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 (Po0.05 versus vehicle). In another cohort, PZ administration increased spared cortical tissue from 86% to 97% (Po0.03). These results suggest that PZ's neuroprotective effect is due to mitochondrial protection by scavenging of LP-derived 4-HNE.

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.

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The efficacy of the amphipathic ketoamide calpain inhibitor SNJ-1945 in attenuating calpain-mediated degradation of the neuronal cytoskeletal protein a-spectrin was examined in the controlled cortical impact (CCI) traumatic brain injury (TBI) model in male CF-1 mice. Using a single early (15 min after CCI-TBI) i.p. bolus administration of SNJ-1945 (6.25, 12.5, 25, or 50-mg/kg), we identified the most effective dose on a-spectrin degradation in the cortical tissue of mice at its 24 h peak after severe CCI-TBI. We then investigated the effects of a pharmacokinetically optimized regimen by examining multiple treatment paradigms that varied in dose and duration of treatment. Finally, using the most effective treatment regimen, the therapeutic window of a-spectrin degradation attenuation was assessed by delaying treatment from 15 min to 1 or 3 h post-injury. The effect of SNJ-1945 on a-spectrin degradation exhibited a U-shaped doseresponse curve when treatment was initiated 15 min post-TBI. The most effective 12.5 mg/kg dose of SNJ-1945 significantly reduced a-spectrin degradation by~60% in cortical tissue. Repeated dosing of SNJ-1945 beginning with a 12.5 mg/kg dose did not achieve a more robust effect compared with a single bolus treatment, and the required treatment initiation was less than 1 h. Although calpain has been firmly established to play a major role in post-traumatic secondary neurodegeneration, these data suggest that even brain and cell-permeable calpain inhibitors, when administered alone, do not show sufficient cytoskeletal protective efficacy or a practical therapeutic window in a mouse model of severe TBI. Such conclusions need to be verified in the human clinical situation.

Ceramides have been implicated in a number of disease processes. However, current means of evaluation with flow infusion analysis (FIA) have been limited primarily due to poor sensitivity within our high-resolution mass spectrometry lipidomics analytical platform. To circumvent this deficiency, we investigated the potential of chloride adducts as an alternative method to improve sensitivity with electrospray ionization. Chloride adducts of ceramides and ceramide subfamilies provided 2- to 50-fold increases in sensitivity both with analytical standards and biological samples. Chloride adducts of a number of other lipids with reactive hydroxy groups were also enhanced. For example, monogalactosyl diacylglycerols (MGDGs), extracted from frontal lobe cortical gray and subcortical white matter of cognitively intact subjects, were not detected as ammonium adducts but were readily detected as chloride adducts. Hydroxy lipids demonstrate a high level of specificity in that phosphoglycerols and phosphoinositols do not form chloride adducts. In the case of choline glycerophospholipids, the fatty acid substituents of these lipids could be monitored by MS2 of the chloride adducts. Monitoring the chloride adducts of a number of key lipids offers enhanced sensitivity and specificity with FIA. In the case of glycerophosphocholines, the chloride adducts also allow determination of fatty acid substituents. The chloride adducts of lipids possessing electrophilic hydrogens of hydroxyl groups provide significant increases in sensitivity. In the case of glycerophosphocholines, chloride attachment to the quaternary ammonium group generates a dominant anion, which provides the identities of the fatty acid substituents under MS2 conditions.