Cellular and subcellular oxidative stress parameters following severe spinal cord injury

Abstract

The present study undertook a comprehensive assessment of the acute biochemical oxidative stress parameters in both cellular and, notably, mitochondrial isolates following severe upper lumbar contusion spinal cord injury (SCI) in adult female Sprague Dawley rats. At 24 h post-injury, spinal cord tissue homogenate and mitochondrial fractions were isolated concurrently and assessed for glutathione (GSH) content and production of nitric oxide (NO ^•), in addition to the presence of oxidative stress markers 3-nitrotyrosine (3-NT), protein carbonyl (PC), 4-hydroxynonenal (4-HNE) and lipid peroxidation (LPO). Moreover, we assessed production of superoxide ( ${\mathrm{O}}_2^{•-}$ ) and hydrogen peroxide (H ₂O ₂) in mitochondrial fractions. Quantitative biochemical analyses showed that compared to sham, SCI significantly lowered GSH content accompanied by increased NO ^• production in both cellular and mitochondrial fractions. SCI also resulted in increased  ${\mathrm{O}}_2^{•-}$ and H ₂O ₂ levels in mitochondrial fractions. Western blot analysis further showed that reactive oxygen/nitrogen species (ROS/RNS) mediated PC and 3-NT production were significantly higher in both fractions after SCI. Conversely, neither 4-HNE levels nor LPO formation were increased at 24 h after injury in either tissue homogenate or mitochondrial fractions. These results indicate that by 24 h post-injury ROS-induced protein oxidation is more prominent compared to lipid oxidation, indicating a critical temporal distinction in secondary pathophysiology that is critical in designing therapeutic approaches to mitigate consequences of oxidative stress.

Graphical abstract

After acute contusion spinal cord injury (SCI), increased free radical production (e.g. ${\mathrm{O}}_2^{•}$ and H ₂O ₂) and simultaneous depletion of endogenous antioxidant glutathione (GSH) leads to increased oxidative stress markers, protein carbonyls (PC) and 3-nitrotyrosine (3-NT), at both cellular as well as mitochondrial levels. This ultimately results in long-term tissue damage and functional deficits (solid arrows). Pharmacological treatment(s) that reduce oxidative stress while maintaining antioxidants to near normal levels after injury have potential to decrease tissue damage and improve functional recovery (dashed arrows) following SCI.

Highlights

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  Acute spinal cord injury (SCI) decreases cellular and mitochondrial glutathione.

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  Protein carbonyls increase in cellular and mitochondrial fractions after SCI.

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  O ₂˙‾, H ₂O ₂ and NO˙ production increases in mitochondrial fractions after SCI.

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  Mitochondrial protein (~95 kDa) is prominently susceptible to 3-NT after SCI.

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  Lipid peroxidation adducts were notably unaltered after acute SCI.