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Ann Thorac Surg 1999;68:143-148
© 1999 The Society of Thoracic Surgeons

Original Articles

Morphology and histology of human and canine internal thoracic arteries

Tadahiro Sasajima, MDa,b, Vishwanath Bhattacharya, MD, FRCSa,b, Moses Hong-De Wu, MDa,b, Qun Shi, MDa,b, Naoki Hayashida, MDa, Lester R. Sauvage, MDa,b,c

a The Hope Heart Institute, University of Washington School of Medicine, Seattle, Washington, USA
b Providence Seattle Medical Center, University of Washington School of Medicine, Seattle, Washington, USA
c Department of Surgery, University of Washington School of Medicine, Seattle, Washington, USA

Address reprint requests to Dr Sauvage, The Hope Heart Institute, 528 18th Ave, Seattle, WA 98122


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 References
 
Background. We evaluated human and canine internal thoracic arteries (ITAs) to determine whether the latter is valid for studies relevant to clinical use.

Methods. We studied 19 human ITAs obtained from 1 female and 14 male victims of recent fatal accidents who had no evidence of cardiovascular disease (mean age = 39 ± 19 years; range = 15 to 79 years), and ITAs of 21 randomly-selected mongrel dogs of both sexes, weighing 18–40 kg (average = 24.3 ± 5.7 kg). Specimens were fixed in formalin at a controlled pressure of 120 mm Hg, before extensive assessment that included intimal thickening, condition of the internal elastic lamina, and number of medial elastic lamellae and vasa vasorum.

Results. The canine morphology and histology were similar to the human ITAs, but there was no intimal hyperplasia, and the media and adventitia were thinner (ITAs of humans older than 40 years had significant increases in medial thickness, as well as in overall length). Morphologically and histologically, the left and right canine ITAs were almost completely the same.

Conclusions. Canine ITAs are valid for bilateral comparative studies and are a useful tissue source and model for clinically-relevant experimental studies.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 References
 
The internal thoracic artery (ITA) has become the conduit of choice for coronary artery bypass, because of its superior 90% patency at 10 or more years with little or no evidence of atherosclerotic change [1–3]. Therefore, numerous in vivo and in vitro animal experiments have been done to study its biologic behavior. Otaki and associates studied bilateral versus single ITA grafting in coronary artery occlusion [4], Harris and associates studied the effects of graded reduction in ITA bypass flow on left ventricular function [5], and Robinson and associates measured the effect of ITA endothelium-derived substances and flow on the myocardial contractile response [6]. We have previously studied the effects of altered blood flow on the caliber and morphology of the ITA [7], and the effect of skeletonizing dissection on the canine ITA [8].

To better appreciate the relation of this vessel’s structure to its function as a coronary graft, several authors have reported detailed histologic findings of the human ITA in a flaccid state [9, 10]. There are, however, no comparative histologic data on the human and canine ITAs fixed in the inflated state at 120 mm Hg. We report such observations in this paper.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 References
 
Specimen procurement and preparation
Human ITAs
Full-length human ITAs harvested from their pedicles were obtained from cadavers through our local tissue bank (Northwest Tissue Center, Seattle, WA). The average length of time from death to procurement of ITAs in humans was 8.4 ± 4.3 hours. The ITAs were then sent to our laboratory in a phosphate-buffered solution. The causes of death were traffic accident, gunshot head injury, and pneumonia. Those with a history of cardiovascular disease or any other chronic illnesses were not included. The 19 ITAs evaluated were taken from 1 female and 14 male cadavers, 15 to 79 years old (mean age = 39 ± 19). Their weight ranged from 63 to 120 kg with an average of 75.4 ± 13.7 kg. All ITAs were inflated and fixed for 3 days in 10% formalin at a constant monitored pressure of 120 mm Hg. All the branches were ligated or sealed with a bipolar cautery to prevent leakage.

Canine ITAs
Twenty one randomly selected mongrel dogs of both sexes, weighing 18–40 kg (average weight 24.3 ± 5.7 kg) were used in the study. These dogs had previously been used for a primary experiment in which a vascular graft was implanted in the abdominal aorta before the procurement of ITAs. However, their chest areas had not been disturbed surgically and the previous experiments would not have affected ITA quality and histology. Their care and use complied with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for Care and Use of Laboratory Animals," Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council (Washington: National Academy Press, 1996).

Following the primary experiments, the dogs were anesthetized, using an induction with 5% to 10% thiamylal intravenously, and then maintained with a combination of 0.5% to 1% halothane and a mixture of nitrous oxide and oxygen in a 2:1 ratio via an endotracheal tube connected to a closed circuit respirator. A supraclavicular incision provided access to the proximal ITAs on both sides. The proximal blood flow rate of each ITA was measured using a Transonic flowmeter (T-208, Transonic System Inc, Ithaca, NY) at a site 2 cm distal to the origin of the vessel.



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  		Fig 2. Comparison of paired left and right canine thoracic arteries (LITA, RITA), at 8 cm from the origin. Cross sections of LITA (A) and RITA (B) (Verhoeff’s and van Gieson’s stains; X 20 before % reduction). Sections of the same canine LITA (C) and RITA (D) at higher magnification (Verhoeff’s and van Gieson’s stains; X 80 before % reduction).

 
After 100 mg of heparin sodium was given intravenously, the dogs were euthanized with an intravenous overdose of potassium chloride. The bilateral subclavian arteries were then ligated and severed 1 cm proximal and distal to the origin of the ITA. A 2-cm length of vertebral artery was left attached to the opposite side of the origin of the ITA. The entire chest wall (20 cm by 25 cm) containing the intact bilateral ITAs was removed as a plate. The distal musculophrenic and superior epigastric arteries were ligated. All the canine ITAs were then cannulated at their origin with an 18 gauge angiocatheter via the vertebral artery and connected to a dynamic perfusion system [11], which was able to provide a stable perfusion pressure at 120 mm Hg during fixation. The entire chest plate, with the ITAs, was perfused and fixed in 10% formalin for 3 days.



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  		Fig 1. (A) Cross section (Verhoeff’s and van Gieson’s stains; x 20 before % reduction) of a human internal thoracic artery, 8 cm from the origin, taken from a 19-year-old. (B) Cross section of the same artery. (Verhoeff’s and van Gieson’s stains; x 80 before % reduction) (C) Cross section (Verhoeff’s and van Gieson’s stains; x 80 before % reduction) of human ITA from a 79-year-old, showing degenerating original internal elastic lamina (arrow a) and intact innermost medial elastic lamina (arrow b).

 
Histologic evaluation
After the ITA lengths were measured, 3 segmental tissue blocks at 2, 8, and 14 cm from the origin were taken from the fixed ITAs in all the specimens. In the canine samples where the left and right ITAs were compared, an additional 2 samples were taken at 2 cm proximal to the bifurcated area and at the beginning of the superior epigastric artery.

The tissue samples were embedded in paraffin, cross-sectioned, and stained with hematoxylin and eosin for general histologic study. Modified Verhoeff’s and von Gieson’s stains (VvG Procedure No. HT 25, Sigma, St. Louis, MO) were used for identification and evaluation of internal elastic lamina, medial elastic lamellae, and external elastic lamina.

Photomicrographs of all sections were taken. A computerized morphometric analysis system (Macintosh, Apple Computer Inc, Cupertino, CA), with the public domain program "Image" (NIH Research Service Branch, NIH, Bethesda, MD), was used to measure the circumference (C) of the internal elastic lamina (IEL) or endothelial lining, external elastic lamina (EEL), and the outer boundary of the adventitia (ADV).

The quantitative morphologic data, including the luminal diameter and thickness of the media and adventitia, were then obtained using the following formulas

where C = circumference, IEL = internal elastic lamina or endothelial lining, EEL = external elastic lamina, and ADV = outer boundary of the adventitia.

The numbers of elastic lamellae in the media and vasa vasorum in the adventitia were also counted. Because of the spiral arrangement of the medial elastic lamellae, their number varied in different locations within the same cross section. Therefore, each cross section was equally divided into 8 sectors of 45 degrees. Only well-stained, uninterrupted medial elastic lamellae present in each region were counted and then averaged for each cross section. Microvessel channels or circles in the adventitia that were lined with endothelial cells were considered to be vasa vasorum, whether or not they contained red blood cells. During the histologic evaluation, special attention was paid to any intimal hyperplasia or degenerative changes of the internal elastic lamina. Intimal hyperplasia was defined as intimal thickness over 20 µm that occupied over a fourth of the circumference of the section. Wherever this was seen, the true luminal diameter was calculated from the circumference of the endothelial lining. Otherwise, it was calculated from the circumference of the internal elastic lamina, because the endothelial lining in these cases was considered too thin to be of any consequence. Degeneration of the internal elastic lamina was defined as disappearance of more than a fourth of the circumference, or frequent disruptions of the lamina.

Statistical analysis
Quantitative data were expressed as mean ± standard deviation. A StatView software package (Abacus Concepts, Inc, Berkeley, CA) was used for statistical evaluation, using either the {chi}2 test, an unpaired t test, or one-way analysis of variance (ANOVA), whenever appropriate. Statistical significance was defined as p < 0.05. Pearson’s correlation was used to evaluate the correlation between age and variables for human ITAs. Significance was defined as r > 0.44.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 References
 
Human ITA
The detailed quantitative morphologic and histologic data are given in Table 1. All 19 human ITAs showed a positive correlation of increased length (r = 0.627) and diameter (r = 0.734) with age (Fig 1).


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  		Table 1. Morphologic and Histologic Comparison of Canine and Human Internal Thoracic Arteries

 
Table 2 shows that there was a significant increase in ITA length (p = 0.014), and microscopically there was a significant increase in medial thickness in those over 40 years old. There was also a tendency toward an increase in internal diameter and adventitial thickness and a decrease in number of medial elastic lamellae in the age group over 40 years (Table 2), though these were not statistically significant.


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  		Table 2. Human Internal Thoracic Arteries: Morphology and Histology at Ages Below and Above 40 Years

 
In the age group over 60 years, intimal hyperplasia/thickening was found in 94% of the cross sections (17 of 18), of which 18% had intimal thickening between 20 µm and 100 µm, 20% had thickening between 100 µm to 200 µm and 61% had intimal thickening of over 200 µm, but still in the microscopic range with no change in gross appearance. Only 13% of the cross sections (2 of 15) in the age group 35 to 60 years had intimal hyperplasia, all of which was of low grade, between 20 µm to 100 µm. There was no intimal thickening in those below 35 years. Endothelial coverage was incomplete on the surface of most of the areas where intimal hyperplasia/thickening was present.

Degenerative changes of the internal elastic lamina, manifested as frequent disruptions or disappearances, were found in 72% (13 of 18) of cross-sections in those above 60 years, 33% (5 of 15) in those 35 to 60 years, and 22% (10 of 45) in those below the age of 35 years. The endothelial lining was intact only on the areas where the IEL was not disrupted. VvG staining showed that in some specimens with IEL degeneration the innermost layer of medial elastic lamina was still intact and distinctive, even though the original IEL had broken into many fragments. There was no atherosclerosis or calcification in any of the human ITAs studied, even in the older age group.

Canine ITA
Basically, the canine and human ITAs had the same histologic structure and composition. A comparison of quantitative histologic data between the canine and human ITAs is shown in Table 1. There was no significant difference between the lengths of the canine and human ITAs, and their overall diameters were very comparable, except at the distal part, where the canine ITA was about 10% smaller. The thickness of the media in the proximal third was very comparable in the two species, although the middle and distal areas of the canine ITA were thinner than the human was. There were significant statistical differences between the thickness of the adventitia, the number of medial elastic lamellae, and the number of vasa vasorum, although the absolute values were not widely different. Intimal hyperplasia/thickening and internal elastic lamina degeneration was not seen in the dogs (Fig 2).

Symmetry of bilateral canine ITAs
Detailed quantitative comparisons of the 6 pairs of left and right canine ITAs studied are given in Table 3. Morphologically and histologically the left and right ITAs were almost completely symmetric in terms of flow rate, length, diameter, and the quantitative histologic measurements.


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  		Table 3. Symmetry of the Left and Right Canine Internal Thoracic Arteries (ITAs)

 

   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
References
 
A readily available tissue source comparable to the human ITA is needed for study of physiobiologic characteristics. This study of human and canine ITAs showed comparable morphologic and histologic findings. There were only minor statistical variations in certain aspects between canine and human ITAs (Table 1). Morphologically, they were very comparable in terms of length, diameter, structure, and composition (Table 1). Seki and associates, using angiographic evaluation of 147 postoperative subjects, found that the average diameter of the human ITA within 1 cm of the anastomotic site is 2.0 ± 0.7 mm [11]. Their data were very similar to ours, for both the canine and human ITAs, when measured at an inflated physiologic pressure (Table 1). In our study, the flow rate data of human ITAs was unavailable because we used cadavers. Mills and Bringaze have reported a mean human flow rate of 18 mL/min, increasing to 51 mL/min after topical papaverine to eliminate spasm before bypass anastomosis [12]. The average flow rate of all canine ITAs measured in our study was 35.6 ± 15.7 mL/min, with a range of 10 to 67 mL/min, which resembles the range in humans. These observations suggest that the study of canine ITAs is clinically relevant.

As mentioned earlier, canine ITAs have been frequently used in both in vivo and in vitro experimental studies [5–8]. Many of these studies used paired ITAs. Our study shows that the left and right canine ITAs were almost completely symmetric (Table 3). These data further validate the use of paired ITAs for comparative study, because one side may be used to assess the effect of surgical manipulation or a pharmacologic agent, whereas the opposite side is used as a control.

This study was not designed to be a complete investigation of the human ITA, but with the limited number of human ITAs, some useful observations were made. There were no major histologic or morphologic differences in the age groups below and above 40 years, except for an increase in length as a result of tortuosity in the older group (Table 2). The incidence and degree of intimal hyperplasia/thickening correlated with increasing age. In the older age group, the intimal layer, although thicker, remained in the microscopic range, without affecting the macroscopic morphology. This may lessen misgivings about using the ITA for bypass in the elderly.

Sims has found that degeneration of the internal elastic lamina is associated with effective reduplication in all species, including the dog, but not in humans, who are unique in their failure to repair this defect [13]. This may explain why we did not observe any internal elastic lamina degeneration in the dog, but noticed the appearance of internal elastic lamina degeneration even in the younger humans. This finding indicates a need for extra care during dissection of the ITA, in order to prevent further damage to the internal elastic lamina. Although we observed some loss of endothelial coverage on human ITAs where there was intimal hyperplasia/ thickening, the possibility of a postmortem proteolytic event as a possible cause for this phenomenon could not be excluded.

Sims has also noted that the internal elastic lamina of the ITA forms a significant obstruction to the diffusion of macromolecules and suggests that the internal elastic lamina is a significant barrier to intimal thickening and atherosclerosis [13, 14]. He observed that in cases with internal elastic lamina degeneration, the innermost layer of the medial elastic lamina was still intact and distinct, presumably taking over the "barrier" function to prevent intimal thickening and atherosclerosis. This may explain the positive, long-term results of ITA bypass regardless of age.

In summary, this study showed that morphologically the canine ITA is generally comparable to the human ITA and histologically there are many similarities between them. The canine ITA is a suitable experimental tissue source and model for studying the ITA. The left and right canine ITAs are almost completely symmetrical and are valid models for paired comparative studies.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 References
 

  1. Galbut D.L., Traad E.A., Dorman M.J., et al. Twelve-year experience with bilateral internal mammary artery graft. Ann Thorac Surg 1985;40:264-270.[Abstract]
  2. Barner H.B., Standeven J.W., Reese J. Twelve-year experience with internal mammary artery for coronary artery bypass. J Thorac Cardiovasc Surg 1985;90:668-675.[Abstract]
  3. Loop F.D., Lytle W.B., Cosgrove D.M., et al. Influence of the internal-mammary-artery graft on 10-year survival and other cardiac events. N Engl J Med 1986;314:1-6.[Abstract]
  4. Otaki M., Lust R.M., Sun Y.S., et al. Bilateral vs single internal thoracic artery grafting for left main coronary artery occlusion. Chest 1994;106:1260-1263.[Abstract/Free Full Text]
  5. Harris L.J., Crooke G.A., LaMendola C.L., Grossi E.A., Baumann F.G., Espito R.A. Effects of graded reductions in internal mammary artery bypass flow on left ventricular function. Ann Thorac Surg 1993;56:1348-1350.[Abstract]
  6. Robinson B.L., Morita T., Fujita K., Chow M., Schaff H.V., Morris J.J. Bypass conduit vessel wall biology substantially influ-ences downstream myocardial contractile response to injury from ischaemia and reperfusion. J Thorac Cardiovasc Surg 1996;111:62-73.[Abstract/Free Full Text]
  7. Kouchi Y., Onuki Y., Wu M.H.-D., Shi Q., Sauvage L.R. Effect of altered blood flow on the caliber and morphology of the internal thoracic artery in the dog. J Thorac Cardiovasc Surg 1997;113:114-120.[Abstract/Free Full Text]
  8. Sasajima T., Wu H.D., Shi Q., Hayashida N., Sauvage L.R. Effect of skeletonizing dissection on the internal thoracic artery. Ann Thorac Surg 1998;65:1009-1013.[Abstract/Free Full Text]
  9. Van Son J.A.M., Smedts F., Vincent J.G., et al. Comparative anatomic studies of various arterial conduits for myocardial revascularization. J Thorac Cardiovasc Surg 1990;99:703-707.[Abstract]
  10. Francel T.J., Dufresne C.R., Baumgartner W.A., O’Kelley J. Anatomic and clinical considerations of an internal mammary artery harvest. Arch Surg 1992;127:1107-1111.[Abstract/Free Full Text]
  11. Seki T., Kitamura S., Kawachi K., Morita R., Kawata T., Mizuguchi K., et al. A quantitative study of postoperative luminal narrowing of the internal thoracic artery in coronary artery bypass surgery. J Thorac Cardiovasc Surg 1992;104:1532-1538.[Abstract]
  12. Mills N.L., Bringaze W.L. Preparation of the internal mammary artery graft. J Thorac Cardiovasc Surg 1989;98:73-79.[Abstract]
  13. Sims F.H. A comparison of structural features of the walls of coronary arteries from 10 different species. Pathology 1989;21:115-124.[Medline]
  14. Sims F.H. Discontinuities in the internal elastic lamina. Artery 1985;13:127-143.[Medline]
Accepted for publication February 15, 1999.




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