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. Author manuscript; available in PMC: 2019 Nov 22.
Published in final edited form as: Neuroscience. 2018 Feb 5;376:13–23. doi: 10.1016/j.neuroscience.2018.01.058

The neurotoxin DSP-4 induces hyperalgesia in rats that is accompanied by spinal oxidative stress and cytokine production

Jillienne C Touchette 1, Joshua W Little 2, Gerald H Wilken 1, Daniela Salvemini 1, Heather Macarthur 1,*
PMCID: PMC6874213  NIHMSID: NIHMS1542834  PMID: 29421433

Abstract

Central neuropathic pain (CNP) a significant problem for many people, is not well-understood and difficult to manage. Dysfunction of the central noradrenergic system originating in the locus coeruleus (LC) may be a causative factor in the development of CNP. The LC is the major noradrenergic nucleus of the brain and plays a significant role in central modulation of nociceptive neurotransmission. Here, we examined CNS pathophysiological changes induced by intraperotineal administration of the neurotoxin DSP-4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride). Administration of DSP-4 decreased levels of norepinephrine in spinal tissue and cerebrospinal fluid (CSF) and led to the development of thermal and mechanical hyperalgesia over 21 days, that was reversible with morphine. Hyperalgesia was accompanied by significant increases in noradrenochrome (oxidized norepinephrine) and expression of 4-hydroxynonenal in CSF and spinal cord tissue respectively at day 21, indicative of oxidative stress. In addition, spinal levels of pro-inflammatory cytokines (interleukins 1β and 6, tumor necrosis factor-α), as well as the the anti-inflammatory cytokine interleukin10 were also significantly elevated at day 21, indicative that an inflammatory response occurred. The inflammatory effect of DSP-4 presented in this study that includes oxidative stress may be particularly useful in elucidating mechanisms of CNP in inflammatory disease states.

Keywords: Central neuropathic pain, locus coeruleus, DSP-4, norepinephrine, oxidative stress, cytokines

Introduction

Central neuropathic pain (CNP) occurs following pathology to the central nervous system (CNS). Physical damage to the CNS, by spinal cord injury, stroke, tumors, or traumatic brain injury (TBI) for example, can lead to CNP, and several disease states are known to cause neuronal damage that can lead to CNP including multiple sclerosis, epilepsy, and Parkinson’s disease (Finnerup, 2008). Clinically, CNP presents itself differently between patients due to the variety of potential causes, and can be described as spontaneous and unexplained sensations of stabbing, burning, scalding, numbness, aching, and/or tingling, as well as a vague overall sensation of discomfort (Finnerup, 2008; Ford, 2010). As the underlying mechanisms of CNP are under investigation, it remains difficult to treat clinically and patients often endure pain without sufficient pain relief. Current treatments are often not successful in fully alleviating pain, and can have deleterious side effects (Finnerup, 2008). Studies in humans (Urakami et al., 1990; Djaldetti et al., 2004; Schestatsky et al., 2007; Mylius et al., 2009) have revealed an association between spontaneous CNP sensations and hyperalgesia and allodynia, although the relationship has not been fully elucidated.

CNP arises following damage to regions of the CNS involved in central pain modulation pathways. Animal CNP models due to spinal cord injury consistently exhibit hyperalgesia and allodynia (Christensen et al., 1996; Hulsebosch et al., 2000; Tanabe et al., 2009; Densmore et al., 2010). Additionally, CNP in rodents has been described in models of thalamic stroke (Wasserman and Koeberle, 2009), multiple sclerosis (experimental autoimmune encephalomyelitis) (Olechowski et al., 2009), Parkinson’s disease (Carey, 1986; Tassorelli et al., 2007), and traumatic brain injury (Feliciano et al., 2014; Macolino et al.). Several regions of the brain are involved in the complex pain modulation neurocircuitry, including the somatosensory cortex, amygdala, thalamus, periaqueductal grey, rostral ventromedial medulla and locus coeruleus (LC) (see reviews (Gebhart, 2004; Ossipov et al., 2010; Kwon et al., 2014)). The LC noradrenergic system plays a critical role in central modulation of pain, in part, due its descending projections to the spinal cord. Descending projections from the LC interact with pain responsive neurons of trigeminal sensory nuclei and the spinal dorsal horn (Hagihira et al., 1990; Clark and Proudfit, 1991), altering neuronal sensitivity in these regions and thereby modulating nociceptive transmission and the experience of pain (Stamford, 1995). The antinociceptive effects of norepinephrine are primarily mediated through α2-adrenergic receptors on the central terminals of nociceptive primary afferent fibers (Howe and Zieglgansberger, 1987), which inhibit the release of excitatory neurotransmitters by primary afferent terminals and the subsequent activation of secondary projection neurons (Kuraishi et al., 1985a; Kamisaki et al., 1993). Because descending noradrenergic projections from the LC can inhibit the sensation of pain (Margalit and Segal, 1979; Reddy and Yaksh, 1980; Fleetwood-Walker et al., 1985; Proudfit, 1988) and pharmacological inhibition of spinal noradrenergic transmission results in decreased nociceptive thresholds (Reddy et al., 1980; Sagen and Proudfit, 1984), it is hypothesized that dysfunctional descending noradrenergic pathways might contribute to CNP.

In this study, we investigated the effect of systemic administration of the neurotoxin N-(−2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) on thermal and mechanical pain sensitivity. DSP-4 is widely described as a selective neurotoxin for LC axons as it readily crosses the blood-brain barrier and acts as an irreversible inhibitor of the norepinephrine uptake transporter, causing long-lasting depletion of norepinephrine from LC axon terminals (Igari et al., 1977; Jasmin et al., 2003). Here, we expand on previous success using DSP-4 to model CNP in rats (Kudo et al., 2010, 2011; Kinoshita et al., 2013), showing that DSP-4 administration depletes spinal norepinephrine leads to a hyperalgesic state that is accompanied by increased spinal oxidative stress and inflammatory cytokine production.

Experimental Procedures

Animals

Adult male Sprague Dawley rats (Charles River Laboratories, MA, USA) weighing 250 – 300g were used for all experiments. Rats were housed in a controlled environment (12hr light/dark cycle) with food and water provided ad libitum in groups of three until surgeries were performed, after which all were singly housed. All animal experiments were conducted in accordance with the guidelines mandated in the National Institutes of Health Guide for the Care and Use of Laboratory Animals and International Association for the Study of Pain and were approved by the Institutional Animal Care and Use Committee of Saint Louis University. A total of 74 rats were used for the behavioral experiments this study. The number of animals used for each separate experiment is indicated below.

Reagent preparation and administration

All compounds were obtained from Sigma-Aldrich (St. Louis, MO) and freshly prepared for each experiment. DSP-4 was weighed and dissolved in sterile 0.9% saline to 50mg/mL. The rats received a single intraperotineal injection of DSP-4 (50mg/kg) or saline on day 0 prior to behavioral testing. Morphine was administered to a subset of rats at 6mg/kg via a subcutaneous injection.

Behavioral analysis

Behavioral testing was performed in prior to drug administration (day 0) and then subsequently every 7 days for 21 days. In 18 rats (8 treated with DSP-4 and 10 treated with saline), thermal hyperalgesia was determined using the Hargreaves method (Hargreaves et al., 1988) and defined as a significant reduction in the withdrawal response time to a noxious heat stimulus applied to the hindpaw. Before thermal stimulation, rats were individually confined to an enclosed plexiglass chamber on a clear glass platform and allowed to habituate for 15min. An infrared bulb (UGO Basile, Italy) was positioned under the glass platform to deliver a thermal stimulus to the plantar surface of the hindpaw. The animal’s withdrawal latencies were recorded electronically, using a cutoff latency of 20 seconds. Because DSP-4 affects the LC noradrenergic system bilaterally, the mean paw withdrawal latency of both hindpaws was reported. In 13 rats (7 DSP-4 and 6 saline), mechanical hyperalgesia was determined using the Randall and Selitto paw pressure test (Randall and Selitto, 1957) using a Ugo-Basile analgesiometer that applies a linearly increasing mechanical force to the dorsum of the rat’s hind paw. The nociceptive threshold was defined as the force (g) at which the rat withdrew its paw (cutoff set at 200g). Animals receiving DSP-4 did not display signs of morbidity at the time of behavior testing. In a subset of animals treated with DSP-4, morphine was administered on day 21 and thermal (n=4) and mechanical (n=4) tests were performed at 30min, 1hr, 2hr, and 5hr post-morphine administration. The behavioral analysis described herein was performed by the experimenter to confirm findings from previous studies (Kudo et al., 2010, 2011; Kinoshita et al., 2013). The morphine experiments were performed in DSP-4-treated animals using the same animals as internal controls and therefore could not be blinded. The new information provided in this manuscript is the effect of DSP-4 on inflammation/oxidative stress, and all experiments as follows were appropriately controlled and measured.

High pressure liquid chromatography

Twenty-one days after administration of DSP-4, cerebrospinal fluid (CSF) (n=8 DSP-4, n=9 saline) was collected as was LC (n=11 DSP-4, n=9 saline) and spinal cord tissue (n=9 DSP-4, n=13 saline), and assayed for norepinephrine levels via high pressure liquid chromatography (HPLC) with electrochemical detection. Additionally, oxidized norephephrine in the form of noradrenochrome was measured in CSF (n=12 DSP-4, n=11 saline) and LC tissue (n=4 DSP-7, n=4 saline) via HPLC with electrochemical detection (Ochs et al., 2005). Tissue levels of both norepinephrine and noradrenochrome (ng) were normalized to protein levels determined by a bicinchoninic acid (BCA) assay and expressed as ng/mg protein. Levels of norepinephrine or noradrenochrome in CSF are reported as total nM concentration. Data was analyzed using independent sampling (one data point per animal) for each individual analysis.

Immunohistochemistry

Twenty-one days after administration of DSP-4 (n=8) or saline (n=4), rats were deeply anesthetized with ketamine/xylazine (87:13 mg/kg; i.p.) and perfused transaortically first with a rinse solution containing 0.01M PBS (pH 7.4), 0.9% sodium chloride and 2.5% sucrose followed by 0.1M PBS (pH 7.4) containing 4% paraformaldehyde and 2.5% sucrose. The brain and lumbar spinal cord were removed and were post-fixed overnight in 4% paraformaldehyde, and then cryoprotected in 30% sucrose solution at 4°C for 3 days. Using a sliding microtome, 4 adjacent series of 40μm thick sections of the pons containing the LC were cut in the coronal plane. Identification of the pons/LC region was confirmed using a brain atlas (Paxinos and Watson, 1998). Series of sections were stored at −20°C prior to processing in a cryoprotectant solution composed of 30% sucrose and 33% ethylene glycol in PBS. For immunohistochemistry, a series of sections was thoroughly rinsed in 0.1M PBS and incubated in 1% sodium borohydride for 15min followed by rinsing with PBS. Sections were incubated at room temperature with a primary antibody mouse anti-tyrosine hydroxylase (TH, 250ng/mL, BD Biosciences) or mouse anti-NeuN (1μg/mL, Millipore) in 0.1M PBS containing 0.1% Triton X-100 (PBS-T) overnight. The next day, sections were incubated with biotinylated goat anti-mouse IgG secondary antibody (1.25μg/mL, BD Biosciences) in PBS-T for 2 hr, then in ABC reagent (1:200, Vector Laboratories) in PBS-T for 2 hr. A DAB reaction was used to visualize immunoreactivity. A black reaction product for staining TH-positive neurons was visualized using a solution of 0.015% 3,3’-diaminobenzidine, 0.4% nickel ammonium sulfate, and 0.0003% hydrogen peroxide in 0.025M Tris buffer (pH 8.0). A brown reaction product for staining neuronal nuclei was visualized using a solution of 0.05% DAB and 0.0003% hydrogen peroxide in 0.05M PBS. After rinsing, sections were mounted on gelatin-coated slides and coverslipped with Permount (Fishers, St. Louis, MO). Representative images from n=1 rat per group are shown. In 4 rats (n=2 DSP-4, n=2 saline), spinal cord sections were incubated in mouse anti-4-hydroxynonenal (4-HNE) (25μg/mL, Percipio Biosciences) and mouse anti-neuronal nuclei (NeuN) (1μg/mL, Millipore) primary antibodies, without prior antigen retrieval. The following day, immunohistochemistry was performed as described above. Images were visualized on an Olympus BX51 microscope with a 10× or 20× objective lens and captured with a DVC 2000C-00-GE-MBF digital camera. Image analysis in the dorsal horn was determined using the NIH freeware program ImageJ (version 1.43). The superficial dorsal horns (laminae I and II) were outlined on images bilaterally on the 10× images using the ImageJ region of interest tool. Mean 4-HNE immunostaining was quantified using the Analyze Particle tools using the MaxEntropy threshold tool. For 4-HNE quantification, 2–3 spinal cord sections from each rat were counted using non-independent sampling; therefore this data applies only to this set of sampled animals and may not apply to the general population of rats.

Cytokine expression

Twenty-one days after administration of DSP-4 (n=8) or saline (n=8), fresh spinal lumbar enlargements (L4–L6) were collected and diluted in lysis buffer (20mM TRIS pH 7.4, 150mM NaCl, 2.5% glycerol, 1% Triton X-100, 1% Chaps, 1mM EDTA, 1mM EGTA, 0.1% SDS, 1% Halt protease and phosphatase inhibitor cocktail, 2mM PMSF) with a 1:10 w/v ratio. Tissues were sonicated using a probe sonicator. Lysates were centrifuged (13,000×g, 15min) at 4°C and supernatants were collected and stored at −20°C. Protein concentration was determined using the BCA protein assay (Pierce, Rockford, IL). A Milliplex Magnetic Bead Panel kit (Milliplex Rat Cytokine/Chemokine magnetic bead panel kit, EMD Millipore, Billerica, MA) was used to quantify the expression levels of TNF-α, IL-1β, IL-6, IL-10, IL-17A, and IFNγ. The beads were evaluated in a Luminex 200 instrument, and the data were collected and analyzed using Milliplex Analyst software (Millipore, Billerica, MA, USA). A minimum of 50 beads were analyzed. Results were expressed as picograms per milligram of wet tissue (pg/mg) using independent sampling.

Immunofluorescence

Immunofluorescence was performed using modifications of previously described methods (Little et al., 2012a). Briefly, rats were perfusion-fixed, and the spinal cords were removed, post-fixed, and cryoprotected as described above. The spinal lower lumber enlargement (L4–L6) was cut by freezing microtome into 4 adjacent series of sections of 30 μm thickness. Immunofluorescence was performed as described above, for astrocytic glial fibrillary acid protein (GFAP) (in n=8 DSP-4, n=5 saline-treated rats) using a mouse monoclonal anti-GFAP primary antibody (275ng/mL, Sigma) incubated at room temperature overnight. Microglial ionized calcium-binding adaptor molecule (Iba-1) was detected (in n=7 DSP-4, n=6 saline-treated rats) using a rabbit polyclonal anti-Iba-1 primary antibody (0.5ng/μL, WAKO) incubated at room temperature overnight. The secondary antibodies used were Alexa Fluor 488-conjugated Goat Anti-Mouse IgG (8μg/mL, Invitrogen) or Alexa Fluor 568-conjugated goat anti-rabbit IgG secondary antibody (8μg/mL, Invitrogen), incubated at room temperature for 2hr. Image acquisition was performed with a Olympus FV 1000 confocal microscope with a 10× objective (UPLSAPO; NA 0.40). Image analysis and mean fluorescence intensity in the dorsal horn was determined using the NIH freeware program ImageJ (version 1.43). 2–3 sections from different lower lumbar levels were collected from each animal and confirmed using an atlas (Paxinos and Watson, 1998). The superficial dorsal horns (laminae I and II) were outlined on images bilaterally by a blinded experimenter using the ImageJ region of interest tool. MFI was calculated as a combined value for each animal and reported as fold change compared to the vehicle group.

Western analysis

Fresh spinal cord tissue corresponding to the lower lumbar enlargement (L4–L6) was collected 21 days after administration of DSP-4 (n=9) or saline (n=9) and diluted in lysis buffer (20 mM TRIS pH 7.4, 150 mM NaCl, 2.5% glycerol, 1% Triton X-100, 1% Chaps, 1 mM EDTA, 1 mM EGTA, 0.1% SDS, 1% Halt protease and phosphatase inhibitor cocktail, 2mM PMSF) with a 1:10 w/v ratio. Tissues were sonicated using a probe sonicator (Fisher Scientific, Pittsburgh, PA) and incubated on ice. The lysates were centrifuged (13,000 × g, 15min) at 4°C and supernatants were collected. The supernatants were stored at −20°C immediately. Protein concentration was determined using the BCA protein assay (Pierce, Rockford, IL). Lysates were brought to an equal volume with lysis buffer and supplemented with 6× Laemmli buffer containing mercaptoethanol. Lysates were run on a 4–15% Mini Protean TGX gel and transferred to PVDF membrane. The membrane was blocked and incubated with primary antibody against GFAP (1:5000, DAKO), coronin-1α (1:5000, Abcam) and β-actin (1:5000, Sigma) incubated overnight at 4°C. Bound antibodies were visualized with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence. Relative protein expression was quantified by band densitometry with Chemidox XRS+ Documentation System Bio-Rad software. All densitometry data were normalized to β-actin bands.

Statistical analyses

All quantitative results are expressed as mean ± standard error of the means (s.e.m.). Statistical differences between means were evaluated using Student’s t-tests. The Mann-Whitney rank sum test was applied when the samples were not drawn from normally distributed populations with equal variances. Statistical differences in behavior between and within groups were determined using a two-way repeated measures ANOVA with Bonferroni posthoc analysis for multiple comparisons. Differences in behavior after morphine treatment within the DSP-4-treated group were determined using a one-way ANOVA with Dunnett’s comparisons. All statistical analyses were performed using Prism 5 (GraphPad), and significance was defined as p<0.05.

Results

DSP-4 administration decreases spinal and cerebrospinal norepinephrine levels

Twenty one days after administration of DSP-4 (50 mg/kg, i.p.), norepinephrine levels were significantly reduced in the CSF [t15=6.09, P<0.0001] (Fig 1A), and spinal cord tissue [U207,46=1.00, P=0.0001] (Fig 1B) compared to saline-treated rats. In the LC brain tissue, norepinephrine levels were not significantly different in rats treated with DSP-4 compared to saline treatment [U103,107=41.00, P=0.54] (Fig 1C). Immunohistochemical staining of the LC cell bodies at day 21 after DSP-4 administration revealed the presence of neuronal immunoreactivity for TH and NeuN in both DSP-4 and vehicle-treated rats (Fig 1D–G).

Figure 1: CSF and spinal cord, but not LC, norepinephrine levels are reduced in DSP-4-treated rats.

Figure 1:

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Norepinephrine levels measured 21 days after administration of DSP-4 in the (A) CSF (n=8 DSP-4, n=9 vehicle), (B) spinal cord tissue (n=9 DSP-4, n=13 vehicle), and (C) LC tissue (n=11 DSP-4, n=9 vehicle) using HPLC-ED analysis. ***P<0.0001 compared to vehicle treatment. Representative micrograph of 40μm coronal brain sections in a vehicle (D-E) and DSP-4-treated rat (F-G) immunostained for TH (brown) and NeuN (black) on day 21 to visualize double-labeled noradrenergic LC neurons. Scale bar is 200μm. NE=norepinephrine.

DSP-4 administration induces thermal and mechanical hyperalgesia

We tested the effect of DSP-4 (50 mg/kg, i.p.) on result in thermal and mechanical nociception. A Hargreave’s test revealed the development of thermal hyperalgesia in DSP-4-treated rats, as demonstrated by a time-dependent decrease in paw withdrawal latency (PWL; s) [F3,48=12.35, P=0.0007; post-hoc tests vs Day 0 were P<0.05 for Day 14 and P<0.001 for Day 21] that was significantly different from vehicle-treated rats on Day 21 [F3,48=19.46, P=0.011; post-hoc tests were P<0.01 on Day 21] (Fig 2A). The Randall Selitto paw pressure test revealed the development of mechanical hyperalgesia in DSP-4-treated rats as demonstrated by a decrease in paw withdrawal threshold (PWT; g) compared to vehicle-treated rats [F3,33=32.32, P=0.0008; post-hoc tests were P<0.01 on Days 14 and 21] (Fig 2B).

Figure 2: DSP-4-treated rats develop thermal and mechanical hyperalgesia, which is reversible by treatment with morphine.

Figure 2:

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(A) Average paw withdrawal latency to thermal stimulus (n= 8 DSP-4, 10 vehicle). (B) Average paw withdrawal threshold to mechanical stimulus (n=7 DSP-4, 6 vehicle). The Y-axes have been cropped for clarity. **P<0.01 for DSP-4 vs. vehicle and †P<0.05, ††P<0.01, and †††P<0.001 for DSP-4 vs day 0. Effect of morphine (6mg/kg, s.c. administered on Day 21) on average paw withdrawal latency to noxious (C) thermal and (D) mechanical stimulus (n=4 per group) in DSP-4-treated rats.*P<0.05, **P<0.01, ***P<0.001 compared to Day 0 and † P<0.001 compared to Day 21.

In separate groups of rats, morphine (6 mg/kg, s.c.) was administered after the final experimental behavior measurement on day 21 to confirm the behavioral responses observed were related to nociception. A one-way ANOVA of Hargreave’s test results confirmed the development of thermal hyperalgesia after DSP-4 administration [F3,12=14.24, P=0.0003; post-hoc tests vs Day 0 were P<0.01 for Day 14 and P<0.001 for Day 21]. Hyperalgesic behaviors were completely reversed by morphine 30 min post administration, and returned to pre-morphine levels by 5hrs [F3,15=32.79, P<0.0001; post-hoc tests vs Day 21 were P<0.001 for 0.5, 1, and 2 hours after morphine administration] (Fig 2C). In another set of rats, a one-way ANOVA of Randall Selitto test results confirmed the development of mechanical hyperalgesia after DSP-4 administration [F3,12=6.34, P=0.0078; post-hoc tests vs Day 0 were P<0.05 at Day 7 and P<0.01 at Day 14 and Day 21]. Hyperalgesic behaviors were completely reversed by morphine 30 min post administration, and returned to pre-morphine levels by 5 hours [F4,15=36.03, P<0.0001; post-hoc tests vs Day 21 were P<0.001 for 0.5, 1, and 2 hours after morphine administration] (Fig 2D).

DSP-4-induced hyperalgesia is accompanied by spinal oxidative stress

We then sought to determine whether administration of DSP-4 resulted in oxidative stress. On day 21 after administration of DSP-4, we measured noradrenochrome levels in the CSF using HPLC-ED analysis. Noradrenochrome in CSF was significantly increased compared to vehicle-treated rats [t21=2.57, P=0.018] (Fig 3A). In contrast, noradrenochrome levels in the LC tissue was not significantly increased by DSP-4 [U13,23=3.00, P=0.20] (Fig 3B). We also performed an immunohistochemical analysis for the presence of 4-HNE in fixed spinal cord sections through the lower lumbar enlargement (L4–L6), and observed a significant increase in 4-HNE staining in the superficial dorsal horn of DSP-4-treated animals compared to saline treatment [U21,57=0.00, P=0.0022] (Fig 3C–G).

Figure 3: Oxidative stress markers are present in CSF and spinal cord tissue during DSP-4-induced hyperalgesia.

Figure 3:

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Noradrenochrome levels on day 21, measured by HPLC-ED analysis in the (A) CSF (n=12 DSP-4, n=11 vehicle) and (B) LC tissue (n=4 per group). *P<0.05 compared to vehicle treatment. C) Average 4-HNE immunostaining per section in superficial dorsal horn in rats treated with vehicle or DSP-4 (n=2–3 sections from 2 rats per group). The Y-axis has been broken for clarity. D) Representative spinal cord section after immunohistochemical analysis for 4-HNE in a vehicle-treated rat and F) a rat treated with DSP-4 at 10× magnification. Black staining shows location of 4-HNE within the superficial dorsal horn, and brown staining shows the presence of neuronal nuclei (NeuN). Panels E) and G) are 20× magnifications of outlined area in panel D) and F), respectively. Scale bar is 250μm in panels D and F and 125μm in panels E and G.

DSP-4-induced hyperalgesia is associated with enhanced pro-inflammatory cytokine expression in the spinal cord

Finally, we investigated whether DSP-4 administration increases the expression of pro-inflammatory cytokines in the spinal cord. A magnetic bead panel assay was performed on lower lumbar spinal cord tissue on day 21 after administration of DSP-4, and revealed increased levels of TNFα [t14=2.95, P=0.011], IL-17A [t14=2.36, P=0.034] IL-10 [t14=3.01, P=0.0094], and IL-6 [t14=2.61, P=0.021], but not IL-1β [t14=1.99, P=0.067] or IFNγ [t14=1.92, P=0.075] compared to vehicle treatment (Fig 4A). A Western analysis (Fig 4B) on whole lower lumbar spinal tissue did not detect an increase in the expression of microglial coronin-1α [t16=0.51, P=0.62] (Fig 4C) or GFAP [t16=1.27, P=0.22] (Fig 4D). Immunofluorescence staining on lower lumbar sections of the spinal cord revealed no significant changes in the expression of microglial protein Iba-1 [U40,51=19.00, P=0.84] and GFAP [U27,64=12.00, P=0.28] in laminae I and II of the superficial dorsal horn (Fig 4E–J).

Figure 4: DSP-4-induced hyperalgesia is associated with spinal inflammation by 21 days.

Figure 4:

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(A) Results of cytokine magnetic bead panel assay for TNFα, IL-1β, IL-6, IL-10, IL-17A, and IFNγ in (n=8 per group). *P<0.05 compared to vehicle treatment. (B-D) Representative bands from Western analyses for GFAP and coronin-1α on lower lumbar spinal cord tissue (n=9 per group). (E-F) Representative images of the L4 level immunostained for Iba-1. (G) The mean fluorescence intensity of Iba-1 in laminae I and II normalized to vehicle and expressed as fold change (n=6 vehicle, n=7 DSP-4). (H-I) Representative images of the L4 level immunostained for GFAP. (J) The mean fluorescence intensity of GFAP in lamina I and II normalized to vehicle and expressed as fold change (n=5 vehicle, n=8 DSP-4). Scale bars are 100μm.

Discussion

In this study, we show that systemic administration of the noradrenergic neurotoxin DSP-4 reduces central levels of norepinephrine and leads to the development of thermal and mechanical hyperalgesia in rats. We also confirmed the findings by Roczniak et al. (Roczniak et al., 2016) that CNP induced by DSP-4 is reversible by treatment with morphine. Importantly, we demonstrate for the first time that DSP-4-induced hyperalgesia is accompanied by increases in spinal cord oxidative stress and pro-inflammatory cytokines 21 days after administration, which may be a major contribution to the observed hyperalgesic effect of DSP-4. DSP-4 is often described as an LC-specific neurotoxin because of the observed reduction of dopamine-β-hydroxylase activity (Fritschy et al., 1990), TH immunoreactivity (Booze et al., 1988) and presynaptic α2 receptors in LC axon terminals (Prieto and Giralt, 2001), reduced levels of norepinephrine and norepinephrine transporters (Ross, 1976; Jaim-Etcheverry and Zieher, 1980; Jonsson et al., 1981), and decreased LC neuronal firing (Olpe et al., 1983). Consistent with these findings, we report that DSP-4 reduced norepinephrine levels in both spinal cord tissue and CSF. However, the mechanism by which DSP-4 remains controversial as there are several reports that DSP-4 does not reduce NE levels in the LC cell bodies and does not cause degeneration. For example, Booze, et al. reported no evidence of fiber degeneration up to 5 weeks after DSP-4 treatment in rats (Booze et al., 1988). Similarly, Szot et al. published a thorough investigation of the effect of DSP-4 on the rat LC system, and observed a rapid reduction in NE and NE transporter levels in many brain regions innervated by the LC (Szot et al., 2010). Changes in LC neuron firing patterns were described, but this report concluded that DSP-4 does not alter LC noradrenergic neurons themselves, but does affect noradrenergic terminals locally, as evidenced by reductions in transmitter and markers at terminal regions including the prefrontal cortex, hippocampus, cerebellum, and septum/bed nucleus of the stria terminalis up to 3 months after administration. Consistent with this, we observed no substantial decrease in NE levels or TH immunoreactivity in the LC cell bodies 21 days after administration of DSP-4.

The behavioral findings described in this study are consistent with multiple behavioral (Reddy and Yaksh, 1980; Sagen and Proudfit, 1984; Kuraishi et al., 1985b), pharmacological (Reddy et al., 1980; Fleetwood-Walker et al., 1985; Yaksh, 1985; Proudfit, 1988; Tjolsen et al., 1990; Pires et al., 2011), and electrical stimulation studies (Margalit and Segal, 1979; Hammond and Yaksh, 1984; Yeomans et al., 1992) demonstrating the role of the LC in modulating nociception. Descending LC neurons have long been known to inhibit spinal nociceptive transmission (for reviews, see (Millan, 2002; Llorca-Torralba et al., 2016)). Activation of LC neurons reduces stimulus-evoked activity of dorsal horn neurons and inhibits pain transmission (Tsuruoka et al., 2011), particularly in inflammatory pain models (Tsuruoka and Willis, 1996a, b; Martin et al., 1999). Lesion studies have provided conflicting evidence as to whether tonic noradrenergic transmission affects non-pathological pain, as some studies show that electrolytic or noradrenergic lesions of the LC alter baseline responses to noxious in previously uninjured animals (Sasa et al., 1977; Bodnar et al., 1978; Kudo et al., 2010, 2011), while others report no change (Martin et al., 1999; Jasmin et al., 2003). However, recent evidence suggests the existence of distinct but compatible systems of spinal-versus prefrontal-projecting LC neuronal populations that inhibit or facilitate pain, respectively (Llorca-Torralba et al., 2016; Hirschberg et al., 2017; Taylor and Westlund, 2017). The majority of the output from the LC consists of ascending fiber projections that target the hypothalamus (Osaka and Matsumura, 1994), thalamus (Westlund et al., 1991; Voisin et al., 2005), amygdala (Wallace et al., 1989), hippocampus (Pascual et al., 1992), periaqueductal gray (Pickel et al., 1974), and vast regions of the cortex (Seguela et al., 1990) as well as the cerebellum. LC neural pathways targeting the dorsal reticular nucleus, medial prefrontal cortex and trigeminal nucleus caudalis may facilitate pain (Martins et al., 2013; Kaushal et al., 2016). Furthermore, it has been suggested that at later time points after nerve injury, certain LC neuronal subpopulations become pro-nociceptive (Brightwell and Taylor, 2009; Kaushal et al., 2016) and contribute to the maintenance of neuropathic pain (Olson and Fuxe, 1972; Hickey et al., 2014). The hyperalgesic effects observed in the current study were specific to the spinal cord as the spinal NE levels were specifically depleted. The existing literature on DSP-4 does not offer evidence of non-specific side effects of DSP-4 leading to the development of hyperalgesia. Rather, the literature supports DSP-4 as a disrupter of noradrenergic transmission due to axonal disruption (Ross and Renyl, 1976; Grzanna et al., 1989; Fritschy and Grzanna, 1991; Cheetham et al., 1996; Hormigo et al., 2012)

It remains unclear whether increased pain sensitivity in animals treated with DSP-4 is solely due to the loss of descending anti-nociceptive noradrenergic tone and/or a result of chronic injury from to oxidative damage and inflammation in the dorsal horn. Oxidative stress is a widely recognized hallmark of neuropathic pain (Salvemini et al., 2011; Janes et al., 2012; Little et al., 2012a). Here, we show that there was an increase in noradrenochrome in the spinal cord and CSF of rats treated with DSP-4. The presence of noradrenochrome in the CSF is indicative of oxidative stress, as noradrenochrome is the oxidized aminochrome product of norepinephrine (Graham, 1978; Sulzer et al., 2000). Interestingly, we and others have shown that dopaminochrome, the aminochrome species of dopamine oxidation, induces neurotoxicity both in vitro and in vivo (Linsenbardt et al., 2009; Linsenbardt et al., 2012; Touchette et al., 2016), begging the question as to the role noradrenochrome may play in the development of CNP in rats treated with DSP-4. In addition, we observed lipid peroxidation in the form of 4-HNE (Esterbauer et al., 1991) in the spinal dorsal horn of DSP-4-treated animals.

In addition to oxidative stress, we observed increased expression of TNFα, IL-6, IL-10, and IL-17A in rats exposed to DSP-4, indicative of neuroinflammation. These inflammatory mediators are known to be upregulated in neuropathic pain states (for reviews, see (Scholz and Woolf, 2007; Grace et al.)). Neuroinflammation occurs following CNS damage or enhanced nociceptive signaling and can lead to microglia hyperactivation, which may recruit astrocytes and T cells that then release cytokines and chemokines (for reviews, see (Scholz and Woolf, 2007; Grace et al., 2014)). These inflammatory mediators, along with reactive oxygen and nitrogen species, can directly modulate excitatory and inhibitory synaptic transmission in the spinal dorsal horn (Salvemini et al., 2011; Little et al., 2012b). The combination of neuroinflammation, increased neuronal excitability, and disrupted spinothalamic neurocircuitry is thought to be the cause of CNP (Watkins et al., 2001; Ossipov et al., 2010; Grace et al., 2014). Indeed, intrathecal administration of cytokines including TNFα have been reported to elicit thermal and mechanical hypersensitivity (Reeve et al., 2000; Narita et al., 2008; Gao et al., 2009). Additionally, neuronal norepinephrine can inhibit inflammation by activating adrenergic receptors on glial cells and suppressing the release of inflammatory mediators (Feinstein et al., 2002; Heneka et al., 2003). Thus, we speculate that DSP-4 has a dual mechanism of upregulating inflammatory mediators by both damaging neurons and neuronal damage and disinhibiting glia.

In contrast to elevated cytokine levels, we did not observe microglial and astrocytic hyperactivation in the form of changes in spinal cord coronin-1α, Iba-1 or GFAP. A limitation of our study is that all of the biochemical and immunohistochemical results are taken from day 21 after administration of DSP-4. This time point was chosen based on studies investigating the time-course of norepinephrine depletion after administration of DSP-4 in rats (Ross et al., 1973; Ross, 1976; Jaim-Etcheverry and Zieher, 1980), however it does not account for the dynamics of glia activation and cytokine release that occur in response to neuronal damage induced by DSP-4. It is possible that glial are involved earlier in the onset of hyperalgesic responses and by day 21 are no longer morphologically hyperactivated, but are still promoting pro-inflammatory cytokine levels (DeLeo et al., 2004; Salter, 2004; Guo et al., 2007). However, glia can promote pro-inflammatory states without clear indication of glial hyperactivation (e.g., morphological changes) (Hains and Waxman, 2006; Ji and Suter, 2007; Clark et al., 2010). Microglia demonstrate hyperactivation within 1–3 days following nerve injury; however, microglia-dependent signaling pathways, especially the p38 mitogen-activated protein kinase, contribute to the maintenance of neuropathic pain through 21 days post nerve injury (Jin et al., 2003; Ji and Suter, 2007). This p38-mediated process occurs through the upregulation of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, etc.) (Ji and Suter, 2007), in agreement with the findings of this study. Thus, it is possible that microglia are contributing to neuroinflammation during DSP-4-induced CNP without demonstrating hyperactivation. It is also possible that the increase in cytokines observed in DSP-4-treated rats were released by inflammatory cells other than astrocytes or microglia, such as T cells, which are known to migrate across the blood-brain barrier and release cytokines in response to neuronal signals and chemokines (Costigan et al., 2009; Grace et al., 2014). Additional shorter-term studies need to be carried out to correlate the precise time course of hyperalgesia to inflammation after treatment with DSP-4.

In conclusion, we have provided evidence that oxidative stress and inflammatory responses occur in the spinal cord after noradrenergic disruption with DSP-4. These processes, in addition to disruption in noradrenergic signaling, may lead to hyperexcitability and the development of CNP behaviors. Although noradrenergic reuptake inhibitors can be effective for treating CNP, targeting the central noradrenergic system itself has not been successful therapeutically as currently available α2 adrenergic agonists result in side effects such as sedation and hypotension (Giovannoni et al., 2009). If neuroinflammation and oxidative stress are indeed required for the development of CNP, patients with CNP etiology originating in the CNS may benefit from the drug development efforts for peripheral neuropathies that are currently aimed at normalizing pathological oxidative and neuroimmune transmission.

Highlights.

  • DSP-4 reduces norepinephrine levels in rat spinal cord

  • DSP-4 induces oxidative stress and increased inflammatory cytokine levels in the spinal cord

  • DSP-4 treated rats develop thermal and mechanical hypersensitivity

Acknowledgements

We wish to thank Drs. Timothy Doyle and Kali Janes for their helpful discussions and technical suggestions. This work was supported by the National Institutes of Health [NIGMS008306].

Abbreviations:

CNP

central neuropathic pain

CNS

central nervous system

DSP-4

N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine

LC

locus coeruleus

SC

Spinal Cord

CSF

Cerebrospinal Fluid

TH

tyrosine hydroxylase

References

  1. Bodnar RJ, Ackermann RF, Kelly DD, Glusman M (1978), Elevations in nociceptive thresholds following locus coeruleus lesions. Brain Res Bull. 3:125–130. [DOI] [PubMed] [Google Scholar]
  2. Booze RM, Hall JA, Cress NM, Miller GD, Davis JN (1988), DSP-4 treatment produces abnormal tyrosine hydroxylase immunoreactive fibers in rat hippocampus. Exp Neurol. 101:75–86. [DOI] [PubMed] [Google Scholar]
  3. Brightwell JJ, Taylor BK (2009), Noradrenergic neurons in the locus coeruleus contribute to neuropathic pain. Neuroscience. 160:174–185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carey RJ (1986), Acute ipsilateral hyperalgesia and chronic contralateral hypoalgesia after unilateral 6-hydroxydopamine lesions of the substantia nigra. Exp Neurol. 91:277–284. [DOI] [PubMed] [Google Scholar]
  5. Cheetham SC, Viggers JA, Butler SA, Prow MR, Heal DJ (1996), [3H]nisoxetine--a radioligand for noradrenaline reuptake sites: correlation with inhibition of [3H]noradrenaline uptake and effect of DSP-4 lesioning and antidepressant treatments. Neuropharmacology. 35:63–70. [DOI] [PubMed] [Google Scholar]
  6. Christensen MD, Everhart AW, Pickelman JT, Hulsebosch CE (1996), Mechanical and thermal allodynia in chronic central pain following spinal cord injury. Pain. 68:97–107. [DOI] [PubMed] [Google Scholar]
  7. Clark AK, Staniland AA, Marchand F, Kaan TK, McMahon SB, Malcangio M (2010), P2X7-dependent release of interleukin-1beta and nociception in the spinal cord following lipopolysaccharide. J Neurosci. 30:573–582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clark FM, Proudfit HK (1991), The projection of locus coeruleus neurons to the spinal cord in the rat determined by anterograde tracing combined with immunocytochemistry. Brain Res. 538:231–245. [DOI] [PubMed] [Google Scholar]
  9. Costigan M, Moss A, Latremoliere A, Johnston C, Verma-Gandhu M, Herbert TA, Barrett L, Brenner GJ, Vardeh D, Woolf CJ, Fitzgerald M (2009), T-cell infiltration and signaling in the adult dorsal spinal cord is a major contributor to neuropathic pain-like hypersensitivity. J Neurosci. 29:14415–14422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. DeLeo JA, Tanga FY, Tawfik VL (2004), Neuroimmune activation and neuroinflammation in chronic pain and opioid tolerance/hyperalgesia. Neuroscientist. 10:40–52. [DOI] [PubMed] [Google Scholar]
  11. Densmore VS, Kalous A, Keast JR, Osborne PB (2010), Above-level mechanical hyperalgesia in rats develops after incomplete spinal cord injury but not after cord transection, and is reversed by amitriptyline, morphine and gabapentin. Pain. 151:184–193. [DOI] [PubMed] [Google Scholar]
  12. Djaldetti R, Shifrin A, Rogowski Z, Sprecher E, Melamed E, Yarnitsky D (2004), Quantitative measurement of pain sensation in patients with Parkinson disease. Neurology. 62:2171–2175. [DOI] [PubMed] [Google Scholar]
  13. Esterbauer H, Schaur RJ, Zollner H (1991), Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic Biol Med. 11:81–128. [DOI] [PubMed] [Google Scholar]
  14. Feinstein DL, Heneka MT, Gavrilyuk V, Dello Russo C, Weinberg G, Galea E (2002), Noradrenergic regulation of inflammatory gene expression in brain. Neurochem Int. 41:357–365. [DOI] [PubMed] [Google Scholar]
  15. Feliciano DP, Sahbaie P, Shi X, Klukinov M, Clark JD, Yeomans DC (2014), Nociceptive sensitization and BDNF up-regulation in a rat model of traumatic brain injury. Neurosci Lett. 583:55–59. [DOI] [PubMed] [Google Scholar]
  16. Finnerup NB (2008), A review of central neuropathic pain states. Curr Opin Anaesthesiol. 21:586–589. [DOI] [PubMed] [Google Scholar]
  17. Fleetwood-Walker SM, Mitchell R, Hope PJ, Molony V, Iggo A (1985), An alpha 2 receptor mediates the selective inhibition by noradrenaline of nociceptive responses of identified dorsal horn neurones. Brain Res. 334:243–254. [DOI] [PubMed] [Google Scholar]
  18. Ford B (2010), Pain in Parkinson’s disease. Mov Disord. 25 Suppl 1:S98–103. [DOI] [PubMed] [Google Scholar]
  19. Fritschy JM, Geffard M, Grzanna R (1990), The response of noradrenergic axons to systemically administered DSP-4 in the rat: an immunohistochemical study using antibodies to noradrenaline and dopamine-beta-hydroxylase. J Chem Neuroanat. 3:309–321. [PubMed] [Google Scholar]
  20. Fritschy JM, Grzanna R (1991), Selective effects of DSP-4 on locus coeruleus axons: are there pharmacologically different types of noradrenergic axons in the central nervous system? Prog Brain Res. 88:257–268. [DOI] [PubMed] [Google Scholar]
  21. Gao YJ, Zhang L, Samad OA, Suter MR, Yasuhiko K, Xu ZZ, Park JY, Lind AL, Ma Q, Ji RR (2009), JNK-induced MCP-1 production in spinal cord astrocytes contributes to central sensitization and neuropathic pain. J Neurosci. 29:4096–4108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gebhart GF (2004), Descending modulation of pain. Neurosci Biobehav Rev. 27:729–737. [DOI] [PubMed] [Google Scholar]
  23. Giovannoni MP, Ghelardini C, Vergelli C, Dal Piaz V (2009), Alpha2-agonists as analgesic agents. Med Res Rev. 29:339–368. [DOI] [PubMed] [Google Scholar]
  24. Grace PM, Hutchinson MR, Maier SF, Watkins LR (2014), Pathological pain and the neuroimmune interface. Nat Rev Immunol. 14:217–231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Graham DG (1978), Oxidative pathways for catecholamines in the genesis of neuromelanin and cytotoxic quinones. Mol Pharmacol. 14:633–643. [PubMed] [Google Scholar]
  26. Grzanna R, Berger U, Fritschy JM, Geffard M (1989), Acute action of DSP-4 on central norepinephrine axons: biochemical and immunohistochemical evidence for differential effects. J Histochem Cytochem. 37:1435–1442. [DOI] [PubMed] [Google Scholar]
  27. Guo W, Wang H, Watanabe M, Shimizu K, Zou S, LaGraize SC, Wei F, Dubner R, Ren K (2007), Glial-cytokine-neuronal interactions underlying the mechanisms of persistent pain. J Neurosci. 27:6006–6018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hagihira S, Senba E, Yoshida S, Tohyama M, Yoshiya I (1990), Fine structure of noradrenergic terminals and their synapses in the rat spinal dorsal horn: an immunohistochemical study. Brain Res. 526:73–80. [DOI] [PubMed] [Google Scholar]
  29. Hains BC, Waxman SG (2006), Activated microglia contribute to the maintenance of chronic pain after spinal cord injury. J Neurosci. 26:4308–4317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Hammond DL, Yaksh TL (1984), Antagonism of stimulation-produced antinociception by intrathecal administration of methysergide or phentolamine. Brain Res. 298:329–337. [DOI] [PubMed] [Google Scholar]
  31. Hargreaves K, Dubner R, Brown F, Flores C, Joris J (1988), A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain. 32:77–88. [DOI] [PubMed] [Google Scholar]
  32. Heneka MT, Gavrilyuk V, Landreth GE, O’Banion MK, Weinberg G, Feinstein DL (2003), Noradrenergic depletion increases inflammatory responses in brain: effects on IkappaB and HSP70 expression. J Neurochem. 85:387–398. [DOI] [PubMed] [Google Scholar]
  33. Hickey L, Li Y, Fyson SJ, Watson TC, Perrins R, Hewinson J, Teschemacher AG, Furue H, Lumb BM, Pickering AE (2014), Optoactivation of locus ceruleus neurons evokes bidirectional changes in thermal nociception in rats. J Neurosci. 34:4148–4160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Hirschberg S, Li Y, Randall A, Kremer EJ, Pickering AE (2017), Functional dichotomy in spinal- vs prefrontal-projecting locus coeruleus modules splits descending noradrenergic analgesia from ascending aversion and anxiety in rats. Elife. 6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Hormigo S, Horta Junior Jde A, Gomez-Nieto R, Lopez DE (2012), The selective neurotoxin DSP-4 impairs the noradrenergic projections from the locus coeruleus to the inferior colliculus in rats. Front Neural Circuits. 6:41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Howe JR, Zieglgansberger W (1987), Responses of rat dorsal horn neurons to natural stimulation and to iontophoretically applied norepinephrine. J Comp Neurol. 255:1–17. [DOI] [PubMed] [Google Scholar]
  37. Hulsebosch CE, Xu GY, Perez-Polo JR, Westlund KN, Taylor CP, McAdoo DJ (2000), Rodent model of chronic central pain after spinal cord contusion injury and effects of gabapentin. J Neurotrauma. 17:1205–1217. [DOI] [PubMed] [Google Scholar]
  38. Igari T, Takeda M, Obara K, Ono S (1977), Catecholamine metabolism in the patients with rheumatoid arthritis. Tohoku J Exp Med. 122:9–20. [DOI] [PubMed] [Google Scholar]
  39. Jaim-Etcheverry G, Zieher LM (1980), DSP-4: a novel compound with neurotoxic effects on noradrenergic neurons of adult and developing rats. Brain Res. 188:513–523. [DOI] [PubMed] [Google Scholar]
  40. Janes K, Neumann WL, Salvemini D (2012), Anti-superoxide and anti-peroxynitrite strategies in pain suppression. Biochim Biophys Acta. 1822:815–821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Jasmin L, Boudah A, Ohara PT (2003), Long-term effects of decreased noradrenergic central nervous system innervation on pain behavior and opioid antinociception. J Comp Neurol. 460:38–55. [DOI] [PubMed] [Google Scholar]
  42. Ji RR, Suter MR (2007), p38 MAPK, microglial signaling, and neuropathic pain. Mol Pain. 3:33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Jin SX, Zhuang ZY, Woolf CJ, Ji RR (2003), p38 mitogen-activated protein kinase is activated after a spinal nerve ligation in spinal cord microglia and dorsal root ganglion neurons and contributes to the generation of neuropathic pain. J Neurosci. 23:4017–4022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Jonsson G, Hallman H, Ponzio F, Ross S (1981), DSP4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine)--a useful denervation tool for central and peripheral noradrenaline neurons. Eur J Pharmacol. 72:173–188. [DOI] [PubMed] [Google Scholar]
  45. Kamisaki Y, Hamada T, Maeda K, Ishimura M, Itoh T (1993), Presynaptic alpha 2 adrenoceptors inhibit glutamate release from rat spinal cord synaptosomes. J Neurochem. 60:522–526. [DOI] [PubMed] [Google Scholar]
  46. Kaushal R, Taylor BK, Jamal AB, Zhang L, Ma F, Donahue R, Westlund KN (2016), GABA-A receptor activity in the noradrenergic locus coeruleus drives trigeminal neuropathic pain in the rat; contribution of NAalpha1 receptors in the medial prefrontal cortex. Neuroscience. 334:148–159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Kinoshita J, Takahashi Y, Watabe AM, Utsunomiya K, Kato F (2013), Impaired noradrenaline homeostasis in rats with painful diabetic neuropathy as a target of duloxetine analgesia. Mol Pain. 9:59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Kudo T, Kushikata T, Kudo M, Kudo T, Hirota K (2010), A central neuropathic pain model by DSP-4 induced lesion of noradrenergic neurons: preliminary report. Neurosci Lett. 481:102–104. [DOI] [PubMed] [Google Scholar]
  49. Kudo T, Kushikata T, Kudo M, Kudo T, Hirota K (2011), Antinociceptive effects of neurotropin in a rat model of central neuropathic pain: DSP-4 induced noradrenergic lesion. Neurosci Lett. 503:20–22. [DOI] [PubMed] [Google Scholar]
  50. Kuraishi Y, Hirota N, Sato Y, Kaneko S, Satoh M, Takagi H (1985a), Noradrenergic inhibition of the release of substance P from the primary afferents in the rabbit spinal dorsal horn. Brain Res. 359:177–182. [DOI] [PubMed] [Google Scholar]
  51. Kuraishi Y, Hirota N, Satoh M, Takagi H (1985b), Antinociceptive effects of intrathecal opioids, noradrenaline and serotonin in rats: mechanical and thermal algesic tests. Brain Res. 326:168–171. [DOI] [PubMed] [Google Scholar]
  52. Kwon M, Altin M, Duenas H, Alev L (2014), The role of descending inhibitory pathways on chronic pain modulation and clinical implications. Pain Pract. 14:656–667. [DOI] [PubMed] [Google Scholar]
  53. Linsenbardt AJ, Breckenridge JM, Wilken GH, Macarthur H (2012), Dopaminochrome induces caspase-independent apoptosis in the mesencephalic cell line, MN9D. J Neurochem. 122:175–184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Linsenbardt AJ, Wilken GH, Westfall TC, Macarthur H (2009), Cytotoxicity of dopaminochrome in the mesencephalic cell line, MN9D, is dependent upon oxidative stress. Neurotoxicology. 30:1030–1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Little JW, Chen Z, Doyle T, Porreca F, Ghaffari M, Bryant L, Neumann WL, Salvemini D (2012a), Supraspinal peroxynitrite modulates pain signaling by suppressing the endogenous opioid pathway. J Neurosci. 32:10797–10808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Little JW, Doyle T, Salvemini D (2012b), Reactive nitroxidative species and nociceptive processing: determining the roles for nitric oxide, superoxide, and peroxynitrite in pain. Amino Acids. 42:75–94. [DOI] [PubMed] [Google Scholar]
  57. Llorca-Torralba M, Borges G, Neto F, Mico JA, Berrocoso E (2016), Noradrenergic Locus Coeruleus pathways in pain modulation. Neuroscience. 338:93–113. [DOI] [PubMed] [Google Scholar]
  58. Macolino CM, Daiutolo BV, Albertson BK, Elliott MB (2014), Mechanical alloydnia induced by traumatic brain injury is independent of restraint stress. J Neurosci Methods. 226:139–146. [DOI] [PubMed] [Google Scholar]
  59. Margalit D, Segal M (1979), A pharmacologic study of analgesia produced by stimulation of the nucleus locus coeruleus. Psychopharmacology (Berl). 62:169–173. [DOI] [PubMed] [Google Scholar]
  60. Martin WJ, Gupta NK, Loo CM, Rohde DS, Basbaum AI (1999), Differential effects of neurotoxic destruction of descending noradrenergic pathways on acute and persistent nociceptive processing. Pain. 80:57–65. [DOI] [PubMed] [Google Scholar]
  61. Martins I, de Vries MG, Teixeira-Pinto A, Fadel J, Wilson SP, Westerink BH, Tavares I (2013), Noradrenaline increases pain facilitation from the brain during inflammatory pain. Neuropharmacology. 71:299–307. [DOI] [PubMed] [Google Scholar]
  62. Millan MJ (2002), Descending control of pain. Prog Neurobiol. 66:355–474. [DOI] [PubMed] [Google Scholar]
  63. Mylius V, Engau I, Teepker M, Stiasny-Kolster K, Schepelmann K, Oertel WH, Lautenbacher S, Moller JC (2009), Pain sensitivity and descending inhibition of pain in Parkinson’s disease. J Neurol Neurosurg Psychiatry. 80:24–28. [DOI] [PubMed] [Google Scholar]
  64. Narita M, Shimamura M, Imai S, Kubota C, Yajima Y, Takagi T, Shiokawa M, Inoue T, Suzuki M, Suzuki T (2008), Role of interleukin-1beta and tumor necrosis factor-alpha-dependent expression of cyclooxygenase-2 mRNA in thermal hyperalgesia induced by chronic inflammation in mice. Neuroscience. 152:477–486. [DOI] [PubMed] [Google Scholar]
  65. Ochs SD, Westfall TC, Macarthur H (2005), The separation and quantification of aminochromes using high-pressure liquid chromatography with electrochemical detection. J Neurosci Methods. 142:201–208. [DOI] [PubMed] [Google Scholar]
  66. Olechowski CJ, Truong JJ, Kerr BJ (2009), Neuropathic pain behaviours in a chronic-relapsing model of experimental autoimmune encephalomyelitis (EAE). Pain. 141:156–164. [DOI] [PubMed] [Google Scholar]
  67. Olpe HR, Laszlo J, Dooley DJ, Heid J, Steinmann MW (1983), Decreased activity of locus coeruleus neurons in the rat after DSP-4 treatment. Neurosci Lett. 40:81–84. [DOI] [PubMed] [Google Scholar]
  68. Olson L, Fuxe K (1972), Further mapping out of central noradrenaline neuron systems: projections of the “subcoeruleus” area. Brain Res. 43:289–295. [DOI] [PubMed] [Google Scholar]
  69. Osaka T, Matsumura H (1994), Noradrenergic inputs to sleep-related neurons in the preoptic area from the locus coeruleus and the ventrolateral medulla in the rat. Neurosci Res. 19:39–50. [DOI] [PubMed] [Google Scholar]
  70. Ossipov MH, Dussor GO, Porreca F (2010), Central modulation of pain. J Clin Invest. 120:3779–3787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Pascual J, Grijalba B, Garcia-Sevilla JA, Zarranz JJ, Pazos A (1992), Loss of high-affinity alpha 2-adrenoceptors in Alzheimer’s disease: an autoradiographic study in frontal cortex and hippocampus. Neurosci Lett. 142:36–40. [DOI] [PubMed] [Google Scholar]
  72. Paxinos G, Watson C (1998), The Rat Brain in Stereotaxic Coordinates. Academic Press, San Diego. [Google Scholar]
  73. Pickel VM, Segal M, Bloom FE (1974), A radioautographic study of the efferent pathways of the nucleus locus coeruleus. J Comp Neurol. 155:15–42. [DOI] [PubMed] [Google Scholar]
  74. Pires OC, Ashmawi HA, Constantino E, Pelogia NC, Posso Ide P (2011), Subarachnoid serotonergic and noradrenergic antagonists increase the pain response in rats. Rev Bras Anestesiol. 61:202–205, 206–210, 108–212. [DOI] [PubMed] [Google Scholar]
  75. Prieto M, Giralt MT (2001), Effects of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) on alpha2-adrenoceptors which regulate the synthesis and release of noradrenaline in the rat brain. Pharmacol Toxicol. 88:152–158. [DOI] [PubMed] [Google Scholar]
  76. Proudfit HK (1988), Pharmacologic evidence for the modulation of nociception by noradrenergic neurons. Prog Brain Res. 77:357–370. [DOI] [PubMed] [Google Scholar]
  77. Randall LO, Selitto JJ (1957), A method for measurement of analgesic activity on inflamed tissue. Arch Int Pharmacodyn Ther. 111:409–419. [PubMed] [Google Scholar]
  78. Reddy SV, Maderdrut JL, Yaksh TL (1980), Spinal cord pharmacology of adrenergic agonist-mediated antinociception. J Pharmacol Exp Ther. 213:525–533. [PubMed] [Google Scholar]
  79. Reddy SV, Yaksh TL (1980), Spinal noradrenergic terminal system mediates antinociception. Brain Res. 189:391–401. [DOI] [PubMed] [Google Scholar]
  80. Reeve AJ, Patel S, Fox A, Walker K, Urban L (2000), Intrathecally administered endotoxin or cytokines produce allodynia, hyperalgesia and changes in spinal cord neuronal responses to nociceptive stimuli in the rat. Eur J Pain. 4:247–257. [DOI] [PubMed] [Google Scholar]
  81. Roczniak W, Oswiecimska JM, Brodziak-Dopierala B, Cipora E, Nowak PG, Babuska-Roczniak M (2016), Evaluation of the analgesic effect of morphine on models of acute nociceptive pain in rats with a central noradrenergic system lesion. Neuro Endocrinol Lett. 37:239–244. [PubMed] [Google Scholar]
  82. Ross SB (1976), Long-term effects of N-2-chlorethyl-N-ethyl-2-bromobenzylamine hydrochloride on noradrenergic neurones in the rat brain and heart. Br J Pharmacol. 58:521–527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  83. Ross SB, Johansson JG, Lindborg B, Dahlbom R (1973), Cyclizing compounds. I. Tertiary N-(2-bromobenzyl)-N-haloalkylamines with adrenergic blocking action. Acta Pharm Suec. 10:29–42. [PubMed] [Google Scholar]
  84. Ross SB, Renyl AL (1976), On the long-lasting inhibitory effect of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP 4) on the active uptake of noradrenaline. J Pharm Pharmacol. 28:458–459. [DOI] [PubMed] [Google Scholar]
  85. Sagen J, Proudfit HK (1984), Effect of intrathecally administered noradrenergic antagonists on nociception in the rat. Brain Res. 310:295–301. [DOI] [PubMed] [Google Scholar]
  86. Salter MW (2004), Cellular neuroplasticity mechanisms mediating pain persistence. J Orofac Pain. 18:318–324. [PubMed] [Google Scholar]
  87. Salvemini D, Little JW, Doyle T, Neumann WL (2011), Roles of reactive oxygen and nitrogen species in pain. Free Radic Biol Med. 51:951–966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  88. Sasa M, Munekiyo K, Osumi Y, Takaori S (1977), Attenuation of morphine analgesia in rats with lesions of the locus coeruleus and dorsal raphe nucleus. Eur J Pharmacol. 42:53–62. [DOI] [PubMed] [Google Scholar]
  89. Schestatsky P, Kumru H, Valls-Sole J, Valldeoriola F, Marti MJ, Tolosa E, Chaves ML (2007), Neurophysiologic study of central pain in patients with Parkinson disease. Neurology. 69:2162–2169. [DOI] [PubMed] [Google Scholar]
  90. Scholz J, Woolf CJ (2007), The neuropathic pain triad: neurons, immune cells and glia. Nat Neurosci. 10:1361–1368. [DOI] [PubMed] [Google Scholar]
  91. Seguela P, Watkins KC, Geffard M, Descarries L (1990), Noradrenaline axon terminals in adult rat neocortex: an immunocytochemical analysis in serial thin sections. Neuroscience. 35:249–264. [DOI] [PubMed] [Google Scholar]
  92. Stamford JA (1995), Descending control of pain. Br J Anaesth. 75:217–227. [DOI] [PubMed] [Google Scholar]
  93. Sulzer D, Bogulavsky J, Larsen KE, Behr G, Karatekin E, Kleinman MH, Turro N, Krantz D, Edwards RH, Greene LA, Zecca L (2000), Neuromelanin biosynthesis is driven by excess cytosolic catecholamines not accumulated by synaptic vesicles. Proc Natl Acad Sci U S A. 97:11869–11874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  94. Szot P, Miguelez C, White SS, Franklin A, Sikkema C, Wilkinson CW, Ugedo L, Raskind MA (2010), A comprehensive analysis of the effect of DSP4 on the locus coeruleus noradrenergic system in the rat. Neuroscience. 166:279–291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  95. Tanabe M, Ono K, Honda M, Ono H (2009), Gabapentin and pregabalin ameliorate mechanical hypersensitivity after spinal cord injury in mice. Eur J Pharmacol. 609:65–68. [DOI] [PubMed] [Google Scholar]
  96. Tassorelli C, Armentero MT, Greco R, Fancellu R, Sandrini G, Nappi G, Blandini F (2007), Behavioral responses and Fos activation following painful stimuli in a rodent model of Parkinson’s disease. Brain Res. 1176:53–61. [DOI] [PubMed] [Google Scholar]
  97. Taylor BK, Westlund KN (2017), The noradrenergic locus coeruleus as a chronic pain generator. J Neurosci Res. [DOI] [PMC free article] [PubMed] [Google Scholar]
  98. Tjolsen A, Lund A, Hole K (1990), The role of descending noradrenergic systems in regulation of nociception: the effects of intrathecally administered alpha-adrenoceptor antagonists and clonidine. Pain. 43:113–120. [DOI] [PubMed] [Google Scholar]
  99. Touchette JC, Breckenridge JM, Wilken GH, Macarthur H (2016), Direct intranigral injection of dopaminochrome causes degeneration of dopamine neurons. Neurosci Lett. 612:178–184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  100. Tsuruoka M, Tamaki J, Maeda M, Hayashi B, Inoue T (2011), The nucleus locus coeruleus/subcoeruleus contributes to antinociception during freezing behavior following the air-puff startle in rats. Brain Res. 1393:52–61. [DOI] [PubMed] [Google Scholar]
  101. Tsuruoka M, Willis WD (1996a), Descending modulation from the region of the locus coeruleus on nociceptive sensitivity in a rat model of inflammatory hyperalgesia. Brain Res. 743:86–92. [DOI] [PubMed] [Google Scholar]
  102. Tsuruoka M, Willis WD Jr. (1996b), Bilateral lesions in the area of the nucleus locus coeruleus affect the development of hyperalgesia during carrageenan-induced inflammation. Brain Res. 726:233–236. [PubMed] [Google Scholar]
  103. Urakami K, Takahashi K, Matsushima E, Sano K, Nishikawa S, Takao T (1990), The threshold of pain and neurotransmitter’s change on pain in Parkinson’s disease. Jpn J Psychiatry Neurol. 44:589–593. [DOI] [PubMed] [Google Scholar]
  104. Voisin DL, Guy N, Chalus M, Dallel R (2005), Nociceptive stimulation activates locus coeruleus neurones projecting to the somatosensory thalamus in the rat. J Physiol. 566:929–937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  105. Wallace DM, Magnuson DJ, Gray TS (1989), The amygdalo-brainstem pathway: selective innervation of dopaminergic, noradrenergic and adrenergic cells in the rat. Neurosci Lett. 97:252–258. [DOI] [PubMed] [Google Scholar]
  106. Wasserman JK, Koeberle PD (2009), Development and characterization of a hemorrhagic rat model of central post-stroke pain. Neuroscience. 161:173–183. [DOI] [PubMed] [Google Scholar]
  107. Watkins LR, Milligan ED, Maier SF (2001), Glial activation: a driving force for pathological pain. Trends Neurosci. 24:450–455. [DOI] [PubMed] [Google Scholar]
  108. Westlund KN, Zhang D, Carlton SM, Sorkin LS, Willis WD (1991), Noradrenergic innervation of somatosensory thalamus and spinal cord. Prog Brain Res. 88:77–88. [DOI] [PubMed] [Google Scholar]
  109. Yaksh TL (1985), Pharmacology of spinal adrenergic systems which modulate spinal nociceptive processing. Pharmacol Biochem Behav. 22:845–858. [DOI] [PubMed] [Google Scholar]
  110. Yeomans DC, Clark FM, Paice JA, Proudfit HK (1992), Antinociception induced by electrical stimulation of spinally projecting noradrenergic neurons in the A7 catecholamine cell group of the rat. Pain. 48:449–461. [DOI] [PubMed] [Google Scholar]

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