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Ann Thorac Surg 2002;73:1927-1932
© 2002 The Society of Thoracic Surgeons
a Department of Cardiothoracic Surgery, Wythenshawe Hospital, Manchester, United Kingdom
b Department of Human Anatomy and Cell Biology, University of Liverpool, The Sherrington Buildings, Liverpool, United Kingdom
Accepted for publication March 5, 2002.
* Address reprint requests to Dr Salmons, Department of Human Anatomy and Cell Biology, University of Liverpool, The Sherrington Buildings, Ashton St, Liverpool L69 3GE, UK
e-mail: s.salmons{at}liverpool.ac.uk
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Abstract |
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Methods. Adult pigs were assigned to two experimental groups: an electrically prestimulated group (n = 10) and a vascular delay group (n = 10). In the prestimulated group the left latissimus dorsi muscle was activated in situ at 2 Hz for 24 h/d. In the vascular delay group, the intercostal perforating arteries to the left latissimus dorsi muscle were divided. Two weeks later, hyperemic blood flow was measured by means of fluorescent microspheres immediately before and after mobilizing the latissimus dorsi muscle and again after recovery for a further 2 days.
Results. In the prestimulated group, blood flow was not significantly depressed in any region of the muscle immediately after mobilization, and blood flow increased significantly in proximal (p = 0.01), middle (p = 0.02), and distal (p = 0.007) regions following recovery. In muscles subjected to vascular delay the proximal and middle regions showed no significant changes in blood flow after mobilization or recovery, but flow in the distal region was 50% lower after mobilization (p = 0.003), and it remained significantly depressed even after recovery (p = 0.008).
Conclusions. Prestimulation was significantly more effective than vascular delay in preserving distal blood flow. Because it is also less invasive and initiates metabolic transformation before mobilization, this technique should allow cardiac assistance to be introduced at an earlier postoperative stage without compromising the viability of the grafted muscle.
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Introduction |
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The vascular trees of the thoracodorsal artery and perforating arteries in the LDM are connected by arterial anastomoses [4]. Blood flow through these anastomoses is enhanced by electrical stimulation of the muscle in situ [5]. When such a prestimulated muscle is raised as a graft, blood flow throughout the muscle is better maintained, the distal region is no longer selectively affected, and any initial ischemia is completely reversed within 5 days [6]. Prestimulation therefore offers a potential solution to the problem of graft viability [7].
An alternative approach to the restoration of distal blood flow is the use of vascular delay, a reconstructive surgery procedure in which the perforating arteries are divided but the LDM is left in situ for a predetermined period, usually 2 weeks, before elevating it as a graft [8, 9].
In this study we test the hypothesis that the reduction of distal blood flow following mobilization of the LDM is prevented more effectively by prestimulation than by vascular delay.
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Material and methods |
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Anesthetic technique
Following preanesthetic medication with Stresnil (azaperone; Janssen Animal Health; 400 mg, given intramuscularly), general anesthesia was induced by intravenous injection of propofol (150 mg). The animal was intubated, and a 50:50 mixture of oxygen and nitrous oxide was administered through a rebreathing anesthetic circuit (Ohmeda, Essex, UK). Anesthesia was maintained by intravenous infusion of propofol at a rate estimated to maintain a constant blood concentration of 4 µg · mL-1. The animal breathed spontaneously throughout. Analgesia was provided by continuous intravenous infusion of alfentanil at a rate of 0.5 to 1.5 µg · kg-1 · min-1. Hartman’s solution was given throughout the procedure at a rate of 10 mL · kg-1 · h-1. Blood loss was replaced by a mixture of warmed crystalloid (0.9% saline) and colloid (Gelofusine; Braun Medical, Aylesbury, UK). The electrocardiogram, heart rate, and oxygen saturation were monitored continuously.
Placement of stimulating electrodes
The animal was anesthetized and the surface of the LDM was exposed through a long flank incision. The proximal anterior border of the muscle was carefully reflected to expose the thoracodorsal neurovascular bundle; this could be achieved without disturbing arterial branches entering the costal surface of the muscle. Each stimulating electrode consisted of the deinsulated terminal segment of a stainless steel lead, which was mounted on a silicone rubber plate to limit unwanted stimulus spread. The first electrode was fixed on the deep surface of the muscle and the second on the superficial surface, opposite the first. This arrangement ensured that the stimulating current passed through the nerve and its branches but avoided mechanical interference with the neurovascular bundle. The electrodes were connected to a programmable neuromuscular stimulator (Itrel SP4721, Medtronic, Minneapolis, MN), which was placed subcutaneously on the flank.
Measurement of blood flow
Blood flow was measured by the method of fluorescent microspheres, substantially as described and validated previously [4–6, 10]. In brief, a catheter (multipurpose 5 F cardiac catheter; Cordis, Miami, FL) was passed into the left ventricle through a 7 F introducer sheath (Desivalve, Vygon, Cirencester, UK) in the right internal carotid artery. An 18 G cannula (Venflon, Becton Dickinson, Helsingborg, Sweden) was placed in the femoral artery and connected to a withdrawal pump (55-2226 Harvard Apparatus, Kent, UK). For each measurement, hyperemia was induced by programming the neuromuscular stimulator to deliver trains of supramaximal impulses (amplitude 8 V, duration 210 microseconds) at 30 Hz, for 0.19 seconds on and 1.5 seconds off over a period of 2 minutes. Fluorescent microspheres (20 x 106 blue, blue-green, or yellow-green; FluoSpheres, Molecular Probes, Inc, Oregon) were then delivered into the left ventricle while a reference blood sample was withdrawn at 12 mL · min-1 from the femoral artery.
At the end of the experiment the animal was killed by anesthetic overdose and the LDM removed. The muscle was divided into proximal, middle, and distal thirds, based on the distance from the humeral attachment. These three segments were further divided into samples of 6 to 10 g and digested for 72 hours at room temperature in 35 mL of 4 mol/L KOH containing 2% Tween 80. The solution was heated to 65°C and agitated in a water bath to emulsify solidified fat. The fluorescent microspheres that had been trapped in tissue capillaries were then recovered from the digest by vacuum filtration through a filter of pore size 10 µm (Millipore, Poretics Corp, Livermore, CA). The dye contained within the microspheres was released by placing the filter in a solvent (Cellosolve [diethylene glycol monoethyl ether acetate]; Fluka, Dorset, UK), and the fluorescent signal in the solution was read in a spectrofluorophotometer (Shimadzu RF-540, Kyoto, Japan). There was no overlap between the spectral contributions from the three colors of fluorescent microspheres used.
For each blood flow measurement there was a corresponding 25-mL reference blood sample, which was divided into five 5-mL aliquots. These were digested and dye-extracted as described earlier, and the signals were combined.
Blood flow was calculated from the formula:
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Acute study
A preliminary study was carried out on the left and right LDM of 2 pigs under acute conditions to confirm that the major features of the blood supply were broadly similar to those already established in the sheep.
With the animal anesthetized, electrodes were placed near the thoracodorsal nerves on both sides and connected to separate neuromuscular stimulators. These were activated to induce hyperemia in the two muscles, following which both thoracodorsal arteries were occluded with vascular clamps. Blue fluorescent microspheres (20 x 106) were injected to measure the contribution to LDM blood flow from all sources other than the thoracodorsal artery. After a few minutes the clamps were released, restoring thoracodorsal blood flow. The two muscles were then dissected free from the chest, dividing in the process all truncal attachments and perforating arterial branches, leaving only the thoracodorsal neurovascular bundle intact. The muscles were then reattached to the chest at their anatomical lengths, and the skin was closed to maintain the muscles at body temperature. Hyperemia was induced, and 20 x 106 blue–green fluorescent microspheres were injected to measure the contribution to blood flow from the thoracodorsal artery alone. The animals were killed and the LDM of both sides harvested for digestion and recovery of the microspheres as already described.
Chronic study
For the chronic study, 20 pigs were assigned at random to two experimental groups: an electrical prestimulation group (n = 10) and a vascular delay group (n = 10).
Implantation of neuromuscular stimulators and electrodes
Animals of both groups were anesthetized, and sterile stimulating electrodes were placed in relation to the left thoracodorsal nerve as described earlier. The electrodes were tunneled under the skin and connected to a programmable neuromuscular stimulator (Itrel SP4721, Medtronic), which was placed in a subcutaneous pocket on the lateral abdominal wall. In the vascular delay group the device remained quiescent. In the electrical prestimulation group the device was programmed remotely 1 day later to activate the LDM in situ at 2 Hz for 24 h/d.
Ligation of intercostal perforating arteries
In the vascular delay group of animals only, a second incision was made along the lateral border of the erector spinae muscle. The aponeurosis was divided along the posterior border of the LDM to expose the deep surface and the perforating arteries, all of which were then divided between ligatures or coagulated by diathermy. The aponeurosis was reattached with sutures so as to maintain the muscle at its anatomical length.
This surgical approach differs slightly from that of previously published studies on rodents, rabbits, and dogs, in which the arteries are usually accessed through the ventral border of the LDM. In those species, the skin is loosely bound to the LDM and offers unconstrained surgical access. In the pig the muscle is much more tightly adherent to the thick, adipose, overlying skin, and the perforating arteries are closer to the posterior border. However, both routes achieve the same goal, which is to divide the perforating arteries with minimal disturbance to the muscle.
Muscle mobilization and regional blood flow measurements
Following the procedure just described, hyperemic blood flow to the left LDM was measured by injecting 20 x 106 blue fluorescent microspheres (FluoSpheres, Molecular Probes, Inc) into the left ventricle. This measurement is referred to as "baseline" flow.
The LDM was then freed from its truncal attachments and raised as a unipedicled graft. This process included division of the intercostal perforating vessels in the prestimulation group, in which they were still intact. The humeral insertion of the muscle and the thoracodorsal neurovascular bundle were left undisturbed. The muscle was then reattached to the chest wall at 80% of its anatomic length. All incisions were closed. Regional blood flow in the LDM was measured, and 20 x 106 blue-green fluorescent microspheres were injected at this stage to assess the acute effects of mobilization ("acute phase"). Vascular access devices were withdrawn, except for the Desivalve introducer sheath, which was flushed with heparinized saline and left subcutaneously where it could be accessed again at the neck. The animals were allowed to recover from anesthesia.
Two days later all animals were reanesthetized. Regional blood flow in the LDM was measured by injection of 20 x 106 yellow-green fluorescent microspheres to evaluate the extent of spontaneous recovery of blood flow from any acute vasospasm that had been induced by mobilization and handling ("recovery phase"). Two weeks after the beginning of the chronic study, stimulation was discontinued in animals of the electrical prestimulation group, and all animals were anesthetized as before.
Data handling
For each region the specific flow (mL · min-1 · 100 g-1) was calculated as the sum of the flows determined for individual samples divided by their combined mass. In the preliminary study the results for the right and left LDM were averaged before combining the data from the two animals. In the main study, a repeated measures analysis of variance was used to compare blood flows from the two groups and the three time points. Changes in blood flow with respect to baseline were analyzed by an unpaired Student’s t test.
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Results |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Chronic study: regional blood flow
Measurements of regional blood flow were all made under hyperemic conditions.
In the prestimulated group of muscles, there was no significant shift from baseline values of blood flow in any region immediately after mobilization (Table 1). Following the recovery period there was a significant increase in blood flow over baseline values in all three regions (p = 0.01, 0.02, and 0.007 for the proximal, middle, and distal regions, respectively).
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In all three regions the values for baseline hyperemic blood flow recorded before mobilizing the muscle were lower in the prestimulated muscles than in the vascular delay group (Table 1; p = 0.003, 0.0003, and 0.0009 for the proximal, middle, and distal regions, respectively). This adaptation to chronic stimulation has been observed previously in the sheep [5, 6]. In the context of the present study, which addresses the effect of mobilization on blood flow, the only meaningful way to compare the two pretreatments is to examine the percentage changes in blood flow from baseline values. These data are presented in Figure 2.
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Following the recovery period (Fig 2B), regional blood flow showed little or no improvement in the vascular delay group, whereas it was significantly enhanced in all three regions of the prestimulated group. As a result, the difference between the two groups of muscles became statistically significant in all three regions (Fig 2B; p = 0.01, 0.03, and 0.0006 for the proximal, middle, and distal regions, respectively).
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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To overcome this problem, a so-called vascular delay of several weeks was introduced before commencing stimulation [17], the intention being to provide time for neovascularization, which would expand the territory of the thoracodorsal artery. The clinical cardiomyoplasty protocol that has been most widely adopted includes such a delay, but its effectiveness is questionable [2, 3]. The "true vascular delay" is different: the perforating vessels are divided, but the LDM is left in situ for approximately 2 weeks before elevating it and reconfiguring it as a functional graft [8, 9]. This is the procedure that has been compared in the present study with prestimulation of the otherwise undisturbed muscle, a technique whose effectiveness has been demonstrated in the sheep [5, 6].
It was necessary to confirm at the outset that the main features of the blood supply to the LDM in the pig were the same as had been established in the sheep. The pig differed only in the extent of functional overlap between the arterial territories. For example, the thoracodorsal artery supported only about 15% of total blood flow in the distal region of the LDM, as opposed to 35% in the sheep. This indicates either that the flow capacity of the arterial anastomoses is lower in the pig than in the sheep or that the anastomoses are more sensitive to surgical disturbance. The greater dependence of distal blood flow on the perforating intercostal arteries in the pig should therefore constitute a more critical test of techniques designed to maintain flow when this blood supply is cut off during mobilization of the muscle.
If prestimulation of the pig LDM had had no protective effect, a maximum flow of 2.9 mL · min-1 · 100 g-1 would be expected to remain after mobilization (15% of a distal baseline flow of 19.3 mL · min-1 · 100 g-1). Yet the flow measured was 22.1 mL · min-1 · 100 g-1 immediately after mobilization and 29.9 mL · min-1 · 100 g-1 2 days later. If the vascular delay procedure had had no protective effect, a maximum flow of 5.9 mL · min-1 · 100 g-1 would be expected to remain after mobilization (15% of a distal baseline flow of 39.5 mL · min-1 · 100 g-1). The flow measured was 19.4 mL · min-1 · 100 g-1 immediately after mobilization and 26.4 mL · min-1 · 100 g-1 2 days later. It may be concluded that prestimulation and vascular delay increased the flow after mobilization and recovery by not less than 10.3-fold and 4.5-fold, respectively.
Our earlier studies in the sheep showed that continuous stimulation at 2 Hz, applied to the LDM before any other intervention, reduced regional blood flow by 50% to 60% [6]. In the present study the absolute blood flows in the prestimulated LDMs show evidence of a similar reduction compared with the completely unconditioned muscles of the vascular delay group (Table 1). Because all flows were measured under hyperemic conditions, a decline could represent a reduction in either the resting flow or the hyperemic response [17, 18]. Although the absolute baseline flows in the two groups of muscles are different, both are assumed to be equal to the physiologic needs of the muscles, with the lower blood flow in the prestimulated muscles reflecting the more efficient oxygen extraction that is part of the metabolic adaptation to a sustained increase in activity [18].
Figure 2 shows the departures from these physiologically adequate levels of blood flow brought about by mobilization and subsequent recovery. Evidently both prestimulation and vascular delay had a positive influence on distal blood flow in the mobilized LDM, but whereas blood flow throughout the prestimulated muscles continued to equal or exceed baseline values, muscles of the vascular delay group showed a deficit in distal blood flow of nearly 50%, and flow remained more than 30% below baseline even after 2 days.
In view of published data from rodents, rabbits, and dogs, it may seem surprising that the vascular delay procedure was not more effective. The pig LDM is broader and thicker than that of the other experimental species and more similar in its morphology to that of humans. Furthermore, large animals tend to have less oxidative, and therefore less densely vascular, muscles. It may be that these or other factors delay vascular recovery by neoangiogenesis.
The results of an earlier study in dogs favored vascular delay over prestimulation [8]. However, neither blood flow nor muscle viability were assessed in that study; the outcome measure used was the performance of the muscle as a cardiomyoplasty wrap. The contractile properties of the prestimulated muscle would have been modified by the 4-week stimulation protocol used, and this could account for the difference in mechanical performance that was observed.
Prestimulation restores distal perfusion by enhancing flow through anastomotic connections to preexisting vessels left behind when the perforating arteries are sacrificed [5, 6]. Its protective effects would therefore be expected to emerge sooner than if there were an absolute requirement for neoangiogenesis. There may, however, be an added angiogenetic component to its action, because stimulation is known to enhance capillary formation [19], possibly by upregulating the expression of fibroblast growth factors [20]. Prestimulation has other advantages. The vascular delay procedure involves extensive dissection of the deep surface of the LDM. Prestimulation requires access to only the thoracodorsal nerve and is therefore less likely to create adhesions that would interfere with the subsequent redeployment of the muscle. Prestimulation also initiates the induction of fatigue resistance at a preoperative stage. A muscle that is prestimulated for 6 weeks, for example, should be capable of sustaining cardiac levels of work almost immediately after mobilization.
We conclude that there is a substantial case for prestimulating the LDM before raising it as a graft for use in cardiac assistance. This procedure improves the viability of the graft by maintaining blood flow in all regions of the muscle. It is more effective in this respect, as well as less invasive, than vascular delay. Finally, inclusion of preoperative stimulation in the protocol would enable a patient to receive the benefits of sustainable cardiac assistance at a much earlier postoperative stage.
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Acknowledgments |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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References |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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This article has been cited by other articles:
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  S. Salmons Cardiac assistance from skeletal muscle: a reappraisal Eur. J. Cardiothorac. Surg., February 1, 2009; 35(2): 204 - 213. [Abstract] [Full Text] [PDF]
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 S. Salmons Cardiac Assistance From Skeletal Muscle: Should We Be Downhearted? Ann. Thorac. Surg., April 1, 2005; 79(4): 1101 - 1103. [Full Text] [PDF]
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G. Zakine, E. Martinod, P. Fornes, M. Sapoval, D. Barritault, A. F. Carpentier, and J. C. Chachques Growth factors improve latissimus dorsi muscle vascularization and trophicity after cardiomyoplasty Ann. Thorac. Surg., February 1, 2003; 75(2): 549 - 554. [Abstract] [Full Text] [PDF]
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