Main

In the human neonate GBS sepsis is often associated with pulmonary hypertension, hypoxemia, diminished cardiac output, and altered pulmonary mechanics(1, 2). In addition, the arterial hypoxemia and decreased tissue perfusion associated with changes in microcirculation lead to tissue hypoxia, resulting in cellular abnormalities marked by altered membrane function, impaired calcium regulation, mitochondrial dysfunction, anaerobic glycolysis and decreased ATP production(3–5). Deteriorating cardiac performance, hypotension, and metabolic acidosis may result in multiorgan system damage and death(6). It is therefore likely that therapy directed specifically at reversing these early hemodynamic changes and cellular abnormalities might play an adjunctive role in the prevention of the pathologic consequences of sepsis.

ATP is a high energy purine nucleotide that plays an essential role in intracellular metabolism and membrane function. In addition, it has become evident that extracellular ATP at low concentrations can influence many biologic processes, including vascular tone, cardiac function, bronchodilation, surfactant release, and platelet aggregation(7–9). MgCl2 in combination with ATP enhances the effect of ATP on cellular function and microcirculatory blood flow during shock and ischemia(4). Several studies have shown that ATP-MgCl2 improves cellular function after the ischemic injury, accompanying hemorrhagic shock, bowel ischemia, and endotoxic shock(3, 4, 10). In addition, investigators(11) have shown that ATP-MgCl2 restores cardiac output and decreases cytokine release during hemorrhagic shock and may act during hypoxic and thromboxane-induced pulmonary hypertension as a selective pulmonary vasodilator in low doses secondary to its stimulation of pulmonary purinergic receptors and its rapid metabolism in the pulmonary circulation(12–15). Furthermore, by reducing pulmonary artery pressure ATP-MgCl2 may also increase compliance in the early phase of sepsis(2, 16).

Based on the above properties we hypothesized that the pulmonary hypertension, myocardial and metabolic dysfunction, and decreased compliance and bronchoconstriction observed in the septic model would be ameliorated by an infusion of ATP-MgCl2. The aim of this study was to evaluate the effects of ATP-MgCl2 on the hemodynamic and pulmonary manifestations of GBS sepsis in a young animal model. To date no experiments have been published describing the use of ATP-MgCl2 in septic newborn animals. We chose the piglet because its cardiovascular response to sepsis has been well studied and appears to mimic the clinical presentation of septic shock in the neonate.

METHODS

Animal preparation. Fourteen Yorkshire piglets were anesthetized with sodium pentobarbital (30 mg/kg intraperitoneally). A tracheostomy was performed and a 5-mm endotracheal tube placed. Femoral vessels were cannulated and used for measurement of systemic arterial blood pressure and blood sampling. The left external jugular vein was cannulated and a double lumen catheter advanced into the right atrium for measurement of Pra, injection of ice-cold saline for measurement of cardiac output, and infusion of ATP-MgCl2. A 5 Fr Swan-Ganz thermodilution catheter was introduced into the right external jugular vein and advanced under fluoroscopy into the left pulmonary artery. Cardiac output was measured by thermodilution using a cardiac output computer (95510-A, Edwards Laboratory, Santa Ana, CA). The Swan Ganz catheter was also used to measure Ppa and Pwp. Heparinized normal saline(10 U/mL; maximum of 100 U/kg delivered during experiment) was infused continuously through the pulmonary artery catheter and an infusion of 6 mg/kg/min of 5% dextrose solution through a peripheral vein. Vascular pressures were measured with pressure transducers (model P23XL, Gould Instruments, Cleveland, OH) and recorded on a multichannel recorder.

The animals were ventilated with a timed-cycled, pressure-limited, infant ventilator (Bourns BP 200, Riverside CA). Peak inflation pressure was set at 12 cm H2O, positive end-expiratory pressure at 2 cm H2O, and respiratory rate at 40 breaths/min. Animals were ventilated with room air. Ventilator adjustments were made to keep the pH 7.50-7.65, Paco2 3.3-4.0 kPa, and Pao2 greater than 9.3 kPa, after which ventilator settings were not altered during the study. These values were chosen because of the rapid deterioration in hemodynamic function observed shortly after GBS infusion was begun, resulting in normal to mildly alkalotic values at 15 min. Arterial blood gases (170 pH Blood Gas Analyzer, Corning, Medfield, MA) were measured before and then 15 min after the bacterial infusion was begun and every 30 min until 240 min. Skin temperature was maintained at 38°C by means of a servocontrolled radiant warmer. Rectal temperature was continuously monitored with a thermistor probe (Yellow Springs Instrument Co., Yellow Springs, OH).

Animals were paralyzed with pancuronium bromide using an initial dose of 0.2 mg/kg IV followed by an infusion of 0.4 mg/kg/h after a 60-min stabilization period. Baseline cardiovascular measurements (Psa, Ppa, cardiac output, Pwp, and Pra) and arterial blood gases were obtained before bacterial infusion. These values were referred to as baseline. Chloral hydrate (50 mg/kg every 3 h) given via nasogastric tube shortly after the surgery was completed was used to sedate animals throughout the study period.

Lung mechanics were measured before and then 15 min after the bacterial infusion was begun and every 30 min for 240 min. Respiratory flow was measured using a Fleisch No. 0 pneumotachometer (OEM Medical, Richmond, VA), a differential pressure transducer (model MP45, Validyne Engineering Co., Northridge, CA), and a pressure amplifier (Grass, Quincy, MA). The flow signal was electronically integrated to obtain tidal volume using a Grass integrator amplifier. Calibration of tidal volume was done before and after each study using a calibrated glass syringe. Esophageal pressure was measured using a water filled 8 Fr feeding tube placed in the lower esophagus and attached to a pressure transducer (model P23XL, Gould Instrument) and a Grass pressure amplifier calibrated with a water manometer. Air flow, tidal volume, and airway and esophageal pressures were recorded by way of a multichannel recorder (model 7, Grass Instruments). Cdyn and total RL were calculated from the tracings, using the method of Mead and Whittenberger(17).

Bacterial preparation. Group B β-hemolytic streptococci type Ia/c isolated from an infected neonate cared for in the Neonatal Intensive Care Unit at Jackson Memorial Hospital were cultured in Todd-Hewitt broth for 18 h at 37°C. The organisms were collected by centrifugation, washed twice in pyrogen-free saline, and resuspended in sterile Ringer's lactate solution with 5% dextrose at a concentration determined by OD measurements to be equivalent to 7.9 × 109 colony-forming units/mL. The bacterial cell suspension was free of endotoxin as determined by a Limulus amebocyte lysate test (Associate of Cape Cod, MA) which had a sensitivity of >0.03 endotoxin unit/mL.

Drug preparation. The disodium salt of ATP (Sigma Chemical Co., St. Louis, MO) was dissolved in sterile water and magnesium chloride was added to give an ATP:MgCl2 ratio of 3:1 by weight. The pH of the solution was adjusted to 7.0 ± 0.5 using 0.1 M NaOH(15).

PROTOCOL

A dose-response study tested a range of ATP-MgCl2 (0.2-2.0μmol/kg/min) infusions beginning 15 min after the GBS-induced pulmonary hypertension was noted in animals (n = 10). ATP-MgCl2 infusion rates at or above 0.6 μmol/kg/min resulted in marked reduction in[horizontal bar over]Ppa, [horizontal bar over]Psa, PVR, and SVR, and an increase in cardiac output. Lower rates of infusion had minimal effect on these hemodynamic parameters. As a result of the greater reduction in the PVR/SVR ratio with 0.6 μmol/kg/min of ATP-MgCl2 compared with 2.0μmol/kg/min (-33 ± 8% versus -10 ± 12%), the former dose was chosen for this study.

Bacteria were infused through a femoral vein at a rate calculated to deliver approximately 7.5 × 107 colony-forming units/kg/min. This infusion was continued until the animals died or 240 min had elapsed. Study animals were then randomly assigned to a treatment (n = 7)([horizontal bar over]X ± SD; weight, 2.8 ± 0.8 kg; age, 7 ± 3 d) or control group (n = 7) (weight, 2.7 ± 0.7 kg; age, 7 ± 1 d). The treatment group received a continuous infusion of 0.6 μmol/kg/min ATP-MgCl2 starting 15 min after the onset of bacteria-induced pulmonary hypertension and continued throughout the remainder of the 240-min study period. The control group received a continuous infusion of normal saline at the same rate as the continuous infusion of ATP-MgCl2.

Systemic and pulmonary arterial pressures were measured continuously throughout the baseline and study periods.Cardiac output, Pwp, Pra, arterial blood gases, Cdyn and RL were measured before and during GBS infusion. These measurements were repeated every 15 min for the 1st h and then every 30 min until 240 min.

Handling and care of the animals were in accordance with the guidelines of the National Institute of Health and this study protocol was approved by the Animal Care Committee of the University of Miami School of Medicine.

DATA ANALYSES

Repeated measures analysis of covariance was used to compare the pattern of responses after 15 min of GBS infusion between the ATP-MgCl2 and control groups, both over time (time-treatment interaction) and independent of time (overall group difference) for the dependent variables. The 15-min post-GBS baseline measurement of each variable was the covariate. The Huyn-Feldt correction was used to adjust for the effects of correlation between repeated measures. A Pearson correlation was used to evaluate the relationships between Cdyn and [horizontal bar over]Ppa as well as PVR at different time periods. Data are expressed as mean ± SD. Survival data were analyzed by the Mann-Whitney test as there were no censored cases. Values used in the analysis included the time from bacterial infusion through 120 min. Significant mortality in the control group prevented the analysis of data after this time period. Dependent variables in this study included[horizontal bar over]Ppa, [horizontal bar over]Psa, Pwp, stroke volume, cardiac output, pH, Pao2, Paco2, base deficit, PVR, SVR, PVR/SVR ratio, Cdyn, and RL.

RESULTS

Mean weight and age were not different between control and treatment groups. Baseline hemodynamic measurements before bacteria and after 15 min of bacterial infusion were also comparable between groups (Table 1).

Table 1 Hemodynamic values for the control (C) and ATP-MgCl2 (T) groups
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Mean [horizontal bar over]Ppa rose from a baseline of approximately 10± 2 to 45 ± 2 mm Hg after GBS infusion was begun in both groups. In the ATP-MgCl2-treated animals, [horizontal bar over]Ppa fell to 24± 4 mm Hg and subsequently remained significantly lower over the study period compared with control animals (time-treatment interaction, p< 0.001) (Fig. 1).

Figure 1
figure 1

(Upper) [horizontal bar over]Ppa was significantly lower in ATP-MgCl2 group (n = 7) compared with controls (n = 7) (time-treatment interaction, p < 0.001). (Lower) [horizontal bar over]Psa was significantly lower in ATP-MgCl2 treated group (time-treatment interaction, p < 0.003). Significant p values indicated by horizontal lines refer to time-treatment interactions; p values indicated with vertical lines refer to treatment effects.

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Mean [horizontal bar over]Psa increased from a baseline of approximately 77± 14 to 102 ± 13 mm Hg with bacterial infusion in both groups. In the treated animals [horizontal bar over]Psa decreased to 81 ± 8 mm Hg but then remained stable over baseline, whereas the [horizontal bar over]Psa in the control group declined significantly to 62 ± 27 mm Hg during this time period (time-treatment interaction, p < 0.003) (Fig. 1).

Cardiac output decreased significantly by 37 ± 2% with the onset of bacterial infusion in both groups of animals. Cardiac output in the treatment group increased to baseline values (0.26 ± 0.07 L/min/kg), whereas in the control animals it remained significantly lower during the study period(treatment effect, p < 0.001) (Fig. 2). Similarly, stroke volume decreased with GBS infusion. ATP-MgCl2 treatment resulted in a significantly greater stroke volume compared with placebo (treatment effect, p < 0.01) (Fig. 2). Heart rate initially decreased in the treatment group but was similar to the control group at 120 min (time-treatment interaction, p < 0.01) (Table 1).

Figure 2
figure 2

(Upper) Cardiac output was significantly higher in the ATP-MgCl2-treated group (treatment effect, p< 0.001). (Lower) Stroke volume was significantly higher in ATP-MgCl2-treated group (treatment effect, p < 0.01).

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PVR increased dramatically during GBS infusion from 35 ± 8 to approximately 245 ± 69 mm Hg/L/min/kg. ATP-MgCl2 treatment resulted in an early, precipitous decrease in PVR to 97 ± 27 compared with 197 ± 28 mm Hg/L/min/kg in the control animals (30 min values). Subsequently, PVR remained significantly lower in the treated animals(treatment effect, p < 0.0001) (Fig. 3).

Figure 3
figure 3

(Upper) PVR was significantly lower in ATP-MgCl2 group (treatment effect, p < 0.0001).(Lower) PVR/SVR ratio was significantly lower in ATP-MgCl2 group compared with controls (time-treatment interaction, p < 0.03). Vertical bars are used to denote treatment effect.

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SVR increased approximately 2-fold with GBS infusion in control and treatment groups. ATP-MgCl2 treated animals showed an early decrease to 368 ± 159 compared with 560 ± 66 mm Hg/L/min/kg in control animals (treatment effect, p < 0.01) (Table 1). Subsequently, SVR in the treated animals stabilized above pre-GBS baseline whereas SVR in the control animals showed a steady decline toward baseline, and at 120 min there were no significant differences between groups.

In an effort to compare the relative effects of ATP-MgCl2 treatment on pulmonary and systemic vascular resistances, the ratio of PVR/SVR was calculated. The ratio increased in both groups to approximately 0.39. In the treatment group the ratio of PVR/SVR decreased to 0.27 ± 0.05 compared with 0.36 ± 0.04 in the control animals. PVR/SVR was also significantly lower over time in the treated animals (time-treatment interaction,p < 0.03) (Fig. 3).

Animals developed a significant fall in Pao2 within 15 min of GBS infusion. Pao2 increased toward baseline with ATP-MgCl2 infusion whereas it remained significantly lower in the control animals (time-treatment interaction, p < 0.04) (Table 2). In both groups Paco2 increased after GBS infusion. It continued to increase in the control animals whereas it stabilized with ATP-MgCl2 treatment and at 120 min Paco2 was 7.0 ± 1.4 kPa in control animals compared with 3.9 ± 0.7 kPa in treated animals (time-treatment interaction,p < 0.02). pH was higher (time-treatment interaction, p< 0.001) and base excess significantly lower (time-treatment interaction,p < 0.005) in the treatment group (Table 2).

Table 2 Arterial blood gases and acid-base status for control (C) and ATP-MgCl2 (T) groups
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With GBS infusion, Cdyn decreased from approximately 1.13 to 0.82 mL/cm H2O in control and treatment groups. ATP-MgCl2 infusion was associated with an increase in Cdyn to 0.97 ± 0.086 mL/cm H2O at 30 min, followed by a slow decline to 0.84 ± 0.06 mL/cm H2O at 120 min. However, Cdyn in control animals remained lower(treatment effect, p < 0.001) (Table 3). RL increased with the onset of GBS infusion in both groups of animals. ATP-MgCl2 infusion resulted in a 20% decline in RL which was significantly lower than controls (treatment effect, p < 0.001)(Table 3). There was a negative correlation between Cdyn and [horizontal bar over]Ppa (r90 = -0.72,p < 0.003) as well as between Cdyn and PVR(r90 = -0.81, p < 0.001).

Table 3 Pulmonary function studies for control (C) and ATP-MgCl2 (T) groups
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In control animals, survival significantly declined after 120 min with no animals surviving beyond 180 min. All ATP-MgCl2 treated animals survived until 240 min, when they were killed (p < 001) (Fig. 4).

Figure 4
figure 4

Survival was significantly longer in the ATP-MgCl2 group compared with control animals (p < 0.001).

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DISCUSSION

Purine nucleotides such as ATP are well known vasodilators(18). Their vascular effects are mediated by stimulation of purinoceptors (P1, P2) on vascular endothelial and smooth muscle cells(7, 8, 19). P2y receptors, which are found predominantly in the pulmonary vascular endothelial cells, produce vasodilation by stimulating the release of prostacyclin (prostaglandin I2) and endothelial derived nitric oxide. Endothelial derived nitric oxide is believed to be responsible under most conditions for the vasodilator effect of ATP as prostaglandin I2 release occurs with higher doses of ATP. The decrease in [horizontal bar over]Ppa and PVR observed in our study may be related to an increase in endothelial derived nitric oxide synthesis and release. Previous studies have shown that ATP-induced pulmonary vasodilation in newborn and fetal lambs was reduced by inhibitors of nitric oxide synthase(20, 21). In addition, Bogleet al.(22) reported that ATP stimulates L-arginine uptake and nitric oxide release in vascular endothelial cells.

In this study we noted that the decline in PVR was greater than the fall in SVR, as reflected by a reduction in the PVR/SVR ratio, suggesting that ATP-MgCl2 has a somewhat greater effect on the pulmonary vasculature. This apparent selectivity may be secondary to the rapid metabolism of ATP in the pulmonary circulation and increased numbers of P2y receptors in the lung. The half-life of adenine nucleotides in the pulmonary circulation has been calculated to be less than 0.6 s(23). ATP is rapidly hydrolyzed mainly in the pulmonary circulation by ectoenzymes on the luminal surface of the endothelium to ADP, AMP, and adenosine, which are then taken up by active transport and rephosphorylated intracellularly(24). The exact site of pulmonary vasoconstriction in GBS-induced pulmonary hypertension is not known but is believed to occur in smaller caliber vessels which are the site of ATP activity(25).

GBS infusion resulted in elevated [horizontal bar over]Psa and SVR and decreased cardiac output. With ATP-MgCl2 treatment, [horizontal bar over]Psa and SVR decreased, whereas cardiac output increased. In spite of the early decrease, [horizontal bar over]Psa and SVR remained above pre-GBS baseline and were higher than control animals in the later phase of GBS sepsis. The dose-response experiments revealed that higher doses caused increased systemic vasodilator effects. Similar results were reported when higher doses of ATP-MgCl2 were used during hypoxia and infusion of U-46619, a thromboxane A2 mimetic(12–15). The greater systemic effects with higher doses of ATP-MgCl2 may be related either to saturation of the enzyme system responsible for its metabolism and/or circulation of metabolites of ATP such as ADP, AMP, and adenosine which may result in peripheral vasodilation(26).

Cardiac output increased in ATP-MgCl2 treated animals and was associated with increased stroke volume, decreased heart rate and no significant change in right atrial pressure. Previous studies have demonstrated that ATP-MgCl2 infusion in normal subjects(27), hypoxic lambs and piglets(12, 14, 15), and hemorrhagic shock models(11) resulted in increased cardiac output. ATP is a known positive inotropic and negative chronotropic agent(10). The cause of the increased cardiac output in this model of sepsis may be multifactorial(28), including 1) afterload reduction associated with decreased PVR and SVR(29, 30), 2) increased coronary blood flow(31, 32), and 3) decreased cytokine release (tumor necrosis-α, IL-2, and IL-6)(33).

The profound hypoxemia after GBS infusion was significantly ameliorated by ATP-MgCl2 treatment. Similar increases `in oxygenation were demonstrated with ATP-MgCl2 infusion in piglets and lambs after thromboxane A2 mimetic infusion(14, 15). The exact mechanism of this hypoxemia is not known. Sorenson et al.(34) suggested that hypoxemia develops as the result of decreased cardiac output and mismatching of pulmonary blood flow to alveolar ventilation. In addition, endotoxin(35) and thromboxane A2(36) produce bronchoconstriction which may decrease oxygenation. The observed improvement in oxygenation may be related to the effect of ATP-MgCl2 in producing bronchodilation(7) and increasing cardiac output(32).

Dynamic lung compliance decreased significantly with GBS infusion, whereas RL rose minimally. Treatment with ATP-MgCl2 resulted in early improvement in Cdyn and RL, and delayed the deterioration in pulmonary function. Byrne et al.(16) showed that alteration in lung function occurred before changes in lung water and protein increase. These investigators noted that the early changes in both dynamic and static lung compliance correlated temporally with peripheral neutropenia and increase in [horizontal bar over]Ppa. The results of the present study showing a negative correlation between lung compliance and[horizontal bar over]Ppa as well as with PVR are in agreement with their findings and the data of other investigators(2, 37). The modest increase in RL noted in this model is in contrast to the dramatic rise observed by investigators using septic adult animals(35), but is not inconsistent with this young animal model which has limited bronchoconstrictor tone(2). The observed decrease in RL may in fact be secondary to bronchodilatory properties of ATP.

Another potential benefit of ATP-MgCl2 was the improvement in base deficit and pH. In spite of the lack of well defined evidence for cellular energy deficit in sepsis and multiorgan failure, some investigators(3–5) have suggested that a metabolic abnormality may exist which can theoretically be ameliorated with ATP-MgCl2. However, evidence for the effect of ATP in altering this metabolic abnormality is circumstantial. The improvement in base deficit and pH observed in this study was most likely related to increased microcirculatory blood flow associated with the increase in cardiac output and decrease in SVR. In addition, a recent study by Konduri(31) has shown that ATP-infusion in hypoxic lambs decreased myocardial and systemic lactate levels by improving O2 delivery. Earlier studies have also shown that ATP-MgCl2 infusion during hypovolemic and normovolemic conditions decreased total body and myocardial oxygen consumption(32, 38).

The rationale for using the ATP-MgCl2 complex was its reported increased effectiveness compared with ATP, adenosine, or adenosine-MgCl2 in reducing hypoxic pulmonary hypertension(12) and improving cellular function and survival in animals experiencing shock or ischemic injury(13). MgCl2 possibly inhibits the deamination and dephosphorylation of ATP and prevents it from complexing with other vascular ions(39). In addition, it may replenish cellular magnesium levels in shock(4, 40) and enhance several metabolically important reactions especially those involving ATP(41). Magnesium is a known vasodilator and in relatively high doses has been studied in hypoxic and GBS-induced pulmonary hypertension(42, 43). However, at the dose used in this study we found that MgCl2 had no hemodynamic effects. Similar results have been reported by other investigators using equivalent doses of MgCl2 in lambs and piglets(12, 15).

It is possible that the anesthesia and sedation used in this study may have influenced the cardiovascular changes and survival during GBS infusion. The administration of sodium pentobarbital to adult rats has been shown to decrease basal cardiac output and SV, and increase mean aortic pressure, HR and SVR when compared with awake states. However, the hemodynamic changes observed after endotoxin infusion were not significantly different between conscious and pentobarbital-treated animals(44). Furthermore, the mortality and changes in small bowel pathology were similar in both groups(44). The doses of pentobarbital used in the present study were similar in the control and ATP groups, thereby, minimizing the possible influence of anesthesia on the observed cardiovascular responses.

In summary, the results of the present study suggest that low dose ATP-MgCl2 preferentially reduces pulmonary vascular resistance, increases cardiac output, ameliorates the deterioration in lung function and acid-base status, and prolongs survival during GBS infusion in the piglet. These properties of ATP-MgCl2, and its lack of side effects, may make it attractive as an adjunctive therapeutic modality in sepsis and septic shock in the newborn.