**2. Oxidative stress and multiple sclerosis**

OS is a cellular state where the homeostasis of redox reactions is altered when the production of reactive oxygen (ROS) and reactive nitrogen species (RNS) exceed their elimination. These reactive species are generated, among other causes, by oxidative metabolism. Neurons of the CNS are very active in oxidative metabolism, as they are constantly exposed to low‐to‐ moderate levels of ROS, and these species are removed by antioxidants (melatonin, vitamin D, vitamin E, glutathione) and antioxidant enzymes (superoxide dismutase, catalase, gluta‐ thione peroxidase, etc.). In chronic inflammatory diseases, such as MS, antioxidant defenses are overcome, which leads to oxidative stress [3].

Collectively, the ROS are reactive species derived from oxygen that include the superoxide anion (O- ), hydrogen peroxide (H2O2), and the hydroxyl radical (•OH). The RNS are reactive species derived from nitrogen and include nitric oxide (NO**∙**) and peroxynitrite (ONOO- ). The ROS and RNS are extremely unstable and reactive because they have an unpaired electron in their outer orbital. They take electrons from proteins, lipids, carbohydrates, and nucleic acids, causing damage to biological membranes, genetic material, and other macromolecules. The CNS is particularly vulnerable to oxidative damage since it has a very active mitochondrial metabolism, which leads to high levels of intracellular superoxide anions. Moreover, oligo‐ dendrocytes have low levels of antioxidant enzymes and a high concentration of iron. Unsaturated fatty acids are the most vulnerable to free radicals, and because myelin has a high lipid‐to‐protein ratio, it is a preferred target of ROS [4]. The ROS are generated by a number of cellular oxidative and metabolic processes including activity of the enzymes of the mito‐ chondrial respiratory chain, xanthine oxidase, NADPH oxidase, monoamine oxidases, and metabolism of arachidonic acid (AA) mediated by the activity of lipoxygenases (LOX), and ROS are produced primarily by leakage of electrons in the mitochondrial respiratory chain [3].

**Figure 2.** Oxidative stress levels are directly related to the progression of MS. Pathway Builder Online Tool was used to draw the figure [27].

Numerous studies in MS patients have shown an increase in the production of OS markers (such as cholesteryl ester hydroperoxides) and lower levels of uric acid (a ONOO scavenger). These changes are accompanied by significant deficiencies in antioxidant enzymes compared to healthy subjects. The increase in ROS coupled with decreased antioxidant capacity is not enough to entirely explain the pathogenesis of MS [4, 5]. Other reports suggest that the loss of myelin nerve sheath is possible because the immune system participates in combination with defects in the mitochondria, and these defects cause the generation of ROS and RNS. Macro‐ phages and monocytes release mediators of OS that degrade the unsaturated fatty acids. The ROS have also been implicated as a mediator of demyelination of axonal damage in MS and experimental autoimmune encephalomyelitis (EAE) [6]. It is important to mention that in assessing platelets in MS patients, increased activity of free radicals with decreased levels of important antioxidants such as glutathione and alpha‐tocopherol has been reported [7] (**Figure 2**).

moderate levels of ROS, and these species are removed by antioxidants (melatonin, vitamin D, vitamin E, glutathione) and antioxidant enzymes (superoxide dismutase, catalase, gluta‐ thione peroxidase, etc.). In chronic inflammatory diseases, such as MS, antioxidant defenses

Collectively, the ROS are reactive species derived from oxygen that include the superoxide

ROS and RNS are extremely unstable and reactive because they have an unpaired electron in their outer orbital. They take electrons from proteins, lipids, carbohydrates, and nucleic acids, causing damage to biological membranes, genetic material, and other macromolecules. The CNS is particularly vulnerable to oxidative damage since it has a very active mitochondrial metabolism, which leads to high levels of intracellular superoxide anions. Moreover, oligo‐ dendrocytes have low levels of antioxidant enzymes and a high concentration of iron. Unsaturated fatty acids are the most vulnerable to free radicals, and because myelin has a high lipid‐to‐protein ratio, it is a preferred target of ROS [4]. The ROS are generated by a number of cellular oxidative and metabolic processes including activity of the enzymes of the mito‐ chondrial respiratory chain, xanthine oxidase, NADPH oxidase, monoamine oxidases, and metabolism of arachidonic acid (AA) mediated by the activity of lipoxygenases (LOX), and ROS are produced primarily by leakage of electrons in the mitochondrial respiratory chain [3].

**Figure 2.** Oxidative stress levels are directly related to the progression of MS. Pathway Builder Online Tool was used

species derived from nitrogen and include nitric oxide (NO**∙**) and peroxynitrite (ONOO-

), hydrogen peroxide (H2O2), and the hydroxyl radical (•OH). The RNS are reactive

). The

are overcome, which leads to oxidative stress [3].

anion (O-

152 Trending Topics in Multiple Sclerosis

to draw the figure [27].

**Figure 3.** Damage to axons. Pathway Builder Online Tool was used to draw the figure [27].

The molecular mechanisms proposed to explain how ROS could specifically mediate brain damage are the following: (1) The lower levels of antioxidants can promote increased activity of lipoxygenase in CNS stimulating leukotriene production, thereby increasing the immu‐ noinflammatory processes in the cerebral cortex; (2) the damage to myelin can be caused by activation of T cells that may be activated for the presence of free radicals produced by the synthesis route of AA. Then appear the markers of OS associated with reduced activity of superoxide dismutase and the increase in glutamine, followed by increases of ∙OH and the production of peroxides which ultimately has a negative impact on myelin. After that, the evident changes in mitochondrial activity and finally changes in membrane fluidity (particu‐ larly, mitochondrial membranes) appear [8].

**Figure 4.** Effect of nitric oxide in inflammation. Pathway Builder Online Tool was used to draw the figure [27].

Paraclinical studies have shown an increased metabolism of the RNS in serum, lymphocytes, and cerebrospinal fluid of MS patients, which correlate to pathology studies. ONOO is also closely associated with acute inflammatory lesions [9]. Damage to axons is mediated by the following: (1) failure in mitochondrial energy metabolism due to inhibition of the respiratory chain by nitric oxide, which in turn causes a decrease in Na+ /K+ ATPase activity and alters Na + ‐dependent glutamate transporters, (2) over‐expression of glutamate receptors, (3) oligoden‐ droglial excitotoxicity, (4) massive influx of extracellular Ca++, (5) activation of proteases, and (6) impaired axonal transport. These mechanisms produce glutamate excitotoxicity and increased generation of nitric oxide leading to nitrosative stress. Nitric oxide is a highly toxic element that by itself blocks nerve conduction, especially in demyelinated axons, and stimu‐ lates apoptosis. When nitric oxide is combined with the superoxide anion, it generates a potent free radical, the pro‐oxidant peroxynitrite. Glutamate in turn causes neurodegeneration through the AMPA and NMDA receptors in oligodendrocytes and astrocytes (**Figure 3**). It is possible to explain the role of mediators using an experimental model of autoimmune encephalitis: Protection against the experimental disease occurs after administration of a glutamate antagonist [10].

Under physiologic conditions, nitric oxide is produced from L‐arginine by constitutive nitric oxide synthase (cNOS) and participates in a variety of important biological functions such as immunoregulation of inflammatory reactions, the downregulation of tumor necrosis factor (TNF)‐α production, MHC II expression in macrophages, induction of apoptosis in CD4 cells, physiological regulation of the mitochondrial respiratory chain, inhibition of antigen presen‐ tation, and leukocyte adhesion and migration. However, during inflammatory reactions, exposure of macrophages to interferon (IFN)‐γ and TNF‐α results in the activation of the inducible isoenzyme of NOS (iNOS), which increases up to 10 times the levels of nitric oxide. Nitric oxide facilitates the formation of peroxynitrite radicals. Only cells capable of generating a high flow of NO• have the potential for causing nitrosative stress. The role of nitric oxide in MS is therefore complex, and in fact, peroxynitrite is definitely more toxic than nitric oxide [9] (**Figure 4**).
