Particle-induced coronary vasoconstriction during cardioplegic infusion

J

THoRAc CARDIOVASC SURG

89:428-438, 1985

Particle-induced coronary vasoconstriction during cardioplegic infusion Characterization and possible mechanisms We have characterized an isolated rat heart preparation in which particles induce transient coronary vasoccestriction, Exploiting the fact that aU commerciaUy available intravenous solutions contain permissible levels of contaminant particles (usuaUy 2 to 20 ~m in diameter), we investigated whether these particles have any adverse effect upon coronary flow. A commerciaUy available intravenous solution was modified to produce the St, Thomas' Hospital cardioplegic solution. Constant-pressure infusion of this solution over a 20 minute period caused a 46.2% ± 5.1 % reduction in coronary flow. This flow impairment could be limited to 13.3% ± 3.5% by the incorporation of a 0.8 ~m in-Iine fIlter. In hearts perfused with particle-containing solution foUowed by u1trafdtered solutions, the impairment of coronary flow was reversed within 1 minute. This quick reversal indicates that the particles were impairing flow not by physicalocclusion of vessels but by triggering some form of transient vasoconstriction. In studies with fdters of varying porosity (between 0.8 and 15.0 ~m), the phenomenon was shown to be attributable to relatively smaU numbers of particles greater than 10.0 ~m in diameter. In studies of myocardial protection, it was shown that the impairment of solution delivery and distribution caused by particles could severely reduce the protective properties of a chemical cardioplegic solution; hearts subjected to 180 minutes of hypothermic (200 C) ischemic arrest with multidose (3 minutes every 30 minutes) cardioplegia recovered almost completely upon reperfusion if a filtered (0.8 ~m) solution was used, but failed to recover when unfIltered, commerciaUy prepared solutions were used. In an attempt to defme the mechanisms underlying the particle-induced vasoconstriction, we conducted dose-response studies in which various vasoactive agents were used in an attempt to combat the effects of the particles. At their optimal concentrations, procaine (10.0 mmoljL), nifedipine (0.1 ~moIjL), and adenosine triphosphate (1.0 mmoljL) completely prevented the problem; lidocaine and dipyridamole partially aUeviated the effect; verapamil and isosorbide dinitrate were ineffective. These results indicate that several mechanisms acting at a smaU vessel level might contribute to the particle-induced vasoconstriction.

David J. Hearse, D.Sc., F.A.C.C., Cetin Erol, M.D., Lary A. Robinson, M.D., Miles P. Maxwell, B.Sc., and Mark V. Braimbridge, ERC.S., London, United Kingdom, and Omaha, Neb.

WeI.2

have reported recently that permissible levels (as defined by the United States and British PharmacoFrom The Heart Research Unit, The Rayne Institute, St. Thomas' Hospital, London SEI, United Kingdom, and the Department of Thoracic and Cardiovascular Surgery, University of Nebraska Medical Center, Omaha, Neb. 68105 (Dr. Robinson). Supported in part by grants from the British Heart Foundation and St. Thomas' Hospital Research Endowments Fund. Received for publication Aug. 30, 1984. Accepted for publication Oct. I, 1984. Address for reprints: Dr. D. J. Hearse, The Heart Research Unit, The Rayne Institute, St Thomas' Hospital, London SEI, United Kingdom.

428

poeias) of contaminant particles in commercially available intravenous solutions can cause an impairment of coronary flow when infused intraarterially into the isolated rat heart. These studies indicated that this phenomenon could adversely affect the infusion characteristics of a crystalloid cardioplegic solution and, as a consequence, severely reduce the ability of this solution to protect the myocardium against periods of ischemic cardiac arrest. We then showed that this particleinduced vasoconstriction could be overcome by filtration of solutions through a 0.8 ~m filter immediately prior to their infusion into the coronary vasculature. From recent studies,' it has become apparent that the

Volume 89 Number 3 March, 1985

vasoconstriction induced by contaminant particles is transient and as such might represent some mechanism whereby each particle in some way triggers regional vasoconstriction that reverses very rapidly. The objectives of the present study were fourfold: (1) to determine the reversibility of the effect; (2) to determine the size of particles primarily responsible for the phenomenon; (3) to ascertain the level of the microvasculature at which the effect is most likely to be manifest; and (4) in the context of cardioplegia and in the absence of ideal filters for clinical use, to investigate whether various vasoactive drugs could be used to control the phenomenon. Methods Animals. Hearts were obtained from male Sprague Dawley rats (200 to 250 gm body weight) that had been maintained on a standard laboratory diet. Heart perfusion and experimental time course. For coronary infusion studies, a modified Langendorff" preparation was used that allowed accurate measurement of coronary flow rates under conditions of constant-pressure infusion. Initially, hearts were perfused at a pressure of 100 em of water via the aorta with ultrafiltered (5.0 /-Lm porosity filter) Krebs-Henseleit' bicarbonate buffer containing glucose (11.1 mmol/L) gassed with 95% oxygen plus 5% carbon dioxide (pH 7.4 at 37° C) for a 5 minute equilibration period. During this time, coronary flow was measured at 1 minute intervals. The perfusion line was then clamped, and infusion of the solution under study was initiated via a side arm on the aortic cannula. This solution, which may have been particle-containing, ultrafiltered, or drugcontaining, was then infused at constant pressure (60 em of water) and at 20 ° C (to mimic sugical conditions of hypothermic infusion of a cardioplegic solution for a 20 minute period). During this time, batches of coronary effluent were collected for 1 minute and coronary flow rates were determined. In some studies, hearts were then switched back to perfusion with ultrafiltered bicarbonate buffer, and coronary flow values were recorded for a further period of time. In studies of the effect of contaminant particles upon the anti-ischemic properties of cardioplegic solutions, an isolated working rat heart preparation was used. This preparation has been described in detail previously.s' Briefly, the heart is excised and the aorta and left atrium are cannulated. Oxygenated bicarbonate buffer at 37° C flows into the left atrium from a reservoir located 17 em above the heart. The left ventricle ejects this buffer against a pressure head of 100 cm of water. In the process, the coronary bed is perfused and coronary effluent can be collected from the right heart. In this

Particle-induced coronary vasoconstriction

429

Table I. Limits for particle contamination of large-volume parenteral solutions Maximum permissible No. of particles Particle diameter

2:2 I'm 2:5 I'm 2:10 I'm 2:25 I'm

United States Pharmacopoeia

British Pharmacopoeia

200/5 ml 400/5 ml 250/5 ml 25/5 ml

study, aortic flow, coronary flow, cardiac output (the sum of the previous two indices), peak aortic pressure, rate of rise of left ventricular pressure (dP/dt), heart rate, stroke volume (cardiac output divided by heart rate), and minute work (cardiac output multiplied by peak systolic pressure) were determined during a 20 minute control working period. The heart was then rendered globally ischemic by clamping of the atrial and aortic lines. Next, the heart was subjected to coronary infusion (60 ern of water) with nonoxygenated cardiaplegic solution (unfiltered or 0.8 /-Lm filtered) at 20° C for 3 minutes. Cardioplegic infusion was then terminated and the heart was' subjected to 180 minutes of global ischemia at 20° C (temperature maintained via a thermostatically controlled heart chamber) with reinfusions (3 minutes every 30 minutes) of cardioplegic solution. Cumulative cardioplegic infusion time was 18 minutes. During each cardioplegic infusion the volume of coronary effluent was recorded. At the end of the 3 hour ischemic period, the hearts was reperfused at 37° C initially (15 minutes) in the Langendorff mode, during which time the total coronary effluent was collected and taken for the measurement of creatine kinase leakage." The heart was then perfused in the working mode for a further 20 minutes, during which time the recovery of the indices of cardiac function were recorded and expressed as a percent of the preischemic control value. Particle-containing cardioplegic solutions. These solutions were all formulated from commercially available solutions to which various commercially available concentrates, such as sodium bicarbonate, were added so as to produce the composition of the St. Thomas' Hospital cardioplegic solution (NaCI 110.0 mmol/L; KCl 16.0 mmol/L; MgCl 2 16.0 mmol/L; CaCl 2 1.2 mmol/L; NaHC0 3 10.0 mmol/L, pH 7.8). Since this study was concerned with particulate contaminants in 1 L bags of base solution, all additives were introduced to the bag via a 0.22 /-Lm Millex GS Millipore filter. Filtration. Solutions were always filtered via an in-line filter (43 mm diameter) positioned adjacent to

430

The Journal of Thoracic and Cardiovascular Surgery

Hearse et al.

130

control, and postischemic enzyme leakage was expressed as international units per 15 minutes per gram of dry weight of the heart. At least six hearts were studied for each experiment and all data are expressed as the mean ± the standard error of the mean. Statistical analysis of the results utilized the unpaired Student's t test and statistical significance was assumed when p < 0.05.

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Results and discussion Basic particle-induced impairment of coronary flow. Hearts (n = 6 in each group) were subjected to a

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20 minute period of coronary infusion at 20 C with filtered (0.8 JIom) and unfiltered cardioplegic solution. The coronary flow profiles are shown in Fig. 1 and indicate that, whereas coronary flow decreased by 46.2% ± 5.1% in the unfiltered group, the decline in the filtered group was only 13.3% ± 3.5%. The improvement in coronary flow arising as a consequence of filtration achieved a level of statistical significance at all times beyond the first 2 minutes of cardioplegic infusion. The particle-induced increase in vascular resistance in the unfiltered group resulted in a total infusion volume of 127 ± 8 ml over the 20 minute period, which was 27% less than that (175 ± 10 ml) in the filtered group. Particle counting revealed that, in 5 ml of the unfiltered cardioplegic solution, there were 3,862 ± 706 particles 2 to 5 JIom in diameter, 939 ± 169 particles 5 to 10 JIom in diameter, 34 ± 5 particles 10 to 15 JIom in diameter, 10 ± 2 particles greater than 15 um in diameter By contrast, in the filtered group, the corresponding values were 5 ± 2, 3 ± 1, 0, and particles per 5 ml of solution. Reversibility of particle-induced vasoconstriction. The progressive reduction of coronary flow caused by particles might conceivably arise as a consequence of physical occlusion of vessels or vasoconstriction induced by the particles. To investigate these possibilities, we infused hearts (n = 6) with ultrafiltered (0.8 JIom) bicarbonate buffer for 5 minutes; the next step was perfusion with unfiltered cardioplegic solution for 20 minutes, during which time coronary flow was monitored at 1 minute intervals. At the end of this period, perfusion was switched to an ultrafiltered (0.8 um) solution and coronary flow was monitored for a further 6 minutes. The results (Fig. 2) reveal that, upon the reintroduction of the filtered solution, coronary flow returned to its pre-particle control value within 1 minute. This result demonstrates that the increase in vascular resistance cannot be attributed to the physical obstruction of vessels by particles, but must be due to some transient 0

Perfusion Time I min)

Fig. 1. Particle-induced impairment of coronary flow. Isolated rat hearts were perfused at constant pressure (60 em of water) for a 20 minute period with (D) ultrafiltered (0.8 jlm) and (0) unfiltered, commercially available intravenous solutions. For details see text. Each point represents the mean for six hearts and the bars represent the standard error of the mean. Coronary flow is expressed as a percent of the value recorded during the first I minute collection period.

the aortic cannula. Depending upon the study, the filter porosity was 0.8, 5.0, 8.0, 10.0, or 12.0 JIom. Drug addition. In studies in which various vasoactive agents were added to particle-containing solutions, the drug was always solubilized prior to addition in a small volume of St. Thomas' Hospital cardioplegic solution, which was then introduced into the bag via a 0.22 JIom filter. In studies with nifedipine, photodegradation of the drug was prevented either by working under sodium light or by masking all bags and lines with light-proof tape. Particle counting. After thorough mixing of the appropriate filtered additives with each solution at 20 C, the particle content was measured with an HIAC 420 particle counter that had been calibrated immediately prior to use. The solution under investigation was introduced into the instrument directly from its original plastic bag, and particle counting limits were set to correspond to the size limits defined by the United States Pharmacopoeia? and the British Pharmacopoeia'? (Table I). Five counts were taken from each of three bags of each solution before and after they were passed through the appropriate filter. The first two of each five recordings were discarded according to standard counting procedures. Expression of results. In coronary infusion studies, flow was expressed as a percent of the first 1 minute value during the cardioplegic infusion measurement period. In myocardial protection studies, postischemic recovery was expressed as a percent of its preischemic 0

°

Volume 89 Number 3

Particle-induced coronary vasoconstriction

431

March. 1985

UNFILTERED SOLUTION

130 120 110

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Fig. 2. Reversibility of particle-induced impairment of coronary flow. Isolated rat hearts were perfused at constant pressure (60 em of water) for a 20 minute period with unfiltered solution. This was followed by perfusion at constant pressure (100 em of water) with ultrafiltered (0.8 /Lm) solution for 6 minutes. Coronary flow is expressed as a percent of the value recorded during the first I minutecollection period. Each point represents the mean for six hearts and the bars represent the standard error of the mean.

Table II. Selective filtration of particle-containing solutions Particle content (per 5 ml) Condition

Unfiltered 0.8 /lm filter 5.0/lm filter 8.0 /lm filter 10.0/lm filter 12.0/lm filter

2-5/lm

3,862 5 29 3,105 4,161 4,695

± ± ± ± ±

706 2 10 260 406 ± 354

effect, such as particle-induced coronary vasoconstriction. Two further arguments can be given against physical blockage as a mediator of the effect: First, despite the large number of contaminant particles, this number is small relative to the number of microvessels in the myocardium; second, the profile for coronary flow reduction tends to reach a plateau at later stages of perfusion, possibly indicating the establishment of an equilibrium between particle-induced vasoconstriction and spontaneous relaxation. If physical blockage had been involved, a continuing and linear decline of flow would have been expected. Particle size. As is apparent from the particle counting results in an earlier section, contaminant particles vary greatly in their dimensions and numbers, with large numbers of small «5 /Lm) particles and relatively few large ones (> 15 /Lm). In an attempt to ascertain the size of particles most likely to be responsible for the induction of vasoconstriction and thereby to

5- IO/lm

10-15/lm


939 ± 169 3±1 31 ± 9 599 ± 39 938 ± 96 1,144 ± 64

34 ± 5 0 2±1 I±O 10 ± I 15 ± 3

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obtain some indication as to the likely level of the microvasculature at which the particles exert their effect, we conducted a series of studies with graded filters. Hearts (n = 8 for each group) were perfused for 20 minutes with solutions that were either unfiltered or subject to filtration through membranes with defined porosity limits (0.8, 5.0, 8.0, 10.0, and 12.0 /Lm). Fig. 3 shows the percent reduction in coronary flow in each of these groups. Filtration at a 0.8 or 5.0 /Lm level resulted in the smallest decline in flow (<30%), and there was no significant difference between these two groups. Increasing filter porosity to 8.0 /Lm resulted in a small additional loss of flow. However, it was not until filter porosity was increased to 10.0 /Lm that a major decline was observed. Increasing porosity limits to 12.0 /Lm had no further effect, and there was no significant difference between unfiltered solutions and solutions that had been filtered at the 10.0 or 12.0 /Lm level. Thus, if the

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declared specifications for the filters are correct, these results would indicate that the impairment of coronary perfusion can be attributed primarily to the relatively small number of contaminant particles of 10 urn diameter and larger. In an attempt to validate this conclusion and also to confirm the declared porosity limits of the filters, we subjected batches of prepared cardioplegic solution to selective particle counting before and after filtration with 0.8, 5.0, 8.0, 10.0, and 12.0 urn porosity filters. The results (Table 11), in addition to confirming the high degree of reliability of these filters (removing greater than 95% of the particles above their respective porosity limit), also confirm the claimed cutoff limits of the filters and support our conclusion that particles of 10 /-tm and larger are primarily responsible for the vascular disturbance. Identification of 10 /-tm and greater as the culprit particle size might also be used to support the suggestion that events probably occur at a small vessel level, possibly in the region of the precapillary sphincter, where vessel diameters of the order of 10 to 15 /-tm are found. This suggestion gains further support from studies in a later section, in which a differential pharmacologic responsiveness is identified. Particles and myocardial protection. In order to determine whether the particle-induced impairment of coronary perfusion is critical to the efficacy of cardio-

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Fig. 4. Particle-induced loss of myocardial protection. Hearts were subjected to a 180 minute period of hypothermic (20 C) ischemic arrest with multidose (3 minutes every 30 minutes) cardioplegia with filtered (0.8 /Lm) (open block) and unfiltered (black block) St. Thomas' Hospital cardioplegic solution, which had been prepared from commercially available intravenous solutions. The postischemic recovery of each index of cardiac function is expressed as a percent of its preischemic control At least six hearts were studied in each group, and the bars indicate the standard error of the mean. 0

plegic infusion solutions when used to prevent injury during global ischemia,' we subjected hearts (n = 6 for each group) to 180 minutes of hypothermic (20 C) ischemic arrest with multidose cardioplegia (3 minutes every 30 minutes) using filtered (0.8 /-tm porosity) and unfiltered solutions of commercial origin. The results (Fig. 4) show that in the unfiltered group (in which only one of the six hearts recovered any effective pump function) the mean recovery of all indices was very low. For example, aortic flow recovered to only 0.7% ± 0.3% of control, cardiac output to only 11.8% ± 0.8%, and minute work was only 3.5% ± 0.9%; creatine kinase leakage was high-39.9 ± 5.6 IU/15 min/gm dry weight. In contrast, in the filtered group all hearts recovered to a high functional level. Aortic flow was 86.8% ± 1.6% of control and cardiac output and minute work recovered to 86.5% ± 1.6% and 77.5% ± 1.9%, respectively; enzyme leakage (24.9 ± 0.7 IU/15 mini gm dry weight was 38% less than in the unfiltered group. Thus, the presence of contaminant particles essentially abolished the protective properties of the St. Thomas' Hospital cardioplegic solution. Since the cardioplegic 0

Volume 89 Number 3

March. 1985

infusion rate at the end of the infusion period in the unfiltered group was only 1.8 ± 0.4 rnl/rnin/gm dry weight (compared with 9.9 ± 0.4 ml/rnin/gm dry weight in the filtered group), the most likely mechanism for this loss of protection was inadequate delivery of the solution to the myocardium during ischemia. Pharmacology of particle-induced vasoconstriction. In an attempt to obtain some indication of the likely mechanism and vascular levels of the particleinduced vasoconstriction, we assessed whether certain vasoactive agents are able to prevent the phenomenon. For these studies we selected agents known to act primarily at different levels of the microcirculation. Because of the known complexity of the dose-response characteristics of some of these agents,"!"!' we constructed dose-response curves for each agent studied. Slow calcium-channel blockers. Some of these agents, e.g., nifedipine, possess potent vasodilator or antispasm properties. In our previous study, we2 reported that a single dose of nifedipine (0.1 ,umoljL) was able to reduce substantially the particle effect. We speculated that this might even offer an explanation for the reported" protective effects of nifedipine when added to unfiltered or coarsely filtered cardioplegic solutions. In the present study, we therefore investigated the antiparticle properties of two calcium antagonists, nifedipine and verapamil, over a wide range of concentrations. As detailed in the Methods section, precautions were taken to prevent photodegradation of nifedipine. As shown in Table III, nifedipine afforded a dosedependent protection against particle-induced vasoconstriction. In contrast to control hearts receiving unfiltered solution, in which the decline in flow over a 20 minute period of infusion was 46.2% ± 5.1%, the addition of nifedipine in concentrations of 0.02, 0.05, 0.1, and 0.5 ,umol/L to the unfiltered solution reduced the 20 minute decline in coronary flow to 34.7% ± 2.1%, 32.3% ± 1.9%, 22.0% ± 1.4%, and 27.3% ± 1.9%, respectively. The flow reduction value for the 0.1 ,umol/L group does not differ significantly from that of the filtered controls, in which the decline in flow was 13.3% ± 3.5%. A nifedipine concentration of 0.1 ,umol/L was therefore selected as optimal. Fig. 5, A shows the coronary flow profile for a particle-containing solution plus this concentration of nifedipine, and also shows the curves for filtered and unfiltered nifedipinefree control solutions. It is clear that at every time point, nifedipine at its optimal concentration abolishes the particle-induced vasoconstriction. In contrast to nifedipine, verapamil exerted no significant effect upon particle-induced vasoconstriction. Even at very high concentrations (20 ,umol/L), this drug failed to displace

Particle-induced coronary vasoconstriction

433

Table m. Dose-response studies of anti-particle activity of various vasoactive agents

Agent

Nifedipine

Verapamil

Procaine

Lidocaine

Dipyridamole

Adenosine triphosphate

Isosorbide dinitrate

Dose 0 0.02 0.05 0.1 0.5 0 0.2 4 20 0 0.05 0.1 0.5 I 5 10 20 0 0.1 I 5 . 0 0.1 I 5 0 0.01 0.1 I 0 I 4 40 400

/Lmol/L /Lmol/L

umol/L /Lmol/L

umol/L umol/L umol/L mrnol/L mmol/L

mrnol/L mrnol/L mrnol/L mmol/L

mrnol/L mmol/L mrnol/L mrnol/L

/Lmol/L /Lmol/L I'mol/L mmol/L rnmol/L rnrnol/L

I'mol/L I'mol/L I'mol/L I'mol/L

Percent decline in coronary flow after 20 min perfusion 46 ± 5 35 ± 2 32 ± 2 22 ± I 27 ± 2 46 ± 5 48 ± 2 46 ± 3 41 ± 3 46 ± 5 36 ± 2 39 ± 4 37 ± 2 38 ± 3 24 ± 4 19 ± 3 33 ± 3 46 ± 5 39 ± 2 42 ± I 56 ± I 46 ± 5 41 ± 3 30 ± 4 31 ± 3 46 ± 5 27 ± I 26 ± 4 9 ± 3 46 ± 5 41 ± 2 40 ± 6 46 ± 2 55 ± 4

significantly the flow profile from its unfiltered position (Fig. 5, B, Table III). Local anesthetics. One factor that might have acted to minimize particle-induced injury in clinical cardiaplegic solutions, in Europe at least, is the common practice of including high concentrations (often 1 to 10 mmol/L) of procaine in clinical solutions. In North America, lidocaine is often used. In high concentrations, procaine exerts vasodilator effects; therefore, we investigated the anti-particle properties of this agent. The results (Table III and Fig. 5, C) show that over a very wide range of concentrations procaine acts to limit the particle effect and in high concentrations such as 10 mmoljL it is as effective as filtration. In contrast to procaine, lidocaine (Table III, Fig. 5,

The Journal of

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Thoracic and Cardiovascular Surgery

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Fig. 5. The ability of various vasoactive agents to overcome the particle-induced impairment of coronary flow. Hearts were perfused at constant pressure (60 ern of water) for a 20 minute period with (0) ultrafiltered (0.8 11m) solution; (0) unfiltered solution; (e) unfiltered solution plus the optimal active concentration of a vasoactive drug. Each point represents the mean of six hearts and the bars represent the standard error of the mean. A, Nifedipine (0.1 Ilmol/L). B, Verapamil (20.0 Ilmol/L). C. Procaine (10 rnmol/L). D, Lidocaine (0.1 mrnol/L).

D) was relatively ineffective, exerting a moderate pro-

tective effect at low concentrations (0.1 mmol/L) but exacerbating the effect at high concentrations (5 mmoIj L). Dipyridamole and adenosine triphosphate. Dipyridamole is a potent coronary vasodilator, and dose-response studies (Table III) show that it exerts some anti-particle effects. However, it was not as effective as nifedipine or procaine and, as its optimal concentration (1.0 /-LmoljL), only partially limited the decline in coronary flow induced by particle-containing solutions (Fig. 5, E). Dipyridamole exerts its effect by inhibiting degradation and loss of adenosine triphosphate (ATP) and its catabolites. Since ATP can be a potent vasodilator (when used in low concentration) and also has been shown to provide additional protection to the St. Thomas' Hospital cardioplegic solution," we investigated its anti-particle properties.

Dose-response studies (Table III) revealed ATP to be highly effective over a wide range of concentrations (0.01 to 1.0 mmoljL). At its optimal concentration (l.0 mmoIjL) it completely prevented (Fig. 5, F) the particle effect, and by the end of the 20 minute infusion period it afforded superior protection to that observed with filtration. Nitrates. The results with the preceding pharmacologic agents, together with our studies with selective filtration, would suggest that particle-induced vasoconstriction occurs primarily at the level of the small vessels. If this is the case, then agents that act predominantly at a large vessel level might not be expected to protect against contaminant particles. This theory was confirmed in a study with isosorbide dinitrate, which over a wide range of concentrations (1.0 to 400.0 /-LmoljL) failed to exert any significant effect upon particleinduced vasoconstriction (Table III, Fig. 5, G).

Volume 89 Number 3

435

Particle-induced coronary vasoconstriction

March, 1985

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Fig. 5. Cont'd. E, Dipyridamole (l J.LmoljL). F, Adenosine triphosphate (l mmoljL). G, Isosorbide dinitrate (4 J.LmoljL).

The pharmacologic spectrum of results. Fig. 6 summarizes the relative abilities of all drugs studied (optimal concentration) to reduce particle-induced impairment of coronary flow. It is clear that a wide spectrum of activities exists, sometimes with very differing drugs exerting very similar effects. This observation indicates that several distinct molecular mechanisms may underly the genesis and control of particle-induced vasoconstriction. Dissection of these mechanisms would require extensive pharmacologic investigations.

Conclusion In this study in the isolated perfused rat heart, we have demonstrated that contaminant particles in commercially available intravenous solutions can cause an increase in vascular resistance. We have attributed this impairment of coronary perfusion to repeated episodes of transient vasoconstriction probably occurring at a small vessel leveL Particle-induced coronary vasoconstriction of this type can adversely affect the infusion of

cardioplegic solutions and can result in a decrease in the efficacy of these protective solutions. Our studies indicate that the problem can be completely overcome by in-line filtration designed to remove all particles of 10 ~m or larger. The problem can also be circumvented by the inclusion of certain vasoactive agents such as procaine, ATP, or nifedipine. Our preliminary studies with these and other vasoactive agents indicate that multiple pharmacologic mechanisms may be involved in the initiation and control of particle-induced vasoconstriction. The clinical significance of particulate contamination. More than 150 years have elapsed since the first clinical use of parenteral fluids. However, it is only in the last 35 years that any attention has been directed toward the possible hazards of particulate contamination. The work of Garvan and Gunner l 6, 17 and others":" drew attention to the wide variety of foreign, undissolved substances present in parenteral fluids. Contaminants included rubber, cellulose fibers, starch granules, glass,

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Thoracic and Cardiovascular Surgery

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20 30 40 10 Percent Decline in Coronary Flow after 20 min.

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Fig. 6. Relative potencies of various vasoactive agents in relation to their ability to prevent particle-induced coronary vasoconstriction. Hearts were subjected to 20 minutes of perfusion at constant pressure (60 ern of water) with unfiltered solution containing the optimal concentration (see text and Table III) of a vasoactive compound. Each histogram block represents the percent decline in coronary flow at the end of the 20 minute perfusion period expressed as a percent of the I minute value. Each result represents the mean of six hearts and the bars represent the standard error of the mean. For comparative purposes, also included are the results for drugfree filtered (0.8 /Lm) and unfiltered solutions.

crystals, cotton fibers, talc, asbestos, fungal spores, and diatoms. Subsequent refinements in formulation and improved filtering, along with the introduction of sealed all-plastic containers by the pharmaceutical industry, have greatly reduced the quantity of debris present in the modern fluids. Likewise, improvements in instrumentation for inspection and detection of particulates have led to tighter controls with higher purity standards and the institution of specific limits for permissible number and size by both the United States and British Pharmacopoeias (Table I). In general, pathological conditions of real significance, proven to be the result of particulate contamination in parenteral fluids, have been shown only in animal models, although there has been some circumstantial evidence in man.": 17 Sarrut and N ezelof" reported foreign body macrophage reactions caused by fine cotton fibers in the lungs of premature infants after they had received large maintenance volumes of intravenous solutions. This and other reports have led to the conclusion that particulate contaminants may produce a variety of pathological conditions including pulmonary

microemboli, thrombi, or granulomas.P" This conclusion is strengthened by the demonstration by Rusho and Bair" that inclusion of an in-line 0.45 Mm filter in routine intravenous fluid administration lines in surgical patients reduced the incidence of phlebitis-related intravenous complications. Cardioplegia: Does intra-arterial administration create a unique vulnerability to injury? The general absence of serious tissue damage as a consequence of the intravenous administration of particle-containing solutions is perhaps more a reflection of the usual mode of administration of these solutions than the safety of the contaminants. Intravenous solutions in conventional usage pass through the lungs, which act as an effective filter for most particles of 5 Mm diameter or larger. When the lungs are deliberately bypassed and fluid is administered directly into the arterial circulation, the consequences of particulate contamination might be expected to be more severe. Over 20 years ago, Silberman, Cravioto, and Feigin" detected cotton fibers in the brains of five patients undergoing cerebral angiography, and in two of the cases this was claimed to be the ultimate cause of cerebral injury. Since then there have been other reports, such as that of Dimmick and associates," who detected fiber embolization of renal, cerebral, mesenteric, and pulmonary arteries during autopsies in 14 patients who had undergone operation of angiography. A substantial body of evidence has now accumulated incriminating microemboli as an important cause of tissue damage during cardiopulmonary bypass.":" The recent explosive growth in the use of cardioplegic solutions during cardiac operations adds another important source for potential injury. During routine cardioplegia, 2 L of solution or more, delivering many thousands of contaminant particles, may be infused into the heart. More than 10 years ago, Brown and colleagues" suggested that filtration through a 5 Mm porosity membrane might improve myocardial protection. Recently, we2, 3 have confirmed this in using a rat heart model and four different commercially available solutions. These studies would argue for the use of

filters or appropriate vasoactive agents during all cardioplegic infusions. This conclusion is reinforced by

the fact that successful cardioplegia and myocardial protection is dependent upon the rapid delivery of adequate quantities of cardioplegic solution to all areas of the heart. In diseased hearts with diffuse ischemia or stenoses of major vessels, the distribution of cardioplegic solution may be suboptimal. The further impairment of solution distribution by particle-induced vasoconstriction may seriously exacerbate the problem of inadequate tissue protection.

Volume 89 Number 3

Particle-induced coronary vasoconstriction

March, 1985

Until suitable cardioplegic filters are available for routine use, it might be appropriate to consider the addition of pharmacologic agents to clinical cardioplegic solutions. In this respect, it is perhaps fortuitous that in Europe, at least, procaine in high concentrations is almost invariably included in cardioplegic formulations. In our experiments, lidocaine failed to exert comparable protective effects, an observation that might explain the widespread lack of enthusiasm for the inclusion of local anesthetics in cardioplegic solutions in the United States, where lidocaine is the available agent. Mechanisms and sites of particle-induced injury. The studies reported in this paper identify particles of 10 ~m in diameter or greater as being the primary culprits in the genesis of injury. This observation, coupled with the profile of pharmacologic responsiveness (inability of nitrates to overcome the effect), suggest that the problem occurs at the small vessel level. It is tempting to identify small arterioles as the likely site of injury, although, as will be discussed later, other possibilities such as venous constriction may exist. Physical blockage of vessels by particles can be dismissed as a direct cause of flow reduction for several reasons: (1) The effect is readily and rapidly reversed by switching to perfusion with a filtered solution; (2) the effect can be overcome by a variety of drugs; (3) there are relatively few particles of 10 ~m and greater in the solutions, and even if the effect were caused by the large number of small contaminant particles (5 ~m or less) the numbers of these particles are small in comparison to the vast number of microvessels;(4) there is a plateau in the coronary flow profile that would not occur in the case of physical blockage. The most likely mechanisms for the induction of vasoconstriction must be either the release of some endogenous vasoconstrictor, possibly as a consequence of particle-induced injury to some vascular component, or an alteration in responsiveness to circulating or endogenous vasoactive agents as a consequence of some particle-induced vascular injury. In the first instance, the particle may trigger the release of some vasoconstrictor agent, for example, which may cause localized vasoconstriction downstream from the site of irritation. The release of this agent may be transient, such that the vasoconstriction is rapidly reversed. In the second instance, particle-induced injury to the vascular endothelium, for example, may alter the responsivenessof the vessel to circulating vasocative agents. This reaction would seem less likely, since under these circumstances sustained vasoconstriction responses might be expected. It would be inappropriate to assume that the site of particle-induced injury and the site of vasoconstriction

437

necessarily coincide. Thus, although we would argue that small arterioles are the most probable site of injury (the particles of 10 ~m and larger being unlikely to traverse the capillary bed), the site of vasoconstriction might well be at the level of the venules. Detailed investigation of differential pharmacologic responsiveness might be employed to resolve such a possibility. The preliminary pharmacologic studies reported in this paper do not allow us to identify a single mediator of the observed vasoconstriction. Since it is difficult to define a common link in the actions of the three most effective agents, nifedipine, ATP, and procaine, it may well be that multiple mechanisms may be involved. We are currently undertaking detailed pharmacologic studies aimed at clarifying this situation. In considering the sites of initiation and mechanisms of vasoconstriction, attention should not be restricted to vascular endothelial cells, since vasoconstrictors such as serotonin and histamine may well arise from nervous tissue, mast cells, or residual platelets. Again, detailed pharmacologic investigations should help resolve the problem. Applicability to man? The findings described in this paper are limited, of course, by their observation in the rat. It is now important to ascertain whether these observations can be reproduced in other species and whether they occur in man. Until this is accomplished, we would suggest that serious consideration be given to the use of vasoactive agents or appropriate filters during cardioplegic infusion. If it finally transpires that the human heart is susceptible to these effects, then we believe that limits of purity as defined by various pharmacopoeias should be revised for all intra-arterial applications. The assistance of Mr. F. Curley, Mrs. C. Boles,and Miss C. Gardener is gratefully acknowledged.

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REFERENCES Robinson LA, Hearse OJ, Braimbridge MY: Particulate contamination. A potential hazard of cardioplegia (letter). Lancet 1:995-996, 1983 Robinson LA, Braimbridge MY, Hearse OJ: The potential hazard of particulate contamination of cardioplegic solutions. J THoRAc CARDIOVASC SURG 87:48-58, 1984 Hearse OJ, Erol C, Maxwell MP, Coltart OJ: Particleinduced coronary vasoconstriction. Patterns of amelioration with various vasoactive drugs (abstr). J Mol Cell Cardiol 15:270, 1983 Langendorff 0: Untersuchungen am uberlebeden Saugethierherzen. Pflugers Arch 61:291-332,1895 Krebs HA, Henseleit K: Untersuchungen uber die Harnstoffbildung im Tierkorper. Hoppe Seylers Z Physiol Chern 210:33-66, 1932 Neely JR, Liebermeister H, Battersby EJ, Morgan HE:

The Journal of

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Effect of pressure development on oxygen consumption by isolated rat hearts. Am J Physiol 212:804-814, 1967 7 Yamamoto F, Manning AS, Braimbridge MY, Hearse DJ: Cardioplegia and slow calcium-channel blockers. Studies with verapamil. J THORAC CARDIOVASC SURG 86:252-261, 1983 8 Szaoz GS, Gruber W, Bernt E: Creatine kinase in serum. 1. Determination of optimal reaction conditions. Clin Chern 22-8:50-56, 1976 9 The United States Pharmacopoeia, United States Pharmacopeial Convention, Inc., Rockville, Md., 1980, revision 20, Yol 20, P 863 10 The British Pharmacopoeia, Cambridge University Press, 1980, Yol 11, Appendix XII, p A120 11 Hearse DJ, O'Brien K, Braimbridge MY: Protection of the myocardium during ischemic arrest. Dose-response curves for procaine and lignocaine in cardioplegic solutions. J THoRAc CARDIOVASC SURG 81:873-879, 1981 12 Yamamoto F, Manning AS, Braimbridge MY, Hearse DJ: Calcium antagonists and myocardial protection. Diltiazem during cardioplegic arrest. Thorac Cardiovasc Surg 31:369-373, 1983 13 Yamamoto F, Manning AS, Braimbridge MY, Hearse DJ: Nifedipine and cardioplegia. Rat heart studies with the St. Thomas' cardioplegic solution. Cardiovasc Res 17:719-727, 1983 14 Clarke RE, Christlieb IY, Ferguson TB, Weldon CS, Marbarger JP, Biello DR, Roberts R, Ludbrook PA, Sobel BE: The first American clinical trial of nifedipine in cardioplegia. J THoRAc CARDIOVASC SURG 82:848-859, 1981 15 Hearse DJ, Stewart DA, Braimbridge MY: Cellular protection during myocardial ischemia. The development and characterization of a procedure for the induction of reversible ischemic arrest. Circulation 54:193-202, 1976 16 Garvan JM, Gunner BW: The harmful effects of particles in intravenous fluids. Med J Aust 11:3-6, 1964 17 Garvan JM, Gunner BW: Particulate contamination of intravenous fluids. Br J Clin Prac 25:119-121, 1971 18 Groves MJ: Particulate contamination in intravenous fluids. Pharm J 210:185-187,1973 19 Turco S, Davis NM: Clinical significance of particulate matter. A review of the literature. Hasp Pharm 8: 137-140, 1973

Thoracic and Cardiovascular Surgery

20 Groves MJ, deMalka SR: The relevance of pharmacopoeial particulate matter limit tests. Drug Devel Comm 2:285-324, 1976 21 Rusho WJ, Bair IN: Effect of filtration on complications of post-operative intravenous therapy. Am J Hasp Pharm 36: 1355-1356, 1979 22 Sarrut S, Nezelof C: A complication of intravenous therapy. Giant cellular macrophatic pulmonary arteritis. Presse Med 68:375-377, 1960. 23 Lockhart JD: The medical significance of particulate matter in large volume parenteral solutions. Safety of large volume parenteral solutions. National Symposium Proceedings, Washington, D. C., 1966, Food and Drug Administration 24 Silberman J, Cravioto H, Feigin I: Foreign body emboli following cerebral angiography. Arch Neurol 3:711-717, 1960 25 Dimmick JE, Bove KE, McAdams AJ, Benzing G: Fiber embolization. A hazard of cardiac surgery and catheterization. N Engl J Med 13:685-687, 1975 26 Kessler J, Patterson RH: The production of microemboli by various blood oxygenators. Ann Thorac Surg 9:221228, 1970 27 Brennan RW, Patterson RH, Kessler J: Cerebral blood flow and metabolism during cardiopulmonary bypass. Evidence of microembolic encephalopathy. Neurology 21:665-672, 1971 28 Patterson RH, Twichell JB: Disposable filter for microemboli. Use in cardiopulmonary bypass and massive transfusion. JAMA 215:76-80, 1971 29 Egeblad K, Osborn JJ, Burns W, Hill JD, Gerbode F: Blood filtration during cardiopulmonary bypass. J THORAC CARDIOVASC SURG 63:384-390, 1972 30 Solis RT, Noon GP, Beall AC, DeBakey ME: Particulate microembolism during cardiac operation. Ann Thorac Surg 17:332-344, 1974 31 Brown HA, Niles NR, Braimbridge MY, Austen WG: Preservation of the myocardium by means of cold physiological solutions, as assessed by ventricular function, histochemistry and birefringence. J Surg Res 14:46-57, 1973