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Delayed functional expression of neuronal chemokine receptors following focal nerve demyelination in the rat: a mechanism for the development of chronic sensitization of peripheral nociceptors

Delayed functional expression of neuronal chemokine receptors following focal nerve demyelination... Background: Animal and clinical studies have revealed that focal peripheral nerve axon demyelination is accompanied by nociceptive pain behavior. C-C and C-X-C chemokines and their receptors have been strongly implicated in demyelinating polyneuropathies and persistent pain syndromes. Herein, we studied the degree to which chronic nociceptive pain behavior is correlated with the neuronal expression of chemokines and their receptors following unilateral lysophosphatidylcholine (LPC)-induced focal demyelination of the sciatic nerve in rats. Results: Focal nerve demyelination increased behavioral reflex responsiveness to mechanical stimuli between postoperative day (POD) 3 and POD28 in both the hindpaw ipsilateral and contralateral to the nerve injury. This behavior was accompanied by a bilateral increase in the numbers of primary sensory neurons expressing the chemokine receptors CCR2, CCR5, and CXCR4 by POD14, with no change in the pattern of CXCR3 expression. Significant increases in the numbers of neurons expressing the chemokines monocyte chemoattractant protein-1 (MCP-1/CCL2), Regulated on Activation, Normal T Expressed and Secreted (RANTES/CCL5) and interferon γ-inducing protein-10 (IP- 10/CXCL10) were also evident following nerve injury, although neuronal expression pattern of stromal cell derived factor-1α (SDF1/CXCL12) did not change. Functional studies demonstrated that acutely dissociated sensory neurons 2+ derived from LPC-injured animals responded with increased [Ca ] following exposure to MCP-1, IP-10, SDF1 and RANTES on POD 14 and 28, but these responses were largely absent by POD35. On days 14 and 28, rats received either saline or a CCR2 receptor antagonist isomer (CCR2 RA-[R]) or its inactive enantiomer (CCR2 RA-[S]) by intraperitoneal (i.p.) injection. CCR2 RA-[R] treatment of nerve-injured rats produced stereospecific bilateral reversal of tactile hyperalgesia. Conclusion: These results suggest that the presence of chemokine signaling by both injured and adjacent, uninjured sensory neurons is correlated with the maintenance phase of a persistent pain state, suggesting that chemokine receptor antagonists may be an important therapeutic intervention for chronic pain. Page 1 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 receptor upregulation, the continued expression of neuro- Introduction Inflammatory events induced by nerve injury are thought nal chemokine/receptors appears to correlate with to play a central role in the pathogenesis of inflammatory changes in chronic nociceptive behavior. Furthermore, pain. The production and release of molecules that medi- administration of a CCR2 receptor antagonist produced ate the acute inflammatory response include bradykinin, an attenuation of the nociceptive behavior, further high- tachykinins, serotonin, histamine, ATP and cytokines lighting the potential role of chemokine signaling in states such as tumor necrosis factor-alpha (TNFα), interleukin of neuropathic pain. 1-β (IL-1β), and interleukin-6 (IL-6). Many of these mol- ecules, which are produced in association with acute Parts of this study have been previously published in inflammatory responses, are known to induce hyperalge- abstract form [17,18]. sia [1,2] Methods Chemokines, which also contribute to the development Animals of inflammatory pain states, can directly excite subsets of Pathogen-free, adult female Sprague-Dawley rats sensory neurons [3-8]. This excitation is likely to be due to (150–200 g; Harlan Laboratories, Madison, WI) were transactivation of ion channels, such as TRPV1 and housed in temperature (23 ± 3°C) and light (12-h TRPA1, expressed by sensory nerves [9,10]. As such, it is light:12-h dark cycle; lights on at 07:00 h) controlled quite possible that a prolonged de novo expression of rooms with standard rodent chow and water available ad chemokines and/or their cognate receptors by sensory libitum. Experiments were performed during the light neurons following peripheral nerve injury may be central cycle. Animals were randomly assigned to the treatment to the development and/or maintenance of chronic pain groups. These experiments were approved by the Institu- states. Indeed, we previously demonstrated that in a tional Animal Care and Use Committee of Loyola Univer- rodent model of spinal stenosis, chronic compression of sity, Chicago. All procedures were conducted in the DRG (CCD), produced a delayed but chronic expres- accordance with the Guide for Care and Use of Laboratory sion of both the chemokine receptor CCR2 and its ligand, Animals published by the National Institutes of Health the chemokine MCP-1/CCL2 in lumbar DRGs [8]. Fur- and the ethical guidelines of the International Association thermore, MCP-1/CCL2 depolarized or increased the for the Study of Pain. All animals were randomly assigned excitability of several subpopulations of sensory neurons, to either treatment or control groups. including nociceptors, in both the intact and dissociated DRG [6,8]. Interestingly, mice deficient in the chemokine Sciatic nerve demyelination receptor, CCR2, exhibit an impaired neuropathic pain Animals were anesthetized with 4% isoflurane and main- response following partial nerve ligation [11]. tained on 2% isoflurane (Halocarbon, River Edge, NJ) in O . For all demyelination experiments, lysophosphatidyl- In order to fully understand the extent and significance of choline (LPC), (type V, 99% pure; Sigma-Aldrich, St. neuronal chemokine signaling in states of pain hypersen- Louis, MO) was dissolved in buffered sterile saline (pH sitivity, we examined whether induction of a focal demy- 7.2) to give a final concentration of 10 mg/ml. The right elination of the sciatic nerve, a known rodent model of sciatic nerve of the rat was exposed at the mid-thigh level neuropathic pain [12], produced changes in the neuronal under sterile conditions. A sterile polyvinyl acetal (PVAc) expression of certain key chemokines previously shown to sponge (Ivalon, San Diego, CA), 2-mm × 2-mm soaked in be extensively upregulated in peripheral neuroinflamma- 7 μl of LPC, was placed adjacent to the sciatic nerve. The tory responses [3,13-16]. These chemokines included dermal incision site was closed with 5.0 suture thread. monocyte chemoattractant protein-1 (MCP-1/CCL2), Sham control animals were prepared as described above, interferon γ-inducing protein-10 (IP-10/CXCL10), regu- but buffered sterile saline was used in place of LPC plus lated on activation normal T cell expressed and released saline. Some control rats were also given an intramuscular (RANTES/CCL5) and stromal cell derived factor-1 (SDF1/ injection of LPC (10 ul, 1%) into the gastrocnemius mus- CXCL12) and their cognate receptors (CCR2, CXCR3, cle. CCR5 and CXCR4, respectively). Drugs and method of administration We now demonstrate that focal peripheral nerve demyeli- A CCR2 receptor antagonist and its inactive enantiomer nation in the right thigh of the rat produces chronic bilat- were employed in this study [19]. The CCR2 antagonist eral nociceptive behavior as measured by hindpaw active enantiomer's full name is (R)-4-Acetyl-1-(4-chloro- withdrawal. Together with the ongoing display of nocice- 2-fluorophenyl)-5-cyclohexyl-3-hydroxy-1,5-dihydro- ptive behavior is a delayed upregulation of several C-C 2H-pyrrol-2-one (CCR2 RA [R]). The inactive enantiomer and C-X-C chemokines and their cognate receptors by sen- is (S)-4-Acetyl-1-(4-chloro-2-fluorophenyl)-5-cyclohexyl- sory neurons. Though there is an initial delay in ligand/ 3-hydroxy-1,5-dihydro-2H-pyrrol-2-one (CCR2 RA [S]) Page 2 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 (Additional file 1). Both were employed as Na+ salts. The slopes of the logistic functions from which the PWTs are affinity of CCR2 RA [R] for the rat CCR2 receptor is > derived. The experimenter was blinded to both the injury 4000 that of the S-isomer. Both compounds were freshly condition of the animal and the drugs utilized in all prepared in saline on the day of the experiment (10 mg/ behavioral trials. kg). Active and inactive enantiomer and vehicle-treated groups (n = 8 per group) were given a one-time intraperi- Foot withdrawal to thermal stimulus toneal (i.p.) injection one hour prior to behavioral test- To evaluate the PWT to thermal stimulation, we used the ing. Hargreaves' plantar test apparatus (Ugo Basile, Varese, Italy). Rats were placed on a 2-mm-thick glass floor; a Foot withdrawal to punctate mechanical indentation mobile infrared heat generator with an aperture of 10 mm The incidence of foot withdrawal was measured in was aimed at the rat's hind paw under the floor. Following response to mechanical indentation of the plantar surface activation of the heat source, the reaction time (the with- of each hind paw with sharp, Von Frey-type nylon fila- drawal latency of the hindpaw) of the rat was recorded ments. Mechanical stimuli were applied with seven fila- automatically. A shortening of the withdrawal latency ments, each differing in the bending force delivered (10, indicated thermal hyperalgesia. The temperature of the 20, 40, 60, 80, 100, and 120 mN), but each fitted with the glass floor was kept at 22.5–23.5°C. Measurements of the same metal cylinder with a flat tip and a fixed diameter of withdrawal latency of the paw began after the rats were 0.2 mm [3]. In each behavioral testing sequence, the oper- habituated to the testing environment (IR setting = 70). ator was blinded to the animal treatment condition. The measurements were repeated four times, at 5 min intervals, on each paw, and the initial pair of measure- The rat was placed on a metal mesh floor and covered ments was not used. The averages of the three remaining with a transparent plastic dome. Typically, the animals pairs of measurements taken were employed as data. rest quietly in this situation after an initial few minutes of exploration. Animals were habituated to this testing appa- In situ hybridization ratus for 15 minutes a day, two days prior to the behavio- In situ hybridization histochemistry for chemokine recep- ral assays. Following acclimation, each filament was tors was performed by using digoxigenin-labeled ribo- applied to six spots spaced across the hind paw. The fila- probes. Adult female Sprague-Dawley rats were ments were tested in order of ascending force, with each euthanized using carbon dioxide. L L DRGs ipsi- and 4 5 st th filament delivered in sequence from the 1 to the 6 spot contralateral to LPC nerve injury were rapidly removed, alternating from one hind paw to the other. The duration embedded in OCT compound (Tissue Tek, Ted Pella, Inc., of each stimulus was 1 second and the interstimulus inter- Redding, CA) and frozen. Sections were serially cut at 14 val was 10–15 seconds. A cutoff value of 120 mN was μm. The CCR2 probe was prepared as described [8]. used; animals that did not respond at 120 mN were Briefly, an 848-bp CCR2 cDNA fragment (nucleotides assigned that value [3,20]. 489–1336 of GenBank no. U77349) was cloned by PCR using rat spleen cDNA. The resulting PCR product was The incidence of foot withdrawal was expressed as a per- subcloned into a pGEM-T Easy vector and sequenced to centage of the six applications of each filament as a func- ensure identity for riboprobe use. The CCR2 template was tion of force. A Hill equation was fitted to the function linearized with SacII to generate a probe of 950 bases by (Origin version 6.0, Microcal Software, Northhampton using SP6 polymerase. Signals were visualized by using MA) relating the percentage of indentations eliciting a NBT/BCIP reagents (Roche Diagnostics/Boehringer Man- withdrawal to the force of indentation. From this equa- nheim, Indianapolis, IN) in the dark for 2–20 h depend- tion, the paw withdrawal threshold (PWT) force was ing upon the abundance of the RNA. Images were obtained and defined as the force corresponding to a 50% captured using brightfield or differential interference con- withdrawal. At least a -20 mN difference from the baseline trast optics with a Nikon E600 fluorescent microscope PWT in a given animal is representative of neuropathic (NikonUSA, Melville, NY) fitted with a charge-coupled pain [3]. device camera (Retiga EXi, Q-Imaging Corporation, Van- couver, BC). CCR2 mRNA expression studies were used Measurements were taken on three successive days before for receptor localization because of the failure of immu- surgery. Postoperative testing was performed on one, nocytochemistry to detect neuronal CCR2 protein. three and seven days after surgery and weekly thereafter for the duration of the experiment. PWT values were sta- The RANTES plasmid was a gift from Dr. Richard M. Ran- tistically analyzed for each foot separately and for the sig- sohoff (Cleveland Clinic Foundation). The RANTES plas- nificance of differences between the average of the three mid was sub-cloned into a pGEM vector. The plasmid preoperative tests and the mean obtained for each postop- templates were linearized with restriction enzyme diges- erative test. The same statistical analyses are applied to the tion. Page 3 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 The CXCR4 and SDF-1 probes were generated as described from the total number of Hoescht-positive neuronal previously [21]. For the CXCR3 and CCR5 probes, we nuclei present in a tissue section. The overall diameter and used the CD1 mouse brain cDNA. The CXCR3 cDNA frag- brightness of the Hoescht-positive neuronal nuclei ment was amplified using the forward primer 5'-gag gtt agt allowed for a clear delineation between neurons and non- gaa cgt caa gtg-3' and the reverse primer 5'-tgg aga cca gca neuronal cells in the DRG. At least 5000 neuronal profiles gaa cag cta g-3'. The CCR5 fragment used the forward from six animals (minimum of 625 cells per ganglia) were primer 5'-tgg att atg gta tgt cag cac cc-3'and the reverse quantified for each cell type in the single neuronal marker primer 5'-tcg att atg gta tgt cag cac cc-3'. All PCR fragments study and for each combination of cellular markers. were subcloned into a pCR II-TOPO vector, and were ver- Quantification of cell numbers, degree of colocalization ified by restriction analysis and automated DNA sequenc- and cell diameters was determined using ImagePro Plus ing (Perkin Elmer, Boston MA) (Media Cybernetics, Silver Spring, MD). As noted above, individuals conducting cell quantification were blinded The plasmid templates were linearized by restriction to the treatment conditions. Data are represented as enzyme digestion. Then transcription was labeled by dig- means ± SEM%. oixigenin (Roche Applied Science, Indianapolis, IN). Preparation of acutely dissociated dorsal root ganglion Immunocytochemical labeling neurons Adult female Sprague-Dawley rats were deeply anesthe- The L –L DRG were acutely dissociated using methods 4 5 tized with isoflurane and transcardially perfused with described by Ma and LaMotte [22]. Briefly, L and L DRG 4 5 saline followed by 4% paraformaldehyde. Lumbar ganglia were removed from control or LPC-treated animals at var- associated with the sciatic nerve ipsilateral and contralat- ious post-operative day timepoints. The DRGs were eral to focal nerve demyelination injury (n = 6) or sham treated with collagenase A and collagenase D in HBSS for treatment (n = 6) were immediately removed following 20 minutes (1 mg/ml; Roche Applied Science, Indianapo- behavior on POD 7 or 14 and postfixed for 4 hours. Addi- lis, IN), followed by treatment with papain (30 units/ml, tional lumbar DRGs were removed from naïve, behavio- Worthington Biochemical, Lakewood, NJ) in HBSS con- rally tested rats (n = 6). Lumbar DRGs were encoded at the taining .5 mM EDTA and cysteine at 35°C. The cells were outset and processed in random order. Sagittal sections of then dissociated via mechanical trituration in culture the DRG were serially cut at 14 μm onto SuperFrost micro- media containing 1 mg/ml bovine serum albumin and scope slides (Fisher Scientific, Pittsburgh PA). At least 6 trypsin inhibitor (1 mg/ml, Sigma, St. Louis MO). The cul- sections were obtained for immunocytological analysis ture media was Ham's F12 mixture, supplemented with per DRG. Tissue was processed such that DRG sections on 10% fetal bovine serum, penicillin and streptomycin (100 each slide were at intervals of 80 um. Slides were incu- ug/ml and 100 U/ml) and N2 (Life Technologies). The bated with blocking buffer (3% BSA/3% horse serum/ cells were then plated on coverslips coated with poly-L- 0.4% Triton-X; Fisher Scientific, Pittsburgh PA) for 1 hour, lysine and laminin (1 mg/ml) and incubated for 2 hours followed by overnight incubation with the rabbit polyclo- before more culture media was added to the wells. The nal antisera generated against MCP-1 (1:500; Chemicon, cells were then allowed to sit undisturbed for 12–15 hours Temecula, CA), IP-10 (1:1000, Abcam, Cambridge MA) or to adhere at 37°C (with 5% CO ). CCR2 (1:500; Aviva Systems Biology, San Diego CA) at 2+ Intracellular Ca imaging room temperature. After primary incubation, secondary antibodies (anti-rabbit conjugated to CY3, made in don- The dissociated DRG cells were loaded with fura-2 AM (3 key at 1:800; Jackson ImmunoResearch, West Grove, PA) uM, Molecular Probes/Invitrogen Corporation, Carlsbad were used to visualize cells. Some experiments were aug- CA) for 25 minutes at room temperature in a balanced salt mented with the addition of Griffoniasimplicifolia I-isolec- solution (BSS) [NaCl (140 mM), Hepes (10 mM), CaCl tin B4 (IB4) conjugated with fluorescein (1 mg/1 ml; (2 mM), MgCl (1 mM), Glucose (10 mM), KCl (50 Sigma, St. Louis MO). Slides were washed in PBS for 5 min mM)]. The cells were rinsed with the BSS and mounted each (×3) and coverslipped with a PBS/glycerol solution. onto a chamber that was placed onto the inverted micro- All tissue sections were also stained with Hoechst 33258 scope and continuously perfused with BSS at a rate of 1 nuclear marker (Invitrogen Corporation, Carlsbad CA). ml/min. Intracellular calcium was measured by digital video microfluorometry with an intensified CCD camera Tissue sections were analyzed for the presence of IB -bind- coupled to a microscope and MetaFluor software (Molec- ing neurons and either MCP-1, IP-10 or CCR2. Because a ular Devices Corporation, Downington, PA). Cells were stereological approach was not employed in this study, illuminated with a 150 W xenon arc lamp, and the excita- quantification of the data may represent a biased estimate tion wavelengths of the fura-2 (340/380 nm) were of the actual numbers of immunopositive neurons. The selected by a filter changer. Chemokines were applied proportions of immunoreactive neurons were determined directly into the coverslip bathing solution after the per- Page 4 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 fusion was stopped. If no response was seen within 1 minute, the chemokine was washed out. For all experi- 80 80 ments, MCP-1, SDF1, RANTES and IP10 were added to 70 70 the cells in random order, after which capsaicin, high K+ (50 K) and ATP were added. The chemokines used were 60 60 purchased from R & D Systems (Minneapolis, MN), and * * ** ** * * 50 50 * * all were used at a concentration of 100 nm to ensure max- * * * * imal activation. They were reconstituted in 0.1%BSA/PBS, 40 40 and aliquots were stored at -20°C. * * * * * * * * 30 30 Statistical Analyses 20 20 Data is presented as the mean ± SEM, unless otherwise ip ips siill. . t to o ner nerve in ve injur jury y con cont tra ral. t l. to o ner nerve ve inju injur ry y noted. GB-Stat School Pack software (Dynamic Microsys- 10 10 ip ips siill. . t to o s sham in ham injur jury y con cont tra ral. t l. to o sha sham m in injur jury y tems, Inc. Silver Springs, MD) was used to statistically 0 0 evaluate all data. The significance difference was deter- 00 11 33 77 1142 4212 1288 3355 4422 P Po ostoper stopera ativ tive e Da Day y mined by two-way ANOVA with Bonferroni's post-hoc test for animal behavior. The one way ANOVA with a Dunnett's Multiple Comparison test was used to analyze the differences between naïve, sham and experimental groups. A difference of p < 0.05 was considered signifi- c contr ontra all. . t to o ne nerv rve e in injju ur ry y cant. ip ips sii. to . to nerv nerve e i in nju jury ry 12 12 10 10 Results Mechanical stimuli elicit bilateral tactile hyperalgesia 8 8 following LPC-induced sciatic nerve demyelination 6 6 To study changes in behavioral sensitivity following LPC- induced nerve demyelination, we investigated alterations 4 4 in the paw withdrawal threshold (PWT) force of indenta- tion (produced by von Frey filaments) necessary for elicit- 2 2 ing a flexion hindpaw withdrawal reflex. At POD1, the 0 0 PWT ipsilateral to the LPC-induced sciatic nerve demyeli- 0 da 0 day y 7 7 day day 14 d 14 da ay y nation was significantly reduced when compared to pre- P Po ostoper stopera ativ tive e Da Day y surgical PWTs (Fig. 1A). The force required to elicit a paw withdrawal steadily declined until POD14, before gradu- Mea Frey stimulation ing Figure 1 LP n thresh C-induold force required ced fo at 1, 3, 7 cal nerve , 14 de , 21, 28, 35 a mye for paw wi linationn thdraw d 42 days follow- al to Von ally returning to near pre-surgical levels by POD35. These Mean threshold force required for paw withdrawal to Von PWTs met the pre-determined levels indicative of hyperal- Frey stimulation at 1, 3, 7, 14, 21, 28, 35 and 42 days follow- gesia (-20 mN force) between POD1 and POD28. ing LPC-induced focal nerve demyelination. Each data point is Changes in behavior were also observed in the hind paw the mean threshold (± SE) force on the hindpaw ipsilateral contralateral to the LPC-induced nerve injury (Fig. 1A). (black circle) or contralateral (white circle) to the focal nerve These behavioral changes met the pre-determined PWT injury site eliciting a withdrawal response (n = 10). Reduced behavioral thresholds for the hindpaw ipsilateral to the nerve levels indicative of hyperalgesia between POD3 and lesion were significantly different from pre-operative baseline POD28 (Fig. 1A). Vehicle-treated sham operated rodents on postoperative days 1–28. The threshold force for the did not exhibit a PWT decrease that was significant at any hindpaw contralateral to the nerve lesion did not reach sig- time point up to POD14. Animals given intramuscular nificance until postoperative day 3, and significant differences injections of LPC into the gastrocnemius muscle (10 ul, were observed until postoperative day 28. The time course 1% LPC) did not develop cutaneous hyperalgesia (n = 3, of sham injury (n = 6) is also represented but did not differ data not shown). These data indicate that a unilateral from the uninjured animals. Analysis was performed using LPC-induced nerve demyelination results in bilateral tac- two-way ANOVA followed by the Bonferroni post-hoc pair- tile hyperalgesia. wise comparisons (*p < 0.01).B) LPC-induced focal nerve demyelination did not produce changes in thermal responses Effect of unilateral focal nerve demyelination on thermal as assessed by the Hargreaves test. Each bar is the mean withdrawal latency (± SE) of the hindpaw ipsilateral (white thresholds bar) or contralateral (black bar) to the focal nerve demyeli- In contrast to the effectiveness of LPC-induced focal nerve nation injury at postoperative day 7 and 14 (n = 10). demyelination in altering mechanical PWTs, focal nerve Page 5 of 20 (page number not for citation purposes) Wi Witth h d d ra ra w wa a ll L L a atte e nc ncy y ((se sec) c) M Mean Thr ean Threshold (mN eshold (mN) ) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 demyelination had little if any effect on thermal respon- neuronal expression did not change appreciably follow- sivity from POD0 to POD14 (Fig. 1B). ing vehicle treatment (Fig. 4A and 4C) or focal nerve demyelination at POD14 (Fig. 4B). Despite little change CCR2, CXCR4, CXCR3 and CCR5 upregulation in L –L in non-neuronal cells, there was an increase in the 4 5 DRG after unilateral LPC-induced nerve demyelination number of neurons expressing CXCR4 mRNA transcripts We previously reported upregulation of CCR2 chemokine by POD14 in the lumbar DRG ipsilateral to the nerve receptor signaling in association with chronic compres- lesion (Fig. 4D). This pattern of staining was also sion of the DRG [8]. We therefore examined the state of observed in DRG contralateral to the nerve injury (data CCR2 expression in association with LPC-induced demy- not shown). DRGs taken from injured animals on POD35 elination. L L DRGs removed from naïve (data not exhibited CXCR4 mRNA staining that was similar to that 4 5 shown) and vehicle-treated rats at POD7 did not express seen in naïve and vehicle-treated animals (data not CCR2 mRNA (Fig. 2A) or CCR2 immunoreactivity (Fig. shown). 2B). Small and medium diameter L L neurons ipsilateral 4 5 to the focal nerve lesion exhibited low levels of both Next we used in situ hybridization to examine the expres- CCR2 mRNA transcripts at POD7 (Fig. 2C) and CCR2 sion of the chemokine receptors CXCR3 and CCR5 in the immunoreactivity (Fig. 2D). By POD14, many sensory DRG associated with the LPC-induced focal demyelina- neurons of all diameters exhibited CCR2 mRNA in L L tion. These are the receptors for the chemokines IP-10 and 4 5 DRGs both ipsilateral and contralateral to LPC-induced RANTES (among other chemokines) respectively. Many injury (Figs. 2E, 3). Immunoreactivity for neuronal CCR2 sensory neurons were positive for CXCR3 mRNA in tissues at POD14 (Fig. 2F) was also increased relative to POD7. taken from both naïve (data not shown) and sham-treated Neuronal binding of the plant isolectin, Griffoniasimplici- rodents at POD14 (Fig. 5A). The pattern of neuronal folia B (IB ) in the rat DRG distinguishes a population of expression of CXCR3 mRNA at POD14 following focal 4 4 C-fiber nociceptors [23,24]. We double-labeled the sec- nerve demyelination did not change relative to naïve or tions stained for CCR2 protein with IB . Many IB -bind- sham-treated tissue, but the nerve injury did increase the 4 4 ing neurons were present in lumbar DRG of naïve, sham- intensity of CXCR3 mRNA expression in the DRG ipsilat- operated rats and those subjected to LPC-induced focal eral to the injury (Fig. 5C), as well as in the contralateral nerve demyelination (Fig. 2B, D, F). These neurons dis- DRG (data not shown). played strong labeling of the plasma membrane, as well as perinuclear staining that in all likelihood represents the Unlike CXCR3 expression, lumbar DRG in naïve (data not Golgi apparatus [25]. Quantitative analysis of neurons shown) and sham-treated rats at POD14 (Fig. 5B) were positive for CCR2 revealed that 33.35 ± 2.16% of sensory devoid of CCR5 mRNA transcripts. As with CCR2 expres- neurons ipsilateral and 35.87 ± 3.36% contralateral to the sion in sensory neurons, CCR5 mRNA transcript levels nerve lesion were positive for the chemokine receptor. were strongly increased in many sensory neurons ipsilat- Relatively few (<6%) CCR2-positive neurons were colo- eral and contralateral to the nerve injury at POD14 (Fig. calized with IB -binding neurons ipsilateral (Fig. 2F and 5D). Lumbar DRGs derived from injured animals on Fig. 3) or contralateral to the nerve injury at POD 14 (Fig. POD35 showed no CCR5 mRNA staining (data not 3). CCR2 mRNA was no longer detected in lumbar DRGs shown). taken from injured animals on POD35 (data not shown). Chemokine expression in response to LPC-induced Activation of numerous chemokine receptors in addition demyelination of the sciatic nerve to CCR2 might potentially be involved in the production As the data discussed above indicates strong expression of of sensory neuron hyperexcitability and pain [3,4,26]. The several chemokine receptors by sensory neurons follow- chemokine SDF-1/CXCL12 and its receptor CXCR4 are ing LPC-induced demyelination injury, we sought to constitutively expressed by peripheral nerves [3,27-29] determine whether sensory neurons would also exhibit and chemokines that activate CCR5 and CXCR3 receptors, changes in the expression of chemokines that are possible such as RANTES and IP-10, are synthesized in association ligands for these receptors under the same circumstances. with neuroinflammatory responses [16,30]. To investi- Using immunocyctochemistry, we examined both the gate whether chemokine receptors, in addition to CCR2, nerve lesion site and lumbar DRG associated with the are upregulated following focal nerve demyelination, in injured nerve for MCP-1/CCL2 protein expression. situ hybridization studies were performed on injured rat Despite the multitude of ED-1-immunopositive macro- DRG tissue sections. phages in the injured sciatic nerve, MCP-1/CCL2-immu- noreactive cells were absent from the nerve lesion site on Basal expression of CXCR4 mRNA was predominantly POD1, 3, 7, 14 (data not shown), lumbar DRG removed detected in non-neuronal cells of the lumbar DRG derived from naïve rats (data not shown) and lumbar DRG from from naïve animals (data not shown). This level of non- vehicle-treated rats at POD7 (Fig. 6A). In sharp contrast, Page 6 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 E Figure 2 xpression of CCR2 mRNA and protein immunoreactivity in rat lumbar DRG ipsilateral to focal nerve demyelination Expression of CCR2 mRNA and protein immunoreactivity in rat lumbar DRG ipsilateral to focal nerve demyelination. A) Lum- bar DRG removed from vehicle-treated animals at POD7 did not exhibit CCR2 mRNA expression (n = 5). B) Many lumbar DRG neurons in vehicle-treated rats sensory neurons were positive for isolectin IB , a neuronal phenotype that distinguishes some C-fiber nociceptors (green cells). There was no evidence of CCR2 protein expression in sham animals (n = 5). C) Lum- bar DRG neurons from nerve-injured rats on POD7 exhibited CCR2 mRNA transcripts in some small and medium diameter neurons (black arrows). Open black arrowhead indicates a neuron without CCR2 mRNA transcripts (n = 4). D) Lumbar DRG neurons from a rat subjected to focal nerve demyelination exhibited few CCR2 immunopositive (white arrows) sensory neu- rons (n = 4). E) Many lumbar DRG neurons on POD14 exhibited CCR2 mRNA transcripts (black arrows). Open arrowhead indicates non-labeled neuron. F) CCR2 immunoreactivity was present in an increased number of neurons at POD14 (white arrows; n = 5). Scale bar is; 30 μm (A, C), 50 μm (B, D, F), and 100 μm (E). Page 7 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 60 60 60 60 50 50 50 50 IB IB IB IB4 4 4 4 MCP- MCP- MCP- MCP-1 1 1 1 M M M MC C C CP P P P- - - -1 1 1 1/IB /IB /IB /IB4 4 4 4 CCR2 CCR2 CCR2 CCR2 40 40 40 40 CCR2/ CCR2/ CCR2/ CCR2/IIIIB B B B4 4 4 4 IP IP IP IP- - - -1 1 1 10 0 0 0 I I I IP P P P-10 -10 -10 -10/IB /IB /IB /IB4 4 4 4 30 30 30 30 20 20 20 20 10 10 10 10 0 0 0 0 ip ipsil sila at te er ra al LP l LPC DRG C DRG cont contr ra ala lat te eral ral LP LPC C D DR RG G iips psila ilat te era ral l s sh ham DRG am DRG co cont ntral rala at te er ra al s l sh ham DRG am DRG Figure 3 Percentage of MCP-1, CCR2 and IP-10 immunoreactive neurons with IB -positive neuronal profiles on POD 14 Percentage of MCP-1, CCR2 and IP-10 immunoreactive neurons with IB -positive neuronal profiles on POD 14. MCP-1 expression was increased by LPC-induced nerve injury within the IB -labeled neuronal group in both the DRG ipsilateral and contralateral to the nerve injury. Sham-injury treatment did not produce significant changes in the extent of MCP-1/IB colocal- ization. CCR2 expression was increased by LPC-induced nerve injury within IB -labeled neuronal group in both the DRG ipsi- lateral and contralateral to the nerve injury. Like, MCP-1, sham-injury treatment did not produce significant changes in CCR2/ IB4 colocalization in either DRG ipsi- or contralateral to the sham injury. IP-10 expression was increased by LPC-induced nerve injury within IB -labelled neuronal group in both the DRG ipsilateral and contralateral to the nerve injury, while sham- injury treatment did not produce significant changes in IP-10/IB colocalization. Comparisons of immunoreactive cell percent- ages were made between LPC-treatment and sham-treated animals. Data represent means ± SE. Analysis was performed using two-way ANOVA followed by the Bonferroni post-hoc pair-wise comparisons (*p < 0.01). numerous MCP-1 immunopositive neurons were present cant portion of the MCP-1 positive neurons co-localized in associated lumbar ganglia of the LPC-treated rats by at POD 7 and POD 14 (Fig. 6B, D; Fig. 3). More with IB POD7 (Fig. 6B). The diameter of MCP-1 immunoreactive specifically, just over half of the MCP-1 immunoreactive neurons in the LPC-treated animals averaged 26.49 ± 0.47 neurons present at POD14 in DRG ipsilateral to nerve μm (n = 6). Interestingly, no MCP-1 protein immunoreac- injury were also positive for IB (Fig. 3; 20.12 ± 0.75%; n tivity was detectable in injured DRG non-neuronal cells at = 6). any time point examined. In a previous study, it was shown that in a rodent model Once again, we co-stained DRG sections with IB , a of spinal stenosis, chronic compression of the DRG, there marker for C-fiber nociceptors. Many IB -binding neurons was increased MCP-1 expression and increased excitabil- were present in lumbar DRG of naïve, sham-operated and ity of sensory neurons in injured and adjacent uninjured nerve-injured rats at POD7 and POD 14 (Fig. 6C) and Fig. DRG [8]. Therefore, we wished to see whether MCP-1 3, respectively). The average number of IB neurons activity was altered in the L4/L5 DRG contralateral to the present in the lumbar DRG was 47.87 ± 1.44% (n = 6 per nerve injury. Increased numbers of MCP-1-immunoreac- condition) and did not significantly differ across treat- tive cells were present in ganglia contralateral to nerve ment groups (sham and injured at POD14) (Fig. 3; p > injury (Fig. 3; 34.27 ± 1.17%; n = 6) and 13.32 ± 0.57% 0.1). A series of MCP-1 immunopositive/IB4-binding (n = 6) exhibited IB4 colocalization. Relatively few neu- colocalization experiments demonstrated that a signifi- rons were positive for MCP-1 in ganglia ipsi- or contralat- Page 8 of 20 (page number not for citation purposes) P Pe er rc cen enta tage ge o of f Immu Immunopo noposi siti tive ve Ce Cell lls s Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 Expression of CXCR4 mRNA in rat lumbar DR Figure 4 G ipsilateral to focal nerve demyelination Expression of CXCR4 mRNA in rat lumbar DRG ipsilateral to focal nerve demyelination. Low (A) and high power (C) phot- omicrographs of CXCR4 mRNA transcripts present in lumbar DRG removed from vehicle-treated rodents at POD14 (n = 3). Many non-neuronal cells strongly expressed CXCR4. (C) Some presumptive neurons expressed low levels of CXCR4 mRNA (white arrow). Low (B) and high power (D) photomicrographs of CXCR4 mRNA transcripts present in lumbar DRGs derived from injured rats at POD14 (n = 4). The expression level and number of non-neuronal cells exhibiting CXCR4 mRNA tran- scripts in the lumbar DRG did not change following focal nerve demyelination. However, many neurons upregulated CXCR4 mRNA expression (D; white arrows indicate neurons with low levels of mRNA transcripts; white arrowhead points to a neu- ron lacking CXCR4 mRNA expression). (Scale bar is 1 mm (A and B); 40 μm (C and D). eral to the injury site in sham-treated animals (Fig. 3; SDF-1 is the unique ligand for the CXCR4 receptor <5%) and IB4 colocalization was rare (Fig. 3). [31,32]. It has been reported that Schwann cells in the peripheral nerve express SDF-1 and that its expression As noted above, nearly every neuron in the DRG ipsilat- increases moderately after nerve injury [3,29,33]. Using in eral to the nerve injury upregulated CCR5. We used in situ situ hybridization we examined lumbar DRG associated hybridization to study the levels of RANTES expression, with the injured sciatic nerve for SDF-1 expression at one known ligand for CCR5, in the DRG ipsilateral and POD14. Many lumbar DRG non-neuronal cells present in contralateral to the nerve injury. RANTES expression was both the naïve (data not shown) and vehicle-treated rats absent in the L4/L5 DRG in vehicle treated rats (Fig 7A). were positive for SDF-1 mRNA transcripts (Additional file We found that after nerve injury, RANTES expression was 2A, C). Neither the pattern nor the expression levels of strongly upregulated in DRG neurons ipsilateral and con- SDF-1 mRNA transcripts changed following focal nerve tralateral to the nerve injury, when compared to sham demyelination at POD14 (Additional file 2B, D). control tissue. Like CCR5, RANTES was expressed in small, medium and large sensory neurons (Fig 7B, C). IP-10 is one of three ligands that bind to the CXCR3 recep- tor [34-36]. Little is known, however, concerning a role for IP-10 in sensory neuron function. We demonstrated Page 9 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 Expression of CXCR3 and Figure 5 CCR5 mRNA in rat lumbar DRG following focal nerve demyelination Expression of CXCR3 and CCR5 mRNA in rat lumbar DRG following focal nerve demyelination. (A) Many sensory neurons in the lumbar DRG removed from vehicle-treated rats exhibited CXCR3 mRNA transcripts at POD14 (n = 3). (B) CCR5 mRNA expression was absent from the lumbar DRG of vehicle-treated rats at POD14 (n = 3). (C) CXCR3 mRNA expression pat- terns in sensory neurons subjected to focal nerve demyelination did not differ from vehicle-treated rodents at POD14 (n = 4), but there was an increase in the intensity of CXCR3 mRNA expression. (D) Many neurons in the injured rat lumbar DRG expressed CCR5 transcripts at POD14 (n = 4). Scale bar is 250 μm (A and C); 100 μm (B and D). that the sensory neurons in both vehicle-treated and significantly reduced to 34.49 ± 0.98 μm. By POD14, the injured lumbar DRGs exhibited appreciable levels of number of IP-10 immunoreactive sensory neurons had CXCR3 expression (Fig. 5A, C). Similarly, lumbar DRG increased over three-fold (when compared to the vehicle- from both naïve (data not shown) and vehicle-treated treated rat lumbar DRGs) to 36.8 ± 4.9% of the total neu- rodents displayed constitutive IP-10 immunoreactivity ronal population (Fig. 3, Fig. 8C), and the average neuro- (12.04 ± 1.2%, at least 400 cells/DRG from each of 6 vehi- nal diameter was further reduced to 29.74 ± 1.1 μm (n = cle-treated animals) (Fig. 3, Fig. 8A), both ipsi- and con- 6). tralateral to the site of nerve injury. The mean diameter of IP-10-immunopositive neurons in the vehicle-treated rat A reduction in the average diameter of IP-10-immunopo- was 47.53 ± 1.7 μm. Seven days after LPC-induced focal sitive neurons concurrent with a three-fold increase in the nerve demyelination, the number of IP-10-immunoposi- number of neurons implies upregulated expression of IP- tive neurons in injured DRG had increased over two-fold 10 occurred in cells normally negative for the chemokine. to 30.41 ± 2.9% (n = 6) (Fig. 3, Fig. 8B). The average cell Similar to previous findings in subpopulations of cells diameter of IP-10 immunoreactive neurons on POD7 was positive for the neurotrophin brain-derived neurotrophic Page 10 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 demyel Figure 6 Colocalization ination i of MCP-1 im njury at POD7 munoreactivity (ir) and isolectin B (IB )-binding in the lumbar DRG ipsilateral to LPC-induced 4 4 Colocalization of MCP-1 immunoreactivity (ir) and isolectin B (IB )-binding in the lumbar DRG ipsilateral to LPC-induced 4 4 demyelination injury at POD7. IB -binding in rat DRG neurons distinguishes C-fiber nociceptors. (A) Naïve rat lumbar DRG were completely negative for MCP-1ir. (B) Lumbar DRG ipsilateral to LPC-induced sciatic nerve injury exhibited numerous small diameter neurons that are MCP-1 positive at POD7 (red arrows). (C) Numerous small IB -binding presumptive nocicep- tors are present in the same DRG tissue section (green arrows). (D) Merging panels B and C demonstrates the extent of colo- calization present in lumbar DRG tissue section (yellow arrows). Note not all MCP-1ir neurons were positive for IB at POD7 (red arrow). Scale bar is 100 μm (A, B, C and D). factor (BDNF) by Obata and colleagues [37], this change IP-10 immunoreactivity and IB4-binding (n = 6) (Fig. 3, in neuronal phenotype may be indicative of pathophysio- Fig. 8E, H). However, a two-fold increase in the number logical changes in primary afferent neurons following of neurons positive for both IP-10-immunoreactivity and focal nerve demyelination. To determine the degree to IB4-binding (19.08 ± 2.31%; n = 6) was evident at POD14 which small, presumably nociceptive IB4-binding neu- (p < 0.01) (Fig. 3, Fig. 8F, I). rons, also displayed IP-10 immunoreactivity following 2+ LPC-induced nerve demyelination, we performed a series Chemokines increase [Ca ] in DRG cells subjected to LPC-induced nerve demyelination of colocalization experiments (Fig. 8D–I). Our analysis revealed that generally speaking, IP-10 immunoreactive Activation of chemokine receptors expressed by primary neurons did not co-localize with IB4-binding neurons in sensory neurons results in excitation and in the increase in 2+ vehicle-treated lumbar DRG (Fig. 3, Fig. 8D, G). On the intracellular Ca concentration [4]. To compliment POD7, 9.39 ± 1.64% of the total neurons exhibited both the anatomical observations of upregulated chemokine Page 11 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 E Figure 7 xpression of RANTES mRNA in rat lumbar DRG following focal nerve demyelination Expression of RANTES mRNA in rat lumbar DRG following focal nerve demyelination. (A) RANTES mRNA expression was absent from the lumbar DRG of vehicle-treated rats at POD14 (n = 3). (B) Many sensory neurons in the DRG ipsilateral to the nerve injury at POD 14 were positive for RANTES mRNA (C) Numerous sensory neurons in the DRG contralateral to the nerve injury displayed expression of RANTES mRNA, albeit at a lower level when compared with the DRG ipsilateral to the nerve injury. Scale bar is 250 μm (A and C); 100 μm (B and D). receptor expression, we used fura-2 imaging of chemok- nal cells (Table 2). It was also noted that at POD 14, most 2+ ine-induced increases in [Ca ]i in acutely isolated rat cells responded to only one chemokine. However, at POD DRG neurons as a measure of functional chemokine 28, when upregulated chemokine signaling was at its receptor expression. We acutely isolated the DRG cells greatest, most cells responded to multiple chemokines, from both the ipsilateral and contralateral sides of nerve indicating that single neurons expressed multiple chem- injured animals and sham controls. For all experiments, okine receptors. In the DRG contralateral to the nerve the chemokines MCP-1, SDF-1, RANTES and IP-10 were injury, there was an upward trend in chemokine signaling added in random order to the cells, after which capsaicin, at POD 14–28 (Table 1), but this did not reach signifi- high K+(50 mM) and ATP were added to assess the cells' cance. By POD 35, dissociated DRG cell responses to identity and viability, respectively. A response to high K+ chemokines had returned to baseline levels (Fig. 9D, stimulation indicates the presence of voltage dependent Table 1). Thus, the fura-2 imaging generally confirmed the Ca2+ channels, which is indicative of neurons. Addition- observed upregulated expression and function of chemok- ally, a positive response to capsaicin as well as high K+ ine signaling in DRG cells subjected to a peripheral nerve indicates the cell is a nociceptor expressing the TRPV1 demyelination. channel. A response to ATP, which activates purinergic CCR2 receptor antagonist attenuates bilateral focal nerve receptors, without one to High K+ and/or capsaicin, indi- cates a non neuronal cell, presumably a type of glial cell demyelination induced tactile hyperalgesia such as a satellite glial cell or Schwann cell. The concentra- Using LPC nerve-injured animals, we tested the effect of a tions of chemokines used in these experiments were all single i.p injection of a CCR2 receptor antagonist (CCR2 supramaximal to ensure activation of any expressed recep- RA-[R]) or its inactive enantiomer (CCR2 RA-[S]) on tors. nociceptive behavior at POD14 and POD28 (10 mg/kg). Neither vehicle nor CCR2 RA-[S] administration at day 14 It was evident that an increased number of cells or day 28 had an effect on the bilateral mechanical PWT responded to MCP-1 application from POD 14–28 in the when tested 1 hour later (Fig. 10). In contrast, bilateral DRG cells ipsilateral to the nerve injury (Fig. 9B and 9C), increases in PWT were observed one hour after adminis- (Table 1), when compared to vehicle-treated control ani- tration of CCR2 RA-[R]. These PWTs did not differ from mals (Fig 9A). The number of cells responding went from pre-operative basal threshold levels (Fig. 10). The effects 5.8% to 31.7% by POD 28. The majority of these cells of CCR2 RA-[R] were stereospecific as administration of were characterized as neurons based on their positive an equal dose of the inactive stereoisomer did not inhibit responses to capsaicin and/or high K+ (Table 2). Many pain behavior. All PWTs returned to pre-drug administra- 2+ cells also exhibited increases in [Ca ]i in response to the tion levels within 24 hours (data not shown). other chemokines tested (i.e. SDF1, RANTES and IP-10) and the frequency of these responses was always greatest Discussion by POD 28 in the nerve injured animals. Chemokine- Previous work carried out in our own and other laborato- induced changes in dissociated DRG were not limited to ries has indicated that chemokine signaling may contrib- sensory neurons, but also included occasional non-neuro- ute to the genesis and maintenance of neuropathic pain Page 12 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 Colocalization subject Figure 8 ed to LPC-induced of IP-10 immunoreactiv nerve injuryity (-ir) and isolectin B4 (IB )-binding neurons in the lumbar DRG of naïve rat and rats Colocalization of IP-10 immunoreactivity (-ir) and isolectin B4 (IB )-binding neurons in the lumbar DRG of naïve rat and rats subjected to LPC-induced nerve injury. IB -binding in rat DRG neurons distinguishes a population of C-fiber nociceptors. A) The majority of IP-10-ir cells were limited to medium diameter neurons in the lumbar DRG from vehicle-treated rats (red arrows). D) IB -binding small diameter presumptive nociceptors (green arrows) did not colocalize with IP-10-ir lumbar DRG neurons from vehicle-treated rodents at POD7 (G, merged images). B) Lumbar DRG ipsilateral to focal nerve demyelination exhibited numerous medium and small diameter IP-10-ir neurons at POD7. Limited numbers of neurons were positive for both IP-10-ir (B, red arrows) and IB4-binding (E, green arrows) on POD7 (H, merged images). C) Many IP-10-ir neurons (red arrows) colocalized with IB -binding neurons (F, green arrows) at POD14 (I, merged images). Yellow arrows indicate colocal- ized cells. Scale bar is 100 μm. [11,17,18]. Thus, the present studies were designed to ine receptors and chemokine/receptor signaling increased investigate a potential association between focal nerve significantly. This occurred not only in the DRG directly demyelination, neuropathic pain behavior and chemok- associated with the injured nerve, but also to a lesser ine signaling in DRG neurons. Based on our previous degree, in the DRG directly contralateral to the nerve studies, we hypothesized that the focal demyelination of injury. Bilateral nociceptive behavior was apparent the sciatic nerve, a known rodent model of neuropathic between days 3–28 and could be stereospecifically atten- pain [12], would result in upregulated chemokine expres- uated with a CCR2 receptor antagonist on days 14 and 28. sion and chemokine receptor signaling in DRG neurons. Moreover, five weeks following injury, both the neuro- Indeed, we observed the predicted increases for several pathic pain behavior and the incidence of chemokine sig- chemokines and their receptors. Importantly, as the PWT naling were greatly diminished. Together with the known decreased over 14 days post-injury, chemokines, chemok- cellular effects produced by chemokines on sensory neu- Page 13 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 Table 1: Table illustrates the chemokine response profile of acutely dissociated DRG cells from rats with and without peripheral nerve injury (n = 5/group). The cells were imaged while the various chemokines were added to the bathing solution. A rise in (Ca)i after chemokine application was indicative of the expression of a functional chemokine receptor. After chemokine application, capsaicin, high K+ and ATP were used to characterize the identity of the cells (see text). Note the upregulation of chemokine signaling at 14 and 28 days after peripheral nerve demyelination in isolated DRG cells ipsilateral to the nerve injury. There was also an upward trend in chemokine signaling in the cells isolated from the DRG contralateral to nerve injury at POD 14–28. By POD 35, chemokine responsiveness returned to baseline levels. Control POD 14 POD 28 POD 35 Chemokine Ipsilateral Contralateral Ipsilateral Contralateral Ipsilateral Contralateral Ipsilateral Contralateral MCP-1 5.8% 7.9% 11.6% 2.5% 31.7% ** 15.1% 2.4% 0.0% SDF1 5.8% 7.0% 2.3% 5.0% 25% ** 8.2% 7.3% 6.6% RANTES 5.8% 7.0% 23.2% ** 2.5% 8.3% 13.6% 9.7% 3.3% IP-10 9.6% 8.8% 0.0% 20.0% 21.1%* 8.2% 7.3% 6.6% *p < 0.01 ** p < 0.05 n = 5/age group rons [3,7], these results suggest that the changes in sensory Presumably, chemokines released in this fashion may neuron chemokine/receptor signaling may be central to influence neural cells in the local vicinity eliciting excita- the maintenance phase of neuropathic pain behavior in tion as described above. Such activation would produce particular. further chemokine release and excitation driving the over- all excitability of the chemokine sensitive neurons to new The results of our previous studies, together with the levels. The resulting neuronal behavior may explain cer- present results on LPC-associated neuropathy, point to a tain aspects of pathologically maintained neuronal states significant role for chemokine signaling expressed directly of depolarization or electrical hyperexcitability of periph- by peripheral nerves. For example, we have previously eral sensory neurons [39-41]. In addition to the chronic demonstrated that activation of chemokine receptors maintenance of sensory neuron hyperexcitability, release expressed by cultured or acutely isolated DRG neurons of chemokines such as MCP-1 and fracktalkine from cen- 2+ produces increased (Ca ) , or neural excitation [4,38]. tral axon terminals in the spinal cord may initiate micro- Indeed, following upregulation of CCR2 by sensory neu- glial-mediated neuropathic pain states [7,11,42-44]. rons in whole DRG derived from animals exhibiting neu- However, it is important to note that pharmacological ropathic pain, application of MCP-1 produces powerful therapies which inhibit microglial activation and effec- excitation [8]. The mechanism underlying this response tively attenuate the development of hyperalgesia and allo- probably involves activation of phospholipase C-induced dynia have no effects on preexisting nociceptive pain degradation of PIP2, production and concomitant trans- behavior [45]. activation of TRPV1 and/or TRPA1 together with inhibi- tion of K+ conductance [9,10]. As we have demonstrated, the exact pattern of changes in chemokine signaling observed following focal nerve The experiments reported here suggest a model in which demyelination depends on the particular chemokine focal nerve demyelination produces a concomitant upreg- receptor and ligand examined. There are over 50 known ulation of several chemokines and their receptors in the chemokines and 20 chemokine receptors [32], and it is cell bodies of sensory neurons in the DRG. We have obviously not feasible to study all of these simultane- observed that chemokines expressed by DRG neurons, ously. However, the receptors studied in the present including MCP1, IP10 and SDF1, can be packaged into experiments represent obvious candidates for a role in secretory vesicles and released upon depolarization [9]. peripheral neuropathy. Chemokines that signal via the CCR2, CCR5, CXCR3 and CXCR4 receptors have previ- Table 2: At 28 days, the majority of cells responded to capsaicin ously been shown to influence the behavior of sensory or high K+, indicating that most of the chemokine responsive neurons [3,4,6,8,11,17]. Furthermore, many of these cells were neurons. receptors can be upregulated in leukocytes by mecha- Capsaicin/High K+/ATP positive (TRPV1 39.1% nisms suggesting that regulation of their expression may expressing nociceptor) often be coordinated through the same transcriptional High K+/ATP positive only (Non TRPV1- 39.1% control mechanisms [46]. expressing neuron) ATP positive only (Non-neuronal cell) 21.7% Page 14 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 A A B B C C D D s s Ti Time (s me (sec) ec) Chemoki Figure 9nes increased [Ca2+]i levels in acutely isolated rat DRG cells following focal demyelination injury Chemokines increased [Ca2+]i levels in acutely isolated rat DRG cells following focal demyelination injury. The figure shows examples of responses of cells acutely isolated from rat DRGs ipsilateral to the nerve injury at various days after a focal demy- elination injury. Under normal conditions, cells rarely respond to any chemokine but did respond to other stimuli such as high K or ATP (A). However, there was an increased responsiveness of the cells, the majority of which could be characterized as neurons, between post-operative days 14–28 (B and C, respectively). The frequency of the responses to chemokines returned to approximately the same level as control animals by post-operative day 35 (D). For all experiments, MCP-1 (M), IP- 10 (I), RANTES (R), SDF1 (S) were applied at a concentration of 100 nM. Capsaicin (C), high K (K) and ATP (A) were applied at concentrations of 100 nM, 50 mM and 100 uM, respectively. The four chemokines/chemokine receptors that we stud- DRG. The upregulation of MCP-1 and the CCR2 chemok- ied all displayed different patterns of expression in ine receptor observed in association with focal nerve response to focal nerve demyelination, suggesting differ- demyelination is similar to the pattern we previously ent roles in the genesis of pain or other functions in the observed using a spinal stenosis model of neuropathic Page 15 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 model. The particular effectiveness of CCR2 block could be due to the fact that upregulation of chemokines can be bilaterally expressed in different populations of sensory neurons following nerve injury, as is this case of chole- cystokinin vasoactive inhibitory peptide and neuropep- tide Y [43,47]. In our experiments over 50% of the cells that upregulated MCP-1 also expressed IB-4, which is a marker for C-fiber nociceptors that are responsible for transmitting pain information. This differs from the case of IP-10, where a majority of the neurons upregulating this chemokine did not co-localize with IB-4. As such, it is possible that the population of neurons that upregulates CCR2 signaling is particularly linked to the production of neuronal hyperexcitability. It is also likely that the CCR2R antagonist may impact non-neuronal cells within the !"   !" CNS, such as microglial cells, which are known to express CCR2 in the spinal cord and contribute to the develop- reversed existing n Figure 10 CCR2 receptor antagonist (CCR2 ociceptive behavior RA-[R]) administration ment of chronic pain states [11,48,49]. CCR2 receptor antagonist (CCR2 RA-[R]) administration reversed existing nociceptive behavior. Animals were sub- The precise location of action of the CCR2 antagonist is jected to a nerve demyelination injury on day 0 and nocicep- not known. However, it has been shown that the blood tive behavior was assessed for 28 days. On days 14 and 28 nerve barrier is less restrictive than the blood brain barrier post-surgery, animals received 5 mg/kg CCR2 RA-[R] or 5 [50], with the cell body rich area of the DRG being vulner- mg/kg of its inactive enantiomer, (CCR2 RA-[S], or saline by able to extravascular leakage. Given these studies, it is intraperitoneal injection, and behavioral responses were tested 1 h later. Administration of the CCR2 RA-[R] to focal likely that the CCR2 R antagonist reached the cell bodies nerve demyelination injured rats resulted in a significant bilat- of the DRG. As activation of CCR2 receptors in the DRG is eral increase of mN force required to elicit a paw withdrawal probably of considerable importance in the production of compared with vehicle-treated controls and animals sub- pain behavior it is likely that block of these receptors con- jected to CCR2 RA-[S]. Nociceptive behavior in vehicle- tributes to the antinociceptive effects of the CCR2 antago- treated controls and animals subjected to CCR2 RA-[S] dif- nist. fered significantly from day 0 pre-injury baseline responses (*p < 0.01). Data represent means ± SE. In the face of the effectiveness of CCR2 receptor block either pharmacologically (Fig 9) or genetically [11], the function of other types of upregulated chemokine signal- pain [8]. Indeed, CCR2 receptor deficient mice are resist- ing to chronic pain behavior is not immediately obvious. ant to the induction of some sensory neuropathies, high- Like CCR2, the CCR5 receptor function and its ligand, lighting the potential importance of this chemokine RANTES, were also strongly upregulated in DRG neurons signaling system [11]. In the current experiments, we uti- in response to focal demyelination. It has previously been 2+ imaging paradigm in lieu of electrophysiolog- lized a Ca shown that RANTES may be important in other chronic ical recording, as chemokine-induced increased neuronal pain situations [51]. Our findings in this sciatic nerve excitability would be expected to be correlated with a injury model differ from a report by Taskinen and Royotta 2+ chemokine induced increase in (Ca ) . The observed [16] which demonstrated bilateral upregulation of non- increase and subsequent decrease in MCP-1-induced neuronal RANTES for up to four weeks following sciatic 2+ Ca responsiveness in acutely isolated DRG neurons over nerve transection in the rat. The apparent differences may time generally correlated with the anatomical observa- be due to the nature of the nerve injuries. Alternatively, tions of receptor expression, and both effects returned to CCR5 may also be activated by a number of other chem- baseline by POD 35. Importantly, the ability of the CCR2 okine ligands which we did not measure [52]. Chemokine receptor antagonist to attenuate bilateral nociceptive interactions with CCR5 may also depress the analgesic behavior at both 14 and 28 days after nerve injury strongly action of endogenous opioids and/or sensitize TRPV1 suggests an integral role for MCP-1/CCR2 signaling in [10,53,54] thereby generally promoting hyperalgesia. maintaining this phase of pain hypersensitivity. The signaling pattern of IP-10 and CXCR3 receptors in the Although the CCR2 antagonist was effective in blocking DRG differs in some respects as there is appreciable basal pain hypersensitivity, more than one chemokine or chem- neuronal expression of both CXCR3 receptors and IP-10. 2+ okine receptor was upregulated in this neuropathic pain In spite of this, few Ca neuronal responses were Page 16 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 observed in the naïve or sham animals, perhaps because chemokines/receptors following unilateral sciatic nerve of desensitization resulting from ongoing receptor activa- demyelination by releasing cytokines from activated tion induced by constitutive expression of IP-10. Neuro- microglia in the spinal cord dorsal horn following chronic nal expression of IP-10/CXCR3 under basal conditions activity in injured DRG afferent neurons [70]. Spinal cord- may have a specific role to play that is analogous to the derived cytokines or growth factors such as TNF-α, IL-1β, expression and release of fractalkine by neurons [42,55]. IL-6 and/or BDNF, may also directly signal contralateral Following strong neuronal excitation in the peripheral lumbar DRG neurons through receptors present on pri- nervous system (i.e. trauma or disease), IP-10 may be mary afferent central terminations [71,72]. Perhaps not released within the DRG and/or from central terminals in surprisingly, blockade of the action of TNF-α or inhibition the spinal cord dorsal horn resulting in both local and dis- of glial metabolic activity can attenuate bilateral nocicep- tant glial activation [26,56,57]. tive pain behavior [69,73], while the low dose administra- tion of a gap junction protein decoupler only extinguishes Focal nerve demyelination changes in SDF-1 signaling via only contralateral pain behavior [74]. Although current the CXCR4 receptor show still another pattern. In this trends in pain research favor bilateral spinal cord glial case, the chemokine receptor is not generally expressed in activation as a mechanism of central activation in the spi- neurons, but in satellite glia and Schwann cells. Upregu- nal cord, it does not appear to be central to all pain con- lated CXCR4 expression, however, is primarily restricted ditions [61]. It is also interesting to note in the context of to neurons making them a potential target for the release the present set of investigations that TNF-α can upregulate of SDF-1 from glia. It is interesting to note that a role for MCP-1 expression by sensory neurons [75], further sup- Schwann cell release of SDF-1 and for neuronally- porting the possibility that it may function as an upstream expressed CXCR4 receptors has also been suggested in regulator of chemokine signaling in the DRG. recently proposed models of HIV-1 and NRTI related neu- ropathies [14,51]. Alternatively, aspects of bilateral tactile hyperalgesia may be due to spontaneous ectopic activity in A, but not C-fib- It is clear that focal nerve demyelination injury-induced ers. This type of ongoing ectopic hyperexcitability in pri- behavioral changes are correlated with widespread mary sensory neurons post-injury can occur in both changes in the neuronal expression of chemokine/recep- injured neurons and adjacent, uninjured neurons tors and that the pattern of expression of each chemokine [20,37,76,77]. Changes necessary for this type of mecha- and its receptor is unique, suggesting that the influence of nism implicate modification of the electrical properties of chemokine signaling on rodent nociceptive behavior may the neurons [78-80]. Nerve demyelination in the mid- be complex. It should also be noted that the upregulated thigh may effectively trigger just this type of change in the expression of different chemokines and receptors that we primary sensory neuron (i.e. neurochemistry and physiol- have observed may occur as part of a cytokine "cascade", ogy of primary afferent neurons), which then contribute where the expression of one chemokine or its receptor is to central sensitization and higher levels of nociceptive dependent on previous events. If that is the case it is also sensory processing. Taken together, the evidence of possible that drugs which block several upregulated chronic changes in chemokine/receptor protein expres- chemokine receptors may also prove to be effective if they sion and the ability of certain chemokines to excite neuro- are upstream of CCR2 expression. nal subpopulations [6,8] is suggestive of a potential scenario. In the course of these studies, we also noted that the PWT to mechanical stimulation decreased bilaterally. The Another behavioral component commonly observed with degree of threshold change in the hindpaw contralateral rodent pain models, especially those accompanied by to the nerve injury was qualitatively similar but smaller in robust inflammatory responses, is the presence of thermal magnitude, and briefer in time course, when compared hyperalgesia. Despite a presence of thermal hyperalgesia with the hindpaw ipsilateral to the lesion. This type of in the mouse focal nerve demyelination model [12], the bilateral hyperalgesia has previously been described in rat nerve demyelination model does not exhibit changes other rodent models of neuropathic pain [58-67]. As such, in response to temperature. Thermal hyperalgesia is it is of interest to compare the LPC-induced peripheral largely thought to be a pain-related symptom caused by nerve pain model with previously described rodent peripheral sensitization. The absence of thermal hyperal- inflammatory pain models which demonstrated bilateral gesia would suggest that there is a lack of ongoing inflam- tactile pain behavior [60] and contralateral changes in the matory mediator-initiated sensory neuron signaling. Lack DRG [65,68]. Milligan et al. [69] suggested that this phe- of thermal hyperalgesia in peripheral nerve injury models nomenon is likely due to changes in the spinal dorsal of pain, although not common, include perineural gp-120 horn. This type of spinal mechanism may drive both bila- administration [81]; Bhangoo and White, unpublished tateral pain sensitivity and contralateral DRG changes in observations) acidic saline-induced hyperalgesia [63] and Page 17 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 4. Oh SB, Tran PB, Gillard SE, Hurley RW, Hammond DL, Miller RJ: chronic constriction injury performed in a 5-HT trans- Chemokines and glycoprotein120 produce pain hypersensi- porter knockout mouse [82]. Lack of thermal hyperalgesia tivity by directly exciting primary nociceptive neurons. J Neu- in the model of muscle pain and CCI implicate central rosci 2001, 21:5027-35. 5. Qin X, Wan Y, Wang X: CCL2 and CXCL1 trigger calcitonin descending mechanisms for the display of bilateral hyper- gene-related peptide release by exciting primary nociceptive algesia. It is possible that similar mechanisms are operat- neurons. J Neurosci Res 2005, 82:51-62. 6. Sun JH, Yang B, Donnelly DF, Ma C, LaMotte RH: MCP-1 enhances ing within the rat nerve demyelination injury model. excitability of nociceptive neurons in chronically com- pressed dorsal root ganglia. J Neurophysiol 2006, 96:2189-99. Taken together the data suggest that upregulation of 7. Tanaka T, Minami M, Nakagawa T, Satoh M: Enhanced production of monocyte chemoattractant protein-1 in the dorsal root chemokine signaling by sensory neurons may help to ganglia in a rat model of neuropathic pain: possible involve- integrate several phenomena that account for changes in ment in the development of neuropathic pain. Neurosci Res the properties of peripheral nerves resulting in bilateral 2004, 48:463-9. 8. White FA, Sun J, Waters SM, Ma C, Ren D, Ripsch M, Steflik J, Cor- pain hypersensitivity. The present results, together with tright DN, Lamotte RH, Miller RJ: Excitatory monocyte chem- previous studies [3], suggest that the mode of injury may oattractant protein-1 signaling is up-regulated in sensory neurons after chronic compression of the dorsal root gan- determine which particular chemokines play a central role glion. Proc Natl Acad Sci USA 2005, 102:14092-7. in maintaining the neuropathic pain state. Chemokine 9. Jung H, Toth PT, White FA, Miller RJ: Monocyte chemoattractant receptors may then represent novel targets for therapeutic protein-1 functions as a neuromodulator in dorsal root gan- glia neurons. J Neurochem 2007. intervention in demyelination associated neuropathic 10. Zhang N, Inan S, Cowan A, Sun R, Wang JM, Rogers TJ, Caterina M, pain as well as other chronic pain states. Oppenheim JJ: A proinflammatory chemokine, CCL3, sensi- tizes the heat- and capsaicin-gated ion channel TRPV1. Proc Natl Acad Sci USA 2005, 102:4536-41. Additional material 11. Abbadie C, Lindia JA, Cumiskey AM, Peterson LB, Mudgett JS, Bayne EK, DeMartino JA, MacIntyre DE, Forrest MJ: Impaired neuro- pathic pain responses in mice lacking the chemokine recep- Additional file 1 tor CCR2. Proc Natl Acad Sci USA 2003, 100:7947-52. 12. Wallace VC, Cottrell DF, Brophy PJ, Fleetwood-Walker SM: Focal The chemical structures and full names of the CCR2 antagonist (CCR2- lysolecithin-induced demyelination of peripheral afferents [R]) and its inactive enantiomer (CCR2-[S]). results in neuropathic pain behavior that is attenuated by Click here for file cannabinoids. J Neurosci 2003, 23:3221-33. [http://www.biomedcentral.com/content/supplementary/1744- 13. Cook WJ, Kramer MF, Walker RM, Burwell TJ, Holman HA, Coen 8069-3-38-S1.doc] DM, Knipe DM: Persistent expression of chemokine and chemokine receptor RNAs at primary and latent sites of her- pes simplex virus 1 infection. Virol J 2004, 1:5. Additional file 2 14. Melli G, Keswani SC, Fischer A, Chen W, Hoke A: Spatially distinct Expression of SDF1 mRNA in rat lumbar DRG ipsilateral to focal nerve and functionally independent mechanisms of axonal degen- demyelination. (A) Many cells, both satellite glia and neurons, in the eration in a model of HIV-associated sensory neuropathy. Brain 2006, 129:1330-8. lumbar DRG removed from vehicle-treated rats exhibited SDF1 mRNA 15. Subang MC, Richardson PM: Influence of injury and cytokines on transcripts at POD14 (n = 3). (B) SDF1 mRNA expression did not synthesis of monocyte chemoattractant protein-1 mRNA in change significantly in the lumbar DRG of LPC-treated rats at POD14 peripheral nervous tissue. Eur J Neurosci 2001, 13:521-8. (n = 3). (C) A magnified photomicrograph of the lumbar DRG from a 16. Taskinen HS, Roytta M: Increased expression of chemokines vehicle treated rat. (D) A magnified photomicrograph of the lumbar DRG (MCP-1, MIP-1alpha, RANTES) after peripheral nerve removed from a LPC-treated rat at POD14. transection. J Peripher Nerv Syst 2000, 5:75-81. 17. Bhangoo S, Jung H, Chan DM, Ripsch M, Ren D, Miller RJ, White FA: Click here for file Peripheral demyelination injury induces upregulation of [http://www.biomedcentral.com/content/supplementary/1744- chemokine/receptor expression and neuronal signaling in a 8069-3-38-S2.doc] model of neuropathic pain. In Abstract Viewer/Itinerary Planner Atlanta, GA: Society for Neuroscience Annual Meeting; 2006:250.3. 18. White FA, Ripsch M, Bhangoo S, Ren D, Weiss C, Miller RJ: Regula- tion of chemokines/receptors in the dorsal root ganglion fol- lowing focal demyelination of the sciatic nerve. In Abstract Viewer/Itinerary Planner Washington, DC: Society for Neuroscience Acknowledgements Annual Meeting; 2005:748.9. FAW, NIH Grant NS049136, National Multiple Sclerosis Society Pilot 19. 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Delayed functional expression of neuronal chemokine receptors following focal nerve demyelination in the rat: a mechanism for the development of chronic sensitization of peripheral nociceptors

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Springer Journals
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Copyright © 2007 by Bhangoo et al; licensee BioMed Central Ltd.
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Medicine & Public Health; Pain Medicine; Molecular Medicine; Neurobiology
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1744-8069
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10.1186/1744-8069-3-38
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18076762
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Abstract

Background: Animal and clinical studies have revealed that focal peripheral nerve axon demyelination is accompanied by nociceptive pain behavior. C-C and C-X-C chemokines and their receptors have been strongly implicated in demyelinating polyneuropathies and persistent pain syndromes. Herein, we studied the degree to which chronic nociceptive pain behavior is correlated with the neuronal expression of chemokines and their receptors following unilateral lysophosphatidylcholine (LPC)-induced focal demyelination of the sciatic nerve in rats. Results: Focal nerve demyelination increased behavioral reflex responsiveness to mechanical stimuli between postoperative day (POD) 3 and POD28 in both the hindpaw ipsilateral and contralateral to the nerve injury. This behavior was accompanied by a bilateral increase in the numbers of primary sensory neurons expressing the chemokine receptors CCR2, CCR5, and CXCR4 by POD14, with no change in the pattern of CXCR3 expression. Significant increases in the numbers of neurons expressing the chemokines monocyte chemoattractant protein-1 (MCP-1/CCL2), Regulated on Activation, Normal T Expressed and Secreted (RANTES/CCL5) and interferon γ-inducing protein-10 (IP- 10/CXCL10) were also evident following nerve injury, although neuronal expression pattern of stromal cell derived factor-1α (SDF1/CXCL12) did not change. Functional studies demonstrated that acutely dissociated sensory neurons 2+ derived from LPC-injured animals responded with increased [Ca ] following exposure to MCP-1, IP-10, SDF1 and RANTES on POD 14 and 28, but these responses were largely absent by POD35. On days 14 and 28, rats received either saline or a CCR2 receptor antagonist isomer (CCR2 RA-[R]) or its inactive enantiomer (CCR2 RA-[S]) by intraperitoneal (i.p.) injection. CCR2 RA-[R] treatment of nerve-injured rats produced stereospecific bilateral reversal of tactile hyperalgesia. Conclusion: These results suggest that the presence of chemokine signaling by both injured and adjacent, uninjured sensory neurons is correlated with the maintenance phase of a persistent pain state, suggesting that chemokine receptor antagonists may be an important therapeutic intervention for chronic pain. Page 1 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 receptor upregulation, the continued expression of neuro- Introduction Inflammatory events induced by nerve injury are thought nal chemokine/receptors appears to correlate with to play a central role in the pathogenesis of inflammatory changes in chronic nociceptive behavior. Furthermore, pain. The production and release of molecules that medi- administration of a CCR2 receptor antagonist produced ate the acute inflammatory response include bradykinin, an attenuation of the nociceptive behavior, further high- tachykinins, serotonin, histamine, ATP and cytokines lighting the potential role of chemokine signaling in states such as tumor necrosis factor-alpha (TNFα), interleukin of neuropathic pain. 1-β (IL-1β), and interleukin-6 (IL-6). Many of these mol- ecules, which are produced in association with acute Parts of this study have been previously published in inflammatory responses, are known to induce hyperalge- abstract form [17,18]. sia [1,2] Methods Chemokines, which also contribute to the development Animals of inflammatory pain states, can directly excite subsets of Pathogen-free, adult female Sprague-Dawley rats sensory neurons [3-8]. This excitation is likely to be due to (150–200 g; Harlan Laboratories, Madison, WI) were transactivation of ion channels, such as TRPV1 and housed in temperature (23 ± 3°C) and light (12-h TRPA1, expressed by sensory nerves [9,10]. As such, it is light:12-h dark cycle; lights on at 07:00 h) controlled quite possible that a prolonged de novo expression of rooms with standard rodent chow and water available ad chemokines and/or their cognate receptors by sensory libitum. Experiments were performed during the light neurons following peripheral nerve injury may be central cycle. Animals were randomly assigned to the treatment to the development and/or maintenance of chronic pain groups. These experiments were approved by the Institu- states. Indeed, we previously demonstrated that in a tional Animal Care and Use Committee of Loyola Univer- rodent model of spinal stenosis, chronic compression of sity, Chicago. All procedures were conducted in the DRG (CCD), produced a delayed but chronic expres- accordance with the Guide for Care and Use of Laboratory sion of both the chemokine receptor CCR2 and its ligand, Animals published by the National Institutes of Health the chemokine MCP-1/CCL2 in lumbar DRGs [8]. Fur- and the ethical guidelines of the International Association thermore, MCP-1/CCL2 depolarized or increased the for the Study of Pain. All animals were randomly assigned excitability of several subpopulations of sensory neurons, to either treatment or control groups. including nociceptors, in both the intact and dissociated DRG [6,8]. Interestingly, mice deficient in the chemokine Sciatic nerve demyelination receptor, CCR2, exhibit an impaired neuropathic pain Animals were anesthetized with 4% isoflurane and main- response following partial nerve ligation [11]. tained on 2% isoflurane (Halocarbon, River Edge, NJ) in O . For all demyelination experiments, lysophosphatidyl- In order to fully understand the extent and significance of choline (LPC), (type V, 99% pure; Sigma-Aldrich, St. neuronal chemokine signaling in states of pain hypersen- Louis, MO) was dissolved in buffered sterile saline (pH sitivity, we examined whether induction of a focal demy- 7.2) to give a final concentration of 10 mg/ml. The right elination of the sciatic nerve, a known rodent model of sciatic nerve of the rat was exposed at the mid-thigh level neuropathic pain [12], produced changes in the neuronal under sterile conditions. A sterile polyvinyl acetal (PVAc) expression of certain key chemokines previously shown to sponge (Ivalon, San Diego, CA), 2-mm × 2-mm soaked in be extensively upregulated in peripheral neuroinflamma- 7 μl of LPC, was placed adjacent to the sciatic nerve. The tory responses [3,13-16]. These chemokines included dermal incision site was closed with 5.0 suture thread. monocyte chemoattractant protein-1 (MCP-1/CCL2), Sham control animals were prepared as described above, interferon γ-inducing protein-10 (IP-10/CXCL10), regu- but buffered sterile saline was used in place of LPC plus lated on activation normal T cell expressed and released saline. Some control rats were also given an intramuscular (RANTES/CCL5) and stromal cell derived factor-1 (SDF1/ injection of LPC (10 ul, 1%) into the gastrocnemius mus- CXCL12) and their cognate receptors (CCR2, CXCR3, cle. CCR5 and CXCR4, respectively). Drugs and method of administration We now demonstrate that focal peripheral nerve demyeli- A CCR2 receptor antagonist and its inactive enantiomer nation in the right thigh of the rat produces chronic bilat- were employed in this study [19]. The CCR2 antagonist eral nociceptive behavior as measured by hindpaw active enantiomer's full name is (R)-4-Acetyl-1-(4-chloro- withdrawal. Together with the ongoing display of nocice- 2-fluorophenyl)-5-cyclohexyl-3-hydroxy-1,5-dihydro- ptive behavior is a delayed upregulation of several C-C 2H-pyrrol-2-one (CCR2 RA [R]). The inactive enantiomer and C-X-C chemokines and their cognate receptors by sen- is (S)-4-Acetyl-1-(4-chloro-2-fluorophenyl)-5-cyclohexyl- sory neurons. Though there is an initial delay in ligand/ 3-hydroxy-1,5-dihydro-2H-pyrrol-2-one (CCR2 RA [S]) Page 2 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 (Additional file 1). Both were employed as Na+ salts. The slopes of the logistic functions from which the PWTs are affinity of CCR2 RA [R] for the rat CCR2 receptor is > derived. The experimenter was blinded to both the injury 4000 that of the S-isomer. Both compounds were freshly condition of the animal and the drugs utilized in all prepared in saline on the day of the experiment (10 mg/ behavioral trials. kg). Active and inactive enantiomer and vehicle-treated groups (n = 8 per group) were given a one-time intraperi- Foot withdrawal to thermal stimulus toneal (i.p.) injection one hour prior to behavioral test- To evaluate the PWT to thermal stimulation, we used the ing. Hargreaves' plantar test apparatus (Ugo Basile, Varese, Italy). Rats were placed on a 2-mm-thick glass floor; a Foot withdrawal to punctate mechanical indentation mobile infrared heat generator with an aperture of 10 mm The incidence of foot withdrawal was measured in was aimed at the rat's hind paw under the floor. Following response to mechanical indentation of the plantar surface activation of the heat source, the reaction time (the with- of each hind paw with sharp, Von Frey-type nylon fila- drawal latency of the hindpaw) of the rat was recorded ments. Mechanical stimuli were applied with seven fila- automatically. A shortening of the withdrawal latency ments, each differing in the bending force delivered (10, indicated thermal hyperalgesia. The temperature of the 20, 40, 60, 80, 100, and 120 mN), but each fitted with the glass floor was kept at 22.5–23.5°C. Measurements of the same metal cylinder with a flat tip and a fixed diameter of withdrawal latency of the paw began after the rats were 0.2 mm [3]. In each behavioral testing sequence, the oper- habituated to the testing environment (IR setting = 70). ator was blinded to the animal treatment condition. The measurements were repeated four times, at 5 min intervals, on each paw, and the initial pair of measure- The rat was placed on a metal mesh floor and covered ments was not used. The averages of the three remaining with a transparent plastic dome. Typically, the animals pairs of measurements taken were employed as data. rest quietly in this situation after an initial few minutes of exploration. Animals were habituated to this testing appa- In situ hybridization ratus for 15 minutes a day, two days prior to the behavio- In situ hybridization histochemistry for chemokine recep- ral assays. Following acclimation, each filament was tors was performed by using digoxigenin-labeled ribo- applied to six spots spaced across the hind paw. The fila- probes. Adult female Sprague-Dawley rats were ments were tested in order of ascending force, with each euthanized using carbon dioxide. L L DRGs ipsi- and 4 5 st th filament delivered in sequence from the 1 to the 6 spot contralateral to LPC nerve injury were rapidly removed, alternating from one hind paw to the other. The duration embedded in OCT compound (Tissue Tek, Ted Pella, Inc., of each stimulus was 1 second and the interstimulus inter- Redding, CA) and frozen. Sections were serially cut at 14 val was 10–15 seconds. A cutoff value of 120 mN was μm. The CCR2 probe was prepared as described [8]. used; animals that did not respond at 120 mN were Briefly, an 848-bp CCR2 cDNA fragment (nucleotides assigned that value [3,20]. 489–1336 of GenBank no. U77349) was cloned by PCR using rat spleen cDNA. The resulting PCR product was The incidence of foot withdrawal was expressed as a per- subcloned into a pGEM-T Easy vector and sequenced to centage of the six applications of each filament as a func- ensure identity for riboprobe use. The CCR2 template was tion of force. A Hill equation was fitted to the function linearized with SacII to generate a probe of 950 bases by (Origin version 6.0, Microcal Software, Northhampton using SP6 polymerase. Signals were visualized by using MA) relating the percentage of indentations eliciting a NBT/BCIP reagents (Roche Diagnostics/Boehringer Man- withdrawal to the force of indentation. From this equa- nheim, Indianapolis, IN) in the dark for 2–20 h depend- tion, the paw withdrawal threshold (PWT) force was ing upon the abundance of the RNA. Images were obtained and defined as the force corresponding to a 50% captured using brightfield or differential interference con- withdrawal. At least a -20 mN difference from the baseline trast optics with a Nikon E600 fluorescent microscope PWT in a given animal is representative of neuropathic (NikonUSA, Melville, NY) fitted with a charge-coupled pain [3]. device camera (Retiga EXi, Q-Imaging Corporation, Van- couver, BC). CCR2 mRNA expression studies were used Measurements were taken on three successive days before for receptor localization because of the failure of immu- surgery. Postoperative testing was performed on one, nocytochemistry to detect neuronal CCR2 protein. three and seven days after surgery and weekly thereafter for the duration of the experiment. PWT values were sta- The RANTES plasmid was a gift from Dr. Richard M. Ran- tistically analyzed for each foot separately and for the sig- sohoff (Cleveland Clinic Foundation). The RANTES plas- nificance of differences between the average of the three mid was sub-cloned into a pGEM vector. The plasmid preoperative tests and the mean obtained for each postop- templates were linearized with restriction enzyme diges- erative test. The same statistical analyses are applied to the tion. Page 3 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 The CXCR4 and SDF-1 probes were generated as described from the total number of Hoescht-positive neuronal previously [21]. For the CXCR3 and CCR5 probes, we nuclei present in a tissue section. The overall diameter and used the CD1 mouse brain cDNA. The CXCR3 cDNA frag- brightness of the Hoescht-positive neuronal nuclei ment was amplified using the forward primer 5'-gag gtt agt allowed for a clear delineation between neurons and non- gaa cgt caa gtg-3' and the reverse primer 5'-tgg aga cca gca neuronal cells in the DRG. At least 5000 neuronal profiles gaa cag cta g-3'. The CCR5 fragment used the forward from six animals (minimum of 625 cells per ganglia) were primer 5'-tgg att atg gta tgt cag cac cc-3'and the reverse quantified for each cell type in the single neuronal marker primer 5'-tcg att atg gta tgt cag cac cc-3'. All PCR fragments study and for each combination of cellular markers. were subcloned into a pCR II-TOPO vector, and were ver- Quantification of cell numbers, degree of colocalization ified by restriction analysis and automated DNA sequenc- and cell diameters was determined using ImagePro Plus ing (Perkin Elmer, Boston MA) (Media Cybernetics, Silver Spring, MD). As noted above, individuals conducting cell quantification were blinded The plasmid templates were linearized by restriction to the treatment conditions. Data are represented as enzyme digestion. Then transcription was labeled by dig- means ± SEM%. oixigenin (Roche Applied Science, Indianapolis, IN). Preparation of acutely dissociated dorsal root ganglion Immunocytochemical labeling neurons Adult female Sprague-Dawley rats were deeply anesthe- The L –L DRG were acutely dissociated using methods 4 5 tized with isoflurane and transcardially perfused with described by Ma and LaMotte [22]. Briefly, L and L DRG 4 5 saline followed by 4% paraformaldehyde. Lumbar ganglia were removed from control or LPC-treated animals at var- associated with the sciatic nerve ipsilateral and contralat- ious post-operative day timepoints. The DRGs were eral to focal nerve demyelination injury (n = 6) or sham treated with collagenase A and collagenase D in HBSS for treatment (n = 6) were immediately removed following 20 minutes (1 mg/ml; Roche Applied Science, Indianapo- behavior on POD 7 or 14 and postfixed for 4 hours. Addi- lis, IN), followed by treatment with papain (30 units/ml, tional lumbar DRGs were removed from naïve, behavio- Worthington Biochemical, Lakewood, NJ) in HBSS con- rally tested rats (n = 6). Lumbar DRGs were encoded at the taining .5 mM EDTA and cysteine at 35°C. The cells were outset and processed in random order. Sagittal sections of then dissociated via mechanical trituration in culture the DRG were serially cut at 14 μm onto SuperFrost micro- media containing 1 mg/ml bovine serum albumin and scope slides (Fisher Scientific, Pittsburgh PA). At least 6 trypsin inhibitor (1 mg/ml, Sigma, St. Louis MO). The cul- sections were obtained for immunocytological analysis ture media was Ham's F12 mixture, supplemented with per DRG. Tissue was processed such that DRG sections on 10% fetal bovine serum, penicillin and streptomycin (100 each slide were at intervals of 80 um. Slides were incu- ug/ml and 100 U/ml) and N2 (Life Technologies). The bated with blocking buffer (3% BSA/3% horse serum/ cells were then plated on coverslips coated with poly-L- 0.4% Triton-X; Fisher Scientific, Pittsburgh PA) for 1 hour, lysine and laminin (1 mg/ml) and incubated for 2 hours followed by overnight incubation with the rabbit polyclo- before more culture media was added to the wells. The nal antisera generated against MCP-1 (1:500; Chemicon, cells were then allowed to sit undisturbed for 12–15 hours Temecula, CA), IP-10 (1:1000, Abcam, Cambridge MA) or to adhere at 37°C (with 5% CO ). CCR2 (1:500; Aviva Systems Biology, San Diego CA) at 2+ Intracellular Ca imaging room temperature. After primary incubation, secondary antibodies (anti-rabbit conjugated to CY3, made in don- The dissociated DRG cells were loaded with fura-2 AM (3 key at 1:800; Jackson ImmunoResearch, West Grove, PA) uM, Molecular Probes/Invitrogen Corporation, Carlsbad were used to visualize cells. Some experiments were aug- CA) for 25 minutes at room temperature in a balanced salt mented with the addition of Griffoniasimplicifolia I-isolec- solution (BSS) [NaCl (140 mM), Hepes (10 mM), CaCl tin B4 (IB4) conjugated with fluorescein (1 mg/1 ml; (2 mM), MgCl (1 mM), Glucose (10 mM), KCl (50 Sigma, St. Louis MO). Slides were washed in PBS for 5 min mM)]. The cells were rinsed with the BSS and mounted each (×3) and coverslipped with a PBS/glycerol solution. onto a chamber that was placed onto the inverted micro- All tissue sections were also stained with Hoechst 33258 scope and continuously perfused with BSS at a rate of 1 nuclear marker (Invitrogen Corporation, Carlsbad CA). ml/min. Intracellular calcium was measured by digital video microfluorometry with an intensified CCD camera Tissue sections were analyzed for the presence of IB -bind- coupled to a microscope and MetaFluor software (Molec- ing neurons and either MCP-1, IP-10 or CCR2. Because a ular Devices Corporation, Downington, PA). Cells were stereological approach was not employed in this study, illuminated with a 150 W xenon arc lamp, and the excita- quantification of the data may represent a biased estimate tion wavelengths of the fura-2 (340/380 nm) were of the actual numbers of immunopositive neurons. The selected by a filter changer. Chemokines were applied proportions of immunoreactive neurons were determined directly into the coverslip bathing solution after the per- Page 4 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 fusion was stopped. If no response was seen within 1 minute, the chemokine was washed out. For all experi- 80 80 ments, MCP-1, SDF1, RANTES and IP10 were added to 70 70 the cells in random order, after which capsaicin, high K+ (50 K) and ATP were added. The chemokines used were 60 60 purchased from R & D Systems (Minneapolis, MN), and * * ** ** * * 50 50 * * all were used at a concentration of 100 nm to ensure max- * * * * imal activation. They were reconstituted in 0.1%BSA/PBS, 40 40 and aliquots were stored at -20°C. * * * * * * * * 30 30 Statistical Analyses 20 20 Data is presented as the mean ± SEM, unless otherwise ip ips siill. . t to o ner nerve in ve injur jury y con cont tra ral. t l. to o ner nerve ve inju injur ry y noted. GB-Stat School Pack software (Dynamic Microsys- 10 10 ip ips siill. . t to o s sham in ham injur jury y con cont tra ral. t l. to o sha sham m in injur jury y tems, Inc. Silver Springs, MD) was used to statistically 0 0 evaluate all data. The significance difference was deter- 00 11 33 77 1142 4212 1288 3355 4422 P Po ostoper stopera ativ tive e Da Day y mined by two-way ANOVA with Bonferroni's post-hoc test for animal behavior. The one way ANOVA with a Dunnett's Multiple Comparison test was used to analyze the differences between naïve, sham and experimental groups. A difference of p < 0.05 was considered signifi- c contr ontra all. . t to o ne nerv rve e in injju ur ry y cant. ip ips sii. to . to nerv nerve e i in nju jury ry 12 12 10 10 Results Mechanical stimuli elicit bilateral tactile hyperalgesia 8 8 following LPC-induced sciatic nerve demyelination 6 6 To study changes in behavioral sensitivity following LPC- induced nerve demyelination, we investigated alterations 4 4 in the paw withdrawal threshold (PWT) force of indenta- tion (produced by von Frey filaments) necessary for elicit- 2 2 ing a flexion hindpaw withdrawal reflex. At POD1, the 0 0 PWT ipsilateral to the LPC-induced sciatic nerve demyeli- 0 da 0 day y 7 7 day day 14 d 14 da ay y nation was significantly reduced when compared to pre- P Po ostoper stopera ativ tive e Da Day y surgical PWTs (Fig. 1A). The force required to elicit a paw withdrawal steadily declined until POD14, before gradu- Mea Frey stimulation ing Figure 1 LP n thresh C-induold force required ced fo at 1, 3, 7 cal nerve , 14 de , 21, 28, 35 a mye for paw wi linationn thdraw d 42 days follow- al to Von ally returning to near pre-surgical levels by POD35. These Mean threshold force required for paw withdrawal to Von PWTs met the pre-determined levels indicative of hyperal- Frey stimulation at 1, 3, 7, 14, 21, 28, 35 and 42 days follow- gesia (-20 mN force) between POD1 and POD28. ing LPC-induced focal nerve demyelination. Each data point is Changes in behavior were also observed in the hind paw the mean threshold (± SE) force on the hindpaw ipsilateral contralateral to the LPC-induced nerve injury (Fig. 1A). (black circle) or contralateral (white circle) to the focal nerve These behavioral changes met the pre-determined PWT injury site eliciting a withdrawal response (n = 10). Reduced behavioral thresholds for the hindpaw ipsilateral to the nerve levels indicative of hyperalgesia between POD3 and lesion were significantly different from pre-operative baseline POD28 (Fig. 1A). Vehicle-treated sham operated rodents on postoperative days 1–28. The threshold force for the did not exhibit a PWT decrease that was significant at any hindpaw contralateral to the nerve lesion did not reach sig- time point up to POD14. Animals given intramuscular nificance until postoperative day 3, and significant differences injections of LPC into the gastrocnemius muscle (10 ul, were observed until postoperative day 28. The time course 1% LPC) did not develop cutaneous hyperalgesia (n = 3, of sham injury (n = 6) is also represented but did not differ data not shown). These data indicate that a unilateral from the uninjured animals. Analysis was performed using LPC-induced nerve demyelination results in bilateral tac- two-way ANOVA followed by the Bonferroni post-hoc pair- tile hyperalgesia. wise comparisons (*p < 0.01).B) LPC-induced focal nerve demyelination did not produce changes in thermal responses Effect of unilateral focal nerve demyelination on thermal as assessed by the Hargreaves test. Each bar is the mean withdrawal latency (± SE) of the hindpaw ipsilateral (white thresholds bar) or contralateral (black bar) to the focal nerve demyeli- In contrast to the effectiveness of LPC-induced focal nerve nation injury at postoperative day 7 and 14 (n = 10). demyelination in altering mechanical PWTs, focal nerve Page 5 of 20 (page number not for citation purposes) Wi Witth h d d ra ra w wa a ll L L a atte e nc ncy y ((se sec) c) M Mean Thr ean Threshold (mN eshold (mN) ) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 demyelination had little if any effect on thermal respon- neuronal expression did not change appreciably follow- sivity from POD0 to POD14 (Fig. 1B). ing vehicle treatment (Fig. 4A and 4C) or focal nerve demyelination at POD14 (Fig. 4B). Despite little change CCR2, CXCR4, CXCR3 and CCR5 upregulation in L –L in non-neuronal cells, there was an increase in the 4 5 DRG after unilateral LPC-induced nerve demyelination number of neurons expressing CXCR4 mRNA transcripts We previously reported upregulation of CCR2 chemokine by POD14 in the lumbar DRG ipsilateral to the nerve receptor signaling in association with chronic compres- lesion (Fig. 4D). This pattern of staining was also sion of the DRG [8]. We therefore examined the state of observed in DRG contralateral to the nerve injury (data CCR2 expression in association with LPC-induced demy- not shown). DRGs taken from injured animals on POD35 elination. L L DRGs removed from naïve (data not exhibited CXCR4 mRNA staining that was similar to that 4 5 shown) and vehicle-treated rats at POD7 did not express seen in naïve and vehicle-treated animals (data not CCR2 mRNA (Fig. 2A) or CCR2 immunoreactivity (Fig. shown). 2B). Small and medium diameter L L neurons ipsilateral 4 5 to the focal nerve lesion exhibited low levels of both Next we used in situ hybridization to examine the expres- CCR2 mRNA transcripts at POD7 (Fig. 2C) and CCR2 sion of the chemokine receptors CXCR3 and CCR5 in the immunoreactivity (Fig. 2D). By POD14, many sensory DRG associated with the LPC-induced focal demyelina- neurons of all diameters exhibited CCR2 mRNA in L L tion. These are the receptors for the chemokines IP-10 and 4 5 DRGs both ipsilateral and contralateral to LPC-induced RANTES (among other chemokines) respectively. Many injury (Figs. 2E, 3). Immunoreactivity for neuronal CCR2 sensory neurons were positive for CXCR3 mRNA in tissues at POD14 (Fig. 2F) was also increased relative to POD7. taken from both naïve (data not shown) and sham-treated Neuronal binding of the plant isolectin, Griffoniasimplici- rodents at POD14 (Fig. 5A). The pattern of neuronal folia B (IB ) in the rat DRG distinguishes a population of expression of CXCR3 mRNA at POD14 following focal 4 4 C-fiber nociceptors [23,24]. We double-labeled the sec- nerve demyelination did not change relative to naïve or tions stained for CCR2 protein with IB . Many IB -bind- sham-treated tissue, but the nerve injury did increase the 4 4 ing neurons were present in lumbar DRG of naïve, sham- intensity of CXCR3 mRNA expression in the DRG ipsilat- operated rats and those subjected to LPC-induced focal eral to the injury (Fig. 5C), as well as in the contralateral nerve demyelination (Fig. 2B, D, F). These neurons dis- DRG (data not shown). played strong labeling of the plasma membrane, as well as perinuclear staining that in all likelihood represents the Unlike CXCR3 expression, lumbar DRG in naïve (data not Golgi apparatus [25]. Quantitative analysis of neurons shown) and sham-treated rats at POD14 (Fig. 5B) were positive for CCR2 revealed that 33.35 ± 2.16% of sensory devoid of CCR5 mRNA transcripts. As with CCR2 expres- neurons ipsilateral and 35.87 ± 3.36% contralateral to the sion in sensory neurons, CCR5 mRNA transcript levels nerve lesion were positive for the chemokine receptor. were strongly increased in many sensory neurons ipsilat- Relatively few (<6%) CCR2-positive neurons were colo- eral and contralateral to the nerve injury at POD14 (Fig. calized with IB -binding neurons ipsilateral (Fig. 2F and 5D). Lumbar DRGs derived from injured animals on Fig. 3) or contralateral to the nerve injury at POD 14 (Fig. POD35 showed no CCR5 mRNA staining (data not 3). CCR2 mRNA was no longer detected in lumbar DRGs shown). taken from injured animals on POD35 (data not shown). Chemokine expression in response to LPC-induced Activation of numerous chemokine receptors in addition demyelination of the sciatic nerve to CCR2 might potentially be involved in the production As the data discussed above indicates strong expression of of sensory neuron hyperexcitability and pain [3,4,26]. The several chemokine receptors by sensory neurons follow- chemokine SDF-1/CXCL12 and its receptor CXCR4 are ing LPC-induced demyelination injury, we sought to constitutively expressed by peripheral nerves [3,27-29] determine whether sensory neurons would also exhibit and chemokines that activate CCR5 and CXCR3 receptors, changes in the expression of chemokines that are possible such as RANTES and IP-10, are synthesized in association ligands for these receptors under the same circumstances. with neuroinflammatory responses [16,30]. To investi- Using immunocyctochemistry, we examined both the gate whether chemokine receptors, in addition to CCR2, nerve lesion site and lumbar DRG associated with the are upregulated following focal nerve demyelination, in injured nerve for MCP-1/CCL2 protein expression. situ hybridization studies were performed on injured rat Despite the multitude of ED-1-immunopositive macro- DRG tissue sections. phages in the injured sciatic nerve, MCP-1/CCL2-immu- noreactive cells were absent from the nerve lesion site on Basal expression of CXCR4 mRNA was predominantly POD1, 3, 7, 14 (data not shown), lumbar DRG removed detected in non-neuronal cells of the lumbar DRG derived from naïve rats (data not shown) and lumbar DRG from from naïve animals (data not shown). This level of non- vehicle-treated rats at POD7 (Fig. 6A). In sharp contrast, Page 6 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 E Figure 2 xpression of CCR2 mRNA and protein immunoreactivity in rat lumbar DRG ipsilateral to focal nerve demyelination Expression of CCR2 mRNA and protein immunoreactivity in rat lumbar DRG ipsilateral to focal nerve demyelination. A) Lum- bar DRG removed from vehicle-treated animals at POD7 did not exhibit CCR2 mRNA expression (n = 5). B) Many lumbar DRG neurons in vehicle-treated rats sensory neurons were positive for isolectin IB , a neuronal phenotype that distinguishes some C-fiber nociceptors (green cells). There was no evidence of CCR2 protein expression in sham animals (n = 5). C) Lum- bar DRG neurons from nerve-injured rats on POD7 exhibited CCR2 mRNA transcripts in some small and medium diameter neurons (black arrows). Open black arrowhead indicates a neuron without CCR2 mRNA transcripts (n = 4). D) Lumbar DRG neurons from a rat subjected to focal nerve demyelination exhibited few CCR2 immunopositive (white arrows) sensory neu- rons (n = 4). E) Many lumbar DRG neurons on POD14 exhibited CCR2 mRNA transcripts (black arrows). Open arrowhead indicates non-labeled neuron. F) CCR2 immunoreactivity was present in an increased number of neurons at POD14 (white arrows; n = 5). Scale bar is; 30 μm (A, C), 50 μm (B, D, F), and 100 μm (E). Page 7 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 60 60 60 60 50 50 50 50 IB IB IB IB4 4 4 4 MCP- MCP- MCP- MCP-1 1 1 1 M M M MC C C CP P P P- - - -1 1 1 1/IB /IB /IB /IB4 4 4 4 CCR2 CCR2 CCR2 CCR2 40 40 40 40 CCR2/ CCR2/ CCR2/ CCR2/IIIIB B B B4 4 4 4 IP IP IP IP- - - -1 1 1 10 0 0 0 I I I IP P P P-10 -10 -10 -10/IB /IB /IB /IB4 4 4 4 30 30 30 30 20 20 20 20 10 10 10 10 0 0 0 0 ip ipsil sila at te er ra al LP l LPC DRG C DRG cont contr ra ala lat te eral ral LP LPC C D DR RG G iips psila ilat te era ral l s sh ham DRG am DRG co cont ntral rala at te er ra al s l sh ham DRG am DRG Figure 3 Percentage of MCP-1, CCR2 and IP-10 immunoreactive neurons with IB -positive neuronal profiles on POD 14 Percentage of MCP-1, CCR2 and IP-10 immunoreactive neurons with IB -positive neuronal profiles on POD 14. MCP-1 expression was increased by LPC-induced nerve injury within the IB -labeled neuronal group in both the DRG ipsilateral and contralateral to the nerve injury. Sham-injury treatment did not produce significant changes in the extent of MCP-1/IB colocal- ization. CCR2 expression was increased by LPC-induced nerve injury within IB -labeled neuronal group in both the DRG ipsi- lateral and contralateral to the nerve injury. Like, MCP-1, sham-injury treatment did not produce significant changes in CCR2/ IB4 colocalization in either DRG ipsi- or contralateral to the sham injury. IP-10 expression was increased by LPC-induced nerve injury within IB -labelled neuronal group in both the DRG ipsilateral and contralateral to the nerve injury, while sham- injury treatment did not produce significant changes in IP-10/IB colocalization. Comparisons of immunoreactive cell percent- ages were made between LPC-treatment and sham-treated animals. Data represent means ± SE. Analysis was performed using two-way ANOVA followed by the Bonferroni post-hoc pair-wise comparisons (*p < 0.01). numerous MCP-1 immunopositive neurons were present cant portion of the MCP-1 positive neurons co-localized in associated lumbar ganglia of the LPC-treated rats by at POD 7 and POD 14 (Fig. 6B, D; Fig. 3). More with IB POD7 (Fig. 6B). The diameter of MCP-1 immunoreactive specifically, just over half of the MCP-1 immunoreactive neurons in the LPC-treated animals averaged 26.49 ± 0.47 neurons present at POD14 in DRG ipsilateral to nerve μm (n = 6). Interestingly, no MCP-1 protein immunoreac- injury were also positive for IB (Fig. 3; 20.12 ± 0.75%; n tivity was detectable in injured DRG non-neuronal cells at = 6). any time point examined. In a previous study, it was shown that in a rodent model Once again, we co-stained DRG sections with IB , a of spinal stenosis, chronic compression of the DRG, there marker for C-fiber nociceptors. Many IB -binding neurons was increased MCP-1 expression and increased excitabil- were present in lumbar DRG of naïve, sham-operated and ity of sensory neurons in injured and adjacent uninjured nerve-injured rats at POD7 and POD 14 (Fig. 6C) and Fig. DRG [8]. Therefore, we wished to see whether MCP-1 3, respectively). The average number of IB neurons activity was altered in the L4/L5 DRG contralateral to the present in the lumbar DRG was 47.87 ± 1.44% (n = 6 per nerve injury. Increased numbers of MCP-1-immunoreac- condition) and did not significantly differ across treat- tive cells were present in ganglia contralateral to nerve ment groups (sham and injured at POD14) (Fig. 3; p > injury (Fig. 3; 34.27 ± 1.17%; n = 6) and 13.32 ± 0.57% 0.1). A series of MCP-1 immunopositive/IB4-binding (n = 6) exhibited IB4 colocalization. Relatively few neu- colocalization experiments demonstrated that a signifi- rons were positive for MCP-1 in ganglia ipsi- or contralat- Page 8 of 20 (page number not for citation purposes) P Pe er rc cen enta tage ge o of f Immu Immunopo noposi siti tive ve Ce Cell lls s Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 Expression of CXCR4 mRNA in rat lumbar DR Figure 4 G ipsilateral to focal nerve demyelination Expression of CXCR4 mRNA in rat lumbar DRG ipsilateral to focal nerve demyelination. Low (A) and high power (C) phot- omicrographs of CXCR4 mRNA transcripts present in lumbar DRG removed from vehicle-treated rodents at POD14 (n = 3). Many non-neuronal cells strongly expressed CXCR4. (C) Some presumptive neurons expressed low levels of CXCR4 mRNA (white arrow). Low (B) and high power (D) photomicrographs of CXCR4 mRNA transcripts present in lumbar DRGs derived from injured rats at POD14 (n = 4). The expression level and number of non-neuronal cells exhibiting CXCR4 mRNA tran- scripts in the lumbar DRG did not change following focal nerve demyelination. However, many neurons upregulated CXCR4 mRNA expression (D; white arrows indicate neurons with low levels of mRNA transcripts; white arrowhead points to a neu- ron lacking CXCR4 mRNA expression). (Scale bar is 1 mm (A and B); 40 μm (C and D). eral to the injury site in sham-treated animals (Fig. 3; SDF-1 is the unique ligand for the CXCR4 receptor <5%) and IB4 colocalization was rare (Fig. 3). [31,32]. It has been reported that Schwann cells in the peripheral nerve express SDF-1 and that its expression As noted above, nearly every neuron in the DRG ipsilat- increases moderately after nerve injury [3,29,33]. Using in eral to the nerve injury upregulated CCR5. We used in situ situ hybridization we examined lumbar DRG associated hybridization to study the levels of RANTES expression, with the injured sciatic nerve for SDF-1 expression at one known ligand for CCR5, in the DRG ipsilateral and POD14. Many lumbar DRG non-neuronal cells present in contralateral to the nerve injury. RANTES expression was both the naïve (data not shown) and vehicle-treated rats absent in the L4/L5 DRG in vehicle treated rats (Fig 7A). were positive for SDF-1 mRNA transcripts (Additional file We found that after nerve injury, RANTES expression was 2A, C). Neither the pattern nor the expression levels of strongly upregulated in DRG neurons ipsilateral and con- SDF-1 mRNA transcripts changed following focal nerve tralateral to the nerve injury, when compared to sham demyelination at POD14 (Additional file 2B, D). control tissue. Like CCR5, RANTES was expressed in small, medium and large sensory neurons (Fig 7B, C). IP-10 is one of three ligands that bind to the CXCR3 recep- tor [34-36]. Little is known, however, concerning a role for IP-10 in sensory neuron function. We demonstrated Page 9 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 Expression of CXCR3 and Figure 5 CCR5 mRNA in rat lumbar DRG following focal nerve demyelination Expression of CXCR3 and CCR5 mRNA in rat lumbar DRG following focal nerve demyelination. (A) Many sensory neurons in the lumbar DRG removed from vehicle-treated rats exhibited CXCR3 mRNA transcripts at POD14 (n = 3). (B) CCR5 mRNA expression was absent from the lumbar DRG of vehicle-treated rats at POD14 (n = 3). (C) CXCR3 mRNA expression pat- terns in sensory neurons subjected to focal nerve demyelination did not differ from vehicle-treated rodents at POD14 (n = 4), but there was an increase in the intensity of CXCR3 mRNA expression. (D) Many neurons in the injured rat lumbar DRG expressed CCR5 transcripts at POD14 (n = 4). Scale bar is 250 μm (A and C); 100 μm (B and D). that the sensory neurons in both vehicle-treated and significantly reduced to 34.49 ± 0.98 μm. By POD14, the injured lumbar DRGs exhibited appreciable levels of number of IP-10 immunoreactive sensory neurons had CXCR3 expression (Fig. 5A, C). Similarly, lumbar DRG increased over three-fold (when compared to the vehicle- from both naïve (data not shown) and vehicle-treated treated rat lumbar DRGs) to 36.8 ± 4.9% of the total neu- rodents displayed constitutive IP-10 immunoreactivity ronal population (Fig. 3, Fig. 8C), and the average neuro- (12.04 ± 1.2%, at least 400 cells/DRG from each of 6 vehi- nal diameter was further reduced to 29.74 ± 1.1 μm (n = cle-treated animals) (Fig. 3, Fig. 8A), both ipsi- and con- 6). tralateral to the site of nerve injury. The mean diameter of IP-10-immunopositive neurons in the vehicle-treated rat A reduction in the average diameter of IP-10-immunopo- was 47.53 ± 1.7 μm. Seven days after LPC-induced focal sitive neurons concurrent with a three-fold increase in the nerve demyelination, the number of IP-10-immunoposi- number of neurons implies upregulated expression of IP- tive neurons in injured DRG had increased over two-fold 10 occurred in cells normally negative for the chemokine. to 30.41 ± 2.9% (n = 6) (Fig. 3, Fig. 8B). The average cell Similar to previous findings in subpopulations of cells diameter of IP-10 immunoreactive neurons on POD7 was positive for the neurotrophin brain-derived neurotrophic Page 10 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 demyel Figure 6 Colocalization ination i of MCP-1 im njury at POD7 munoreactivity (ir) and isolectin B (IB )-binding in the lumbar DRG ipsilateral to LPC-induced 4 4 Colocalization of MCP-1 immunoreactivity (ir) and isolectin B (IB )-binding in the lumbar DRG ipsilateral to LPC-induced 4 4 demyelination injury at POD7. IB -binding in rat DRG neurons distinguishes C-fiber nociceptors. (A) Naïve rat lumbar DRG were completely negative for MCP-1ir. (B) Lumbar DRG ipsilateral to LPC-induced sciatic nerve injury exhibited numerous small diameter neurons that are MCP-1 positive at POD7 (red arrows). (C) Numerous small IB -binding presumptive nocicep- tors are present in the same DRG tissue section (green arrows). (D) Merging panels B and C demonstrates the extent of colo- calization present in lumbar DRG tissue section (yellow arrows). Note not all MCP-1ir neurons were positive for IB at POD7 (red arrow). Scale bar is 100 μm (A, B, C and D). factor (BDNF) by Obata and colleagues [37], this change IP-10 immunoreactivity and IB4-binding (n = 6) (Fig. 3, in neuronal phenotype may be indicative of pathophysio- Fig. 8E, H). However, a two-fold increase in the number logical changes in primary afferent neurons following of neurons positive for both IP-10-immunoreactivity and focal nerve demyelination. To determine the degree to IB4-binding (19.08 ± 2.31%; n = 6) was evident at POD14 which small, presumably nociceptive IB4-binding neu- (p < 0.01) (Fig. 3, Fig. 8F, I). rons, also displayed IP-10 immunoreactivity following 2+ LPC-induced nerve demyelination, we performed a series Chemokines increase [Ca ] in DRG cells subjected to LPC-induced nerve demyelination of colocalization experiments (Fig. 8D–I). Our analysis revealed that generally speaking, IP-10 immunoreactive Activation of chemokine receptors expressed by primary neurons did not co-localize with IB4-binding neurons in sensory neurons results in excitation and in the increase in 2+ vehicle-treated lumbar DRG (Fig. 3, Fig. 8D, G). On the intracellular Ca concentration [4]. To compliment POD7, 9.39 ± 1.64% of the total neurons exhibited both the anatomical observations of upregulated chemokine Page 11 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 E Figure 7 xpression of RANTES mRNA in rat lumbar DRG following focal nerve demyelination Expression of RANTES mRNA in rat lumbar DRG following focal nerve demyelination. (A) RANTES mRNA expression was absent from the lumbar DRG of vehicle-treated rats at POD14 (n = 3). (B) Many sensory neurons in the DRG ipsilateral to the nerve injury at POD 14 were positive for RANTES mRNA (C) Numerous sensory neurons in the DRG contralateral to the nerve injury displayed expression of RANTES mRNA, albeit at a lower level when compared with the DRG ipsilateral to the nerve injury. Scale bar is 250 μm (A and C); 100 μm (B and D). receptor expression, we used fura-2 imaging of chemok- nal cells (Table 2). It was also noted that at POD 14, most 2+ ine-induced increases in [Ca ]i in acutely isolated rat cells responded to only one chemokine. However, at POD DRG neurons as a measure of functional chemokine 28, when upregulated chemokine signaling was at its receptor expression. We acutely isolated the DRG cells greatest, most cells responded to multiple chemokines, from both the ipsilateral and contralateral sides of nerve indicating that single neurons expressed multiple chem- injured animals and sham controls. For all experiments, okine receptors. In the DRG contralateral to the nerve the chemokines MCP-1, SDF-1, RANTES and IP-10 were injury, there was an upward trend in chemokine signaling added in random order to the cells, after which capsaicin, at POD 14–28 (Table 1), but this did not reach signifi- high K+(50 mM) and ATP were added to assess the cells' cance. By POD 35, dissociated DRG cell responses to identity and viability, respectively. A response to high K+ chemokines had returned to baseline levels (Fig. 9D, stimulation indicates the presence of voltage dependent Table 1). Thus, the fura-2 imaging generally confirmed the Ca2+ channels, which is indicative of neurons. Addition- observed upregulated expression and function of chemok- ally, a positive response to capsaicin as well as high K+ ine signaling in DRG cells subjected to a peripheral nerve indicates the cell is a nociceptor expressing the TRPV1 demyelination. channel. A response to ATP, which activates purinergic CCR2 receptor antagonist attenuates bilateral focal nerve receptors, without one to High K+ and/or capsaicin, indi- cates a non neuronal cell, presumably a type of glial cell demyelination induced tactile hyperalgesia such as a satellite glial cell or Schwann cell. The concentra- Using LPC nerve-injured animals, we tested the effect of a tions of chemokines used in these experiments were all single i.p injection of a CCR2 receptor antagonist (CCR2 supramaximal to ensure activation of any expressed recep- RA-[R]) or its inactive enantiomer (CCR2 RA-[S]) on tors. nociceptive behavior at POD14 and POD28 (10 mg/kg). Neither vehicle nor CCR2 RA-[S] administration at day 14 It was evident that an increased number of cells or day 28 had an effect on the bilateral mechanical PWT responded to MCP-1 application from POD 14–28 in the when tested 1 hour later (Fig. 10). In contrast, bilateral DRG cells ipsilateral to the nerve injury (Fig. 9B and 9C), increases in PWT were observed one hour after adminis- (Table 1), when compared to vehicle-treated control ani- tration of CCR2 RA-[R]. These PWTs did not differ from mals (Fig 9A). The number of cells responding went from pre-operative basal threshold levels (Fig. 10). The effects 5.8% to 31.7% by POD 28. The majority of these cells of CCR2 RA-[R] were stereospecific as administration of were characterized as neurons based on their positive an equal dose of the inactive stereoisomer did not inhibit responses to capsaicin and/or high K+ (Table 2). Many pain behavior. All PWTs returned to pre-drug administra- 2+ cells also exhibited increases in [Ca ]i in response to the tion levels within 24 hours (data not shown). other chemokines tested (i.e. SDF1, RANTES and IP-10) and the frequency of these responses was always greatest Discussion by POD 28 in the nerve injured animals. Chemokine- Previous work carried out in our own and other laborato- induced changes in dissociated DRG were not limited to ries has indicated that chemokine signaling may contrib- sensory neurons, but also included occasional non-neuro- ute to the genesis and maintenance of neuropathic pain Page 12 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 Colocalization subject Figure 8 ed to LPC-induced of IP-10 immunoreactiv nerve injuryity (-ir) and isolectin B4 (IB )-binding neurons in the lumbar DRG of naïve rat and rats Colocalization of IP-10 immunoreactivity (-ir) and isolectin B4 (IB )-binding neurons in the lumbar DRG of naïve rat and rats subjected to LPC-induced nerve injury. IB -binding in rat DRG neurons distinguishes a population of C-fiber nociceptors. A) The majority of IP-10-ir cells were limited to medium diameter neurons in the lumbar DRG from vehicle-treated rats (red arrows). D) IB -binding small diameter presumptive nociceptors (green arrows) did not colocalize with IP-10-ir lumbar DRG neurons from vehicle-treated rodents at POD7 (G, merged images). B) Lumbar DRG ipsilateral to focal nerve demyelination exhibited numerous medium and small diameter IP-10-ir neurons at POD7. Limited numbers of neurons were positive for both IP-10-ir (B, red arrows) and IB4-binding (E, green arrows) on POD7 (H, merged images). C) Many IP-10-ir neurons (red arrows) colocalized with IB -binding neurons (F, green arrows) at POD14 (I, merged images). Yellow arrows indicate colocal- ized cells. Scale bar is 100 μm. [11,17,18]. Thus, the present studies were designed to ine receptors and chemokine/receptor signaling increased investigate a potential association between focal nerve significantly. This occurred not only in the DRG directly demyelination, neuropathic pain behavior and chemok- associated with the injured nerve, but also to a lesser ine signaling in DRG neurons. Based on our previous degree, in the DRG directly contralateral to the nerve studies, we hypothesized that the focal demyelination of injury. Bilateral nociceptive behavior was apparent the sciatic nerve, a known rodent model of neuropathic between days 3–28 and could be stereospecifically atten- pain [12], would result in upregulated chemokine expres- uated with a CCR2 receptor antagonist on days 14 and 28. sion and chemokine receptor signaling in DRG neurons. Moreover, five weeks following injury, both the neuro- Indeed, we observed the predicted increases for several pathic pain behavior and the incidence of chemokine sig- chemokines and their receptors. Importantly, as the PWT naling were greatly diminished. Together with the known decreased over 14 days post-injury, chemokines, chemok- cellular effects produced by chemokines on sensory neu- Page 13 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 Table 1: Table illustrates the chemokine response profile of acutely dissociated DRG cells from rats with and without peripheral nerve injury (n = 5/group). The cells were imaged while the various chemokines were added to the bathing solution. A rise in (Ca)i after chemokine application was indicative of the expression of a functional chemokine receptor. After chemokine application, capsaicin, high K+ and ATP were used to characterize the identity of the cells (see text). Note the upregulation of chemokine signaling at 14 and 28 days after peripheral nerve demyelination in isolated DRG cells ipsilateral to the nerve injury. There was also an upward trend in chemokine signaling in the cells isolated from the DRG contralateral to nerve injury at POD 14–28. By POD 35, chemokine responsiveness returned to baseline levels. Control POD 14 POD 28 POD 35 Chemokine Ipsilateral Contralateral Ipsilateral Contralateral Ipsilateral Contralateral Ipsilateral Contralateral MCP-1 5.8% 7.9% 11.6% 2.5% 31.7% ** 15.1% 2.4% 0.0% SDF1 5.8% 7.0% 2.3% 5.0% 25% ** 8.2% 7.3% 6.6% RANTES 5.8% 7.0% 23.2% ** 2.5% 8.3% 13.6% 9.7% 3.3% IP-10 9.6% 8.8% 0.0% 20.0% 21.1%* 8.2% 7.3% 6.6% *p < 0.01 ** p < 0.05 n = 5/age group rons [3,7], these results suggest that the changes in sensory Presumably, chemokines released in this fashion may neuron chemokine/receptor signaling may be central to influence neural cells in the local vicinity eliciting excita- the maintenance phase of neuropathic pain behavior in tion as described above. Such activation would produce particular. further chemokine release and excitation driving the over- all excitability of the chemokine sensitive neurons to new The results of our previous studies, together with the levels. The resulting neuronal behavior may explain cer- present results on LPC-associated neuropathy, point to a tain aspects of pathologically maintained neuronal states significant role for chemokine signaling expressed directly of depolarization or electrical hyperexcitability of periph- by peripheral nerves. For example, we have previously eral sensory neurons [39-41]. In addition to the chronic demonstrated that activation of chemokine receptors maintenance of sensory neuron hyperexcitability, release expressed by cultured or acutely isolated DRG neurons of chemokines such as MCP-1 and fracktalkine from cen- 2+ produces increased (Ca ) , or neural excitation [4,38]. tral axon terminals in the spinal cord may initiate micro- Indeed, following upregulation of CCR2 by sensory neu- glial-mediated neuropathic pain states [7,11,42-44]. rons in whole DRG derived from animals exhibiting neu- However, it is important to note that pharmacological ropathic pain, application of MCP-1 produces powerful therapies which inhibit microglial activation and effec- excitation [8]. The mechanism underlying this response tively attenuate the development of hyperalgesia and allo- probably involves activation of phospholipase C-induced dynia have no effects on preexisting nociceptive pain degradation of PIP2, production and concomitant trans- behavior [45]. activation of TRPV1 and/or TRPA1 together with inhibi- tion of K+ conductance [9,10]. As we have demonstrated, the exact pattern of changes in chemokine signaling observed following focal nerve The experiments reported here suggest a model in which demyelination depends on the particular chemokine focal nerve demyelination produces a concomitant upreg- receptor and ligand examined. There are over 50 known ulation of several chemokines and their receptors in the chemokines and 20 chemokine receptors [32], and it is cell bodies of sensory neurons in the DRG. We have obviously not feasible to study all of these simultane- observed that chemokines expressed by DRG neurons, ously. However, the receptors studied in the present including MCP1, IP10 and SDF1, can be packaged into experiments represent obvious candidates for a role in secretory vesicles and released upon depolarization [9]. peripheral neuropathy. Chemokines that signal via the CCR2, CCR5, CXCR3 and CXCR4 receptors have previ- Table 2: At 28 days, the majority of cells responded to capsaicin ously been shown to influence the behavior of sensory or high K+, indicating that most of the chemokine responsive neurons [3,4,6,8,11,17]. Furthermore, many of these cells were neurons. receptors can be upregulated in leukocytes by mecha- Capsaicin/High K+/ATP positive (TRPV1 39.1% nisms suggesting that regulation of their expression may expressing nociceptor) often be coordinated through the same transcriptional High K+/ATP positive only (Non TRPV1- 39.1% control mechanisms [46]. expressing neuron) ATP positive only (Non-neuronal cell) 21.7% Page 14 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 A A B B C C D D s s Ti Time (s me (sec) ec) Chemoki Figure 9nes increased [Ca2+]i levels in acutely isolated rat DRG cells following focal demyelination injury Chemokines increased [Ca2+]i levels in acutely isolated rat DRG cells following focal demyelination injury. The figure shows examples of responses of cells acutely isolated from rat DRGs ipsilateral to the nerve injury at various days after a focal demy- elination injury. Under normal conditions, cells rarely respond to any chemokine but did respond to other stimuli such as high K or ATP (A). However, there was an increased responsiveness of the cells, the majority of which could be characterized as neurons, between post-operative days 14–28 (B and C, respectively). The frequency of the responses to chemokines returned to approximately the same level as control animals by post-operative day 35 (D). For all experiments, MCP-1 (M), IP- 10 (I), RANTES (R), SDF1 (S) were applied at a concentration of 100 nM. Capsaicin (C), high K (K) and ATP (A) were applied at concentrations of 100 nM, 50 mM and 100 uM, respectively. The four chemokines/chemokine receptors that we stud- DRG. The upregulation of MCP-1 and the CCR2 chemok- ied all displayed different patterns of expression in ine receptor observed in association with focal nerve response to focal nerve demyelination, suggesting differ- demyelination is similar to the pattern we previously ent roles in the genesis of pain or other functions in the observed using a spinal stenosis model of neuropathic Page 15 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 model. The particular effectiveness of CCR2 block could be due to the fact that upregulation of chemokines can be bilaterally expressed in different populations of sensory neurons following nerve injury, as is this case of chole- cystokinin vasoactive inhibitory peptide and neuropep- tide Y [43,47]. In our experiments over 50% of the cells that upregulated MCP-1 also expressed IB-4, which is a marker for C-fiber nociceptors that are responsible for transmitting pain information. This differs from the case of IP-10, where a majority of the neurons upregulating this chemokine did not co-localize with IB-4. As such, it is possible that the population of neurons that upregulates CCR2 signaling is particularly linked to the production of neuronal hyperexcitability. It is also likely that the CCR2R antagonist may impact non-neuronal cells within the !"   !" CNS, such as microglial cells, which are known to express CCR2 in the spinal cord and contribute to the develop- reversed existing n Figure 10 CCR2 receptor antagonist (CCR2 ociceptive behavior RA-[R]) administration ment of chronic pain states [11,48,49]. CCR2 receptor antagonist (CCR2 RA-[R]) administration reversed existing nociceptive behavior. Animals were sub- The precise location of action of the CCR2 antagonist is jected to a nerve demyelination injury on day 0 and nocicep- not known. However, it has been shown that the blood tive behavior was assessed for 28 days. On days 14 and 28 nerve barrier is less restrictive than the blood brain barrier post-surgery, animals received 5 mg/kg CCR2 RA-[R] or 5 [50], with the cell body rich area of the DRG being vulner- mg/kg of its inactive enantiomer, (CCR2 RA-[S], or saline by able to extravascular leakage. Given these studies, it is intraperitoneal injection, and behavioral responses were tested 1 h later. Administration of the CCR2 RA-[R] to focal likely that the CCR2 R antagonist reached the cell bodies nerve demyelination injured rats resulted in a significant bilat- of the DRG. As activation of CCR2 receptors in the DRG is eral increase of mN force required to elicit a paw withdrawal probably of considerable importance in the production of compared with vehicle-treated controls and animals sub- pain behavior it is likely that block of these receptors con- jected to CCR2 RA-[S]. Nociceptive behavior in vehicle- tributes to the antinociceptive effects of the CCR2 antago- treated controls and animals subjected to CCR2 RA-[S] dif- nist. fered significantly from day 0 pre-injury baseline responses (*p < 0.01). Data represent means ± SE. In the face of the effectiveness of CCR2 receptor block either pharmacologically (Fig 9) or genetically [11], the function of other types of upregulated chemokine signal- pain [8]. Indeed, CCR2 receptor deficient mice are resist- ing to chronic pain behavior is not immediately obvious. ant to the induction of some sensory neuropathies, high- Like CCR2, the CCR5 receptor function and its ligand, lighting the potential importance of this chemokine RANTES, were also strongly upregulated in DRG neurons signaling system [11]. In the current experiments, we uti- in response to focal demyelination. It has previously been 2+ imaging paradigm in lieu of electrophysiolog- lized a Ca shown that RANTES may be important in other chronic ical recording, as chemokine-induced increased neuronal pain situations [51]. Our findings in this sciatic nerve excitability would be expected to be correlated with a injury model differ from a report by Taskinen and Royotta 2+ chemokine induced increase in (Ca ) . The observed [16] which demonstrated bilateral upregulation of non- increase and subsequent decrease in MCP-1-induced neuronal RANTES for up to four weeks following sciatic 2+ Ca responsiveness in acutely isolated DRG neurons over nerve transection in the rat. The apparent differences may time generally correlated with the anatomical observa- be due to the nature of the nerve injuries. Alternatively, tions of receptor expression, and both effects returned to CCR5 may also be activated by a number of other chem- baseline by POD 35. Importantly, the ability of the CCR2 okine ligands which we did not measure [52]. Chemokine receptor antagonist to attenuate bilateral nociceptive interactions with CCR5 may also depress the analgesic behavior at both 14 and 28 days after nerve injury strongly action of endogenous opioids and/or sensitize TRPV1 suggests an integral role for MCP-1/CCR2 signaling in [10,53,54] thereby generally promoting hyperalgesia. maintaining this phase of pain hypersensitivity. The signaling pattern of IP-10 and CXCR3 receptors in the Although the CCR2 antagonist was effective in blocking DRG differs in some respects as there is appreciable basal pain hypersensitivity, more than one chemokine or chem- neuronal expression of both CXCR3 receptors and IP-10. 2+ okine receptor was upregulated in this neuropathic pain In spite of this, few Ca neuronal responses were Page 16 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 observed in the naïve or sham animals, perhaps because chemokines/receptors following unilateral sciatic nerve of desensitization resulting from ongoing receptor activa- demyelination by releasing cytokines from activated tion induced by constitutive expression of IP-10. Neuro- microglia in the spinal cord dorsal horn following chronic nal expression of IP-10/CXCR3 under basal conditions activity in injured DRG afferent neurons [70]. Spinal cord- may have a specific role to play that is analogous to the derived cytokines or growth factors such as TNF-α, IL-1β, expression and release of fractalkine by neurons [42,55]. IL-6 and/or BDNF, may also directly signal contralateral Following strong neuronal excitation in the peripheral lumbar DRG neurons through receptors present on pri- nervous system (i.e. trauma or disease), IP-10 may be mary afferent central terminations [71,72]. Perhaps not released within the DRG and/or from central terminals in surprisingly, blockade of the action of TNF-α or inhibition the spinal cord dorsal horn resulting in both local and dis- of glial metabolic activity can attenuate bilateral nocicep- tant glial activation [26,56,57]. tive pain behavior [69,73], while the low dose administra- tion of a gap junction protein decoupler only extinguishes Focal nerve demyelination changes in SDF-1 signaling via only contralateral pain behavior [74]. Although current the CXCR4 receptor show still another pattern. In this trends in pain research favor bilateral spinal cord glial case, the chemokine receptor is not generally expressed in activation as a mechanism of central activation in the spi- neurons, but in satellite glia and Schwann cells. Upregu- nal cord, it does not appear to be central to all pain con- lated CXCR4 expression, however, is primarily restricted ditions [61]. It is also interesting to note in the context of to neurons making them a potential target for the release the present set of investigations that TNF-α can upregulate of SDF-1 from glia. It is interesting to note that a role for MCP-1 expression by sensory neurons [75], further sup- Schwann cell release of SDF-1 and for neuronally- porting the possibility that it may function as an upstream expressed CXCR4 receptors has also been suggested in regulator of chemokine signaling in the DRG. recently proposed models of HIV-1 and NRTI related neu- ropathies [14,51]. Alternatively, aspects of bilateral tactile hyperalgesia may be due to spontaneous ectopic activity in A, but not C-fib- It is clear that focal nerve demyelination injury-induced ers. This type of ongoing ectopic hyperexcitability in pri- behavioral changes are correlated with widespread mary sensory neurons post-injury can occur in both changes in the neuronal expression of chemokine/recep- injured neurons and adjacent, uninjured neurons tors and that the pattern of expression of each chemokine [20,37,76,77]. Changes necessary for this type of mecha- and its receptor is unique, suggesting that the influence of nism implicate modification of the electrical properties of chemokine signaling on rodent nociceptive behavior may the neurons [78-80]. Nerve demyelination in the mid- be complex. It should also be noted that the upregulated thigh may effectively trigger just this type of change in the expression of different chemokines and receptors that we primary sensory neuron (i.e. neurochemistry and physiol- have observed may occur as part of a cytokine "cascade", ogy of primary afferent neurons), which then contribute where the expression of one chemokine or its receptor is to central sensitization and higher levels of nociceptive dependent on previous events. If that is the case it is also sensory processing. Taken together, the evidence of possible that drugs which block several upregulated chronic changes in chemokine/receptor protein expres- chemokine receptors may also prove to be effective if they sion and the ability of certain chemokines to excite neuro- are upstream of CCR2 expression. nal subpopulations [6,8] is suggestive of a potential scenario. In the course of these studies, we also noted that the PWT to mechanical stimulation decreased bilaterally. The Another behavioral component commonly observed with degree of threshold change in the hindpaw contralateral rodent pain models, especially those accompanied by to the nerve injury was qualitatively similar but smaller in robust inflammatory responses, is the presence of thermal magnitude, and briefer in time course, when compared hyperalgesia. Despite a presence of thermal hyperalgesia with the hindpaw ipsilateral to the lesion. This type of in the mouse focal nerve demyelination model [12], the bilateral hyperalgesia has previously been described in rat nerve demyelination model does not exhibit changes other rodent models of neuropathic pain [58-67]. As such, in response to temperature. Thermal hyperalgesia is it is of interest to compare the LPC-induced peripheral largely thought to be a pain-related symptom caused by nerve pain model with previously described rodent peripheral sensitization. The absence of thermal hyperal- inflammatory pain models which demonstrated bilateral gesia would suggest that there is a lack of ongoing inflam- tactile pain behavior [60] and contralateral changes in the matory mediator-initiated sensory neuron signaling. Lack DRG [65,68]. Milligan et al. [69] suggested that this phe- of thermal hyperalgesia in peripheral nerve injury models nomenon is likely due to changes in the spinal dorsal of pain, although not common, include perineural gp-120 horn. This type of spinal mechanism may drive both bila- administration [81]; Bhangoo and White, unpublished tateral pain sensitivity and contralateral DRG changes in observations) acidic saline-induced hyperalgesia [63] and Page 17 of 20 (page number not for citation purposes) Molecular Pain 2007, 3:38 http://www.molecularpain.com/content/3/1/38 4. Oh SB, Tran PB, Gillard SE, Hurley RW, Hammond DL, Miller RJ: chronic constriction injury performed in a 5-HT trans- Chemokines and glycoprotein120 produce pain hypersensi- porter knockout mouse [82]. Lack of thermal hyperalgesia tivity by directly exciting primary nociceptive neurons. J Neu- in the model of muscle pain and CCI implicate central rosci 2001, 21:5027-35. 5. Qin X, Wan Y, Wang X: CCL2 and CXCL1 trigger calcitonin descending mechanisms for the display of bilateral hyper- gene-related peptide release by exciting primary nociceptive algesia. It is possible that similar mechanisms are operat- neurons. J Neurosci Res 2005, 82:51-62. 6. Sun JH, Yang B, Donnelly DF, Ma C, LaMotte RH: MCP-1 enhances ing within the rat nerve demyelination injury model. excitability of nociceptive neurons in chronically com- pressed dorsal root ganglia. J Neurophysiol 2006, 96:2189-99. Taken together the data suggest that upregulation of 7. Tanaka T, Minami M, Nakagawa T, Satoh M: Enhanced production of monocyte chemoattractant protein-1 in the dorsal root chemokine signaling by sensory neurons may help to ganglia in a rat model of neuropathic pain: possible involve- integrate several phenomena that account for changes in ment in the development of neuropathic pain. Neurosci Res the properties of peripheral nerves resulting in bilateral 2004, 48:463-9. 8. White FA, Sun J, Waters SM, Ma C, Ren D, Ripsch M, Steflik J, Cor- pain hypersensitivity. The present results, together with tright DN, Lamotte RH, Miller RJ: Excitatory monocyte chem- previous studies [3], suggest that the mode of injury may oattractant protein-1 signaling is up-regulated in sensory neurons after chronic compression of the dorsal root gan- determine which particular chemokines play a central role glion. Proc Natl Acad Sci USA 2005, 102:14092-7. in maintaining the neuropathic pain state. Chemokine 9. Jung H, Toth PT, White FA, Miller RJ: Monocyte chemoattractant receptors may then represent novel targets for therapeutic protein-1 functions as a neuromodulator in dorsal root gan- glia neurons. J Neurochem 2007. intervention in demyelination associated neuropathic 10. Zhang N, Inan S, Cowan A, Sun R, Wang JM, Rogers TJ, Caterina M, pain as well as other chronic pain states. Oppenheim JJ: A proinflammatory chemokine, CCL3, sensi- tizes the heat- and capsaicin-gated ion channel TRPV1. Proc Natl Acad Sci USA 2005, 102:4536-41. Additional material 11. Abbadie C, Lindia JA, Cumiskey AM, Peterson LB, Mudgett JS, Bayne EK, DeMartino JA, MacIntyre DE, Forrest MJ: Impaired neuro- pathic pain responses in mice lacking the chemokine recep- Additional file 1 tor CCR2. Proc Natl Acad Sci USA 2003, 100:7947-52. 12. Wallace VC, Cottrell DF, Brophy PJ, Fleetwood-Walker SM: Focal The chemical structures and full names of the CCR2 antagonist (CCR2- lysolecithin-induced demyelination of peripheral afferents [R]) and its inactive enantiomer (CCR2-[S]). results in neuropathic pain behavior that is attenuated by Click here for file cannabinoids. J Neurosci 2003, 23:3221-33. [http://www.biomedcentral.com/content/supplementary/1744- 13. Cook WJ, Kramer MF, Walker RM, Burwell TJ, Holman HA, Coen 8069-3-38-S1.doc] DM, Knipe DM: Persistent expression of chemokine and chemokine receptor RNAs at primary and latent sites of her- pes simplex virus 1 infection. Virol J 2004, 1:5. Additional file 2 14. Melli G, Keswani SC, Fischer A, Chen W, Hoke A: Spatially distinct Expression of SDF1 mRNA in rat lumbar DRG ipsilateral to focal nerve and functionally independent mechanisms of axonal degen- demyelination. (A) Many cells, both satellite glia and neurons, in the eration in a model of HIV-associated sensory neuropathy. Brain 2006, 129:1330-8. lumbar DRG removed from vehicle-treated rats exhibited SDF1 mRNA 15. Subang MC, Richardson PM: Influence of injury and cytokines on transcripts at POD14 (n = 3). (B) SDF1 mRNA expression did not synthesis of monocyte chemoattractant protein-1 mRNA in change significantly in the lumbar DRG of LPC-treated rats at POD14 peripheral nervous tissue. Eur J Neurosci 2001, 13:521-8. (n = 3). (C) A magnified photomicrograph of the lumbar DRG from a 16. Taskinen HS, Roytta M: Increased expression of chemokines vehicle treated rat. (D) A magnified photomicrograph of the lumbar DRG (MCP-1, MIP-1alpha, RANTES) after peripheral nerve removed from a LPC-treated rat at POD14. transection. J Peripher Nerv Syst 2000, 5:75-81. 17. Bhangoo S, Jung H, Chan DM, Ripsch M, Ren D, Miller RJ, White FA: Click here for file Peripheral demyelination injury induces upregulation of [http://www.biomedcentral.com/content/supplementary/1744- chemokine/receptor expression and neuronal signaling in a 8069-3-38-S2.doc] model of neuropathic pain. In Abstract Viewer/Itinerary Planner Atlanta, GA: Society for Neuroscience Annual Meeting; 2006:250.3. 18. White FA, Ripsch M, Bhangoo S, Ren D, Weiss C, Miller RJ: Regula- tion of chemokines/receptors in the dorsal root ganglion fol- lowing focal demyelination of the sciatic nerve. In Abstract Viewer/Itinerary Planner Washington, DC: Society for Neuroscience Acknowledgements Annual Meeting; 2005:748.9. FAW, NIH Grant NS049136, National Multiple Sclerosis Society Pilot 19. 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Molecular Pain – Springer Journals

Published: Dec 12, 2007

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