Early administration of umbilical cord blood cells following brief high tidal volume ventilation in preterm sheep: a cautionary tale.

BACKGROUND: Umbilical cord blood (UCB) cells are a promising treatment for preterm brain injury. Access to allogeneic sources of UCB cells offer the potential for early administration to optimise their therapeutic capacities. As preterm infants often require ventilatory support, which can contribute to preterm brain injury, we investigated the efficacy of early UCB cell administration following ventilation to reduce white matter inflammation and injury. METHODS: Preterm fetal sheep (0.85 gestation) were randomly allocated to no ventilation (SHAM; n = 5) or 15 min ex utero high tidal volume ventilation. One hour following ventilation, fetuses were randomly allocated to i.v. administration of saline (VENT; n = 7) or allogeneic term-derived UCB cells (24.5 ± 5.0 million cells/kg; VENT + UCB; n = 7). Twenty-four hours after ventilation, lambs were delivered for magnetic resonance imaging and post-mortem brain tissue collected. Arterial plasma was collected throughout the experiment for cytokine analyses. To further investigate the results from the in vivo study, mononuclear cells (MNCs) isolated from human UCB were subjected to in vitro cytokine-spiked culture medium (TNFα and/or IFNγ; 10 ng/mL; n = 3/group) for 16 h then supernatant and cells collected for protein and mRNA assessments respectively. RESULTS: In VENT + UCB lambs, systemic IFNγ levels increased and by 24 h, there was white matter neuroglial activation, vascular damage, reduced oligodendrocytes, and increased average, radial and mean diffusivity compared to VENT and SHAM. No evidence of white matter inflammation or injury was present in VENT lambs, except for mRNA downregulation of OCLN and CLDN1 compared to SHAM. In vitro, MNCs subjected to TNFα and/or IFNγ displayed both pro- and anti-inflammatory characteristics indicated by changes in cytokine (IL-18 & IL-10) and growth factor (BDNF & VEGF) gene and protein expression compared to controls. CONCLUSIONS: UCB cells administered early after brief high tidal volume ventilation in preterm fetal sheep causes white matter injury, and the mechanisms underlying these changes are likely dysregulated responses of the UCB cells to the degree of injury/inflammation already present. If immunomodulatory therapies such as UCB cells are to become a therapeutic strategy for preterm brain injury, especially after ventilation, our study suggests that the inflammatory state of the preterm infant should be considered when timing UCB cells administration.

Influence of the chest wall on respiratory function at birth in near-term lambs.

Airway liquid is cleared into lung tissue after birth, which becomes edematous and forces the chest wall to expand to accommodate both the cleared liquid and incoming air. This study investigated how changing chest wall mechanics affects respiratory function after birth in near-term lambs with different airway liquid volumes. Surgically instrumented near-term lambs (139 ± 2 days) were randomized into Control (n = 7) or Elevated Liquid (EL; n = 6) groups. Control lambs had lung liquid drained to simulate expected volumes following vaginal delivery. EL lambs had airway liquid drained and 30 mL/kg liquid returned to simulate expected airway liquid volumes after elective cesarean section. Lambs were delivered, transferred to a Perspex box, and ventilated (30 min). Pressure in the box was adjusted to apply positive (7-8 cmH2O above atmospheric pressure) or negative (7-8 cmH2O below atmospheric pressure) pressures for 30 min before pressures were reversed. External negative pressures expanded the chest wall, reduced chest wall compliance (CCW) and increased lung compliance (CL) in Control and EL lambs. External positive pressures compressed the chest wall, increased CCW and reduced CL in Control and EL lambs. External negative pressure improved pulmonary oxygen exchange, reducing the alveolar-arterial difference in oxygen (AaDO2) by 69 mmHg (95% CI [13, 125]; P = 0.016) in Control lambs and by 300 mmHg (95% CI [233, 367]; P < 0.001) in EL lambs. In contrast, external positive pressures impaired pulmonary gas exchange, increasing the AaDO2 by 179 mmHg (95% CI [73, 285]; P = 0.002) in Control and by 215 mmHg (95% CI [89, 343]; P < 0.001) in EL lambs. The application of external thoracic pressures influences respiratory function after birth.NEW & NOTEWORTHY This study investigated how changes in chest wall mechanics influence respiratory function after birth. Our data indicate that the application of continuous external subatmospheric pressure greatly improves respiratory function in near-term lambs with respiratory distress, whereas external positive pressures impair respiratory function. Our findings indicate that, during neonatal resuscitation at birth, the forces applied to the chest wall should not be ignored as they can have a major impact on neonatal respiratory function.

In utero ventilation induces lung parenchymal and vascular alterations in extremely preterm fetal sheep.

Extremely preterm infants are often exposed to long durations of mechanical ventilation to facilitate gas exchange, resulting in ventilation-induced lung injury (VILI). New lung protective strategies utilizing noninvasive ventilation or low tidal volumes are now common but have not reduced rates of bronchopulmonary dysplasia. We aimed to determine the effect of 24 h of low tidal volume ventilation on the immature lung by ventilating preterm fetal sheep in utero. Preterm fetal sheep at 110 ± 1(SD) days' gestation underwent sterile surgery for instrumentation with a tracheal loop to enable in utero mechanical ventilation (IUV). At 112 ± 1 days' gestation, fetuses received either in utero mechanical ventilation (IUV, n = 10) targeting 3-5 mL/kg for 24 h, or no ventilation (CONT, n = 9). At necropsy, fetal lungs were collected to assess molecular and histological markers of lung inflammation and injury. IUV significantly increased lung mRNA expression of interleukin (IL)-1ß, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF) compared with CONT, and increased surfactant protein (SP)-A1, SP-B, and SP-C mRNA expression compared with CONT. IUV produced modest structural changes to the airways, including reduced parenchymal collagen and myofibroblast density. IUV increased pulmonary arteriole thickness compared with CONT but did not alter overall elastin or collagen content within the vasculature. In utero ventilation of an extremely preterm lung, even at low tidal volumes, induces lung inflammation and injury to the airways and vasculature. In utero ventilation may be an important model to isolate the confounding mechanisms of VILI to develop effective therapies for preterm infants requiring prolonged respiratory support.NEW & NOTEWORTHY Preterm infants often require prolonged respiratory support, but the relative contribution of ventilation to the development of lung injury is difficult to isolate. In utero mechanical ventilation allows for mechanistic investigations into ventilation-induced lung injury without confounding factors associated with sustaining extremely preterm lambs ex utero. Twenty-four hours of in utero ventilation, even at low tidal volumes, increased lung inflammation and surfactant protein expression and produced structural changes to the lung parenchyma and vasculature.

Mechanical ventilation induces brainstem inflammation in preterm fetal sheep.

Background: Preterm infants have immature respiratory drive and often require prolonged periods of mechanical ventilation. Prolonged mechanical ventilation induces systemic inflammation resulting in ventilation-induced brain injury, however its effect on brainstem respiratory centers is unknown. We aimed to determine the effects of 24 h of mechanical ventilation on inflammation and injury in brainstem respiratory centres of preterm fetal sheep. Methods: Preterm fetal sheep at 110 ± 1 days (d) gestation were instrumented to provide mechanical ventilation in utero. At 112 ± 1 d gestation, fetuses received either mechanical ventilation (VENT; n = 7; 3 ml/kg) for 24 h, or no ventilation (CONT; n = 6). At post-mortem, fetal brainstems were collected for assessment of mRNA and histological markers of inflammation and injury. Results: In utero ventilation (IUV) did not alter any blood-gas parameters. IUV significantly increased systemic IL-6 and IL-8 concentrations over the 24 h period compared to CONT. The number of ameboid microglia within the nucleus tractus solitarius and the raphe nucleus increased in VENT fetuses (p < 0.05 for both vs. control). The % area fraction of GFAP + staining was not significantly higher within the preBötzinger complex (p = 0.067) and retrotrapezoid nucleus and parafacial respiratory group (p = 0.057) in VENT fetuses compared to CONT. Numbers of caspase-3 and TUNEL-positive cells were similar between groups. Gene expression (mRNA) levels of inflammation, injury, cell death and prostaglandin synthesis within the brainstem were similar between groups. Conclusion: Mechanical ventilation induces a systemic inflammatory response with only moderate inflammatory effects within the brainstem respiratory centres of preterm fetal sheep.

Progressive inflammation reduces high-frequency EEG activity and cortical dendritic arborisation in late gestation fetal sheep.

BACKGROUND: Antenatal infection/inflammation is associated with disturbances in neuronal connectivity, impaired cortical growth and poor neurodevelopmental outcomes. The pathophysiological substrate that underpins these changes is poorly understood. We tested the hypothesis that progressive inflammation in late gestation fetal sheep would alter cortical neuronal microstructure and neural function assessed using electroencephalogram band power analysis. METHODS: Fetal sheep (0.85 of gestation) were surgically instrumented for continuous electroencephalogram (EEG) recording and randomly assigned to repeated saline (control; n = 9) or LPS (0 h = 300 ng, 24 h = 600 ng, 48 h = 1200 ng; n = 8) infusions to induce inflammation. Sheep were euthanised 4 days after the first LPS infusion for assessment of inflammatory gene expression, histopathology and neuronal dendritic morphology in the somatosensory cortex. RESULTS: LPS infusions increased delta power between 8 and 50 h, with reduced beta power from 18 to 96 h (P < 0.05 vs. control). Basal dendritic length, numbers of dendritic terminals, dendritic arborisation and numbers of dendritic spines were reduced in LPS-exposed fetuses (P < 0.05 vs. control) within the somatosensory cortex. Numbers of microglia and interleukin (IL)-1ß immunoreactivity were increased in LPS-exposed fetuses compared with controls (P < 0.05). There were no differences in total numbers of cortical NeuN + neurons or cortical area between the groups. CONCLUSIONS: Exposure to antenatal infection/inflammation was associated with impaired dendritic arborisation, spine number and loss of high-frequency EEG activity, despite normal numbers of neurons, that may contribute to disturbed cortical development and connectivity.

Lung aeration reduces blood pressure surges caused by umbilical cord milking in preterm lambs.

Background: Umbilical cord milking (UCM) at birth causes surges in arterial blood pressure and blood flow to the brain, which may explain the high risk of intraventricular haemorrhage (IVH) in extremely preterm infants receiving UCM. This high risk of IVH has not been reported in older infants. Objective: We hypothesized that lung aeration before UCM, reduces the surge in blood pressure and blood flow induced by UCM. Methods: At 126 days' gestation, fetal lambs (N = 8) were exteriorised, intubated and instrumented to measure umbilical, pulmonary, cerebral blood flows, and arterial pressures. Prior to ventilation onset, the umbilical cord was briefly (2-3 s) occluded (8 times), which was followed by 8 consecutive UCMs when all physiological parameters had returned to baseline. Lambs were then ventilated. After diastolic pulmonary blood flow markedly increased in response to ventilation, the lambs received a further 8 consecutive UCMs. Ovine umbilical cord is shorter than the human umbilical cord, with ∼10 cm available for UCMs. Therefore, 8 UCMs/occlusions were done to match the volume reported in the human studies. Umbilical cord clamping occurred after the final milk. Results: Both umbilical cord occlusions and UCM caused significant increases in carotid arterial blood flow and pressure. However, the increases in systolic and mean arterial blood pressure (10 ± 3 mmHg vs. 3 ± 2 mmHg, p = 0.01 and 10 ± 4 mmHg vs. 6 ± 2 mmHg, p = 0.048, respectively) and carotid artery blood flow (17 ± 6 ml/min vs. 10 ± 6 ml/min, p = 0.02) were significantly greater when UCM occurred before ventilation onset compared with UCM after ventilation. Conclusions: UCM after ventilation onset significantly reduces the increases in carotid blood flow and blood pressure caused by UCM.

Effect of prenatal diaphragmatic hernia on pulmonary arterial morphology.

Congenital diaphragmatic hernia (CDH) is a major cause of severe lung hypoplasia and pulmonary hypertension in the newborn. While the pulmonary hypertension is thought to result from abnormal vascular development and arterial vasoreactivity, the anatomical changes in vascular development are unclear. We have examined the 3D structure of the pulmonary arterial tree in rabbits with a surgically induced diaphragmatic hernia (DH). Fetal rabbits (n = 6) had a left-sided DH created at gestational day 23 (GD23), delivered at GD30, and briefly ventilated; sham-operated litter mates (n = 5) acted as controls. At postmortem the pulmonary arteries were filled with a radio-opaque resin before the lungs were scanned using computed tomography (CT). The 3D reconstructed images were analyzed based on vascular branching hierarchy using the software Avizo 2020.2. DH significantly reduced median number of arteries (2,579 (8440) versus 576 (442), p = .017), artery numbers per arterial generation, mean total arterial volume (43.5 ± 8.4 vs. 19.9 ± 3.1 µl, p = .020) and mean total arterial cross-sectional area (82.5 ± 2.3 vs. 28.2 ± 6.2 mm2 , p =.036). Mean arterial radius was increased in DH kittens between the eighth and sixth branching generation and mean arterial length between the sixth and 28th branching generation. A DH in kittens resulted in threefold reduction in pulmonary arterial cross-sectional area, primarily due to reduced arterial branching. Thus, the reduction in arterial cross-sectional area could be a major contributor to pulmonary hypertension infants with CDH.

Role of platelet-derived growth factor-B, vascular endothelial growth factor, insulin-like growth factor-II, mitogen-activated protein kinase and transforming growth factor-beta1 in expansion-induced lung growth in fetal sheep.

Increased fetal lung expansion induces lung growth, cell differentiation and extracellular matrix remodelling, although the mechanisms involved are unknown. Platelet-derived growth factor (PDGF)-B, vascular endothelial growth factor (VEGF) and insulin-like growth factor (IGF)-II are mitogens activating the mitogen-activated protein kinase (MAPK) pathway, whereas transforming growth factor (TGF)-beta1 induces differentiation and extracellular matrix remodelling. In the present study, we investigated the mRNA levels of PDGF-B, VEGF, IGF-II and TGF-beta1, as well as active MAPK levels, during increased fetal lung expansion induced by tracheal obstruction (TO) in sheep for 0 (controls), 36 h or 2, 4, or 10 days (n = 5 in each group). The 3.7-kb VEGF transcript increased by 30% (P < 0.05) at 36 h TO. The expression of PDGF-B decreased by approximately 25% (P < 0.01) at 2-10 days TO. In contrast, TGF-beta1 mRNA increased by 96% (P < 0.05) at 10 days TO, when bioactive TGF-beta1 decreased by 55% (P < 0.05). Insulin-like growth factor-II mRNA tended to increase at 10 days TO (37% above controls; P = 0.07), whereas mRNA for its receptor, IGF1R, was reduced by TO. There was no change in active MAPK levels preceding or at the time of a TO-induced 800% increase in cell proliferation. We conclude that VEGF is likely to promote expansion-induced endothelial cell proliferation, but the mechanisms underlying expansion-induced proliferation of fibroblasts and alveolar epithelial cells are unlikely to be mediated by increases in PDGF-B or IGF-II expression or activation of the MAPK pathway.