Phenotype of early-onset fetal growth restriction in sheep.

Introduction: Fetal growth restriction (FGR) is a common pregnancy complication, caused by placental insufficiency, with serious adverse consequences for development in utero and postnatal wellbeing. There are no antenatal treatments to improve growth or organ development in FGR, and animal models are essential to mimic the physiological adaptations in FGR and to assess potential interventions. This study aimed to identify the temporal nature of reduced developmental trajectory in fetuses with FGR, and to examine the effects of common factors that may mediate differential growth such as glucocorticoid treatment. We hypothesised that the trajectory of growth would be adversely impacted by FGR. Methods: FGR was induced via surgical placental insufficiency in fetal sheep (89 days gestation/0.6 gestation; n=135) and compared to age-matched controls over the last third of gestation and into neonatal life (n=153). Results: Body weight of FGR fetuses/lambs was significantly reduced compared to controls (p<0.0001) from 127 days of gestation (term is 148 days), with increased brain:body weight ratio (p<0.0001) indicative of brain sparing. All biometric measures of body size were reduced in the FGR group with the exception of biparietal (head) diameter. The trajectory of body growth in the last trimester of sheep pregnancy was significantly reduced in the FGR group compared to controls, and stillbirth rate increased with longer gestation. Discussion: This work provides a well characterised FGR animal model that mimics the known physiological adaptations in human pregnancy and can be used to determine the efficacy of potential interventions.

The cerebral haemodynamic response to somatosensory stimulation in preterm newborn lambs is reduced following intrauterine inflammation and dopamine infusion.

BACKGROUND: Neurovascular coupling leads to an increase in local cerebral blood flow and oxygenation in response to increased neural activity. Reduced cerebral functional responses may predispose to tissue hypoxia when neural activity is increased. Intrauterine inflammation, identified clinically as chorioamnionitis, is a major contributor to the neuropathology arising after preterm birth. The impact of chorioamnionitis on the preterm cerebral functional haemodynamic response is unknown. Previously, we have reported that somatosensory stimulation produces predominantly positive cerebral haemodynamic responses (i.e., increased cerebral oxygenation) in preterm lambs, which are reduced with dopamine treatment. As preterm infants born after chorioamnionitis often suffer from hypotension and are treated with dopamine, we aimed to investigate how chorioamnionitis with and without dopamine treatment affect the cerebral haemodynamic response in preterm lambs. METHODS: At 119 days of gestation, intrauterine inflammation was induced by intra-amniotic injection of lipopolysaccharide (LPS) in pregnant ewes. At 126-7 days of gestation (term is ~147 days), these LPS-exposed lambs were delivered and mechanically ventilated. The cerebral functional response was assessed by near infrared spectroscopy as changes in cerebral oxy- and deoxyhaemoglobin (ΔoxyHb, ΔdeoxyHb), following left median nerve stimulation of 1.8, 4.8 and 7.8 s durations without dopamine; and 4.8 and 7.8 s stimulations with intravenous dopamine infusion. RESULTS: Stimulation for 1.8, 4.8 and 7.8 s durations led to negative functional responses (decreased ΔoxyHb) in 5 (62.5%), 5 (62.5%) and 4 (50%) of 8 preterm lambs respectively, while other lambs showed positive responses (increased ∆oxyHb). Dopamine infusion increased baseline tissue oxygenation index (TOI), oxyHb and total Hb. In lambs with a positive functional response, dopamine decreased the evoked ΔoxyHb response, increasing the overall incidence of negative cerebral haemodynamic responses. CONCLUSIONS: Somatosensory stimulation produced mostly negative responses with decreased cerebral oxygenation in preterm lambs exposed to intrauterine inflammation, contrasting with our previous findings of predominantly positive responses in non-inflamed, control, preterm lambs. Dopamine increased baseline cerebral oxygenation, but further increased the incidence of negative functional responses. Impaired neurovascular coupling leading to intermittent localised tissue hypoxia may therefore contribute to the neuropathy in infants with chorioamnionitis, with the risk of injury exacerbated with dopamine treatment.

Effect of 2D and 3D Culture Microenvironments on Mesenchymal Stem Cell-Derived Extracellular Vesicles Potencies.

Therapeutic benefits of mesenchymal stem cells (MSCs) are now widely believed to come from their paracrine signalling, i.e. secreted factors such as cytokines, chemokines, and extracellular vesicles (EVs). Cell-free therapy using EVs is an active and emerging field in regenerative medicine. Typical 2D cultures on tissue culture plastic is far removed from the physiological environment of MSCs. The application of 3D cell culture allows MSCs to adapt to their cellular environment which, in turn, influences their paracrine signalling activity. In this study we evaluated the impact of 3D MSCs culture on EVs secretion, cargo proteome composition, and functional assessment in immunomodulatory, anti-inflammatory and anti-fibrotic properties. MSC-EVs from 2D and 3D cultures expressed classical EV markers CD81, CD63, and CD9 with particle diameter of <100 nm. There were distinct changes in immunomodulatory potencies where 3D cultures exhibited reduced indoleamine 2,3-dioxygenase (IDO) activity and significantly reduced macrophage phagocytosis. Administration of 2D and 3D EVs following double dose bleomycin challenge in aged mice showed a marked increase of bodyweight loss in 3D group throughout days 7-28. Histopathological observations of lung tissues in 3D group showed increased collagen deposition, myofibroblast differentiation and leukocytes infiltrations. Assessment of lung mechanics showed 3D group did not improve lung function and instead exhibited increased resistance and tissue damping. Proteome profiling of MSC-EV composition revealed molecular enrichment of EV markers (compared to parental cells) and differential proteome between EVs from 2D and 3D culture condition associated with immune-based and fibrosis/extracellular matrix/membrane organization associated function. This study provides insight into distinct variation in EV protein composition dependent on the cellular microenvironment of the parental cells, which could have implications in their therapeutic effect and potency. Overall, this work suggests that EVs produced from 3D MSC cultures did not enhance typical MSC-EV properties expected from 2D cultures (immunomodulation, anti-fibrotic, anti-inflammatory). The outcome highlights critical differences between MSC-EVs obtained from different culture microenvironments, which should be considered when scaling up MSC culture for clinical manufacturing.

Cerebral haemodynamic response to somatosensory stimulation in preterm lambs and 7-10-day old lambs born at term: Direct synchrotron microangiography assessment.

Neurovascular coupling has been well-defined in the adult brain, but variable and inconsistent responses have been observed in the neonatal brain. The mechanisms that underlie functional haemodynamic responses in the developing brain are unknown. Synchrotron radiation (SR) microangiography enables in vivo high-resolution imaging of the cerebral vasculature. We exploited SR microangiography to investigate the microvascular changes underlying the cerebral haemodynamic response in preterm (n = 7) and 7-10-day old term lambs (n = 4), following median nerve stimulation of 1.8, 4.8 and 7.8 sec durations.Increasing durations of somatosensory stimulation significantly increased the number of cortical microvessels of ≤200 µm diameter in 7-10-day old term lambs (p < 0.05) but not preterm lambs where, in contrast, stimulation increased the diameter of cerebral microvessels with a baseline diameter of ≤200 µm. Preterm lambs demonstrated positive functional responses with increased oxyhaemoglobin measured by near infrared spectroscopy, while 7-10-day old term lambs demonstrated both positive and negative responses. Our findings suggest the vascular mechanisms underlying the functional haemodynamic response differ between the preterm and 7-10-day old term brain. The preterm brain depends on vasodilatation of microvessels without recruitment of additional vessels, suggesting a limited capacity to mount higher cerebral haemodynamic responses when faced with prolonged or stronger neural stimulation.

The cerebral haemodynamic response to somatosensory stimulation in preterm newborn lambs is reduced with dopamine or dobutamine infusion.

BACKGROUND: In the adult brain, increases in neural activity lead to increases in local blood flow. However, in the preterm neonate, studies of cerebral functional haemodynamics have yielded inconsistent results, including negative responses suggesting decreased perfusion and localised tissue hypoxia, probably due to immature neurovascular coupling. Furthermore, the impact of vasoactive medications, such as dopamine and dobutamine used as inotropic therapies in preterm neonates, on cerebrovascular responses to somatosensory input is unknown. We aimed to characterise the cerebral haemodynamic functional response after somatosensory stimulation in the preterm newborn brain, with and without dopamine or dobutamine treatment. METHODS: We studied the cerebral haemodynamic functional response in 13 anaesthetised preterm lambs, using near infrared spectroscopy to measure changes in cerebral oxy- and deoxyhaemoglobin (ΔoxyHb, ΔdeoxyHb) following left median nerve stimulation using stimulus trains of 1.8, 4.8 and 7.8 s. The 4.8 and 7.8 s stimulations were repeated during dopamine or dobutamine infusion. RESULTS: Stimulation always produced a somatosensory evoked response. Majority of preterm lambs demonstrated positive functional responses (i.e. increased ΔoxyHb) in the contralateral cortex following stimulus trains of all durations. Dopamine increased baseline oxyHb and total Hb, whereas dobutamine increased baseline deoxyHb. Both dopamine and dobutamine reduced the evoked ΔoxyHb responses to 4.8 and 7.8 s stimulations. CONCLUSIONS: Somatosensory stimulation increases cerebral oxygenation in the preterm brain, consistent with increased cerebral blood flow due to neurovascular coupling. Notably, our results show that dopamine/dobutamine reduces oxygen delivery relative to consumption in the preterm brain during somatosensory stimulations, suggesting there may be a risk of intermittent localised tissue hypoxia which has clear implications for clinical practice and warrants further investigation.

Maternal sildenafil impairs the cardiovascular adaptations to chronic hypoxaemia in fetal sheep.

KEY POINTS: Fetal growth restriction induces a haemodynamic response that aims to maintain blood flow to vital organs such as the brain, in the face of chronic hypoxaemia Maternal sildenafil treatment impairs the hypoxaemia-driven haemodynamic response and potentially compromises fetal development. ABSTRACT: Inadequate substrate delivery to a fetus results in hypoxaemia and fetal growth restriction (FGR). In response, fetal cardiovascular adaptations redirect cardiac output to essential organs to maintain oxygen delivery and sustain development. However, FGR infants remain at risk for cardiovascular and neurological sequelae. Sildenafil citrate (SC) has been examined as a clinical therapy for FGR, but also crosses the placenta and may exert direct effects on the fetus. We investigated the effects of maternal SC administration on maternal and fetal cardiovascular physiology in growth-restricted fetal sheep. Fetal sheep (0.7 gestation) underwent sterile surgery to induce growth restriction by single umbilical artery ligation (SUAL) or sham surgery (control, AG). Fetal catheters and flow probes were implanted to measure carotid and femoral arterial blood flows. Ewes containing SUAL fetuses were randomized to receive either maternal administration of saline or SC (36 mg i.v. per day) beginning 4 days after surgery, and continuing for 20 days. Physiological recordings were obtained throughout the study. Antenatal SC treatment reduced body weight by 32% and oxygenation by 18% in SUAL compared to AG. SC did not alter maternal or fetal heart rate or blood pressure. Femoral blood flow and peripheral oxygen delivery were increased by 49% and 30% respectively in SUALSC compared to SUAL, indicating impaired cardiovascular adaptation to chronic hypoxaemia. Antenatal SC directly impairs the fetal haemodynamic response to chronic hypoxaemia. Consideration of the consequences upon the fetus should be paramount when administering interventions to the mother during pregnancy.

Effects of Maternal Sildenafil Treatment on Vascular Function in Growth-Restricted Fetal Sheep.

Objective- The objective of this study was to investigate the effect of intravenous maternal sildenafil citrate (SC) administration on vascular function in growth-restricted fetal sheep. Approach and Results- Fetal growth restriction (FGR) results in cardiovascular adaptations that redistribute cardiac output to optimize suboptimal intrauterine conditions. These adaptations result in structural and functional cardiovascular changes, which may underlie postnatal neurological and cardiovascular sequelae. Evidence suggests SC, a potent vasodilator, may improve FGR. In contrast, recent clinical evidence suggests potential for adverse fetal consequence. Currently, there is limited data on SC effects in the developing fetus. We hypothesized that SC in utero would improve vascular development and function in an ovine model of FGR. Preterm lambs (0.6 gestation) underwent sterile surgery for single umbilical artery ligation or sham (control, appropriately grown) surgery to replicate FGR. Ewes received continuous intravenous SC (36 mg/24 h) or saline from surgery until 0.83 gestation. Fetuses were delivered and immediately euthanized for collection of femoral and middle cerebral artery vessels. Vessel function was assessed via in vitro wire myography. SC exacerbated growth restriction in growth-restricted fetuses and resulted in endothelial dysfunction in the cerebral and femoral vasculature, irrespective of growth status. Dysfunction in the cerebral circulation is endothelial, whereas smooth muscle in the periphery is the origin of the deficit. Conclusions- SC crosses the placenta and alters key fetal vascular development. Extensive studies are required to investigate the effects of SC on fetal development to address safety before additional use of SC as a treatment.

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.

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.

Lung ultrasound accurately detects pneumothorax in a preterm newborn lamb model.

AIM: Pneumothorax is a common emergency affecting extremely preterm. In adult studies, lung ultrasound has performed better than chest x-ray in the diagnosis of pneumothorax. The purpose of this study was to determine the efficacy of lung ultrasound (LUS) examination to detect pneumothorax using a preterm animal model. METHODS: This was a prospective, observational study using newborn Border-Leicester lambs at gestational age = 126 days (equivalent to gestational age = 26 weeks in humans) receiving mechanical ventilation from birth to 2 h of life. At the conclusion of the experiment, LUS was performed, the lambs were then euthanised and a post-mortem exam was immediately performed. We used previously published ultrasound techniques to identify pneumothorax. Test characteristics of LUS to detect pneumothorax were calculated, using the post-mortem exam as the 'gold standard' test. RESULTS: Nine lambs (18 lungs) were examined. Four lambs had a unilateral pneumothorax, all of which were identified by LUS with no false positives. CONCLUSIONS: This was the first study to use post-mortem findings to test the efficacy of LUS to detect pneumothorax in a newborn animal model. Lung ultrasound accurately detected pneumothorax, verified by post-mortem exam, in premature, newborn lambs.