Reperfusion injury

Editor-In-Chief: C. Michael Gibson, M.S., M.D. [1] Associate Editor(s)-in-Chief: Shivam Singla, M.D.[2]

Reperfusion injury, also known as ischemia-reperfusion injury (IRI) or re-oxygenation injury, is the tissue damage which results from the restoration of blood supply to the tissue after a period of ischemia, anoxia or hypoxia from different pathologies. During the period of absence of blood to the tissues a condition is created in which the resulting reperfusion will result in inflammation and oxidative damage through the involvement of various mechanisms mainly involving oxidation, free radical formation and complement activation which ultimately leads to cell death, rather than restoration of normal function. Various intracellular or extracellular changes during ischemia leads to increased intracellular calcium and ATP depletion that will ultimately land up in the cell death if the ongoing process does not stopped. Reperfusion forms reactive oxygen species . This leads to Increased mitochondrial pore permeability, complement activation & cytochrome release, inflammation and edema formation, Neutrophil platelet adhesion and thrombosis leading to progressive tissue death. In Heart reperfusion injury is attributed to oxidative stress which in turn leads to arrhythmias, Infarction and Myocardial stunning. In case of trauma the resulting restoration of blood flow to the tissue after prolonged ischemia aggravates tissue damage by either directly causing additional injury or by unmasking the injury sustained during the ischemic period. Reperfusion injury can occur in any organ of body mainly seen in the heart, intestine, kidney, lung, and muscle, and is due to microvascular damage.

The component playing a major role in the pathophysiology of Ischemia-reperfusion injury is Reactive oxygen species (ROS) causing damage to cellular and biological membranes. Neutrophils also play an important role in initiating and propagating much of the damage involved in the process of Ischemia-reperfusion injury. Ischemia is the phase that precedes the restoration of blood flow to that organ or tissue, resulting in the built-up of xanthine oxidase and hypoxanthine that upon the restoration of blood flow leads to the formation of ROS. Neutrophils also potentiate the effect of Ischemia-reperfusion injury through microvascular injury by releasing various proteolytic enzymes and ROS. Most of the experimental studies carried out in helping understand the mechanism of Ischemia reperfusion injury are mainly on the cat, dog, and horses.

Ischemia reperfusion injury is a complex disorder associated with various cardiovascular and other risk factors mainly including Hypertension, hyperlipidemia, Diabetes, Insulin resistance, aging, and defects with coronary artery circulation. Although the exact mechanism about how these causes injuries are still not clear but studies have done so far best explains their role in mediating oxidative stress and endothelial cell dysfunctions, the two most important pathophysiological processes involved in the mediation of injury.

Reperfusion injury mechanism is mostly studied by scientists in cats and dogs with the first experimental study done in 1955 by Sewell and later different studies were done to understand more about the reperfusion injury mechanisms. Most of the complications associated with reperfusion injury are mainly due to the formation of reactive oxygen species and neutrophil activation ultimately resulting in tissue damage-causing Ischemia, Infarction, Haemorrhage, and edema. Prognosis, in general, is poor with Ischemia-reperfusion injury resulting in tissue injury and damage. The most important determinant of prognosis is the time taken to reperfuse the ischemic tissue. More the delay in time to reperfusion, worse the prognosis is.

The most common myth about the ischemia-reperfusion injury is itself related to blood flow. One can easily think like if everything is happening due to ischemia and with the restoration of blood flow, the injury should heal. Here is the trick, reperfusion in turn further exacerbates the injury mainly due to the formation of free radicals. There are few approaches that are studied widely and do play a major role in controlling the injury related to ischemia-reperfusion injury

• Prevent generation of free radicals( Oxidative stress) or Increase the tissue’s capacity to trap the free radicals
• Controlling the neutrophil activation and infiltration of ischemic tissue
• Hypoxic pre-conditioning

Hyperbaric oxygen therapy is also studied widely and best suited when used within 6 hrs of hypoxia as it helps in the reduction of local and systemic hypoxia and in turn, increases the survival of affected tissue.

A lot of studies done in the past three decades helped a lot in understanding the molecular mechanisms associated with Ischemia-reperfusion injury. Also, these studies helped in evaluating various strategies to decrease the incidence and severity associated with Ischemia-reperfusion injury. Existing therapies for Ischemia-reperfusion injury can be divided into Pharmacological and non-pharmacological interventions. A lot of promising studies and clinical trials are still under the pipeline. Till the date, the most encouraging results are associated with ischemic preconditioning and postconditioning, adenosine, and exenatide. A lot of studies have demonstrated the combined effect of pharmacological and nonpharmacological approach as together to be used as a multifactorial approach to improve the clinical outcomes.

Editor-In-Chief: C. Michael Gibson, M.S., M.D. [4] Associate Editor(s)-in-Chief: Anjan K. Chakrabarti, M.D. [5] Shivam Singla, M.D.[6] Kashish Goel,M.D.,

The component playing a major role in the pathophysiology of Ischemia-reperfusion injury is Reactive oxygen species (ROS) causing damage to cellular and biological membranes. Neutrophils also play an important role in initiating and propagating much of the damage involved in the process of Ischemia-reperfusion injury. Ischemia is the phase that precedes the restoration of blood flow to that organ or tissue, resulting in the built-up of xanthine oxidase and hypoxanthine that upon the restoration of blood flow leads to the formation of ROS. Neutrophils also potentiate the effect of Ischemia-reperfusion injury through microvascular injury by releasing various proteolytic enzymes and ROS. Most of the experimental studies carried out in helping understand the mechanism of Ischemia reperfusion injury are mainly on the cat, dog, and horses.

1) Ischemic phase

2) Reperfusion Phase

Reperfusion injury ( Ischemic Phase) During this phase mainly the dysregulation of metabolic pathways occurs and in the reperfusion phase there will be a generation of free radicals.

• Ischemia when the blood supply to the tissues decreases with respect to the demand required to function properly. This results in deficiency in oxygen, glucose and various other substrates required for cellular metabolism. As previously dais the derangement or dysregulation of metabolic function begins in this phase^[1]. Due to less oxygen supply cellular metabolism shifts to anaerobic glycolysis causing the glycogen to breakdown resulting in the production of 2 ATP and a lactic acid. This decrease in tissue PH starts further inhibits the ATP generation by negative feed back mechanism. ATP gets broken down into ADP, AMP and IMP. This finally gets converted to adenosine, inosine, hypoxanthine and xanthine.

• Lack of ATP at the cellular level causes impairment in the function of ionic pumps – Na+/K+ and Ca²+ pumps. As a result cytosolic sodium rises which in turn withdraws water to maintain the osmotic equilibrium consequently resulting in the cellular swelling^[2]. To maintain ionic balance potassium ion escape from the cell. Calcium is released from the mitochondria to the cytoplasm and into extracellular spaces resulting in the activation of Mitochondrial calcium- dependent cytosolic proteases. These converts the enzyme xanthine dehydrogenase to xanthine oxidase. Phospholipases activated during ischemia promotes membrane degradation and increases level of free fatty acids

• Ischemia also induces expression of a large number of genes and transcription factors, which play a major role in the damage to the tissues^[3][4].
  • Transcription factors
    • Activating protein-1 (AP-1)
    • Hypoxia-inducible factor-1 (HIF-1) which in turn activates transcription of VEGF, Erythropoietin and Glucose transporter-1
    • Nuclear factor-kappa b (NF-kb)
    • Activation of NF-kb occurs during both the ischemic and reperfusion phases

The ROS play major role in the tissue damage related to ischemia reperfusion injury. Once the ischemic tissue is reperfused the molecular oxygen catalyzes the conversion of hypoxanthine to uric acid and liberating the superoxide anion (O₂^–). This superoxide gets further converted to (H₂O₂) and the hydroxyl radical (OH^•). This OH ion causes the peroxidation lipids in the cell membranes resulting in the production and release of proinflammatory eicosanoids and ultimately cell death^[5]. Reperfusion Injury

During the Ischemia-reperfusion injury ROS also activate endothelial cells, which further produces numerous adhesion molecules.^[6]

• E-selectin
• VCAM-1 (vascular cell adhesion molecule-1)
• ICAM-1 (intercellular adhesion molecule-1)
• EMLMl Am -1 ( endothelial-leukocyte adhesion molecule)
• PAi-1 (plasminogen activator inhibitor-1 ), and
• Interleukin-8 (il-8)

ROS causes lipid peroxidation of cell membranes resulting in the release of:

• Arachidonic acid (substrate for prostaglandins)
  • Prostaglandins usually have a vasodilatory effect hat provides protective effect during Ischemia reperfusion injury. But they have a short life so their fast depletion leads to vasoconstriction ultimately leading to reduced blood flow and exacerbation of ischemia^[7].
• Thromboxane
  • Plasma thromboxane A₂ level rises within minutes after reperfusion, resulting in vasoconstriction and platelet aggregation. This usually coincide with rapid rise in pulmonary artery pressure and a subsequent increase in pulmonary microvascular permeability^[8].

• Leukotrienes
  • Leukotrienes are also synthesized from arachidonic acid. Leukotrienes acts directly in the endothelial cells, smooth muscle and indirectly on the neutrophils. The leukotrienes C₄, D_4, and E₄ alters the endothelial cytoskeleton, resulting in increased vascular permeability and smooth muscle contraction, and finally leading to vasoconstriction^[9][10].

L-arginine is the substrate for the synthesis of Nitric oxide with the help of nitric oxide synthase enzyme. The nitric oxide synthase enzyme is usually of 3 types^[11]

• CNOS- Constitutive nitric oxide synthase enzyme
• INO S- Inducible nitric oxide synthase enzyme
• ENO S- Endothelial nitric oxide synthase enzyme

In the first 15 minutes of ischemia NO level rises due to transient ENOS activation. As said this elevation is transient so ultimately after a few minutes there will be a general decline in endothelial function resulting in the fall of NO production. The reduction in ENOS levels during ischemia reperfusion injury are also predisposed to vasoconstriction, the response mainly seen in IRI^[12].

These are peptide vasoconstrictors mainly produced from the endothelium. They mainly mediate vasoconstriction through Ca²⁺-mediated vasoconstriction. Endothelin -1 levels increase during ischemia reperfusion injury in both the phases of ischemia as well as reperfusion, that mainly help in capillary vasoconstriction. Endothelin – 1 inhibitors are studied widespread regarding their role in inhibiting vasoconstriction and increasing vascular permeability^[13].

Ischemia and reperfusion phase of ischemia reperfusion injury induces expression of numerous cytokines mainly:

• TNF-a
  • Elevated levels detected during cerebral and skeletal IRI^[14]. it can also induce the generation of ROS and enhance the susceptibility of vascular endothelium to neutrophil-mediated injury by increasing the expression of ICAM-1 which helps in binding of neutrophils to the endothelium^[15].
• IL-1, IL-6, IL-8
  • IL-6 is a proinflammatory cytokine produces in large amounts in hypo perfused tissues.
  • IL-8 is a neutrophil chemotactic and activating factor and mainly results in the diapedesis of activated neutrophils through the endothelium.
• PAF
  • It enhances the binding of neutrophils to the endothelial cells^[16][17].

These cytokines mainly generate systemic inflammatory response ultimately leads to multi organ failure.

Neutrophils plays Important role in the tissue damage^[18]. Activated neutrophils secrete proteases, metalloproteinase, that results in the degradation of basement membrane and contributes to tissue damage. Selectins^[19] are expressed on the surface of leucocytes, endothelial cells and platelets. Selectins^[20] play important role in the initiation of neutrophil–endothelial cell interactions (rolling) which is essential for their subsequent adhesion and extravasation. L-selectin are present on surface of neutrophils and help in the reversible attachment of neutrophils to endothelial cells. Antibody-mediated blocking of L-selectin studied widely and is one of the important treatment option under consideration.

Contributes in the pathogenesis of IRI. Reperfusion is usually associated with depletion of complement proteins, factor B that will indicates the turning on of alternate complement pathway^[21]. The C5b-9^[22] also gets deposited into the endothelial cell after ischemia leading to osmotic lysis.

• Central Nervous System
  • Reperfusion injury is a major pathophysiological mechanism involved in ischemia related injury to the central nervous system consequently resulting in the patients landing up with complications of a stroke, TIA, and other neurological problems. A lot of studies regarding this are still under the pipeline.

• Cardiovascular system
  • In the cardiovascular system, the most common complications studied are arrhythmias, and myocardial stunning and myocardial cells death also. According to various studies done so far, Impaired microvascular function is the main reason behind the myocardial stunning.

Editor-In-Chief: C. Michael Gibson, M.S., M.D. [2] Associate Editor(s)-in-Chief: Anjan K. Chakrabarti, M.D. [3]Shivam Singla, M.D.[4] Kashish Goel, M.D.

Ischemia reperfusion injury is a complex disorder associated with various cardiovascular and other risk factors mainly including Hypertension, hyperlipidemia, Diabetes, Insulin resistance, aging, and defects with coronary artery circulation. Although the exact mechanism about how these causes injuries are still not clear but studies have done so far best explains their role in mediating oxidative stress and endothelial cell dysfunctions, the two most important pathophysiological processes involved in the mediation of injury.

Risk factors for reperfusion injury include^[1]

• Hypertension with left ventricular hypertrophy,
• Congestive heart failure
• Increased age^[2],
• Diabetes, and
• Hyperlipidemia

It is important to identify the risk factors associated with worse reperfusion injury in STEMI patients. This may help in early risk stratification and develop therapeutic targets to reduce the infarct size associated with reperfusion injury. These risk factors also increase the risk of a first cardiac event and emphasize the importance of secondary prevention. Most of these associations are based on animal studies and includes:

• Left Ventricular hypertrophy:
  • Long-standing hypertension leads to pressure overload hypertrophy. This is associated with metabolic and biochemical changes, predisposing the myocardium to severe reperfusion injury.
  • Moreover, an increase in lactate dehydrogenase and creatine kinase release after reperfusion increases the susceptibility of the hypertrophied heart to ischemia/reperfusion injury.
• Heart failure: Left ventricular dysfunction may predispose the heart to reperfusion injury.
• Age: Aging is associated with^[3]
  • Oxidative stress as well as
  • Impaired systolic and diastolic function,Both the mech. increases the risk related to reperfusion injury.

• Diabetes: The data from preclinical studies in diabetic patients is not clear, however, there is clearly increased susceptibility of the heart to ischemic stress and possibly reperfusion.
• Hyperlipidemia: Hyperlipidemia is a known risk factor for ischemic heart disease.
  • Animal and human studies have shown that the presence of hyperlipidemia increases the risk of reperfusion injury and may also attenuate the protective effect of ischemic preconditioning.

Editor-In-Chief: C. Michael Gibson, M.S., M.D. [4] Associate Editor(s)-in-Chief: Shivam Singla, M.D.[5] Anjan K. Chakrabarti, M.D. [6] Kashish Goel, M.D.

Reperfusion injury mechanism is mostly studied by scientists in cats and dogs with the first experimental study done in 1955 by Sewell and later different studies were done to understand more about the reperfusion injury mechanisms. Most of the complications associated with reperfusion injury are mainly due to the formation of reactive oxygen species and neutrophil activation ultimately resulting in tissue damage-causing Ischemia, Infarction, Haemorrhage, and edema. Prognosis, in general, is poor with Ischemia-reperfusion injury resulting in tissue injury and damage. The most important determinant of prognosis is the time taken to reperfuse the ischemic tissue. More the delay in time to reperfusion, worse the prognosis is.

The Ischemia–reperfusion injury was first seen in 1955 by Sewell while performing ligation of Dog’s coronary arteries

Then later Jennings gave the term Myocardial IRI in 1960. He is the first person to name the term Ischemia Reperfusion Injury(IRI) on the basis of histological changes in canine myocardium.

Later various terms were given by the scientist depending upon the various organ system involved:

• In 1968 Ames gave the term Brain IRI

• 1972 Flore gave the term Kidney IRI

• Then Modry gave the term Lung IRI in 1978

• Greenberg gave the term Intestinal IRI in 1981 by his experiment showing reperfusion induced damage in the intestinal mucosal cells of a cat after long hours of ischemia.

Ischemic preconditioning was first explained by Murry et al. In 1986 he performed a study in dog explaining the importance of ischemia reperfusion in dogs on infarct size reduction^[1].

Ischemic post-conditioning was given by Zhao et al in 2003 and explained this with the help of an experiment showing a small episode of Ischemia or reperfusion performed immediately after the resumption of flow following a period of ischemia. They found that this helped in the reduction of infarct size in dogs by up to 40%^[2].

• Myocardial stunning: It is mainly defined as an abnormality in the contractile function of myocardium that sometimes persists even after the return of reperfusion and resolution of ischemia. It is mainly due to the release of
  • Reactive oxygen species
  • Intracellular calcium overload.

• Myocardial infarction: Irreversible myocyte cell death secondary to reduced oxygen delivery for more than 20-30 minutes will eventually leads to infarction. Reperfusion helps prevent complete loss of the involved area, however oxidative stress due to this may prevent complete resolution^[3].

• Acute heart failure: Loss of myocardial contractility and systolic dysfunction associated with ischemia/reperfusion injury may lead to the development of acute heart failure.

  

• Early reperfusion in the course of STEMI prevents myocardial necrosis and may lead to complete recovery of function resulting in a better prognosis as compared to a situation where there is a delay in perfusion^[4].

• Ventricular arrhythmias: Reperfusion of the blocked coronary artery can also precipitate arrhythmias ranging from ventricular premature beats to life-threatening ventricular fibrillation.

• Intestinal ischemia reperfusion is associated with the development of necrotizing enterocolitis, hypotension, and thrombosis.
  • It is mainly seen as a consequence of acute mesenteric ischemia that mainly results due to impaired blood flow to the intestines through the mesenteric vessels^[5].

Prognosis in CNS patients

• Those patients who are identified and treated early, the prognosis is better along with the decreased incidence of intracranial hemorrhage. Outcomes usually depend on the timely recognition and prevention of precipitating factors. Hypertension management is most important before it can inflict damage in the form of edema or hemorrhage^[6]
  • The prognosis following hemorrhagic transformation is poor. Mortality in such cases is 63%, and 80% of survivors have significant morbidity.
• In the case of Central nervous system the brain is a very sensitive organ to ischemia and results in death within 5 minutes of the onset of ischemia. Reperfusion is usually beneficial if it is conducted within a very short period of time after the onset of ischemia but in most cases, reperfusion leads to the development of cerebral ischemia and hemorrhage resulting in a bad prognosis.
• Ischemia reperfusion injury in kidneys is mainly associated with the development of high morbidity and mortality, worse prognosis with the involvement of corticomedullary junction.
• In CVS patients reperfusion injury is mainly associated with Arrhythmias, myocardial stunning, and myocyte death, which mainly results in the occurrence of Myocardial Infarction with a worse prognosis.