INTRODUCTION

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Severe infant respiratory distress syndrome (IRDS) and other pulmonary diseases in infancy and childhood with surfactant deficiency, induce an increase in pulmonary vascular resistance (1, 2). It has been suggested that cardiac output () is decreased during ventilatory support primarily as a result of reduced systemic venous return. Only at very high ventilatory pressures would be affected because of direct effects on pulmonary vascular resistance and myocardial performance (3, 4). In the acute phase of the adult respiratory distress syndrome (ARDS) with pulmonary hypertension, cardiac performance is affected through several interacting mechanisms such as an increase in the right ventricular (RV) preload (dilatation of the thin-walled right ventricle), followed by a reduced RV pump function in the descending limb of the Frank-Starling curve, resulting in a reduced left ventricular (LV) preload and a reduced LV compliance due to a leftward shift of the septum, together accountable for a lower with reduced myocardial perfusion (5). However, these findings may not be applicable in IRDS in the newborn, where the RV has a relatively much thicker wall than in the adult.

One of the new, promising techniques for the treatment of severe IRDS is partial liquid ventilation (PLV). However, this technique can cause a further rise in pulmonary vascular resistance (6). Also, it has been shown that during the early phases of liquid ventilation in adult cats, is reduced (7). Other investigators, however, found that is readily maintained during PLV in properly hydrated newborn piglets (8) or during PLV in an acute lung injury model in adult pigs (9), both during the acute phases after starting treatment. The relative importance of interacting factors on RV and LV pump function in the clinical setting of IRDS followed by PLV in the newborn is still obscure. It is therefore important to detect how the effects on (and stroke volume) are related to changes in chronotropy, inotropy, and preload (Frank-Starling mechanism).

The aims of the present study were to investigate the effects of an artificially induced IRDS and the influence of subsequent PLV on RV and LV systolic pump function and on pulmonary and systemic hemodynamics in the newborn lamb. Pump function of either the RV or LV was assessed by constructing pressure-volume (P-V) loops on-line and, from those, end-systolic P-V relations (ESPVR) by transient inflow reduction. The ESPVR has been shown previously to reflect the contractile state of the ventricle and to be relatively independent of afterload conditions (10). Ventricular pressures were measured by micromanometer-tip catheters, ventricular volumes by the conductance catheter method (11).

	METHODS

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Animal Preparation

Nine newborn lambs, age 5 to 10 d, weighing 3.2 to 6.4 kg, were used. For obtaining P-V loops, four lambs were studied for LV measurements (LV subgroup), four lambs were studied for RV measurements (RV subgroup), and in one lamb both LV and RV measurements were performed simultaneously. Remaining hemodynamic and respiratory parameters were obtained in all nine lambs. After induction with ketamine hydrochloride (3 mg/kg, intravenously), general anesthesia was maintained using continuous infusion with ketamine hydrochloride (7 to 30 mg/kg/h) and xylazine (1 mg/kg, intramuscularly). After intubation, animals were ventilated with the Babylog 8000, a pressure-controlled ventilator (Dräger Werke AB, Lübeck, Germany). Upon ventilation, pancuronium (0.2 mg/kg, intravenously) was administered for muscle paralysis. Ventilator settings and oxygen supply were adjusted to maintain aortic Pa_O2 and Pa_CO2 within the normal range. An intravenous infusion of 5% glucose in 0.45% NaCl solution was continued throughout the study (10 ml/kg/h). Sodium bicarbonate was supplemented if the arterial pH was lower than 7.30 and the base-deficit more than 5 mmol/L. Arterial blood gases and pH were measured using a Corning 178 pH/blood gas analyzer (Corning, Halstead, UK).

Instrumentation

Self-sealing sheaths, 5-French or 6-French, were placed in both left and right femoral arteries and veins. All catheters were positioned under fluoroscopic guidance. A 5-French Berman angiographic catheter (American Edwards Laboratories, Irvine, CA) was advanced into the midthoracic aorta to measure aortic pressure (Pao). An inflatable balloon catheter was placed in the inferior vena cava via the left or right femoral vein to reduce inflow to the heart transiently. Via an incision in the neck a 5-French micromanometer catheter (Millar Instruments, Houston, TX) was introduced into either the LV or the RV via the left carotid artery or the right jugular vein respectively. For measuring ventricular volume an eight-electrode conductance catheter (size 5-French, custom made by Webster Labs, Baldwin Park, CA) was placed in either the LV or the RV via a 5-French sheath in the left femoral artery or the left jugular vein respectively. For the LV a pigtailed catheter was used, for the RV a catheter without pigtail. The catheter was connected to a Leycom-Sigma-5 signal-conditioner-processor (CardioDynamics, Zoetermeer, The Netherlands) for assessment of ventricular volume (11). With single ventricular volume measurement, the normal 20-kHz electrical field was applied to the current-carrying electrodes of the conductance catheter. When biventricular volumes were measured, a 20-kHz field was used for one catheter whereas the other was operated with lower excitation frequency to avoid cross talk between the two catheters. Pulmonary artery pressure (Ppa) was measured using a balloon flotation catheter placed in situ via the left jugular vein or via the right femoral vein.

Assessment of Myocardial Contractility and Pump Performance

Right or left ventricular contractility was quantified by the ESPVR, represented by a straight line connecting the upper left hand corners of the P-V loops when volume is reduced by transient inflow restriction. The ESPVRs were quantified by their slope (E_ES) (10) and by the volume intercepts of the relation at a fixed pressure: 5 kPa (RV-V₅) for the right ventricle and 10 kPa (LV-V₁₀) for the left ventricle, respectively (12). These values for fixed pressures were chosen such that they fell into the actual end-systolic pressure range found during inflow restriction in these experiments. From each inflow intervention we also calculated the ventricular dP/dt_max-end-diastolic volume relationship (dP/dt_max-EDV) (15). The position of the dP/dt_max-EDV at a fixed EDV, henceforth called preload-corrected dP/dt_max, was used as an additional index of myocardial performance. Both an increase in the latter index and in E_ES, as well as a leftward shift of the ESPVR (i.e., reduced volume intercept) have been shown to reflect an increase in ventricular contractility (10, 13, 14, 16, 17). The method to measure LV volume by means of the conductance catheter in the (newborn) lamb has been described earlier (14, 18). Likewise, measurements of RV volume by this technique have been evaluated in rabbits (19) and swine (20). In short, a multielectrode catheter is employed to measure electrical conductances at five levels in the ventricle. Total conductance is calculated as the sum of the segmental conductances. Only segments located in the ventricle were included. The instantaneous conductance signals, G(t), are converted to volume signals, V(t), by: V(t) = (1/) ·  · L² · G(t)  V_c. Here,  is a dimensionless slope factor, L is the distance between the sensing electrodes,  is the resistivity of blood, and V_c is the correction volume for the conductance of the surrounding tissue (commonly referred to as parallel conductance).

To determine parallel conductance, necessary to obtain absolute ventricular volumes (11), a small bolus (0.5 to 1.0 ml) of hypertonic saline (6 M) was injected into either the inferior vena cava for RV measurements or the pulmonary artery for LV measurements. This calibration was repeated at every stage in the experimental protocol. Because we were mainly interested in within-animal effects, the slope factor for the left ventricle, _L, was not assessed and assumed to be 1. For the right ventricle we corrected the measurements by estimating its slope factor, _R. For this purpose, we assumed per kg body mass in RV and LV subgroups to be identical so that the _R/_L ratio could be calculated. This ratio was found to be 1.47 and hence was used to correct right ventricular volume data.

Hemodynamic Measurements

Pao and Ppa were monitored continuously using Statham P23Db strain-gauge transducers, and a Beckman R 612 (Beckman Instruments, Palo Alto, CA) or a Gould 2800s (Gould, Cleveland, OH) polygraph. Stroke volume (SV) and were analyzed from the conductance catheter signal in a beat-to-beat fashion, using dedicated software. was calculated by multiplying heart rate (HR) and SV. Subsequently, systemic and pulmonary vascular resistances were calculated by dividing mean Pao () or mean Ppa (), respectively, by , thus neglecting the (small) influence of systemic and pulmonary venous pressures. Reported data represent averaged values over the first 10 steady-state beats of each run.

Experimental Protocol

After instrumentation, 30 to 40 min were allowed for the animals to reach hemodynamic steady state (as judged by monitoring Pao, Ppa, and HR). Then IRDS was induced by multiple lung lavages with 50 to 60 ml/kg warm saline over 30 min. After 1 h of ventilatory and hemodynamic steady state, PLV was started and continued for 1 h as follows: After disconnecting the lambs from the ventilator, 25 to 35 ml/ kg body weight of preoxygenated and warmed (37° C) Rimar 101 perfluorocarbon (Miteni, Milan, Italy) was instilled into the trachea as a bolus in 30 s. Immediately thereafter, animals were reconnected and PLV was carried out with the same ventilator as used for gas respiration. Perfluorocarbon liquid was replenished as necessary to maintain the level of the expiratory meniscus in the endotracheal tube (23). At four stages ventilator settings, blood gases, Pao, Ppa, and ventricular pressure and volume as well as dP/dt_max were measured: at baseline conditions just before induction of IRDS, 1 h after the induction of steady-state IRDS (just before starting PLV), and at 30 min and 60 min after starting PLV. At these time points, ESPVRs were obtained by inferior vena cava occlusion and the following variables were calculated: ratio of arterial oxygen pressure to fraction of inspired oxygen (Pa_O2/FI_O2), SV, , systemic and pulmonary vascular resistances, RV-E_ES, OR LV-E_ES, RV-V₅ or LV-V₁₀, and preload corrected RV-dP/ dt_max or LV-P/dt_max.

Statistical Analysis

Data are summarized as means ± 1 SD. Differences between the RV and LV subgroups were tested using an unpaired Student's t test. The newborn lamb served as its own control. Analysis of variance (ANOVA) for repeated measurements was used to evaluate if there were significant changes in ventilatory, hemodynamic, and pulmonary parameters between the subsequent conditions. When a significant difference was found, ANOVA was followed by the Scheffe's procedure for comparison between the groups. Finally, differences between the various conditions for myocardial contractility of RV and LV, RV-E_ES or LV-E_ES, RV-V₅ or LV-V₁₀, and preload-corrected RV-dP/ dt_max or LV-P/dt_max were evaluated by multiple linear regression analysis. A p value of less than 0.05 was considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

No significant differences were detected for pH, blood gases, FI_O2, Pao, and Ppa between the RV and LV subgroups. We therefore used the pooled means of these two groups as baseline values in Table 1 and in Figure 1 for statistical analysis. Moreover, these findings provided justification for our assumption that normalized values were similar for the two groups (which was used to calibrate RV volumes; see METHODS). Autopsy showed a closed ductus arteriosus in all lambs. Table 1 shows the results of pH and blood gases during all conditions. No significant changes in these parameters were found between the subsequent conditions.

View larger version (20K): [in this window] [in a new window]   Figure 1.   Mean value (± SD) of PaO2/FIO2 ratio during baseline conditions, IRDS, 30 min after starting subsequent PLV (PLV-30), and 60 min after starting PLV (PLV-60). *p < 0.05 versus baseline; #p < 0.05 versus IRDS.

Figure 1 shows the results for the Pa_O2/FI_O2 ratio. The Pa_O2/ FI_O2 ratio showed a sharp decrease with the induction of IRDS (p < 0.05) from a mean (± SD) of 0.60 ± 0.13 at baseline to 0.10 ± 0.06 during IRDS, but a recovery toward baseline conditions during PLV (0.35 ± 0.13 at 30 min PLV and 0.26 ± 0.16 at 60 min PLV). Ventilator pressures increased considerably and significantly with the induction of IRDS and remained high during PLV. The peak inspiratory pressure showed a significant increase during IRDS (39 ± 6 cm H₂O) compared with the baseline conditions (15 ± 4 cm H₂O) (p < 0.0001). We were able to lower the peak inspiratory pressure slightly during subsequent PLV (30 min and 60 min) albeit not significantly versus IRDS.

Positive end-expiratory pressure (PEEP) was also significantly higher during IRDS (6 ± 3 cm H₂O) compared with baseline conditions (2 ± 1 cm H₂O). The decrease in the PEEP setting was significant during PLV (3 ± 2 cm H₂O at 30 min and 3 ± 2 cm H₂O at 60 min) as compared with IRDS conditions.

The effects of IRDS and subsequent PLV on Ppa and Pao are shown in Figure 2. Pao did not change significantly during IRDS or during subsequent treatment with PLV compared with baseline conditions. Ppa increased significantly during IRDS from 2.22 ± 0.58 to 3.60 ± 1.32 kPa during IRDS, and remained significantly increased during PLV compared with baseline conditions (p < 0.05) with 3.40 ± 0.86 kPa at 30 min PLV and 3.52 ± 0.84 kPa at 60 min PLV. Pulmonary vascular resistance was 8.72 ± 7.74 kPa/ml/min at baseline conditions, increased during IRDS to 12.18 ± 7.48 kPa/ml/min (not significant), and remained high, significantly when compared with baseline conditions, with 19.60 ± 21.31 kPa/ml/min at 30 min PLV and 16.39 ± 13.84 kPa/ml/min at 60 min PLV (both p < 0.05). Systemic vascular resistance did not change significantly during IRDS or during subsequent treatment with PLV compared with baseline conditions.

View larger version (44K): [in this window] [in a new window]   Figure 2.   Mean value (± SD) of Ppa and Pao during baseline conditions, IRDS, 30 min after starting subsequent PLV (PLV-30), and 60 min after starting PLV (PLV-60). *p < 0.05 versus baseline.

Table 2 gives the results for HR, SV, and . Neither of the parameters showed a change during the subsequent conditions. End-diastolic volume (EDV) of the RV did not change significantly during any of the conditions.

Assessment of Myocardial Performance

Figure 3 shows the RV-E_ES, RV-V₅, and RV-dP/dt_max as measures of ventricular contractility for the right ventricle. After the induction of IRDS there was a significant rise in RV-E_ES (from 1.22 ± 0.58 to 1.88 ± 0.64 kPa/ml during IRDS) as well as a significant lowering of RV-V₅ (from 1.88 ± 0.64 at baseline to 0.91 ± 0.46 ml during IRDS), both indicating an increase in RV contractility. During subsequent PLV, RV contractility remained higher as shown by a significantly higher RV-E_ES of 2.07 ± 0.71 kPa/ml at 30 min PLV and 1.69 ± 0.86 kPa/ml at 60 min PLV and a significantly lower RV-V₅ of 1.15 ± 0.35 ml at 30 min PLV and 1.01 ± 0.63 ml at 60 min PLV, consistent with a sustained increase in myocardial contractility. Preload-corrected RV-dP/dt_max also tended to increase during IRDS (from 112 ± 59 kPa/s to 172 ± 87 kPa/s during IRDS), whereas it returned toward baseline values during PLV, but these changes failed to reach statistical significance. Figure 4 shows the typical P-V loops for the right ventricle as found in baseline conditions and during IRDS. Compared with control, the IRDS loops, with unchanged preload, show a higher stroke work, higher end-systolic pressure, and higher peak developed pressure, whereas SV remained the same.

View larger version (31K): [in this window] [in a new window]   Figure 3.   Mean value (± SD) of RV-EES, RV-V5, and preload corrected RV-dPdtmax during baseline conditions, IRDS, PLV-30, and PLV-60. *p < 0.05 versus baseline.

View larger version (14K): [in this window] [in a new window]   Figure 4.   P-V loops for the RV at baseline conditions and during IRDS (dotted lines). Loops were selected from inferior vena cava occlusions with the same EDV. Lines indicate ESPVR in both conditions. Compared with control, the IRDS loops, with unchanged preload, show a higher stroke work, higher end-systolic pressure, and higher peak developed pressure, whereas SV remained the same.

Results for the LV contractility are shown in Figure 5. LV-E_ES, LV-V₁₀, and LV-dP/dt_max did not change significantly after the induction of IRDS or during subsequent PLV for 30 or 60 min.

View larger version (31K): [in this window] [in a new window]   Figure 5.   Mean value (± SD) of LV-EES, LV-V10, and preload corrected LV-dP/dtmax during baseline conditions, IRDS, PLV-30, and PLV-60.

Analysis of RV Parallel Conductance

Because intervention-related changes in parallel conductance, especially for the RV, were anticipated, we statistically analyzed RV-V_c values. Table 3 gives the results for right ventricular V_c (RV-V_c) and contractility (RV-E_ES, the R² of the ESPVR and RV-V₅) for all animals in the RV subgroup. Mean values of RV-V_c were 6.56 ± 2.28 ml at baseline, 6.59 ± 2.68 ml during IRDS, 7.27 ± 3.31 ml at 30 min PLV, and 7.44 ± 3.14 ml at 60 min PLV. Although there is a tendency to rise with the induction of PLV, these values were not statistically different from baseline conditions. Nevertheless, parallel conductance volumes of the RV increased during PLV in three animals, as would be expected, given the fact that perfluorocarbon liquid is conductive. However, it should be kept in mind that V_c is also dependent on , which generally dropped during the course of the experiments. This explains why the increase is not seen in two animals.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This study shows that IRDS deteriorated pulmonary function and increased pulmonary vascular resistance. Subsequent PLV had a considerable beneficial effect on pulmonary function with an increase in the Pa_O2/FI_O2 ratio as has been found before (23). However, the pulmonary vascular resistance remained high during PLV. Interestingly, myocardial contractility in the RV increased substantially with IRDS, whereas , SV, and EDV did not change significantly. In the LV there was only a tendency for an increase in contractility with IRDS. Although subsequent PLV had a beneficial effect on pulmonary function, there were no further changes in myocardial contractility in both RV and LV.

Our study specifically aimed at mimicking the clinical situation and the promising intervention, from the ventilatory point of view, in which the lungs are filled up to FRC with perfluorocarbon liquid. In studies with incremental dosages of perfluorocarbon liquid in rabbits (24) and sheep (25), it has been shown that filling of the lungs near FRC has maximal effect on pulmonary function. Filling of the lungs up to FRC should presumably create a maximal increase in pulmonary vascular resistance with consequent maximal effect on RV function.

Earlier studies of our group have indicated that the heart of the newborn lamb shows homeometric autoregulation (HAR or the Anrep effect) (26) for the LV with an increase in contractility after a rise in afterload (14). Our current results in the newborn lamb demonstrate that the RV shows the same pattern of HAR in response to increased pulmonary pressure. This is illustrated in Figure 4 where typical P-V loops for the RV at baseline conditions and during IRDS are shown. At matched EDV, systolic Ppa and thus, afterload for the RV, was substantially higher during IRDS whereas SV did not change. This is the classic picture of HAR: the ability to maintain SV at increased afterload without a change in preload conditions (26).

For the LV, the effect of increased afterloadaortic pressurein the intact circulation is by necessity accompanied by a similar increase in coronary perfusion pressure. It has often been assumed that this concomitant factor is responsible for the increased contractile performance of the LV (27), and likewise, increased coronary perfusion pressure invokes an increase in RV contractile performance in isolated canine hearts, albeit to a much lesser extent than for the LV (28). However, increase of RV afterload by raising Ppa does not entail an increase in coronary perfusion pressure and thus, the mechanism for the HAR effect, at least for the RV, must be sought to lie elsewhere. Our findings are consistent with a recent study of Szabo and coworkers (29) in which increased RV systolic function in response to pulmonary afterload increase by pulmonary artery constriction was found. However, their study was based on local measurement of segmental wall dimensions and, thus, does not directly show the classic picture of constant SV at constant EDV with increased pressure. By plotting pressure against segmental wall dimension with increasing Ppa, these investigators found that the pressure- dimension (P-D) loops displayed an almost vertical end-systolic P-D relation, whereas end-diastolic dimension increased little. The phenomenon was abolished by brain death (29). Also using dimensional measurements but converting them to approximate RV volume, P-V relations were investigated in the canine right ventricle by Karunanithi and coworkers, studying pulmonary constriction, among other factors (30). Whereas in their study no mention is made of HAR, it was shown that the slope of the ESPVR of the right ventricle increased by 45% in response to pulmonary artery constriction, i.e., similar to the increase in RV-E_ES found in our present study.

Whereas it has been hypothesized that HAR is caused by an increase in coronary perfusion, it is clear that this effect is not present in our experiments because Pao, and thus coronary perfusion pressure, did not change. Another explanation for the improved RV contractile performance might lie in the direct effect of increased pressure upon the RV wall, i.e., at the endocardial interface. Because of the extensive trabeculation, this interface area is large, which implies a realistic possibility for deformation of the myocardial cell membranes in close proximity to this surface. Such deformation may affect the so-called "stretch-activated channels" (31) sufficiently to invoke an increase in intracellular calcium activation: Even an increase of the transmembrane pressure gradient was shown by Morris (31) to cause the aforementioned effect. Alternatively, the endocardial endothelium, abundantly present in the same interface, might influence contractility in a positive way (32): It has been shown that mechanical stimulation of aortic endothelial cells can cause an endothelium-mediated increase in calcium concentrations in neighboring cells (33). A third possibility lies in a release of catecholamines in response to the stress caused by the injury. However, if this were the case we would have observed at least a similar effect on the contractile performance of the LV, especially since the ESPVRs of the LV have been shown to respond more strongly to low-dose dobutamine than the RV ESPVRs (13).

Our finding that volume-corrected dP/dt_max does not increase significantly with pulmonary afterload deserves attention because it is generally believed that E_ES and dP/dt_max behave similarly (15). This observation is corroborated by the study of Karunanithi and coworkers who also found no effect on dP/dt_max whereas E_ES increased with pulmonary constriction, but these investigators explained it tentatively by the noise in their data, exacerbated by the procedure of differentiating RV pressure (30). However, in our data noise in the pressure signal could be excluded. The discrepancy in behavior of dP/dt_max and the ESPVR for the RV as observed, may lie in the circumstance that the former quantity reflects an early-systolic, preejection parameter of contractile performance whereas the ESPVR is a parameter determined, by definition, at end-systole. Supposedly, the early systolic parameter dP/dt_max reflects the initial slope of the intracellular calcium concentration (34), whereas parameters later during systole reflect the amplitude and the total amount of calcium available for actin-myosin interaction. Indeed, the mechanism of the HAR effect has been argued to be similar to the mechanism involved in increased resting length, based on enhancement of calcium influx and/or myofilament sensitivity (35). If these analogies are valid, our data, as well as Karunanithi's (30), indicate that, at least in the RV, the initial speed of calcium influx is not affected during HAR.

Even though LV contractility did not change significantly in our model of IRDS, we cannot exclude that other factors also play a role in affecting RV (and possibly LV) contractility. In the clinical setting of ARDS there are more factors (i.e., myocardial perfusion, myocardial oxygenation, ventricular dimensions) that affect ventricular contractility (36). It has been shown that ventilatory support in IRDS (4) and filling of normal lungs with perfluorocarbon liquid can influence negatively (7). In the present study there was no change in , which is consistent with the findings of other investigators that is readily maintained during liquid ventilation in properly hydrated newborn piglets (8) or during PLV in an acute lung injury model in adult pigs (9), both in the acute phase after starting treatment.

The finding that PLV improves pulmonary function in IRDS, but that Ppa remains high will be of clinical significance. RV pump function was not compromised with IRDS and subsequent PLV in our acute study in the newborn animal, where the RV maintains its SV (and ) through the HAR effect, i.e., without having to invoke its Frank-Starling mechanism. Specifically, this means that no dilatation of the RVand especially its compliant free walloccurs, which, if it were present, might lead to remodeling and (eventually) RV failure. It is still uncertain whether RV contractility remains adequate through the positive inotropic HAR mechanism for longer duration. Further studies are required to investigate this aspect.

In summary, IRDS increased Ppa and RV contractility without significantly changing LV contractility. The RV maintained its output, accomplished by employing the HAR effect. Subsequent PLV improved lung function and decreased Pa_O2/ FI_O2 but cardiac performance was unaltered. In view of treatment modalities in acute pulmonary diseases with an increase in pulmonary vascular resistance, the direct role of increased RV afterload on biventricular performance is an important issue for future studies.