Pharmacological Therapies

**39**

**Chapter 4**

**Abstract**

limitations.

nitric oxide

**1. Introduction**

Cord Injury

Current Developments in

*Jonathan Vilchis Villa, Dulce M. Parra Villamar,* 

*Juan Herrera García and Raúl Silva García*

*José Alberto Toscano Zapien, Liliana Blancas Espinoza,* 

When spinal cord injury (SCI) occurs, numerous sources of reactive oxygen species and nitrogen species may be active within first minutes or hours and even reactivate few days later. Free radical formation and lipid peroxidation (LP) have been described as an important mechanism in the beginning and accelerated progress in the development of diverse pathologies, importantly in those related to central nervous system. The compromise of molecules and cellular structures due to the oxidative state of microenvironment in SCI may determinate survival or apoptosis of resident and infiltrating cells and polarization toward an inflammatory response, which lead to an extension of damaged tissue and loss of neuronal function, or a regulatory/regenerative response. The investigation of new antioxidant agents and their action at a molecular level begins to reveal mechanisms that, if correctly modulated, promise an improvement in recovery of functions with respect to conventional pharmacological therapies. In this chapter, we will review the general mechanisms of oxidative stress and lipid peroxidation, those antioxidant treatments in experimental development and clinical phase, as well as their achievements and

**Keywords:** antioxidant therapy, lipid peroxidation,free radicals, spinal cord injury,

Among the different pharmacological strategies for treating spinal cord injury (SCI), it has been observed that the quick intervention after the injury results in a better outcome for the patients [1]. This can be explained by the biochemical processes occurring at a cellular level that develop immediately after the mechanical damage, which define the subsequent physiological chain of events determining the evolution of pathophysiology of the SCI and, therefore, the degree of functional loss or recovery. One of the most important processes participating in the balance between the prevalence of damage or protection of tissue structure and the function in the central nervous system (CNS) is the generation of diverse reactive molecules by oxidative stress that target mainly lipids. This process is known as lipid

Antioxidant Therapies for Spinal

#### **Chapter 4**

## Current Developments in Antioxidant Therapies for Spinal Cord Injury

*Jonathan Vilchis Villa, Dulce M. Parra Villamar, José Alberto Toscano Zapien, Liliana Blancas Espinoza, Juan Herrera García and Raúl Silva García*

### **Abstract**

When spinal cord injury (SCI) occurs, numerous sources of reactive oxygen species and nitrogen species may be active within first minutes or hours and even reactivate few days later. Free radical formation and lipid peroxidation (LP) have been described as an important mechanism in the beginning and accelerated progress in the development of diverse pathologies, importantly in those related to central nervous system. The compromise of molecules and cellular structures due to the oxidative state of microenvironment in SCI may determinate survival or apoptosis of resident and infiltrating cells and polarization toward an inflammatory response, which lead to an extension of damaged tissue and loss of neuronal function, or a regulatory/regenerative response. The investigation of new antioxidant agents and their action at a molecular level begins to reveal mechanisms that, if correctly modulated, promise an improvement in recovery of functions with respect to conventional pharmacological therapies. In this chapter, we will review the general mechanisms of oxidative stress and lipid peroxidation, those antioxidant treatments in experimental development and clinical phase, as well as their achievements and limitations.

**Keywords:** antioxidant therapy, lipid peroxidation,free radicals, spinal cord injury, nitric oxide

#### **1. Introduction**

Among the different pharmacological strategies for treating spinal cord injury (SCI), it has been observed that the quick intervention after the injury results in a better outcome for the patients [1]. This can be explained by the biochemical processes occurring at a cellular level that develop immediately after the mechanical damage, which define the subsequent physiological chain of events determining the evolution of pathophysiology of the SCI and, therefore, the degree of functional loss or recovery. One of the most important processes participating in the balance between the prevalence of damage or protection of tissue structure and the function in the central nervous system (CNS) is the generation of diverse reactive molecules by oxidative stress that target mainly lipids. This process is known as lipid peroxidation (LP), and its end products could modify proteins and DNA present in cellular structures, causing cell death and a lower probability of regeneration [2]. SCI is a highly disabling and irreversible condition that causes physiological complications (bowel, cardiac, urinary, respiratory) and it has a social-economic impact in patients. The research of new agents targeting degenerative processes such as oxidative stress and LP is important especially due to the lack of efficacy and safety of conventional therapies on patients with SCI [1]. Here, we review the efforts to discover new compounds aimed to offer an option in antioxidant treatments and the use of some in combination or in an innovative way, both in experimental and in clinical trials. We would like to mention that there is a wide range of antioxidant therapies in study, and we are only briefly mentioning some of them at this time.

#### **2. Acute spinal cord injury mechanisms**

The pathophysiology of the SCI has been divided in primary and secondary injury, the latter generally described in acute and chronic phases. The mechanisms involved in the secondary injury include biochemical degenerative processes that exacerbates damage, such as the loss of blood-spinal cord barrier (BSCB) integrity, ischemia/reperfusion, hypoxia, loss of ionic homeostasis, Ca2+ overload, glutamatergic excitotoxicity, immune cell invasion, inflammation, release of cytokines, free radical (FR) production, LP, and excessive production of nitric oxide (NO• ). All these events occur in the acute SCI and may be clinically targeted due to their times of action, different from the unexpected primary injury [3] (**Figure 1**). It has also been demonstrated that these mechanisms are related in a way that exacerbates when the levels of oxidative stress and LP molecules are increased and that attenuates its effects when the antioxidant treatment is immediately given after SCI [4].

#### **2.1 Mechanism of oxidative stress and free radical's generation**

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are molecules that participate in oxidative stress. They are endogenously produced under physiological conditions, and in low amounts, they are essential for biological and immune process [4]. Oxidative stress could be defined as a disturbance in the pro-/antioxidant equilibrium, for the presence of high levels of ROS and RNS that exceeds the endogenous antioxidative defense mechanisms, and they are associated with damage to a wide range of molecular species, such as lipids, proteins, and nucleic acids, contributing to the pathophysiology of SCI [3].

ROS are oxygen-derived compounds that include radicals (unstable molecules with a single unpaired electron), such as superoxide (O2 •−), hydroxyl (HO• ) and peroxyl (RO2 • /HO2 • ) radicals, and non-radicals such as hydrogen peroxide (H2O2). Within the first minutes and hours post-injury, different sources of O2 •− such as arachidonic acid cascade, mitochondrial leak, and enzymes systems [nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, myeloperoxidases, cyclooxygenase (COX), and xanthine oxidase], present in activated microglia and infiltrating cells (macrophages and neutrophils), may act providing O2 •− [5], derived from the reduction of oxygen molecules (O2) with a single electron (e− ). Although O2 •− itself is reactive, its direct oxidative reactivity toward biological substrates in aqueous environments is relatively weak, but it distinguishes itself as an active nucleophile and oxidizing agent that can react with hydrogen donors (e.g., ascorbate and tocopherol) [4–6]. On one hand, superoxide dismutase (SOD) rapidly catalyzes the dismutation of O2 •− into H2O2 and O2 (2O2 •− + 2H+ → H2O2 + O2), and at low pH, O2 •− can dismutate spontaneously.

**41**

**Figure 1.**

ing species such as HO•

*recover its motor and sensory functions [155].*

*Current Developments in Antioxidant Therapies for Spinal Cord Injury*

In oxidative stress, this H2O2 can react with transition metal cations to form oxidiz-

presence of iron (Fe) and cooper (Cu) ions. The central nervous system (CNS) is rich in ferric iron (Fe3+), contained in transferrin in plasma, and ferritin intracellularly. This iron can be released from its transporters at pH values of 6 or less, like the one reached in hypoxia and accumulation of lactic acid in SCI, and become

), and this occurs mainly in the

and hydroxyl anion (HO−

*Progression of the inflammatory response in spinal cord parenchyma. The condition produced by the mechanical injury induces the activation of the damage targeting mechanisms, initially propitiated by the resident microglia, which secretes pro-inflammaory cytokines. Astrocytes and endothelial cells allow the permeabilization of BSCB and express chemoattractants to facilitate the admission of immune cells from the periphery, increasing the response at site. Collateral injuries can occur, largely due to the low antioxidant capacity of neural tissue to counteract the ROS produced by inflammatory cells, spreading the damage to other uninvolved cells. The extent of the initial damage is proportional to the final capacity of the organism to* 

*DOI: http://dx.doi.org/10.5772/intechopen.85424*

#### **Figure 1.**

*Spinal Cord Injury Therapy*

**2. Acute spinal cord injury mechanisms**

peroxidation (LP), and its end products could modify proteins and DNA present in cellular structures, causing cell death and a lower probability of regeneration [2]. SCI is a highly disabling and irreversible condition that causes physiological complications (bowel, cardiac, urinary, respiratory) and it has a social-economic impact in patients. The research of new agents targeting degenerative processes such as oxidative stress and LP is important especially due to the lack of efficacy and safety of conventional therapies on patients with SCI [1]. Here, we review the efforts to discover new compounds aimed to offer an option in antioxidant treatments and the use of some in combination or in an innovative way, both in experimental and in clinical trials. We would like to mention that there is a wide range of antioxidant therapies in study, and we are only briefly mentioning some of them at this time.

The pathophysiology of the SCI has been divided in primary and secondary injury, the latter generally described in acute and chronic phases. The mechanisms involved in the secondary injury include biochemical degenerative processes that exacerbates damage, such as the loss of blood-spinal cord barrier (BSCB) integrity, ischemia/reperfusion, hypoxia, loss of ionic homeostasis, Ca2+ overload, glutamatergic excitotoxicity, immune cell invasion, inflammation, release of cytokines, free radical (FR) production, LP, and excessive production of nitric oxide (NO•

All these events occur in the acute SCI and may be clinically targeted due to their times of action, different from the unexpected primary injury [3] (**Figure 1**). It has also been demonstrated that these mechanisms are related in a way that exacerbates when the levels of oxidative stress and LP molecules are increased and that attenuates its effects when the antioxidant treatment is immediately given after SCI [4].

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are molecules that participate in oxidative stress. They are endogenously produced under physiological conditions, and in low amounts, they are essential for biological and immune process [4]. Oxidative stress could be defined as a disturbance in the pro-/antioxidant equilibrium, for the presence of high levels of ROS and RNS that exceeds the endogenous antioxidative defense mechanisms, and they are associated with damage to a wide range of molecular species, such as lipids, proteins, and

ROS are oxygen-derived compounds that include radicals (unstable molecules

) radicals, and non-radicals such as hydrogen peroxide (H2O2).

•−), hydroxyl (HO•

•− into H2O2 and O2

•− can dismutate spontaneously.

) and

•− [5],

).

•− such as

**2.1 Mechanism of oxidative stress and free radical's generation**

nucleic acids, contributing to the pathophysiology of SCI [3].

Within the first minutes and hours post-injury, different sources of O2

(e.g., ascorbate and tocopherol) [4–6]. On one hand, superoxide dis-

arachidonic acid cascade, mitochondrial leak, and enzymes systems [nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, myeloperoxidases, cyclooxygenase (COX), and xanthine oxidase], present in activated microglia and infiltrating cells (macrophages and neutrophils), may act providing O2

derived from the reduction of oxygen molecules (O2) with a single electron (e−

substrates in aqueous environments is relatively weak, but it distinguishes itself as an active nucleophile and oxidizing agent that can react with hydrogen donors

•− itself is reactive, its direct oxidative reactivity toward biological

with a single unpaired electron), such as superoxide (O2

mutase (SOD) rapidly catalyzes the dismutation of O2

•− + 2H+ → H2O2 + O2), and at low pH, O2

).

**40**

(2O2

peroxyl (RO2

Although O2

• /HO2 •

*Progression of the inflammatory response in spinal cord parenchyma. The condition produced by the mechanical injury induces the activation of the damage targeting mechanisms, initially propitiated by the resident microglia, which secretes pro-inflammaory cytokines. Astrocytes and endothelial cells allow the permeabilization of BSCB and express chemoattractants to facilitate the admission of immune cells from the periphery, increasing the response at site. Collateral injuries can occur, largely due to the low antioxidant capacity of neural tissue to counteract the ROS produced by inflammatory cells, spreading the damage to other uninvolved cells. The extent of the initial damage is proportional to the final capacity of the organism to recover its motor and sensory functions [155].*

In oxidative stress, this H2O2 can react with transition metal cations to form oxidizing species such as HO• and hydroxyl anion (HO− ), and this occurs mainly in the presence of iron (Fe) and cooper (Cu) ions. The central nervous system (CNS) is rich in ferric iron (Fe3+), contained in transferrin in plasma, and ferritin intracellularly. This iron can be released from its transporters at pH values of 6 or less, like the one reached in hypoxia and accumulation of lactic acid in SCI, and become

catalytic; a second source for Fe comes from the hemoglobin released after mechanical-induced hemorrhage. O2 •− acts donating an electron to Fe3+, and the ferrous iron (Fe2+) catalyzes the conversion of H2O2 to HO• and HO− . Therefore, O2 •− and H2O2 react in the presence of Fe3+/Fe2+ and promote the formation of HO• and HO− [2].

On the other hand, O2 •− can interact with NO• , a hydrophobic and mildly reactive radical generated enzymatically from L-arginine by nitric oxide synthase (NOS) isoforms, and give rise to one of the most important RNS, peroxynitrite ONOO− (NO• + O2 •− → ONOO− ), a potent oxidizing and nitrating agent in vivo, either for direct oxidation reactions, in which it reacts with targets of low molecular weight and proteins (with thiols and metal centers), and carbon dioxide, or by derived radicals from homolytic cleavage, secondary to the reaction with carbon dioxide or protonation, included in RNS [2, 7]. Under biological conditions, ONOO− exists in equilibrium with its acidic form, the peroxynitrous acid (ONOOH), which decays rapidly by homolysis to give place to highly reactive nitrogen dioxide radical (NO2 •−) and HO• favored by the low pH in SCI [8]. Among the different direct reactions of ONOO− , one of the most relevant is this with CO2 (from bicarbonate buffer system), to form nitrosoperoxocarbonate (ONOOCO2), forming by cleavage strong oxidant agents, such as nitrogen dioxide (NO2 •−) and carbonate (CO3•−) radicals [7, 8].

#### **2.2 Lipid peroxidation (LP)**

Lipids are the most susceptible class of biomolecules to undergo oxidation; polyunsaturated fatty acids (PUFAs) are long-chain fatty acids with two or more double bonds in *cis* configuration, each separated by a methylene bridge (–CH2–) at their carbon backbone, and the hydrogen attached to the methylene bridge is very easy to remove. The LP is defined as an oxidative degradation and decomposition of lipids in an uncontrolled manner by nonenzymatic pathway and occurs when ROS react with PUFAs, leading to the modification of its physicochemical properties, disrupting the cellular membrane integrity. The enzymatic pathway produces lipid mediators such as prostanoids, leukotrienes, lipoxins, resolvins, and maresins by the action of COX or lipoxygenases (LOX), among others, causing dysregulation of blood flow, BSCB damage, inflammatory response, and programmed cell death pathway [9]. The CNS is particularly vulnerable to LP by various factors: it has high oxidative metabolic activity, PUFA content, and transition metal cations. In contrast, it has low antioxidant defenses and neuron-glia replication [8, 10].

The LP is a chain process that involves the participation of ROS, RNS, PUFAs, and oxidative systems, among others, where therapeutic intervention has been proposed with molecules that can both prevent FR formation and prevent those already formed from reacting with biomolecules. Because the peak of ROS production occurs within the first 24 h after the injury, or during ischemia-reperfusion, the drugs that can be used for this "first FR production" are limited by their time of intervention. However, the phases in which LP develops may persist as long as there are oxidizable substrates, so knowing the reactions involved allows the design of strategies and drugs with a greater therapeutic window [11, 12]. The nonenzymatic peroxidation of PUFAs is the principal pathway of oxidative stress; HO• participates as one of the starts of LP due to its solubility and the lack of an enzymatic system to eliminate it. This and other radicals remove an H• radical inside a lipid (LH), which provides a lipid radical (L• ) [11, 12] (**Figure 2**). The resonance stabilization of L• produces a conjugated diene that reacts with O2 to form a lipid peroxyl radical (LOO• ) and generates a lipid hydroperoxide (LOOH) when it withdraws hydrogen from an adjacent PUFA, producing a second L• [2, 12]. The LOOH are regarded as the initial product of LP, but these compounds are unstable and can be discomposed with the participation of Fe3+ or Fe2+ again in LOO• or

**43**

alkoxyl (LO•

*lipid hydroperoxide; LO•*

**Figure 2.**

+ Fe3+ → LOO•

Fe2+ (LOOH + Fe2+ → LO•

too, such as the LOO•

) radicals, respectively. Both, the reduction of the LOO•

*The three steps of nonenzymatic lipid peroxidation of PUFAs. In the initiation step, a hydrogen atom at a bis allylic position is removed using either a radical or a redox active metal to generate a resonance-stabilized alkyl radical. The radical isomerizes to form the more stable conjugated diene, prior to reacting with molecular oxygen. In the propagation step, radicals are able to react with new substrates, forming lipid hydroperoxides (LOOH), which can react with iron creating new radicals. This step repeats until the termination step, where radicals are "quenched" by antioxidants or react with another radical. The decomposition of LOOH* 

reactions describe above. Thus, one HO•

number of LOOH through a series of chain reactions. Finally, termination of chain reactions occurs by the stabilization of the radicals reacting between themselves,

+ Fe3+) and its conversion back to LOO•

*, alkyl/lipid radical; LOO•*

*, lipid alkoxyl radical; HNE: 4-hydroxy-2-nonenal; MDA: malondialdehyde; HHE:* 

+ Fe2+) reactions, have acidic pH optimal conditions and are more

+ HO•

likely to occur in SCI tissue environment [5]. The LO•

*4-hydroxy-2-hexenal. Modified from Gaschler and Stockwell [12].*

*generates species such as MDA, HNE, etc. LH, lipid; L•*

to an LO•

*, peroxyl radical; LOOH,* 

can generate a high

can initiate chain reactions

by

(LOOH

*Current Developments in Antioxidant Therapies for Spinal Cord Injury*

*DOI: http://dx.doi.org/10.5772/intechopen.85424*

*Current Developments in Antioxidant Therapies for Spinal Cord Injury DOI: http://dx.doi.org/10.5772/intechopen.85424*

#### **Figure 2.**

*Spinal Cord Injury Therapy*

ical-induced hemorrhage. O2

On the other hand, O2

•− → ONOO−

agents, such as nitrogen dioxide (NO2

**2.2 Lipid peroxidation (LP)**

(NO•

and HO•

ONOO−

+ O2

(Fe2+) catalyzes the conversion of H2O2 to HO•

catalytic; a second source for Fe comes from the hemoglobin released after mechan-

react in the presence of Fe3+/Fe2+ and promote the formation of HO•

•− can interact with NO•

protonation, included in RNS [2, 7]. Under biological conditions, ONOO−

radical generated enzymatically from L-arginine by nitric oxide synthase (NOS) isoforms, and give rise to one of the most important RNS, peroxynitrite ONOO−

direct oxidation reactions, in which it reacts with targets of low molecular weight and proteins (with thiols and metal centers), and carbon dioxide, or by derived radicals from homolytic cleavage, secondary to the reaction with carbon dioxide or

equilibrium with its acidic form, the peroxynitrous acid (ONOOH), which decays rapidly by homolysis to give place to highly reactive nitrogen dioxide radical (NO2

to form nitrosoperoxocarbonate (ONOOCO2), forming by cleavage strong oxidant

Lipids are the most susceptible class of biomolecules to undergo oxidation; polyunsaturated fatty acids (PUFAs) are long-chain fatty acids with two or more double bonds in *cis* configuration, each separated by a methylene bridge (–CH2–) at their carbon backbone, and the hydrogen attached to the methylene bridge is very easy to remove. The LP is defined as an oxidative degradation and decomposition of lipids in an uncontrolled manner by nonenzymatic pathway and occurs when ROS react with PUFAs, leading to the modification of its physicochemical properties, disrupting the cellular membrane integrity. The enzymatic pathway produces lipid mediators such as prostanoids, leukotrienes, lipoxins, resolvins, and maresins by the action of COX or lipoxygenases (LOX), among others, causing dysregulation of blood flow, BSCB damage, inflammatory response, and programmed cell death pathway [9]. The CNS is particularly vulnerable to LP by various factors: it has high oxidative metabolic activity, PUFA content, and transition metal cations. In con-

trast, it has low antioxidant defenses and neuron-glia replication [8, 10].

The LP is a chain process that involves the participation of ROS, RNS, PUFAs, and oxidative systems, among others, where therapeutic intervention has been proposed with molecules that can both prevent FR formation and prevent those already formed from reacting with biomolecules. Because the peak of ROS production occurs within the first 24 h after the injury, or during ischemia-reperfusion, the drugs that can be used for this "first FR production" are limited by their time of intervention. However, the phases in which LP develops may persist as long as there are oxidizable substrates, so knowing the reactions involved allows the design of strategies and drugs with a greater therapeutic window [11, 12]. The nonenzymatic peroxidation of PUFAs is the principal pathway of oxidative stress;

participates as one of the starts of LP due to its solubility and the lack of an

LOOH are regarded as the initial product of LP, but these compounds are unstable and can be discomposed with the participation of Fe3+ or Fe2+ again in LOO•

produces a conjugated diene that reacts with O2 to form a lipid

) and generates a lipid hydroperoxide (LOOH) when it

enzymatic system to eliminate it. This and other radicals remove an H•

withdraws hydrogen from an adjacent PUFA, producing a second L•

a lipid (LH), which provides a lipid radical (L•

favored by the low pH in SCI [8]. Among the different direct reactions of

, one of the most relevant is this with CO2 (from bicarbonate buffer system),

•− acts donating an electron to Fe3+, and the ferrous iron

•−) and carbonate (CO3•−) radicals [7, 8].

. Therefore, O2

, a hydrophobic and mildly reactive

•− and H2O2

[2].

exists in

radical inside

or

[2, 12]. The

) [11, 12] (**Figure 2**). The resonance

•−)

and HO−

and HO−

), a potent oxidizing and nitrating agent in vivo, either for

**42**

HO•

stabilization of L•

peroxyl radical (LOO•

*The three steps of nonenzymatic lipid peroxidation of PUFAs. In the initiation step, a hydrogen atom at a bis allylic position is removed using either a radical or a redox active metal to generate a resonance-stabilized alkyl radical. The radical isomerizes to form the more stable conjugated diene, prior to reacting with molecular oxygen. In the propagation step, radicals are able to react with new substrates, forming lipid hydroperoxides (LOOH), which can react with iron creating new radicals. This step repeats until the termination step, where radicals are "quenched" by antioxidants or react with another radical. The decomposition of LOOH generates species such as MDA, HNE, etc. LH, lipid; L• , alkyl/lipid radical; LOO• , peroxyl radical; LOOH, lipid hydroperoxide; LO• , lipid alkoxyl radical; HNE: 4-hydroxy-2-nonenal; MDA: malondialdehyde; HHE: 4-hydroxy-2-hexenal. Modified from Gaschler and Stockwell [12].*

alkoxyl (LO• ) radicals, respectively. Both, the reduction of the LOO• to an LO• by Fe2+ (LOOH + Fe2+ → LO• + HO• + Fe3+) and its conversion back to LOO• (LOOH + Fe3+ → LOO• + Fe2+) reactions, have acidic pH optimal conditions and are more likely to occur in SCI tissue environment [5]. The LO• can initiate chain reactions too, such as the LOO• reactions describe above. Thus, one HO• can generate a high number of LOOH through a series of chain reactions. Finally, termination of chain reactions occurs by the stabilization of the radicals reacting between themselves,

forming a new bond and eliminating the radical, or by donating electrons (generally H• ) to the radicals by compounds, without turning into radicals. In the case of LOOH provided in the previous LP reactions, these undergo fragmentation in which oxidized PUFAs give rise to short-chain secondary products, such as hydroxy-alkenals (neurotoxic aldehydes) relatively stable like malondialdehyde (MDA), 4-hydroxy-2-nonenal (HNE), 4-hydroxy-2-hexenal (HHE), and 2-propenal (acrolein), that can diffuse within or even escape from the cell and attack targets far from the site of the original event [13] (**Figure 2**). In general, the LOOH can react in different ways that lead to a cleavage of the C-C bond and formation of hydroxy-alkenals by means of different mechanisms [13].

While the LP compromises the integrity of the cell membrane, the highly reactive secondary products can be covalently bound to proteins and DNA, compromising their structure and function. Regarding the HNE as the most studied product of LP, it must be mentioned that the HNE physiological concentration inside the cell ranges from 0.1 to 3 μM. Moreover, under oxidative stress conditions, HNE can accumulate at concentrations that range from 5 μM to 10 mM [14]. It has been demonstrated that HNE can play an important role as a signaling molecule, enhancing cellular antioxidant capacity and adaptive response at low concentrations; can promote protein and DNA damage in organelles, leading to the induction of autophagy, senescence, or cell cycle arrest; and finally can induce apoptosis or necrosis programmed cell death at a high or very high level [13, 15, 16].

#### **2.3 Proteins as target of oxidation**

The oxidation of proteins for ROS can lead to the hydroxylation of aromatic groups and aliphatic amino acid (aa) side chains, nitration of aromatic aa residues, reversible nitrosylation of sulfhydryl groups, sulfoxidation of Met residues, conversion of some aa residues to carbonyl derivatives, cleavage of the polypeptide chain, and formation of cross-linked protein aggregates. Furthermore, functional groups of proteins can react with products of LP and carbohydrate derivatives (glycation/ glycoxidation) to produce inactive derivatives [17], where the irreversible protein oxidation is described by four pathways: peptide bond rupture, carbonylation, formation of protein-protein bonds, and nitration [18]. The initial oxidation can form a carbon-centered radical, which can react with O2 to form a ROO• , to cleave protein backbone by either α-amidation or diamide pathways.

The cleavage of side chains (glutamyl, aspartyl, and probably prolyl side chains) may occur directly or by metal-catalyzed oxidation (proline [Pro], arginine [Arg], lysine [Lys], and threonine [Thr] residues), yielding carbonyl derivatives [17, 18]. One of the most important of irreversible oxidation processes is by protein carbonylation. It involves the previous protein and aa carbonyl derivatives ,CO3 •− oxidation (reacting preferentially on tryptophan [Trp], Thr, cysteine [Cys], methionine [Met], and histidine [His] residues), ketones and aldehyde reactions over Cys, Lys, His, and by glycation/glycoxidation of Lys amino groups, etc. [2, 8, 17, 18].

The modification of the protein structure after oxidation can also give rise to intra- or inter-protein cross-linked derivatives by several different mechanisms. For example, the protein-protein bond may be due to the interaction of two carboncentered radicals or two aromatic aa residues radicals, formed by direct attack of ROS [17]. Final products of LP, such as HNE and MDA, can cause cross-linked proteins, as reactions of both MDA aldehyde groups with two different residues in the same protein or two different proteins [17]. Another protein-protein bond is disulfide bridge (RSSR) that results from the oxidation of thiols (RSH) forming sulfenic acid (RSOH) as the last intermediate and reacting with another thiol, forming RSSR. This can be promoted in the presence of OONO<sup>−</sup> or driven by ROS

**45**

*Current Developments in Antioxidant Therapies for Spinal Cord Injury*

and RNS, with the possibility of chain reactions [8]. Regarding this, some enzymes containing Cys, in its catalytic site, can act as scavengers, by direct interaction or consuming glutathione (GSH), due to the reversible modification of the RSSR bond [8]. HNE possess three functional groups in its structure (**Figure 2**), making this electrophilic molecule highly reactive toward nucleophilic groups such as thiol (–SH) and amino (–NH2). Thus, aa such as Cys, Lys, His, and Arg are HNE targets, whose modification inhibits the functions of a variety of enzymatic and structural cellular proteins [19]. MDA with enhanced reactivity in low pH and existing as β-hydroxyacrolein is strongly reactive to nucleophiles such as Lys, His, or Arg residues [20]. Protein modifications by RNS act over aromatic, Cys, and Met

such as GSH, albumin, and metalloproteins (heme, myeloperoxidase, cytochrome

hydrogen in the position 3 of the phenolic ring, produces 3-nitrotyrosine (NT-3)

modifications, diverse molecules can be identified both in cerebrospinal fluid and blood, both in humans and in animals, and they have been proposed as biomarkers to diagnose the severity of SCI. Some of those biomarkers derived from proteins are neurofilament proteins, glial fibrillary acidic protein (GFAP), tau, neuron-specific enolase, and S100 calcium-binding protein β (S100β), being part of the components of neurons, oligodendrocytes, and reactive astrocytes. A more detailed list can be found in the works of Lubieniecka et al. and the Hulme et al. review [21, 22].

The ROS/RNS produced in oxidative stress and LP can damage the nucleic acids of DNA; cause DNA-protein cross-links, strand breaks, and modification of purine and pyridine bases; and lead to DNA mutations. More than 20 DNA adducts have been identified, such as 8-hydroxy-2′-guanosine (8-OHdG), increased in patients in whom the antioxidant systems are suspected to be deficient [23]. MDA is an important contributor to DNA damage and mutations that can react with several nucleosides (deoxyguanosine and cytidine) to form adducts, and the major resulting product is a pyrimido-purinone called M1dG [24]. HNE can also react with deoxyguanosine to form two pairs of diastereomer adducts (4-HNE-dG 1,2 and 3,4) or etheno-DNA adducts in the presence of peroxides that could further induce DNA cross-link or DNA-protein conjugates [25, 26]. Other markers of oxidative damage

The cellular antioxidant systems are composed by antioxidant enzymes and nonenzymatic molecules able to donate electrons to different radical chemical structures. In the CNS, they are present in lower concentrations than the oxidizable substrate and are responsible of maintaining the pro-/antioxidant equilibrium, relieving oxidative stress, and reducing or interrupting uncontrolled LP, DNA mutations, protein oxidation/degradation, as well as other cell damage features. The essential endogenous components of the enzymatic antioxidant defense are SOD, catalase (CAT), glutathione peroxidases (GPx), glutathione reductases (GR), and glutathione S-transferases (GST), while the nonenzymatic antioxidants include GSH, proteins (ferritin, transferrin, ceruloplasmin, metallothionein, thioredoxin (Trx), albumin), vitamins C and E (tocopherol), trace elements, and low molecular weight scavengers, such as uric acid, coenzyme Q,

reacts directly with thiol groups present in a variety of proteins

), nitrate (NO3

−

•−, with substitution of a

), or NO2

[2, 8]. From all these

•− [8].

−

*DOI: http://dx.doi.org/10.5772/intechopen.85424*

P450, SOD isoforms, etc.) forming nitrite (NO2

Finally, irreversible protein tyrosine nitration by NO2

as a specific footprint of induced cellular damage by OONO−

in DNA, among other biomolecules, were reviewed in [23].

**2.5 Enzymatic and nonenzymatic antioxidant systems**

residues; OONO−

**2.4 DNA damage**

*Current Developments in Antioxidant Therapies for Spinal Cord Injury DOI: http://dx.doi.org/10.5772/intechopen.85424*

and RNS, with the possibility of chain reactions [8]. Regarding this, some enzymes containing Cys, in its catalytic site, can act as scavengers, by direct interaction or consuming glutathione (GSH), due to the reversible modification of the RSSR bond [8]. HNE possess three functional groups in its structure (**Figure 2**), making this electrophilic molecule highly reactive toward nucleophilic groups such as thiol (–SH) and amino (–NH2). Thus, aa such as Cys, Lys, His, and Arg are HNE targets, whose modification inhibits the functions of a variety of enzymatic and structural cellular proteins [19]. MDA with enhanced reactivity in low pH and existing as β-hydroxyacrolein is strongly reactive to nucleophiles such as Lys, His, or Arg residues [20]. Protein modifications by RNS act over aromatic, Cys, and Met residues; OONO− reacts directly with thiol groups present in a variety of proteins such as GSH, albumin, and metalloproteins (heme, myeloperoxidase, cytochrome P450, SOD isoforms, etc.) forming nitrite (NO2 − ), nitrate (NO3 − ), or NO2 •− [8]. Finally, irreversible protein tyrosine nitration by NO2 •−, with substitution of a hydrogen in the position 3 of the phenolic ring, produces 3-nitrotyrosine (NT-3) as a specific footprint of induced cellular damage by OONO− [2, 8]. From all these modifications, diverse molecules can be identified both in cerebrospinal fluid and blood, both in humans and in animals, and they have been proposed as biomarkers to diagnose the severity of SCI. Some of those biomarkers derived from proteins are neurofilament proteins, glial fibrillary acidic protein (GFAP), tau, neuron-specific enolase, and S100 calcium-binding protein β (S100β), being part of the components of neurons, oligodendrocytes, and reactive astrocytes. A more detailed list can be found in the works of Lubieniecka et al. and the Hulme et al. review [21, 22].

#### **2.4 DNA damage**

*Spinal Cord Injury Therapy*

ally H•

forming a new bond and eliminating the radical, or by donating electrons (gener-

of LOOH provided in the previous LP reactions, these undergo fragmentation in which oxidized PUFAs give rise to short-chain secondary products, such as hydroxy-alkenals (neurotoxic aldehydes) relatively stable like malondialdehyde (MDA), 4-hydroxy-2-nonenal (HNE), 4-hydroxy-2-hexenal (HHE), and 2-propenal (acrolein), that can diffuse within or even escape from the cell and attack targets far from the site of the original event [13] (**Figure 2**). In general, the LOOH can react in different ways that lead to a cleavage of the C-C bond and formation of

While the LP compromises the integrity of the cell membrane, the highly reactive secondary products can be covalently bound to proteins and DNA, compromising their structure and function. Regarding the HNE as the most studied product of LP, it must be mentioned that the HNE physiological concentration inside the cell ranges from 0.1 to 3 μM. Moreover, under oxidative stress conditions, HNE can accumulate at concentrations that range from 5 μM to 10 mM [14]. It has been demonstrated that HNE can play an important role as a signaling molecule, enhancing cellular antioxidant capacity and adaptive response at low concentrations; can promote protein and DNA damage in organelles, leading to the induction of autophagy, senescence, or cell cycle arrest; and finally can induce apoptosis or

necrosis programmed cell death at a high or very high level [13, 15, 16].

form a carbon-centered radical, which can react with O2 to form a ROO•

ylation. It involves the previous protein and aa carbonyl derivatives ,CO3

His, and by glycation/glycoxidation of Lys amino groups, etc. [2, 8, 17, 18].

forming RSSR. This can be promoted in the presence of OONO<sup>−</sup>

(reacting preferentially on tryptophan [Trp], Thr, cysteine [Cys], methionine [Met], and histidine [His] residues), ketones and aldehyde reactions over Cys, Lys,

The modification of the protein structure after oxidation can also give rise to intra- or inter-protein cross-linked derivatives by several different mechanisms. For example, the protein-protein bond may be due to the interaction of two carboncentered radicals or two aromatic aa residues radicals, formed by direct attack of ROS [17]. Final products of LP, such as HNE and MDA, can cause cross-linked proteins, as reactions of both MDA aldehyde groups with two different residues in the same protein or two different proteins [17]. Another protein-protein bond is disulfide bridge (RSSR) that results from the oxidation of thiols (RSH) forming sulfenic acid (RSOH) as the last intermediate and reacting with another thiol,

protein backbone by either α-amidation or diamide pathways.

The oxidation of proteins for ROS can lead to the hydroxylation of aromatic groups and aliphatic amino acid (aa) side chains, nitration of aromatic aa residues, reversible nitrosylation of sulfhydryl groups, sulfoxidation of Met residues, conversion of some aa residues to carbonyl derivatives, cleavage of the polypeptide chain, and formation of cross-linked protein aggregates. Furthermore, functional groups of proteins can react with products of LP and carbohydrate derivatives (glycation/ glycoxidation) to produce inactive derivatives [17], where the irreversible protein oxidation is described by four pathways: peptide bond rupture, carbonylation, formation of protein-protein bonds, and nitration [18]. The initial oxidation can

The cleavage of side chains (glutamyl, aspartyl, and probably prolyl side chains) may occur directly or by metal-catalyzed oxidation (proline [Pro], arginine [Arg], lysine [Lys], and threonine [Thr] residues), yielding carbonyl derivatives [17, 18]. One of the most important of irreversible oxidation processes is by protein carbon-

, to cleave

•− oxidation

or driven by ROS

hydroxy-alkenals by means of different mechanisms [13].

**2.3 Proteins as target of oxidation**

) to the radicals by compounds, without turning into radicals. In the case

**44**

The ROS/RNS produced in oxidative stress and LP can damage the nucleic acids of DNA; cause DNA-protein cross-links, strand breaks, and modification of purine and pyridine bases; and lead to DNA mutations. More than 20 DNA adducts have been identified, such as 8-hydroxy-2′-guanosine (8-OHdG), increased in patients in whom the antioxidant systems are suspected to be deficient [23]. MDA is an important contributor to DNA damage and mutations that can react with several nucleosides (deoxyguanosine and cytidine) to form adducts, and the major resulting product is a pyrimido-purinone called M1dG [24]. HNE can also react with deoxyguanosine to form two pairs of diastereomer adducts (4-HNE-dG 1,2 and 3,4) or etheno-DNA adducts in the presence of peroxides that could further induce DNA cross-link or DNA-protein conjugates [25, 26]. Other markers of oxidative damage in DNA, among other biomolecules, were reviewed in [23].

#### **2.5 Enzymatic and nonenzymatic antioxidant systems**

The cellular antioxidant systems are composed by antioxidant enzymes and nonenzymatic molecules able to donate electrons to different radical chemical structures. In the CNS, they are present in lower concentrations than the oxidizable substrate and are responsible of maintaining the pro-/antioxidant equilibrium, relieving oxidative stress, and reducing or interrupting uncontrolled LP, DNA mutations, protein oxidation/degradation, as well as other cell damage features. The essential endogenous components of the enzymatic antioxidant defense are SOD, catalase (CAT), glutathione peroxidases (GPx), glutathione reductases (GR), and glutathione S-transferases (GST), while the nonenzymatic antioxidants include GSH, proteins (ferritin, transferrin, ceruloplasmin, metallothionein, thioredoxin (Trx), albumin), vitamins C and E (tocopherol), trace elements, and low molecular weight scavengers, such as uric acid, coenzyme Q,

and lipoic acid [4, 6, 23], which act by depleting molecular O2 or decreasing its local concentration; removing pro-oxidative metal ions; trapping aggressive ROS, such as O2 •− or H2O2; scavenging chain-initiating radicals like HO• , RO2 • /HO2 • , or LO• ; or breaking the chain of a radical sequence [4]. There are also important exogenous nonenzymatic antioxidants (vitamins A, C, E, flavonoids, carotenoids, phenolics, acetylcysteine, exogenous selenium, zinc), acquired through diet, which are being studied. A table of these enzymatic and nonenzymatic antioxidants important in the CNS was reviewed in [23]. Preventing the formation of ROS, or at least its accumulation, and blocking or capturing those radicals already formed is the first defense against oxidative stress. The O2 •− generated by various sources can be converted to H2O2 by SODs [4]. The O2 •− intracellularly produced in the mitochondria can be converted into H2O2 by MnSOD (SOD3) [18]. Once generated, H2O2 (but not other peroxides) is decomposed to water and oxygen O2 (2H2O2 + 2GHS → H2O + O2) by the action of CAT, a ferrihemecontaining enzyme. However, small amounts of ROS escape from the antioxidant defense and can be converted to HO• , which may be scavenged by low molecular mass nonenzymatic antioxidants, such as ascorbate, tocopherol, GSH, etc. [27]. H2O2 is also reduced by the action of different peroxidases, such as GPx (H2O2 + 2GHS → H2O + GSSG), which, additionally, can reduce lipid hydroperoxides (LOOH + 2GSH → LOH + GSSG) [11, 12]. Other enzymes that catalyze this reaction include peroxiredoxin and thioredoxin reductase [4]. Some enzymes that participate in the detoxification of LP products by oxidation, reduction, and glutathione conjugation, the latter being a mechanism also used to reverse the effects of RNS, are aldehyde dehydrogenases (ALDH), alcohol dehydrogenase (ADH), aldo-keto reductase (AKR), and the aforementioned GST, GPx, and GR [28].

In SCI, the primary injury causes disruption of blood flow and vascular insult, such as ischemia-reperfusion, which conducts to the loss of metabolic function of cells in gray matter with decrease of ATP, causing depolarization of membranes due to the inhibition of Na<sup>+</sup> /K<sup>+</sup> and Ca2+ ATPases function. Ca2+ overload and glutamate excitotoxicity compromise the function and integrity of mitochondria through the activation of proteases and inactivation of important enzymes. Due to the low ratio of antioxidant systems' oxidizable substrate in acute SCI, the mitochondrial antioxidant reserves decrease and are incapable of restoring the redox equilibrium, giving place to an increase of mitochondrial concentration of O2 •− and an increase and leak of free radicals formed downstream including ONOO<sup>−</sup> , initiating LP. The damage produced by this excess of radicals or end products of LP over proteins and membranes of the mitochondria and endoplasmic reticulum potentiates the processes of secondary injury mentioned here to the local and adjacent cells to SCI [4].

#### **3. Antioxidant therapy strategies**

The early therapeutic intervention for SCI is crucial to improve the chances of maximum possible recovery. This was observed in clinical trials where the current treatment of choice, methylprednisolone sodium succinate (MP or MPSS), was effective only when administered within the first 8 h after injury, at high doses (5.4 mg/kg/h). In 48-h regimens, however, it increases the incidence of complications from infections (severe sepsis and pneumonia), while the 24-h safe regimen is not effective in the long term, at least after 3 h [1]. Being an ineffective treatment, there are no alternative therapeutic treatments that offer safety and certainty regarding the recovery of the motor function. The research of new

**47**

**3.2 Minocycline**

*Current Developments in Antioxidant Therapies for Spinal Cord Injury*

pharmacological agents for the treatment of SCI focuses on the processes of secondary injury, being antioxidant therapies the most important. The main goals of drug therapies for SCI can be classified in neuroprotection and neuroregeneration; antioxidant therapies are cataloged within the first. Here we present some of the agents that are in experimental phase and others when mentioned, in clinical trials, either because their efficacy has been demonstrated in animal models or because of their use already approved in other pathologies. Regarding the diverse SCI models, they have been used to simulate SCI with high relevance and validity to preclinical evaluation due to the replication of human traumatic injuries. The rational use of animals is strongly controlled, and the possibility of pain and distress must be considered and minimized by veterinary staff through the appropriate use of

Melatonin (N-acetyl-5-methoxytryptamine) is a pleiotropic compound that works mainly in the regulation of circadian rhythms and sleep. When reacting with

It stimulates the expression and activity of SOD, GPx, CAT, and GR and inhibits or decreases the expression of pro-oxidative NOs, different signaling pathways, transcription factors, and pro-inflammatory cytokines [29–31]. Decreased melatonin production has been linked to various CNS disorders, and the neuroprotective activity was detected in rat models of traumatic brain injury ischemic stroke and SCI [29, 30]. To cite only some SCI examples, in a study in Sprague-Dawley rats of 250 g with moderate lesion, 10 mg/kg of melatonin was applied subcutaneously twice a day for 4 weeks, and an increase in motor recovery and decrease in inducible nitric oxide synthase (iNOS) expression were observed. Intravenously, it decreased the synthesis of MDA and increased the synthesis of GSH and angiopoietin 1, and in mice with severe lesion, it decreased the expression of interleukin 1 beta (IL-1β) and NG-2 (neuron/glial antigen 2) [30, 32, 33]. In a model with lesion with vascular clips, the administration of 30 mg/kg alleviated post-traumatic injury associated with SCI by binding the PPARα-receptor; the administration of 50 mg/kg in moderate lesion decreased the BSCB permeability modulating the expression of brainderived neurotrophic factor (BDNF), growth-associated protein 43 (GAP-43), and caspase-3 [33–35]. In combination therapy with dexamethasone (10–0.025 mg/kg), it showed significant anti-inflammatory effects, attenuating the synthesis of tumor necrosis factor alpha (TNF-α) and iNOS and the nitration of tyrosine residues, increasing tissue recovery and motor capacity in an experimental SCI model of mouse [36], while the combination with methylprednisolone favored neurological recovery and decreased LP; its administration with zinc activated the internal

Minocycline hydrochloride is an available semisynthetic tetracycline antibiotic with potent anti-inflammatory (regulation of phospholipase A2 and MAPK/PIK3 pathways) and neuroprotective (protecting against glutamate-induced inflammation) activities; it also inhibits matrix metalloproteinases and mitochondrial Ca2+ influx. Minocycline has antioxidant and antiapoptotic properties, probably acting at high doses as a direct radical scavenger, like vitamin E, due to its phenolic ring structure [40]. In rats with SCI, minocycline given at oral doses of 3, 30, and 90 mg/kg 1 and 24 h after the lesion reduced MDA concentration and increased

, it is converted to cyclic 3-hydroxymelatonin.

*DOI: http://dx.doi.org/10.5772/intechopen.85424*

analgesics and animal care.

, H2O•

, and LOO•

antioxidant system and also decreased the LP [37–39].

**3.1 Melatonin**

ROS, such as HO−

*Current Developments in Antioxidant Therapies for Spinal Cord Injury DOI: http://dx.doi.org/10.5772/intechopen.85424*

pharmacological agents for the treatment of SCI focuses on the processes of secondary injury, being antioxidant therapies the most important. The main goals of drug therapies for SCI can be classified in neuroprotection and neuroregeneration; antioxidant therapies are cataloged within the first. Here we present some of the agents that are in experimental phase and others when mentioned, in clinical trials, either because their efficacy has been demonstrated in animal models or because of their use already approved in other pathologies. Regarding the diverse SCI models, they have been used to simulate SCI with high relevance and validity to preclinical evaluation due to the replication of human traumatic injuries. The rational use of animals is strongly controlled, and the possibility of pain and distress must be considered and minimized by veterinary staff through the appropriate use of analgesics and animal care.

#### **3.1 Melatonin**

*Spinal Cord Injury Therapy*

such as O2

or LO•

and lipoic acid [4, 6, 23], which act by depleting molecular O2 or decreasing its local concentration; removing pro-oxidative metal ions; trapping aggressive ROS,

; or breaking the chain of a radical sequence [4]. There are also important exogenous nonenzymatic antioxidants (vitamins A, C, E, flavonoids, carotenoids, phenolics, acetylcysteine, exogenous selenium, zinc), acquired through diet, which are being studied. A table of these enzymatic and nonenzymatic antioxidants important in the CNS was reviewed in [23]. Preventing the formation of ROS, or at least its accumulation, and blocking or capturing those radicals

, RO2 • /HO2 • ,

•− generated by

•− intracellularly

, which may be scavenged by low molecular

and Ca2+ ATPases function. Ca2+ overload and

•− or H2O2; scavenging chain-initiating radicals like HO•

produced in the mitochondria can be converted into H2O2 by MnSOD (SOD3) [18]. Once generated, H2O2 (but not other peroxides) is decomposed to water and oxygen O2 (2H2O2 + 2GHS → H2O + O2) by the action of CAT, a ferrihemecontaining enzyme. However, small amounts of ROS escape from the antioxidant

mass nonenzymatic antioxidants, such as ascorbate, tocopherol, GSH, etc. [27]. H2O2 is also reduced by the action of different peroxidases, such as GPx (H2O2 + 2GHS → H2O + GSSG), which, additionally, can reduce lipid hydroperoxides (LOOH + 2GSH → LOH + GSSG) [11, 12]. Other enzymes that catalyze this reaction include peroxiredoxin and thioredoxin reductase [4]. Some enzymes that participate in the detoxification of LP products by oxidation, reduction, and glutathione conjugation, the latter being a mechanism also used to reverse the effects of RNS, are aldehyde dehydrogenases (ALDH), alcohol dehydrogenase (ADH), aldo-keto reductase (AKR), and the aforementioned GST, GPx, and GR [28].

In SCI, the primary injury causes disruption of blood flow and vascular insult, such as ischemia-reperfusion, which conducts to the loss of metabolic function of cells in gray matter with decrease of ATP, causing depolarization of membranes

glutamate excitotoxicity compromise the function and integrity of mitochondria through the activation of proteases and inactivation of important enzymes. Due to the low ratio of antioxidant systems' oxidizable substrate in acute SCI, the mitochondrial antioxidant reserves decrease and are incapable of restoring the redox equilibrium, giving place to an increase of mitochondrial concentration

•− and an increase and leak of free radicals formed downstream including

, initiating LP. The damage produced by this excess of radicals or end products of LP over proteins and membranes of the mitochondria and endoplasmic reticulum potentiates the processes of secondary injury mentioned here to the

The early therapeutic intervention for SCI is crucial to improve the chances of maximum possible recovery. This was observed in clinical trials where the current treatment of choice, methylprednisolone sodium succinate (MP or MPSS), was effective only when administered within the first 8 h after injury, at high doses (5.4 mg/kg/h). In 48-h regimens, however, it increases the incidence of complications from infections (severe sepsis and pneumonia), while the 24-h safe regimen is not effective in the long term, at least after 3 h [1]. Being an ineffective treatment, there are no alternative therapeutic treatments that offer safety and certainty regarding the recovery of the motor function. The research of new

already formed is the first defense against oxidative stress. The O2

various sources can be converted to H2O2 by SODs [4]. The O2

/K<sup>+</sup>

defense and can be converted to HO•

due to the inhibition of Na<sup>+</sup>

local and adjacent cells to SCI [4].

**3. Antioxidant therapy strategies**

**46**

of O2

ONOO<sup>−</sup>

Melatonin (N-acetyl-5-methoxytryptamine) is a pleiotropic compound that works mainly in the regulation of circadian rhythms and sleep. When reacting with ROS, such as HO− , H2O• , and LOO• , it is converted to cyclic 3-hydroxymelatonin. It stimulates the expression and activity of SOD, GPx, CAT, and GR and inhibits or decreases the expression of pro-oxidative NOs, different signaling pathways, transcription factors, and pro-inflammatory cytokines [29–31]. Decreased melatonin production has been linked to various CNS disorders, and the neuroprotective activity was detected in rat models of traumatic brain injury ischemic stroke and SCI [29, 30]. To cite only some SCI examples, in a study in Sprague-Dawley rats of 250 g with moderate lesion, 10 mg/kg of melatonin was applied subcutaneously twice a day for 4 weeks, and an increase in motor recovery and decrease in inducible nitric oxide synthase (iNOS) expression were observed. Intravenously, it decreased the synthesis of MDA and increased the synthesis of GSH and angiopoietin 1, and in mice with severe lesion, it decreased the expression of interleukin 1 beta (IL-1β) and NG-2 (neuron/glial antigen 2) [30, 32, 33]. In a model with lesion with vascular clips, the administration of 30 mg/kg alleviated post-traumatic injury associated with SCI by binding the PPARα-receptor; the administration of 50 mg/kg in moderate lesion decreased the BSCB permeability modulating the expression of brainderived neurotrophic factor (BDNF), growth-associated protein 43 (GAP-43), and caspase-3 [33–35]. In combination therapy with dexamethasone (10–0.025 mg/kg), it showed significant anti-inflammatory effects, attenuating the synthesis of tumor necrosis factor alpha (TNF-α) and iNOS and the nitration of tyrosine residues, increasing tissue recovery and motor capacity in an experimental SCI model of mouse [36], while the combination with methylprednisolone favored neurological recovery and decreased LP; its administration with zinc activated the internal antioxidant system and also decreased the LP [37–39].

#### **3.2 Minocycline**

Minocycline hydrochloride is an available semisynthetic tetracycline antibiotic with potent anti-inflammatory (regulation of phospholipase A2 and MAPK/PIK3 pathways) and neuroprotective (protecting against glutamate-induced inflammation) activities; it also inhibits matrix metalloproteinases and mitochondrial Ca2+ influx. Minocycline has antioxidant and antiapoptotic properties, probably acting at high doses as a direct radical scavenger, like vitamin E, due to its phenolic ring structure [40]. In rats with SCI, minocycline given at oral doses of 3, 30, and 90 mg/kg 1 and 24 h after the lesion reduced MDA concentration and increased

GPx and SOD activity in a dose-dependent manner [41]. Minocycline decreased pro-inflammatory cytokines and the chemokines release from microglia and their activation, including their levels of enzymes that regulate LP and NO production [42]. A recovery difference between treatment and placebo, approaching to statistical significance in patients with cervical injury, was shown in a phase II clinical trial. The trail determined safety and dose optimization, within 12 h of SCI and for 7 days, with steady-state concentrations of 12.7 µg/mL in serum and 2.3 µg/mL in in cerebrospinal fluid (ClinicalTrials.gov number NCT00559494) [43].

#### **3.3 Estrogen**

Treatment with gonadal steroid hormones (estradiol, testosterone, estrogen) has resulted in motor recovery with a reduction of the lesion volume in animal models. Through its receptors (ERα and ERβ), estrogen exerts neuroprotection at physiological concentrations, and it exerts better neuroprotection as an antioxidant at high concentrations. Estrogen modulates gene expression; promotes angiogenesis; inhibits inflammation, blocking microglia from releasing inflammatory molecules such as TNF-α, ROS, prostaglandin E2, etc.; regulates the expression of antioxidant enzymes; and induces mitochondrial GSH production [44]. Different low doses and times of administration (between 10 and 100 μg/ kg/day/7 days to 4 mg/kg/15 min and 24 h, i.v.) appear to be effective, suggesting that pre-treatment or immediate posttreatment at either physiological or supraphysiological dose could minimize secondary injury in SCI and promote functional recovery, reflected in both acute and chronic stages [44, 45]. Additionally, the development of selective agonists of ER with higher affinity for ERα, ERβ, or both, such as tamoxifen, looks promising in SCI treatment, when applied in subdermal implants 7 days before, immediately, or 24 h post-injury; with an immediate release of 0.71 mg/day for 21 days, it provided motor recovery and preservation of white matter, dorsal and ventral horn neurons, with a decrease of O2 •− production [46].

#### **3.4 Omega-3 fatty acids**

The omega-3 fatty acids: α-linolenic acid, eicosapentaenoic acid (EPA, with five unsaturated bonds), and docosahexaenoic acid (DHA, with six unsaturated bonds) are part of the triacylglycerols that are consumed in the diet. DHA is a primary structural component of human brain, cerebral cortex, and retina. The lack of DHA may affect the fluidity and integrity of the membrane in synaptosomes; additionally, it affects the architecture of proteins that act as receptors and channels. Several studies have studied the effects of DHA in SCI, with treatments that include intravenous bolus, nutritional supplementation, and the use of transgenic [47]. In SCI in rats, a single application of DHA (250 nmol/kg, i.v., 30 min after injury) showed an improvement in motor recovery, smaller lesion size, greater survival of neurons and oligodendrocytes, and lower oxidation of DNA/RNA in comparison to rats without treatment [48]. More details of the application of DHA in SCI are mentioned in the chapter on Samaddar [47], as well as interesting effects on molecules involved in the repair and conservation of axonal integrity.

#### **3.5 Endogenous antioxidants (vitamins C, D, and E and ubiquinol)**

Several molecules that already act as endogenous antioxidants have been studied as candidates for application in antioxidant therapies for SCI. Vitamin C, or ascorbic acid, is a small water-soluble molecule that has a double bond and participates in

**49**

compared with the controls [57, 58].

*Current Developments in Antioxidant Therapies for Spinal Cord Injury*

preventing LP and reducing the size of the lesions [55].

The use of antibodies in the treatment of SCI is diverse and is directed to the functions of immune cells involved in inflammation and the pathological process. The initial invasion of leukocytes depends on the interaction of CD11d/ CD18 (cluster of differentiation; CD) integrin with vascular cell adhesion molecule-1 (VCAM-1). In the case of the use of anti-CD11d monoclonal antibody administered in rats to determinate the therapeutic window with 1 mg/kg doses i.v. on groups at different times of application (2, 6, 12, 24, or 48 h post-lesion), it was shown that the treatment beginning even up to 6 h after the lesion resulted in an attenuation of infiltrating leukocytes (neutrophils and macrophages, sources of ROS and RNS), lowered the expression of COX-2 and iNOS, and lowered the amounts of HNE, NT-3, and dinitrophenyl (DNP) (used for the detection of protein carbonylation) therefore acting as an indirect antioxidant. This treatment also showed improvement in motor recovery vs. a control antibody [56]. Another important integrin is the dimer α4β1 also known as very late antigen 4 (VLA-4), and treatments with anti-α4 blocking monoclonal antibodies (2.5 mg/kg/2 and 24 h/i.v.) or small molecule blocker BIO5192 (10 mg/kg/2 h/ continuous i.v. infusion for assessment of oxidative damage) showed a decreased influx of neutrophils/macrophages, reduced oxidant activity (COX-2, NO or iNOS, MDA), preserved white and gray matter, improved motor function in different evaluations, and decreased mechanical allodynia after SCI, when

**3.6 Immunotherapy**

various metabolic processes as a reducing agent. It is considered nontoxic because it does not accumulate and its concentration declines during SCI. In rats, it decreases tissue inflammation and necrosis and only at high doses (200 mg/kg i.p. 1-h postinjury, daily, until they were sacrificed, 4th week) showed improvements in motor evaluations [49]. Vitamin D (1,25-dihydroxyvitamin D3, VDH, active form) is a molecule with cholesterol skeleton and acts similarly to hormones and steroids on several systems. Its receptor (VDR) is widely distributed in the CNS, and it apparently acts on the same targets as progesterone through similar pathways. Its use in CNS damage models in vivo and in vitro has shown promising results on several aspects. The prolongation or exacerbation of inflammation also gives way to greater damage by oxidative stress; therefore, the effect of VDH in vivo on the inhibition of iNOS and increase of IL-4 and TGF-β and in vitro modulating the production of molecules involved in oxidative stress, neurotoxic damage, and axonal growth on various cells are of interest for being use in SCI [50]. Tocopherols are a group of four fat-soluble phenolic compounds designated α, β, γ, and δ, which are found in vegetable oils, being alpha (α-T, considered the classic vitamin E) the one with the highest proportion in blood and tissues. All tocopherols are strong chain breaking antioxidants by effectively scavenging ROS and RNS. α-T significantly reduces the activity of iNOS and COX-2 [51]; in addition, the effect of extracts or synthetic derivatives has been evaluated, decreasing cell death due to excitotoxicity and oxidative stress in astrocytes [52] and accelerating remyelination of focal demyelinated lesions chemically induced [53]. In rats with SCI, the use of α-T (600 mg/kg i.m., twice weekly, for 6 weeks) decreased the damage caused by ischemia-reperfusion, improving the levels of motor and sensory recovery and the level of oxidative stress [54]. Ubiquinol (reduced form) or coenzyme Q10 is among the antioxidants that decrease their concentration after SCI. It is a fat-soluble cofactor present in the inner mitochondrial membrane acting as an antioxidant in the respiratory chain. Previously, the effect on ischemia-reperfusion damage in the CNS has been proven,

*DOI: http://dx.doi.org/10.5772/intechopen.85424*

#### *Current Developments in Antioxidant Therapies for Spinal Cord Injury DOI: http://dx.doi.org/10.5772/intechopen.85424*

various metabolic processes as a reducing agent. It is considered nontoxic because it does not accumulate and its concentration declines during SCI. In rats, it decreases tissue inflammation and necrosis and only at high doses (200 mg/kg i.p. 1-h postinjury, daily, until they were sacrificed, 4th week) showed improvements in motor evaluations [49]. Vitamin D (1,25-dihydroxyvitamin D3, VDH, active form) is a molecule with cholesterol skeleton and acts similarly to hormones and steroids on several systems. Its receptor (VDR) is widely distributed in the CNS, and it apparently acts on the same targets as progesterone through similar pathways. Its use in CNS damage models in vivo and in vitro has shown promising results on several aspects. The prolongation or exacerbation of inflammation also gives way to greater damage by oxidative stress; therefore, the effect of VDH in vivo on the inhibition of iNOS and increase of IL-4 and TGF-β and in vitro modulating the production of molecules involved in oxidative stress, neurotoxic damage, and axonal growth on various cells are of interest for being use in SCI [50]. Tocopherols are a group of four fat-soluble phenolic compounds designated α, β, γ, and δ, which are found in vegetable oils, being alpha (α-T, considered the classic vitamin E) the one with the highest proportion in blood and tissues. All tocopherols are strong chain breaking antioxidants by effectively scavenging ROS and RNS. α-T significantly reduces the activity of iNOS and COX-2 [51]; in addition, the effect of extracts or synthetic derivatives has been evaluated, decreasing cell death due to excitotoxicity and oxidative stress in astrocytes [52] and accelerating remyelination of focal demyelinated lesions chemically induced [53]. In rats with SCI, the use of α-T (600 mg/kg i.m., twice weekly, for 6 weeks) decreased the damage caused by ischemia-reperfusion, improving the levels of motor and sensory recovery and the level of oxidative stress [54]. Ubiquinol (reduced form) or coenzyme Q10 is among the antioxidants that decrease their concentration after SCI. It is a fat-soluble cofactor present in the inner mitochondrial membrane acting as an antioxidant in the respiratory chain. Previously, the effect on ischemia-reperfusion damage in the CNS has been proven, preventing LP and reducing the size of the lesions [55].

#### **3.6 Immunotherapy**

*Spinal Cord Injury Therapy*

**3.3 Estrogen**

GPx and SOD activity in a dose-dependent manner [41]. Minocycline decreased pro-inflammatory cytokines and the chemokines release from microglia and their activation, including their levels of enzymes that regulate LP and NO production [42]. A recovery difference between treatment and placebo, approaching to statistical significance in patients with cervical injury, was shown in a phase II clinical trial. The trail determined safety and dose optimization, within 12 h of SCI and for 7 days, with steady-state concentrations of 12.7 µg/mL in serum and 2.3 µg/mL in in

Treatment with gonadal steroid hormones (estradiol, testosterone, estrogen) has resulted in motor recovery with a reduction of the lesion volume in animal models. Through its receptors (ERα and ERβ), estrogen exerts neuroprotection at physiological concentrations, and it exerts better neuroprotection as an antioxidant at high concentrations. Estrogen modulates gene expression; promotes angiogenesis; inhibits inflammation, blocking microglia from releasing inflammatory molecules such as TNF-α, ROS, prostaglandin E2, etc.; regulates the expression of antioxidant enzymes; and induces mitochondrial GSH production [44]. Different low doses and times of administration (between 10 and 100 μg/ kg/day/7 days to 4 mg/kg/15 min and 24 h, i.v.) appear to be effective, suggesting that pre-treatment or immediate posttreatment at either physiological or supraphysiological dose could minimize secondary injury in SCI and promote functional recovery, reflected in both acute and chronic stages [44, 45]. Additionally, the development of selective agonists of ER with higher affinity for ERα, ERβ, or both, such as tamoxifen, looks promising in SCI treatment, when applied in subdermal implants 7 days before, immediately, or 24 h post-injury; with an immediate release of 0.71 mg/day for 21 days, it provided motor recovery and preservation of white matter, dorsal and ventral horn neurons, with a decrease of

The omega-3 fatty acids: α-linolenic acid, eicosapentaenoic acid (EPA, with five unsaturated bonds), and docosahexaenoic acid (DHA, with six unsaturated bonds) are part of the triacylglycerols that are consumed in the diet. DHA is a primary structural component of human brain, cerebral cortex, and retina. The lack of DHA may affect the fluidity and integrity of the membrane in synaptosomes; additionally, it affects the architecture of proteins that act as receptors and channels. Several studies have studied the effects of DHA in SCI, with treatments that include intravenous bolus, nutritional supplementation, and the use of transgenic [47]. In SCI in rats, a single application of DHA (250 nmol/kg, i.v., 30 min after injury) showed an improvement in motor recovery, smaller lesion size, greater survival of neurons and oligodendrocytes, and lower oxidation of DNA/RNA in comparison to rats without treatment [48]. More details of the application of DHA in SCI are mentioned in the chapter on Samaddar [47], as well as interesting effects on molecules involved in the

Several molecules that already act as endogenous antioxidants have been studied as candidates for application in antioxidant therapies for SCI. Vitamin C, or ascorbic acid, is a small water-soluble molecule that has a double bond and participates in

cerebrospinal fluid (ClinicalTrials.gov number NCT00559494) [43].

**48**

O2

•− production [46].

**3.4 Omega-3 fatty acids**

repair and conservation of axonal integrity.

**3.5 Endogenous antioxidants (vitamins C, D, and E and ubiquinol)**

The use of antibodies in the treatment of SCI is diverse and is directed to the functions of immune cells involved in inflammation and the pathological process. The initial invasion of leukocytes depends on the interaction of CD11d/ CD18 (cluster of differentiation; CD) integrin with vascular cell adhesion molecule-1 (VCAM-1). In the case of the use of anti-CD11d monoclonal antibody administered in rats to determinate the therapeutic window with 1 mg/kg doses i.v. on groups at different times of application (2, 6, 12, 24, or 48 h post-lesion), it was shown that the treatment beginning even up to 6 h after the lesion resulted in an attenuation of infiltrating leukocytes (neutrophils and macrophages, sources of ROS and RNS), lowered the expression of COX-2 and iNOS, and lowered the amounts of HNE, NT-3, and dinitrophenyl (DNP) (used for the detection of protein carbonylation) therefore acting as an indirect antioxidant. This treatment also showed improvement in motor recovery vs. a control antibody [56]. Another important integrin is the dimer α4β1 also known as very late antigen 4 (VLA-4), and treatments with anti-α4 blocking monoclonal antibodies (2.5 mg/kg/2 and 24 h/i.v.) or small molecule blocker BIO5192 (10 mg/kg/2 h/ continuous i.v. infusion for assessment of oxidative damage) showed a decreased influx of neutrophils/macrophages, reduced oxidant activity (COX-2, NO or iNOS, MDA), preserved white and gray matter, improved motor function in different evaluations, and decreased mechanical allodynia after SCI, when compared with the controls [57, 58].

#### **3.7 Antioxidant peptides**

#### *3.7.1 A91 peptide*

Modified neural peptides are peptide analogs of the myelin basic protein (MBP) epitopes that possess one or more aa substitutions and that have a partial agonist or antagonist action when in contact with the T lymphocyte (TL) receptor [59, 60].

Schwartz and Hauben tested the administration of non-encephalitogenic peptides of different aa sequences associated with MBP, which are named according to the position of the aa substitution that is performed: A96, G91, and A91, among others. A91 showed the best results after a traumatic injury, both in the optic nerve and in spinal cord, without showing clinical signs of autoimmune disease, hypersensitivity, immunosuppression, and controlling the destructive action of autoreactive TL [61, 62].

A91 is a peptide belonging to the aa 87–99 sequence of MBP with the substitution of an aa at position 91 of a lysine (VHFFKNIVTPRTP) by an alanine (VHFFANIVTPRTP), functioning as a partial agonist peptide and promoting a change of the profile of cytokines produced by TL reactive against the 87–99 sequence of the MBP of a Th1 phenotype (interferon gamma [IFN-γ], TNF, IL-2) to a Th2 (IL-4, IL-10) and decreasing the action and synthesis of the FR, among other effects [63]. A91 allows activating the microglia with a phenotype producing neurotrophic factors, which together with the release of factors produced by other cells such as monocytes (MN) and TL reduce secondary neuronal degeneration [64–66].

The beneficial effect of subcutaneous immunization at the base of the tail has been demonstrated with A91 at a single dose (150–200 μg/kg) after SCI due to moderate contusion. This immunization, among various factors and effects, promotes neuroprotection and motor recovery by decreasing the expression of iNOS and production of NO• , LP, caspase 3, and pro-inflammatory cytokines and increasing the release of neurotrophic factors such as BDNF and NT-3. The effect of the immunization is preserved in the chronic stage of the lesion and as a prophylactic treatment or up to 72 h after the SCI; however, it diminishes when applied to lesions due to severe contusion or complete medullar cut and is eliminated with a double immunization. It has also been determined that the severity of the lesion determines the profile of genetic expression in the lesion after immunization and that immunization plus the removal of the fibroglial scar and/or the implant of a scaffold as support for mesenchymal stem cells favors a permissive microenvironment for motor recovery and improves the electrophysiological activity in the chronic stage after a complete section of the spinal cord [67–73]. The protective response of A91 is between 4 and 6 days, indicating that it acts on subsequent mechanisms to the acute stage. During this time, the oxidative processes are not completely modulated. Regarding this, it has been shown that the therapeutic combination of A91 peptide with peptides acting at shorter times, such as glutathione monoethyl ester (GSH-MEE) or the monocyte locomotion inhibitory factor (MLIF), reduces FR and LP and induces better motor recovery, neural survival, presence of myelinated axons, and tissue protection. In the same way, it was demonstrated that the combination of A91 with GSH-MEE retains the effect if applied until 72 hrs after the lesion [68, 74, 75].

#### *3.7.2 Monocyte locomotion inhibitory factor (MLIF) peptide*

MLIF is a pentapeptide (Met-Gln-Cys-Asn-Ser). In vitro studies showed that MLIF decreases MN locomotion, the production of ROS (H2O2, O2 •-, HO• ), NO• , and cGMP, and it induces an increase of microtubules associated to the centriole and the concentration of cAMP [76–78]. The pharmacophore group of the MLIF is integrated

**51**

**Figure 3.**

*Current Developments in Antioxidant Therapies for Spinal Cord Injury*

rats when applied directly to the site of injury after surgery [85].

underway to determine the concentration of MLIF in plasma [88].

decrease in the LP, the concentration of NO•

by the Cys-Asn-Ser tripeptide, which retains the same biological activities of the

Studies in cerebral ischemia showed that the penetrating, antioxidant, antiinflammatory, and neuroprotective capacity of the pharmacophore group is favored in analogs when the N-terminal end is modified by adding one of the following aa: Asp, His, Try, or Arg. In the same way, cardioprotective effects have been seen in myocardial ischemia [86, 87]. On the other hand, pharmacokinetic studies are

In base studies of our group, rats were subjected to a moderate SCI, and a dose of 200 μg of MLIF was applied directly to the site of the lesion. The animals treated with the factor presented a greater motor recovery than the non-treated, and a

increase in the expression of the IL-10 and TGF-β (Transforming Growth Factor beta) genes was observed at 3 h and 7 days post-injury, favoring the survival of the ventral horn neurons [75]. Subsequent studies showed that four doses of the MLIF at the same concentration immediately initiating direct administration at the site of injury and subsequently one dose every 24 h for 3 days by i.p. administration are sufficient to improve motor recovery in rats subjected to SCI. In the same way, therapeutic combinations of MLIF, at different times and doses, have favored the effect of the MLIF in the experimental model of SCI modulating the synthesis of

GSH (**Figure 3**) is a tripeptide (L-γ-glutamyl-L-cysteinyl-glycine), nonprotein thiol. It is synthesized in the cellular cytoplasm by the consecutive action of two enzymes. The first, γ-glutamylcysteinyl ligase, is regulated by the nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or Nrf2), which is sensitive to oxidative stress. This enzyme uses glutamic acid (Glu) and Cys aa, glutamic acid (Glu) and Cys aa, as a substrate to form the γ-glutamylcysteine dipeptide (γ-GluCys), which

*Condensed structural chemical formula of glutathione (IUPAC name: (2S)-2-amino-4-{[(1R)-1-*

*[(carboxymethyl) carbomoyl]-2-sulfanylethyl] carbomyl1} butanoic acid). Modified from Gaucher et al. [89].*

, and the expression of the iNOS. An

The MLIF favors the Th2 response; modulates the synthesis of pro-/anti-inflammatory cytokines and the expression of genes involved in inflammation, proliferation, angiogenesis, synthesis/degradation of extracellular matrix, angiogenesis, and axonal guidance, among others; and acts mainly through the signaling pathways: NF-kB, MAPKinases, and eEF1A1/endothelial nitric oxide synthase [81–84]. In vivo, the factor retards the arrival of MN in Rebuck windows and inhibits cutaneous delayed hypersensitivity to dinitrochlorobenzene, while in guinea pigs, it lobs down the expression of VLA-4 and VCAM-1 adhesion molecules in postcapillary vascular endothelium and decreases the formation of pericardial adhesions in

*DOI: http://dx.doi.org/10.5772/intechopen.85424*

factor [79, 80].

the FR and ROS.

*3.7.3 Glutathione (GSH) peptide*

#### *Current Developments in Antioxidant Therapies for Spinal Cord Injury DOI: http://dx.doi.org/10.5772/intechopen.85424*

*Spinal Cord Injury Therapy*

**3.7 Antioxidant peptides**

Modified neural peptides are peptide analogs of the myelin basic protein (MBP) epitopes that possess one or more aa substitutions and that have a partial agonist or antagonist action when in contact with the T lymphocyte (TL) receptor [59, 60]. Schwartz and Hauben tested the administration of non-encephalitogenic peptides of different aa sequences associated with MBP, which are named according to the position of the aa substitution that is performed: A96, G91, and A91, among others. A91 showed the best results after a traumatic injury, both in the optic nerve and in spinal cord, without showing clinical signs of autoimmune disease, hypersensitivity, immunosuppression, and controlling the destructive action of autoreactive TL [61, 62]. A91 is a peptide belonging to the aa 87–99 sequence of MBP with the substitution of an aa at position 91 of a lysine (VHFFKNIVTPRTP) by an alanine (VHFFANIVTPRTP), functioning as a partial agonist peptide and promoting a change of the profile of cytokines produced by TL reactive against the 87–99 sequence of the MBP of a Th1 phenotype (interferon gamma [IFN-γ], TNF, IL-2) to a Th2 (IL-4, IL-10) and decreasing the action and synthesis of the FR, among other effects [63]. A91 allows activating the microglia with a phenotype producing neurotrophic factors, which together with the release of factors produced by other cells such as monocytes (MN) and TL reduce secondary neuronal degeneration [64–66]. The beneficial effect of subcutaneous immunization at the base of the tail has been demonstrated with A91 at a single dose (150–200 μg/kg) after SCI due to moderate contusion. This immunization, among various factors and effects, promotes neuroprotection and motor recovery by decreasing the expression of iNOS and pro-

, LP, caspase 3, and pro-inflammatory cytokines and increasing the

release of neurotrophic factors such as BDNF and NT-3. The effect of the immunization is preserved in the chronic stage of the lesion and as a prophylactic treatment or up to 72 h after the SCI; however, it diminishes when applied to lesions due to severe contusion or complete medullar cut and is eliminated with a double immunization. It has also been determined that the severity of the lesion determines the profile of genetic expression in the lesion after immunization and that immunization plus the removal of the fibroglial scar and/or the implant of a scaffold as support for mesenchymal stem cells favors a permissive microenvironment for motor recovery and improves the electrophysiological activity in the chronic stage after a complete section of the spinal cord [67–73]. The protective response of A91 is between 4 and 6 days, indicating that it acts on subsequent mechanisms to the acute stage. During this time, the oxidative processes are not completely modulated. Regarding this, it has been shown that the therapeutic combination of A91 peptide with peptides acting at shorter times, such as glutathione monoethyl ester (GSH-MEE) or the monocyte locomotion inhibitory factor (MLIF), reduces FR and LP and induces better motor recovery, neural survival, presence of myelinated axons, and tissue protection. In the same way, it was demonstrated that the combination of A91 with GSH-MEE retains

MLIF is a pentapeptide (Met-Gln-Cys-Asn-Ser). In vitro studies showed that

cGMP, and it induces an increase of microtubules associated to the centriole and the concentration of cAMP [76–78]. The pharmacophore group of the MLIF is integrated

•-, HO•

), NO•

, and

the effect if applied until 72 hrs after the lesion [68, 74, 75].

*3.7.2 Monocyte locomotion inhibitory factor (MLIF) peptide*

MLIF decreases MN locomotion, the production of ROS (H2O2, O2

*3.7.1 A91 peptide*

duction of NO•

**50**

by the Cys-Asn-Ser tripeptide, which retains the same biological activities of the factor [79, 80].

The MLIF favors the Th2 response; modulates the synthesis of pro-/anti-inflammatory cytokines and the expression of genes involved in inflammation, proliferation, angiogenesis, synthesis/degradation of extracellular matrix, angiogenesis, and axonal guidance, among others; and acts mainly through the signaling pathways: NF-kB, MAPKinases, and eEF1A1/endothelial nitric oxide synthase [81–84].

In vivo, the factor retards the arrival of MN in Rebuck windows and inhibits cutaneous delayed hypersensitivity to dinitrochlorobenzene, while in guinea pigs, it lobs down the expression of VLA-4 and VCAM-1 adhesion molecules in postcapillary vascular endothelium and decreases the formation of pericardial adhesions in rats when applied directly to the site of injury after surgery [85].

Studies in cerebral ischemia showed that the penetrating, antioxidant, antiinflammatory, and neuroprotective capacity of the pharmacophore group is favored in analogs when the N-terminal end is modified by adding one of the following aa: Asp, His, Try, or Arg. In the same way, cardioprotective effects have been seen in myocardial ischemia [86, 87]. On the other hand, pharmacokinetic studies are underway to determine the concentration of MLIF in plasma [88].

In base studies of our group, rats were subjected to a moderate SCI, and a dose of 200 μg of MLIF was applied directly to the site of the lesion. The animals treated with the factor presented a greater motor recovery than the non-treated, and a decrease in the LP, the concentration of NO• , and the expression of the iNOS. An increase in the expression of the IL-10 and TGF-β (Transforming Growth Factor beta) genes was observed at 3 h and 7 days post-injury, favoring the survival of the ventral horn neurons [75]. Subsequent studies showed that four doses of the MLIF at the same concentration immediately initiating direct administration at the site of injury and subsequently one dose every 24 h for 3 days by i.p. administration are sufficient to improve motor recovery in rats subjected to SCI. In the same way, therapeutic combinations of MLIF, at different times and doses, have favored the effect of the MLIF in the experimental model of SCI modulating the synthesis of the FR and ROS.

#### *3.7.3 Glutathione (GSH) peptide*

GSH (**Figure 3**) is a tripeptide (L-γ-glutamyl-L-cysteinyl-glycine), nonprotein thiol. It is synthesized in the cellular cytoplasm by the consecutive action of two enzymes. The first, γ-glutamylcysteinyl ligase, is regulated by the nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or Nrf2), which is sensitive to oxidative stress. This enzyme uses glutamic acid (Glu) and Cys aa, glutamic acid (Glu) and Cys aa, as a substrate to form the γ-glutamylcysteine dipeptide (γ-GluCys), which

**Figure 3.**

*Condensed structural chemical formula of glutathione (IUPAC name: (2S)-2-amino-4-{[(1R)-1- [(carboxymethyl) carbomoyl]-2-sulfanylethyl] carbomyl1} butanoic acid). Modified from Gaucher et al. [89].*

is combined with glycine (Gly) in a reaction catalyzed by the second enzyme (glutathione synthase) to form GSH, whose concentration is regulated by the nhibition of γ-GluCys ligase, the cellular content of L-cysteine, and the final concentration of GSH. Thus, the intracellular and extracellular concentrations of GSH are determined by the balance among its synthesis, catabolism, and transport between cytosol and the different organelles [89].

GSH, by itself, is not transported effectively into the cells, and under normal physiological conditions, it is in a reduced form. During its oxidation (where the thiol group of Cys is responsible for the redox reactions) by ROS and RNS, it involves two types of reactions, a nonenzymatic reaction with the NO• , HO− , and O2 •− radicals and an enzymatic one providing an electron for the reduction of peroxides in the reaction, catalyzed by GPx to form the oxidized glutathione GSSG (two GSH molecules bound by the disulfide bridge), which is regenerated by Gr, an enzyme that transfers electrons from NADPH to GSSG by reducing it [90, 91]. Thus, the redox state of GSH activates the activator protein 1 (AP-1) responsible for the expression of cytokine genes, TGF-β, and collagenase and AP-2 responsible for the activation of c-Jun-N-terminal kinases (JNK), stress-activated protein kinases (SAPK), protein kinase c (PK-C), and tyrosine kinase, while the decrease in the GSH level stimulates the activation of NF-κB, protein kinase B, c-Jun N-terminal Kinase, and mitogen-activated protein kinase with the subsequent increase in synthesis of pro-inflammatory cytokines and caspases. In suitable concentrations, GSH increases the activation, proliferation, and cellular differentiation and regulates the Ca2+ homeostasis [91], granting a fundamental role in cellular homeostasis and pathologies related to patient's age and oxidative stress states, such as neurodegenerative, neuroinflammatory, cardiovascular diseases, and cerebral ischemia, among others [92, 93]. To increase the intracellular GSH concentration levels, GSH precursors have been used, without modifying the Cys that is critical for the functioning of the peptide. GSH precursor molecules such as N-acetyl cysteine (NAC) stimulate the biosynthesis of GSH that acts directly on ROS, RNS [89, 93, 94], and glutathione esters, mainly mono- and dimethyl esters such as glutathione monoethyl ester [γ- Glu-Cys-Gly-OEt (GSH-MEE)], where the carboxyl group of Gly is esterified and, due to its high hydrophobicity, increases its permeability to the cell membrane and facilitates its transport in brain-spinal fluid [95–97]. Once GSH-MEE is located in the cellular cytoplasm, it is hydrolyzed by the intracellular esterases to release and cause the intracellular increase in the GSH concentration and react with the FR without enzymatic intervention or it reduces the peroxides by means of GPx through its oxidation to GSSG [89, 91, 98, 99].

GSH-MEE has been used effectively to protect cells from oxidizing agents and various toxic compounds in various cell lines and animal models with neurodegenerative and inflammatory processes [92, 99, 100]. Studies of our group and collaborators have shown that the i.p. administration of 12 mg/kg of GSH-MEE divided into four doses in the first 24 h post-lesion in rats subjected to a moderate SCI contributes to the reduction of oxidative stress, significantly improves motor function and survival of red core neurons, and stabilizes spinal cord blood flow [100], while a therapeutic combination of GSH-MEE (at the same dose and under the same scheme) with intradermal application (i.d.) of the A91 peptide at the base of the tail at a dose of 600 μg/kg immediately after the injury promotes a better neurological recovery and morphological preservation. This combination is able to maintain its neuroprotective action even if it starts 72 h after the injury [68, 74]. In the same way, our group has demonstrated that the therapeutic combination of GSH-MEE and MLIF promotes greater motor recovery and maintains several morphological aspects on the site of lesion in rats subjected to moderate SCI.

**53**

**Figure 4.**

*Current Developments in Antioxidant Therapies for Spinal Cord Injury*

A variety of ingredients and active ingredients derived from herbal extracts, known for their antioxidant and anti-inflammatory activity, have also called the attention to complement SCI treatments. Among all these, ingredients such as curcumin, resveratrol, epigallocatechin gallate, ligustrazine, quercitrin, and puerarin and herbs such as Dashen, *Ginkgo biloba*, Ginseng, Notoginseng, and Astragali Radix are outlined as candidates for various experimental studies. In the review by Zhang et al. [101], the molecular structures, application, and dose are listed, as well as the results found at a molecular level on SCI. In particular, the compound curcumin is a polyphenol substance isolated from the yellow extract from rhizome of *Curcuma longa*, and it has been widely used for medicinal purposes due to its potent effect in inhibiting acute and chronic inflammation. Regarding its antioxidant action, its application in SCI (300 mg/kg in DMSO, single dose i.p. after injury) has shown a decrease in MDA and an increase in SOD at 24 h [102], and at a lower dose, it has also increased the concentration and induction of GSH, GPx, and

Nrf2 and decreased the expression of NF-κB, TNF-α, and IL-1β [101].

A highly studied antioxidant strategy consists of scavengers of FR that include, among others, thiols (lipoic acid), GSH precursors, NAC, polyphenolic compounds, hydroxyl stilbenes, nitrones, and spin trappings (noncyclic and cyclic nitrones); we will only review the latter. Most spin trappings have a nitronate or nitroxide nucleus and are chemical agents that react with FR, forming stable products (adducts), and were originally developed as a tool to detect and stabilize the FR in chemistry and later in biological oxidation processes [103–105]. The first spin trappings had short

sion of heterocyclic rings (pyrrolines or phenol, generating Imidazolyl-nitrones, Furil-nitrones, Arylnitrons, and others) toxicity was reduced, improving its neuroprotective, anti-inflammatory, functionality, stability, bioavailability, and trapping different types of FR centered on O2, carbon, and sulfur derivatives. In turn, this increases their solubility in high concentrations in a large number of solvents (~0.1M), producing a positive effect when administered in a varied-dose scheme before or after a traumatic event [103, 106]. A basic example of the nitrones is phenyl N-tert-butylnitrone (PBN), an arylnitrone with general formula X-CN = NO-Y,

•− and/or HO−

is formed, the radical is inactivated and unable to damage the cell tissue [104, 107]. The general reaction is that of the formation of adduct, schematized in **Figure 4**. In general, it is indicated that PBN is not toxic and the suitable concentration to form adducts is 10–15 mg/100 g of weight, while the estimated lethal dose is 10 times higher (100–150 mg/100 g of weight) [108]. The first neuroprotective evidence was in neurodegenerative models administered at low doses after injury and in the

*Basic reaction of a nitrone with FR to produce a stable spin product (adduct). Modified from Refs. [105, 106].*

. By designing the spin trappings with the inclu-

to produce adducts. Once the adduct

*DOI: http://dx.doi.org/10.5772/intechopen.85424*

**3.8 Natural extracts as antioxidants**

**3.9 Spin trappings**

half-lives and generated toxic HO•

which acts by reacting with O2

#### **3.8 Natural extracts as antioxidants**

*Spinal Cord Injury Therapy*

is combined with glycine (Gly) in a reaction catalyzed by the second enzyme (glutathione synthase) to form GSH, whose concentration is regulated by the nhibition of γ-GluCys ligase, the cellular content of L-cysteine, and the final concentration of GSH. Thus, the intracellular and extracellular concentrations of GSH are determined by the balance among its synthesis, catabolism, and trans-

GSH, by itself, is not transported effectively into the cells, and under normal physiological conditions, it is in a reduced form. During its oxidation (where the thiol group of Cys is responsible for the redox reactions) by ROS and RNS, it involves two

an enzymatic one providing an electron for the reduction of peroxides in the reaction, catalyzed by GPx to form the oxidized glutathione GSSG (two GSH molecules bound by the disulfide bridge), which is regenerated by Gr, an enzyme that transfers electrons from NADPH to GSSG by reducing it [90, 91]. Thus, the redox state of GSH activates the activator protein 1 (AP-1) responsible for the expression of cytokine genes, TGF-β, and collagenase and AP-2 responsible for the activation of c-Jun-N-terminal kinases (JNK), stress-activated protein kinases (SAPK), protein kinase c (PK-C), and tyrosine kinase, while the decrease in the GSH level stimulates the activation of NF-κB, protein kinase B, c-Jun N-terminal Kinase, and mitogen-activated protein kinase with the subsequent increase in synthesis of pro-inflammatory cytokines and caspases. In suitable concentrations, GSH increases the activation, proliferation, and cellular differentiation and regulates the Ca2+ homeostasis [91], granting a fundamental role in cellular homeostasis and pathologies related to patient's age and oxidative stress states, such as neurodegenerative, neuroinflammatory, cardiovascular diseases, and cerebral ischemia, among others [92, 93]. To increase the intracellular GSH concentration levels, GSH precursors have been used, without modifying the Cys that is critical for the functioning of the peptide. GSH precursor molecules such as N-acetyl cysteine (NAC) stimulate the biosynthesis of GSH that acts directly on ROS, RNS [89, 93, 94], and glutathione esters, mainly mono- and dimethyl esters such as glutathione monoethyl ester [γ- Glu-Cys-Gly-OEt (GSH-MEE)], where the carboxyl group of Gly is esterified and, due to its high hydrophobicity, increases its permeability to the cell membrane and facilitates its transport in brain-spinal fluid [95–97]. Once GSH-MEE is located in the cellular cytoplasm, it is hydrolyzed by the intracellular esterases to release and cause the intracellular increase in the GSH concentration and react with the FR without enzymatic intervention or it reduces the peroxides by means of GPx

GSH-MEE has been used effectively to protect cells from oxidizing agents and various toxic compounds in various cell lines and animal models with neurodegenerative and inflammatory processes [92, 99, 100]. Studies of our group and collaborators have shown that the i.p. administration of 12 mg/kg of GSH-MEE divided into four doses in the first 24 h post-lesion in rats subjected to a moderate SCI contributes to the reduction of oxidative stress, significantly improves motor function and survival of red core neurons, and stabilizes spinal cord blood flow [100], while a therapeutic combination of GSH-MEE (at the same dose and under the same scheme) with intradermal application (i.d.) of the A91 peptide at the base of the tail at a dose of 600 μg/kg immediately after the injury promotes a better neurological recovery and morphological preservation. This combination is able to maintain its neuroprotective action even if it starts 72 h after the injury [68, 74]. In the same way, our group has demonstrated that the therapeutic combination of GSH-MEE and MLIF promotes greater motor recovery and maintains several morphological

, HO−

, and O2

•− radicals and

port between cytosol and the different organelles [89].

types of reactions, a nonenzymatic reaction with the NO•

through its oxidation to GSSG [89, 91, 98, 99].

aspects on the site of lesion in rats subjected to moderate SCI.

**52**

A variety of ingredients and active ingredients derived from herbal extracts, known for their antioxidant and anti-inflammatory activity, have also called the attention to complement SCI treatments. Among all these, ingredients such as curcumin, resveratrol, epigallocatechin gallate, ligustrazine, quercitrin, and puerarin and herbs such as Dashen, *Ginkgo biloba*, Ginseng, Notoginseng, and Astragali Radix are outlined as candidates for various experimental studies. In the review by Zhang et al. [101], the molecular structures, application, and dose are listed, as well as the results found at a molecular level on SCI. In particular, the compound curcumin is a polyphenol substance isolated from the yellow extract from rhizome of *Curcuma longa*, and it has been widely used for medicinal purposes due to its potent effect in inhibiting acute and chronic inflammation. Regarding its antioxidant action, its application in SCI (300 mg/kg in DMSO, single dose i.p. after injury) has shown a decrease in MDA and an increase in SOD at 24 h [102], and at a lower dose, it has also increased the concentration and induction of GSH, GPx, and Nrf2 and decreased the expression of NF-κB, TNF-α, and IL-1β [101].

#### **3.9 Spin trappings**

A highly studied antioxidant strategy consists of scavengers of FR that include, among others, thiols (lipoic acid), GSH precursors, NAC, polyphenolic compounds, hydroxyl stilbenes, nitrones, and spin trappings (noncyclic and cyclic nitrones); we will only review the latter. Most spin trappings have a nitronate or nitroxide nucleus and are chemical agents that react with FR, forming stable products (adducts), and were originally developed as a tool to detect and stabilize the FR in chemistry and later in biological oxidation processes [103–105]. The first spin trappings had short half-lives and generated toxic HO• . By designing the spin trappings with the inclusion of heterocyclic rings (pyrrolines or phenol, generating Imidazolyl-nitrones, Furil-nitrones, Arylnitrons, and others) toxicity was reduced, improving its neuroprotective, anti-inflammatory, functionality, stability, bioavailability, and trapping different types of FR centered on O2, carbon, and sulfur derivatives. In turn, this increases their solubility in high concentrations in a large number of solvents (~0.1M), producing a positive effect when administered in a varied-dose scheme before or after a traumatic event [103, 106]. A basic example of the nitrones is phenyl N-tert-butylnitrone (PBN), an arylnitrone with general formula X-CN = NO-Y, which acts by reacting with O2 •− and/or HO− to produce adducts. Once the adduct is formed, the radical is inactivated and unable to damage the cell tissue [104, 107]. The general reaction is that of the formation of adduct, schematized in **Figure 4**. In general, it is indicated that PBN is not toxic and the suitable concentration to form adducts is 10–15 mg/100 g of weight, while the estimated lethal dose is 10 times higher (100–150 mg/100 g of weight) [108]. The first neuroprotective evidence was in neurodegenerative models administered at low doses after injury and in the

$$\underbrace{\begin{array}{c} \text{\raisebox{-0.0pt}{ $\mathop{\text{\raisebox{-0.0pt}{$ \mathop{\text{\raisebox{-0.0pt}{ $\mathop{\text{\raisebox{-0.0pt}{$ \mathop{\text{\raisebox{-0.0pt}{ $\mathop{\text{\raisebox{-0.0pt}{$ \mathop{\text{\raisebox{-0.0pt}{ $\mathop{\text{\raisebox{-0.0pt}{$ \mathop{\text{\raisebox{-0.0pt}{ $\mathop{\text{\raisebox{-0.0pt}{$ \mathop{\text{\raisebox{-0.0pt}{ $\mathop{\text{\raisebox{-0.0pt}{$ \mathop{\text{\raisebox{-0.0pt}{ $\cdots$ 0}}{ $\rightlef{I}}{$ \mathop{\text{\raisebox{-0.0pt}{ $\mathop{\text{\raisebox{-0.0pt}{$ \cdots $0}}{$ \rightlef{I}}{ $\cdot$ }}}}}}}}}}}}}} \x}} \x \x \x \x \x \x \x \x \x \x \x \x \x \x \x \x \x \x \x \x \x \x \x} $$

**Figure 4.**

*Basic reaction of a nitrone with FR to produce a stable spin product (adduct). Modified from Refs. [105, 106].*

prevention of stroke-induced mortality in models of ischemia in gerbils [103, 104, 109–113]. The pharmacological effects of PBN in animal models are extensive, protecting against death after endotoxic shock, bacterial meningitis, teratogenicity induced by thalidomide, diabetogenesis, hepatocarcinogenesis, etc. Many studies have reported a neuroprotective effect in SCI and the brain (the most studied) decreasing the expression of genes associated with apoptosis, inflammation, and iNOS by decreasing the activation of MAP p-38 NF-κB nitrogen kinase and synthesis of NO• [114]. In a process of ischemia or perfusion, PBN reduces the size of the infarct by increasing ischemic reperfusion and decreasing neurodegeneration, excitotoxicity, and the activation of microglia; it also induces neurite growth through indirect activation of the Ras-ERK pathway, increasing animal survival [106, 115–117]. The neuroprotective effect of PBN is attributed to its ability to quickly and easily penetrate the membranes and the blood-brain barrier with a half-life of 3 h in plasma; decrease the levels of oxidized proteins, 8-isoprostane, HNE, IL-1β, TNF-α, IFN-γ, c-fos, IL-3, IL-4, IL-5, and H2O2; and favor an increase of GHS and IL-10, among others [106, 117–119]. In a model of cortical contusion in rats, it was demonstrated that pre-treatment with PBN with a single intravenous dose of 30 mg/ kg 30 min before the injury reduces the cognitive deficit and its volume; it has shown to have a wide therapeutic window in focal ischemia rodent models, reducing the infarct volume when administered up to 12 h after the beginning of the stroke and reducing the loss of tissue when administered by fluid percussion 30 min. After injury in rats [120]. Currently, the nitrones derived from PBN [102] are being widely studied as neuroprotective in different CNS pathologies and in traumatic lesions. For example, 2,4-disulfophenyl-N-tert-butylnitrone (NXY-059) has neuroprotective effects when applied 4–5 hr post-occlusion at equimolar doses to PBN and reduced infarct volume from 37.2 to 12.5% when 30 mg/kg was administered i.v. 1 h after reperfusion in Wistar rats [121–124]. Meanwhile, stilbazulenyl nitrone (STAZN) exerts similar effects at lower doses than the one used for NXY-059; in fact, the tolerability and safety of NXY-059 were studied in patients with acute stroke in clinical trials [103, 124]. Although not all compounds have demonstrated their neuroprotective effect when administered 24 h after the traumatic event, some of them have allowed favoring the therapeutic window at repeated doses [103].

Other derivatives are 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and diesterified nitrone (EMEPO), which have shown similarities to the action of PBN but with some other advantages, such as being less toxic and increasing the levels of antiapoptotic proteins such as Bcl2 and p-Bad and decreasing the synthesis of pro-apoptotic ones such as caspase 3, p53, and Bax [125–127]. In addition, (2, 2, 6, 6-tetramethylpiperidin-1-yl)oxyl (Tempo) and (4-hydroxy-2,2,6,6-tetramethylpiperidin-1-yl)oxyl (Tempol) have shown antioxidant properties in radiation damage and injury [128, 129]. In a traumatic brain injury mouse model, Tempol reduced post-traumatic LP and oxidative damage induced by protein nitration, decreasing mitochondrial damage, cytoskeletal damage, and neurodegeneration and improving motor function [128, 130, 131].

Despite the results observed with the nitrones and the wide range of studies performed for therapeutic uses at different doses and times, their action is attributed to their ability to form adducts, but not before indicating the possible participation of other mechanisms that favor their neuroprotective activity, thus expanding the information on antioxidant therapy strategies in the clinical area.

#### **3.10 Polyethylene glycol (PEG)-superoxide dismutase (SOD)**

Polyethylene glycol (PEG) is a surfactant that due to its hydrophilic nature allows the fusion and fluidity of the cell membrane that reduces the oxidative

**55**

**3.11 Mannitol**

*Current Developments in Antioxidant Therapies for Spinal Cord Injury*

effects of the secondary stage and that during the acute phase of SCI, it may inhibit nerve fiber degeneration and create a favorable microenvironment for the regeneration of nerve filaments that can stimulate angiogenesis and reduce glial scar, promoting the regeneration of axonal guidance and motor recovery. PEG has been widely used as a scaffold for a large variety of molecules in treatment for SCI [132–136], while the SOD enzyme has antioxidant properties, as mentioned previously. The combination of SOD with PEG (PEG-SOD) allows an increase of the enzyme intracellularly and its antioxidant activity, and it may have an important role in vascular relaxation by reducing the concentration of O2•− and limiting the LP [132]. It has been used in myocardial ischemia and in lung injury models, proposed

In a controlled phase II study in patients in a coma who suffered a stroke and received a single i.v. dose of 2000, 5000, or 10,000 IU/kg 4 h after the injury, its recovery was better in comparison to the group that received placebo (44% were in a vegetative state or died); no side effects were observed in this study due to the

In a study in a cerebral ischemia model performed in rats, 10,000 IU/kg of PSG-SOD were i.v. administered, and the group presented a significant reduction in infarct size in comparison to the control group [138]. In other study with Sprague-Dawley male rats (300-350g of weight), an occlusion of the hepatic artery was performed and reperfusion was performed after 90 min to generate liver damage. A group of animals received i.v. 5000 U/kg of PEG-SOD before vascular occlusion and immediately after reperfusion, while the control group only received a saline solution following the same scheme. In the group treated with PEG-SOD, hepatic ischemia and LP were attenuated. Meanwhile, another study examined the effect of PEG-SOD on focal cerebral ischemia/ reperfusion in rats; the results showed that the effect is variable, depending on the dosage [132, 139]. In a dog experiment, thoracic aortic cross-clamping was performed; a dose of 5000 U/kg of PEG-SOD was i.v. administered to one group 15–20 min before clamping, and the other group only received a saline solution. Delayed paraplegia was avoided in the group of dogs that received the conjugate, unlike the groups that did not receive it [140]. Edward et al. conducted an important review of the use of PEG-SOD in

When mannitol is used for medical purposes, it is administered intravenously. Mannitol can be found in varying concentrations, dissolved in 100 mL of fluid (5, 20, and 25% mannitol). A common solution is 20% mannitol. Cruz and colleagues described the dose-response effect of preoperative mannitol on acute subdural hematomas in traumatic brain injury in which mannitol therapy has been classically directed, establishing and maintaining an osmotic gradient between the blood and brain [142, 143]. Maintaining an adequate spinal cord perfusion pressure is crucial after SCI. Intramedullary edema within the spinal cord and consecutively raised intrathecal pressure at the injury are important secondary injury mechanisms in the pathobiology after traumatic SCI. Increased intraspinal pressure reduces spinal cord

perfusion pressure, which leads to worsen post-traumatic ischemia [144].

Mannitol allows the control of blood flow patterns in the spinal cord; it has been used experimentally in some studies in rats that have suffered a controlled SCI and in dogs/cats that suffer an SCI within the clinical area. Mannitol is recommended to reduce the effect of inflammation and edema, an effect that has been corroborated with microangiographic and electrophysiological studies. One hour after the application of a 3 g/kg dose, an improved intramedullary vascular pattern was detected among the animals treated with mannitol compared to those that were not treated,

*DOI: http://dx.doi.org/10.5772/intechopen.85424*

as a treatment vs. oxidative stress [132].

phase II and III studies in traumatic brain injury [141].

administration of the drug [137].

#### *Current Developments in Antioxidant Therapies for Spinal Cord Injury DOI: http://dx.doi.org/10.5772/intechopen.85424*

effects of the secondary stage and that during the acute phase of SCI, it may inhibit nerve fiber degeneration and create a favorable microenvironment for the regeneration of nerve filaments that can stimulate angiogenesis and reduce glial scar, promoting the regeneration of axonal guidance and motor recovery. PEG has been widely used as a scaffold for a large variety of molecules in treatment for SCI [132–136], while the SOD enzyme has antioxidant properties, as mentioned previously. The combination of SOD with PEG (PEG-SOD) allows an increase of the enzyme intracellularly and its antioxidant activity, and it may have an important role in vascular relaxation by reducing the concentration of O2•− and limiting the LP [132]. It has been used in myocardial ischemia and in lung injury models, proposed as a treatment vs. oxidative stress [132].

In a controlled phase II study in patients in a coma who suffered a stroke and received a single i.v. dose of 2000, 5000, or 10,000 IU/kg 4 h after the injury, its recovery was better in comparison to the group that received placebo (44% were in a vegetative state or died); no side effects were observed in this study due to the administration of the drug [137].

In a study in a cerebral ischemia model performed in rats, 10,000 IU/kg of PSG-SOD were i.v. administered, and the group presented a significant reduction in infarct size in comparison to the control group [138]. In other study with Sprague-Dawley male rats (300-350g of weight), an occlusion of the hepatic artery was performed and reperfusion was performed after 90 min to generate liver damage. A group of animals received i.v. 5000 U/kg of PEG-SOD before vascular occlusion and immediately after reperfusion, while the control group only received a saline solution following the same scheme. In the group treated with PEG-SOD, hepatic ischemia and LP were attenuated. Meanwhile, another study examined the effect of PEG-SOD on focal cerebral ischemia/ reperfusion in rats; the results showed that the effect is variable, depending on the dosage [132, 139]. In a dog experiment, thoracic aortic cross-clamping was performed; a dose of 5000 U/kg of PEG-SOD was i.v. administered to one group 15–20 min before clamping, and the other group only received a saline solution. Delayed paraplegia was avoided in the group of dogs that received the conjugate, unlike the groups that did not receive it [140]. Edward et al. conducted an important review of the use of PEG-SOD in phase II and III studies in traumatic brain injury [141].

#### **3.11 Mannitol**

*Spinal Cord Injury Therapy*

thesis of NO•

prevention of stroke-induced mortality in models of ischemia in gerbils [103, 104, 109–113]. The pharmacological effects of PBN in animal models are extensive, protecting against death after endotoxic shock, bacterial meningitis, teratogenicity induced by thalidomide, diabetogenesis, hepatocarcinogenesis, etc. Many studies have reported a neuroprotective effect in SCI and the brain (the most studied) decreasing the expression of genes associated with apoptosis, inflammation, and iNOS by decreasing the activation of MAP p-38 NF-κB nitrogen kinase and syn-

the infarct by increasing ischemic reperfusion and decreasing neurodegeneration, excitotoxicity, and the activation of microglia; it also induces neurite growth through

indirect activation of the Ras-ERK pathway, increasing animal survival [106, 115–117]. The neuroprotective effect of PBN is attributed to its ability to quickly and easily penetrate the membranes and the blood-brain barrier with a half-life of 3 h in plasma; decrease the levels of oxidized proteins, 8-isoprostane, HNE, IL-1β, TNF-α, IFN-γ, c-fos, IL-3, IL-4, IL-5, and H2O2; and favor an increase of GHS and IL-10, among others [106, 117–119]. In a model of cortical contusion in rats, it was demonstrated that pre-treatment with PBN with a single intravenous dose of 30 mg/ kg 30 min before the injury reduces the cognitive deficit and its volume; it has shown to have a wide therapeutic window in focal ischemia rodent models, reducing the infarct volume when administered up to 12 h after the beginning of the stroke and reducing the loss of tissue when administered by fluid percussion 30 min. After injury in rats [120]. Currently, the nitrones derived from PBN [102] are being widely studied as neuroprotective in different CNS pathologies and in traumatic lesions. For example, 2,4-disulfophenyl-N-tert-butylnitrone (NXY-059) has neuroprotective effects when applied 4–5 hr post-occlusion at equimolar doses to PBN and reduced infarct volume from 37.2 to 12.5% when 30 mg/kg was administered i.v. 1 h after reperfusion in Wistar rats [121–124]. Meanwhile, stilbazulenyl nitrone (STAZN) exerts similar effects at lower doses than the one used for NXY-059; in fact, the tolerability and safety of NXY-059 were studied in patients with acute stroke in clinical trials [103, 124]. Although not all compounds have demonstrated their neuroprotective effect when administered 24 h after the traumatic event, some of them have

allowed favoring the therapeutic window at repeated doses [103].

information on antioxidant therapy strategies in the clinical area.

**3.10 Polyethylene glycol (PEG)-superoxide dismutase (SOD)**

ing motor function [128, 130, 131].

Other derivatives are 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and diesterified nitrone (EMEPO), which have shown similarities to the action of PBN but with some other advantages, such as being less toxic and increasing the levels of antiapoptotic proteins such as Bcl2 and p-Bad and decreasing the synthesis of pro-apoptotic ones such as caspase 3, p53, and Bax [125–127]. In addition, (2, 2, 6, 6-tetramethylpiperidin-1-yl)oxyl (Tempo) and (4-hydroxy-2,2,6,6-tetramethylpiperidin-1-yl)oxyl (Tempol) have shown antioxidant properties in radiation damage and injury [128, 129]. In a traumatic brain injury mouse model, Tempol reduced post-traumatic LP and oxidative damage induced by protein nitration, decreasing mitochondrial damage, cytoskeletal damage, and neurodegeneration and improv-

Despite the results observed with the nitrones and the wide range of studies performed for therapeutic uses at different doses and times, their action is attributed to their ability to form adducts, but not before indicating the possible participation of other mechanisms that favor their neuroprotective activity, thus expanding the

Polyethylene glycol (PEG) is a surfactant that due to its hydrophilic nature allows the fusion and fluidity of the cell membrane that reduces the oxidative

[114]. In a process of ischemia or perfusion, PBN reduces the size of

**54**

When mannitol is used for medical purposes, it is administered intravenously. Mannitol can be found in varying concentrations, dissolved in 100 mL of fluid (5, 20, and 25% mannitol). A common solution is 20% mannitol. Cruz and colleagues described the dose-response effect of preoperative mannitol on acute subdural hematomas in traumatic brain injury in which mannitol therapy has been classically directed, establishing and maintaining an osmotic gradient between the blood and brain [142, 143].

Maintaining an adequate spinal cord perfusion pressure is crucial after SCI. Intramedullary edema within the spinal cord and consecutively raised intrathecal pressure at the injury are important secondary injury mechanisms in the pathobiology after traumatic SCI. Increased intraspinal pressure reduces spinal cord perfusion pressure, which leads to worsen post-traumatic ischemia [144].

Mannitol allows the control of blood flow patterns in the spinal cord; it has been used experimentally in some studies in rats that have suffered a controlled SCI and in dogs/cats that suffer an SCI within the clinical area. Mannitol is recommended to reduce the effect of inflammation and edema, an effect that has been corroborated with microangiographic and electrophysiological studies. One hour after the application of a 3 g/kg dose, an improved intramedullary vascular pattern was detected among the animals treated with mannitol compared to those that were not treated,

and 4 h after the perfusion, many areas of the lateral white matter of the spinal cord were almost normal [145]. In a study in dogs, an SCI was experimentally induced, and it was reported that mannitol alone did not help to reverse the paralysis of these animals [146]; however, another study stated that the i.v. administration of mannitol at a dose of 2 g/kg had a good effect on the white matter of the spinal cord and areas of the brain [147]. In a retrospective study with Sprague-Dawley rats, a group with SCI by compression by means of a clamp, 2 g/kg mannitol were administered immediately after the injury, while the control group was given 0.9% saline solution; all groups underwent structural and electrophysiological studies. The group treated with mannitol obtained excellent results, finding significant improvement in neural structures and protection of the spinal cord after SCI [148]. In a study in dogs to which an edema was induced by severe external spinal cord trauma, 3 g/kg of mannitol was i.v. administered, and they were neurologically evaluated, and a myelography study was performed after 2 h of the treatment, to identify the edema, showing that there was reduction of it [149].

#### **3.12 Combinatory therapies and results in symptoms of SCI**

In addition to its independent use, several studies have evaluated the use of one or more antioxidants together by themselves or in addition to other existing therapies for SCI, such as rehabilitation exercise or cell transplantation, expecting a synergism to enhance the recovery. Moreover, some therapies not only aim to improve the immediate treatment of SCI but also improve the effects it has on relieving the most common complications in patients. To mention some, the combination of vitamin C as antioxidant (100 mg/kg/1 h and daily/28d, i.p.) together with the transplantation of bone marrow mesenchymal stem cells (BMMSC) (3 × 106 cells) induced improvements in motor recovery in rats when compared with methylprednisolone (MP), vitamin C, or BMMSC alone in SCI [150]; simultaneous administration of vitamin D (5 μg/kg/twice daily) and progesterone (0.5 mg/kg/ twice daily i.m.) for 5 days demonstrated a higher efficacy in reducing neuroinflammation in comparison to when they were administered separately, and when they were administrated early (first 4 h) in SCI patients receiving MP, there was improvement in the motor and sensory functions 6 months after starting therapy [151]. Applying once a day a combination of low-dose fluoxetine (1 mg/kg/i.p.) and vitamin C (100 mg/kg/i.p.) immediately after the event and for 14 days had a protective effect on the BSCB integrity, improving the functional recovery, showing inhibition of the expression and activation of the matrix metalloproteinase, and decreasing the infiltration of leukocytes and the expression of inflammatory and oxidizing molecules, but not when they were applied separately in rats [152]. In SCI patients, dietary supplementation for 3 months, which included three 750 mg per day of omega-3 fatty acids and antioxidants (400 mg of mixed tocopherols, coenzyme Q10, curcumin, etc.), caused a decrease of inflammatory cytokines with reduction in neuropathic pain [153]; 2 months vitamin E dietary supplementation 765–1020 IU/day in rats before SCI showed accelerated bladder recovery, significant motor improvement, and a high number of oligodendrocytes compared to the controls [154].

#### **4. Conclusion**

After a primary injury occurs on the spinal cord, destructive biochemical mechanisms are initiated (secondary injury) that play a fundamental role in the pathophysiology of spinal cord injury. Within these, oxidative stress and lipid

**57**

*Current Developments in Antioxidant Therapies for Spinal Cord Injury*

The authors declare no competing financial interests.

BMMSC bone marrow mesenchymal stem cells

peroxidation exacerbate the biochemical mechanisms once initiated and propagate neurodegenerative damage, so the degree of loss of long-term motor and sensory functions depends largely on their intensity. This damage suffered during the acute phase and that may be irreversible requires a timely intervention. To guarantee the antioxidant effect that will render better results, it is important to consider the new agents and therapies in the SCI treatment at the appropriate times. There is no fully restorative therapy for SCI, but strategies for the modulation of this damage contribute to neuroprotection and, although partially, to functional recovery.

We are grateful for the support given to the line of research related to the evaluation of immunomodulatory peptides in oxidative stress under the support No. FIS/IMSS/PROT/G16/1605 and FIS /IMSS/PROT/G17/1676 and the scholarship granted to the students of Master's degree Dulce M. Parra and Jonathan Vilchis by

*DOI: http://dx.doi.org/10.5772/intechopen.85424*

**Acknowledgements**

CONACYT and IMSS.

**Conflict of interest**

**Acronyms and abbreviations**

ADH alcohol dehydrogenase ALDH aldehyde dehydrogenase BSCB blood-spinal cord barrier

CD cluster of differentiation CNS central nervous system

<sup>⦁</sup><sup>−</sup> carbonate radical COX cyclooxygenase DHA docosahexaenoic acid

EPA eicosapentaenoic acid ER estrogen receptor FR free radicals

GAP-43 growth-associated protein 43 GFAP glial fibrillary acidic protein GPx glutathione peroxidases GR glutathione reductases GSH glutathione, reduced

GSH-MEE glutathione monoethyl ester GSSG glutathione, oxidized GST glutathione S-transferases HNE 4-hydroxy-2-nonenal H2O2 hydrogen peroxide HO<sup>⦁</sup> hydroxyl radical HO<sup>−</sup> hydroxyl anion

DNP dinitrophenyl

aa amino acids

CAT catalase

CO3

*Current Developments in Antioxidant Therapies for Spinal Cord Injury DOI: http://dx.doi.org/10.5772/intechopen.85424*

peroxidation exacerbate the biochemical mechanisms once initiated and propagate neurodegenerative damage, so the degree of loss of long-term motor and sensory functions depends largely on their intensity. This damage suffered during the acute phase and that may be irreversible requires a timely intervention. To guarantee the antioxidant effect that will render better results, it is important to consider the new agents and therapies in the SCI treatment at the appropriate times. There is no fully restorative therapy for SCI, but strategies for the modulation of this damage contribute to neuroprotection and, although partially, to functional recovery.

#### **Acknowledgements**

*Spinal Cord Injury Therapy*

showing that there was reduction of it [149].

**3.12 Combinatory therapies and results in symptoms of SCI**

In addition to its independent use, several studies have evaluated the use of one or more antioxidants together by themselves or in addition to other existing therapies for SCI, such as rehabilitation exercise or cell transplantation, expecting a synergism to enhance the recovery. Moreover, some therapies not only aim to improve the immediate treatment of SCI but also improve the effects it has on relieving the most common complications in patients. To mention some, the combination of vitamin C as antioxidant (100 mg/kg/1 h and daily/28d, i.p.) together with the transplantation of bone marrow mesenchymal stem cells (BMMSC) (3 ×

 cells) induced improvements in motor recovery in rats when compared with methylprednisolone (MP), vitamin C, or BMMSC alone in SCI [150]; simultaneous administration of vitamin D (5 μg/kg/twice daily) and progesterone (0.5 mg/kg/ twice daily i.m.) for 5 days demonstrated a higher efficacy in reducing neuroinflammation in comparison to when they were administered separately, and when they were administrated early (first 4 h) in SCI patients receiving MP, there was improvement in the motor and sensory functions 6 months after starting therapy [151]. Applying once a day a combination of low-dose fluoxetine (1 mg/kg/i.p.) and vitamin C (100 mg/kg/i.p.) immediately after the event and for 14 days had a protective effect on the BSCB integrity, improving the functional recovery, showing inhibition of the expression and activation of the matrix metalloproteinase, and decreasing the infiltration of leukocytes and the expression of inflammatory and oxidizing molecules, but not when they were applied separately in rats [152]. In SCI patients, dietary supplementation for 3 months, which included three 750 mg per day of omega-3 fatty acids and antioxidants (400 mg of mixed tocopherols, coenzyme Q10, curcumin, etc.), caused a decrease of inflammatory cytokines with reduction in neuropathic pain [153]; 2 months vitamin E dietary supplementation 765–1020 IU/day in rats before SCI showed accelerated bladder recovery, significant motor improvement, and a high number of oligodendrocytes compared to the

After a primary injury occurs on the spinal cord, destructive biochemical mechanisms are initiated (secondary injury) that play a fundamental role in the pathophysiology of spinal cord injury. Within these, oxidative stress and lipid

and 4 h after the perfusion, many areas of the lateral white matter of the spinal cord were almost normal [145]. In a study in dogs, an SCI was experimentally induced, and it was reported that mannitol alone did not help to reverse the paralysis of these animals [146]; however, another study stated that the i.v. administration of mannitol at a dose of 2 g/kg had a good effect on the white matter of the spinal cord and areas of the brain [147]. In a retrospective study with Sprague-Dawley rats, a group with SCI by compression by means of a clamp, 2 g/kg mannitol were administered immediately after the injury, while the control group was given 0.9% saline solution; all groups underwent structural and electrophysiological studies. The group treated with mannitol obtained excellent results, finding significant improvement in neural structures and protection of the spinal cord after SCI [148]. In a study in dogs to which an edema was induced by severe external spinal cord trauma, 3 g/kg of mannitol was i.v. administered, and they were neurologically evaluated, and a myelography study was performed after 2 h of the treatment, to identify the edema,

**56**

controls [154].

**4. Conclusion**

106

We are grateful for the support given to the line of research related to the evaluation of immunomodulatory peptides in oxidative stress under the support No. FIS/IMSS/PROT/G16/1605 and FIS /IMSS/PROT/G17/1676 and the scholarship granted to the students of Master's degree Dulce M. Parra and Jonathan Vilchis by CONACYT and IMSS.

#### **Conflict of interest**

The authors declare no competing financial interests.

#### **Acronyms and abbreviations**



### **Author details**

Jonathan Vilchis Villa, Dulce M. Parra Villamar, José Alberto Toscano Zapien, Liliana Blancas Espinoza, Juan Herrera García and Raúl Silva García\* Medical Research Unit in Immunology, Hospital of Pediatric "Dr. Silvestre Frenk Freund"—CMN, SXXI, IMSS, Mexico City, Mexico

\*Address all correspondence to: silgarrul@yahoo.com.mx

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**59**

*Current Developments in Antioxidant Therapies for Spinal Cord Injury*

[9] Anthonymuthu T, Kenny EM, Bayir H. Therapies targeting lipid peroxidation in traumatic brain injury. Brain Research. 2016;**1640**(1

[10] Salem N, Lin M, Moriguchi Y, et al. Distribution of omega-6 and omega-3 polyunsaturated fatty acids in the whole rat body and 25 compartments. Prostaglandins, Leukotrienes, and Essential Fatty Acids. 2015;**100**:13-20. DOI: 10.1016/j.plefa.2015.06.002

[11] Lushchak V. Free radicals, reactive oxygen species, oxidative stress and its classification. Chemico-Biological Interactions. 2014;**224**:164-175. DOI:

10.1016/j.cbi.2014.10.016

[12] Gaschler MM, Stockwell BR. Lipid peroxidation in cell death. Biochemical and Biophysical Research Communications. 2017;**482**(3):419-425.

DOI: 10.1016/j.bbrc.2016.10.086

in chemistry and analysis. Redox Biology. 2013;**1**:145-152. DOI: 10.1016/j.

[14] Singhal S, Singh S, Singhal P, et al. Antioxidant role of glutathione S-Transferases: 4-Hydroxynonenal, a key molecule in stress-mediated Signaling. Toxicology and Applied Pharmacology. 2015;**289**(3):361-370. DOI: 10.1016/j.taap.2015.10.006

[15] Ji Y, Dai Z, Wu G, et al. 4-Hydroxy-2 nonenal induces apoptosis by activating

ERK1/2 signaling and depleting intracellular glutathione in intestinal epithelial cells. Scientific Reports. 2016;**6**:32929. DOI: 10.1038/srep32929

[16] Ayala A, Muñoz MF, Argüelles S. Lipid peroxidation: production,

metabolism, and signaling

redox.2013.01.007

[13] Spickett C. The lipid peroxidation product 4-hydroxy-2-nonenal: Advances

Pt A):57-76. DOI: 10.1016/j. brainres.2016.02.006

*DOI: http://dx.doi.org/10.5772/intechopen.85424*

[1] Fehlings MG, Wilson JR, Harrop JS. Efficacy and safety of methylprednisolone sodium succinate in acute spinal cord injury: A systematic review. Global Spine Journal. 2017;**7**(3S):116S-137S. DOI:

10.1177/2192568217706366

s10863-015-9600-5

[2] Hall E, Wang JA, Bosken JM, et al. Lipid peroxidation in brain or spinal cord mitochondria after injury. Journal of Bioenergetics and Biomembranes. 2016;**48**(2):169-174. DOI: 10.1007/

[3] Oyimbo C. Secondary injury mechanisms in traumatic spinal cord injury: A nugget of this multiply cascade. Acta Neurobiologiae Experimentalis. 2011;**71**:281-299

[4] Pisoschi A, Pop A. The role of

10.1016/j.ejmech.2015.04.040

[5] Hall E. Antioxidant therapies for acute spinal cord injury.

protection. Chemico-Biological Interactions. 1994;**9**:133-140

[7] Kohen R, Ayska A. Oxidation of biological systems: Oxidative stress phenomena, antioxidants, redox reactions, and methods for their quantification. Toxicologic Pathology. 2002;**30**(6):620-650. DOI:

10.1080/01926230290166724

[8] Carballal S, Bartesaghi S, Radi R. Kinetic and mechanistic considerations to assess the biological fate of peroxynitrite. Biochimica et Biophysica Acta. 2014;**1840**(2):768-780. DOI: 10.1016/j.bbagen.2013.07.005

Neurotherapeutics. 2011;**8**(2):152-167. DOI: 10.1007/s13311-011-0026-4

[6] Gutteridge J. Biological origin of free radicals, and mechanisms of antioxidant

antioxidants in the chemistry of oxidative stress: A review. European Journal of Medicinal Chemistry. 2015;**97**:55-74. DOI:

**References**

*Current Developments in Antioxidant Therapies for Spinal Cord Injury DOI: http://dx.doi.org/10.5772/intechopen.85424*

#### **References**

*Spinal Cord Injury Therapy*

IFN-γ interferon gamma IL interleukin

LP lipid peroxidation LOO<sup>⦁</sup> lipid peroxyl radical LO<sup>⦁</sup> lipid alkoxyl radical LOOH lipid hydroperoxide

MDA malondialdehyde

NAC N-acetyl cysteine

NOS nitric oxide synthase NT-3 3-nitrotyrosine

PEG polyethylene glycol

VLA-4 very late antigen 4 XO xanthine oxidase

**Author details**

•− nitrogen dioxide radical

PBN phenyl N-tert-butylnitrone

PUFA polyunsaturated fatty acid ROS reactive oxygen species RNS reactive nitrogen species

<sup>⦁</sup> peroxyl radical SCI spinal cord injury SOD superoxide dismutase

TGF-β transforming growth factor beta TNF-α tumor necrosis factor alpha VCAM-1 vascular cell adhesion molecule-1 VDH vitamin D: 1,25-dihydroxyvitamin D3

Freund"—CMN, SXXI, IMSS, Mexico City, Mexico

\*Address all correspondence to: silgarrul@yahoo.com.mx

NO<sup>⦁</sup> nitric oxide

<sup>⦁</sup><sup>−</sup> superoxide OONO− peroxynitrite

NO2

O2

RO2 ⦁ /HO2

L<sup>⦁</sup> lipid radical

LH lipid

iNOS inducible nitric synthase

MLIF monocyte locomotion inhibitory factor

MP or MPSS methylprednisolone sodium succinate

PEG-SOD polyethylene glycol-superoxide dismutase

NADPH nicotinamide-adenine dinucleotide phosphate, reduced

**58**

provided the original work is properly cited.

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Jonathan Vilchis Villa, Dulce M. Parra Villamar, José Alberto Toscano Zapien,

Medical Research Unit in Immunology, Hospital of Pediatric "Dr. Silvestre Frenk

Liliana Blancas Espinoza, Juan Herrera García and Raúl Silva García\*

[1] Fehlings MG, Wilson JR, Harrop JS. Efficacy and safety of methylprednisolone sodium succinate in acute spinal cord injury: A systematic review. Global Spine Journal. 2017;**7**(3S):116S-137S. DOI: 10.1177/2192568217706366

[2] Hall E, Wang JA, Bosken JM, et al. Lipid peroxidation in brain or spinal cord mitochondria after injury. Journal of Bioenergetics and Biomembranes. 2016;**48**(2):169-174. DOI: 10.1007/ s10863-015-9600-5

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[130] Deng Y, Singh I, Carrico K, et al. Neuroprotective effects of tempol, a catalytic scavenger of peroxynitritederived free radicals, in a mouse traumatic brain injury model. Journal of Cerebral Blood Flow and Metabolism. 2008;**28**:1114-1126. DOI: 10.1038/ jcbfm.2008.10

[131] Hillard V, Peng H, Zhang Y, et al. Tempol, a nitroxide antioxidant, improves locomotor and histological outcomes after spinal cord contusion

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[133] Shi R. Polyethylene glycol repairs membrane damage and enhances functional recovery: A tissue engineering approach to spinal cord injury. Neuroscience Bulletin. 2013;**29**(4):460-466. DOI: 10.1007/ s12264-013-1364-5

[134] Kong X, Tang Q, Chen X, et al. Polyethylene glycol as a promising synthetic material for repair of spinal cord injury. Neural Regeneration Research. 2017;**12**(6):1003-1008. DOI: 10.4103/1673-5374.208597: 10.4103/1673-5374.208597

[135] Lu X, Perera T, Aria A, et al. Polyethylene glycol in spinal cord injury repairs: A critical review. Journal of Experimental Pharmacology. 2018;**10**:37- 49. DOI: 10.2147/JEP.S148944

[136] Luo J, Borgens R, Shi R. Polyethylene glycol improves function and reduces oxidative stress in synaptosomal preparations following spinal cord injury. Journal of Neurotrauma. 2004;**21**(8):994-1007. DOI: 10.1089/0897715041651097

[137] Muizelaar J, Marmarou A, Young H, et al. Improving the outcome of severe head injury with the oxygen radical scavenger polyethylene glycolconjugated superoxide dismutase: A phase II trial. Journal of Neurosurgery. 1993;**78**(3):375-382. DOI: 10.3171/ jns.1993.78.3.0375

[138] He Y, Hsu C, Ezrin A, et al. Polyethylene glycol conjugated superoxide

**69**

*Current Developments in Antioxidant Therapies for Spinal Cord Injury*

flow patterns, and electrophysiology.

[146] De la Torre J, Johnson C, Goode D, et al. Pharmacologic treatment and evaluation of permanent experimental

spinal cord trauma. Neurology.

[147] Feldman Z, Reichenthal E, Zachari Z, et al. Mannitol, intracranial pressure, and Vasogenic Edema. Neurosurgery. 1995;**36**(6):1236-1237. DOI:

[148] Baysefer A, Erdogan E, Kahraman S, et al. Effect of mannitol in experimental spinal cord injury: An ultrastructural and electrophysiological study. Neurology

India. 2003;**51**(3):350-354

[149] Parker A, Park R, Stowater J. Reduction of trauma-induced edema of spinal cord in dogs given mannitol. American Journal of Veterinary Research. 1973;**34**(10):1355-1357

et al. Does vitamin C have the ability to augment the therapeutic effect of bone marrow-derived mesenchymal stem cells on spinal cord injury? Neural Regeneration Research. 2017;**12**(12):2050-2058. DOI:

10.4103/1673-5374.221163

[151] Aminmansour B, Asnaashari A, Rezvani M, et al. Effects of progesterone and vitamin D on outcome of patients with acute traumatic spinal cord injury: A randomized, double-blind, placebo controlled study. The Journal of Spinal Cord Medicine. 2016;**39**(3):272-280. DOI: 10.1080/10790268.2015.1114224

[152] Lee JY, Choi HY, Yune TY.

Fluoxetine and vitamin C synergistically inhibits blood-spinal cord barrier disruption and improves functional recovery after spinal cord injury.

[150] Salem N, Salem MY, Elmaghrabi MM,

10.1227/00006123-199506000-00040

Spine. 1979;**4**(5):391-397

1975;**25**(6):508-514

*DOI: http://dx.doi.org/10.5772/intechopen.85424*

dismutase in focal cerebral ischemiareperfusion. American Journal of Physiology. 1993;**265**(1 Pt 2):H252-H256. DOI: 10.1152/ajpheart.1993.265.1.H252

[139] Wyn DN, Donald HK, Hasan BA, et al. Polyethylene glycol-superoxide dismutase inhibits lipid peroxidation in hepatic ischemia/reperfusion injury. Critical Care. 1999;**3**(5):127-130. DOI:

[140] Granke K, Hollier L, Zdrahal P, et al. Longitudinal study of cerebral spinal fluid drainage in polyethylene glycol-conjugated superoxide dismutase in paraplegia associated with thoracic aortic cross-clamping. Journal of Vascular Surgery. 1991;**13**(5):615-621

[141] Edward D, Wang J, Miller D, et al. Newer pharmacological approach for antioxidant neuroprotection in traumatic brain injury. Neuropharmacology. 2018;**18**:30473-30478. DOI: 10.1016/j.

[142] Cruz J, Minoja G, Okuchi K. Improving clinical outcomes from acute subdural hematomas with the emergency preoperative administration

[143] Tenny S, Thorell W. Mannitol. Treasure Island (FL): StatPearls Publishing LLC; 2018. Bookshelf ID:

[144] Grasner L, Winkler P, Strowitzki M, et al. Increased intrathecal pressure after traumatic spinal cord injury: An illustrative case presentation and a review of the literature. European Spine Journal. 2017;**26**(1):20-25. DOI: 10.1007/

[145] Reed J, Allen W, Dohrmann G. Effect of mannitol on the traumatized spinal cord. Microangiography, blood

of high doses of mannitol: A randomized trial. Neurosurgery.

2001;**49**:864-871

NBK470392

s00586-016-4769-9

neuropharm.2018.08.005

10.1186/cc358

*Current Developments in Antioxidant Therapies for Spinal Cord Injury DOI: http://dx.doi.org/10.5772/intechopen.85424*

dismutase in focal cerebral ischemiareperfusion. American Journal of Physiology. 1993;**265**(1 Pt 2):H252-H256. DOI: 10.1152/ajpheart.1993.265.1.H252

*Spinal Cord Injury Therapy*

s10753-012-9552-4

DOI: 10.1039/B616076J

journal.pone.0052005

trap 5,5-dimethyl-1-pyrroline N-oxide affects stress response and fate of lipopolysaccharide-primed RAW264.7 macrophage cells. Inflammation. 2013;**36**:346-354. DOI: 10.1007/

in rats. Journal of Neurotrauma. 2004;**21**:1405-1414. DOI: 10.1089/

[133] Shi R. Polyethylene glycol repairs membrane damage and enhances functional recovery: A tissue engineering approach to spinal cord injury. Neuroscience Bulletin. 2013;**29**(4):460-466. DOI: 10.1007/

[134] Kong X, Tang Q, Chen X, et al. Polyethylene glycol as a promising synthetic material for repair of spinal cord injury. Neural Regeneration Research. 2017;**12**(6):1003-1008. DOI: 10.4103/1673-5374.208597: 10.4103/1673-5374.208597

[135] Lu X, Perera T, Aria A, et al. Polyethylene glycol in spinal cord injury repairs: A critical review. Journal of Experimental Pharmacology. 2018;**10**:37-

49. DOI: 10.2147/JEP.S148944

[136] Luo J, Borgens R, Shi R. Polyethylene glycol improves function and reduces oxidative stress in synaptosomal preparations following spinal cord injury. Journal of Neurotrauma. 2004;**21**(8):994-1007. DOI: 10.1089/0897715041651097

[137] Muizelaar J, Marmarou A,

[138] He Y, Hsu C, Ezrin A, et al.

Polyethylene glycol conjugated superoxide

jns.1993.78.3.0375

Young H, et al. Improving the outcome of severe head injury with the oxygen radical scavenger polyethylene glycolconjugated superoxide dismutase: A phase II trial. Journal of Neurosurgery. 1993;**78**(3):375-382. DOI: 10.3171/

[132] Francesco M, Paolo C, Oddose S, et al. Polyethylene glycol-superoxide dismutase, a conjugate in search of exploitation. Advanced Drug Delivery Reviews. 2002;**54**:587-606. DOI: 10.1016/S0169-409X(02)00029-7

neu.2004.21.1405

s12264-013-1364-5

[126] Hardy M, Chalier F, Ouari O, et al. Mito-DEPMPO synthesized from a novel NH2-reactive DEPMPO spin trap: A new and improved trap for the detection of superoxide. Chemical Communications. 2007;**10**:1083-1085.

[127] Traynham C, Roof S, Wang H, et al. Diesterified nitrone rescues nitroso-redox levels and increases myocyte contraction via increased SR Ca(2+) handling. PLoS One. 2012;**7**(12):e52005. DOI: 10.1371/

[128] Ban S, Nakagawa H, Suzuki T, et al. Novel mitochondria-localizing TEMPO derivative for measurement of cellular oxidative stress in mitochondria. Bioorganic & Medicinal Chemistry Letters. 2007;**17**:2055-2058. DOI: 10.1016/j.bmcl.2007.01.011

[129] Lee C, Yu L, Wang J. Nitroxide antioxidant as a potential strategy to attenuate the oxidative/nitrosative stress induced by hydrogen peroxide plus nitric oxide in cultured neurons. Nitric Oxide. 2016;**54**:38-50. DOI:

[130] Deng Y, Singh I, Carrico K, et al. Neuroprotective effects of tempol, a catalytic scavenger of peroxynitritederived free radicals, in a mouse

traumatic brain injury model. Journal of Cerebral Blood Flow and Metabolism. 2008;**28**:1114-1126. DOI: 10.1038/

[131] Hillard V, Peng H, Zhang Y, et al. Tempol, a nitroxide antioxidant, improves locomotor and histological outcomes after spinal cord contusion

10.1016/j.niox.2016.02.001

**68**

jcbfm.2008.10

[139] Wyn DN, Donald HK, Hasan BA, et al. Polyethylene glycol-superoxide dismutase inhibits lipid peroxidation in hepatic ischemia/reperfusion injury. Critical Care. 1999;**3**(5):127-130. DOI: 10.1186/cc358

[140] Granke K, Hollier L, Zdrahal P, et al. Longitudinal study of cerebral spinal fluid drainage in polyethylene glycol-conjugated superoxide dismutase in paraplegia associated with thoracic aortic cross-clamping. Journal of Vascular Surgery. 1991;**13**(5):615-621

[141] Edward D, Wang J, Miller D, et al. Newer pharmacological approach for antioxidant neuroprotection in traumatic brain injury. Neuropharmacology. 2018;**18**:30473-30478. DOI: 10.1016/j. neuropharm.2018.08.005

[142] Cruz J, Minoja G, Okuchi K. Improving clinical outcomes from acute subdural hematomas with the emergency preoperative administration of high doses of mannitol: A randomized trial. Neurosurgery. 2001;**49**:864-871

[143] Tenny S, Thorell W. Mannitol. Treasure Island (FL): StatPearls Publishing LLC; 2018. Bookshelf ID: NBK470392

[144] Grasner L, Winkler P, Strowitzki M, et al. Increased intrathecal pressure after traumatic spinal cord injury: An illustrative case presentation and a review of the literature. European Spine Journal. 2017;**26**(1):20-25. DOI: 10.1007/ s00586-016-4769-9

[145] Reed J, Allen W, Dohrmann G. Effect of mannitol on the traumatized spinal cord. Microangiography, blood

flow patterns, and electrophysiology. Spine. 1979;**4**(5):391-397

[146] De la Torre J, Johnson C, Goode D, et al. Pharmacologic treatment and evaluation of permanent experimental spinal cord trauma. Neurology. 1975;**25**(6):508-514

[147] Feldman Z, Reichenthal E, Zachari Z, et al. Mannitol, intracranial pressure, and Vasogenic Edema. Neurosurgery. 1995;**36**(6):1236-1237. DOI: 10.1227/00006123-199506000-00040

[148] Baysefer A, Erdogan E, Kahraman S, et al. Effect of mannitol in experimental spinal cord injury: An ultrastructural and electrophysiological study. Neurology India. 2003;**51**(3):350-354

[149] Parker A, Park R, Stowater J. Reduction of trauma-induced edema of spinal cord in dogs given mannitol. American Journal of Veterinary Research. 1973;**34**(10):1355-1357

[150] Salem N, Salem MY, Elmaghrabi MM, et al. Does vitamin C have the ability to augment the therapeutic effect of bone marrow-derived mesenchymal stem cells on spinal cord injury? Neural Regeneration Research. 2017;**12**(12):2050-2058. DOI: 10.4103/1673-5374.221163

[151] Aminmansour B, Asnaashari A, Rezvani M, et al. Effects of progesterone and vitamin D on outcome of patients with acute traumatic spinal cord injury: A randomized, double-blind, placebo controlled study. The Journal of Spinal Cord Medicine. 2016;**39**(3):272-280. DOI: 10.1080/10790268.2015.1114224

[152] Lee JY, Choi HY, Yune TY. Fluoxetine and vitamin C synergistically inhibits blood-spinal cord barrier disruption and improves functional recovery after spinal cord injury.

Neuropharmacology. 2016;**109**:78-87. DOI: 10.1016/j.neuropharm.2016.05.018

[153] Allison DJ, Thomas A, Beaudry K, et al. Targeting inflammation as a treatment modality for neuropathic pain in spinal cord injury: A randomized clinical trial. Journal of Neuroinflammation. 2016;**13**(1):152. DOI: 10.1186/s12974-016-0625-4

[154] Cordero K, Coronel GG, Serrano-Illán M, et al. Effects of dietary vitamin E supplementation in bladder function and spasticity during spinal cord injury. Brain Sciences. 2018;**8**(38):1-20. DOI: 10.3390/brainsci8030038

[155] Herrera García JS. Evaluation of motor recovery and gene expression from acute phase in rats with spinal cord injury [Master's thesis]. Mexico City, MX: National Autonomous University of Mexico; 2017

**71**

**Chapter 5**

**Abstract**

Effects of Cyclosporin-A,

Spinal Cord Injury in Rats

**Keywords:** cyclosporin-A, minocycline, tacrolimus (FK506), rats,

spinal cord injury, behavior, oxidative stress

**1. Introduction**

Minocycline, and Tacrolimus

(FK506) on Enhanced Behavioral

and Biochemical Recovery from

*Mohammad Ahmad and Abdualrahman Saeed Alshehri*

Spinal cord injury (SCI) results into an immediate primary injury (physical damages) followed by secondary damages (prolonged posttraumatic inflammatory disorder) resulting into severe motor dysfunction including paralysis. The present chapter discusses and investigates the neuroprotective effects of cyclosporin-A (CsA), minocycline, and tacrolimus (FK506) and their therapeutic effectiveness in recovery from the animal model of SCI. Based on the available recent literature on these three drugs, as well as in perspective of the results obtained on some experimental behavioral, biochemical, and oxidative stress parameters in the present study, the therapeutical potential of these three drugs has been discussed. Furthermore, the animal model of SCI used herein has been reviewed and compared with other reported animal models for understanding the utility, suitability, and reproducibility of the methodology of the present model for screening purposes in quest of searching ideal therapeutic compounds for maximum recovery from SCI.

Spinal cord injury (SCI) is prevalent worldwide [1, 2] and often incapacitates the victims for life resulting in disability. Injury to the spinal cord results in processes that occur in three phases: the first phase is immediate physical phase also known as acute phase comprising affected spinal shock and initial trauma (primary injury) followed by the second phase known as secondary phase which is a prolonged cascade of damaging processes over a time period of minutes to weeks after the injury (secondary injury). Such damages include ischemia, vascular alterations, biochemical alterations, and cellular responses that lead to peripheral posttraumatic inflammatory cell infiltration and cell death (secondary injury) [1, 3–5]. The third phase that sustains between days and years after SCI trauma is characterized by proapoptotic degeneration and scarring that establishes permanent functional impairment [6, 7]. Secondary injury leads to the key pathophysiological response

#### **Chapter 5**

*Spinal Cord Injury Therapy*

Neuropharmacology. 2016;**109**:78-87. DOI: 10.1016/j.neuropharm.2016.05.018

[153] Allison DJ, Thomas A, Beaudry K, et al. Targeting inflammation as a treatment modality for neuropathic

randomized clinical trial. Journal of Neuroinflammation. 2016;**13**(1):152. DOI: 10.1186/s12974-016-0625-4

[154] Cordero K, Coronel GG, Serrano-Illán M, et al. Effects of dietary vitamin E supplementation in bladder function and spasticity during spinal cord injury. Brain Sciences. 2018;**8**(38):1-20. DOI:

[155] Herrera García JS. Evaluation of motor recovery and gene expression from acute phase in rats with spinal cord injury [Master's thesis]. Mexico City, MX: National Autonomous University

pain in spinal cord injury: A

10.3390/brainsci8030038

of Mexico; 2017

**70**

## Effects of Cyclosporin-A, Minocycline, and Tacrolimus (FK506) on Enhanced Behavioral and Biochemical Recovery from Spinal Cord Injury in Rats

*Mohammad Ahmad and Abdualrahman Saeed Alshehri*

### **Abstract**

Spinal cord injury (SCI) results into an immediate primary injury (physical damages) followed by secondary damages (prolonged posttraumatic inflammatory disorder) resulting into severe motor dysfunction including paralysis. The present chapter discusses and investigates the neuroprotective effects of cyclosporin-A (CsA), minocycline, and tacrolimus (FK506) and their therapeutic effectiveness in recovery from the animal model of SCI. Based on the available recent literature on these three drugs, as well as in perspective of the results obtained on some experimental behavioral, biochemical, and oxidative stress parameters in the present study, the therapeutical potential of these three drugs has been discussed. Furthermore, the animal model of SCI used herein has been reviewed and compared with other reported animal models for understanding the utility, suitability, and reproducibility of the methodology of the present model for screening purposes in quest of searching ideal therapeutic compounds for maximum recovery from SCI.

**Keywords:** cyclosporin-A, minocycline, tacrolimus (FK506), rats, spinal cord injury, behavior, oxidative stress

#### **1. Introduction**

Spinal cord injury (SCI) is prevalent worldwide [1, 2] and often incapacitates the victims for life resulting in disability. Injury to the spinal cord results in processes that occur in three phases: the first phase is immediate physical phase also known as acute phase comprising affected spinal shock and initial trauma (primary injury) followed by the second phase known as secondary phase which is a prolonged cascade of damaging processes over a time period of minutes to weeks after the injury (secondary injury). Such damages include ischemia, vascular alterations, biochemical alterations, and cellular responses that lead to peripheral posttraumatic inflammatory cell infiltration and cell death (secondary injury) [1, 3–5]. The third phase that sustains between days and years after SCI trauma is characterized by proapoptotic degeneration and scarring that establishes permanent functional impairment [6, 7]. Secondary injury leads to the key pathophysiological response

to SCI causing severe and permanent functional deficits. Most of the clinical trials and experimental studies are conducted for intense research to unfold the underlying pathophysiological processes and for searching ideal and potential therapy for recovery from secondary SCI injuries [8]. Besides motor dysfunction, some of the other important SCI-related biochemical and immunological impairments that get involved are serum tumor necrosis factor (TNF)-α, interleukin (IL)-1β, interleukin-6, nuclear factor (NF)-κB p65, p38 mitogen-activated protein kinase (MAPK), inducible nitric oxide synthase (iNOS), caspase-3, superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px) [9]. SCI trauma causes a devastating effect not only to the individual patient, but it also incurs heavy expensive burden to the society in general and to the family members, due to substantial long-term healthcare expenditures [10].

Despite considerable therapeutic studies, no proven drugs or techniques are available for satisfactory treatment of SCI. Much of these therapeutic studies have been reported from animal models, and it needs to be understood that a successful clinical trial in humans can only be initiated based on previously available preclinical data reported from animal model studies that closely mimic the losses as in human SCI functions [11]. Although rats are the animal model of choice for SCI studies, the major anatomical differences in axonal tracts and sensory motor pathways between quadrupeds and bipeds need to be taken into careful account to improve the targets of human SCI treatments [12].

Currently, methylprednisolone is the only recognized treatment for human SCI; however, it has significant adverse effects, including respiratory complications, sepsis, and gastrointestinal hemorrhage [13]. Furthermore, other important evidence-based therapies that have potential neuroprotective and neural reparative therapeutic properties and are undergoing clinical trials for human SCI include surgical decompression, blood pressure augmentation, riluzole, granulocyte colonystimulating factor, minocycline, glibenclamide, cerebrospinal fluid drainage, magnesium, therapeutic hypothermia, Cethrin (VX-210), anti-NOGO antibody, cell-based approaches, and bioengineered biomaterials [5, 14, 15].

Some other experimental drugs that have been studied for therapeutical use in animal SCI are recombinant human erythropoietin [10], tetrodotoxin [16], BCL-2 [17], cyclosporin-A [18], edaravone [19], atorvastatin [20], calpain inhibitors [21] FK506, and minocycline [22]. Also, some natural products like eugenol oil [9], curcumin [23], and melatonin [24] have shown promising effects in animal SCI functional recovery. It sounds reasonable that instead of using a highly selective treatment that targets a specific molecule or pathway, a compound with multifunctional properties that targets several mediators involved in spinal cord pathology may be more effective for recovery from SCI [25]. In our earlier study [22], the promising potential of FK506 and minocycline has been reported for their effectiveness in rat SCI model. Thus, in the present study, besides these two multifactorial effective compounds minocycline and FK506, a third compound cyclosporin-A (CsA) was also included, and all the three compounds minocycline, FK506 (tacrolimus), and cyclosporine-A were chosen to evaluate in a comparative manner for their therapeutical potential using some important and reliable parameters that are most commonly used in rat SCI model [22]. Before discussing the outcome of our present results, we review the multifactorial effects of these three compounds that have been reported in literature using rat SCI model.

#### **1.1 Minocycline**

Minocycline, a semisynthetic second-generation tetracycline, has robust neuroprotective effects in rodent models of neurodegenerative diseases [26] and provides

**73**

**1.2 Cyclosporin-A**

*Effects of Cyclosporin-A, Minocycline, and Tacrolimus (FK506) on Enhanced Behavioral…*

neuroprotection in experimental models of neurological diseases, including SCI [27]. In a broad range of secondary injury mechanisms via its anti-inflammatory, antioxidant, and antiapoptotic properties, minocycline is effective in reducing secondary injury and promoting locomotor functional recovery [28–31]. Minocycline prevents N-methyl-d-aspartate (NMDA)-induced excitotoxicity by diminishing NMDA-induced Ca2+ influx and mitochondrial Ca2+ uptake [32] and protects gray and white matter from SCI [33]. Minocycline also inhibits p38 mitogen-activated protein kinase (p38 MAPK) activation and microglial pro-nerve growth factor (proNGF) expression resulting from inflammatory reactions due to SCI and improves oligodendrocyte survival [34]. Inflammation due to SCI also upregulates and activates a class of enzymes like phospholipase A2s (PLA2s), and minocycline reduces cPLA2s [35]. It also inhibits monocyte and microglial expression of cyclooxygenase 2 (COX2) and production of proinflammatory prostaglandins E2 [36] and suppresses 5-lipoxygenase (5-LOX) action in SCI tissue [37]. Minocycline also eliminates free radicals in the post-SCI microenvironment and protects from oxidative stress [38]. It inhibits malondialdehyde, a by-product of lipid peroxidation [39, 40], and increases glutathione (GSH) [39], superoxide dismutase, and glutathione peroxidase [40], suggesting the powerful antioxidative mechanisms of minocycline to recover from secondary injury in SCI. Minocycline is reported to inhibit matrix metalloproteinases (MMPs) that are upregulated following SCI and are involved in injury and recovery processes [41, 42]. Furthermore, minocycline improves functional outcome, reduces lesion size and cell death, and alters cytokine expression after SCI [43–45]. Minocycline reduces the lesion area, increases the number of descending sympathoexcitatory axons traversing the injury site, and ultimately reduces the severity of autonomic dysreflexia [46]. In a murine model of SCI, minocycline treatment was superior to methylprednisolone in promoting functional improvement [44] and had neuroprotective effects on the SCI epicenter [47], motor neuron recovery, and neuropathic pain [48]. Minocycline has recently been reported to be effective in reducing secondary injury and promoting locomo-

*DOI: http://dx.doi.org/10.5772/intechopen.85212*

tor functional recovery in experimental SCI [28].

It has also been reported to attenuate reactive astrocytosis in SCI which directly damages cell bodies and triggers endogenous processes including neuroinflammation and reactive astrocytosis [49, 50]. In combination studies also, minocycline has been reported for better recovery from SCI when used in combination with other drugs like FK506 [22] and bone marrow mesenchymal cells (BMSCs) [51] showing a very significant recovery in behavioral function, oxidative stress, and reduction in

Cyclosporin-A is an immunosuppressive cyclic undecapeptide that inhibits T cells and depresses both cellular and humoral immune responses to prevent graft rejection and reduces the inflammatory responses [52]. CsA significantly decreases the expression levels of interleukin-10, tumor necrosis factor-α, cyclophilin-D (Cyp-D), and apoptosis-inducing factor (AIF) [53]. CsA does not readily cross the blood-spinal cord barrier (BSCB), which restricts the clinical application of CsA for SCI treatment. Thus, polyethylene glycol (PEG)-transactivating-transduction protein (TAT)-modified CsA-loaded cationic multifunctional polymeric liposomepoly (lactic-co-glycolic acid) (PLGA) core/shell nanoparticles (PLGA/CsA NPs) to transport and deliver CsA across the BSCB have a new potential to treat SCI [54]. CsA inhibits primarily the inflammatory reaction and the synthesis of constitutive nitric oxide (NO) and inducible nitric oxide synthases (NOS), well-known neurotoxic agents for SCI diminishing overproduction of free radicals, and secondarily

lesion size from SCI in rats warranting further research on this drug.

*Effects of Cyclosporin-A, Minocycline, and Tacrolimus (FK506) on Enhanced Behavioral… DOI: http://dx.doi.org/10.5772/intechopen.85212*

neuroprotection in experimental models of neurological diseases, including SCI [27]. In a broad range of secondary injury mechanisms via its anti-inflammatory, antioxidant, and antiapoptotic properties, minocycline is effective in reducing secondary injury and promoting locomotor functional recovery [28–31]. Minocycline prevents N-methyl-d-aspartate (NMDA)-induced excitotoxicity by diminishing NMDA-induced Ca2+ influx and mitochondrial Ca2+ uptake [32] and protects gray and white matter from SCI [33]. Minocycline also inhibits p38 mitogen-activated protein kinase (p38 MAPK) activation and microglial pro-nerve growth factor (proNGF) expression resulting from inflammatory reactions due to SCI and improves oligodendrocyte survival [34]. Inflammation due to SCI also upregulates and activates a class of enzymes like phospholipase A2s (PLA2s), and minocycline reduces cPLA2s [35]. It also inhibits monocyte and microglial expression of cyclooxygenase 2 (COX2) and production of proinflammatory prostaglandins E2 [36] and suppresses 5-lipoxygenase (5-LOX) action in SCI tissue [37]. Minocycline also eliminates free radicals in the post-SCI microenvironment and protects from oxidative stress [38]. It inhibits malondialdehyde, a by-product of lipid peroxidation [39, 40], and increases glutathione (GSH) [39], superoxide dismutase, and glutathione peroxidase [40], suggesting the powerful antioxidative mechanisms of minocycline to recover from secondary injury in SCI. Minocycline is reported to inhibit matrix metalloproteinases (MMPs) that are upregulated following SCI and are involved in injury and recovery processes [41, 42]. Furthermore, minocycline improves functional outcome, reduces lesion size and cell death, and alters cytokine expression after SCI [43–45]. Minocycline reduces the lesion area, increases the number of descending sympathoexcitatory axons traversing the injury site, and ultimately reduces the severity of autonomic dysreflexia [46]. In a murine model of SCI, minocycline treatment was superior to methylprednisolone in promoting functional improvement [44] and had neuroprotective effects on the SCI epicenter [47], motor neuron recovery, and neuropathic pain [48]. Minocycline has recently been reported to be effective in reducing secondary injury and promoting locomotor functional recovery in experimental SCI [28].

It has also been reported to attenuate reactive astrocytosis in SCI which directly damages cell bodies and triggers endogenous processes including neuroinflammation and reactive astrocytosis [49, 50]. In combination studies also, minocycline has been reported for better recovery from SCI when used in combination with other drugs like FK506 [22] and bone marrow mesenchymal cells (BMSCs) [51] showing a very significant recovery in behavioral function, oxidative stress, and reduction in lesion size from SCI in rats warranting further research on this drug.

#### **1.2 Cyclosporin-A**

*Spinal Cord Injury Therapy*

to SCI causing severe and permanent functional deficits. Most of the clinical trials and experimental studies are conducted for intense research to unfold the underlying pathophysiological processes and for searching ideal and potential therapy for recovery from secondary SCI injuries [8]. Besides motor dysfunction, some of the other important SCI-related biochemical and immunological impairments that get involved are serum tumor necrosis factor (TNF)-α, interleukin (IL)-1β, interleukin-6, nuclear factor (NF)-κB p65, p38 mitogen-activated protein kinase (MAPK), inducible nitric oxide synthase (iNOS), caspase-3, superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px) [9]. SCI trauma causes a devastating effect not only to the individual patient, but it also incurs heavy expensive burden to the society in general and to the family

Despite considerable therapeutic studies, no proven drugs or techniques are available for satisfactory treatment of SCI. Much of these therapeutic studies have been reported from animal models, and it needs to be understood that a successful clinical trial in humans can only be initiated based on previously available preclinical data reported from animal model studies that closely mimic the losses as in human SCI functions [11]. Although rats are the animal model of choice for SCI studies, the major anatomical differences in axonal tracts and sensory motor pathways between quadrupeds and bipeds need to be taken into careful account to

Currently, methylprednisolone is the only recognized treatment for human SCI; however, it has significant adverse effects, including respiratory complications, sepsis, and gastrointestinal hemorrhage [13]. Furthermore, other important evidence-based therapies that have potential neuroprotective and neural reparative therapeutic properties and are undergoing clinical trials for human SCI include surgical decompression, blood pressure augmentation, riluzole, granulocyte colonystimulating factor, minocycline, glibenclamide, cerebrospinal fluid drainage, magnesium, therapeutic hypothermia, Cethrin (VX-210), anti-NOGO antibody,

Some other experimental drugs that have been studied for therapeutical use in animal SCI are recombinant human erythropoietin [10], tetrodotoxin [16], BCL-2 [17], cyclosporin-A [18], edaravone [19], atorvastatin [20], calpain inhibitors [21] FK506, and minocycline [22]. Also, some natural products like eugenol oil [9], curcumin [23], and melatonin [24] have shown promising effects in animal SCI functional recovery. It sounds reasonable that instead of using a highly selective treatment that targets a specific molecule or pathway, a compound with multifunctional properties that targets several mediators involved in spinal cord pathology may be more effective for recovery from SCI [25]. In our earlier study [22], the promising potential of FK506 and minocycline has been reported for their effectiveness in rat SCI model. Thus, in the present study, besides these two multifactorial effective compounds minocycline and FK506, a third compound cyclosporin-A (CsA) was also included, and all the three compounds minocycline, FK506 (tacrolimus), and cyclosporine-A were chosen to evaluate in a comparative manner for their therapeutical potential using some important and reliable parameters that are most commonly used in rat SCI model [22]. Before discussing the outcome of our present results, we review the multifactorial effects of these three compounds that

Minocycline, a semisynthetic second-generation tetracycline, has robust neuroprotective effects in rodent models of neurodegenerative diseases [26] and provides

members, due to substantial long-term healthcare expenditures [10].

cell-based approaches, and bioengineered biomaterials [5, 14, 15].

improve the targets of human SCI treatments [12].

have been reported in literature using rat SCI model.

**72**

**1.1 Minocycline**

Cyclosporin-A is an immunosuppressive cyclic undecapeptide that inhibits T cells and depresses both cellular and humoral immune responses to prevent graft rejection and reduces the inflammatory responses [52]. CsA significantly decreases the expression levels of interleukin-10, tumor necrosis factor-α, cyclophilin-D (Cyp-D), and apoptosis-inducing factor (AIF) [53]. CsA does not readily cross the blood-spinal cord barrier (BSCB), which restricts the clinical application of CsA for SCI treatment. Thus, polyethylene glycol (PEG)-transactivating-transduction protein (TAT)-modified CsA-loaded cationic multifunctional polymeric liposomepoly (lactic-co-glycolic acid) (PLGA) core/shell nanoparticles (PLGA/CsA NPs) to transport and deliver CsA across the BSCB have a new potential to treat SCI [54]. CsA inhibits primarily the inflammatory reaction and the synthesis of constitutive nitric oxide (NO) and inducible nitric oxide synthases (NOS), well-known neurotoxic agents for SCI diminishing overproduction of free radicals, and secondarily

lipid peroxidation (LP) observed after SCI [55, 56]. CsA may also induce other non-immunological effects that could be beneficial for treatment of neurological disorders [57]. CsA has been widely used in the treatment of various diseases including aplastic anemia, nephritic syndrome, rheumatoid arthritis, psoriasis, and cerebral ischemic injuries [53]. CsA promotes neuroprotection by diminishing both demyelination and neuronal cell death, resulting in a better motor outcome after SCI [52, 58, 59]. CsA in combination with FK506 had a neuroprotective treatment against SCI hypoxia-induced damage mediated via their antioxidant actions on mitochondrial ATP, tissue-reduced glutathione, tissue LPO level, and myeloperoxidase (MPO) activity [60]. Administration of CsA in combination with olfactory ensheathing cell (OEC) transplantation results in augmented functional improvements and promotes axon regeneration after SCI [61].

#### **1.3 FK506**

FK506 (tacrolimus), a macrolide lactane antibiotic, was introduced as an immunosuppressive agent [62] with virtually no side effects [63]. FK506, a potent calcineurin inhibitor, exhibits neuroprotective actions in several experimental models of central nervous system trauma, including stroke, and improved neurological recovery following peripheral and spinal cord injuries [47, 63–67]. It is reported that FK506 has beneficial effects in SCI recovery involving various mechanisms such as neuroregeneration and neuroprotection [67], promotion of axonal outgrowth [68], and suppression of oxidative stress [60]. FK506 improves the functional outcome of SCI [67–69] and has an in vivo neurotrophic action, whereby it enhances the rate of axon regeneration, leading to more rapid neurological recovery [70–73]. Significant functional recovery from SCI due to FK506 treatment has been reported in rat models [22, 67, 74]. Activation of NF-κB and proinflammatory cytokines (TNF-a, IL-1b, and IL-6) expression levels in SCI animals is reversed by FK506 treatment involving microglial activation after SCI [7]. FK506 upregulates epidermal growth factor (EGF)-level expression of astrocytes that have an important role as mediators for SCI functional recovery promoting axonal regeneration [74]. FK506 in combination as a cocktail with other drugs like minocycline [22], CSA [60], RhoA inhibitors [75], nerve growth factor (NFG) [76], and methylprednisolone (MP) [77] has shown significant therapeutic recovery from SCI in rats.

Considering the above-discussed multifactorial effects of CsA, minocycline, and FK506, the present study was undertaken to investigate the neuroprotective effects of these three compounds in a comparative manner on recovery from experimental SCI, as these three drugs target multiple processes involved in mediating cell death and the development of secondary injury in SCI. Furthermore, our earlier findings on FK506 and minocycline [22] prompted us to include CsA (another promising drug for SCI recovery) and compare their effectiveness in rat model of SCI, using the behavioral and biochemical parameters as in earlier [22].

#### **2. Utility of experimental animal models for SCI studies**

For SCI studies, animal models are used because of their easy accessibility, convenience, and capability of the researchers to explore them at several levels (simulated to human clinical SCI levels) for motor functional, biochemical, and oxidative stress and genetic, therapeutic, and pathophysiological evaluations [78]. Over the last decade, a variety of animal models have been used for experimental SCI studies, including rats, mice, gerbils, guinea pigs, hamsters, rabbits, dogs, goats, pigs, and nonhuman primates [79]. Among these animals, rodents in general

**75**

*Effects of Cyclosporin-A, Minocycline, and Tacrolimus (FK506) on Enhanced Behavioral…*

and rats in particular are the most widely and commonly studied SCI models [80]. In the present study also, we have used young adult male Sprague-Dawley rats with all similar specifications of breeding, housing facilities, and experimental han-

To establish an ideal SCI animal model for research purposes, various models have been tried and reported till to date in quest of searching methodology to obtain maximum recovery from SCI. These experimental animal models include spinal cord traumatic injury model [81], photochemical-induced SCI model [82], spinal cord transection model [83], bidirectional distraction SCI model [84], and the spinal cord ischemiareperfusion injury model [85]. For traumatic injury model, the contusive SCI model is used by inducing contusion on the dorsal spinal cord by dropping a desired weight either from a computer-controlled impact device [86] or from a customized impact device [87]. Another traumatic injury model known as compressive SCI model is also very commonly used where instead of dropping the weight, it is placed on the exposed spinal cord segment in the dorsoventral direction to induce a compressive SCI [88, 89]. However, since SCI caused by impact and compression is more common in clinical patients [79], in the present study also, we have used the compressed SCI model induced in the rats as described in our earlier study [22]. Briefly, the SCI was induced in the rats following the modified method of Nystrom and Berglund [89]. Laminectomy was performed at the T 7–8 level, and spinal cord compression injury was produced by placing a load with a total

All experimental rats were randomly divided into the following six groups with

Group I: The normal control group without laminectomy or compression injury Group II: Sham group with laminectomy alone but no spinal compression injury Group III: SCI control group with laminectomy and spinal compression injury SCI-treated groups were the same as the SCI control group (Group III) and consisted of three groups in which the effect on the recovery from SCI using the same parameters is mentioned in our earlier study [22]. Doses of the three drugs CSA, minocycline, and FK506 were selected on the basis of our pilot screening of these drugs at low, medium, and high doses, and the best effective dose in each was

All protocols for the drug administration, follow-ups, care, and experimental

To analyze the therapeutic recovery from induced SCI in animal models, several behavioral outcome measures have been developed and widely used, such as the catwalk [90], the Basso-Beattie-Bresnahan (BBB) locomotor scale [91], the horizontal ladder test, and the cylinder rearing test [92]. From the literature review of the recent years, it is found that BBB locomotor scale has been most widely used in SCI rat models to evaluate motor functional recovery from SCI [9, 12, 22–24, 53, 58, 74, 77, 93]. However, in the present study, besides BBB locomotor scale [94], a battery of some more behavioral motor functions was included like Tarlov scoring [95], inclined plane test [96], and some functional deficit scorings like toe spread, platform hang, wire mesh descent, and hind foot bar grab [97, 98]. Our pilot study showed that a naive control group of animals treated with CsA, minocycline, and FK506 without SCI

handlings of the animals for various evaluation parameters were the same as

*DOI: http://dx.doi.org/10.5772/intechopen.85212*

dlings, as described in our earlier study [22].

weight of 35 g, for 5 min over the exposed extradural area.

eight animals in each as described earlier [22]:

used in the present study as follows: Group IV: Cyclosporin-A 5 mg/kg Group V: Minocycline 50 mg/kg Group VI: FK506 (tacrolimus) 1 mg/kg

**3. Behavioral evaluations in SCI animals**

described earlier [22].

#### *Effects of Cyclosporin-A, Minocycline, and Tacrolimus (FK506) on Enhanced Behavioral… DOI: http://dx.doi.org/10.5772/intechopen.85212*

and rats in particular are the most widely and commonly studied SCI models [80]. In the present study also, we have used young adult male Sprague-Dawley rats with all similar specifications of breeding, housing facilities, and experimental handlings, as described in our earlier study [22].

To establish an ideal SCI animal model for research purposes, various models have been tried and reported till to date in quest of searching methodology to obtain maximum recovery from SCI. These experimental animal models include spinal cord traumatic injury model [81], photochemical-induced SCI model [82], spinal cord transection model [83], bidirectional distraction SCI model [84], and the spinal cord ischemiareperfusion injury model [85]. For traumatic injury model, the contusive SCI model is used by inducing contusion on the dorsal spinal cord by dropping a desired weight either from a computer-controlled impact device [86] or from a customized impact device [87]. Another traumatic injury model known as compressive SCI model is also very commonly used where instead of dropping the weight, it is placed on the exposed spinal cord segment in the dorsoventral direction to induce a compressive SCI [88, 89]. However, since SCI caused by impact and compression is more common in clinical patients [79], in the present study also, we have used the compressed SCI model induced in the rats as described in our earlier study [22]. Briefly, the SCI was induced in the rats following the modified method of Nystrom and Berglund [89]. Laminectomy was performed at the T 7–8 level, and spinal cord compression injury was produced by placing a load with a total weight of 35 g, for 5 min over the exposed extradural area.

All experimental rats were randomly divided into the following six groups with eight animals in each as described earlier [22]:

Group I: The normal control group without laminectomy or compression injury Group II: Sham group with laminectomy alone but no spinal compression injury Group III: SCI control group with laminectomy and spinal compression injury

SCI-treated groups were the same as the SCI control group (Group III) and consisted of three groups in which the effect on the recovery from SCI using the same parameters is mentioned in our earlier study [22]. Doses of the three drugs CSA, minocycline, and FK506 were selected on the basis of our pilot screening of these drugs at low, medium, and high doses, and the best effective dose in each was used in the present study as follows:

Group IV: Cyclosporin-A 5 mg/kg

Group V: Minocycline 50 mg/kg

Group VI: FK506 (tacrolimus) 1 mg/kg

All protocols for the drug administration, follow-ups, care, and experimental handlings of the animals for various evaluation parameters were the same as described earlier [22].

#### **3. Behavioral evaluations in SCI animals**

To analyze the therapeutic recovery from induced SCI in animal models, several behavioral outcome measures have been developed and widely used, such as the catwalk [90], the Basso-Beattie-Bresnahan (BBB) locomotor scale [91], the horizontal ladder test, and the cylinder rearing test [92]. From the literature review of the recent years, it is found that BBB locomotor scale has been most widely used in SCI rat models to evaluate motor functional recovery from SCI [9, 12, 22–24, 53, 58, 74, 77, 93]. However, in the present study, besides BBB locomotor scale [94], a battery of some more behavioral motor functions was included like Tarlov scoring [95], inclined plane test [96], and some functional deficit scorings like toe spread, platform hang, wire mesh descent, and hind foot bar grab [97, 98]. Our pilot study showed that a naive control group of animals treated with CsA, minocycline, and FK506 without SCI

*Spinal Cord Injury Therapy*

**1.3 FK506**

lipid peroxidation (LP) observed after SCI [55, 56]. CsA may also induce other non-immunological effects that could be beneficial for treatment of neurological disorders [57]. CsA has been widely used in the treatment of various diseases including aplastic anemia, nephritic syndrome, rheumatoid arthritis, psoriasis, and cerebral ischemic injuries [53]. CsA promotes neuroprotection by diminishing both demyelination and neuronal cell death, resulting in a better motor outcome after SCI [52, 58, 59]. CsA in combination with FK506 had a neuroprotective treatment against SCI hypoxia-induced damage mediated via their antioxidant actions on mitochondrial ATP, tissue-reduced glutathione, tissue LPO level, and myeloperoxidase (MPO) activity [60]. Administration of CsA in combination with olfactory ensheathing cell (OEC) transplantation results in augmented functional improve-

FK506 (tacrolimus), a macrolide lactane antibiotic, was introduced as an immunosuppressive agent [62] with virtually no side effects [63]. FK506, a potent calcineurin inhibitor, exhibits neuroprotective actions in several experimental models of central nervous system trauma, including stroke, and improved neurological recovery following peripheral and spinal cord injuries [47, 63–67]. It is reported that FK506 has beneficial effects in SCI recovery involving various mechanisms such as neuroregeneration and neuroprotection [67], promotion of axonal outgrowth [68], and suppression of oxidative stress [60]. FK506 improves the functional outcome of SCI [67–69] and has an in vivo neurotrophic action, whereby it enhances the rate of axon regeneration, leading to more rapid neurological recovery [70–73]. Significant functional recovery from SCI due to FK506 treatment has been reported in rat models [22, 67, 74]. Activation of NF-κB and proinflammatory cytokines (TNF-a, IL-1b, and IL-6) expression levels in SCI animals is reversed by FK506 treatment involving microglial activation after SCI [7]. FK506 upregulates epidermal growth factor (EGF)-level expression of astrocytes that have an important role as mediators for SCI functional recovery promoting axonal regeneration [74]. FK506 in combination as a cocktail with other drugs like minocycline [22], CSA [60], RhoA inhibitors [75], nerve growth factor (NFG) [76], and methylprednisolone (MP) [77] has

Considering the above-discussed multifactorial effects of CsA, minocycline, and FK506, the present study was undertaken to investigate the neuroprotective effects of these three compounds in a comparative manner on recovery from experimental SCI, as these three drugs target multiple processes involved in mediating cell death and the development of secondary injury in SCI. Furthermore, our earlier findings on FK506 and minocycline [22] prompted us to include CsA (another promising drug for SCI recovery) and compare their effectiveness in rat model of SCI, using

ments and promotes axon regeneration after SCI [61].

shown significant therapeutic recovery from SCI in rats.

the behavioral and biochemical parameters as in earlier [22].

**2. Utility of experimental animal models for SCI studies**

For SCI studies, animal models are used because of their easy accessibility, convenience, and capability of the researchers to explore them at several levels (simulated to human clinical SCI levels) for motor functional, biochemical, and oxidative stress and genetic, therapeutic, and pathophysiological evaluations [78]. Over the last decade, a variety of animal models have been used for experimental SCI studies, including rats, mice, gerbils, guinea pigs, hamsters, rabbits, dogs, goats, pigs, and nonhuman primates [79]. Among these animals, rodents in general

**74**

showed no different behaviors than the naïve control untreated groups (data not shown in behavioral results) for all the observed behavioral parameters. Thus, the results of the drug treatments alone were not included in all behavioral results (**Figures 1**–**4**).

#### **Figure 1.**

*Effect of CSA, FK506, and minocycline on gait performance tunnel (GPT) behavioral motor performance activities (BBB Score) of hind limbs of rats subjected to SCI. The graph shows the comparative functional recovery from SCI over a period of 29 days. Animals were treated with the drugs daily after SCI for 3 weeks. Abbreviations: CSA, cyclosporin-A; FK506, tacrolimus; SCI, spinal cord injury; BBB, Basso, Beattie, and Bresnahan. Drug doses used are cyclosporin (5 mg/kg), FK506 (1 mg/kg), and minocycline (50 mg/kg); the drugs are effective in the order FK506 > minocycline > cyclosporin-A. # shows the SCI group is significantly (p < 0.001) different from the SCI uninjured control group. \*, \*\*, and \*\*\* represent the SCI-treated groups are significantly different at p < 0.05, p < 0.01, and p < 0.001, respectively, compared to the SCI group by ANOVA with post hoc testing using Tukey-Kramer or Student-Newman-Keuls Multiple Comparison Tests.*

#### **Figure 2.**

*The effect of cyclosporin-A, FK506, and minocycline on the behavioral motor performance activity (Tarlov's Score) of hind limbs of rats subjected to SCI. The graph shows the comparative functional recovery from SCI over a period of 29 days. Animals were treated with drugs daily after SCI for 3 weeks. Abbreviations, drugs used and their doses, and all statistical significances are the same as in* **Figure 1***.*

#### **Figure 3.**

*The effect of cyclosporin-A, FK506, and minocycline on the behavioral motor performance activity (Inclined Plane Test) of hind limbs (HL) of rats subjected to SCI. The graph shows the comparative functional recovery from SCI over a period of 29 days. Animals were treated with drugs daily after SCI for 3 weeks. Abbreviations, drugs used and their doses, and all statistical significances are the same as in* **Figure 1***.*

**77**

**Figure 4.**

*significances are the same as in* **Figure 1***.*

*Effects of Cyclosporin-A, Minocycline, and Tacrolimus (FK506) on Enhanced Behavioral…*

The present results of behavioral observations indicated that treatment with all the three drugs in this study induced significant recovery from SCI with respect to time in all behavioral activities compared to the SCI control group, and the drugs were effective in the order of FK506 > minocycline > CsA (F = 13.49, F = 5.82, and F = 3.14; df = 3;

*(A–D) The effect of cyclosporin-A, FK506, and minocycline on the behavioral motor functional scoring of toe spread (A), platform hanging (B), hind foot bar grab (C), and wire mesh decent (D) of rats subjected to SCI. The graph shows the comparative functional recovery from SCI over a period of 29 days. Animals were treated with drugs daily after SCI for 3 weeks. Abbreviations, drugs used and their doses, and all statistical* 

Biochemical evaluations have a vast list of parameters that exist as biomarkers for assessing recovery from SCI in animal models. Some of the most important biochemical parameters include oxidative stress indices like lipid peroxidation and total glutathione, nitric oxide synthase, myeloperoxidase, mitochondrial permeability, inflammatory responses, autonomic dysreflexia, cerebrospinal fluid biomarkers, immune responses, astrocyte modulations, etc., and all of these have been reviewed in detail earlier in this chapter, especially for the three drugs, CsA,

The biochemical parameters evaluated in this study included determination of monoamines 5-hydroxy-indoleacetic acid (5-HIAA) and serotonin or 5-hydroxy

p < 0.001, p < 0.01, and p < 0.05, respectively) throughout (**Figures 1**–**4**).

minocycline, and FK506, that have been evaluated in the present study.

**4. Biochemical evaluations in SCI animal models**

*DOI: http://dx.doi.org/10.5772/intechopen.85212*

*Effects of Cyclosporin-A, Minocycline, and Tacrolimus (FK506) on Enhanced Behavioral… DOI: http://dx.doi.org/10.5772/intechopen.85212*

#### **Figure 4.**

*Spinal Cord Injury Therapy*

showed no different behaviors than the naïve control untreated groups (data not shown in behavioral results) for all the observed behavioral parameters. Thus, the results of the drug treatments alone were not included in all behavioral results (**Figures 1**–**4**).

*Effect of CSA, FK506, and minocycline on gait performance tunnel (GPT) behavioral motor performance activities (BBB Score) of hind limbs of rats subjected to SCI. The graph shows the comparative functional recovery from SCI over a period of 29 days. Animals were treated with the drugs daily after SCI for 3 weeks. Abbreviations: CSA, cyclosporin-A; FK506, tacrolimus; SCI, spinal cord injury; BBB, Basso, Beattie, and Bresnahan. Drug doses used are cyclosporin (5 mg/kg), FK506 (1 mg/kg), and minocycline (50 mg/kg); the drugs are effective in the order FK506 > minocycline > cyclosporin-A. # shows the SCI group is significantly (p < 0.001) different from the SCI uninjured control group. \*, \*\*, and \*\*\* represent the SCI-treated groups are significantly different at p < 0.05, p < 0.01, and p < 0.001, respectively, compared to the SCI group by ANOVA* 

*with post hoc testing using Tukey-Kramer or Student-Newman-Keuls Multiple Comparison Tests.*

*The effect of cyclosporin-A, FK506, and minocycline on the behavioral motor performance activity (Tarlov's Score) of hind limbs of rats subjected to SCI. The graph shows the comparative functional recovery from SCI over a period of 29 days. Animals were treated with drugs daily after SCI for 3 weeks. Abbreviations, drugs* 

*The effect of cyclosporin-A, FK506, and minocycline on the behavioral motor performance activity (Inclined Plane Test) of hind limbs (HL) of rats subjected to SCI. The graph shows the comparative functional recovery from SCI over a period of 29 days. Animals were treated with drugs daily after SCI for 3 weeks. Abbreviations,* 

*drugs used and their doses, and all statistical significances are the same as in* **Figure 1***.*

*used and their doses, and all statistical significances are the same as in* **Figure 1***.*

**76**

**Figure 3.**

**Figure 2.**

**Figure 1.**

*(A–D) The effect of cyclosporin-A, FK506, and minocycline on the behavioral motor functional scoring of toe spread (A), platform hanging (B), hind foot bar grab (C), and wire mesh decent (D) of rats subjected to SCI. The graph shows the comparative functional recovery from SCI over a period of 29 days. Animals were treated with drugs daily after SCI for 3 weeks. Abbreviations, drugs used and their doses, and all statistical significances are the same as in* **Figure 1***.*

The present results of behavioral observations indicated that treatment with all the three drugs in this study induced significant recovery from SCI with respect to time in all behavioral activities compared to the SCI control group, and the drugs were effective in the order of FK506 > minocycline > CsA (F = 13.49, F = 5.82, and F = 3.14; df = 3; p < 0.001, p < 0.01, and p < 0.05, respectively) throughout (**Figures 1**–**4**).

#### **4. Biochemical evaluations in SCI animal models**

Biochemical evaluations have a vast list of parameters that exist as biomarkers for assessing recovery from SCI in animal models. Some of the most important biochemical parameters include oxidative stress indices like lipid peroxidation and total glutathione, nitric oxide synthase, myeloperoxidase, mitochondrial permeability, inflammatory responses, autonomic dysreflexia, cerebrospinal fluid biomarkers, immune responses, astrocyte modulations, etc., and all of these have been reviewed in detail earlier in this chapter, especially for the three drugs, CsA, minocycline, and FK506, that have been evaluated in the present study.

The biochemical parameters evaluated in this study included determination of monoamines 5-hydroxy-indoleacetic acid (5-HIAA) and serotonin or 5-hydroxy

tryptamine (5-HT) [99], lipid peroxides determined as thiobarbituric acid-reactive substances (TBARS) [100, 101], total glutathione [102, 103], and myeloperoxidase [104] and have been described for their methods in our earlier study [22].

The present biochemical results showed significant ameliorating effect of all three drugs on the levels of 5-HT (**Figure 5A**, 5-HIAA; **Figure 5B**, on the ratio of 5-HIAA; and **Figure 5C**, 5-HT). TBARS was significantly stimulated (**Figure 6A**), whereas GSH was significantly inhibited (**Figure 6B**), and MPO level was significantly diminished toward the normal level (**Figure 6C**). Overall, the entire biochemical parameters evaluated in the present study were significantly affected by the three drugs effectively in the order FK506 > minocycline > CsA throughout.

#### **Figure 5.**

*(A–C) Levels of (A) 5-HT (5-hydroxytryptamine), (B) 5-HIAA (5-hydroxy-indoleacetic acid), and (C) the ratio of 5-HIAA and 5-HT activities in the spinal cord tissue of rats 29 days post-SCI and the effects of treatment with various drugs. Abbreviations, drugs used and their doses, and all statistical significances are the same as in* **Figure 1***.*

**79**

**5. Discussion**

**Figure 6.**

[18, 22, 52, 106].

*Effects of Cyclosporin-A, Minocycline, and Tacrolimus (FK506) on Enhanced Behavioral…*

SCI leads to persistent pain and motor dysfunction, both of which lack effective therapeutics [105]. Therapeutic approaches that promote both neuroprotection and neuroregeneration are valuable for SCI therapies [52]. From the present discussed literature, the potential agents that have generated interest in SCI studies in the recent past include the multifactorial drugs minocycline, FK506, and CsA

*Levels of (A) thiobarbituric acid, (B) glutathione, and (C) myeloperoxidase activities in the injured spinal cord tissue of rats 29 days post-SCI and the effects of treatment with various drugs. Abbreviations, drugs used* 

*and their doses, and all statistical significances are the same as in* **Figure 1***.*

In the present chapter also, the treated rats showed recovery in their hind limb reflexes rapidly regaining responses comparable with those of uninjured control rats (**Figure 1**). Although all drug-treated groups showed improved recovery in BBB and all behavioral activities, the best and the most significant recovery was observed with FK506 treatment. The drugs were effective in the order FK506 > minocycline > CsA throughout. Earlier studies have also used BBB scoring along with other behavioral parameters and have shown significant behavioral functional outcome in the

*DOI: http://dx.doi.org/10.5772/intechopen.85212*

*Effects of Cyclosporin-A, Minocycline, and Tacrolimus (FK506) on Enhanced Behavioral… DOI: http://dx.doi.org/10.5772/intechopen.85212*

#### **Figure 6.**

*Spinal Cord Injury Therapy*

tryptamine (5-HT) [99], lipid peroxides determined as thiobarbituric acid-reactive substances (TBARS) [100, 101], total glutathione [102, 103], and myeloperoxidase

*(A–C) Levels of (A) 5-HT (5-hydroxytryptamine), (B) 5-HIAA (5-hydroxy-indoleacetic acid), and (C) the ratio of 5-HIAA and 5-HT activities in the spinal cord tissue of rats 29 days post-SCI and the effects of treatment with various drugs. Abbreviations, drugs used and their doses, and all statistical significances are the same as in* **Figure 1***.*

The present biochemical results showed significant ameliorating effect of all three drugs on the levels of 5-HT (**Figure 5A**, 5-HIAA; **Figure 5B**, on the ratio of 5-HIAA; and **Figure 5C**, 5-HT). TBARS was significantly stimulated (**Figure 6A**), whereas GSH was significantly inhibited (**Figure 6B**), and MPO level was significantly diminished toward the normal level (**Figure 6C**). Overall, the entire biochemical parameters evaluated in the present study were significantly affected by the three

[104] and have been described for their methods in our earlier study [22].

drugs effectively in the order FK506 > minocycline > CsA throughout.

**78**

**Figure 5.**

*Levels of (A) thiobarbituric acid, (B) glutathione, and (C) myeloperoxidase activities in the injured spinal cord tissue of rats 29 days post-SCI and the effects of treatment with various drugs. Abbreviations, drugs used and their doses, and all statistical significances are the same as in* **Figure 1***.*

#### **5. Discussion**

SCI leads to persistent pain and motor dysfunction, both of which lack effective therapeutics [105]. Therapeutic approaches that promote both neuroprotection and neuroregeneration are valuable for SCI therapies [52]. From the present discussed literature, the potential agents that have generated interest in SCI studies in the recent past include the multifactorial drugs minocycline, FK506, and CsA [18, 22, 52, 106].

In the present chapter also, the treated rats showed recovery in their hind limb reflexes rapidly regaining responses comparable with those of uninjured control rats (**Figure 1**). Although all drug-treated groups showed improved recovery in BBB and all behavioral activities, the best and the most significant recovery was observed with FK506 treatment. The drugs were effective in the order FK506 > minocycline > CsA throughout. Earlier studies have also used BBB scoring along with other behavioral parameters and have shown significant behavioral functional outcome in the

SCI animals treated with FK506 [7, 22, 60, 67, 69, 70, 74, 76], minocycline [22, 28, 43–45], and CsA [18, 52, 53, 58, 61]. In addition to the therapeutic effects of the three drugs, it has been also suggested that the daily routine behavioral assessment procedures may also assist the animals as equivalent to their exercises that help them to recover from SCI [107]. However, more studies are required to confirm this presumption. Additionally, no notable side effects were noted at the dosing regimen of the drugs (selected from the pilot studies) used in the present chapter. However, it has been suggested particularly for FK506 [63] that the therapeutic dosing regimen is a key factor that can affect efficacy as a neuroprotectant for CNS injuries.

FK506 has been reported by others also for being a potential therapy for SCI recovery through various mechanisms [7, 60, 63, 68, 108]. It is evidently proven that FK506 prevents the activation of NF-κB in microglia which reduces production of proinflammatory cytokines like TNF-a, IL-1b, and IL-6 in the SCI responses for effective recovery [7, 109]. Furthermore, it is suggested that inhibition of inflammatory reaction in SCI by FK506 could be due to its inhibitory action on decreasing the free radical formation and lipid peroxidation preventing calcineurin-mediated dephosphorylation of NOS activity in a Ca2+-dependent manner [77]. FK506 also enhances neurite outgrowth and improves functional recovery from SCI by stimulating astrocytes to secrete epidermal growth factor (EGF) for neural repair [74].

CsA has also been reviewed and reported as a potent neuroprotectant for functional recovery from SCI [5, 55]. The significant protective role of CsA has been reported for recovery from SCI through inhibiting the apoptosis of spinal cord cells [53], improving locomotor function [58], increasing mean arterial pressure [110], inhibiting NOS [56], diminishing demyelination and neuronal cell death [60], attenuating reactive astrocytosis due to injury improved neurologic outcome [50], and reducing pain [111].

Minocycline has also been reviewed recently for its effectiveness through multiple mechanisms for functional recovery from SCI [38]. The multiple targets that minocycline works for SCI functional recovery include upregulation of the protein VEGF and BDNF expressions; downregulation of protein p-38MAPK, proNGF, p75NTR, and RhoA expressions and suppressed caspase-3 activity [51]; and improved antioxidant activity through amelioration in oxidative stress in the SCI tissue [40].

Monoamines such as norepinephrine (NE), dopamine (DA), and serotonin (5-HT) can activate the spinal neurons involved in walking [112–114]. Thus, the decrease in the level of 5-HT and 5-HIAA in the SCI animals in the present chapter clearly indicates that SCI inevitably affects the normal functioning of these spinal neurotransmitters involved in locomotor function. SCI-injured animals treated with the drugs herein improved levels of 5-HT and 5-HIAA (**Figure 5A–C**, respectively). Our present behavioral findings also showed an overall correlation and significant improvement in the functional deficits of the hind limbs after treatment with these drugs, indicating the presence of potential mechanisms of serotonergic agents in these drugs, as present in indorenate (5-methoxytryptamine, beta-methyl carboxylate hydrochloride), a 5-HT1A agonist that improved motor function in rats with chronic SCI [115].

The oxidant/antioxidant balance was clearly reflected by the increased level of TBARS (**Figure 6A**) and decreased level of GSH (**Figure 6B**) in the contused tissue of SCI control animals. However, treatment of SCI animals with the drugs interfered with the formation of free radicals following traumatic SCI. The comparative behavioral restorative effects of these drugs in the formation of free radicals in injured SC were in the order of FK506 > minocycline > CsA.

Spinal cord injury in mice results in severe trauma characterized by edema and neutrophil infiltration (measured as an increase in myeloperoxidase activity), and

**81**

*Effects of Cyclosporin-A, Minocycline, and Tacrolimus (FK506) on Enhanced Behavioral…*

these neutrophils are thought to be involved in tissue injury through the release of various inflammatory mediators [116, 117]. The MPO levels in the present SCI animals were also significantly increased in the injured spinal cord tissue (**Figure 6C**). However, administration of minocycline, FK506, and CsA interfered significantly with the formation of MPO following traumatic SCI. The comparative restorative effects of these drugs in the formation of MPO in injured SC were in the order of

The pathophysiological events resulting from SCI are reported to involve free radical production; lipid peroxidation; excitotoxic molecules such as glutamate, eicosanoid, and prostaglandin production; protease activity; and intracellular increases in Ca2+ [118]. Furthermore, the primary auto-destructive event is initiated by the hydrolysis of fatty acids from membrane phospholipids, leading to cellular damage [119], and microglia becomes activated [120], which in turn may release

The hind limb functional deficits in the model of SCI (like the one as in the present chapter) are largely due to the loss of white matter axonal tracts [16, 122]. The white matter degeneration is caused by the primary injury (i.e., mechanical lesion), and there is also evidence that post-SCI demyelination caused by oligodendrocyte death/ malfunction contributes significantly to chronic SCI functional deficits [123, 124]. The secondary injury is reported to result from several proposed auto-destructive events, including reactive oxygen species-induced lipid peroxidation [125], activation of non-NMDA ionotropic glutamate receptors [126], and caspase-3

 accumulation leading to proteinase activation, which destroys the cytoskeleton [16, 129], as well as the induction of oligodendroglial apoptosis with subsequent demyelination of the surviving axons [79, 130]. Lipid peroxidation is one of the main pathological mechanisms involved in secondary damage after SCI [79]. Another key factor in the secondary injury mechanism is Ca2+ ions. Following trauma or ischemia, Ca2+ influx plays an important role in the pathogenesis of neural injury [130, 131]. Many drugs, including steroids, gangliosides, ion channel blockers, antioxidants, and free radical scavengers, have mild therapeutic effectiveness in experimental spinal cord injury [74, 119]. Another mechanism to promote functional recovery after spinal cord injury is enhancing axonal regeneration. Several strategies, including blocking myelin or glial scar inhibitors, delivery of neurotrophic factors, and cell transplantation, induce axonal outgrowth after experimental spinal cord injury. Among them, olfactory ensheathing cell grafts promote neuroprotection, axonal regeneration, and functional recovery after incomplete spinal cord injury [132, 133]. Furthermore, a regular enforced movement activity may additionally help provide faster functional restoration and

Studies on combinatorial effects of CsA, minocycline, and FK506 in various combinations with each other or with other compounds may prove to be more effective in recovery from SCI. Earlier combined treatments like FK506 and NGF [76], FK506 and minocycline [22], FK506 and methylprednisolone [77], FK506 and RhoA inhibitor [76, 77], minocycline and bone marrow mesenchymal stem cells [51], and CsA with PEG-TAT [54] have all shown significant functional recovery

From the overall literature review on the multifactorial effects of CsA, minocycline, and FK506 and from the discussion of the present findings, it can be

influx-mediated intra-

neurotoxic molecules that further damage nearby neurons [121].

activation [127, 128]. Secondary injury events include Na<sup>+</sup>

from SCI as compared to these compounds individually.

*DOI: http://dx.doi.org/10.5772/intechopen.85212*

FK506 > minocycline > CsA.

axonal Ca<sup>+</sup>

recovery after SCI [134].

**6. Conclusions**

#### *Effects of Cyclosporin-A, Minocycline, and Tacrolimus (FK506) on Enhanced Behavioral… DOI: http://dx.doi.org/10.5772/intechopen.85212*

these neutrophils are thought to be involved in tissue injury through the release of various inflammatory mediators [116, 117]. The MPO levels in the present SCI animals were also significantly increased in the injured spinal cord tissue (**Figure 6C**). However, administration of minocycline, FK506, and CsA interfered significantly with the formation of MPO following traumatic SCI. The comparative restorative effects of these drugs in the formation of MPO in injured SC were in the order of FK506 > minocycline > CsA.

The pathophysiological events resulting from SCI are reported to involve free radical production; lipid peroxidation; excitotoxic molecules such as glutamate, eicosanoid, and prostaglandin production; protease activity; and intracellular increases in Ca2+ [118]. Furthermore, the primary auto-destructive event is initiated by the hydrolysis of fatty acids from membrane phospholipids, leading to cellular damage [119], and microglia becomes activated [120], which in turn may release neurotoxic molecules that further damage nearby neurons [121].

The hind limb functional deficits in the model of SCI (like the one as in the present chapter) are largely due to the loss of white matter axonal tracts [16, 122]. The white matter degeneration is caused by the primary injury (i.e., mechanical lesion), and there is also evidence that post-SCI demyelination caused by oligodendrocyte death/ malfunction contributes significantly to chronic SCI functional deficits [123, 124].

The secondary injury is reported to result from several proposed auto-destructive events, including reactive oxygen species-induced lipid peroxidation [125], activation of non-NMDA ionotropic glutamate receptors [126], and caspase-3 activation [127, 128]. Secondary injury events include Na<sup>+</sup> influx-mediated intraaxonal Ca<sup>+</sup> accumulation leading to proteinase activation, which destroys the cytoskeleton [16, 129], as well as the induction of oligodendroglial apoptosis with subsequent demyelination of the surviving axons [79, 130]. Lipid peroxidation is one of the main pathological mechanisms involved in secondary damage after SCI [79]. Another key factor in the secondary injury mechanism is Ca2+ ions. Following trauma or ischemia, Ca2+ influx plays an important role in the pathogenesis of neural injury [130, 131]. Many drugs, including steroids, gangliosides, ion channel blockers, antioxidants, and free radical scavengers, have mild therapeutic effectiveness in experimental spinal cord injury [74, 119]. Another mechanism to promote functional recovery after spinal cord injury is enhancing axonal regeneration. Several strategies, including blocking myelin or glial scar inhibitors, delivery of neurotrophic factors, and cell transplantation, induce axonal outgrowth after experimental spinal cord injury. Among them, olfactory ensheathing cell grafts promote neuroprotection, axonal regeneration, and functional recovery after incomplete spinal cord injury [132, 133]. Furthermore, a regular enforced movement activity may additionally help provide faster functional restoration and recovery after SCI [134].

Studies on combinatorial effects of CsA, minocycline, and FK506 in various combinations with each other or with other compounds may prove to be more effective in recovery from SCI. Earlier combined treatments like FK506 and NGF [76], FK506 and minocycline [22], FK506 and methylprednisolone [77], FK506 and RhoA inhibitor [76, 77], minocycline and bone marrow mesenchymal stem cells [51], and CsA with PEG-TAT [54] have all shown significant functional recovery from SCI as compared to these compounds individually.

#### **6. Conclusions**

From the overall literature review on the multifactorial effects of CsA, minocycline, and FK506 and from the discussion of the present findings, it can be

*Spinal Cord Injury Therapy*

outcome [50], and reducing pain [111].

SCI tissue [40].

with chronic SCI [115].

SCI animals treated with FK506 [7, 22, 60, 67, 69, 70, 74, 76], minocycline

is a key factor that can affect efficacy as a neuroprotectant for CNS injuries.

[22, 28, 43–45], and CsA [18, 52, 53, 58, 61]. In addition to the therapeutic effects of the three drugs, it has been also suggested that the daily routine behavioral assessment procedures may also assist the animals as equivalent to their exercises that help them to recover from SCI [107]. However, more studies are required to confirm this presumption. Additionally, no notable side effects were noted at the dosing regimen of the drugs (selected from the pilot studies) used in the present chapter. However, it has been suggested particularly for FK506 [63] that the therapeutic dosing regimen

FK506 has been reported by others also for being a potential therapy for SCI recovery through various mechanisms [7, 60, 63, 68, 108]. It is evidently proven that FK506 prevents the activation of NF-κB in microglia which reduces production of proinflammatory cytokines like TNF-a, IL-1b, and IL-6 in the SCI responses for effective recovery [7, 109]. Furthermore, it is suggested that inhibition of inflammatory reaction in SCI by FK506 could be due to its inhibitory action on decreasing the free radical formation and lipid peroxidation preventing calcineurin-mediated dephosphorylation of NOS activity in a Ca2+-dependent manner [77]. FK506 also enhances neurite outgrowth and improves functional recovery from SCI by stimulating astrocytes to secrete epidermal growth factor (EGF) for neural repair [74]. CsA has also been reviewed and reported as a potent neuroprotectant for functional recovery from SCI [5, 55]. The significant protective role of CsA has been reported for recovery from SCI through inhibiting the apoptosis of spinal cord cells [53], improving locomotor function [58], increasing mean arterial pressure [110], inhibiting NOS [56], diminishing demyelination and neuronal cell death [60], attenuating reactive astrocytosis due to injury improved neurologic

Minocycline has also been reviewed recently for its effectiveness through multiple mechanisms for functional recovery from SCI [38]. The multiple targets that minocycline works for SCI functional recovery include upregulation of the protein VEGF and BDNF expressions; downregulation of protein p-38MAPK, proNGF, p75NTR, and RhoA expressions and suppressed caspase-3 activity [51]; and improved antioxidant activity through amelioration in oxidative stress in the

Monoamines such as norepinephrine (NE), dopamine (DA), and serotonin (5-HT) can activate the spinal neurons involved in walking [112–114]. Thus, the decrease in the level of 5-HT and 5-HIAA in the SCI animals in the present chapter clearly indicates that SCI inevitably affects the normal functioning of these spinal neurotransmitters involved in locomotor function. SCI-injured animals treated with the drugs herein improved levels of 5-HT and 5-HIAA (**Figure 5A–C**, respectively). Our present behavioral findings also showed an overall correlation and significant improvement in the functional deficits of the hind limbs after treatment with these drugs, indicating the presence of potential mechanisms of serotonergic agents in these drugs, as present in indorenate (5-methoxytryptamine, beta-methyl carboxylate hydrochloride), a 5-HT1A agonist that improved motor function in rats

The oxidant/antioxidant balance was clearly reflected by the increased level of TBARS (**Figure 6A**) and decreased level of GSH (**Figure 6B**) in the contused tissue of SCI control animals. However, treatment of SCI animals with the drugs interfered with the formation of free radicals following traumatic SCI. The comparative behavioral restorative effects of these drugs in the formation of free radicals in

Spinal cord injury in mice results in severe trauma characterized by edema and neutrophil infiltration (measured as an increase in myeloperoxidase activity), and

injured SC were in the order of FK506 > minocycline > CsA.

**80**

concluded that the drugs CsA, minocycline, and FK506 induce good recovery from experimentally induced SCI in rats. However, these drugs significantly improve functional restoration, replenish 5-HT and 5-HIAA levels, and restore the oxidant/ antioxidant balance in the contused tissue after moderate SCI in rats in the order FK506 < minocycline < CsA. Furthermore, it is suggested that the present compressive SCI model of rats could still serve as the most convenient model for therapeutic screenings of various drugs in search of ideal therapy for SCI. CsA, minocycline, and FK506 appear to have gained support in a multifactorial effective manner through ample research work and should be considered as ideal therapeutical agents for the treatment of acute SCI. These drugs should be supported for clinical trials with further studies and tests. Although FK506 appears to be the most promising among the three drugs, more work is needed to screen all three compounds as cocktails in various combinations with better expected outcomes in SCI recovery possibly due to their cumulative multifactorial beneficial effects.

### **Acknowledgements**

The authors are thankful to the Deanship of Scientific Research, College of Nursing Research Centre at King Saud University, for funding this research.

### **Conflict of interest**

The authors have no conflict of interests.

### **Author details**

Mohammad Ahmad\* and Abdualrahman Saeed Alshehri Department of Medical Surgical Nursing, College of Nursing, King Saud University, Riyadh, Saudi Arabia

\*Address all correspondence to: mbadshah@ksu.edu.sa

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**83**

*Effects of Cyclosporin-A, Minocycline, and Tacrolimus (FK506) on Enhanced Behavioral…*

Neuroscience. 2006;**7**:628-643. DOI:

[10] Gorio A, Gokmen N, Erbayraktar S, Yilmaz O, Madaschi L, Cichetti C, et al. Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma. Proceedings of the National Academy of Sciences of the United States of America. 2002;**99**:9450-9455.

[9] Ma L, Mu Y, Zhang Z, Sun Q. Eugenol promotes functional recovery and alleviates inflammation, oxidative stress, and neural apoptosis

in a rat model of spinal cord injury. Restorative Neurology and Neuroscience. 2018;**36**:659-668. DOI:

DOI: 10.1073/pnas.142287899

10.1038/srep09640

[13] Fu ES, Tummala

[11] Lukovic D, Moreno-Manzano V, Lopez-Moncholi E, Rodriguez-Jime'nez FJ, Jendelova P, Sykova E, et al. Complete rat spinal cord transection as a faithful model of spinal cord injury for translational cell transplantation. Scientific Reports. 2015;**5**:9640. DOI:

[12] Filipp ME, Travis BJ, Henry SS, Idzikowski EC, Magnuson SA, Loh MY, et al. Differences in neuroplasticity after spinal cord injury in varying animal models and humans. Neural Regeneration Research. 2019;**14**:7-19. DOI: 10.4103/1673-5374.243694

RP. Neuroprotection in brain and spinal cord trauma. Current Opinion in Anaesthesiology. 2005;**18**:181-187. DOI: 10.1097/01.aco.0000162838.56344.88

[14] Witiw CD, Fehlings MG. Acute Spinal Cord Injury. Journal of Spinal Disorders & Techniques. 2015;**28**:202-210. DOI: 10.1097/ BSD.0000000000000287

10.3233/RNN-180826

10.1038/nrn1955

*DOI: http://dx.doi.org/10.5772/intechopen.85212*

[1] Sekhon LH, Fehlings MG. Epidemiology, demographics, and pathophysiology of acute spinal injury. Spine (Phila Pa 1976). 2001;**26**:S2-S12

10.1016/j.apmr.2014.11.019

1994;**19**:2321-2329

[3] O'Brien MF, Lenke LG, Lou J, Bridwell KH, Joyce ME. Astrocyte response and transforming growth factor-beta localization in acute spinal cord injury. Spine (Phila Pa 1976).

[4] Gorman PH. The review of systems in spinal cord injury and dysfunction. Continuum (Minneapolis, Minn.). 2001;**17**:630-634. DOI: 10.1212/01. CON.0000399092.88866.1c

[5] Ahuja CS, Nori S, Tetreault L, Wilson J, Kwon B, Harrop J, et al. Traumatic spinal cord injury-repair and regeneration. Neurosurgery. 2017;**80**: 9-22. DOI: 10.1093/neuros/nyw080

[6] Profyris C, Cheema SS, Zang D, Azari MF, Boyle K, Petratos S. Degenerative and regenerative mechanisms governing spinal cord injury. Neurobiology of Disease. 2004;**15**:415-436. DOI: 10.1016/j.

[7] Liu G, Fan G, Guo G, Kang W, Wang D, Xu B, et al. FK506 attenuates the inflammation in rat spinal cord injury by inhibiting the activation of NF-κB in microglia cells. Cellular and Molecular Neurobiology. 2017;**37**:843-855. DOI:

10.1007/s10571-016-0422-8

[8] Thuret S, Moon LD, Gage FH. Therapeutic interventions after spinal cord injury. Nature Reviews.

nbd.2003.11.015

[2] Saunders LL, Clarke A, Tate DG, Forchheimer M, Krause JS. Lifetime prevalence of chronic health conditions among persons with spinal cord injury. Archives of Physical Medicine and Rehabilitation. 2015;**96**:673-679. DOI:

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*Effects of Cyclosporin-A, Minocycline, and Tacrolimus (FK506) on Enhanced Behavioral… DOI: http://dx.doi.org/10.5772/intechopen.85212*

#### **References**

*Spinal Cord Injury Therapy*

**Acknowledgements**

**Conflict of interest**

**82**

**Author details**

provided the original work is properly cited.

King Saud University, Riyadh, Saudi Arabia

The authors have no conflict of interests.

Mohammad Ahmad\* and Abdualrahman Saeed Alshehri Department of Medical Surgical Nursing, College of Nursing,

\*Address all correspondence to: mbadshah@ksu.edu.sa

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

concluded that the drugs CsA, minocycline, and FK506 induce good recovery from experimentally induced SCI in rats. However, these drugs significantly improve functional restoration, replenish 5-HT and 5-HIAA levels, and restore the oxidant/ antioxidant balance in the contused tissue after moderate SCI in rats in the order FK506 < minocycline < CsA. Furthermore, it is suggested that the present compressive SCI model of rats could still serve as the most convenient model for therapeutic screenings of various drugs in search of ideal therapy for SCI. CsA, minocycline, and FK506 appear to have gained support in a multifactorial effective manner through ample research work and should be considered as ideal therapeutical agents for the treatment of acute SCI. These drugs should be supported for clinical trials with further studies and tests. Although FK506 appears to be the most promising among the three drugs, more work is needed to screen all three compounds as cocktails in various combinations with better expected outcomes in SCI recovery

The authors are thankful to the Deanship of Scientific Research, College of Nursing Research Centre at King Saud University, for funding this research.

possibly due to their cumulative multifactorial beneficial effects.

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[10] Gorio A, Gokmen N, Erbayraktar S, Yilmaz O, Madaschi L, Cichetti C, et al. Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma. Proceedings of the National Academy of Sciences of the United States of America. 2002;**99**:9450-9455. DOI: 10.1073/pnas.142287899

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**93**

Section 3

Non-Pharmacological

Therapies

### Section 3
