Protective Effects Of Erythropoietin On Bronchopulmonary Dysplasia Induced By Hyperoxia In Newborn Rats

IntroductionBronchopulmonary dysplasia(BPD) is a multifactorial disease resulting from the impact of injury(including oxygen toxicity, barotrauma, volutrauma, and infection) on the immature lung. It is one of the most common and significant medical complications associated with preterm birth. Advances in perinatal medicine have resulted in increasing numbers of very low birth weight preterm infants who are at risk of BPD, and the incidence of BPD has increased. The histopathologic changes of severe airway injury and alternating sites of overinflation and fibrosis which used to be seen in older forms of BPD have been replaced by a milder form characterized by alveolar and capillary hypoplasia and variable interstitial cellularity and/or fibroproliferation.Oxygen toxicity is thought to be a major contributing factor in the pathogenesis in BPD. Previous animal studies show that exposure to hyperoxia in the neonatal period caused lung structural changes that are similar to the histology seen in human infants with BPD. The treatment goals of BPD focus on minimizing interstitial fibroproliferation and facilitating lung growth. However, there has not been any improvement in these therapies. Therefore, investigating the effectiveness of new strategies in the treatment of BPD is of great interest.Vascular endothelial growth factor(VEGF) is a potent angiogenic growth factor which has played an important role in the developing lung. Recent studies show that vascular endothelial growth factor gene therapy and recombinant human VEGF promote lung angiogenesis, and prevent alveolar damage in hyperoxia-induced lung injury. It seems possible that by augmenting or restoring vascular growth, overall lung growth and ultimately lung function can be restored.Pulmonary inflammation is a key feature of fibrosis in the pathogenesis of fibrosis in BPD. An imbalance between pro-inflammatory and anti-inflammatory factors might result in lung injury that affects normal aveolarization and pulmonary vascular development in preterm infants with BPD. Gene expressions of monocyte chemoattractant protein-1(MCP-1) and cytokine-induced neutrophil chemoattractant-1(CINC-1) have been assoiated with BPD. Anti-chemokine treatment may prevent neutrophil and macrophage influx in hyperoxia-exposed newborn rat lung.Erythropoietin(EPO) has pleiotropic actions including anti-oxidative, anti-apoptosis, anti-inflammation, and angiogenic effect.Few animal experiments revealed that EPO may have protective effects on hyperoxic lung injury, but the mechanisms remain unknown. So far, the interactive action between EPO and VEGF has not resulted in a conclusion. Researches show EPO may play a protective role in inflammatory reaction, just like the anti-inflammatory effect of steroid.The aim of the study is, therefore, to evaluate the angiogenic and anti-inflammation effects of EPO treatment on the histopathologic characteristics of hyperoxia-induced lung injury in BPD. Moreover, we try to verify the relationship between EPO and VEGF, and the effects of EPO on expressions of MCP-1 and CINC-1 in BPD model induced by hyperoxia.Material and Methods1. Animal modelsSeveral litters of Wistar pups were pooled together within 12 hours after birth and randomly divided into four groups:â… . air-exposed group,â…¡. air-exposed recombinant human erythropoietin(rhEPO)-treated group,â…¢. hyperoxia-exposed group andâ…£. hyperoxia-exposed rhEPO-treated group. Rats in groupâ…¢andâ…£were placed in an oxygen chamber into which oxygen was continuously delivered(FiO₂= 0.85±0.02)(OM-25ME oxygen monitor, USA). CO₂ were kept below 0.5%(Dapex Gas monitor, USA). Groupâ…¡andâ…£received 1200 IU/kg rhEPO subcutaneously at the lower back on postnatal days 0 and 2. Groupâ… andâ…¢received 0.9% saline in the same way. Temperature and humidity were maintained at 22～25℃and 60～70%, respectively. The chamber was opened for <15min daily to switch dams between air and O₂ environment to avoid the dams from oxygen toxicity.2. Preparation of Lung SamplesPups from each group were killed on days 3,7, and 14, and a tracheal cannula was placed. An abdominal incision was made, the diaphragm was punctured carefully to collapse the lungs. Left lung about 1cm³ was fixed in 2.5 glutaral for ultrastructure observation. Right lung was inflation-fixed via tracheal cannula using 10% neutral formaldehyde solution for morphology observation and immunohistochemistry study. In some cases, after midline thoracotomy, blood was collected from right ventricle for blood cell count, then the pulmonary artery was cannulated and the left atrial appendage was clipped and the lungs were gently perfused with 10 ml of 0.9% saline to remove blood. Then put lungs in RNase-free Eppendorf tubes and stored at-80℃freezer for RT-PCR,Western blotting or biolchemistry analysis. In some animals, both lungs were slowly lavaged via the tracheal cannula four times with 0.5 ml 0.9% saline and bronchoalveolar lavage fluid(BALF) was collected and stored at-80℃freezer for biolchemistry analysis.3. Experimental methods3.1 The appearance and weight were monitored everyday, life table was used for survival analysis.3.2 Red blood cell count, hemoglobin concentration, hematocrit and platelet count were determined automatically by blood cell analyzer.3.3 Morphology observation: histological study, radical alveolar counts(RAC), Masson staining and observation of ultrastructure by transmission electron microscope.3.4 Immunohistochemistry: measurement the expression levels of platelet endothelial cell adhensive-1(PECAM-1), erythropoietin receptor(EPOR), VEGF and MCP-1.3.5 Biochemistry assay: detection of total protein content in BALF and myeloperoxidase(MPO) in lung tissue and BALF.3.6 Western blotting: measurement of PECAM-1, EPOR, VEGF and MCP-1 protein expression3.7 RT-PCR: measurement of EPOR, MCP-1 and CINC-1 mRNA expression.4. Statistical analysisSPSS 13.0 statistical software was used to perform statistical analysis. All dada were expressed as((?)±s). Life table was used for survival analysis and survival rate was compared by Wilconxon(Gehan) test. Significant differences in the mean values among multiple groups were analyzed by one-way ANOVA. SNK and LSD were used to determine significance between two groups. Correlation between two variables was analyzed with Spearman analysis. Statistical significance was achieved at P<0.05.Results1. General status and survival curveIn groupâ…¢, dyspnea and cyanosis appeared on days 7～10 and became more severe after detached from oxygen champer on days 10～14. Those signs can be relieved gradually when they were put into the chamber again. The rats in groupâ…¢showed a significant decrease in body weight gain on day 7 and the difference was more evident on day 14(P<0.01, P<0.001). Body weights of groupâ…£were not different from those of groupâ… . The survival rate of groupâ…£was higher than those of groupâ…¢.2. Changes of red blood cell, hemoglobin concentration, hematocrit and plateletIn groupâ…¡, red blood cell counts, hemoglobin concentration and hematocrit became higher on day 7 and 14(p<0.05, p<0.01, p<0.001) compared with groupâ… . In groupâ…¢, they were significantly lower on day 14 than those of groupâ… (p<0.01, P<0.001). Red blood cell counts, hemoglobin concentration and hematocrit were significantly higher in groupâ…£compared with groupâ…¢(p<0.01, p<0.05). There was no difference in platelet counts between four groups.3. Lung morphology3.1 Changes of lung pathologyIn groupâ… , the size of alveolus was equal and alveolar septum was thinner on day 14. In groupâ…¡, capillary proliferation was obvious on day 14. In groupâ…¢, there were a few inflammatory cells infiltration in interstitial on day 3. Inflammatory response worsen on day 7, and hemorrhage and exudation could be seen. On day 14 of groupâ…¢, alveolar numbers became more fewer and terminal air space became even much larger compared with those of groupâ… and interstitial fibrosis was evident. The pathological changes were ameliorated greatly in groupâ…£.3.2 Changes of RACRAC decreased from day 7 in groupâ…¢compared with groupâ… (P<0.05), which was more evident on day 14(P<0.001). There were no difference in RAC on day 7 between groupâ…£and groupâ… , RAC increased in groupâ…£on day 14 compared with groupâ…¢, but still were lower than that of groupâ… (P<0.001).3.3 Masson stainingLungs in groupâ… andâ…¡had thin alveolar septa and minimal collagen staining. Red blood cells are noted in subepithelial capillaries. Lungs in groupâ…¢had fine strands or thick bundles of collagen in the thick alveolar septa on day 14. There was no apparent more collagen staining in groupâ…£.3.4 Changes of ultrastructureCapillary inflammation occurred on day 3 in groupâ…¢, and there were increased thickness and rupture on the basement membrane of capillaries. On day 7, inflammation was much more prominent in groupâ…¢. Capillaries were occluded by inflammation cells and necrosis endothelial cells, and the number of capillaries reduced. Apoptosis body was evident in alveolar epithelia typeâ…¡cells. On day 14, capillaries became rare and collagen fibers deposited in interstitum in groupâ…¢. Blood-gas barrier thickened and necrosis could be seen in alveolar epithelia typeâ…¡cells on day 14 in groupâ…¢. There were more capillaries in groupâ…£than in groupâ…¢. There were enlarged alveolar epithelia typeâ… cells, shrunk alveolar epithelia typeâ…¡cells and proliferated fibroblasts on day 14 in groupâ…£.4. Changes of total protein content in BALF and MPO in lung tissue and BALFIn groupâ…¢, the total protein content of BALF increased on day 3 and reached peak on day 7(P<0.001) compared with groupâ… which was greatly depressed in groupâ…£(P<0. 05, P<0.001). Changes of MPO in lung tissue and BALF were similar to that of protein content. The MPO content in BALF was positively related to the MPO of lung covering all groups on days 3, 7 and 14, respectively(r=0.561, p<0.001; r=0.882, p<0.001; r=0.656, p<0.001).5. Changes of PECAM-1,EPOR,VEGF and MCP-1 protein expressionExpression of PECAM-1 protein in groupâ…¡was slightly increased compared with groupâ… on day 14(P<0. 05).Expression of PECAM-1 protein in groupâ…¢decreased on day 7 and 14(P<0.001) compared with groupâ… which was relieved to some degree in groupâ…£(P<0.001). Expression of EPOR protein in groupâ…¢started to decrease on day 3(P<0. 05) and became more evident on day 7 and day 14 compared with groupâ… (P<0.001). This reduction could be attenuated in groupâ…£(P<0.001). Expression of VEGF protein in groupâ…¢was depressed on day 7 and 14 compared with groupâ… (P<0. 05, P<0.001) but was increased partly in groupâ…£(P<0. 05, P<0.001). Expression of MCP-1 protein increased in groupâ…¢compared with groupâ… especially on day 7(P<0.001) which was relieved significantly in groupâ…£ (P<0.001).6. Changes of EPOR,MCP-1 and CINC-1 mRNA expressionThe changes of EPOR and MCP-1 mRNA expression were in accordance with their protein changes. MCP-1 and CINC-1 mRNA expression was much higher on day 7 in groupâ…¢(P<0.001) compared with all groups which was relieved significantly in groupâ…£(P<0. 05, P<0. 01, P<0.001). The lung MPO content was positively related to the MCP-1 and CINC-1 mRNA expression covering all groups on days 3, 7 and 14, respectively(r=0.628, P<0.001; r=0.841, P<0.001; r=0.72, P<0.001)(r=0.723, P<0.001; r=0.884, P<0.001; r=0.697, P<0.001).Conclusions1.The histopathological changes of lung injury induced by hyperoxia(FiO₂=0.85) are similar to the characteristics of BPD in preterm infants.2. EPO treatment prevents the negative effects of hyperoxia resulting in improved development of alvelarization and angiogenesis.3. Upregulation of EPOR expression by EPO treatment may be associated with the vascular remodeling in hyperoxic induced lung injury.4. Depressed expression of VEGF play a pivotal role in the pathogenesis of BPD. The protective effect of EPO on BPD may be the interaction between EPO and VEGF.5. MPO content in BALF may be a marker that reflects the early inflammation injury. EPO could attenuate inflammation response of BPD at early stage.6. The expressions of MCP-1 and CINC-1 regulate the neutrophil and macrophage influx into the lungs of BPD at inflammtion process. EPO could down-regulate the expression of MCP-1 and CINC-1 to prevent hyperoxic lung injury.