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, 333, 13-26

Oxidative Stress in the Development, Maintenance and Resolution of Paclitaxel-Induced Painful Neuropathy

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Oxidative Stress in the Development, Maintenance and Resolution of Paclitaxel-Induced Painful Neuropathy

Natalie A Duggett et al. Neuroscience.

Abstract

Paclitaxel is a first-line chemotherapeutic with the major dose-limiting side effect of painful neuropathy. Previous preclinical studies indicate mitochondrial dysfunction and oxidative stress are associated with this disorder; however no direct assessment of reactive oxygen species (ROS) levels and antioxidant enzyme activity in sensory neurons following paclitaxel has been undertaken. As expected, repeated low doses of systemic paclitaxel in rats induced long-lasting pain behaviour with a delayed onset, akin to the clinical scenario. To elucidate the role of ROSinthe development and maintenance ofpaclitaxel-inducedpainful neuropathy, we have assessed ROS and antioxidant enzyme activity levels in the nociceptive system in vivo at three key behavioural time-points; prior to pain onset (day 7), peak pain severity and pain resolution. In isolated dorsal root ganglia (DRG) neurons, ROS levels were unchanged following paclitaxel-exposure in vitro or in vivo. ROS levels were further assessed in DRG and spinal cord in vivo following intrathecal MitoTracker®RedCM-H2XRos administration in paclitaxel-/vehicle-treated rats. ROS levels were increased at day 7, specifically in non-peptidergic DRG neurons. In the spinal cord, neuronally-derived ROS was increased at day 7, yet ROS levels in microglia and astrocytes were unaltered. In DRG, CuZnSOD and glutathione peroxidase (GPx) activity were increased at day 7 and peak pain time-points, respectively. In peripheral sensory nerves, CuZnSOD activity was increased at day 7, and at peak pain, MnSOD, CuZnSOD and GPx activity were increased. Catalase activity was unaltered in DRG and saphenous nerves. These data suggest that neuronally-derived mitochondrial ROS, accompanied with an inadequate endogenous antioxidant enzyme response, are contributory factors in paclitaxel-induced painful neuropathy.

Keywords: Taxol; chemotherapy; chemotherapy-induced neuropathy; mitochondria; neurotoxicity; reactive oxygen species.

Figures

Fig. 1
Fig. 1
Time course of paclitaxel-induced mechanical hypersensitivity. Graphs show the mean ± SEM of the number of withdrawal responses to (A) 4g, (B) 8g and (C) 15g von Frey filaments at; baseline, day 7, peak pain (day 23–31) and pain resolution (day 173–220), following paclitaxel/vehicle administration at days 0, 2, 4 and 6. *p < 0.05, two-tailed multiple comparison unpaired t-tests with Holm-Sidak correction. Day 7 n = 108 vehicle, n = 114 paclitaxel; peak pain n = 73 vehicle, n = 75 paclitaxel; resolution n = 30 vehicle, n = 30 paclitaxel. NB: These data are compiled from several cohorts of animals used to generate tissues for these studies. Data from each individual cohort show no significant difference in mechanical hypersensitivity, between vehicle and paclitaxel-treated animals, at day 7 and pain resolution.
Fig. 2
Fig. 2
Effect of in vivo paclitaxel on total ROS and superoxide production in isolated DRG neurons. (A) The mean ± SEM of MTRed fluorescence intensity in small, medium and large isolated DRG neurons from paclitaxel-/vehicle-treated rats at day 7, peak pain (day 23–31) and pain resolution (day 173–194), n = 7–8 animals per treatment group. Neuronal categories were based on cell body size; small (10–25 μm), medium (25–50 μm) and large (>50 μm). Numbers in brackets indicate total number of neurons analysed in each category from paclitaxel- and vehicle-treated rats combined. Dotted line indicates the fluorescence of the vehicle-control group set as 100%. (B) Representative image of MTRed staining in an isolated DRG preparation. (C) MTRed colocalisation in non-neuronal DRG cells; DAPI stained nuclei are shown in blue, MitoTracker Red and MitoTracker Green are shown in red and green, respectively. Scale bar = 10 μm. (D) The mean ± SEM of MitoSOX fluorescence intensity of intensity in small, medium and large isolated DRG neurons from paclitaxel-/vehicle-treated rats at day 7, peak pain (day 24–31) and pain resolution (day 173–194), n = 7–8 per treatment group. Numbers in brackets indicate total number of neurons analysed in each category from paclitaxel- and vehicle-treated rats combined. Dotted line indicates the fluorescence of the vehicle-control group set as 100%. (E) Representative image of MitoSOX staining in an isolated DRG preparation. (F) MitoSOX Red colocalisation in non-neuronal DRG cells; DAPI stained nuclei are shown in blue, MitoSOX Red and MitoTracker Green are shown in red and green, respectively. Scale bar = 10 μm.
Fig. 3
Fig. 3
In vivo ROS levels in DRG neurons during the time-course of paclitaxel-induced painful neuropathy. (A–C) The mean ± SEM of MTRed fluorescence intensity of small, medium and large DRG neurons in paclitaxel- and vehicle- treated rats in vivo at (A) day 7, (B) peak pain – day 27–30 and (C) pain resolution – day 195–220. Neuronal categories were based on cell body size; small (10–23 μm), medium (23–32 μm) and large (>32 μm). Numbers in brackets indicate total number of neurons counted in each category, n = 6 animals per treatment group, per time point. (D–F) Example images of cut DRG from each treatment group; (D) day 7, (E) peak pain and (F) pain resolution. White boxed areas are highlighted to show DAPI, MTRed and merged image channels. Scale bar = 50 μm. Areas of intense MTRed staining are due to proximity of tissue to intrathecal injection site and were avoided during analysis.
Fig. 4
Fig. 4
In vivo ROS levels in IB4+ DRG neurons during the time-course of paclitaxel-induced painful neuropathy. (A–C) The mean ± SEM of MTRed fluorescence intensity of small and medium IB4+ DRG neurons in paclitaxel- and vehicle-treated rats in vivo at (A) day 7, (B) peak pain – day 27–30 and (C) pain resolution – day 195–220. Neuronal categories were based on cell body size; small (10–23 μm) and medium (23–32 μm). Numbers in brackets indicate total number of neurons counted in each category, n = 5–6 animals per treatment group, per time point. *p < 0.05 unpaired, one-tailed t-tests. (D–F) Example images of cut DRG from each treatment group; (D) day 7, (E) peak pain and (F) pain resolution. White boxed areas are highlighted to show DAPI, IB4+, MTRed and merged image channels. Scale bar = 50 μm. Areas of intense MTRed staining are due to proximity of tissue to intrathecal injection site and were avoided during analysis.
Fig. 5
Fig. 5
In vivo ROS levels in the superficial dorsal horn of the spinal cord during the time-course of paclitaxel-induced painful neuropathy. (A) Example images of superficial dorsal horn in sections of L4/5 spinal cord, following intrathecal injection of MTRed. Scale bar = 50 μm. (B) The mean ± SEM of MTRed fluorescence intensity in laminae I–III of L4/5 spinal cord in paclitaxel-treated animals, expressed as a percentage of vehicle-treated controls at day 7, peak pain (day 27–30) and pain resolution (day 195–220). n = 6–9 animals per treatment group, per time-point. (C) The mean ± SEM of MTRed fluorescence intensity of NeuN positive neurons in the dorsal horn of vehicle- and paclitaxel-treated rats at day 7. *p < 0.05 unpaired, one-tailed t-test, n = 5–7 animals per group. No significant difference in ROS levels of spinal microglia or astrocytes in paclitaxel-treated rats compared to vehicle-treated rats was evident at day 7, n = 4 animals per group (data not shown).
Fig. 6
Fig. 6
Superoxide dismutase activity in the DRG and saphenous nerve during the time-course of paclitaxel-induced painful neuropathy. MnSOD activity in (A) DRG and (C) saphenous nerves of paclitaxel- and vehicle-treated rats at day 7, peak pain (day 28) and pain resolution (day 182–218), n = 4–6 animals per group, *p < 0.05 unpaired, two-tailed t-tests. CuZnSOD activity in (B) DRG and (D) saphenous nerves of paclitaxel- and vehicle-treated rats at day 7, peak pain (day 28) and pain resolution (day 182–218), n = 4–6 animals per group, *p < 0.05 unpaired, two-tailed t-test. Adjacent panels display scans of activity gels used in analysis, which show significantly altered activity between groups. Black line on peak pain gel due to small tear.
Fig. 7
Fig. 7
Glutathione peroxidase (GPx) and catalase activity in the DRG and saphenous nerve during the time-course of paclitaxel-induced painful neuropathy. GPx activity in (A) DRG and (C) saphenous nerves of paclitaxel- and vehicle-treated rats at day 7, peak pain (day 28) and pain resolution (day 182–218), n = 4–6 animals per group. (B) Catalase activity in the DRG of paclitaxel- and vehicle-treated rats at day 7 and peak pain (day 28), n = 3–5 animals per group. (D) Catalase activity in saphenous nerves of paclitaxel- and vehicle-treated rats at day 7 and peak pain (day 28), n = 5 animals per group. *p < 0.05 unpaired, two-tailed t-tests. Adjacent panels display scans of activity gels used in analysis, which show significantly altered activity between groups.

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