animal before surfactant administration, after exposure of the foetal head, and 1 (T1), 2 (T2), 4 (T4), and 6 (T6) hr after surfactant administration. In each bronchial aspirate sample, we measured the total hydroperoxide (TH) concentration, the advanced oxidation protein products (AOPPs), and the non-protein bound iron (NPBI). TH production was measured with a d-ROMs Kit (Diacron srl, Grosseto, Italy) by using a spectrophotometric procedure.11 The results were expressed in conventional units, (Carr units: the value of 1 Carr unit is equal to a concentration of 0.08 mg/dl of hydrogen peroxide). AOPPs were measured by the method of Witko-Sarsat et al.,12 using spectrophotometry on a microplate reader. The AOPP concentration was expressed as mmol/L chloramine-T equivalents. NPBI levels were determined by HPLC using the method described by Kime et al.,13 partially modified. The results were expressed in nmol/ml. Since no uniformly accepted correction factor is currently available for bronchial aspirate samples in both human and animal neonates, oxidative marker concentrations are expressed per volume of lavage fluid, as recommended by the European Respiratory Society.14
Time-course evolution of studied parameters in the different groups was compared using repeated-measures ANOVA, followed by Bonferroni’s multiple comparison tests. Comparisons between different treatment groups were tested with paired Student’s t-tests, as data were normally distributed. P value of
Natural surfactant combined with superoxide dismutase and catalase decreases oxidative lung injury in the preterm lamb Carlo Dani, MD,1* Iuri Corsini, MD,1 Mariangela Longini, DVM,2 Silvia Burchielli, Giulia Dichiara, DVM,3 Carlo Cantile, MD,4 and Giuseppe Buonocore, MD2
3 DVM,
Summary. We performed a randomized study in preterm lambs to assess the hypothesis that treatment with a natural surfactant combined with superoxide dismutase (SOD) and catalase (CAT) might decrease pulmonary oxidative stress in an animal model of respiratory distress syndrome (RDS). Animals received 200 mg/kg of porcine natural surfactant or 200 mg/kg of natural surfactant combined with 2 mg/ml of SOD and 3,000 U/ml of CAT. Lung tissue oxidation was studied by measuring total hydroperoxide (TH), advanced oxidation protein products (AOPP), and non-protein bound iron (NPBI) in bronchial aspirate samples. In addition, the animal’s lung mechanics were evaluated. TH, AOPP, and NPBI were lower in the groups treated with surfactant plus SOD and CAT than in the surfactant group, while lung mechanics did not vary. We concluded that natural surfactant combined with SOD and CAT is effective in reducing the oxidative lung stress in an animal model of RDS. Pediatr Pulmonol. 2014; 49:898–904. ß 2013 Wiley Periodicals, Inc.
Key words: oxidative stress; superoxide dismutase; catalase; surfactant; respiratory distress syndrome; preterm lamb. Funding source: Chiesi Farmaceutici Srl
INTRODUCTION
Preterm infants are frequently affected by lung disorders such as respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD). Several investigators have assessed the role of reactive oxygen species (ROS) in the development of these disorders and the importance of antioxidant enzymes (AOEs) in preventing them.1,2 It is interesting that surfactant, a cornerstones of RDS treatment, has been found to be effective in decreasing patients’ mortality, but without consistently reducing the incidence of BPD.3 Actually, an important aspect of increased oxidant stress to the lung is injury to pulmonary surfactant, with surfactant replacement mitigating the pathophysiology of oxygen toxicity.4 Since surfactant metabolism and function is damaged by hyperoxia, it seems reasonable to supplement commercial surfactant preparations with antioxidant enzymes, which are often deficient in the premature lung.5,6 Moreover, because surfactant and associated proteins are continuously taken up by type II cells, it may serve as effective vehicle for the delivery of antioxidant enzymes to the lung epithelium. Matalon et al.7 demonstrated that natural calf lung surfactant contains significant amounts of superoxide dismutase (SOD) and catalase (CAT), and we recently ß 2013 Wiley Periodicals, Inc.
observed that commercial natural surfactants show measurable SOD and CAT activity, which differs between surfactants likely due their different origins and preparation procedures.8 These commercial surfactants exhibit scavenger activity against H2O2 and addition of SOD and CAT induces a remarkable increase in their antioxidant effects in vitro.8 It has also been demonstrated that intratracheal instillation of natural surfactant plus SOD
1 Department of Neuroscience, Psychology, Drug Research and Child Health, Careggi University Hospital of Florence, Florence, Italy. 2 Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena, Siena, Italy. 3 Institute of Clinical Physiology, Consiglio Nazionale delle Ricerche, Pisa, Italy. 4
Department of Animal Pathology, University of Pisa, Pisa, Italy.
Correspondence to: Carlo Dani, MD, Division of Neonatology, Careggi University Hospital, University of Florence School of Medicine, Viale Morgagni, 85 Firenze, Italy. E-mail: [email protected] Received 13 May 2013; Revised 1 August 2013; Accepted 13 August 2013. DOI 10.1002/ppul.22955 Published online 13 December 2013 in Wiley Online Library (wileyonlinelibrary.com).
Surfactant Plus SOD and CAT for RDS
and CAT results in a significant increase in antioxidant enzymes in the lung of RDS animal models.9,10 On the basis of these considerations, we hypothesized that the treatment with natural surfactant combined with SOD and CAT might decrease the lung tissue oxidative stress in preterm animals with RDS in comparison with treatment with surfactant alone. To assess this hypothesis, we evaluated the effect of the intratracheal administration of a porcine surfactant combined with SOD and CAT on pulmonary oxidative stress in preterm lambs with RDS and compared the results with a group of preterm lambs administered only surfactant. MATERIALS AND METHODS
The experimental protocol was designed in compliance with the recommendations of the European Economic Community (86/609/CEE) for the care and use of laboratory animals and was approved by the animal care committee of the University of Florence. Animals and Instrumentation
All animals were delivered by caesarean section at 125 1.3 days of gestational age from Massa ewes (term 145 days of gestational age). After exposure of the fetal head and neck, a carotid artery catheter was inserted for continuous blood pressure monitoring and blood sampling; the catheter was connected to pressure transducers, zero-referenced to mid-chest level. A venous catheter was inserted in the right internal jugular vein for continuous infusion of fluids (dextrose 10%, 100 ml/kg/day) and medications and an endotracheal tube was tied into the trachea. The fetal lung fluid that could be easily aspirated by syringe was recovered, and the lambs were delivered and weighed (2,430 320 g). A standard limb lead electrocardiogram (ECG) was used. Rectal temperature was kept within a normal range using electric warming pads. Before the first breath the lambs received randomly 200 mg/kg of natural surfactant (Curosurf1, Chiesi Spa, Parma, Italy), followed by 10 ml air given into the airways by syringe, or 200 mg/kg of natural surfactant combined with 2 mg/ml of SOD (Superoxide Dismutase, Sigma Chemical Co., St. Louis, MO), and 3,000 U/ml of CAT (Sigma Chemical Co.). The amount of SOD and CAT was decided on the basis of a previous study in which we
ABBREVIATIONS: AOEs antioxidant enzymes BPD bronchopulmonary dysplasia CAT catalase RDS respiratory distress syndrome ROS reactive oxygen species SOD superoxide dismutase
899
demonstrated in vitro that they were effective in increasing significantly the surfactant’s scavenger activity against H2O2.8 Randomization was performed using sealed opaque envelopes before the delivery. All animals were ventilated for 6 h with time-cycled and pressure-limited infant ventilators (Newport Breeze Ventilator, Soma Technology, Inc., Bloomfield, CT) using similar ventilation strategies. The initial ventilator settings included positive endexpiratory pressure (PEEP) of 4 cm H2O and a peak inspiratory pressure (PIP) sufficient to obtain a target tidal volume (VT) of about 10 ml/kg. The inspired oxygen fraction was adjusted to keep a target pO2 (100– 150 mm Hg). These ventilation parameters can injure the lung, thus evidencing the therapeutic effects of surfactant with or without SOD and CAT. To exclude the possibility that changes in ventilator setting might affect the results of the study, researchers responsible for ventilator adjustments were masked to the treatment assignment. Blood gases, pH, and base excess (BE) were analyzed by a blood gas, electrolyte, and metabolite system (Radiometer Copenhagen USA, West Lake, OH) at least every 30 min or when ventilatory status changed as indicated by changes in chest movement and tidal volumes. During the whole experiment the plasma expander polygeline (Emagel1, Novaselect, Potenza, Italy) and/or dopamine, 10–20 mg/kg/min were administered as needed to maintain a mean systemic arterial pressure above 30 mm Hg. Metabolic acidosis (pH < 7.25 and BE < 8 mmol/L) was corrected with sodium bicarbonate or THAM infusion (in case of hypercapnia: PaCO2 > 45 mm Hg). The pH, pO2, and pCO2, of each animal were recorded as soon as possible after delivery (baseline), and 1 (T1), 2 (T2), 4 (T4), and 6 (T6) hr after surfactant administration. Lung Mechanics Monitoring
Mean airway pressure (MAP), dynamic lung compliance (Cdyn), exhaled tidal volume (TV), and expiratory resistance (Raw) were measured with a neonatal respiratory monitor (Florian Neonatal Respiration MonitorTM, Acutronic, Hirzel, Switzerland) and their values at baseline, T1, T2, T4, and T6 were recorded. Experimental Procedures
Bronchial aspirate samples from animals were obtained with the following technique: 1 ml/kg sterile 0.9% saline was instilled using a 10 ml syringe through a 8F gauge feeding catheter that had been placed in the endotracheal tube so that the tip extended 1 cm beyond the distal end of the tube. The saline was instilled and immediately aspirated back into the syringe. The mean volume of saline returned was 0.8 (0.2) ml/kg. Pediatric Pulmonology
900
Dani et al.
Lung Oxidative Stress Assays
Statistical Analysis
All samples were clarified by centrifugation (1,000 rpm 5 min) and the supernatant was immediately frozen at 708C and stored for subsequent analysis. Bronchial aspirate samples were collected from each animal before surfactant administration, after exposure of the foetal head, and 1 (T1), 2 (T2), 4 (T4), and 6 (T6) hr after surfactant administration. In each bronchial aspirate sample, we measured the total hydroperoxide (TH) concentration, the advanced oxidation protein products (AOPPs), and the non-protein bound iron (NPBI). TH production was measured with a d-ROMs Kit (Diacron srl, Grosseto, Italy) by using a spectrophotometric procedure.11 The results were expressed in conventional units, (Carr units: the value of 1 Carr unit is equal to a concentration of 0.08 mg/dl of hydrogen peroxide). AOPPs were measured by the method of Witko-Sarsat et al.,12 using spectrophotometry on a microplate reader. The AOPP concentration was expressed as mmol/L chloramine-T equivalents. NPBI levels were determined by HPLC using the method described by Kime et al.,13 partially modified. The results were expressed in nmol/ml. Since no uniformly accepted correction factor is currently available for bronchial aspirate samples in both human and animal neonates, oxidative marker concentrations are expressed per volume of lavage fluid, as recommended by the European Respiratory Society.14
Time-course evolution of studied parameters in the different groups was compared using repeated-measures ANOVA, followed by Bonferroni’s multiple comparison tests. Comparisons between different treatment groups were tested with paired Student’s t-tests, as data were normally distributed. P value of
