Fluorescent vs. radioactive microsphere measurement of regional myocardial blood flow.

OBJECTIVES: This study compared simultaneous regional myocardial blood flow (RMBF) measurements using fluorescent microspheres (FM) and radiolabeled microspheres (RM). The utility of an internal standard during processing was also examined. METHODS: Paired FM and RM were injected into the left atrium of 9 anaesthetised rabbits. RMBF was altered by use of either regional ischaemia or (-)-N6-(2-phenylisopropyl)-adenosine. Radioactivity of blood reference and tissue samples was quantitated using standard methods. Samples were then digested with potassium hydroxide and microspheres recovered by vacuum filtration, with an additional label of FM as the internal standard. FM labels were extracted using Carbitol acetate and quantitated using fluorescence spectroscopy. Agreement between the fluorescent and radioactive methods was assessed using both orthogonal regression and difference-against-mean analyses. RESULTS: Using recovery-uncorrected data, the slope of the orthogonal regression of RM and FM-determined RMBF was not statistically different from 1, but the intercept was statistically different from 0 [-0.03(0.01), P = 0.005] and the mean RMBF by each method differed from one another [1.24(0.08) vs. 1.17(0.08) ml.min-1.g-1, P = 0.0002]. The mean +/- 2 s.d. of the differences of RMBF (RM minus FM) was +0.07 +/- 0.30 ml.min-1.g-1. Although recovery of FM from tissue averaged 97.6(1.2)%, use of the internal standard to correct for losses substantially improved the agreement between RM and FM-determined RMBF: the orthogonal regression slope was not statistically different from 1, the intercept was not statistically different from 0, and the means of the flows were not different. The mean +/- 2 s.d. of the differences of RMBF was -0.01 +/- 0.22 ml.min-1.g-1. The internal standard also improved RMBF estimates from samples with simulated large spillage during processing. CONCLUSION: Fluorescent microspheres are an equivalent alternative to radiolabeled microspheres for the estimation of RMBF. Although the overall recovery of microspheres using this technique was high, use of an internal standard is recommended for correction of random losses.

Metabolic adaptation to a gradual reduction in myocardial blood flow.

BACKGROUND: Studies during 20% to 50% reductions in regional coronary blood flow have revealed a number of metabolic and functional adaptations that suggest the heart downregulates energy requirements and contractility in response to ischemia. In contrast to prior studies of sudden changes in coronary blood flow, we tested whether the heart could reduce ATP consumption commensurate with a gradual decrease in coronary blood flow or whether transient metabolic abnormalities are a necessary trigger in this process. METHODS AND RESULTS: From 0 to 35 minutes, mean left anterior descending coronary artery blood flow was reduced by approximately 1% per minute in 10 acutely anesthetized and instrumented swine. Coronary blood flow then was held constant between 35 and 60 minutes at the resulting 35% net blood flow reduction. Although systemic hemodynamics remained stable, a significant decrease in regional left ventricular systolic wall thickening developed (from control value of 45 +/- 11% to 18 +/- 11% at 60 minutes, P < .001) without a sustained decrease in the phosphorylation potential (as assessed by a < 2% decrease in either the transmural or subendocardial phosphocreatine-to-ATP ratio) and with minimal myocardial lactate production (4 +/- 44 mumol.min-1 x 100 g-1). CONCLUSIONS: Metabolic markers of ischemia such as ratio of phosphocreatine to ATP, ATP content, lactate content, and lactate production were blunted during this protocol of gradually worsening ischemia. Thus, contractile abnormalities of mild ischemia can develop with minimal metabolic evidence of ischemia. The downregulation of myocardial energy requirements can almost keep pace with the gradual decline in coronary blood flow.

Energy metabolism and contractile function after 15 beats of moderate myocardial ischemia.

Difficulties in studying myocardial metabolism with adequate time resolution have led to contradictory conclusions regarding the mechanisms causing contractile abnormalities during the early stages of ischemia. In acutely instrumented swine, we investigated whether abnormalities in subendocardial ATP, phosphocreatine, or lactate content develop rapidly enough during the first few heart beats after onset of partial myocardial ischemia to contribute to contractile failure. Within the first 15 beats of a 40-50% reduction in left anterior descending coronary artery blood flow, regional myocardial function was significantly reduced but continuing to deteriorate. Rapidly frozen transmural left ventricular biopsies obtained on the 15th heart beat (+/- 1.5 beats) after the onset of ischemia revealed significant decrements in subendocardial phosphocreatine and ATP levels to 77% (p less than 0.05) and 84% (p less than 0.005) of control values, respectively, but minimal change in lactate content. Metabolic effects as assessed by transmural averages took longer to become detectable; thus, there was a tendency to underestimate the importance of subendocardial metabolic effects on myocardial function. When left ventricular preload was assessed during this early time period, left ventricular end-diastolic wall thickness only decreased by 3%, and left ventricular end-diastolic pressure did not change significantly despite a large fall in coronary perfusion pressure. Thus, in an in vivo pig model with techniques optimized to detect subendocardial metabolic changes within the period of very early moderate myocardial ischemia, abnormalities in high energy phosphate compounds occurred rapidly enough to contribute to developing myocardial dysfunction, whereas preload-mediated mechanisms related to vascular distending pressure could not explain the functional deterioration under these conditions.

Active downregulation of myocardial energy requirements during prolonged moderate ischemia in swine.

We studied the effects of rapid atrial pacing during the final 10 minutes of a 70-minute, 31% reduction in coronary blood flow in anesthetized swine to understand the significance of apparent metabolic improvements during the initial 60 minutes of segmental ischemia. Within 5-10 minutes of ischemia, subendocardial phosphocreatine (PCr) and ATP were depleted to 47% and 63% of control, respectively; lactate accumulated within the subendocardium to 300% of control; and net arteriovenous lactate production occurred. Despite continued ischemia and no significant changes in the external determinants of myocardial oxygen consumption, by 60 minutes subendocardial PCr and lactate contents returned to near control levels and there was net arteriovenous lactate consumption. Ischemic left ventricular wall thickening and ATP levels remained depressed throughout the experiment. Atrial pacing during the final 10 minutes of ischemia again resulted in depletion of PCr and lactate production. Since the myocardium was capable of hydrolyzing PCr in response to atrial pacing at 60 minutes of ischemia, we conclude it was capable of hydrolyzing PCr during the period of constant ischemia when instead it was accumulating PCr. We propose the ischemic myocardium downregulates regional energy requirements below blood flow-limited rates of energy production during ischemia. This appears to be an active adaptation to ischemia and not a result of passive damage or cellular injury.

Regeneration of myocardial phosphocreatine in pigs despite continued moderate ischemia.

The effects of 1 hour of mild and moderate reductions in coronary blood flow on myocardial high-energy phosphate levels were evaluated. Thirty anesthetized pigs were instrumented with left anterior descending arterial and venous catheters, crystals for instantaneous wall thickness, and a fluid-filled occluder. Measurement of myocardial blood flow was performed with microspheres, and a series of myocardial biopsies also was performed. In 10 pigs, overall coronary blood flow was lowered by 22%, with a fall in subendocardial-to-subepicardial flow ratio from 1.11 to 0.54 and in wall thickening from 33% to 15%. Subendocardial flow fell 48%. Coronary blood flow and thickening were constant during 1 hour of ischemia. Phosphocreatine (mumol/g wet wt) in the subendocardial third of the ischemic zone fell from 7.6 to 3.8 at 5 minutes of ischemia (p less than 0.005 versus control) and returned to normal (7.9) at 60 minutes (p = NS), despite ongoing ischemia. Subendocardial ATP (mumol/g wet wt) fell slowly from 4.3 and leveled off at 2.1 at 60 minutes of ischemia (p less than 0.001 versus control). Similar regeneration of phosphocreatine was found in seven additional pigs, with a 43% transmural reduction in coronary blood flow and a 66% reduction in subendocardial flow. No significant changes in ATP and phosphocreatine were noted in two different control groups (n = 13 pigs). The regeneration of phosphocreatine despite ongoing ischemia and low ATP levels was not related to changes in myocardial oxygen demand or consumption, or in regional function during the period of ischemia. This may reflect 1) a successful downregulation of the energy needs of the ischemic myocardium to maintain cell viability, or 2) a metabolic abnormality in the ability of the cells to produce ATP primarily or by use of phosphocreatine.

Importance of vasomotor tone to myocardial function and regional metabolism during constant flow ischaemia in swine.

STUDY OBJECTIVE: The aim was to test the hypothesis that the release of vascular tone with adenosine during constant flow ischaemia alters both transmural function and regional metabolism in a detrimental way. DESIGN: In one group of anaesthetised swine, the effects of graded reductions of flow on segmental left ventricular function, myocardial oxygen consumption (MVO2), and lactate production in the distribution of the left anterior descending coronary artery (LAD) were determined. In a second group, a model of constant flow ischaemia was induced to test how altering vascular tone with adenosine changed the relationship of flow, function, and metabolism. EXPERIMENTAL MATERIAL: The experiments were performed in 20 open chest, anaesthetised swine. Protocol A consisted of 11 animals and protocol B of nine animals. MEASUREMENTS AND MAIN RESULTS: In protocol A, during graded ischaemia, reductions in flow, % systolic wall thickening (WTh), normalised MVO2 and % lactate extraction (%LE) correlated well with reductions in coronary perfusion pressure when fitted with 3rd order polynominal curves (r = 0.78, 0.87, 0.85 and 0.81 respectively; p less than 0.00001). In protocol B, during constant flow ischaemia, at control, % WTh was 33 (SD 11)%, mean coronary artery pressure was 72(10) mm Hg, mean LAD transmural flow was 0.99(0.43) ml.min-1.g-1, and % LE was +14(9)%. With inflation of a hydraulic occluder on the LAD, perfusion pressure was lowered to 38(5) mm Hg and transmural flow dropped to 0.76(0.31) ml.min-1.g-1 (intact vasomotion). During an infusion of intracoronary adenosine with flow held constant (absent vasomotion), %WTh was further reduced from 27(9) to 13(10) (p less than 0.001), and %LE from -18(42) to -70(61) (p less than 0.05). MVO2 with and without vasomotion did not differ significantly at 3.14(0.75) and 3.18(0.86) ml.min-1.g-1 respectively. CONCLUSION: In swine coronary circulation, reductions in regional function, MVO2 and lactate production correlate well with reductions in flow and perfusion pressure during ischaemia with vasomotor tone intact. The effect of adenosine on vascular tone during constant flow ischaemia caused dramatic reductions in function and lactate extraction without altering MVO2. This emphasises the important role of vascular tone in protecting both transmural function and regional metabolism during moderate ischaemia.

No reflow and extent of infarction during maximal vasodilation in the porcine heart.

To explore the relation between myocardial and vascular injury in the generation of the no-reflow phenomenon, the pressure-flow relation during maximal vasodilation after coronary artery reperfusion was studied in the open-chest porcine model. During both endogenous and maximal vasodilation with intracoronary adenosine, pressure-flow (P/Q) plots were constructed before and after 20-minute (n = 9) or 40-minute (n = 17) circumflex artery occlusions. Decreases in circumflex vascular bed conductance were represented by downward shifts in P/Q plot regression lines. No significant change occurred in P/Q line slope or pressure at zero flow 30 minutes after release of the 20-minute occlusion, and no infarction was found. After release of the 40-minute occlusion, a small but insignificant decrease in P/Q line slope occurred during endogenous vasodilation. However, during maximal vasodilation, a significant (p less than 0.01) decrease in P/Q line slope was present during reperfusion compared with preocclusion corresponding to a decrease in vasodilatory reserve (P/Q line slope = 1.52 +/- 0.14 ml/min/mm Hg preocclusion vs. 1.03 +/- 0.13 at 15 minutes reperfusion). Pretreatment with aspirin did not prevent this decrease in vascular conductance during maximal vasodilation. Total circumflex, as well as subendocardial, midmyocardial, and subepicardial blood flows, was measured with radioactive microspheres. There was a good correlation between the extent of infarction measured by triphenyltetrazolium chloride staining and the decrease in vascular conductance during maximal vasodilation for all three myocardial layers as well as for the total circumflex vascular bed. Hence, the degree of no-reflow correlates closely with the extent of infarction during maximal vasodilation (but not during endogenous vasodilation) and is not altered by aspirin therapy.

Effect of coronary sinus occlusion on coronary flow, resistance, and zero flow pressure during maximum vasodilatation in swine.

The effects of coronary sinus occlusion on the relation between coronary artery pressure and flow during maximum vasodilatation were studied in seven swine. The left anterior descending (LAD) coronary artery was instrumented with two catheters, a hydraulic occluder, and a flowprobe. Mean flow was measured at a series of pressures produced by partial LAD occlusion during maximum vasodilatation induced by an intracoronary infusion of adenosine. Observations were made under control conditions and during occlusion of the coronary sinus produced by inflating the balloon on the catheter positioned in the coronary sinus. Systemic haemodynamic variables did not change significantly after the coronary sinus was occluded. The mean right atrial pressure was 4 mmHg. At any given LAD perfusion pressure mean flow during coronary sinus occlusion was always less than during the control state: at LAD pressure 30 mmHg, control flow was 53 ml.min-1 vs occluded flow 24 ml.min-1; at LAD pressure 40 mmHg, control flow 79 ml.min-1 vs occluded flow 49 ml.min-1; and at LAD pressure 50 mmHg, control flow 105 ml.min-1 vs occluded flow 74 ml.min-1; p less than 0.001 for all comparisons. The mean (SD) LAD pressure at which flow stopped (Pzf) when the coronary sinus was unobstructed was 10(2) mmHg. The Pzf during occlusion of the coronary sinus was significantly higher at 20(4) mmHg (p less than 0.001). The slopes of the mean pressure-flow relations were not significantly different during the control state (2.62(0.65) ml.min-1 per mmHg) vs the occluded state (2.47(0.63) ml.min-1 per mmHg), indicating no change in vascular resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased vascular resistance due to a reduction in red cell deformability in the isolated hind limb of swine.

This study investigated whether red cells with reduced deformability impeded flow through the microcirculation. Red cells were made less deformable in their normal biconcave disc shape by incubation with 2% formaldehyde (fRBCs). The blood supply to the right hind limb was isolated in 26 swine and the femoral artery was instrumented with two fine catheters, a flow probe, and an inflatable occluder. Flow was measured over a range of different perfusion pressures during adenosine-induced vasodilation under control conditions (C) and during an infusion of fRBCs at 1 ml/kg per minute (not to exceed 20 ml) into the femoral artery. At the same perfusion pressure (P), flow was significantly reduced 5 min after the fRBC infusion: Flow at P = 20 mm Hg, C = 41 ml/min vs fRBC = 10 ml/min; at P = 40, C = 160 ml/min vs fRBC = 79 ml/min; and at P = 60, C = 278 ml/min vs fRBC = 147 ml/min, with P less than 0.02 for all comparisons. Flow was still significantly reduced 15 min after the fRBCs, but by 30 min, it had returned to the control value. Chromium-51-labeling of red cells revealed that about one-third of fRBCs was trapped in the microcirculation compared to less than 3% of normal cells. This reduction in flow with fRBC infusion was not altered by alpha blockade, indicating that adrenergically mediated spasm was not responsible for the reduced flow. Aspirin 35 mg/kg iv completely prevented the reduction in flow despite an absence of change in the percentage of fRBCs trapped. Thus, red cells with reduced deformability infused into the circulation caused a significant, but transient, reduction in flow. The reduction in flow was not primarily related to entrapment of the abnormal RBCs, but may be mediated through platelet aggregation or release of potent vasoconstrictor substances from platelets or endothelial cells.

Relation of lactate production to postischaemic reduction in function and myocardial oxygen consumption after partial coronary occlusion in swine.

Postischaemic myocardial dysfunction (stunning) induced by partial occlusion of the left anterior descending coronary artery and its relation to lactate production during reperfusion were studied in nine swine. A 40% reduction in regional left ventricular wall thickening, as measured by ultrasonic crystals, was prospectively defined as stunning. A perfusion pressure of 20 mmHg was maintained with a hydraulic occluder for each ischaemic period and was monitored by a distal arterial catheter. To achieve a 40% reduction in function, four animals required three ischaemic periods (mean ischaemic flow reduction 73%), four two (86% flow reduction), and one one (93% flow reduction). At 25 min of reperfusion transmural flow was slightly reduced from 0.67 ml.g-1.min-1 at control to 0.58 ml.g-1.min-1 (p less than 0.05), whereas regional flow endocardial to epicardial flow ratio was unchanged. At 60 min reperfusion, percentage systolic wall thickening was reduced to 25% from a control of 39% (p less than 0.01) and parallel reductions in regional myocardial oxygen consumption from 4.3 ml.min-1 to 2.7 ml.min-1 occurred (p less than 0.01). Lactate extraction was depressed at 15 min reperfusion (-4.0% compared with control +18.0% (p less than 0.05)) but returned to control values by 30 min. It is concluded that postischaemic myocardial dysfunction (stunning) can be induced by partial coronary occlusions and that the extent of dysfunction depends on the degree of flow reduction. The reductions in myocardial oxygen consumption parallel those of wall thickening during reperfusion after stunning. Finally, lactate production occurs during early reperfusion but does not persist with the postischaemic reductions in function and myocardial oxygen consumption.

Coronary vasodilator reserve persists despite tachycardia and myocardial ischemia.

During myocardial ischemia, we tested whether coronary blood flow would increase in response to tachycardia, thereby employing known coronary flow reserve. We instrumented the left anterior descending (LAD) coronary circulation in anesthetized pigs and performed three sets of experiments while coronary pressure was controlled and several heart rate increases were produced. Pacing-induced tachycardia at normal LAD pressure was characterized by increased LAD flow and myocardial oxygen consumption, without production of lactate. Tachycardia at a mean LAD pressure of 38 mmHg was associated with a lower, fixed coronary flow and oxygen consumption. At average heart rates of 90 and 150 beats/min, LAD flow was 19.6 and 19.4 ml/min and corresponding myocardial blood flows were 0.59 and 0.54 ml X g-1 X min-1. Lactate was produced at all rates and local myocardial function declined progressively. Coronary flow at low LAD pressure doubled during tachycardia when intracoronary adenosine was added. The increase to the subepicardium was greater than 100%, whereas subendocardial flow changed little. There is persistent coronary flow reserve during moderately severe myocardial ischemia, even when metabolic demand is increased by tachycardia. This reserve, however, is predominantly subepicardial.

Increased vascular resistance during complement-activated plasma infusion in swine.

To investigate the acute effects of complement activation on blood flow, we infused complement-activated plasma into the femoral artery of the isolated hindlimb of 19 anesthetized swine. Femoral artery blood flow decreased abruptly, was lowest at 1 min of the infusion, and thereafter slowly increased despite continued infusion. There was no significant change in femoral artery pressure or femoral vein pressure, confirming an acute increase in vascular resistance. Control infusion of heat-decomplemented-activated plasma caused no change in pressure or flow. Slope of the femoral artery pressure-flow relationship during maximal vasodilation with adenosine was significantly lower after infusion of complement-activated plasma, confirming a persistent increase in vascular resistance. Neither the acute nor the persistent increase in vascular resistance was prevented by alpha-adrenergic blockade with phentolamine or granulocytopenia produced by cyclophosphamide. We conclude that complement-activated plasma infusion in the femoral circulation causes an abrupt increase in vascular resistance that persists during pharmacologically maximal vasodilation, is not due to alpha-mediated vasoconstriction, and is not altered by severe granulocytopenia.

Incomplete coronary vasodilation during myocardial ischemia in swine.

To determine if endogenous (ENDG) vasodilation was maximum during myocardial ischemia, left anterior descending (LAD) mean pressure (P) was reduced for 20 min in 13 swine. At LAD P of 45 mmHg (LAD P45) flow fell during ENDG = 25 but rose to 44 ml/min during adenosine (AD) infusion (P less than 0.01). Flow increased to subendocardium (ENDG 0.65 vs. AD 1.04 ml X min-1 X g-1) and to subepicardium (ENDG 0.99 vs. AD 1.83 ml X min-1 X g-1; P less than 0.05). No significant change occurred in myocardial O2 consumption (MVO2; ENDG 2.91 vs. AD 3.18 ml X min-1 X g-1), lactate extraction (ENDG = -5 vs. AD-1%), and wall thickening (WTh; ENDG + 16 vs. AD + 17%). At LAD P35, flow during ENDG was 12 but rose to 19 ml/min during AD (P less than 0.01). Flow increased to subendocardium (ENDG 0.24 vs. AD 0.46 ml X min-1 X g-1; P less than 0.02) and subepicardium (ENDG 0.51 vs. AD 0.87 ml X min-1 X g-1; P less than 0.01). No significant change occurred in MVo2 (ENDG 1.38 vs. AD 1.59 ml/min), lactate extraction (ENDG -38 vs. AD -22%), WTh (ENDG -1 vs. AD + 1%). Thus endogenous vasodilation reserve was not used fully during ischemia. AD increased flow but did not improve abnormalities in myocardial function or metabolism.

Vasopressin-induced coronary constriction at low perfusion pressures.

We studied the effects of intracoronary vasopressin on the relationship between pressure and flow in the coronary circulation of anaesthetised swine. In addition to measurements at control levels, diastolic pressure-flow relationships were constructed from steady-state points below a coronary pressure of 50 mmHg, where endogenous vasodilatation is strongly stimulated. At baseline pressures, flow fell 28% with maximal vasopressin effect. At all levels of diastolic pressure below 50 mmHg vasopressin also decreased flow, eg, at 30 mmHg flow was depressed by 40%. The slope of the steady-state pressure-flow relationship fell from 1.21 to 0.75 ml.min-1.mmHg-1. The diastolic pressure at which coronary flow ceased rose slightly from 13 to 15 mmHg. Intracoronary adenosine completely prevented vasopressin's effect, and the vasodilator response to adenosine was not attenuated by simultaneous administration of vasopressin. The porcine coronary circulation will constrict in response to vasopressin, not only at normal perfusion pressure, but also at low levels when metabolic vasodilatation is intense. Our study has implications about the therapeutic use of vasopressin, and demonstrates interaction of vasoactive stimuli in the coronary circulation.