Newer pharmacological approaches for antioxidant neuroprotection in traumatic brain injury.

Reactive oxygen species-induced oxidative damage remains an extensively validated secondary injury mechanism in traumatic brain injury (TBI) as demonstrated by the efficacy of various pharmacological antioxidants agents in decreasing post-traumatic free radical-induced lipid peroxidation (LP) and protein oxidative damage in preclinical TBI models. Based upon strong preclinical efficacy results, two antioxidant agents, the superoxide radical scavenger polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) and the 21-aminosteroid LP inhibitor tirilazad, which inhibits lipid peroxidation, (LP) were evaluated in large phase III trials in moderately- and severely-injured TBI patients. Both failed to improve 6 month survival and neurological recovery. However, in the case of tirilazad, a post hoc analysis revealed that the drug significantly improved survival of male TBI patients who exhibited traumatic subarachnoid hemorrhage (tSAH) that occurs in half of severe TBIs. In addition to reviewing the clinical trial results with PEG-SOD and tirilazad, newer antioxidant approaches which appear to improve neuroprotective efficacy and provide a longer therapeutic window in rodent TBI models will be presented. The first approach involves pharmacological enhancement of the multi-mechanistic Nrf2-antioxidant response element (ARE) pathway. The second involves scavenging of the neurotoxic LP-derived carbonyl compounds 4-hydroxynonenal (4-HNE) and acrolein which are highly damaging to neural protein and stimulate additional free radical generation. A third approach combines mechanistically complimentary antioxidants to interrupt post-TBI oxidative neurodegeneration at multiple points in the secondary injury cascade. These newer strategies appear to decrease variability in the neuroprotective effect which should improve the feasibility of achieving successful translation of antioxidant therapy to TBI patients.

Nrf2-ARE activator carnosic acid decreases mitochondrial dysfunction, oxidative damage and neuronal cytoskeletal degradation following traumatic brain injury in mice.

The importance of free radical-induced oxidative damage after traumatic brain injury (TBI) has been well documented. Despite multiple clinical trials with radical-scavenging antioxidants that are neuroprotective in TBI models, none is approved for acute TBI patients. As an alternative antioxidant target, Nrf2 is a transcription factor that activates expression of antioxidant and cytoprotective genes by binding to antioxidant response elements (AREs) within DNA. Previous research has shown that neuronal mitochondria are susceptible to oxidative damage post-TBI, and thus the current study investigates whether Nrf2-ARE activation protects mitochondrial function when activated post-TBI. It was hypothesized that administration of carnosic acid (CA) would reduce oxidative damage biomarkers in the brain tissue and also preserve cortical mitochondrial respiratory function post-TBI. A mouse controlled cortical impact (CCI) model was employed with a 1.0mm cortical deformation injury. Administration of CA at 15 min post-TBI reduced cortical lipid peroxidation, protein nitration, and cytoskeletal breakdown markers in a dose-dependent manner at 48 h post-injury. Moreover, CA preserved mitochondrial respiratory function compared to vehicle animals. This was accompanied by decreased oxidative damage to mitochondrial proteins, suggesting the mechanistic connection of the two effects. Lastly, delaying the initial administration of CA up to 8h post-TBI was still capable of reducing cytoskeletal breakdown, thereby demonstrating a clinically relevant therapeutic window for this approach. This study demonstrates that pharmacological Nrf2-ARE induction is capable of neuroprotective efficacy when administered after TBI.

Temporal and spatial dynamics of nrf2-antioxidant response elements mediated gene targets in cortex and hippocampus after controlled cortical impact traumatic brain injury in mice.

The pathophysiological importance of oxidative damage after traumatic brain injury (TBI) has been extensively demonstrated. The transcription factor nuclear factor erythoid related factor 2 (Nrf2) mediates antioxidant and cytoprotective genes by binding to antioxidant response elements (ARE) present in nuclear DNA. In this study, we characterized the time course of Nrf2-ARE-mediated expression in the cortex and hippocampus using a unilateral controlled cortical impact model of focal TBI. Ipsilateral hippocampal and cortical tissue was collected for Western-blot protein analysis (n=6/group) or quantitative reverse transcription-polymerase chain reaction for mRNA (n=3/group) at 3, 6, 12, 24, 48, and 72 h or 1 week post-injury. Multiple genes mediated by Nrf2-ARE were altered post-TBI. Specifically, Nrf2 mRNA increased significantly post-TBI at 48 and 72 h in the cortex and at 48 and 72 h and 1 week in the hippocampus with a coincident increase in glial fibrillary acidic protein mRNA, thereby implying this response is likely occurring in astrocytes. Presumably linked to Nrf2 activation, heme-oxygenase-1, nicotinamide adenine dinucleotide phosphate-quinone-oxidoreductase 1, glutathione reductase, and catalase mRNA overlap throughout the post-injury time course. This study demonstrates the first evidence of such changes during the first week after focal TBI and that increases in expression of some Nrf2-ARE-mediated cytoprotective genes are not observed until 24-48 h post-injury. Unfortunately, this does not precede, but rather coincides with, the occurrence of lipid peroxidative damage. This is the first known comparison between the time course of peroxidative damage and that of Nrf2-ARE activation during the first week post-TBI. These results underscore the necessity to discover pharmacological agents to accelerate and amplify Nrf2-ARE-mediated expression early post-TBI.

Administration of the Nrf2-ARE activators sulforaphane and carnosic acid attenuates 4-hydroxy-2-nonenal-induced mitochondrial dysfunction ex vivo.

The transcription factor NF-E2-related factor 2 (Nrf2) mediates transcription of antioxidant/cytoprotective genes by binding to the antioxidant-response element (ARE) within DNA. Upregulation of these genes constitutes a pleiotropic cytoprotective defense pathway, which has been shown to produce neuroprotection in numerous models by decreasing lipid peroxidation (LP) as measured by the neurotoxic LP by-product 4-hydroxynonenal (4-HNE). As neuronal mitochondria have previously been shown to be susceptible to insult-induced LP-mediated oxidative damage, we sought to mechanistically investigate whether Nrf2-ARE activation in vivo could protect mitochondria from subsequent 4-HNE exposure ex vivo. Young adult male CF-1 mice were administered one of two known Nrf2-ARE activators as single intraperitoneal doses-sulforaphane (SFP; 5.0mg/kg) or carnosic acid (CA; 1.0mg/kg)-or their respective vehicles 48 h before Ficoll isolation of rat cerebral cortical mitochondria. Purified mitochondria were then exposed ex vivo to 4-HNE for 15 min at 37 °C, which we showed to cause a concentration-related inhibition of mitochondrial respiration together with covalent binding of 4-HNE to mitochondrial proteins. We chose a 30 µM concentration of 4-HNE, which produced an approximately 50% inhibition of complex I- or complex II-driven respiration, to assess whether prior in vivo Nrf2-ARE-activating compounds would increase the resistance of the isolated cortical mitochondria to 4-HNE's mitotoxic effects. Administration of either compound significantly increased (p < 0.05) expression of heme oxygenase-1 mRNA in cortical tissue 48 h postadministration, verifying that both compounds were capable of inducing the Nrf2-ARE pathway. Moreover, the prior in vivo administration of SFP and CA significantly (p < 0.05) attenuated 4-HNE-induced inhibition of mitochondrial respiration for complex I, but only carnosic acid acted to protect complex II. Furthermore, both CA and SFP significantly (p < 0.05) reduced the amount of 4-HNE bound to mitochondrial proteins as determined by Western blot. These results demonstrate the capability of in vivo Nrf2-ARE induction to protect from 4-HNE toxicity to cortical mitochondria ex vivo. Ongoing studies will determine the therapeutic efficacy of Nrf2-ARE activators to attenuate traumatic brain injury-induced pathophysiology.

Phenelzine mitochondrial functional preservation and neuroprotection after traumatic brain injury related to scavenging of the lipid peroxidation-derived aldehyde 4-hydroxy-2-nonenal.

Phenelzine (PZ) is a scavenger of the lipid peroxidation (LP)-derived reactive aldehyde 4-hydroxynonenal (4-HNE) due to its hydrazine functional group, which can covalently react with 4-HNE. In this study, we first examined the ability of PZ to prevent the respiratory depressant effects of 4-HNE on normal isolated brain cortical mitochondria. Second, in rats subjected to controlled cortical impact traumatic brain injury (CCI-TBI), we evaluated PZ (10 mg/kg subcutaneously at 15 minutes after CCI-TBI) to attenuate 3-hour post-TBI mitochondrial respiratory dysfunction, and in separate animals, to improve cortical tissue sparing at 14 days. While 4-HNE exposure inhibited mitochondrial complex I and II respiration in a concentration-dependent manner, pretreatment with equimolar concentrations of PZ antagonized these effects. Western blot analysis demonstrated a PZ decrease in 4-HNE in mitochondrial proteins. Mitochondria isolated from peri-contusional brain tissue of CCI-TBI rats treated with vehicle at 15 minutes after injury showed a 37% decrease in the respiratory control ratio (RCR) relative to noninjured mitochondria. In PZ-treated rats, RCR suppression was prevented (P<0.05 versus vehicle). In another cohort, PZ administration increased spared cortical tissue from 86% to 97% (P<0.03). These results suggest that PZ's neuroprotective effect is due to mitochondrial protection by scavenging of LP-derived 4-HNE.

Relationship of nitric oxide synthase induction to peroxynitrite-mediated oxidative damage during the first week after experimental traumatic brain injury.

We have previously shown the pathophysiological importance of the reactive nitrogen species peroxynitrite (PN) formed from the reaction of nitric oxide (•NO) and superoxide (O(2)(•-)) radicals and its involvement in lipid peroxidation (LP) and protein nitration damage in brain tissue following traumatic brain injury (TBI). Nitric oxide is produced by at least three isoforms of the enzyme nitric oxide synthase (NOS) including: endothelial NOS (eNOS) in the CNS vasculature, neuronal NOS (nNOS), and inducible NOS (iNOS) in macrophages/microglia. In view of the requirement of •NO synthesis for PN formation, we sought to address the time course of NOS expression (mRNA by real time quantitative PCR and protein by western blot) after TBI in comparison with the time course of PN-mediated protein nitration (3-nitrotyrosine, 3-NT) in ipsilateral cortex (CTX) and hippocampus (HIPP) between 3 hours and 1 week post-injury using a controlled cortical impact (CCI) mouse model of TBI in young adult CF-1 mice. Protein nitration showed a progressive posttraumatic increase that became significant in CTX at 24 hours and then peaked at 72 hours in both CTX and HIPP. During the increase in PN-derived 3-NT, there was no increase in either CTX or HIPP eNOS mRNA levels, whereas eNOS protein levels were significantly (p<0.05) increased at 48 and 72 hours in both brain regions. There was a significant decrease in HIPP, but not CTX nNOS mRNA; however, nNOS protein did not change except for a significant increase in CTX at 1 week. There was significantly increased CTX and HIPP iNOS mRNA levels at 24, 48, and 72 hours (p<.05) post-injury. In contrast, no change was seen in CTX or HIPP iNOS protein at any timepoint. Taken together, eNOS protein expression and iNOS mRNA appear to bear a coincident temporal relationship to the time course of PN-mediated protein nitrative damage after CCI-TBI suggesting that both constitutive and inducible NOS isoforms contribute •NO for PN formation and 3-NT protein modification after TBI.

Extensive spontaneous plasticity of corticospinal projections after primate spinal cord injury.

Although axonal regeneration after CNS injury is limited, partial injury is frequently accompanied by extensive functional recovery. To investigate mechanisms underlying spontaneous recovery after incomplete spinal cord injury, we administered C7 spinal cord hemisections to adult rhesus monkeys and analyzed behavioral, electrophysiological and anatomical adaptations. We found marked spontaneous plasticity of corticospinal projections, with reconstitution of fully 60% of pre-lesion axon density arising from sprouting of spinal cord midline-crossing axons. This extensive anatomical recovery was associated with improvement in coordinated muscle recruitment, hand function and locomotion. These findings identify what may be the most extensive natural recovery of mammalian axonal projections after nervous system injury observed to date, highlighting an important role for primate models in translational disease research.

Lipid peroxidation-derived reactive aldehydes directly and differentially impair spinal cord and brain mitochondrial function.

Mitochondrial bioenergetic dysfunction in traumatic spinal cord and brain injury is associated with post-traumatic free radical-mediated oxidative damage to proteins and lipids. Lipid peroxidation by-products, such as 4-hydroxy-2-nonenal and acrolein, can form adducts with proteins and exacerbate the effects of direct free radical-induced protein oxidation. The aim of the present investigation was to determine and compare the direct contribution of 4-hydroxy-2-nonenal and acrolein to spinal cord and brain mitochondrial dysfunction. Ficoll gradient-isolated mitochondria from normal rat spinal cords and brains were treated with carefully selected doses of 4-hydroxy-2-nonenal or acrolein, followed by measurement of complex I- and complex II-driven respiratory rates. Both compounds were potent inhibitors of mitochondrial respiration in a dose-dependent manner. 4-Hydroxy-2-nonenal significantly compromised spinal cord mitochondrial respiration at a 0.1-muM concentration, whereas 10-fold greater concentrations produced a similar effect in brain. Acrolein was more potent than 4-hydroxy-2-nonenal, significantly decreasing spinal cord and brain mitochondrial respiration at 0.01 muM and 0.1 muM concentrations, respectively. The results of this study show that 4-hydroxy-2-nonenal and acrolein can directly and differentially impair spinal cord and brain mitochondrial function, and that the targets for the toxic effects of aldehydes appear to include pyruvate dehydrogenase and complex I-associated proteins. Furthermore, they suggest that protein modification by these lipid peroxidation products may directly contribute to post-traumatic mitochondrial damage, with spinal cord mitochondria showing a greater sensitivity than those in brain.

Progesterone improves acute recovery after traumatic brain injury in the aged rat.

Recent evidence has demonstrated that treatment with progesterone can attenuate many of the pathophysiological events following traumatic brain injury (TBI) in young adult rats, but this effect has not been investigated in aged animals. In this study, 20-month-old male Fischer 344 rats with bilateral contusions of the frontal cortex (n = 4 per group) or sham operations received 8, 16, or 32 mg/kg of progesterone or vehicle. Locomotor activity was measured at 72 h to assess behavioral recovery. Brain tissue was harvested at 24, 48, and 72 h, and Western blotting was performed for inflammatory and apoptotic factors. Edema was assessed at 48 h by measuring brain water content. Injured animals treated with 8 and 16 mg/kg progesterone showed decreased expression of COX-2, IL-6, and NFkappaB at all time points, indicating a reduction in the acute inflammatory process compared to vehicle. The 16 mg/kg group also showed reduced apoptosis at all time points as well as decreased edema and improved locomotor outcomes. Thus, in aged male rats, treatment with 16 mg/kg progesterone improves short-term motor recovery and attenuates edema, secondary inflammation, and cell death after TBI.