Direct-to-PCR tissue preservation for DNA profiling.

Disaster victim identification (DVI) often occurs in remote locations with extremes of temperatures and humidities. Access to mortuary facilities and refrigeration are not always available. An effective and robust DNA sampling and preservation procedure would increase the probability of successful DNA profiling and allow faster repatriation of bodies and body parts. If the act of tissue preservation also released DNA into solution, ready for polymerase chain reaction (PCR), the DVI process could be further streamlined. In this study, we explored the possibility of obtaining DNA profiles without DNA extraction, by adding aliquots of preservative solutions surrounding fresh human muscle and decomposing human muscle and skin tissue samples directly to PCR. The preservatives consisted of two custom preparations and two proprietary solutions. The custom preparations were a salt-saturated solution of dimethyl sulfoxide (DMSO) with ethylenediaminetetraacetic (EDTA) and TENT buffer (Tris, EDTA, NaCl, Tween 20). The proprietary preservatives were DNAgard (Biomatrica(®)) and Tissue Stabilising Kit (DNA Genotek). We obtained full PowerPlex(®) 21 (Promega) and GlobalFiler(®) (Life Technologies) DNA profiles from fresh and decomposed tissue preserved at 35 °C for up to 28 days for all four preservatives. The preservative aliquots removed from the fresh muscle tissue samples had been stored at -80 °C for 4 years, indicating that long-term archival does not diminish the probability of successful DNA typing. Rather, storage at -80 °C seems to reduce PCR inhibition.

Pressure- versus volume-limited sustained inflations at resuscitation of premature newborn lambs.

BACKGROUND: Sustained inflations (SI) are advocated for the rapid establishment of FRC after birth in preterm and term infants requiring resuscitation. However, the most appropriate way to deliver a SI is poorly understood. We investigated whether a volume-limited SI improved the establishment of FRC and ventilation homogeneity and reduced lung inflammation/injury compared to a pressure-limited SI. METHODS: 131 d gestation lambs were resuscitated with either: i) pressure-limited SI (PressSI: 0-40 cmH2O over 5 s, maintained until 20 s); or ii) volume-limited SI (VolSI: 0-15 mL/kg over 5 s, maintained until 20 s). Following the SI, all lambs were ventilated using volume-controlled ventilation (7 mL/kg tidal volume) for 15 min. Lung mechanics, regional ventilation distribution (electrical impedance tomography), cerebral tissue oxygenation index (near infrared spectroscopy), arterial pressures and blood gas values were recorded regularly. Pressure-volume curves were performed in-situ post-mortem and early markers of lung injury were assessed. RESULTS: Compared to a pressure-limited SI, a volume-limited SI had increased pressure variability but reduced volume variability. Each SI strategy achieved similar end-inflation lung volumes and regional ventilation homogeneity. Volume-limited SI increased heart-rate and arterial pressure faster than pressure-limited SI lambs, but no differences were observed after 30 s. Volume-limited SI had increased arterial-alveolar oxygen difference due to higher FiO2 at 15 min (p = 0.01 and p = 0.02 respectively). No other inter-group differences in arterial or cerebral oxygenation, blood pressures or early markers of lung injury were evident. CONCLUSION: With the exception of inferior oxygenation, a sustained inflation targeting delivery to preterm lambs of 15 mL/kg volume by 5 s did not influence physiological variables or early markers of lung inflammation and injury at 15 min compared to a standard pressure-limited sustained inflation.

In utero LPS exposure impairs preterm diaphragm contractility.

Preterm birth is associated with inflammation of the fetal membranes (chorioamnionitis). We aimed to establish how chorioamnionitis affects the contractile function and phenotype of the preterm diaphragm. Pregnant ewes received intra-amniotic injections of saline or 10 mg LPS, 2 days or 7 days before delivery at 121 days of gestation (term = 150 d). Diaphragm strips were dissected for the assessment of contractile function after terminal anesthesia. The inflammatory cytokine response, myosin heavy chain (MHC) fibers, proteolytic pathways, and intracellular molecular signaling were analyzed using quantitative PCR, ELISA, immunofluorescence staining, biochemical assays, and Western blotting. Diaphragm peak twitch force and maximal tetanic force were approximately 30% lower than control values in the 2-day and 7-day LPS groups. Activation of the NF-κB pathway, an inflammatory response, and increased proteasome activity were observed in the 2-day LPS group relative to the control or 7-day LPS group. No inflammatory response was evident after a 7-day LPS exposure. Seven-day LPS exposure markedly decreased p70S6K phosphorylation, but no effect on other signaling pathways was evident. The proportion of MHC IIa fibers was lower than that for control samples in the 7-day LPS group. MHC I fiber proportions did not differ between groups. These results demonstrate that intrauterine LPS impairs preterm diaphragmatic contractility after 2-day and 7-day exposures. Diaphragm dysfunction, resulting from 2-day LPS exposure, was associated with a transient activation of proinflammatory signaling, with subsequent increased atrophic gene expression and enhanced proteasome activity. Persistently impaired contractility for the 7-day LPS exposure was associated with the down-regulation of a key component of the protein synthetic signaling pathway and a reduction in the proportions of MHC IIa fibers.

Chronic fetal exposure to Ureaplasma parvum suppresses innate immune responses in sheep.

The chorioamnionitis associated with preterm delivery is often polymicrobial with ureaplasma being the most common isolate. To evaluate interactions between the different proinflammatory mediators, we hypothesized that ureaplasma exposure would increase fetal responsiveness to LPS. Fetal sheep were given intra-amniotic (IA) injections of media (control) or Ureaplasma parvum serovar 3 either 7 or 70 d before preterm delivery. Another group received an IA injection of Escherichia coli LPS 2 d prior to delivery. To test for interactions, IA U. parvum-exposed animals were challenged with IA LPS and delivered 2 d later. All animals were delivered at 124 ± 1-d gestation (term = 150 d). Compared with the 2-d LPS exposure group, the U. parvum 70 d + LPS group had 1) decreased lung pro- and anti-inflammatory cytokine expression and 2) fewer CD3(+) T lymphocytes, CCL2(+), myeloperoxidase(+), and PU.1(+) cells in the lung. Interestingly, exposure to U. parvum for 7 d did not change responses to a subsequent IA LPS challenge, and exposure to IA U. parvum alone induced mild lung inflammation. Exposure to U. parvum increased pulmonary TGF-ß1 expression but did not change mRNA expression of either the receptor TLR4 or some of the downstream mediators in the lung. Monocytes from fetal blood and lung isolated from U. parvum 70 d + LPS but not U. parvum 7 d + LPS animals had decreased in vitro responsiveness to LPS. These results are consistent with the novel finding of downregulation of LPS responses by chronic but not acute fetal exposures to U. parvum. The findings increase our understanding of how chorioamnionitis-exposed preterm infants may respond to lung injury and postnatal nosocomial infections.

Variable ventilation enhances ventilation without exacerbating injury in preterm lambs with respiratory distress syndrome.

BACKGROUND: As compared with constant respiratory rate (RR) and tidal volume (V(T)) during controlled conventional mechanical ventilation (CV), variable ventilation (VV) using the same breath-to-breath minute volume but variable V(T) and RRs enhances ventilation efficiency in preterm lambs. We hypothesized that if V(T) was adjusted to target permissive hypercarbia, VV would result in more efficient gas exchange without increasing inflammatory and injurious responses in the lung. METHODS: Preterm lambs at 129 d gestation were anesthetized, tracheotomized, and randomized to either CV (n = 8) or VV (n = 8) using the same initial average V(T) and RR. Lung mechanics and gas exchange were measured intermittently, and average V(T) was adjusted to target partial pressure of arterial carbon dioxide (PaCO2) of 40-50 mm Hg for 3 h. Lung injury and inflammation were assessed from bronchoalveolar lavage fluid, lung tissue, and peripheral blood. RESULTS: VV achieved permissive hypercarbia using a lower average V(T), peak inspiratory pressure, and elastance (increased compliance) as compared with CV. Oxygenation and markers of lung tissue inflammation or injury were not different apart from a lower wet:dry tissue ratio in the VV lungs. CONCLUSIONS: VV improves ventilation efficiency and in vivo lung compliance in the ovine preterm lung without increasing lung inflammation or lung injury.

Positive end-expiratory pressure and surfactant decrease lung injury during initiation of ventilation in fetal sheep.

The initiation of ventilation in preterm, surfactant-deficient sheep without positive end-expiratory pressure (PEEP) causes airway injury and lung inflammation. We hypothesized that PEEP and surfactant treatment would decrease the lung injury from initiation of ventilation with high tidal volumes. Fetal sheep at 128-day gestational age were randomized to ventilation with: 1) no PEEP, no surfactant; 2) 8-cmH(2)O PEEP, no surfactant; 3) no PEEP + surfactant; 4) 8-cmH(2)O PEEP + surfactant; or 5) control (2-cmH(2)O continuous positive airway pressure) (n = 6-7/group). After maternal anesthesia and hysterotomy, the head and chest were exteriorized, and the fetus was intubated. While maintaining placental circulation, the fetus was ventilated for 15 min with a tidal volume escalating to 15 ml/kg using heated, humidified, 100% nitrogen. The fetus then was returned to the uterus, and tissue was collected after 30 min for evaluation of early markers of lung injury. Lambs receiving both surfactant and PEEP had increased dynamic compliance, increased static lung volumes, and decreased total protein and heat shock proteins 70 and 60 in bronchoalveolar lavage fluid compared with other groups. Ventilation, independent of PEEP or surfactant, increased mRNA expression of acute phase response genes and proinflammatory cytokine mRNA in the lung tissue compared with controls. PEEP decreased mRNA for cytokines (2-fold) compared with groups receiving no PEEP. Surfactant administration further decreased some cytokine mRNAs and changed the distribution of early growth response protein-1 expression. The use of PEEP during initiation of ventilation at birth decreased early mediators of lung injury. Surfactant administration changed the distribution of injury and had a moderate additive protective effect.

Nebulised surfactant to reduce severity of respiratory distress: a blinded, parallel, randomised controlled trial.

OBJECTIVE: To evaluate if nebulised surfactant reduces intubation requirement in preterm infants with respiratory distress treated with nasal continuous positive airway pressure (nCPAP). DESIGN: Double blind, parallel, stratified, randomised control trial. SETTING: Sole tertiary neonatal unit in West Australia. PATIENTS: Preterm infants (290-336 weeks' gestational age, GA) less than 4 hours of age requiring 22%-30% supplemental oxygen, with informed parental written consent. INTERVENTIONS: Infants were randomised within strata (290-316 and 320-336 weeks' GA) to bubble nCPAP or bubble nCPAP and nebulised surfactant (200 mg/kg: poractant alfa) using a customised vibrating membrane nebuliser (eFlow neonatal). Surfactant nebulisation (100 mg/kg) was repeated after 12 hours for persistent supplemental oxygen requirement. MAIN OUTCOME MEASURES: The primary outcomes were requirement for intubation and duration of mechanical ventilation at 72 hours. Data analysis followed the intention-to-treat principle. RESULTS: 360 of 606 assessed infants were eligible; 64 of 360 infants were enrolled and randomised (n=32/group). Surfactant nebulisation reduced the requirement for intubation within 72 hours: 11 of 32 infants were intubated after continuous positive airway pressure (CPAP) and nebulised surfactant compared with 22 of 32 infants receiving CPAP alone (relative risk (95% CI)=0.526 (0.292 to 0.950)). The reduced requirement for intubation was limited to the 320-336 weeks' GA stratum. The median (range) duration of ventilation in the first 72 hours was not different between the intervention (0 (0-62) hours) and control (9 (0-64) hours; p=0.220) groups. There were no major adverse events. CONCLUSIONS: Early postnatal nebulised surfactant may reduce the need for intubation in the first 3 days of life compared with nCPAP alone in infants born at 290-336 weeks' GA with mild respiratory distress syndrome. Confirmation requires further adequately powered studies. TRIAL REGISTRATION NUMBER: ACTRN12610000857000.

The bioactive nature of human breastmilk.

Human breastmilk is widely accepted to be the optimal source for nutrition for the newborn infant, containing all the proteins, lipids, carbohydrates, micronutrients and trace elements required for growth, development and immune protection. In addition human breastmilk plays a significant role in the prevention of medical conditions such as childhood cancers, obesity and gastrointestinal diseases. The importance of breastmilk and its superiority as a source of infant nutrition is highlighted in the recent overhaul of teh World Health Organization growth charts that are now based on exclusively breastfed infants. Human breastmilk also contains a population of cells, the importance of which is often not considered when the bioactivity of breastmilk is discussed.

Pressure-limited sustained inflation vs. gradual tidal inflations for resuscitation in preterm lambs.

Support of the mechanically complex preterm lung needs to facilitate aeration while avoiding ventilation heterogeneities: whether to achieve this gradually or quickly remains unclear. We compared the effect of gradual vs. constant tidal inflations and a pressure-limited sustained inflation (SI) at birth on gas exchange, lung mechanics, gravity-dependent lung volume distribution, and lung injury in 131-day gestation preterm lambs. Lambs were resuscitated with either 1) a 20-s, 40-cmH2O pressure-limited SI (PressSI), 2) a gradual increase in tidal volume (Vt) over 5-min from 3 ml/kg to 7 ml/kg (IncrVt), or 3) 7 ml/kg Vt from birth. All lambs were subsequently ventilated for 15 min with 7 ml/kg Vt with the same end-expiratory pressure. Lung mechanics, gas exchange and spatial distribution of end-expiratory volume (EEV), and tidal ventilation (electrical impedance tomography) were recorded regularly. At 15 min, early mRNA tissue markers of lung injury were assessed. The IncrVt group resulted in greater tissue hysteresivity at 5 min (P = 0.017; two-way ANOVA), higher alveolar-arterial oxygen difference from 10 min (P < 0.01), and least uniform gravity-dependent distribution of EEV. There were no other differences in lung mechanics between groups, and the PressSI and 7 ml/kg Vt groups behaved similarly throughout. EEV was more uniformly distributed, but Vt least so, in the PressSI group. There were no differences in mRNA markers of lung injury. A gradual increase in Vt from birth resulted in less recruitment of the gravity-dependent lung with worse oxygenation. There was no benefit of a SI at birth over mechanical ventilation with 7 ml/kg Vt.

The development and evaluation of a medical imaging training immersive environment.

INTRODUCTION: A novel realistic 3D virtual reality (VR) application has been developed to allow medical imaging students at Queensland University of Technology to practice radiographic techniques independently outside the usual radiography laboratory. METHODS: A flexible agile development methodology was used to create the software rapidly and effectively. A 3D gaming environment and realistic models were used to engender presence in the software while tutor-determined gold standards enabled students to compare their performance and learn in a problem-based learning pedagogy. RESULTS: Students reported high levels of satisfaction and perceived value and the software enabled up to 40 concurrent users to prepare for clinical practice. Student feedback also indicated that they found 3D to be of limited value in the desktop version compared to the usual 2D approach. A randomised comparison between groups receiving software-based and traditional practice measured performance in a formative role play with real equipment. The results of this work indicated superior performance with the equipment for the VR trained students (P = 0.0366) and confirmed the value of VR for enhancing 3D equipment-based problem-solving skills. CONCLUSIONS: Students practising projection techniques virtually performed better at role play assessments than students practising in a traditional radiography laboratory only. The application particularly helped with 3D equipment configuration, suggesting that teaching 3D problem solving is an ideal use of such medical equipment simulators. Ongoing development work aims to establish the role of VR software in preparing students for clinical practice with a range of medical imaging equipment.

Lipopolysaccharide-induced weakness in the preterm diaphragm is associated with mitochondrial electron transport chain dysfunction and oxidative stress.

Diaphragmatic contractility is reduced in preterm lambs after lipopolysaccharide (LPS) exposure in utero. The mechanism of impaired fetal diaphragm contractility after LPS exposure is unknown. We hypothesise that in utero exposure to LPS induces a deficiency of mitochondrial complex activity and oxidative damage in the fetal diaphragm. To test this hypothesis, we used a well-established preterm ovine model of chorioamnionitis: Pregnant ewes received intra-amniotic (IA) saline or 10 mg LPS, at 2 d or 7 d prior to surgical delivery at 121 d GA (term = 150 d). The fetus was killed humanely immediately after delivery for tissue sampling. Mitochondrial fractions were prepared from the isolated diaphragm and mitochondrial electron transfer chain activities were evaluated using enzymatic assays. Oxidative stress was investigated by quantifying mitochondrial oxidative protein levels and determining antioxidant gene and protein (catalase, superoxide dismutase 2 and glutathione peroxidase 1) expression. The activity of the erythroid 2-related factor 2 (Nrf2)-mediated antioxidant signalling pathway was examined by quantifying the Nrf2 protein content of cell lysate and nuclear extract. A 2 d LPS exposure in utero significantly decreased electron transfer chain complex II and IV activity (p<0.05). A 7 d LPS exposure inhibited superoxide dismutase 2 and catalase expression at gene and protein levels, and Nrf2 pathway activity (p<0.05) compared with control and 2 d LPS groups, respectively. Diaphragm mitochondria accumulated oxidised protein after a 7 d LPS exposure. We conclude that intrauterine exposure to LPS induces mitochondrial oxidative stress and electron chain dysfunction in the fetal diaphragm, that is further exacerbated by impairment of the antioxidant signalling pathway and decreased antioxidant activity.

Interleukin-1 in lipopolysaccharide induced chorioamnionitis in the fetal sheep.

We tested the hypothesis that interleukin 1 (IL-1) mediates intra-amniotic lipopolysaccharide (LPS)-induced chorioamnionitis in preterm fetal sheep. Time-mated Merino ewes with singleton fetuses received IL-1α, LPS, or saline (control) by intra-amniotic injection 1 to 2 days before operative delivery at 124 ± 1 days gestational age (N = 5-9/group; term = 150 days). Recombinant human IL-1 receptor antagonist (rhIL-1ra) was given into the amniotic fluid 3 hours before intra-amniotic LPS or saline to block IL-1 signaling. Inflammation in the chorioamnion was determined by histology, cytokine messenger RNA (mRNA), protein expression, and by quantitation of activated inflammatory cells. Intra-amniotic IL-1 and LPS both induced chorioamnionitis. However, IL-1 blockade with IL-1ra did not decrease intra-amniotic LPS-induced increases in pro-inflammatory cytokine mRNAs, numbers of inflammatory cells, myeloperoxidase, or monocyte chemotactic protein-1-expressing cells in the chorioamnion. We conclude that IL-1 and LPS both can cause chorioamnionitis, but IL-1 is not an important mediator of LPS-induced chorioamnionitis in fetal sheep.