Role of nitric oxide in cerebral ischemia/reperfusion injury: A biomolecular overview

Abstract

Nitric oxide (NO) is a gaseous molecule produced by 3 different NO synthase (NOS) isoforms: Neural/brain NOS (nNOS/bNOS, type 1), endothelial NOS (eNOS, type 3) and inducible NOS (type 2). Type 1 and 3 NOS are constitutively expressed. NO can serve different purposes: As a vasoactive molecule, as a neurotransmitter or as an immunomodulator. It plays a key role in cerebral ischemia/reperfusion injury (CIRI). Hypoxic episodes simulate the production of oxygen free radicals, leading to mitochondrial and phospholipid damage. Upon reperfusion, increased levels of oxygen trigger oxide synthases; whose products are associated with neuronal damage by promoting lipid peroxidation, nitrosylation and excitotoxicity. Molecular pathways in CIRI can be altered by NOS. Neuroprotective effects are observed with eNOS activity. While nNOS interplay is prone to endothelial inflammation, oxidative stress and apoptosis. Therefore, nNOS appears to be detrimental. The interaction between NO and other free radicals develops peroxynitrite; which is a cytotoxic agent. It plays a main role in the likelihood of hemorrhagic events by tissue plasminogen activator (t-PA). Peroxynitrite scavengers are currently being studied as potential targets to prevent hemorrhagic transformation in CIRI.

Key Words: Nitric oxide; Cerebral ischemia/reperfusion injury; Nitric oxide synthase; Reactive nitrogen species; Nitrosylation

Core Tip: Nitric oxide (NO) plays a key role in cerebral ischemia/reperfusion injury (CIRI). Ischemic episodes lead to mitochondrial and phospholipid damage. While reperfusion is associated with neuronal damage by promoting lipid peroxidation, nitrosylation and excitotoxicity. Neural NO synthase (nNOS) interplay is prone to endothelial inflammation, oxidative stress and apoptosis. Therefore, nNOS appears to be detrimental. The interaction between NO and other free radicals develops peroxynitrite. And, limiting the negative effects of NO-derived compounds has been implemented as an important strategy to improve neurological outcomes after CIRI.

INTRODUCTION

Tissue ischemia/reperfusion triggers a biomolecular response that involves reactive oxygen species. In physiological conditions, antioxidant mechanisms are sufficient to protect the cells from free radicals[1,2]. However, under oxidative stress, reactive nitrogen species (RNS) are one of the primary effectors of cerebral ischemia/reperfusion injury (CIRI), by triggering calcium-dependent nitric oxide synthases (NOS). Nitric oxide (NO) is a gaseous molecule produced by 3 different NOS isoforms: Neural/brain NOS (nNOS/bNOS, type 1), endothelial NOS (eNOS, type 3) and inducible NOS (iNOS, type 2). Type 1 and 3 NOS are constitutively expressed[3]. nNOS produces different types of RNS; such as nitrogen dioxide (NO₂) and peroxynitrite (ONOO-)[4]. Specifically, this ion is highly reactive with biological molecules in the respiratory complex III and IV. These reactions result in cytotoxic effects due to a significant reduction in ATP levels. Therefore, altered resting membrane potential and osmotic moves take place. The presence of these enzymes is associated with three major events: Mitochondrial membrane damage, mitochondrial membrane permeability and protein nitrosylation[5,6].

Membrane damage is mediated by free radicals upon linoleic and arachidonic acid, a process called lipid peroxidation. As a result, there is an increased mitochondrial membrane permeability. Therefore, free radical outflow from the mitochondria into the cytoplasm ensues[7]. Excessive oxidative conditions can overcome glutathione, nicotinamide adenine dinucleotide phosphate and lysosomal iron sequestration, which are protective mechanisms against oxytosis/ferroptosis. Ferroptosis is a type of programmed cell death (PCD) mediated by iron lipid peroxidation (Figure 1). This PCD is genetically and biochemically different from other types of apoptosis[8]. NO is incorporated to another molecule through a chemical process called Nitrosylation. This usually occurs when an organic molecule “R” receives a NO molecule through a covalent bond: “R - N = O”. This reaction gives rise to nitroso compounds. And there are multiple nitroso compounds. However, the most common interaction is between sulfur and NO, which results in S-nitroso compounds. Eventually, NO₂ reacts with biological molecules in a process called nitration. And Tyrosine residues, present in proteins, are prone to nitration. This reaction leads to 3-nitrotyrosine, which is a well-documented marker of NO-dependent oxidative damage[9].

Figure 1 The biomolecular pathways of nitric oxide in brain damage after cerebral ischemia/reperfusion injury. eNOS: Endothelial nitric oxide synthase or type 3; nNOS: Neuronal nitric oxide synthase or type 1; iNOS: Inducible nitric oxide synthase or type 2); NO: Nitric oxide; NO₂: Nitrogen dioxide; ONOO^-: Peroxynitrite; PCD: Programmed cell death.

It has been documented that eNOS-induced NO plays a key role as brain parenchyma blood flow regulator. Adequate blood flow reduces oxidative, procoagulant and inflammatory pathways[10,11]. Cerebrovascular accident is one of the main causes of morbidity and mortality worldwide. Pathophysiology of acute stroke can be either ischemic or hemorrhagic. Ischemic type represents 85% of the cases[12,13]. Upon reperfusion, restoration of oxygen levels results in detrimental effects. In the central nervous system, this phenomenon is referred to as CIRI[14]. The relationship between NO and CIRI depends on both, brain region and the source of NO. During CIRI there is eNOS downregulation, while nNOS is upregulated. Animal models of CIRI have demonstrated that RNS-mediated damage is due to nNOS upregulation[15-17]. Neural NOS appears to play a major role in CIRI because its activity is the highest 1 hour after reperfusion[16,18].

NO

NO higher levels are associated with oxidative stress, excitotoxicity and neural apoptosis[19]. With reperfusion, extracellular hydrogen excess and Calcium (Ca⁺) ions are moved back into the cell. It is well known that mitochondria suffer continuous swelling in order to meet metabolic demands. However, calcium excess alters mitochondrial ion homeostasis and mitochondrial matrix structure, eventually leading to pathological swelling[20,21]. This alteration results in an increased membrane permeability and cytochrome cleakage. Cytochrome c is a protein that transports electrons between Complex III and IV and triggers PCD through caspase activation[20,21]. Nitrosylation and nitration can further modify cytochrome c. There is, however, controversial information about reduced proapoptotic properties of cytochrome c, due to specific tyrosine nitration residues. Nevertheless, evidence indicates that cytochrome cnitration modifies correct electron transport[22]. Current knowledge indicates that nNOS downregulation is associated with less neurological damage[18,23,24]. While more deleterious effects are shown when iNOS reaches significant activity. This usually occurs 12 hours after reoxygenation/reperfusion process[18,24,25].

Limiting the negative effects of NO-derived compounds has been implemented as an important strategy to improve neurological outcomes. Although the specific mechanism has not yet been clarified, Edaravone^Ò is used as treatment for amyotrophic lateral sclerosis and stroke due to its antioxidant properties[3]. The use of Edaravone^Ò in mice models of CIRI has not demonstrated significant differences in nitrite (NO_2-) levels, cerebral blood flow and blood pressure. However, treated animals show a significant reduction in both nNOS and hydroxyl radicals. Therefore, better outcomes observed with Edaravone^Ò obey to neuroprotective effects[26].

Tissue plasminogen activator is another drug indicated within the first 4.5 hours of ischemic stroke[27]. Hemorrhagic transformation is more likely to occur when administered after this time frame[27,28]. This effect is the result of Matrix Metalloproteinases triggering by RNS such as peroxynitrite. Baicalin is being studied as a RNS scavenger for different diseases. However, its role remains controversial[27].

CONCLUSION

RNS downregulation appears to be a promising practice to minimize CIRI. Drugs currently under study show significant antioxidant and neuroprotective effects. However, measuring the efficacy of such drugs remains a challenge.