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Inhibition of Mitochondrial Fission Protein Reduced Mechanical Allodynia and Suppressed Spinal Mitochondrial Superoxide Induced by Perineural Human Immunodeficiency Virus gp120 in Rats

Kanda, Hirotsugu MD*†; Liu, Shue BS*; Iida, Takafumi MD*; Yi, Hyun BS*; Huang, Wan MD, PhD*; Levitt, Roy C. MD*‡§; Lubarsky, David A. MD, MBA*; Candiotti, Keith A. MD*; Hao, Shuanglin MD, PhD*

doi: 10.1213/ANE.0000000000000962
Pain and Analgesic Mechanisms: Research Report

BACKGROUND: Mitochondria play an important role in many cellular and physiologic functions. Mitochondria are dynamic organelles, and their fusion and fission regulate cellular signaling, development, and mitochondrial homeostasis. The most common complaint of human immunodeficiency virus (HIV)-sensory neuropathy is pain on the soles in patients with HIV, but the exact molecular mechanisms of HIV neuropathic pain are not clear. In the present study, we investigated the role of mitochondrial dynamin-related protein 1 (Drp1, a GTPase that mediates mitochondrial fission) in the perineural HIV coat glycoprotein gp120-induced neuropathic pain state.

METHODS: Neuropathic pain was induced by the application of recombinant HIV-1 envelope protein gp120 into the sciatic nerve. Mechanical threshold was tested using von Frey filaments. The mechanical threshold response was assessed over time using the area under curves. Intrathecal administration of antisense oligodeoxynucleotide (ODN) against Drp1, mitochondrial division inhibitor-1 (mdivi-1), or phenyl-N-tert-butylnitrone (a reactive oxygen species scavenger) was given. The expression of spinal Drp1 was examined using western blots. The expression of mitochondrial superoxide in the spinal dorsal horn was examined using MitoSox imaging.

RESULTS: Intrathecal administration of either antisense ODN against Drp1 or mdivi-1 decreased mechanical allodynia (a sensation of pain evoked by nonpainful stimuli) in the gp120 model. Intrathecal ODN or mdivi-1 did not change basic mechanical threshold in sham surgery rats. Intrathecal Drp1 antisense ODN decreased the spinal expression of increased Drp1 protein induced by peripheral gp120 application. Intrathecal phenyl-N-tert-butylnitrone reduced mechanical allodynia. Furthermore, both intrathecal Drp1 antisense ODN and mdivi-1 reversed the upregulation of mitochondrial superoxide in the spinal dorsal horn in the gp120 neuropathic pain state.

CONCLUSIONS: These data suggest that mitochondrial division plays a substantial role in the HIV gp120-related neuropathic pain state through mitochondrial reactive oxygen species and provides evidence for a novel approach to treating chronic pain in patients with HIV.

Published ahead of print September 28, 2015

From the *Department of Anesthesiology, University of Miami Miller School of Medicine, Miami, Florida; Department of Anesthesiology, Asahikawa Medical University, Asahikawa, Japan; Hussman Institute of Human Genomics, University of Miami Miller School of Medicine, Miami, Florida; and §Veterans Affairs Medical Center, Miami, Florida.

Accepted for publication June 30, 2015.

Published ahead of print September 28, 2015

Funding: The study was supported by the National Institutes of Health (NIH) DA026734 (SH), DA025527 (SH), NS066792 (SH), and DA34749 (SH). RCL was supported by NIH DE022903. HK was supported greatly by Professor and Chair, Dr. Hiroshi Iwasaki (Department of Anesthesiology, Asahikawa Medical University, Japan).

The authors declare no conflicts of interest.

Reprints will not be available from the authors.

Address correspondence to Shuanglin Hao, MD, PhD, Department of Anesthesiology, University of Miami, Miller School of Medicine, 1550 NW 10th Ave., Miami, FL 33136. Address e-mail to shao@med.miami.edu.

The most common complaint of human immunodeficiency virus-sensory neuropathy (HIV-SN) is peripheral pain on the soles in patients with HIV. Many conventional drugs used as pharmacologic therapies for neuropathic pain are not effective for providing satisfactory analgesia in painful HIV-related distal sensory polyneuropathy because the exact molecular mechanisms of the HIV neuropathic pain are not clear. Mitochondria play an important role in many cellular and physiologic functions.1 Mitochondrial division is critical for the maintenance of mitochondrial structure, and alterations in this process are linked to many human diseases, including peripheral neuropathies and central age-related neurodegenerative disorders.1,2 Dynamin-related protein 1 (Drp1) is involved in mitochondria division and biogenesis,1 and recent studies have shown that it is involved in the neuropathic pain induced by antiretroviral and anticancer chemotherapy.3

HIV coat protein gp120 is a viral exterior envelope glycoprotein. HIV gp120 exerts both direct and indirect neurotoxic effects in the nervous system through the release of proinflammatory factors and neurotransmitters.4 A host of evidence has demonstrated that free radicals act as mediators of chronic pain.5–10 HIV gp120 has been implicated in the initiation and/or intensification of reactive oxygen species (ROS) and disruption of mitochondrial transmembrane potential, and HIV-induced ROS regulates apoptosis signaling.11 Mitochondrial ROS is significantly involved in gp120-induced neuropathic pain model.12 Furthermore, hydrogen peroxide administered intradermally into the dorsum of the hindpaws induces mechanical hyperalgesia; and intrathecal mitochondrial division inhibitor-1 (mdivi-1)13 administered into the dorsum of the hindpaws attenuates mechanical hyperalgesia.3 However, it is not clear whether spinal Drp1 is involved in the HIV gp120-induced neuropathic pain state. In the present study, we evaluated the role of mitochondrial fission in neuropathic pain induced by HIV gp120 and examined whether ROS was involved in the role of spinal mitochondrial fission in the gp120-induced pain state.

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METHODS

Animals

Male Sprague-Dawley rats weighing 225 to 250 g were housed 1 to 3 per cage for approximately 7 days before the beginning of the study. Rats were maintained with free access to food and water and were on a 12:12 light/dark schedule at 21°C and 60% humidity. All housing conditions and experimental procedures were approved by the University Animal Care and Use Committee and were conducted in accordance with the ethical guidelines of the International Association for the Study of Pain.

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Perineural gp120 Neuropathic Pain Model

Under anesthesia, the rat’s left sciatic nerve was exposed in the popliteal fossa without damaging the nerve construction. A 2- × 6-mm strip of oxidized regenerated cellulose was previously soaked in 250 µL of a 0.1% rat serum albumin (RSA) in saline, containing 400 ng gp120 (ImmunoDiagnostics, Bedford, MA) or 0.1% RSA without gp120 in saline for the sham surgery. A length of 3 to 4 mm of the sciatic nerve was wrapped loosely with the previously soaked cellulose, proximal to the trifurcation so as not to cause any nerve constriction damage and left in situ, as described previously.14,15 The incision was closed with 4/0 sutures.

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Mechanical Threshold

The mechanical threshold was determined using calibrated von Frey filaments (Stoelting, Wood Dale, IL) introduced serially to the hindpaws in ascending order of strength, and animals were placed in nontransparent plastic cubicles on a mesh floor for an acclimatization period of at least 30 minutes in the morning of the test day. A positive response was defined as a rapid withdrawal and/or licking of the paw immediately on application of the stimulus. Whenever a positive response to a stimulus occurred, the next smaller von Frey hair was applied, and whenever a negative response occurred, the next higher force was applied. In the absence of a response at a pressure of 15.1 grams, animals were assigned to this cutoff value. The mechanical threshold was determined according to the method described previously, with a tactile stimulus producing a 50% likelihood of withdrawal determined by using the up-and-down method.16,17

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Intrathecal Catheter Implantation

For studying the effect of intrathecal administration of drugs on neuropathic pain, chronic intrathecal catheters were implanted under isoflurane anesthesia.18 Briefly, through an incision in the atlanto-occipital membrane, a polyethylene catheter, filled with 0.9% saline, was advanced 8.5 cm caudally to position its tip at the level of the lumbar enlargement. The rostral tip of the catheter was passed subcutaneously, externalized on top of the skull, and sealed with a stainless steel plug. Animals showing neurologic deficits after implantation were excluded. Mdivi-1 and phenyl-N-tert-butylnitrone (PBN) were purchased from Sigma (St Louis, MO), dissolved in saline. Spinal agents were delivered over 60 seconds in a volume of 10-μL solution. Drug injection was immediately followed by a 10-μL physiologic saline to flush the catheter.

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Oligodeoxynucleotide Antisense to Drp1

The oligodeoxynucleotide (ODN) antisense (AS) to Drp1 mRNA, 5′CCA CTA CGA CAA TCT GAG GC3′ (Invitrogen, Carlsbad, CA), was directed against a unique region of the rat mRNA sequence, as described previously.3 The mismatch (MM) ODN sequence 5′ACA CTT CTA CAT TCG GAC GC3′ corresponds to the Drp1 AS sequence with 6 bases mismatched. ODNs were reconstituted in nuclease-free saline to a concentration of 10 μg/μL and stored at −80°C until use. For each injection, rats were briefly anesthetized with 1.0% isoflurane. A dose of 40 μg (injection volume 20 μL) of AS-Drp1 or MM-ODN was administered followed by 10 μL of saline using intrathecal catheter, once daily for 3 consecutive days.

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Western Blots

Under deep anesthesia, the L4-5 dorsal root ganglion (DRG) or spinal dorsal horn ipsilateral to the gp120 application was removed rapidly, frozen on dry ice, and stored at −80°C, as described previously.17 These tissues were homogenized in protein lysis buffer (150 mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris, pH 8.0) containing protease inhibitors (Sigma) and phosphatase inhibitors (Phosphatase Inhibitor Cocktails, Sigma). The homogenate was centrifuged at 18,000g for 20 minutes at 4°C. The supernatant was collected and assayed for protein concentration using the DC protein assay (Bio-Rad, Hercules, CA). Aliquots containing 30 μg of protein were dissolved in Laemmli buffer and denatured at 95°C for 5 minutes; proteins were separated by 10% to 12% Tris-glycine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membranes were blocked with rapid block solution (Amresco LLC, Solon, OH) and then incubated with primary antibodies for overnight at 4°C, including rabbit anti-Drp1 (1:4000; Novus Biotech, Littleton, CO) and mouse anti-β-actin (1:10000; Sigma). The blots were incubated with secondary antibodies (Santa Cruz Biotechnology, Inc., Dallas, TX) and developed in a chemiluminescence solution (Pierce Biotechnology, Rockford, IL). Quantification of western blots was performed using the obtained chemiluminescence values (Bio-Rad ChemiDoc, Bio-Rad). Target protein bands were normalized using the amount of β-actin.

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Mitochondrial Superoxide Imaging in the Spinal Dorsal Horn

MitoSox Red (a mitochondrial superoxide indicator; Invitrogen) was dissolved in a 1:1 mixture of dimethylsulfoxide and saline to a final concentration of 33 μM, as described previously.10 MitoSox (30 μL) was injected intrathecally. Approximately 70 minutes after injection of MitoSox, rats were perfused intracardially with 4% paraformaldehyde in 0.1 M phosphate buffer, and the L4-5 segments of the spinal cord were removed, postfixed in the same solution for overnight, and cryoprotected with 30% sucrose in PBS for 2 days. The 35 μm sections were examined under a fluorescent microscope with a rhodamine filter. Two different regions of the dorsal horn were photographed from 5 randomly selected sections from each animal: the lateral part of laminas I to II and laminas III to V. The number of MitoSox-positive cellular profiles with distinctive nuclei (dark oval-shaped space surrounded by red granules) was counted from the pictures, as described previously.10,19

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Data Analysis and Statistics

The results describing behavioral responsiveness to von Frey stimulation indicating mechanical sensitivity were given as a mechanical threshold. Differences in mean changes at the same time points were evaluated individually for the assessment of treatment effects on hypersensitivity with a 2-tailed t test. The area under the time-effect curves (AUC) data, depicting the mechanical threshold (g) over time, were calculated by the trapezoidal rule to express the overall magnitude and duration of effect and analyzed between 2 different treatments using a 2-tailed t test. Data for the effects of Drp1 inhibition on the neurochemical changes among 3 groups were compared with 1-way analysis of variance (ANOVA) with post hoc Fisher-protected least significant difference test (software StatView5; SAS Institute Inc., Cary, NC). The values from each test group were graphed as mean ± SEM. The actual P values were shown in the figures. Because of the small samples and multiple comparison in some studies, we considered the P ≤ 0.01 as statistically significant and 0.01 < P < 0.05 as having very great trends.20

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RESULTS

The Antiallodynic Effect of Knockdown of Drp1 by AS-ODN Against Drp1 in Rats with Perineural gp120 Neuropathic Pain

Figure 1

Figure 1

We have reported that rats exposed to perineural HIV-1 gp120 application developed a persistent, mechanical allodynia of the ipsilateral hind paw compared with sham surgery (the same procedure without gp120 but RSA).15 Previous studies show that intrathecal administration of an AS-ODN to Drp1 mRNA reverses mechanical hyperalgesia induced by antiretroviral therapy.3 To further examine the role of Drp1 in neuropathic pain, we tested the antiallodynic effect of knockdown of Drp1 by AS-ODN in HIV gp120-induced pain state, which is different from that induced by antiretroviral drugs. Drp1 AS-ODN was administered intrathecally at 40 µg/d for 3 consecutive days in rats with gp120-induced neuropathic pain. Drp1 AS-ODN administration increased mechanical threshold compared with MM-ODN (Fig. 1). For the comparison of the differences at individual time points between 2 groups, we used a t test (Fig. 1A) and actual P values were shown in Figure 1A. The AUC in Drp1 AS-ODN group was higher than that in the control group ([MM-ODN], P = 0.010, t test, n = 6; Fig. 1B).

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The Antiallodynic Effect of Intrathecal mdivi-1 in Rats with Perineural gp120 Neuropathic Pain

Figure 2

Figure 2

Mdivi-1, a specific inhibitor of Drp1, modulates mitochondrial morphology.13 Ferrari et al.3 report that intradermal mdivi-1 reduces mechanical hyperalgesia induced by 2′-3′-dideoxycytidine (ddC, an anti-HIV drug) and oxaliplatin (an anticancer drug). Here, we examined the effect of intrathecal mdivi-1 on the neuropathic pain state induced by gp120. We found that mdivi-1 dose-dependently increased mechanical threshold. The antiallodynic effect of a large dose (3 μg) lasted approximately 2 hours. For the comparison of the differences at individual time points between drug and saline, we used a 1-way ANOVA (actual P values were shown in Fig. 2A). The AUC with mdivi-1 was dose-dependently higher than that in the vehicle group ([saline], 1-way ANOVA, n = 4–6, actual P values are shown in Fig. 2B).

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The Effect of Knockdown of Drp1 by AS-ODN Against Drp1 or mdivi-1 on Mechanical Threshold in Rats with Sham Surgery

Figure 3

Figure 3

To test whether knockdown of Drp1 by AS-ODN against Drp1 or mdivi-1 affects mechanical threshold in sham surgery rats, we intrathecally injected AS-ODNs or mdivi-1 and observed mechanical thresholds as described earlier. Mechanical thresholds between AS-Drp1 and MM-Drp1 groups were similar in their time course (Fig. 3A). Mechanical threshold in the intrathecal mdivi-1 group was similar to the saline group (Fig. 3B).

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The Expression of Drp1 Was Reduced by Intrathecal Drp1 AS-ODN in the Spinal Dorsal Horn

Figure 4

Figure 4

Previous studies demonstrate that intrathecal injection of ODN AS for mRNA encoding Drp1 produced a decrease in the level of Drp1 in the saphenous nerve.3 In this study, we investigated whether knockdown of Drp1 decreased the expression of Drp1 protein in the spinal dorsal horn in the gp120 pain state. Neuropathic animals received Drp1 AS or MM-ODN at day 12 to 14 after gp120. Rats in the sham group received MM-ODN. The spinal cord dorsal horn (SDH) was harvested at 14 days after gp120. Drp1 protein was measured using western blots. There was an increase in Drp1 in the gp120 + MM-Drp1 group compared with that in the sham + MM-Drp1 (P = 0.0002, 1-way ANOVA, n = 4–5; Fig. 4). The expression of Drp1 in the gp120 + AS-Drp1 group was lower than that in the gp120 + MM-Drp1 group (P = 0.0009, 1-way ANOVA, n = 4–5; Fig. 4).

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The Antiallodynic Effect of Intrathecal ROS Scavenger in Rats with Perineural gp120 Neuropathic Pain

Figure 5

Figure 5

Previous evidence shows that spinal ROS scavenger, PBN, reduces chronic pain in animals.21 Here, we examined the effect of intrathecal PBN (1 mg) on the neuropathic pain state induced by gp120. We found that PBN increased mechanical threshold. The antiallodynic effect of PBN lasted approximately 4 hours. For comparing the differences at individual time points between drug and saline, we used t test (actual P values were shown in Fig. 5A). The AUC with PBN was higher than that in the vehicle group ([saline], P < 0.0001, t test, n = 7; Fig. 5B).

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The Effect of Intrathecal Drp1 AS-ODN or mdivi-1 on Mitochondrial Superoxide Expression

Previous studies demonstrated that mitochondrial ROS is involved in the pain state.10,19,22 Kim et al.23 have reported that ROS production is increased in cultured melanocytes and melanoma cells with fragmented mitochondria and that inhibition of ROS suppresses the antimelanogenic effect of mitochondrial fission. Administration of mdivi-1 reduced oxidative stress and attenuated cell apoptosis in cultured rat primary hippocampal cells.24 In this study, we investigated whether knockdown of Drp1 decreased mitochondrial superoxide in the spinal dorsal horn associated with the gp120-induced pain state. Neuropathic animals received Drp1 AS or MM-ODN at day 12 to 14 after gp120 and were perfused at 14 days after gp120. MitoSox Red was intrathecally administered 70 minutes before perfusion. MitoSox-positive imaging was detected using a fluorescent microscope with a rhodamine filter, as described previously.19,22 The number of MitoSox-positive neurons was accounted in the spinal dorsal horn. MitoSox-positive imaging in sham with MM-ODN of Drp1 (MM-Drp1), neuropathic rats with MM-Drp1, and neuropathic rats with AS-ODN of Drp1 (AS-Drp1) is shown in Figure 6A, B, and C, respectively. The number of MitoSox-positive cells in the gp120 + MM-Drp1 group was more than that in the sham + MM-Drp1 (P < 0.0001, 1-way ANOVA, n = 5–6; Fig. 6D). There was a decrease in the MitoSox-positive cell number in the gp120 + AS-Drp1 group compared with that in the gp120 + MM-Drp1 (P < 0.0001, 1-way ANOVA, n = 5–6; Fig. 6D).

Figure 6

Figure 6

Figure 7

Figure 7

Meanwhile, to investigate the effect of intrathecal mdivi-1 on mitochondrial superoxide expression at 14 days after gp120, neuropathic animals received intrathecal saline or mdivi-1 60 minutes before perfusion. MitoSox Red was intrathecally administered 70 minutes before perfusion. MitoSox-positive imaging in sham with saline, neuropathic rats with saline, and neuropathic rats with mdivi-1 is shown in Figure 7A, B, and C, respectively. The number of MitoSox-positive cells in the gp120 + saline group was more than that in the sham + saline (P < 0.0001, 1-way ANOVA, n = 5–6; Fig. 7D). There was a decrease in the MitoSox-positive cell number in the gp120 + mdivi-1 group compared with that in the gp120+ saline (P < 0.0001, 1-way ANOVA, n = 5–6; Fig. 7D).

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DISCUSSION

Mitochondrial division is critical for the maintenance of mitochondrial structure and function. In the current study, we demonstrated that (1) intrathecal administration of Drp1 AS-ODN or mdivi-1 reversed the lowered mechanical threshold in the gp120-induced painful peripheral neuropathy model, (2) intrathecal Drp1 AS-ODN reduced the expression of Drp1 in the spinal dorsal horn, (3) intrathecal PBN reduced mechanical allodynia in the gp120 neuropathic pain model, and (4) intrathecal Drp1 AS-ODN or mdivi-1 reduced mitochondrial superoxide in the spinal dorsal horn in the gp120-induced neuropathic pain model.

The histologic feature of HIV-SN is characterized by loss of DRG sensory neurons, Wallerian degeneration of the long axons in distal regions, DRG infiltration by HIV-infected macrophages, a “dying back” SN, and loss of unmyelinated sensory fibers25–27; in the spinal cord of HIV painful patients, there is a spinal gliosis of glial activity and inflammatory cytokine release.28 Herzberg and Sagen14 report that the gp120-exposed sciatic nerve exhibits early pathology, notably axonal swelling and increased tumor necrosis factor-α within the nerve trunk; intense astrocytic and microglial activation is observed in the spinal cord, and this gliosis persists in parallel with neuropathic pain behaviors. Wallace et al.29 and other groups have shown that the model of gp120 application into sciatic nerve can induce neuropathic pain behavior, inflammatory cell infiltration into DRG, spinal gliosis, and reduction in intraepidermal nerve fiber density,14,15 which correlates well with the clinical scenario27,30; therefore, the gp120 model is considered as HIV-related painful neuropathy state.

Tissue damage or inflammation is often accompanied by the accumulation of endogenous factors (e.g., tumor necrosis factor-α) released from activated nociceptors or non-neural cells (e.g., macrophages) that reside within, or infiltrate into, the injured area.31,32 Primary afferent nerve fibers project to the dorsal horn of the spinal cord, which is organized into anatomically and electrophysiologically distinct laminae to induce central sensitization.32 Central sensitization refers to the process through which a state of hyperexcitability is established in the central nervous system, leading to enhanced processing of nociceptive (painful) messages.33 Central sensitization in the SDH stimulates astrocytosis to release proinflammatory cytokines/chemokines in patients with HIV and animals with chronic pain.28,34 Overactivation of cytokines/chemokine receptor activity induces neuronal injury in the SDH in the HIV neuropathic pain state.26 The exact molecular mechanisms of HIV-induced neuropathic pain are not yet clear. In the current study, we focus on the neurochemical changes in the SDH and investigated the role of mitochondrial division protein Drp1.

Drp1 is a member of the conserved dynamin GTPase superfamily. Drp1 mainly localizes in the cytosol and, during mitochondrial fission, translocates from the cytosol to prospective fission sites on the mitochondria.35 In addition to controlling mitochondrial number and morphology, mitochondrial dynamics is critical for maintaining various mitochondrial functions.36 However, it remains unclear how excessive fission or fusion affect mitochondrial function. It is reported that mitochondrial Ca2+ uptake and intramitochondrial Ca2+ diffusion are impaired in cells with fragmented mitochondria, suggesting that an imbalance between mitochondrial fission and fusion can disrupt normal Ca2+ ion homeostasis.36 Mitochondrial fragmentation mediated by the fission process is a necessary component for high glucose-induced ROS overproduction; inhibition of mitochondrial fission prevented periodic fluctuation of ROS production during high glucose exposure.37 Recent studies demonstrated that mitochondrial Ca2+ uptake, with consequent production of ROS, is essential for the synaptic plasticity underlying chronic pain.22

Oxidative stress causes activation of a number of complex and interrelated signaling events.38 ROS are critically involved in the spinal sensitization.8,39 Evidence has demonstrated that free radicals are implicated as mediators of chronic pain.5–10 Mitochondrial oxidative stress causes activation of a number of complex and interrelated signaling events in the pathogenesis of chronic pain.40 Furthermore, ROS accumulation is observed primarily in the mitochondria of SDH neurons in different pain models.10,19,22,41 A significant analgesic effect of ROS scavengers is reported in capsaicin-induced secondary hyperalgesia.42 Superoxide generated from mitochondrial oxidative phosphorylation is a major source of neuronal ROS.43 Capsaicin-induced pain represents a model for identifying the site of action for ROS.10 An analgesic effect of ROS scavengers is observed in capsaicin-induced secondary hyperalgesia,42 suggesting ROS involvement in the spinal cord. Recent work shows that ROS are involved in the development and maintenance of neuropathic pain.10,19,22 HIV gp120 has been implicated in initiation and/or intensification of ROS.11 Mitochondrial fission may be necessary for the overproduction of ROS.36 In the present study, we found that inhibition of spinal mitochondrial fission function regulated by Drp1 reduced neuropathic pain and mitochondrial superoxide, suggesting that mitochondrial fission plays a critical role in the neuropathic pain symptoms induced by HIV gp120.

It is known that oxidative stress induces mitochondrial fragmentation by inhibiting fusion and enhancing fission.44 Kim et al.23 report that ROS production is increased in cultured melanocytes and melanoma cells with fragmented mitochondria and that mitochondrial dynamics may regulate melanogenesis by modulating ROS signaling pathway. H2O2 administered intradermally into the dorsum of the hindpaws induces mechanical hyperalgesia, which is reversed by intradermal pretreatment with mdivi-1.3 However, in vitro studies show that mdivi-1 reduced oxidative stress and attenuated cell apoptosis in the cultured rat primary hippocampal cells.24 Further evidence that Drp1 is critical in mitochondrial ROS is seen with P110, a novel and selective peptide inhibitor of Drp1 enzyme activity, which also inhibits mitochondrial fission, leading to reduced mitochondrial fragmentation and ROS production, improving mitochondrial membrane potential and enhancing mitochondrial integrity.45,46 Mitochondrial fission was increased after seizures, and the inhibition of mitochondrial fission by mdivi-1 significantly attenuated oxidative stress and reduced neuronal loss after seizures.47 In the present study, we found that either knockdown of Drp1 protein or inhibition of mitochondrial fission function by mdivi-1 reduced spinal mitochondrial superoxide in the gp120-induced neuropathic pain model, suggesting that mitochondrial fission through mitochondrial ROS is involved in chronic pain.

In summary, mitochondrial division is critical for the maintenance of the structure and function of neurons. Although the mechanisms underlying HIV-related neuropathic pain are poorly understood, the current results provide important insights into the pathogenesis of mitochondrial division in the HIV-related neuropathic pain state.

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DISCLOSURES

Name: Hirotsugu Kanda, MD.

Contribution: This author helped conduct the study and analyze the data.

Attestation: Hirotsugu Kanda reviewed the original data and the analysis of the data and approved the final manuscript.

Name: Shue Liu, BS.

Contribution: This author helped conduct the study and analyze the data.

Attestation: Shue Liu approved the final manuscript.

Name: Takafumi Iida, MD.

Contribution: This author helped conduct the study and analyze the data and participated in writing the manuscript.

Attestation: Takafumi Iida reviewed the original data and the analysis of the data and approved the final manuscript.

Name: Hyun Yi, BS.

Contribution: This author helped conduct the study and analyze the data and participated in writing the manuscript.

Attestation: Hyun Yi reviewed the original data and the analysis of the data and approved the final manuscript.

Name: Wan Huang, MD, PhD.

Contribution: This author helped conduct the study and analyze the data and participated in writing the manuscript.

Attestation: Wan Huang reviewed the original data and the analysis of the data and approved the final manuscript.

Name: Roy C. Levitt, MD.

Contribution: This author participated in writing the manuscript.

Attestation: Roy C. Levitt approved the final manuscript.

Name: David A. Lubarsky, MD, MBA.

Contribution: This author helped write the manuscript.

Attestation: David A. Lubarsky approved the final manuscript.

Name: Keith A. Candiotti, MD.

Contribution: This author helped write the manuscript.

Attestation: Keith A. Candiotti approved the final manuscript.

Name: Shuanglin Hao, MD, PhD.

Contribution: This author helped design the whole study and analyze the data and wrote the manuscript.

Attestation: Shuanglin Hao was the archival author/corresponding author and approved the final manuscript.

This manuscript was handled by: Jianren Mao, MD, PhD.

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ACKNOWLEDGMENTS

The authors thank Dr. Chuanhui Dong (Associate Professor and Biostatistician of Neurology, University of Miami Miller School of Medicine, Miami, FL) for statistics assistance.

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