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Ann Thorac Surg 1999;68:46-51
© 1999 The Society of Thoracic Surgeons
a Department of Human Anatomy and Cell Biology, University of Liverpool, Liverpool, United Kingdom
b Department of Cardiothoracic Surgery, Wythenshawe Hospital, Manchester, United Kingdom
Address reprint requests to Dr Salmons, British Heart Foundation Skeletal Muscle Assist Research Group, Department of Human Anatomy and Cell Biology, University of Liverpool, Liverpool, L69 3GE UK
e-mail: s.salmons{at}liverpool.ac.uk
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Abstract |
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Methods. Ten sheep were randomly assigned to two equal groups. In one group the latissimus dorsi muscle was stimulated continuously in situ at 2 Hz for 2 weeks; in the other group the muscle was not stimulated. Regional blood flows in the muscle were determined sequentially (1) under baseline conditions, (2) immediately after surgical mobilization, handling, and reattachment at 80% of the resting length, and (3) after 5 days.
Results. Manipulation of the unstimulated muscle resulted in an acute global reduction in blood flow with no improvement after 5 days. The distal region was most severely affected (26.2% ± 4.2% of baseline blood flow). Electrical prestimulation significantly reduced regional blood flow under baseline conditions but rendered the whole muscle more resistant to the surgical manipulations; blood flow was significantly better-preserved immediately afterwards, and there was complete recovery to baseline levels after 5 days.
Conclusions. Electrical prestimulation of the latissimus dorsi muscle in situ reduces the acute distal ischemia caused by surgical manipulations, and promotes subsequent recovery of blood flow to baseline levels after a few days. Use of a prestimulated graft may therefore improve the outcome of skeletal muscle cardiac assistance.
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Introduction |
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Recently we have studied the blood supply to the LD muscle in some detail, focusing in particular on the role of vessels that connect the two major arterial networks within the muscle [8]. Based on a knowledge of these structures, and in particular their response to electrical stimulation [9], we proposed that an LD muscle that had been stimulated in situ would be less likely to show distal ischemia when it was mobilized under conditions that simulated its surgical use as a graft.
In the present study we have tested this hypothesis. We mobilized, handled, and cooled the LD muscle and reattached it at a reduced resting length to simulate the disturbance caused by surgical redeployment of the muscle in cardiac assistance. We used a method based on fluorescent microspheres to examine changes in the distribution of blood flow immediately after manipulating the muscle in this way. We then reexamined regional flows after 5 days to determine the extent to which changes observed immediately after intervention might subside over a short period. These measurements were performed with and without prior electrical stimulation of the muscles.
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Material and methods |
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Blood flow measurements
Blood flow was measured by the dye extraction method with the use of fluorescent microspheres. Polystyrene microspheres with a diameter of 15.0 ± 0.5 µm (mean ± SD) and loaded with a known quantity of fluorescent dye (FluoSpheres, Triton Technology Inc, San Diego, CA) were introduced systemically. Microspheres were trapped in the muscle capillaries in proportion to the blood flow, and this number could be accurately determined by measurement of extracted fluorescent dye. Blood flow could then be derived by reference to the microsphere content of a blood sample withdrawn at a known rate during systemic injection [10].
Anesthesia and monitoring
Animals were premedicated with intramuscular xylazine (0.5 mg/kg), and after 1 hour general anesthesia was induced with intravenous thiopentone (4 mg/kg). After endotracheal intubation, anesthesia was maintained with a mixture of equal parts of nitrous oxide and oxygen supplemented with halothane (1% to 2%). Intramuscular buprenorphine (4 µg/kg) was given for analgesia and supplemented by further 12-hourly doses as required. Gas exchange was monitored with continuous pulse oximetry (Minimon 7137B, Kontron, Watford, UK) at the tongue. Respiratory assistance was provided where necessary by a volume-controlled ventilator (OAV 7710, Ohmeda, Essex, UK) and adjusted to maintain oxygen saturation greater than 90%. A tube was placed in the rumen to avoid gastric dilatation and consequent respiratory embarrassment. Fluid and blood losses were compensated by infusion of warmed crystalloid (Ringer’s lactate solution) at a rate of 10 mL · kg-1 · h-1, and a mixture of crystalloid (0.9% saline) and colloid (Gelofusine; Braun Medical Ltd, Aylesbury, UK), respectively. Throughout the blood flow experiments the cardiovascular status of the animal was monitored by electrocardiography and measurement of left ventricular pressure. Core body temperature was monitored by an esophageal thermometer and maintained constant at 38.5°C by a warming system based on the circulation of heated air. In recovery procedures antimicrobial prophylaxis was provided by a single intravenous dose (1 g) of cefotaxime (Roussel, Uxbridge, UK).
Operative procedures
In a preliminary aseptic procedure, the anterior border of the left LD muscle was exposed through a limited flank incision with minimum disturbance to its blood supply. Within the proximal hilum on the costal surface of the muscle, a bipolar electrode (SP5528; Medtronic Inc, Minneapolis, MN) was gently secured across the main trunk of the thoracodorsal nerve. The electrode lead was tunneled to a subcutaneous pocket over the upper abdomen and connected to a bipolar stimulator (Itrel SP4721, Medtronic Inc). After testing the assembly for satisfactory operation and ensuring adequate hemostasis, wounds were closed in layers and the animal was allowed to recover. After 1 week, the stimulators in group 2 were programmed to activate the muscle to contract continuously at 2 Hz; the stimulators in group 1 remained switched off.
Three weeks after the first procedure, the animals were anesthetized as described previously, and blood flow measurements were performed under aseptic conditions. An introducer sheath (Desivalve 6F, Vygon, Cirencester, UK) was inserted into the left carotid artery through a cut-down to allow the passage of a cardiac catheter (Supertorque Castillo II 5.2F, Cordis, Miami, FL) into the left ventricle. The tip of the catheter was placed in the left ventricle just below the aortic valve by monitoring the pressure waveform recorded by a pressure transducer (Gaeltec, Dunvegan, Scotland) and displayed by real-time data acquisition software (CODAS, Dataq Instruments, Inc, Akron, OH). A cannula (LeaderCath 18G, Vygon, Cirencester, UK) in the right femoral artery was connected to a syringe pump (55-2226 Harvard Apparatus, Kent, UK) to permit the withdrawal of reference blood samples at a rate of 12 mL/min during the systemic injections of microspheres. The left LD muscle was exposed through a full-length incision in the flank, taking care to divide all the cutaneous vessels arising from the muscle. Before each injection of microspheres, the parameters in the stimulator were altered to 30 Hz, on for 0.19 seconds and off for 1.5 seconds, pulse duration 210 microseconds at supramaximal amplitude, and used to elicit intermittent tetanic contractions of the LD muscle for 2 minutes. As soon as this tetanic stimulation stopped, the microspheres were introduced systemically through the left ventricular catheter with simultaneous blood sampling from the right femoral artery. This period of tetanic contraction induced functional hyperemia in the muscle so that the blood flow measurements would reflect the maximum capacity for blood flow in each of the corresponding states [9]. The first injection of 20 x 106 blue FluoSpheres was given to measure baseline blood flow before any disturbance of the blood supply of the LD muscle. After the perforating arteries had been divided, the LD muscle was freed from its truncal attachments, raised as a pedicled graft, subjected to moderate handling and cooling, and then resutured in place at 80% of its normal physiologic length; under these conditions there was essentially no resting tension in the muscle. The second injection of 20 x 106 blue-green FluoSpheres was given to measure muscle blood flow, now sustained exclusively by the thoracodorsal artery, in the acute phase immediately after mobilization. After the animals had regained consciousness they were returned to the holding facilities with indwelling cannulas. After a further 5 days they were again anesthetized, the cannulas were accessed, and the left LD muscle was reexposed. The third injection of 20 x 106 yellow-green FluoSpheres was given at this time to measure longer-term changes in muscle blood flow derived from the thoracodorsal artery. The animal was killed by an overdose of anesthetic, and the entire LD muscle was excised.
Recovery of microspheres and dye extraction
The LD muscle was divided in its long axis into three regions: proximal, middle, and distal (Fig 1). To facilitate digestion, each region was further divided into segments, each weighing at least 6 g. Muscle and blood samples were digested in a mixture of 4 mol/L potassium hydroxide and 2% Tween 80 at 60°C for at least 72 hours. The microsphere content of each digested sample was recovered on a 10-µm-diameter-pore filter by vacuum-filtration (Millipore, Poretics Corp, Livermore, CA). A fixed quantity (3 mL) of diethylene glycol monoether ethyl acetate (Fluka, Dorset, UK) was added to each filtrate in a conical tube to dissolve the polystyrene microspheres, releasing the fluorescent dye. Each sample was vortexed briefly and centrifuged for 3 minutes at 900g (Mistral 3000i, MSE, Crawley, Sussex, UK) to minimize scatter from debris during fluorescence measurement. One milliliter of the supernatant was transferred to a microcuvette and read in a spectrofluorophotometer (Shimadzu RF-540, Kyoto, Japan) set at the optimal excitation-emission wavelengths for the corresponding dye. When emission of a sample exceeded the linear range, it was diluted by adding more dissolution agent until the reading was within range. Samples with an emission corresponding to less than 400 microspheres were excluded for statistical reasons [11]. To avoid fluorescent decay, the samples from a given experiment were shielded from direct light and analyzed immediately as a single batch.
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The specific flow (milliliters per minute per gram) for the whole region was calculated as the sum of the flows determined for individual segments divided by their combined mass. The second (acute phase) and the third (5 days’ recovery) measurements of blood flow were also expressed as percentages of the first (baseline) measurement.
Statistical analysis
The blood flow data were analyzed by three-factor repeated measures analysis of variance. From examination of residuals, the statistical model provided a good fit to the data. Statistical significance was defined as p < 0.05, and for each significant finding, multiple comparisons were made using the Tukey post hoc test. In the figures, the results are displayed as the mean values with the corresponding upper and lower 95% confidence intervals (CI). Computations were performed using GLIM 3.77 statistical software (Royal Statistical Society, London, UK).
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Results |
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1 for each corresponding region).
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Comment |
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To date, attempts to deal with this problem have depended on a somewhat simplified picture of the blood supply to the LD muscle. According to this view, the thoracodorsal artery supplies the proximal part of the muscle and the collateral perforating arteries supply the distal portion. Loss of the collateral supply during mobilization of the muscle renders the distal portion vulnerable to ischemia. To prevent damage the onset of stimulation is delayed to allow neovascularization of the distal portion by the thoracodorsal artery—the so-called vascular delay. This approach has two shortcomings. First, it delays by at least 2 weeks the benefit that the patient might otherwise derive from a highly invasive operation; in practice this restricts the operation to candidates who still have an adequate cardiovascular reserve. Second, its effectiveness is by no means certain: even after such a vascular delay it is still possible to demonstrate stimulation-induced damage on a scale that would not be observed in an undisturbed muscle [6, 15].
The simplified view is actually deficient in a major respect: it fails to take account of vessels that link the two major arterial supplies to the LD muscle at a precapillary level. The existence of these vascular anastomoses has been established in man [16–20], dog [16, 18], and in the rabbit and rat [21] by resin injection and angiographic techniques. A potential criticism of these findings is that the pressure needed to inject the viscous materials might have created artifactual channels. This issue was addressed in a recent study, in which we injected fluorescent microspheres into the normal systemic circulation of anesthetized sheep and were able to show unequivocally that the anastomotic connections are functional under physiologic conditions of pressure and flow [8]. The significance of this observation is that after the loss of the perforating arteries, the distal portion of the mobilized muscle could, in principle, continue to be perfused from the thoracodorsal artery—through an existing vascular network.
This being so, why should muscles be damaged at all by mobilization and stimulation? The answer would seem to be that there are several ways in which the arterial anastomoses could be rendered ineffective. Cooling and handling of the LD muscle and use of electrocautery during surgical mobilization could induce vasospasm. Reduction in resting tension could bring about distortion or collapse of vessels, which would explain the apparently ischemic nature of the damage that results when the muscle is fixed at less than its physiologic resting length. The normal blood-carrying capacity of the thoracodorsal artery could be insufficient to perfuse the entire vascular network in every case, or it could be insufficient to reperfuse vessels that have constricted or collapsed through one of the mechanisms already mentioned.
We examined these propositions by making sequential measurements of regional blood flow in the same muscles at different stages of surgical intervention. In a previous study [9] we introduced the techniques for measuring blood flow from the thoracodorsal artery and perforating arteries individually in sheep LD muscles. We showed then that a global reduction in blood flow took place immediately after mobilizing the muscle on the thoracodorsal neurovascular pedicle and reattaching it at physiologic length. In the present study, the mobilized muscle was reattached at reduced length, and the resultant reduction in blood flow was greater still. Although the reduction in blood flow took place throughout the muscle, it was the distal region that was most affected. No significant improvement was seen after resting the muscle for 5 days. At first sight this would suggest that acute vasoconstrictive events were not an important component of the loss of distal flow.
A moderate regimen of electrical stimulation, applied to the LD muscle before any other intervention, produced a reduction of some 50% to 60% in regional blood flow. It should be borne in mind that all flows were measured under hyperemic conditions, so that a decline could represent a reduction in either the resting flow or the hyperemic response of a muscle that is adapted for more efficient oxygen extraction [22]. Some previous work would suggest that both resting and hyperemic flow could have been affected [23]. At the same time, the proximodistal gradient that was a conspicuous feature of regional blood flow in the unstimulated control muscles was largely abolished. We had seen this combination of a reduced, but more uniformly distributed, blood flow in stimulated muscles in the previous study [9]. It points to a reduction in vascular resistance in the central part of the muscle, which would be consistent with enlargement of the anastomotic vessels bridging the two arterial territories.
The changes caused by prestimulation had a profound influence on the response of the muscles to mobilization and reattachment at reduced muscle length. The acute reduction in flow was much less than in the unstimulated control muscles, and the distal region was no longer selectively affected. Even more remarkably, flow in all regions of the muscle had returned to baseline values by 5 days. This last result cannot easily be explained in terms of angiogenetic changes after surgical mobilization, which would take weeks rather than days to accomplish. Rather it suggests that the procedures associated with mobilization do bring about some acute generalized collapse or constriction of vessels but that vascular changes in the prestimulated muscles moderate these effects and enable them to be reversed.
The sheep LD muscle is considered to be a clinically relevant model for the human LD muscle in applications such as dynamic cardiomyoplasty [24]. The present findings therefore lead to the following predictions in relation to the clinical use of this muscle. If the LD muscle is prestimulated, blood flow will recover to baseline levels after raising the graft, and this will occur in a far shorter time than for current protocols. On this basis it is reasonable to suppose—although it needs to be formally tested—that prestimulated LD muscles would be less vulnerable to ischemic damage, and possibly subsequent reperfusion injury, resulting from surgical mobilization. Patient surveys, such as that of Moreira and colleagues [4], indicate that any improvement in graft viability would be reflected in an improved clinical outcome. Moreover the prestimulated muscle will already have undergone some degree of metabolic transformation, and would require only a little more conditioning to become adapted to cardiac levels of work. This would allow earlier introduction of cardiac assistance, with the potential for extending the operation to patients who, in current practice, would be considered a poor risk.
In the past, we and others have discussed the possible benefits of stimulating the LD muscle before raising it as a graft [23, 25, 26]. The major drawback of this approach is the need for a preliminary invasive procedure under general anesthesia, but it may be possible to overcome this problem through the use of noninvasive or minimally invasive techniques of stimulation. Thus the results of this study begin to make a case for prestimulation as the basis of future protocols for dynamic cardiomyoplasty.
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Acknowledgments |
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This article has been cited by other articles:
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  I. R. Ramnarine, M. Capoccia, Z. Ashley, H. Sutherland, M. Russold, N. Summerfield, S. Salmons, and J. C. Jarvis Counterpulsation From the Skeletal Muscle Ventricle and the Intraaortic Balloon Pump in the Normal and Failing Circulations Circulation, July 4, 2006; 114(1_suppl): I-10 - I-15. [Abstract] [Full Text] [PDF]
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 E. B.C. Woo, J. C. Jarvis, T. L. Hooper, and S. Salmons Avoiding ischemia in latissimus dorsi muscle grafts: electrical prestimulation versus vascular delay Ann. Thorac. Surg., June 1, 2002; 73(6): 1927 - 1932. [Abstract] [Full Text] [PDF]
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G. A. Thomas, R. L. Hammond, K. Greer, H. Lu, J. C. Jarvis, A. P. Shortland, D. M. Pullan, S. Salmons, and L. W. Stephenson Functional assessment of skeletal muscle ventricles after pumping for up to four years in circulation Ann. Thorac. Surg., October 1, 2000; 70(4): 1281 - 1289. [Abstract] [Full Text] [PDF]
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