Dedja, Arben (2012) Administration of L-citrulline in an animal model of perinatal lung damage. [Tesi di dottorato]
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Moderate hyperoxia and induced chorioamnionitis by intrauterine administration of endotoxin LPS into near-term pregnant rats cause alveolar and vascular lung derangement in the newborns. Endogenous nitric oxide (NO), which promotes lung growth, is produced from the metabolism of L-arginine to L-citrulline in endothelial cells. We investigated whether administering L-citrulline by raising the serum levels of L-arginine and enhancing NO endogenous synthesis, attenuates lung injury in a chorioamnionitis and/or moderate hyperoxia-induced model. Material and Methods. Newborn rats (receiving or not intrauterine LPS) were exposed to FiO2=0.6 or room air till 14 days after birth and were administered L-citrulline. Serum and lung tissues were collected for further analysis. The lung sections were subsequently stained with H&E and photomicrographs were obtained at 10X magnification. For vessel density assessment, sections were stained to reveal the presence of von Willebrand Factor Antigen. VEGF and eNOS protein expression was examined by Western blot. High performance liquid chromatography-mass spectrometry was used for simultaneous determination and quantification of ADMA, SDMA, L-arginine, L-citrulline, NMMA and homo-arginine in the serum. Results. The lung histopathology analysis of the Hyperoxia group showed a pattern typically emphysematous, similar to the LPS exposure group, when compared to controls. Exposure to hyperoxia was associated with an arrested alveolarization, inducing a change in lung morphology with patchy areas of parenchymal thickening interspersed with areas of enlarged air spaces. The lung sections of the CITR+hyperoxia and LPS+CITR rats contained smaller and more numerous air spaces, and were more similar to the control lungs. The mean alveolar size was higher in Hyperoxia group vs. controls, or LPS+CITR, in a post hoc comparison unchanged with respect to CITR+hyperoxia, or LPS, or CITR. The secondary crests were higher in the Control and CITR+hyperoxia and LPS+CITR groups than in the Hyperoxia only, or LPS only groups. VEGF gene expression evaluated by real-time quantitative PCR was lower in the Hyperoxia group, than in the CITR+hyperoxia or Control groups. Also, lung sections from Control and CITR+hyperoxia animals showed a similar vWF expression, whereas staining was weaker in the Hyperoxia group. In the CITR+hyperoxia sections there was also evidence of a better organization of the vessel network than in animals exposed to hyperoxia. The amount of eNOS protein normalized in the lung tissue from the L-citrulline treated animals was higher than in the tissues from the Hyperoxia group. Serum assessment with mass spectrometry did not show major differences in the time course and treatment groups. Conclusions. Our main findings were that: (i) administering L-citrulline proved effective in improving alveolar growth after oxygen-induced and antenatal endotoxin exposure lung damage; (ii) VEGF gene and protein were over-expressed in the group treated with L-citrulline. There may have been further protective effects on the alveolar vascular network and, consequently, on matrix maturation in our model and this may be promising with a view to BPD prevention strategies.
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La corioamnionite indotta dalla somministrazione intrauterina dell’endotossina LPS e da una moderata iperossia nei primi giorni di vita causano uno squilibrio alveolare e vascolare del polmone nel ratto neonato. L’ossido nitrico (NO) endogeno, che promuove la crescita polmonare, viene prodotto nelle cellule endoteliali dal metabolismo del L-arginina verso il suo prodotto, la L-citrullina. Abbiamo studiato l’efficacia della somministrazione di L-citrullina in un modello di danno indotto da corioamnionite e/o da iperossia nei ratti neonati nell’attenuare il danno polmonare intervenendo sulla sintesi del NO endogeno aumentando i livelli di L-arginina.
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