Patients with neuropathic pain often experience exaggerated pain response and aversive emotion.7 Central sensitization has been linked with the development of neuropathic pain.24,38 Among several cortical regions, the anterior cingulate cortex (ACC) is assumed to contribute to sensory perception and emotional responses.40,58,77,95,96 Compelling experimental and clinical evidence has demonstrated that neurons in the ACC are activated by nerve injury, and inhibiting central plasticity in the ACC produces analgesic and anxiolytic effects.2,4,35,95,96 However, intervention of the central nervous system (CNS) is associated with unwanted central side effects, eg, cognitive impairment and psychiatric disorders.53,78 Thus, for clinical exploitation of analgesic therapeutics, a major challenge is to devise strategies that diminish or abolish adverse effects without sacrificing analgesic effects.
Previous studies report that central sensitization after peripheral neuropathies causes ectopic firing of central neurons, which can autonomously trigger pain hypersensitivity and is independent on ongoing peripheral afferent inputs.33,79 Conversely, emerging preclinical and clinical evidence has accumulated that peripheral nociceptor-driven sensory input to the CNS is required for neuropathic pain.12,14,27,28,50,76,89 Once the afferent input from the affected area is removed, pain hypersensitivity and associated aversive disorders caused by nerve injury are abolished.28,76 This has led to the view that peripheral nociceptors, the first-order neurons in nociceptive pathway, are critical generators for the development of neuropathic pain and possibly associated emotional comorbidity. However, whether central plasticity in the ACC after nerve injury is dependent on peripheral nociceptor activity and which peripheral candidates are involved in this process has remained elusive.
The cyclic nucleotide signaling, such as cyclic guanosine monophosphate (cGMP), is well known to play crucial roles in pain sensitization.46,47,61,62,68 This process involves activation of N-methyl-D-aspartic acid (NMDA) receptors, NO release, and synthesis of cGMP. Among multiple cGMP downstream targets,30,32 cGMP-dependent protein kinase I (PKG-I) is predominantly expressed in nociceptors and shows upregulation upon injury.57,60,66,74 Previous studies have documented the key significance of nociceptor-localized PKG-I in nociceptor hyperexcitability, spinal synaptic plasticity, as well as inflammatory pain.41,44,47,48,65,66,67,93 Recently, a more selective PKG-I inhibitor, N46, has been synthesized and shows a powerful antinociception on inflammatory pain.67 More importantly, our recent study demonstrated that deleting PKG-I in nociceptors is capable of depressing Fos upregulation in several pain-related cortical regions in capsaicin and muscle pain models.22 These data suggest that PKG-I expressed in nociceptors is not only a key determinant of peripheral sensitization but also an important modulator of central sensitization after inflammation. However, whether and how central plasticity in the ACC as well as exaggerated pain and anxiety after peripheral neuropathies is dependent on PKG-I in nociceptors remains elusive.
Here, we demonstrated that activation of PKG-I in nociceptors is associated with the development of synaptic potentiation in the ACC after neuropathy through a presynaptic mechanism involving brain-derived neurotropic factor (BDNF) signaling. Moreover, behavioral surveys demonstrated that PKG-I in nociceptors is sufficient and necessary for exaggerated pain and associated anxiety upon nerve injury. This study presents a strong basis for PKG-I as a novel therapeutic target in the periphery for treating comorbidity of neuropathic pain and anxiety with least side effects.
2. Materials and methods
Homozygous mice carrying the floxed allele of the mouse prkg1 gene, which encodes the cGMP-dependent kinase1 (PKG-Ifl/fl), have been described previously in details.82 PKG-Ifl/fl mice were obtained from Prof. Robert Feil (Universität Tübingen, Tübingen, Germany). PKG-Ifl/fl mice were crossed with sensory neuron specific (SNS)-Cre mice, which express the Cre recombinase under the influence of the mouse Scn10a promoter (encoding Nav1.8)1 to obtain litters consisting of PKG-Ifl/fl, SNS-Cre+ mice (referred to SNS-PKG-I−/− mice) and PKG-Ifl/fl mice (control littermates). Mice of all genotypes were individually backcrossed into the C57BL6 background for more than 8 generations before being crossed with each other. Experiments were conducted in mice (4-10 weeks old) of either sex, and littermates were strictly used to control for genetic effects of the background.
SNS-Cre mice were gifted from Prof. Rohini Kuner at the University of Heidelberg (Germany). C57BL6 mice were purchased from the Experimental Animal Center of Fourth Military Medical University (Xi'an, China). Mice were housed in standard 12-hour cycle lighting and were allowed ad libitum access to food and water before and throughout the experimental protocols. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University. All testing was performed in a double-blinded manner. The experimenter was blind to treatment and/or genotype of mice throughout.
2.2. Spared nerve injury
To model clinical traumatic painful peripheral neuropathy, we performed a spared nerve injury (SNI) model, a well-established model of peripheral nerve injury as described previously.15 In brief, animals were anesthetized using 1% pentobarbitone. Under anesthesia, the left sciatic nerve was isolated under aseptic surgical conditions by blunt dissection of the femoral biceps muscle. The sciatic nerve and its 3 branches were isolated (sural, common peroneal, and tibial nerves), and both tibial nerve and peroneal nerve were tightly ligated and transected distal to the ligation, leaving the sural nerve intact. The overlying muscle and skin were then sutured after surgery. Sham-operated mice were subjected to all preceding procedures without nerve ligation and transection.
2.3. Behavioral analyses
All mice were habituated to the testing environment for 3 days and in individual test compartments for at least 1 hour before each testing. All testing was performed in a blinded fashion. The experimenter was blind to treatment and/or genotype of mice throughout. Stimuli were applied to the lateral margin of the plantar aspect of the foot in the sural area of innervation.
2.3.1 Mechanical allodynia
Mechanical withdrawal threshold testing (von Frey test) was performed using calibrated monofilaments (0.008, 0.02, 0.04, 0.07, 0.16, 0.40, 0.60, 1.0, 1.4, and 2.0 g) (Danmic Global, Campbell, CA). Animals were habituated in individual test compartments in an elevated mesh-bottomed platform with a grid for at least 1 hour before testing. Beginning with the 0.008-g filament, filaments were applied to the plantar skin with just enough force to bend the fiber and held for 1 second. A “response” to the von Frey stimuli was defined as an abrupt foot lift upon application of the von Frey filament. Each filament was applied 10 times, and the paw withdrawal response frequency (the percentage of positive responses to the stimulus) was recorded. The force of a particular filament required to elicit 50% frequency of paw withdrawal was expressed as the mechanical threshold.
2.3.2 Thermal nociception
This was performed using a device designed for purpose of identifying heat sensitivity (IITC model 400, Woodland Hills, CA). Mice were placed on a temperature-regulated glass platform heated to 30°C, and the lateral plantar surface of hind paws stimulated with a radiant heat source (50-W halogen bulb) directed through an aperture. The time elapsed from initiation of the stimulus until paw withdrawal was recorded as paw withdrawal thermal latency. Each hind paw was tested 4 times with 5 minutes in between, and the withdrawal latency values averaged. To avoid tissue damage by prolonged thermal stimuli, cut-off latency was set as 20 seconds.
2.3.3 Elevated plus maze test
This test was performed at day 7 after SNI in a light- and sound-controlled room using an elevated plus maze (EPM) apparatus (Anhui zhenghua biologic apparatus facilities, China) elevated 70 cm above the floor. The EPM was constructed of black Plexiglas, consisting of 2 opposing open arms (60 × 5 cm), 2 opposing closed arms (60 × 5 × 25 cm), and a central area (5 × 5 cm). The walls of the closed arms were constructed of clear acrylic. The distance the mice spent in open arms were calculated to evaluate anxiety levels.
2.4. Injection of lidocaine, PKG-I antagonist, and NS398
C57BL/6 mice were anaesthetized with 2% isoflurane. Injection of lidocaine and KT5823 (Abcam, Cambridge, United Kingdom) or vehicle (saline, control) took place as follows. The procedure for intraneural injection of lidocaine was the same as described previously.34 Lidocaine at concentration of 1% was used, which was diluted from the stock solution of lidocaine hydrochloride injection (5 mL:0.1 g). In brief, the left sciatic nerve was exposed at midthigh level and 1% lidocaine (5 μL) or vehicle was injected into the nerve using a pulled micropipette. Either KT5823 or NS398 was suspended in a 1% DMSO/saline mix. Delivery of KT5823 was performed through intervertebral foramen route procedures as described in a previous report.49 The bilateral iliac spines were exposed to locate the L3 and L4 vertebrae of mice. The 26-gauge needle mated to a Hamilton syringe (Hamilton, Reno, NV) was inserted at a 45° angle at the intersection of the lower edge of the ipsilateral L3 and L4 vertebrae and the paravertebral line, infiltrating the intervertebral foramen with KT5823 (1 μg/50 μL). NS398 (2.5 μg/50 μL) was also injected following the procedures. There was a sense of restriction when the needle entered the transverse foramen, and the paw retraction reaction of mice was the sign of the needle entering the transverse foramen.
2.5. Western blotting
Dorsal root ganglia (DRG) and ACC tissues were collected and homogenised in ice-cold lysis buffer containing 50-mM Tris-HCl, pH 7.4, 150-mM NaCl, 5-mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and standard protease inhibitors. Insoluble material was removed by centrifugation (13,000 r/minute × 10 minutes), and supernatant was collected. Protein concentration for each sample was determined by the bicinchoninic acid method using the MICRO bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Proteins were loaded on a polyacrylamide gel and separated by electrophoresis. The membrane blots were blocked with 10% nonfat dry milk for 12 hours and incubated with primary antibodies: rabbit anti-PKG-I (1:1000; Abcam) and rabbit anti-BDNF (1:1000; Novus Biologicals, Littleton, CO) overnight at 4°C. The membranes were then incubated with horseradish peroxidase–conjugated goat anti-rabbit secondary antibody (1:3000; Abcam) for 2 hours at room temperature. To normalize the loaded samples, mouse monoclonal anti-β-actin antibody (1:2000; Sigma Aldrich, St. Louis, MO) was used, followed by incubation with horseradish peroxidase–conjugated goat anti-mouse IgG (1:3000; Abcam). Membranes were incubated with enhanced chemiluminescence reagents (Pierce, Rockford, IL), and images of the membrane were acquired with the CHEMIL-MAGER chemiluminescence imaging system. The intensity of each sample's PKG-I, BDNF band and its respective β-actin was measured using ImageJ software (NIH). For the analysis, intensity of each sample's PKG-I, BDNF band was normalized to its respective β-actin and represented as a percent of the average intensity of naive bands.
2.6. Immunofluorescence labelling
At 7 days after SNI, mice were anesthetized with 1% pentobarbital sodium and transcardially perfused with saline followed by 4% paraformaldehyde. Brain was removed, postfixed overnight in 4% paraformaldehyde, and cryoprotected in 30% sucrose at 4°C until the tissue sank to the bottom of the container. Anterior cingulate cortex sections (50 μm) were cut on a cryostat and were immunostained with rabbit anti-c-Fos antibody (1: 300; Santa cruz, CA) or rabbit anti-phospho-ERK1/2 antibody (1: 300; Cell signaling Technology, MA). Immunoreactive cells in lamina II and III of the ACC were microscopically counted in 5 to 6 sections per mouse from 4 mice per treatment group.
To visualize Fos and phospho-ERK1/2 induction, immunofluorescence labeling methods were used. Briefly, the sections were incubated with a solution containing 0.1% Triton X-100 and 5% bovine serum albumin for 1 hour at room temperature. The sections then were incubated separately with rabbit anti-c-Fos and anti-pERK antibody in PBST for 24 hours at 4°C. After 3 washes with PBST, the sections were further incubated with the secondary antibodies Alexa Fluor 488 (goat anti-rabbit IgG, 1:1000) (Abcam) for 2 hours at room temperature. All images were captured with an Olympus confocal microscope (FV1200, Olympus, Japan).
2.7. Brain slice patch clamp recording
Mice (4-6 weeks old) were anesthetized with 1% pentobarbital sodium and transcardially perfused with ice-cold oxygenated (95% O2, 5% CO2) incubation solution (in mM: NaCl, 95; KCl, 1.8; KH2PO4, 1.2; CaCl2, 0.5; MgSO4, 7; NaHCO3, 26; glucose, 15; sucrose, 50; pH 7.4). The brain was removed and mounted on a vibratome (Leica1200s, Wetzlar, Germany), and coronal slices (300 μm thick) containing the ACC (bregma 1.42-0.50 mm, determined by the shapes of lateral ventricles and corpus callosum) were prepared using the vibratome and stored in an incubation solution at room temperature. A slice was then transferred into a recording chamber and superfused with oxygenated recording solution at 2 mL/minute at room temperature. The recording solution was identical to the incubation solution except for (in mM): NaCl 127, CaCl2 2.4, MgSO4 1.3, and sucrose 0. Standard whole-cell patch clamp recordings were performed with glass pipettes having a resistance of 3 to 5 MΩ in pyramidal neurons in lamina II and III of the ACC contralateral to nerve injury. Neurons were visualized with “Dodt” infrared optics using X40 water-immersion objective on an Olympus BX51 microscope equipped with a CCD camera. Recorded pyramidal neurons were identified based on their morphological properties and their ability to show spike frequency adaptation in response to the prolonged depolarizing-current injection.73 The pipette solution consisted of (in mM): K-gluconate, 135; KCl, 5; CaCl2, 0.5; MgCl2, 2; EGTA, 5; HEPES, 5; and Mg-ATP, 5, pH 7.4 with KOH, measured osmolarity 300 mOsm. The electrophysiological properties of the recorded neurons were acquired with an Axon700B amplifier (Molecular Devices Corporation, San Jose, CA) and pCLAMP10.0 software. The membrane potential was held at −70 mV. For miniature excitatory postsynaptic current (mEPSC) recording, 0.5-μM TTX (Abcam) and 100-μM Picrotoxin (Tocris Bioscience, Bristol, United Kingdom) were added in the perfusion solution. The mEPSCs were detected and analyzed using Mini Analysis program (Synaptosoft Inc, Decatur, GA). Evoked EPSCs were recorded from layer II/III neurons, and the stimulations were delivered by a field stimulating electrode placed in layer V/VI of the ACC. α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated eEPSCs were induced by repetitive stimulations at 0.05 Hz in the presence of AP5 (50 μM, Abcam), an antagonist at NMDA receptors and picrotoxin (100 μM), blocker for inhibitory synaptic transmission. In some experiments for presynaptic mechanism analysis, paired-pulse paradigm with paired stimulation pulse at an interval of 50 ms was used to evoke paired eEPSCs. Long-term potentiation (LTP) was induced by 80 paired presynaptic pulses at 2 Hz coupled with postsynaptic depolarization at +30 mV, as reported previously.71 Except for AP recording, QX-314 (5 mM, Abcam) was added to the pipette solution to prevent discharge of action potentials (AP). Data were excluded when the resting membrane potential of neurons were positive than −55 mV and action potentials did not have overshoot. Signals were low-pass filtered at 5 kHz, sampled at 10 kHz, and analysed offline.
2.8. Data analysis and statistics
All statistical analyses were performed in Prism v.8.0 (GraphPad Software, Inc) and were presented as mean ± SEM. For comparisons of multiple groups, one-way or two-way analysis of variance of random measures followed by (the Tukey, Dunnett, and Bonferroni) multiple comparisons test was performed to determine statistically significant differences. Unless otherwise specified, the P values shown in figures and text are derived from analysis of variance. P < 0.05 was considered significant.
3.1. Ongoing primary afferent inputs are critical for maintaining exaggerated pain response in peripheral neuropathy
To determine whether nerve injury–induced exaggerated pain is dependent upon ongoing primary afferent input, we performed the experiments in which nerve conduction was blocked with lidocaine in the sciatic nerve when stable pain hypersensitivity was established. At 7 days after SNI, wildtype mice demonstrated the characteristic leftward and upward shift in the stimulus–response curve over basal state reflecting mechanical allodynia in the ipsilateral hind paw (Fig. 1A, n = 6 mice). Analysis of mechanical threshold to von Frey filaments revealed a dramatic drop after SNI as compared to basal level (Fig. 1B, P < 0.0001, n = 6 mice). In comparison with the vehicle group, intraneural injection of lidocaine (1%, 5 μL) in the ipsilateral sciatic nerve at 7 days after SNI almost abolished the established mechanical allodynia in SNI mice (Figs. 1A and B, P < 0.0001, n = 6 mice). Lidocaine treatment shifted the deviated stimulus–response curves after SNI back to basal level and reversed the reduction of mechanical threshold induced by SNI (Figs. 1A and B, P < 0.0001, n = 6 mice). Similarly, blockade of peripheral primary afferent inputs with lidocaine strongly prevented the ipsilateral thermal hyperalgesia at 7 days after SNI (Fig. 1C, P < 0.0001, n = 6 mice).
In addition to primary hyperalgesia and allodynia observed in the ipsilateral hind paw, SNI induced an obvious secondary hyperalgesia and allodynia observed in the contralateral hind paw as well (Figs. 1D–F, n = 6 mice). Although some studies report the absence of contralateral hyperalgesia,15,26 there is increasing evidence showing that unilateral nerve injury induces contralateral hypersensitivity in both clinical conditions37 and animal models of neuropathic pain.3,9,11,21,29,54,81,86,88 Central sensitization, which involves structures in the spinal cord and brain, is deemed essential for secondary hyperalgesia and allodynia.23,36,51,64 Interestingly, we demonstrated that peripheral blockade of nociceptive signals ipsilaterally was able to relieve the established secondary mechanical allodynia and thermal hyperalgesia in the contralateral hind paw (Figs. 1D–F, P < 0.0001, n = 6 mice). Thus, we infer from the above that ongoing primary afferent inputs are critical for maintaining primary and secondary pain hypersensitivity after peripheral neuropathy.
3.2. Peripheral nerve injury alters PKG-I expression in the dorsal root ganglion
We next sought to determine which candidates in primary afferent inputs are involved in the development of neuropathic pain. Previous studies have reported that cyclic GMP-dependent protein kinase I (PKG-I) is predominantly expressed in peripheral nociceptors and PKG-I specifically localized in nociceptors is essential for inflammatory pain.39,47,48 However, whether PKG-I in nociceptors contributes to SNI-induced neuropathic pain remains elusive. To this end, we first examined the changes of PKG-I expression in DRG after SNI in wildtype mice. In this SNI procedure in mice, it is reported that L3 DRG is enriched in injured fibers, L4 DRG has a mixture of injured and noninjured fibers, and L5 DRG is enriched in noninjured fibers as described previously.39,59,69 PKG-I protein expression levels in each of L3, L4, and L5 DRG were assessed in naive, sham, and SNI groups at 7 days and 28 days after SNI by Western blotting. In the ipsilateral L3 DRG, PKG-I expression displayed significant upregulation at 7 days after SNI as compared to the sham group, which tended to return to control level at 28 days after SNI (typical example in Fig. 2A, quantitative summary in Fig. 2B, P < 0.05, n = 12 mice per group). In the ipsilateral L4 DRG, no significant change in PKG-I expression was seen in SNI-treated mice compared with the sham group (typical example in Fig. 2C, quantitative summary in Fig. 2D, P > 0.05, n = 12 mice per group). On the other hand, in the L5 DRG ipsilateral to injury, PKG-I expression decreased significantly at 7 days and 28 days after SNI (Figs. 2E and F, P < 0.01 at 7 days, P < 0.0001 at 28 days, n = 12 mice per group). By contrast, in the ipsilateral L6 DRG which does not innervate the plantar surface of mice hind paw, PKG-I expression was comparable in different groups (Figs. 2G and H, P > 0.05, n = 12 mice per group). Consistent with our results, a similar upregulation of PKG-I was observed in the compressed DRG in rats subjected to chronic compression of DRG.31 These results suggest that PKG-I in the DRG may be relevant to the development of neuropathic pain.
3.3. Silencing PKG-I specifically in nociceptors produces analgesic and anxiolytic effects
To delineate whether PKG-I in nociceptors is involved in the development of neuropathic pain and pain-related anxiety, we conducted experiments using PKG-Ifl/fl and SNS-PKG-I−/− mice in both sexes. The basal nociception to mechanical and thermal stimulation was comparable in PKG-Ifl/fl and SNS-PKG-I−/− mice (Figs. 3A–C, P > 0.05, n = 8-12 mice). After SNI surgery, PKG-Ifl/fl mice displayed a persistent exaggerated pain over 4 weeks in bilateral hind paws, as characterized by leftward and upward shift in the stimulus–response curve over basal curve, reflecting mechanical allodynia and hyperalgesia (Fig. 3A, n = 12 mice). Analysis of mechanical threshold to von Frey filaments revealed a prominent reduction in PKG-Ifl/fl mice in bilateral hind paws as compared to controls (Fig. 3B, P < 0.0001, n = 12 mice). In striking contrast, SNS-PKG-I−/− mice showed very little deviation upon SNI operation over basal curves (Fig. 3A, P > 0.5, n = 8 mice). Spared nerve injury–induced reduction in mechanical threshold was not developed in SNS-PKG-I−/− mice (Fig. 3B, P < 0.0001, n = 8 mice). In parallel, a slight, but significant thermal hyperalgesia was observed in PKG-Ifl/fl mice upon SNI in bilateral hind paws as well (Fig. 3C, P < 0.05, n = 12 mice). This thermal hyperalgesia was absent in SNS-PKG-I−/− mice (Fig. 3C, P < 0.001, n = 8 mice). These results suggest that the development of neuropathic pain is dependent upon activation of PKG-I in nociceptors. To examine whether there is sex difference in PKG-I role in neuropathic pain, we analyzed the male and female mice separately. We observed that specific ablation of PKG-I in nociceptors produced similar analgesic effect in both sexes (Fig. S1A, B, n = 6 for female, n = 4 for male; available online as supplemental digital content at http://links.lww.com/PAIN/B136). It is noteworthy to point out that the loss of pain hypersensitivity observed in SNS-PKG-I−/− mice is not due to development defects of nociceptors because normal development of nociceptors as well as its peripheral and central targeting was seen in SNS-PKG-I−/− mice as reported previously.47
To further confirm the maintenance of neuropathic pain is dependent on PKG-I activation in nociceptors, we delivered PKG-inhibitor KT5823 (1 μg) into the L3 and L4 ganglia within the intervertebral foramen in wildtype mice at 7 days after SNI. As compared to the vehicle group, intervertebral foramen injection of KT5823 greatly attenuated the magnitude of bilateral mechanical and thermal pain hypersensitivity at 7 days after SNI (Figs. 3D–I, P < 0.001, n = 13 mice). The basal nociception to mechanical and thermal stimulation was comparable in the vehicle group and the KT5823 group (Fig. S2, P > 0.05, n = 6 mice, available online as supplemental digital content at http://links.lww.com/PAIN/B136). By contrast, intraforaminal injection of nonsteroid anti-inflammatory drugs, NS398 (2.5 μg), was not able to relieve the maintenance of bilateral mechanical and thermal hypersensitivity (Fig. S3, P > 0.05, n = 10 mice, available online as supplemental digital content at http://links.lww.com/PAIN/B136). Consistent with nonsteroid anti-inflammatory drugs, a previous study reported that intraforaminal injection of morphine or fentanyl did not alter the maintenance of hyperalgesia and allodynia induced by spinal nerve ligation.91 These results suggest that the maintenance of neuropathic pain is dependent upon activation of PKG-I in nociceptors.
Neuropathic pain is frequently associated with aversive emotion, such as anxiety.6 We next assessed whether PKG-I in nociceptors influences neuropathic pain–related anxiety with EPM paradigm. In the EPM, sham PKG-Ifl/fl mice (n = 15 mice) showed comparable general locomotor ability and traveled equally in the open arm with sham SNS-PKG-I−/− mice (typical example in Fig. 4A, quantitative summary in Fig. 4B, P > 0.05, n = 14 mice). After SNI, PKG-Ifl/fl mice travelled less distance in the open arm as compared to the sham group, indicative of anxiety-like behaviors (Figs. 4A and B, P < 0.0001, n = 15 mice). Ablation of PKG-I in nociceptors enabled the mice to travel frequently in the open arm, indicative of relief from pain-related anxiety (Figs. 4A and B, P < 0.05, n = 14 mice). The total distance in the EPM was comparable in both genotypes (Fig. 4B, P > 0.05). In addition, we did not observe the sex difference of nociceptor-localized PKG-I on the development of pain-related anxiety, since both male and female SNS-PKG-I−/− mice showed comparable depression of anxiety induced by SNI (Fig. S4A, B, n = 7-8 mice per sex per genotype, available online as supplemental digital content at http://links.lww.com/PAIN/B136). We further confirmed PKG-I in nociceptors is involved in the maintenance of pain-related anxiety by pharmacological blockade of PKG-I in nociceptors. After SNI, wildtype mice travelled less distance in the open arm as compared to the sham group, indicative of anxiety-like behaviors (Figs. 4C and D, P < 0.0001, n = 8 mice). Intervertebral foramen injection of KT5823 in nociceptors significantly increased the distance that SNI mice travelled in the open arm, indicating that pain relief in the periphery by KT58223 greatly relieved the associated comorbidity symptoms, such as anxiety (Figs. 4C and D, P < 0.01, n = 9 mice). The total distance in the EPM was comparable in the vehicle and KT5823 group (Fig. 4D, P > 0.05, n = 8-9 mice). Taken together, these results suggest that the development and maintenance of pain-related anxiety are dependent upon activation of PKG-I in nociceptors.
3.4. Deletion of PKG-I in nociceptors eliminates spared nerve injury–induced neuronal activity in the anterior cingulate cortex
We went on to address by which mechanisms PKG-I in nociceptors modulates neuropathic pain and pain-related anxiety. The anterior cingulate cortex is assumed to play crucial roles in perception of pain and pain-related emotions.6,8,13,77,95,96 Previous studies have shown that phosphorylation of ERK1/2 (extracellular receptor-activated MAP Kinases 1/2) (pERK1/2) can be stably activated in the ACC by noxious stimuli and is required for ACC LTP as well as behavioral sensitization.8,72,83 We therefore chose to examine whether and to what extent PKG-I in nociceptors affect the populated neuronal activity in the ACC after peripheral nerve injury. In SNI-operated PKG-Ifl/fl mice, a significant increase was observed in the immunoreactivity for pERK1/2 over neurons in the ACC as compared to sham PKG-Ifl/fl mice, as judged by the number of pERK1/2-immunoreactive neurons (Figs. 5A and B, P < 0.0001, n = 4 mice). This upregulation of pERK1/2 after nerve injury was largely decreased in SNS-PKG-I−/− mice (Figs. 5A and B, P < 0.0001, n = 4 mice). No significant difference for pERK1/2 immunoreactivity was seen in the sham group between 2 genotypes (Figs. 5A and B, P > 0.05, n = 4 mice). In parallel, another neuronal activity marker c-Fos was observed. Similarly, SNI induced a robust enhancement for Fos expression in the ACC neurons derived from PKG-Ifl/fl mice, which was almost prevented by the deficiency of PKG-I in nociceptors (Figs. 5C and D, P < 0.001, n = 4 mice). SNS-PKG-I−/− and its littermates floxed controls showed very few and comparable c-Fos immunoreactivity in the sham group (Figs. 5C and D, P > 0.05, n = 4 mice). These data indicate that after nerve injury, activation of PKG-I is responsible for the increased nociceptor activity, which leads to the increased neuronal activity in the ACC.
3.5. Nociceptor-specific ablation of PKG-I reduces hyperexcitability of anterior cingulate cortex neurons after nerve injury
Peripheral injury induces central sensitization, which is reflected in neuronal hyperexcitability in central neurons, ie, ACC.90 Neuronal excitability depends on 2 components, the synaptic inputs and the intrinsic excitability. We then tested whether PKG-I in nociceptors influenced the intrinsic excitability of pyramidal neurons in layer II/III of the ACC neurons after nerve injury by using whole-cell patch-clamp recordings. The pyramidal neurons were identified based on their morphological properties and their ability to show spike frequency adaptation in response to the prolonged depolarizing-current injection73 (Fig. 6A). The passive membrane properties including membrane capacitance (Cm), membrane resistance (Rm), and resting membrane potential (RMP) were comparable between PKG-Ifl/fl and SNS-PKG-I−/− mice in both sham and SNI groups (Fig. 6B, P > 0.05, n = 17 neurons/6 mice). No sex difference was also observed in the passive membrane properties between PKG-Ifl/fl and SNS-PKG-I−/− mice (Fig. S5A, n = 9 neurons for male, n = 8 neurons for female, available online as supplemental digital content at http://links.lww.com/PAIN/B136). However, the active membrane properties of ACC pyramidal neurons represented significant differences between PKG-Ifl/fl and SNS-PKG-I−/− mice after SNI. A detailed input (current intensity)–output (AP frequency) curve in response to a depolarizing current step was drawn in both genotypes from the sham and SNI group (Figs. 6C and D, left panels). The mean firing frequency induced by depolarizing current was not significantly different in PKG-Ifl/fl and SNS-PKG-I−/− mice in the sham group (Fig. 6C, P > 0.05, n = 17 neurons/6 mice). Upon SNI surgery, ACC neurons from PKG-Ifl/fl mice displayed a dramatic augmentation in firing frequency, as characterized by a leftward and upward shift of the I-O curve over sham curve (Figs. 6C and D). This deviation of the I-O curve and increase in firing frequency was reversed after deletion of PKG-I in nociceptors (Fig. 6D, P < 0.0001, n = 17 neurons/6 mice). In addition, SNI surgery produced a lowered rheobase in PKG-Ifl/fl mice as compared to sham treatment (Figs. 6E and F, P < 0.01, n = 17 neurons/6 mice), which was normalized in SNS-PKG-I−/− mice (Figs. 6E and F, P < 0.001, n = 17 neurons/6 mice). The other parameters such as AP threshold, amplitude, as well as half-width were not altered after SNI and showed similarity in both genotypes (Figs. 6E and F, P > 0.05, n = 17 neurons/6 mice). Detailed analysis of female and male mice revealed that no sex difference was observed for the role of nociceptor-localized PKG-I in the neuronal hyperexcitability of the ACC (Fig. S5B, C, http://links.lww.com/PAIN/B136, n = 9 neurons for male, n = 8 neurons for female). Taken together, the above results indicate that PKG-I activation in nociceptors leads to nociceptor hyperexcitability after peripheral neuropathies, which contributes to neuronal hyperexcitability in the ACC.
3.6. Silence of PKG-I in nociceptors depresses excitatory transmission through presynaptic mechanisms in layer II/III neurons in the anterior cingulate cortex after nerve injury
Enhanced synaptic transmission in the ACC after nerve injury is crucial for the central sensitization.2,4,35,95,96 We further explored whether PKG-I in nociceptors contributed to enhanced synaptic transmission in the ACC after nerve injury. To address this, we recorded AMPA receptor-mediated eEPSCs in pyramidal neurons of layer II/III in the ACC at a holding potential of −70 mV by applying local stimulation in layer V/VI in the presence of inhibitory synaptic transmission antagonist, picrotoxin (100 μM), and NMDA receptor antagonist, AP5 (50 μM). The input (stimulation intensity)–output (eEPSC amplitude) curve of eEPSCs was drawn in response to different presynaptic stimulation intensity in both genotypes from the sham and SNI group. In basal state, PKG-Ifl/fl mice showed comparable basal synaptic transmission with SNS-PKG-I−/− mice (Fig. 7A for typical traces, Fig. 7B for quantitative summary, P > 0.05, n = 12-13 neurons). At 7 days after SNI surgery, PKG-Ifl/fl mice displayed enhanced synaptic transmission, manifesting as a leftward and upward shift in the I-O curve of eEPSCs and increased magnitude of eEPSCs (Fig. 7C for typical traces, Fig. 7D for quantitative summary, P < 0.01, n = 14-16 neurons). By contrast, deficiency of PKG-I in nociceptors prevented this potentiation of synaptic transmission (Figs. 7C and D, P < 0.0001, n = 14 neurons). Separate analysis of female and male mice revealed that both sexes of PKG-Ifl/fl mice developed comparable degree of synaptic potentiation upon SNI, which was similarly eliminated by deletion of PKG-I in nociceptors (Fig. S6, n = 6-7 neurons for male, n = 6-9 neurons for female, available online as supplemental digital content at http://links.lww.com/PAIN/B136). These results suggest that activation of PKG-I in nociceptors sensitizes nociceptor, which is necessary for the enhanced excitatory synaptic transmission in the ACC after peripheral nerve injury.
To examine whether presynaptic or postsynaptic mechanisms mediate the reduced excitatory synaptic transmission in the ACC in SNS-PKG-I−/− mice with neuropathic pain, we used 2 protocols which are commonly used as a measure for presynaptic function.97 We first focused on the analysis of paired-pulse facilitation (PPF), which represents a short-lasting increase in the second eEPSCs when it follows shortly after the first and is well accepted as an indication of presynaptic mechanisms of LTP in the hippocampus.63 In the control group, we recorded a comparable PPF in ACC neurons derived from PKG-Ifl/fl and SNS-PKG-I−/− mice (representative examples in Fig. 7E, quantitative summary in Fig. 7F, P > 0.05, n = 14-15 neurons). Upon SNI surgery, there was a significant reduction in PPF in ACC neurons from PKG-Ifl/fl mice (Figs. 7E and F, P < 0.001, n = 15 neurons), indicating an increase in the neurotransmitter release probability. By contrast, this drop in PPF did not come about in neurons from SNS-PKG-I−/− mice (Figs. 7E and F, P < 0.0001, n = 14 neurons). These results infer that after nerve injury, PKG-I enables a state of hyperexcitability in nociceptors, which leads to elevated synaptic transmission in the ACC through a presynaptic mechanism. This inference was further consolidated by miniature EPSC (mEPSC) analysis. As shown in Figures 7G–I, SNI induced a robust increase in the frequency of mEPSCs, but no change in the amplitude in ACC neurons derived from PKG-Ifl/fl mice (Fig. 7G for typical traces, Figs. 7H and I for quantitative summary, P < 0.0001 for frequency, P > 0.05 for amplitude, n = 14 neurons). This facilitation in mEPSC frequency induced by nerve injury was eliminated in nociceptor-specific PKG-I-deficient mice (Figs. 7G and H, P < 0.0001, n = 14 neurons). No sex difference was observed in the PPF and mEPSC analysis (Fig. S6C, D, n = 7-8 neurons for male, n = 7-8 neurons for female, available online as supplemental digital content at http://links.lww.com/PAIN/B136). Taken together, these 2 lines of evidence collectively strengthened the involvement of hyperexcitability of nociceptors by PKG-I in the cortical synaptic potentiation after nerve injury through a presynaptic mechanism involving transmitter release.
3.7. PKG-I deficiency in nociceptors abolished long-term potentiation in layer II/III neurons in the anterior cingulate cortex
Long-term potentiation is a major form of synaptic plasticity, and cortical LTP of excitatory synaptic transmission in the ACC is believed to be a key cellular mechanism for chronic pain. Next, we explored the possible involvement of PKG-I localized in nociceptors in the cortical LTP. We used a typical LTP inducing protocol to trigger LTP in ACC slices (Fig. 8A), which contained 80 presynaptic pulses at 2 Hz with postsynaptic depolarization at +30 mV (referred to as the pairing training conditioning stimulus) as described previously.71 We induced LTP within 12 minutes after establishing the whole-cell configuration to avoid washout of intracellular contents that are critical for the establishment of synaptic plasticity. Long-term potentiation was induced by conditioning stimulus which produced a significant, long-lasting potentiation of eEPSCs in slices of PKG-Ifl/fl mice compared with baseline responses (Fig. 8B for time course and Fig. 8C for typical examples, Figs. 8D and E for quantitative summary, P < 0.01, n = 14-16 neurons). This LTP showed comparable potency in male and female mice (Fig. S7A, B, n = 6-7 neurons for male, n = 8-9 neurons for female, available online as supplemental digital content at http://links.lww.com/PAIN/B136), which is consistent with a previous study by Liu et al.45 By contrast, this synaptic potentiation was abolished in slices from SNS-PKG-I−/−mice in both sexes (Figs. 8B–E, P < 0.001, n = 14-16 neurons; Fig. S7A, B, n = 6-7 neurons for male, n = 8-9 neurons for female, available online as supplemental digital content at http://links.lww.com/PAIN/B136). These results provide the first evidence that nociceptor hyperexcitability caused by PKG-I activation is critical for cortical LTP in the ACC.
3.8. Deletion of PKG-I in nociceptors prevents brain-derived neurotropic factor upregulation in the anterior cingulate cortex after nerve injury
Finally, we attempted to further elucidate the molecular basis with which nociceptor hyperexcitability caused by PKG-I activation enhances synaptic potentiation in the ACC after nerve injury. Given the crucial role of BDNF in mediation of ACC synaptic potentiation, pain hypersensitivity, and pain-related aversion,70,80,92 we examined changes of BDNF expression in the ACC of PKG-Ifl/fl and SNS-PKG-I−/− mice after nerve injury. Lysates from the ACC in PKG-Ifl/fl mice revealed that BDNF expression was upregulated at 7 days after SNI and returned back to basal level at 28 days after injury (Fig. 9A for typical blot, Fig. 9B for quantitative summary, P < 0.001, n = 4 mice). This upregulation was excluded with the deficiency of PKG-I in nociceptors (Figs. 9A and B, P < 0.05, n = 4 mice). Consistent with BDNF upregulation, SNI induced a dramatic increase in PKG-I expression in the ACC as well in wildtype mice (Fig. 9C for typical blot, Fig. 9D for quantitative summary, P < 0.01 for 7 days after SNI, P < 0.001 for 28 days after SNI, n = 4 mice). However, this SNI-induced increase of PKG-I expression in the ACC is not dependent upon PKG-I in nociceptors since PKG-Ifl/fl and SNS-PKG-I−/− mice demonstrated similar PKG-I level in the ACC after SNI (Fig. 9E for typical blot, Fig. 9F for quantitative summary, P > 0.05, n = 4 mice). It can thus be concluded that peripheral PKG-I is critical for nociceptor hyperexcitability, which is necessary for BDNF upregulation in the ACC and hence contributes to cingulate synaptic potentiation and pain hypersensitivity as well as pain-related aversions.
Peripheral nerve damage often leads to maladaptive changes in the injured sensory neurons (peripheral sensitization) and along the entire nociceptive pathway within the CNS (central sensitization). However, there has been an ongoing and unresolved debate about the relative role of peripheral and central mechanisms driving neuropathic pain and pain-related aversions.20,25,84 Some preclinical evidence has suggested that central sensitization may become an autonomous pain generator independent upon peripheral inputs after nerve injury,5,33,79 while other clinical cases indicate that ongoing nociceptive afferent input from a peripheral focus maintains altered central processing that accounts for pain hypersensitivity and affective aversions in patients with neuropathic pain.28,76 Given this discrepancy, this study provides a strong experimental support for the decisive role of ongoing primary afferent inputs in maintaining the central sensitization to mediate neuropathic pain and pain-related aversions. More importantly, we unravel a peripheral target, PKG-I localized in primary afferent nociceptors, which influences central sensitization by enhancing cortical plasticity in the ACC through regulation of BDNF function after nerve injury. Developing analgesic therapeutics against peripheral PKG-I may pave the new way for treatment of neuropathic pain with least side effects.
4.1. Peripherally localized PKG-I is a critical generator in maintaining central sensitization that accounts for neuropathic pain and pain-related aversions
The novel and most important finding of this study is that we identify a peripheral target, PKG-I expressed in nociceptors as a critical generator in maintaining central sensitization that accounts for pain hypersensitivity and pain-related anxiety after peripheral neuropathy. Patients with peripheral neuropathy is characterized by persistent pain hypersensitivity and aversive disorders. This includes primary hyperalgesia and allodynia (pain experienced in the injured site), secondary hyperalgesia and allodynia (pain experiences arising from adjacent uninjured site), as well as anxiety, depression, etc.7,24 Peripheral sensitization has been linked with primary hyperalgesia, whereas central sensitization is assumed to underlie allodynia, secondary hyperalgesia, as well as pain-related aversive emotions.7,24,38,85 However, medications interfering with the CNS in humans often produce unwanted side effects, ie, cognitive dysfunction, psychiatric disorders, defects in motor coordination, etc.53,78 Thus, to seek the analgesic therapeutics with least side effects without reducing analgesic efficiency is a major challenge. Our study extends previous findings27,28,91 and finds that SNI-induced ipsilateral hyperalgesia and allodynia as well as contralateral hyperalgesia and allodynia is relieved by infiltration of lidocaine in the ipsilateral sciatic nerve at 7 days after nerve injury, indicating that central sensitization associated with neuropathic pain is initiated and maintained by ongoing nociceptive primary afferent inputs from peripheral nociceptors. Taken together with the fact that peripheral nociceptors lie outside of the blood–brain barrier,16 it can be inferred that targeting nociceptors represent a promising strategy for fulfillment of optimal analgesic therapeutics with least side effects.
PKG-I is well known to be predominantly expressed in nociceptors22,47,57,60 and the spinal cord. Several brain regions also express PKG-I.17,18 Previous pharmacological and genetic studies have linked PKG-I to the development of pain hypersensitivity associated with tissue inflammation.67,68 However, given the extensive role of PKG-I in the smooth muscle and platelet function,30 global knockout mice are typically demonstrated lethality in the first few weeks, which makes systemic intervention of PKG-I for antinociception infeasible. To keep analgesic effect and minimize side effects against PKG-I target, our previous studies have dissected the role of PKG-I exclusively expressed in nociceptors and demonstrated its prominent contribution to nociceptor hyperexcitability, spinal LTP, and hyperalgesia after tissue inflammation.47,48 However, specific role of PKG-I localized in nociceptors in the maintenance of neuropathic pain and pain-related aversions as well as its underlying mechanisms has not been fully characterized. This study combined genetic (SNS-PKG-I−/− mice) and pharmacological (KT5823, a PKG-I antagonist) evidence demonstrates for the first time that PKG-I expressed in nociceptors is a key determinant for the development and maintenance of primary, secondary hyperalgesia and allodynia as well as pain-related anxiety after nerve injury. This action is supported by the obvious changes of PKG-I expression in different lumbar DRG after SNI. These results strongly indicate that in addition to peripheral sensitization which is reported to require PKG-I,41,44,65,93 central sensitization is further initiated and maintained by activation of PKG-I in nociceptors in neuropathic pain states. This extends a previous study in inflammatory pain conditions for PKG-I action.22 These evidences enable exploration of peripheral PKG-I target against neuropathic pain practicable. The potential beneficial effect with anti-PKG-I strategy against chronic pain including neuropathic pain is also supported by previous reports.41,44,46,65,67,93 The only inconsistency with our study is from a recent study showing that PKG-I deficiency in injured neurons impairs regeneration of the sciatic nerve after injury leading to an enhancement of neuropathic pain behavior. Injury-evoked redox imbalances were reported to be involved in this process.75 This discrepancy remains to be further investigated in the future study. Then what could be the potential mechanisms by which PKG-I in nociceptors is capable to mediate central sensitization?
4.2. PKG-I localized in nociceptors mediates central sensitization through enhancement of cortical synaptic transmission in the anterior cingulate cortex after nerve injury
Compelling evidence shows that synaptic transmission in the cortical area, mainly in the ACC, displays activity-dependent potentiation under peripheral neuropathy.2,4,35,87,95,96 A number of candidates, i.e., NMDA receptors, Ca2+-permeable AMPA receptors, kainate receptors, adenylyl cyclase type 1 (AC1), PKM£, pERK, and hyperpolarization-activated cyclic nucleotide-gated channels (HCNs), in the ACC has been revealed to underlie this synaptic potentiation.4,10,35,42,43,87 However, unwanted side effects for these central targets hamper their practical clinical translation. Therefore, we are interested to know whether this cortical synaptic potentiation is dependent upon ongoing peripheral nociceptive inputs after nerve injury, and if so, which peripheral targets underlie this cortical potentiation. This is exactly another intriguing finding that we obtain in the present study. We demonstrate that plastic changes of cortical neurons in the intrinsic properties and synaptic transmission upon nerve injury is diminished by silence of PKG-I in nociceptors. PPR and mEPSC analysis reveal that activation of PKG-I induced by nerve injury increases transmitter release in the ACC through a presynaptic mechanism. Induction of synaptic potentiation at some glutamatergic synapses including cortical synapses depends on the action of BDNF.19,48,55,70,94 Our observation that nociceptor-specific deletion of PKG-I attenuates SNI-induced BDNF upregulation in the ACC suggests that PKG-I in nociceptors modulates cortical synaptic plasticity through BDNF action in the ACC. This is supported by previous studies showing that BDNF is involved in the mediation of ACC synaptic potentiation, pain hypersensitivity, as well as pain-related aversions after injury.70,80,92 No alteration of cingulate PKG-I upregulation seen in SNS-PKG-I−/− mice after SNI surgery further suggests the specific regulation of BDNF-dependent plasticity in the ACC by peripheral PKG-I. However, whether BDNF is derived presynaptically or postsynaptically and how BDNF controls synaptic plasticity at cingulate synapses remains to be further investigated. Meanwhile, it is also interesting to know the exact role of enhanced PKG-I expression in the ACC after nerve injury in the future study.
4.3. Targeting nociceptor-localized PKG-I as a potential therapeutic intervention for comorbidity of neuropathic pain and anxiety
There is abundant clinical evidence showing that lidocaine administration at the injured site suppresses pain in a variety of chronic neuropathic pain conditions.27,28,52,56,76 This provides a strong hint that persistent peripheral nociceptive information inflow is able to trigger and maintain both peripheral and central sensitization. However, since sodium channels are expressed in all neurons and fibers, lidocaine application may block normal sensory and motor function along with pain. To overcome this shortcoming, in this study, we identify a nociceptor-specific target, PKG-I, with the capability of maintaining nerve injury-induced exaggerated pain and pain-related aversions. More importantly, ablation of PKG-I function in nociceptors using genetic and pharmacological approaches indicates that it is not involved in the basal nociception, but selectively interfering with the abnormal neuropathic pain. This action was also observed in inflammatory pain conditions.47,48 So far, several PKG-I inhibitors such as Rp-8-pCPT-cGMPS, KT5823 have been available and showed significant analgesic effect in different pain models.46,47,65 In particular, a new selective inhibitor for PKG-Iα subunit that nociceptive sensory neurons predominantly express, N46, is synthesized and characterized to possess a potent antinociceptive effect in inflammatory and osteoarthritic pain.67 Last but not least, nociceptors are located in the periphery; the inhibitors for PKG-I need not enter the CNS and can hence minimize central side effects without sacrificing analgesic efficacy.
In summary, our results demonstrate that PKG-I expressed in nociceptors is a key determinant for the initiation and especially maintenance of neuropathic pain and pain-related anxiety. Further mechanistic analysis reveals that PKG-I in nociceptors is associated with enhancement of cortical synaptic transmission in the ACC through a presynaptic mechanism involving BDNF signaling after nerve injury. This study presents a strong basis for opening up a novel therapeutic target, PKG-I, in nociceptors for treatment of a variety of neuropathic pain and pain-related affective disorders.
Conflict of interest statement
The authors have no conflicts of interest to declare.
Appendix A. Supplemental digital content
Supplemental digital content associated with this article can be found online at http://links.lww.com/PAIN/B136
Supplemental video content
A video abstract associated with this article can be found at http://links.lww.com/PAIN/B137.
The authors are grateful to Robert Feil (Universität Tübingen, Tübingen, Germany) and Rohini Kuner (University of Heidelberg, Germany) for the kind gift of genetic mice. This work was supported by Natural Science Foundation of China (NSFC) grants (No. 31671088) to C.L.; grants from Natural Science Foundation of Shaanxi province (No. 2017ZDJC-01) to C.L.; NSFC grant (No. 81730035) and Innovation Teams in Priority Areas Accredited by the Ministry of Science and Technology (No. 2014RA4029) to S.-X. Wu; and NSFC grant to R.-G. Xie (No. 81870867).
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