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Ann Thorac Surg 2004;77:1257-1261
© 2004 The Society of Thoracic Surgeons
a Department of Cardiac Surgery, Catholic University, Rome, Italy
b Institute of Human Anatomy, Catholic University, Rome, Italy
Accepted for publication September 8, 2003.
* Address reprint requests to Dr Gaudino, Divisione di Cardiochirurgia, Policlinico Universitario A. Gemelli, Largo A. Gemelli 8, 00168 Rome, Italy
e-mail: mgaudino{at}tiscali.it
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Abstract |
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 Top Abstract IntroductionPatients and methodsResultsCommentAcknowledgmentsReferences |
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METHODS: Nineteen patients who underwent primary isolated coronary artery bypass grafting were randomly assigned to receive a skeletonized (n = 9) or pedicled (n = 10) internal thoracic artery graft. Immunohistochemical nerve localization using anti–S-100 protein, anti–160-kd neurofilament polypeptide and anti–tyrosine hydroxylase antibodies was performed on distal specimens of arteries to study vascular innervation. Moreover, endovascular vasoactive challenges using serotonin and methylergometrine were performed at early angiographic control to evaluate the eventual presence of denervation supersensitivity.
RESULTS: Quantitative analysis of immunohistochemical specimens revealed lack of difference in the number of positive cells between skeletonized and pedicled arteries for all the antibodies used. No difference in the reaction to serotonin and methylergometrine was found between skeletonized and pedicled arteries.
CONCLUSIONS: Skeletonization does not influence internal thoracic artery innervation.
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Introduction |
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TopAbstractIntroductionPatients and methodsResultsCommentAcknowledgmentsReferences |
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This study was designed to compare the effect of surgical harvesting on ITA innervation in pedicled versus skeletonized arteries and to assess the eventual presence of denervation supersensitivity in skeletonized ITA grafts. All observations were performed during the period in which the functional consequences of the different type of surgical harvesting are likely to be more evident and the clinical implications of denervation supersensitivity would be maximal, the first postoperative days.
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Patients and methods |
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Preoperative inclusion criteria for enrollment were as follows:
After enrollment, patients were randomly assigned to receive a skeletonized (n = 9) or pedicled (n = 10) ITA graft. The main preoperative and intraoperative characteristics of the entire series of patients and of the two subgroups are summarized in Table 1.
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Briefly, when the ITA was harvested as a pedicled conduit, the left pleura was opened for its entire length and the internal thoracic vessels visualized; a longitudinal incision of the internal thoracic fascia (parallel and medial to the medial mammary vein) was then performed along the entire course of the vessel. The ITA branches were clipped proximally and transected using the electrocautery, and the artery and its pedicle (including the endothoracic fascia and the two satellite veins) were isolated from the origin to the epigastric bifurcation.
When the ITA was isolated in a skeletonized fashion, the pleura was left intact. The longitudinal incision of the fascia was performed as for the pedicled ITA. Using the distal end of the electrocautery as a blunt dissector, the surgeon exposed the branches of the ITA, clipped them distally and proximally, and transected them using Pott's forceps, and the artery was isolated alone, without any other surrounding structure.
The coronary artery bypass grafting procedure was then performed in standard fashion with cardiopulmonary bypass, aortic cross-clamping, and cardioplegic arrest. The ITA was always anastomosed to the left anterior descending coronary artery by using 8-0 polypropylene suture, and complementary venous or radial artery grafts were used for target vessels other than the left anterior descending coronary artery. At the end of the procedure, normal functioning of the anastomosis was assessed with an intraoperative flowmeter.
Immunohistochemical analysis
For immunohistochemistry samples of ITA were obtained from the distal portion of the conduits immediately after harvesting and before surgical dilatation. According to a described immunohistochemistry protocol [7], tissue samples were immediately fixed in 4% paraformaldehyde at room temperature for 24 hours. They were then rinsed in phosphate-buffered saline (pH 7.4), dehydrated in an ascending series of alcohol, and embedded in paraffin by means of xylene. Five-micrometer serial transverse sections were then cut and processed for Mallory's trichrome method modified according to Azan, hematoxylin and eosin, Unna-Tanzer-Livini stain for elastic fibers staining, or immunohistochemical studies. For immunohistochemical experiments, endogenous peroxidase activity was quenched with 0.5% H2O2 in absolute methanol for 30 minutes at room temperature; slides were then permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (20 minutes at room temperature) and incubated with 3% normal goat serum in phosphate-buffered saline (30 minutes at room temperature) to block nonspecific binding. Sections were then incubated overnight at 4°C with the following primary antibodies: rabbit polyclonal anti-S100 protein (1:8000; Dako, Italy), rabbit polyclonal anti–tyrosine hydroxylase (1:6000; Chemicon, CA), monoclonal anti–160-kd neurofilament polypeptide (1:500; Amersham, UK).
S-100 protein is a widely used marker for Schwann cells in the peripheral nervous system [8]. The 160-kd neurofilament polypeptide is one of the major components of the neuronal cytoskeleton for most mature neurons [9]. Tyrosine hydroxylase is a marker of all catecholaminergic neurons [10, 11].
Control sections were treated with normal rabbit or mouse immunoglobulins at the same concentration used for primary antibodies.
Reactions were revealed by standard avidin-biotin peroxidase complex procedure (Vectastain Elite ABC kit; Vector, Burlingame, CA) using 3,3' diaminobenzidine tetrahydrochloride (Sigma, Italy) as chromogen. Sections were examined under a Zeiss Axiophot (Germany) light microscope.
Quantitative analysis of the immunohistochemical data was performed following a previously described methodology [7]. For quantitative analysis, positive cells for the different antibodies that fell into 35 randomly selected 55-µm squares for each patient were counted using a computerized system. For this purpose, images from immunostained sections were captured using a Sony XC77CCD (Sony Corp, Cypress, CA) camera coupled to a Macintosh IIvX (Apple Computer Inc, Cupertino, CA) computer by means of a Scion LG3 (Scion Corp, Frederick, MD) frame grabber. The number of cells marked was then evaluated on the captured images using the public domain software NIH IMAGE 1.59 (developed at the US National Institutes of Health, Bethesda, MD, available from the Internet).
Angiographic protocol
Postoperative angiography was performed on the second postoperative day. No patient received inotropic or vasoactive medications during the 24 hours before the procedure. Patients were studied in a fasting state after medication with diazepam (10 mg orally). Selective left ITA angiography was performed by percutaneous left radial or right femoral approach. Internal thoracic 5F or 6F catheters (Boston Scientific Corp, Boston, MA) were used as appropriate to obtain optimal ITA visualization and selective contrast medium injection.
Multiple angiographic views were obtained to detect significant stenosis at any ITA or left anterior descending coronary artery level. The Thrombolysis in Myocardial Infarction Study flow grade was visually estimated separately by two different observers who were blinded to ITA harvesting technique.
Pharmacologic ITA stimulation was then started with a four-point protocol according to a previously described method [12]:
Serotonin hydrochloride at 10−5 mol/L (ICN Pharmaceuticals, Inc, Costa Mesa, CA) was selectively injected into the ITA graft at a rate of 3 mL/min for 3 minutes. At the end of the serotonin challenge, 2 mg of isosorbide dinitrate was injected into the conduit. After a 20-minute period, methylergometrine maleate 0.2 mg (Methergin; Novartis Farma, Varese, Italy) was administered intravenously. Again, at the end of the methylergometrine infusion, 2 mg of isosorbide dinitrate was injected into the graft. Drug infusion was always performed under electrocardiographic and invasive blood pressure monitoring.
At the end of each step of the protocol, a cineangiographic run was performed, keeping a fixed angiographic view. Digital angiograms were then analyzed with computerized quantitative angiography (Medis, Neuen, The Netherlands). For each step of the protocol, two vascular segments were measured in end-diastolic frames after proper catheter calibration: proximal left ITA (just distal to the ostium) and preanastomosis left ITA (proximal to the distal anastomosis).
Statistical analysis
Quantitative angiographic data, expressed in millimeters, were normally distributed and are expressed as mean ± standard deviation. Analysis of variance for repeated measures was used to test differences between steps; post hoc comparison was performed by Newman-Keuls test. The Student's t test was used to compare the two groups. Relative (percentage) changes in diameter between the skeletonized and pedicled groups were compared by Fisher's exact test. Analysis was conducted with the software Statistica for Windows 4.1 (StatSoft Inc, Tulsa, OK).
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Results |
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TopAbstractIntroductionPatients and methodsResultsCommentAcknowledgmentsReferences |
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Microscopic analysis
All transverse sections from each specimen stained by histochemical techniques exhibited similar tissue architecture, and no pathologic changes were noted.
S-100 protein immunostaining revealed the presence of myelinated nerve fibers in all examined specimens. These fibers were usually located in the outer third of the adventitia. Tyrosine hydroxylase–positive and 160-kd neurofilament polypeptide–positive nerves were detected in all arteries, independently from the technique of surgical preparation; these fibers were mainly located in the inner two thirds of the adventitia and in the outer media, below myelinated fibers (Figs 1 and 2).
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There was no difference in the reaction to serotonin, isosorbide dinitrate, and methylergometrine between skeletonized and pedicled ITAs (Table 3).
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Comment |
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TopAbstractIntroductionPatients and methodsResultsCommentAcknowledgmentsReferences |
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This hypothesis, if proven true, can have major clinical implications as the use of inotropic and vasoactive drugs is often required in the early postoperative period and could then be particularly dangerous in presence of "hypersensitive" skeletonized conduits.
To clarify this issue we designed the present protocol and decided to perform our observations during the period when the effect of surgical harvesting is more evident (as no healing process has had time to take place) and, at the same time, the eventual presence of denervation supersensitivity would be more detrimental—the first postoperative days.
Immunohistochemical data denied any influence of skeletonization on ITA innervation, and vasoactive challenges ruled out the possibility of denervation supersensitivity among skeletonized ITAs. The vascular innervation appeared well represented in both skeletonized and pedicled arteries, and, in particular, sympathetic nerve fibers were not affected by the type of surgical harvesting (Figs 1 and 2 and Table 2). Moreover, skeletonized and pedicled grafts exhibited similar reaction to the vasoconstrictive stimuli used in the early postoperative period (Table 3), confirming our previous observations [6] and denying the presence of denervation supersensitivity in skeletonized arteries.
As in every in vivo study on the vasoreactivity of coronary artery bypass graft conduits, it is not possible to rule out the possibility that the observed ITA diameter changes after vasoactive drug administration are the consequences of modifications of the resistance of the grafted coronary artery; however, if this is the case, this adaptive effect is likely to have been present in both the skeletonized and pedicled group and does not seem to have the potential to affect the reliability of our observations. Moreover, our immunohistochemical analysis obviously documents the innervation status at the time of surgery and cannot provide any information on the long-term consequences of the harvesting trauma (although the fact that even the results of the vasoactive tests that were performed some days later showed no differences is somewhat reassuring in this regard). Another possible limitation of our protocol is related to the statistical power; however, despite the fact that with 19 cases the possibility of a type II statistical error cannot be excluded, the fact that all p values were far from statistically significant and the reproducibility of the observations in the different patients seems to reduce to a minimum the possibility of this type of bias.
In conclusion, skeletonization does not influence ITA innervation or induce denervation supersensitivity (at least to the vasoconstrictive stimuli we tested). These observations further confirm the absence of detrimental consequences of ITA skeletonization. Skeletonized ITA can be safely used when judged appropriate.
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Acknowledgments |
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TopAbstractIntroductionPatients and methodsResultsCommentAcknowledgmentsReferences |
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References |
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TopAbstractIntroductionPatients and methodsResultsCommentAcknowledgmentsReferences |
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This article has been cited by other articles:
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  M. A. Deja, K. S. Golba, M. Malinowski, S. Wos, M. Kolowca, J. Biernat, M. Kajor, and T. J. Spyt Skeletonization of internal thoracic artery affects its innervation and reactivity Eur. J. Cardiothorac. Surg., October 1, 2005; 28(4): 551 - 557. [Abstract] [Full Text] [PDF]
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