Spinal mitochondrial-derived peroxynitrite enhances neuroimmune activation during morphine hyperalgesia and antinociceptive tolerance

Abstract

Treatment of severe pain by morphine, the gold-standard opioid and a potent drug in our arsenal of analgesic medications, is limited by the eventual development of hyperalgesia and analgesic tolerance. We recently reported that systemic administration of a peroxynitrite (PN) decomposition catalyst (PNDC) or superoxide dismutase mimetic attenuates morphine hyperalgesia and antinociceptive tolerance and reduces PN-mediated mitochondrial nitroxidative stress in the spinal cord. These results suggest the potential involvement of spinal PN signaling in this setting; which was examined in the present study. PN removal with intrathecal delivery of manganese porphyrin-based dual-activity superoxide/PNDCs, MnTE-2-PyP(5+) and the more lipophilic MnTnHex-2-PyP(5+), blocked hyperalgesia and antinociceptive tolerance in rats. Noteworthy is that intrathecal MnTnHex-2-PyP(5+) prevented nitration and inactivation of mitochondrial manganese superoxide dismutase. Mitochondrial manganese superoxide dismutase inactivation enhances the superoxide-to-PN pathway by preventing the dismutation of superoxide to hydrogen peroxide, thus providing an important enzymatic source for PN formation. Additionally, intrathecal MnTnHex-2-PyP(5+) attenuated neuroimmune activation by preventing the activation of nuclear factor kappa B, extracellular-signal-regulated kinase and p38 mitogen activated protein kinases, and the enhanced levels of proinflammatory cytokines, interleukin (IL)-1β and IL-6, while increasing anti-inflammatory cytokines, IL-4 and IL-10. The role of PN was further confirmed using intrathecal or oral delivery of the superoxide-sparing PNDC, SRI-110. These results suggest that mitochondrial-derived PN triggers the activation of several biochemical pathways engaged in the development of neuroinflammation in the spinal cord that are critical to morphine hyperalgesia and tolerance, further supporting the potential of targeting PN as an adjunct to opiates to maintain pain relief.

Fig. 1

Intrathecal injections of peroxynitrite decomposition catalyst (PNDCs) prevent morphine-induced hyperalgesia and antinociceptive tolerance. When compared with rats that received subcutaneous infusions of saline (Veh-Sal), chronic morphine infusion in rats (Veh-Mor) resulted in significant decreases in hindpaw withdrawal latency (A, B) and percentage maximal possible antinociceptive effect (%MPE) (C, D) on day 6. Daily intrathecal administration of MnTE-2-PyP⁵⁺ (0.1 to 1 nmol/d; MnTE-2-PyP-Mor) (A, C) or MnTnHex-2-PyP⁵⁺ (0.01 to 0.1 nmol/d; MnTnHex-2-PyP-Mor) (B, D) blocked morphine-induced hyperalgesia and retained %MPE in a dose-dependent manner. Results are expressed as mean ± SD for n = 6 animals. Data were analyzed by analysis of variance with the Dunnett post hoc test. *P < .001 vs Veh -Sal; ^†P < .001 vs Veh-Mor.

Fig. 2

Dose-response curves for peroxynitrite decomposition catalysts. Intrathecal SRI-110 (■) and MnTnHex-2-PyP⁵⁺ (◇) were nearly equipotent at reducing antinociceptive tolerance. Both SRI-110 and MnTnHex-2-PyP⁵⁺ were more potent than MnTE-2-PyP (○). Differences in dose-response curves when compared with SRI-110 were analyzed using the extra sum-of-squares F-test. #P < .05; ##P < .001 vs SRI-110.

Fig. 3

Nitration and inactivation of spinal manganese superoxide dismutase (MnSOD) is abrogated by intrathecal MnTnHex-2-PyP⁵⁺. In subcutaneous morphine infused rats (Veh-Mor), the levels of spinal nitrated MnSOD (A, B) increased as MnSOD activity (C) decreased on day 6 when compared with subcutaneous saline (Veh-Sal). Daily intrathecal MnTnHex-2-PyP⁵⁺ (0.1 nmol/d; MnTnHex-2-PyP-Mor) prevented MnSOD nitration (A, B) and inactivation (C). Intrathecal injection of MnTnHex-2-PyP⁵⁺ had no effect on MnSOD activity in rats receiving subcutaneous saline (MnTnHex-2-PyP-Sal). Spinal copper/zinc superoxide dismutase (Cu,Zn SOD) activity was unaltered by any treatment (D). Results are expressed as mean ± SD for n = 5 animals. Composite densitometric analyses for gels of nitrated proteins of 5 rats are expressed as %β-actin (B). Data were analyzed by analysis of variance with the Dunnett post hoc test. *P < .001 vs subcutaneous infusion of saline (Veh-Sal); ^†P < .01 and ^††P < .001 vs Veh-Mor.

Fig. 4

Intrathecal MnTnHex-2-PyP⁵⁺ blocks nuclear factor kappa B (NF-κB) activation. On day 6, when compared with rats that received chronic infusion of vehicle (Veh-Sal), chronic morphine (Veh-Mor) was associated with a significant decrease in spinal cytosolic IκBα (A), and significant increases in levels of spinal cytosolic phosphorylated NF-κB subunit p65 (B) and nuclear p65 (C). These events were abrogated with daily intrathecal MnTnHex-2-PyP⁵⁺ (0.1 nmol/d; MnTnHex-2-PyP-Mor) (A-C). Results are expressed as mean ± SD for n = 5 animals. Composite densitometric analyses for gels of proteins of 5 rats are expressed as %β-actin (A, B) or %lamin A/C (C). Data were analyzed by analysis of variance with the Dunnett post hoc test. *P < .01 and **P < .001 vs subcutaneous infusion of saline (Veh-Sal); ^†P < .001 vs Veh-Mor.

Fig. 5

The spinal peroxynitrite (PN) pathway is required for activation of mitogen activating protein kinases (MAPK) signaling pathways. Chronic morphine (Veh-Mor) was associated with a significant increase in phosphorylation of both extracellular-signal-regulated kinase (ERK)1/2 subunits 42 and 44 (A) and p38 MAPK (B) compared with rats receiving daily subcutaneous saline (Veh-Sal). Daily intrathecal MnTnHex-2-PyP⁵⁺ (0.1 nmol/d; MnTnHex-2-PyP-Mor) (A, B) attenuated phosphorylation of ERK1/2 and p38. Results are expressed as mean ± SD for n = 5 animals. Composite densitometric analyses for gels of proteins of 5 rats are expressed as %β-actin. Data were analyzed by analysis of variance with the Dunnett post hoc test. *P < .001 vs Veh-Sal; ^†P < .001 vs Veh-Mor.

Fig. 6

Intrathecal MnTnHex-2-PyP⁵⁺ decreases proinflammatory and increases anti-inflammatory cytokines. Rats receiving chronic morphine (Veh-Mor) had significantly increased the expressions of the proinflammatory tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 (A) and the anti-inflammatory IL-10 with no change in IL-4 levels (B) compared with subcutaneous infusion of saline (Veh-Sal) on day 6. Intrathecal MnTnHex-2-PyP⁵⁺ (0.1 nmol/d; MnTnHex-2-PyP-Mor) significantly increased the anti-inflammatory cytokines, IL-10 and IL-4 (B), while preventing the enhanced expression of IL-1β and IL-6 (A). Results are expressed as mean ± SD for n = 6 animals. Data were analyzed by analysis of variance with the Dunnett post hoc test. *P < .05 and **P < .001 vs Veh-Sal; ^†P < .05 and ^††P < .01 vs Veh-Mor.

Fig. 7

Spinal SRI-110 attenuates hyperalgesia and antinociceptive tolerance. Morphine-induced thermal hypersensitivity (A) and antinociceptive tolerance (B) were blocked in a dose-dependent manner with daily intrathecal administration of SRI-110 (0.03 to 1.0 nmol/d; SRI-110-Mor), compared with intrathecal saline (Veh-Mor) by day 6. SRI-110 (intrathecal 1.0 nmol/d) in rats receiving subcutaneous infusion of saline (SRI-110-Sal) had no effect on paw withdrawal latencies or percentage maximal possible antinociceptive effect (%MPE). Results are expressed as mean ± SD for n = 6 animals. Data were analyzed by analysis of variance with the Dunnett post hoc test *P < .001 vs subcutaneous infusion of saline (Veh-Sal); ^†P < .001 vs Veh-Mor.

Fig. 8

Oral administration of SRI-110 prevents morphine-induced hyperalgesia and antinociceptive tolerance. When compared with rats receiving subcutaneous saline (○), chronic morphine (●) resulted in a significant decrease in paw withdrawal latency (PWL) (A) and percentage maximal possible antinociceptive effect (%MPE) (B) by day 6. Daily oral administration of MnTnHex-2-PyP (30 mg/kg/d; ▲) had no effect on the PWL or %MPE; however, SRI-110 (30 mg/kg/d; ■) attenuated the development of hyperalgesia and maintained acute morphine analgesia. Oral administration of SRI-110 in rats receiving subcutaneous saline (□) had no effect on PWL or %MPE. Results are expressed as mean ± SD for n = 6 animals. Data were analyzed by analysis of variance with the Bonferroni post hoc test. *P < .01 and **P < .001 vs t₀; ^†P < .001 vs subcutaneous morphine (Veh-Mor).