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1.  Influence of intrathecal delivery of bone marrow-derived mesenchymal stem cells on spinal inflammation and pain hypersensitivity in a rat model of peripheral nerve injury 
Background
Multipotent mesenchymal stem (stromal) cells (MSCs) have been credited with immunomodulative properties, supporting beneficial outcomes when transplanted into a variety of disease models involving inflammation. Potential mechanisms include the secretion of paracrine factors and the establishment of a neurotrophic microenvironment. To test the hypothesis that MSCs release soluble mediators that can attenuate local inflammation, we here analysed the influence of MSCs on the activation of microglia cells, as well as on inflammatory parameters and pain behaviour in a surgical rat model of neuropathic pain.
Methods
We focussed on an experimental model of partial sciatic nerve ligation (PSNL), characterised by a rapid and persistent inflammation in the dorsal lumbar spinal cord where sensory inputs from the sciatic nerve are processed. Via indwelling intrathecal catheters, MSCs were repetitively grafted into the intrathecal lumbar space. Animals were evaluated for mechanical and thermal hypersensitivity over a period of 21 days after PSNL. Afterwards, spinal cords were processed for immunohistochemical analysis of the microglial marker ionized calcium-binding adapter molecule 1 (Iba1) and quantification of inflammatory markers in ipsilateral dorsal horns. We hypothesised that injections on postsurgical days 2 to 4 would interfere with microglial activation, leading to a reduced production of pro-inflammatory cytokines and amelioration of pain behaviour.
Results
PSNL-induced mechanical allodynia or heat hyperalgesia were not influenced by MSC transplantation, and spinal cord inflammatory processes remained largely unaffected. Indeed, the early microglial response to PSNL characterised by increased Iba1 expression in the lumbar dorsal horn was not significantly altered and cytokine levels in the spinal cord at 21 days after surgery were similar to those found in vehicle-injected animals. Grafted MSCs were detected close to the pia mater, but were absent within the spinal cord parenchyma.
Conclusions
We conclude that intrathecal administration is not an appropriate route to deliver cells for treatment of acute spinal cord inflammation as it leads to entrapment of grafted cells within the pia mater. We propose that the early inflammatory response triggered by PSNL in the lumbar spinal cord failed to effectively recruit MSCs or was insufficient to disturb the tissue integrity so as to allow MSCs to penetrate the spinal cord parenchyma.
doi:10.1186/s12974-014-0157-8
PMCID: PMC4172959  PMID: 25212534
Mesenchymal stem cells; Neuroinflammation; Microglia; PSNL; Pain
2.  Enhanced neuroinflammation and pain hypersensitivity after peripheral nerve injury in rats expressing mutated superoxide dismutase 1 
Background
Neuroinflammation and nitroxidative stress are implicated in the pathophysiology of neuropathic pain. In view of both processes, microglial and astroglial activation in the spinal dorsal horn play a predominant role. The present study investigated the severity of neuropathic pain and the degree of glial activation in an inflammatory- and nitroxidative-prone animal model.
Methods
Transgenic rats expressing mutated superoxide dismutase 1 (hSOD1G93A) are classically used as a model for amyotrophic lateral sclerosis (ALS). Because of the associated inflammatory- and nitroxidative-prone properties, this model was used to study thermal and mechanical hypersensitivity following partial sciatic nerve ligation (PSNL). Next to pain hypersensitivity assessment, microglial and astroglial activation states were moreover characterized, as well as inflammatory marker gene expression and the glutamate clearance system.
Results
PSNL induced thermal and mechanical hypersensitivity in both wild-type (WT) and transgenic rats. However, the degree of thermal hypersensitivity was found to be exacerbated in transgenic rats while mechanical hypersensitivity was only slightly and not significantly increased. Microglial Iba1 expression was found to be increased in the ipsilateral dorsal horn of the lumbar spinal cord after PSNL but such Iba1 up-regulation was enhanced in transgenic rats as compared WT rats, both at 3 days and at 21 days after injury. Moreover, mRNA levels of Nox2, a key enzyme in microglial activation, but also of pro-inflammatory markers (IL-1β and TLR4) were not modified in WT ligated rats at 21 days after PSNL as compared to WT sham group while transgenic ligated rats showed up-regulated gene expression of these 3 targets. On the other hand, the PSNL-induced increase in GFAP immunoreactivity spreading that was evidenced in WT rats was unexpectedly found to be attenuated in transgenic ligated rats. Finally, GLT-1 gene expression and uptake activity were shown to be similar between WT sham and WT ligated rats at 21 days after injury, while both parameters were significantly increased in the ipsilateral dorsal region of the lumbar spinal cord of hSOD1G93A rats.
Conclusions
Taken together, our findings show that exacerbated microglial activation and subsequent inflammatory and nitroxidative processes are associated with the severity of neuropathic pain symptoms.
doi:10.1186/1742-2094-8-33
PMCID: PMC3090736  PMID: 21489258
3.  Neuropathic Pain in Rats with a Partial Sciatic Nerve Ligation Is Alleviated by Intravenous Injection of Monoclonal Antibody to High Mobility Group Box-1 
PLoS ONE  2013;8(8):e73640.
High mobility group box-1 (HMGB1) is associated with the pathogenesis of inflammatory diseases. A previous study reported that intravenous injection of anti-HMGB1 monoclonal antibody significantly attenuated brain edema in a rat model of stroke, possibly by attenuating glial activation. Peripheral nerve injury leads to increased activity of glia in the spinal cord dorsal horn. Thus, it is possible that the anti-HMGB1 antibody could also be efficacious in attenuating peripheral nerve injury-induced pain. Following partial sciatic nerve ligation (PSNL), rats were treated with either anti-HMGB1 or control IgG. Intravenous treatment with anti-HMGB1 monoclonal antibody (2 mg/kg) significantly ameliorated PSNL-induced hind paw tactile hypersensitivity at 7, 14 and 21 days, but not 3 days, after ligation, whereas control IgG had no effect on tactile hypersensitivity. The expression of HMGB1 protein in the spinal dorsal horn was significantly increased 7, 14 and 21 days after PSNL; the efficacy of the anti-HMGB1 antibody is likely related to the presence of HMGB1 protein. Also, the injury-induced translocation of HMGB1 from the nucleus to the cytosol occurred mainly in dorsal horn neurons and not in astrocytes and microglia, indicating a neuronal source of HMGB1. Markers of astrocyte (glial fibrillary acidic protein (GFAP)), microglia (ionized calcium binding adaptor molecule 1 (Iba1)) and spinal neuron (cFos) activity were greatly increased in the ipsilateral dorsal horn side compared to the sham-operated side 21 days after PSNL. Anti-HMGB1 monoclonal antibody treatment significantly decreased the injury-induced expression of cFos and Iba1, but not GFAP. The results demonstrate that nerve injury evokes the synthesis and release of HMGB1 from spinal neurons, facilitating the activity of both microglia and neurons, which in turn leads to symptoms of neuropathic pain. Thus, the targeting of HMGB1 could be a useful therapeutic strategy in the treatment of chronic pain.
doi:10.1371/journal.pone.0073640
PMCID: PMC3749159  PMID: 23991202
4.  α2δ-1 Gene Deletion Affects Somatosensory Neuron Function and Delays Mechanical Hypersensitivity in Response to Peripheral Nerve Damage 
The Journal of Neuroscience  2013;33(42):16412-16426.
The α2δ-1 subunit of voltage-gated calcium channels is upregulated after sensory nerve injury and is also the therapeutic target of gabapentinoid drugs. It is therefore likely to play a key role in the development of neuropathic pain. In this study, we have examined mice in which α2δ-1 gene expression is disrupted, to determine whether α2δ-1 is involved in various modalities of nociception, and for the development of behavioral hypersensitivity after partial sciatic nerve ligation (PSNL). We find that naive α2δ-1−/− mice show a marked behavioral deficit in mechanical and cold sensitivity, but no change in thermal nociception threshold. The lower mechanical sensitivity is mirrored by a reduced in vivo electrophysiological response of dorsal horn wide dynamic range neurons. The CaV2.2 level is reduced in brain and spinal cord synaptosomes from α2δ-1−/− mice, and α2δ-1−/− DRG neurons exhibit lower calcium channel current density. Furthermore, a significantly smaller number of DRG neurons respond to the TRPM8 agonist menthol. After PSNL, α2δ-1−/− mice show delayed mechanical hypersensitivity, which only develops at 11 d after surgery, whereas in wild-type littermates it is maximal at the earliest time point measured (3 d). There is no compensatory upregulation of α2δ-2 or α2δ-3 after PSNL in α2δ-1−/− mice, and other transcripts, including neuropeptide Y and activating transcription factor-3, are upregulated normally. Furthermore, the ability of pregabalin to alleviate mechanical hypersensitivity is lost in PSNL α2δ-1−/− mice. Thus, α2δ-1 is essential for rapid development of mechanical hypersensitivity in a nerve injury model of neuropathic pain.
doi:10.1523/JNEUROSCI.1026-13.2013
PMCID: PMC3797367  PMID: 24133248
5.  Neuropeptide Y acts at Y1 receptors in the Rostral Ventral Medulla To Inhibit Neuropathic Pain 
Pain  2007;131(1-2):83-95.
Brain microinjection studies in the rat using local anesthetics suggest that the rostral ventral medulla (RVM) contributes to the facilitation of neuropathic pain. However, these studies were restricted to a single model of neuropathic pain (the spinal nerve ligation model) and to just two stimulus modalities (non-noxious tactile stimulus and heat). Also, few neurotransmitter systems have been shown to modulate descending facilitation. After either partial sciatic nerve ligation (PSNL) or spared nerve injury (SNI), we found that unilateral or bilateral microinjection of lidocaine into the RVM reduced not only mechanical allodynia (decreased threshold to von Frey hairs and/or an automated device) and mechanical hyperalgesia (increased paw lifting in response to a noxious pin), but also cold hypersensitivity (increased lifting in response to the hindpaw application of a drop of acetone). Application of a drop of water did not elicit paw withdrawal, indicating that the acetone test is indeed a measure of cold hypersensitivity. We found significant neuropeptide Y Y1-like immunoreactivity within, and lateral to, the midline RVM. Intra-RVM injection of neuropeptide Y (NPY) dose-dependently inhibited the mechanical and cold hypersensitivity associated with PSNL or SNI, an effect that could be blocked by the Y1 receptor antagonist BIBO 3304. We conclude that medullary facilitation spans multiple behavioral signs of allodynia and hyperalgesia in multiple models of neuropathic pain. Furthermore, NPY inhibits behavioural signs of neuropathic pain, possibly by acting at Y1 receptors in the RVM.
doi:10.1016/j.pain.2006.12.018
PMCID: PMC2077302  PMID: 17276005
descending facilitation; allodynia; hyperalgesia; spared nerve injury; lidocaine; rat
6.  A preconditioning nerve lesion inhibits mechanical pain hypersensitivity following subsequent neuropathic injury 
Molecular Pain  2011;7:1.
Background
A preconditioning stimulus can trigger a neuroprotective phenotype in the nervous system - a preconditioning nerve lesion causes a significant increase in axonal regeneration, and cerebral preconditioning protects against subsequent ischemia. We hypothesized that a preconditioning nerve lesion induces gene/protein modifications, neuronal changes, and immune activation that may affect pain sensation following subsequent nerve injury. We examined whether a preconditioning lesion affects neuropathic pain and neuroinflammation after peripheral nerve injury.
Results
We found that a preconditioning crush injury to a terminal branch of the sciatic nerve seven days before partial ligation of the sciatic nerve (PSNL; a model of neuropathic pain) induced a significant attenuation of pain hypersensitivity, particularly mechanical allodynia. A preconditioning lesion of the tibial nerve induced a long-term significant increase in paw-withdrawal threshold to mechanical stimuli and paw-withdrawal latency to thermal stimuli, after PSNL. A preconditioning lesion of the common peroneal induced a smaller but significant short-term increase in paw-withdrawal threshold to mechanical stimuli, after PSNL. There was no difference between preconditioned and unconditioned animals in neuronal damage and macrophage and T-cell infiltration into the dorsal root ganglia (DRGs) or in astrocyte and microglia activation in the spinal dorsal and ventral horns.
Conclusions
These results suggest that prior exposure to a mild nerve lesion protects against adverse effects of subsequent neuropathic injury, and that this conditioning-induced inhibition of pain hypersensitivity is not dependent on neuroinflammation in DRGs and spinal cord. Identifying the underlying mechanisms may have important implications for the understanding of neuropathic pain due to nerve injury.
doi:10.1186/1744-8069-7-1
PMCID: PMC3022745  PMID: 21205324
7.  Alleviation of chronic neuropathic pain by environmental enrichment in mice well after the establishment of chronic pain 
Background
In animal models, the impact of social and environmental manipulations on chronic pain have been investigated in short term studies where enrichment was implemented prior to or concurrently with the injury. The focus of this study was to evaluate the impact of environmental enrichment or impoverishment in mice three months after induction of chronic neuropathic pain.
Methods
Thirty-four CD-1 seven to eight week-old male mice were used. Mice underwent surgery on the left leg under isoflurane anesthesia to induce the spared nerve injury model of neuropathic pain or sham condition. Mice were then randomly assigned to one of four groups: nerve injury with enriched environment (n = 9), nerve injury with impoverished environment (n = 8), sham surgery with enriched environment (n = 9), or sham surgery with impoverished environment (n = 8). The effects of environmental manipulations on mechanical (von Frey filaments) heat (hot plate) and cold (acetone test) cutaneous hypersensitivities, motor impairment (Rotarod), spontaneous exploratory behavior (open field test), anxiety-like behavior (elevated plus maze) and depression-like phenotype (tail suspension test) were assessed in neuropathic and control mice 1 and 2 months post-environmental change. Finally, the effect of the environment on spinal expression of the pro-nociceptive neuropeptides substance P and CGRP form the lumbar spinal cord collected at the end of the study was evaluated by tandem liquid chromatography mass spectrometry.
Results
Environmental enrichment attenuated nerve injury-induced hypersensitivity to mechanical and cold stimuli. In contrast, an impoverished environment exacerbated mechanical hypersensitivity. No antidepressant effects of enrichment were observed in animals with chronic neuropathic pain. Finally, environmental enrichment resulted lower SP and CGRP concentrations in neuropathic animals compared to impoverishment. These effects were all observed in animals that had been neuropathic for several months prior to intervention.
Conclusions
These results suggest that environmental factors could play an important role in the rehabilitation of chronic pain patients well after the establishment of chronic pain. Enrichment is a potentially inexpensive, safe and easily implemented non-pharmacological intervention for the treatment of chronic pain.
doi:10.1186/1744-9081-9-22
PMCID: PMC3679946  PMID: 24025218
Chronic neuropathic pain; Environmental enrichment; Environmental impoverishment; Animal model; Spared nerve injury
8.  Phospholipase C-related but catalytically inactive protein modulates pain behavior in a neuropathic pain model in mice 
Molecular Pain  2013;9:23.
Background
An inositol 1,4,5-trisphosphate binding protein, comprising 2 isoforms termed PRIP-1 and PRIP-2, was identified as a novel modulator for GABAA receptor trafficking. It has been reported that naive PRIP-1 knockout mice have hyperalgesic responses.
Findings
To determine the involvement of PRIP in pain sensation, a hind paw withdrawal test was performed before and after partial sciatic nerve ligation (PSNL) in PRIP-1 and PRIP-2 double knockout (DKO) mice. We found that naive DKO mice exhibited normal pain sensitivity. However, DKO mice that underwent PSNL surgery showed increased ipsilateral paw withdrawal threshold. To further investigate the inverse phenotype in PRIP-1 KO and DKO mice, we produced mice with specific siRNA-mediated knockdown of PRIPs in the spinal cord. Consistent with the phenotypes of KO mice, PRIP-1 knockdown mice showed allodynia, while PRIP double knockdown (DKD) mice with PSNL showed decreased pain-related behavior. This indicates that reduced expression of both PRIPs in the spinal cord induces resistance towards a painful sensation. GABAA receptor subunit expression pattern was similar between PRIP-1 KO and DKO spinal cord, while expression of K+-Cl--cotransporter-2 (KCC2), which controls the balance of neuronal excitation and inhibition, was significantly upregulated in DKO mice. Furthermore, in the DKD PSNL model, an inhibitor-induced KCC2 inhibition exhibited an altered phenotype from painless to painful sensations.
Conclusions
Suppressed expression of PRIPs induces an elevated expression of KCC2 in the spinal cord, resulting in inhibition of nociception and amelioration of neuropathic pain in DKO mice.
doi:10.1186/1744-8069-9-23
PMCID: PMC3651726  PMID: 23639135
KCC2; GABAA Receptor; Neuropathic pain; Partial sciatic nerve ligation; PRIP
9.  Influence of the vanilloid receptor TRPV1 on the activation of spinal cord glia in mouse models of pain 
Experimental neurology  2009;220(2):383-390.
Although activation of spinal glia has been implicated in the development of pathological pain, the mechanisms underlying glial activation are not fully understood. One such mechanism may be triggered by reaction to neuroactive substances released from central axons of sensory afferents. The vanilloid receptor TRPV1, a nonselective cation channel in nociceptive sensory afferents, mediates the release of neurotransmitters, such as glutamate and CGRP in the dorsal horn, which can subsequently activate glia. To test the hypothesis that activation of spinal glia is mediated, at least in part, by TRPV1, we studied the expression of markers for microglia (Ionized calcium-binding adapter molecule 1, Iba1) and astrocytes (Glial Fibrillary Acidic Protein, GFAP) in the spinal cord of TRPV1 knockout mice (KO) vs. wild-type mice (WT) in models of acute (intraplantar capsaicin), inflammatory (Adjuvant-Induced Arthritis, AIA), and neuropathic pain (Partial Sciatic Nerve Ligation, PSNL). We found that i) naïve KO mice had denser immunostaining for both Iba1 and GFAP than naïve WT mice, ii) the immunostaining for Iba1 increased significantly in treated mice, compared to naïve mice, 3 days after capsaicin and 7–14 days after AIA or PSNL, and was significantly greater in WT than in KO mice 3 days after capsaicin, 7–14 days after AIA, and 7 days after PSNL, iii) the immunostaining for GFAP increased significantly in treated mice, compared to naïve mice, 3 days after capsaicin and 14–21 days after AIA or PSNL, and was significantly greater in WT than in KO mice 14 days after AIA or PSNL. Our results suggest that TRPV1 plays a role in the activation of spinal glia in mice with nociceptive, inflammatory, and neuropathic pain.
doi:10.1016/j.expneurol.2009.09.030
PMCID: PMC2784271  PMID: 19815011
10.  Further characterisation of a rat model of varicella zoster virus (VZV)-associated pain 
Neuroscience  2006;144(4):1495-1508.
Persistent herpes zoster-associated pain is a significant clinical problem and an area of largely unmet therapeutic need. Progress in elucidating the underlying pathophysiology of zoster-associated pain and related co-morbidity behaviour, in addition to appropriately targeted drug development has been hindered by the lack of an appropriate animal model. This study further characterises a recently developed rat model of zoster-associated hypersensitivity and investigates (a) response to different viral strains; (b) relationship between viral inoculum concentration (‘dose’) and mechanical hypersensitivity (‘response’); (c) attenuation of virus-associated mechanical hypersensitivity by clinically useful analgesic drugs; and (d) measurement of pain co-morbidity (anxiety-like behaviour) and pharmacological intervention in the open field paradigm (in parallel with models of traumatic peripheral nerve injury). VZV was propagated on fibroblast cells before subcutaneous injection into the glabrous footpad of the left hind limb of adult male Wistar rats. Control animals received injection of uninfected fibroblast cells. Hind-limb reflex withdrawal thresholds to mechanical, noxious thermal and cooling stimuli were recorded at specified intervals post-infection. Infection with all viral strains was associated with a dose-dependent mechanical hypersensitivity but not a thermal or cool hypersensitivity. Systemic treatment with intraperitoneal (i.p.) morphine (2.5mg/kg), amitriptyline (10mg/kg), gabapentin (30mg/kg), (S)-(+)-ibuprofen (20mg/kg) and the cannnabinoid WIN55,212-2 (2mg/kg) but not the antiviral, acyclovir (50mg/kg), was associated with a reversal of mechanical paw withdrawal thresholds. In the open field paradigm, virus-infected and nerve-injured animals demonstrated an anxiety-like pattern of ambulation (reduced entry into the central area of the open arena) which was positively correlated with mechanical hypersensitivity. This may reflect pain-related comorbidity. Further, anxiety-like behaviour was attenuated by acute i.p. administration of gabapentin (30mg/kg) in nerve-injured, but not virus-infected animals. This model will prove useful in elucidating the pathophysiology of zoster-associated pain and provide a tool for pre-clinical screening of analgesic drugs.
doi:10.1016/j.neuroscience.2006.11.029
PMCID: PMC2394505  PMID: 17197105
zoster-associated pain; postherpetic neuralgia; neuropathy; analgesia; open field; anxiety-like behaviour
11.  Neuropathic Pain Activates the Endogenous κ Opioid System in Mouse Spinal Cord and Induces Opioid Receptor Tolerance 
Release of endogenous dynorphin opioids within the spinal cord after partial sciatic nerve ligation (pSNL) is known to contribute to the neuropathic pain processes. Using a phosphoselective antibody [κ opioid receptor (KOR-P)] able to detect the serine 369 phosphorylated form of the KOR, we determined possible sites of dynorphin action within the spinal cord after pSNL. KOR-P immunoreactivity (IR) was markedly increased in the L4 –L5 spinal dorsal horn of wild-type C57BL/6 mice (7–21 d) after lesion, but not in mice pretreated with the KOR antagonist nor-binaltorphimine (norBNI). In addition, knock-out mice lacking prodynorphin, KOR, or G-protein receptor kinase 3 (GRK3) did not show significant increases in KOR-P IR after pSNL. KOR-P IR was colocalized in both GABAergic neurons and GFAP-positive astrocytes in both ipsilateral and contralateral spinal dorsal horn. Consistent with sustained opioid release, KOR knock-out mice developed significantly increased tactile allodynia and thermal hyperalgesia in both the early (first week) and late (third week) interval after lesion. Similarly, mice pretreated with norBNI showed enhanced hyperalgesia and allodynia during the 3 weeks after pSNL. Because sustained activation of opioid receptors might induce tolerance, we measured the antinociceptive effect of the κ agonist U50,488 using radiant heat applied to the ipsilateral hindpaw, and we found that agonist potency was significantly decreased 7 d after pSNL. In contrast, neither prodynorphin nor GRK3 knock-out mice showed U50,488 tolerance after pSNL. These findings suggest that pSNL induced a sustained release of endogenous prodynorphin-derived opioid peptides that activated an anti-nociceptive KOR system in mouse spinal cord. Thus, endogenous dynorphin had both pronociceptive and antinociceptive actions after nerve injury and induced GRK3-mediated opioid tolerance.
doi:10.1523/JNEUROSCI.5552-03.2004
PMCID: PMC2376823  PMID: 15140929
κ opioid receptor; opiate; dynorphin; receptor phosphorylation; desensitization; allodynia; hyperalgesia
12.  Central Administration of C-X-C Chemokine Receptor Type 4 Antagonist Alleviates the Development and Maintenance of Peripheral Neuropathic Pain in Mice 
PLoS ONE  2014;9(8):e104860.
Aim
To explore the roles of C-X-C chemokine receptor type 4 (CXCR4) in spinal processing of neuropathic pain at the central nervous system (CNS).
Methods
Peripheral neuropathic pain (PNP) induced by partial sciatic nerve ligation (pSNL) model was assessed in mice. Effects of a single intrathecal (central) administration of AMD3100 (intrathecal AMD3100), a CXCR4 antagonist, on pain behavior and pain-related spinal pathways and molecules in the L3-L5 spinal cord segment was studied compare to saline treatment.
Results
Rotarod test showed that intrathecal AMD3100 did not impair mice motor function. In pSNL-induced mice, intrathecal AMD3100 delayed the development of mechanical allodynia and reversed the established mechanical allodynia in a dose-dependent way. Moreover, intrathecal AMD3100 downregulated the activation of JNK1 and p38 pathways and the protein expression of p65 as assessed by western blotting. Real-time PCR test also demonstrated that substance P mRNA was decreased, while adrenomedullin and intercellular adhesion molecule mRNA was increased following AMD3100 treatment.
Conclusion
Our results suggest that central (spinal) CXCR4 is involved in the development and maintenance of PNP and the regulation of multiple spinal molecular events under pain condition, implicating that CXCR4 would potentially be a therapeutic target for chronic neuropathic pain.
doi:10.1371/journal.pone.0104860
PMCID: PMC4132096  PMID: 25119456
13.  Behavioral and anatomical characterization of the bilateral sciatic nerve chronic constriction (bCCI) injury: correlation of anatomic changes and responses to cold stimuli 
Molecular Pain  2010;6:7.
Background
Unilateral constrictive sciatic nerve injury (uCCI) is a common neuropathic pain model. However, the bilateral constrictive injury (bCCI) model is less well studied, and shows unique characteristics. In the present study, we sought to correlate effects of bCCI on nocifensive responses to cold and mechanical stimuli with selected dorsal horn anatomic markers. bCCI or sham ligation of both rat sciatic nerves were followed up to 90 days of behavioural testing. Additional rats sacrificed at 15, 30 and 90 days were used for anatomic analyses. Behavioural tests included hindpaw withdrawal responses to topical acetone, cold plate testing, an operant thermal preference task and hindpaw withdrawal thresholds to mechanical probing.
Results
All nocifensive responses to cold increased and remained enhanced for >45 days. Mechanical withdrawal thresholds decreased for 25 days only. Densitometric analyses of immunoperoxidase staining in the superficial dorsal horn at L4-5 revealed decreased cholecystokinin (CCK) staining at all times after bCCI, decreased mu opiate receptor (MOR) staining, maximal at 15 days, increased neuropeptide Y (NPY) staining only at days 15 and 30, and increased neurokinin-1 receptor (NK-1R) staining at all time points, maximal at 15 days. Correlation analyses at 45 days post-bCCI, were significant for individual rat nocifensive responses in each cold test and CCK and NK-1R, but not for MOR or NPY.
Conclusions
These results confirm the usefulness of cold testing in bCCI rats, a new approach using CCI to model neuropathic pain, and suggest a potential value of studying the roles of dorsal horn CCK and substance P in chronic neuropathic pain. Compared to human subjects with neuropathic pain, responses to cold stimuli in rats with bCCI may be a useful model of neuropathic pain.
doi:10.1186/1744-8069-6-7
PMCID: PMC2825192  PMID: 20105332
14.  Autotaxin, a synthetic enzyme of lysophosphatidic acid (LPA), mediates the induction of nerve-injured neuropathic pain 
Molecular Pain  2008;4:6.
Recently, we reported that lysophosphatidic acid (LPA) induces long-lasting mechanical allodynia and thermal hyperalgesia as well as demyelination and upregulation of pain-related proteins through one of its cognate receptors, LPA1. In addition, mice lacking the LPA1 receptor gene (lpa1-/- mice) lost these nerve injury-induced neuropathic pain behaviors and phenomena. However, since lpa1-/- mice did not exhibit any effects on the basal nociceptive threshold, it is possible that nerve injury-induced neuropathic pain and its machineries are initiated by LPA via defined biosynthetic pathways that involve multiple enzymes. Here, we attempted to clarify the involvement of a single synthetic enzyme of LPA known as autotaxin (ATX) in nerve injury-induced neuropathic pain. Wild-type mice with partial sciatic nerve injury showed robust mechanical allodynia starting from day 3 after the nerve injury and persisting for at least 14 days, along with thermal hyperalgesia. On the other hand, heterozygous mutant mice for the autotaxin gene (atx+/-), which have 50% ATX protein and 50% lysophospholipase D activity compared with wild-type mice, showed approximately 50% recovery of nerve injury-induced neuropathic pain. In addition, hypersensitization of myelinated Aβ˜- or Aδ-fiber function following nerve injury was observed in electrical stimuli-induced paw withdrawal tests using a Neurometer®. The hyperalgesia was completely abolished in lpa1-/- mice, and reduced by 50% in atx+/- mice. Taken together, these findings suggest that LPA biosynthesis through ATX is the source of LPA for LPA1 receptor-mediated neuropathic pain. Therefore, targeted inhibition of ATX-mediated LPA biosynthesis as well as LPA1 receptor and its downstream pathways may represent a novel way to prevent nerve injury-induced neuropathic pain.
doi:10.1186/1744-8069-4-6
PMCID: PMC2277392  PMID: 18261210
15.  A Novel Tool for the Assessment of Pain: Validation in Low Back Pain 
PLoS Medicine  2009;6(4):e1000047.
Joachim Scholz and colleagues develop and validate an assessment tool that distinguishes between radicular and axial low back pain.
Background
Adequate pain assessment is critical for evaluating the efficacy of analgesic treatment in clinical practice and during the development of new therapies. Yet the currently used scores of global pain intensity fail to reflect the diversity of pain manifestations and the complexity of underlying biological mechanisms. We have developed a tool for a standardized assessment of pain-related symptoms and signs that differentiates pain phenotypes independent of etiology.
Methods and Findings
Using a structured interview (16 questions) and a standardized bedside examination (23 tests), we prospectively assessed symptoms and signs in 130 patients with peripheral neuropathic pain caused by diabetic polyneuropathy, postherpetic neuralgia, or radicular low back pain (LBP), and in 57 patients with non-neuropathic (axial) LBP. A hierarchical cluster analysis revealed distinct association patterns of symptoms and signs (pain subtypes) that characterized six subgroups of patients with neuropathic pain and two subgroups of patients with non-neuropathic pain. Using a classification tree analysis, we identified the most discriminatory assessment items for the identification of pain subtypes. We combined these six interview questions and ten physical tests in a pain assessment tool that we named Standardized Evaluation of Pain (StEP). We validated StEP for the distinction between radicular and axial LBP in an independent group of 137 patients. StEP identified patients with radicular pain with high sensitivity (92%; 95% confidence interval [CI] 83%–97%) and specificity (97%; 95% CI 89%–100%). The diagnostic accuracy of StEP exceeded that of a dedicated screening tool for neuropathic pain and spinal magnetic resonance imaging. In addition, we were able to reproduce subtypes of radicular and axial LBP, underscoring the utility of StEP for discerning distinct constellations of symptoms and signs.
Conclusions
We present a novel method of identifying pain subtypes that we believe reflect underlying pain mechanisms. We demonstrate that this new approach to pain assessment helps separate radicular from axial back pain. Beyond diagnostic utility, a standardized differentiation of pain subtypes that is independent of disease etiology may offer a unique opportunity to improve targeted analgesic treatment.
Editors' Summary
Background
Pain, although unpleasant, is essential for survival. Whenever the body is damaged, nerve cells detecting the injury send an electrical message via the spinal cord to the brain and, as a result, action is taken to prevent further damage. Usually pain is short-lived, but sometimes it continues for weeks, months, or years. Long-lasting (chronic) pain can be caused by an ongoing, often inflammatory condition (for example, arthritis) or by damage to the nervous system itself—experts call this “neuropathic” pain. Damage to the brain or spinal cord causes central neuropathic pain; damage to the nerves that convey information from distant parts of the body to the spinal cord causes peripheral neuropathic pain. One example of peripheral neuropathic pain is “radicular” low back pain (also called sciatica). This is pain that radiates from the back into the legs. By contrast, axial back pain (the most common type of low back pain) is confined to the lower back and is non-neuropathic.
Why Was This Study Done?
Chronic pain is very common—nearly 10% of American adults have frequent back pain, for example—and there are many treatments for it, including rest, regulated exercise (physical therapy), pain-killing drugs (analgesics), and surgery. However, the best treatment for any individual depends on the exact nature of their pain, so it is important to assess their pain carefully before starting treatment. This is usually done by scoring overall pain intensity, but this assessment does not reflect the characteristics of the pain (for example, whether it occurs spontaneously or in response to external stimuli) or the complex biological processes involved in pain generation. An assessment designed to take such factors into account might improve treatment outcomes and could be useful in the development of new therapies. In this study, the researchers develop and test a new, standardized tool for the assessment of chronic pain that, by examining many symptoms and signs, aims to distinguish between pain subtypes.
What Did the Researchers Do and Find?
One hundred thirty patients with several types of peripheral neuropathic pain and 57 patients with non-neuropathic (axial) low back pain completed a structured interview of 16 questions and a standardized bedside examination of 23 tests. Patients were asked, for example, to choose words that described their pain from a list provided by the researchers and to grade the intensity of particular aspects of their pain from zero (no pain) to ten (the maximum imaginable pain). Bedside tests included measurements of responses to light touch, pinprick, and vibration—chronic pain often alters responses to harmless stimuli. Using “hierarchical cluster analysis,” the researchers identified six subgroups of patients with neuropathic pain and two subgroups of patients with non-neuropathic pain based on the patterns of symptoms and signs revealed by the interviews and physical tests. They then used “classification tree analysis” to identify the six questions and ten physical tests that discriminated best between pain subtypes and combined these items into a tool for a Standardized Evaluation of Pain (StEP). Finally, the researchers asked whether StEP, which took 10–15 minutes, could identify patients with radicular back pain and discriminate them from those with axial back pain in an independent group of 137 patients with chronic low back pain. StEP, they report, accurately diagnosed these two conditions and was well accepted by the patients.
What Do These Findings Mean?
These findings indicate that a standardized assessment of pain-related signs and symptoms can provide a simple, quick diagnostic procedure that distinguishes between radicular (neuropathic) and axial (non-neuropathic) low back pain. This distinction is crucial because these types of back pain are best treated in different ways. In addition, the findings suggest that it might be possible to identify additional pain subtypes using StEP. Because these subtypes may represent conditions in which different pain mechanisms are acting, classifying patients in this way might eventually enable physicians to tailor treatments for chronic pain to the specific needs of individual patients rather than, as at present, largely guessing which of the available treatments is likely to work best.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000047.
This study is further discussed in a PLoS Medicine Perspective by Giorgio Cruccu and and Andrea Truini
The US National Institute of Neurological Disorders and Stroke provides a primer on pain in English and Spanish
In its 2006 report on the health status of the US, the National Center for Health Statistics provides a special feature on the epidemiology of pain, including back pain
The Pain Treatment Topics Web site is a resource, sponsored partly by associations and manufacturers, that provides information on all aspects of pain and its treatment for health care professionals and their patients
Medline Plus provides a brief description of pain and of back pain and links to further information on both topics (in English and Spanish)
The MedlinePlus Medical Encyclopedia also has a page on low back pain (in English and Spanish)
doi:10.1371/journal.pmed.1000047
PMCID: PMC2661253  PMID: 19360087
16.  THE ABSENCE OF ENDOGENOUS β-ENDORPHIN SELECTIVELY BLOCKS PHOSPHORYLATION AND DESENSITIZATION OF MU OPIOID RECEPTORS FOLLOWING PARTIAL SCIATIC NERVE LIGATION 
Neuroscience  2007;146(4):1795-1807.
Phosphorylation of specific sites in the 2nd intracellular loop and in the C-terminal domain have previously been suggested to cause desensitization and internalization of the mu-opioid receptor (MOP-R). To assess sites of MOP-R phosphorylation in vivo, affinity-purified, phosphoselective antibodies were raised against either phosphothreonine-180 in the 2nd intracellular loop (MOR-P1) or the C-terminal domain of MOP-R containing phosphothreonine-370 and phosphoserine-375 (MOR-P2). We found that MOR-P2-immunoreactivity (IR) was significantly increased within the striatum of wild-type C57BL/6 mice after injection of the agonist fentanyl. Pretreatment with the antagonist naloxone blocked the fentanyl-induced increase. Furthermore, mutant mice lacking MOP-R showed only non-specific nuclear MOR-P2-IR before or after fentanyl treatment, confirming the specificity of the MOR-P2 antibodies. To assess whether MOP-R phosphorylation occurs following endogenous opioid release, we induced chronic neuropathic pain by partial sciatic nerve ligation (pSNL), which caused a significant increase in MOR-P2-IR in the striatum. pSNL also induced signs of mu opioid receptor tolerance demonstrated by a rightward shift in the morphine dose response in the tail withdrawal assay and by a reduction in morphine conditioned place preference (CPP). Mutant mice selectively lacking all forms of the β-endorphin peptides derived from the Pomc gene did not show increased MOR-P2-IR, decreased morphine antinociception, or reduced morphine CPP following pSNL. In contrast gene deletion of either proenkephalin or prodynorphin opioids did not block the effects of pSNL. These results suggest that neuropathic pain caused by pSNL in wild-type mice activates the release of the endogenous opioid β-endorphin, which subsequently induces MOP-R phosphorylation and opiate tolerance.
doi:10.1016/j.neuroscience.2007.03.029
PMCID: PMC2012364  PMID: 17467916
Opiate Tolerance; Opioid peptides; neuropathic pain; enkephalins; dynorphins; antinociception
17.  Transcriptional repression of the M channel subunit Kv7.2 in chronic nerve injury 
Pain  2011;152(4):742-754.
Neuropathic pain is a severe health problem for which there is a lack of effective therapy. A frequent underlying condition of neuropathic pain is a sustained overexcitability of pain-sensing (nociceptive) sensory fibres. Therefore, the identification of mechanisms for such abnormal neuronal excitability is of utmost importance for understanding neuropathic pain. Despite much effort, an inclusive model explaining peripheral overexcitability is missing. We investigated transcriptional regulation of the Kcnq2 gene, which encodes the Kv7.2 subunit of membrane potential-stabilizing M channel, in peripheral sensory neurons in a model of neuropathic pain—partial sciatic nerve ligation (PSNL). We show that Kcnq2 is the major Kcnq gene transcript in dorsal root ganglion (DRG); immunostaining and patch-clamp recordings from acute ganglionic slices verified functional expression of Kv7.2 in small-diameter nociceptive DRG neurons. Neuropathic injury induced substantial downregulation of Kv7.2 expression. Levels of repressor element 1–silencing transcription factor (REST), which is known to suppress Kcnq2 expression, were upregulated in response to neuropathic injury identifying the likely mechanism of Kcnq2 regulation. Behavioural experiments demonstrated that neuropathic hyperalgesia following PSNL developed faster than the downregulation of Kcnq2 expression could be detected, suggesting that this transcriptional mechanism may contribute to the maintenance rather than the initiation of neuropathic pain. Importantly, the decrease in the peripheral M channel abundance could be functionally compensated by peripherally applied M channel opener flupirtine, which alleviated neuropathic hyperalgesia. Our work suggests a novel mechanism for neuropathic overexcitability and brings focus on M channels and REST as peripheral targets for the treatment of neuropathic pain.
Neuropathic injury induces transcriptional downregulation of the Kcnq2 potassium channel gene by the transcriptional suppressor repressor element 1–silencing transcription factor; this mechanism contributes to peripheral sensitization of the afferent fibres.
doi:10.1016/j.pain.2010.12.028
PMCID: PMC3071978  PMID: 21345591
KCNQ; M current; REST; Sensory neuron; DRG; Neuropathic pain
18.  Effectiveness of anodal transcranial direct current stimulation in patients with chronic low back pain: Design, method and protocol for a randomised controlled trial 
Background
Electrical stimulation of central nervous system areas with surgically implanted stimulators has been shown to result in pain relief. To avoid the risks and side effects of surgery, transcranial direct current stimulation is an option to electrically stimulate the motor cortex through the skull. Previous research has shown that transcranial direct current stimulation relieves pain in patients with fibromyalgia, chronic neuropathic pain and chronic pelvic pain. Evidence indicates that the method is pain free, safe and inexpensive.
Methods/Design
A randomised controlled trial has been designed to evaluate the effect of transcranial direct current stimulation over the motor cortex for pain reduction in patients with chronic low back pain. It will also investigate whether transcranial direct current stimulation as a prior treatment enhances the symptom reduction achieved by a cognitive-behavioural group intervention. Participants will be randomised to receive a series of 5 days of transcranial direct current stimulation (2 mA, 20 mins) or 20 mins of sham stimulation; followed by a cognitive-behavioural group programme. The primary outcome parameters will measure pain (Visual Analog Scale) and disability (Oswestry Disability Index). Secondary outcome parameters will include the Fear Avoidance Beliefs Questionnaire, the Funktionsfragebogen Hannover (perceived function), Hospital Anxiety Depression Scale, bothersomeness and Health Related Quality of Life (SF 36), as well as Patient-Perceived Satisfactory Improvement. Assessments will take place immediately prior to the first application of transcranial direct current stimulation or sham, after 5 consecutive days of stimulation, immediately after the cognitive-behavioural group programme and at 4 weeks, 12 weeks and 24 weeks follow-up.
Discussion
This trial will help to determine, whether transcranial direct current stimulation is an effective treatment for patients with chronic low back pain and whether it can further enhance the effects of a cognitive behavioural pain management programme. Trial registration: Current Controlled Trials ISRCTN89874874.
doi:10.1186/1471-2474-12-290
PMCID: PMC3339335  PMID: 22204615
19.  Trigeminal Nerve Injury ErbB3/ErbB2 Promotes Mechanical Hypersensitivity 
Anesthesiology  2012;117(2):381-388.
BACKGROUND
Chronic constriction injury of trigeminal infraorbital nerve results in transient analgesia followed by whisker pad mechanical allodynia in rats. Neuregulin1 expressed on axonal membranes binds receptor tyrosine kinase ErbB promoting Schwann cell development and remyelination. This study investigated whether orofacial mechanical allodynia is signaled by ErbB3/ErbB2 heterodimers in injured nerves.
METHODS
Whisker pad mechanical allodynia (von Frey stimuli) was quantified in wild type rats and in transgenic rats with Sleeping Beauty transposon mutation for neuregulin1 transgene. Pain-related behavior was retested after intraperitoneal injection of ErbB2 inhibitor, Lapatinib, an agent shown by others to reduce breast cancer pain. Infraorbital nerve injury was evaluated histologically with myelin and neuronal biomarkers. ErbB3 changes over time were measured with western blots.
RESULTS
Whisker pad mechanical hypersensitivity began in week 2 in wild type rats (3.11 ± 5.93 vs. 18.72 ± 0.00g after sham surgery, n = 9, p < 0.001) indicating trigeminal neuropathic pain, but was not evident in transgenic rats (odds ratio: 1.12, 95% confidence interval: 0.38-3.35). Initiation of statistically significant mechano-hypersensitivity was delayed until week 6 after surgery in transgenic rats (3.44 ± 4.60g vs. 18.72 ± 0.00g, n = 4, p < 0.001). Mechanical allodynia which persisted 8 weeks in wild type rats was alleviated by Lapatinib (15 ± 3.89g vs. 2.45 ± 1.13g, n = 6, p < 0.001). Infraorbital nerve damage was verified histologically. Statistically significant ErbB3 increases (weeks 5 and 10) in wild type and transgenic rats (week 10) coincided with time points when mechanical hypersensitivity was present.
CONCLUSION
Neuregulin1/ErbB3/ErbB2 is a causal mechanism in nerve injury induced trigeminal neuropathic pain. Understanding peripheral glial mechanisms after nerve injury will improve neuropathic pain treatment.
doi:10.1097/ALN.0b013e3182604b2b
PMCID: PMC3406246  PMID: 22705569
20.  Minocycline prevents impaired glial glutamate uptake in the spinal sensory synapses of neuropathic rats 
Neuroscience  2010;170(3):901-912.
Activation of glutamate receptors and glial cells in the spinal dorsal horn are two fundamental processes involved in the pathogenesis of various pain conditions, including neuropathic pain induced by injury to the peripheral or central nervous systems. Numerous studies have demonstrated that minocycline treatment attenuates allodynic and hyperalgesic behaviors induced by tissue inflammation or nerve injury. However, the synaptic mechanisms by which minocycline prevents hyperalgesia are not fully understood. We recently reported that deficient glutamate uptake by glial glutamate transporters (GTs) is key for the enhanced activation of N-methyl-D-aspartate (NMDA) receptors in the spinal sensory synapses of rats receiving partial sciatic nerve ligation (pSNL). In this study, we investigated how minocycline affects activation of NMDA receptors in the spinal sensory synapses in rats with pSNL by whole cell recordings of NMDA currents in spinal laminea I and II neurons from spinal slices. The effects of minocycline treatments on the dorsal horn expression of glial GTs and astrocyte marker glial fibrillary acidic protein (GFAP) were analyzed by immunohistochemistry. We demonstrated that normalized activation of NMDA receptors in synapses activated by both weak and strong peripheral input in the spinal dorsal horn is temporally associated with attenuated mechanical allodynia in rats with pSNL receiving intraperitoneal injection of minocycline. Minocycline ameliorated both the downregulation of glial GT expression and the activation of astrocytes induced by pSNL in the spinal dorsal horn. We further revealed that preventing deficient glial glutamate uptake at the synapse is crucial for preserving the normalized activation of NMDA receptors in the spinal sensory synapses in pSNL rats treated with minocycline. Our studies suggest that glial GTs may be a potential target for the development of analgesics.
doi:10.1016/j.neuroscience.2010.07.049
PMCID: PMC2939184  PMID: 20678556
glutamate transporters; glutamate receptors; spinal sensory processing; nociception; glia; pain
21.  Anxiety-like behaviour is attenuated by gabapentin, morphine and diazepam in a rodent model of HIV anti-retroviral-associated neuropathic pain 
Neuroscience Letters  2008;448(1-9):153-156.
Neuropathic pain is commonly associated with affective disorders such as anxiety and depression. We have previously characterised a rodent model of HIV, anti-retroviral-associated neuropathy in which rats develop hypersensitivity to a punctate mechanical stimulus and display anxiety-like behaviour in the open field paradigm. To assess the potential of this behavioural paradigm for the assessment of pain related co-morbidities in rodent models of pain, here we test the sensitivity of this anxiety-like behaviour to the analgesic agents gabapentin and morphine in comparison to the known anxiolytic drug diazepam. We found that gabapentin (30 mg/kg, i.p.) and morphine (2.5 mg/kg, i.p.), which reduce mechanical hypersensitivity in these rats, significantly reduces measures of thigmotaxis in the open field. The effect of gabapentin and morphine did not differ significantly from diazepam (1 mg/kg, i.p.). This study highlights the potential use of this rodent model and behavioural paradigm in the validation of the affective component of novel analgesic pharmacological targets and elucidation of underlying pathophysiological mechanisms.
doi:10.1016/j.neulet.2008.10.005
PMCID: PMC2706951  PMID: 18926876
Neuropathic pain; Pain comorbidity; Open field activity; HIV neuropathy; Gabapentin
22.  Peripheral effects of morphine and expression of μ-opioid receptors in the dorsal root ganglia during neuropathic pain: nitric oxide signaling 
Molecular Pain  2011;7:25.
Background
The local administration of μ-opioid receptor (MOR) agonists attenuates neuropathic pain but the precise mechanism implicated in this effect is not completely elucidated. We investigated if nitric oxide synthesized by neuronal (NOS1) or inducible (NOS2) nitric oxide synthases could modulate the local antiallodynic effects of morphine through the peripheral nitric oxide-cGMP-protein kinase G (PKG)-ATP-sensitive K+ (KATP) channels signaling pathway activation and affect the dorsal root ganglia MOR expression during neuropathic pain.
Results
In wild type (WT) mice, the subplantar administration of morphine dose-dependently decreased the mechanical and thermal allodynia induced by the chronic constriction of the sciatic nerve (CCI), which effects were significantly diminished after their co-administration with different subanalgesic doses of a selective NOS1 (N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N'-nitroguanidine tris(trifluoroacetate) salt; NANT), NOS2 (L-N(6)-(1-iminoethyl)-lysine; L-NIL), L-guanylate cyclase (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; ODQ), PKG ((Rp)-8-(para-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate; Rp-8-pCPT-cGMPs) inhibitor or a KATP channel blocker (glibenclamide). The evaluation of the expression of MOR in the dorsal root ganglia from sham-operated and sciatic nerve-injured WT, NOS1 knockout (KO) and NOS2-KO mice at 21 days after surgery demonstrated that, although the basal mRNA and protein levels of MOR were similar between WT and both NOS-KO animals, nerve injury only decreased their expression in WT mice.
Conclusions
These results suggest that the peripheral nitric oxide-cGMP-PKG-KATP signaling pathway activation participates in the local antiallodynic effects of morphine after sciatic nerve injury and that nitric oxide, synthesized by NOS1 and NOS2, is implicated in the dorsal root ganglia down-regulation of MOR during neuropathic pain.
doi:10.1186/1744-8069-7-25
PMCID: PMC3094254  PMID: 21486477
23.  Bilateral elevation of interleukin-6 protein and mRNA in both lumbar and cervical dorsal root ganglia following unilateral chronic compression injury of the sciatic nerve 
Background
Current research implicates interleukin (IL)-6 as a key component of the nervous-system response to injury with various effects.
Methods
We used unilateral chronic constriction injury (CCI) of rat sciatic nerve as a model for neuropathic pain. Immunofluorescence, ELISA, western blotting and in situ hybridization were used to investigate bilateral changes in IL-6 protein and mRNA in both lumbar (L4-L5) and cervical (C7-C8) dorsal root ganglia (DRG) following CCI. The operated (CCI) and sham-operated (sham) rats were assessed after 1, 3, 7, and 14 days. Withdrawal thresholds for mechanical hyperalgesia and latencies for thermal hyperalgesia were measured in both ipsilateral and contralateral hind and fore paws.
Results
The ipsilateral hind paws of all CCI rats displayed a decreased threshold of mechanical hyperalgesia and withdrawal latency of thermal hyperalgesia, while the contralateral hind and fore paws of both sides exhibited no significant changes in mechanical or thermal sensitivity. No significant behavioral changes were found in the hind and fore paws on either side of the sham rats, except for thermal hypersensitivity, which was present bilaterally at 3 days. Unilateral CCI of the sciatic nerve induced a bilateral increase in IL-6 immunostaining in the neuronal bodies and satellite glial cells (SGC) surrounding neurons of both lumbar and cervical DRG, compared with those of naive control rats. This bilateral increase in IL-6 protein levels was confirmed by ELISA and western blotting. More intense staining for IL-6 mRNA was detected in lumbar and cervical DRG from both sides of rats following CCI. The DRG removed from sham rats displayed a similar pattern of staining for IL-6 protein and mRNA as found in naive DRG, but there was a higher staining intensity in SGC.
Conclusions
Bilateral elevation of IL-6 protein and mRNA is not limited to DRG homonymous to the injured nerve, but also extended to DRG that are heteronymous to the injured nerve. The results for IL-6 suggest that the neuroinflammatory reaction of DRG to nerve injury is propagated alongside the neuroaxis from the lumbar to the remote cervical segments. This is probably related to conditioning of cervical DRG neurons to injury.
doi:10.1186/1742-2094-10-55
PMCID: PMC3657546  PMID: 23634725
Unilateral nerve injury; Contralateral reaction; Remote ganglia; Neuroinflammation; Cytokines; Conditioning
24.  Local Loperamide Injection Reduces Mechanosensitivity of Rat Cutaneous, Nociceptive C-Fibers 
PLoS ONE  2012;7(7):e42105.
Loperamide reverses signs of mechanical hypersensitivity in an animal model of neuropathic pain suggesting that peripheral opioid receptors may be suitable targets for the treatment of neuropathic pain. Since little is known about loperamide effects on the responsiveness of primary afferent nerve fibers, in vivo electrophysiological recordings from unmyelinated afferents innervating the glabrous skin of the hind paw were performed in rats with an L5 spinal nerve lesion or sham surgery. Mechanical threshold and responsiveness to suprathreshold stimulation were tested before and after loperamide (1.25, 2.5 and 5 µg in 10 µl) or vehicle injection into the cutaneous receptive field. Loperamide dose-dependently decreased mechanosensitivity in unmyelinated afferents of nerve-injured and sham animals, and this effect was not blocked by naloxone pretreatment. We then investigated loperamide effects on nerve conduction by recording compound action potentials in vitro during incubation of the sciatic nerve with increasing loperamide concentrations. Loperamide dose-dependently decreased compound action potentials of myelinated and unmyelinated fibers (ED50 = 8 and 4 µg/10 µl, respectively). This blockade was not prevented by pre-incubation with naloxone. These results suggest that loperamide reversal of behavioral signs of neuropathic pain may be mediated, at least in part, by mechanisms independent of opioid receptors, most probably by local anesthetic actions.
doi:10.1371/journal.pone.0042105
PMCID: PMC3405035  PMID: 22848720
25.  The Spinal Cord Expression of Neuronal and Inducible Nitric Oxide Synthases and Their Contribution in the Maintenance of Neuropathic Pain in Mice 
PLoS ONE  2010;5(12):e14321.
Background
Nitric oxide generated by neuronal (NOS1), inducible (NOS2) or endothelial (NOS3) nitric oxide synthases contributes to pain processing, but the exact role of NOS1 and NOS2 in the maintenance of chronic peripheral neuropathic pain as well as the possible compensatory changes in their expression in the spinal cord of wild type (WT) and NOS knockout (KO) mice at 21 days after total sciatic nerve ligation remains unknown.
Methodology/Principal Findings
The mechanical and thermal allodynia as well as thermal hyperalgesia induced by sciatic nerve injury was evaluated in WT, NOS1-KO and NOS2-KO mice from 1 to 21 days after surgery. The mRNA and protein levels of NOS1, NOS2 and NOS3 in the spinal cord of WT and KO mice, at 21 days after surgery, were also assessed. Sciatic nerve injury led to a neuropathic syndrome in WT mice, in contrast to the abolished mechanical allodynia and thermal hyperalgesia as well as the decreased or suppressed thermal allodynia observed in NOS1-KO and NOS2-KO animals, respectively. Sciatic nerve injury also increases the spinal cord expression of NOS1 and NOS2 isoforms, but not of NOS3, in WT and NOS1-KO mice respectively. Moreover, the presence of NOS2 is required to increase the spinal cord expression of NOS1 whereas an increased NOS1 expression might avoid the up-regulation of NOS2 in the spinal cord of nerve injured WT mice.
Conclusions/Significance
These data suggest that the increased spinal cord expression of NOS1, regulated by NOS2, might be responsible for the maintenance of chronic peripheral neuropathic pain in mice and propose these enzymes as interesting therapeutic targets for their treatment.
doi:10.1371/journal.pone.0014321
PMCID: PMC3001461  PMID: 21179208

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