Brian C. Cooley, Kevin L. Tadych, and John S. Gould
PERFUSION OF FREE FLAPS WITH HEPARINIZED WHOLE BLOOD DURING ISCHEMIC STORAGE Downloaded by: Universite Laval. Copyrighted material.
ABSTRACT This study was designed to investigate the use of heparinized whole blood as a perfusate for extending the tolerable ischemic interval of free flaps. Unilateral free groin flaps stored at 20° for 30 hr were used in four groups of 12 rats each. One group served as a control while a second involved systemic heparinization immediately prior to raising the flap for storage. The third and fourth groups underwent a brief perfusion (1030 min) with anticoagulated whole blood midway through the ischemic interval (15 hr); one of these groups involved temporary revascularization to the arterial system of a donor heparinized rat. The fourth group received hand-perfused anticoagulated (with heparin and citrate) whole blood administered through a cannula placed in the flap artery. After the 30-hr interval, flaps were replanted to the original groin site with standard microvascular technique. Percentages of the numbers of surviving flaps were: 8 percent for controls, 58 percent for both preheparinized and donor-perfused flaps, and 92 percent for the hand-perfused group. The authors conclude that anticoagulated blood within the flap vasculature during ischemic storage can improve the chance for viable reperfusion after replantation, and may offer a safeguard against the noreflow phenomenon after extended ischemia.
Replantation of amputated extremities has become a frequently practiced emergency trauma procedure in many world medical centers.1-2 However, not all patients are good candidates for primary repair, as acute health problems and/or related trauma from the incident may contraindicate exposure to the often lengthy anesthetic period associated with the attempt at replantation.3 There is a need for the development of a procedure that would permit extension of the interval between the initial amputation incident and the replantation effort, without an associated compromise to extremity survival or function.4 As an initial step in the development of such a procedure, this study has used the rat free groin flap. Cooley et al5 have defined the maximum period of ischemia for this free flap model at different storage temperatures. This study explores the use of anticoagulated whole blood as a perfusion solution for flaps stored at room temperature.
MATERIALS AND METHODS
Adult male Sprague-Dawley rats, weighing 200 to 300 gm were anesthetized with pentobarbital, 50 mg/ kg body weight. Each rat underwent dissection of a unilateral island groin flap ( 3 x 6 cm) based on the epigastric vessels. The femoral vessels were dissected free on both sides of the epigastric branchpoint. All vessels distal to the femoral-epigastric branchpoint were cauterized, leaving the common femoral circulation exclusively for the flap. The femoral vessels were individually ligated with 10-0 nylon midway between the inguinal ligament and epigastric branchpoint, then severed distally adjacent to the ligatures. The created free flap was placed in a saline-soaked gauze sponge within a sealed plastic bag, and stored at room temperature (20°C). The groin wound was closed with 4-0 49
Department of Orthopaedic Surgery, Medical College of Wisconsin, Milwaukee Materials in this paper were presented at the American Society for Reconstructive Microsurgery, Fourth Annual Meeting, September 17-19,1988, in Baltimore, Maryland Reprint requests: Mr. Cooley, Dept. of Orthopaedic Surgery, Medical College of Wisconsin, 8700 W. Wisconsin Ave., Milwaukee, Wl 53226 Accepted for publication October 6, 1989 Copyright © 1990 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
WVOLUl
RESULTS Only three rats died from the procedure, all from Group 1, within one day postoperatively. Another two animals from Group 1 and one from Group 4 had early signs of infection. All of these animals were excluded
30 hours
Grc u p l : Unh eparinized
\ Grc up 2: Heparinized
-
\ Grc up 3:
10 - 30 minutes
Donor Rat-Perfused
Grc up 4:
10 - 30 minutes
Syringe/Hand-Perfused
Flap into Storage
Flap Replanted
Pe rfusion Period
Figure 1. Thirty total hours of room-temperature ischemia per group: Group 1 (control); Group 2 received heparin, 120 U/kg body weight immediately prior to raising the flap and placing it in storage; Groups 3 and 4 were treated initially as Group 1, with a brief (10 to 30 min) period of removal from storage and perfusion midway through the ischemic episode, either with arterial anastomosis to a heparinized rat (Group 3) or by hand through a 6-cc syringe and 25-gauge blunt needle cannula, using heparinized, citrated blood. All flaps were replanted to their original animals after the 30-hr interval.
from the study, and replacements were used to balance the numbers. The percentage of the number of flaps per group with better than 50 percent viable tissue at seven days is plotted for each group in Figure 2. The control group had a 7-day survival rate of 8 percent, with only one flap remaining viable (Table 1). The systemically heparinized series (Group 2) had a 58 percent (7/12) success, as did the donor-perfused (Group 3) series. These success rates were significantly better than that of the control group (p < .05; Fisher exact test). The handperfused group (Group 4) had the highest success rate, 92 percent (11/12). This was significant at the p < 0.001 level, in comparison to the controls, but not significant in comparison to Groups 2 or 3 [p — 0.07). Of the "surviving" flaps, 11 displayed complete survival of the skin surface (100 percent), 11 had a 75 to 100 percent survival, and four had 50 to 75 percent survival. The remainder (n = 22), considered replantation failures, had less than 20 percent survival at seven days. Although three of the four flaps in the 50 to 75 percent range were from the hand-perfused group,
Table 1. Results of Free Flap Survival Following Different Methods of Heparinization
50
Croup
n
Preoperative Systemic Heparin
1 2 3 4
12 12 12 12
— 120 U/kg b.w. — —
Midischemia Perfusion
7-Day Flap Survival
10-30 min heparinized donor rat 10-30 min syringe/hand perfusion
1/12 7/12 7/12 11/12
(8%) (58%) (58%) (92%)
Downloaded by: Universite Laval. Copyrighted material.
nylon, and the animal resuscitated, with free access to food and water. After a 30-hr storage interval, anesthetization was reinduced and the flap wound reopened. The stored flap was returned to the site and placed in proximity to the ligated femoral vessels. After proximal clamping and removal of the ligatures, the femoral vessels were reconnected with end-to-end anastomoses using 10-0 nylon suture. Vessel patency was monitored for 20 min after clamp release, and the flap was sewn into its original bed. Survival was assessed through daily observation of skin color and feel, with an endpoint of seven days and at least 50 percent flap skin survival considered as a successful replantation. Flap specimens were harvested at the 7-day period for histologic evaluation. Four groups of 12 rats each were used (Table 1). Group 1 served as the control group, following the protocol without change. Group 2 served as a systemic heparin control, using a single bolus of intravenous heparin (120 U/kg body weight) 5 to 10 min prior to raising the flap. Groups 3 and 4 followed the protocol, except that at the midpoint of flap storage (after 15 hr of ischemia), each flap was removed from storage and given blood perfusion treatment for 10 to 30 min. Group 3 had an arterial anastomotic connection to the carotid artery of another rat which served as a heparinized blood donor. The anastomosed portion was resected after the perfusion period. Group 4 involved hand perfusion of the flap with citrated, heparinized whole blood through a cannula placed in the femoral artery of the flap, using a 25-gauge blunt-tipped needle with a 6 cc syringe. Unhindered venous efflux of perfused blood was allowed for both methods, with free flow occurring from the femoral vein of the flap. Flaps were returned to the same storage conditions for 15 additional hours after the perfusion procedure, then replanted in the same manner as the first two groups. Thus, the total ischemic time was 30 hr (Fig. 1).
6, NUMBER 1 JANUARY 1990
PERFUSION OF ISCHEMIC FLAPS/COOLER TADYCH, GOULD
% Flaps Surviving 7 Days
n
50
Control Unhcparinized
He Perfused
Perfused
Figure 2. Percentage of surviving flaps per group. Statistical comparisons with the control group (Group 1):* p < 0.05; ** p < 0.001.
there was no clear correlation of surviving flap area with the technique of flap treatment. The histologic appearance of the 7-day nonischemically-stored flap shows a normal arrangement of healthy dermis, fat cells, panniculus, and pilatory muscle tissue, with localized lymphoid cells, glands, and ducts within fibrofatty tissue (Fig. 3). A mild inflammatory response is found, with histiocytic and lymphocytic invasion in the subcutaneous space, and the creation of fibroblastic fibrous tissue elements. The necrotic regions of 7-day flaps were clearly nonviable and, in many cases, had undergone partial or complete autocannibalism. Only the viable portions were evaluated histologically.
The experimental surviving flaps from all groups displayed predominantly healthy tissue layers, with some evidence of mild muscle and glandular atrophy and commencement of recovery (Figs. 4 and 5). Mild inflammation and cellular invasion were similar to nonischemic control flaps. The appearance of the hand-perfused flap histology was not considerably altered. However, evidence of localized vascular breakdown was seen, with residual extravasated elements. Since the hand-perfusion technique was not designed for regulation of the perfusion pressure, the intraarterial pressure generated with this procedure may have been excessive, resulting in vascular disruption, apparently in evidence histologically. In a separate investigation, it was found through manometric recordings that this hand-perfusion method resulted in pressures in excess of 300 mmHg.
DISCUSSION The no-reflow phenomenon was initially described for its occurrence after cerebral ischemia.6 The concept was readily applied to other organ/tissue systems that also experienced this inability to perfuse after ischemia. 78 Although tissues such as the rat groin flap, with substantial cutaneous elements, are able to withstand much longer ischemic intervals, 59 " 12 the no-
.
J »
* ' ,
•
.
..-•
Figure 3. Cross-section through normal rat groin skin, showing healthy structures in all layers (m = muscle) (H&E, X20).
Downloaded by: Universite Laval. Copyrighted material.
100
,
.
-
•
"
•
-
•
Figure 4. Cross-section through a 7-day surviving flap from Group 2, demonstrating general integrity and viability, with clear evidence of muscle atrophy in the panniculus carnosus (m = muscle) (H&E, x20).
51
Figure 5. Higher magnification of inner region of a 7-day postoperative flap from Group 3, showing atrophied muscle (m) nearapairof small vessels (a = artery, v = vein) (H&E, X120).
52
reflow phenomenon is believed to apply to these tissues as well. The exact course of the development of detrimental irreversible changes associated with this process is still unknown. Most theories focus on a concept of endothelial disruption caused by lack of oxygen replenishment to ongoing cellular activities; shutdown of the active sodium pump from the metabolic deficit leads to osmotic imbalances, resulting in cellular swelling. 7 '' 3 In the extreme, this swelling causes vascular cell rupture, with resultant coagulation on exposed subendothelial surfaces. Additionally, the cellular swelling may include red blood cells, resulting in the clogging of capillaries. Although the association of critical ischemia to no-reflow has been established, 5 9 1 0 1 4 ischemia may not be a prerequisite for the no-reflow occurrence. Clinical transfers of free flaps have met with a significant percentage of failures, generally 5 to 15 percent, even when the ischemic interval is minimal.' 5~17 These incidences of failure create a perplexing issue in the reconstructive field. Experimental studies for investigating techniques to extend the safe ischemic time have focused on the use of perfusion solutions for replacing intravascular blood elements. Following the research findings in kidney transplantation, investigators have attempted
JANUARY 1990
to wash the blood components from the flap, both before and after the ischemic interval, but with little success. 1 0 1 8 1 9 By adding such agents as thrombolytics20"22 or oxygen free-radical scavengers,23 modest improvements have been reported. Following these research findings, the clinical use of thrombolytics has been attempted in a few cases, with impressive results. 1524 Recent studies by Rosen and colleagues 25 " 27 have indicated that by mimicking the salt and small molecule composition of plasma in a perfusate, experimental free flaps can withstand longer ischemic intervals after blood washout, with assessment based on survival following revascularization. Our results are complementary to those reported by Rosen et al, and suggest that prevention of coagulation while maintaining hematologic ionic concentration is more important than replenishment of oxygen and other metabolic factors in moderately extended ischemia. The simple use of heparinization prior to flap isolation, resulting in significant improvement in flap survival, strongly supports this anticoagulative influence. The hand-perfused flaps resulted in an increase in survival over the donor-perfused flaps, although this did not reach a significant level. The two apparent differences offered by this group were a higher perfusion pressure and the presence of citrate in the perfusate. If the former was a responsible factor, it was clearly at the cost of microvascular compromise from an associated perfusion injury, as seen on histologic examination. Such damage may be even more severe in flaps with more delicate tissue components (e.g., nerve) and, as such, would not be a clinically beneficial technique. Other than as an additional anticoagulant, the influence of citrate on flap survival can only be conjectural at this time. It is interesting that the short duration of the flap perfusion procedure (10 to 30 min) allowed for a functionally complete effect in Groups 3 and 4. This brief reperfusion stimulus may function by replacing the stagnant, metabolically depleted blood in the flap, or by simply moving the intravascular blood components enough to allow a diffusional exchange of metabolites to the endothelial surface. Clearly the results in Group 2 indicate the benefit of heparinization, a presumed anticoagulant effect, on viability after extended ischemia. The success in Groups 3 and 4 may be due solely to the exchange of anticoagulated blood, preventing thrombotic events at late ischemia times. The findings of this study suggest that an initial perfusion with an anticoagulant-containing blood solution may lead to a higher incidence of successful flap transfer. Additionally, with lengthy ischemic-storage involvement, as with extremity amputations, periodic perfusion with oxygenated, anticoagulated blood may enhance the probability for a favorable outcome in replantation.
Downloaded by: Universite Laval. Copyrighted material.
JOURNAL OF RECONSTRUCTIVE MICROSURGERY/VOLUME 6, NUMBER 1
PERFUSION OF ISCHEMIC FLAPS/COOLEY, TADYCH, GOULD
1. Urbaniak )R: Digit and hand replantation: Current status. Neurosurg 4:551, 1979 2. Tamai S: Twenty years' experience of limb replantation— Review of 293 upper extremity replants. I Hand Surg 7:549, 1982 3. Kaufman HH, Huchton ID, Patten BM, et ai: Limb preservation for reimplantation—A review. I Microsurg 2:36, 1980 4. Godina M, Bajec I, Baraga A: Salvage of the mutilated upper extremity with temporary ectopic implantation of the undamaged part. Plast Reconstr Surg 78:295, 1986 5. Cooley BC, Hansen FC, Dellon AL: The effect of temperature on tolerance to ischemia in experimental free flaps. J Microsurg 3:11, 1981 6. Ames A III, Wright RL, Kowada M, et al.\ Cerebral ischemia—II. The no-reflow phenomenon. Am | Path 52:437, 1968 7. Summers WK, lamison RL: The no-reflow phenomenon in renal ischemia. Lab Invest 25:635, 1971 8. Kloner RA, Ganote CE, lennings RB: The no-reflow phenomenon after temporary coronary occlusion in the dog. I Clin Invest 54:1496, 1974 9. May IW, Chait LA, O'Brien BM, Hurley IV: The no-reflow phenomenon in experimental free flaps. Plast Reconstr Surg 61:256, 1978 10. Chait LA, May JW, O'Brien BM, Hurley IV: The effects of the perfusion of various solutions on the no-reflow phenomenon in experimental free flaps. Plast Reconstr Surg 61:421, 1978 11. Van Giesen P|, Seaber AV, Urbaniak JR: Storage of amputated parts prior to replantation—An experimental study with rabbit ears. ) Hand Surg 8:60, 1983 12. Weinberg H, Song Y, Silverman DG, et ai: Vascular island skinflap tolerance to warm ischemia: An analysis by perfusion fluorometry. Plast Reconstr Surg 73:949, 1984 13. Leaf A: Cell swelling: A factor in ischemic tissue injury. Circulation 48:458, 1973 14 Zdeblick TA, Shaffer JW, Field GA: An ischemia-induced model of revascularization failure of replanted limbs.) Hand Surg
15. 16. 17. 18. 19 20. 21. 22. 23.
24.
25. 26. 27.
1OA:125, 1985 Lipton HA, Jupiter )B: Streptokinase salvage of a free-tissue transfer: Case report and review of the literature. Plast Reconstr Surg 79:977, 1987 Russell RC, O'Brien BM, Morrison WA, et ai: The late functional results of upper limb revascularization and replantation. J Hand Surg 9A:623, 1984 Daniel RK, WeilandA): Free tissue transfers for upper extremity reconstruction. ) Hand Surg 7A:66, 1982 Harashina T, Buncke HI: Study of washout solutions for microvascular replantation and transplantation. Plast Reconstr Surg 56:542, 1975 Gould |S, Sully L, O'Brien BM, et ai: The effects of combined cooling and perfusion on experimental free-flap survival in rabbits. Plast Reconstr Surg 76:104, 1985 Cooley BC, Morgan RF, Dellon AL: Thrombolytic reversal of noreflow phenomenon in rat free flap model. Surg Forum 34: 638, 1983 Zdeblick TA, Shaffer )W, Field GA: The use of urokinase in ischemic replanted extremities in rats. I Bone Joint Surg 69A:442, 1987 Puckett CL, Misholy H, Reinisch )F: The effects of streptokinase on ischemic flaps. I Hand Surg 8:101, 1983 Angel MF, Narayanan K, Swartz WM, et ai: Deferoxamine increases skin flap survival: Additional evidence of free radical involvement in ischaemic flap surgery. Brit J Plast Surg 39:469, 1986 Schubert W, Hunter DW, Guzman-Stein G, et ai: Use of streptokinase for the salvage of a free flap: Case report and review of the use of thrombolytic therapy. Microsurgery 8: 117, 1987 Rosen HM, Slivjak MJ, McBreartry FX: Preischemic flap washout and its effect on the no-reflow phenomenon. Plast Reconstr Surg 76:737, 1985 Rosen HM, Slivjak MJ, McBreartry FX: Delayed microcirculatory hyperpermeability following perfusion washout. Plast Reconstr Surg 79:102, 1987 Rosen HM, Slivjak MJ, McBrearty FX: The role of perfusion washout in limb revascularization procedures. Plast Reconstr Surg 80:595, 1987
Downloaded by: Universite Laval. Copyrighted material.
REFERENCES
53
PERFUSION OF FREE FLAPS WITH HEPARINIZED WHOLE BLOOD DURING ISCHEMIC STORAGE Downloaded by: Universite Laval. Copyrighted material.
ABSTRACT This study was designed to investigate the use of heparinized whole blood as a perfusate for extending the tolerable ischemic interval of free flaps. Unilateral free groin flaps stored at 20° for 30 hr were used in four groups of 12 rats each. One group served as a control while a second involved systemic heparinization immediately prior to raising the flap for storage. The third and fourth groups underwent a brief perfusion (1030 min) with anticoagulated whole blood midway through the ischemic interval (15 hr); one of these groups involved temporary revascularization to the arterial system of a donor heparinized rat. The fourth group received hand-perfused anticoagulated (with heparin and citrate) whole blood administered through a cannula placed in the flap artery. After the 30-hr interval, flaps were replanted to the original groin site with standard microvascular technique. Percentages of the numbers of surviving flaps were: 8 percent for controls, 58 percent for both preheparinized and donor-perfused flaps, and 92 percent for the hand-perfused group. The authors conclude that anticoagulated blood within the flap vasculature during ischemic storage can improve the chance for viable reperfusion after replantation, and may offer a safeguard against the noreflow phenomenon after extended ischemia.
Replantation of amputated extremities has become a frequently practiced emergency trauma procedure in many world medical centers.1-2 However, not all patients are good candidates for primary repair, as acute health problems and/or related trauma from the incident may contraindicate exposure to the often lengthy anesthetic period associated with the attempt at replantation.3 There is a need for the development of a procedure that would permit extension of the interval between the initial amputation incident and the replantation effort, without an associated compromise to extremity survival or function.4 As an initial step in the development of such a procedure, this study has used the rat free groin flap. Cooley et al5 have defined the maximum period of ischemia for this free flap model at different storage temperatures. This study explores the use of anticoagulated whole blood as a perfusion solution for flaps stored at room temperature.
MATERIALS AND METHODS
Adult male Sprague-Dawley rats, weighing 200 to 300 gm were anesthetized with pentobarbital, 50 mg/ kg body weight. Each rat underwent dissection of a unilateral island groin flap ( 3 x 6 cm) based on the epigastric vessels. The femoral vessels were dissected free on both sides of the epigastric branchpoint. All vessels distal to the femoral-epigastric branchpoint were cauterized, leaving the common femoral circulation exclusively for the flap. The femoral vessels were individually ligated with 10-0 nylon midway between the inguinal ligament and epigastric branchpoint, then severed distally adjacent to the ligatures. The created free flap was placed in a saline-soaked gauze sponge within a sealed plastic bag, and stored at room temperature (20°C). The groin wound was closed with 4-0 49
Department of Orthopaedic Surgery, Medical College of Wisconsin, Milwaukee Materials in this paper were presented at the American Society for Reconstructive Microsurgery, Fourth Annual Meeting, September 17-19,1988, in Baltimore, Maryland Reprint requests: Mr. Cooley, Dept. of Orthopaedic Surgery, Medical College of Wisconsin, 8700 W. Wisconsin Ave., Milwaukee, Wl 53226 Accepted for publication October 6, 1989 Copyright © 1990 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
WVOLUl
RESULTS Only three rats died from the procedure, all from Group 1, within one day postoperatively. Another two animals from Group 1 and one from Group 4 had early signs of infection. All of these animals were excluded
30 hours
Grc u p l : Unh eparinized
\ Grc up 2: Heparinized
-
\ Grc up 3:
10 - 30 minutes
Donor Rat-Perfused
Grc up 4:
10 - 30 minutes
Syringe/Hand-Perfused
Flap into Storage
Flap Replanted
Pe rfusion Period
Figure 1. Thirty total hours of room-temperature ischemia per group: Group 1 (control); Group 2 received heparin, 120 U/kg body weight immediately prior to raising the flap and placing it in storage; Groups 3 and 4 were treated initially as Group 1, with a brief (10 to 30 min) period of removal from storage and perfusion midway through the ischemic episode, either with arterial anastomosis to a heparinized rat (Group 3) or by hand through a 6-cc syringe and 25-gauge blunt needle cannula, using heparinized, citrated blood. All flaps were replanted to their original animals after the 30-hr interval.
from the study, and replacements were used to balance the numbers. The percentage of the number of flaps per group with better than 50 percent viable tissue at seven days is plotted for each group in Figure 2. The control group had a 7-day survival rate of 8 percent, with only one flap remaining viable (Table 1). The systemically heparinized series (Group 2) had a 58 percent (7/12) success, as did the donor-perfused (Group 3) series. These success rates were significantly better than that of the control group (p < .05; Fisher exact test). The handperfused group (Group 4) had the highest success rate, 92 percent (11/12). This was significant at the p < 0.001 level, in comparison to the controls, but not significant in comparison to Groups 2 or 3 [p — 0.07). Of the "surviving" flaps, 11 displayed complete survival of the skin surface (100 percent), 11 had a 75 to 100 percent survival, and four had 50 to 75 percent survival. The remainder (n = 22), considered replantation failures, had less than 20 percent survival at seven days. Although three of the four flaps in the 50 to 75 percent range were from the hand-perfused group,
Table 1. Results of Free Flap Survival Following Different Methods of Heparinization
50
Croup
n
Preoperative Systemic Heparin
1 2 3 4
12 12 12 12
— 120 U/kg b.w. — —
Midischemia Perfusion
7-Day Flap Survival
10-30 min heparinized donor rat 10-30 min syringe/hand perfusion
1/12 7/12 7/12 11/12
(8%) (58%) (58%) (92%)
Downloaded by: Universite Laval. Copyrighted material.
nylon, and the animal resuscitated, with free access to food and water. After a 30-hr storage interval, anesthetization was reinduced and the flap wound reopened. The stored flap was returned to the site and placed in proximity to the ligated femoral vessels. After proximal clamping and removal of the ligatures, the femoral vessels were reconnected with end-to-end anastomoses using 10-0 nylon suture. Vessel patency was monitored for 20 min after clamp release, and the flap was sewn into its original bed. Survival was assessed through daily observation of skin color and feel, with an endpoint of seven days and at least 50 percent flap skin survival considered as a successful replantation. Flap specimens were harvested at the 7-day period for histologic evaluation. Four groups of 12 rats each were used (Table 1). Group 1 served as the control group, following the protocol without change. Group 2 served as a systemic heparin control, using a single bolus of intravenous heparin (120 U/kg body weight) 5 to 10 min prior to raising the flap. Groups 3 and 4 followed the protocol, except that at the midpoint of flap storage (after 15 hr of ischemia), each flap was removed from storage and given blood perfusion treatment for 10 to 30 min. Group 3 had an arterial anastomotic connection to the carotid artery of another rat which served as a heparinized blood donor. The anastomosed portion was resected after the perfusion period. Group 4 involved hand perfusion of the flap with citrated, heparinized whole blood through a cannula placed in the femoral artery of the flap, using a 25-gauge blunt-tipped needle with a 6 cc syringe. Unhindered venous efflux of perfused blood was allowed for both methods, with free flow occurring from the femoral vein of the flap. Flaps were returned to the same storage conditions for 15 additional hours after the perfusion procedure, then replanted in the same manner as the first two groups. Thus, the total ischemic time was 30 hr (Fig. 1).
6, NUMBER 1 JANUARY 1990
PERFUSION OF ISCHEMIC FLAPS/COOLER TADYCH, GOULD
% Flaps Surviving 7 Days
n
50
Control Unhcparinized
He Perfused
Perfused
Figure 2. Percentage of surviving flaps per group. Statistical comparisons with the control group (Group 1):* p < 0.05; ** p < 0.001.
there was no clear correlation of surviving flap area with the technique of flap treatment. The histologic appearance of the 7-day nonischemically-stored flap shows a normal arrangement of healthy dermis, fat cells, panniculus, and pilatory muscle tissue, with localized lymphoid cells, glands, and ducts within fibrofatty tissue (Fig. 3). A mild inflammatory response is found, with histiocytic and lymphocytic invasion in the subcutaneous space, and the creation of fibroblastic fibrous tissue elements. The necrotic regions of 7-day flaps were clearly nonviable and, in many cases, had undergone partial or complete autocannibalism. Only the viable portions were evaluated histologically.
The experimental surviving flaps from all groups displayed predominantly healthy tissue layers, with some evidence of mild muscle and glandular atrophy and commencement of recovery (Figs. 4 and 5). Mild inflammation and cellular invasion were similar to nonischemic control flaps. The appearance of the hand-perfused flap histology was not considerably altered. However, evidence of localized vascular breakdown was seen, with residual extravasated elements. Since the hand-perfusion technique was not designed for regulation of the perfusion pressure, the intraarterial pressure generated with this procedure may have been excessive, resulting in vascular disruption, apparently in evidence histologically. In a separate investigation, it was found through manometric recordings that this hand-perfusion method resulted in pressures in excess of 300 mmHg.
DISCUSSION The no-reflow phenomenon was initially described for its occurrence after cerebral ischemia.6 The concept was readily applied to other organ/tissue systems that also experienced this inability to perfuse after ischemia. 78 Although tissues such as the rat groin flap, with substantial cutaneous elements, are able to withstand much longer ischemic intervals, 59 " 12 the no-
.
J »
* ' ,
•
.
..-•
Figure 3. Cross-section through normal rat groin skin, showing healthy structures in all layers (m = muscle) (H&E, X20).
Downloaded by: Universite Laval. Copyrighted material.
100
,
.
-
•
"
•
-
•
Figure 4. Cross-section through a 7-day surviving flap from Group 2, demonstrating general integrity and viability, with clear evidence of muscle atrophy in the panniculus carnosus (m = muscle) (H&E, x20).
51
Figure 5. Higher magnification of inner region of a 7-day postoperative flap from Group 3, showing atrophied muscle (m) nearapairof small vessels (a = artery, v = vein) (H&E, X120).
52
reflow phenomenon is believed to apply to these tissues as well. The exact course of the development of detrimental irreversible changes associated with this process is still unknown. Most theories focus on a concept of endothelial disruption caused by lack of oxygen replenishment to ongoing cellular activities; shutdown of the active sodium pump from the metabolic deficit leads to osmotic imbalances, resulting in cellular swelling. 7 '' 3 In the extreme, this swelling causes vascular cell rupture, with resultant coagulation on exposed subendothelial surfaces. Additionally, the cellular swelling may include red blood cells, resulting in the clogging of capillaries. Although the association of critical ischemia to no-reflow has been established, 5 9 1 0 1 4 ischemia may not be a prerequisite for the no-reflow occurrence. Clinical transfers of free flaps have met with a significant percentage of failures, generally 5 to 15 percent, even when the ischemic interval is minimal.' 5~17 These incidences of failure create a perplexing issue in the reconstructive field. Experimental studies for investigating techniques to extend the safe ischemic time have focused on the use of perfusion solutions for replacing intravascular blood elements. Following the research findings in kidney transplantation, investigators have attempted
JANUARY 1990
to wash the blood components from the flap, both before and after the ischemic interval, but with little success. 1 0 1 8 1 9 By adding such agents as thrombolytics20"22 or oxygen free-radical scavengers,23 modest improvements have been reported. Following these research findings, the clinical use of thrombolytics has been attempted in a few cases, with impressive results. 1524 Recent studies by Rosen and colleagues 25 " 27 have indicated that by mimicking the salt and small molecule composition of plasma in a perfusate, experimental free flaps can withstand longer ischemic intervals after blood washout, with assessment based on survival following revascularization. Our results are complementary to those reported by Rosen et al, and suggest that prevention of coagulation while maintaining hematologic ionic concentration is more important than replenishment of oxygen and other metabolic factors in moderately extended ischemia. The simple use of heparinization prior to flap isolation, resulting in significant improvement in flap survival, strongly supports this anticoagulative influence. The hand-perfused flaps resulted in an increase in survival over the donor-perfused flaps, although this did not reach a significant level. The two apparent differences offered by this group were a higher perfusion pressure and the presence of citrate in the perfusate. If the former was a responsible factor, it was clearly at the cost of microvascular compromise from an associated perfusion injury, as seen on histologic examination. Such damage may be even more severe in flaps with more delicate tissue components (e.g., nerve) and, as such, would not be a clinically beneficial technique. Other than as an additional anticoagulant, the influence of citrate on flap survival can only be conjectural at this time. It is interesting that the short duration of the flap perfusion procedure (10 to 30 min) allowed for a functionally complete effect in Groups 3 and 4. This brief reperfusion stimulus may function by replacing the stagnant, metabolically depleted blood in the flap, or by simply moving the intravascular blood components enough to allow a diffusional exchange of metabolites to the endothelial surface. Clearly the results in Group 2 indicate the benefit of heparinization, a presumed anticoagulant effect, on viability after extended ischemia. The success in Groups 3 and 4 may be due solely to the exchange of anticoagulated blood, preventing thrombotic events at late ischemia times. The findings of this study suggest that an initial perfusion with an anticoagulant-containing blood solution may lead to a higher incidence of successful flap transfer. Additionally, with lengthy ischemic-storage involvement, as with extremity amputations, periodic perfusion with oxygenated, anticoagulated blood may enhance the probability for a favorable outcome in replantation.
Downloaded by: Universite Laval. Copyrighted material.
JOURNAL OF RECONSTRUCTIVE MICROSURGERY/VOLUME 6, NUMBER 1
PERFUSION OF ISCHEMIC FLAPS/COOLEY, TADYCH, GOULD
1. Urbaniak )R: Digit and hand replantation: Current status. Neurosurg 4:551, 1979 2. Tamai S: Twenty years' experience of limb replantation— Review of 293 upper extremity replants. I Hand Surg 7:549, 1982 3. Kaufman HH, Huchton ID, Patten BM, et ai: Limb preservation for reimplantation—A review. I Microsurg 2:36, 1980 4. Godina M, Bajec I, Baraga A: Salvage of the mutilated upper extremity with temporary ectopic implantation of the undamaged part. Plast Reconstr Surg 78:295, 1986 5. Cooley BC, Hansen FC, Dellon AL: The effect of temperature on tolerance to ischemia in experimental free flaps. J Microsurg 3:11, 1981 6. Ames A III, Wright RL, Kowada M, et al.\ Cerebral ischemia—II. The no-reflow phenomenon. Am | Path 52:437, 1968 7. Summers WK, lamison RL: The no-reflow phenomenon in renal ischemia. Lab Invest 25:635, 1971 8. Kloner RA, Ganote CE, lennings RB: The no-reflow phenomenon after temporary coronary occlusion in the dog. I Clin Invest 54:1496, 1974 9. May IW, Chait LA, O'Brien BM, Hurley IV: The no-reflow phenomenon in experimental free flaps. Plast Reconstr Surg 61:256, 1978 10. Chait LA, May JW, O'Brien BM, Hurley IV: The effects of the perfusion of various solutions on the no-reflow phenomenon in experimental free flaps. Plast Reconstr Surg 61:421, 1978 11. Van Giesen P|, Seaber AV, Urbaniak JR: Storage of amputated parts prior to replantation—An experimental study with rabbit ears. ) Hand Surg 8:60, 1983 12. Weinberg H, Song Y, Silverman DG, et ai: Vascular island skinflap tolerance to warm ischemia: An analysis by perfusion fluorometry. Plast Reconstr Surg 73:949, 1984 13. Leaf A: Cell swelling: A factor in ischemic tissue injury. Circulation 48:458, 1973 14 Zdeblick TA, Shaffer JW, Field GA: An ischemia-induced model of revascularization failure of replanted limbs.) Hand Surg
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1OA:125, 1985 Lipton HA, Jupiter )B: Streptokinase salvage of a free-tissue transfer: Case report and review of the literature. Plast Reconstr Surg 79:977, 1987 Russell RC, O'Brien BM, Morrison WA, et ai: The late functional results of upper limb revascularization and replantation. J Hand Surg 9A:623, 1984 Daniel RK, WeilandA): Free tissue transfers for upper extremity reconstruction. ) Hand Surg 7A:66, 1982 Harashina T, Buncke HI: Study of washout solutions for microvascular replantation and transplantation. Plast Reconstr Surg 56:542, 1975 Gould |S, Sully L, O'Brien BM, et ai: The effects of combined cooling and perfusion on experimental free-flap survival in rabbits. Plast Reconstr Surg 76:104, 1985 Cooley BC, Morgan RF, Dellon AL: Thrombolytic reversal of noreflow phenomenon in rat free flap model. Surg Forum 34: 638, 1983 Zdeblick TA, Shaffer )W, Field GA: The use of urokinase in ischemic replanted extremities in rats. I Bone Joint Surg 69A:442, 1987 Puckett CL, Misholy H, Reinisch )F: The effects of streptokinase on ischemic flaps. I Hand Surg 8:101, 1983 Angel MF, Narayanan K, Swartz WM, et ai: Deferoxamine increases skin flap survival: Additional evidence of free radical involvement in ischaemic flap surgery. Brit J Plast Surg 39:469, 1986 Schubert W, Hunter DW, Guzman-Stein G, et ai: Use of streptokinase for the salvage of a free flap: Case report and review of the use of thrombolytic therapy. Microsurgery 8: 117, 1987 Rosen HM, Slivjak MJ, McBreartry FX: Preischemic flap washout and its effect on the no-reflow phenomenon. Plast Reconstr Surg 76:737, 1985 Rosen HM, Slivjak MJ, McBreartry FX: Delayed microcirculatory hyperpermeability following perfusion washout. Plast Reconstr Surg 79:102, 1987 Rosen HM, Slivjak MJ, McBrearty FX: The role of perfusion washout in limb revascularization procedures. Plast Reconstr Surg 80:595, 1987
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