Animal Preparation

The experiments were conducted in 14 Noroc pigs of either sex (mean weight, 52.4 ± 5.1 kg), initially sedated with intramuscular injection of azaperone 3 mg/kg, ketamine 20 mg/kg, and atropine 0.02 mg/kg. Anesthesia was then induced with intravenous pentobarbital (2–3 mg/kg) and boluses of morphine (0.5 mg/kg). Tracheotomy was performed with a neck midline incision, and the animals were mechanically ventilated (Servo ventilator 900C; Siemens Healthcare, Erlangen, Germany) with an air mixture containing 35% oxygen. Ventilation was adjusted to maintain an arterial partial pressure of carbon dioxide of about 5.3 kPa. The animals received inhaled isoflurane at an end-tidal concentration of 1% and a continuous intravenous infusion of morphine at 1 to 2 mg/kg/h. Preceding median sternotomy, lidocaine was given intravenously as a bolus (2 mg/kg), followed by continuous infusion (1 mg/kg/h), to prevent ventricular arrhythmias. The pericardium was opened and the heart suspended in a cradle. The left internal mammary artery (LIMA) was isolated from the thoracic wall and grafted to the left anterior descending coronary artery (LAD), distal to the first diagonal branch. The grafting was performed using the off-pump technique with an intracoronary shunt (ClearView; Medtronic, Minneapolis, MN) ( Figure 1 ). The median duration of coronary artery occlusion during grafting was 57 sec (45–130 sec). Heparin was given to maintain an activating clotting time > 200 sec and intravenous nitroglycerin 5 mg/kg/min to prevent spasm of the LIMA and distal LAD during preparation and grafting of the LIMA to the LAD. When a successful anastomosis was established, the native blood supply from the LAD to the intervention area was interrupted by a silicon snare proximal to the anastomosis.

Schematic illustration of the experimental model and the ultrasonic transducer. (A) The LIMA was grafted to the LAD. Miniaturized ultrasonic transducers and microdialysis catheters were positioned in the supply areas of the LAD and Cx, and intracavitary pressures were measured by micromanometers (see text for details). (B) The ultrasonic transducer was fixed to the epicardium by sutures in the three eyes. It was therefore ensured that the ultrasonic beam was perpendicular to the myocardium independent to cardiac movements. Cx , Circumflex coronary artery; LAD , left anterior descending coronary artery; LIMA , left internal mammary artery.

Among the initial 14 animals, one animal was excluded because of fibrinous pericarditis. Fatal ventricular fibrillation emerged in three animals during coronary surgery. Thus, the remaining 10 animals entered the experimental protocol.

Pressure and Flow Measurements

The internal carotid arteries on both sides were cannulated with 8-Fr sheath introducers. Two micromanometer-tipped 5-Fr high-fidelity catheters (Millar Instruments, Houston, TX) were introduced and advanced to the left ventricle and the ascending aorta. Before catheter insertion, each transducer was calibrated against air pressure as a zero reference and after insertion with a standard signal (1 mV/100 mm Hg). The right jugular vein was cannulated, and a fluid-filled 5-Fr catheter was used to obtain central venous pressure. A 5-Fr arterial thermodilution catheter (PV 2025L20, PULSIOCATH; Pulsion Medical Systems, Munich, Germany) was inserted in the femoral artery and connected to a pressure transducer (PV8115; PULSION Medical Systems), and the signals were computed in a PiCCO monitor (Pulsion Plus version 5.1; PULSION Medical Systems). Calibration was performed by three consecutive thermodilutions with cold 5% glucose. Blood flow measurements in the LIMA graft were done with an ultrasonic flow probe (Medistim, Oslo, Norway), and continuous measurements were registered.

Tissue Velocities from the Miniaturized Epicardial Ultrasound Transducer

Subendocardial tissue velocities were obtained from two miniaturized (Ø = 3 mm, height = 2.5 mm) 10-MHz ultrasonic transducers (Imasonic SA, Besançon, France) ( Figure 1 ). The epicardial positioning of the transducers, using three eyes for suture fixation to the epicardium, ensured a perpendicular insonation angle on the myocardium, enabling the assessment of regional myocardial function unaffected by global heart movements. Each transducer acted as an ultrasonic transmitter-receiver; the signals gave real-time M-mode images from each region, with corresponding wall thickening and thinning velocities. The method has been validated against speckle-tracking echocardiography, demonstrating a very good ability to detect regional myocardial dysfunction, with excellent interobserver reproducibility. It is unaffected by moderate changes in preload but has the sensitivity to detect changes in contractility. One transducer was positioned in the anteroapical LAD region supplied via the LIMA graft and the other in a midlateral region corresponding to the supply area of the circumflex coronary artery (Cx). Continuous M-mode monitoring was performed from these transducers and displayed on a monitor ( Figures 1 and 2 , top). Intermittent recordings were obtained for post hoc analysis. Time-velocity traces were obtained with dedicated software from a range-gated volume sample at a fixed depth in the subendocardium ( Figure 2 ). From these traces, the myocardial thickening velocities were calculated.

M-mode traces, regional myocardial function, and hemodynamic parameters during increasing ischemia. ( Top ) M-mode pictures obtained from the miniaturized ultrasound transducer. In this typical example, the systolic thickening of the myocardium decreased when increased flow reduction was applied. Please also note the increasing postsystolic shortening. The yellow line in the M-mode traces demonstrates the fixed depth from which the velocity curves ( bottom ) were achieved. ( Bottom ) The computed velocity traces demonstrated systolic ( red arrows ) and postsystolic velocities ( green arrows ) at baseline and during increasing levels of flow reduction. As the coronary flow decreased, there was a corresponding decrease in systolic velocities. A similar but inverse pattern was seen in the postsystolic velocities. There was, however, no further increase in postsystolic velocities from moderate to severe flow reduction. Beneath the velocity traces, LV ( red line ) and aortic ( green line ) pressures were continually measured from a Millar catheter, allowing calculation of the time derivative of the LV pressure (LV d P /d t ). The start and end of systole are defined by the electrocardiogram and peak negative LV d P /d t , respectively. LV , Left ventricular; V _post , peak postsystolic velocity; V _sys , peak velocity in systole.

Myocardial Tissue Lactate

Microdialysis catheters were positioned 2 to 5 mm subepicardially in close relation to the ultrasonic transducer. Tissue lactate was dialyzed at a perfusion rate of 1 μL/min from the LAD and Cx areas using microdialysis catheters (CMA 71 [100 kDa]; CMA Microdialysis AB, Solna, Sweden). Samples were taken at baseline and at the end of partial occlusion. Lactate was analyzed with a colorimetric method in an ISCUS analyzer (CMA Microdialysis AB).

Experimental Protocol

The animal was allowed to stabilize for 60 min after completed preparation, with the chest and pericardium remaining open. Patency of the LIMA graft was checked before and between the interventions by measuring LIMA blood flow, LV contractions by epicardial ultrasound, and myocardial lactate. To experimentally induce incremental degrees of regional myocardial ischemia, blood flow in the LIMA graft was reduced by manual inflation of the occluder. Thereby, flow was reduced stepwise by 25% (mild), 50% (moderate), and 75% (severe) of initial values. The reduced magnitude of flow was maintained for 18 min at each level. A recovery period with fully restored LIMA flow for ≥30 min was performed between the three interventions.

Recordings were taken at baseline and at 5, 10, and 15 min at each level of flow reduction. During severe flow impairment (75% reduction), two of the 10 animals developed intractable ventricular fibrillation. Recordings from this intervention in these two animals were not included.

Calculations

Statistical Analysis

All values were obtained by means of three consecutive heartbeats. All data are presented as mean ± SD, unless otherwise stated. Multiple comparisons of repeated measurements were analyzed with a linear mixed model. Taking into consideration that values vary among different individuals, a random-intercept model, with subject as the random effect, was chosen. Post hoc all pairwise comparisons of the main effects were performed, and significance was adjusted for multiple comparisons with the Bonferroni method. P values < .05 were considered statistically significant. Pearson correlation coefficients were calculated to quantify the association between two continuous (and normally distributed) variables. Statistical analysis was performed using PASW version 18 (SPSS, Inc, Chicago, IL).

Regional Function: Tissue Velocities in Systole and Diastole

Overall, two main findings appeared in the LAD region when flow was reduced. First, the myocardial tissue velocity trace obtained from the miniaturized ultrasound transducer demonstrated a typical pattern, with decreased systolic velocity followed by a simultaneous increase in postsystolic velocity ( Figure 2 ). These findings were present even during mild flow reduction but were most pronounced when a higher degree of coronary stenosis was imposed. Second, when the flow reduction was held constant for an additional 10 min, no further changes in the velocity pattern appeared during either of the levels of flow reduction ( Figure 3 ).

Regional systolic and postsystolic velocities during three levels of flow reduction. ( Top ) Tissue velocity in the LAD region during mild, moderate, and severe flow reduction. Data are presented as mean and standard deviation from baseline, 5, 10, and 15 min of reduced flow. The changes in systolic and postsystolic velocities appeared during the first 5 min of flow reduction. Prolonged, continuous impairment of flow for additional 10 min did not lead to further aggravation of myocardial function. ( Bottom ) Flow reduction was kept constant for 15 min at each level. Thick line represents mean flow and gray zone the standard deviation. Vpost, postsystolic shortening. * P < .01 and # P < .05 versus before occlusion. LAD , Left anterior descending coronary artery; V _post , peak postsystolic velocity; V _sys , peak velocity in systole.

A slight but significant reduction in systolic velocity (V _sys ) was seen during 25% flow reduction, with further reductions during 50% and 75% impairment of coronary flow ( Table 1 ). Concomitant changes were also seen in postsystolic velocities ( Table 1 ).

In three of 10 animals, there was fusion of the e′ and a′ waves, precluding e′ amplitude analysis in the LAD region. In the remainder, the e′ wave showed a tendency toward reduced amplitude from baseline to 75% flow reduction (−1.8 ± 1.0 to −1.6 ± 0.9 cm/sec, P = .06). In the nonischemic Cx area, there were no changes in either the systolic or the postsystolic velocities during the interventions ( Table 1 ). Tau was analyzed in all animals and did not change (31 ± 4 msec at baseline vs 32 ± 3 msec at 75% flow reduction, P = .29). However, slight reductions in LV d P /d t _min were seen at 50% and 75% flow reduction ( Table 2 ), implying global diastolic dysfunction.

Table 2

Hemodynamic variables at baseline and at three levels of partial LAD occlusion

Variable	Flow reduction	Baseline	Effect			P
			5 min	10 min	15 min
Heart rate (beats/min)	25%	105 ± 11	108 ± 12	108 ± 12	108 ± 14	.24
	50%	110 ± 16	109 ± 15	108 ± 15	111 ± 15	.62
	75%	103 ± 9	103 ± 12	105 ± 13	106 ± 14	.56
Peak LVP (mm Hg)	25%	84 ± 6	84 ± 7	84 ± 8	84 ± 8	.82
	50%	84 ± 8	81 ± 7 ∗	80 ± 7 ∗	81 ± 7 ∗	<.01
	75%	85 ± 6	79 ± 7 ∗	79 ± 7 ∗	78 ± 7 ∗	<.01
LV EDP (mm Hg)	25%	11.4 ± 4.1	12 ± 3.9	12.1 ± 3.9	11.7 ± 4.2	.57
	50%	10.8 ± 5.1	12.1 ± 5.7	11.7 ± 4.9	11.8 ± 5.2	.05
	75%	12.8 ± 6	16.4 ± 7.3 ∗	16.1 ± 8.1 †	15.4 ± 7.1	<.01
LV d P /d t max (mm Hg/sec)	25%	1,372 ± 273	1,343 ± 271	1,344 ± 273	1,392 ± 313	.29
	50%	1,413 ± 365	1,271 ± 305 ∗	1,265 ± 288 ∗	1,310 ± 321 †	<.01
	75%	1,369 ± 213	1,224 ± 249 ∗	1,228 ± 243 †	1,235 ± 237 †	<.01
LV d P /d t min (mm Hg/sec)	25%	−1,185 ± 209	−1,144 ± 225	−1,155 ± 215	−1,150 ± 245	.44
	50%	−1,197 ± 330	−1,081 ± 274 ∗	−1,077 ± 331 ∗	−1,108 ± 313 †	<.01
	75%	−1,251 ± 219	−1,103 ± 304 ∗	−1,102 ± 245 ∗	−1,120 ± 276 ∗	<.01
Cardiac index (L/min)	25%	4.5 ± 0.7			4.7 ± 0.7	.15
	50%	4.5 ± 0.7			4.4 ± 0.7	.39
	75%	4.5 ± 0.8			4.2 ± 0.6	.14
LVEF (%)	25%	61 ± 7			58 ± 7	.10
	50%	58 ± 5			54 ± 3	.06
	75%	59 ± 10			48 ± 4	<.01

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