Research Of Lung Injury And Alveolization Impairment Meidiated By NMDA Receptor Activation In Newborn Rat After Hyperoxia Exposure And The Related Mechanisms

BackgroundOxygen therapy is an important measure to improve the state of hypoxia.But prolonged inhalation of high concentrations of oxygen (hyperoxia) will lead to varying degrees of acute and chronic lung injury. Neonatal period is alveolar mature critical period,long-term inhalation of high concentrations of oxygen could lead to diffuse alveolar damage associated with infiltration of inflammatory cells characterized by acute lung injury and alveolization impairment and fibrosis mainly characterized chronic lung injury.Glutamate is one of the major excitatory neurotransmitters and is abundantly present in the mammalian central nervous system(CNS).It plays key roles in brain development,learning and memory,and synaptic plasticity.On the other hand,glutamate may also be lethal to neurons, through over-activation of N-methyl-D-aspartate receptors (NMDAR).This results in an influx of a large quantity of calcium,which triggers a series of toxic events and ultimately leads to cell death. Over-stimulation of NMDA receptors can lead to neuronal cell death under many acute and chronic conditions,and has been found responsible for neural loss in ischemia,epilepsy,Parkinson's disease,Alzheimer's disease,Huntington's chorea,and AIDS encephalopathy.The earlier stage of our examination had found that hyperoxia can induce a high level of the releasing of Glu and the expressing of NMDAR, and MK-801 could decrease the hyperoxia-associated lung injury.For the first time,we conclude that Glu may play an important role in hyperoxia-induced lung injury by activation of NMDA receptor. However,it remains indistinct of the NMDA receptor effects on the alveolar development and lung collagen deposition after hyperoxic exposure,and also unclear that the relationship between NMDA receptor and lung fibroblasts.Our investigation could provide a new way to solve this contradictive clinical trouble.Chapterâ… Effect of Different Prolong Hyperoxia Exposure Days on Newborn RatObjectiveTo establish more reasonable animal model of CLD in newborn rats by comparing the effect of different prolong hyperoxia exposure days on hyperoxia-induced lung injury.MethodsThe 22-day gestation full term rats in 2-4 hours after birth were randomly marked with a diferent number and assigned to three groups:air control group,hyperoxia group with 95%O₂ for 7 days,followed by 60%O₂ 14 days and hyperoxia group with 95%O₂ for 3 days,followed by air condition 14 days.Postnatal 3,7,14 and 21 days,the neonatal rats were sacrificed with an overdose of intra-peritoneal pentobarbital,and exsanguinated by severing the abdominal aorta.The counts of inflammatory cell,the content of protein,LDH in bronchoalveolar lavage fluid(BALF);body weight,lung weight,lung dry weight,lung index,the ratio of lung wet weight to lung dry weight(W/D),the content of hydroxyproline in the Lung homogenates and histological changes, including staining of HE,and radical alveolar count(RAC) were measured respectively.All data were expressed as means±SD and analyzed by one-way ANOVA and LSD-t test with SPSS 13.0 statistical software.Values at P＜0.05 were considered statistical significant.Result1.Lung index,lung W/D and BALF analyse:After having 3days of exposure,the hyperoxia group has been saw a significant increase in lung W/D and BALF inflammatory cell counts,protein and LDH.If stopping hyperoxia exposure at this time,above-mentioned index decline to normal except W/D and lung index,and no obvious change in the next days.After having 7 days of exposure,above-mentioned index further increase compared with 3^rd day and air control group. Postnatal 14 day,they have declined and lower than the 7^th day,but still higher than air control group.2.The content of HYP in lung homogenates have increased after 7days of exposure compared with the air control group,with the days of exposure increasing,the content continue increased and much higher than air control group.If stopping hyperoxia exposure at postnatal 3 day,it does not increase significantly until postnatal 14 day,and going further at 21 day.3.Lung histology and RAC:The hyperoxia group showed dilated and congestive capillary vessels,erythrocyte extravasation,and leukocyte infiltration in alveolar,pulmonary interstitial inflammation cell infiltration and a little collagen deposition.The pathologic changes were more and more severe with days of exposure increasing that broadened pulmonary interstitial tissue and disruption of the alveolar structure were saw in the lung tissures.At the same time,RAC has decreased dramatically in the hyperoxia group.If stopping hyperoxia exposure at postnatal 3 day,pulmonary alveolus inflammation cell infiltrations disappear at 7 day.At 14 and 21 day,fibroelastosis and fewer RAC were found compared with the control group.Conclusion1.High concentration of≥95%oxygen can cause acute inflammatory lung injury in newborn rats after 3 days exposure,and aggravated with the prolong of exposure days.Lung injury change from early alveolitis acute damage to the alveolization impairment and lung tissue fibrosis with a lot of collagen deposition.2.3 days of hyperoxia exposure can induced CLD characteristiced by alveolization impairment,lung collagen deposition and fibroelastosis. Chapterâ…¡Effect of MK-801on 3 Days Hyperoxia Induced Alveolization Impairment and Lung Injury inNewborn Rat at Different Stages of Hyperoxia ExposureOjectiveTo research the direct role of NMDAR in hyperoxia induced BPD by comparing the effect of applications of MK-801 at different times.MethodsThe 22-day gestation full term rats in 2-4 hours after birth were randomly marked with a diferent number and assigned to six groups:air control group,air +MK-801 group with injection MK-801 at postnatal 1-3 days, air +MK-801 group with injection MK-801 at postnatal 8-10 days, hyperoxia group with 95%O₂ for 3 days,hyperoxia +MK-801 group with injection MK-801 at postnatal 1-3 days and hyperoxia +MK-801 group with injection MK-801 at postnatal 8-10-3 days.At postnatal 3,7,14 and 21 days,the neonatal rats were sacrificed with an overdose of intra-peritoneal pentobarbital,and exsanguinated by severing the abdominal aorta.The counts of inflammatory cell,the content of protein, LDH in bronchoalveolar lavage fluid(BALF);body weight,lung weight, lung dry weight,lung index,the ratio of lung wet weight to lung dry weight(W/D),the content of hydroxyproline in the Lung homogenates and histological changes,including staining of HE,and radical alveolar count(RAC) were measured respectively.Pulmonary respiratory function was measure too at 21st day.All data were expressed as means±SD and analyzed by one-way ANOVA and LSD-t test with SPSS 13.0 statistical software.Values at P＜0.05 were considered statistical significant.Result1.Application of MK-801 in hyperoxia exposure days,W/D and BALF inflammatory cell counts,protein and LDH were significantly decreased at postnatal 3 day.HYP and RAC were lower than air control at postnatal 14 and 21 day.Cdyn was declined compared with air control at postnatal 21 day.The degree of pulmonary fibrosis and alveolization impairment were reduced.2.Application of MK-801 in post hyperoxia exposure,HYP,Cdyn and RAC were lower than air control at postnatal 21 day although their higher than use of MK-801 in hyperoxia exposure days.The degree of pulmonary fibrosis and alveolization impairment were also reduced.ConclusionThe role of NMDAR on hyperoxia-induced CLD is not only the inhibition of early cell injury and inflammation,but also the direct relationship between the NMDAR and the progress of chronic lung injury. Which suggest that NMDAR may immediacy take part in the alveolar development of newborn rat and fibroelastosis induced by hyperoxia. Chapterâ…¢Collagen Secretion and Self-degradation Mediated by NMDR Activation in Human Fetal Lung FibroblastsObjectiveTo detect the expression of NMDA receptors in human fetal lung fibroblasts,and observe the role of NMDAR in the secretion and self-degradation of collagen.Methods1.NMDAR1 and NR2D expression was analyzed by Immunohistochemistry and immunofluorescence,2.Expression of NMDAR1 and four NR2 subunit(NR2A,NR2B,NR2C and N2D) mRNA were examined by real-time PCR.3.Cell proliferations were measured by cell counting and MTT in different concentrations of glutamate or NMDA.4.Detection of HYP in cell supernatant at different concentrations of glutamate or NMDA.5.â… ,â…¢collagen and MMP-1,TIMP-1 secretion in cell supematant were measured by ELISA.Result1.Immunohistochemistry and immunofluorescence indicated the expression of NR1 and NR2D. 2.real time PCR detected the expression of NR1 and four NR2D subtypes(A,B,C and D) mRNA,the strongest two subtypes are NR2A and NR2D,followed by N2C,N2B and NAR1.3.1mM glutamate and NMDA can promote cell proliferation,1mM glutamate was significantly higher than 1mM NMDA group,10mM glutamate group inhibit the proliferation.4.1mM glutamate and NMDA can promote the secretion of HYP,1mM NMDA was significantly higher than 1mM glutamate group,MK-801 can inhibit the NMDA or glutamate-induced increase in HYP.5.1mM NMDA induced secretion increase inâ… /â…¢collagen and TIMP-1, secretion of MMP-1 have no significant effect.0.5mM MK-801 could significantly inhibit the NMDA-induced increase ofâ… /â…¢collagen and TIMP- 1.Conclusion1.We found Glutamate can promote human fetal lung fibroblasts proliferation for the first time.2.NR1 and four NR2D subtypes(A,B,C and D) all express in human fetal lung fibroblasts.3.NMDA can promote the secretion of collagen,and inhibit collagen self-degradation in human fetal lung fibroblasts. Chapterâ…£Cell Signaling Pathways of NMDA-mediated Collagen Secretion and Self-degradation in Human Fetal Lung FibroblastsObjective To study the cell signaling pathways in human fetal lung fibroblast collagen secretion and self-degradation though NMDAR.Methods1.Measurement of phosphorylated ERK by Western Blot in human fetal lung fibroblasts2.Detection ofâ… /â…¢collagen,MMP1/TIMP1 by ELISA after inhibiting NMDAR,ERK phosphorylation and PKC.3.Change of expression for NMDAR1 and four NR2 subunits(NR2A, NR2B,NR2C and N2D ) rnRNA after being induced by NMDA were examined by real-time PCR.Result1.NMDA can enhance ERK phosphorylation;MK-801 can reduce NMDA-induced phosphorylation of ERK.Phosphorylated ERK inhibitor U0126 significantly reduced ERK phosphorylation in normal cells,and blocks the increase of NMDA-induced phosphorylation of ERK.PKC inhibitor H7 show a significant reduction in the normal cells,and block the increase of NMDA-induced phosphorylation of ERK. 2.NMDA induced the increase ofâ… /â…¢collagen and TIMP-1 secretion were significantly inhibited by ERK inhibitor U0126 and PKC inhibitor H73.NR1 no signifcant change in expression after application of NMDA. NR2D expression markedly increased more than six-fold,NR2B and NR2C expression was signifcantly enhanced compared with the control group.NR2A expression decreased significantly.Conclusion.1.The secretion and self-degradation of collagen induced by NMDA in human fetal lung fibroblasts was through NMDA-mediated ERK phosphorylation.2.NMDAR-PKC-ERK_1/2 pathway was one of the cell signaling pathways in NMDA-mediated collagen secretion and self degradation of human fetal lung fibroblasts.3.NR2D may play an important role in NMDAR-mediated collagen secretion and self-degradation in human fetal lung fibroblasts.