| BackgroundPulmonary surfactant (PS) is a mixture of phospholipids, neutral lipids and associated proteins. It reduces surface tension at air-fluid interface, thereby facilitating alveolar aeration during breathing, lung fluid absorption, and pulmonary vascular perfusion. The principal surface tension-reducing component of PS is dipalmitoylphosphatidylcholine (DPPC). Incorporation of surfactant protein (SP)-A, B or SP-C results in formation of a stable surfactant film with biophysical properties of rapid adsorption and insertion of phospholipids into the surface film, low surface tension upon film compression and rapid respreading of phospholipids during alveolar expansion. Neonatal respiratory distress syndrome (RDS) is routinely treated with modified surfactants for primary PS deficiency and structural immaturity of the lungs. Beneficial effects of exogenous surfactant may be impaired by lung injury from continuing mechanical ventilation, and by serum proteins leaking into alveolar spaces when damage to alveolar septae occurs. Exogenous surfactant therapy is sometimes ineffective in patients with surfactant inhibition or dysfunction in acute lung injury in those with severe hypoxic respiratory failure due to RDS, or in those with meconium aspiration syndrome or pneumonia. Several reports indicate that ionic and nonionic polymers may improve surfactant activity when used to treat lung injury. One of these ionic polymers, hyaluronic acid (HA, hyaluronan), is a linear, repeating polymer of N-acetyl-glucosamine linked β 1, 4 to glucuronic acid which is linked β 1, 3 to N-acetylglucosamine. It is ubiquitous in the extra cellular matrix of higher species animals. The molecular weight of HA varies with the tissue sources and is about 2.2 ×10~5 Da in the alveolar subphase. Addition of HA to surfactants could decrease inactivation caused by serum in vitro and HA added to Survanta could decrease inactivation caused by meconium in vitro and improves gas exchange and pulmonary mechanics of animals with meconium-induced acute lung injury (ALI).The fetus and newborns have a lower antioxidant and anti-inflammatory capacitythan infants and adults who have mature lungs and antioxidant system. The main mechanism of oxidative lung injury in premature infants is lipid peroxidation with increased production of organic hydroperoxides and malondialdehyde (MDA). Oxidation of surfactant phospholipids may interfere with normal surface activity of endogenous surfactant. Myeloperoxidase (MPO) is a marker of accumulated polymorphonuclear granulocytes (PMN) in lung tissue and alveolar lining fluid. Both MDA and MPO are considered as markers for lung injury. Induction of pro-inflammatory cytokines in premature lung tissue and cells in the airways increases risk for development of bronchopulmonary dysplasia (BPD) and chronic lung disease (CLD). Tumor necrosis factor (TNF)-ct, interleukin (IL)-lp\ 6 and 8 in the lungs are known to amplify inflammatory injury and may also cause extrapulmonary organ dysfunction and failure. Some authors suggested that an inability of preterm infants to counterbalance this inflammation by production of the down-regulatory cytokine IL-10 might also be an important factor. They were rarely able to detect either IL-10 protein or IL-10 mRNA in airway aspirates from ventilated preterm infants with hyaline membrane disease. It may be very important for decreasing the morbility of CLD if we can block the oxidative and inflammatory injury in the early stage of RDS.Objective1. Adding hyaluronan to PS to estimate the improvement and anti-inhibition effects in vitro and then select an optimal composition of PS for in vivo study.2. In the in vivo study we investigated treatment with PS with or without HA to find whether it altered ventilation efficiency, surface activity of recovered surfactant, surfactant proteins, inflammatory indicators, or oxidant protection in preterm neonatal piglets with RDS subjected to mechanical ventilation.Methods1 In vitro study.Measure the minimum surface tension (ymin) of different concentration PS with or without HA or/and meconium or albumin with a pulsating bubble surfactometer.2 In vivo study.2.1 The establishment of RDS model with preterm neonatal piglets.Preterm newborn piglets of various gestation age (GA) were delivered by cesarean section. Analysis of general condition, total phospholipids (TPL) inbronchoalveolar lavage (BALF), lamellar body in type II alveolar epithelial cells (AEC II) under electron microscope and alveolar aeration under microscope were made, all of which revealed evidences characteristic of surfactant deficiency in RDS. Six term newborn piglets were considered as a control for maturity. 2.2 The therapeutic effect of HA-fortified PS on RDS piglets.The normal gestational term for pig is about 114 days. Correspongding to 85% gestation piglets with GA 94-97 days were used in this study.A dose of 5-10 mg/kg ketamine was injected intravenously, the pregnant pig was placed on the operation table and anesthesia was maintained with 1-2 mg/kg ketamine when needed. After a local anesthesia with ledocaine subcostal right-side incision of the pregnant pig was made and the piglet's head was exteriorized. Immediately dried with towels and suctioned and gave oxygen with face-mask. When all the piglets were taken out we transported them to the laboratory by bus (about 45 min). No treatment was made in this period except oxygen supplement with face-mask. The umbilical artery and vein were catheterized for blood sampling and fluid and drug administration. A longitudinal anterior incision was made on the neck, the trachea exposed, and intubated with a tube with ID of 2.5-3 mm then connected to the ventilators. Arterial blood gases were analysed before ventilation as the baseline. All ventilated animals were divided into 5 groups and treated with ventilation alone (Cont), 50 mg/kg Curosurf (C50), 50 mg/kg HA-Surfactant (CH50), 100 mg/kg Curosurf (C100), 100 mg/kg HA-Surfactant (CHI00). An additional control group from the same littermates was served as non-ventilated group (Non). Eight piglets enrolled in each ventilated-group and 12 piglets enrolled in Non group.The initial settings were fraction of inspired oxygen (FiCh) 0.8-1.0, peak inspiratory pressure (PIP) 20-30 cmH2O, positive end expiratory pressure (PEEP) 6-8 cmH20, inspiratory: expiratory ratio (I:E) 1:1.5-2, ventilatory rate (RR) 40-50/min and inspiratory time (Ti) 0.35-0.45 sec. The PIP was changed to maintain a tidal volume (Vj) of 6-8 ml/kg. After ventilation for about 15 minutes, the blood gas and pulmonary function were measured as time zero (0 h) and then PS was instilled. The blood gas and pulmonary function were measured at each hour. During the treatment adnephrin was used when heart rate less than 100/min and sodium bicarbonate (NaHCCh) was used if there was evidence of metabolic acidosis.At the end of the experiment, the piglets were killed with 10% KC1 given intravenously. The abdomen was opened and the diaphragms inspected for signs ofpneumothorax. The lungs and heart were excised. About 1 gram of right mid-lobe were weighed and then dried in oven to get the wet-dry ratio and the remainding right mid-lobe was stored in the liquid nitrogen for analysis of mRNA expression of inflammatory mediators and cytokines related to ALL Four animal lungs were perfused with 4% paraformaldehyde and stored in the same fixative to get the morphology information. Four others of each group were subjected to bronchoalveolar lavage (BAL) for biochemical, biophysical and cytological analyses. BAL was performed with sterile 0.9% NaCl at room temperature delivered into the lungs via intratracheal tube. Each volume of the fluid was washed 3 times and retrieved, and the first volume was 30 ml/kg body weight, and subsequent volumes were adjusted according to the previous ones retrieved. Totally four volumes of fluid were applied (12 times wash). Pooled BAL fluid (BALF) was centrifuged immediately at 1000 rpm for 10 min at room temperature, and its supernatant stored under -20 °C for further analysis of TPL, disaturated phosphatidylcholine (DSPC), total protein (TP). The total number of white blood cell counts (WCC) in sediment of the BALF was determined with a standard hemocytometer.Results1 In vitro study.The concentrations of HA from 1.25% to 20% all improved the physical property of Curosurf and 2.5% was the lowest concentration that had a striking effect to decrease the mean ymin from 15 mN/m to 5 mN/m of 2.5 mg/ml Curosurf. More HA in the PS, lower the ymjn of it. When added 10% HA the Ymin of 2.5 mg/ml Curosurf was near to 0 mN/m.Meconium and albumin both inhibited the property of Curosurf and adding HA resisted or restored the inactivation by them. The anti-inhibition of HA was improved by increasing the concentration of HA or Curosurf itself. The anti-inhibition of HA to meconium was more significant than that to albumin.2 In vivo study.2.1 The establishment of RDS model with preterm neonatal piglets.The gestation of piglets with RDS was about 94 or 97 days. They had evidences of respiratory failure due to surfactant insufficiency: dyspnea and cyanosis shortly after birth; the TPL in BALF was about 10 mg/kg which was much lower than that of the term piglets; little lamellar body in AEC II was seen and most of the alveoli wasatelectatic.2.2 The effect on physiology and histopathology.Baseline values for blood-gas analysis were similar among the 6 groups. At the treatment time 0 h, there were no significant differences among the ventilated-groups in FiC>2 (0.8-1), mean airway pressure (MAP, 12-14 cmfyO), resistance of airway (Raw, 130-150 cmH2O/L.s), dynamic compliance (Cdyn, 0.3-0.5 ml/cmH2O/kg), PaO2/FiC>2 (100 mmHg) and oxygenation index (OI, 20). During the treatment period, mean levels of Vj of each group were maintained within 6-8 ml/kg, mean levels of PaCO2 and pH were kept at 30-50 mmHg and 7.40-7.50, respectively. After treatment with various dose of PS, the FiO2 and MAP were significantly decreased compared to the Cont group. Though there were no significant differences among the four PS-treated groups, CH50, C100 and CHI00 groups had better ventilation efficiency. Mean values of Cdyn were significantly improved from 0.3-0.5 ml/cmH2O/kg to 0.8-1.0 ml/cmH2O/kg in the above three groups compared with the Cont and C50 groups for which values remained at 0.4-0.6 ml/cmH20/kg. At 2 h, there was a significant difference in Cdyn between CHI00 and CH50 or C100 groups (PO.01 and P<0.05 respectively). Sustained improvement in PaCh/FiCh was found in the three surfactant groups after treatment in contrast to the Cont and C50 groups that had OI's of 40 and PaO2/FiO2 less than 100 mmHg (PO.01 versus other groups). In both CH50 and CHI 00 groups, levels of PaO2/FiO2 were significantly higher and the values of OI were significantly lower at 2 and 4 h than their levels at 0 h (P<0.05), respectively, as measured with repeated within-group tests. Similarly, Cdyn was also improved significantly among the 3 PS-treated groups at 2, 4 and 6 h compared to 0 h, but these alteration were not found for Raw. Most PS-treated animals were alive at 6 hour, though the Cont and Non animals died at about 3 and 1 hour, respectively.Most of the alveoli were atelectatic in the lungs of the Non group. In the Cont and C50 group alveolar aeration was modestly improved but irregular. In the other three PS-treated groups, most of the lungs were homogenously aerated, with thin alveolar septa and intact small airways. All ventilated groups had lower W/D than the Non group (p<0.01), and the CHI00 group tended to have the lowest value (p<0.05 versus Cont).2.3 The effect on pulmonary surfactantTPL were about 10-15 mg/kg and 20-25 mg/kg in the Non and Cont groups, respectively. TPL in the 4 PS-treated groups were 3-4 fold higher than those in bothCont and Non groups. TP was about the same in the five ventilated groups, however, in the surfactant treated groups DSPC/TP was 2-3 fold higher than that in the Cont group. Ymjn and Ymaxof TPL from BALF of Non group were around 10 and 35 mN/m, and the 3 PS-treated groups about 7 and 33 mN/m, respectively. Cont group had higher surface tension values than the other groups. There was a trend for enhanced expression of SP-A mRNA in both CH50 and CHI00 groups, and SP-B mRNA was clearly increased in CHI00. 2.4 The protection on oxidative and/or inflammatory lung injury.No significant difference for WCC was found among the ventilated groups, although the CH50 and C50 groups tended to have higher values than the Non group. All five mechanically ventilated groups had higher levels of MPO and MDA compared to the Non group, suggesting mechanical ventilation with high oxygen treatment may have a potential to induce inflammatory and hyperoxic injury in the lungs. The C100 group had a moderate increase of MPO (P<0.01 vs. Non), whereas CHI 00 modestly decreased MDA of lung tissue (PO.05 vs Cont).The mechanical ventilation mildly enhanced the levels of NF-kB in preterm lung tissue at 6 hour after birth. And exogenous PS had a deregulated trend though there was no significant difference among the 6 groups. There were no significant differences among the five ventilated groups for pro-inflammatory cytokine mRNA expression in IL-ip and IL-6. Significantly increased IL-8 mRNA expression in the lung tissue was found in the Cont compared to the Non group. While enhanced expression of IL-8 and TNF-a mRNA were found in C100 and C50 group, CHI00 and CH50 lessened this trend. The expression of IL-10 mRNA was low and there was no difference among the PS-treated and control groups.Conclusions1. Adding HA improved surface property of Curosurf at a low concentration and 2.5 percent concentration was the lowest one that had a significant improvement effect.2. Meconium and albumin both inhibited the property of Curosurf and adding HA resisted or restored the inactivation.3. It exerted that HA anti-inhibition to meconium more significantly than that to albumin.4. HA-PS markedly improved gas exchange and pulmonary mechanics of RDS... |
PDF Full Text Request