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. 2011 Aug 3;31(31):11404-10.
doi: 10.1523/JNEUROSCI.2223-11.2011.

Role of Drp1, a key mitochondrial fission protein, in neuropathic pain

Luiz F Ferrari  1 , Adrienne Chum, Oliver Bogen, David B Reichling, Jon D Levine
Affiliations

Role of Drp1, a key mitochondrial fission protein, in neuropathic pain

Luiz F Ferrari et al. J Neurosci. .

Abstract

While oxidative stress has been implicated in small-fiber painful peripheral neuropathies, antioxidants are only partially effective to treat patients. We have tested the hypothesis that Drp1 (dynamin-related protein 1), a GTPase that catalyzes the process of mitochondrial fission, which is a mechanism central for the effect and production of reactive oxygen species (ROS), plays a central role in these neuropathic pain syndromes. Intrathecal administration of oligodeoxynucleotide antisense against Drp1 produced a decrease in its expression in peripheral nerve and markedly attenuated neuropathic mechanical hyperalgesia caused by HIV/AIDS antiretroviral [ddC (2',3'-dideoxycytidine)] and anticancer (oxaliplatin) chemotherapy in male Sprague Dawley rats. To confirm the role of Drp1 in these models of neuropathic pain, as well as to demonstrate its contribution at the site of sensory transduction, we injected a highly selective Drp1 inhibitor, mdivi-1, at the site of nociceptive testing on the dorsum of the rat's hindpaw. mdivi-1 attenuated both forms of neuropathic pain. To evaluate the role of Drp1 in hyperalgesia induced by ROS, we demonstrated that intradermal hydrogen peroxide produced dose-dependent hyperalgesia that was inhibited by mdivi-1. Finally, mechanical hyperalgesia induced by diverse pronociceptive mediators involved in inflammatory and neuropathic pain-tumor necrosis factor α, glial-derived neurotrophic factor, and nitric oxide-was also inhibited by mdivi-1. These studies provide support for a substantial role of mitochondrial fission in preclinical models of inflammatory and neuropathic pain.

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Figures

Figure 1.
Figure 1.
Role of Drp1 in ddC-induced neuropathic pain. A, Knockdown of Drp1. Analyzing the expression of Drp1 in rats treated with ODN MM or AS for Drp1 mRNA demonstrated a significant reduction of Drp1 protein levels in the saphenous nerve; MM = 87 ± 7; AS = 66 ± 7 arbitrary units normalized to the reference protein β-actin (N = 6; **p = 0.027, unpaired Student's t test) showed a 24% decrease in Drp1 protein. Of note, the calculated molecular weights of Drp1 and β-actin are 84 and 42 kDa, respectively. B, Attenuation of chemotherapy-induced hyperalgesia by Drp1 ODN antisense. Rats were treated with a single intravenous injection of ddC (50 mg/kg). 2 d later, ODN antisense (DRP1 AS, ▴) or mismatch (DRP1 MM, ▵) (40 μg) for Drp1 mRNA was injected intrathecally on 3 consecutive days. Mechanical nociceptive threshold was measured, by the Randall–Selitto paw-withdrawal test, on the second and fifth day post-ddC injection. N = 6 paws for both groups. One-way ANOVA showed a significant difference between the AS and MM groups on the fifth day (F(1,10) = 141.960; *p < 0.001).
Figure 2.
Figure 2.
Effect of local injection of Drp1 inhibitor mdivi-1 on ddC-induced hyperalgesia. A, Dose–response curve for the effect of intradermal mdivi-1 on the hyperalgesia induced by intravenous injection of ddC. Rats received a single injection of ddC (50 mg/kg). Ten days later mdivi-1, (0, 0.1, 1, 3, and 10 μg) was administered in separate groups. Mechanical thresholds were tested 30 min after mdivi-1 administration. One-way ANOVA showed a significant difference (F(4,25) = 1287.841; p < 0.001). Scheffé's post hoc tests showed that the effects of all doses except 0.1 μg were significantly different (*p < 0.001) from the control group (0 μg, vehicle); however, there was no significant difference in the effects of doses between 3 and 10 μg (p = 0.151 and 0.941, respectively); therefore, we chose the 3 μg dose for this study. B, Attenuation of ddC-induced hyperalgesia by mdivi-1. For this experiment, the maximal dose of mdivi-1 (10 μg; n = 6) or vehicle (n = 6) was administered 10 d after ddC injection in separate groups of rats. Mechanical thresholds were tested before and 30 min after injections. As shown in A, mdivi-1 (■) attenuated ddC-induced hyperalgesia, but vehicle (□) had no effect. The two-way repeated-measures ANOVA showed a significant main effect of group (F(1,10) = 60.503; *p < 0.001), a significant group × time interaction (F(1,10) = 35.133; *p < 0.001), as well as a significant main effect of time (F(1,10) = 31.385; *p < 0.001).
Figure 3.
Figure 3.
Effect of the Drp1 inhibitor on early and late phases of oxaliplatin-induced neuropathy. A single dose of oxaliplatin (2 mg/kg) was administered intravenously. Intradermal injection of mdivi-1 (3 μg, n = 6) or vehicle (n = 6) was performed on the sixth day (first phase, left) or on the 15th day (second phase, right) post-oxaliplatin injection. Mechanical thresholds were tested before and 30 min after mdivi-1 (• and ■) or vehicle (○ and □) administration. mdivi-1 significantly attenuated both phases of oxaliplatin-induced hyperalgesia. For early-phase hyperalgesia, a two-way repeated-measures ANOVA showed a significant main effect of group (F(1,20) = 64.590; *p < 0.001), a significant group × time interaction (F(1,20) = 50.641; *p < 0.001), and a significant main effect of time (F(1,20) = 48.010; *p < 0.001). For late-phase hyperalgesia, there was also a significant main effect of group (F(1,8) = 35.666; *p < 0.001), a significant group × time interaction (F(1,8) = 41.183; *p < 0.001), and a significant main effect of time (F(1,8) = 49.882; *p < 0.001).
Figure 4.
Figure 4.
Hydrogen peroxide (H2O2)-induced mechanical hyperalgesia is dependent on Drp1 activation. A, Increasing doses of H2O2 (control, 0.1, 0.3, 1, 3, 10%) were administered intradermally into the dorsum of the hindpaws in separate groups of rats. Testing was performed 5, 15, and 30 min later. Higher doses of H2O2 induced significant hyperalgesia at all three time points. The two-way repeated-measures ANOVA showed a significant main effect of group (F(5,28) = 21.351; p < 0.001), but neither the main effect of time (F(2,56) = 775; p = 0.466) nor the group × time interaction (F(2,56) = 1.885; p < 0.067) were significant. Scheffé's post hoc tests showed a significant difference between the control and the three highest H2O2 groups (*p < 0.001 for all three) but not the group that received 0.1% H2O2 (p = 0.115). B, Effect of mdivi-1 (3 μg/paw) on H2O2-induced hyperalgesia. mdivi-1 (•, 3 μg, n = 6) or vehicle (○, n = 6) was administered 5 min before H2O2 in separate groups of rats. Testing was performed 5, 15, and 30 min after H2O2 (1%) administration into the same site. mdivi-1 attenuated H2O2-induced hyperalgesia. A two-way repeated-measures ANOVA showed a significant main effect of group (F(6,5) = 11.126; #p = 0.009), a significant group × time interaction (F(12,10) = 5.578; p = 0.013), and a significant main effect of time (F(2,10) = 17.522; p = 0.002).
Figure 5.
Figure 5.
Involvement of Drp1 in the mechanical hyperalgesia induced by mediators implicated in inflammatory and neuropathic pain. The effect of mdivi-1 (3 μg) was evaluated in five different models of acute hyperalgesia, including NO (A), TNFα (B), GDNF (C), NGF (D), and epinephrine (EPI) (E). mdivi-1 (full symbols) or vehicle (empty symbols) was injected 10 min before the nitric oxide donor NOR-3 (1 μg), TNFα (0.1 μg), or EPI (0.1 μg). Rats treated with GDNF (10 ng) or NGF (1 μg) received mdivi-1 or vehicle 1 or 24 h later, respectively. Mechanical thresholds were evaluated 30 min after mdivi-1/vehicle injection by the Randall–Selitto paw-withdrawal test. Student's t tests showed significant attenuation of the mechanical hyperalgesia induced by NOR-3 (♦, t(10) = 3.902; **p = 0.003), TNFα (•, t(10) = 8.474; *p < 0.001), and GDNF (▾, t(10) = 10.953; *p < 0.001) in the groups treated with mdivi-1. No significant effect of mdivi-1 was observed on NGF- or EPI-induced hyperalgesia (■, t(10) = 1.307; p = 0.220 and ▴, t(10) = 1.281; p = 0.229, respectively). N = 6 for all groups.
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