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INTRODUCTION
Central sensitization is considered a key mechanism underlying the development and maintenance of neuropathic pain and is primarily associated with neuroinflammation in the central nervous system (CNS) [1]. During the process of neuroinflammation, activated microglia and astrocytes release various inflammatory mediators that promote central sensitization, thereby excessively amplifying pain signal transmission and contributing to the persistence of chronic pain [2]. Recently, central sensitization driven by neuroinflammation is suggested as an important mechanism for conditions like fibromyalgia and complex regional pain syndrome [3,4].
Most animal models of neuropathic pain are established through intense and persistent stimulation of peripheral nerves, typically via direct nerve injury [5,6]. However, it has been reported that neuroinflammation and pain hypersensitivity can also be induced in the absence of peripheral nerve injury through immune activation of the CNS using toll-like receptor (TLR) agonists [7–9]. Additionally, selective stimulation of spinal astrocytes utilizing optogenetic techniques can induce mechanical pain hypersensitivity [10].
Mincle (macrophage-inducible C-type lectin), a member of the C-type lectin receptor family, belongs to membranous pattern recognition receptor along with TLR, and plays a crucial role in innate immune signaling [11]. While TLR is well known for its role in pain signaling, the role of Mincle in neuropathic pain is still unclear. However, Mincle has been shown to be involved in the process of neuroinflammation in animal models of subarachnoid hemorrhage, traumatic brain injury, and ischemic stroke [12–14], suggesting the possibility of involvement of Mincle in pathologic neuroinflammatory processing in neuropathic pain. In a peripheral nerve injury model, increased Mincle mRNA expression was observed in the dorsal root ganglion [15]. Recent studies showed that single intrathecal (i.t.) injection of Mincle ligand induced mechanical allodynia in naïve rats [16], and administration of antibody to Mincle attenuated the pain behavior in a spinal nerve ligation (SNL) model [17]. However, little is known about long-term pain patterns and neuroglial reactions in Mincle activation-induced pain and any difference from established pain models.
The present study aimed to establish an optimized model for inducing neuroinflammation and pain hypersensitivity through Mincle activation, and to compare its characteristics with those of the conventional SNL model of neuropathic pain.
MATERIALS AND METHODS
1. Experiment animals
All experiments were performed in accordance with the International Association for the Study of Pain guidelines for the Use of Animals in Research. The protocol was approved by the Institutional Animal Care and Ethical Use Committee (CNUHIACUC-24024) of the Chonnam National University Hospital. Male Sprague-Dawley rats (250–270 g) were housed in a temperature-controlled room (22°C–23°C) under a 12-hour light/dark cycle with free access to food and water.
2. Intrathecal administration of Mincle ligand for inducing pain
All experimental agents were administered using a polyethylene catheter implanted into the i.t. space [18]. Under general anesthesia with sevoflurane, the catheter was inserted through the atlanto-occipital membrane and advanced 8.5 cm caudally to the lumbar enlargement. Rats exhibiting neurological deficits following catheter implantation were immediately euthanized with an overdose of inhaled anesthetic. After surgery, all rats were housed individually and allowed a 5-day recovery period before i.t. administration of trehalose-6,6'-dibehenate (TDB; InvivoGen), a synthetic Mincle ligand. TDB was dissolved in 10% (v/v) dimethyl sulfoxide and delivered to the i.t. space in a volume of 10 μL, followed by an additional 10 μL normal saline to flush the catheter using a hand-driven, geared syringe pump.
TDB was administered at varying doses and injection frequencies across two sets of animals. On the first day of the experiment (day 0), rats received a single i.t. injection of TDB at one of three doses (0.1 µg, 1 µg, and 10 µg; n = 8 per dose). In a separate set of animals, TDB (10 µg) was administered either as a single injection on day 0 (single injection group, S-TDB; n = 8) or as repeated injections on days 0 and 1 (repeated injection group, R-TDB; n = 8). The dose of 10 µg for the R-TDB group was selected based on previous studies demonstrating the effect of i.t. injection to fully saturate the Mincle receptor and ensure the reproducibility and consistency of the experiment.
3. Neuropathic pain model induced by spinal nerve ligation
To establish a peripheral nerve injury-induced neuropathic pain, the surgery for SNL was performed [19]. Under sevoflurane anesthesia, the left L5 and L6 spinal nerves were isolated near the vertebral column and tightly ligated distal to the dorsal root ganglion using a 6-0 silk suture. The sham group (n = 8) underwent the same surgical procedure as the SNL group (n = 8), except that the L5 and L6 spinal nerves were not ligated.
4. Behavioral pain assessment
Behavioral assessment for mechanical allodynia and thermal hyperalgesia was conducted on days 0, 1, 3, 5, 7, 10, 14, 21, and 28. To evaluate the paw withdrawal threshold (PWT), von Frey filaments (0.41–15.2 g) were applied perpendicularly to the central plantar surface of the hind paw through a mesh floor. Each stimulus was applied for 5 seconds or until the paw was withdrawn or licked, which was recorded as a positive response. The up-down method was used to calculate thresholds, with a cutoff value of 15 g recorded if no withdrawal or licking response was observed.
For the paw withdrawal latency (PWL) test, animals were placed in transparent acrylic boxes for acclimation. A focused heat beam was applied to the plantar surface of the hind paw using a Hargreaves apparatus, and the time taken for the animal to withdraw its paw was recorded as the PWL. If neither a withdrawal nor a licking response was observed, the cutoff time (20 seconds) was recorded. The average of two repeated measurements was used to quantify hypersensitivity. All behavioral experiments were performed by the same researcher, who was blind to the injectate.
The hyperalgesic response was quantified by calculating the area under the curve (AUC) of percent change from baseline. For PWT, the percent hyperalgesic effect at each time point was defined as (baseline PWT – post-treatment PWT) / baseline PWT × 100 [20]. Time (days after TDB injection or SNL) was plotted against this percentage, and the enclosed area was summed to yield the AUC. PWL data were processed using the same method. In this analysis, the AUC represents the cumulative deviation below baseline, such that larger values indicate a greater overall magnitude of mechanical allodynia (for PWT) or thermal hyperalgesia (for PWL) over the observation period.
5. Immunofluorescence study
The spinal cords of animals of the R-TDB (n = 4) and SNL (n = 4) groups were subjected to immunofluorescence study. Samples were collected on days 1, 3, 7, 14, and 28. After deep anesthesia with sevoflurane, rats were transcardially perfused with 0.9% sterile saline, followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4). The lumbar enlargement of the spinal cord was dissected and fixed overnight at 4°C in 4% paraformaldehyde. The tissues were then immersed in 30% sucrose in 0.1 M PBS until they sank, embedded in optimal cutting temperature (OCT) compound on dry ice, and cryosectioned to a thickness of 10 μm.
For immunofluorescence staining, sections were blocked for 1 hour at room temperature with 3% (v/v) normal chicken serum containing 0.3% Triton X-100 and incubated overnight at 4°C with the following primary antibodies: rabbit anti-Iba1 polyclonal antibody (1:100; Wako Chemicals) or rabbit anti-GFAP polyclonal antibody (1:100; Dako). Antibody binding was visualized by incubating the sections with chicken anti-rabbit IgG conjugated to Alexa Fluor 488 (1:1,000; Invitrogen) for 1 hour at room temperature. After washing with 0.01 M PBS, the sections were coverslipped, and immunofluorescence images were captured at 20× magnification using a fluorescence microscope (EVOS M5000; Thermo Fisher Scientific). Immunoreactivity was quantified using ImageJ (version 1.53) by measuring density and calculating the percentage of the stained target area within the defined spinal dorsal horn.
6. Statistical analysis
Statistical analyses were performed using IBM SPSS (version 29.0.1; IBM). Data obtained from the von Frey test and Hargreaves test were expressed as mean ± standard error of the mean. Differences in the time course data for PWT or PWL were analyzed using repeated measures analysis of variance (ANOVA) followed by Bonferroni post hoc tests. In addition, comparisons between the SNL group and the R-TDB group at each time point were performed using independent t-tests. The AUC for hyperalgesia of PWT or PWL were analyzed using independent t -test or a one-way ANOVA followed by Bonferroni post hoc tests. The immunofluorescence intensity of Iba1 and GFAP was analyzed using one-way ANOVA followed by Bonferroni post hoc tests. A P value < 0.05 was considered statistically significant.
RESULTS
To evaluate behavioral responses based on concentration, S-TDB group (single injection on day 0) was administered i.t. TDB at three different concentrations (0.1 µg, 1 µg, and 10 µg). As a result, the PWT (Fig. 1A) and PWL (Fig. 1B) of the left hind paw significantly decreased from day 1 to day 28 in all the doses. A similar pattern was observed in the right hind paw (data not shown). However, no significant differences among the doses were observed in the AUC for hyperalgesia of PWT (369 ± 68 [0.1 µg], 390 ± 62 [1 µg], and 405 ± 65 [10 µg]; Fig. 1C) and PWL (199 ± 20 [0.1 µg], 200 ± 10 [1 µg], and 197 ± 13 [10 µg]; Fig. 1D).
In another experiment using the S-TDB and R-TDB group (repeated injection on day 0 and 1), PWT and PWL began to decrease from day 1 after TDB administration in both groups (Fig. 2A, B). On day 3 after the first TDB administration, the R-TDB group showed a significantly greater reduction in PWT compared to the S-TDB group, and this difference was maintained until day 28 (Fig. 2A). The AUC for hyperalgesia of PWT was significantly higher in the R-TDB group (552 ± 29) than in the S-TDB group (320 ± 51; Fig. 2C). However, no significant differences in PWL (Fig. 2B) or the AUC for hyperalgesia were observed between the two groups (223 ± 25 [R-TDB] vs. 206 ± 19 [S-TDB]; Fig. 2D).
In the SNL group, PWT and PWL of the left hind paw significantly decreased starting on day 1 after SNL (Fig. 3A, B). The AUC for hyperalgesia was 490 ± 51 for PWT (Fig. 3C) and 144 ± 31 for PWL (Fig. 3D), respectively. In comparison with the R-TDB group, the SNL group exhibited a significantly lower PWT on day 1 after surgery, but no difference in PWL was observed (see Supplementary Table 1). In addition, no significant differences were observed between the two groups in both PWT and PWL at subsequent time points (days 3–28).
The expression of microglia and astrocytes in the spinal cord was compared between the R-TDB and SNL groups using immunofluorescence staining on days 1, 3, 7 (Fig. 4). The immunoreactivity for Iba1 and GFAP increased earlier in the R-TDB group than the SNL group (Fig. 4A, B). Iba1 immunoreactivity in the R-TDB group was significantly increased on day 1 compared to vehicle. In contrast, in the SNL group, it began to significantly rise on day 3 compared to the sham group (Fig. 4C). GFAP immunoreactivity started to increase on day 3 in the R-TDB group, but on day 7 in the SNL group (Fig. 4D). In both groups, however, the immunoreactivity (GFAP, Iba1) remained elevated until day 28. The immunoreactivity levels of Iba1 and GFAP on days 14 and 28 showed no significant differences compared to day 7 (data not shown in Fig. 4, see Supplementary Fig. 1).
DISCUSSION
Neuroinflammation and central sensitization in the CNS play a crucial role in the development and maintenance of neuropathic pain. Animal models such as SNL or carrageenan inflammation are regarded as essential tools for pain research to understand the mechanisms of neuroinflammation induced by peripheral nerve injury or tissue inflammation [21]. These models replicate key aspects of pain responses by inducing direct peripheral nerve injury or intense tissue inflammation, which subsequently triggers neuroinflammation in the CNS, central sensitization, and maladaptive neural plasticity as pain persists [22,23]. In these models, however, the changes such as sensitization or neuroinflammation can occur simultaneously in both the peripheral and CNS because peripheral injury or inflammation does not resolve. As a result, it is difficult to clearly determine whether the persistence of pain is primarily due to ongoing peripheral injury or neuroinflammatory changes within the CNS.
Mincle is a key pattern recognition receptor involved in innate immune responses and is primarily expressed in myeloid cells including macrophages [11]. Its expression has also been detected in various cell types within the CNS, including neurons, microglia, and CNS-resident macrophage-like cells [12–14]. In the spinal cord, Mincle is distributed throughout the gray matter, including both the dorsal and ventral horns [16]. While the precise mechanisms by which Mincle activation leads to neuroglial activation and pain hypersensitivity remain unclear, previous studies have identified the Mincle–spleen tyrosine kinase (Syk) signaling pathway as a critical downstream mediator of its immune functions [24,25]. The Mincle/Syk axis has been implicated in several neuroinflammatory disease models and is known to contribute to microglial activation and the production of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β [12,14]. It is plausible that these inflammatory processes enhance the excitability of dorsal horn neurons in the spinal cord, thereby contributing to the development and maintenance of neuropathic pain, as observed in the present study.
Prior work demonstrated that spinal Mincle activation can induce mechanical allodynia and microglial activation [16], and that Mincle antibodies alleviate pain behavior in the SNL model [17]. However, these studies did not examine long-term behavioral changes, glial activation patterns, or compare Mincle activation with a peripheral nerve injury model. In contrast, our study systematically assessed both mechanical and thermal hypersensitivity over 28 days and evaluated glial activation (Iba1, GFAP) over the same period. We also provide a head-to-head comparison with the SNL model, demonstrating that Mincle activation can replicate several features of nerve injury-induced neuroinflammation.
The activation of Mincle by i.t. TDB is mechanistically comparable to TLR activation, which leads to NF-κB activation via the MyD88-dependent pathway [26]. Similar to Mincle ligand, i.t injection of a TLR agonist such as lipopolysaccharide (LPS) has been shown to induce mechanical allodynia [8,9,27]. However, in these TLR-related studies, the onset of mechanical allodynia occurred within 1 hour and resolved within 48 hours and thermal hyperalgesia was not assessed. In those studies, a dose-dependent relationship between i.t. LPS concentration and allodynia severity was not consistently observed, in which the effect response showed a plateau or inverted U shape [8,9]. Another study also showed similar results, in which intraneural injection of IL-1β and TNF at an intermediate dose produced significant hypersensitivity, but at a low or high dose lost these effects [28]. The current study also observed no dose-response relationship between Mincle ligand concentration and pain hypersensitivity. These findings may reflect the complex mechanisms underlying physiologic immune reaction and pathologic neuropathic pain.
Repeated TDB administration (group R-TDB) resulted in a greater intensity of mechanical allodynia compared to a single injection (group S-TDB), even though no dose-dependent effect was observed among the different doses in the S-TDB groups. This enhanced pain response may reflect the central sensitization at the spinal level, which shows that repeated or intense nociceptive input induces neuroplastic changes in the CNS [29,30]. Similar phenomena were also found in inflammatory pain models, in which peripheral stimulation can exacerbate hypersensitivity or induce secondary hypersensitivity, especially following an initial sensitization by agents such as carrageenan or complete Freund’s adjuvant [31,32].
Interestingly, thermal hyperalgesia did not differ significantly between the R-TDB and S-TDB groups. This dissociation may reflect differences in the underlying pain mechanisms: microglial activation, indicated by increased Iba1 expression, is more closely linked to mechanical allodynia [33], while thermal hyperalgesia is thought to be driven primarily by proinflammatory cytokines such as IL-1β and TNF-α and sensitization of the TRPV1 channel [34]. One possible explanation is that thermal pathways were already maximally activated by the initial TDB injection, preventing further enhancement upon repeated exposure.
Compared to the SNL group, the R-TDB group exhibited a similar pattern in the timing of pain onset, pain threshold, and the progression of pain responses over time—features consistent with those typically observed in the SNL model. Notably, however, the R-TDB group showed more rapid activation of microglia and astrocytes than the SNL group. This difference in the timing of glial activation suggests that distinct underlying mechanisms are involved in the two models. In both models, immune cell activation within the CNS amplifies and sustains inflammatory signaling, which promotes long term changes in neural circuits and contributes to central sensitization [35,36]. In the SNL group, however, the inflammatory response develops gradually as a secondary consequence of peripheral nerve injury. The early pain response may be caused by acute changes of the injured nerve such as altered nerve conduction and the release of inflammatory mediators at the lesion site. In contrast, in the R-TDB group, inflammation is triggered by the direct activation of Mincle within the spinal cord, and pain behaviors could be indirectly elicited through glial activation. These differences in pain onset and immune cell activation may lie in the distinct underlying mechanisms of each model.
The present study demonstrates that the R-TDB model elicits pain behaviors and neuroinflammatory responses comparable to those observed in the SNL model. Given previous findings showing an anti-allodynic effect of i.t. Mincle antibody administration in the SNL model [17], as well as increased Mincle expression in the L4 spinal nerve transection model [15], it is plausible that peripheral nerve injury and spinal Mincle activation may share common mechanistic pathways underlying neuropathic pain. However, whether Mincle operates through mechanisms analogous to those triggered by nerve injury—and the specific molecular cascades involved—remains to be elucidated in future studies.
In conclusion, activation of spinal Mincle receptors induces robust pain hypersensitivity and accelerated neuroinflammation in the absence of peripheral nerve injury, supporting its utility as an experimental model for studying neuroinflammation and neuropathic pain.
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