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Ann Thorac Surg 1998;66:1246-1249
© 1998 The Society of Thoracic Surgeons

Original articles: cardiovascular

High-flow gas insufflation to facilitate MIDCABG: effects on coronary endothelium

William R. Burfeind, Jr, MDa, Francis G. Duhaylongsod, MDa, Brian H. Annex, MDb, David Samuelson, BAa

a Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA
b Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA

Accepted for publication April 24, 1998.

Address reprint requests to Dr Duhaylongsod, Duke University Medical Center, DUMC-3457, Durham, NC 27710
e-mail: (duhay001{at}mc.duke.edu)

Presented at the Seventieth Scientific Session of the American Heart Association, Orlando, FL, Nov 10–13, 1997.


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Background. During less invasive coronary bypass operations on the beating heart, as well as conventional operations using continuous warm cardioplegia, a precise anastomosis is facilitated by a bloodless field. To maintain a clear field, many surgeons use high-flow gas insufflation. However, the potentially damaging effects of gas insufflation on coronary endothelium have not been elucidated.

Methods. Seven pigs underwent median sternotomy. Between two coronary occluders, an arteriotomy in the mid left anterior descending coronary artery (LAD) was performed. In the experimental group (n = 5), the operative field was kept clear by exposing the arteriotomy to a catheter-directed stream of carbon dioxide at 15 L/min. In the control group (n = 2), the arteriotomy was left open to room air. After 20 minutes, the segments of LAD exposed to carbon dioxide or room air, and the unexposed proximal LAD and right coronary artery, were processed, sectioned, and stained together. A murine anti-human tie-2 monoclonal antibody was used to identify endothelium.

Results. All unexposed LAD and right coronary artery segments and all LAD segments exposed only to room air demonstrated normal, contiguous staining of endothelium with the murine anti-human tie-2 monoclonal antibody. In contrast, all LAD segments exposed to high-flow carbon dioxide gas insufflation demonstrated near-complete loss of endothelium.

Conclusions. These data demonstrate that high-flow carbon dioxide gas insufflation denudes the coronary artery of its endothelium. This exposes blood elements to the subendothelium and promotes clotting, and endothelial loss may promote smooth muscle cell migration and proliferation. These events set the stage for early and late graft failure.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Minimally invasive techniques for myocardial revascularization are emerging as an alternative to standard coronary artery bypass grafting in selected cases [1]. A major obstacle to the safe and accurate performance of coronary bypass grafting on the beating heart is bleeding arising from the coronary arteriotomy. Although the optimal method of arteriotomy visualization has not been identified, it has been suggested that a catheter-directed stream of air, oxygen, or carbon dioxide is helpful [2]. Carbon dioxide is the most common gas used because of its high solubility in blood, which decreases the risk of gaseous embolization [3]. However, the potentially adverse effects on coronary endothelium of brief exposure to high-flow gas insufflation are unknown.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 Acknowledgments
 References
 
All animals received humane care in compliance with the Institutional Animal Care and Use Committee at Duke University and the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication no. 85-23, revised 1985).

Seven male crossbred pigs weighing 20 to 25 kg were sedated using 10 mg/kg intramuscular ketamine. An ear vein was cannulated using an 18-gauge catheter for fluid and drug administration. Twenty minutes later anesthesia was induced with 10 mg/kg intravenous thiopental sodium, and the animals were endotracheally intubated. Anesthesia was maintained with 1% halothane using an Ohio V5 mechanical ventilator and anesthesia machine (Ohio Medical Products, Madison, WI). Respiratory rate, end-tidal CO2, inspired percentage O2, and inspired and expired halothane concentrations were monitored using a Capnomac infrared spectrophotometer (Datex, Helsinki, Finland). Frequent arterial blood gas samplings were obtained to guide ventilator settings. Electrocardiogram was monitored continuously. Before further surgical manipulation, 0.2 mg/kg intravenous pancuronium bromide, 1 mg/kg intravenous lidocaine, and 5 mg/kg intravenous bretylium tosylate were administered. The left femoral artery was cannulated with a 7F Fast-Cath (Diag Corp, Minnetonka, MN) through which arterial blood samples were obtained and a Millar pressure transducer was placed to monitor central aortic blood pressure (Millar Instruments, Inc., Houston, TX). The heart was exposed through a median sternotomy and suspended in a pericardial cradle. A segment of mid left anterior descending coronary artery (LAD) was isolated by encircling it proximal and distal to the proposed arteriotomy site with silicone elastomer surgical tapes (Quest Medical, Inc., Dallas, TX). A mechanical cardiac stabilizer (USSC, Norwalk, CT) was used to immobilize a region of the anterior heart. A 1-cm longitudinal arteriotomy was then performed. To expose the arteriotomy, epicardial fat was retracted using fine monofilament suture. Figure 1 illustrates the surgical preparation.



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  		Fig 1. Surgical preparation. The stabilizing foot-plate was used to facilitate coronary artery dissection.

 
In the control group, the arteriotomy was exposed to room air for 20 minutes before the vessel was harvested. In the experimental group, the arteriotomy was exposed to 20 minutes of continuous nonhumidified carbon dioxide at a flow rate of 15 L/min. The gas jet emerged from a flexible plastic catheter affixed to the retractor at a standardized distance of 4 to 6 cm from the arteriotomy site. After 20 minutes, a 2-cm segment of LAD centered around the arteriotomy was carefully harvested. In addition, 2-cm segments of proximal LAD and right coronary artery were harvested and served as internal controls.

To identify endothelial cells in serial sections, immunostaining with a murine anti-human tie-2 (TEK) monoclonal antibody was used. The receptor tyrosine kinase, TEK, is expressed exclusively on endothelial cell membranes. The TEK antibody specifically targeted the soluble extracellular domain of tyrosine kinase, as described by Wong and colleagues [4]. In sections of porcine coronary artery, this antibody reproducibly stained the endothelium of both the arteries and the adventitial blood vessels (unpublished results).

All fresh tissue specimens were kept in 30% sucrose–phosphate-buffered saline solution (PBS) at 4°C, for 2 to 3 hours immediately after harvest. The samples were cross-sectioned in OCT (Optimal Cutting Temperature, Miles Pharmaceutical, West Haven, CT) and snap-frozen in liquid nitrogen. Frozen sections were made in a cryostat on microscope slides for histologic and immunochemical studies. Slides were removed from the freezer, warmed to room temperature, placed in ice-cold acetone for 2 minutes, and immersed in PBS. To examine the internal elastic lamina, elastin staining was performed using standard techniques. For immunohistochemical analysis, the slides were placed in blocking solution (10% horse serum in PBS) for 1 hour at room temperature. The TEK monoclonal antibody was diluted (1:1,000) in blocking solution and exposed to tissue sections for 1 hour. Incubation with the primary antibody was followed by sequential incubation with biotinylated anti-mouse IgG and ABC reagent, in accordance with the manufacturer’s specifications (Vectastain ABC kit, Vector Laboratories, Burlingame, CA). Levamisole was added to block endogenous alkaline phosphatase activity and immune complexes were localized using the chromogenic alkaline phosphatase substrate Vector Red (Vector Laboratories). The sections were counterstained with hematoxylin, dehydrated, and mounted with Permount (Fischer Scientific, Pittsburgh, PA). When using this method the antigen will appear red against a light blue background. For each sample, a serial section was incubated with a nonsense murine IgG monoclonal antibody that served as a negative control.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 Acknowledgments
 References
 
All animals survived the surgical procedure. The segments of LAD exposed to carbon dioxide (n = 5) or room air (n = 2) and the unexposed proximal LAD and right coronary artery were processed, sectioned, and stained. The specimens from the unexposed LAD and right coronary artery displayed contiguous immunostaining with TEK antibody throughout the endothelium (Fig 2).



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  		Fig 2. Photomicrograph, immunohistochemically stained with a mouse anti-human tie-2 monoclonal antibody, depicts the appearance of an unexposed control segment of left anterior descending coronary artery. All three layers of vessel wall are visible. Note the dark staining of the endothelium. (x13.2 before 35% reduction.)

 
In LAD segments exposed to carbon dioxide, an elastin stain was used to determine the depth of injury. The uniform uptake of stain in all specimens confirmed the presence of an intact internal elastic lamina (Fig 3C). In contrast, these same specimens demonstrated significant loss of endothelial staining (Figs 3A, 3B). The proportion of internal elastic lamina denuded of overlying endothelium ranged between 50% and 100% of the vessel’s circumference and extended for the length of the arteriotomy. To differentiate between the effects of high-flow gas insufflation and direct surgical trauma, control animals underwent arteriotomy but were not exposed to high-flow carbon dioxide gas. These LAD segments demonstrated intact endothelium that was indistinguishable from specimens prepared from unexposed proximal LAD or right coronary artery (Fig 4).



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  		Fig 3. (A) Photomicrograph, immunohistochemically stained with a mouse anti-human tie-2 monoclonal antibody, depicts the appearance of a segment of left anterior descending coronary artery that underwent arteriotomy and was exposed to high-flow gas insufflation at 15 L/min for 20 minutes. (B) Higher power magnification of box marked in A. This demonstrates the near-complete loss of endothelium after gas insufflation. The arrow in A points to an adventitial blood vessel endothelium that is staining red, providing an internal control. (C) Elastin stain of previous section demonstrating an intact internal elastic lamina. (A, x25; B, x66; C, x66; all before 31% reduction.)

 


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  		Fig 4. Photomicrograph, immunohistochemically stained with a mouse anti-human tie-2 monoclonal antibody, depicts the appearance of a segment of left anterior descending coronary artery that underwent an arteriotomy and was exposed to room air for 20 minutes. (x33 before 46% reduction.)

 

   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
Acknowledgments
 References
 
A precise and effective coronary artery anastomosis is the primary objective of surgical revascularization for ischemic heart disease. With the resurgence of popularity for coronary bypass operations on the beating heart, the importance of this principle cannot be overemphasized. Bleeding from the coronary arteriotomy site hinders precise suture placement in the often small and diffusely diseased vessel. Several techniques have been introduced to improve visualization of the anastomotic site. These have included frequent blotting of the arteriotomy with an absorbent material, intermittently irrigating with saline solution, intraluminal coronary shunts or occluders, and the use of high-flow gas insufflation. Each technique has its limitations. Blotting provides only transient and incomplete improvement in hemostasis, usually requires a second surgical assistant, and may directly traumatize endothelium. Irrigating with saline solution has the same disadvantages as blotting, but may also produce visual parallax errors when suturing and may contribute to hemodilution [2]. Intraluminal coronary shunts or occluders are somewhat cumbersome to use, and may cause endothelial injury if the device significantly stretches the arterial wall (unpublished observation). Although high-flow gas insufflation is effective in maintaining a bloodless field, the most commonly used gases (oxygen, carbon dioxide, and air) are each associated with hypothetical risks. Oxygen is flammable, carbon dioxide may promote hypercarbia if scavenged by the cardiotomy suction circuit, and air may produce gaseous emboli [5].

In the present study, carbon dioxide gas directed at the arteriotomy at a flow rate of 15 L/min for 20 minutes significantly denuded the endothelial surface of the LAD. Despite the prominent endothelial loss, however, the internal elastic lamina was preserved. Ip and associates [6] classified vascular wall injuries into three broad categories: (1) functional alterations without significant morphologic changes (type I); (2) denuded endothelium with intact internal elastic lamina and media (type II); and (3) denuded endothelium with intimal and medial damage (type III). Thus, the arterial exposure to high-flow carbon dioxide gas can produce a type II vascular injury. Pilot studies were performed to establish the minimum flow rate of gas needed to sufficiently clear blood from an anastomotic site. These studies yielded a value of 15 L/min, which is in agreement with the report by Teoh and coworkers [2]. The minimum flow rate to achieve adequate blood clearance varies, however, with the diameter of the catheter opening from which the gas emerges and with the distance between the catheter tip and target site.

Endothelial cells serve several roles in maintaining vascular homeostasis. In addition to their role in governing membrane permeability, endothelial cells modulate vascular tone by producing nitric oxide, as well as other endothelial-derived relaxing factors [7]. Endothelial cells also elaborate an array of potent growth promoters and inhibitors that primarily affect vascular smooth muscle cells [8]. Finally, endothelial cells function as a physical barrier between blood elements and procoagulants in the subendothelium, such as tissue factor. Injury to endothelial cells may disrupt this delicate balance. The exposure of blood elements to procoagulants within the subendothelium promotes platelet adhesion, aggregation, and degranulation [9]. In the presence of diminished blood flow this predisposes to coagulation and vascular thrombosis. Furthermore, because platelets produce potent mitogens that regulate smooth muscle cell migration [10], such as platelet-derived growth factor, this leads to smooth muscle cell proliferation and migration into the intima [11]. The extent of intimal thickening appears to correlate with the severity of vessel wall injury [12]. Fingerle and colleagues [12] demonstrated, in a rat carotid model of a type II arterial injury, a significant increase in smooth muscle cell proliferation and the formation of intimal lesions when the endothelial loss persisted for several days. Moreover, when arterial injury involved both the endothelium and medial smooth muscle (ie, a type III injury), the magnitude of the proliferative response was significantly greater. These findings suggest that destruction of coronary endothelium to the extent observed with high-flow gas insufflation will produce disruption of vascular homeostasis, setting the stage for early and late graft failure.

A potential limitation to this study was the use of normal coronary arteries rather than atherosclerotic vessels. Although we suggest that normal endothelium is more resistant to physical trauma than the abnormal endothelium associated with atherosclerotic disease, this is unproved. A second limitation is that the arteriotomy was exposed to 20 minutes of continuous gas insufflation. Although this time interval was chosen as a reasonable approximation of the time required to complete a distal coronary anastomosis on the beating heart, we concede that technical ability varies. Nevertheless, this does not detract from the central conclusion that high-flow gas insufflation can produce significant endothelial destruction. Insofar as endothelial injury may be temporally dependent, we suggest that rather than restricting the duration of exposure, high-flow gas insufflation be avoided altogether. Alternatively, the addition of humidification may cause less tissue desiccation and is an area of future study.

In summary, this experimental study demonstrates that high-flow carbon dioxide insufflation, when used to maintain a bloodless operative field during bypass grafting, can cause profound endothelial loss. Loss of this endothelial barrier exposes the subendothelium to the blood elements and promotes clotting. Furthermore, endothelial loss may promote smooth muscle cell migration and proliferation. These events set the stage for early and late bypass graft failure.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
References
 
Supported in part by grant HL09696-01 from the National Heart, Lung, and Blood Institute.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 

  1. Buffolo E., Silva de Andrade J.C., Branco Rodrigues J.N., Teles C.A., Aguiar L.F., Gomes W.J. Coronary artery bypass grafting without cardiopulmonary bypass. Ann Thorac Surg 1996;61:63-66.[Abstract/Free Full Text]
  2. Teoh K.H., Panos A.L., Harmantas A.A., Lichtenstein S.V., Salerno T.A. Optimal visualization of coronary artery anastomoses by gas jet. Ann Thorac Surg 1991;52:564.[Abstract]
  3. Sasaguri S., Hosoda Y., Yamamoto S. Carbon dioxide gas blow for the safe visualization of coronary artery anastomosis [Letter]. Ann Thorac Surg 1995;60:1861.[Free Full Text]
  4. Wong A.L., Haroon Z.A., Werner S., Dewhirst M.W., Greenberg C.S., Peters K.G. Tie2 expression and phosphorylation in angiogenic and quiescent adult tissues. Circ Res 1997;81:567-574.[Abstract/Free Full Text]
  5. Poulton T.J. Visualization of coronary artery anastomoses by gas jet [Letter]. Ann Thorac Surg 1992;54:598-599.
  6. Ip J.H., Fuster V., Badimon L., Badimon J., Taubman M.B., Chesebro J.H. Syndromes of accelerated atherosclerosis: role of vascular injury and smooth muscle cell proliferation. J Am Coll Cardiol 1990;15:1667-1687.[Abstract]
  7. Billiar T.R. Nitric oxide: novel biology with clinical relevance. Ann Surg 1995;221:339-349.[Medline]
  8. Luscher T.F., Tanner F.C., Tschudi M.R., Noll G. Endothelial dysfunction in coronary artery disease. Annu Rev Med 1993;44:395-418.[Medline]
  9. Wilentz J.R., Sanborn T.A., Haudenschild C.C., Valeri C.R., Ryan T.J., Faxon D.P. Platelet accumulation in experimental angioplasty: time course and relation to vascular injury. Circulation 1987;75:636-642.[Abstract/Free Full Text]
  10. Fingerle J., Johnson R., Clowes A.W., Majesky M.W., Reidy M.A. Role of platelets in smooth muscle cell proliferation and migration after vascular injury in rat carotid artery. Proc Natl Acad Sci USA 1989;86:8412-8416.[Abstract/Free Full Text]
  11. Davies M.G., Hagen P.O. Pathobiology of intimal hyperplasia. Br J Surg 1994;81:1254-1269.[Medline]
  12. Fingerle J., Tina Au Y.P., Clowes A.W., Reidy M.A. Intimal lesion formation in rat carotid arteries after endothelial denudation in absence of medial injury. Arteriosclerosis 1990;10:1082-1087.[Abstract/Free Full Text]



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