1. Changes in regional sympathetic nerve distribution and pain level in TMJ-OA mice
TMJ-OA was induced using the UAC method25–27. Three weeks after UAC induction, histological analyses including hematoxylin-eosin (HE) staining, safranin o-fast green staining and SEM observations revealed notable osteoarthritic features in the mice of the UAC group compared to the CON group (Fig. 1a). Specifically, there was a reduction in cartilage thickness, an increase in microfractures and fissures at the cartilage-bone interface, and the presence of resorption pits in the subchondral bone. Consistent with these structural changes, the Osteoarthritis Research Society International (OARSI) scores were significantly higher in the UAC-induced mice compared to the CON group. Behavioral tests are frequently employed to delineate the pain status of mice with osteoarthritis. Accordingly, we utilized the von-frey test to assess the pain sensitivity in mice, which demonstrated a decreased pain threshold in the UAC group compared to the CON group (Fig. 1b), indicative of exacerbated pain. Research has evidenced that mice in chronic pain, besides exhibiting pain-related behaviors, also display signs of depression28. Consequently, we conducted two behavioral tests associated with depression: the elevated plus maze test and the open field test. The results showed that, relative to the CON group, the mice of UAC group made fewer entries into the open arms, spent less time there, and primarily confined their movements in the open field to the periphery, with a reduction in total distance traveled and average velocity, manifesting depressive-like behaviors (Fig. 1c).
Studies have revealed that pain in the TMJ is hierarchically modulated by the primary somatosensory barrel field (S1BF) cortex29, the caudal part of the trigeminal subnucleus caudalis (Vc), and the trigeminal ganglion (TG)30. Consequently, we employed electroencephalography (EEG) to examine cortical electrophysiological activity in the S1BF region and utilized immunofluorescence staining to quantify the levels of c-fos protein, a marker of neuronal activation31–33, in Vc and TG. The EEG results demonstrated that, compared to the CON group, power spectral energy across all frequency bands were elevated in the S1BF cortex of the UAC group (Fig. 1d). Immunofluorescence findings further indicated a significant increase in c-fos expression in both the Vc and TG of the mice of UAC group relative to the CON group (Fig. 1e). Collectively, these outcomes illustrate that the neural activity in the various levels of pain-modulating centers is significantly enhanced in the mice of UAC group, reflecting an exacerbation of the pain phenotype. Moreover, previous studies have established a close association between locally elevated levels of netrin-1 and PGE2 with bone-originated pain34–37. Consequently, we employed immunofluorescence staining and ELISA assays to measure the contents of netrin-1 and PGE2 in the subchondral bone. Our findings revealed that compared to the CON group, the mice of UAC group showed a significant increase in netrin-1 content in the subchondral bone, which prominently co-localized with the osteoclast marker tartrate-resistant acid phosphatas (TRAP) (Fig. 1e). Additionally, the concentration of PGE2 was also significantly increased (Fig. 1f), suggesting an exacerbation of pain in the mice of UAC group.
In addition to pain manifestations, we assessed the sympathetic nerve innervation in the subchondral bone of mice through tyrosine hydroxylase (TH) immunofluorescence staining (Fig. 1g) and viral anterograde tracing (Fig. 1h). The results demonstrated a marked increase in sympathetic nerve terminals in the subchondral bone of osteoarthritic mice, with the newly added terminals predominantly clustered near the bone marrow cavity and extending toward the cartilage layer, even approaching or penetrating the tidemark demarcating the bone-cartilage boundary. Notably, however, ELISA results showed no statistically significant difference in serum norepinephrine (NE) concentrations between the UAC and CON groups (Fig. S1). These findings indicate that UAC induction had augmented regional sympathetic nerve innervation within the subchondral bone in osteoarthritic mice, but with no alteration in changing their overall sympathetic nervous system activity.
Collectively, these results suggest elevated sympathetic nerves in the subchondral bone area is concurrent with augmented pain expression in TMJ-OA mice. However, whether the sympathetic nervous system is associated with the pain experienced by the mice needs to be further explored.
2. Effect of sympathetic nerves on TMJ-OA pain
To elucidate the association between the sympathetic nervous system and pain modulation, we built upon the UAC model by establishing the CIS model to activate sympathetic nerves, and employed superior cervical ganglionectomy (SCG)38, as well as intraperitoneal injections of 6-hydroxydopamine (6-OHDA)27, to downregulate sympathetic signaling activity in UAC mice (Fig. 2a), followed by assessing the degree of pain in mice across these different models. For SCG-treated mice, presence of ptosis (Horner's syndrome) after ganglionectomy was indicative of successful removal of the sympathetic nerves (Fig. S2). For 6-OHDA-treated mice, bilateral ptosis observed within 6 h after the first injection was indicative of successful suppression of sympathetic signals (Fig. S2). Immunofluorescence analysis revealed that, those subjected to CIS + UAC treatment displayed a significant increase in sympathetic nerve density in the condylar subchondral bone compared to mice in the UAC group, while those subjected to SCG + UAC and 6-OHDA + UAC showed a substantial decrease in sympathetic innervation in the condylar subchondral bone (Fig. S3). These findings were indicative of the successful establishment of a murine model for sympathetic nerve activation and blockade.
Subsequently, we examined the pain conditions of mice across different groups. Von Frey tests indicated that, compared to the mice of UAC group, the mice of CIS + UAC group had a significantly lower pain threshold, whereas the mice of 6-OHDA + UAC and SCG + UAC groups showed a marked increase in pain threshold (Fig. 2b). Results from the open field and elevated plus maze tests (Fig. 2c) demonstrated that, compared to the mice of UAC group, mice of CIS + UAC group had a significantly reduced total movement distance and average velocity in the open field, concentrating their activities at the edges. In the elevated plus maze, they showed a decreased frequency of entries into open arms and shorter stay times, exhibiting evident depressive-like behavior. Conversely, the mice of 6-OHDA + UAC and SCG + UAC groups displayed a significant alleviation of depression. Electroencephalogram (EEG) results showed that, compared to the mice of UAC group, the mice of CIS + UAC group had significantly elevated electrophysiological signals in the S1BF cortex, whereas the mice of 6-OHDA + UAC and SCG + UAC groups exhibited reduced cortical signals (Fig. 2d). Immunofluorescence staining results (Fig. 2e) revealed that, in comparison to the mice of UAC group, the mice of CIS + UAC group had increased c-fos expression in both the Vc and TG, indicating enhanced neuronal activity, whereas the mice of 6-OHDA + UAC and SCG + UAC groups showed reduced c-fos expression in the Vc and TG, pointing to decreased neuronal activity. Moreover, compared to the mice of UAC group, the mice of CIS + UAC group had higher levels of pain-related substances including netrin-1 and PGE2 in the subchondral bone, whereas the mice of 6-OHDA + UAC and SCG + UAC groups had lower levels of netrin-1 (Fig. 2e) and PGE2 (Fig. 2f) in their subchondral bone. These findings collectively suggest that pain is exacerbated when the sympathetic nervous system is activated and relieved when sympathetic signal is inhibited.
Collectively, these findings indicate that the sympathetic nervous system plays a regulatory role in osteoarthritic pain. However, the specific mechanisms by which the sympathetic nervous system regulates osteoarthritic pain remain unclear and require further investigation.
3. Spatiotemporal relation between sympathetic and sensory nerves in TMJ-OA
Research indicates that TMJ-OA pain largely originates from an increase in sensory nerve terminals and their sensitization within the subchondral bone of the joint8. Thus, the regulation process of sympathetic nerves on osteoarthritic pain raises our interest in whether it is related to sensory nerves. To explore this, we employed immunofluorescence staining and viral anterograde tracing techniques to investigate the temporal and spatial relationship between sympathetic and sensory nerves during the course of osteoarthritis development.
By using immunofluorescence staining for TH, NPY, CGRP and PGP9.5 the sympathetic (Fig. 3a, b, d) and sensory (Fig. 3c, e) nerve innervation in the subchondral bone during the early stages of TMJ-OA was observed. The results showed that in the mice of CON group, the content of sympathetic and sensory nerve terminals in the subchondral bone remained consistently low and did not exhibit a significant increase over time. In contrast, in mice of UAC group, just three days after TMJ-OA induction, the distribution of sympathetic nerve terminals in subchondral bone exceeded that of the mice of CON group, while the sensory nerve terminals in the subchondral bone were significantly more abundant on day 7 post-UAC induction compared to those in the mice of UAC group. As TMJ-OA progressed, sympathetic and sensory nerve innervation persistently intensified, demonstrating a tendency to progressively extend towards the interface between bone and cartilage, even breaching the tidemark. This finding implies that, during the development of TMJ-OA, the increase in sympathetic nerve innervation in the subchondral bone precedes that of sensory nerves. From a temporal standpoint, the results suggested that sympathetic nerves may exert a modulatory influence on sensory nerves. Of particular note, the close distribution of sympathetic and sensory nerves suggests a possible direct interaction between them.
To delve deeper into the possibility of sympathetic regulation of sensory nerves, we employed immunofluorescence staining (Fig. 4a-c) and viral anterograde tracing (Fig. 4d-g) to examine the spatial relationship between these two neural systems. Immunofluorescence staining results revealed that, compared to the mice of CON group, the sympathetic and sensory nerves in the mice of UAC group were closely intertwined. With the progression of osteoarthritis, innervation by both types of nerves significantly increased, extending progressively towards the cartilage-bone interface and even penetrating the tidemark. The viral anterograde tracing confirmed this trend, indicating a high degree of congruency in their spatial positioning. These findings provide compelling morphological evidence of a tightly correlated spatial arrangement between the two neural systems, which paves the way for further exploration into their mutual interactions and potential regulatory mechanisms in disease progression.
These findings suggest that, during the progression of TMJ-OA, sympathetic nerves infiltrate the subchondral bone earlier than sensory nerves and run in close proximity to them. Based on their distribution patterns and sequential growth over time, we speculate that, in the course of TMJ-OA, sympathetic nerves might modulate the pain by regulating the growth of sensory nerve terminals in the subchondral bone, thereby exerting control over TMJ-OA pain.
4. Influence of regional sympathetic nerves on sensory nerves in TMJ-OA
To further clarify whether the sympathetic nervous system modulates TMJ-OA pain via regulation of sensory nerves, we employed immunofluorescence staining to analyze the distribution of sensory nerves in the subchondral bone in mice under sympathetic nerve activation and blockade conditions. Compared to the mice of UAC group, in the mice of CIS + UAC group where sympathetic nerves were activated, sensory nerves closely accompanied sympathetic nerves, and the number of sensory nerve terminals significantly increased alongside the elevation of sympathetic nerves. Conversely, in the mice of 6-OHDA + UAC and SCG + UAC groups, where sympathetic nerves were blocked, the sensory nerves decreased along with the decline in sympathetic nerves (Fig. 5a). We then established a co-culture system of trigeminal ganglion neurons (TGN) and superior cervical ganglion neurons (SCGN) to investigate the direct regulatory effect of sympathetic neurons on sensory neurons in vitro. Scanning electron microscopy (Fig. 5b) and β-tubulin immunofluorescence staining (Fig. 5c) were employed to observe the morphology and axonal extension of TGNs. The results showed that, starting from 6 hours of co-culture, TGNs grown together with sympathetic neurons displayed a markedly accelerated increase in axon elongation and expansion compared to those cultured alone. This facilitative effect became increasingly pronounced as the culture duration extended.
These findings confirm a regulatory influence of sympathetic nerves on sensory nerves. However, the precise mechanisms by which sympathetic nerves regulate sensory nerves remain unclear and warrant further investigation.
5. How sympathetic nerves regulate sensory nerves
Previous studies indicated that sensory nerves are modulated by sympathetic nerves through neurotransmitter release. Among the neurotransmitters, norepinephrine (NE) and neuropeptide Y (NPY) are particularly implicated in pain regulation39–43. Accordingly, ELISA was used to measure NE and NPY levels in the subchondral bone. A significant increase in NE levels was found in the UAC group, compared to the CON group (Fig. 6a). No significant difference was noted for NPY between the two groups (Fig. 6b). This finding suggests that NE may play a role in the crosstalk between sympathetic and sensory nerves in TMJ-OA.
Guanethidine (GUA) is commonly used to block NE release from sympathetic nerves44–46. Propranolol (Pro), a non-selective β-receptor antagonist, is frequently employed to inhibit β-receptors47,48. To elucidate the regulatory effect of NE on sensory neurons, we introduced guanethidine (GUA) and propranolol (Pro) into the TGN + SCGN co-culture system in vitro, with the aim of inhibiting NE secretion and blocking β-receptors on the sensory neurons, respectively. Under equal cultivation periods, our experimental results showed that, compared to the TGN group, the TGN + SCGN group exhibited a significantly greater axonal expansion area of sensory neurons. However, axonal growth of sensory neurons was notably reduced when added with NE blockers in the groups of GUA (TGN + SCGN + GUA) or Pro (TGN + SCGN + Pro), similar to the growth state observed in the TGN group (Fig. 6c). This finding robustly demonstrates that suppressing NE production or its engagement with β-receptors on sensory neurons can effectively reverse the axon growth advantage induced by co-culture. Consequently, this confirms that sympathetic neurons activate sensory neurons through NE release, playing a crucial role in facilitating axonal elongation. To further validate the regulatory role of NE on sensory neurons, we directly treated TGNs with NE and employed netrin-1 and PGE2, substances previously shown to modulate sensory neurons34,36,37, as positive controls and the objective was to observe the effect of NE on sensory neuron regulation. Our results demonstrated that NE significantly promoted axonal growth of sensory neurons (Fig. 6d). Furthermore, calcium imaging revealed that NE notably activated these neurons (Fig. 6E and Video. S1-4), leading to heightened intracellular calcium signal and obvious calcium oscillations activity after adding NE49. Additionally, we uncovered that when NE acts in conjunction with netrin-1 or PGE2 on sensory neurons, it synergistically amplifies the effects of netrin-1 or PGE2 on these neurons (Fig. 6d, e and Video. S1-4). Collectively, these findings indicate that NE can directly or cooperatively with netrin-1 and PGE2 facilitate the growth and activation of sensory neurons, contributing to aberrant pain sensations.
6. The effect of norepinephrine on TMJ-OA pain
To verify if sympathetic nerves regulate pain in TMJ-OA via NE secretion, intra-articular injections of guanethidine and NE were administered to mice in the UAC group. Changes in pain status were observed compared to mice with TMJ-OA that were injected with sterile physiological saline (Fig. 7a).
The von-frey results indicated that, compared to the mice of UAC group, the pain threshold was significantly reduced in the mice of NE + UAC group, whereas it was notably elevated in the mice of GUA + UAC group (Fig. 7b). In the open field test and elevated plus maze assessment (Fig. 7c), the mice of NE + UAC group manifested clear depressive-like behaviors. Conversely, the mice of GUA + UAC group displayed exploratory behaviors and a marked reduction in depressive symptoms. Electrophysiological analyses revealed that, compared to the mice of UAC group, the mice of NE + UAC group showed significantly heightened cortical electrical activity in the S1BF region, whereas the mice of GUA + UAC group exhibited a decrease in such signals (Fig. 7d). Immunofluorescence staining results (Fig. 7e) demonstrated that, compared to the mice of UAC group, the mice of NE + UAC group exhibited increased c-fos expression in the Vc and TG regions, suggesting enhanced neuronal activity. Conversely, the mice of GUA + UAC group showed reduced c-fos expression in the Vc and TG, indicating lowered neural activity. Moreover, in comparison to the mice of UAC group, the mice of NE + UAC group had higher levels of pain-related substances netrin-1 and PGE2 in their subchondral bone, whereas the mice of GUA + UAC group displayed lower concentrations of netrin-1 (Fig. 7e) and PGE2 (Fig. 7f) in the subchondral bone. These findings collectively imply that a local increase in NE exacerbates pain, whereas the blockade of NE alleviates pain symptoms.
These findings indicate that sympathetic nerves have a regulatory effect on TMJ-OA pain, and this regulation is mediated through the release of NE by sympathetic nerves, which acts on sensory nerves to promote the growth and activation of sensory nerve terminals, thereby modulating TMJ-OA pain.