An old dream revitalised: preconditioning strategies to protect surgical flaps from critical ischaemia and ischaemia-reperfusion injury

The prevention of ischaemia and the adequate restitution of blood flow to ischaemic tissue are pivotal to halt the progression of cellular injury associated with decreased oxygen and nutrient supply. Accordingly, the search for novel strategies which aim at preventing ischaemia-reperfusion-induced tissue damage is still of major interest in flap surgery. Preconditioning represents an elegant approach to render the tissue more resistant against deleterious ischaemic insults. For many decades, 'surgical delay' has been the standard method of tissue preconditioning. During the last 10 years, ischaemic preconditioning was added to the repertoire of plastic surgeons to protect flaps from ischaemic necrosis. The invasiveness and expenditure of time of these procedures, however, have always been major drawbacks, hindering a wide distribution in clinical practice. Consequently, the motivation has all along been to further refine and simplify protective strategies. Recent experimental studies have now shown that efficient protection from ischaemic necrosis can also be achieved by remote preconditioning or pretreatment with chemical agents and growth factors, which mimic the action of surgical delay and ischaemic preconditioning. In addition, the local application of unspecific stressors, including both heating and cooling, have been shown to effectively improve flap microcirculation and, thus, tissue survival. In view of successful translational research, it is now time that the efficacy of these novel preconditioning procedures is proven in prospective randomised clinical trials.

Successful lower extremity angioplasty improves brachial artery flow-mediated dilation in patients with peripheral arterial disease

INTRODUCTION: Peripheral arterial disease (PAD) is associated with systemic impaired flow-mediated dilation (FMD) and increased risk for cardiovascular events. Decreased FMD may be caused by a decrease in arterial shear stress due to claudication and inflammation due to muscle ischemia and reperfusion. We assumed that endovascular revascularization of lower limb arterial obstructions ameliorates FMD and lowers inflammation through improvement of peripheral perfusion. METHODS: The study was a prospective, open, randomized, controlled, single-center follow-up evaluation assessing the effect of endovascular revascularization on brachial artery reactivity (FMD) measured by ultrasound, white blood cell (WBC) count, high-sensitive C-reactive protein (hs-CRP), and fibrinogen. We investigated 33 patients (23 men) with chronic and stable PAD (Rutherford 2 to 3) due to femoropopliteal obstruction. Variables were assessed at baseline and after 4 weeks in 17 patients (group A) who underwent endovascular revascularization and best medical treatment, and in 16 patients (group B) who received best medical treatment only. RESULTS: FMD did not differ between group A and B (4.96% +/- 1.86% vs 4.60% +/- 2.95%; P = .87) at baseline. It significantly improved after revascularization in group A (6.44% +/- 2.88%; P = .02) compared with group B at 4 weeks of follow-up (4.53% +/- 3.17%; P = .92), where it remained unchanged. The baseline ankle-brachial index (ABI) was similar for group A and B (0.63 +/- 0.15 vs 0.66 +/- 0.10; P = .36). At 4 weeks of follow-up, ABI was significantly increased in group A (1.05 +/- 0.15; P = .0004) but remained unchanged in group B (0.62 +/- 0.1). WBC counts of the two groups were comparable at baseline (group A: 7.6 +/- 2.26 x 10(6)/mL and group B: 7.8 +/- 2.02 x 10(6)/mL, P = .81). In group A, the leukocyte count significantly decreased after angioplasty from 7.6 +/- 2.26 to 6.89 +/- 1.35 x 10(6)/mL (P = .03). For group B, WBC count did not differ significantly compared with baseline (7.76 +/- 2.64 x 10(6)/mL; P = .94). No effects were observed on hs-CRP or fibrinogen from endovascular therapy. CONCLUSION: Endovascular revascularization with reestablishment of peripheral arterial perfusion improves FMD and reduces WBC count in patients with claudication. Revascularization may therefore have clinical implications beyond relief of symptoms, for example, reducing oxidative stress caused by repeated muscle ischemia or increased shear stress due to improved ambulatory activity.

Therapeutic angiogenesis with intramuscular NV1FGF improves amputation-free survival in patients with critical limb ischemia

This study evaluated the efficacy and safety of intramuscular administration of NV1FGF, a plasmid-based angiogenic gene delivery system for local expression of fibroblast growth factor 1 (FGF-1), versus placebo, in patients with critical limb ischemia (CLI). In a double-blind, randomized, placebo-controlled, European, multinational study, 125 patients in whom revascularization was not considered to be a suitable option, presenting with nonhealing ulcer(s), were randomized to receive eight intramuscular injections of placebo or 2.5 ml of NV1FGF at 0.2 mg/ml on days 1, 15, 30, and 45 (total 16 mg: 4 x 4 mg). The primary end point was occurrence of complete healing of at least one ulcer in the treated limb at week 25. Secondary end points included ankle brachial index (ABI), amputation, and death. There were 107 patients eligible for evaluation. Improvements in ulcer healing were similar for use of NV1FGF (19.6%) and placebo (14.3%; P = 0.514). However, the use of NV1FGF significantly reduced (by twofold) the risk of all amputations [hazard ratio (HR) 0.498; P = 0.015] and major amputations (HR 0.371; P = 0.015). Furthermore, there was a trend for reduced risk of death with the use of NV1FGF (HR 0.460; P = 0.105). The adverse event incidence was high, and similar between the groups. In patients with CLI, plasmid-based NV1FGF gene transfer was well tolerated, and resulted in a significantly reduced risk of major amputation when compared with placebo.

Tolerance of human placental tissue to severe hypoxia and its relevance for dual ex vivo perfusion

In the dual ex vivo perfusion of an isolated human placental cotyledon it takes on average 20-30 min to set up stable perfusion circuits for the maternal and fetal vascular compartments. In vivo placental tissue of all species maintains a highly active metabolism and it continues to puzzle investigators how this tissue can survive 30 min of ischemia with more or less complete anoxia following expulsion of the organ from the uterus and do so without severe damage. There seem to be parallels between "depressed metabolism" seen in the fetus and the immature neonate in the peripartum period and survival strategies described in mammals with increased tolerance of severe hypoxia like hibernators in the state of torpor or deep sea diving turtles. Increased tolerance of hypoxia in both is explained by "partial metabolic arrest" in the sense of a temporary suspension of Kleiber's rule. Furthermore the fetus can react to major changes in surrounding oxygen tension by decreasing or increasing the rate of specific basal metabolism, providing protection against severe hypoxia as well as oxidative stress. There is some evidence that adaptive mechanisms allowing increased tolerance of severe hypoxia in the fetus or immature neonate can also be found in placental tissue, of which at least the villous portion is of fetal origin. A better understanding of the molecular details of reprogramming of fetal and placental tissues in late pregnancy may be of clinical relevance for an improved risk assessment of the individual fetus during the critical transition from intrauterine life to the outside and for the development of potential prophylactic measures against severe ante- or intrapartum hypoxia. Responses of the tissue to reperfusion deserve intensive study, since they may provide a rational basis for preventive measures against reperfusion injury and related oxidative stress. Modification of the handling of placental tissue during postpartum ischemia, and adaptation of the artificial reperfusion, may lead to an improvement of the ex vivo perfusion technique.

Ischemia-induced up-regulation of heme oxygenase-1 protects from apoptotic cell death and tissue necrosis

BACKGROUND: Tissues are endowed with protective mechanisms to counteract chronic ischemia. Previous studies have demonstrated that endogenous heme oxygenase (HO)-1 may protect parenchymal tissue from inflammation- and reoxygenation-induced injury. Nothing is known, however, on whether endogenous HO-1 also plays a role in chronic ischemia to protect from development of tissue necrosis. The aim of this study is, therefore, to evaluate in vivo whether endogenous HO-1 exerts protection on chronically ischemic musculocutaneous tissue, and whether this protection is mediated by an attenuation of the microcirculatory dysfunction. MATERIALS AND METHODS: In C57BL/6-mice, a chronically ischemic flap was elevated and fixed into a dorsal skinfold chamber. In a second group, tin-protoporphyrin-IX was administrated to competitively block the action of HO-1. Animals without flap elevation served as controls. With the use of intravital fluorescence microscopy, microcirculation, apoptotic cell death, and tissue necrosis were analyzed over a 10-day observation period. The time course of HO-1 expression was determined by Western blotting. RESULTS: Chronic ischemia induced an increase of HO-1 expression, particularly at day 1 and 3. This was associated with arteriolar dilation and hyperperfusion, which was capable of maintaining an adequate capillary perfusion density in the critically perfused central part of the flap, demarcating the distal necrosis. Inhibition of endogenous HO-1 by tin-protoporphyrin-IX completely abrogated arteriolar dilation (44.6 +/- 6.2 microm versus untreated flaps: 71.3 +/- 7.3 microm; P < 0.05) and hyperperfusion (3.13 +/- 1.29 nL/s versus 8.55 +/- 3.56 nL/s; P < 0.05). This resulted in a dramatic decrease of functional capillary density (16 +/- 16 cm/cm(2)versus 84 +/- 31 cm/cm(2); P < 0.05) and a significant increase of apoptotic cell death (585 +/- 51 cells/mm(2)versus 365 +/- 53 cells/mm(2); P < 0.05), and tissue necrosis (73% +/- 5% versus 51% +/- 5%; P < 0.001). CONCLUSION: Thus, our results suggest that chronic ischemia-induced endogenous HO-1 protects ischemically endangered tissue, probably by the vasodilatory action of the HO-1-associated carbon monoxide.

Long-term outcome of acute spinal cord ischemia syndrome

BACKGROUND AND PURPOSE: Current knowledge of long-term outcome in patients with acute spinal cord ischemia syndrome (ASCIS) is based on few studies with small sample sizes and <2 years' follow-up. Therefore, we analyzed clinical features and outcome of all types of ASCIS to define predictors of recovery. METHODS: From January 1990 through October 2002, 57 patients with ASCIS were admitted to our center. Follow-up data were available for 54. Neurological syndrome and initial degree of impairment were defined according to American Spinal Injury Association (ASIA)/International Medical Society of Paraplegia criteria. Functional outcome was assessed by walking ability and bladder control. RESULTS: Mean age was 59.4 years; 29 were women; and mean follow-up was 4.5 years. The origin was atherosclerosis in 33.3%, aortic pathology in 15.8%, degenerative spine disease in 15.8%, cardiac embolism in 3.5%, systemic hypotension in 1.8%, epidural anesthesia in 1.8%, and cryptogenic in 28%. The initial motor deficit was severe in 30% (ASIA grades A and B), moderate in 28% (ASIA C), and mild in 42% (ASIA D). At follow-up, 41% had regained full walking ability, 30% were able to walk with aids, 20% were wheelchair bound, and 9% had died. Severe initial impairment (ASIA A and B) and female sex were independent predictors of unfavorable outcome (P=0.012 and P=0.043). CONCLUSIONS: Considering a broad spectrum of clinical presentations and origins, the outcome in our study was more favorable than in previous studies reporting on ASCIS subgroups with more severe initial deficits.

Velocity recovery cycles of human muscle action potentials and their sensitivity to ischemia

This study was undertaken to test whether recovery cycle measurements can provide useful information about the membrane potential of human muscle fibers. Multifiber responses to direct muscle stimulation through needle electrodes were recorded from the brachioradialis of healthy volunteers, and the latency changes measured as conditioning stimuli were applied at interstimulus intervals of 2-1000 ms. In all subjects, the relative refractory period (RRP), which lasted 3.27 +/- 0.45 ms (mean +/- SD, n = 12), was followed by a phase of supernormality, in which the velocity increased by 9.3 +/- 3.4% at 6.1 +/- 1.3 ms, and recovered over 1 s. A broad hump of additional supernormality was seen at around 100 ms. Extra conditioning stimuli had little effect on the early supernormality but increased the later component. The two phases of supernormality resembled early and late afterpotentials, attributable respectively to the passive decay of membrane charge and potassium accumulation in the t-tubules. Five minutes of ischemia progressively prolonged the RRP and reduced supernormality, confirming that these parameters are sensitive to membrane depolarization. Velocity recovery cycles may provide useful information about altered muscle membrane potential and t-tubule function in muscle disease. Muscle Nerve, 2008.

C1-esterase inhibitor reduces reperfusion injury after lung transplantation

BACKGROUND: Activation of the complement system and polymorphonuclear neutrophilic leukocytes plays a major role in mediating reperfusion injury after lung transplantation. We hypothesized that early interference with complement activation would reduce lung reperfusion injury after transplantation. METHODS: Unilateral left lung autotransplantation was performed in 6 sheep. After hilar stripping the left lung was flushed with Euro-Collins solution and preserved for 2 hours in situ at 15 degrees C. After reperfusion the right main bronchus and pulmonary artery were occluded, leaving the animal dependent on the reperfused lung (reperfused group). C1-esterase inhibitor group animals (n = 6) received 200 U/kg body weight of C1-esterase inhibitor as a short infusion, half 10 minutes before, the other half 10 minutes after reperfusion. Controls (n = 6) underwent hilar preparation only. Pulmonary function was assessed by alveolar-arterial oxygen difference and pulmonary vascular resistance. The release of beta-N-acetylglucosaminidase served as indicator of polymorphonuclear neutrophilic leukocyte activation. Extravascular lung water was an indicator for pulmonary edema formation. Biopsy specimens were taken from all groups 3 hours after reperfusion for light and electron microscopy. RESULTS: In the reperfused group, alveolar-arterial oxygen difference and pulmonary vascular resistance were significantly elevated after reperfusion. All animals developed frank alveolar edema. The biochemical marker beta-N-acetylglucosaminidase showed significant leukocyte activation. In the C1-esterase inhibitor group, alveolar-arterial oxygen difference, pulmonary vascular resistance, and the level of polymorphonuclear neutrophilic leukocyte activation were significantly lower. CONCLUSIONS: Treatment with C1-esterase inhibitor reduces reperfusion injury and improves pulmonary function in this experimental model.

Ascorbic acid for amelioration of reperfusion injury in a lung autotransplantation model in sheep

BACKGROUND: Reperfusion injury is the leading cause of early graft dysfunction after lung transplantation. Activation of neutrophilic granulocytes with generation of free oxygen radicals appears to play a key role in this process. The efficacy of ascorbic acid as an antioxidant in the amelioration of reperfusion injury after lung transplantation has not been studied yet. METHODS: An in situ autotransplantation model in sheep is presented. The left lung was flushed (Euro-Collins solution) and reperfused; after 2 hours of cold storage, the right hilus was then clamped (group R [reference], n = 6). Group AA animals (n = 6) were treated with 1 g/kg ascorbic acid before reperfusion. Controls (group C, n = 6) underwent hilar preparation and instrumentation only. RESULTS: In group R, arterio-alveolar oxygen difference (AaDO2) and pulmonary vascular resistance (PVR) were significantly elevated after reperfusion. Five of 6 animals developed frank alveolar edema. All biochemical parameters showed significant PMN activation. In group AA, AaDO2, PVR, work of breathing, and the level of PMN activation were significantly lower. CONCLUSIONS: The experimental model reproduces all aspects of lung reperfusion injury reliably. Ascorbic acid was able to weaken reperfusion injury in this experimental setup.

Amelioration of lung reperfusion injury by L- and E- selectin blockade

OBJECTIVE: Reperfusion injury is the main reason for early graft failure after lung transplantation. Inhibition of the adherence of polymorphonuclear leukocytes to activated endothelium by blocking L- and E-selectins (antibody EL-246) could potentially inhibit reperfusion injury. METHODS: Reperfusion injury was induced in a left lung autotransplant model in sheep. After hilar stripping the left lung was flushed with Euro-Collins solution and preserved for 2 h in situ at 15 degrees C. After reperfusion right main bronchus and pulmonary artery were occluded leaving the animal dependent on the reperfused lung (control, n = 6). Pulmonary function was assessed by alveolo-arterial oxygen difference (AaDO2) and pulmonary vascular resistance (PVR), the chemiluminescence of isolated neutrophils, as well as the release of beta-N-acetyl-glucosaminidase (beta-NAG) served as indicator of neutrophilic activation. Extravascular lung water was an indicator for pulmonary edema formation. EL-246 group animals (n = 6) were treated additionally with 1 mg/kg BW of EL-246 given prior and during reperfusion. RESULTS: After 3 h of reperfusion five control animals developed alveolar edema compared to one animal in the EL-246 group (P = 0.08). AaDO2 (mm Hg) was significantly higher in the control compared to the EL-246 group (510 +/- 148 vs. 214 +/- 86). PVR (dyn x s x cm(-5)) was significantly increased in the control compared to the EL-246 group (656 +/- 240 vs. 317 +/- 87). Neutrophilic activation was significantly lower in the EL-246 group. Extravascular lung water was significantly lower compared to control (6.88 +/- 1.0 vs. 13.4 +/- 2.8 g/g blood-free lung weight). CONCLUSIONS: Treatment with EL-246 results in improved pulmonary function and less in vivo PMN activation in this experimental model. Further studies are necessary to evaluate the possible role of selectin blockade in amelioration of reperfusion injury in human lung transplantation.

Comparison of low potassium Euro-Collins solution and standard Euro-Collins solution in an extracorporeal rat heart-lung model

OBJECTIVE: Euro-Collins solution (EC) is routinely used in lung transplantation. The high potassium of EC, however, may damage the vascular endothelium, thereby contributing to postischemic reperfusion injury. To assess the influence of the potassium concentration on lung preservation, we evaluated the effect of a "low potassium Euro-Collins solution" (LPEC), in which the sodium and potassium concentrations were reversed. METHODS: In an extracorporeal rat heart-lung model lungs were preserved with EC and LPEC. The heart-lung blocks (HLB) were perfused with Krebs-Henseleit solution containing washed bovine red blood cells and ventilated with room air. The lungs were perfused via the working right ventricle with deoxygenated perfusate. Oxygenation and pulmonary vascular resistance (PVR) were monitored. After baseline measurements, hearts were arrested with St. Thomas' solution and the lungs were perfused with EC or LPEC, or were not perfused (controls). The HLBs were stored for 5 min or 2 h ischemic time at 4 degrees C. Reperfusion and ventilation was performed for 40 min. At the end of the trial the wet/dry ratio of the lungs was calculated and light microscopic assessment of the degree of edema was performed. RESULTS: After 5 min of ischemia oxygenation was significantly better in both preserved groups compared to the controls. Pulmonary vascular resistance was elevated in all three groups after 30 min reperfusion at both ischemic times. After 2 h of ischemia PVR of the group preserved with LPEC was significantly lower than those of the EC and controls (LPEC-5 min: 184 +/- 65 dynes * sec * cm-5, EC-5 min: 275 +/- 119 dynes * sec * cm * cm-5, LPEC-2 h: 324 +/- 47 dynes * sec * m-5, EC-2 h: 507 +/- 83 dynes * sec * cm-5). Oxygenation after 2 h of ischemia and 30 min reperfusion was significantly better in the LPEC group compared to EC and controls (LPEC: 70 +/- 17 mmHg, EC: 44 +/- 3 mmHg). The wet/dry ratio was significantly lower in the two preserved groups compared to controls (LPEC-5 min: 5.7 +/- 0.7, EC-5 min: 5.8 +/- 1.2, controls-5 min: 7.5 +/- 1.8, LPEC-2 h: 6.7 +/- 0.4, EC: 6.9 +/- 0.4, controls-2 h: 7.3 +/- 0.4). CONCLUSIONS: We thus conclude that LPEC results in better oxygenation and lower PVR in this lung preservation model. A low potassium concentration in lung preservation solutions may help in reducing the incidence of early graft dysfunction following lung transplantation.

University of Wisconsin versus St. Thomas' Hospital solution for human donor heart preservation

Prolongation of the safe period of ischemia of the heart is an efficient way to overcome donor organ shortage, as demonstrated in renal and hepatic transplantation. We present the results of a prospective, randomized study comparing preservation with University of Wisconsin solution (UWS) versus St. Thomas' Hospital solution (STS) in clinical heart transplantation. A total of 39 patients were enrolled in the study (n = 20 for UWS and n = 19 for STS). Hemodynamic, electron microscopic, and biochemical evaluation did not reveal any significant differences in postoperative myocardial performance. Only the number of intraoperative defibrillations (0.82 for UWS versus 1.7 for STS) and the rhythm stability after reperfusion (13/20 UWS hearts versus 6/19 STS hearts in sinus rhythm) were significantly different. Heart preservation with UWS and STS appears to be of comparable efficacy at mean ischemic times of less than 4 hours.