The Mitochondrial Uncoupling Agent 2,4-Dinitrophenol Improves Mitochondrial Function, Attenuates Oxidative Damage, and Increases White Matter Sparing in the Contused Spinal Cord

Abstract

The purpose of this study was to investigate the potential neuroprotective efficacy of the mitochondrial uncoupler 2,4-dinitrophenol (DNP) in rats following a mild to moderate spinal cord contusion injury. Animals received intraperitoneal injections of vehicle (DMSO) or 5 mg/mL of DNP prior to injury. Twenty-four hours following surgery, mitochondrial function was assessed in mitochondria isolated from spinal cord synaptosomes. In addition, synaptosomes were used to measure indicators of reactive oxygen species formation, lipid peroxidation, and protein oxidation. Relative to vehicle-treated animals, pretreatment with DNP maintained mitochondrial bioenergetics and significantly decreased reactive oxygen species levels, lipid peroxidation, and protein carbonyl content following spinal cord injury. Furthermore, pretreatment with DNP significantly increased the amount of remaining white matter at the injury epicenter 6 weeks after injury. These results indicate that treatment with mitochondrial uncoupling agents may provide a novel approach for the treatment of secondary injury following spinal cord contusion.

Targeting Mitochondrial Function for the Treatment of Acute Spinal Cord Injury

“…Briefly, the oxygen consumption of isolated mitochondria is first measured at baseline (state I) and then following the addition of various compounds to the respiratory chamber to induce different respiratory states. The type of substrates and the order of their administration may vary slightly among laboratories, but the following sequence has worked well in our hands: 1) pyruvate plus malate, which are complex I substrates (state II); 2) ADP (state III); 3) oligomycin, an ATP synthase inhibitor (state IV); 4) carbonyl cyanide 4-(trifluoromethoxy) phenylhydrozone (FCCP), a mitochondrial uncoupler (state V, complex I-driven respiration); and (5) rotenone, a complex I inhibitor, followed by succinate, a complex II substrate (state V, complex IIdriven respiration) [43][44][45][46][47]. Mitochondrial uncoupling, produced by FCCP, refers to a condition in which electron transport is functionally disconnected from the production of ATP [48,49].…”

“…Specifically, state III respiration reflects the maximal rate of coupled respiration, whereas state IV respiration reflects the rate of proton leakage from the mitochondrial intermembrane space into the matrix. Reports indicate that RCR values are comparable to control levels at 6 hours [47], but decreased at 12 hours [47] and 24 hours [43][44][45][46][47] following SCI, primarily due to a decrease in the rate of oxygen consumption following the addition of ADP (state III). Further investigation revealed that both complex I-driven [43][44][45][46][47] and complex II-driven [43,44] respiration was impaired postinjury and that mitochondrial oxidative stress was elevated [43,44,46,47].…”

“…Reliable and reproducible measures of mitochondrial function and respiration can be obtained in mitochondria isolated from the intact and injured spinal cord [43,44,46,47]. In an initial study, the effects of NIM811 treatment on mitochondrial bioenergetics and ROS production following acute SCI were examined [44].…”

Early Mitochondrial Dysfunction after Cortical Contusion Injury

“…Both complex 1 and 2 maximum respiration have been shown to drop by approximately 50% following a moderate unilateral CCI in SD rats (Opii et al, 2007). Significant changes in both NAD-linked and succinate-driven respiration have also been reported following ischemic=reperfusion injury (Sciamanna et al, 1992;Sciamanna and Lee, 1993), as well as spinal cord contusion injury ( Jin et al, 2004).…”

Novel neuroprotective, neuritogenic and anti-amyloidogenic properties of 2,4-dinitrophenol: The gentle face of Janus

“…That same study also showed that neuronal death induced by oxygen and glucose deprivation in cultured cortical neurons was reduced in the presence of DNP under conditions that caused mild mitochondrial uncoupling. In a different experimental paradigm, intraperitoneal injection of DNP in rats was shown to improve mitochondrial function, to attenuate oxidative damage and to increase white matter sparing in the contused spinal cord (20), suggesting that this might constitute a novel approach for the treatment of spinal cord contusion damage. Taken together, these studies suggest that, contrary to the uncontrolled and deleterious mitochondrial uncoupling that takes place in the presence of high concentrations of DNP, mild uncoupling induced by low doses of DNP may actually be beneficial in terms of reducing oxidative neuronal damage in different pathological conditions.…”