Tissue oxygen exchange and reactive hyperemia following microembolization

JOURNALOFSURGICALRESEARCH32,38-43

Tissue

(1982)

Oxygen Exchange and Reactive Following Microembolization

Hyperemia

STEVEN E. LANDAU, M.D., ROBERT S. ALEXANDER, PH.D., SAMUEL R.PowERs,JR., M.D.,D.Sc., HOWARD H. STRATTON, PH.D., ROY D. GOLDFARB, PH.D. Departments

AND

of Physiology and Surgery, Neil Hellman Medical Research Building, AIbany Medical of Union University, Albany, New York 12208, and Department of Mathematics, State University of New York, Albany, New York Submitted

for publication

College

December 23, 1980

We tested the hypothesis that microembolization of canine skeletal muscle causes a defect in tissue oxygen transport leading to reduced oxygen consumption at low blood flow rates. Before embolization, oxygen consumption was proportional to flow at flow levels less than 2.8 ml mitt-’ 100 g-‘. Embolization with up to 90 million 1S-am polystyrene spheres caused oxygen consumption to be proportional to flow at flows less than 4.0 ml min- ’ 100 g-’ (P -Z 0.05). At higher flow rates, oxygen consumption was independent of flow rate and the same before and after microembolization. The increase in vascular conductance in response to 2 min arterial occlusion or 1 ml 10 mM adenosine were progressively diminished by increasing doses of microspheres. Both responses were attenuated to the same degree. These data support the hypothesis that microembolization diminishes the ability of the vascular bed to recruit capillaries and restricts tissue oxygen transport, reducing oxygen consumption. Therefore, microembolization may model the defect in tissue oxygen utilization and reactive hyperemia that have been observed in the clinical states of trauma and sepsis suggesting that microembolization may be the mechanism for these defects.

seldom limits oxygen consumption. During shock conditions, however, microaggregates could be deposited in the microvasculature [ 1 l] and it is possible that they could limit tissue oxygen delivery and thereby restrict oxygen consumption in these circumstances. Such capillary plugging would he produced by platelet aggregation [ 121, or the appearance of other blood-borne particulate matter [ 11. Significant capillary microembolization might redistribute microvascular blood flow from nutritional to functionally nonnutritional channels, as defined by Renkin [lo]. It could also reduce or abolish the reactive hyperemic response [5, 131. A shift in blood flow toward nonnutrition pathways would impede the transport of oxygen from the capillary to the cell and induce anaerobic glycolysis. Such a shift in perfusion has been reported by Gaehtgens et al. [5] in skeletal muscles. Following partial microembolization with latex microspheres they observed

INTRODUCTION

Observations of resuscitated trauma patients suggested an impairment of peripheral oxygen transport, since oxygen consumption increased with increasing oxygen delivery [ 141. These findings occurred in conjunction with normal or elevated venous oxygen tensions and cardiac indices. In addition, these patients often exhibited deficient autoregulation as indicated by a reduced reactive hyperemic response in the limb. Similar observations have been made by other investigators in patients following shock, trauma, and sepsis [3, 6, 71. Therefore, the reduced oxygen consumption in severe hemorrhagic and traumatic shock is well established. The cause for this reduction could be due to either a reduced delivery of oxygen or a paralysis of normal cellular metabolic activity or both. In normal circumstances, inadequate oxygen delivery 0022-4804/82/010038-06501.00 Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form rcservrd.

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ET AL.: O2 TRANSPORT

an increase in venous oxygen saturation despite a decrease in total blood flow. Clearance of 4-aminoantipyrine (a diffusion-limited substance) and capillary transport coefficients also decreased. These results implicated a shift in blood flow toward functionally nonnutritive channels. Since microembolization blocks some capillaries directly and may impede flow through other capillaries, a redistribution of blood flow may be expected. If such a redistribution results in the appearance of channels which do not provide nutritional flow to meet metabolic requirements of the adjacent cells, the oxygen consumption of these cells will become blood flow limited. When viewed at an organ level, this would then decrease oxygen consumption. A second direct effect of microembolization may be to reduce the number of capillaries available for recruitment. This would then be reflected in a reduced response to a vasodilatory stimulus such as bolus infusion of adenosine or a 2min arterial occlusion. In this study we sought to examine whether partial microembolization could limit oxygen transport and capillary recruitment capacity of the canine skeletal muscle vasculature. METHODS

We studied nine mongrel dogs weighing about 20 kg. The dogs were anesthetized with 30 mg/kg sodium pentobarbital iv supplemented as necessary. Intravenous saline was infused at a rate of 50 ml/hr to prevent dehydration. A tracheostomy was performed, and the animals breathed spontaneously. Two dogs required ventilatory support to maintain adequate arterial Pco2 levels. The muscles of the leg were isolated by tourniquets. The leg was skinned, wrapped in cellophane, and a tourniquet was placed at the ankle. Catheters were introduced through side branches of the femoral artery and vein for pressure measurements, microsphere or adenosine injection, and venous blood sampling. Arterial blood samples were obtained from the carotid artery.

AFTER

MICROEMBOLIZATION

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Blood gas tensions and pH were measured using standard electrodes (Corning 161 blood gas analyzer) and adjusted for rectal temperature by standard nomograms. Hemoglobin concentrations were measured spectrophotometrically using the cyanmethemoglobin method. Oxygen contents were measured directly on an oxygen analyzer ( Lex-02-Con, Lexington Instruments) and checked against values calculated from the blood gases using a standard Pso of 28.8 Torr. Arterial blood flow was measured by a noncannulating electromagnetic flow probe (Biotronix Laboratories) with mean and phasic tracings recorded on a polygraph. Oxygen consumption (ri,,) was calculated as the product of mean stead-state flow per 100 g wet wt times the arteriovenous (A-V) oxygen content difference. Vascular conductance was calculated as flow divided by the arteriovenous blood pressure difference. Reactive hyperemia of the isolated vascular bed was evaluated by observing changes in blood flow following a 2-min occlusion of the femoral artery. The hyperemic response was quantitated by calculating vascular conductance at the time of peak reactive hyperemic flow. The time required to reach peak reactive hyperemic flow from occlusion release was also recorded. Another estimate of the dilatory capacity of the vascular bed was obtained by calculating the peak conductance of the bed following an injection of 1 ml of 10 mM adenosine (Sigma) into the femoral artery. Microembolization was performed by injections of 20, 30, or 60 million 15 f 3-pm polystyrene microspheres (3M Co.) in sequential boli during the peak conductive period following a 2-min arterial occlusion. The spheres were suspended in saline and 0.05% Tween 80 at a concentration of 30 million spheres/ml. When the surgical preparation was complete, the animals were allowed to stabilize for 30 min. Baseline measurements, taken 30 min apart, included arterial and venous blood gases, oxygen content, venous hemoglobin concentration, intravascular pres-

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sures, arterial flow with occlusive zero flow, and the reactive hyperemic response to both arterial occlusion and to adenosine injection. In later experiments, in order to study a wider range of flows, flow was decreased to 50-75s of the prevailing spontaneous flow by partial arterial occlusion. These parameters were measured at steady state following each of several sequential administrations of microspheres; the experiments were terminated when the flow had been reduced to 1 ml min-’ 100 g-‘. The muscle was then excised from the bone and weighed. Postmortem lung and muscle biopsies were taken for microscopic examination. The relationship between limb O2 consumption and limb blood flow was modeled by fitting a least-squares linear-linear spline wherein the line on the left passed through the origin and the line on the right was parallel to the x axis (see Fig. 1). This treatment of the data approximated two physiological conditions: the first where O2 consumption was dependent on flow (at the left) and the second where O2 consumption was independent of the flow (at the right, Fig. 1). The general principles of the permutation test [2] were employed to statistically test the significance of the differencee between the spline generated by the data groups before and after microembolization. The control values of all parameters were obtained by averaging the measurement made at the two baseline periods and this value was statistically compared to the subsequent measurement. All other data were analyzed with Student’s paired t test, with significance being attributed to a P value less than 0.05. Values are expressed as the mean +- SEM.

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1982

thermore, at high flows (above 4.0 ml min-’ 100 g-‘) the vo, was not changed by microembolization. Before microembolization, the vascular bed exhibited an appropriate reactive hyperemic response to a 2-min arterial occlusion since mean blood flow increased markedly after the release of the arterial occlusion (Fig. 2, top). The peak reactive hyperemic flow occurred at 5.0 -t 0.9 set after the release of the occlusion. After embolization with 60 million spheres, the reactive hyperemit response was markedly reduced (Fig. 2, bottom panel). Before microembolization the instantaneous flow exceeded the baseline by over 10 ml min-’ 100 g-’ for a duration of at least 30 sec. In contrast, following 60 million microspheres embolization, the excess flow was decreased almost to baseline levels along a single exponential curve within 15 set (Fig. 3). The peak hyperemic response to 1 ml of 10 mM adenosine was almost identical to peak hyperemic response to a 2-min arterial occlusion (Fig. 4). Peak conductance in responseto the 2-min arterial occlusion or to adenosine infusion decreased as the number of injected microspheres increased (Fig. 5). When approximately 60 million microspheres had been infused per hindlimb, the increase in conductance in response to either adenosine or 2 min arterial occlusion was abolished (Fig. 5). Muscle biopsies showed single spheres in capillaries, groups of two or three in arterioles and occasional clumps in larger vessels. Lung biopsies revealed that very few microspheres had passed the muscle capillary network. DISCUSSION

RESULTS

Prior to embolization, voZ was found to be dependent on flow below 2.85 ml min-’ 100 g-’ and was independent of flow at higher flows (Fig. 1). After embolization, voZ was found to be dependent on flows below 4.00 ml min-’ 100 g-’ and similarly independent of flow at higher flow rates. Fur-

Although initial blood flow was high in some of these preparations, oxygen consumptions were in the normal range for resting skeletal muscle [4]. This fortuitously provided a broad plateau of the flow values to demonstrate that tissue oxygen utilization was independent of the rate of blood flow. As has been reported by others [4, 91, we

LANDAU

ET AL.: O2 TRANSPORT

AFTER

41

MICROEMBOLIZATION

0 Pre - Embolizotion l Post - Embolization

0

I2345

IO I5 20 BLOOD FLOW ml/min/lOO g

25

FIG. 1. Microembolization effects on oxygen consumption. Oxygen consumption was found to be dependent upon flow at low flows and independent at high Rows. The break between these conditions occurred at 2.85 ml min-’ 100 g-’ prior to embolization. Embolization caused this break to increase significantly (P i 0.01) to 3.92 ml min-’ 100 g-‘.

found that in the normal preparation blood flow had to be artificially restricted to values of less than 3 ml min-’ 100 g-’ for tissue oxygen utilization to become a function of oxygen delivery. Microembolization at dosages up to 90 million microspheres per hindlimb was found to have no significant effect

200 J-2-

IWASK

EMBOLIZED FIG. 2. Phasic and mean flow recordings of a typical reactive hyperemic response to 2 min arterial occlusion before (top) and after microembolization (bottom). Before embolization, peak flow occurred 5 set after release and recorded flow was in excess of baseline flow for greater than 30 set (top). After microembolization, peak flows were recorded immediately after release. The decline in the Row was rapid and decayed exponentially.

on oxygen utilization when flow remained in the normal flow range. This suggests that cellular metabolic mechanisms remained functional. Yet, when flow was reduced artificially after microspheres, dk’urbcd function became evident in that the inflection from the plateau occurred at a higher flow value of almost 4 ml min-’ 100 g-’ with a corresponding shallower slope of the flowdependent segment of oxygen utilization. Accompanying this evidence of disturbed oxygen delivery to the tissues was a loss of the normal reactive hyperemic response as well as a deficit in the vasodilator response to adenosine. The correlation between these two deficiencies as a function of dosage of microspheres provides strong support for the hypothesis that normal blood flows are maintained after microembolization by the recruitment of previously nonperfused segments of the microcirculatory bed. This recruitment would obviously diminish the capacity of the vascular bed to be dilated by adenosine or by the metabolites accumulated during a transient ischemia. The steep slope

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JOURNAL OF SURGICAL

RESEARCH: VOL. 32, NO. 1, JANUARY

1982

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Peak Adenosine Conductance

TIME (seconds)

FIG. 3. The amount by which the flow during reactive hyperemia exceeded the baseline (excess reactive hyperemia flow) declined monoexponentially and rapidly following microsphere injection. Before embolization this flow remained elevated for 30 set of more following release of the arterial occlusion. The rapid monoexponential decline suggests that the response was limited to capacity filling of the vascular bed.

of the reactive hyperemia plot following microembolization (Fig. 3) appears to be due to the refilling of the vasculature following ischemia. This refilling occurs in conduit vessels which are not involved in recruitment [8]. All of these observations are in agreement with the hypothesis that microembolization, by blocking off segments of the microcirculatory bed, creates potential problems for oxygen diffusion despite the capacity of the vascular bed to autoregulate by recruitment of new capillary channels. If oxygen delivery to the tissues is compromised by other factors leading to reduced perfusion or deficient arterial oxygen content, the compensatory ability of the microcirculation will be inadequate and oxygen diffusion to tissue cells may fail to meet metabolic requirements. In summary, this study determined the microembolization of the peripheral vasculature mimics the defects in oxygen utili-

FIG. 4. Correlation of peak conductances induced by 2 min arterial occlusion and adenosine injection. Peak conductances induced by either method are identical. Microembolization shifted peak conductances to the lower left-hand corner.

zation in the apparently resuscitated trauma patient. We found that graded doses of microspheres impaired oxygen transport to the tissues of the hindlimb at low flows even though capillary recruitment could compensate for the deficiency at normal flows. Our I loo + 102 0 103 : o d o b

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FIG. 5. Change in reactive hyperemic conductance with increasing dosesof microspheres. The reactive hyperemic conductance was reduced markedly by increasing microembolization. The reduction followed an exponential decay (r = 0.81). Each symbol represents a separate experiment.

LANDAU

ET AL.: O2 TRANSPORT

studies provide quantitative rationale for the clinical use of reactive hyperemia or a vasodilator drug in order to identify when microembolization has impaired circulatory function. ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of Health: GM-15426 and HL-19977. Drs. Goldfarb and Stratton are recipients of Research Career Development Awards (HL-00947 and GM-00082, respectively). Dr. Powers is now deceased. The other authors of this manuscript hereby acknowledge his invaluable contribution and enrichment not only to this study but to our lives, professional and personal.

REFERENCES 1. Appelgren, K. D., and Lewis, D. H. Capillary flow and capillary transport in dog skeletal muscle after induced intravascular RBC aggregation and disaggregation. Eur. Surg. Res. 2: 161, 1970. 2. Cox, D. R., and Hinkley, D. V. Theoretical Starisks. London: Chapman & Hall, 1974. 3. Dab&i, R. P., Fantini, F., CappeBi, G., and Grandonico, F. Relationship between reactive hyperemia and response to vasodilatory drugs in patients with obliterating arterial disease of the lower limb. Minerva Cardioangiol. 18: 287, 1970. 4. Duran, W. N., and Renkin, E. M. Oxygen consumption and blood flow in resting mammalian skeleta1 muscle. Amer. J. Physiol. 226: 173, 1974.

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5. Gaehtgens, P., Benner, K. U., and Schickendantz, S. Nutritive and nonnutritive blood flow in canine skeletal muscle after partial microembolization. Ppuegers Arch. 361: 183, 1976. 6. Gump, F. E., Butler, H., and Kinney, J. M. Oxygen transport and consumption during acute hemodilution. Ann. Surg. 168: 54, 1968. 7. Gump, R. D., Kinney, J. M., and Price, J. B., Jr. Energy metabolism in surgical patients: Oxygen consumption and blood flow. J. Surg. Res. 10: 613, 1970. 8. Johnson, P. C., Burton, K. S., Henrich, H., and Henrich, U. Effect of occlusion duration on reactive hyperemia in sartorius muscle capillaries. Amer. J. Physiol. 230: 715, 1976. 9. Pappenheimer, J. R. Blood flow, arterial oxygen saturation and oxygen consumption in the isolated perfused hindlimb of the dog. J. Physiol. 99: 283, 1941. 10. Renkin, E. H. Effects of blood flow on diffusion kinetics in isolated, perfused hind legs of cats. Amer. J. Physiol. 183: 125, 1955. 11. Saldeen, T. The microembolization syndrome. Microvasc. Res. 11: 277, 1976. 12. Saldeen, T., Busch, C., and Lindquist, 0. The effect of fibrin and platelets on the microembolization syndrome in the dog. Microvasc. Rex 6: 250, 1973. 13. Selkurt, E. E., Rothe, C. F., and Richardson, D. Characteristics of reactive hyperemia in the canine intestine. Circ. Res. 15: 532, 1964. 14. Shah, D. M., Dutton, R. E., Newell, J. C., and Powers, S. R. Jr. Vascular autoregulatory failure following trauma and shock. Surg. Forum 28: 11, 1977.