Protective effects of phenelzine administration on synaptic and non-synaptic cortical mitochondrial function and lipid peroxidation-mediated oxidative damage following TBI in young adult male rats.

Traumatic brain injury (TBI) results in mitochondrial dysfunction and induction of lipid peroxidation (LP). Lipid peroxidation-derived neurotoxic aldehydes such as 4-HNE and acrolein bind to mitochondrial proteins, inducing additional oxidative damage and further exacerbating mitochondrial dysfunction and LP. Mitochondria are heterogeneous, consisting of both synaptic and non-synaptic populations, with synaptic mitochondria being more vulnerable to injury-dependent consequences. The goal of these studies was to explore the hypothesis that interrupting secondary oxidative damage following TBI using phenelzine (PZ), an aldehyde scavenger, would preferentially protect synaptic mitochondria against LP-mediated damage in a dose- and time-dependent manner. Male Sprague-Dawley rats received a severe (2.2 mm) controlled cortical impact (CCI)-TBI. PZ (3-30 mg/kg) was administered subcutaneously (subQ) at different times post-injury. We found PZ treatment preserves both synaptic and non-synaptic mitochondrial bioenergetics at 24 h and that this protection is partially maintained out to 72 h post-injury using various dosing regimens. The results from these studies indicate that the therapeutic window for the first dose of PZ is likely within the first hour after injury, and the window for administration of the second dose seems to fall between 12 and 24 h. Administration of PZ was able to significantly improve mitochondrial respiration compared to vehicle-treated animals across various states of respiration for both the non-synaptic and synaptic mitochondria. The synaptic mitochondria appear to respond more robustly to PZ treatment than the non-synaptic, and further experimentation will need to be done to further understand these effects in the context of TBI.

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.

Effects of Phenelzine Administration on Mitochondrial Function, Calcium Handling, and Cytoskeletal Degradation after Experimental Traumatic Brain Injury.

Traumatic brain injury (TBI) results in the production of peroxynitrite (PN), leading to oxidative damage of lipids and protein. PN-mediated lipid peroxidation (LP) results in production of reactive aldehydes 4-hydroxynonenal (4-HNE) and acrolein. The goal of these studies was to explore the hypothesis that interrupting secondary oxidative damage following a TBI via phenelzine (PZ), analdehyde scavenger, would protect against LP-mediated mitochondrial and neuronal damage. Male Sprague-Dawley rats received a severe (2.2 mm) controlled cortical impact (CCI)-TBI. PZ was administered subcutaneously (s.c.) at 15 min (10 mg/kg) and 12 h (5 mg/kg) post-injury and for the therapeutic window/delay study, PZ was administered at 1 h (10 mg/kg) and 24 h (5 mg/kg). Mitochondrial and cellular protein samples were obtained at 24 and 72 h post-injury (hpi). Administration of PZ significantly improved mitochondrial respiration at 24 and 72 h compared with vehicle-treated animals. These results demonstrate that PZ administration preserves mitochondrial bioenergetics at 24 h and that this protection is maintained out to 72 hpi. Additionally, delaying the administration still elicited significant protective effects. PZ administration also improved mitochondrial Ca2+ buffering (CB) capacity and mitochondrial membrane potential parameters compared with vehicle-treated animals at 24 h. Although PZ treatment attenuated aldehyde accumulation post-injury, the effects were insignificant. The amount of α-spectrin breakdown in cortical tissue was reduced by PZ administration at 24 h, but not at 72 hpi compared with vehicle-treated animals. In conclusion, these results indicate that acute PZ treatment successfully attenuates LP-mediated oxidative damage eliciting multiple neuroprotective effects following TBI.

Synaptic Mitochondria are More Susceptible to Traumatic Brain Injury-induced Oxidative Damage and Respiratory Dysfunction than Non-synaptic Mitochondria.

Traumatic brain injury (TBI) results in mitochondrial dysfunction and induction of lipid peroxidation (LP). Lipid peroxidation-derived neurotoxic aldehydes such as 4-HNE and acrolein bind to mitochondrial proteins, inducing additional oxidative damage and further exacerbating mitochondrial dysfunction and LP. Mitochondria are heterogeneous, consisting of both synaptic and non-synaptic populations. Synaptic mitochondria are reported to be more vulnerable to injury; however, this is the first study to characterize the temporal profile of synaptic and non-synaptic mitochondria following TBI, including investigation of respiratory dysfunction and oxidative damage to mitochondrial proteins between 3 and 120 h following injury. These results indicate that synaptic mitochondria are indeed the more vulnerable population, showing both more rapid and severe impairments than non-synaptic mitochondria. By 24 h, synaptic respiration is significantly impaired compared to synaptic sham, whereas non-synaptic respiration does not decline significantly until 48 h. Decreases in respiration are associated with increases in oxidative damage to synaptic and non-synaptic mitochondrial proteins at 48 h and 72 h, respectively. These results indicate that the therapeutic window for mitochondria-targeted pharmacological neuroprotectants to prevent respiratory dysfunction is shorter for the more vulnerable synaptic mitochondria than for the non-synaptic population.

Continuous Infusion of Phenelzine, Cyclosporine A, or Their Combination: Evaluation of Mitochondrial Bioenergetics, Oxidative Damage, and Cytoskeletal Degradation following Severe Controlled Cortical Impact Traumatic Brain Injury in Rats.

To date, all monotherapy clinical traumatic brain injury (TBI) trials have failed, and there are currently no Food and Drug Administration (FDA)-approved pharmacotherapies for the acute treatment of severe TBI. Due to the complex secondary injury cascade following injury, there is a need to develop multi-mechanistic combinational neuroprotective approaches for the treatment of acute TBI. As central mediators of the TBI secondary injury cascade, both mitochondria and lipid peroxidation-derived aldehydes make promising therapeutic targets. Cyclosporine A (CsA), an FDA-approved immunosuppressant capable of inhibiting the mitochondrial permeability transition pore, and phenelzine (PZ), an FDA-approved monoamine oxidase inhibitor capable of scavenging neurotoxic lipid peroxidation-derived aldehydes, have both been shown to be partially neuroprotective following experimental TBI. Therefore, it follows that the combination of PZ and CsA may enhance neuroprotection over either agent alone through the combining of distinct but complementary mechanisms of action. Additionally, as the first 72 h represents a critical time period following injury, it follows that continuous drug infusion over the first 72 h following injury may also lead to optimal neuroprotective effects. This is the first study to examine the effects of a 72 h subcutaneous continuous infusion of PZ, CsA, and the combination of these two agents on mitochondrial respiration, mitochondrial bound 4-hydroxynonenal (4-HNE), and acrolein, and α-spectrin degradation 72 h following a severe controlled cortical impact injury in rats. Our results indicate that individually, both CsA and PZ are able to attenuate mitochondrial 4-HNE and acrolein, PZ is able to maintain mitochondrial respiratory control ratio and cytoskeletal integrity but together, PZ and CsA are unable to maintain neuroprotective effects.

Time courses of post-injury mitochondrial oxidative damage and respiratory dysfunction and neuronal cytoskeletal degradation in a rat model of focal traumatic brain injury.

Traumatic brain injury (TBI) results in rapid reactive oxygen species (ROS) production and oxidative damage to essential brain cellular components leading to neuronal dysfunction and cell death. It is increasingly appreciated that a major player in TBI-induced oxidative damage is the reactive nitrogen species (RNS) peroxynitrite (PN) which is produced in large part in injured brain mitochondria. Once formed, PN decomposes into highly reactive free radicals that trigger membrane lipid peroxidation (LP) of polyunsaturated fatty acids (e.g. arachidonic acid) and protein nitration (3-nitrotyrosine, 3-NT) in mitochondria and other cellular membranes causing various functional impairments to mitochondrial oxidative phosphorylation and calcium (Ca2+) buffering capacity. The LP also results in the formation of neurotoxic reactive aldehyde byproducts including 4-hydroxynonenal (4-HNE) and propenal (acrolein) which exacerbates ROS/RNS production and oxidative protein damage in the injured brain. Ultimately, this results in intracellular Ca2+ overload that activates proteolytic degradation of α-spectrin, a neuronal cytoskeletal protein. Therefore, the aim of this study was to establish the temporal evolution of mitochondrial dysfunction, oxidative damage and cytoskeletal degradation in the brain following a severe controlled cortical impact (CCI) TBI in young male adult rats. In mitochondria isolated from an 8 mm diameter cortical punch including the 5 mm wide impact site and their respiratory function studied ex vivo, we observed an initial decrease in complex I and II mitochondrial bioenergetics within 3 h (h). For complex I bioenergetics, this partially recovered by 12-16 h, whereas for complex II respiration the recovery was complete by 12 h. During the first 24 h, there was no evidence of an injury-induced increase in LP or protein nitration in mitochondrial or cellular homogenates. However, beginning at 24 h, there was a gradual secondary decline in complex I and II respiration that peaked at 72 h. post-TBI that coincided with progressive peroxidation of mitochondrial and cellular lipids, protein nitration and protein modification by 4-HNE and acrolein. The oxidative damage and respiratory failure paralleled an increase in Ca2+-induced proteolytic degradation of the neuronal cytoskeletal protein α-spectrin indicating a failure of intracellular Ca2+ homeostasis. These findings of a surprisingly delayed peak in secondary injury, suggest that the therapeutic window and needed treatment duration for certain antioxidant treatment strategies following CCI-TBI in rodents may be longer than previously believed.

Synaptic Mitochondria Sustain More Damage than Non-Synaptic Mitochondria after Traumatic Brain Injury and Are Protected by Cyclosporine A.

Currently, there are no Food and Drug Administration (FDA)-approved pharmacotherapies for the treatment of those with traumatic brain injury (TBI). As central mediators of the secondary injury cascade, mitochondria are promising therapeutic targets for prevention of cellular death and dysfunction after TBI. One of the most promising and extensively studied mitochondrial targeted TBI therapies is inhibition of the mitochondrial permeability transition pore (mPTP) by the FDA-approved drug, cyclosporine A (CsA). A number of studies have evaluated the effects of CsA on total brain mitochondria after TBI; however, no study has investigated the effects of CsA on isolated synaptic and non-synaptic mitochondria. Synaptic mitochondria are considered essential for proper neurotransmission and synaptic plasticity, and their dysfunction has been implicated in neurodegeneration. Synaptic and non-synaptic mitochondria have heterogeneous characteristics, but their heterogeneity can be masked in total mitochondrial (synaptic and non-synaptic) preparations. Therefore, it is essential that mitochondria targeted pharmacotherapies, such as CsA, be evaluated in both populations. This is the first study to examine the effects of CsA on isolated synaptic and non-synaptic mitochondria after experimental TBI. We conclude that synaptic mitochondria sustain more damage than non-synaptic mitochondria 24 h after severe controlled cortical impact injury (CCI), and that intraperitoneal administration of CsA (20 mg/kg) 15 min after injury improves synaptic and non-synaptic respiration, with a significant improvement being seen in the more severely impaired synaptic population. As such, CsA remains a promising neuroprotective candidate for the treatment of those with TBI.

Phenelzine Protects Brain Mitochondrial Function In Vitro and In Vivo following Traumatic Brain Injury by Scavenging the Reactive Carbonyls 4-Hydroxynonenal and Acrolein Leading to Cortical Histological Neuroprotection.

Lipid peroxidation (LP) is a key contributor to the pathophysiology of traumatic brain injury (TBI). Traditional antioxidant therapies are intended to scavenge the free radicals responsible for either initiation or propagation of LP. A more recently explored approach involves scavenging the terminal LP breakdown products that are highly reactive and neurotoxic carbonyl compounds, 4-hydroxynonenal (4-HNE) and acrolein (ACR), to prevent their covalent modification and rendering of cellular proteins nonfunctional leading to loss of ionic homeostasis, mitochondrial failure, and subsequent neuronal death. Phenelzine (PZ) is a U.S. Food and Drug Administration-approved monoamine oxidase (MAO) inhibitor (MAO-I) used for treatment of refractory depression that possesses a hydrazine functional group recently discovered by other investigators to scavenge reactive carbonyls. We hypothesized that PZ will protect mitochondrial function and reduce markers of oxidative damage by scavenging LP-derived aldehydes. In a first set of in vitro studies, we found that exogenous application of 4-HNE or ACR significantly reduced respiratory function and increased markers of oxidative damage (p < 0.05) in isolated noninjured rat brain cortical mitochondria, whereas PZ pre-treatment significantly prevented mitochondrial dysfunction and oxidative modification of mitochondrial proteins in a concentration-related manner (p < 0.05). This effect was not shared by a structurally similar MAO-I, pargyline, which lacks the hydrazine group, confirming that the mitochondrial protective effects of PZ were related to its carbonyl scavenging and not to MAO inhibition. In subsequent in vivo studies, we documented that PZ treatment begun at 15 min after controlled cortical impact TBI significantly attenuated 72-h post-injury mitochondrial respiratory dysfunction. The cortical mitochondrial respiratory protection occurred together with a significant increase in cortical tissue sparing.

Lipid peroxidation in brain or spinal cord mitochondria after injury.

Extensive evidence has demonstrated an important role of oxygen radical formation (i.e., oxidative stress) as a mediator of the secondary injury process that occurs following primary mechanical injury to the brain or spinal cord. The predominant form of oxygen radical-induced oxidative damage that occurs in injured nervous tissue is lipid peroxidation (LP). Much of the oxidative stress in injured nerve cells initially begins in mitochondria via the generation of the reactive nitrogen species peroxynitrite (PN) which then can generate multiple highly reactive free radicals including nitrogen dioxide (•NO2), hydroxyl radical (•OH) and carbonate radical (•CO3). Each can readily induce LP within the phospholipid membranes of the mitochondrion leading to respiratory dysfunction, calcium buffering impairment, mitochondrial permeability transition and cell death. Validation of the role of LP in central nervous system secondary injury has been provided by the mitochondrial and neuroprotective effects of multiple antioxidant agents which are briefly reviewed.

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.

Pharmacological inhibition of lipid peroxidation attenuates calpain-mediated cytoskeletal degradation after traumatic brain injury.

Free radical-induced lipid peroxidation (LP) is critical in the evolution of secondary injury following traumatic brain injury (TBI). Previous studies in our laboratory demonstrated that U-83836E, a potent LP inhibitor, can reduce post-TBI LP along with an improved maintenance of mouse cortical mitochondrial bioenergetics and calcium (Ca(2+)) buffering following severe (1.0 mm; 3.5 m/s) controlled cortical impact TBI (CCI-TBI). Based upon this preservation of a major Ca(2+) homeostatic mechanism, we have now performed dose-response and therapeutic window analyses of the ability of U-83836E to reduce post-traumatic calpain-mediated cytoskeletal (α-spectrin) proteolysis in ipsilateral cortical homogenates at its 24 h post-TBI peak. In the dose-response analysis, mice were treated with a single i.v. dose of vehicle or U-83836E (0.1, 0.3, 1.3, 3.0, 10.0 or 30.0 mg/kg) at 15 min after injury. U-83836E produced a dose-related attenuation of α-spectrin degradation with the maximal decrease being achieved at 3.0 mg/kg. Next, the therapeutic window was tested by delaying the single 3 mg/kg i.v. dose from 15 min post-injury out to 1, 3, 6 or 12 h. No reduction in α-spectrin degradation was observed when the treatment delay was 1 h or longer. However, in a third experiment, we re-examined the window with repeated U-83836E dosing (3.0 mg/kg i.v. followed by 10 mg/kg i.p. maintenance doses at 1 and 3 h after the initial i.v. dose) which significantly reduced 24 h α-α-spectrin degradation even when treatment initiation was withheld until 12 h post-TBI. These results demonstrate the relationship between post-TBI LP, disruptions in neuronal Ca(2+) homeostasis and calpain-mediated cytoskeletal damage.