Brief ex vivo perfusion with heparinized and/or citrated whole blood enhances tolerance of free muscle flaps to prolonged ischemia

This study investigated the use of heparinized and/or citrated whole blood as a perfusate for enhancing muscle tolerance to warm ischemia. Unilateral cutaneous trunci muscle flaps were harvested from Sprague-Dawley rats and stored for 10 hr at 22-24 degrees C prior to transplantation to the groin. One group served as a non-perfused control. In three experimental groups, the flaps were hand-perfused ex vivo with 1.0 ml of heparinized, citrated, or heparinized and citrated autogenous whole blood at physiological pressures. Perfusion was administered over a 10-min period 5 hr into the ischemic period. Flaps were revascularized on the femoral vessels and then harvested 48 hr following revascularization. Tissue injury was assessed by calculation of flap weight change (indicator of tissue edema), histochemical evaluation of muscle dehydrogenase activity (nitroblue tetrazolium assay), and light microscopy. All perfused groups had significantly higher muscle dehydrogenase activity compared with non-perfused controls (P < 0.005). Perfusion with combined heparin-citrated blood was significantly more protective than perfusion with either anticoagulant alone (P < 0.025). The only statistically significant reduction in percent flap edema was seen in the combined heparin-citrate perfusion of flaps compared with nonperfused controls (P < 0.05). Histologic evaluation confirmed a reduction in tissue edema in the perfused flaps. We conclude that mid-ischemic perfusion with heparinized and/or citrated blood limits the deleterious effects of extended warm ischemia.     

The beneficial effect of heparin in preischemic perfusion solutions for cold-stored skin flaps

Storage of skin flaps in the cold before replantation increases their tolerance to ischemic damage. Rat epigastric skin flaps were perfused immediately before 2 days of cold ischemia with 3 ml of normal saline containing either 10 U per milliliter of heparin (group 1, N = 11) or normal saline (group 2, N = 10), or stored without perfusion (group 3, N = 6), and replanted. Flap viability was assessed 7 days later. The mean flap survival in groups 1, 2, and 3 was 73% (p<0.01 compared with group 2), 10%, and 33% respectively. Intravascular fibrin deposits were detected histochemically 5 minutes before reperfusion in nonperfused flaps and 5 minutes after reperfusion in saline-perfused flaps, but not in flaps perfused with heparinized saline. Angiography revealed evidence of no reflow in the first 5 minutes of reperfusion in saline-perfused flaps, but normal blood flow in heparinized saline-perfused flaps. Tissue water content, myeloperoxidase activity, and hydroperoxide levels after 1 and 24 hours of reperfusion were not significantly different in flaps perfused with heparinized saline and normal saline. These findings indicate that in skin flaps perfused before ischemia, flaps perfused with heparinized saline survive significantly better than flaps perfused with normal saline. They also survive better than nonperfused flaps but the improvement is not significant.     

Heparin decreases ischemia-reperfusion injury in isolated canine gracilis model

The mechanisms of ischemia-reperfusion injury in skeletal muscle remain controversial. Some investigators have demonstrated that heparin can ameliorate ischemic injury to heart, brain, and renal tissue. We investigated the ability of heparin sodium to decrease ischemia-reperfusion injury in an isolated gracilis muscle model in ten anesthetized mongrel dogs. One gracilis muscle was perfused normally while the contralateral muscle was subjected to six hours of ischemia followed by one hour of reperfusion. Five dogs were given a preischemic bolus of heparin sodium (200 U/kg, intravenously followed by a continuous infusion (15 U/kg/h, intravenously), and five control dogs received no heparin. Quantitation of skeletal muscle ischemia-reperfusion injury was determined by histochemical staining with triphenyl tetrazolium-chloride and computerized planimetry of the infarct size. Results from the ischemic muscle demonstrate a significant beneficial effect of heparinization. The nonheparinized dogs had a 72% +/- 5% infarct size, which was significantly reduced to 24% +/- 8% in the heparinized dogs. The mechanism of this protective effect may be due to heparin's anticoagulant, antiplatelet, or anti-inflammatory action.     

Perfusion of free flaps with heparinized whole blood during ischemic storage

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 degrees 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 (10-30 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 no-reflow phenomenon after extended ischemia.     

The influence of skin flap ischemia on serum nitric oxide concentrations

Nitric oxide (NO), identified as a mediator of endothelium-dependent relaxation of vascular smooth muscle, is known to cause a number of inflammatory diseases, especially ischemia-reperfusion injury. This experimental study, using a rabbit epigastric island flap, was designed to investigate whether skin flap ischemia followed by reperfusion influences serum NO concentrations. In addition, the author investigated the effects of NO synthase inhibitors and heparin on skin flap ischemia. Serum NO concentrations after 15, 30, 45, and 60 minutes of ischemia followed by reperfusion were significantly increased compared with non-ischemic controls and elevated flaps. On the other hand, serum NO concentrations were suppressed in nitro-amino-methyl-L-arginine- and aminoguanidine-treated animals. Furthermore, administration of heparin increased serum NO concentrations in controls and animals with elevated flaps, but decreased serum NO concentrations in ischemic flaps with subsequent reperfusion. These results suggest that NO is one of the factors responsible for ischemia-reperfusion injury and that NO synthase inhibitors and heparin may protect against such injury. © 1997 Wiley-Liss, Inc. MICROSURGERY 17:191–197 1996     

Thromboxane A2 release in ischemia and reperfusion of free flaps in rats, studied by microdialysis

Several studies have implicated enhanced eicosanoid production in reperfusion injury. The reported study investigated the use of microdialysis in the in vivo measurement of thromboxane levels during reperfusion in ischemic and reperfused experimental free muscle flaps. Microdialysis probes were inserted, via a guide, into the gracilis and semitendinosus free flap in the rat. The probe was perfused at a flow of 5 microl/min, with samples collected at intervals of 20 min, and analyzed by the ELISA technique. Animals were randomly distributed into three groups. After ischemic periods of 2, 4, and 6 hr, respectively, the free muscle flaps were revascularized on the contralateral femoral vessels. The mean thromboxane level during ischemia was 1785.34 +/- 124.81 pg/ml. The mean levels of thromboxane rose significantly (p < 0.05), compared to base level, with 151.65 percent in the 2-hr ischemia group, 192.33 percent in the 4-hr ischemia group, and 294.69 percent in the 6-hr ischemia group, and correlated well with histologic observations. The results suggest that a microdialysis technique, combined with a sensitive assay for measuring thromboxane, is a useful method for in vivo monitoring of inflammatory processes during ischemia and reperfusion. The evolution of thromboxane release following 6 hr of ischemia indicates that this mediator may be involved in facilitation of cell death, following ischemia and reperfusion, since its tissue level correlates with histologic tissue damage.     

Circulatory changes after prolonged ischemia in the epigastric flap

The circulation system seems to have early encounters with pathophysiologic processes during ischemia and reperfusion, such as overproduction of oxygen radicals, nitric oxide depletion, and leukocyte plugging. The aim of this study was to determine the superficial perfusion and vessel distribution of the epigastric flap with a laser Doppler Imaging (LDI) system during ischemia/reperfusion, and to observe the clinical outcomes 7 days after reperfusion in a separate set of animals. An epigastric flap from male Sprague-Dawley rats (320 to 380 g) was used to assess perfusion in sham animals (n=6) or in 12 hr-ischemia animals (12 hr of ischemia and 3 hr of reperfusion, n = 10) with the LDI system. In a separate experiment, flap size, flap failure index, and histologic sections of the flap from sham animals (n=6) and 12-hr ischemia animals (n=6) were evaluated 7 days after reperfusion. Evaluation of the vessel distribution demonstrated a diffuse picture of flap perfusion after clamp release. Moreover, in the distal portion of the flap, circulation stopped immediately, resulting in a significantly decreased perfusion in the 12-hr ischemia animals during reperfusion, when compared with pre-surgical values (100 percent) or sham animals (77 +/- 26.5 vs. 108 +/- 9.6 percent PU). On day 7, the flaps of animals after ischemia and reperfusion showed significant shrinkage, an increase in flap failure index, as well as necrosis, edema, and leukocyte infiltration. Based on the findings, the authors propose that, after prolonged ischemia, the circulation becomes diffuse, and "no-reflow" occurs in the distal portions of the myocutaneous flap during reperfusion. Perfused areas, assessed with the LDI early during reperfusion, might still become necrotic after several days. In the authors' flap model, edema formation and leukocyte infiltration seem to be related more to ischemia reperfusion damage than to thrombus formation.     

In vivo visualization of platelet/endothelium cell interaction in muscle flaps

Increasing evidence underlines the substantial pathophysiological impact of platelets on the development of ischemia/reperfusion injury (I/R) in flaps. Methods for studying dynamic platelet mechanisms in flaps in vivo are not available. The aim of this study was to develop a model enabling quantitative analysis of platelet kinetics and platelet-endothelium cell interaction within the microcirculation of muscle flaps in vivo. Balb/c mice (n = 16) were anesthetized, and an epigastric muscle flap was prepared. Autologous platelets were separated from blood donor animals (n = 16) and labeled ex vivo by means of rhodamine-6-G. After I/R (90 minutes' clamping, 10 minutes' reperfusion), the platelets were administered intra-arterially (i.a.). Microhemodynamics and kinetics of platelets were investigated by intravital fluorescence microscopy. I/R of muscle flaps induced disturbances in microcirculation. The number of rolling platelets, as well as platelets adhering to the inner vessel wall of venules, was increased in the ischemia group. Using intravital fluorescence microscopy, platelet kinetics were analyzed directly in flap microcirculation in vivo for the first time. Since platelet/endothelial cell interaction is a key event in the pathophysiology after microsurgical procedures, this model will help to understand basic molecular mechanisms of platelet behavior during I/R.     

Evaluation of fructose 1,6 diphosphate for salvage of ischemic gracilis flaps in rats

Fructose 1, 6 diphosphate (FDP), a metabolic intermediate, provides an alternative mechanism to circumvent the rate-limiting step in the Kreb's cycle. This agent has been observed to prevent the effects of ischemia on heart tissue and kidney function and the effects of endotoxic shock. It has been shown conclusively to minimize the adverse effects of ischemia-reperfusion injury in experimental pedicled skin flaps in animals. The present study was done to evaluate the effect of intra-arterial administration of FDP on salvage of ischemic microvascular transfer of gracilis muscle flaps in rats, with the premise that it might prolong the ischemia time of muscle flaps at room temperature, thus increasing chances of flap survival. Irrigation with FDP did not change the quantitative survival of the flaps, but there was qualitative improvement on histologic evaluation and DNA analysis. Decreased inflammatory damage and DNA fragmentation were seen at the 2.5-hr period. Histologic staining for mitochondrial oxygenation in gracilis muscle also showed increased uptake in the FDP-treated group vs. control at the 2.5-hr ischemia period. Further experiments with different modes of FDP administration should be carried out to identify more effective means of amelioration of flap ischemia.     

Effects of VEGF administration following ischemia on survival of the gracilis muscle flap in the rat.

The incidence of free flap transplantation failure is only 3% to 5%, yet still occurs in cases in which the flap suffers prolonged ischemia. The purpose of the current study was to determine the effects of vascular endothelial growth factor (VEGF)--a potent angiogenic agent with a suspected role in the protection of endothelium--on flap survival in a model of ischemia-reperfusion injury. The model chosen was the rat gracilis muscle flap. A total of 36 adult male Sprague-Dawley rats were divided into three groups (N = 12). One experimental group received VEGF treatment and the other received heparin. A third group was treated with saline and served as the control. The gracilis muscle flap was dissected and isolated based on a vascular pedicle originating at the femoral vessels. Following 3.75 hours of ischemia, induced by clamping the femoral vessels, either VEGF, heparin, or saline was infused directly into the pedicle of the flap via a cannula. The flaps were evaluated both grossly and histologically after 72 hours of reperfusion. Eleven of the 12 flaps from the VEGF group survived, whereas the survival rate was 6 of 12 and 5 of 12 flaps for the heparin- and saline-treated groups respectively. Flap survival was significantly greater in the VEGF-treated group compared with the heparin- and saline-treated groups (p < 0.025, p < 0.01 respectively). Furthermore, there was no significant difference between the heparin and saline groups. These results indicate that VEGF plays a role in reducing the damage that occurs in ischemia-reperfusion injury, and that the use of VEGF holds promise as a potential therapy for increasing flap survival.     

Effects of University of Wisconsin and lactated Ringer's solutions to ischemia-reperfusion injury in isolated cremaster flap

Ischemia-reperfusion (I/R) injury is a topic that has been much-discussed by various researchers during the last decade in plastic surgery. Though much progress has occurred, the problem is not totally solved yet. In particular, the pathophysiology of reperfusion injury in skeletal muscle has not been clearly elucidated. The aims of this study are to assess the effects of a variety of perfusants on the microcirculation after reperfusion injury and to better understand the pathophysiology of reperfusion injury. Isolated cremaster flaps were performed in 44 rats, preserving the femoral artery and vein in order to cannulate with microtubes. There were 2 control and 2 experiment groups. In one of the control groups and in both experimental groups, 2 h of ischemia were applied by clamping the iliac vessels. Immediately after this, the muscle was locally perfused and washed with lactated Ringer's (LR) and University of Wisconsin (UW) solutions, given from the femoral artery and drained by the femoral vein in the two respective experimental groups. The effects of these solutions to I/R injury were shown at the microcirculatory level via measuring and determining preischemic and postischemic diameters of arterioles and venules, tissue perfusion, capillary density, velocity of red blood cells, and leukocyte sticking. Both tested perfusion solutions were found to be harmful in all parameters. This study demonstrates that both LR and UW solutions aggravate I/R injury.     

Heparinization reduces endothelial permeability and hydrogen ion accumulation in a canine skeletal muscle ischemia-reperfusion model

Skeletal muscle injury after revascularization (ischemia-reperfusion) continues to be a major clinical problem. Although heparinization has been recommended, its action in an experimental model of I-R has not been evaluated. We investigated the ability of heparinization to decrease I-R injury in 10 anesthetized dogs (nonheparinized, n = 5; heparinized, n = 5), subjecting one gracilis muscle to 6 hours of ischemia followed by 1 hour of reperfusion while the identically prepared contralateral muscle served as a nonischemic control. Skeletal muscle infarction was determined by Tc-PYP uptake. Endothelial permeability was quantified by measurement of skeletal muscle 125I-Alb activity after intravenous injection. Interstitial hydrogen ion (H+) accumulation was determined by a miniature pH electrode inserted into the gracilis muscle. Isotopic activities from the ischemic muscle were calculated as a percentage of the contralateral nonischemic muscle (mean +/- SEM). Nonheparinized ischemic muscles had an increase in the activities of Tc-PYP and 125I-Alb of 684% +/- 149% and 742% +/- 130%, which were reduced to 218% +/- 54% and 378% +/- 85% by heparinization, respectively (p less than 0.05). During ischemia, the nonheparinized muscles accumulated 1223 +/- 121 nmol of H+ compared with 785 +/- 95 nmol in the heparinized animals (p less than 0.01). This significant reduction in I-R injury may be causally related to diminished endothelial permeability and H+ accumulation.     

Postischemic flap washout with hydroxyethyl starch (HAES) and its beneficial effect on the no-reflow phenomenon in rat skin island flaps

This study investigated the possible effect of hydroxyethyl starch (HAES) as a postischemic perfusion washout on survival of skin flaps. Forty-eight, male, Sprague-Dawley rats were used in this experiment. A 4×6 cm unilateral island skin flap based on the superficial inferior epigastric artery and vein was raised. The femoral neurovascular bundle supplying the flap pedicle was also dissected free. The flaps were divided into four groups, each consisting of 12 rats. Group 1 (Nonishemic control); Group 2 (Control) – no perfusion washout; Group 3 (Saline) – postischemic washout with normal saline solution; Group 4 (HAES) – postischemic washout with hydroxyethyl starch 10% (HAES) solution. The flaps were subjected to 11 hours of warm ischemia. Thirty minutes prior to the completion of the ischemic period, the flaps were perfused with normal saline solution in group 3 and with HAES in group 4. The percentage of flap survival was assessed on postoperative day 7. Statistical analysis was performed using the Student’s t-test. Flap survival rates for Group 4 (HAES) were significantly greater than Group 2 (Control nonperfusion washout) and Group 3 (saline solution) (p<0.001). This is the first study to investigate the effect of HAES on skin flap survival based on the results, HAES solution may be useful in clinical practice. Received: 6 August 1997 / Accepted: 11 December 1997     

Six-day kidney preservation in a canine model. Influence of a one-to-four-hour ex vivo perfusion interval

Continuous hypothermic perfusion (CHP) was improved when interrupted by an interval of ex vivo perfusion. The influence of the length (1-4 hr) of this interval of ex vivo perfusion was studied in a dog model. Kidneys were subjected to CHP for 72 hr, then connected with the donor for 1, 2, 3, and 4 hr, then returned to CHP and, after a total preservation time of 144 hr, autotransplanted. Simultaneous contralateral nephrectomy was performed. Life-sustaining kidney function was obtained in all of the 6 animals in the 3-hr group and in 5 of the 6 in the 4-hr group. Shorter ex vivo perfusion intervals (1-hr and 2-hr) resulted in no survivors and 2 survivors out of 6, respectively. Creatinine levels of animals that received autotransplants of 3-hr and 4-hr ex vivo perfused kidneys returned to near normal values within 10 days after transplantation.     

Decrease in core liver temperature with 10 degrees C by in situ hypothermic perfusion under total hepatic vascular exclusion reduces liver ischemia and reperfusion injury during partial hepatectomy in pigs

OBJECTIVE: We attempted to assess liver ischemia/reperfusion injury under a mild decrease in core liver temperature of 10 degrees C by in situ hypothermic perfusion during ischemia. METHODS: Liver ischemia was induced in pigs by total hepatic vascular exclusion with concomitant in situ perfusion with hypothermic (4 degrees C) Ringer-glucose (cold perfused group, core liver temperature maintained at 28 degrees C), with normothermic (38 degrees C) Ringer-glucose (warm perfused group) or without in situ perfusion (control group). RESULTS: In the cold perfused, warm perfused, and control groups, 24-hour survival was 5/5, 0/5, and 3/5, respectively. Hemodynamic parameters in the cold perfused group remained stable, whereas pigs in both other groups required circulatory support. Plasma AST and interleukin-6 levels were lower in the cold perfused group than in both other groups. Hepatocellular function was best preserved in the cold perfused group as indicated by complete recovery of bile production during reperfusion and no loss of indocyanine green clearance capacity. In both other groups, bile production and indocyanine green clearance capacity were reduced significantly. The hyaluronic acid uptake capacity of pigs in the cold perfused group or control group did not differ, indicating preserved sinusoidal endothelial cell function. Histopathologic injury scores during reperfusion were significantly lower in the cold perfused group when compared to both other groups. CONCLUSIONS: A mild decrease in core liver temperature of 10 degrees C by in situ hypothermic liver perfusion during ischemia protects the liver from ischemia/reperfusion injury. This protection appears to be related to cooling of the liver rather than to the washout of blood during perfusion.     

Extracorporeal perfusion of free muscle flaps in a porcine model using a miniaturized perfusion system

Introduction  

The development of techniques in transplantation medicine—including various aspects—has made extraordinary progress within the past three decades. However, the transplantation of free tissue flaps with the common problem of limited ischemia time frames remains an area in which the understanding of mechanism during ischemia and reperfusion is still limited. Thus, similar to other organ transplantations, the prolongation of ischemic time and the possibility to perform an ex vivo perfusion is desirable. The purpose of this study was to create a closed and steady ex vivo perfusion system in order to analyze the possibility of using a miniaturized perfusion system for free muscle flaps that could also be clinically used for other solid organ transplantation.     

Local Cooling Provides Muscle Flaps Protection from Ischemia-Reperfusion Injury in the Event of Venous Occlusion During the Early Reperfusion Period

Clinicians often place patients in heated rooms following muscle flap transfers. We hypothesize that exposure of flaps to heated room temperatures could result in an unnecessary hyperthermic ischemic insult if the flaps were to be compromised by venous outflow obstruction, while exposure of elective flaps to local cooling during early perfusion may provide protection in the event of venous occlusion. The rat rectus femoris muscle flap was elevated and clamped for 1 h. The muscle was then exposed to various temperatures for 1 h of perfusion followed by complete venous occlusion for 3 h. Occlusion clamps were removed and flaps were allowed to reperfuse for 24 h. Flaps were assessed for muscle necrosis and edema. Venous occluded muscles demonstrated decreased muscle necrosis and edema in the locally cooled group (8.5 +/- 6.7%, 3.06 +/- 0.14; P < 0.001) compared to the room temperature group (76.2 +/- 23.0%, 3.73 +/- 0.13), and the local warming group (97.3 +/- 1.4%, 3.84 +/- 0.29) respectively. No difference was noted in muscle necrosis nor edema amongst non-ischemic muscles irrespective of temperature exposure. These results suggest a beneficial role for exposure of elective flaps to local cooling during the early perfusion period in order to provide protection from ischemia reperfusion injury in the event of a venous occlusion insult. The prophylactic exposure of flaps to local cooling is further supported by the lack of a harmful effect when flaps were not compromised by venous occlusion.     

Effect of superoxide dismutase for improvement of survival of ischemic reperfused island skin flap]

Using a rat model, we evaluated the effect of SOD on the survival of ischemic reperfused island skin flaps. In experiment 1, the oxygen free radical concentration in the flaps was measured by the technique of ESR. The results showed that the oxygen free radical concentration in ischemic reperfused flaps was significantly higher than in the corresponding control flaps (P less than 0.001). In experiment 2, the flaps were perfused with SOD (2000 U in 1 ml saline) before reperfusion after 8 hours of ischemia. Seven days after operation, the area of flap survived in the test group was significantly larger than in the control group (P less than 0.0005). The obtained data demonstrated that the generation of oxygen free radical increases with time during ischemia reperfusion in island skin flap and the role of oxygen free radical in tissue injury following ischemia and reperfusion. The use of SOD can enhance the survival of ischemic island skin flap.     

Perfusion washout: increasing a microvascular free flap tolerance to ischemia

Thirty-five Sprague-Dawley rats were divided into control (non-exsanguinated) groups of 8 and 10 hr of ischemia, and exsanguinated groups of 8, 10, 14, 16, and 18 hr of ischemia. Free flaps based on the superficial inferior epigastric artery were anastomosed to the contralateral femoral vessels after their designated ischemia period. Reperfusion was assessed by measuring fluorescein uptake in the free flaps after clamp release. In the exsanguinated groups, 100 percent (5/5) of the 8-hr, 10-hr, and 14-hr ischemia groups survived; 80 percent (4/5) of the 16-hr ischemia group free flaps survived; and none (0/5) of the 18-hr ischemia flaps survived. In the control groups, 100 percent (5/5) of the 8-hr ischemia flaps survived, while none (0/5) of the control 10-hr ischemia flaps survived. Fluorescein uptake correlated well with flap survival. By exsanguinating an experimental microvascular free flap prior to its ischemia period, it is possible to dramatically increase flap tolerance to ischemia.     

The effects of cardioplegia washout on myocardial preservation

Preservation of the myocardium in vitro is more effective than preservation in vitro when preservation conditions are apparently the same. The washout of cardioplegia from the myocardium by noncoronary blood flow has been implicated as a probable cause of the poorer myocardial preservation seen in vivo. Isolated dog hearts were used to study the effects of cardioplegia washout by low flow perfusion (0.05 ml/min/g LV weight) during a 2-hr preservation period. Six experimental groups of five hearts each underwent 2 hr of myocardial preservation at temperatures of 20 degrees C (three groups) or 30 degrees C (three groups). The three groups for each temperature consisted of retrograde coronary sinus perfusion (to simulate cardioplegia washout by collateral flow) with blood cardioplegia (BC), normal blood (NB), or no perfusion at all (No CSP). The quality of preservation in each group was assessed by measuring recovery of left ventricular function, tissue water and electrolyte content, and myocardial high-energy phosphate and adenylate content prior to, during, and following preservation. In hearts maintained at 20 degrees C, cardioplegia washout did not significantly affect left ventricular function or myocardial levels of H2O, Na, K, ATP, or total adenylates. When myocardial temperature was allowed to increase to 30 degrees C, preservation was better with low flow perfusion during the preservation period. Hearts warmed to 30 degrees C with no cardioplegia washout experienced a 50% loss of tissue ATP levels and recovered less than 30% of normal left ventricular function during a 150-min reperfusion period. In contrast, hearts slowly perfused during the preservation period maintained nearly normal levels of ATP and returned to normal function by 150 min of reperfusion. Although myocardial warming by noncoronary perfusion has a detrimental effect on myocardial preservation, the slow washout of cardioplegia per se has no apparent detrimental effect on preservation.