Maternal diabetes induces changes in the umbilical cord gene expression.

INTRODUCTION: Since maternal diabetes may affect fetal development and the umbilical cord provides an extension of the fetal vasculature, we decided to investigate cords' biological responses to maternal diabetic milieu. METHODS: Using microarray analysis, we determined the gene expression profiles in the umbilical cords of six neonates born to type 1 diabetic mothers and in six control cords. Umbilical cord tissue was collected immediately after elective cesarean section. Expression data were confirmed by real-time polymerase chain reaction (11 genes). Additionally, the same umbilical cords were analyzed histologically. RESULTS: Two hundred eighty six genes were differentially expressed in the umbilical cords from diabetic pregnancies compared to the controls (fold change ±1.5 and P < 0.01). Maternal diabetes had a major effect on the expression of genes involved in vascular development (Bone morphogenetic protein 4, Delta-like 1, and Notch homolog 4), vessel wall integrity (Collagen type VIII alpha 1, Myocyte enhancer factor 2C, and Matrix metalloproteinase 2), and vascular function (Natriuretic peptide precursor B, Endothelin 1, Endothelin receptor B, Cyclooxygenase 1, and Phosphodiesterase 5A). Maternal diabetes was associated with thicker umbilical vein intima-media layers and larger umbilical vein and artery intima-media areas compared to the controls. DISCUSSION: Maternal diabetic environment seems to alter umbilical cord expression of genes involved in the regulation of vascular development and function with simultaneous umbilical vessel muscle layer thickening. These alterations suggest vascular phenotypic modifications, which in turn may lead to long-term vascular consequences in various tissues in infants of diabetic mothers.

Phospholipase A2 in meconium-induced lung injury.

Although the triggering mechanisms of tissue inflammation and injury in meconium-contaminated lungs are still unclear, there is increasing evidence to suggest a central role for phospholipase A(2)'s (PLA(2)). In fact, elevated PLA(2) activities together with high enzyme concentrations, especially the amount of pancreatic (group I) secretory PLA(2) (PLA(2)-I), have been detected in human meconium and in meconium-contaminated lungs. Recent data from our laboratory further indicate that human pancreatic PLA(2), introduced in high amounts within aspirated particulate meconium, is a potent inducer of lung tissue inflammatory injury. Our finding of elevated human PLA(2)-I concentrations in plasma during the first hours after intratracheal meconium administration in newborn piglets further suggests that intrapulmonary aspiration of meconium could also have systemic inflammatory and injurious effects. This, however, remains to be studied in further detail.

Gene expression of pulmonary cytokines after sevoflurane or thiopentone anaesthesia in pigs.

BACKGROUND: Volatile anaesthetics have diverse inflammatory effects on the lungs. They increase gene expression of some pro-inflammatory cytokines in alveolar macrophages whereas in alveolar type II cells they seem to decrease secretion and gene expression of pro-inflammatory cytokines. We have previously detected increased leukotriene C4, nitrate and nitrite concentrations in bronchoalveolar lavage fluid after sevoflurane anaesthesia. In the current study, we measured gene expression of inflammatory cytokines in the lung tissue and plasma concentrations of cytokines in pigs after thiopentone or sevoflurane anaesthesia. METHODS: Sixteen pigs were randomly selected to receive either a continuous thiopentone infusion (control group, n = 8) or sevoflurane (n = 8) at 4.0% inspiratory concentration (1.5 MAC) in air for 6 h. Tissue samples were collected at the end of the study for measurement of gene expression of inflammatory cytokines. Blood samples were collected during anaesthesia for measurement of plasma cytokine concentrations. RESULTS: Compared with thiopentone anaesthesia, lower gene expression of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in lung tissue was observed after sevoflurane anaesthesia. Of measured cytokines IL-1beta, TNF-alpha, IL-6, IL-8 and IL-10 only plasma concentrations of IL-6 could be measured during the study without a difference between the groups. CONCLUSION: Lower gene expression of TNF-alpha and IL-1beta was found in the intact porcine lung tissue after sevoflurane anaesthesia compared with thiopentone anaesthesia. Clinical significance of this finding is unknown.

Intravenous immunoglobulin g attenuates pulmonary hypertension but induces local neutrophil influx in meconium aspiration in piglets.

BACKGROUND: Pulmonary hypertension and inflammation are well-identified pathogenetic features in meconium aspiration syndrome of newborns, but current approaches to their treatment or prevention are still often unsatisfactory. OBJECTIVES: To investigate the possible protective effects of human intravenous immunoglobulin G (IVIG) on the hypertensive and inflammatory lung injury in severe neonatal meconium aspiration. METHODS: Eleven newborn (10-12 days old) ventilated and catheterized piglets that received an intratracheal bolus (3 ml/kg) of a 65-mg/ml mixture of human meconium were studied for 6 h. IVIG was infused in 5 piglets 30 min before meconium administration, and 6 piglets served as controls and received the vehicle only. RESULTS: Meconium instillation induced a biphasic pulmonary hypertensive response, which was significantly diminished by IVIG pretreatment. Similarly, IVIG improved the oxygenation of the piglets, but the intrapulmonary shunt fraction or systemic hemodynamic parameters did not differ between the study groups, except of a minor decrease in the mean arterial blood pressure caused by IVIG. The blood leukocyte count was comparable in the 2 groups. The lung tissue ultrastructural and histological changes, number of apoptotic cells and phospholipase A2 activity were similar in the 2 groups. The amount of neutrophil accumulation, assessed by myeloperoxidase activity, was however significantly increased in macroscopically damaged lung tissue after IVIG administration. CONCLUSIONS: Our results thus indicate that IVIG treatment of newborns with severe meconium aspiration significantly diminishes the pulmonary hypertensive response and improves oxygenation, but the effects do not extend to protection of lung cellular injury.

Pulmonary inflammatory mediators after sevoflurane and thiopentone anaesthesia in pigs.

BACKGROUND: Volatile anaesthetics have been shown to affect the release of pulmonary inflammatory mediators and exacerbate pulmonary injury after experimental aspiration. Thus, in theory, volatile anaesthetics may worsen inflammatory pulmonary injury and disease. We have previously described that no significant changes in alveolar ultrastructure are seen after sevoflurane anaesthesia. However, this does not exclude any possible physiological alterations. The aim of our study was to evaluate pulmonary inflammatory mediators in bronchoalveolar lavage (BAL) after sevoflurane and thiopentone anaesthesia in pigs with intact lungs. METHODS: Sixteen pigs were randomly selected to receive either a continuous thiopentone infusion (control group, n = 8) or sevoflurane (n = 8) at 4.0% inspiratory concentration (1.5 MAC) in air for 6 h. Bronchoalveolar lavage samples were collected at the end of the study to determine pulmonary inflammatory markers. RESULTS: Compared with thiopentone anaesthesia, significant increases in BAL leukotriene C4 (LTC4), NO3-, and NO2- levels were observed after sevoflurane anaesthesia. In addition, there was a significant decrease in total blood leukocyte count in sevoflurane-treated animals. CONCLUSION: We conclude that sevoflurane increases pulmonary LTC4, NO3-, and NO2- production in pigs, indicating an inflammatory response.

Alveolar integrity and ultrastructure in pigs remain undamaged after exposure to sevoflurane.

Previous studies have shown that both halothane and isoflurane have adverse but reversible effects on alveolar physiology. The present study was designed to test the hypothesis that also sevoflurane may affect alveolar integrity. Fifteen pigs were randomly selected to receive either thiopentone infusion (control group, n=8) or sevoflurane (n=7) at 4.0% inspiratory concentration (1.5 MAC) in air for 6 h. Tissue samples from the lungs were obtained at the end of the experiment. Both histopathological light microscopy and electron microscopy were used to assess the structural integrity of the alveoli. Pulmonary hemodynamics were comparable in both groups. Light microscopy showed no difference between the groups in the amount of alveolar macrophages, red blood cells or edema. Electron microscopy showed minor changes such as moderate local swelling of alveolar epithelium in both study groups. Alveolar type II cells were ultrastructurally unaltered in both study groups. We conclude that long-term, high concentration exposure to sevoflurane has no detrimental effect on the alveolar integrity in pigs.

Human meconium has high phospholipase A2 activity and induces cellular injury and apoptosis in piglet lungs.

Aspiration of meconium produces an inflammatory reaction resulting in necrotic changes in lung tissue. To further investigate the mechanisms of the meconium-induced early pulmonary injury, twenty 10-12-d-old piglets were studied for lung tissue ultrastructural and apoptotic changes and phospholipase A2 activity. Twelve piglets received an intratracheal bolus (3 mL/kg) of a 20-mg/mL (thin, n = 6) or 65-mg/mL (thick, n = 6) mixture of human meconium, and control piglets (n = 5) received the same amount of intratracheal saline. Three ventilated piglets with no aspiration were also studied. Pulmonary hemodynamics and systemic oxygenation were followed for 6 h after meconium or saline insufflation. In the control groups, the pulmonary tissue showed open alveolar spaces and intact vascular walls, whereas meconium administration resulted in severe pneumonitis, with alveolar spaces filled with inflammatory exudate. Meconium instillation additionally resulted in edematous changes in the vascular walls and alveolar epithelium, whereas type II pneumocytes were intact. The amount of apoptotic cells was increased, especially in the respiratory epithelium, and the catalytic activity of phospholipase A2 in lung tissue samples was significantly elevated after thick meconium instillation. This activity rise proved to be mainly because of human group I phospholipase A2, introduced by meconium. Our data thus show that aspiration of meconium leads to severe lung tissue inflammation with early ultrastructural changes in the pulmonary alveolar walls and is associated with apoptotic cell death in the epithelium, already during the first hours after the insult. These results further suggest that high phospholipase A2 activity, mainly introduced into the lungs within the meconium, may have an important role in the initiation of these alterations in neonatal lungs.

Involvement of thromboxane A2 and prostacyclin in the early pulmonary hypertension after porcine meconium aspiration.

Severe perinatal aspiration of meconium is frequently complicated by unsuccessful neonatal adaptation with associated pulmonary hypertension. This vascular complication is supposedly related to pulmonary release of vasoconstrictory agents, including metabolites of arachidonic acid. Thus, to investigate the role of prostanoids on these meconium-induced circulatory changes in the lungs, the hemodynamic response to meconium instillation was studied in acetylsalicylic acid-pretreated juvenile pigs. Twelve 10-wk-old pigs with adapted lung circulation received 3 mL/kg of 65 mg/mL human meconium via the endotracheal tube. Six of them were medicated with 10 mg/kg acetylsalicylic acid 30 min before meconium insufflation. Hemodynamic parameters and urinary excretion of stable metabolites of thromboxane A2 and prostacyclin were measured serially for 6 h after the insult. Meconium administration induced a biphasic increase in mean pulmonary artery pressure and pulmonary vascular resistance, and a rapid rise in urinary levels of prostanoid metabolites. Acetylsalicylic acid pretreatment prevented the initial (0-1 h) pulmonary hypertensive response and increase in prostanoid excretion. During the second phase (1-6 h), acetylsalicylic acid did not attenuate the progressive increase in mean pulmonary artery pressure and pulmonary vascular resistance nor did it affect the longitudinal distribution of the pulmonary resistances. Our results thus show that in adapted porcine lungs, arachidonic acid metabolites contribute to the early hypertensive response, but have only minor effects during the second phase vascular hypertension.

Endothelin-1, atrial natriuretic peptide and pathophysiology of pulmonary hypertension in porcine meconium aspiration.

To evaluate the role of endothelin-1 (ET-1) and atrial natriuretic peptide (ANP) in the development of meconium aspiration-induced pulmonary hypertension, plasma ET-1 and ANP levels were measured serially for 6h after meconium instillation in juvenile pigs. Eleven 10-week-old, anaesthetized and catheterized pigs received intratracheally a bolus of 3 ml kg(-1) 20% human meconium, and five of them were premedicated with 30 mg kg(-1) methylprednisolone i.v. Another six pigs served as controls and were given 3 ml kg(-1) sterile saline intratracheally. Meconium instillation resulted in an increase in plasma ET-1 levels with a significant correlation to the simultaneously increasing PVR (r = 0.72). Methylprednisolone had no effect on the early (0-1 h) ET-1 increase, but prevented significantly the second phase (1-6 h) rise with a concomitant attenuation of the progressive pulmonary hypertension. ANP concentrations were higher in the meconium than in the control group throughout the study and further increased after steroid treatment with a good correlation to ET-1 (r = 0.86). Thus, the postinjury rise in circulating vasoactive peptides, together with the pulmonary hypertensive response, and modulation of the peptide balance and pressor reaction by steroids, suggest a contributory role for ET-1 and ANP in the development of pulmonary hypertension after meconium aspiration.

Meconium aspiration induces a concentration-dependent pulmonary hypertensive response in newborn piglets.

To investigate the effects of aspirating different meconium concentrations on the pulmonary circulation in 10- to 12-day-old piglets, 30 catheterized animals were studied. The piglets received an intratracheal bolus of 3 ml/kg of a mixture of human meconium in saline with concentrations of 20 mg/ml (light, n = 7), 40 mg/ml (moderate, n = 6), or 65 mg/ml (thick, n = 10) meconium in saline. Control piglets (n = 7) received 3 ml/kg of intratracheal saline. Pulmonary and systemic pressures were measured and vascular resistances calculated at baseline and serially for 4 hours after instillation. Four of the piglets died early and were excluded from the study. In addition, 23 samples of human meconium-stained amniotic fluid were collected at delivery for determination of their meconium concentration. After an initial rise in pulmonary artery pressure and vascular resistance after meconium and saline instillation, pulmonary artery pressure and resistance increased progressively and concentration-dependently in the meconium groups, but returned to baseline in the control group. The saline and meconium-induced initial increases, and the subsequent meconium-stimulated progressive rise in vascular resistance occurred mainly in the postarterial segment. There were no significant changes in systemic hemodynamics. Mean airway pressure increased and oxygenation deteriorated after meconium instillation. The impairment of oxygenation depended on the meconium concentration in the instilled bolus and persisted throughout the study after moderate and thick meconium instillation. Similarly, the intrapulmonary shunt fraction increased initially and remained elevated in the moderate and thick meconium groups. Meconium concentrations in the human amniotic fluid samples were in the same range as concentrations used in the present experimental study. These results indicate that aspirated meconium at concentrations found in light to moderate meconium-stained human amniotic fluid has significant effects on pulmonary hemodynamic and oxygenation in newborn piglets.

Methylprednisolone attenuates the pulmonary hypertensive response in porcine meconium aspiration.

Severe neonatal aspiration of meconium is frequently complicated by fatal pulmonary hypertension. The protective effect of an i.v. bolus of methylprednisolone on meconium aspiration-induced hypertensive lung injury was studied in anesthetized pigs with adapted lung circulation. Eleven 10-wk-old pigs received 3 mL/kg 20% human meconium via the endotracheal tube. Five of them were pretreated with 30 mg/kg methylprednisolone 30 min before aspiration. Ventilator settings were adjusted to keep arterial PO2 above 8 kPa and arterial PCO2 below 5 kPa. Meconium insufflation induced a biphasic pulmonary pressor response during the 6 h follow-up. Methylprednisolone tended to prevent the early (0-1 h) increase in pulmonary artery pressure and inhibited significantly the second phase (1-6 h) progressive rise in pulmonary artery pressure and pulmonary vascular resistance. This inhibition of resistance increase was most profound in the postarterial segment of the lung circulation, as determined by pulmonary artery occlusion. Additionally, the methylprednisolone pretreated group demonstrated a significant decrease in venous admixture together with improved oxygenation during the late phase after the insult, and further showed evidence of diminished lung edema formation. Although meconium aspiration-induced fall in blood leukocyte concentration was inhibited by methylprednisolone pretreatment, no histologic difference was found in pulmonary leukocyte sequestration. Our results thus show that in adapted porcine lungs methylprednisolone pretreatment improves oxygenation and attenuates the meconium aspiration-induced pulmonary hypertensive response by preventing the increase in the postarterial resistance.

Meconium aspiration induces ARDS-like pulmonary response in lungs of ten-week-old pigs.

To investigate whether aspiration of meconium induces a hemodynamic and histologic pulmonary response similar to that frequently seen in experimental acute respiratory distress syndrome, twelve 10-week-old pigs with postnatally adapted lungs were studied. Six 10-week-old pigs received 3 ml/kg 20% human meconium via the endotracheal tube. Six control pigs of the same age were given sterile saline. Ventilator settings were adjusted to keep PaO2 above 8 kPa and PaCO2 below 5 kPa. The pulmonary hemodynamic response to aspiration consisted of two separate hypertensive components. An initial peak in pulmonary artery pressure (PAP) and pulmonary vascular resistance (PVR) was followed by a progressive increase in PAP and PVR in the meconium group, whereas in the saline group these parameters returned to baseline levels. The distribution of PVR, determined by pulmonary artery occlusion, was characterized by an increase in the postarterial resistance immediately after meconium aspiration and a progressive increase in both arterial and postarterial resistance during the later phase. On histological examination, marked neutrophil sequestration was seen in the meconium lungs. In addition, lung edema formation was significantly enhanced in the meconium group, as shown by an increased lung wet/dry weight ratio. Thus, meconium aspiration resulted in a biphasic pulmonary pressor response and severe pulmonary inflammation. This response resembled that of models of experimental acute respiratory distress syndrome following diverse types of precipitating insults; this suggests that similar pathophysiologic mechanisms are elicited and cause similar pulmonary dysfunction following different forms of lung injury.

Human meconium has potent antioxidative properties.

In vitro the antioxidative capacity of pooled and lyophilized human meconium, measured by chemiluminescence, was compared to that of three potent antioxidants: vitamin C, a vitamin E analogue and a synthetic antioxidant, butylated hydroxytoluene. Meconium showed a significant superoxide trapping and peroxidation prevention capacity, but its capacity to trap peroxyl radicals was minor. These effects of meconium were possibly due to bilirubin and ubiquinol-10, both found in high concentrations in meconium. It is speculated that human meconium may have a physiological role as an important endogenous antioxidant during perinatal transition.