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Contribution of afferent pathways to nerve injury-induced spontaneous pain and evoked hypersensitivity

King, Tamara; Qu, Chaoling1; Okun, Alec; Mercado, Ramon; Ren, Jiyang; Brion, Triza; Lai, Josephine; Porreca, Frank*

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doi: 10.1016/j.pain.2011.04.020
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Abstract

1. Introduction

A common symptom of patients with neuropathic pain is spontaneous pain that is often described as burning [1,2]. Some neuropathic pain patients also suffer from pain that is elicited by normally innocuous touch or cold, that is, allodynia [12,28]. Preclinical studies of experimental neuropathic pain have generally relied on enhanced withdrawal responses to normally innocuous tactile stimuli (ie, von Frey filaments or brushing) or to noxious thermal stimuli. After nerve injury, an enhanced response to a noxious thermal stimulus is a measure of hyperalgesia. However, whether an exaggerated response to a normally innocuous tactile stimulus is an indication of pain, that is, allodynia, has been questioned [13].

Systemic administration of resiniferatoxin (RTX), an ultrapotent TRPV1 agonist, to adult rats produces desensitization of TRPV1-positive fibers, resulting in a long-lasting elimination of nerve injury-induced thermal hyperalgesia as well as thresholds to noxious heat; this treatment, however, does not affect nerve injury-induced tactile hypersensitivity [25]. Small unmyelinated fibers, including TRPV1-positive fibers, synapse within the superficial dorsal horn to lamina I NK-1-positive cells, and these have been demonstrated to be critical in expression of nerve injury-induced thermal and tactile hypersensitivity [21,24,32]. However, whether these mechanisms contribute to spontaneous pain is not known.

Nerve injury-induced tactile allodynia is thought to be mediated by large-diameter Aβ fibers [6,15,40]. In addition to synapses in the spinal cord, however, these large-diameter cells also project, via the dorsal column pathway, to brainstem nuclei including nucleus gracilis and nucleus cuneatus (for hindpaw and forepaw projections, respectively). After spinal nerve ligation (SNL) injury, large-diameter afferents selectively upregulate neuropeptide Y (NPY) [26]. Microinjection of NPY into the nucleus gracilis of uninjured rats elicits tactile but not thermal hypersensitivity, and inactivation of the nucleus gracilis after nerve injury with lidocaine or anti-NPY antiserum reverses tactile but not thermal hypersensitivity [26,31,42]. Although the dorsal column pathway to nucleus gracilis contributes to enhanced withdrawal response after nerve injury, it is not known whether this pathway contributes to neuropathic allodynia.

Although gain-of-function responses to evoked stimuli are taken as the main translation feature of experimental neuropathic models [30], nonevoked, or spontaneous, pain is the most prominent feature of the neuropathic state [1]. Such pain has been difficult to measure preclinically [4,18,30,39]. We recently demonstrated that nerve injury-induced spontaneous pain can be unmasked using the principle of negative reinforcement in models of partial nerve injury pain [18]. The present study explored the relative contribution of afferent and ascending sensory tracts to nerve injury-induced spontaneous pain and evoked hypersensitivity.

2. Materials and methods

Male Sprague–Dawley rats (Harlan, Indianapolis, IN), 250 to 350g at the time of testing, were maintained in a climate-controlled room on a 12-hour light-dark cycle (lights on at 7:00 am) with food and water available ad libitum. All testing was performed in accordance with the policies and recommendations of the International Association for the Study of Pain and the National Institutes of Health guidelines for the handling and use of laboratory animals, and received approval from the Institutional Animal Care and Use Committee of the University of Arizona.

2.1. Rostral ventromedial medulla (RVM) and nucleus gracilis cannulation

Animals were anesthetized with injection of ketamine–xylazine (100mg/kg, intraperitoneally [i.p.]) and placed in a stereotaxic apparatus. Bilateral cannulation of the RVM was performed as previously described [3]. Two 26-gauge guide cannulas separated by 1.2mm (Plastics One, Roanoke, VA) were directed toward the lateral portions of the RVM (anteroposterior, −11.0mm from bregma; lateral, ±0.6mm; dorsoventral, −7.5mm from the dura mater [27]). Cannulation of the nucleus gracilis was performed as previously described [26]. A 26-gauge guide cannula (Plastics One) was directed toward the ipsilateral nucleus gracilis (anteroposterior, −5.5mm from the interaural line, mediolateral 0.5mm from midline, and 2mm above the interaural line [27]). Both RVM and nucleus gracilis guide cannulas were cemented in place and secured to the skull by small stainless steel machine screws. Rats then received gentamicin and were allowed to recover 7days before any behavioral testing and SNL or sham surgeries. Shorter recovery periods were avoided to prevent possible behavioral impairment in place conditioning assays.

2.2. Sar9Met(O2)11 substance P-saporin (SSP-SAP) administration

Rats underwent surgery for implantation of an intrathecal catheter under halothane anesthesia (polyethylene-10 tubing, 7.5cm) as described previously [41] for drug administration at the level of the lumbar cord. Rats were anesthetized with ketamine–xylazine (Sigma, St. Louis, Mo 80mg/kg:20mg/kg), and the atlanto-occipital membrane was exposed and punctured. A section of polyethylene −10 tubing 8cm in length was passed caudally from the cisterna magna to the lumbar enlargement. Intrathecal injections of blank-SAP (Advanced Targeting Systems, San Diego, CA 0.1μM, 16.5pg/5μL) or the substituted SP analog, SSP-SAP (Advanced Targeting Systems, 0.1μM, 16.5pg/5μL) were made in a volume of 5μL followed by a 9-μL flush of saline. This dose is 10-fold lower than previously published data with unsubstituted SP-SAP [21], as SSP-SAP shows approximately 10-fold greater potency at the NK-1 receptor in addition to resistance to peptidase digestion [23]. Progress of the injection was monitored by the movement of an air bubble placed between the drug solution and the saline flush. Immediately after injection, catheters were slowly removed from the spinal cord and the wound was closed. All rats received gentamicin after surgery. Any animals displaying motor impairment or paralysis during recovery (<10% total rats) were immediately euthanized. Animals were kept in home cages for 28days before further testing to allow for elimination of NK-1 receptor expressing cells in the spinal cord as described previously [38]. Animals were routinely checked across the 28days to monitor health.

2.3. Spinal nerve ligation (SNL)

After a 1-week recovery following RVM or nucleus gracilis surgeries, baseline sensory thresholds were measured for evoked pain behaviors. The surgical procedure for L5/L6 SNL was performed according to that described previously [17]. Sham-operated control rats were prepared in an identical manner except that the L5/L6 spinal nerves were not ligated. The behavior of the rats was monitored carefully for any visual indication of motor disorders or change in weight or general health.

2.4. Behavioral testing

Tactile and thermal hypersensitivity and place preference assays were determined 14days after SNL or sham surgery by experimenters blinded to the treatment groups of the rats.

2.5. Tactile hypersensitivity

Tactile hypersensitivity was determined 14days after SNL or sham surgery by measuring the withdrawal threshold of the ipsilateral hindpaw in response to probing with a series of 8 calibrated von Frey filaments by sequentially increasing and decreasing the stimulus strength (up and down method), analyzed using a Dixon nonparametric test [5,7].

2.6. Thermal hypersensitivity

Thermal hypersensitivity was determined 14days after SNL or sham surgery by measuring the withdrawal latency of the ipsilateral hindpaw in response to infrared radiant heat. A motion detector halted both lamp and timer when the paw was withdrawn. Baseline latencies were established at 17 to 25seconds to allow a sufficient window for the detection of possible hyperalgesia. A maximal cutoff of 33seconds was used to prevent tissue damage.

2.7. Conditioned place pairing

The single-trial conditioned place preference protocol was performed as previously described [18], with conditioning day 10days after SNL or sham surgeries. Starting 7days post-SNL/sham surgery, all rats underwent a 3-day preconditioning period with behavior recorded on day 3 to verify no preconditioning chamber preference. All animals are exposed to the environment with full access to all chambers across 30minutes each day. On day 3, behavior was recorded for 15minutes and analyzed to verify absence of preconditioning chamber preference. The next day (day 4), rats received the appropriate control (ie, vehicle) and immediately were placed in the appropriate conditioning chamber for 30minutes. Four hours later, rats received the appropriate drug treatment and immediately were placed in the opposite conditioning chamber for 30minutes. Chamber pairings were counterbalanced. On test day 5, 20hours after the afternoon pairing, rats were placed in the conditioned place preference (CPP) box with access to all chambers, and their behavior was recorded for 15minutes for analysis for chamber preference. Increased postconditioning time spent in the drug-paired chamber, as compared with preconditioning time, indicates conditioned place preference. Decreased postconditioning time spent in the drug-paired chamber, as compared with preconditioning time, indicates conditioned place aversion. No change between time spent in the drug-paired chamber, as compared with preconditioning time, indicates no conditioned place preference or aversion.

2.8. RTX injection

RTX (Tocris Bioscience, Ellisville, MO), an ultrapotent TRPV1 receptor agonist, was administered systemically (0.1mg/kg, i.p.) in a dose previously demonstrated to abrogate thermal responses across a period of 40days, the longest time point tested [25]. RTX was dissolved in a 95% ethanol mixture, which was used as the vehicle control.

2.9. RVM and nucleus gracilis microinjection

Saline or lidocaine (4% w/v) was administered by slowly expelling 0.5μL through a 33-gauge injection cannula inserted through the guide cannula and protruding an additional 1mm into fresh brain tissue to prevent backflow of drug into the guide cannula. Drug administration into the nucleus gracilis was performed by slowly expelling 0.5μL of the anti-NPY antiserum, NPY (1nmol, 2.66μg/0.5μL) or appropriate vehicle through a 33-gauge injection cannula inserted an additional 2mm into fresh brain tissue to prevent backflow of drug into the guide cannula. Anti-NPY antiserum (Peninsula Laboratories, Inc., San Carlos, CA) was dissolved in 50μL distilled water. Preadsorbed serum was prepared from the anti-NPY antiserum as a control. The preadsorbed serum was prepared by incubating 0.2mL of the antiserum (40mg protein/mL) with 0.8mL of agarose-bound protein A (Vector Laboratories, Burlingame, CA) for 24hours at 4°C. The suspension was pelleted by low-speed centrifugation (5000g for 5minutes). The supernatant, which is devoid of anti-NPY immunoglobulin G, was used to define the immunoglobulin G-independent effects of the antiserum administration. Doses of the anti-NPY antiserum and NPY were based on previously published data [26].

2.10. Tissue verification

After the termination of all of the behavioral testing, 0.5 μL India ink was injected into the bilateral RVM or the nucleus gracilis. Rats were perfused transcardially with 0.9% 100 to 150mL saline, followed by 10% formalin solution. Subsequent to perfusion, the brains were rapidly removed and placed into the same, fresh fixative to be fixed for additional 4hours at 4°C. Subsequently, the brains were soaked in 25% sucrose solution overnight at 4°C and then cut serially into 30-μm-thick coronal sections on a freezing microtome (Microm, HM 525; International GmbH, Germany). Sections were stained with cresyl violet to verify the RVM injection. Data from animals with incorrectly placed cannula were not included within the data analysis.

2.11. Immunohistochemistry

Rats were deeply anesthetized with a mixture of ketamine-xylazine and perfused transcardially with 100mL saline, followed by 500mL cold 4% paraformaldehyde. After perfusion the spinal cords were removed, postfixed overnight in 4% paraformaldehyde, and cryoprotected with 25% sucrose in phosphate-buffered saline (PBS) overnight at 4°C. Coronal frozen sections (20μm) were cut from the lumbar enlargement of the spinal cord. Spinal cord sections were immunolabeled with a rabbit antiserum against the NK-1 receptor (1:4000, Millipore, Billerica, MA). The sections were washed 3 times for 10minutes each in PBS and then preincubated with PBS containing 5% normal goat serum and 0.3% Triton X-100 for 30minutes at room temperature. The sections were then incubated overnight in the primary antiserum diluted in 2% normal goat serum. The next day, the sections were washed 3 times in PBS for 10minutes each, followed by incubation with secondary antibody (fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit immunoglobulin G; 1:2000; Invitrogen, Carlsbad, CA) for 2 hours. The sections were rinsed and mounted in Vectashield (Vector Laboratories). Fluorescent digital images were captured using an Olympus BX51 microscope using a Hamamatsu CCD digital camera. The person taking the digital pictures was blinded to the experimental conditions.

2.12. Statistical analysis

For analysis of evoked pain behaviors, significant changes from presurgery baseline control values were detected by analysis of variance, followed by Bonferroni posttest. These evaluations were all performed using GraphPad Prism 5.03 (GraphPad Software, San Diego, CA, www.graphpad.com). For CPP experiments, data were analyzed before conditioning (baseline) and after conditioning using 2-factor analysis of variance (chambers vs treatment) followed by a Bonferroni test of postconditioning compared with preconditioning time spent in the drug-paired chamber to determine conditioned place preference (increase in postconditioning time vs preconditioning time) or conditioned place aversion (decrease in postconditioning time vs preconditioning time). If significant conditioned place preference or conditioned place aversion was determined, group differences were analyzed using the difference from baseline scores were calculated for each rat using the formula: test time in chamber − preconditioning time spent in chamber. Difference scores from baseline for the drug-paired chamber between SNL and sham-operated rats were analyzed using paired t-tests. For all analyses, significance was set at P<.05.

3. Results

3.1. RTX desensitization of TRPV1-positive afferent fibers blocks SNL-induced spontaneous pain

Systemic administration of RTX has been demonstrated to produce long-term desensitization of TRPV1-positive fibers [34]. Rats showed thermal hyperalgesia (Fig. 1A) and tactile hypersensitivity (Fig. 1B) 5days after SNL surgery. Consistent with our previous study [25], systemic administration of RTX (0.1mg/kg, i.p.) 3days before testing-evoked behaviors eliminated responses to noxious thermal stimulation in sham- as well as SNL-treated rats (Fig. 1A; ∗∗∗P<.001 vs sham vehicle). Moreover, systemic RTX administration at the same dose failed to alter SNL-induced tactile hypersensitivity (Fig. 1B; ∗∗∗P<.001 vs sham vehicle).

F1-13
Fig. 1:
Resiniferatoxin (RTX) eliminated spinal nerve ligation (SNL)-induced thermal hyperalgesia and spontaneous pain, but not tactile hypersensitivity. (A) Rats showed significant reduction in paw-flick latencies 7 days after SNL surgery (postsurgery). Systemic administration of RTX (0.1 mg/kg, i.p.) produced robust analgesia, increasing paw-flick latencies of both sham- and SNL-treated rats to near cutoff (32 seconds) values (post-RTX). P < .05 vs presurgery, n = 8. (B) Rats showed significant reduction in paw withdrawal thresholds to probing with von Frey filaments 7 days after SNL surgery (postsurgery). Systemic administration of RTX failed to alter SNL-induced tactile hypersensitivity (post-RTX). P < .05 vs presurgery, n = 8. (C) All rats showed equivalent preconditioning time spent in the conditioning chambers. Because no differences were observed between treatment groups, preconditioning values were pooled for graphical representation. SNL rats treated with vehicle (SNL-vehicle) showed increased time spent in the RVM lidocaine-paired chamber after conditioning. Treatment with RTX before the conditioned place preference (CPP) protocol blocked the lidocaine-induced CPP in SNL treated rats. P < .05 vs preconditioning, n = 5 to 7. (D) Difference from baseline scores confirms that only SNL rats treated with vehicle demonstrated CPP to the RVM lidocaine-paired chamber, and that RTX treatment 3 days before the start of CPP blocked the RVM lidocaine-induced CPP. P < .05 vs vehicle sham-treated rats, n = 5 to 7.

Systemic RTX administration (0.1mg/kg, i.p.) 5days after SNL and 3days before habituation blocked RVM lidocaine-induced place preference in SNL-treated rats. Preconditioning times spent in the saline- or lidocaine-paired chambers were equivalent across all treatment groups (P>.05). Because no group differences were observed, data were pooled across groups for graphical representation (Fig. 1C). SNL rats that received vehicle injection showed place preference to the chamber paired with RVM lidocaine (Fig. 1C, ∗∗P<.01 vs preconditioning) consistent with previous observations [18]. In contrast, SNL rats that were treated with systemic RTX showed equivalent time spent in the lidocaine- and saline-paired chambers (Fig. 1C). The difference from baseline scores confirmed that only the SNL rats treated with vehicle showed increased time spent in the RVM lidocaine-paired chamber, and that RTX treatment blocked preference for the RVM lidocaine-paired chamber (Fig. 1D, ∗∗∗P<.001 vs sham vehicle).

3.2. SSP-SAP decreases the number of NK-1 receptor expressing cells in the spinal cord

SSP-SAP (1nM, 1.65pg/0.5μL) resulted in an almost total loss of NK-1 receptor staining in the spinal dorsal horn 28 to 30days after intrathecal administration (Fig. 2A). Spinal administration of equivalent concentration of blank-SAP (1nM, 1.65pg/0.5μL) did not alter NK-1 receptor expression (Fig. 2A), indicating ablation of the NK-1 receptor expressing cells was specific to the SAP-conjugated selective NK-1 receptor agonist as previously demonstrated [23].

F2-13
Fig. 2:
Spinal Sar9Met(O2)11 substance p-saporin (SSP-SAP) ablation of NK-1 receptor expressing cells blocks SNL-induced evoked pain. (A) Representative image showing immunofluorescent staining of NK-1 receptors within the spinal dorsal horn 40 days after spinal administration of SSP-SAP or control injection (blank-SAP). SSP-SAP-treated rats showed greatly diminished NK-1 immunopositive staining. Animals that received the control injection (blank-SAP) show clear labeling of the NK-1 receptor. (B) SNL-induced thermal hyperalgesia is established within 7 days in rats that received the control spinal SAP injection (blank-SAP) 28 days before SNL surgery. In contrast, SNL rats treated with SSP-SAP 28 days before SNL surgery failed to develop thermal hypersensitivity. SSP-SAP failed to alter paw withdrawal latencies of sham rats. (C) SNL induced tactile hypersensitivity within 7 days in rats that received spinal blank SAP 28 days before the SNL surgery. In contrast, SNL rats treated with SSP-SAP 28 days before SNL surgery failed to develop tactile hypersensitivity. All graphs are mean ± SEM, ∗∗∗ P < .001 vs presurgery values, n = 12 to 18.

3.3. SSP-SAP blocks SNL-induced thermal hyperalgesia and tactile hypersensitivity

The paw withdrawal thresholds to probing with von Frey filaments and the withdrawal latencies from noxious radiant heat were determined 7days after SNL or sham surgeries in animals treated 35days previously with blank-SAP or SSP-SAP. Consistent with previous studies, presurgery thermal and tactile thresholds did not differ irrespective of treatment group (P>.05, data not shown) [21]. Postsurgery sensory thresholds of sham rats did not differ from presurgical baselines irrespective of treatment group (P>.05). In SNL rats treated with blank-SAP, robust thermal hyperalgesia and tactile hypersensitivity was observed (Figs. 2B–D; ∗∗∗P<.001 vs pre-SNL). In contrast, thermal and tactile hypersensitivity failed to develop in SNL rats that had received spinal SSP-SAP treatment (Fig. 2B and C; P>.05, vs pre-SNL), indicating that ablation of spinal NK-1 receptor-expressing neurons blocked SNL-induced thermal hyperalgesia and tactile hypersensitivity as previously reported [24]. No differences in responses to thermal or tactile stimulation were observed in sham-operated animals irrespective of treatment group.

3.4. SSP-SAP blocks spontaneous pain in the SNL rats

To determine whether ablation of NK-1 receptor-expressing neurons within the spinal cord also blocks SNL-induced spontaneous pain, rats previously treated with blank-SAP (control) or SSP-SAP underwent single-trial conditioning with microinjection of lidocaine into the RVM, previously demonstrated to induce CPP in SNL rats [18]. Preconditioning times spent in the saline- or lidocaine-paired chambers were equivalent across all treatment groups (P>.05). Because no group differences were observed, data were pooled across groups for graphical representation (Fig. 3A). Rats with SNL that had received intrathecal injection of blank-SAP showed a significant preference for the RVM lidocaine-paired chamber (Fig. 3A, P<.05 vs before lidocaine). In contrast, SNL-treated rats with SSP-SAP showed no preference for the RVM lidocaine-paired chamber. Sham SNL rats had equivalent postconditioning times spent in the saline- and lidocaine-paired chambers irrespective of whether they received intrathecal blank-SAP or SSP-SAP. The difference from baseline scores verified that SSP-SAP blocked RVM lidocaine-induced CPP in the SNL rats because only SNL rats with blank-SAP demonstrated increased time spent in the lidocaine-paired chamber (Fig. 3B, P<.05 vs SSP-SAP+SNL group).

F3-13
Fig. 3:
Spinal Sar9Met(O2)11 substance p-saporin (SSP-SAP) ablation of NK-1 receptor expressing cells blocks SNL-induced spontaneous pain. (A) Preconditioning time spent in the conditioning chambers did not differ across treatment groups, therefore data were pooled for graphical representation. RVM lidocaine did not produced CPP in sham-operated rats irrespective of treatment group. spinal nerve ligation (SNL) rats that received spinal control injection (blank-SAP) 28 days before SNL surgery showed increased time spent in the lidocaine-paired chamber, P < .05 compared with preconditioning values. SNL rats that received spinal SSP-SAP injection 28 days before SNL surgery failed to show CPP to the lidocaine-paired chamber. (B) Difference scores confirm that only SNL rats that received spinal injection of the control (blank-SAP) showed CPP to the lidocaine-paired chamber, P < .05 vs sham-blank control rats. All graphs are mean ± SEM, n = 6 to 8.

3.5. NPY into the nucleus gracilis induces tactile hypersensitivity, but not CPA in uninjured rats

Consistent with previous reports [26], microinjection of NPY (2.66μg/0.5μL) into the nucleus gracilis induced robust tactile hypersensitivity that peaked within 20minutes of administration and dissipated within 40minutes postadministration (Fig. 4A, ∗∗∗P<.001, ∗∗P<.01 vs predrug baseline (BL)). Administration of this same dose of NPY into nucleus gracilis, however, failed to induce conditioned place aversion (Fig. 4B). These data indicate that administration of NPY into the nucleus gracilis of uninjured rats enhances paw withdrawal responses to tactile stimulation but is not aversive.

F4-13
Fig. 4:
Neuropeptide Y (NPY) administration into the nucleus gracilis induced tactile hypersensitivity, but did not induce conditioned place aversion in naïve rats. (A) Administration of NPY (2.66 μg/0.5 μL) into the ipsilateral nucleus gracilis induced robust tactile hypersensitivity within 20 minutes of administration that resolved by 40 minutes postadministration. Administration of saline failed to alter paw withdrawal thresholds. P < .05 vs baseline (BL), n = 9. (B) Administration of the same dose of NPY into the nucleus gracilis failed to alter time spent in conditioning chambers.

3.6. Anti-NPY into the nucleus gracilis reverses tactile hypersensitivity, but not spontaneous pain

Consistent with previous reports [26], microinjection of anti-NPY antiserum (20μg/0.5μL) reversed SNL-induced tactile hypersensitivity within 10minutes, lasting through 60minutes postinjection and dissipating within 90minutes postinjection (Fig. 5A, P<.05 vs postsurgery). Microinjection of the same dose of anti-NPY antiserum failed to induce CPP in nerve-injured rats. Preconditioning times spent in the saline- or anti-NPY antiserum-paired chambers were equivalent (Fig. 5B, P>.05). after conditioning, both sham- and SNL-treated rats spent equivalent time in the saline- and anti-NPY-paired chambers, indicating no conditioned place preference to the anti-NPY-paired chamber (Fig. 5B, P>.05).

F5-13
Fig. 5:
Anti-neuropeptide Y (NPY) administration into the Nucleus gracilis reversed SNL-induced tactile hypersensitivity, but not spontaneous pain. (A) Administration of anti-NPY (20 μg/0.5 μL) into the ipsilateral nucleus gracilis fully reversed SNL-induced tactile hypersensitivity within 20 minutes, with thresholds returning to pre-anti-NPY values within 40 minutes of administration. P < .05 vs postsurgery, n = 5 to 6. (B) All rats showed equivalent preconditioning time spent in the conditioning chambers. Because no differences were observed between treatment groups, preconditioning values were pooled for graphical representation. Administration of the same dose of anti-NPY into the ipsilateral nucleus gracilis failed to alter the time spent in the conditioning chambers in sham- or SNL-treated rats, n = 5 to 10.

4. Discussion

The present study investigated the possible contribution of specific classes of afferent fibers and ascending pathways in spontaneous and evoked components of pain after experimental nerve injury. Our findings demonstrate that (1) TRPV1-positive afferent fibers are critical for SNL-induced spontaneous pain and thermal hyperalgesia but may not be essential for hypersensitivity to a tactile stimulus; (2) spinal NK-1-positive projections mediate nerve injury-induced spontaneous pain as well as thermal hyperalgesia and tactile hypersensitivity; and (3) NPY-positive signaling within the nucleus gracilis mediates SNL-induced tactile hypersensitivity, but not spontaneous pain.

Consistent with previous studies, desensitization of TRPV1-positive fibers after systemic administration of RTX eliminated the behavioral response to noxious thermal stimulation in both sham- and SNL-treated rats but failed to alter responses to tactile stimulation [25]. Here, functional blockade of TRPV1-positive fibers also blocked nerve injury-induced spontaneous pain. The possibility that spontaneous pain and touch-evoked allodynia are mediated by different fiber classes is supported clinically. Koltzenburg et al. found that compression of the radial nerve resulted in progressive block of large myelinated (Aβ) followed by thin myelinated (Aδ) fibers and resulted in an inhibition of brush-evoked pain without eliminating ongoing pain in patients with chronic neuralgia [20]. Notably, the brush-evoked pain was eliminated before loss of cold sensation indicating transmission by Aβ fibers, which normally encode nonpainful tactile stimuli. Additionally, pressure cuff blocks have demonstrated that abolishing Aβ fiber function blocked allodynia while maintaining thermal sensation [12].

Human psychophysical studies indicate that activation of touch fibers produces sensations of pain when TRPV1-positive C-fibers are driven by capsaicin [37]. Additionally, touch can elicit pain in the setting of nerve injury [12,20]. Patients with postherpetic neuralgia have been classified into subgroups characterized by those with prominent allodynia as well as others with impaired responses to touch and thermal stimuli and spontaneous pain [11]. In patients who have profound loss of thermosensory and nociceptor function, intradermal lidocaine failed to alleviate pain, leading to the proposal that pain in these patients is mediated by increased spontaneous activity in deafferented central neurons and/or reorganization of central connections [11]. However, in patients with preserved sensation, application of intradermal local anesthetic produced pain relief [9,27], leading to the proposal that this group of patients has neuropathic pain driven by “irritable nociceptors” [11]. Other studies show that selective stimulation of TRPV1-positive nociceptive fibers by application of capsaicin to an area of postherpetic neuralgia pain increased pain and allodynia [28]. Similarly, local anesthetic block of painful foci in patients with reflex sympathetic dystrophy abolished mechanoallodynia, cold allodynia, and spontaneous pain, while tactile perception was preserved [12]. Our studies are consistent with these observations in humans in suggesting that TRPV1-positive fibers likely mediate spontaneous neuropathic pain, often described clinically as burning pain, as well as thermal responses. In neuropathic pain questionnaires, one of the strongest predictors of nerve injury pain is the description of pain as burning [2], an observation that implicates C-nociceptors in spite of the fact that not all patients experience thermal hyperalgesia [20]. It should be noted that the burning aspect of pain may reflect either dysfunction of injured nerves as would occur in a neuropathic state or physiological function of nerves in an inflammatory setting [2,10,16]. Preclinical studies have suggested that tactile allodynia may be mediated by Aβ fiber inputs to a sensitized spinal cord [6]. Our studies show that tactile hypersensitivity is present in SNL-rats even after functional desensitization of TRPV1-positive fibers, a finding that argues against interpretation of this behavioral response as allodynia.

Nociceptive fibers, such as TRPV1-positive fibers, have been demonstrated to form synaptic connections with NK-1-positive cells in spinal cord lamina I [36]. In the present studies, ablation of NK-1 receptor expressing cells was shown to eliminate all components of nerve injury-induced pain including thermal and tactile hypersensitivity as well as spontaneous pain. The NK-1 receptor has been suggested to be expressed on approximately 80% of the projection neurons in lamina I of the spinal cord that play a crucial role in the transmission of ascending nociceptive information to regions including the parabrachial area and thalamus [22,35,36]. Additionally, nerve injury has been suggested to engage spinal NK-1 receptor expressing cells as the ascending limb of a pronociceptive spinal-bulbo-spinal loop proposed to maintain spinal sensitization [33]. This pathway is also believed to drive descending pain facilitatory pathways from the RVM that are critical to the expression of nerve injury-induced tactile and thermal hypersensitivity as well as spontaneous pain [3,18,29,38]. Previous reports have shown that ablation of spinal NK-1 receptor expressing cells reliably block nerve injury-induced tactile and thermal responses [14,21,24] as well as an SNL-induced increase in evoked firing of spinal neurons [32]. Importantly, ablation of spinal NK-1 expressing cells blocked an SNL-induced increase in spontaneous activity of spinal dorsal horn neurons, suggesting a role for these neurons in SNL-induced spontaneous pain [32]; our present findings agree with this conclusion. Thus, converging evidence supports the importance of NK-1 receptor expressing cells in neuropathic spontaneous pain as well as in allodynia and thermal hyperalgesia.

Large-diameter fibers encode touch and vibration through the ascending dorsal column pathway. Multiple studies have demonstrated that interference with this pathway blocks nerve injury-induced tactile, but not thermal, hypersensitivity. Spinal hemisection or dorsal column lesion blocked tactile hypersensitivity when performed ipsilateral but not contralateral to the nerve injury [31]. Moreover, inactivation of the nucleus gracilis, the projection site of low-threshold myelinated fibers from the hindpaw, blocks nerve injury-induced tactile hypersensitivity, but not thermal hyperalgesia [26,31]. NPY is selectively upregulated in large-diameter fibers and in the nucleus gracilis after nerve injury [26]. Consistent with our previous studies, blockade of NPY signaling in nucleus gracilis reversed nerve injury-induced tactile hypersensitivity. However, anti-NPY antiserum in nucleus gracilis at a dose that effectively blocked tactile hypersensitivity failed to produce place preference in nerve-injured rats, suggesting that this pathway does not contribute to spontaneous pain. Additionally, microinjection of NPY intonucleus gracilis at a dose that produces robust tactile but not thermal hypersensitivity failed to produce conditioned place aversion. It should be noted that neuropathic pain patients often report unfamiliar sensations that are described as dysesthesias but that are not identified as pain [9]. Additionally, such patients have reported unpleasant feelings in response to tactile stimulation, but notably the sensation is not referred to as pain [9,19,20]. Thus, although enhancement of tactile responses through this pathway can be elicited pharmacologically with microinjection of NPY, this manipulation does not produce a state that is sufficiently aversive to be detected in this assay, as would be expected in pain. Nevertheless, the stimulation of signaling at this site might be hypothesized to contribute to unpleasant sensations as reported in patients.

As noted earlier, however, interference with spinal NK-1-positive pathways, as well as many other spinal manipulations (eg, spinal administration of clonidine or ω-conotoxin [18]) blocks nerve injury-induced enhanced behavioral responses to tactile stimulation. Clinical studies have shown that spinal administration of adenosine can block allodynia without affecting ongoing pain [8]. Thus, enhanced behaviors to tactile stimulation after nerve injury can be modulated either through a spinal pathway or by interfering with the dorsal column (ie, touch) pathway. These observations create confusion regarding whether enhanced behavioral sensitivity to tactile stimulation can be interpreted as allodynia. Hogan et al. [13] propose that stimulation by von Frey filaments produces sensations in the form of itch or tickle, which have been demonstrated to be highly motivating without being painful. These conclusions are consistent with our findings that interference with, or stimulating, signaling within nucleus gracilis does not elicit preference in nerve-injured animals or promote aversion in uninjured animals. Collectively, these observations suggest that the enhanced evoked hypersensitivity to touch stimuli is a complex behavior, possibly reflecting both aversive and nonaversive components that are associated with the neuropathic state.

Although our data and clinical observations support the role of TRPV1-positive fibers in neuropathic pain, it is not clear whether additional contributions may arise from TRPV1-negative fibers or whether there is an additional contribution of mechanosensation to allodynia from desensitized TRPV1-positive fibers. The present study highlights the uncertainty of nerve injury-induced tactile hypersensitivity as a measure of clinical painful allodynia. Whereas spinal manipulations that reverse SNL-induced tactile hypersensitivity are likely to reflect painful allodynia, it is impossible to discern whether changes in threshold responses to tactile stimuli resulting from systemic treatments reflects reduction in painful allodynia or nonpainful sensations. These studies also highlight the need for increased understanding of the mechanisms by which spinal NK-1-positive cells drive pain. Increased understanding of such mechanisms may offer translational opportunities for the discovery of novel therapeutic strategies for neuropathic pain.

Conflict of interest statement

The authors have no conflicts of interest to declare.

Acknowledgements

The authors thank Professor Howard Fields for helpful discussions. This work was funded by R01 NS066958 (F.P.).

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Keywords:

Ongoing pain; Nerve injury; NPY; TRPV1; Tactile allodynia; Thermal hyperalgesia

© 2011 Lippincott Williams & Wilkins, Inc.