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. Author manuscript; available in PMC: 2024 Jan 1.
Published in final edited form as: Brain Behav Immun. 2022 Oct 21;107:215–224. doi: 10.1016/j.bbi.2022.10.013

Spinal GABAergic disinhibition allows microglial activation mediating the development of nociplastic pain in male mice

Kathleen E McDonough a, Regan Hammond a, Jigong Wang a, Jessica Tierney a, Kali Hankerd a, Jin Mo Chung a, Jun-Ho La a,*
PMCID: PMC9855286  NIHMSID: NIHMS1865431  PMID: 36273650

Abstract

Previously we developed a murine model in which postinjury stimulation of an injured area triggers a transition to a nociplastic pain state manifesting as persistent mechanical hypersensitivity outside of the previously injured area. This hypersensitivity was maintained by sex-specific mechanisms; specifically, activated spinal microglia maintained the hypersensitivity only in males. Here we investigated whether spinal microglia drive the transition from acute injury-induced pain to nociplastic pain in males, and if so, how they are activated by normally innocuous stimulation after peripheral injury. Using intraplantar capsaicin injection as an acute peripheral injury and vibration of the injured paw as postinjury stimulation, we found that inhibition of spinal microglia prevents the vibration-induced transition to a nociplastic pain state. The transition was mediated by the ATP-P2X4 pathway, but not BDNF-TrkB signaling. Intrathecally injected GABA receptor agonists after intraplantar capsaicin injection prevented the vibration-induced transition to a nociplastic pain state. Conversely, in the absence of intraplantar capsaicin injection, intrathecally injected GABA receptor antagonists allowed the vibration stimulation of a normal paw to trigger the transition to a spinal microglia-mediated nociplastic pain state only in males. At the spinal level, TNF-α, IL-1β, and IL-6, but not prostaglandins, contributed to the maintenance of the nociplastic pain state in males. These results demonstrate that in males, the transition from acute injury-induced pain to nociplastic pain is driven by spinal microglia causing neuroinflammation and that peripheral injury-induced spinal GABAergic disinhibition is pivotal for normally innocuous stimulation to activate spinal microglia.

Keywords: Nociplastic pain, microglia, disinhibition, GABA, ATP, P2X4, pro-inflammatory cytokines

1. Introduction

Nociplastic pain encompasses a multitude of chronic pain conditions with pain hypersensitivity, including, but not limited to, complex regional pain syndrome type I (CRPS I), non-neuropathic chronic post-surgical pain, and fibromyalgia, (Kosek et al., 2016). These conditions are often incited by an injury which subsequently heals (McCabe and Blake, 2008; Steegers et al., 2008), but pain persists despite no detectable tissue or nerve damage in progress, distinguishing nociplastic pain from chronic inflammatory and neuropathic pain. Previously, we developed a murine model of nociplastic pain which allows investigation into the mechanisms of this key aspect of nociplastic pain: namely, the transition to this chronic pain state from acute injury-induced pain (Hankerd et al., 2021). This transition may be triggered during the inciting injury-induced pain hypersensitivity that normally resolves as the injury heals. This notion is supported by our nociplastic pain model in which stimulating an injured area at normally innocuous intensity triggers such transition only during the timeframe of substantial pain hypersensitivity after the inciting acute injury (Hankerd et al., 2021). When considering that an acute injury (e.g., capsaicin injection)-produced nociceptor inputs inhibit spinal inhibitory neurons to allow innocuous stimuli to activate nociceptive circuits (Pernía-Andrade et al., 2009), as envisaged in the gate control theory of pain (Melzack and Wall, 1965), it begs the question of whether this spinal disinhibition is a pivotal factor for the transition to a nociplastic pain state.

In the abovementioned murine model of nociplastic pain, we found that a nociplastic pain state is maintained by sexually dimorphic mechanisms (Hankerd et al., 2021). Specifically, spinal microglia maintain a nociplastic pain state only in males, as in chronic inflammatory and neuropathic pain models (Mapplebeck et al., 2018; Sorge et al., 2015; Taves et al., 2016). While this suggests that reactive spinal microglia are a common culprit for chronic pain in males, it raises a question of whether spinal microglia activation ‘drives’ the transition to the nociplastic pain state, and if so, whether this activation also utilizes a critical molecular pathway identified in other chronic pain conditions. In addition, considering that nociplastic pain differs from chronic inflammatory or neuropathic pain, it is important to understand how spinal microglia can be activated without chronic tissue injury or direct nerve damage. Therefore, in this study, we investigated whether spinal microglia activation drives the transition to the nociplastic pain state, and if so, how they are activated by normally innocuous stimuli after an acute tissue injury in the context of spinal disinhibition. Portions of these studies have been reported in abstract form (McDonough et al., 2021a, 2021b).

2. Methods

2.1. Animals

Adult male and female C57BL/6N mice (aged 7–9 weeks) were purchased from Charles River (Houston, TX, USA) or bred in-house. Mice were group-housed with up to 5 mice per cage on a 12-to-12-hour light-dark cycle and fed ad libitum. All experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Texas Medical Branch and in accordance with the National Institutes of Health (NIH) guidelines.

2.2. Animal Models of Pain

We utilized a model of nociplastic pain described previously (Hankerd et al., 2021). In brief, unilateral intraplantar capsaicin (0.1% in 10% ethanol, 10% Tween-20 in saline; Sigma-Aldrich, St. Louis, MO, USA) injection using a 30-G needle was followed two hours later by unilateral vibration stimulation (92 Hz) of the same hind paw. The vibration stimulation was applied focally to the hind paw using a Hitachi HV-1 mini massager (2.25mm2 contact surface with 13–22 mN/mm2 of pressure) repeatedly applied for 10 seconds at 30-second intervals (10 seconds on, 20 seconds off) over 10 minutes. Other experiments utilized nociplastic pain models lacking direct peripheral injury. Specifically, (+)-bicuculline (30 ng/5 μL in 10% DMSO, 1% Tween-20 in saline; Sigma-Aldrich) or CGP 52432 (342.5 ng/5 μL in saline; Tocris, Bristol, UK) was delivered intrathecally and followed 1 or 1.5 hours later by vibration stimulation as described above. Additionally, an animal model of intrathecal Brain-derived neurotrophic factor (BDNF) injection (Marcos et al., 2017; M’Dahoma et al., 2015; Zhou et al., 2011) was used, in which mice received a single intrathecal injection of human BDNF (10 ng/kg in saline, Alomone Labs, Jerusalem, Israel).

2.3. Drug Administration

For all injections, mice were anesthetized by 1.5–2.5% isoflurane, and injections were administered using a 30-G needle. Intraplantar injections were given at the base of the third and fourth digits of a hind paw. Intrathecal injections were given percutaneously, following the removal of fur from the injection site using clippers and sterilization of the area using ethanol and betadine; the needle was inserted between the L5 and L6 vertebrae (lumbar puncture), and its proper placement was confirmed by a brief tail-flick response upon penetration.

For experiments investigating the mechanisms of transition from acute to nociplastic pain, mice received a single intrathecal injection of (1) unconjugated saporin or Mac-1-saporin (8.85 μM, 5 μL; Advanced Targeting Systems, San Diego, CA, USA) immediately after intraplantar capsaicin injection or together with intrathecal GABAB receptor antagonist CGP 52432; (2) minocycline hydrochloride (0.15 mg, 5 μL, 10% DMSO, 0.5% Tween-20 in saline; Sigma-Aldrich) 1.5 hours after capsaicin or together with intrathecal GABAA receptor antagonist bicuculline; (3) the P2X4-selective antagonist PSB-12062 (70 ng, 5 μL, in 10% DMSO, Sigma-Aldrich) 1.5 hours after capsaicin; (4) recombinant human TrkB Fc chimera (2 μg, 5 μL, in saline; R&D Systems, Minneapolis, MN, USA) 1.5 hours after capsaicin or 30 minutes before intrathecal BDNF; or (5) GABA receptor agonists [muscimol (0.1 μg/ 5 μL in saline; Tocris) or (+)-baclofen (60 ng/ 5 μL in saline; Sigma-Aldrich)] 1.5 hours after capsaicin.

For experiments studying the mechanisms of nociplastic pain maintenance, 7–10 days after capsaicin injection, mice received a single intrathecal injection of (1) indomethacin (20 ug/ 5uL, 1% DMSO, 1% Tween-80 in saline; Sigma-Aldrich); (2) minocycline hydrochloride (150 μg, 5 μL, 10% DMSO, 0.5% Tween-20 in saline; Sigma-Aldrich); or (3) a cocktail of neutralizing antibodies against tumor necrosis factor (TNF)-α, interleukin (IL)-1 β, and IL-6 (0.167 μg each anti-TNF-α, anti-IL-1β, and anti-IL-6 in a 5 μL of phosphate buffered saline, R&D Systems) (Echeverry et al., 2017).

2.4. Behavioral test

Prior to conducting behavioral procedures, mice were habituated to behavioral test conditions and experimenters for 4 days. Mice were placed in acrylic chambers (14 cm length X 5 cm width X 4.5 cm height) on a raised metal grid-floor platform and were acclimated for 30 minutes prior to testing on the day of the experiment. Mechanical sensitivity of the hind paw was tested using a von Frey filament (0.98 mN) that evokes only 0–20% withdrawal responses in naïve mice. In mice that received intraplantar capsaicin injection, the hind paw which had been injected with capsaicin was tested, with probing occurring 4–5 mm proximal to the initially injured area (mid-hind paw). In mice receiving intrathecal GABA receptor antagonist injection, the mid-hind paw was probed. The percent of withdrawal responses out of 10 probing trials was recorded.

2.5. Immunohistochemistry

Seven to ten days after capsaicin plus vibration, bicuculline plus vibration, or CGP 52432 plus vibration, mice were perfusion fixed using phosphate-buffered saline followed by Lana’s fixative (4% paraformaldehyde and 14% picric acid in phosphate-buffered saline). The lumbar spinal cord (L3–L5) was resected and postfixed in Lana’s fixative for 4 hours and then dehydrated in 30% sucrose solution overnight. Samples were embedded in Tissue-Plus Optimal Cutting Temperature Compound (Fisher Health Care, Norwich, United Kingdom). Lumbar spinal cord sections (12 μm) were stained for Iba1 using an anti-Iba1 primary antibody (1:2,000, rabbit; Wako, Japan), followed by AlexaFluor 488-conjugated anti-rabbit secondary antibody (1:300; Thermo Fisher, Waltham, MA, USA). Stained sections were mounted with DAPI-containing media (Vectashield; Vector Laboratories, Burlingame, CA, USA). Confocal microscope images were analyzed to obtain the number of microglia per mm3 (i.e., cell density) within the dorsal horn by dividing the number of DAPI-positive, Iba1-immunoreactive cells by the volume of the region of interest. Cell count analysis was completed using ImageJ.

2.6. Statistical analyses

Data from behavioral experiments assessing mechanical sensitivity by counting the number of paw withdrawals out of 10 trials at multiple time points were presented as mean with standard error of the mean (SEM) and analyzed using a generalized linear mixed model (GLMM including random intercepts) with a logit link function for binomial distribution and the AR1 covariance structure for repeated measures. The Sequential Sidak test was used for multiple comparisons between groups at each time point or between time points (e.g., pre-drug vs. post-drug) within a group; the Satterthwaite approximation was used for effective degrees of freedom (SPSS ver. 25, IBM, Armonk, NY, USA).

Iba1-immunoreactive microglia density was obtained from each dorsal horn side of multiple sections from each mouse and presented in the figures as the mean of the multiple sections per mouse. For statistical comparisons, a multilevel analysis (with random intercepts) was used for this nested design (multiple sections nested within each mouse) with the scaled identity covariance type for repeated measures (dorsal horn side). We took an a priori approach for predetermining comparison pairs (e.g., 3 comparisons: ipsilateral vs. contralateral sides within each treatment, and ipsilateral sides between treatments). P-values were adjusted accordingly using the Bonferroni correction.

To perform simulation-based power analysis for mixed models, lme4 (Bates et al., 2012) and simr (Green and MacLeod, 2016) R packages were used, and sample size (i.e., the number of mice per group) was determined for ≥80% statistical power at alpha=0.05.

3. Results

Intraplantar capsaicin injection, as an experimental acute injury, normally produces secondary mechanical hypersensitivity (manifested as increased % paw withdrawals from von Frey filament probing of an area outside of the injection site) that is no longer present by 7 days post-injection in mice. Previously, we have shown that this acute injury-induced secondary mechanical hypersensitivity becomes persistent beyond the normal resolution time when the injured area is stimulated at normally innocuous intensity (e.g., warmth or vibration stimulation). This persistent mechanical hypersensitivity, observed ≥ 7 days after the inciting injury in our model, is regarded as nociplastic as it arises from altered nociception without evidence of nociceptor-activating tissue injury at the time of observation or nerve damage causing the hypersensitivity (Hankerd et al., 2021; Supplemental Fig. 1A). It should be noted that the vibration stimulation itself, which normally activates low-threshold mechanoreceptors (LTMRs), does not produce any changes in the hind paw mechanosensitivity in normal mice (Supplemental Fig. 1A). Importantly, as reported in the capsaicin plus warmth version of the nociplastic pain model (Hankerd et al., 2021), the nociplastic mechanical hypersensitivity 7–10 days after the capsaicin plus vibration was also mediated by activated spinal microglia in males (Supplemental Fig. 1B–D).

3.1. Proinflammatory cytokines, but not prostaglandins, maintain prolonged mechanical hypersensitivity

Activated microglia are known to release a variety of proinflammatory factors which contribute to pain hypersensitivity; these include proinflammatory cytokines such as IL-6, IL-1β, and TNF-α, as well as prostaglandins generated from the cyclooxygenase (COX) pathway, all of which have been implicated in neuropathic pain models (Echeverry et al., 2017; Ma et al., 2002). Having identified microglia as a key mediator of persistent mechanical hypersensitivity in our male nociplastic pain model, we investigated whether these inflammatory factors played a role in the maintenance of male nociplastic pain. Seven days after capsaicin plus vibration, we intrathecally injected either a cocktail of neutralizing antibodies to inhibit IL-6, IL-1β, and TNF-α, or the COX-inhibitor indomethacin. Mice treated with the cocktail of neutralizing antibodies showed significant attenuation of persistent mechanical hypersensitivity (Fig. 1A: F(1,8)=6.68, p=0.03). However, mice treated with indomethacin showed no change (Fig. 1B: F(1,9)=0.88; p=0.37). These data indicated that proinflammatory cytokines, but not prostaglandins, play a key role in maintaining prolonged mechanical hypersensitivity in our male model of nociplastic pain.

Fig. 1: Inflammatory cytokines, but not the cyclooxygenase (COX) pathway, mediate the nociplastic pain state in males.

Fig. 1:

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(A) Intrathecal injection of a cocktail of antibodies sequestering TNF-α, IL-1β, and IL-6 delivered 7–10 days after capsaicin plus vibration significantly attenuated persistent mechanical hypersensitivity. n=5 in each group. (B) However, intrathecal injection of indomethacin, an inhibitor of the COX pathway, did not alter the hypersensitivity. n=8 in vehicle group, n=7 in indomethacin group. *p<0.05, **p<0.01 vs. pre-drug level in each group by sequential Sidak multiple comparison tests.

3.2. Microglial inhibition prevents the transition to a nociplastic pain state in males

While our results indicate that activated spinal microglia ‘maintain’ the nociplastic pain state in male mice, it is unknown whether the ‘transition’ to this nociplastic pain state is driven by spinal microglia activation. To address this question, we inhibited spinal microglia activation during the transition phase by intrathecally injecting Mac-1-saporin (Fig. 2) or minocycline (Supplemental Fig. 2) after capsaicin injection but before the postinjury vibration stimulation. This transition phase-targeting approach prevented the development of nociplastic mechanical hypersensitivity (Fig. 2A: F(1,10)=18.66; p<0.01).

Fig. 2: Inhibiting spinal microglia before postinjury vibration stimulation prevents the transition from acute injury-induced pain to nociplastic pain.

Fig. 2:

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(A) Intrathecal injection of Mac-1-saporin (black arrow) after an experimental acute injury (capsaicin injection, red arrow) but prior to postinjury vibration stimulation (orange arrow) prevented the transition to persistent mechanical hypersensitivity. n=6 in each group; * p<0.05, **p<0.01 vs. vehicle at each timepoint by sequential Sidak multiple comparison tests. (B and C) Compared to the contralateral side of the spinal cord dorsal horn, the ipsilateral (to the capsaicin injected/vibrated hind paw) side showed significantly increased microglia density in the male capsaicin plus vibration model on day 7. This increase was not detected in mice treated with intrathecal Mac-1-saporin before vibration on day 0. n=5 in saporin control group, n=6 in Mac-1-saporin group; **adjusted p<0.01.

We previously found increased immunoreactivity of Iba1, a protein which is upregulated in activated microglia (Ito et al., 1998), in the ipsilateral (to the capsaicin-injected hind paw) dorsal horn during the maintenance phase of nociplastic pain state (≥ day 7) in males (Hankerd et al., 2021). In the present experiment, we likewise found that control mice (no microglia inhibitor before the postinjury stimulation) displayed the expected increase in the density of Iba1-expressing microglia in the ipsilateral dorsal horn compared to the contralateral side on day 7 post-capsaicin plus vibration. However, mice treated with Mac-1-saporin before the postinjury vibration stimulation did not show a significant difference in microglia density between the ipsilateral and contralateral dorsal horns on day 7 (Fig. 2B and C: Saporin: F(1,16)=19.76, p<0.01; Mac-1-saporin: F(1,63)=0.56, p=1), showing that spinal microglia drive the postinjury stimulation-triggered transition to a nociplastic pain state maintained by activated spinal microglia in males.

3.3. The ATP-P2X4 pathway, but not BDNF-TrkB signaling, mediates the transition to a nociplastic pain state in males

Having found that spinal microglial activation is necessary for postinjury vibration stimulation to trigger the transition to a nociplastic pain state in males, we next sought to identify the molecular mechanism(s) underlying this microglia-driven transition. In animal models of neuropathic pain, microglial activation has been shown to require the activation of P2X4 receptors (Inoue et al., 2007; Trang et al., 2012). P2X4 receptor binding with ATP increases BDNF secretion from microglia, and this microglia-derived BDNF, in turn, prolongs microglia activation through the activation of its receptor TrkB (Zhang et al., 2014). The BDNF-TrkB signaling has been also shown to long-term potentiate C-fiber synaptic responses in a microglia-dependent manner (Zhou et al., 2011) and mediate chronic inflammatory and neuropathic pain in animal models (Coull et al., 2005; Ding et al., 2020; Obata and Noguchi, 2006; Renn et al., 2011). Therefore, we examined if the spinal microglia-driven transition to a nociplastic pain state is mediated by the ATP-P2X4 and BDNF-TrkB pathways. To this end, we first inhibited the P2X4 receptors with an intrathecal injection of the antagonist PSB 12062 following capsaicin injection but prior to vibration. This was able to block the transition to a nociplastic pain state in male mice (Fig. 3: t(48)=11.14, p<0.001 at 7 days post-capsaicin by sequential Sidak test following GLMM). Having found that the ATP-P2X4 pathway was involved in the transition to a nociplastic pain state, we next examined the involvement of the BDNF-TrkB pathway. To do so, we intrathecally injected TrkB Fc chimera to sequester BDNF following capsaicin injection but prior to vibration stimulation. This inhibition of the BDNF-TrkB pathway did not prevent the development of nociplastic mechanical hypersensitivity (Fig. 4A). We validated our experimental approach of inhibiting the BDNF-TrkB pathway by confirming the efficacy of intrathecal TrkB Fc chimera pretreatment against intrathecal BDNF-induced mechanical hypersensitivity (Fig. 4B: F(1,22)=8.10; p<0.01). Taken together, these results indicated that the microglia-driven transition to a nociplastic pain state in males involves the ATP-P2X4 pathway, but not the BDNF-TrkB signaling.

Fig. 3: The ATP-P2X4 pathway mediates the transition to a nociplastic pain state in males.

Fig. 3:

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Intrathecal injection of PSB 12062 (purple arrow), a P2X4 receptor antagonist, prevented the prolongation of capsaicin (red arrow)-induced mechanical hypersensitivity by vibration (orange arrow). n=4 in each group. *p<0.05, **p<0.01 vs vehicle control by sequential Sidak multiple comparison tests.

Fig. 4: The TrkB-BDNF pathway does not mediate the transition to a nociplastic pain state in males.

Fig. 4:

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(A) Intrathecal injection of TrkB Fc chimera (blue arrow), which sequesters BDNF, following capsaicin injection but before vibration failed to prevent the transition to persistent mechanical hypersensitivity. n=5 in each group. (B) The same dose of TrkB Fc chimera injection as in (B) was able to significantly attenuate BDNF (black arrow)-induced mechanical hypersensitivity. n=8 in each group. **p<0.01 vs vehicle control by sequential Sidak multiple comparison tests.

3.4. GABAergic disinhibition underlies the transition to a nociplastic pain state in males

The gate-control theory of pain highlights how injury-evoked nociceptor inputs cause spinal disinhibition, allowing LTMRs to activate transmission neurons leading to the perception of pain (Melzack, 1996; Torsney and MacDermott, 2006). As our vibration stimulation normally activates LTMRs, we hypothesized that injury-induced spinal GABAergic disinhibition is critical for postinjury vibration stimulation to trigger the spinal microglia-driven transition to a nociplastic pain state. To test this hypothesis, we increased spinal GABAergic inhibition during the transition phase by intrathecally injecting muscimol (GABAA receptor agonist) or baclofen (GABAB receptor agonist) after capsaicin injection but before postinjury vibration stimulation. The GABA receptor agonists prevented the development of nociplastic mechanical hypersensitivity (Fig. 5A: Muscimol: t(15)=4.08, p<0.01 by sequential Sidak test; Baclofen: t(15)=3.55, p<0.01 by sequential Sidak test) and an increase in microglia density in the ipsilateral dorsal horn on day 7 post-capsaicin plus vibration (Fig. 5B and C: Vehicle: F(1,92)=26.04, p<0.01; Muscimol: F(1,83)=0.06, p=1; Baclofen: F(1,83)=0.20, p=1). Conversely, in the next experiment, we investigated if spinal GABAergic disinhibition alone (i.e., without a direct peripheral injury such as intraplantar capsaicin injection) would be sufficient for vibration stimulation to trigger a transition to a nociplastic pain state. Similar to previous reports (Lee and Lim, 2010; Malan et al., 2002; Minami et al., 1994), we found that a single intrathecal injection of the GABAA receptor antagonist bicuculline or the GABAB receptor antagonist CGP 52432 produced transient mechanical hypersensitivity in both male and female mice, which peaks at 1 hour post-injection in males, and 1.5 hours in females (data not shown). We applied vibration stimulation at these peak-hypersensitivity timepoints following intrathecal bicuculline injection for each sex. This procedure produced persistent mechanical hypersensitivity lasting at least 21 days in males (Fig. 6A: F(1,12)=96.18, p<0.01) only in the hind paw ipsilateral to the vibration stimulation (Supplemental Fig. 3). In contrast, bicuculline-induced mechanical hypersensitivity was not prolonged by the vibration stimulation in females (Fig. 6B: F(1,12)=0.00, p=0.98), indicating a dramatic sex-difference in the spinal GABAergic disinhibition-associated mechanisms for producing the transition from acute pain to nociplastic pain.

Fig. 5: Increasing spinal GABAergic inhibition before postinjury vibration stimulation prevents the transition from acute injury-induced pain to nociplastic pain.

Fig. 5:

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(A) Intrathecal injection (black arrow) of the GABAA receptor agonist muscimol or the GABAB receptor agonist baclofen after capsaicin injection (red arrow) but prior to vibration (orange arrow) prevented the transition to persistent mechanical hypersensitivity in males. n=6 in vehicle group, n=5 in muscimol group, and n=5 in baclofen group; ++p<0.01 vs. vehicle in baclofen-treated group, **p<0.01 vs. vehicle in muscimol-treated group by sequential Sidak multiple comparisons test. (B and C) Compared to the contralateral side of the spinal cord dorsal horn, the ipsilateral (to the capsaicin injected/vibrated hind paw) side showed significantly increased microglia density in vehicle-treated mice on day 7. This increase was not detected in mice treated with intrathecal muscimol or baclofen before vibration on day 0. n=5 in each group; **adjusted p<0.01.

Fig. 6: Without direct peripheral injury, nociplastic pain develops by paw vibration stimulation under the acute impairment of GABAA-mediated spinal inhibition only in males.

Fig. 6:

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Chronification of bicuculline (a GABAA receptor antagonist, black arrow)-induced mechanical hypersensitivity by vibration stimulation (orange arrow) occurred in (A) males, but not in (B) females. The vibration was applied at the time of peak mechanical hypersensitivity response to bicuculline injection: 1 h post-injection in males, 1.5 h post-injection in females. Males: n=5 in each group, females: n=5 in each group. *p<0.05, **p<0.01 vs bicuculline control by sequential Sidak multiple comparison tests.

Likewise, after intrathecal injection of CGP 52432, unilateral hind paw vibration produced persistent mechanical hypersensitivity in males, lasting at least 21 days (Fig. 7A: F(1,8)=84.39, p<0.01). Interestingly, unlike in female mice that received intrathecal bicuculline, CGP 52432-treated females also developed prolonged mechanical hypersensitivity after vibration stimulation (Fig. 7B: F(1,11)=15.14, p<0.01), indicating that the sex-differences in the spinal disinhibition-associated mechanisms for the transition to a nociplastic pain state are GABA receptor type-specific.

Fig. 7: Without direct peripheral injury, nociplastic pain develops by paw vibration stimulation under the acute impairment of GABAB-mediated spinal inhibition in both sexes.

Fig. 7:

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Chronification of CGP 52432 (a GABAB receptor antagonist, black arrow)-induced mechanical hypersensitivity by vibration stimulation (orange arrow) occurred in both (A) males and (B) females, lasting at least 21 days. The vibration was applied at 1 h and 1.5 h post-injection in males and females, respectively. Males: n=5 in each group, females: n=5 in each group. *p<0.05, **p<0.01 vs CGP 52432 control by sequential Sidak multiple comparison tests.

Having determined that a state of nociplastic mechanical hypersensitivity could be produced by spinal disinhibition followed by normally innocuous peripheral stimulation in the absence of an actual injury in the periphery, we further assessed whether the nociplastic mechanical hypersensitivity produced by bicuculline plus vibration was also maintained by activated spinal microglia. Thus, 7–10 days after bicuculline plus vibration, we intrathecally injected Mac-1-saporin. This spinal microglia inhibition significantly attenuated the persistent mechanical hypersensitivity in male mice (Fig. 8A: F(1,8)=7.10; p=0.03), indicating that spinal microglia contribute to the maintenance of the nociplastic pain state in this bicuculline plus vibration model similar to that in the capsaicin plus vibration model. In the spinal cord obtained 7–10 days after bicuculline plus vibration, microglia density was found to be increased in the ipsilateral (to the vibrated hind paw) dorsal horn, which was not found in the group treated with Mac-1-saporin on days 7–10 post-bicuculline plus vibration (Fig. 8B and C: Saporin ipsi vs. contra: F(1,37)=31.84, p<0.01; Mac-1-sap ipsi vs. contra: F(1,58)=0.11, p=1).

Fig. 8: Spinal microglia maintain nociplastic pain that has developed by paw vibration stimulation under the acute impairment of GABAA-mediated spinal inhibition in males.

Fig. 8:

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(A) Intrathecal injection of Mac-1-saporin 7–10 days after bicuculline plus vibration attenuated persistent mechanical hypersensitivity in males. n=7 in Mac-1-saporin treated group, n=6 in saporin treated group. *p<0.05 vs. pre-drug level in each group by sequential Sidak multiple comparison tests. (B and C) Compared to the contralateral side of the spinal cord dorsal horn, the density of microglia in the ipsilateral (to the vibrated hind paw) side was significantly increased in the male bicuculline plus vibration nociplastic pain model. This increase was not detected in mice treated with intrathecal Mac-1-saporin. n=6 in Mac-1-saporin group, n=5 in saporin group; **adjusted p<0.01.

In the CGP 52432 plus vibration model, the maintenance of the nociplastic pain state was likewise mediated by activated spinal microglia in males (Fig. 9A: F(1,8)=15.26, p<0.01), but not females (Fig. 9D: F(1,8)=0.59, p=0.47). In the male spinal cord obtained 7–10 days after CGP 52432 plus vibration, we found a corresponding increase in microglia density in the ipsilateral dorsal horn, which was not found in the group treated with Mac-1-saporin on days 7–10 post-CGP 52432 plus vibration (Fig. 9B and C: Saporin ipsi vs.contra: F(1, 51)=55.66, p<0.01; Mac-1-sap ipsi vs. contra: F(1,52)=0.07, p=1). By contrast, in the female spinal cord obtained 7–10 days after CGP 52432 plus vibration, no increase in microglia density was detected in the ipsilateral dorsal horn compared with the contralateral side (Fig. 9E and F).

Fig. 9: Spinal microglia maintain nociplastic pain that has developed by paw vibration stimulation under acute impairment of GABAB-mediated spinal inhibition only in males.

Fig. 9:

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(A) Intrathecal injection of Mac-1-saporin 7–10 days post-CGP 52432 plus vibration attenuated persistent mechanical hypersensitivity in males. Males: n=5 in each group, females: n=5 in each group. *p<0.05, **p<0.01 vs. pre-drug level in each group by sequential Sidak multiple comparison tests. (B and C) Compared to the contralateral side of the spinal cord dorsal horn, the density of microglia in the ipsilateral (to the vibrated hind paw) side was significantly increased in the male CGP 52432 plus vibration nociplastic pain model. This increase was not detected in mice treated with intrathecal Mac-1-saporin. n=5 in each group; **adjusted p<0.01. (D) Intrathecal injection of Mac-1-saporin 7–10 days post-CGP 52432 plus vibration did not attenuate persistent mechanical hypersensitivity in females. (E and F) Females did not display a significant difference in microglia density between the ipsilateral and contralateral spinal cord dorsal horns.

Having determined that spinal microglia contributed to the maintenance of the nociplastic pain state induced by (intrathecal) GABA receptor antagonist plus (normal hind paw) vibration in males, we next examined whether the transition to this pain state is also mediated by spinal microglia as in the capsaicin plus vibration model. To this end, we intrathecally injected GABA antagonist together with a microglia inhibitor; bicuculline with minocycline (Mac-1-saporin could not be used as it was not compatible with the vehicle of bicuculline), and CGP 52432 with Mac-1-saporin. Vibration stimulation was applied to one hind paw 1 hour later. As shown in Fig. 10, while vibration exacerbated the GABA receptor antagonist-produced mechanical hypersensitivity in these mice, the hypersensitivity completely resolved by day 7, indicating a failure to transition to a nociplastic pain state.

Fig. 10: Spinal microglia mediate the transition to a nociplastic pain state by paw vibration stimulation under acute impairment of spinal GABAergic inhibition in males.

Fig. 10:

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When microglia inhibitors were given before vibration stimulation, the vibration transiently exacerbated the mechanical hypersensitivity induced by intrathecal (A) bicuculline or (B) CGP 52432 but failed to prolong the hypersensitivity beyond its normal resolution time in male mice (c.f., Figs. 6A and 7A). n=5 in each group. **p<0.01 vs. control (i.e., no vibration) by sequential Sidak multiple comparison tests.

4. Discussion

This study demonstrates that spinal microglia is necessary for the transition from acute injury-induced pain to chronic nociplastic pain in males (Fig. 2) and that the ATP-P2X4 pathway mediates this transition (Fig. 3). These findings are in line with the body of literature reporting a significant role of this purinergic signaling pathway in microglia activation mediating chronic neuropathic and inflammatory pain (Guo et al., 2005; Inoue et al., 2007; Trang et al., 2012). The ATP-P2X4 pathway activates microglia in a manner that increases BDNF release from microglia, prolonging microglial activation (Zhang et al., 2014). However, while BDNF sequestration via TrkB-Fc chimera inhibited ATP-induced microglia activation in the same study (Zhang et al., 2014), we were unable to prevent the microglia-driven transition to nociplastic pain in our model using the same approach (Fig. 4A). Therefore, it appears that while activation of spinal microglia for/during the transition to a chronic pain state depends on the ATP-P2X4 pathway as in other chronic pain models, this process does not lead to or involve the BDNF-TrkB pathway during the transition phase in our nociplastic pain model.

The literature reports that suppression of spinal GABAergic inhibition (i.e. disinhibition) allows Aβ-fibers to activate dorsal horn nociceptive circuitry, resulting in Aβ-fiber-mediated mechanical allodynia (Duan et al., 2014; Melzack and Wall, 1965; Peirs et al., 2015). However, whether these Aβ-fibers are also capable of activating spinal microglia within the context of disinhibition remains unknown. Interestingly, it has been shown that in male rats, a brief intense electrical stimulation of C-fibers, but not Aβ- or Aδ-fibers, activates dorsal horn microglia, and induces long-lasting hypersensitivity (Hathway et al., 2009). However, in our model, even vibration that normally activates Aβ-fibers triggers a transition to long-lasting mechanical hypersensitivity, with a key difference being that the pain circuit is already in a sensitized state by C-fiber inputs due to the inciting capsaicin injury (in the capsaicin plus vibration model). Considering that intense nociceptor inputs cause disinhibition in the dorsal horn to result in the activation of nociceptive circuitry by normally innocuous stimuli (Pernía-Andrade et al., 2009), it is possible that acute injury-induced disinhibition in the dorsal horn plays a key role in the spinal microglial activation by low-threshold afferent inputs and consequently, in the transition from an acute to a nociplastic pain state in males. Here, we provided support for this hypothesis by showing that boosting spinal GABAergic inhibition before the postinjury vibration stimulation prevented the prolongation of mechanical hypersensitivity and the associated activation of spinal microglia (Fig. 5). We further demonstrated that, even in the absence of an acute peripheral injury, inhibiting either GABAA or GABAB receptors at the spinal level and applying vibration stimulation was sufficient for producing a nociplastic pain state in males (Figs. 6A and 7A), which is similarly mediated by activated microglia (Figs. 8, 9A–C, and 10). Collectively, these findings suggest that low-threshold afferent inputs can activate spinal microglia in the dorsal horn in males, thus producing a prolonged state of mechanical hypersensitivity; however, spinal GABAergic inhibition functions as a gate that normally blocks such microglia activation in males.

The present findings may seem to be at odds with previous studies showing that activation of low-threshold Aβ-fibers (clinically by spinal cord stimulation, for example) inhibits C-fiber synaptic strengths on dorsal horn neurons and relieves chronic neuropathic pain (Sdrulla et al., 2015; Yang et al., 2016). According to the gate-control theory of pain, the role of Aβ afferent inputs in pain exacerbation vs. relief is critically determined by the status of the “gate.” In pain conditions where a gate-opening drive prevails by intense nociceptor inputs (e.g., intraplantar capsaicin) or a gate-closing drive by low-threshold Aβ-fiber inputs does not produce results (e.g., intrathecal GABA receptor antagonists), vibration-generated Aβ-fiber inputs will produce/exacerbate pain. What is most notable in this study is that when the gate is open, Aβ-fiber inputs not only exacerbate pain but also prolong it to become nociplastic pain because the inputs end up activating spinal microglia in males (but not in females as discussed further on). In chronic pain conditions where stimulating Aβ-fibers relieves pain, it must be that the gate properly responds to Aβ-fiber inputs which are still able to function as an effective ‘gate-closing’ drive. Therapeutic approaches such as spinal cord stimulation to activate Aβ-fibers are likely to boost the drive to enhance the inhibitory tone in spinal nociceptive circuits.

Interestingly, in females, we saw that inhibiting GABAB receptors (with CGP 52432), but not GABAA receptors (with bicuculline), prior to vibration was sufficient to produce a nociplastic pain state. While the cause of this receptor-specific sex difference requires further investigation, this study clearly demonstrates that in the female CGP 52432 plus vibration model, the nociplastic pain state is not mediated by spinal microglia unlike in its male counterpart (Fig. 9). The male-specific spinal microglia activation by LTMR inputs in the spinal GABAergic disinhibition state begs a question of whether spinal microglia activation is gated by a different type of inhibitory control in females. It is our immediate interest in a future study to examine whether spinal ‘glycinergic’ disinhibition is different from the ‘GABAergic’ one in terms of sex-dependent spinal microglia activation by LTMR inputs.

As mentioned above, it remains to be elucidated by what mechanisms the nociplastic pain is induced and maintained in the female CGP 52432 plus vibration model. In our previous study, ongoing primary afferent activity at the previously capsaicin-injected area was found to maintain the nociplastic pain state in the female capsaicin plus warmth model (Hankerd et al., 2021). Considering that GABAB receptor activity modulates the release of L-glutamate from C-fiber central terminals (Ataka et al., 2000; Wang et al., 2007), it could be that inhibition of GABAB receptors potentially allow ongoing activity of central terminals of primary afferents in females, driving the nociplastic pain state.

Activated microglia are known to cause neuroinflammation in the central nervous system. In line with this, we found that the nociplastic pain state in males, shown to be microglia-driven in both our present and previous studies, was maintained by proinflammatory cytokines TNF-α, IL-1β, and IL-6 (Fig. 1A), all of which are known to be released from microglia and have been shown to play a key role in nociception, including chronic neuropathic pain (Echeverry et al., 2017; Ji et al., 2014). Interestingly, prostaglandins also play a key role in other pain conditions and are also released by microglia (Kanda et al., 2013; Ma et al., 2002), but were not found to play a significant role in our nociplastic pain model. Considering that IL-1β upregulates COX-2, prostaglandin E2, and its receptor EP4 in a cell culture system (Endo et al., 1995; Watanabe et al., 2009), it seems counterintuitive that indomethacin, a non-selective COX inhibitor, had no significant effect while proinflammatory cytokines effectively alleviated nociplastic mechanical hypersensitivity in our model. It could be that TNF-α and/or IL-6 have a greater functional contribution to mechanical hypersensitivity than IL-1β in our nociplastic pain model, which cannot be distinguished because of our ‘cocktail approach’ toward inhibiting these cytokines. It is also important to note that the effect of COX inhibitors, including indomethacin, on neuropathic pain (known to be cytokine-mediated (Gui et al., 2016; Hung et al., 2017) is inconsistent across animal studies. For instance, in the chronic constriction injury model, indomethacin effectively alleviated mechanical hypersensitivity only in the early phase (1–4 days after the nerve injury) but not in the late phase (≥ 7 days) (Medeiros et al., 2020). Furthermore, while an intrathecally injected inhibitor selective to either COX-1 or COX-2 attenuated neuropathic mechanical hypersensitivity in the partial sciatic nerve ligation model, a non-selective COX inhibitor was without effect (Ma et al., 2002).

In conclusion, we have shown that both the transition to and maintenance of male nociplastic pain are mediated by the complex interplay between the spinal GABAergic inhibitory system and spinal microglia. In males, GABAergic disinhibition at the level of the spinal cord is pivotal for a transition to a nociplastic pain state when followed by postinjury activation of low-threshold afferents (e.g., by vibration stimulation), suggesting that microglia activation by low-threshold afferent inputs is gated by the spinal GABAergic inhibitory system in males. These findings indicate that preemptively inhibiting spinal microglia or increasing spinal GABAergic inhibitory tone during acute nociceptive pain may help prevent the development of chronic nociplastic pain in males.

Supplementary Material

Supple Fig 3
Supple Fig 2
Supple Fig 1

Acknowledgments

The authors thank the UTMB Optical Microscopy Core for use of their facilities. This study was supported by NIH R01 NS112344 and Supplement to Promote Diversity (J.H.L.), F99 NS120636 (K.E.M.), the Jeane B. Kempner Predoctoral Fellowship (K.M.H.), and R01 DA050530 (J.M.C).

Footnotes

Declaration of interest: none

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Supplementary Materials

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Supple Fig 2
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Supple Fig 1
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