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J Thorac Cardiovasc Surg 1995;109:21-29
© 1995 Mosby, Inc.
SURGERY FOR ACQUIRED HEART DISEASE |
Athens and Augusta, Ga.
Supported by the Georgia Heart Association and the American Foundation for Pharmaceutical Education.
Received for publication Dec. 23, 1993. Accepted for publication May 10, 1994. Address for reprints: Randall Tackett, PhD, Associate Professor and Head, Pharmacology & Toxicology, College of Pharmacy, University of Georgia, Athens, GA 30602-2356.
Abstract
Methylene blue is occasionally applied to the adventitia of blood vessels during coronary artery bypass and other vascular procedures to assist in the orientation of the vessel. Inherent in this method is the assumption that extravascular application of methylene blue is innocuous with regard to vascular function. In the first part of this study, the in vitro vascular reactivity of methylene blue-labeled saphenous veins was compared with that of veins that were not marked with methylene blue. The vasoactive agents tested were designed to examine multiple pathways. They included potassium chloride, prostaglandin F2a , phenylephrine, serotonin, angiotensin II, BHT-933 (
2 -adrenergic agonist), sodium nitroprusside, acetylcholine, isoproterenol, and verapamil. Compared with unmarked veins, those marked with methylene blue demonstrated a significant impairment of both vasoconstrictor and vasodilator function. These observations were made on a relatively small number of patients and could therefore be attributed to inherent differences between patients or surgical procedures. In the second part of this study, these variables were eliminated by dividing a single vein from one patient into three segments for a 45-minute exposure to external only methylene blue, internal and external methylene blue, or no methylene blue. The segments were then evaluated for vasoreactivity in vitro. Externally applied methylene blue reduced vasoconstriction regardless of the agonist. Further, both endothelium-dependent and -independent vasodilation was diminished by external methylene blue exposure. In veins exposed to methylene blue both internally and externally the results were similar but the magnitude of impairment greater. It is concluded that surgical marking of blood vessels with methylene blue has the potential to adversely affect vascular reactivity and therefore the use of alternative dyes should be considered. (J THORACIC CARDIOVASC SURG 1995;109:21-9.)
Methylene blue (MB) is occasionally applied extravascularly during a variety of vascular procedures to assist in the orientation of the blood vessel. The use of marking dyes serves to facilitate isolation and may therefore reduce surgical trauma. This is particularly true with microvascular operations in which vascular torsion can be reduced by the use of marking dyes.
1 Procedural trauma to blood vessels is a legitimate practical concern, especially when considering the functional importance of the delicate endothelium. The use of MB as a marking dye is thought to be innocuous on the basis of the assumption that application to the adventitia is unlikely to significantly penetrate the smooth muscle or endothelium. However, a recent report by Mazmanian and associates 2 suggests that MB applied to the adventitia has significant pharmacologic actions in rat tail arteries at doses 100 times lower than the conventional 1% MB stock solution used for surgical marking. Further, Bentz and coworkers 3 used an in vitro human arterial model to demonstrate that topical MB may increase platelet deposition. Therefore, careful reevaluation of MB as a surgical marker seems prudent. In this study, we evaluate the potential of MB to affect the in vitro vascular reactivity of human saphenous veins using two independent approaches.
MATERIALS AND METHODS
Vascular procurement
Sections of saphenous vein were randomly obtained from 23 patients undergoing coronary artery bypass grafting. The vessels were immediately transported to the laboratory in a modified Krebs-Henseleit buffer of the following composition: NaCl, 118.0 mmol/L; KCl, 4.7 mmol/L; MgSO 4 , 1.2 mmol/L; KH2 PO 4 , 1.1 mmol/L; CaCl2 , 2.5 mmol/L; NaHCO3 , 25.0 mmol/L; dextrose, 10.0 mmol/L; indomethacin, 2.8 µmol/L; and sodium ethylenediaminetetraacetic acid, 10 µmol/L; 25° C, pH 7.4, previously aerated with 95% oxygen and 5% carbon dioxide, and sealed. The vein was carefully cleaned of all surrounding fat and connective tissue and cut into 4 mm rings. The vascular rings were suspended between two metal hooks in a 10 ml tissue bath containing identical buffer at 37 º C continuously aerated with 95% oxygen and 5% carbon dioxide. The vascular isometric tension was measured with a Grass FT 03 force-displacement transducer (Grass Instrument Co., Quincy, Mass.) integrated to a low-level direct-current amplifier recording on a paper-charted polygraph (Beckman R611; Beckman Instruments, Inc., Fullerton, Calif.). The rings were stretched to optimal diastolic resting tension as determined in pilot studies (2 gm) and allowed 45 minutes to equilibrate before initiation of protocols. Bath solutions were changed every 20 minutes throughout all experiments. The time between tissue harvest and initiation of protocols was less than 3 hours.
Protocols
For the first set of experiments, responses of saphenous veins that had been marked with MB (n = 5/24) in the operating room were compared with unmarked veins (n = 12/72). After equilibration, all rings were constricted with KCl at a concentration of 100 mmol/L and then washed. Vasoconstrictor dose response curves were obtained by exposing each ring to increasing concentrations of only one of the following: phenylephrine, serotonin, angiotensin II, or the 2 -adrenergic agonist BHT-933 (BHT). After washout, the vessels were preconstricted with prostaglandin F2 (PGF2) 3 µmol/L. Vasodilator dose response curves were then obtained by exposing each vascular ring to increasing concentrations of only one of the following: sodium nitroprusside, acetylcholine, isoproterenol, or verapamil. According to this design, each ring was exposed to only one vasoconstrictor and one vasodilator (plus KCl and PGF2 ). The order of drug administration was randomized.
For the second set of experiments, an unmarked saphenous vein was cut into three segments, each 3 to 4 cm long (n = 6/36). One segment was tightly ligated with suture at both ends. The ligated segment and an unligated segment were placed in continuously aerated Krebs buffer containing 0.1% MB (2.7 mmol/L). This dose of MB is 10 times lower than the conventional MB solution used to mark veins in the operating room. The remaining venous segment was placed in an identical buffer without MB. After 45 minutes, the segments were removed and extensively washed with Krebs solution. The exposure time was based on the estimated average time that a vein is exposed to MB from the time of marking to transplantation during a bypass procedure. Two vascular rings from each segment were cut and suspended in the tissue bath as described earlier. The ligated vessels that had been placed in the MB buffer served as the "external MB" group. The unligated vessels exposed to MB buffer and the unligated vessels exposed to MB-free buffer served as the "internal/external MB" group and the "MB-free" group (control), respectively. After equilibration, all rings were constricted with KCl 100 mmol/L and then washed. Vasoconstrictor dose response curves were obtained by exposing each ring to increasing concentrations of either phenylephrine or serotonin. After washout, the vessels were preconstricted with PGF2 3 µmol/L and vasodilator dose response curves were obtained by exposing each ring to increasing concentrations of either sodium nitroprusside or acetylcholine. As with the previous design, each ring was exposed to only one vasodilator. Gross and histologic examination at the conclusion of the experiment showed no visible MB on the endothelial surface of the ligated vessels.
Data analysis
When enough tissue was available, the rings were run in pairs and the responses averaged to yield a single value. Therefore, all n values are presented as the number of patients followed by the number of ring preparations. In all cases, statistical analysis was performed with the number of patients used as the n value. MB impaired the ability of veins to constrict to both KCl and PGF2 . This impairment can potentially skew any data normalized to KCl or PGF2 responses. Therefore, vasoconstrictor responses are reported both in grams of tension and as a percentage of response to KCl. Likewise, vasodilator responses are presented as both a percentage of preconstriction and as grams of relaxation. Dose response curves were evaluated by an analysis of variance with a Scheffe's adjustment for multiple comparisons. For the first set of experiments in which the veins were from different patients, an unpaired t test was used to assess single KCl and PGF2 responses. In the second set of experiments in which veins for each test group were from the same patient, a paired t test was used to compare the single KCl and PGF2 responses to control responses. Effective concentrations for half-maximal responses (EC 50 ) were calculated by performing a curve fit on each individual vascular ring's dose response. The equation chosen for curve fitting was based on receptor theory and is detailed elsewhere. 4 Log-normal distribution is expected for equieffective doses; therefore, mean logs were used in comparing EC50 values as previously described. 5 The EC50 values were compared by an unpaired t test in Table I and an analysis of variance with Scheffe's test in Table II. All data are presented as means plus or minus standard error of the mean and only those differences with a p value of less than 0.05 were considered statistically significant.
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Veins marked with MB during the operation
A total of 12 of 72 unmarked (from 8 men and 4 women) and 5 of 24 MB-marked (from 3 men and 2 women) saphenous vein preparations were evaluated for vascular reactivity. Compared with unmarked vessels, those marked with MB showed a significant impairment in their ability to contract to all vasoconstrictor agents used (Figs. 1 and 2). Vasoconstriction to KCl (Fig. 1, A) and PGF2 (Fig. 1, B) in MB-marked versus unmarked vessels was reduced by 30% ± 10% and 22% ± 8%, respectively. Compared with unmarked vessels, the maximal contraction of MB-marked saphenous vein, expressed in grams of tension, was reduced by 73% ± 8%, 53% ± 17%, 58% ± 13%, and 38% ± 12% for phenylephrine (Fig. 2, A), serotonin (Fig. 2, B), angiotensin II (Fig. 2, C), and BHT (Fig. 2, D), respectively. The same data, expressed as a percentage of maximal response to KCl, showed a reduction of 51% ± 21%, 45% ± 22%, 53% ± 14%, and 36% ± 20% for phenylephrine, serotonin, angiotensin II, and BHT, respectively. Compared with unmarked vessels, those marked with MB showed a significant impairment in their ability to relax to all vasodilator agents used (Fig. 3). MB-marked veins demonstrated a significant rightward shift in EC50 values for sodium nitroprusside and isoproterenol (see 
Table I). Evaluation of the nadir of the dilatory dose response curves demonstrated that relaxation was reduced by 54% ± 17%, 46% ± 17%, 53% ± 14%, and 78% ± 18% for sodium nitroprusside (Fig. 3, A), acetylcholine (Fig. 3, B), isoproterenol (Fig. 3, C), and verapamil (Fig. 3, D), respectively, when data are expressed as a percentage of PGF2 preconstriction. The same data expressed as grams of relaxation showed a similar reduction in the MB-marked vessels of 68% ± 12%, 68% ± 10%, 60% ± 7%, and 82% ± 6% at the same point of sodium nitroprusside, acetylcholine, isoproterenol, and verapamil, respectively.
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and KCl with the greatest impairment seen in the external/internal exposure group. Compared with control vessels, vessels in the external MB group showed a 36% ± 16% (Fig. 4, A) impairment of KCl constriction and a 33% ± 19% (Fig. 4, B) impairment of PGF2 constriction. Vessels in the internal/external MB group showed an even greater impairment of both KCl (40% ± 16%, Fig. 4, A) and PGF2 constriction (59% ± 17%, Fig. 4, B). Phenylephrine and serotonin dose response curves comparing the three experimental groups indicate that internal/external MB exposure significantly attenuates the vasoconstrictor ability of saphenous veins (Fig. 5). Phenylephrine vasoconstriction in the external MB group was not significantly lower than in the control group. However, a trend toward a blunted response is evident (Fig. 5, A). For serotonin constriction, the external MB group was significantly impaired at 10-7 mol/L (Fig. 5, B). Compared with the control group, the maximal response of the external MB group to phenylephrine and serotonin was reduced by 28% ± 19% and 48% ± 13%, respectively. For the internal/external MB group, the maximal response to phenylephrine and serotonin was reduced by 70% ± 6% and 58% ± 16%, respectively.
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preconstriction, MB did not significantly change acetylcholine relaxation although a clear trend is evident (Fig. 6, C). Expressed as a percentage of PGF2 preconstriction, the internal/external MB group demonstrated significant attenuation of sodium nitroprusside relaxation by 18% ± 11% (Fig. 6, A) at the nadir of the dilator response curves. The EC50 value was also significantly shifted rightward, which indicates a decreased sensitivity of the internal/external veins to sodium nitroprusside (see Table II). When the data are expressed in grams of relaxation, both the internal/external and external MB groups are significantly inhibited from relaxing to sodium nitroprusside by 75% ± 6% and 45% ± 12%, respectively (Fig. 6, B). Significant impairment (67% ± 16%) of acetylcholine vasorelaxation in the internal/external group was also evident when the data were expressed in grams of tension (Fig. 6, D).
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The present study demonstrates that the use of MB as an extravascular surgical marker has the potential to impair vascular function, particularly when MB has access to both the adventitia and the endothelium. In practice, MB can access the endothelium when the isolated MB-marked vein is briefly placed in a storage solution until the time of transplantation. Leaching of MB on the adventitia into the storage solution allows for free diffusion of MB into the vessel lumen and hence the endothelium. The concentration of MB leached into the storage solution is not insignificant because the conventional 1% stock solution of MB is in excess of 500 times the dose required to irreversibly inhibit endothelium-derived relaxing factor. 6 Further, results from this study indicate that in addition to the well documented inhibition of vasodilators, 7 MB also diminishes the effects of other vasodilators and vasoconstrictors irrespective of the route of application. The current study was designed to evaluate numerous possible MB pathways through the use of a variety of vasoactive agents, but because MB caused an impairment of all agents tested, isolation of specific mechanisms is difficult. From a practical aspect, the mechanism of MB impairment of vascular function is perhaps less important than the clear documentation of an effect of MB at clinically relevant doses and exposure times.
The pharmacologic actions of MB have been extensively studied but remain poorly understood MB is best known as a direct inhibitor of soluble guanylate cyclase, presumably via MB oxidation of the guanylate cyclase heme moiety. 8 The ability of MB to inhibit those vasodilators that are thought to act by activation of guanylate cyclase is indirect evidence of inhibition of the enzyme by MB. 7 This includes all endothelium-dependent and -independent vasodilators that elicit vascular smooth muscle relaxation by either nitric oxide formation or nitric oxide release. 7 Such a mechanism is consistent with the change in EC50 values and inhibition of sodium nitroprusside and acetylcholine vasorelaxation observed in this study. However, an inhibition of guanylate cyclase by MB does not explain the impairment of isoproterenol or verapamil vasodilation. Nor does it explain the MB-mediated attenuation of vasoconstrictor responses seen in this study. The impairment of vascular reactivity in the current study seems to be global rather than specific for a given transduction system. MB-mediated reduction in the receptor-independent KCl vasoconstriction is evidence of the global nature of the impairment of vascular reactivity. This is not to say that inhibition of guanylate cyclase is an irrelevant mechanism contributing to the vascular impairment seen in this study. Inhibition of this enzyme is probably occurring as evidenced by the shift in EC50 values for sodium nitroprusside. However, other MB-mediated mechanisms of vascular impairment are likely dwarfing any guanylate cyclase effects.
MB is a more potent inhibitor of endothelium-dependent vasodilation mediated by acetylcholine than it is of endothelium-independent nitrodilators 9,10 This suggests that MB acts to inhibit vasodilation by mechanisms independent of guanylate cyclase. A recent study by Marczin, Ryan, and Catravas 11 suggests that MB inactivation of guanylate cyclase is only a minor pathway for the effects of MB. Rather, MB generates superoxide anion, which rapidly reacts with and inactivates nitric oxide as a vasodilator. 12 Marshall,Wei, and Kontos 13 were the first to demonstrate that MB inhibits acetylcholine dilation via the production of a reactive oxygen species. Subsequently, Wolin and coworkers 14 used the cremaster microcirculation as a model to demonstrated that suffusion of superoxide dismutase readily inhibits the effects of MB on acetylcholine and nitric oxide vasodilation. MB is hypothesized to generate superoxide when the reduced form of MB autoxidizes 13 or via an interaction withvarious dehydrogenases. 15 Regardless of the exact mechanism, MB appears to produce significant amounts of superoxide in blood vessels. Moreover, a recent study demonstrated that MB generated superoxide in a dose- and time-dependent manner in the presence, but not absence, of cultured vascular smooth muscle cells. 10
The generation of superoxide by MB is a potential mechanism of the vascular impairment seen in the present study In vascular tissue, superoxide is converted to a variety of other potentially harmful reactive oxygen species including hydrogen peroxide, hydroxyl ion, and peroxynitrite. 16 Countless studies have identified oxygen-derivedfree radicals as mediators of cellular damage, 17,18 including damage to vascular tissue. 19 MB-induced oxidative damage has the potential to affect any number of pathways controlling smooth muscle or endothelial function 20 and would likely lead to a global impairment of vascular function as seen in this study. Whether this is the mechanism of MB impairment remains to be determined.
MB increases the spontaneous outflow and alters the storage of catecholamines from sympathetic nerves in vascular tissue In vitro, MB causes a rise in vascular tone that is readily blocked with -adrenergic agonists. Further, MB inhibits responses to both tyramine and electrical stimulation of adrenergic nerves of rabbit perfused ear arteries. 21 Soares-Da-Silva and Caramona 22 used canine mesenteric arteries to demonstrate that MB releases norepinephrine and dopamine, decreases accumulation of norepinephrine, and inhibits monoamine oxidase and catechol-O-methyl transferase. Collectively, these studies demonstrate that MB produces a prolonged sympathomimetic effect in vascular tissue. The MB-mediated reduction of adrenergic 1 , 2 , and ß2 responses in the present study could be explained by an adrenergic desensitization resulting from the prolonged sympathomimetic effect of MB. However, the magnitude of MB impairment was not greater in adrenergic than in nonadrenergic responses, suggesting other, more global mechanisms. Whether the effects of MB on the vascular adrenergic system are clinically relevant when MB is used as a marker dye is unknown, but in light of the results of the current study, it seems that MB has any number of potential mechanisms by which to adversely affect blood vessels.
In conclusion, the current study has demonstrated that MB applied either luminally or abluminally can decrease both the vasoconstrictor and vasodilatory function of human saphenous veins by an unknown mechanism These results, when coupled with other studies that have shown that MB produces superoxide, inhibits guanylate cyclase, increases platelet deposition, 23 and elicits a sympathomimetic response, suggest that the use of MB as a surgical marker is inappropriate until further studies can evaluate the potentially detrimental effects of MB.
Acknowledgments
We thank the nursing staff at University Hospital and at the Medical College of Georgia for their kind assistance in coordinating and procuring the vascular tissue. Special gratitude is paid to George Shears for his devotion to the project.
Footnotes
From the University of Georgia, aCollege of Pharmacy, Department of Pharmacology and Toxicology, Athens, Ga., the Medical College of Georgiab Section of Thoracic and Cardiac Surgery, Augusta, Ga., and University Hospital, Department of Surgery, Augusta, Ga. 
References
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