Ischaemia is defined as inadequate blood supply to a part of the body, usually caused by partial or total blockage of an artery. Reperfusion injury occurs when tissue perfusion and oxygenation are restored to an affected area after an ischaemic event.
Ischaemia/reperfusion (I/R) injury is a complex cascade of events resulting in devastating effects to the body, sometimes including death. Despite more than 70 years of research, I/R injury is not fully understood. Events such as gastric dilatation-volvulus (GDV) (Figure 1), mesenteric torsion, or where circulation of the limb is cut off (Figure 2) (e.g. rubber band around paw) all lead to I/R injury. It is important for all veterinary personnel to understand I/R injury so treatment and prevention can begin as early as possible.
The ischaemic cascade
The chain of events involved in I/R injury can be broken down into the ischaemic cascade (Box 1) and reperfusion injury (Box 2) An ischaemic episode involves a series of events called the ischaemic cascade. Within 5 minutes of the development of ischaemia, the electrolyte balance within cells becomes disturbed (Grace and Mathie, 1999). The ischaemic cascade usually continues for 2 or 3 hours, but can last for days, even after perfusion is restored to the affected area (Sege, 2006). The term cascade suggests that events follow a sequential pattern, which is not true of the ischaemic cascade. Events can occur simultaneously and do not always occur in a linear pattern (Grace and Mathie, 1999).
To fully understand the ischaemic cascade, it is important to consider adenosine triphosphate (ATP) and how it functions. ATP is a multifunctional nucleotide (a structural factor of DNA and RNA) that is considered to be the most important nucleotide responsible for transporting energy for metabolism within cells (Guyton and Hall, 2010). One of the fastest ways that ATP is produced is by oxidative phosphorylation, implying that ATP production requires oxygen (Guyton and Hall, 2010). Despite the importance of ATP, cells do not stockpile ATP (Guyton and Hall, 2010). They only make what they need for a particular time. When ischaemia occurs, oxygenation of cells ceases, resulting in anaerobic ATP production, which is less efficient (Guyton and Hall, 2010).
When oxygen becomes unavailable to cells, anaerobic glycolysis starts. This can be a lifesaving way for cells to obtain energy; however, this process is extremely wasteful. During the anaerobic process, pyruvic acid and hydrogen atoms combine with nicotinamide adenine dinucleotide (NAD) to form NADH and H+ (Guyton and Hall, 2010). If the buildup of NADH and H+ becomes too great, the anaerobic process stops, thus terminating energy production to maintain cells (Guyton and Hall, 2010). However, NADH and H+ combine to form lactic acid, which diffuses from cells rapidly so that the process can continue (Guyton and Hall, 2010). Although this is not ideal, the body can safely continue this process for several minutes. If the process continues for too long, as in ischaemia, lactic acid can build up, indicating worsening illness. As a consequence of lactic acidosis, pH decreases, injuring and inactivating mitochondria. Some researchers think that lactic acid may also interfere with the recovery of aerobic ATP production after ischaemia (Guyton and Hall, 2010). For all ischaemic patients, a lactate level should be obtained. Values <2 mmol/litre are normal.
When ATP fails to form, cells become depolarised, allowing calcium and sodium (normal extracellular electrolytes) to enter cells (McMichael, 2006). Potassium, which is normally found in cells, leaks rapidly into the extracellular space (McMichael, 2006). Excessive calcium over excites cells, creating free radicals and many calcium-dependent enzymes. The extent of ischaemic damage is related to the amount of calcium that enters cells and the duration of time that the intracellular calcium level remains elevated (Kamada et al, 1996). The longer calcium stays in cells, the greater the amount of harmful chemicals will be created.
One of the most important events involving calcium is the conversion of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) (Wingfield and Raffe, 2002). XO requires oxygen for activation. During ischaemia, oxygen is not present, so XO accumulates without getting used. Later, during reperfusion, XO can damage cells.
As the mitochondria break down, they release toxins, causing apoptosis. Apoptosis is the body's way of safely disposing of dead cell parts by autolysis (self-destruction) of cells. Another important event during ischaemia is the activation of nuclear factor–kB (NFkB), leading to the production of inflammatory mediators (Grace and Mathie, 1999). NFkB becomes activated during stress (Guyton and Hall, 2010). NFkB activates inflammatory cytokines and their receptors as well as platelet-activating factor (Grace and Mathie, 1999). This allows neutrophils to enter through the vascular endothelium. Activated neutrophils are generally more rigid and stiff because of hypoxia and acidosis, which accompany ischaemia. Because of the alteration of the cell membrane and the high number of neutrophils, capillaries may become plugged or clogged by neutrophils (Ambrosio and Tritto, 2002). Even after reperfusion, the redistribution of blood to affected areas may not produce enough force to clear clogs (Ambrosio and Tritto, 2002). The full pathway of NFkB is still not understood (Grace and Mathie, 1999).
Reperfusion injury
It would seem that simply reintroducing oxygen into the affected area would be beneficial. In patients with GDV, oxygen is restored when the stomach is decompressed or untwisted, allowing oxygen and blood to flow back into the stomach wall. However, the reintroduction of oxygen into affected areas initiates a complex chain of events. Despite the harsh effects of ischaemia alone, they do not cause nearly as much damage as reperfusion does (Grace and Mathie, 1999). The longer the onset of the ischaemic event, the greater the insult from reperfusion injury (Grace and Mathie, 1999).
One of the first events in reperfusion is that oxygen finally binds with XO that has built up during ischaemia. XO combines with oxygen and hypoxanthine to form superoxide (O2-), a radical (McMichael, 2006). Superoxide is not that damaging but can inactivate iron–sulphur–containing enzymes, liberating free iron and generating highly reactive hydroxyl (-OH) radicals. Hydroxyl is considered to be a reactive oxygen species (ROS).
Free radicals are radicals that move from where they were created. They are highly reactive and are usually involved in chemical reactions. An ROS is an oxygen-containing molecule that is very chemically reactive even though it may not be classified as a free radical. ROS molecules react quickly with other molecules. If present in high levels, they can damage cellular macromolecules such as DNA and RNA or cause endothelial injury, microvascular dysfunction, and apoptosis (cell death) (McMichael and Moore, 2004). ROS can form within 10 to 30 seconds after the onset of reperfusion (McMichael, 2006).
During ischaemia, neutrophils leak into the endothelium because of the activation of NFkB and XO. Reperfusion accelerates the influx of neutrophils to the affected area (Das, 1994). They respond because of the inflammatory response that takes place. Neutrophil activation alone can lead to even more ROS formation (McMichael, 2006). The inflammatory cascade accelerates during reperfusion. In short, neutrophils and macrophages attack reperfused tissues. Inflammatory cytokines are released as neutrophils are activated (Grace and Mathie, 1999). They are released by activated monocytes, macrophages, and neutrophils. When the body becomes over-whelmed with inflammatory cells, cytokines can be overproduced, resulting in massive cytokine influx (hypercytokinemia) into the affected tissue (Grace and Mathie, 1999). The exact mechanism behind this phenomenon is not fully understood (Grace and Mathie, 1999).
Complications
The ischaemic insult sets up the body for a damaging chain of events that is initiated by reperfusion. Although some researchers debate the exact relationship between I/R injury and these events, the end result can be the death of the patient. The most common complications include disseminated intravascular coagulation, systemic inflammatory response syndrome, multiple organ dysfunction syndrome and, if the I/R injury occurred to the muscles, rhabdomyolysis.
Treatment
There is no known definitive cure for I/R injury. Many doctors and scientists agree that stopping the cascade at the earliest possible point would produce the best results.
ROS formation
In recent years, human and veterinary researchers have conducted several promising studies of I/R injury using N-acetylcysteine, which is a powerful scavenger of the hydroxyl radical (McMichael M, 2006). While most studies involving rats, rabbits, and mice have produced promising results with this drug, the most recent veterinary-related study showed that the use of N-acetylcysteine failed to decrease I/R injury of the liver in a canine model (Baumann et al, 2008). One of the most recent studies concluded that N-acetylcysteine protects lung tissue from the effects of I/R (Takhtfooladi et al, 2016). It is clear that more research is needed.
Deferoxamine has been studied for more than 20 years in humans and is now being studied in animals. The research in humans and animals has been promising; in a study in 2009, deferoxamine significantly protected rats with ischaemic stroke from I/R injury (Marin et al, 1998; Hanson et al, 2009). This is because deferoxamine is an iron chelator and, therefore, inhibits hydroxyl radical formation (White, 2007). A recent study showed that the use of N-acetylcysteine and deferoxamine, but not their isolated use, prevented an increase in creatinine following an I/R injury event in the kidneys (Bernardi et al, 2012).
Allopurinol can inhibit XO formation and neutrophil infiltration during reperfusion (Zhou et al, 2016). The best results have been obtained when allopurinol has been used as a pretreatment against I/R injury. Because predicting when I/R injury will occur is almost impossible, it would be difficult to obtain optimal results on the use of allopurinol in a clinical setting (Zhou et al, 2016).
Other options
Ketamine may also prove to be a cost-effective and safer treatment for I/R injury. Ketamine inhibits N-methyl-D-aspartate receptors, reduces neutrophil adhesion, and decreases cytokine production (Szekely et al, 1999; Ersek, 2004). A study in 2009 concluded that the use of ketamine as an anaesthetic reduced intestinal I/R injury in rats (Cámara et al, 2008). Ketamine has analgesic properties and is frequently used in veterinary medicine in post-surgical constant rate infusion analgesic combinations such as morphine–lidocaine–ketamine.
The use of colloids (hydroxyethyl starches) may have some benefit in reducing the effects of I/R injury (White, 2007). This is likely because high-molecular-weight colloids can help to decrease microvascular permeability. A 2008 study concluded that hydroxyethyl starches were superior to other colloids when used as a pretreatment for protection against I/R injury effects (Varga et al, 2008).
Therapeutic hypothermia has been shown to help minimise harmful effects of the inflammatory cascade and decrease ROS production during reperfusion (Adler, 2007). One of the most common I/R injuries in humans follows cardiac arrest, which cuts off oxygen to the heart. In 2007, Dr Lance Becker at the University of Pennsylvania, showed that cooling the body after cardiac arrest increases the chance of survival by 16% (Vanden et al, 2006; Adler, 2007). This prompted the American Heart Association to recommend cooling of every cardiac arrest patient (American Heart Association, 2007).
Since 2007, an injectable ice–salt mixture has allowed emergency personnel to quickly cool humans to help slow or even prevent I/R injury. In the United States paramedics carry cool sodium chloride fluids in their ambulances so when a person has a heart attack they can drop their body temperature immediately to decrease I/R injury later (American Heart Association, 2007).
Therapeutic hypothermia has started to be used in veterinary medicine during surgical procedures that induce a I/R injury effect. The most common use being tried in veterinary teaching hospitals is with cardiac surgery. A 2015 published case report found that therapeutic hypothermia helped to protect the brain during cardiopulmonary bypass surgery. The report went on to state that ‘for most cardiac surgical procedures, mild to modest (32–36°C) hypothermia will be sufficient to assure neuroprotection’ (Otto, 2015). While still in its infancy in veterinary medicine, therapeutic hypothermia is used widely in human surgical cases as well as in the emergency room.
Conclusion
The pathology of I/R injury is extensive and not fully understood, even in humans. More research is needed to help develop tests and treatments for I/R injury. To improve medical and veterinary knowledge, it is important to identify I/R injury and record treatments used.