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Kobeissy FH, editor. Brain Neurotrauma: Molecular, Neuropsychological, and Rehabilitation Aspects. Boca Raton (FL): CRC Press/Taylor & Francis; 2015.

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Brain Neurotrauma: Molecular, Neuropsychological, and Rehabilitation Aspects.

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Chapter 6The Contributing Role of Lipid Peroxidation and Protein Oxidation in the Course of CNS Injury Neurodegeneration and Neuroprotection

An Overview

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6.1. INTRODUCTION

At present, there are no FDA-approved pharmacological therapies for acute treatment of traumatic brain injury (TBI) patients that are conclusively proven to mitigate the often devastating neurological effects of their injuries. Nevertheless, the possibility of an effective neuroprotective treatment is based upon the fact that even though some of the neural injury is due to the primary mechanical events (i.e., shearing of nerve cells and blood vessels), the majority of post-traumatic neurodegeneration is due to a pathomolecular and pathophysiological secondary cascade that occurs during the first minutes, hours and days following the injury which exacerbates the damaging effects of the primary injury. One of the most validated “secondary injury” mechanisms revealed in experimental TBI studies involves oxygen radical-induced oxidative damage to brain cell lipids and proteins. This chapter outlines the key sources of reactive oxygen species (ROS) including highly reactive (i.e., rapidly oxidizing) free radicals, the mechanisms associated with their neural damage, pharmacological scavenging antioxidants that have been shown to produce neuroprotective effect in TBI models and brief mention of the most widely used methods for studying the extent of lipid and protein oxidative damage in TBI models.

6.1.1. Reactive Oxygen Species and Reactive Nitrogen Species

The term reactive oxygen species (ROS) includes oxygen-derived radicals such as the modestly reactive superoxide radical (O2·−) and the highly reactive hydroxyl (•OH) radicals as well as nonradicals such as hydrogen peroxide (H2O2) and peroxynitrite (ONOO–), the latter often referred to as a reactive nitrogen species (RNS). The cascade of posttraumatic oxygen radical reactions begins in response to rapid elevations in intracellular Ca2+ immediately after the primary mechanical injury to the brain with the single electron (e–) reduction of an oxygen molecule (O2) to produce O2·–, which is considered to be a modestly reactive primordial radical that can potentially react with other molecules to give rise to much more reactive, and thus more potentially damaging radical species. The reason that O2·– is only modestly reactive is that it can act as either an oxidant stealing an electron from another oxidizable molecule or it can act as a reductant by which it donates its unpaired electron to another radical species, thus acting as a reductant or antioxidant. However, if O2·– reacts with a proton (H+) to form a hydroperoxyl radical (HO2–), this results in a superoxide form that is much more likely to cause oxidation (i.e., act as an electron stealer).

One of the most important endogenous antioxidants is the enzyme superoxide dismutase (SOD) that rapidly catalyzes the dismutation of O2·– into H2O2 and oxygen. At low pH, O2·– can dismutate spontaneously. The formation of highly reactive oxygen radicals, which have unpaired electron(s) in their outer molecular orbitals and the propagation of chain reactions, are fueled by nonradical ROS, which do not have unpaired electron(s), but are chemically reactive. For example, •OH radicals are generated in the iron-catalyzed Fenton reaction in which ferrous iron (Fe2+) is oxidized to form •OH in the presence of H2O2 (Fe2+ + H2O2 → Fe3+ + •OH + OH–). Superoxide acting as a reducing agent can actually donate its unpaired electron to ferric iron (Fe3+), cycling it back to the ferrous state in the Haber-Weiss reaction, thus driving subsequent Fenton reactions and increased production of •OH (O2·– + Fe3+ → Fe2+ + O2). Under physiological conditions, iron is tightly regulated by its transport protein, transferrin, and storage protein, ferritin, both of which bind the ferric (Fe3+) form. This reversible bond of transferrin and ferritin with iron decreases with declining pH (below pH7), as is the case after central nervous system (CNS) injury, resulting in the release of iron and initiation of iron-dependent oxygen radical production. A second source of iron comes from hemoglobin on its release after mechanical-induced hemorrhage.

Although O2·– itself is less reactive than an •OH radical, its reaction with a nitric oxide (•NO) radical forms the highly reactive oxidizing agent, peroxynitrite (PN: ONOO–). This reaction (O2·– + •NO → ONOO–) occurs at a very high rate constant that outcompetes SOD’s ability to convert O2·– into H2O2. Subsequently, at physiological pH, ONOO– will largely undergo protonation to form peroxynitrous acid (ONOOH) or it can react with carbon dioxide (CO2) to form nitrosoperoxocarbonate (ONOOCO2). The ONOOH can break down to form highly reactive nitrogen dioxide (•NO2) and •OH (ONOOH → •NO2 + •OH). Alternatively, the ONOOCO2 can decompose into •NO2 and carbonate radical (•CO3) (ONOOCO2 → •NO2 + •CO3).

6.2. LIPID PEROXIDATION

Increased production of reactive free radicals (i.e., “oxidative stress”) in the injured brain has been shown to cause “oxidative damage” to cellular lipids and proteins, leading to functional compromise and possibly cell death in both the brain microvascular and parenchymal compartments. Extensive research shows that a major form of radical-induced oxidative damage involves oxidative attack on cell membrane polyunsaturated fatty acids triggering the process of lipid peroxidation (LP). The LP is initiated when highly reactive oxygen radicals (e.g., •OH, •NO2, •CO3) react with polyunsaturated fatty acids such as arachidonic acid, linoleic acid, eicosapentaenoic acid, or docosahexaenoic acid, resulting in disruptions in cellular and membrane integrity. The process sets off a radical chain reaction characterized by three distinct steps: initiation, propagation, and termination (Gutteridge, 1995).

Initiation of LP begins with an ROS-induced hydrogen atom (H+); its one associated electron is abstracted from an allylic carbon. The basis for the susceptibility of the allylic carbon of the polyunsaturated fatty acid having one of its electrons stolen by a highly electrophilic free radical is that the carbon is surrounded by two relatively electronegative double bonds that tend to pull one of the electrons from the carbon. Consequently, a reactive free radical has an easy time pulling the hydrogen electron off of the carbon because the commitment of the carbon electron to staying paired with it has been weakened by the surrounding double bonds. This results in the original radical being quenched while the polyunsaturated fatty acid (L) becomes a lipid radical (L•) because of its having lost an electron.

In the subsequent propagation step, the unstable L• reacts with O2 to form a lipid peroxyl radical (LOO•). The LOO• in turn abstracts a hydrogen atom from an adjacent polyunsaturated fatty acid yielding a lipid hydroperoxide (LOOH) and a second L•, which sets off a series of propagation “chain” reactions. These propagation reactions are terminated in the third step when the substrate becomes depleted and a lipid radical reacts with another radical or radical scavenger to yield potentially neurotoxic nonradical end-products. One of those end-products that is often used to measure LP is the 3-carbon-containing malondialdehyde, which is mainly a stable nontoxic compound that, when measured, represents an LP “tombstone.”

In contrast, two highly toxic products of LP are 4-hydroxynonenal (4-HNE), the formation of which is illustrated in Figure 6.1, or 2-propenal (acrolein), as shown in Figure 6.2, both of which have been well characterized in CNS injury experimental models (Bains and Hall, 2012; Hall et al., 2010; Hamann and Shi, 2009). These latter two aldehydic LP end-products covalently bind to proteins and amino acids (lysine, histidine, arginine, cysteine), altering their structure and functional properties, the chemistry of which is illustrated in Figure 6.3. Immunohistochemical and immunoblotting (Western, slot, dot) techniques are commonly used to measure 4-HNE or acrolein-modified proteins in the injured brain. For details on a variety of analytical techniques for measurement of various markers of LP, the reader is referred to the following review (Hall and Bosken, 2009).

FIGURE 6.1. Chemistry involved in the initiation, propagation, and termination reactions of arachidonic acid during lipid peroxidation with the resulting formation of the aldehydic end-product 4-hydroxynonenal (4-HNE).

FIGURE 6.1

Chemistry involved in the initiation, propagation, and termination reactions of arachidonic acid during lipid peroxidation with the resulting formation of the aldehydic end-product 4-hydroxynonenal (4-HNE).

FIGURE 6.2. Chemistry involved in the initiation, propagation, and termination reactions of arachidonic acid during lipid peroxidation with the resulting formation of the aldehydic end-product 2-propenal (acrolein).

FIGURE 6.2

Chemistry involved in the initiation, propagation, and termination reactions of arachidonic acid during lipid peroxidation with the resulting formation of the aldehydic end-product 2-propenal (acrolein).

FIGURE 6.3. Chemical reactions of 4-HNE or acrolein with amino acids that lead to impairment of protein structure and function.

FIGURE 6.3

Chemical reactions of 4-HNE or acrolein with amino acids that lead to impairment of protein structure and function.

6.3. PROTEIN CARBONYLATION AND NITRATION

Free radicals can cause various forms of oxidative protein damage. A major mechanism involves carbonylation by reaction of various free radicals with susceptible amino acids. Figure 6.4 shows the chemistry of •OH-induced histidine oxidation, which results in carbonylation of the 2-position of histidine’s imidazole ring. The protein carbonyls thus formed are measurable through immunoblotting after derivatization of the carbonyl groups with diphenylhydrazine. Although the measurement of protein carbonyls by the so-called diphenylhydrazine assay has long been used, a commercially available protein carbonyl Oxyblot assay is widely used by present-day investigators. It should be noted that the carbonyl assay also picks up protein carbonyls that are present because of covalent binding of LP-derived 4-HNE and acrolein to cysteine residues (see Figure 6.3), in addition to those resulting from direct free radical–induced amino acid oxidation. Thus, as a result, the carbonyl assay is as much an indirect index of LP as it is of primary direct protein oxidation.

FIGURE 6.4. Chemical reaction of hydroxyl radical (•OH) with a histidine residue resulting in histidine carbonylation.

FIGURE 6.4

Chemical reaction of hydroxyl radical (•OH) with a histidine residue resulting in histidine carbonylation.

Second, •NO2 can nitrate the 3-position of tyrosine residues in proteins; 3-NT is a specific footprint of PN-induced cellular damage (see Figure 6.5). Similarly, LOO• can promote tyrosine nitration by producing initial oxidation (loss of an electron), which would enhance the ability of •NO2 to nitrate the phenyl ring as also explained in Figure 6.5. Multiple commercially available polyclonal and monoclonal antibodies are available for immunoblot or immunohistochemical measurement of proteins that have been nitrated by PN.

FIGURE 6.5. Mechanism of peroxynitrite-induced nitration of tyrosine residues.

FIGURE 6.5

Mechanism of peroxynitrite-induced nitration of tyrosine residues. The first step involves free radical attack on the phenyl ring pulling away an electron by peroxynitrite-derived radicals or a lipid peroxyl radical (LOO•) that converts tyrosine (more...)

6.4. INTERACTION OF OXIDATIVE DAMAGE WITH OTHER SECONDARY INJURY MECHANISMS

The impact of ROS/RNS production is heightened when oxygen radicals feed back and amplify other secondary injury pathways, creating a continuous cycle of ion imbalance, Ca2+ buffering impairment, mitochondrial dysfunction, glutamate-induced excitotoxicity, and microvascular disruption. One example of ROS-induced ionic disruption arises from LP-induced damage to the plasma membrane adenosine triphosphate (ATP)-driven Ca2+ pump (Ca++-ATPase) and Na+ pump (Na+/K+-ATPase), which contributes to increases in intracellular Ca2+ concentrations, mitochondrial dysfunction and additional ROS production. Both Ca2+ pump and Na+/K+-ATPase disruptions result in further increases in intracellular Ca2+ and Na+ accumulation, respectively (Bains and Hall, 2012), the latter causing reversal of the Na+/Ca++ exchanger (Rohn et al., 1993, 1996).

PN formed from mitochondrial Ca2+ overload contribute to mitochondrial dysfunction. Specifically, nitric oxide (•NO), formed from mitochondrial NOS, in turn reacts with O2·– to produce the highly toxic PN, which impairs respiratory and Ca2+ buffering capacity via its derived free radicals (Bringold et al., 2000). Indeed, increased PN-derived 3NT and 4HNE has been detected during the time of mitochondrial dysfunction and correlates with respiratory and Ca2+ buffering impairment (Sullivan et al., 2007). Increased synaptosomal 4-HNE content is associated with impaired synaptosomal glutamate and amino acid uptake (Springer et al., 1997; Zhang et al., 1996). Glutamate and N-methyl-D-aspartate induced damage in neuronal cultures is attenuated with LP inhibition, confirming LP and oxidative damage as mediators of glutamate excitotoxicity (Monyer et al., 1990; Pellegrini-Giampietro et al., 1990).

6.5. MECHANISMS FOR PHARMACOLOGICAL INHIBITION OF OXIDATIVE DAMAGE IN TBI

Based on the previous discussion concerning oxidative stress (increased ROS/RNS) and oxidative damage (LP, protein oxidation and nitration), several potential mechanisms for its inhibition are apparent, which fall into three categories. The first category includes compounds that inhibit the initiation of LP and other forms of oxidative damage by preventing the formation of ROS or RNS species. For instance, NOS inhibitors, discussed previously, exert an indirect antioxidant effect by limiting •NO production and thus PN formation. However, they also have the potential to interfere with the physiological roles that •NO is responsible for. These include antioxidant effects that are due to its important role as a scavenger of lipid peroxyl radicals (e.g., LOO• + •NO →LOONO) (Hummel et al., 2006). Another approach to blocking posttraumatic radical formation is the inhibition of the enzymatic (e.g., cyclooxygenase, 5-lipoxygenase) arachidonic acid cascade during which the formation of O2·– is produced as a by-product of prostanoid and leukotriene synthesis. Kontos and colleagues (Kontos, 1989; Kontos and Wei, 1986) and Hall (Hall, 1986) have shown that cyclooxygenase inhibiting nonsteroidal anti-inflammatory agents (e.g., indomethacin, ibuprofen) are vaso- and neuro-protective in TBI models.

A second indirect LP inhibitory approach involves chemically scavenging the radical species (e.g., O2·–, •OH, •NO2, •CO3) before it has a chance to steal an electron from a polyunsaturated fatty acid and thus initiate LP. The use of pharmacologically administered SOD represents an example of this strategy. Another example concerns the use of the nitroxide antioxidant tempol, which has been shown to catalytically scavenge the PN-derived free radicals •NO2 and •CO3 (Carroll et al., 2000). In either case, a general limitation to these first two approaches is that they would be expected to have a short therapeutic window and would have to be administered rapidly to have a chance to interfere with the initial posttraumatic “burst” of free radical production that has been documented in TBI models (Hall et al., 1993; Kontos and Wei, 1986). Although it is believed that ROS, including PN production, persists several hours after injury, the major portion is an early event that peaks in the first 60 minutes after injury, making it clinically impractical to pharmacologically inhibit, unless the antioxidant compound is already “on board” when the injury occurs or is available for administration immediately after TBI.

In contrast to these indirect-acting antioxidant mechanisms, the third category involves stopping the “chain reaction” propagation of LP once it has begun. The most demonstrated way to accomplish this is by scavenging of LOO• or lipid alkoxyl (LO•) radicals. The prototype scavenger of these lipid radicals is alpha tocopherol or vitamin E (Vit E) which can donate an electron from its phenolic hydroxyl moiety to quench a LOO•. However, the scavenging process is stoichiometric (1 Vit E can only quench 1 LOO•) and in the process Vit E loses its antioxidant efficacy and becomes Vit E radical (LOO• + Vit E → LOOH + Vit E•). Although Vit E• is relatively unreactive (i.e., harmless), it also cannot scavenge another LOO• until it is reduced back to its active form by receiving an electron from other endogenous antioxidant-reducing agents such as ascorbic acid (vitamin C) or glutathione (GSH). Although this tripartite LOO• antioxidant defense system (Vit E, vitamin C, GSH) works fairly effectively in the absence of a major oxidative stress, numerous studies have shown that each of these antioxidants are rapidly consumed during the early minutes and hours after CNS injury (Hall et al., 1989, 1992). Thus, it has long been recognized that more effective pharmacological LOO• and LO• scavengers are needed. Furthermore, it is expected that compounds that could interrupt the LP process after it has begun would be able to exert a more practical neuroprotective effect (i.e., possess a longer antioxidant therapeutic window).

A second approach to inhibiting the propagation of LP reactions is to chelate free iron, either ferrous (Fe++) or ferric (Fe+++), which potently catalyzes the breakdown of LOOHs, an essential event in the continuation of LP chain reactions in cellular membranes. The prototypical iron-chelating drug that chelates Fe+++ is the bacterially (streptomyces pilosus)-derived tri-hydroxamic acid compound deferoxamine.

6.6. NEUROPROTECTIVE EFFECTS OF PHARMACOLOGICAL ANTIOXIDANTS

6.6.1. TBI Clinical Trial Results with PEG-SOD and Tirilazad

During the past 25 years, there has been an intense effort to discover and develop pharmacological agents for acute treatment of TBI. This has included two compounds that possess free radical scavenging/antioxidant properties polyethylene glycol–conjugated SOD (PEG-SOD) (Muizelaar et al., 1995) and the LP inhibitor tirilazad (Langham et al., 2000; Marshall et al., 1998). However, each of these trials was a therapeutic failure in that no overall benefit was documented in severe and/or moderate TBI patient populations, which was the primary goal in each of these trials. These TBI clinical trial failures can be attributed to several factors. Perhaps most importantly, the preclinical assessment of compounds destined for acute TBI trials has often been woefully inadequate in regard to the definition of neuroprotective dose-response relationships, pharmacokinetic–pharmacodynamic correlations, therapeutic window, and optimum dosing regimen and treatment duration. However, a number of other issues related to design of the clinical trials are also believed to be involved (Narayan et al., 2002). The following sections briefly review the TBI histories of PEG-SOD and tirilazad.

6.6.2. PEG-SOD

As mentioned earlier, the earliest studies of free radical scavenging compounds in TBI models were carried out with Cu/Zn SOD based on the work of Kontos and colleagues who showed that posttraumatic microvascular dysfunction was initiated by O2·– generated as a by-product of the arachidonic acid cascade, which is massively activated during the first minutes and hours after TBI (Kontos, 1989; Kontos and Povlishock, 1986; Kontos and Wei, 1986). Their work showed that administration of SOD prevented the posttraumatic microvascular dysfunction. This led to clinical trials in which the more metabolically stable PEG-conjugated SOD was examined in moderate and severe TBI patients when administered within the first 8 hours after injury. Although an initial small phase II study showed a positive trend, subsequent multicenter phase III studies failed to show a significant benefit in terms of increased survival or improved neurological outcomes (Muizelaar et al., 1995).

Although many explanations for these negative results may be postulated, one reason may be that a large protein like SOD is unlikely to have much brain penetrability and therefore its radical scavenging effects may be limited to the microvasculature. A second reason may be that attempting to scavenge the short-lived inorganic radical O2·– may be associated with a very short therapeutic window, as suggested previously. As pointed out earlier, the time course of measurable posttraumatic •OH formation in the injured rodent brain has been shown to largely run its course by the end of the first hour after TBI (Hall et al., 1993; Smith et al., 1994).

A more rational strategy would be to inhibit the LP that is triggered by the initial burst of inorganic radicals. A comparison of the time course of LP with that of posttraumatic •OH shows that LP reactions continue to build beyond the first posttraumatic hour (Smith et al., 1994) and may continue for 3 or 4 days (Du et al., 2004; Hall et al., 2012; Miller et al., 2014). Despite the failure of PEG-SOD in human TBI, experimental studies have shown that transgenic mice that overexpress Cu/Zn SOD are significantly protected against post-TBI pathophysiology and neurodegeneration (Chan et al., 1995; Gladstone et al., 2002; Lewen et al., 2000; Mikawa et al., 1996; Xiong et al., 2005). This fully supports the importance of posttraumatic O2·– in posttraumatic secondary injury, despite the fact that targeting this primordial radical is probably not the best antioxidant strategy for acute TBI compared with trying to stop the downstream LP process that is initiated by the early increases in •OH, •NO2, and •CO3.

6.6.3. Tirilazad

Consistent with that rationale, the 21-aminosteroid LP inhibitor tirilazad (i.e., U74006F) was discovered, which inhibits free radical–induced LP by a combination of LOO• scavenging and a membrane-stabilizing action that limits the propagation of LP reactions between an LOO• and an adjacent polyunsaturated fatty acid. The protective efficacy of tirilazad has been demonstrated in multiple animal models of acute TBI in mice (Hall et al., 1988), rats (McIntosh et al., 1992) and cats (Dimlich et al., 1990). Although the compound is largely localized in the microvascular endothelium, the posttraumatic disruption of the BBB is known to allow the successful penetration of tirilazad into the brain parenchyma, as noted earlier (Hall et al., 1992). Other mechanistic data derived from the rat-controlled cortical impact and the mouse diffuse concussive head injury models have definitively shown that a major effect of tirilazad is to lessen posttraumatic microvascular damage, including BBB opening (Hall et al., 1992; Smith et al., 1994).

Tirilazad was taken into clinical development in the early 1990s and following a small phase II dose-escalation study that demonstrated the drug’s safety in TBI patients it was evaluated in two phase III multicenter clinical trials for its ability to improve neurological recovery in moderately and severely injured closed TBI patients. One trial was conducted in North America and the other in Europe. In both trials, TBI patients were treated within 4 hours after injury with either vehicle or tirilazad (2.5 mg/kg intravenously every 6 hours for 5 days). The North American trial was never published because of a major confounding imbalance in the randomization of the patients to placebo or tirilazad in regards to injury severity and pretreatment neurological status. In contrast, the European trial had much better randomization balance and was published (Marshall et al., 1998). The results failed to show a significant beneficial effect of tirilazad in either moderate (Glasgow Coma Scale = 9–12) or severe (Glasgow Coma Scale = 4–8) patient categories. However, a post hoc analysis showed that moderately injured male TBI patients with traumatic subarachnoid hemorrhage (SAH) had significantly less mortality after treatment with tirilazad (6%) compared with placebo (24%, p < 0.026). In severely injured males with traumatic SAH tirilazad also lessened mortality from 43% in placebo treated to 34% (p < 0.071) Marshall et al., 1998). This result is consistent with the fact that this compound is also highly effective in animal models of SAH (Hall et al., 1994). Nevertheless, additional trials would have been required to establish the neuroprotective utility of tirilazad in certain human TBI subgroups and to gain Food and Drug Administration approval in the United States. However, the sponsoring company, Pharmacia & Upjohn, opted not to continue the compound’s development for TBI although tirilazad was successfully approved and marketed for use in aneurysmal SAH in several western European and Australasian countries based on its demonstrated efficacy in phase III SAH trials (Kassell et al., 1996; Lanzino and Kassell, 1999).

6.6.4. Effects of Other Direct and Indirect-Acting Lipid Peroxidation Inhibitors

In addition to tirilazad, several other LP inhibitors have been reported to be effective neuroprotectants in TBI models. These include the LOO• scavenging 2-methylaminochromans U-78517F and U-83836E (Hall et al., 1991), the pyrrolopyrimidine U-101033E (Hall, 1995a, 1995b), OPC-14117 (Mori et al., 1998), the naturally occurring LOO• scavengers curcumin (Sharma et al., 2009; Wu et al., 2006) and resveratrol (Ates et al., 2007; Sonmez et al., 2007), the indoleamine melatonin (Beni et al., 2004; Cirak et al., 1999; Mesenge et al., 1998; Ozdemir et al., 2005a, 2005b), and last, the endogenous antioxidant lipoic acid (Toklu et al., 2009). In the case of curcumin and resveratrol, these are potent LOO• scavengers because they possess multiple phenolic hydroxyl groups that can donate electrons to LOO• radicals. Melatonin also has LOO• scavenging capability (Longoni et al., 1998), but in addition appears to react with PN (Zhang et al., 1998, 1999). Lipoic acid may also have LOO• scavenging effects, but these are more likely to be indirect via the regeneration (i.e., re-reduction) of other endogenous electron-donating antioxidants including Vit E, GSH, and vitamin C.

Among these LP inhibitors, arguably the most potent and effective LOO• scavenging LP inhibitor yet discovered is the 2-methylaminochroman compound U-83836E that combines the LOO• scavenging antioxidant chroman ring structure of Vit E with the bis-pyrrolopyrimidine moiety of tirilazad. The phenolic chroman antioxidant moiety can be rereduced by endogenous ascorbic acid (vitamin C) or GSH after it has donated its phenolic electron to an initial LOO•, making it able to quench a second and then a third LOO•, etc. The bis-pyrrolopyrimidine moiety, on the other hand, can also scavenge multiple moles of LOO• by a true catalytic mechanism (Hall, 1995b; Hall et al., 1991). Thus, U-83836E is a dual functionality LOO• scavenger that is understandably more effective than either Vit E, tirilazad (Hall et al., 1991), and possibly the other naturally occurring LOO• scavengers such as curcumin, resveratrol, melatonin, and lipoic acid. Furthermore, U-83836E possesses a high degree of lipophilicity endowing it with a high affinity for membrane phospholipids where LP takes place. Recent studies from the author’s laboratory in the mouse controlled cortical impact TBI model have shown that U-83836E is able to reduce posttraumatic LP and protein nitration and preserve mitochondrial respiratory function in injured cortical tissue and mitochondria (Mustafa et al., 2009).

6.6.5. Effects of Nitroxide Antioxidants and Peroxynitrite Scavengers

In addition to the LOO• radical scavengers, the neuroprotective effects of a family of nitroxide-containing antioxidants have also been examined in experimental TBI models. These are sometimes referred to as “spin-trapping agents” and include α-phenyl-tert-butyl nitrone and its thiol analog NXY-059 and tempol. Both α-phenyl-tert-butyl nitrone and tempol have been shown to be protective in rodent TBI paradigms (Awasthi et al., 1997; Marklund et al., 2001). As mentioned earlier, tempol has been shown by the author and colleagues to catalytically scavenge PN-derived •NO2 and •CO3 (Bonini et al., 2002; Carroll et al., 2000) and to reduce posttraumatic oxidative damage (both LP and protein nitration), preserve mitochondrial function, decrease calcium-activated, calpain-mediated cytoskeletal damage, and reduce neurodegeneration in mice subjected to a severe controlled cortical impact-induced focal TBI (Deng-Bryant et al., 2008). Another laboratory has reported that tempol can reduce posttraumatic brain edema and improve neurological recovery in rat contusion injury model (Beit-Yannai et al., 1996; Zhang et al., 1998). However, the neuroprotective effect of tempol, administered alone, is associated with a therapeutic window of an hour or less in the mouse controlled cortical impact TBI model. Moreover, tempol is not effective at directly inhibiting LP in the latter model (Deng-Bryant et al., 2008).

6.6.6. Effects of the Iron Chelator Deferoxamine

The prototype iron chelator deferoxamine, which binds ferric (Fe+++) iron and thereby would lessen the catalytic effects of iron on LP, has also been reported to have beneficial actions in preclinical TBI or TBI-related models (Gu et al., 2009; Long et al., 1996). However, deferoxamine is hindered by its lack of brain penetration and rapid plasma elimination rate. To partially counter the latter limitation, a dextran-coupled deferoxamine has been synthesized that has been reported to significantly improve early neurological recovery in a mouse diffuse TBI model (Panter et al., 1992). Much of this activity, however, is probably due to microvascular antioxidant protection because of limited brain penetrability. Another caveat to the iron-chelation antioxidant neuroprotective approach that is at least relevant to the ferric iron chelators such as deferoxamine is that they can cause a prooxidant effect in that their binding of Fe+++ can actually drive the oxidation of ferrous to ferric iron, which can increase superoxide radical formation in the process (Fe++ + O2 → Fe+++ + O2·–).

6.6.7. Carbonyl Scavenging as an Approach to Inhibit 4-HNE and Acrolein Binding to Proteins

As pointed out previously (Figure 6.3), the LP-derived aldehydic (carbonyl-containing) breakdown products 4-HNE and acrolein have high affinity for binding to selected protein amino acid residues including histidine, lysine, arginine, and cysteine. These modifications have been shown to inhibit the activities of a variety of enzymatic proteins (Halliwell and Gutteridge, 2008). Several compounds have been recently identified that are able to antagonize this “carbonyl stress” by covalently binding to reactive LP-derived aldehydes. For instance, D-penicillamine has been demonstrated to form an irreversible bond to primary aldehydes. We have previously demonstrated that penicillamine is able to scavenge PN (Althaus et al., 1994) and to protect brain mitochondria from PN-induced respiratory dysfunction in isolated rat brain mitochondria (Singh et al., 2007). This latter action was observed along with an attenuation of 4-HNE–modified mitochondrial proteins (Singh et al., 2007). The PN scavenging action of penicillamine along with its carbonyl scavenging capability may jointly explain our previous findings that acutely administered penicillamine can improve early neurological recovery of mice subjected to moderately severe concussive TBI (Hall et al., 1999).

More recently, it has been demonstrated that a variety of hydrazine-containing compounds such as the antihypertensive agent hydralazine, the antidepressant phenelzine (Figure 6.6), and the antitubercular agent iproniazid can react with the carbonyl moieties of 4-HNE or acrolein that prevents the latter from binding to susceptible amino acids in proteins (Galvani et al., 2008). Consistent with this effect being neuroprotective, others have shown that hydralazine inhibits either compression or acrolein-mediated injuries to ex vivo spinal cord (Hamann et al., 2008). Hydralazine, which is a potent vasodilator, would be difficult to administer in vivo after either spinal cord injury or TBI in which hypotension is already a common pathophysiological problem. However, other hydrazine-containing compounds such as phenelzine and iproniazid do not compromise blood pressure as readily as hydralazine and have a long history of clinical use, although they have never been examined in acute neurotrauma models. Most impressive is that the application of hydrazines can rescue cultured cells from 4-HNE toxicity even when administered after 4-HNE has already covalently bound to cellular proteins (Galvani et al., 2008). Such an effect could be associated with a favorable neuroprotective therapeutic window.

FIGURE 6.6. Chemical scavenging mechanism involved in the reactivity of the hydrazine-containing compound phenelzine with a mole of either 4-HNE or acrolein.

FIGURE 6.6

Chemical scavenging mechanism involved in the reactivity of the hydrazine-containing compound phenelzine with a mole of either 4-HNE or acrolein.

Recently published in vitro studies in our laboratory have documented the ability of the hydrazine-containing phenelzine to protect isolated rat brain mitochondria from the respiratory depressant effects of 4-HNE together with a concentration-related attenuation of the accumulation of 4-HNE–modified mitochondrial proteins. Subsequent in vivo studies in the rat controlled cortical impact TBI model have found that a single 10 mg/kg subcutaneous dose of phenelzine can also reduce early (3 hours) posttraumatic mitochondrial respiratory failure as well as reduce cortical lesion volume at 14 days postinjury (Singh et al., 2013).

6.6.8. Small Molecule Nrf2/ARE Signaling Activators

The body’s endogenous antioxidant defense system is largely regulated by nuclear factor E2-related factor 2/antioxidant response element (Nrf2/ARE) signaling at the transcriptional level (Kensler et al., 2007; Zhang, 2006). Nrf2 activation and the upregulation of antioxidant and antiinflammatory genes represents a valid neurotherapeutic intervention in CNS injury and has been previously described in various experimental models of stroke and neurodegenerative diseases (Shih et al., 2003). More recently, the role of Nrf2/ARE activation has been explored as a neuroprotective strategy for TBI.

The messenger RNA levels of Nrf2-regulated antioxidant enzymes, heme oxygenase (HO-1) and NAD(P)H:quinonereductase-1 (NQO1) are upregulated 24 hours after TBI (Yan et al., 2008). In addition, Nrf2-knockout mice are susceptible to increased oxidative stress and neurologic deficits after TBI compared with their wild-type counterparts (Hong et al., 2010). Administration of the Nrf2 activator sulforaphane is neuroprotective in an animal model of TBI, reducing cerebral edema and oxidative stress, and improving BBB function and cognitive deficits (Dash et al., 2009). Studies by Chen et al. (2011) demonstrated increased expression of Nrf2 and HO-1 in the cortex of the rat subarachnoid hemorrhage model. Treatment with sulforaphane further increased the expression of Nrf2, HO-1, NQ01, and glutathione S-transferase-α1, resulting in the reduction of brain edema, cortical neuronal death, and motor deficits (Chen et al., 2011). Tert-butyl hydroquinone, another activator of Nrf2, protects against TBI-induced inflammation and damage via reduction in nuclear factor-KB activation and tumor necrosis factor-α and interleukin-1β production after injury in the mouse closed-head injury model (Jin et al., 2011). Collectively, these studies demonstrate a significant neuroprotective role of Nrf2 signaling through the activation of antioxidant enzymes and reduction oxidative secondary injury responses after brain injury. Thus, Nrf2 activation may be a prime candidate for the attenuation of oxidative stress and subsequent neurotoxicity in TBI via the development of small-molecule activators of the Nrf2/ARE pathway.

Recent work in our laboratory has revealed that after controlled cortical impact TBI in mice, there is indeed a progressive activation of the Nrf2-ARE system in the traumatically injured brain as evidenced by an increase in HO-1 messenger RNA and protein that peaks at 72 hours after TBI. However, this effect does not precede, but rather is coincident with, the postinjury increase in LP-related 4-HNE (Miller et al., 2014). Therefore, it is apparent that this endogenous neuroprotective antioxidant response needs to be pharmacologically enhanced and/or sped up if it is to be capable of exerting acute post-TBI neuroprotection. Our laboratory is currently studying another Nrf2-ARE activator natural product, carnosic acid, which has been shown by others to more effectively induce this antioxidant defense system than the prototype sulforaphane (Satoh et al., 2008). We have shown that administration of carnosic acid to non-TBI mice is able to significantly increase the resistance of cortical mitochondria harvested 48 hours later to the respiratory depressant effects of the in vitro applied 4-HNE together with a decrease in 4-HNE modification of mitochondrial proteins (Miller et al., 2013a). Subsequently, we have administered a single dose of carnosic acid to mice at 15 minutes after controlled cortical impact TBI and observed that it is able to significantly reduce the level of 4-HNE in cortical tissue surrounding the injury site (Miller et al., 2013b). Ongoing studies are evaluating the behavioral recovery and tissue protective effects of carnosic acid.

6.7. RATIONALE FOR COMBINATION ANTIOXIDANT TREATMENT OF TBI

Antioxidant neuroprotective therapeutic discovery directed at acute TBI has consistently been focused on attempting to inhibit the secondary injury cascade by pharmacological targeting of a single oxidative damage mechanism. As presented previously, these efforts have included either enzymatic scavenging of superoxide radicals with SOD (Muizelaar et al., 1995) or inhibition of LP with tirilazad (Marshall et al., 1998). Although each of these strategies has shown protective efficacy in animal models of TBI, phase III clinical trials with either compound failed to demonstrate a statistically significant positive effect, although post hoc subgroup analysis suggests that the microvascularly localized tirilazad may have efficacy in moderate and severe TBI patients with traumatic SAH (Marshall et al., 1998). Although many reasons have been identified as possible contributors to the failure, one logical explanation has to with the possible need to interfere at multiple points in the oxidative damage portion of the secondary injury cascade either simultaneously or in a phased manner to achieve a clinically demonstrable level of neuroprotection.

Figure 6.7 summarizes the overall rationale for a multimechanistic antioxidant therapy for TBI. It is anticipated that the combination of two or three antioxidant mechanistic strategies may improve the extent of neuroprotective efficacy, lessen the variability of the effect, and possibly provide a longer therapeutic window of opportunity compared with the window for the individual strategies. If these theoretical combinatorial benefits are confirmed in preclinical TBI models, this should greatly enhance the chance of neuroprotective success in future clinical trials in contrast to previous failures with single antioxidant agents.

FIGURE 6.7. Rationale for the combination of two or more antioxidant strategies for the more effective protection of the injured brain.

FIGURE 6.7

Rationale for the combination of two or more antioxidant strategies for the more effective protection of the injured brain. Included in the figure, but not discussed in the text, is the immunosuppressant cyclosporin A and its nonimmunosuppressant analog (more...)

ACKNOWLEDGMENTS

Portions of the work reviewed in this article were supported by funding from 5R01 NS046566, 5P30 NS051220, and 5P01 NS58484 and from the Kentucky Spinal Cord and Head Injury Research Trust.

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