Dose-dependent exacerbation of ventilation-induced lung injury by erythropoietin in preterm newborn lambs.

Erythropoietin (EPO) is being trialled in preterm infants to reduce brain injury, but high doses increase lung injury in ventilated preterm lambs. We aimed to determine whether early administration of lower doses of EPO could reduce ventilation-induced lung injury and systemic inflammation in preterm lambs. Ventilation was initiated in anaesthetized preterm lambs [125 ± 1 (SD) days gestation] using an injurious strategy for the first 15 min. Lambs were subsequently ventilated with a protective strategy for a total of 2 h. Lambs were randomized to receive either intravenous saline (Vent; n = 7) or intravenous 300 ( n = 5), 1,000 (EPO1000; n = 5), or 3,000 (EPO3000; n = 5) IU/kg of human recombinant EPO via an umbilical vein. Lung tissue was collected for molecular and histological assessment of inflammation and injury and compared with unventilated control lambs (UVC; n = 8). All ventilated groups had similar blood gas and ventilation parameters, but EPO1000 lambs had a lower fraction of inspired oxygen requirement and lower alveolar-arterial difference in oxygen. Vent and EPO lambs had increased lung interleukin (IL)-1ß, IL-6, and IL-8 mRNA, early lung injury genes connective tissue growth factor, early growth response protein 1, and cysteine-rich 61, and liver serum amyloid A3 mRNA compared with UVCs; no difference was observed between Vent and EPO groups. Histological lung injury was increased in Vent and EPO groups compared with UVCs, but EPO3000 lambs had increased lung injury scores compared with VENT only. Early low-doses of EPO do not exacerbate ventilation-induced lung inflammation and injury and do not provide any short-term respiratory benefit. High doses (≥3,000 IU/kg) likely exacerbate lung inflammation and injury in ventilated preterm lambs. NEW & NOTEWORTHY Trials are ongoing to assess the efficacy of erythropoietin (EPO) to provide neuroprotection for preterm infants. However, high doses of EPO increase ventilation-induced lung injury (VILI) in preterm lambs. We investigated whether early lower doses of EPO may reduce VILI. We found that lower doses did not reduce, but did not increase, VILI, while high doses (≥3,000 IU/kg) increase VILI. Therefore, lower doses of EPO should be used in preterm infants, particularly those receiving respiratory support.

Unbiased analysis, enrichment and purification of thymic stromal cells.

The microenvironment of the thymus consists of functionally discrete niches composed of distinct stromal cell subsets. Clinically relevant changes affecting T-cell differentiation occur within these niches with age and injury caused by irradiation and chemotherapy treatments. The study of thymic stromal cells has been hampered by the technical difficulty in isolating significant numbers of this important population. Here we present an improved protocol for enzymatic isolation of stromal cells that enables comparative flow cytometric analyses and their purification for downstream cellular or molecular analysis. Fractions analyzed throughout enzymatic digestion of the thymus revealed that various stromal subsets are isolated at characteristic intervals. This highlights the importance of pooling all cells isolated from the thymus for numerical and phenotypic analysis to avoid biased representation of subpopulations. We also describe refined magnetic bead separation techniques that yield almost pure preparations of CD45(-) stroma. Sorting of these suspensions using defined markers enabled purification of the major epithelial subsets, confirmed by keratin staining and PCR analysis. This three-step procedure represents a rapid, reproducible method for the unbiased purification of the stromal cells that direct thymic T-cell differentiation.

Effects of antenatal melatonin therapy on lung structure in growth-restricted newborn lambs.

Oxidative stress arising from suboptimal placental function contributes to a multitude of pathologies in infants compromised by fetal growth restriction (FGR). FGR infants are at high risk for respiratory dysfunction after birth and poor long-term lung function. Our objective was to investigate the contribution of oxidative stress to adverse lung development and the effects of melatonin administration, a powerful antioxidant, on lung structure in FGR lambs. Placental insufficiency and FGR was surgically induced in 13 fetal sheep at ∼105 days of gestation by ligation of a single umbilical artery. Maternal intravenous melatonin infusion was commenced in seven of the ewes 4 h after surgery and continued until birth. Lambs delivered normally at term and lungs were collected 24 h after birth for histological assessment of lung structure and injury and compared with appropriately grown control lambs (n = 8). FGR fetuses were hypoxic and had lower glucose during gestation compared with controls. Melatonin administration prevented chronic hypoxia. Within the lung, FGR caused reduced secondary septal crest density and altered elastin deposition compared with controls. Melatonin administration had no effect on the changes to lung structure induced by FGR. We conclude that chronic FGR disrupts septation of the developing alveoli, which is not altered by melatonin administration. These findings suggest that oxidative stress is not the mechanism driving altered lung structure in FGR neonates. Melatonin administration did not prevent disrupted airway development but also had no apparent adverse effects on fetal lung development.NEW & NOTEWORTHY Fetal growth restriction (FGR) results in poor respiratory outcomes, which may be caused by oxidation in utero. We investigated the contribution of oxidative stress to adverse lung development and the effects of melatonin administration, a powerful antioxidant, on lung structure in FGR lambs. FGR disrupted septation of the developing alveoli, which is not altered by melatonin administration. Oxidative stress may not be the mechanism driving altered lung structure in FGR neonates.

Exacerbation of Ventilation-Induced Lung Injury and Inflammation in Preterm Lambs by High-Dose Nanoparticles.

Mechanical ventilation of preterm neonates causes lung inflammation and injury, with potential life-long consequences. Inert 50-nm polystyrene nanoparticles (PS50G) reduce allergic inflammation in the lungs of adult mice. We aimed to confirm the anti-inflammatory effects of PS50G in a sheep asthma model, and investigate the effects of prophylactic administration of PS50G on ventilation-induced lung injury (VILI) in preterm lambs. We assessed lung inflammatory cell infiltration, with and without PS50G, after airway allergen challenge in ewes sensitised to house dust mite. Preterm lambs (0.83 gestation) were delivered by caesarean section for immediate tissue collection (n = 5) or ventilation either with (n = 6) or without (n = 5) prophylactic intra-tracheal administration of PS50G nanoparticles (3% in 2 ml). Ventilation was continued for a total of 2 h before tissue collection for histological and biomolecular assessment of lung injury and inflammation. In ewes with experimental asthma, PS50G decreased eosinophilic infiltration of the lungs. Ventilated preterm lambs showed molecular and histological signs of lung injury and inflammation, which were exacerbated in lambs that received PSG50G. PS50G treatment decreased established inflammation in the lungs of asthmatic sheep. However, prophylactic administration of PSG50 exacerbated ventilation-induced lung injury and lung inflammation in preterm lambs.

Diffusion tensor imaging detects ventilation-induced brain injury in preterm lambs.

PURPOSE: Injurious mechanical ventilation causes white matter (WM) injury in preterm infants through inflammatory and haemodynamic pathways. The relative contribution of each of these pathways is not known. We hypothesised that in vivo magnetic resonance imaging (MRI) can detect WM brain injury resulting from mechanical ventilation 24 h after preterm delivery. Further we hypothesised that the combination of inflammatory and haemodynamic pathways, induced by umbilical cord occlusion (UCO) increases brain injury at 24 h. METHODS: Fetuses at 124±2 days gestation were exposed, instrumented and either ventilated for 15 min using a high tidal-volume (VT) injurious strategy with the umbilical cord intact (INJ; inflammatory pathway only), or occluded (INJ+UCO; inflammatory and haemodynamic pathway). The ventilation groups were compared to lambs that underwent surgery but were not ventilated (Sham), and lambs that did not undergo surgery (unoperated control; Cont). Fetuses were placed back in utero after the 15 min intervention and ewes recovered. Twenty-four hours later, lambs were delivered, placed on a protective ventilation strategy, and underwent MRI of the brain using structural, diffusion tensor imaging (DTI) and magnetic resonance spectroscopy (MRS) techniques. RESULTS: Absolute MRS concentrations of creatine and choline were significantly decreased in INJ+UCO compared to Cont lambs (P = 0.03, P = 0.009, respectively); no significant differences were detected between the INJ or Sham groups and the Cont group. Axial diffusivities in the internal capsule and frontal WM were lower in INJ and INJ+UCO compared to Cont lambs (P = 0.05, P = 0.04, respectively). Lambs in the INJ and INJ+UCO groups had lower mean diffusivities in the frontal WM compared to Cont group (P = 0.04). DTI colour mapping revealed lower diffusivity in specific WM regions in the Sham, INJ, and INJ+UCO groups compared to the Cont group, but the differences did not reach significance. INJ+UCO lambs more likely to exhibit lower WM diffusivity than INJ lambs. CONCLUSIONS: Twenty-four hours after injurious ventilation, DTI and MRS showed increased brain injury in the injuriously ventilated lambs compared to controls. DTI colour mapping threshold approach provides evidence that the haemodynamic and inflammatory pathways have additive effects on the progression of brain injury compared to the inflammatory pathway alone.