Anesthesia was performed according to our previously published protocol ₂ , except that intramuscular premedication was performed with 1.5–2.0 mg/kg xylazine hydrochloride (Rompun; Bayer AG, Leverkusen, Germany). Arterial oxygen saturation was monitored by continuous-pulse oximetry at the shaved ear of the sheep, and arterial blood gases were controlled periodically (995-AVL; Medizintechnik GmbH, Bad Homburg, Germany).

Figure 1 illustrates the placement of the probe, lines, and catheters, and explains which catheters were used to monitor which parameters. Under X-ray guidance a conductance catheter (Zoetermeer, The Netherlands) was placed into the LV cavity, via the left femoral artery, along the long axis to permit continuous measurement of intraventricular pressure and volume. A polyethylene catheter was advanced 15 cm into the descending aorta from the right femoral artery, and another was advanced into the inferior vena cava from the femoral vein. A thermodilution catheter was advanced into the pulmonary artery via the left internal jugular vein to the superior vena cava to measure stroke volume. A Fogarty catheter was advanced into the inferior vena cava from the right femoral vein to achieve temporary rapid preload reduction by inflating the terminal balloon. Via a left-sided thoracotomy, a 5-MHz echoprobe (T6210, Hewlett Packard, Andover, MA) was sutured onto the myocardial wall to acquire the cross-sectional view of the left ventricle. A U-shaped Doppler flow probe was placed around the exposed right carotid artery. A pacemaker was not applied to maintain a constant heart rate during the sequential periods.

After craniotomy, a laser Doppler flowmeter ₅ was fixed in the brain cortex to measure relative flux transdurally in the right frontal area. A ti-pO₂ catheter (Licox Systems, GMS Kiel-Mielkendorf, Germany) and a microdialysis-catheter (CMA 70 Microdialysis Brain Catheter, Solna, Sweden ) were inserted subcortically in the right frontal area. The ti-pO₂ and microdialysis catheters were fixed at 10 mm subdurally in the white matter of the frontal region. Care was taken to ensure that the distance between both catheters was at least 1 cm. After the surgical procedure of approximately 1.5–2 h, the animals were observed for 60 min to document hemodynamic stability.

Figure 1

Figure 1: Placement of probes, lines, and catheters

To determine E_es, we continuously calculated LV volume from the specific conductivity of the blood ₆,₇ , as well as the offset of the conductivity and fluids surrounding the LV cavity ₈ . The contribution of parallel conductance to the total conductance signal was derived from a measurement of the conductance signal during passage of the bolus through the LV. For absolute volume measurements, we determined the slope factor α, which was introduced to account for the finding that the relationship between “true” volume and conductance did not fall on the line of identity, even after correcting for parallel conductance ₇ . This may result from the fact that equipotential planes are not exactly parallel in the cavity, indicating a nonhomogeneous current distribution. In addition, it may partly result from a mismatch between the electrode distances and the ventricular long axis ₉ . There is no simple way to determine α; its value can be estimated by comparing conductance-derived volume with an independent measure, such as changes in cardiac output (CO) following thermodilution ₁₀ .

Global contractility of the LV was determined by applying the time-varying elastance model ₁₁ . Time-varying elastance was carried out during rapid preload reduction by occlusion of the inferior vena cava every 7 ms of the cardiac cycle, beginning with the end-diastolic point and continuing past the end-systolic point. Maximal elastance was the elastance connecting the zero point on the coordinate cross with the end-systolic point. Linear regression analysis of the end-systolic values during occlusion of the inferior vena cava by rapid preload reduction was used to obtain the slope of LV E_es. The amount of the decrease in LV end-systolic pressure-volume relationship (LVESPV) as a response to the rapid preload reduction depends on the myocardial contractility as expressed by the index E_es. The equation for calculating E_es is:

Figure 2

A gently inclined linear curve implies a substantial decrease in LV volume and a concomitant smooth decrease in LV pressure, which characterizes compromised contractility.

At each time point, a segmental signal was defined as dyssynchronous if its change (i.e., dVseg/dt) was opposite to the simultaneous change in the total LV volume (dVLV/dt). Segmental dyssynchrony is quantified by calculating the percentage of time within the cardiac cycle when a segment is dyssynchronous. Overall LV dyssynchrony (DYS) was calculated as the mean of the segmental dyssynchronies ₁₂ . DYS can be calculated within each specified time interval: we determined DYS during systole (DYSS) and diastole (DYSD), with systole defined as the period between the moments of dP/dtmax and dP/dtmin.

Nonuniform contraction and filling is associated with ineffective shifting of blood volume within the LV. This internal flow (IF) is quantified by calculating the sum of the absolute volume changes of all segments and subtracting the absolute total volume change: IF(t) = [|dVseg,i(t)/dt | – | dVLV(t)/dt|]/2. Note that dVLV(t)/dt represents the effective flow into or out of the LV. Thus, IF measures the segment-to-segment blood volume shifts, which do not result in effective filling or ejection. Division by two takes into account that any noneffective segmental volume change is balanced by an equal and opposite volume change in the remaining segments. Systolic IF fraction (IFFS) is calculated by integrating IF(t) over the full cardiac cycle and dividing by the integrated absolute effective flow (28).

Continuous ultrasound-mediated estimation of LV volume for determining E'_es was performed using an automated detection technique as previously described ₁,₂ . To obtain the time-varying elastance E'_es from the blood-flow velocity-area loops, we used two-dimensional echocardiograms of the LV cavity in short-axis view recorded simultaneously with measurements of blood-flow velocity in the carotid artery, as previously performed ₁ . We plotted arterial blood-flow velocity against the corresponding area. The end of systole was defined as the time at which the elastance reached a maximal value, termed E'_max, for every loading beat synchronized in time ₁₁ . These points on the end-systolic blood-flow velocity area loops were obtained during rapid LV preload reduction induced by inferior caval vein occlusion. The slope of the regression curve calculated from the end-systolic blood-flow velocity area points was defined as elastance E'_{es 13} . Analogous to E_es, the decrease in LV end-systolic flow-area relationship (LVESFA) as a response to the rapid preload reduction equals the myocardial contractility as expressed by the index E'_es. The equation for calculating E'_es is:

Figure 3

Compromised contractility is characterized by a gently inclined slope, which implies a substantial decrease in LV area and a concomitant smooth decrease in LV blood flow velocity.

We monitored cerebral microcirculation by laser Doppler flowmetry, an established technique for real-time measurement of microvascular red blood cell perfusion in tissue ₅ . Perfusion is also referred to as microvascular blood flow or red blood cell flux and is proportional to the product of the average speed of the blood cells and their number concentration (often referred to as blood volume). This value is expressed in arbitrary “perfusion units” and is calculated using the first moment of the power spectral density. Although such measurements are proportional to perfusion, the factor of proportionality varies for different tissues. A calibration of the Doppler-flow measurement using the conductance catheter is not yet available. Therefore, we calculated the relative flux by dividing the flux obtained during the interventions by their corresponding baseline values. The laser Doppler imager recorded cerebral perfusion in the temporal domain and presented the data throughout the experiment. The relative flux served as a record of the cerebral microcirculation, whereas the blood-flow velocity of the carotid artery established the blood-flow velocity-area relationship to calculate E'_es.

Cerebral autoregulation is locally heterogeneous due to the anatomical variation of the brain cortex ₁₄,₁₅ . This heterogeneity necessitated a sequential procedure that excluded alternative methods of perfusion measurement, such as the tracer microsphere technique. This technique delivers absolute measures but only allows documentation of the status quo without disturbing microcirculation. Consequently, we excluded relative flux from the multiple testing procedures because all other parameters were absolute values.

Cerebral microdialysis was used to record whether the lower border of cerebral autoregulation was passed. Changes in p_(ti)O₂ provide the earliest sign of hypoxia/ischemia ₁₆ ; the microdialysis assays of metabolism provide additional information about the consequences of reduced tissue p_(ti)O_{2 17} . Hypoxia occurs below the p_(ti)O₂ threshold of 15–10 mmHg ₁₇,₁₈ . Baseline levels of the lactate-to-pyruvate ratio (L/P) can vary between 15–22; an increase above 30 indicates cerebral cell damage, as supported by the decrease in p_(ti)O_{2 19} .

We carefully inserted a microdialysis probe (CMA 70 brain microdialysis catheter; 1-cm membrane tip, 0.62-mm diameter; CMA Microdialysis) into the brain tissue next to a p_(ti)O₂ probe. Perfusion (CMA107 Microdialysis Pump) of the catheter with sterile modified Ringer's solution was started before implantation. The flow rate was 2 µL/min and fractions were collected every 15 min. Online analysis of samples was performed using the CMA 600 microdialysis analyzer (CMA Microdialysis). Lactate, pyruvate, and p_(ti)O₂ concentrations were determined using extracellular fluid probes.

To observe the autoregulation pattern, height, and area, we positioned the probe far from large vessels in a relatively avascular region of the cortex; however, the sensitive region measured by the laser Doppler flow probe approaches the 1- to 2-mm spacing in oxygen heterogeneity observed in the normal cat cortex ₂₀ . This distance approached the 300-700 µm spacing between two of Bär's medium vascular nodules ₂₁ , and recent work by Lübbers et al. ₂₂ showed oxygen heterogeneity with the same spacing. Several studies support the interpretation that interanimal variations in laser Doppler flow and p_(ti)O₂ are caused by variations within the cortex of each animal ₁₅,₂₃ .

We determined all hemodynamic parameters during apnea prior to rapid preload reduction by occlusion on the inferior vena cava.

Esmolol was used to acutely reduce MAP to 50 mmHg (when compared with the corresponding baseline levels) by impairing myocardial contractility. To exclude influences of cardiac load on E'_es, we sequentially induced a similar hypotension in the same animal using the arterial vasodilator SNP. The two hemodynamic stages were induced in random sequence for each sheep; either SNP (4 µg/kg/min) or esmolol (2–5 mg/kg) was infused and held constant for 15 min. Baseline measurements were taken prior to each stage (baselines 1 and 2). SNP decomposes into pharmacologically inactive metabolites within a few minutes of infusion ₂₄ . Esmolol is an ultra short-acting cardioselective beta-antagonist with an extremely short elimination half-life (mean, 9 min; range, 4–16 min) that has a total body clearance [285 ml/min/kg (17.1 L/h/kg)] approaching three times CO and 14 times hepatic blood flow ₂₅ . We waited 60 min after the end of each intervention before initiating the next intervention.

Data are expressed as mean ± standard deviation (SD). Direct comparisons were made using either the Student's t-test or the Mann Whitney U test. Statistical significance was inferred when p < 0.05.

The relative changes (ς) of elastance in both systems (ς_Ees and ς_E'es) that followed each intervention were calculated by the formulae:

eq3

Positive and negative values of ς_Ees indicated an increase or decrease in LV contractility, respectively. The agreement between ς_Ees and ς_E'es was tested according to Bland and Altman ₂₆ : differences and arithmetic means were plotted and the SD of the differences was calculated.

All hemodynamic parameters are shown in Table 1. MAP decreased significantly from the baseline following both SNP (50 ± 11 mmHg) and esmolol (48 ± 6 mmHg) infusions, reaching intended levels. There was no significant difference between the baseline values. Esmolol infusion caused heart rate to decrease significantly (73 ± 14 min ^-1 ) as compared with baseline (90 ± 21 min ^-1 ) and SNP (94 ± 23 min ^-1 ) values. CO decreased significantly as a result of esmolol infusion, as compared with the baseline (2.4 ± 1.0 ml·min ^-1 ) and following SNP infusion (3.2 ± 2.0 ml·min ^-1 ).

Figure 4

Table 1: Hemodynamic and microdialysis data.

Abbreviations: CO, cardiac output; DYS_sys, LV dyssynchrony during systole; E_es, end-systolic elastance; E´_e,_s end-systolic elastance of the flow velocity-area relationship; EF, ejection fraction; HR, heart rate; IFF_sys, LV internal flow fraction during systole; L/P-ratio, ratio of lactate and pyruvate values; LVEDV, LV end-diastolic volume; LVEDP, end-diastolic pressure; LVESV, end-systolic volume; LVESP, end-systolic pressure; MAP, mean arterial pressure; p_(ti)O₂, partial oxygen pressure in the tissue; SNP, sodium nitroprusside; SW, stroke work; ς_Ees – relative change of the elastance E_es; ς_Ees – relative change of the elastance E´_es.

^# p < 0.05 compared with baseline.

⁺ p < 0.05 compared with SNP.

The blood-flow velocity-area relationship in a single animal during the sequential pharmacological interventions is displayed in Figure 2. The E_es of each relationship indicated a significant decrease in LV contractility attributable to esmolol (1.0 ± 0.2 mmHg·cm ^-3 ) as compared with baseline (3.0 ± 0.9 mmHg·cm ^-3 ) and SNP (3.0 ± 0.7 mmHg·cm ^-3 ) values. The E'_es decreased following esmolol infusion (0.36 ± 0.21 sec ^-1 ·cm ^-1 ) as compared with baseline values (0.88 ± 0.26 sec ^-1 ·cm ^- ) and the value following SNP infusion (0.76 ± 0.27 sec ^-1 ·cm ^-1 ).

Figure 2. Loops of the flow-area and pressure-volume relationship during sequential periods. Pressure-volume and velocity–LV area data from one animal at baseline, during sodium nitroprusside SNP administration, and during esmolol administration. End-systolic points and corresponding slopes are shown, demonstrating increased and decreased end-systolic elastance.

The parameter flux acquired by laser Doppler flowmetry is shown in Table 1. The decrease of Flux following SNP infusion was within the range of 15% deviation from baseline and therefore within the limits of autoregulation. The decrease following esmolol was significantly stronger than the decrease following SNP infusion averaging at 37% deviation from baseline values and therefore ranging below cerebral autoregulation.

Cerebral perfusion. The parameters obtained by microdialysis are given in Table 1. In two cases, we could not analyze lactate and pyruvate levels because of a defective microcatheter. The p_(ti)O₂ was unchanged following SNP infusion and significantly decreased by 50% following esmolol infusion as compared with the baseline and SNP values. L/P reflects the degree of anaerobic glycolysis and indicates the degree of cellular tissue damage due to cerebral hypoxia ₂₇ . According to our predefinition, we would expect an increase in L/P to indicate that the lower limit of autoregulation processes had been passed over which should be the case when 15% of Flux. L/P increased significantly from the baseline following esmolol application, and L/P was significantly higher after esmolol infusion than after SNP infusion.

p(tiO2 vs. L/P. The data pairs from seven animals during the four sequential periods were sampled for correlations. Of these, 28 data pairs were used for analysis; the results are depicted in Figure 3. The curve fitting revealed evidence of a power function for overall data pairs (Figure 3):

p_(ti)O₂ = 69.3 LP ^-0.5 (1)

with R = 0.82, R ² = 0.67, and p < 0.0001. The striking variance revealed by the regression analysis relating p_tiO₂ and L/P can be attributed to the heterogeneous time delay of the induced metabolic response ₁₈,₂₈ .

Figure 5

Figure 3: L/P vs. pO2. Plots of the 28 pooled data pairs demonstrating the relationship of indices derived from the cerebral tissue oxygen and the cerebral metabolites. Results for regression analysis of pO from cerebral tissue oxygen vs. L/P calculated from cerebral metabolites) are shown; = 0.90, = 0.81, and < 0.0001.

Myocardial perfusion. The parameters indicating mechanical dyssynchrony are supposed to represent myocardial perfusion and summarized in Table 1. The increases in IFFS and DYSS due to SNP infusion were not significant whereas esmolol infusion was followed by a significant increase in IFFS and DYSS.

Analysis of the changes in E_es and E'_es are presented in Table 1. Sharing common units, we forwarded ς_Ees and ς_E'es to a bias plot as described by Bland-Altman (Figure 4A and 4B).

The effects of altered LV contractility on E'_es and E_es of the left ventricle are shown in Table 1, and are expressed by the relative contractility changes and . The analysis of the SNP and baseline values to estimate the changes in contractility revealed a systematic bias (mean ± SE) of 0.029 (2.9%) ± 0.064 (6.4%) in estimating E_es by E'_es. The 95% confidence interval was ± 1.96 SE, which equals 13%. The response to SNP infusion resulted in good agreement between E_es and E'_es, as indicated by regression analysis, and Bland-Altman analysis demonstrated an acceptable agreement between E_es and E'_es in indicating contractility changes (Figure 4A).

The bias plot of the esmolol values of the parameters ς_Ees and ς_E'es used to detect changes in contractility revealed a systematic bias (mean ± SE) in estimating E_es by E'_es of 0.43 (43%) ± 0.3 (30%). The 95% confidence interval was ± 1.96 SE, which equals 59% (Figure 4B). This is a striking decrease in the agreement of E'_es with E_es.

Figure 4. Plot of the difference against the mean for relative contractility change (ς_Ees and ς_E'es ) data. The thick line represents the mean, and the broken lines indicate ± 2 standard deviations (SD).

(A) Values for SNP vs. baseline before SNP, SNP vs. baseline before esmolol, and baseline before esmolol vs. baseline before SNP.

(B) Values for esmolol vs. baseline before esmolol and esmolol vs. baseline before SNP.