Dorsal root ganglion stimulation of injured sensory neurons in rats rapidly eliminates their spontaneous activity and relieves spontaneous pain

Introduction

Neuropathic pain often presents with hypersensitivity distal to a nerve injury, including allodynia (pain from stimuli that are below the normal threshold for producing pain) or hyperalgesia (greater pain than expected from normally noxious stimuli). Although understanding the pathogenic roles of axotomized sensory neurons versus intact units is a fundamental issue in explaining neuropathic pain, their comparative contribution is still unresolved [23,52,59,72]. On the one hand, a shift in the functional phenotype of intact units may result in hyperalgesia [3,16]. Alternatively, ongoing spontaneous activity (SA) originating in axotomized units may be critical in sensitizing dorsal horn (DH) pain networks, resulting in normal afferent input from intact units causing pain [50]. To address the relative contribution of injured and uninjured afferents in the pain associated with nerve injury, investigators have used the spinal nerve ligation (SNL) model, in which the 5^th lumbar (L5) nerve is ligated and transected. In contrast to other widely used models of traumatic nerve injury, the SNL model largely segregates injured neurons to the L5 DRG and root, and their uninjured neighbors to the L4 DRG which gives rise to axons that travel in the same nerves (e.g. the sciatic nerve) as those arising from the injured L5. Using this model, some studies have suggested that intact L4 neurons contribute to the observed pain behavior due to neuroinflammation triggered by degenerating L5 axons in fascicles shared by the L4 distal axonal processes [16,66,67]. Alternatively, other reports point to the injured L5 DRG neurons as the main source of neuropathic pain [22,34,54,73]. Although these axotomized neurons lack receptive fields, it has been proposed that injury induced changes in the expression of peptides such as colony-stimulating factor 1 (CSF1) in the injured neurons mediates changes in the DH responsible for the pain induced by normal afferent neuronal traffic in the L4 pathway [16,27,50,73]. These peptides are presumably released in association with SA in the injured neurons.

Dorsal root ganglion field stimulation (GFS) is a recently established clinical approach for controlling chronic neuropathic and possibly non-neuropathic pain [1,14,15,18,31,33,40,58] that has the desirable features of avoiding risks of addiction and overdose that accompany opioid use. GFS is achieved with electrodes placed adjacent to the DRG in the intervertebral foramen, and produces analgesia without anesthesia. In a preclinical rat model, we have shown that GFS relieves neuropathic pain [74] while segmentally interrupting action potential (AP) traffic going through the T-junction of sensory neurons in the stimulated DRG, with nearly complete blockade of the C-type population but minimal effects on Aβ units [11]. Since the onset of GFS action is within seconds [11], this tool enables selectively testing L4 vs. L5 contributions to spontaneous pain using conditioned place preference (CPP) paradigm, and allows examination of underlying electrophysiological roles of sensory neuron SA and DH excitability.

Materials and methods

Animals

Male Sprague Dawley rats weighing 200–250g obtained from the Taconic Farms Biosciences (Rensselaer, NY) were maintained and used according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin (animal protocol AUA0454). Animals were housed in a pathogen-free facility, 2 animals per ventilated cage, in a room maintained at 25 ± 1 °C at 35 to 45% humidity, with a 12/12-hour day/night cycle. Animals had free access to food and water, and bedding was aspen wood chips. At the termination of the study, euthanasia was performed by decapitation during deep isoflurane anesthesia.

Neuropathic pain model

Spinal nerve ligation (SNL) was performed as described in our previous report [75]. During isoflurane anesthesia, the L6 transverse process was removed to expose the L5 spinal nerve, which was ligated and transected, followed by wound closure. Sham SNL control rats had anesthesia and skin incision alone. During surgery, rectal temperature of rats was maintained at 37°C with a thermostatically controlled heating pad. Animals were given a prophylactic dose of enrofloxan (25mg/ml, 0.1ml) after surgery and for the following three days to prevent infection. These animals were used 12–22 days after the surgery for behavioral testing and electrophysiological recording.

GFS electrode implantation

Bipolar GFS electrodes were prepared from 2 platinum-iridium wires (0.010 inch and 0.005 inch), such that two conducting surfaces 1mm apart beside the DRG, as described in our previous report [51], where their implantation is also described. Briefly, during isoflurane anesthesia, a dorsal paramedian incision was made to expose the external aspect of the intervertebral foramen at the required level for GFS, which included L4 or L5. This requires removal of the small, overhanging accessory process at the L4 level. A blunt probe with 0.4-mm diameter was inserted into the intervertebral foramen dorsolateral to the DRG, to create a passage into which the electrode was inserted in juxtaposition to the DRG at that level. A stainless-steel wire was used to affix the electrode to a screw inserted into the transverse process caudal to the foramen. The leads, which were contained in flexible plastic tubing for protection from excess flexion, were tunneled to the head, where the connection hub was secured to the skull with screws and dental cement.

GFS

Animals received GFS while awake and freely moving. In the clinic setting, the intensity commonly used is that which can produce paresthesias [36,39,40,61], indicating a current level that activates low-threshold mechanoreceptors without producing motor activity. Therefore, in these studies on rats, we used current at 80% of the motor threshold, which was determined as the current at which any further increase resulted in perceptible hind limb movement for GFS during stimulation at 2Hz with a pulse width of 200μs with a passive recovery phase, and a frequency of 20Hz, which is a frequency used in previous animal studies and comparable to clinical settings used to obtain optimal GFS analgesia [18,48,51,56,74]. A stimulator and an isolator (Master-9 and Iso-Flex, A.M.P.I., Jerusalem, Israel) were used for this purpose. In awake animals, initiation of this current level fails to produce an alarm response, suggesting that it is subthreshold for pain or arousal, although this may be a level that nonetheless produces paresthesias. We have previously found that stimulation at this intensity level does not produce aversion when measured by conditioned place preference testing [74], indicating that it is not an intensity that produces discomfort.

Behavioral tests

Sensory testing of the plantar skin included eliciting reflexive behaviors evoked by punctate mechanical stimulation at threshold intensity (von Frey test) and at noxious intensity (pin test), by dynamic non-noxious mechanical stimulation (brush test), and by cold stimulation (acetone test). Sensitivity to heat was not tested as we have previously observed no consistent changes after SNL [29]. The presence of spontaneous pain was determined using conditioned place preference (CPP). The experimenters performing these tests were blinded to the animal’s treatment.

von Frey test

The von Frey test was performed using calibrated monofilaments (Patterson Medical, Bolingbrook, IL). Beginning with the 2.8-gram filament, the tip of the filament was applied perpendicularly to the glabrous skin on the central portion of the plantar aspect of the hind paw for 1s, with just enough force to bend the fiber. If a paw removal response was observed, then the next weaker filament was applied, and if no response was observed, then the next stiffer fiber was applied, until a reversal in the sequence occurred. After a reversal event, 4 more stimulations were performed following the same pattern. Applications were separated by intervals of at least 10s. The forces of the filaments before and after the reversal, and the 4 filaments applied after the reversal, were used to calculate the withdrawal threshold [12]. Rats not responding to any filament were assigned a score of 25 g. Since the magnitude of mechanical sense is perceived in a logarithmic scale, as described by Weber’s law, von Frey paw withdrawal threshold data were log transformed [49].

In the absence of a hypersensitivity state, animals often default to the 25g score. In the experiments indicated, to have a dynamic range that would allow identification of changes in baseline sensitivity to mechanical stimulation, we standardized the ends of the von Frey fibers to a smaller contact area by attaching tungsten tips of 100 μm diameter with squared ends to avoid points [29,57].

Noxious punctate mechanical stimulation (pin test)

Noxious punctate mechanical stimulation was performed using the point of a 22-gauge spinal anesthesia needle that was applied to the central portion of the hind paw with enough force to indent the skin but not puncture it. This was repeated for 5 times, with an inter-stimulus interval of at least 10s. This stimulation protocol was repeated after 1min, for a total of 10 stimuli. Each application evoked one of two types of behavior. One type of behavior, typically observed in uninjured rats, consists of a very brief (<1s) withdrawal, with immediate return of the foot to the cage floor. The second type of behavior, that we term a hyperalgesic response, consists of a complex event with sustained elevation of at least 1s, variably combined with grooming that included licking and chewing of the paw, and with shaking of the limb [29]. We have shown that this hyperalgesic behavior is specifically associated with place avoidance [68], indicating that it represents an aversive experience. Hyperalgesia was quantified by tabulating the number of hyperalgesic responses as a percentage of the 10 stimuli delivered.

Cold stimulation (acetone test)

The acetone test was assessed using application of acetone, which was expelled through tubing to form a convex meniscus on the end of the tubing, which was touched to the lateral plantar skin without contact of the tubing with the skin [13]. The response was scored as positive if the paw was removed, and 3 repetitions were spaced at least 1min apart.

Dynamic mechanical stimulation (brush test)

A camel hair brush (4mm wide) was applied to the lateral plantar skin of the hind paw by light stroking in the direction from heel to toe over the span of 2s [29]. The response was scored as either positive if the paw was removed or none in the absence of movement. The test was applied three times to each paw, separated by intervals of at least 10s.

Conditioned place preference (CPP)

This was performed as described previously [51], using a 3-chamber CPP apparatus (Med associates, Fairfax, VT). On the preconditioning day, animals were allowed to explore both sides of chambers for 15min and the time spent in each side was recorded. Animals showing a preference for one chamber (≥67% of total time) were excluded from further study. On the 4 days immediately following the preconditioning day, place conditioning was conducted using an unbiased design. Specifically, on each day, animals received two 30min sessions separated by 6hr in which either GFS or sham GFS without current, or in other experiments in which either gabapentin (GBP) or saline, were administered. The chamber paired with GFS or GBP was consistent on all 4 days for a given animal but was randomly assigned for different animals. In experiments in which we tested if prior administration of GBP can occlude CPP induced by GFS, GBP was intraperitoneally injected 60min before sessions in which GFS or sham GFS were administered. Acquisition of CPP was tested on the day after the last conditioning session. At these final sessions, after each animal was placed in the central chamber, it was allowed to freely explore the chambers for 15min, and the time spent on each side was recorded. A preference score was calculated as the total time spent in the chamber paired with GFS minus the total time spent in the other chamber paired with sham GFS. Each rat had only a single CPP test.

Intra-ganglion injection

Saline, lidocaine (Sigma-Aldrich), flagellin (InvivoGen, San Diego, CA), and QX-314 (Sigma-Aldrich) were microinjected into the right L5 DRG using a previously described technique [11,20]. Briefly, the intervertebral foramen was exposed surgically and then minimal foraminotomy was performed to expose the dorsal aspect of the distal pole of the DRG. Injection was performed with a microinjector (Nanoliter 2000, World Precision Instruments, Sarasota, FL, USA). A glass micropipette filled with solution was advanced ~100μm into the ganglion, followed by injection of 3μl of solution with drugs, a volume that has been shown to fully fill the rat L4 and L5 DRG [20]. Injection was performed over a 5-min period and removal of the pipette was delayed for an additional 5min to minimize the extrusion of the injectate. Overlying muscle and skin were closed. Rats fully recovered from anesthetics in 30 minutes after the surgery and examination of evoked behaviors were initiated 45min after the injection.

Immunohistochemistry

Immunohistochemical procedures were based on our previous study with minor modifications [51]. Rats were deeply anesthetized with 3–4% isoflurane and perfused through the aorta with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS), pH 7.4. After perfusion, DRGs were removed and fixed in the same fixative overnight at 4°C. The DRGs were then cryoprotected in increasing concentrations of sucrose (10, 20, and 30%) in 0.1 M phosphate-buffered saline (PBS) at 4°C, frozen on dry ice, and stored at −80°C until use. DRG sections were cut at 10 μm thickness with a cryostat and mounted on glass slides. After rinsing three times in PBS, those slices were blocked for 1 h at room temperature with blocking solution (1% bovine serum albumin, 5% normal goat serum, and 1% Triton X-100 in 0.1 M PBS, pH 7.4). Sections were then incubated with 1:500 mouse anti-activating transcription factor 3 (ATF-3) antibody, and 1:500 rabbit anti-β3-tubulin antibody at 4°C for 24h. After rinsing three times, 5 min each, in PBS, sections were incubated in the secondary antibodies: 1:800 goat anti-mouse IgG (Alexa Fluor® 594), 1:800 goat anti-rabbit IgG (Alexa Fluor® 488) for 1 h at room temperature. All antibodies were purchased from Cell Signaling Technology, Danvers, MA. Negative control sections were processed with a nonimmune serum in place of the primary antibodies. Sections were analyzed by using a fluorescence microscope (BZ-X800, Keyence Corporation of USA, Itasca, IL). The experimenters performing slides scanning and analyzing were blinded to the animal’s treatment.

Protocol design

In SNL experiments, implantation of GFS electrodes and SNL surgery were performed after baseline (day 0) sensory testing. Fourteen days thereafter, the effect of a 30min GFS administration on sensory behavior was determined. Specifically, first baseline values were measured for the ensemble of evoked pain behavior tests (von Frey, brush, cold, pin test), which were performed over a time span of 5min. Then, after a 15min rest interval, GFS was administered for 30min. Evoked behavior testing was performed again during stimulation at 15min and 30min after the initiation of GFS, and again 15min and 30min after ending GFS stimulation.

CPP testing was performed on days 17 through 25 after SNL surgery (at least 1d after pain behavior testing during electrical or sham stimulation). This consisted of one preconditioning test day, 4 days conditioning (starting 2d after the preconditioning test), and 1 test day. Animals without nerve injury (sham SNL) but with GFS, and animals with nerve injury but without GFS (sham GFS), served as controls.

In vivo electrophysiological recording

Electrophysiological signals were collected with an Axon Axoclamp 900A microelectrode amplifier (Molecular Devices, San Jose, CA), filtered at 1 kHz, and sampled at 10 kHz using a digitizer (DigiData 1440A, Molecular Devices). Individual action potentials were isolated and amplified using either a window discriminator (Clampfit, Molecular Devices) or by template matching using Spike2 (Cambridge Electronic Design Limited, Cambridge, UK).

Animal preparation:

Anesthesia was induced with 2% isoflurane, after which rats were given urethane (100mg/kg, subcutaneous injection), followed 30 min later by progressively reducing isoflurane to 0.2% such that the rats remained immobile but metabolically and hemodynamically stable, confirmed by arterial blood analysis with a blood gas analysis (ABL800FLEX, Radiometer, Copenhagen, Denmark), and continuous intra-arterial blood pressure monitoring via a carotid artery cannula. A heating pad was used to maintain body temperature at 37 °C.

For sciatic nerve stimulation, the nerve was exposed at the midthigh and was wrapped and insulated from surrounding tissues with a nerve cuff electrode (World Precision Instruments, Sarasota, FL). The wound was closed after the implantation. For GFS during electrophysiology recordings, the electrode was implanted beside the DRG as described above and was secured by sutures to nearby tissues. Motor threshold for GFS was determined as described above.

Teased dorsal root single unit recording:

The spinal cord was exposed by a laminectomy at the level of vertebrae T13 to L3 and covered with warm mineral oil (36°C), and bone wax was used to stop bleeding from the bone. The dura was removed, and rats were mounted on a spinal frame and the vertebral column was stabilized by vertebral clamps applied to the spinal process rostral/caudal to the exposure. The loosened skin from the midline incision was sutured to a metal rectangle ring (3 × 4 cm) to form a basin that was filled with warm mineral oil. A fine glass probe with a rounded tip was used to gently release the L4 or L5 dorsal root from connective tissues, after which they were transected at the point where they divide into rootlets. The dorsal root was then placed on a glass platform, upon which the distal portion of the dorsal root was repeatedly teased to create fine neuronal bundles. These were placed on a platinum/iridium recording electrode for recording single-unit activity. A reference electrode was attached to the adjacent muscle tissue. Signals were collected with an Axoclamp 900 A microelectrode amplifier (Molecular Devices, San Jose, CA) and the headstage (HS-9A-x0.1U with feedback resistance of 100MΩ) was served as a preamplifier with gain setting of 500 or higher, filtered at 1 kHz, and sampled at 10 kHz using a digitizer (DigiData 1440 A, Molecular Devices). Action potentials were isolated by setting the threshold above background noise, and individual units were identified by template matching using Spike2 (Cambridge Electronic Design Limited, Cambridge, UK) or pClamp 11 (Molecular Devices). Initially, SA was sought during a 3min observation period, and recorded for 3–4 min if present. To identify unit types, responses evoked by electrical stimulation of either the DRG or sciatic nerve every 5s were used to calculate conduction velocity (CV) by dividing the distance between stimulation and recording sites by the response latency in the recording [11]. We used the following CV standards to distinguish different fiber types for recordings from the dorsal root with DRG stimulation, units with CV>6m/s are Aβ, 1<CV<6m/s for Aδ, CV<1m/s for C-type [11], which are similar to the definitions used by others [19].

The unit’s receptor field (RF) was identified by low intensity mechanical stimulation of the glabrous plantar skin of the hind paw with a small glass probe (with 1 mm round tip), and a von Frey monofilament (29g). Only units with a cutaneous rather than deep RFs were used for the study, which was confirmed by the induction of APs when the skin in the RF was gently pinched with a forceps. This allows comparison with the pain behaviors we evoked by plantar stimulation, and damage of these cutaneous units have been linked with development of neuropathic pain [5,30]. Muscle spindles or joint receptor units that responded to slow extension/flexion of the ankle or knee joint were also excluded.

To characterize response properties, the mechanical threshold was examined with graded von Frey monofilaments. The mechanical threshold was defined as the minimal von Frey hair force that evoked firing.

DH neuron single unit recording:

A laminectomy was performed from the T13 to the L3 vertebrae to expose the mid-lumbar spinal cord, and a stabilizing stereotaxic clamp was applied to the spinal process rostral to the exposure. The dura was opened, and the cord was covered in warm mineral oil. A single-barreled glass micropipette filled with solution containing 1 M NaCl (with resistance of 15–20 MΩ) was advanced into the spinal cord at the L4 to L5 level using a microdrive (David Kopf Instruments, Tujunga, CA) at 2μm per step. Wide dynamic range neurons (WDR) of lamina IV to VI at depths of 400–700 μm from the cord surface were targeted, as those neurons encode the discrimination of noxious from non-noxious stimuli and represent an important component in central sensitization in pain [17,26]. Data were obtained using electronic systems comparable to that described above for dorsal root single unit recording. At 50 μm intervals of electrode advancement, the ipsilateral paw was mechanically stimulated to identify units with RFs in this area. Specifically, sequential mechanical stimuli were applied to the ipsilateral hindpaw using a standard set of graded von Frey fibers applied for 1 s to determine the threshold (i.e. weakest force capable of producing recorded activity) for response to punctate mechanical stimulation, then stroking with a brush to evoke dynamic mechanical stimulation, then application of von Frey fibers (8, 29, 40g) for 10 s each to determine firing rates (averaged over 10s) at these specified stimulation intensities, then application of a 29 g modified von Frey fiber with a tungsten tip for 10 s to determine firing rate. Inclusion criteria for the WDR type were response to innocuous (brush, von Frey filament) and noxious (modified von Frey filament with tungsten tip) stimulation in a graded manner. After the completion of baseline measurement (SA, evoked response, mechanical threshold etc.), GFS (20Hz, 0.2ms duration, 80% of MT) was applied for 180s. Responses were then determined in response to the same activity as baseline measurement was recorded after GFS.

Statistical analysis

Significance testing was performed with Prism 8 (GraphPad Software, La Jolla, CA), unless otherwise specified. To compare pain behavior during and after treatments (GFS or sham GFS) to baseline (i.e. the value prior to treatment), responses to pin, brush and cold were evaluated nonparametrically using three-way repeated measures ANOVA with post hoc Dunn’s test, whereas responses to von Frey across time were evaluated using a parametric repeated measures ANOVA with post hoc comparisons using Dunnett’s test. In order to additionally compare effects of treatment (GFS vs. Sham GFS) and anatomic level of treatment (L4 vs. L5), two approaches were used. Frist, a parametric three-way ANOVA with repeated measures was performed in order to identify main effects of Level of electrode insertion (L4 vs. L5), GFS activation (Sham vs. Active), and Time for all of the behavioral tests, including Pin, Brush, and Cold, since there is no true nonparametric version of a multi-way ANOVA with repeated measures. For this reason, we additionally compared differences between treatments using a second approach by calculating the area under the curve (AUC) for behavior values normalized to the baseline just before the initiation of GFS, which were then compared using a two-way ANOVA (main effects for anatomic level and for active vs. sham GFS), with post hoc comparison using Bonferroni test. A similar approach was used for analysis of pain behavior changes in response to local anesthetic injection, except that there was only one between factor (the injected agent), so a two-way ANOVA with repeated measures was used initially, while the AUC data were analyzed by one-way ANOVA. CPP scores were analyzed with paired t-tests, and two-way ANOVA with post hoc Bonferroni test was used for comparison between treatments. The incidence of SA in dorsal root and dorsal horn was grouped per animal and analyzed parametrically (two-way ANOVA). P<0.05 was considered significant. Data are reported as mean ± SEM. For all experiments, detailed statistical data are presented in Supplementary Table.

Results

GFS activates sensory neurons only at the level of stimulator placement

Using dorsal root teased fiber electrophysiological recording, we first confirmed that GFS selectively affects only the DRG at the level where it is inserted, while having no effects on neighboring DRGs. Specifically, while recording single units with receptive fields in the plantar skin from bundles teased from L5 dorsal roots, stimulation via a GFS electrode placed adjacent to the L4 DRG consistently failed to produce AP trains, and vice versa when recording from the L4 dorsal root during L5 DRG stimulation (33 units from 4 naïve rats), whereas recording from the L5 DRG during stimulation by a GFS electrode placed at the same level always produces APs [11]. Furthermore, L4 DRG stimulation (20Hz, 80%MT) had no effect on propagation of AP trains induced by sciatic nerve stimulation (2Hz, 10s) during recordings from the L5 dorsal root of 7 C-type units from 2 naïve rats. This selectivity therefore allows us to use single level GFS to distinguish the analgesic contribution of treating different DRG levels selectively, in order to infer the relative contributions of afferent activity from each of these two pathways to the pain caused by L5 SNL.

SNL produces minimal axotomy in the L4 population

Depending on the skill of the surgeon [16], the SNL procedure may cause unintended injury to the neurons of L4, resulting in a small population (typically from 2–7%) of activating transcription factor-3 (ATF3) positive neurons in the L4 DRG, similar to the effect of sham SNL surgery [22,38,46]. Since others have reported higher rates [16], we evaluated this factor. As ATF-3 is also expressed in neurons and non-neuronal cells like oligodendrocyte in central nervous system [24], neuronal marker β3-tubulin was used to detect expression of ATF-3 in neurons after injury. ATF3 in 3% of neuronal somata was found in one rat and no ATF-positive neurons were found in two other rats at the L4 level after L5 SNL, indicating a low level of injury to L4 neurons in our hands. In contrast, ATF3 was detected in 92–96% of L5 neurons from the same animals (n=3 rats; Supple. Fig. 1).

GFS at L4 and L5 relieve evoked pain-like behaviors in SNL rats

After SNL, rats developed hyperalgesic responses to noxious mechanical stimulation (Pin, Fig. 1A, B) and showed mechanical and thermal allodynia (Fig. 1C–H). GFS for 30 min (20Hz, 80% motor threshold, 0.2ms in width) at either L4 or L5 increased mechanical thresholds and lowered response rates to tactile and noxious mechanical stimuli, but not to cold stimuli (Fig. 1A, C, E, and G; Supple. Table. 1A). AUC confirmed these differences between groups of the effects of GFS (Fig. 1B, D, F, and H; Supple. Table. 1B). Of particular importance, there was a significant interaction between level of treatment (L4 or L5) and injury for pin-evoked hyperalgesia, due to the larger antinociceptive effect of L5 GFS than L4 GFS in the presence of injury (Fig. 1E). These findings suggest that GFS at L4 reduces but does not completely eliminate noxious afferent traffic in these anatomically intact units, as is expected from our prior findings [11] that showed complete conduction block of C-type units but only partial block of myelinated units. Additionally, it appears that ongoing GFS-sensitive activity conveyed by the axotomized L5 units contributes to pain behavior evoked by activation of intact L4 sensory pathways. All rats developed hyperalgesia and allodynia following SNL surgery.

Figure 1.

Effects of GFS on evoked pain behaviors of rats with SNL. A panel of sensory tests was performed immediately before surgery for electrode implantation and performance of Sham or actual SNL, and again 14 days later to determine effects of GFS. At that time, values were determined immediately before GFS, during GFS at times 15 and 30 minutes after starting, and again 15min and 30min after GFS was terminated. Time course for effects on the testing day are shown in the left panels, and AUC analysis in the right panels, for sensitivity to noxious mechanical stimuli (pin, A, B), threshold mechanical stimuli (von Frey, C, D), cold (E, F), and brush (G, H). The Sham treatment group consisted of animals with GFS electrodes that were inserted but not activated. Results are means ± SEM. The variance at the baseline timepoint is small such that the SEM is smaller than the radius of the data symbol. “1” P<0.05, “2” P<0.01, “3” P<0.001 compared to data immediately before GFS by the Dunnett’s test following three-way repeated-measures ANOVA. * P<0.05, *** P<0.001 by the Bonferroni test following two-way ANOVA. n represents animal numbers.

GFS at L5, but not L4, relieves spontaneous pain after SNL

To determine if GFS analgesia is effective in providing analgesia against ongoing spontaneous pain, we measured the ability of GFS to drive a conditioned place preference. We first tested our CPP design with a positive control using GBP, which at the dose of 100mg/kg i.p. has been shown to be analgesic in our prior study [74]. Initial examination of analgesia onset (Fig. 2A, B, C) showed that this dose of GBP had an onset of reduced pin hyperalgesia and von Frey allodynia at 30 min after administration. Using this timepoint for conditioning, CPP testing showed that rats developed a preference for the chamber associated with the onset of GBP analgesia (Fig. 2D), supporting the existence of spontaneous pain after SNL and our ability to identify its relief using the CPP design. We additionally have previously confirmed that GBP in the absence of a pain condition does not itself produced CPP [11]. For testing GFS, 4 sequential days of conditioning after a 1-day preconditioning phase showed that rats with sham surgery didn’t develop preference for the chamber paired with L5 GFS (Fig. 2E) and SNL rats developed a strong preference for the chamber paired with L5 GFS, but L4 GFS lacked this effect (Fig. 2F; Supple. Table. 1C). Furthermore, removal of the SNL-induced ongoing pain by administration of GBP prior to conditioning with GFS preempted the effectiveness of L5 GFS (Fig. 2G), indicating that pain relief from L5 GFS was the active element producing conditioning in our CPP test. We have also previously shown that GFS in the absence of a painful state does not produce a conditioned place preference [74]. These observations provide evidence of a specific contribution from injured L5 neurons in generating spontaneous pain after SNL, in contrast to evoked pain behaviors that show contributions from both L4 and L5 levels (Fig. 1).

Figure 2.

Effects of GFS on Conditioned Place Preference test in rats. (A) Time course of the test design. The time course for onset of analgesia by gabapentin (GBP, 100 mg/kg intraperitoneally) in SNL animals is shown for Pin (B) and von Frey test (C, n=8). (D) Effects of GBP on preference at baseline (Pre-test) and after treatment. (E) Effects of L5 GFS on preference at baseline (Pre-test) and after treatment in rats with sham surgery. (F) Effects of L4 or L5 GFS on preference at baseline (Pre-test) and after treatment in rats with SNL. (G) Effects of pretreatment of GBP on L5 GFS induced preference in CPP test. Results are means ± SEM. * P<0.05, ** P<0.01, *** P<0.001 by the Dunn’s test following one-way repeated-measures ANOVA (B, C), the paired t-test (D, E, G), and two-way repeated-measures ANOVA (F). n represents animal numbers.

Plantar receptive fields have unchanged mechanical thresholds in L4 units and are absent in L5 units

To examine activity of sensory neurons, we chose to record single units from the dorsal root as this allowed us to identify the ganglion level but without traumatizing the DRG by exposure or manipulation as is necessary for direct recordings from sensory neuron somata. Although it has been reported that regeneration of the transected L5 spinal nerve can result in establishing functional reconnection with its distal RFs [69], mechanical stimulation of the ipsilateral plantar skin failed to evoke any AP firing during our recordings from single units in the L5 dorsal root identified by activation with pulses from the GFS stimulator (65 recordings from 8 rats with SNL, 12–14 days following transection). In contrast, we readily identified RFs for single units recorded from the L4 dorsal root (Fig. 3A). Specifically, of 86 units identified by GFS stimulation, 80 (93%) were found to have RFs in the distal hindlimb. Consistent with our prior findings that animals withdraw from punctate tactile stimulation throughout the plantar surface even after L5 SNL [29], RFs were identified for L4 units throughout the plantar surface (Fig. 3B). The mechanical force threshold for initiating AP firing of these units showed no change after SNL compared to the Sham SNL group (Fig. 3A–D), and mechanical forces above threshold evoked AP firing rates in the SNL group comparable to those in the Sham SNL group (Fig. 3E). These observations suggest that altered transduction properties of the sensory neurons are not the likely cause for our observed reduction in the force threshold for inducing withdrawal during von Frey behavioral testing (Fig. 1C, D).

Figure 3.

Effects of L5 injury in SNL on the mechanical threshold to initiate AP firing in L4 dorsal root. (A) L4 dorsal root is teased and recorded after L5 SNL (or Sham) during plantar mechanical stimulation using von Frey fibers to initiate AP firing by touching the receptive field. (B) Points represent the center of the receptive fields of recordings in the hind paw plantar glabrous skin. (C) Comparison of von Frey threshold before and after GFS in rats with sham surgery and rats with SNL. (D) Conduction velocities of those recordings. (E) von Frey force/response curve in rats with sham surgery and rats with SNL (Sham: Aδ n=5 units, C-type n=6 units; SNL: Aβ n=2 units, Aδ n=8 units, C-type n=12 units). n= 5 rats with sham surgery and n = 5 rats with SNL surgery.

GFS blocks spontaneous activity (SA) in sensory neurons after injury

Since GFS at the level of L5, but not L4, alleviates spontaneous pain after SNL (Fig. 2), it is possible that the injured L5 spinal nerve and DRG may be sources of sensitizing afferent activity. It is known that SA in injured nerves, especially by C-type units, contributes to spontaneous pain and hypersensitivity in both preclinical models and patients [35,50,55]. We therefore hypothesized that SA from the axotomized L5 neurons may be a factor contributing to pain behaviors. In recordings from L4 and L5 dorsal roots of naïve rats and SNL rats (Fig. 4A–D), no difference was observed in the prevalence of SA in bundles teased from the L4 dorsal root in SNL rats vs. naïve rats (Fig. 4E). In contrast, bundles from the L5 dorsal root showed an incidence of SA in SNL animals that was markedly increased in comparison to naïve animals and animals with sham surgery (Fig. 4E; Supple. Table. 1D), which is consistent with previous reports [28,43]. This difference was found to be attributable to an increase in the incidence of spontaneously active Aβ, Aδ and C-type units (Fig. 4F; Supple. Table. 1D), for which the conduction velocity was determined by test pulses from DRG stimulation. Firing rates of spontaneously active L5 Aδ units were also higher after SNL compared to those from sham surgery rats (Fig. 4G; Supple. Table. 1D). The temporal pattern of AP discharge may in part regulate resulting evoked sensations [65], so we categorized the SA to types based on inter-spike interval (Supple. Fig.2). This showed that most of the SA was of the irregular type at L5 level (Fig. 4H).

Figure 4.

Effects of GFS on spontaneous firing from L4 and L5 DRGs. (A) L4 or L5 dorsal roots are teased and recorded following L5 SNL (or Sham or naïve). Sample traces of different patterns of SA, regular (B), irregular (C), and burst (D). (E) Incidence of SA in L4 and L5 dorsal root bundles teased from naïve (10 bundles with SA out of 57 bundles at L4; 11 with SA of 58 at L5), sham (13 with SA of 69 at L4; 14 with SA of 64 at L5) and SNL rats (31 with SA of 133 at L4; 36 with SA of 65 at L5) is shown as the average for each animal. (F) Incidence of SA in units of different CV categories in bundles teased from L5 dorsal root in sham and naïve rats together (12 of 58 Aβ units, 11 of 31 Aδ units, and 12 of 33 C-type units) and from L5 dorsal root from SNL rats (24 of 38 Aβ units, 11 of 14 Aδ units, and 10 of 13 C-type units), shown as average for each animal. (G) Comparison of firing rates of individual units with SA. Data in E, F, and G were analyzed by two-way ANOVA with Bonferroni post hoc paired comparisons. (H) Percentage of spontaneous firing with different patterns, overall Chi-square P< 0.0001; paired Chi-square comparisons shown are Bonferroni corrected. For all panels, red bars represent mean, ** P<0.01, *** P<0.001.

Since SA was more common in dorsal root units at the level of L5 than L4 after SNL, we chose the L5 dorsal root level to test how GFS (90s, at 20 Hz, 80% motor threshold, 0.2ms in width) affects SA (Fig. 5A, B). In Aβ units, the firing rate of SA was on average reduced by 73.5±10.3% during GFS and SA was eliminated during GFS in 3 of 11 units (27.3%, Fig. 5C). In Aδ units, SA was also eliminated by GFS in 2 of 3 (Fig. 5D), while SA in C-type units was eliminated in all of 5 units (100%) during GFS (Fig. 5E), with a significant difference between groups for complete blockade rate (Chi Square P=0.023). In recordings without GFS, SA firing rates were stable across the same time span (Fig. 5F).

Figure 5.

Effects of GFS on spontaneous firing from L5 dorsal root recording. (A) In SNL animals, the GFS lead was implanted besides the L5 DRG, and the L5 dorsal root was teased and recorded. (B) Representative traces from a recorded C-type unit showing SA before and after GFS, but none during GFS. Solid black arrow points to the stimulation artifact from 20Hz GFS. Effects of GFS on SA in Aβ units (C), C-type units (D), and Aδ units (E). (F) SA without GFS. Gray circles and lines represent individual recordings, and solid circles and line represent means ± SEM (C, D, E, F). * P<0.05, ** P<0.01, *** P<0.001 compared to baseline by the Dunnett’s test following one-way repeated-measures ANOVA.

SA from both Aβ and C-type units contributes to pain-like behavior after injury

To further test the hypothesis that SA from injured L5 sensory neurons contributes to mechanical hypersensitivity of uninjured L4 neurons, local blockade of SA from L5 was achieved by intra-DRG injection of lidocaine (3μl, 173mM), which reversibly blocked plantar hypersensitivity to mechanical and cold stimulation (Fig. 6A–H). Since GFS blocks SA from both Aβ and C-type units in L5 (Fig. 5), flagellin coinjected with QX-314 was used to selectively block activity conveyed by Aβ fibers [70]. L5 DRG injection of 3μl solution containing flagellin (0.6μg) + QX-314 (60mM) blocked evoked behaviors but with less effectiveness than lidocaine (Fig. 6A–H; Supple. Table. 1E, F), which indicates that SA from both Aβ and C-type units at L5 may contribute to the hypersensitivity.

Figure 6.

Effects of blockade of SA from L5 by intra-ganglion injection of lidocaine, and flagelllin+QX-314 on evoked pain behaviors of rats with SNL, which was performed with electrode implantation 14d before the sensory testing. Time course for effects on the testing day are shown in the left panels, and AUC analysis in the right panels, for sensitivity to noxious mechanical stimuli (pin, A, B), threshold mechanical stimuli (von Frey, C, D), cold (E, F), and brush (G, H). Results are means ± SEM. The variance at the baseline timepoint is such that the SEM is smaller than the radius of the data symbol. “1” P<0.05, “2” P<0.01, “3” P<0.001 compared to data immediately before GFS; “a” P<0.05, “b” P<0.01, “c” P<0.001 compared to saline injection control; “#” P<0.05 compared to lidocaine injection group by the Dunnett’s test following two-way repeated-measures ANOVA. * P<0.05, ** P<0.01, *** P<0.001 by the Tukey test following one-way ANOVA. n represents animal numbers.

Impulses from the L5 level drive SA of DH WDR neurons

Considering that afferent activity from the plantar skin cannot reach the spinal cord through the transected L5 spinal nerve, and that the L4 units are not sensitized (Fig. 3), combined with our observation that GFS blockade of neuronal activity of L5 DRG neurons relieved both spontaneous pain (Fig. 2) and evoked hyperalgesia and allodynia after SNL (Fig. 6), we hypothesized that SA originating in the injured L5 spinal nerve or DRG may drive hyperalgesia and allodynia behaviors by sensitizing signal processing in spinal cord DH. To test this directly, we recorded SA of DH wide dynamic range (WDR) neurons located in laminae IV and V (Fig. 7A), as these neurons are a site of convergent sensory processing that result in encoding intensity of noxious stimulation, and project to the spinothalamic tract [41]. WDR neurons were sought 12–14 days after surgery (SNL n=12 rats, sham SNL n=4), and in naïve rats (n=7). To allow comparison between different groups, each animal was recorded in a stereotyped fashion with the same number of electrode insertions, lengths of the search path, and uniform incremental electrode advancements (50 μm) that are long enough to avoid duplicate recordings from the same neuron. WDR status was identified using graded mechanical stimulation of the RF, with limitation of recordings to units with RFs in the ipsilateral plantar skin. To identify WDR neurons with SA (Fig. 7B), an initial 5 min recording was obtained. We first examined the possible effect of GFS electrode insertion on generation of SA, and learned that GFS electrode insertion alone did not change the incidence (Fig. 7C) or firing rates (Fig. 7D) of SA in rats with or without SNL. In contrast, nerve injury by L5 SNL increased the incidence of recordings with SA (Fig. 7C; Supple. Table. 1G) and their firing rates (Fig. 7D; Supple. Table. 1G). Next, we examined the effect of GFS (20Hz, 90s) on SA of DH WDR neurons. Baseline SA firing rates were similar for rats with GFS stimulators inserted at L4 (Fig. 7E) and L5 (Fig. 7F). Although SA was unaffected by GFS at the L4 level (Fig. 7E), GFS at the L5 level eliminated SA in 19 of 24 neurons (79%) and decreased SA in the remaining 5 (21%) (Fig. 7F). The effect of GFS upon SA was consistently observed within a few seconds (Fig. 7B).

Figure 7.

Effects of GFS on spontaneous firing of WDR neurons in spinal cord DH. (A) In SNL or Sham animals, the GFS electrode was implanted besides either the L4 or L5 DRG, and activity of DH WDR neurons was recorded. (B) Representative trace showing spontaneous firing of WDR neurons and the immediate blocking effect of L5 GFS. Red arrows represent first 5 of the stimulating artifacts from GFS. (C) Incidence of SA in recorded WDR neurons categorized by injury (SNL vs. without injury, which includes n=7 naive and n=4 sham surgery) and electrode insertion (either L4 or L5), averaged for each animal. The total number of neurons in each group are n=33 for no injury/no electrode, n=27 for no injury/with electrode, n=34 for SNL/no electrode, and n=125 for SNL/with electrode. (D) Firing rates of SA recorded in different injury conditions with and without electrode insertion, shown for individual neurons. Effects of L4 GFS (E) and L5 GFS (F) on firing rates of SA of WDR neurons. For C and D, analysis was by two-way ANOVA with Tukey test for post hoc comparisons. For E and F, analysis was by one-way repeated measures ANOVA with Tukey test for post hoc comparisons * P<0.05, ** P<0.01, *** P<0.001.

Impulses from the L5 level cause hypersensitivity in the L4 cutaneous sensory pathway

In order to identify whether SA input from injured L5 neurons also drives plantar hypersensitivity that follows SNL (Fig. 1), we examined the effects of L5 GFS (20Hz, 90s) on threshold of mechanical force necessary to evoke AP firing and the firing rates during suprathreshold mechanical and noxious stimulation of DH neuronal receptive fields in the glabrous plantar skin after L5 SNL. During recordings from DH neurons (Fig. 8A, B), we first compared baseline firing threshold in Sham and SNL animals to application of graded von Frey fibers, 12–14 days after SNL surgery. This showed lower forces necessary to evoke firing after SNL (Fig. 8C). Immediately following GFS applied for 90sec to the intact, conducting neurons of the L4 DRG, we observed that the threshold for AP firing was unaffected, whereas GFS applied to the axotomized units of the L5 DRG increased firing thresholds for the L4 pathway (Fig. 8D, E; Supple. Table. 1H). To further characterize the effect of GFS on sensory signaling, we evaluated how GFS affects DH neuron firing rates in response to standardized cutaneous force applied using 8g, 29g, 40g standard von Frey fibers and 29g modified von Frey fiber (100μm tungsten tip; n=10 rats, Fig. 9A, B). L5 GFS, but not L4 GFS, reduced the rate of AP firing evoked by mechanical force application with low intensity von Frey fibers of 8g (Fig. 9C; Supple. Table. 1I) and 29g (Fig. 9D; Supple. Table. 1I). During noxious mechanical force application, L4 GFS reduced the rate of WDR neuron firing (Fig. 9E, F; Supple. Table. 1I), consistent with the partial blockade by GFS of afferent traffic [11]. In comparison, the effect size of L5 GFS on evoked WDR firing rate was greater (Fig. 9G; Supple. Table. 1J), indicating that SA in L5 after injury drives central hypersensitivity of WDR neurons to input generated by mechanical stimulation of uninjured L4 afferent units.

Figure 8.

Effects of GFS on mechanical threshold of WDR neurons using von Frey fibers to initiate AP firing. (A) In SNL animals, the GFS electrode was implanted besides either L4 or L5 DRG, and activity of WDR neurons was evoked using von Frey fiber mechanical stimulation to initiate AP firing. (B) Dots represent the center of the receptive fields of recordings in the hind paw plantar glabrous skin. (C) The force threshold to induce AP firing of individual neuronal units was reduced in rats with SNL. Effects of 20Hz L4 GFS and L5 GFS (D) on the force threshold for von Frey stimulation to initiate AP firing of individual units in rats with SNL, compared by two -way repeated measure ANOVA, showing increased threshold only by GFS at L5. (E) Comparison of effect size of L4 GFS and L5 GFS on threshold of individual units, analyzed by t-test. Data are from n=5 rats with sham surgery and n=6 rats with SNL surgery. *** P<0.001.

Figure 9.

Effects of 20Hz GFS on AP firing by mechanical stimulation. (A) In SNL animals, the GFS electrode was implanted besides either L4 or L5 DRG, and evoked activity of WDR neurons was recorded using von Frey fiber mechanical stimulation to initiate AP firing. (B) Time course of procedure (top) and representative traces showing AP firing before and immediately after GFS in an SNL rat (bottom). Solid arrows represent the start point of the 10 sec mechanical stimulation with von Frey fibers. Effects of 20Hz L4 or L5 GFS on AP firing in response to 8g (C), 29g (D), 40g (E), and sharp (F) punctate stimulation. (G) Summarized effects of L4 and L5 GFS on AP firing of WDR neurons evoked by mechanical stimulation. These recordings were from 5 rats with sham surgery and 5 rats with SNL surgery. For C-F, analysis was by two-way repeated measures ANOVA, and simple two-way ANOVA for G, with post hoc paired comparisons by Bonferroni. Red bars represent Mean. * P<0.05, ** P<0.01, *** P<0.01.

Discussion

Numerous changes underlie the neuronal dysfunction that leads to neuropathic pain after peripheral nerve injury. Here, in order to address the unresolved fundamental questions of the relative contributions from directly injured units vs. those exposed to resulting inflammation [23], and the role of sensory neuron SA [50], we employed GFS as a new tool for rapidly controlling AP propagation in a segmentally selective fashion [51,74]. Our findings in the context of the SNL model support a mechanistic model (Fig. 10) in which SA arising in injured peripheral nociceptive neurons at the L5 level directly generates spontaneous pain, which can be relieved by GFS at that level. Additionally, this SA from injured units also causes the DH to generate an elevated response by putative projection neurons in response to afferent AP trains conveyed by intact neurons at the L4 level. Thus, controlling this SA is a potential means for treating neuropathic pain. In contrast, blocking afferent activity at the L4 level has no effect on spontaneous pain, although blockade by GFS of transmission through the intact afferents at the L4 level, particularly of C-type units [11], can reduce evoked pain.

Figure 10.

L5 spinal nerve transection and ligation (1) results in SA (2) that produces spontaneous pain as well as sensitization of the dorsal horn local network (3). Stimulation of the peripheral tissues (4) produces nociceptive activity in the L4 pathway (5), which results in evoked pain that can be partially blocked by GFS at the L4 level (6), whereas GFS at the L5 level (7), by eliminating SA in nociceptive sensory neurons, can stop spontaneous pain and block dorsal horn sensitization that produces excessive response to activity from the L4 pathway.

Contribution of injured vs. non-injured neurons to pain after injury

Our mechanistic model (Fig. 10) suggests that both L4 and L5 afferents contribute to pain after SNL, but in different ways. Previously, it had been difficult to distinguish the relative contribution of injured vs. uninjured afferents to nerve injury-induced hypersensitivity because of the need for L4 afferents to remain intact to mediate evoked responses to stimuli applied to the glabrous skin of the hindpaw. Thus, this question has been largely addressed with manipulations of the injured L5 neurons despite the fact that it is not possible to perform comparable experiments in the L4 ganglia. That is, investigators have concluded a primary role for L5 neurons in the hypersensitivity if it is reversed with manipulations designed to eliminate afferent activity to the spinal cord by L5 neurons, but results from such experiments have produced contradictory results [23,37,73]. Arguments against a role for L4 afferents in producing hypersensitivity behaviors have been, by necessity, indirect, and have relied on the extremely low level of SA observed in L4 neurons (median 0.02Hz 1d after SNL, 0.04Hz at 7d) [66], as well as evidence that hypersensitivity associated with the model is most fully manifest with irritation to the L4 root [16]. Nevertheless, results presented here suggest that hypersensitivity may evolve even in the absence of overt injury to L4 neurons, as manifested by minimal ATF3 expression.

We found that GFS at the L4 level also reduced behavioral responses to noxious stimulation as well as to innocuous stimulation (Fig. 1), and that L4 GFS reduced WDR neuron firing in response to high intensity mechanical stimulation, although to a lesser extent than GFS at L5 (Fig. 9). Effects of L4 GFS on evoked behaviors after SNL are expected based on GFS blocking normal AP propagation of nociceptive traffic in uninjured fibers before reaching hypersensitized DH, as GFS 20Hz and 80% MT can predictably block AP propagation of C-type units in naïve animals and after nerve injury [11].

SA after injury

Here we observed SA originating predominantly in injured neurons of the L5 DRG (Fig. 4E). Various locations of injured sensory neurons distal to the DRGs may generate SA, including active growth cones of regenerating axons following axotomy [69], neuromas [63], and ectopic expression of transducers that create sensitivity to catecholamine and mechanical stimulation [4,7]. Additionally, SA can originate within the DRG after peripheral nerve injury [62], or from direct damage to the DRG itself [44], although our data show that insertion of the GFS electrode does not contribute to this SA. Such ectopic impulse generation can arise within 24hr after injury [42] and persist for many weeks in various rodent nerve injury models [25,71]. This injury-induced afferent traffic may directly contribute to spontaneous pain behaviors [16], but also can sensitize spinal cord local networks [64], such that normally innocuous stimuli applied to uninjured fibers may produce pain.

SA after nerve injury is prevalent in Aβ and Aδ units, but is also present in C-type units [9,16,25,32], although the type of injury appears to influence the fiber(s) that become spontaneously active. Specifically, in the majority of studies using the SNL model in which this issue has been addressed, SA is restricted to A-type units [6,42,45], although at least one reported SA in C-type units [66]. Our experiments were not designed to determine the incidence of spontaneously active units as a fraction of total neurons at each level, but our findings from L5 dorsal root recordings nonetheless indicate that SAs is found both in directly injured A-type units as well as C-type units (Fig. 4). The particular effectiveness of analgesic GFS in blocking C-type SA (Fig. 5) may indicate a dominant contribution by these fibers to spontaneous pain in this model.

Mechanism of pain facilitation by sensory neuron SA

After nerve injury, we observed an increased incidence of SA in DH WDR neurons as well as higher evoked firing rates, which were controlled by SA from injured L5 DRG (Fig. 7). A central question in the field remains whether SA alone, particularly in low threshold afferents that regularly fire at high frequencies for extended periods of time, is sufficient to induce changes in the spinal cord DH that may result in allodynia or ongoing pain. Alternatively investigators have sought to identify the basis for the apparent phenotypic switch in normally low threshold afferents, such as injury-induced increase in the expression of pro-nociceptive neuropeptides such as SP and CGRP [60], and more recently CSF1, which may drive changes in microglia responsible for the loss of inhibition needed for the manifestation of allodynia [27]. In the case of C-type input, even brief (16 APs) and low frequency (0.5Hz) trains can induce posttranslational modifications such as kinase phosphorylation in superficial and deep laminae [21]. However, here we provide evidence that elimination of SA originating in axotomized neurons reduces firing of DH neurons within seconds (Fig. 7B), reverses DH sensitization within minutes (Fig. 7B), reduces evoked pain behaviors at the earliest time tested (15min, Fig. 1), and produces place conditioning within 30min (Fig. 2). Reversing a shifted sensory neuron phenotype or neuroinflammation is unlikely to occur at this pace, which is likewise too fast for reversal of other pain mechanisms such as novel synaptogenesis [34], postsynaptic changes such as long-term potentiation or elevated intrinsic hyperexcitability of the WDR neurons, [53,64], or excitatory drive from supraspinal sites. Our findings therefore point towards a highly labile mechanism such as underlying convergence of ongoing SA that summates with evoked input from intact fibers. We cannot eliminate a possible contributing factor from GFS-induced LTMR activation [11], since LTMR input may modulate DH neuronal activity and inhibit pain behavior [2,8,47]. However, initiation of APs in L4 LTMRs by GFS had no effect on SA of WDR neurons (Fig. 7E), while lidocaine injection into the L5 DRG did produce analgesia (Fig. 6), so LTMR activation is not likely a substantial underlying analgesic factor.

Translational Relevance

Overall, our findings indicate that SA from injured neurons contributes to ongoing pain that follows peripheral nerve injury. It is encouraging that GFS treatment of the traumatized neurons at the level of their DRG may interrupt this pain process, making it a suitable consideration for clinical treatment of neuropathic pain. Furthermore, as novel therapies are being devised for treatments with highly specific anatomical targeting using surgical, neuromodulation, genetic, and imaging approaches [10], it is increasingly relevant to identify the dominant anatomic pathway that contributes to a patient’s pain. Our findings may aid in distinguishing the locations of pathology that generate different aspects of neuropathic pain, possibly allowing therapies that preferentially target spontaneous ongoing pain versus hypersensitivity to evoked pain.