Postnatal steroids as lung protective and anti-inflammatory in preterm lambs exposed to antenatal inflammation.

BACKGROUND: Lung inflammation and impaired alveolarization precede bronchopulmonary dysplasia (BPD). Glucocorticoids are anti-inflammatory and reduce ventilator requirements in preterm infants. However, high-dose glucocorticoids inhibit alveolarization. The effect of glucocorticoids on lung function and structure in preterm newborns exposed to antenatal inflammation is unknown. We hypothesise that postnatal low-dose dexamethasone reduces ventilator requirements, prevents inflammation and BPD-like lung pathology, following antenatal inflammation. METHODS: Pregnant ewes received intra-amniotic LPS (E.coli, 4 mg/mL) or saline at 126 days gestation; preterm lambs were delivered 48 h later. Lambs were randomised to receive either tapered intravenous dexamethasone (LPS/Dex, n = 9) or saline (LPS/Sal, n = 10; Sal/Sal, n = 9) commencing <3 h after birth. Respiratory support was gradually de-escalated, using a standardised protocol aimed at weaning from ventilation towards unassisted respiration. Tissues were collected at day 7. RESULTS: Lung morphology and mRNA levels for inflammatory mediators were measured. Respiratory support requirements were not different between groups. Histological analyses revealed higher tissue content and unchanged alveolarization in LPS/Sal compared to other groups. LPS/Dex lambs exhibited decreased markers of pulmonary inflammation compared to LPS/Sal. CONCLUSION: Tapered low-dose dexamethasone reduces the impact of antenatal LPS on ventilation requirements throughout the first week of life and reduces inflammation and pathological thickening of the preterm lung IMPACT: We are the first to investigate the combination of antenatal inflammation and postnatal dexamethasone therapy in a pragmatic study design, akin to contemporary neonatal care. We show that antenatal inflammation with postnatal dexamethasone therapy does not reduce ventilator requirements, but has beneficial maturational impacts on the lungs of preterm lambs at 7 days of life. Appropriate tapered postnatal dexamethasone dosing should be explored for extuabtion of oxygen-dependant neonates.

Dose-related cardiac outcomes in response to postnatal dexamethasone treatment in premature lambs.

BACKGROUND: Postnatal corticosteroids are used in the critical care of preterm infants for the prevention and treatment of bronchopulmonary dysplasia. We aimed to investigate the effects of early postnatal dexamethasone therapy and dose on cardiac maturation and morphology in preterm lambs. METHODS: Lambs were delivered prematurely at ~128 days of gestational age and managed postnatally according to best clinical practice. Preterm lambs were administered dexamethasone daily at either a low-dose (n = 9) or a high-dose (n = 7), or were naïve to steroid treatment and administered saline (n = 9), over a 7-day time-course. Hearts were studied at postnatal Day 7 for gene expression and assessment of myocardial structure. RESULTS: High-dose dexamethasone treatment in the early postnatal period led to marked differences in cardiac gene expression, altered cardiomyocyte maturation and reduced cardiomyocyte endowment in the right ventricle, as well as increased inflammatory infiltrates into the left ventricle. Low-dose exposure had minimal effects on the preterm heart. CONCLUSION: Neonatal dexamethasone treatment led to adverse effects in the preterm heart in a dose-dependent manner within the first week of life. The observed cardiac changes associated with high-dose postnatal dexamethasone treatment may influence postnatal growth and remodeling of the preterm heart and subsequent long-term cardiac function.

Anesthesia and analgesia for common research models of adult mice.

Anesthesia and analgesia are major components of many interventional studies on laboratory animals. However, various studies have shown improper reporting or use of anesthetics/analgesics in research proposals and published articles. In many cases, it seems "anesthesia" and "analgesia" are used interchangeably, while they are referring to two different concepts. Not only this is an unethical practice, but also it may be one of the reasons for the proven suboptimal quality of many animal researches. This is a widespread problem among investigations on various species of animals. However, it could be imagined that it may be more prevalent for the most common species of laboratory animals, such as the laboratory mice. In this review, proper anesthetic/analgesic methods for routine procedures on laboratory mice are discussed. We considered the available literature and critically reviewed their anesthetic/analgesic methods. Detailed dosing and pharmacological information for the relevant drugs are provided and some of the drugs' side effects are discussed. This paper provides the necessary data for an informed choice of anesthetic/analgesic methods in some routine procedures on laboratory mice.

Airway smooth muscle thickness and contraction are enhanced by intra-amniotic lipopolysaccharide in an ovine model of premature birth.

Abnormalities of the airway smooth muscle (ASM) layer in asthma may develop before birth. We hypothesize that antenatal inflammation causes physiological abnormalities of the ASM that predisposes asthma. This study determined the short-term effects of antenatal inflammation on the developing ASM. Fourteen pregnant ewes were randomly assigned to one of three groups. Fetal lambs were exposed to intra-amniotic injections of lipopolysaccharide (LPS, n = 4) or saline (controls; n = 5) at 127 days' gestational age (GA). Preterm lambs were surgically delivered at 129 days' GA and received intensive care for 7 days before euthanasia. Naïve fetal controls (n = 5) were delivered and euthanized at 136 days' GA. ASM force to acetylcholine was measured in bronchial rings and normalized to ring length (tension) and ASM cross-sectional area (stress). Airway narrowing (% volume) to acetylcholine was assessed in bronchial segments. Fetal controls were structurally and functionally similar to saline-exposed lambs. Compared with saline, LPS-exposed lambs had increased macrophages in lung tissue (P = 0.0002) and interleukin-8 in alveolar wash (P = 0.003). LPS exposure increased ASM thickness (P = 0.005), airway narrowing (P = 0.003), ASM tension (P = 0.0002), and contractile stress (P < 0.0001). Notably, LPS-exposed lambs were more dependent on mechanical ventilation, and both LPS (P < 0.001) and ventilation (P = 0.012) were independent factors in increasing ASM stress. Only LPS independently increased ASM thickness (P = 0.045). Results indicate that antenatal exposure to LPS and subsequent mechanical ventilation promotes intrinsic changes to the ASM that enhances bronchoconstriction. If persistent into postnatal life, these developmental abnormalities may contribute to the known association between chorioamnionitis and asthma.NEW & NOTEWORTHY Abnormalities of the airway smooth muscle (ASM) layer in asthma may develop before birth. Using an ovine model of antenatal inflammation, we demonstrate thickening and increased contraction of the premature ASM layer. If such physiological abnormalities persist throughout postnatal life, this represents a predisposition to an asthma diagnosis.

A proposed framework for holding intensive 3Rs workshops in laboratory animal science.

Principles of 3Rs are the backbone of today's animal research. Applying 3Rs in practice requires proper education and training. Although this could be achieved via long-term courses ranging from several weeks to years, in some circumstances a short-term course may be the only viable option. In this paper we define scientific and ethical objectives for running short-term 3Rs workshops. To meet these objectives, we propose a 2-day workshop curriculum and an ethical framework. The curriculum comprises theoretical and practical sessions and covers Replacement, Reduction, and Refinement strategies. The ethical framework defines the responsibilities of lecturers and attendees, and proposes the animals and facilities requirements to run the proposed workshop curriculum. The attendees will be assessed at the end of the workshop and they receive certificates for working under supervision until they are deemed competent at their intended functions. The proposed curriculum and framework are not subscriptive, instead they share the experience gained through running more than 60 laboratory animal 3Rs workshops.

Intrauterine inflammation exacerbates maladaptive remodeling of the immature myocardium after preterm birth in lambs.

BACKGROUND: Antenatal conditions that are linked with preterm birth, such as intrauterine inflammation, can influence fetal cardiac development thereby rendering the heart more vulnerable to the effects of prematurity. We aimed to investigate the effect of intrauterine inflammation, consequent to lipopolysaccharide exposure, on postnatal cardiac growth and maturation in preterm lambs. METHODS: Preterm lambs (~129 days gestational age) exposed antenatally to lipopolysaccharide or saline were managed according to contemporary neonatal care and studied at postnatal day 7. Age-matched fetal controls were studied at ~136 days gestational age. Cardiac tissue was sampled for molecular analyses and assessment of cardiac structure and cardiomyocyte maturation. RESULTS: Lambs delivered preterm showed distinct ventricular differences in cardiomyocyte growth and maturation trajectories as well as remodeling of the left ventricular myocardium compared to fetal controls. Antenatal exposure to lipopolysaccharide resulted in further collagen deposition in the left ventricle and a greater presence of immune cells in the preterm heart. CONCLUSIONS: Adverse impacts of preterm birth on cardiac structure and cardiomyocyte growth kinetics within the first week of postnatal life are exacerbated by intrauterine inflammation. The maladaptive remodeling of the cardiac structure and perturbed cardiomyocyte growth likely contribute to the increased vulnerability to cardiac dysfunction following preterm birth. IMPACT: Preterm birth induces maladaptive cardiac remodeling and adversely impacts cardiomyocyte growth kinetics within the first week of life in sheep. These effects of prematurity on the heart are exacerbated when preterm birth is preceded by exposure to intrauterine inflammation, a common antecedent of preterm birth. Inflammatory injury to the fetal heart coupled with preterm birth consequently alters neonatal cardiac growth and maturation and thus, may potentially influence long-term cardiac function and health.

Computed tomographic assessment of lung aeration at different positive end-expiratory pressures in a porcine model of intra-abdominal hypertension and lung injury.

BACKGROUND: Intra-abdominal hypertension (IAH) is common in critically ill patients and is associated with increased morbidity and mortality. High positive end-expiratory pressures (PEEP) can reverse lung volume and oxygenation decline caused by IAH, but its impact on alveolar overdistension is less clear. We aimed to find a PEEP range that would be high enough to reduce atelectasis, while low enough to minimize alveolar overdistention in the presence of IAH and lung injury. METHODS: Five anesthetized pigs received standardized anesthesia and mechanical ventilation. Peritoneal insufflation of air was used to generate intra-abdominal pressure of 27 cmH2O. Lung injury was created by intravenous oleic acid. PEEP levels of 5, 12, 17, 22, and 27 cmH2O were applied. We performed computed tomography and measured arterial oxygen levels, respiratory mechanics, and cardiac output 5 min after each new PEEP level. The proportion of overdistended, normally aerated, poorly aerated, and non-aerated atelectatic lung tissue was calculated based on Hounsfield units. RESULTS: PEEP decreased the proportion of poorly aerated and atelectatic lung, while increasing normally aerated lung. Overdistension increased with each incremental increase in applied PEEP. "Best PEEP" (respiratory mechanics or oxygenation) was higher than the "optimal CT inflation PEEP range" (difference between lower inflection points of atelectatic and overdistended lung) in healthy and injured lungs. CONCLUSIONS: Our findings in a large animal model suggest that titrating a PEEP to respiratory mechanics or oxygenation in the presence of IAH is associated with increased alveolar overdistension.

The effect of human amnion epithelial cells on lung development and inflammation in preterm lambs exposed to antenatal inflammation.

BACKGROUND: Lung inflammation and impaired alveolarization are hallmarks of bronchopulmonary dysplasia (BPD). We hypothesize that human amnion epithelial cells (hAECs) are anti-inflammatory and reduce lung injury in preterm lambs born after antenatal exposure to inflammation. METHODS: Pregnant ewes received either intra-amniotic lipopolysaccharide (LPS, from E.coli 055:B5; 4mg) or saline (Sal) on day 126 of gestation. Lambs were delivered by cesarean section at 128 d gestation (term ~150 d). Lambs received intravenous hAECs (LPS/hAECs: n = 7; 30x106 cells) or equivalent volumes of saline (LPS/Sal, n = 10; or Sal/Sal, n = 9) immediately after birth. Respiratory support was gradually de-escalated, aimed at early weaning from mechanical ventilation towards unassisted respiration. Lung tissue was collected 1 week after birth. Lung morphology was assessed and mRNA levels for inflammatory mediators were measured. RESULTS: Respiratory support required by LPS/hAEC lambs was not different to Sal/Sal or LPS/Sal lambs. Lung tissue:airspace ratio was lower in the LPS/Sal compared to Sal/Sal lambs (P<0.05), but not LPS/hAEC lambs. LPS/hAEC lambs tended to have increased septation in their lungs versus LPS/Sal (P = 0.08). Expression of inflammatory cytokines was highest in LPS/hAECs lambs. CONCLUSIONS: Postnatal administration of a single dose of hAECs stimulates a pulmonary immune response without changing ventilator requirements in preterm lambs born after intrauterine inflammation.

The role of abdominal compliance, the neglected parameter in critically ill patients - a consensus review of 16. Part 1: definitions and pathophysiology.

Over the last few decades, increasing attention has been paid to understanding the pathophysiology, aetiology, prognosis, and treatment of elevated intra-abdominal pressure (IAP) in trauma, surgical, and medical patients. However, there is presently a relatively poor understanding of intra-abdominal volume (IAV) and the relationship between IAV and IAP (i.e. abdominal compliance). Consensus definitions on Cab were discussed during the 5th World Congress on Abdominal Compartment Syndrome and a writing committee was formed to develop this article. During the writing process, a systematic and structured Medline and PubMed search was conducted to identify relevant studies relating to the topic. According to the recently updated consensus definitions of the World Society on Abdominal Compartment Syndrome (WSACS), abdominal compliance (Cab) is defined as a measure of the ease of abdominal expansion, which is determined by the elasticity of the abdominal wall and diaphragm. It should be expressed as the change in IAV per change in IAP (mL [mm Hg]⁻¹). Importantly, Cab is measured differently than IAP and the abdominal wall (and its compliance) is only a part of the total abdominal pressure-volume (PV) relationship. During an increase in IAV, different phases are encountered: the reshaping, stretching, and pressurisation phases. The first part of this review article starts with a comprehensive list of the different definitions related to IAP (at baseline, during respiratory variations, at maximal IAV), IAV (at baseline, additional volume, abdominal workspace, maximal and unadapted volume), and abdominal compliance and elastance (i.e. the relationship between IAV and IAP). An historical background on the pathophysiology related to IAP, IAV and Cab follows this. Measurement of Cab is difficult at the bedside and can only be done in a case of change (removal or addition) in IAV. The Cab is one of the most neglected parameters in critically ill patients, although it plays a key role in understanding the deleterious effects of unadapted IAV on IAP and end-organ perfusion. The definitions presented herein will help to understand the key mechanisms in relation to Cab and clinical conditions and should be used for future clinical and basic science research. Specific measurement methods, guidelines and recommendations for clinical management of patients with low Cab are published in a separate review.

The role of abdominal compliance, the neglected parameter in critically ill patients - a consensus review of 16. Part 2: measurement techniques and management recommendations.

The recent definitions on intra-abdominal pressure (IAP), intra-abdominal volume (IAV) and abdominal compliance (Cab) are a step forward in understanding these important concepts. They help our understanding of the pathophysiology, aetiology, prognosis, and treatment of patients with low Cab. However, there is still a relatively poor understanding of the different methods used to measure IAP, IAV and Cab and how certain conditions may affect the results. This review will give a concise overview of the different methods to assess and estimate Cab; it will list important conditions that may affect baseline values and suggest some therapeutic options. Abdominal compliance (Cab), defined as a measure of the ease of abdominal expansion, is measured differently than IAP. The compliance of the abdominal wall is only a part of the total abdominal pressure-volume (PV) relationship. Measurement or estimation of Cab is difficult at the bedside and can only be done in a case of change (removal or addition) in IAV. The different measurement techniques will be discussed in relation to decreases (ascites drainage, haematoma evacuation, gastric suctioning) or increases in IAV (gastric insufflation, laparoscopy with CO2 pneumoperitoneum, peritoneal dialysis). More specific techniques using the interactions between the thoracic and abdominal compartment during positive pressure ventilation will also be discussed (low flow PV loop, respiratory IAP variations, respiratory abdominal variation test, mean IAP and abdominal pressure variation), together with the concept of the polycompartment model. The relation between IAV and IAP is linear at low IAV and becomes curvilinear and exponential at higher volumes. Specific conditions in relation to increased (previous pregnancy or laparoscopy, gynoid fat distribution, ellipse-shaped internal abdominal perimeter) or decreased Cab (obesity, fluid overload, android fat distribution, sphere-shaped internal abdominal perimeter) will be discussed as well as their impact on baseline IAV, IAP, reshaping capacity and abdominal workspace volume. Finally, we suggest possible treatment options in situations of unadapted IAV according to existing Cab, which results in high IAP. A large overlap exists between the treatment of patients with abdominal hypertension and those with low Cab. The Cab plays a key role in understanding the deleterious effects of unadapted IAV on IAP and end-organ perfusion and function. If we can identify patients with low Cab, we can anticipate and select the most appropriate surgical treatment to avoid complications such as IAH or ACS.

Hyperpressure intraperitoneal fluid administration for control of bleeding after liver injury.

BACKGROUND: Acute hemorrhage is the principal cause of death in trauma patients, with most fatalities occurring during the pre-hospital phase. Recently, intra-abdominal insufflation by carbon dioxide has been shown to drastically reduce bleeding in vascular and splanchnic hemorrhagic animal models simulating the pre-hospital phase. Here, we propose that using dialysate fluid for increasing intra-abdominal pressure is at least as effective as gas with some potential advantages. MATERIALS AND METHODS: A novel method of inducing liver trauma was used in 24 White New Zealand rabbits randomized into three groups: intra-abdominal carbon dioxide insufflation (GAS) with 15 cm H(2)O pressure; intra-abdominal infusion of type III dialysate solution (DIAL) with the same pressure; no change in intra-abdominal pressure (CTRL). All groups received intravenous resuscitation when their mean arterial pressure was below 30 mmHg. Physiologic parameters were recorded during 20 min of bleeding. RESULTS: Red blood cell (RBC) volume loss in the DIAL and GAS was 45% and 48% lower than that in the CTRL, respectively (P < 0.0005). Similar trends were observed for losses in RBC count and hemoglobin (Hb). Final mean arterial pressure, arterial RBC, Hb, and hematocrit were higher in the DIAL and GAS than in the CTRL; glucose concentration in the DIAL group was significantly higher than that in the GAS and CTRL groups. No intravenous fluid therapy was needed in the DIAL group. CONCLUSIONS: Hyperpressure intraperitoneal dialysate administration successfully reduced bleeding after severe liver injury in rabbits. This method can potentially be used as an adjunct to increase patient survival during pre-hospital cares.

Integration of inspiratory and expiratory intra-abdominal pressure: a novel concept looking at mean intra-abdominal pressure.

BACKGROUND: The intra-abdominal pressure (IAP) is an important clinical parameter that can significantly change during respiration. Currently, IAP is recorded at end-expiration (IAPee), while continuous IAP changes during respiration (ΔIAP) are ignored. Herein, a novel concept of considering continuous IAP changes during respiration is presented. METHODS: Based on the geometric mean of the IAP waveform (MIAP), a mathematical model was developed for calculating respiratory-integrated MIAP (i.e. MIAPri=IAPee+i⋅ΔIAP), where 'i' is the decimal fraction of the inspiratory time, and where ΔIAP can be calculated as the difference between the IAP at end-inspiration (IAPei) minus IAPee. The effect of various parameters on IAPee and MIAPri was evaluated with a mathematical model and validated afterwards in six mechanically ventilated patients. The MIAP of the patients was also calculated using a CiMON monitor (Pulsion Medical Systems, Munich, Germany). Several other parameters were recorded and used for comparison. RESULTS: The human study confirmed the mathematical modelling, showing that MIAPri correlates well with MIAP (R2 = 0.99); MIAPri was significantly higher than IAPee under all conditions that were used to examine the effects of changes in IAPee, the inspiratory/expiratory (I:E) ratio, and ΔIAP (P <0.001). Univariate Pearson regression analysis showed significant correlations between MIAPri and IAPei (R = 0.99), IAPee (R = 0.99), and ΔIAP (R = 0.78) (P <0.001); multivariate regression analysis confirmed that IAPee (mainly affected by the level of positive end-expiratory pressure, PEEP), ΔIAP, and the I:E ratio are independent variables (P <0.001) determining MIAP. According to the results of a regression analysis, MIAP can also be calculated asMIAP=-0.3+IAPee+0.4⋅ΔIAP+0.5⋅IE. CONCLUSIONS: We believe that the novel concept of MIAP is a better representation of IAP (especially in mechanically ventilated patients) because MIAP takes into account the IAP changes during respiration. The MIAP can be estimated by the MIAPri equation. Since MIAPri is almost always greater than the classic IAP, this may have implications on end-organ function during intra-abdominal hypertension. Further clinical studies are necessary to evaluate the physiological effects of MIAP.

Blood oxygenation during hyperpressure intraperitoneal fluid administration in a rabbit model of severe liver injury: Evaluation of a novel concept for control of pre-hospital liver bleeding.

Oxygen is an essential part of the most important metabolic pathways in aerobic organisms. Oxygen delivery is merely dependent on blood, rendering blood loss a devastating event. Traumatic pre-hospital liver bleeding is a major cause of early trauma deaths in human and animals, with no established therapeutic method yet. Increasing intra-abdominal pressure (IAP) has been shown to reduce liver bleeding by half. Although reduction of blood loss could be in favor of blood oxygen delivery, however, the complex interaction between increased IAP and respiratory mechanics during severe hemorrhagic shock remained unclear. We used a novel model of liver trauma in 16 rabbits and randomly assigned them to either normotensive abdomen group or increased IAP by fluid infusion (HA) groups (n=8 each). Liver size and the amount of liver injury were evaluated. Various blood oxygenation parameters were recorded. Both groups were identical in terms of the liver size and injury. The HA group had significantly lower shock index. Arterial oxygen capacity and oxygen content were higher in the HA group. No significant statistical difference was seen between groups in terms of abdominal perfusion pressure; alveolar pressure of oxygen; dissolved oxygen in blood plasma; alveolar to arterial oxygen tension gradient; arterial to alveolar oxygen pressure ratio; the ratio between partial pressure of arterial oxygen and fraction of inspired oxygen; and respiratory index. In conclusion, the novel therapeutic method of increasing IAP by fluid infusion in a rabbit model of liver hemorrhage preserved blood oxygenation better than the classic therapeutic method.