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Arterial Blood Flow and Microcirculatory Changes in the Rat Groin Flap after Ischemia Provocation by Electrical Stimulation of the Artery
Microvascular Research 62, 243–251 (2001) doi:10.1006/mvre.2001.2339, available online at http://www.idealibrary.com on
Arterial Blood Flow and Microcirculatory Changes in the Rat Groin Flap after Ischemia Provocation by Electrical Stimulation of the Artery Y. Qi,* ,‡ B. Gazelius,* ,§ ,1 B. Linderoth,§ O. Lo¨fgren,* ,‡ O. Gribbe,* ,‡ and T. Lundeberg* ,‡ *Department of Physiology and Pharmacology, Karolinska Institutet; and §Department of Neurosurgery and ‡Department of Plastic and Orthopedic Surgery, Karolinska Hospital, S-171 77 Stockholm, Sweden Received April 17, 2001; published online August 21, 2001
An island groin flap based on the inferior epigastric vessels was raised in 10 rats in order to monitor simultaneous ischemic changes in arterial blood flow and skin microcirculation induced by electrical stimulation of the feeding artery. A modified laser Doppler perfusion system recorded blood flow in the epigastric artery and in the skin microcirculation of the flap before and for 40 min after the experimentally induced ischemia. Sections of the stimulated segment of the vessel were obtained at the end of the experimental procedure for histological analysis to determine the extent of endothelial changes, if any. Artery blood flow and the flap microcirculation decreased significantly immediately after stimulation, both slowly increasing to prestimulation levels after 30 min. Artery perfusion was quicker than microcirculation to recover to the baseline value, indicating that reperfusion of larger vessels could involve mechanisms fundamentally different from those active in the resolution of ischemia at the capillary level. Histological artery examination revealed no significant endothelial damage at the stimulation site, thus demonstrating that electrical stimulation induces reproducible ischemia without visible endothelial damage. The differential effects on the feed1
Deceased December 6, 1999.
0026-2862/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
ing artery and on capillary perfusion indicate recruitment of several different mechanisms. © 2001 Academic Press Key Words: flap; laser Doppler perfusion monitor; microcirculation; electrical stimulation.
INTRODUCTION “Flaps” used for centuries in reconstructive surgery have in the past decade generated research dedicated to the understanding of the basic physiologic and metabolic events regulating flap perfusion and survival. Free or pedicled tissue transfer involves exposure to a long period of ischemia or hypoperfusion. Arterial and venous constriction are common problems in microvascular surgery, where flap survival seems to be related to tissue perfusion during surgery. The length of the ischemic period and reperfusion hyperemia seem to be critical factors [3, 6, 7, 13, 15, 23, 26, 29]. To increase flap survival, several pharmacological treatments have been tested, but beneficial reports of many drugs are contradictory. New treatments, reducing the failure rate after flap surgery, demand increased knowledge of circulatory and metabolic events to provide the necessary future guidelines [10, 12, 13, 16, 20, 21]. Generally, the selection of
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an experimental model is a critical step in any research project, where it may provide data suitable for the clinical situation and allow further experimentation to be justified. The rat groin island flap is the most commonly used model for the exploration of the effects of various agents, such as superoxide dismutase [24] and heparin [30] on microcirculation. Many investigators have attempted to solve the problem of flap failure by applying strategies directly to the ischemic process [7, 8, 13]. There is still a great interest in this field for reliable, easy-to-handle, and noninvasive methods to measure arterial blood flow and flap microcirculation. Laser Doppler perfusion monitoring (LDPM) is probably the most useful technique currently available in the study of arterial vasoconstriction (vasospasm) [1, 25]. Previous studies focused on flow either in the feeding vessels or at the capillary level, but the relationship between the blood flow in the feeding artery and flap microcirculation at the time of flap ischemia has not been investigated in detail. A clarification of this relationship and the factors affecting microcirculatory insufficiency after gross arterial flow is restored may provide important guidelines for the improvement of clinical procedures. The LDPM technique greatly facilitates the study of the epigastric island or free flap, and in the present study vascular spasm initiated by electrical stimulation is used as a model in rats. The effects of acute blood flow reduction on the flap microcirculation, as well as in the feeding vessel per se, are simultaneously studied using laser Doppler perfusion monitoring.
MATERIALS AND METHODS Animals and Flap Preparation All experiments were performed on 10 male Sprague–Dawley rats (300 –350 g, ALAB, Stockholm, Sweden) housed in cages with free access to food and water and maintained at a room temperature of 24 ⫾ 1° with a 12-h light/dark cycle. For anesthesia sodium pentobarbital (50 mg/kg) was used ip with complementary doses of 10 mg/kg/h as required. The groins were shaved with an electric razor and then treated
Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Qi et al.
with a depilatory cream to obtain hairless skin. The rats were kept on a warm pad to avoid temperature loss and the body temperature was thermostatically controlled. A adipocutaneous neurovascular island flap, 2.5 cm in diameter, fed by the epigastric vessels was raised, exposing the feeding vessels under the operating microscope. The flap was then resutured to its anatomical bed by two stitches, allowing direct observation of the superficial epigastric vessels [7]. Immediately after the experiments the rats receive a lethal dose of barbiturate. The experimental procedures and the protocol were examined and approved by the local ethical committee for animal research at Karolinska Institutet.
Acute Artery Blood Flow and Flap Microcirculation Observation Flap microcirculation including the corresponding feeding artery was monitored by a LDPM system, PeriFlux PF 4001 Master, and PeriFlux PF 3 with an extended bandwidth up to 24 kHz for the artery measurements (Perimed AB, Ja¨rfa¨lla, Sweden). The laser Doppler probe holder for the skin measurements was fixed to the flap with double adhesive tape before the incision and a standard probe PF408 was attached to the probe holder. For the recording of artery blood perfusion, a needle probe (PF402) with a custom-made vascular fixation device (a metallic semi-tube into which the laser probe ended) was used to record the arterial blood flow. The vessel probe was firmly kept in place and immobilized by a micromanipulator. Artery and skin laser Doppler readings were performed simultaneously. The systemic blood pressure was monitored with a pressure transducer (Statham, Inc., Halo Rey, Puerto Rico) via a catheter introduced into the left common carotid artery. Both LDPM readings and blood pressure data were stored in a Toshiba T3200SXC desktop computer using a specially designed software (Perisoft, version V5.10; Perimed AB) for data processing.
Electrical Stimulation A bipolar stimulating electrode using platinum electrodes (electrode diameter 0.5 mm, electrode length 5
245
Microcirculatory Changes in the Rat Groin Flap
FIG. 1. Percentage changes of laser Doppler flow values as related to the baseline value (100%). The electrical stimulation was produced with 1, 6, 8, and 10 impulses. Using 1 impulse induced a small short-lasting perfusion decrease. Six, 8, and 10 impulses induced a perfusion decreased more pronounced in the flap than in the artery, but without any significant difference compared to baseline.
mm, electrode separation 1 mm) was connected to a Grass stimulator (Grass SD9, Quincy, MA). Stimulation was delivered to the artery from the Grass stimulator connected to a stimulation isolation unit and a constant current unit (Grass CCU1A) delivering impulses with 100-ms duration and 100 mA at 4 Hz.
Flap Arterial Diameter Measurements of flap arterial diameter were taken from the video image of the vessels using a video cursor (Sony SVO-9500MDP and Panasonic BT-H 1450). This device places a cursor over the image to be measured, and the operator adjusts the size of the cursor to exactly match the size of that image. The
video cursor was calibrated periodically using a micrometer. The lumen diameters of the arterial were monitored before electrical stimulation and at 2, 5, 15, 25, and 40 min after stimulation.
Histological Examination Flap artery specimens were removed from stimulated and unstimulated sections and perfused with 2.5% glutaraldehyde in a 0.1 M sodium phosphate buffer containing 2 mM MgCl 2. The specimens were postfixed in glutaraldehyde, osmicated (2% osmium tetroxide in phosphate buffer) and rinsed for 15 min in buffer, dehydrated in ethanol, and embedded in agar 100. Thin cross-sectional slices were cut on an LKB
Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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FIG. 2. Original recording on-line from one rat showing laser Doppler flow in arbitrary units (perfusion units). Electrical stimulation of the feeding artery with 12 impulses. Stimulus (1) produced a rapid decrease of the LDF readings, both in the artery (top) and in the flap (middle). The systemic blood pressure is also shown (bottom). The X-axis (time) is compressed 64 times.
ultratome, stained with toluidine blue, and examined by light microscopy (Zeiss FM 109, Oberkochem, West Germany) [22].
Experimental Protocols Thirty minutes after flap elevation when the blood perfusion was stabilized a 10-min baseline recording was made, followed by stimulation of the superficial epigastric artery proximal to the LDF reading area. Stimulation impulses were 1, 6, 8, 10 (n ⫽ 6), and 12 (n ⫽ 10). The recording was commenced 30 min prior to stimulation and continued for 40 min afterward.
Statistical Analysis Laser Doppler values are given in percentage changes from baseline and expressed as the mean values ⫾ SE. The baseline recordings were set to 100%.
Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
For statistical analyses the Kruskal–Wallis test with Dunn’s multiple comparisons test was applied to compare percentage changes in blood flow and skin microcirculation at different points following stimulation. P ⬍ 0.05 was considered significant [28].
RESULTS Electrical Stimulation of the Feeding Artery Using 1, 6, 8, and 10 Impulses Electrical stimulation using a single impulse induced a brief perfusion decrease. Six, 8, and 10 impulses induced a decrease greater than the singlepulse-induced one, but it was not significantly different from the baseline. Artery and skin perfusion recovered within 10 min (Fig. 1).
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Microcirculatory Changes in the Rat Groin Flap
FIG. 3. Percentage change of laser Doppler flow values as related to the baseline value (100%). Stimulus was single mode, 12 impulses. In the artery, after electrical stimulation for 1, 2, 5, 10, and 15 min the blood flow was significantly reduced compared to baseline (**P ⬍ 0.01; ***P ⬍ 0.001). After 25 min the blood flow returned to baseline level. In the skin electrical stimulation for 1, 2, 5, 10, 15, and 20 min significantly decreased the microcirculation, but full recovery was delayed 5 min longer than in the artery (*P ⬍ 0.05; ***P ⬍ 0.001).
Electrical Stimulation Using 12 Impulses Stimulation of the epigastric artery with 12 impulses [S (1)] produced a rapid lowering of the LDPM readings, both in the artery and in the flap microcirculation. The decreases in arterial blood flow and flap microcirculation were fairly parallell, indicating that the stimulation induced a vasoconstriction producing ischemia (Fig. 2). In the artery, perfusion was reduced by 68.51% (mean ⫾ SE) (P ⬍ 0.001) 2 min after stimulation, but then recovered slowly recovered to the prestimulatory level within 25 min (mean ⫾ SE). Flap blood perfusion decreased about 59.96% (mean ⫾ SE) (P ⬍ 0.001) 5 min after stimulation, but the recovery was slower than in the artery and took about 30 min to reach the prestimulation level (mean ⫾ SE) (Fig. 3). During the recovery period of blood flow restoration two brief falls in perfusion rate were observed (Fig. 2,
arrows), probably indicating the formation of microthrombi in response to the stimulation of the vessel wall and the released vasoconstriction.
Videoscopic Examinations The magnitude of vasoconstriction was measured by the video camera system prior to and after the 12-impulse stimulation. The lumen diameter of the artery was significantly reduced, from 301.67 ⫾ 38.84 m (mean ⫾ SE) (before stimulation) to 51.04 ⫾ 5.03, 65.42 ⫾ 8.35, and 148.75 ⫾ 10.56 m, at 2, 5, and 15 min after stimulation, respectively. In parallell with this reduction of the lumen diameters of the arteries there was a decrease in blood perfusion (Fig. 4). The video camera facilitates direct observation of all arterial changes as they occur.
Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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FIG. 4. Lumen diameter changes in the stimulated arteries. Before electrical stimulation, the diameter (mean ⫾ SE) was 301.67 ⫾ 38.84 m (n ⫽ 6), and after electrical stimulation for 2, 5, and 15 min the lumen diameters were significantly reduced to 51.04 ⫾ 5.03 (***P ⬍ 0.001), 65.42 ⫾ 8.36 (**P ⬍ 0.01), and 148.75 ⫾ 10.56 m (*P ⬍ 0.05), respectively. In parallell with this reduction in the lumen diameters of arteries there was a decrease in the blood perfusion.
Blood Pressure There was no change in systemic blood pressure during electrical stimulation (Fig. 5) or the period of reduced blood flow in the artery and flap microcirculation.
Histological Examination Histological analysis using light microscopy of the arterial sections showed no significant architectural or endothelial changes in the stimulated and unstimulated sections (Fig. 6).
DISCUSSION In this study, a rat groin flap was used as a model to investigate blood flow changes both in the skin flap and in its feeding vessel following electrical stimulation of the vessel. Parallel reversible decreases of the
Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
LDPM reading of both the skin microcirculation and the flap artery were observed, following exposure to electrical stimulation. Extreme arterial vasoconstriction (vasospasm), a common problem encountered in microvascular surgery, can compromise flap integrity [29]. Agents which prevent the formation of free radicals or scavenge them have been suggested to minimize reperfusion injury. Repeatable, noninvasive measurement of artery and skin blood flow is needed to monitor this and other aspects of flap circulatory changes. Establishment of a reproducible vasospasm model is of cardinal importance for further exploration of this problem. Earlier models used pharmacological agents, particularly epinephrine, to induce vasoconstriction without successfully [17, 23] mimicking the clinical situation of mechanical vasospasm. Our studies on the rat groin flap suggest that electrical stimulation induced ischemia is a reproducible model for temporary ischemia without visible endothelial damage compared to the pinching technique [5, 9].
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FIG. 5. Percentage changes of systemic artery blood pressure compared to the baseline value (100%). No significant change occurred during the electrical stimulation.
The LDF measurements in our model reveal reduced flow (59 – 68%) after 12 impulses of electrical stimulation (Fig. 3) compared to the premanipulatory baseline flow in the artery and skin. There was a significant delay in the reperfusion of the capillary bed. This demonstrates that ischemia is the first component of flap injury, being responsible for a number of metabolic changes resulting in microcirculatory and tissue damage [11]. Lack of oxygen and eventual mechanical endothelial damage can produce an imbalance in the release of the endothelial-derived relaxing and constricting factors, impairing the flap microcirculation [4]. In this study, no endothelial damage at the stimulated site was evident, suggesting that factors released from local endothelial cells in the artery may be responsible for the impaired microcirculation distally in the flap. When a skin flap is rendered ischemic, hypoxia causes tissue depletion of ATP levels available for the Na ⫹–K ⫹ pump, resulting in cell swelling and accumulation of lactate and hypoxanthine. These resultant reactions can cause platelet aggregation and thrombosis in major arteries and microvessels, necrosis of en-
dothelium, and neutrophil-mediated cellular destruction and vasoconstriction, leading to flap necrosis [2, 4, 27]. Clinical research on microsurgical procedures in the past 10 years focuses mostly on microvascular patency mainly involving local thrombus formation and platelet aggregation [18, 19] and including the duration of ischemia as the dominating factor for the endothelial and microcirculation impairment [3, 4, 11, 13]. In our experiments with the 12-impulse stimulation of the feeding artery, short perfusion decreases were frequently observed (Fig. 2), and these were probably due to the formation of microthrombi, which occurs following vasoconstriction. This observation constitutes an important clue for future research. The mechanisms involved in the creation of microthrombi will be examined in a later study.
CONCLUSION This methodological study shows that the lowering in the LDPM readings in the microcirculation of an
Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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FIG. 6. Histological analysis by light microscopy. Sections collected from stimulated (a1, original magnification ⫻16; a2, original magnification ⫻40) and unstimulated (b1, original magnification ⫻16; b2, original magnification ⫻40) artery sections.
experimental flap correlates with the electrically induced vasospasm of the feeding artery. When the stimulation ceased, the feeding artery blood flow was more quickly reestablished than the flap microcirculation. This could indicate that a release of local endothelial and metabolic substances from the stimulated artery wall is responsible for the more long-lasting microcirculatory impairment. Finally, the study demonstrates that the rat groin flap is a feasible model for future investigations concerning ischemia–reperfusion injury phenomena.
Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
REFERENCES 1. Arnold, F., He, C. Y., Jia, C. Y., and Cherry, G. W. (1995). Perfusion imaging of skin island flap blood flow by a scanning laser-Doppler technique. Br. J. Plast. Surg. 48, 280 –285. 2. Baker, J. H., Anderson, G. L., Gu, J-M., Wyllie, F., and Acland, R. D. (1993). Experimental study of the relationship between alterations in tissue perfusion and anastomotic patency. Microsurgery 14, 409 – 415. 3. Bodor, R., Yoleri, L., Zhang, F., Buncke, G. M., Lineaweaver, W. C., and Buncke, H. J. (1997). Blood-flow velocity as a factor in postoperative microvascular patency. J. Reconstr. Microsurg. 13, 463– 470.
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4. D’Ambra, M. N., Akins, C. W., Blackstone, E. H., et al. (1996). Aprotinin in primary valve replacement and reconstruction: A multicenter, double-blind, placebo-controlled trial. J. Thorac. Cardiovasc. Surg. 112, 1081–1089. 5. Gazelius, B., Qi, Y., Gheradini, G., Linderoth, B., and Lundeberg, T. The relation between feeding artery perfusion and skin microcirculation after ischemic provocation in a rat groin flap. Unpublished. 6. Geter, R. K., Winters, R. R., and Puckett, C. L. (1986). Resolution of experimental microvascular spasm and improvement in anastomotic patency by direct topical agent application. Plast. Reconstr. Surg. 77, 105–115. 7. Gherardini, G., Lundeberg, T., Matarasso, A., Michaels, B., Cazelius, B., Brodda-Jansen, G., and Samuelson, U. (1995). Calcitonin gene-related peptide increases microcirculation after mechanically induced ischemia in an experimental island flap. Ann. Plast. Surg. 35, 178 –183. 8. Gherardini, G., Gurlek, A., Milner, S. M., Matarasso, A., Evans, G. R. D., Jernbeck, J., and Lundeberg, T. (1998). Calcitonin gene-related peptide improves skin flap survival and tissue inflammation. Neuropeptides 32, 269 –273. 9. Grulek, A., Schusterman, M. A., Evans, G. R. D., and Gherardini, G. (1997). Blood flow and microcirculatory changes in a ischemia–reperfusion injury model. Experimental study in the rabbit. J. Reconstr. Microsurg. 15, 345–349. 10. Heden, P., Jernbeck, J., Kjartansson, J., and Samuelson, U. E. (1989). Increased skin flap survival and arterial dilatation by calcitonin gene-related peptide. A study in the pig. Scand. J. Plast. Reconstr. Surg. 23, 11–16. 11. Heden, P., Sollevi, A., and Hamberger, B. (1989). Early circulatory and metabolic events in island skin flaps in pigs. Plast. Reconstr. Surg. 84, 468 – 483. 12. Jurell, G., and Jonsson, C-E. (1976). Increased survival of experimental skin flaps in rats following treatment with antiadrenergic drugs. Scand. J. Plast. Reconstr. Surg. 10, 169 –172. 13. Kartansson, J., and Dalsgaard, C. J. (1978). Calcitonin generelated peptide increases survival of a musculocutaneous critical flap in the rat. Eur. J. Pharmacol. 142, 355–358. 14. Kerrigan, C. L., and Stotland, M. A. (1993). Ischemia reperfusion injury: A review. Microsurgery 14, 165–175. 15. Kerrigan, C. L., Wizman, P., Hjortdal, V. E., and Sampalis, J. (1994). Global flap ischemia: A comparison of arterial versus venous etiology. Plast. Reconstr. Surg. 93, 1485–1497. 16. Kerrigan, C. L., and Daniel, R. K. (1982). Pharmacologic treatment of the failing skin flap. Plast. Reconstr. Surg. 70, 541–548. 17. Knight, K. R., Lepore, D. A., Ritz, M., et al. (1993). Infusions of a novel calcitonin gene-related peptide (CGRP) derivative at the
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time of reperfusion to salvage ischaemic rabbit skin flaps. Int. J. Tissue React. 15, 185–193. Kurose, I., Wolf, R., Grisham, M. B., Aw, T. Y., Specian, R. D., and Granger, D. N. (1995). Microvascular responses to inhibition of nitric oxide production. Circ. Res. 76, 30 –39. Lawson, D. L., Mehta, J. L., Nichols, W. W., Mehta, P., and Donnelly, W. H. (1990). Superoxide radical-mediated endothelial injury and vasoconstriction of rat thoracic aortic rings. J. Lab. Clin. Med. 115, 541–548. Lepore, D. A., Knight, K. R., Bhattacharya, S., Ritz, M., Robbins, S., Sieg, P., Morrison, W. A., and O’Brien, B. M. (1994). Drug mixture which improves survival of ischemic rabbit epigastric skin flaps. Microsurgery 15, 685– 692. Mendelson, B. C., and Woods, J. E. (1978). Effect of corticosteroids on the surviving length of skin flaps in pigs. Br. J. Plast. Surg. 31, 293–294. Nordling, L., Lundeberg, T., Liedberg, H., Theodorsson, E., and Ekman, P. (1992). The contribution of epinephrine to experimentally induced urethral inflammation in the rat. Neurosci. Lett. 139, 169 –172. Puckett, C. L., Winters, R. R., and Geter, R. K. (1985). Studies of pathologic vasoconstriction (vasospasm) in microvascular surgery. J. Hand Surg. 10A, 343–349. Sagi, A., Ferder, M., Yu, H. L., Gordon, N. J. V., and Strauch, B. (1990). pH-balanced solutions with superoxide dismutase (SOD): An attempt to increase island groin flap survival in rats. Ann. Plast. Surg. 24, 521–523. Schabauer, A. M. A., and Rooke, T. W. (1994). Cutaneous laser doppler flowmetry: Applications and findings. Mayo Clin. Proc. 69, 564 –574. Seaber, A. V. (1987). Experimental vasospasm. Microsurgery 8, 234 –241. Stadelmanm, W. K., Hess, D. B., Robson, M. C., and Greenwald, D. P. (1998). Aprotinin in ischemia–reperfusion injury: Flap survival and neutrophil response in a rat skin flap model. Microsurgery 18, 354 –361. Theodorsson-Norheim, E. Kruskal–Wallis test: (1986). BASIC computer program to perform nonparametric one-way analysis of variance and multiple comparisons on ranks of several independent samples. Comp. Methods Programs Biomed. 23, 57– 62. Wadstro¨m, J. (1990). Studies on traumatic vasospasm in the central ear artery of the rabbit. Scand. J. Plast. Reconstr. Hand Surg. (Suppl.) 21, 1– 42. Wong, L., Im, M. J., and Hoopes, J. E. (1991). Increased survival of island skin flaps by systemic heparin in rats. Ann. Plast. Surg. 26, 221–226.
Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Arterial Blood Flow and Microcirculatory Changes in the Rat Groin Flap after Ischemia Provocation by Electrical Stimulation of the Artery Y. Qi,* ,‡ B. Gazelius,* ,§ ,1 B. Linderoth,§ O. Lo¨fgren,* ,‡ O. Gribbe,* ,‡ and T. Lundeberg* ,‡ *Department of Physiology and Pharmacology, Karolinska Institutet; and §Department of Neurosurgery and ‡Department of Plastic and Orthopedic Surgery, Karolinska Hospital, S-171 77 Stockholm, Sweden Received April 17, 2001; published online August 21, 2001
An island groin flap based on the inferior epigastric vessels was raised in 10 rats in order to monitor simultaneous ischemic changes in arterial blood flow and skin microcirculation induced by electrical stimulation of the feeding artery. A modified laser Doppler perfusion system recorded blood flow in the epigastric artery and in the skin microcirculation of the flap before and for 40 min after the experimentally induced ischemia. Sections of the stimulated segment of the vessel were obtained at the end of the experimental procedure for histological analysis to determine the extent of endothelial changes, if any. Artery blood flow and the flap microcirculation decreased significantly immediately after stimulation, both slowly increasing to prestimulation levels after 30 min. Artery perfusion was quicker than microcirculation to recover to the baseline value, indicating that reperfusion of larger vessels could involve mechanisms fundamentally different from those active in the resolution of ischemia at the capillary level. Histological artery examination revealed no significant endothelial damage at the stimulation site, thus demonstrating that electrical stimulation induces reproducible ischemia without visible endothelial damage. The differential effects on the feed1
Deceased December 6, 1999.
0026-2862/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
ing artery and on capillary perfusion indicate recruitment of several different mechanisms. © 2001 Academic Press Key Words: flap; laser Doppler perfusion monitor; microcirculation; electrical stimulation.
INTRODUCTION “Flaps” used for centuries in reconstructive surgery have in the past decade generated research dedicated to the understanding of the basic physiologic and metabolic events regulating flap perfusion and survival. Free or pedicled tissue transfer involves exposure to a long period of ischemia or hypoperfusion. Arterial and venous constriction are common problems in microvascular surgery, where flap survival seems to be related to tissue perfusion during surgery. The length of the ischemic period and reperfusion hyperemia seem to be critical factors [3, 6, 7, 13, 15, 23, 26, 29]. To increase flap survival, several pharmacological treatments have been tested, but beneficial reports of many drugs are contradictory. New treatments, reducing the failure rate after flap surgery, demand increased knowledge of circulatory and metabolic events to provide the necessary future guidelines [10, 12, 13, 16, 20, 21]. Generally, the selection of
243
244
an experimental model is a critical step in any research project, where it may provide data suitable for the clinical situation and allow further experimentation to be justified. The rat groin island flap is the most commonly used model for the exploration of the effects of various agents, such as superoxide dismutase [24] and heparin [30] on microcirculation. Many investigators have attempted to solve the problem of flap failure by applying strategies directly to the ischemic process [7, 8, 13]. There is still a great interest in this field for reliable, easy-to-handle, and noninvasive methods to measure arterial blood flow and flap microcirculation. Laser Doppler perfusion monitoring (LDPM) is probably the most useful technique currently available in the study of arterial vasoconstriction (vasospasm) [1, 25]. Previous studies focused on flow either in the feeding vessels or at the capillary level, but the relationship between the blood flow in the feeding artery and flap microcirculation at the time of flap ischemia has not been investigated in detail. A clarification of this relationship and the factors affecting microcirculatory insufficiency after gross arterial flow is restored may provide important guidelines for the improvement of clinical procedures. The LDPM technique greatly facilitates the study of the epigastric island or free flap, and in the present study vascular spasm initiated by electrical stimulation is used as a model in rats. The effects of acute blood flow reduction on the flap microcirculation, as well as in the feeding vessel per se, are simultaneously studied using laser Doppler perfusion monitoring.
MATERIALS AND METHODS Animals and Flap Preparation All experiments were performed on 10 male Sprague–Dawley rats (300 –350 g, ALAB, Stockholm, Sweden) housed in cages with free access to food and water and maintained at a room temperature of 24 ⫾ 1° with a 12-h light/dark cycle. For anesthesia sodium pentobarbital (50 mg/kg) was used ip with complementary doses of 10 mg/kg/h as required. The groins were shaved with an electric razor and then treated
Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Qi et al.
with a depilatory cream to obtain hairless skin. The rats were kept on a warm pad to avoid temperature loss and the body temperature was thermostatically controlled. A adipocutaneous neurovascular island flap, 2.5 cm in diameter, fed by the epigastric vessels was raised, exposing the feeding vessels under the operating microscope. The flap was then resutured to its anatomical bed by two stitches, allowing direct observation of the superficial epigastric vessels [7]. Immediately after the experiments the rats receive a lethal dose of barbiturate. The experimental procedures and the protocol were examined and approved by the local ethical committee for animal research at Karolinska Institutet.
Acute Artery Blood Flow and Flap Microcirculation Observation Flap microcirculation including the corresponding feeding artery was monitored by a LDPM system, PeriFlux PF 4001 Master, and PeriFlux PF 3 with an extended bandwidth up to 24 kHz for the artery measurements (Perimed AB, Ja¨rfa¨lla, Sweden). The laser Doppler probe holder for the skin measurements was fixed to the flap with double adhesive tape before the incision and a standard probe PF408 was attached to the probe holder. For the recording of artery blood perfusion, a needle probe (PF402) with a custom-made vascular fixation device (a metallic semi-tube into which the laser probe ended) was used to record the arterial blood flow. The vessel probe was firmly kept in place and immobilized by a micromanipulator. Artery and skin laser Doppler readings were performed simultaneously. The systemic blood pressure was monitored with a pressure transducer (Statham, Inc., Halo Rey, Puerto Rico) via a catheter introduced into the left common carotid artery. Both LDPM readings and blood pressure data were stored in a Toshiba T3200SXC desktop computer using a specially designed software (Perisoft, version V5.10; Perimed AB) for data processing.
Electrical Stimulation A bipolar stimulating electrode using platinum electrodes (electrode diameter 0.5 mm, electrode length 5
245
Microcirculatory Changes in the Rat Groin Flap
FIG. 1. Percentage changes of laser Doppler flow values as related to the baseline value (100%). The electrical stimulation was produced with 1, 6, 8, and 10 impulses. Using 1 impulse induced a small short-lasting perfusion decrease. Six, 8, and 10 impulses induced a perfusion decreased more pronounced in the flap than in the artery, but without any significant difference compared to baseline.
mm, electrode separation 1 mm) was connected to a Grass stimulator (Grass SD9, Quincy, MA). Stimulation was delivered to the artery from the Grass stimulator connected to a stimulation isolation unit and a constant current unit (Grass CCU1A) delivering impulses with 100-ms duration and 100 mA at 4 Hz.
Flap Arterial Diameter Measurements of flap arterial diameter were taken from the video image of the vessels using a video cursor (Sony SVO-9500MDP and Panasonic BT-H 1450). This device places a cursor over the image to be measured, and the operator adjusts the size of the cursor to exactly match the size of that image. The
video cursor was calibrated periodically using a micrometer. The lumen diameters of the arterial were monitored before electrical stimulation and at 2, 5, 15, 25, and 40 min after stimulation.
Histological Examination Flap artery specimens were removed from stimulated and unstimulated sections and perfused with 2.5% glutaraldehyde in a 0.1 M sodium phosphate buffer containing 2 mM MgCl 2. The specimens were postfixed in glutaraldehyde, osmicated (2% osmium tetroxide in phosphate buffer) and rinsed for 15 min in buffer, dehydrated in ethanol, and embedded in agar 100. Thin cross-sectional slices were cut on an LKB
Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
246
Qi et al.
FIG. 2. Original recording on-line from one rat showing laser Doppler flow in arbitrary units (perfusion units). Electrical stimulation of the feeding artery with 12 impulses. Stimulus (1) produced a rapid decrease of the LDF readings, both in the artery (top) and in the flap (middle). The systemic blood pressure is also shown (bottom). The X-axis (time) is compressed 64 times.
ultratome, stained with toluidine blue, and examined by light microscopy (Zeiss FM 109, Oberkochem, West Germany) [22].
Experimental Protocols Thirty minutes after flap elevation when the blood perfusion was stabilized a 10-min baseline recording was made, followed by stimulation of the superficial epigastric artery proximal to the LDF reading area. Stimulation impulses were 1, 6, 8, 10 (n ⫽ 6), and 12 (n ⫽ 10). The recording was commenced 30 min prior to stimulation and continued for 40 min afterward.
Statistical Analysis Laser Doppler values are given in percentage changes from baseline and expressed as the mean values ⫾ SE. The baseline recordings were set to 100%.
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For statistical analyses the Kruskal–Wallis test with Dunn’s multiple comparisons test was applied to compare percentage changes in blood flow and skin microcirculation at different points following stimulation. P ⬍ 0.05 was considered significant [28].
RESULTS Electrical Stimulation of the Feeding Artery Using 1, 6, 8, and 10 Impulses Electrical stimulation using a single impulse induced a brief perfusion decrease. Six, 8, and 10 impulses induced a decrease greater than the singlepulse-induced one, but it was not significantly different from the baseline. Artery and skin perfusion recovered within 10 min (Fig. 1).
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FIG. 3. Percentage change of laser Doppler flow values as related to the baseline value (100%). Stimulus was single mode, 12 impulses. In the artery, after electrical stimulation for 1, 2, 5, 10, and 15 min the blood flow was significantly reduced compared to baseline (**P ⬍ 0.01; ***P ⬍ 0.001). After 25 min the blood flow returned to baseline level. In the skin electrical stimulation for 1, 2, 5, 10, 15, and 20 min significantly decreased the microcirculation, but full recovery was delayed 5 min longer than in the artery (*P ⬍ 0.05; ***P ⬍ 0.001).
Electrical Stimulation Using 12 Impulses Stimulation of the epigastric artery with 12 impulses [S (1)] produced a rapid lowering of the LDPM readings, both in the artery and in the flap microcirculation. The decreases in arterial blood flow and flap microcirculation were fairly parallell, indicating that the stimulation induced a vasoconstriction producing ischemia (Fig. 2). In the artery, perfusion was reduced by 68.51% (mean ⫾ SE) (P ⬍ 0.001) 2 min after stimulation, but then recovered slowly recovered to the prestimulatory level within 25 min (mean ⫾ SE). Flap blood perfusion decreased about 59.96% (mean ⫾ SE) (P ⬍ 0.001) 5 min after stimulation, but the recovery was slower than in the artery and took about 30 min to reach the prestimulation level (mean ⫾ SE) (Fig. 3). During the recovery period of blood flow restoration two brief falls in perfusion rate were observed (Fig. 2,
arrows), probably indicating the formation of microthrombi in response to the stimulation of the vessel wall and the released vasoconstriction.
Videoscopic Examinations The magnitude of vasoconstriction was measured by the video camera system prior to and after the 12-impulse stimulation. The lumen diameter of the artery was significantly reduced, from 301.67 ⫾ 38.84 m (mean ⫾ SE) (before stimulation) to 51.04 ⫾ 5.03, 65.42 ⫾ 8.35, and 148.75 ⫾ 10.56 m, at 2, 5, and 15 min after stimulation, respectively. In parallell with this reduction of the lumen diameters of the arteries there was a decrease in blood perfusion (Fig. 4). The video camera facilitates direct observation of all arterial changes as they occur.
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FIG. 4. Lumen diameter changes in the stimulated arteries. Before electrical stimulation, the diameter (mean ⫾ SE) was 301.67 ⫾ 38.84 m (n ⫽ 6), and after electrical stimulation for 2, 5, and 15 min the lumen diameters were significantly reduced to 51.04 ⫾ 5.03 (***P ⬍ 0.001), 65.42 ⫾ 8.36 (**P ⬍ 0.01), and 148.75 ⫾ 10.56 m (*P ⬍ 0.05), respectively. In parallell with this reduction in the lumen diameters of arteries there was a decrease in the blood perfusion.
Blood Pressure There was no change in systemic blood pressure during electrical stimulation (Fig. 5) or the period of reduced blood flow in the artery and flap microcirculation.
Histological Examination Histological analysis using light microscopy of the arterial sections showed no significant architectural or endothelial changes in the stimulated and unstimulated sections (Fig. 6).
DISCUSSION In this study, a rat groin flap was used as a model to investigate blood flow changes both in the skin flap and in its feeding vessel following electrical stimulation of the vessel. Parallel reversible decreases of the
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LDPM reading of both the skin microcirculation and the flap artery were observed, following exposure to electrical stimulation. Extreme arterial vasoconstriction (vasospasm), a common problem encountered in microvascular surgery, can compromise flap integrity [29]. Agents which prevent the formation of free radicals or scavenge them have been suggested to minimize reperfusion injury. Repeatable, noninvasive measurement of artery and skin blood flow is needed to monitor this and other aspects of flap circulatory changes. Establishment of a reproducible vasospasm model is of cardinal importance for further exploration of this problem. Earlier models used pharmacological agents, particularly epinephrine, to induce vasoconstriction without successfully [17, 23] mimicking the clinical situation of mechanical vasospasm. Our studies on the rat groin flap suggest that electrical stimulation induced ischemia is a reproducible model for temporary ischemia without visible endothelial damage compared to the pinching technique [5, 9].
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FIG. 5. Percentage changes of systemic artery blood pressure compared to the baseline value (100%). No significant change occurred during the electrical stimulation.
The LDF measurements in our model reveal reduced flow (59 – 68%) after 12 impulses of electrical stimulation (Fig. 3) compared to the premanipulatory baseline flow in the artery and skin. There was a significant delay in the reperfusion of the capillary bed. This demonstrates that ischemia is the first component of flap injury, being responsible for a number of metabolic changes resulting in microcirculatory and tissue damage [11]. Lack of oxygen and eventual mechanical endothelial damage can produce an imbalance in the release of the endothelial-derived relaxing and constricting factors, impairing the flap microcirculation [4]. In this study, no endothelial damage at the stimulated site was evident, suggesting that factors released from local endothelial cells in the artery may be responsible for the impaired microcirculation distally in the flap. When a skin flap is rendered ischemic, hypoxia causes tissue depletion of ATP levels available for the Na ⫹–K ⫹ pump, resulting in cell swelling and accumulation of lactate and hypoxanthine. These resultant reactions can cause platelet aggregation and thrombosis in major arteries and microvessels, necrosis of en-
dothelium, and neutrophil-mediated cellular destruction and vasoconstriction, leading to flap necrosis [2, 4, 27]. Clinical research on microsurgical procedures in the past 10 years focuses mostly on microvascular patency mainly involving local thrombus formation and platelet aggregation [18, 19] and including the duration of ischemia as the dominating factor for the endothelial and microcirculation impairment [3, 4, 11, 13]. In our experiments with the 12-impulse stimulation of the feeding artery, short perfusion decreases were frequently observed (Fig. 2), and these were probably due to the formation of microthrombi, which occurs following vasoconstriction. This observation constitutes an important clue for future research. The mechanisms involved in the creation of microthrombi will be examined in a later study.
CONCLUSION This methodological study shows that the lowering in the LDPM readings in the microcirculation of an
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FIG. 6. Histological analysis by light microscopy. Sections collected from stimulated (a1, original magnification ⫻16; a2, original magnification ⫻40) and unstimulated (b1, original magnification ⫻16; b2, original magnification ⫻40) artery sections.
experimental flap correlates with the electrically induced vasospasm of the feeding artery. When the stimulation ceased, the feeding artery blood flow was more quickly reestablished than the flap microcirculation. This could indicate that a release of local endothelial and metabolic substances from the stimulated artery wall is responsible for the more long-lasting microcirculatory impairment. Finally, the study demonstrates that the rat groin flap is a feasible model for future investigations concerning ischemia–reperfusion injury phenomena.
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