Cerebrovascular Relaxation Responses to Endothelium-dependent and -independent Vasodilators after Normothermic and Hypothermic Cardiopulmonary Bypass in the Rabbit 

Experimental protocols were approved by the Animal Care and Use Committee of the University of Iowa in accordance with the Guide for the Care and Use of Laboratory Animals.

Eighteen New Zealand White rabbits weighing 4.0 - 4.9 kg were assigned randomly to one of two groups, according to whether they would undergo normothermic or hypothermic CPB. Anesthesia was induced by inhalation of 3 - 5% isoflurane in oxygen. After local infiltration with 1% lidocaine, a tracheotomy was performed and the trachea was intubated with a 3-mm cuffed endotracheal tube. Thereafter, the lungs were mechanically ventilated to achieve normocarbia, and anesthesia was maintained with 2% isoflurane in oxygen, monitored by a calibrated agent analyzer (Datex; Puritan-Bennett, Helsinki, Finland). Animals were paralyzed with a succinylcholine/lactated Ringer's infusion (2 mg/ml, 4 ml [middle dot] kg^-1[middle dot] h^-1) via a 22-gauge ear vein catheter and positioned prone. After a midline sagittal scalp incision, a 2-mm burr hole was drilled over the right frontoparietal cortex, and a calibrated, rapid-response 1-mm thermocouple (K-type, L-08419–02; Cole Parmer, Chicago, IL) was introduced under the cranium to rest on the dural surface overlying cerebral cortex. The thermocouple was secured with bone was and fast-drying cyanoacrylate cement, and the animals were placed supine.

The tip of a saline-filled catheter (PE-90; Intramedic, Parsippany, NJ), introduced via the right external jugular vein, was advanced to the superior vena cava to measure central venous pressure. The right subclavian artery was cannulated (saline-filled PE-160) for continuous blood pressure monitoring and intermittent blood sampling. Baseline physiologic measurements were then made: mean arterial pressure, central venous pressure, epidural temperature, arterial blood gases and acid-base state (IL1304; Instrumentation Laboratory, Lexington, MA), hemoglobin concentration (OSM3; rabbit absorption coefficients; Radiometer, Copenhagen, Denmark), and concentration of leukocytes in blood (Neubauer hemocytometer; Reichert Scientific Instruments, Buffalo, NY).

A midline abdominal incision was made. Viscera were packed away with saline-soaked gauze, and the distal abdominal aorta was isolated. The sternum was divided in the midline, the thymus was retracted, and a pledgeted 4 - 0 silk pursestring suture was placed in the right atrium. After systemic anticoagulation with heparin (300 IU/kg given intravenously), the distal aorta was ligated at its bifurcation and cannulated retrograde with a 10-French pediatric arterial perfusion cannula (Biomedicus, Eden Prairie, MN) 7 - 10 mm superior to the distal aortic bifurcation. A 21-French venous cannula (Polystan, Ballerup, Denmark) was placed in the right atrium. The aortic and right atrial cannulas were connected to the perfusion circuit, and CPB was initiated as described subsequently. Just before the start of CPB, the succinylcholine infusion was discontinued and paralysis maintained with pancuronium (0.2 mg/kg).

The CPB circuit consisted of a venous reservoir, membrane oxygenator/heat exchanger (Caplox 308; Terumo, Piscataway, NJ), variable temperature water pump (VWR Scientific, San Francisco, CA), and a centrifugal pump (model 540, BP-50 pump head; Biomedicus). Circuit priming fluid consisted of 300 ml 6.5%(weight/volume) high molecular weight hydroxyethyl starch (McGaw, Irvine, CA) in 0.72 N sodium chloride, 18 mEq sodium bicarbonate, 250 mg CaCl², and 1,000 IU heparin. The priming fluid was circulated through a 40-[micro sign]m filter for 15 - 20 min before addition of [almost equal to] 150 ml filtered packed rabbit erythrocytes, achieving a priming hemoglobin concentration of 6.8 - 9.0 g/dl. Cardiopulmonary bypass was initiated and maintained at a systemic flow rate of 100 ml [middle dot] kg^-1[middle dot] min -1, monitored with a calibrated in-line electromagnetic flow meter (TX-40P; Biomedicus). The pulmonary artery was clamped to ensure total venous outflow to the CPB circuit. To prevent left ventricular ejection or distention, the tip of a 14-gauge catheter was placed transapically in the left ventricle to permit drainage to the venous reservoir. The oxygenator was ventilated with a variable mixture of oxygen and nitrogen to maintain arterial carbon dioxide tension at [almost equal to] 40 mmHg and arterial oxygen tension at [almost equal to] 250 mmHg when measured at an electrode temperature of 37 [degree sign]C. In animals undergoing hypothermic CPB, this constitutes [Greek small letter alpha]-stat acid-base management. Blood from the surgical field was returned to the venous reservoir after passing through a 40-[micro sign]m filter. Sodium bicarbonate was given to maintain a base excess greater than -4 mEq/l, calculated at 37 [degree sign]C. Rabbit erythrocytes were given to maintain hemoglobin concentration at [almost equal to] 7 g/dl. Anticoagulation was maintained with heparin (100 IU/kg) every 30 min. No pharmacologic or mechanical means were used to control arterial pressure.

In animals assigned to hypothermic CPB (n = 9), heat exchanger water temperature was preset at 27 [degree sign]C and systemic cooling began with onset of CPB. At 50 min of CPB, heat exhanger water lines were clamped and water temperature was increased to 40 [degree sign]C. At 60 min of CPB, rewarming was initiated by release of clamps from the heat exchanger water lines. Cardiopulmonary bypass was maintained for 30 min after initiation of rewarming, for a total CPB duration of 90 min. Water bath temperature was decreased whenever brain temperature exceeded 38 [degree sign]C. In animals assigned to normothermic CPB (n = 9), heat exchanger water temperature was set at 36.5 [degree sign]C for the duration of CPB (90 min).

In both groups, anesthesia was maintained during CPB by addition of isoflurane vapor to oxygenator inflow gas. Concentration of isoflurane in exhaust gas was monitored continuously by a calibrated agent analyzer. In animals assigned to hypothermic CPB, concentration of inspired isoflurane was set at 2% at initiation of CPB but was decreased to 1% once a brain temperature of <28 [degree sign]C was achieved. With onset of rewarming, concentration of inspired isoflurane was restored to 2%. Anesthesia in animals undergoing normothermic CPB was maintained with 2% isoflurane throughout. After 90 min of CPB, the experiment was considered complete. Animals were killed by discontinuation of CPB and exsanguination into the venous resorvoir.

With cessation of CPB, descending thoracic aorta and brain (with basilar artery in situ) were rapidly removed and submerged in gas-saturated (95% O^2/5% CO²) room temperature Krebs-bicarbonate buffer (118 mM NaCl, 4.7 mM potassium chloride [KCl], 1.2 mM MgSO⁴, 1.9 mM CaCl², 1.2 mM KH²PO⁴, 24 mM NaHCO³, 11 mM glucose; pH 7.4). Using an operating microscope, the basilar artery was isolated, and extravascular connective tissue was removed from both vessels. Exposure to air was avoided in all steps of vessel preparation. From each animal, two ring segments of aorta (each 3.5 mm long, [almost equal to] 4 mm OD) and two ring segments of basilar artery (each 4.0 mm long, [almost equal to] 450 [micro sign]m OD) were each mounted onto two rigid triangle clips made from 0.29-mm mm and 0.12-mm diameter stainless steel wire, respectively. Each of the four vessel rings was suspended horizontally in a separate 25-ml vessel chamber maintained at 37 [degree sign]C with gas-saturated Krebs-bicarbonate buffer. A mixture of 95% O^2/5% CO²was continuously bubbled through each chamber. In each vessel chamber, one vessel wire was attached to a high-fidelity tension transducer (Grass Instruments, Quincy, MA), and the other wire was attached to a fixed post. Isometric vessel tensions were recorded continuously on a strip-chart recorder (Gould Incorporated, Cleveland, OH). Suspended vessel rings were stretched via an adjustable micrometer to achieve optimal resting tensions (thoracic aorta, 5,000 mg; basilar artery, 1,000 mg) and were equilibrated for 30 min.

Before measurement of vessel relaxation responses to acetylcholine and nitrorprusside, initials screens of vascular smooth muscle and endothelial integrity were performed. KCl was added to each perfusion chamber to achieve a bath concentration of 40 mM. Developed tension (contraction) was recorded. Vessel rings failing to contract would have been considered to have severe smooth muscle injury and would have been discarded. No vessel failed to contract. Thereafter, all vessel chambers underwent one change of fresh gas-saturated buffer, and resting tensions were reestablished. To screen endothelial function, aortic and basilar rings were then contracted with phenylephrine, 3.0 x 10^-7and 3.0 x 10^-3M, respectively.[section] After measurement of developed tension, acetylcholine was added to achieve bath concentrations of 1.0 x 10^-6and 3.0 x 10^-6M, respectively. Vessel rings failing to relax in response to acetylcholine were considered to have severe endothelial injury and were discarded. No vessels failed to relax in response to acetylcholine. Thereafter, all vessel baths underwent one change of fresh gas-saturated buffer, and optimal resting tensions were reestablished.

Dose - response curves to acetylcholine and nitroprusside were then generated. Aortic rings were precontracted with 3.0 x 10^-7M phenylephrine. Basilar rings were precontracted with 3.0 x 10^-3M phenylephrine. In each vessel ring, developed tension in response to phenylephrine was recorded and was designated as initial contraction. Thereafter, vessel relaxation responses to increasing bath concentrations of acetylcholine or nitroprusside were simultaneously assessed in the four respective perfusion chambers (aorta - acetylcholine, aorta - nitroprusside, basilar - acetylcholine, basilar - nitroprusside). Acetylcholine and nitroprusside titrations started at 1 x 10^-9M and increased stepwise to a maximum of 1 x 10^-3M. A complete relaxation response was obtained over 5 - 10 min.

Contrary to our hypotheses, vessel responses to acetylcholine and nitroprusside did not differ between normothermic and hypothermic CPB groups (see Results). To assess whether vessel responses of CPB animals might differ from those of normal animals, a third, non-CPB control group was studied post hoc. Eight animals (4.1 - 5.2 kg) were anesthetized and prepared as described previously, except that after isolation of the abdominal aorta and sternotomy, no additional procedures (e.g., cannulation, CPB) were performed nor medications (e.g., heparin, pancuronium) given. This defined time zero, corresponding to the start of CPB in the CPB groups. For the next 90 min, animals remained ventilated and anesthetized with 2% isoflurane in oxygen and continued to receive a succinylcholine/lactated Ringer's infusion. After this time, animals were killed by clamping the ascending aorta. Vessels were harvested, prepared, ant studied exactly as described previously.

Statistical Analysis. Data are expressed as mean +/- standard deviation. Data from three CPB animals were excluded before analysis. In two animals (one from each CPB group), basilar artery segments had either no response or contracted when exposed to nitroprusside. No other vessel segments behaved in this fashion. Given the known action and mechanisms of nitroprusside, we interpreted these responses as an indication of mechanical vessel injury. Data from additional hypothermic CPB animal were excluded because of mechanical failure of the strip-chart recorder. Hence, data were analyzed from eight normothermic CPB animals, seven hypothermic CPB animals, and eight non-CPB controls.

Developed tension in response to KCl and phenylephrine was compared among groups and between vessel segments exposed to acetylcholine or nitroprusside using repeated measures analysis of variance. Vessel tension at each step of the acetylcholine and nitroprusside titration was expressed as a percentage of the initial contraction. Hotelling's T²was used to test whether mean vasodilatory response was equal among groups at all concentrations of acetylcholine or nitroprusside. [22]This multivariate test gives an overall P value over all concentrations of vasodilator.

Systemic physiologic variables are summarized in Table 1. There were no important differences between groups or over time in mean arterial pressure, central venous pressure, pH^a, or arterial carbon dioxide tension. Non-CPB control animals were 1 - 2 [degree sign]C warmer than normothermic CPB animals. As expected, at 30 and 60 min of CPB, hypothermic CPB animals were approximately 10 [degree sign]C colder than normothermic CPB animals. At 90 min of CPB, hypothermic CPB animals were completely rewarmed. Cardiopulmonary bypass groups did not differ in either arterial oxygen tension or concentrations of hemoglobin but, as expected, were anemic compared with controls. As expected, non-CPB controls had higher arterial oxygen tension than CPB animals.

As shown in Table 2, in descending thoracic aorta, developed tension in response to 40 mM KCl did not differ among groups, nor between vessel segments exposed to acetylcholine or nitroprusside. Likewise, developed tension in response to 3 x 10^-7M phenylephrine did not differ between groups, nor between vessel segments exposed to acetylcholine or nitroprusside. Therefore, the initial contraction ([almost equal to] 7,000 mg higher than resting tension) or aortic vessel segments subsequently exposed to acetylcholine or nitroprusside was equivalent among groups and vessel segments. Relaxation responses to acetylcholine and nitroprusside are shown in Figure 1. There were no differences in aortic relaxation to either acetylcholine or nitroprusside between the two CPB groups (P = 0.44 and 0.83, respectively). Therefore, vessel data from CBP animals were pooled for comparison with control animals. Aortic relaxation in response to acetylcholine did not differ between CPB animals and non-CPB controls (P = 0.10). In contrast, aortic relaxation in response to nitroprusside was slightly greater in CPB animals than in non-CPB controls (P = 0.04).[double vertical bar]

As shown in Table 2, in basilar artery, developed tension in response to 40 mM KCl was significantly less in CPB animals compared with non-CPB control animals (P = 0.002). In contrast, developed tension in response to 3 x 10^-3M phenylephrine did not differ among groups nor between vessel segments exposed to acetylcholine or nitroprusside. Therefore, the initial contraction ([almost equal to] 2,000 mg higher than resting tension) of basilar artery segments subsequently exposed to acetylcholine or nitroprusside was equivalent among groups and vessel segments. Relaxation responses to acetylcholine and nitroprusside are shown in Figure 2. There were no differences in basilar artery relaxation to either acetylcholine or nitroprusside between CPB groups (P = 0.92 and 0.73, respectively). Vessel data from CPB animals were pooled and compared with non-CPB control animals. In contrast to thoracic aorta, basilar arteries of non-CPB animals had greater relaxation than CPB animals in response to both acetylcholine (P = 0.004) and nitroprusside (P = 0.031).#

Hypothermia, independent of CPB, alters endothelial and smooth muscle responses. [22,23]In clinical practice, however, patients are always rewarmed before separation from CPB. To model clinical practice, we incorporated a rewarming phase in the hypothermic CPB group. Cooling and rewarming of vessels in a blood-free medium does not alter endothelial or smooth muscle function. [23,24]Therefore, we hypothesized that if endothelial function was better after hypothermic compared with normothermic CPB, it was probably the result of hypothermic inhibition of inflammatory-mediated endothelial injury. Contrary to our expectation, we did not detect any difference in vessel responses between hypothermic or normothermic CPB. Vessel contraction in response to KCl and phenylephrine and relaxation in response to acetylcholine and nitroprusside did not differ between CPB groups in either test vessel. Therefore, we observed no evidence that hypothermic CPB resulted in better preservation of endothelial or vascular smooth muscle function compared with normothermic CPB.

Although concentrations of proinflammatory mediators may be relatively lower during hypothermic compared with normothermic CPB, they often increase markedly with rewarming. Hence, shortly after separation from CPB, circulating concentrations of inflammatory mediators are often equivalent in patients undergoing normothermic or hypothermic CPB. [5,9,25,26]Recently, Johnson et al. demonstrated that although persistent hypothermia inhibited endothelial inflammatory responses, transient hypothermia did not. [27]Therefore, inflammatory stimuli associated with normothermic and hypothermic CPB may not differ sufficiently in duration or magnitude to differentially affect vascular physiology.

Because of these unexpected results, we wondered whether CPB had any effect on endothelium or smooth muscle compared with its normal state. As a first step toward addressing this question, we studied the vascular responses of a non-CPB group. Because non-CPB animals were studied post hoc, observed differences in vessel responses between non-CPB and CPB animals should be considered tentative. Therefore, it is with reservation that we note that CPB did appear to alter cerebrovascular physiology. First, CPB decreased KCl-induced basilar artery contraction but did not affect subsequent phenylephrine-induced contraction. Second, and more notably, CPB decreased basilar artery relaxation in response to both acetylcholine and nitroprusside. These changes in basilar artery are in contrast to thoracic aorta, where the only difference between CPB and non-CPB groups was a slight increase in the relaxation response to nitroprusside in CPB animals. Thus, CPB-induced alterations of vascular physiology in one vascular bed do not necessarily take place in another.

When comparing basilar artery responses between CPB and non-CPB groups (Figure 2), it appears that, in CPB animals, reduced relaxation responses to acetylcholine and nitroprusside were nearly equal. If CPB had impaired both endothelial and smooth muscle function, one might have expected greater overall impairment in response to acetylcholine (the result of endothelial and smooth muscle dysfunction) compared with nitroprusside (smooth muscle alone). This was not the case. This suggests that, in CPB animals, impaired relaxation in response to acetylcholine was probably not primarily attributable to endothelial dysfunction but rather was largely the result of decreased smooth muscle response to endothelial-derived NO. Use of a different endothelium-dependent vasodilator (e.g., bradykinin, serotonin) might have allowed detection of abnormal endothelial function, but, clearly, the effect of CPB on endothelium was not marked. Another CPB study observed decreased vascular smooth muscle relaxation in response to NO. Isolated pulmonary arteries of transplanted dog lungs were found to have impaired vasodilation in response to nitroprusside when exposed to 30 min of CPB. [28]The effect was specific to CPB because lung arteries not exposed to CPB had no such impairment.

What aspect(s) of CPB could be responsible for the apparent decrease in cerebrovascular smooth muscle relaxation in response to NO? Current evidence does not support nonpulsatile flow during CPB as a likely mechanism. Using a cascade bioassay system with rabbit carotid arteries, Ryan et al. observed increasing pulse pressure (i.e., greater pulsatility) decreased vessel relaxation in response to acetylcholine (reversible with superoxide dismutase), but there was no effect on smooth muscle relaxation to nitroprusside. [14]This is not consistent with our findings; specifically, increased pulse pressure (spontaneous pulsatile flow) was associated with increased vessel relaxation in response to acetylcholine and nitroprusside. Further, if nonpulsatile conditions decreased basilar artery responsiveness to NO, one might expect lesser cerebral blood flow with nonpulsatile compared with pulsatile CPB. In prior studies with this rabbit CPB model, we have observed no difference in either cerebral blood flow or oxygen metabolism between pulsatile and nonpulsatile CPB, at either 27 [degree sign]C or 37 [degree sign]C. [29,30]Therefore, nonpulsatile flow during CPB does not seem likely as the cause of the apparent changes in rabbit basilar artery after CPB.

We speculate that endotoxin or proinflammatory cytokines present during CPB could be responsible for the observed changes in basilar artery. For example, administration of endotoxin decreases rat pulmonary vascular smooth muscle relaxation to nitroprusside. [31]This effect appears to be largely attributable to decreased sensitivity of guanylate cyclase to NO, because vessel relaxation in response to a cyclic guanosine monophosphate analogue (8-bromo-cGMP) is normal. We did not measure concentrations of inflammatory mediators in these experiments. In subsequent studies with this preparation (n = 6), however, we have found concentrations of endotoxin in plasma after 90 min of CPB to range from 5 - 20 endotoxin units/ml (0.5 - 2.0 ng/ml), with no obvious difference between normothermic and hypothermic CPB. In human CPB studies, concentrations of endotoxin approximate 90 pg/ml. [1,2]Given the effect of endotoxin on vascular smooth muscle (described previously), endotoxemia during CPB may have contributed to the decrease in basilar artery responsiveness to NO. Additional experiments are needed to confirm this hypothesis and to establish whether the apparent CPB-induced reduction in basilar NO response is attributable to altered guanylate cyclase or to other changes in vascular smooth muscle.

In the brain, flow-metabolism coupling is mediated in part by NO. [32]In clinical studies [33]and this animal model of CPB, [34]increases in brain metabolism have been shown to temporarily exceed increases in cerebral blood flow during rewarming, resulting in cerebral venous hemoglobin desaturation. Our tentative observation of a CPB-induced impairment of cerebrovascular smooth muscle response to NO could provide a mechanistic basis for this phenomenon.

Our tentative observations regarding the effect of CPB on cerebrovascular endothelium and smooth muscle differ markedly from the only other reports that specifically study cerebral vessels in this context. [17,35]Also using in vitro techniques, Sellke et al. studied cerebral microvessels (100 - 175 [micro sign]m ID) from pigs that underwent 2 h of normothermic CPB. [17]Compared with control microvessels, CPB microvessels had decreased vasodilatory responses to acetylcholine but no change in smooth muscle response to nitroprusside - exactly the opposite of what we observed. In a subsequent report, this group reported less endothelial dysfunction in pigs that underwent hypothermic (25 [degree sign]C) versus normothermic (37 [degree sign]C) CPB. [35]In contrast, we observed no difference in endothelial or smooth muscle function between normothermic and hypothermic CPB groups. Species differences in vascular responses to inflammatory stimuli may be partly responsible for the disparate results. For example, activated complement inhibits endothelium-mediated vasodilation in both species. [13,36]In rabbits, however, this inhibition disappears on removal of activated complement, [13]whereas in pigs, it may not. [36]Therefore, when assessed in vitro, complement-mediated alterations in vascular function may be detectable in porcine vessels but not in rabbit vessels. Another methodologic difference between our study and those of Sellke et al. relates to vessel size. Microvascular endothelium may be more susceptible to the effects of proinflammatory processes than endothelium in larger (conducting) arteries. [37,38]Impaired endothelial function in microvessels with preserved endothelial function in macrovessels has been observed in studies of endotoxemia [37]and neutrophil activation/ischemia. [38]For this reason, endothelial injury may be easier to detect in microvessels than in macrovessels.

Alternatively, the endothelial dysfunction observed in the Sellke et al. studies may relate to their use of bubble oxygenation. Although these investigators included an arterial filter in their CPB circuits, filters only partially reduce cerebral arterial gas embolism; they do not eliminate it. [39]Microbubbles produced by bubble oxygenators (which range from 100 - 200 [micro sign]m in diameter) obstruct vasculature of equivalent diameter. Cranial window studies show that arterioles that have undergone transient air embolism have reduced in vivo vasodilation in response to carbachol, an acetylcholine receptor agonist. [40]Therefore, the impaired acetylcholine-induced vasodilation of cerebral arterioles observed by Sellke et al. is consistent with the effect of microscopic gas emboli. Our preparation used membrane oxygenation, which has been shown to result in vastly fewer cerebral microbubbles. [39]The opposing findings of our experiment and those of Sellke et al. indicate additional studies are needed to:(1) resolve species issues;(2) learn how in vitro vascular responses differ from those in vivo;(3) differentiate brain micro- versus macrovessel responses to CPB; and (4) better characterize the mechanisms of cerebral endothelial and vascular smooth muscle dysfunction during CPB, including inflammatory phenomena and the effects of gas embolism.

In rabbits that underwent 90 min of CPB, in vitro endothelial and vascular smooth muscle function did not differ between normothermic and hypothermic CPB in either basilar artery or descending thoracic aorta. In basilar artery, CPB decreased in vitro relaxation responses to both acetylcholine (endogenous NO) and nitroprusside (exogenous NO) compared with post hoc non-CPB controls. Cardiopulmonary bypass-induced impairment of NO-mediated cerebrovascular relaxation may underlie disturbances in cerebral blood flow-metabolism relationships observed during the warming phase of CPB.

[section] Phenylephrine-induced vessel contraction is independent of endothelium. [12]In pilot aortic rings (n = 12), 3.0 x 10^-7M phenylephrine resulted in vessel contraction that was 82 +/- 7% of maximum (induced by 80 mM KCl). In pilot basilar artery rings (n = 5), 3.0 x 10^-3M phenylephrine resulted in vessel contraction that was 81 +/- 13% of maximum (induced by 80 mM KCl).

[double vertical bar] The concentration of nitroprusside with the greatest difference between groups (3 x 10^-7M) had a P value of 0.005 (two-sided Student's t test).

# The concentration of acetylcholine with the greatest difference between groups (3 x 10^-7M) had a P value of 0.0004. The concentration of nitroprusside with the greatest difference between groups (1 x 10^-7M) had a P value of 0.0008.