Perfusion Washout Research Articles

The Ile164 β 2-adrenoceptor polymorphism alters salmeterol exosite binding and conventional agonist coupling to G s

β 2-adrenoceptors (β 2AR) are polymorphic at amino acid 164 (Thr or Ile) of the fourth transmembrane domain. In transfected fibroblasts, six agonists commonly used in the treatment of bronchospasm were studied. Isoproterenol, albuterol, metaproterenol, terbutaline, formoterol, and salmeterol displayed decreased binding affinities ( K is were 1.2–3.0-fold higher) and a significant degree of impaired maximal stimulation of adenylyl cyclase (∼40%), was observed with all agonists for the Ile164 receptor. The ratios of signal transduction efficiencies (Tau function, Ile164/Thr164) varied from a low of 0.17 for terbutaline to 0.49 for salmeterol. In addition, Ile164 bound salmeterol at the exosite, as delineated in perfusion washout studies, at a decreased level (31±4.8% vs. 49±4.4% retained salmeterol, respectively, P=0.02). In cAMP production studies under perfusion conditions, this decreased exosite binding caused a ∼50% decrease in the duration of action of salmeterol at Ile164 ( t 1/2=21.0±3.6 vs. 46.8±4.1 min for Thr164, P=0.001). The durations of action for isoproterenol and formoterol under similar perfusion conditions were not different between the two receptors. These in vitro results indicate the Ile164 polymorphic receptor represents a pharmacogenetic locus for the most commonly utilized agonists in the treatment of asthma with a unique phenotype for salmeterol.

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.

Combination therapy for salvaging a failing, experimental skin flap.

The failing free flap remains a major problem for the reconstructive surgeon. Many and varied pharmacologic agents have been utilized to reverse the effects of ischemia in these flaps. Treatments have been aimed at inhibiting presumed causative factors in the no-reflow phenomenon. Therapy has generally been single in nature and designed to affect only one of these presumed factors. In this study, several pharmacologic agents were utilized individually or in combination therapy as postischemic washouts, in an effort to attack the multiple causative factors in the no-reflow phenomenon and to improve flap survival in a rat abdominal skin flap model. The treatment agents included lactated Ringer's, superoxide dismutase, and urokinase, with each used independently as a postischemic perfusion washout. Combination therapy utilized an initial postischemic perfusion with urokinase, followed by a second perfusion washout with superoxide dismutase. After 18 hr of primary ischemia, there was increased flap survival in the animals undergoing perfusion washout with either superoxide dismutase alone or with combined urokinase and superoxide dismutase washouts, compared to all other treatments (p < 0.001). It was found that flaps undergoing combined urokinase and superoxide dismutase postischemic perfusion washouts demonstrated significantly improved survival after 20 hr of primary ischemia, compared to all other therapies (p < 0.05). By demonstrating improved survival when a thrombolytic agent is used in conjunction with an oxygen free radical scavenger, these findings may have implications in the treatment of clinically failing free flaps.

Evaluation of a continuous systemic infusion of iloprost, a stable PGI-2 analog, on the survival of experimental skin flaps.

Numerous investigators have attempted to enhance the survival of ischemic experimental skin flaps using various pharmacologic manipulations. Recently, the authors' laboratory demonstrated the beneficial effect of iloprost, a stable PGI2 analogue, as a post-ischemic perfusion washout, in improving the survival of ischemic skin flaps. The rat unilateral abdominal skin flap, based on the superficial epigastric vessels, was utilized in this study involving 30 animals. The animals were divided into three different treatment groups, with ischemic periods of 16 and 18 hr. Perfusion washouts were performed at the completion of the various ischemic periods. Alzet osmotic pumps were used to deliver a continuous systemic infusion of iloprost for 7 days postoperatively. The groups consisted of the following: Group 1 (single ILO)--perfusion washout with iloprost only; Group 2 (continuous LD ILO)--low-dose systemic iloprost infusion (0.066 mcg/kg/min) and perfusion washout with iloprost; and Group 3 (continuous HD ILO)--high-dose systemic iloprost infusion (0.1 mcg/kg/min) and perfusion washout with iloprost. The percentage of flap survival was assessed on postoperative day 7. Skin flaps of the animals receiving the continuous systemic infusion of iloprost were noted to have varying percentages of survival, while skin flaps undergoing perfusion washout only were found to have either complete survival using a continuous systemic infusion of iloprost, compared to iloprost perfusion washout alone. In addition, the hypotensive side effects of systemic iloprost infusion limit its use in the rat skin-flap model.

Use of urokinase during secondary ischemia in experimental skin flaps.

The ability of urokinase to salvage experimental flaps after a secondary ischemic insult was investigated in a rat model. Unilateral abdominal island skin flaps based on the superficial inferior epigastric vessels were raised and subjected to either 4 or 6 hours of primary ischemia followed by 12 hours of reperfusion and varying lengths of secondary ischemia. At the conclusion of secondary ischemia, the flaps were perfused with either lactated Ringer's solution or urokinase. One group of flaps served as a control and received no postischemic perfusion washout. The secondary critical ischemia time at which 50% of the flaps failed clinically was greater for flaps perfused with urokinase. Furthermore, the survival rates for all flaps perfused with urokinase were significantly greater than either control flaps or flaps perfused with lactated Ringer's solution (p < 0.05). Flap survival decreased significantly in all groups with increasing primary and/or secondary ischemia time (p < 0.05).

Enhancement of fluorescein perfusion in experimental skin flaps following postischemic washout with iloprost, urokinase, verapamil, and University of Wisconsin solution.

The enhancement of blood flow in experimental skin flaps following postischemic perfusion washout was investigated in rats. Unilateral island skin flaps based on the superficial epigastric vessels were raised and subjected to 6 hr of primary ischemia. Group 1 was designated as a control and did not undergo postischemic perfusion washout. In the remaining rats, postischemic washout was performed with one of five agents: Group 2--lactated Ringer's solution; Group 3--University of Wisconsin solution, an organ preservation medium; Group 4--verapamil, a calcium channel blocker; Group 5--urokinase, a thrombolytic agent; Group 6--iloprost, a stable prostacyclin analog. Two hours following perfusion washout, fluorometric analysis revealed a statistically significant enhancement of blood flow in Groups 4, 5, and 6, compared to Groups 2 and 3 (p < 0.05). Furthermore, a significant increase in skin surface fluorescence was demonstrated in all the flaps that underwent perfusion washout, compared to the control flaps (p < 0.05). By analyzing skin surface fluorescence, the enhancement of nutritive blood flow in flaps, following postischemic perfusion washout, was evaluated. This is the first study in which the above pharmacologic agents were compared in a quantitative manner.

The Additive Beneficial Effect of Uw Solution and Urokinase on Experimental Microvascular Free-Flap Survival

Pharmacologic manipulation of free flaps to enhance tolerance to ischemia has become a subject of great interest in the research literature. In an effort to improve survival, perfusion washout of experimental free flaps was performed following an episode of primary ischemia. The perfusates utilized were lactated Ringer's solution (LR), University of Wisconsin solution (UW), a high-molecular-weight medium used in organ preservation, and urokinase, a thrombolytic agent. Seventy-five rats were used in this study and divided into groups of 5 each. A 3 x 6-cm abdominal free flap based on the superficial inferior epigastric vessels was raised in each rat. The free flaps were subjected to either 12 or 18 hr of primary ischemia. Following the period of ischemia, perfusion washout was performed with either LR, UW solution, or urokinase at increasing concentrations alone or in combination with UW solution. Urokinase was first evaluated as a perfusate alone at increasing concentrations. In the 12-hr ischemia group, free-flap survival was shown to increase from 0 percent in the LR-perfused flaps to 20 percent, 60 percent, and 80 percent in flaps perfused with 12,500, 25,000, and 100,000 U of urokinase, respectively (p < 0.05). A similar increase in survival was demonstrated in the 18-hr ischemia group, where 0 percent, 20 percent, and 40 percent of flaps survived following perfusion with 12,500, 25,000, and 100,000 U of urokinase, respectively (p < 0.05). Urokinase was then perfused along with UW solution to evaluate the combined effect on flap survival.(ABSTRACT TRUNCATED AT 250 WORDS)

Prolongation of Secondary Critical Ischemia Time of Experimental Skin Flaps Using UW Solution as a Normothermic Perfusate

A myriad of investigations have been published on the pharmacologic manipulation of flaps to enhance tolerance to ischemia. We recently reported a threefold increase in ischemic tolerance of the rat abdominal skin flap pedicle after 6 hours of primary ischemia and 12 hours of reperfusion. Flaps underwent normothermic perfusion washout with lactated Ringer's or U.W. solution, a newly developed organ preservation medium. Perfusion washouts were performed at one of three different points in the protocol: (1) onset of primary ischemia; (2) onset of secondary ischemia; or (3) 2 hours after onset of secondary ischemia. The last group was used to simulate the clinical situation in which flaps are discovered and salvage procedures instituted at a delayed time interval. This is the longest normothermic ischemic interval reported. We undertook the present study to determine the utility of the U.W solution in prolonging the tolerance of the flap to a second ischemic insult after a period of reperfusion. Seventy-five unilateral rat abdominal skin flaps were raised. Secondary ischemia was produced by placing a microvascular clamp across the inferior epigastric pedicle. Flap survival was assessed at 1 week postoperatively. While none of the nonperfused flaps survived 8 hours of secondary ischemia, at least 50% of the U.W. perfused flaps survived an average of 14 hours of secondary ischemia. Lactated Ringer's perfusion washout only modestly increased the ischemic tolerance. Perfusion washout in the secondary ischemic phase improved the ischemic tolerance to a significantly greater degree than in the primary ischemic interval.

The Use of a New Perfusate in Experimental Microvascular Flaps: A Threefold Increase in Ischemic Tolerance

The benefit of perfusion washout in both experimental and clinical skin flaps has long been debated. By perfusing ischemic rat pedicled flaps with UW solution, a recently developed, high-molecular-weight, organ-preservation medium, a 170 percent increase in the critical ischemia time of treated versus untreated control flaps was demonstrated. Sixty rats were used in this study. A 3- x 6-cm unilateral abdominal skin flap based on the superficial inferior epigastric artery and vein was raised. The flaps were divided into three groups: Group 1 (control--no perfusion washout (n = 15); Group 2 (LR)--perfusion washout with lactated Ringer's solution (n = 15); Group 3 (UW)--perfusion washout with UW solution (n = 30). Flaps were subjected to varying periods of ischemia, ranging between 8 and 30 hr. The primary ischemia time at which 50 percent of the flaps survived clinically was 10 hr for Group 1, 15 hr for Group 2, and 27 hr for Group 3. The differences between the survival rates for flaps in Groups 1, 2, and 3 were statistically significant (p less than .0005). By bathing the vascular and parenchymal cells in an impermeant preservation solution, it was hypothesized that cellular swelling would be inhibited, thereby significantly improving a skin flap's tolerance to warm ischemia. Furthermore, after reviewing the pertinent literature, it is evident that the primary critical ischemia time of 27 hr is the highest reported to date for the normothermic experimental rat pedicled flap. Clinical application of these findings, as well as the need for further studies, are discussed.

The role of perfusion washout in limb revascularization procedures.

Amputated rat hindlimbs were subjected to either normothermic (26 degrees C) or hypothermic (4 degrees C) ischemia. Experimental limbs had their microcirculation washed out (either before or after the ischemic insult) with a physiologic acellular plasma substitute previously reported to enhance flap survival following extended periods of warm ischemia. Control limbs were not washed out; i.e., stagnant blood remained in these limbs. Following the ischemic interval, amputated limbs were replanted. Monastral blue B, a colloidal pigment capable of labeling leaky blood vessels, was administered systemically to all rats just prior to vascular declamping. Limb biopsies of skin and muscle were harvested 30 minutes following revascularization in order to assess Monastral labeling and, therefore, the functional integrity of the microcirculation. Results confirm that stagnant blood under conditions of warm ischemia is detrimental to the functionality of the microcirculation in both skin (p less than 0.03) and muscle (p less than 0.007). Accordingly, perfusion washout, when performed prior to the ischemic period, enhances limb survival following 6 hours of warm ischemia (p less than 0.01). Hypothermia protects against the detrimental effects of stagnant blood; perfusion offers no benefit if hypothermic conditions prevail. Physiologic mechanisms responsible for these findings are discussed.

The Role of Perfusion Washout in Limb Revascularization Procedures

The Role of Perfusion Washout in Limb Revascularization Procedures

Delayed microcirculatory hyperpermeability following perfusion washout.

Recent experimental data have demonstrated improved flap survival following perfusion washout with a synthetic, chemically defined, mammalian plasma. In an effort to define the physiology responsible for the efficacy of perfusion, the method of "labeling" hyperpermeable blood vessels with Monastral blue B in rat epigastric vascular island flaps was utilized. Results confirmed that capillary and venular hyperpermeability is an early and progressive pathophysiologic event in ischemic flap tissue and one which is reversible prior to a critical ischemic period. Perfusion washout with a physiologic, acellular plasma substitute delays the onset of vascular hyperpermeability. This may be a mechanism responsible for improving tissue survival following extended periods of warm ischemia (12 hours). It is implied that stagnant blood and products of hemolysis in the microcirculation may be detrimental to the functional and anatomic integrity of the endothelial wall.

Preischemic Flap Washout and Its Effect on the No-Reflow Phenomenon

Preischemic perfusion washout with an acellular physiologic solution delays the no-reflow phenomenon and improves tissue survival in rat epigastric free flaps following 18 and 24 hours of normothermic ischemia. This implies that stagnating blood may be a causative agent in the no-reflow phenomenon. A possible mechanism for this is capillary endothelial damage secondary to the presence of formed blood cells or their products of hemolysis. Perfusion washout may improve ischemic tolerance by preventing this blood cell-induced endothelial damage and by the prevention of sludge and thrombus. Whether any of the metabolic components of the perfusate actively enhance ischemic tolerance cannot be definitively stated.

Release of 14C-norepinephrine into the lateral cerebroventricle of rats by exposure to a conditioned aversive stimulus

Rats chronically implanted with push-pull cannulas were injected with a pulse of 14C-norepinephrine (NE) into the lateral cerebroventricle under a variety of pretreatment and behavioral conditions. Animals pretreated intraventricularly with 6-hydroxydopamine (Group A) or ascorbic acid vehicle (Group B) were subsequently perfused under four conditions: (1) presentation of a novel, visual stimulus in a one-way avoidance chamber; (2) presentation of the light (CS) followed by shock; (3) training to a high level of avoidance behavior, after which the CS was presented in the absence of opportunity for an avoidance response and in the absence of shock; and (4) after forced extinction, followed by CS without opportunity to avoid and without presentation of shock. Samples of perfusate from rats subjected to the four test conditions were analyzed by thin-layer chromatography for total 14C in a scintillation counter and for proportion of NE and normetanephrine (NM). During Tests 1 and 4 the 14C perfusion wash-out did not differ from control values for either Group A or B. During Test 2, total radioactivity as well as the proportions of NE and NM increased in the perfusate for both Groups A and B. Presenting the CS without shock (3) resulted in an increase in 14C and NE and NM for Group B (vehicle), but not for Group A (6-OHDA). To test for non-specific release unrelated to a brain catecholaminergic function, another group of rats was subjected to identical treatments with the exception that 14C-urea replaced 14C-NE as a pulse-label. In these animals Test 2 (shock) induced an increase in 14C in the perfusate, while Tests 1, 3 and 4 yielded wash-out curves essentially identical to controls.