Mariangela Albertini

Carbon monoxide pretreatment prevents respiratory derangement and ameliorates hyperacute endotoxic shock in pigs

Endotoxic shock, one of the most prominent causes of mortality in intensive care units, is characterized by pulmonary hypertension, systemic hypotension, heart failure, widespread endothelial activation/injury, and clotting culminating in disseminated intravascular coagulation and multi‐organ system failure. In the last few years, studies in rodents have shown that administration of low concentrations of carbon monoxide (CO) exerts potent therapeutic effects in a variety of diseases/disorders. In this study, we have administered CO (one our pretreatment at 250 ppm) in a clinically relevant, well‐characterized model of LPS‐induced acute lung injury in pigs. Pretreatment only with inhaled CO significantly ameliorated several of the acute pathological changes induced by endotoxic shock. In terms of lung physiology, CO pretreatment corrected the LPS‐induced changes in resistance and compliance and improved the derangement in pulmonary gas exchange. In terms of coagulation and inflammation, CO reduced the development of disseminated intravascular coagulation and completely suppressed serum levels of the proinflammatory IL‐1β in response to LPS, while augmenting the anti‐inflammatory cytokine IL‐10. Moreover, the effects of CO blunted the deterioration of kidney and liver function, suggesting a beneficial effect in terms of end organ damage associated with endotoxic shock. Lastly, CO pretreatment prevents LPS‐induced ICAM expression on lung endothelium and inhibits leukocyte marginalization on lung parenchyma.

Respiratory Electrodialysis. A Novel, Highly Efficient Extracorporeal CO2 Removal Technique

RATIONALE We developed an innovative, minimally invasive, highly efficient extracorporeal CO2 removal (ECCO2R) technique called respiratory electrodialysis (R-ED). OBJECTIVES To evaluate the efficacy of R-ED in controlling ventilation compared with conventional ECCO2R technology. METHODS Five mechanically ventilated swine were connected to a custom-made circuit optimized for R-ED, consisting of a hemofilter, a membrane lung, and an electrodialysis cell. Electrodialysis regionally modulates blood electrolyte concentration to convert bicarbonate to CO2 before entering the membrane lung, enhancing membrane lung CO2 extraction. All animals underwent three repeated experimental sequences, consisting of four steps: baseline (1 h), conventional ECCO2R (2 h), R-ED (2 h), and final NO-ECCO2R (1 h). Blood and gas flow were 250 ml/min and 10 L/min, respectively. Tidal volume was set at 8 ml/kg, and respiratory rate was adjusted to maintain arterial Pco2 at 50 mm Hg. MEASUREMENTS AND MAIN RESULTS During R-ED, chloride and H(+) concentration increased in blood entering the membrane lung, almost doubling CO2 extraction compared with ECCO2R (112 ± 6 vs. 64 ± 5 ml/min, P < 0.001). Compared with baseline, R-ED and ECCO2R reduced minute ventilation by 50% and 27%, respectively. Systemic arterial gas analyses remained stable during the experimental phases. No major complication occurred, but there was an increase in creatinine level. CONCLUSIONS In this first in vivo application, we proved electrodialysis feasible and effective in increasing membrane lung CO2 extraction. R-ED was more effective than conventional ECCO2R technology in controlling ventilation. Further studies are warranted to assess the safety profile of R-ED, especially regarding kidney function.

Expression of endothelin-1 system in a pig model of endotoxic shock

Endothelin (ET)-1 is a potent vasoconstrictive peptide and it is involved in the pathogenesis of septic shock. Blockade of ET-1 receptors abolishes the LPS-induced pulmonary hypertension and worsens the LPS-dependent systemic hypotension, but the role of ET-1 in sepsis remains uncertain. To determine the role of ET-1 in cardiovascular and respiratory derangement in a porcine model of endotoxemic shock we evaluated ET-1 plasma levels and ET-1 mRNA and protein levels in lung, liver, and heart as well as Endothelin Converting Enzyme-1, ET(A) and ET(B) receptors mRNA in the same tissues. Twelve piglets were randomised to sham operated or to LPS-treated (40 microg/kg/h for 4 h) groups. During the experiment, respiratory and circulatory parameters have been recorded and blood samples collected. At the end of the experiment the animals were sacrificed and tissue samples collected for real-time quantitative PCR and ELISA test. LPS infusion evokes a large increase in ET-1 plasma concentration, and in tissues mRNA levels, associated with an increase in pulmonary arterial pressure, as well as in pulmonary and systemic vascular resistances, and a decrease in stroke volume. LPS infusion caused also a derangement of respiratory mechanics, evidenced by an increase in resistance and a decrease in compliance of the respiratory system. ET(A) and ET(B) receptor mRNA levels were markedly decreased in liver and lung and slightly increased in heart, evidencing that ET receptor subtypes were differentially regulated in the major organs of endotoxin treated pigs. In conclusion our data show the presence of a continuative and differentially regulated stimulating mechanism of ET-1 expression during pig endotoxaemia as well as a fundamental role of ET-1 system in the cardiovascular and respiratory derangement.

Regional blood acidification enhances extracorporeal carbon dioxide removal: a 48-hour animal study

Background:Extracorporeal carbon dioxide removal has been proposed to achieve protective ventilation in patients at risk for ventilator-induced lung injury. In an acute study, the authors previously described an extracorporeal carbon dioxide removal technique enhanced by regional extracorporeal blood acidification. The current study evaluates efficacy and feasibility of such technology applied for 48 h. Methods:Ten pigs were connected to a low-flow veno-venous extracorporeal circuit (blood flow rate, 0.25 l/min) including a membrane lung. Blood acidification was achieved in eight pigs by continuous infusion of 2.5 mEq/min of lactic acid at the membrane lung inlet. The acid infusion was interrupted for 1 h at the 24 and 48 h. Two control pigs did not receive acidification. At baseline and every 8 h thereafter, the authors measured blood lactate, gases, chemistry, and the amount of carbon dioxide removed by the membrane lung (VCO2ML). The authors also measured erythrocyte metabolites and selected cytokines. Histological and metalloproteinases analyses were performed on selected organs. Results:Blood acidification consistently increased VCO2ML by 62 to 78%, from 79 ± 13 to 128 ± 22 ml/min at baseline, from 60 ± 8 to 101 ± 16 ml/min at 24 h, and from 54 ± 6 to 96 ± 16 ml/min at 48 h. During regional acidification, arterial pH decreased slightly (average reduction, 0.04), whereas arterial lactate remained lower than 4 mEq/l. No sign of organ and erythrocyte damage was recorded. Conclusion:Infusion of lactic acid at the membrane lung inlet consistently increased VCO2ML providing a safe removal of carbon dioxide from only 250 ml/min extracorporeal blood flow in amounts equivalent to 50% production of an adult man.

Effects of nitric oxide on diaphragmatic muscle endurance and strength in pigs

The aim of the study was to evaluate the effects of nitric oxide (NO) on diaphragmatic fatigue in fifteen anaesthetized, mechanically ventilated pigs, divided into three groups. The animals were pre‐treated with indomethacin (3 mg kg‐1, i.v.) to block the cyclo‐oxygenase pathway. To group 1 pigs (n = 6) NG‐nitro‐L‐arginine methyl ester (L‐NAME, 5 mg kg‐1 i.v.) was administered as a bolus to block endogenous NO production, while group 2 pigs (n = 6) were infused with sodium nitroprusside (SNP, 0.023 mg kg‐1, i.v.), a donor of NO. Group 3 pigs (n = 3) were used as the controls. We evaluated diaphragmatic strength by measuring the transdiaphragmatic pressure (P di) generated during bilateral phrenic nerve stimulation at 10, 20, 30 and 50 Hz, 15 V, while the diaphragmatic endurance was assessed by a 30s stimulation at 10 Hz, 15 V. Diaphragmatic index was assessed as the ratio of peak force between single twitches performed before and after the 30 s stimulation west. We also evaluated mean systemic (MAP) and pulmonary (MPAP) arterial pressures, pulmonary wedge pressure (PW), systemic (SVR) and pulmonary vascular resistance (PVR) and cardiac output (CO). L‐NAME increased MAP, MPAP, PW, SVR and PVR, but decreased CO. SNP caused a decrease in MAP, MPAP, PW and SVR, while PVR and CO did not change. The main finding of this study was that diaphragmatic strength was not significantly weakened after L‐NAME administration, except at 10 Hz, while it did not change after SNP infusion. However, both L‐NAME and SNP caused significant decreases in diaphragmatic endurance capacity. The fatigue appearing after L‐NAME is probably correlated with a decline in diaphragmatic blood flow, as evidenced by the increase in SVR and the decrease in CO, and consequently in oxygen supply. In contrast, the decrease in endurance capacity after SNP infusion can be attributed to a direct action of NO on skeletal muscle.

Extracorporeal carbon dioxide removal through ventilation of acidified dialysate: an experimental study.

BACKGROUND Extracorporeal (EC) carbon dioxide (CO(2)) removal (ECCO(2)R) may be a powerful alternative to ventilation, possibly avoiding the need for mechanical ventilation and endotracheal intubation. We previously reported how an infusion of lactic acid before a membrane lung (ML) effectively enhances ECCO(2)R. We evaluated an innovative ECCO(2)R technique based on ventilation of acidified dialysate. METHODS Four swine were sedated, mechanically ventilated, and connected to a venovenous dialysis circuit (blood flow, 250 ml/min). The dialysate was recirculated in a closed loop circuit including a ML (gas flow, 10 liters/min) and then returned to the dialyzer. In each animal, 4 different dialysis flows (DF) of 200, 400, 600, and 800 ml/min were evaluated with and without lactic acid infusion (2.5 mEq/min); the sequence was completed 3 times. At the end of each step, we measured the volume of CO(2)R by the ML (V(co2)ML) and collected blood and dialysate samples for gas analyses. RESULTS Acid infusion substantially increased V(co2)ML, from 33 ± 6 ml/min to 86 ± 7 ml/min. Different DFs had little effect on V(co2)ML, which was only slightly reduced at DF 200 ml/min. The partial pressure of CO(2) of blood passing through the dialysis filter changed from 60.9 ± 3.6 to 37.1 ± 4.8 mm Hg without acidification and to 32.5 ± 5.3 mm Hg with acidification, corresponding to a pH increase of 0.18 ± 0.03 and 0.03 ± 0.04 units, respectively. CONCLUSIONS Ventilation of acidified dialysate efficiently increased ECCO(2)R of an amount corresponding to 35% to 45% of the total CO(2) production of an adult man from a blood flow as low as 250 ml/min.

Role of endothelin ETA receptors in sepsis-induced mortality, vascular leakage, and tissue injury in rats

The role of endothelin ETA receptors in sepsis-induced mortality and edema formation was evaluated with a selective antagonist ABT-627 [2-(4-methoxyphenyl)-4-(1,3-benzodioxol-5-yl)-1-(N,N-di(n-butyl)amino carbonylmethyl)-pyrrolidine-3-carboxylic acid]. Sprague-Dawley rats received saline (control group), Escherichia coli endotoxin (10 mg/kg, sepsis group) or infusion of ABT-627 prior and immediately after saline and endotoxin injection. Mortality, edema formation (wet/dry ratios), and multiple tissue injury (indicated by serum concentrations of creatinine, urea, bilirubin, creatine kinase, lactate dehydrogenase, and aspartate aminotransferase) were monitored within 5 h. Endotoxin injection elicited 64% mortality, significantly augmented edema formation in liver, heart, lung, and kidney, and raised serum levels of tissue injury markers. Pretreatment with ABT-627 completely reversed endotoxin-induced mortality, significantly attenuated wet/dry ratios of the heart, liver, and kidney, but not lungs, and reduced serum levels of creatine kinase, creatinine, aspartate aminotransferase, and lactate dehydrogenase, but not that of urea and bilirubin. These results suggest that endothelin ETA receptors play a significant role in promoting mortality, edema formation (except in the lungs), and tissue injury in animals with severe sepsis.

Differential release of prostacyclin and nitric oxide evoked from pulmonary and systemic vascular beds of the pig by endothelin-1

The vascular effects of endothelin-1 (ET-1) and the release of prostacyclin and nitric oxide (NO) evoked by this peptide were analyzed in anesthetized, mechanically ventilated pigs. ET-1 induced biphasic responses in both the pulmonary and systemic vascular beds characterized by a transient hypotension followed by a long-lasting hypertension. To evaluate the involvement of prostacyclin and NO in the ET-1-dependent vascular response, we used indomethacin to block cyclooxygenase and NG-nitro-L-arginine methyl ester (L-NAME) to block NO synthase. The results show that the systemic hypotensive response to ET-1 is mediated by the release of prostanoids and NO, but these are not responsible for the pulmonary hypotension. Indomethacin reduced the hypertensive effect of ET-1, showing that this peptide can also activate release of vasoconstrictor cyclooxygenase metabolites. When L-NAME was administered after indomethacin, the pulmonary vasoconstrictor activity of ET-1 was counterbalanced by NO. By contrast, in pigs pretreated with indomethacin plus L-NAME ET-1 caused transient systemic vasoconstriction, followed by progressive reduction of vascular tone, probably because of release of vasodilator agents other than prostanoids or NO.

PGI2 and nitric oxide involvement in the regulation of systemic and pulmonary basal vascular tone in the pig.

In anesthetized, ventilated pigs we analyzed the involvement of nitric oxide (NO) and prostaglandins (PGs) in the regulation of systemic and pulmonary basal vascular tone. Endogenous release of NO was blocked by NG-nitro-L-arginine methyl ester (L-NAME) and prostanoid biosynthesis by indomethacin. Blocking NO raised pulmonary and systemic arterial pressure and vascular resistances. These effects show that in the pig there is continuous release of minute amounts of NO. Blocking prostanoid biosynthesis did not affect the vasoconstrictor effect of L-NAME on the pulmonary vascular bed, but significantly strengthened the hypertensive effect of L-NAME on the systemic vascular bed. These data show that different mechanisms regulate pulmonary and systemic vascular tone. Administration of a stable analogue of PGI2 to pigs pretreated with indomethacin reversed the systemic vasoconstrictor effect of L-NAME. In conclusion, our data show that NO especially modulates pulmonary vascular tone, while PGI2 preferentially modulates systemic vascular tone.

Hypoxic pulmonary vasoconstriction in pigs : role of endothelin-1, prostanoids and ATP-dependent potassium channels

This study investigated the mechanisms that may contribute to the hypoxic pulmonary vasoconstriction and compared the effects of hypoxia on pulmonary and systemic vascular beds. Six anesthetized spontaneously breathing pigs inhaled a hypoxic mixture (10% O2 in air) in control conditions and after pre-treatment with Indomethacin (3 mg kg(-1) i.v.) to block the cyclooxygenase pathway. During hypoxia, the Indomethacin pre-treated pigs were given Cromakalim (80 microg kg(-1) i.v.) to activate K+(ATP) channels. Bosentan (5 mg kg(-1) i.v.) was administered to block endothelin-1 receptors and then during hypoxia Cromakalim was administered as before. In all experimental conditions we recorded breathing pattern and vascular parameters: mean systemic and pulmonary arterial pressures; systemic and pulmonary vascular resistances; cardiac output; and heart rate. Vascular and respiratory responses to hypoxia were determined when PaO2 was reduced to 50 +/- 5 mmHg. The main finding was that in spontaneously breathing pigs, hypoxia induces pulmonary vasoconstriction and an increase in mean systemic arterial pressure, which are cyclooxygenase-independent. A role of endothelin-1 appears in both vascular districts, but pulmonary vasoconstriction may also be due to ET-1-dependent inhibition of K+(ATP) channels.