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Ann Thorac Surg 1999;68:1623-1627
© 1999 The Society of Thoracic Surgeons

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Original Articles

Effect of skeletonization of the internal thoracic artery on vessel wall integrity

^a Department of Cardiac Surgery, Catholic University, Rome, Italy
^b Department of Anatomy, Catholic University, Rome, Italy

Address reprint requests to Dr Gaudino, Divisione di Cardiochirurgia, Policlinico Universitario A. Gemelli, Largo A. Gemelli 8, 00168 Rome, Italy
e-mail: mgaudino{at}pelagus.it

	Abstract

Top Abstract Introduction Patients and methods Results Comment References

 
Background. This study was conceived to evaluate the effect of internal thoracic artery (ITA) skeletonization on vessel wall integrity.

Methods. Forty consecutive patients undergoing coronary artery bypass were randomized to receive a skeletonized (n = 22) or a pedicled (n = 18) ITA graft. ITA harvesting was performed by 2 experienced surgeons using the same instrumentation and technique. Specimens were examined by light and electron microscope in order to assess vascular wall integrity. A specific immunohistochemical staining and a computerized method were used to quantify the degree of endothelial integrity after surgical preparation.

Results. Morphologic analysis revealed 2 cases of limited subadventitial hemorrhage (one for each group) and no case of major arterial damage. Immunohistochemical staining demonstrated an extremely high degree of maintenance of the endothelial integrity in both groups (97.2% ± 1.9% in the skeletonized and 96.8% ± 2.1% in the pedicled one; p = 0.53).

Conclusions. Skeletonization does not affect ITA wall integrity in humans submitted to coronary artery bypass procedures.

	Introduction

Top Abstract Introduction Patients and methods Results Comment References

 
Skeletonization of the internal thoracic artery (ITA) allows the preparation of longer conduits and consents a wider use of pedicled ITA grafts during coronary artery bypass procedures [1]. However, the effect of this type of surgical harvesting on the integrity of ITA wall has not been studied in detail in humans and some authors have expressed concern about the possible detrimental effect of skeletonization on the endothelial integrity and function [2].

This study was conceived to elucidate the effect of ITA skeletonization on the arterial wall and in particular on the endothelial layer by using light microscopy, immunohistochemistry, and electron microscopy examination.

	Patients and methods

Top Abstract Introduction Patients and methods Results Comment References

 
Fourteen consecutive patients (whose main preoperative characteristics are shown in Table 1) submitted to elective coronary artery bypass grafting (CABG) at our institution from October 1 to November 15, 1997 and were randomly assigned to receive a skeletonized (22 cases) or a pedicled (18 cases) left ITA graft.

Surgical technique
All ITAs were dissected by 2 experienced surgeons who used the same operative technique and who were blinded to the aim of the study and to the microscopic results.

After median sternotomy the left pleura was opened for all its length and the internal thoracic vessels were visualized for their entire course.

When the ITA was harvested as a pedicled conduit, a longitudinal incision of the internal thoracic fascia, parallel and medial to the medial mammary vein, was performed all along the course of the artery; after blunt dissection the intercostal and sternal ITA branches were clipped distally and transected using an electrocautery and the artery and its pedicle were isolated from the origin to the epigastric bifurcation.

When the ITA was isolated in a skeletonized fashion, the longitudinal incision of the fascia was performed at the limit between the artery and the medial satellite vein; this incision was carried out with very low voltage electrocoagulation to avoid any thermal damage to the arterial wall. Using the distal end of the electrocautery as a dissector, the branches of the ITA were exposed, clipped distally and proximally, and transected using Pott’s forceps. The artery was isolated from any other surrounding structure. A bipolar electrocautery was always used for ITA harvesting.

After ITA preparation and section, the part of the conduit judged unnecessary to myocardial revascularization by the operating surgeon was collected for histological and ultrastructural examination.

The last 5 mm of the artery were not used for microscopic evaluation. (As this part is usually submitted to the highest degree of surgical manipulation during section of the artery and measurement of the length necessary to reach the target vessel and in fact is usually discarded when performing CABG procedures.)

Microscopic evaluation
All specimens were submitted for both light and electron microscope analysis to 2 experienced observers blinded to each other and to the harvesting technique used. After review of all data, disagreements were resolved after common re-evaluation.

Light microscopy evaluation
For light microscopy study, specimens were fixed in Bouin’s solution at room temperature (RT) for 12–24 hours, depending on thickness. They were then rinsed in phosphate buffered saline (PBS) (pH 7.4), dehydrated in an ascending series of alcohols, and embedded in paraffin via xylene. Five-micrometer serial transverse sections were then cut and processed for histochemical (Mallory’s trichrome method modified according to Azan, hematoxylin and eosin, Unna-Tanzer-Livini stain for elastic fibers) or immunohistochemical studies.

For immunohistochemistry, endogenous peroxidase activity was quenched with 0.5% H₂O₂ in absolute methanol for 30 minutes at RT; slides were then permeabilized with 0.2% Triton X-100 in PBS (20 min at RT) and incubated with 3% normal goat serum in PBS (30 min at RT) to block nonspecific binding. Sections were then incubated overnight at 4°C with primary polyclonal antibody to factor VIII (YLEM, Milan, Italy, diluted 1:150) a specific marker of endothelial cells [3] used to assess the integrity of the endothelial layer after surgical preparation. Control sections were treated with normal rabbit immunoglobulins at the same concentration used for primary antibody. The reaction was revealed by standard avidin-biotin peroxidase complex procedure (Vectastain Elite ABC kit, Vector, Burlingame, CA) using 3,3’ diaminobenzidine (DAB) (Sigma, Milan, Italy) as chromogen.

For quantitative analysis, endothelial cells positive for factor VIII antibody falling 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 Corporation, Cypress, CA) camera coupled to a Macintosh IIvX (Apple Computer Inc, Cupertino, CA) computer by means of a Scion LG3 (Scion Corporation, Frederick, MD) frame grabber. The percentage of inner surface of the ITA 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).

Electron microscopy evaluation
Specimens 1 cm in length were immediately processed for electron microscopy. All the samples were pinned onto a wax surface to avoid curling and fixed for 12 hours at +4°C with 2.5% glutaraldehyde +4% paraformaldehyde in 0.12mol/L PBS. Smaller samples were trimmed out and postfixed in the same fixative for 2 hours at +4°C. After a thorough washing in PBS, the samples were postfixed with 1% osmium tetroxide 0.1mol/L in cacodylate buffer pH 7.4 and processed for embedding in epoxy Agar 100 resin (Agar Scientific Ltd, Stansted, Essex, UK). Ultrathin 80–90 nm sections were collected on nickel 100 mesh grids and routinely stained with uranyl acetate and lead citrate. Electron microscopy was performed with a Philips EM 208 (Philips, Eindhoven, The Netherlands).

Statistical analysis
Data are expressed as mean ± 1 standard deviation. For statistical analysis the Student’s t test or the ² test were used as appropriate. A p value less than or equal to 0.05 was considered significant.

	Results

Top Abstract Introduction Patients and methods Results Comment References

 
All harvested ITAs were judged of acceptable flow and quality by the operating surgeon and used for myocardial revascularization.

The mean length of ITA used for microscopic analysis was 3.1 ± 1.7 cm in the skeletonized group and 2.7 ± 1.3 cm in the pedicled series (p = 0.41).

Light microscope morphologic analysis demonstrated 2 cases of limited subadventitial hemorrhagia (1 for each group: p = 0.55). No case of major arterial damage (disruption, dissection or thrombosis) were reported in the two groups (Figs 1 and 2).

View larger version (104K): [in this window] [in a new window]   Fig 1. (A, B). Morphologic light microscopic evaluation of a pedicled mammary graft shows good maintenance of the arterial wall architecture (A = adventitia, E = endothelium, EEL = external elastic lamina, IEL = internal elastic lamina, M = media).

View larger version (99K): [in this window] [in a new window]   Fig 2. (A, B). Morphologic light microscopic evaluation of a skeletonized mammary graft shows good maintenance of the arterial wall architecture (A = adventitia, E = endothelium, EEL = external elastic lamina, IEL = internal elastic lamina, M = media).

View larger version (110K): [in this window] [in a new window]   Fig 3. Immunohistochemical staining with anti-factor VIII antibody (stained in brown) demonstrates optimal preservation of the endothelial layer in a pedicled mammary graft (A = 10 x, B = detail of A 64 x).

View larger version (112K): [in this window] [in a new window]   Fig 4. Immunohistochemical staining with anti-factor VIII antibody (stained in brown) demonstrates optimal preservation of the endothelial layer in a skeletonized mammary graft (A = 10 x, B = detail of A 64 x).

Finally, transmission electron microscope examination showed evidence of optimal conservation of the endothelial layer in both the skeletonized and pedicled groups, with no sign of major cellular alteration and good adherence of the endothelium to the basal lamina and preservation of the intercellular junctions of between endothelial cells (Figs 5 and 6). However, in 2 cases (both in the skeletonized series) small microthrombi (formed mainly by red blood cells and adherent to the endothelial layer) without hemodynamic importance were found in one of the examined sections (Fig 7).

View larger version (92K): [in this window] [in a new window]   Fig 5. Morphological electron microscopic evaluation demonstrates good preservation of the ultrastructural aspect of both pedicled (A) and skeletonized (B) mammary graft (BL = basal lamina, E = endothelium).

View larger version (177K): [in this window] [in a new window]   Fig 6. Electron microscope analysis of a skeletonized mammary graft shows good preservation of the intercellular junction between endothelial cells (G = gap junction).

View larger version (159K): [in this window] [in a new window]   Fig 7. Electron microscope imaging of a microthrombus in a skeletonized mammary graft (E = endothelium, L = vessel lumen, RBC = red blood cell).

	Comment

Top Abstract Introduction Patients and methods Results Comment References

This type of preparation has been shown to provide significant advantages in terms of reduction of sternal wound infections and pulmonary complications, especially in high risk patients [4, 5]; moreover, the removal of the endothoracic fascia allows a considerable dilatation of the ITA, so that the free flow in skeletonized arteries is superior to that of conventionally-prepared (pedicled) conduits [6].

On the other hand, skeletonization is certainly more traumatic for the arterial wall than conventional pedicled preparation, and the possibility that the mechanical peeling of the adventitia combined with the repeated stretching of the artery can affect the ITA integrity has never been clearly denied [2].

In a classic experimental study on a canine model, Daly and coauthors demonstrated that stripped ITA (free of all surrounding tissue, including the satellite vein and the thoracic fascia) directly anastomosed to the ascending aorta had a higher incidence of thrombosis, intimal thickening, and medial injury than the pedicled (in situ and free) ITA [7]. In contrast, Noera and coworkers, comparing the histological results of patients in whom the ITA was harvested as a skeletonized graft with those of traditionally prepared ITAs, found no evidence of superior damage in the skeletonized group [8]; however, this investigation was performed using only routine histological techniques (quite insensitive in the detection of endothelial damage) and the use of different methods of ITA dilatation in the skeletonized group (intraluminal vs topically applied papaverine) complicates the data analysis and limits the statistical power of this series.

In the only other investigation published on this subject, Sasajita and colleagues investigated the effect of ITA skeletonization in 6 mongrel dogs in whom the histological aspect of the skeletonized ITA was compared to that of the contralateral untouched artery [9]. Although these authors concluded that ITA skeletonization is not detrimental to vessel wall integrity, the small number of examined cases and the lack of a sensitive method of detection of the endothelial damage limit the liberal application of the conclusions drawn from this experimental model to humans undergoing coronary artery bypass grafting.

Our study demonstrates the excellent preservation of the structural and ultrastructural integrity of human skeletonized ITAs; the large number of cases and the use of specific immunohistochemical and electron microscope techniques to assess even minimal alterations of the vessel wall (with particular regard to the endothelium) constitute strong arguments in favor of the reliability of our observations.

A minor limitation of our investigation relies on the fact that (for obvious reasons) we limited our analysis to the distal tract of the ITA and have no data on the more proximal part of the artery; however, there is no theoretical reason to hypothesize that the high degree of structural preservation that we observed in the distal ITA should not be present even in the proximal part of the artery.

A note of caution should probably arise from the reported electron microscope observation of small microthrombi in 2 cases of skeletonized (but not pedicled) ITA grafts. Although these thrombotic formations were always limited and with no hemodynamic effect, their possible role in promoting chronic graft alterations (intimal hyperplasia, atherosclerosis or thrombosis) can not be denied. In this regard, further investigation on the long-term fate and endothelial function of skeletonized versus pedicled ITA grafts would be a welcome addition to the current literature.

In conclusion, we have demonstrated that skeletonization does not affect ITA wall integrity in humans submitted to coronary artery bypass procedures; skeletonized ITA grafts can thus be liberally used when judged technically appropriate.

	References

Top Abstract Introduction Patients and methods Results Comment References

 

1. Cunningham J.M., Gharavi M.A., Fardin R., Meek R.A. Considerations in the skeletonization technique of internal thoracic artery dissection. Ann Thorac Surg 1992;54:947-951.[Abstract]
2. Yacoub M.H. Reply to Cunningham JM. Skeletonization of internal thoracic artery grafts. J Thorac Cardiovasc Surg 1996;111:489-490.[Free Full Text]
3. Wagner D.D., Marder W.J. Biosynthesis of von Willebrand protein by human endothelial cells. J Cell Biol 1984;99:2123-2130.[Abstract/Free Full Text]
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5. Matsumoto M., Konishi Y., Miwa S., Minakata K. Effect of different methods of internal thoracic artery harvest on pulmonary function. Ann Thorac Surg 1997;63:653-655.[Abstract/Free Full Text]
6. Choi J.B., Lee S.Y. Skeletonized and pedicled internal thoracic artery grafts. Ann Thorac Surg 1996;61:909-913.[Abstract/Free Full Text]
7. Daly R.C., McCarthy P.M., Orszulak T.A., Schaff H.V., Edwards W.D. Histologic comparison of experimental coronary artery bypass grafts. J Thorac Cardiovasc Surg 1988;96:19-29.[Abstract]
8. Noera G., Pensa P., Lodi R., Lamarra M., Biagi B., Guelfi P. Influence of different harvesting techniques on the arterial wall of the internal mammary artery graft. Thorac Cardiovasc Surg 1993;41:16-20.[Medline]
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