Advanced Age and Neurotrauma Diminish Glutathione and Impair Antioxidant Defense after Spinal Cord Injury

Abstract

Advanced age at the time of spinal cord injury (SCI) exacerbates damage from reactive oxygen species (ROS). Mechanisms underlying this age-dependent response are not well understood and may arise from decreased antioxidant defense. We investigated how spinal cord levels of the antioxidant glutathione (GSH), and its regulation, change with age and SCI. GSH is used by GSH peroxidase to sequester ROS and is recycled by GSH reductase. Male and female, 4- and 14-month-old (MO) mice received a 60 kDyn contusion SCI, and the levels of GSH and its regulatory enzymes were evaluated at one and three days post-injury (dpi). The mice with SCI were treated with N-acetylcysteine-amide (NACA; 150 mg/kg), a cysteine supplement that increases GSH, to determine effects on functional and histological outcomes. GSH was decreased with older age in sham mice, and an SCI-dependent depletion was observed in 4-MO mice by three dpi. Neither age nor injury affected the abundance of proteins regulating GSH synthesis or recycling. GSH peroxidase activity, however, increased after SCI only in 4-MO mice. In contrast, GSH peroxidase activity was increased in 14-MO sham mice, indicating that spinal cords of older mice have an elevated oxidative state. Indeed, 14-MO sham mice had more oxidized protein (3-nitrotyrosine [3-NT]) within their spinal cords compared with 4-MO sham mice. Only 4-MO mice had significant injury-induced increases in 3-NT at three dpi. NACA treatment restored GSH and improved the redox environment in injured 4- and 14-MO mice at one dpi; however, three days of NACA delivery did not improve motor, sensory, or anatomical deficits at 28 dpi in 4-MO mice and trended toward toxicity in all outcomes in 14-MO mice. Our observation suggests that GSH levels at acute stages of SCI play a minimal role in age-dependent outcomes reported after SCI in mice. Collective results implicate elements of injury occurring after three dpi, such as inflammation, as key regulators of age-dependent effects.

Keywords: Redox; aging; neuroprotection; neurotrauma; oxidative stress; sex differences.

FIG. 1.

Glutathione synthesis and recycling. Glutathione is a recyclable tripeptide synthesized in two reactions. First, Glutamate-Cysteine Ligase ligates glutamate and cysteine, followed by glutathione synthetase, which adds glycine to finish the tripeptide structure. Two reduced glutathiones (GSH) are used as substrates for glutathione peroxidase (GPx) during the sequestration of reactive oxygen species (ROS). This oxidized glutathione (GSSG) is a dimer formed as a disulfide bridge that can be reduced by glutathione reductase (GR) back into two independent glutathione compounds using nicotinamide adenine dinucleotide phosphate (NADPH) as a substrate. GCLC, glutamate cysteine ligase catalytic domain.

FIG. 2.

Age and spinal cord injury (SCI) diminish spinal glutathione and increase oxidative stress. (A) Both total and free reduced glutathione (GSH) are diminished in 4-month-old (MO) mice three days after SCI, while total, free, and oxidized GSH are diminished in 14-MO sham-injured mice. (B) 3-Nitrotyrosine (3-NT) as a measure of oxidative stress was evaluated using dot blot (C) and was increased in 4-MO mice after SCI, as well as increased in 14-MO, relative to 4-MO, sham-injured mice. Dot densities are representative of group means. All assessments were performed using two-way analysis of variance with Sidak pair-wise comparisons as post hoc. n = 10/group. Graphs represent mean ± standard error of the mean. *p < 0.05, ** p < 0.01. dpi, days post-injury; GSSG, oxidized glutathione.

FIG. 3.

The glutathione synthesis enzymes, GCLC and GSH synthetase, are not affected by age or spinal cord injury (SCI). Both enzymes involved in GSH synthesis were evaluated using Western blots (A–F) to determine how age and injury effect protein expression. (A,C) The GSH synthetase was not affected by age, sex, or SCI at either one or three days post-injury (dpi). (B,D) GCLC expression was not affected by age, sex, or SCI at either one or three dpi. Assessments were performed using two-way analysis of variance. n = 9–10/group. Graphs represent mean ± standard error of the mean. *p < 0.05, **p < 0.01. GCLC, glutamate cysteine ligase catalytic domain; GSH, reduced glutathione.

FIG. 4.

Glutathione peroxidase (GPx) activity is increased after spinal cord injury (SCI). Two enzymes that use and recycle reduced glutathione (GSH) to sequester reactive oxygen species (ROS) were evaluated using activity assays (A–D) and Western blots (E–J) to determine how age and injury effect protein function and expression. (A,B) The GPx activity is significantly increased with age between sham-mice at one day post-injury (dpi) but only significantly increased after SCI in 4-month-old (MO) mice at one and here dpi. (C,D) Glutathione reductase (GR) was not increased after SCI relative to sham controls but was higher in 14- compared with 4-MO mice after SCI. (E–H) While increases in enzyme activity were found for GPx and GR, no differences were found at the level of protein expression at either day. Assessments were performed using two-way analysis of variance with Sidak pair-wise comparisons as post hoc. n = 9–10/group. Graphs represent mean ± standard error of the mean. *p < 0.05.

FIG. 5.

Cysteine supplementation using N-acetylcysteine amide (NACA) increases spinal levels of reduced glutathione (GSH) and improves the redox environment after SCI. (A) NACA (150 mg/kg) was given to naïve female mice to determine the efficacy of increasing spinal levels of GSH at 1.5 h post-treatment, which significantly elevated levels to approximately 170% of vehicle-treated controls. (B) When NACA (150 mg/kg/day) was delivered to mice for 24 h after spinal cord injury (SCI), both total and free GSH as well as the redox ratio (GSH/GSSG) are increased relative to vehicle-treated controls. (C,D) Dot blots performed against 3-nitrotyrosine (3-NT) and 4-hydroxy-nonenol (4-HNE) were used as measures of oxidative stress accumulation and revealed no significant effects of NACA on 3-NT or 4-HNE. (A) Assessment using two-tailed t test. (B,C) Assessments were performed using two-way analysis of variance with Sidak pair-wise comparisons as post-hoc. (A) n = 4/group (B,C) n = 6–7/group. Graphs represent mean ± standard error of the mean. *p < 0.05.

FIG. 6.

Treatment with N-acetylcysteine amide (NACA) does not improve locomotor or sensory outcomes after spinal cord injury (SCI). (A-C) NACA (150 mg/kg/day) was given to 4- and 14-MO mice after SCI and locomotor outcomes were assessed on the Basso Mouse Scale (BMS) (A) and BMS subscore (B), and horizontal ladder at 28 days post-injury (dpi) (C). No significant effects were observed between groups on any outcome. (D) Thermal hypersensitivity was evaluated using the Hargreaves testonce before and once at 28 days after SCI. No effects were found after NACA treatment, but significant hypersensitivity was observed after injury in both groups, with older mice having more hypersensitivity before SCI. (A,B) Assessments were performed using two-way repeated measures analysis of variance (ANOVA) for each age separately. (C) Assessment were performed using two-way ANOVA. (D) Assessment were performed using three-way ANOVA before combining treatments, which followed using a two-way ANOVA; Sidak pair-wise comparisons were used as post hoc. n = 9-10/group. Graphs represent mean ± standard error of the mean. *p < 0.05, **p < 0.01, ***p < 0.001.

FIG. 7.

Treatment with N-acetylcysteine amide (NACA) does not affect tissue sparing nor neuron survival in 4- or 14-month-old (MO) mice after spinal cord injury (SCI). (A–C) NACA (150 mg/kg/day) was given to 4- and 14-MO mice after SCI, and tissue sparing and neuron survival around the lesion epicenter was assessed at 28 days post-injury (dpi). No significant effects were observed for any group on any outcome. (A) Tissue sparing was assessed throughout the lesion, and spared tissue was normalized to total tissue area. (B,C) Effects of NACA on sparing at the lesion epicenter were analyzed separately. (B,C) Neuron counts from ventral horns on sections from every 100 μm up to 500 μm rostral and caudal to the lesion epicenter were quantified and summed for analysis. (C) Images were stained with eriochrome cyanine and neurofilament 200 kD to assess tissue sparing. Images for tissue sparing were representative of group means at the lesion epicenter. Images for neuronal nuclear protein (NeuN) counts were representative of 500 μm rostral to the lesion epicenter and are representative of group means. (A) Assessments were performed using two-way repeated measures analysis of variance (ANOVA) for each age separately. (B) Assessments were performed using two-way ANOVA. n = 8–10/group. Graphs represent mean ± standard error of the mean. Scale bars = 500 μm for tissue sparing and μm for NeuN stained sections.