Neutrophils and inflammatory cytokines have been implicated in ischemia/reperfusion injury. These observations bridge two fundamental areas of biology, cytokines, and free radical reactions.
Ischemic injury occurs when the blood supply to an area of tissue is cut off. The incidence of ischemic injury is vast: myocardial infarction, stroke, and other thrombotic events affect more than 1.3 million individuals each year in the USA alone. Ischemic injury also occurs during surgery when blood vessels are cross-clamped, and in organs for transplant.
The length of time a tissue can survive oxygen deprivation varies, but eventually all ischemic tissue becomes necrotic. Restoration of the blood supply should minimize the damage, but means to reperfuse tissues after thrombosis (e.g. "clot busting" drugs and angioplasty techniques) led to awareness that the extent of injury often increased when the blood supply was restored. It is widely accepted that this additional damage, reperfusion injury, is due to the activity of free radicals.
Why does reperfusion cause such damage? An outline of the likely sequence of events is illustrated in the figure to the left. Lack of oxygen leads to accumulation of metabolic intermediates. With reperfusion these reactions proceed with a sudden increase in oxygen radicals. Experiments with a variety of animal models and observations on patients undergoing surgery support this hypothesis.1-3 This increase may overwhelm the cells' usual defenses leading to uncontrolled oxidation of vital cell components.
There are two opportunities to reduce this damage. The first is to prevent generation of free radicals and hydrogen peroxide directly. Allopurinol, which inhibits the production of superoxide by xanthine oxidase, has been shown to reduce infarct size in animal models and in some clinical trials, but the outcomes have not been universally successful. Parmley et al.4 found more infarct extensions with allopurinol than with a placebo, contrary to expectations. In the more standardized environment of elective coronary artery bypass grafting (CABG), however, lipid peroxidation (a marker of oxidative cell damage) was reduced by allopurinol5, and in other studies the incidence of complications following surgery was reduced by 70% by administering the drug both before and after the operation.6
A second option is to enhance the tissues’ capacity to trap the free radicals. A number of antioxidants and free radical scavengers have been investigated. Recombinant superoxide dismutase (SOD), an enzyme that detoxifies O2-•, has shown promise in some animal models of reperfusion injury1, but it was ineffective in others.7,8 Clinical trial results have also been variable. Pollak et al.9 administered SOD or placebo as a bolus before reperfusion of transplanted kidneys and as an infusion for an additional hour, but there was no difference in post-operative renal function. In another study a similar dose of SOD was administered as a single rapid infusion before reperfusion of renal transplants.10 The incidence of acute rejection was greatly reduced and long term graft survival was enhanced. This was attributed to a reduction in free radical damage and consequently less stimulation of the immune system by the graft. Conversely a trial of SOD in 120 patients undergoing angioplasty for acute myocardial infarction11 found no beneficial effect of the enzyme on cardiac function.
Neutrophil activity is also implicated in reperfusion injury. Neutrophils use the destructive capabilities of free radicals to attack bacteria, and there is evidence that they also act on reperfused tissue. Formigli et al.12 studied the effects of ischemia/reperfusion of the legs in patients undergoing temporary aortic occlusion during surgery. Biopsies of the muscles showed infiltration with granulocytes, mostly neutrophils, during ischemia and reperfusion and an increase in circulating neutrophils. Morphological injury to the muscle fibers was evident only after reperfusion. Fabiani et al.13 demonstrated that neutrophils entered the myocardium during CABG and degranulated on reperfusion. Neutrophils also contribute to ischemia when they accumulate in such numbers that they block the microvasculature.12
Neutrophil activity seems to be associated with endothelial damage and is part of the inflammatory response, coordinated by a network of inflammatory cytokines and chemokines that is not completely understood. Seekamp et al.14 used a rat leg model of ischemia/reperfusion to investigate the kinetics of cytokine production. IL-1, TNF-alpha and IL-6 all increased dramatically on reperfusion. TNF-alpha and IL-1 peaked after two hours after blood flow was restored and IL-6 continued to rise throughout the four hour recording period. Furthermore, the amount of injury to both the leg muscles and the lungs was reduced significantly by antibodies to IL-1 and TNF, by IL-1 receptor antagonist and by soluble TNF receptor. Where large volumes of tissue are reperfused, as in this study, post-ischemic syndrome may develop. This has many features in common with the systemic inflammatory response syndrome (SIRS) that occurs in sepsis, including acute respiratory distress and organ failure, and it is similarly associated with large amounts of inflammatory cytokines in circulation.
Another approach to reducing neutrophil mediated reperfusion injury is to inhibit neutrophil infiltration of the ischemic tissue. Adhesion molecules are needed for neutrophils to extravasate, and several studies have shown that the levels do indeed increase during ischemia/reperfusion. Consequently antibodies to adhesion molecules, including ICAM-115,16 and P-selectin17, have been tried as potential therapies in animals, with considerable success. A selectin blocker that binds to the neutrophil ligand for both E- and P-selectin also reduced infarct size.18
Studies of reperfusion injury and attempts to control it present no clear cut solutions as yet. Possibly because of the variety of experimental protocols, none of the potential therapies to limit the generation of free radicals on reperfusion has been consistently beneficial in controlled trials. The involvement of inflammatory cytokines and their effects on neutrophils is a comparatively recent discovery, and it opens new avenues for investigation. Perhaps, in the longer term, a combination of better techniques for rapid reperfusion and use of anti-oxidants and neutrophil inhibitors will reduce the enormous cost, both human and economic, of ischemia/reperfusion injury.