The effect of capsaicin on expression patterns of CGRP in trigeminal ganglion and trigeminal nucleus caudalis following experimental tooth movement in rats

ABSTRACT Objectives The aim of this study was to explore the effect of capsaicin on expression patterns of calcitonin gene-related peptide (CGRP) in the trigeminal ganglion (TG) and trigeminal subnucleus caudalis (Vc) following experimental tooth movement. Material and Methods Male Sprague-Dawley rats were used in this study and divided into small-dose capsaicin+force group, large-dose capsaicin+force group, saline+force group, and no force group. Closed coil springs were used to mimic orthodontic forces in all groups except for the no force group, in which springs were inactivated. Capsaicin and saline were injected into periodontal tissues. Rats were euthanized at 0 h, 12 h, 1 d, 3 d, 5 d, and 7 d following experimental tooth movement. Then, TG and Vc were obtained for immunohistochemical staining and western blotting against CGRP. Results Immunohistochemical results indicated that CGRP positive neurons were located in the TG, and CGRP immunoreactive fibers were distributed in the Vc. Immunohistochemical semiquantitative analysis and western blotting analysis demonstrated that CGRP expression levels both in TG and Vc were elevated at 12 h, 1 d, 3 d, 5 d, and 7 d in the saline + force group. However, both small-dose and large-dose capsaicin could decrease CGRP expression in TG and Vc at 1 d and 3 d following experimental tooth movement, as compared with the saline + force group. Conclusions These results suggest that capsaicin could regulate CGRP expression in TG and Vc following experimental tooth movement in rats.


INTRODUCTION
Orthodontic pain induced by tooth movement is widely considered as a negative consequence of orthodontic treatments 19 . Although the perception of pain is a subjective experience and might vary in individuals, almost all patients complain the discomfort during orthodontic treatment 3 . Therefore, how to alleviate orthodontic pain and elucidate its underlying mechanisms clearly represent one of the major concerns for both patients and orthodontists. Orofacial pain signals induced by tooth movement are received by peripheral nociceptors, transmitted (TG), conveyed to the trigeminal subnucleus caudalis (Vc) located in the caudal part of medulla oblongata, then relayed to the third-order neurons in the thalamus, and finally perceived by the cortex 19 . Well-grounded experimental observations have documented that both TG and Vc play a crucial role in the transmission of orthodontic pain 16,30 .
It is well known that the perception of orthodontic localized in periodontal tissues that involves the 28 . In particular, calcitonin gene-related peptide (CGRP), a representative neuropeptide, is evidenced to participate in the initiation and maintenance 2016;24(6):597-606 of inflammatory pain 2 . Moreover, it has been demonstrated that CGRP participates in orthodontic pain following experimental tooth movement 18 . Capsaicin, the main pungent ingredient in hot chili peppers, could elicit heat sensation and stimulate the release of sensory neuropeptide through selectively binding to transient receptor potential vanilloid 1 (TRPV1, a non-selective cation channel) 6 . CGRP could be synthesized and released from a subset of capsaicin-sensitive primary afferent neurons in the TG 12 . However, the relationship between capsaicin and CGRP release following tooth movement is still poorly understood. Although previous studies have indicated that capsaicin-sensitized TRPV1 could evoke the CGRP release from peripheral nerve axons 13 , the effect of capsaicin on CGRP expressions in TG and Vc following experimental tooth movement remains largely unknown. Furthermore, administration of capsaicin does not affect the rate of orthodontic tooth movement in rats 9 .
Therefore, the purpose of the present study was to investigate the effect of capsaicin on expression patterns of CGRP in TG and Vc following experimental tooth movement in rats and to explore its underlying mechanisms.

MATERIAL AND METHODS
All experimental procedures were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and were approved by the Ethical Committee of West China School of Stomatology, Sichuan University and State Key Laboratory of Oral Diseases. Efforts were made to minimize both the number of animals and their discomfort.

Animals
Male Sprague-Dawley rats (age: 8 weeks; weight: 200-250 g) were used in this study. These rats were provided by the Animal Experimental Center, Sichuan University, Chengdu, China. They were housed in a temperature-regulated room at 25±2°C with standard rat chow and water ad libitum, and maintained on a 12/12 day-night cycle the experiment.

Experiment design
In order to examine the effects of capsaicin on CGRP expression following experimental tooth movement, rats were randomly divided into small-dose capsaicin+force group, large-dose capsaicin+force group, saline+force group, and no force group. Fixed and stainless steel closed-coil and the ipsilateral upper incisors to delivery a mesial force of 40 g as previously described 23 in the force groups while springs inactivated in the no force group (0 g). Rats were fed with soft food after force application. They were euthanized through cervical dislocation following general anesthesia at 0 h, 12 h, 1 d, 3 d, 5 d, and 7 d (six rats at 0 h, 12 h, 1 d, 3 d, 5 d, and 7 d in each group). In particular, in all groups, the rats euthanized at 0 h without any intervention were taken as the baseline control for each group.
The rats of the small-dose capsaicin+force (3×10 -2 mol/L; Sigma-Aldrich, St. Louis, MO, USA) 15 into periodontal tissues around upper left molars study has been shown to be effective in a previous study 31 was used in the saline+force group. Periodontal injections were performed 30 min before force application.

Tissue sample preparations
Following cervical dislocation, the maxillary portion of TG at the ipsilateral side of force application and the caudal part of medulla oblongata were obtained for immunohistochemistry and western blot.

Immunohistochemistry
The expressions of CGRP in TG and Vc were detected by immunohistochemistry at six time points (0 h, 12 h, 1 d, 3 d, 5 d, and 7 d) after experimental tooth movement in rats (n=6 per group). Tissue washed in phosphate-buffer saline (PBS) three citrate acid buffer), the sections were washed in PBS, blocked with goat serum, and incubated ab47027; Abcam, Cambridge, MA, USA) at 37°C for 45 min. After that, sections were rinsed with PBS for 10 min and incubated with EnVision TM (K500711; DAKO, Carpinteria, CA, USA) at 37°C for 45 min. Following washing with PBS for 10 min, they were visualized with 3,3'-diaminobenzidine (DAB) and washed with distilled water. After counterstained with hematoxylin and dehydrated with ethanol, immunostained sections were mounted on coverslips. All tissues followed the same immunohistochemical procedures to minimize the variability in laboratory.
Visualization of immunoreactive tissues was obtained through a light microscope (Axio Imager 2, Carl Zeiss, Oberkochen, German). The positive stained cytoplasm for TG and yellow-brown stained reticular formation for Vc. For each rat, integrated optical density (IOD/Area) for TG and Vc were calculated (Image-Pro Plus 6.0, Media Cybernetics, Rockville, MD, USA) in each of five randomly expression level.

Western blotting
The CGRP protein expression in TG and Vc were studied by western blotting at six time points after experimental tooth movement in rats (n=6 per group). Following mechanical grinding, tissue samples were disintegrated with RIPA lysis buffer on ice for 30 min and centrifuged at 4°C Then, the supernatants were extracted and stored at -70°C. After determining the total protein concentration, samples were separated by 15% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. They were washed four times and blocked with 5% defatted milk powder for 2 h, antibody (1:200; ab47027, Abcam, Cambridge, MA, USA). The membranes were then washed in PBS and incubated with secondary antibody, Dylight 680 conjugated goat anti-rabbit IgG (1:10000, protein. Band intensity was computer analyzed by a densitometer (Quantity One, Bio-Rad, Hercules, CA, USA). Each band was analyzed repeatedly three employed as the CGRP expression level.

Statistical analyses
Data were expressed as mean±standard error (SEM). One-way analysis of variance (ANOVA) (Tukey post hoc test) was applied to analyze differences of CGRP expression levels among different time points in each group and to compare the differences of CGRP expression levels among the

RESULTS
The effects of capsaicin on CGRP expression in the TG following experimental tooth movement As displayed in Figure 1A, bands of CGRP were 42 kDa.
For the saline+force group, when compared with the baseline level (0.628±0.041), as shown in Figure 1B, experimental tooth movement induced an up-regulation of CGRP expression in the saline+force group at 12 h (0.988±0.019, p<0.001) and 1 d (1.216±0.011, p<0.001), peaked at 3 d (1.599±0.034, p<0.001), then decreased gradually at 5 d (0.962±0.026, p<0.001) and  Interestingly, regarding the comparison between the two groups with different capsaicin dosages, we higher in the large-dose capsaicin+force group than in the small-dose capsaicin+force group at 12 h (p<0.05). Conversely, following the similar CGRP expression levels between these two groups at 1 d, 3 d, and 5 d (all p>0.05), CGRP expression capsaicin+force group than in the large-dose capsaicin+force group (p<0.05).
As shown in Figure 2, CGRP was basically expressed in trigeminal neurons and CGRPpositive cells could also be observed at baseline (as indicated by the black arrow). As displayed in Figure 3, trends in CGRP expression levels (quantified through immunostaining intensity) for all the four groups were similar with those of capsaicin+force group, CGRP expression levels increased rapidly at 12 h, declined at 1 d, 3 d, and 5 d, and rebounded at 7 d. In contrast, for the largedose capsaicin+force group, CGRP expression levels peaked at 12 h and decreased thereafter without rebounding at 7 d. Moreover, for the saline+force group, CGRP expression levels started to increase at 12 h, peaked at 3 d, and gradually decreased thereafter. In contrast, for the no force group, CGRP expression levels only increased at 3 d.

The effects of capsaicin on CGRP expression in Vc following experimental tooth movement
We could also observe that bands of CGRP were 42 kDa ( Figure 4A). Compared with the baseline level (0.406±0.004), as displayed in Figure 4B, CGRP expression levels in the saline+force group increased at 12 h (1.187±0.041, p<0.001) and As shown in Figure 5, CGRP-like immunoreactivity was observed in Vc at baseline, which displayed were the main structures. As presented in Figure  6, following experimental tooth movement, the chronological changes in CGRP expression levels were similar with those of western blotting. group, CGRP expression levels increased steadily from 12 h and peaked at 7 d. In contrast, for the large-dose capsaicin+force group, CGRP expression levels peaked at 12 h, declined thereafter until 5 d, and rebounded at 7 d. Moreover, for the saline+force group, CGRP expression levels began to increase at 12 h, peaked at 3 d, and dropped thereafter. For the no force group, CGRP expression levels increased and reached a plateau from 12 h to 5 d, and returned to baseline at 7 d. Figure 7-When orthodontic force was exerted on a tooth, nociceptive stimulus would be induced and perceived by peripheral nociceptors in periodontal tissues, where it could then generate pain signals that propagate to the TG. Once receiving these signals, TG would synthesize and release CGRP to periodontal tissues and Vc via bidirectional transport. After local injection of capsaicin, small-dose capsaicin could recruit more CGRP released from TG into periodontal tissues than Vc compared with large-dose capsaicin In this study, we found that capsaicin could regulate CGRP release both in TG and Vc following experimental tooth movement. Moreover, both small-dose and large-dose capsaicin could cause downregulation of CGRP expression at 1 d and 3 d, whereas a large CGRP release was observed in the saline+force group.

DISCUSSION
It is well-known that most patients could perceive orthodontic pain from 1 d to 3 d and pain sensation gradually subsides thereafter 22 , which has been the main adverse effect of orthodontic treatment. During experimental tooth movement, the force exerted on periodontal tissues could initiate local in the orofacial region, TG could receive the nociceptive signal from peripheral nerve terminals located in periodontal tissues, and transmit it to the Vc. CGRP is exclusively synthesized in primary afferent neurons of the TG 12 . Previous studies have indicated that CGRP plays an important role in the underlying pathogenesis of pain conditions 4 . Our results showed that CGRP expression levels both increased at 1 d and peaked at 3 d, then gradually decreased at 5 d and 7 d. This change trend was consistent with a previous study, which indicated that CGRP was involved in the transmission and modulation of pain following experimental tooth movement 18 . Accordingly, we suggest that increased on activation of trigeminal system and subsequent transmission and processing of this information in the brainstem 25 . However, ironically, CGRP expression levels increased in the no force group both in TG and Vc. Actually, since no force was exerted in this group, CGRP expression levels should be similar with those at baseline. We attribute it to discomforts and the buccal peculiar sense that might be caused by inactivated intraoral springs in rats 17 .
Our results showed that both small-dose capsaicin and large-dose capsaicin had an obvious impact on CGRP expression levels in TG and Vc following experimental tooth movement when compared with the saline+force group. It has been well-documented that capsaicin could selectively TRPV1 6 calcium and CGRP release from peripheral nerve axons 13,29 . In our study, local periodontal injections of capsaicin caused a large CGRP release both in TG and Vc at 12 h following experimental tooth the stimulatory effect of capsaicin, which is known to activate sensory neurons and stimulate CGRP release 5 . It is worth mentioning that these two groups with different capsaicin dosages showed some differences in the CGRP expression levels at 12 h. The mechanism behind this effect might be the dose-dependent release of CGRP induced by excitation of TRPV1 24 . This could also be supported by the observation that application of capsaicin caused a dose-dependent release of salivary trigeminal system 27 . Moreover, in our study, we found that following experimental tooth movement, CGRP expression levels both in TG and Vc at 1 d saline+force group. One possible explanation for this phenomenon is based on the evidence that there is another effect of capsaicin, which is the inhibitory effect. 20 that when recurrent application of capsaicin or administration of a single high dose, it could desensitize the voltage-gated calcium channels on sensory neurons 10 and thereby could reduce the release of neurotransmitters. (This might be explained by that when recurrent or high dose of capsaicin is applied, it could desensitize the voltagegated calcium channels on sensory neurons 10 . Subsequently, the release of neurotransmitters would be inhibited. Another explanation might be the large depletion of CGRP both in TG and Vc following injections of capsaicin and experimental tooth movement.
In addition, CGRP expression levels began to increase both in TG and Vc at 7 d after experimental tooth movement. We attribute this phenomenon to the beginning of CGRP synthesis in TG neurons and subsequent CGRP release in Vc. Regarding the difference of CGRP expression levels between small-dose+force group and large-dose+force group at the aforementioned time point, there are some evidence that might account for this. Previous studies have reported that after prolonged exposure to capsaicin or high-concentration capsaicin applied, it might initiate a process described as defunctionalization of voltage-gated calcium channels 1 , which is mainly due to the activation of calcium-dependent proteases 7 . This process could induce degeneration of capsaicinsensitive nociceptive nerve endings 8 . We hereby hypothesized that large-dose capsaicin might cause a slower recovery of the TRPV1 activity, resulting in lower CGRP expression levels than small-dose capsaicin at 7 d.
Interestingly, in our study, we also found that the change trend of CGRP expression levels in Vc was not exactly the same as that in TG, especially in small-dose+force group. As previously mentioned, CGRP is mainly synthesized in TG and released one hand, peripheral released CGRP around blood vessels in periodontal ligament (PDL) originates The effect of capsaicin on expression patterns of CGRP in trigeminal ganglion and trigeminal nucleus caudalis following experimental tooth movement in rats 2016;24(6):597-606 from the TG 11 , which could also be supported by inferior alveolar nerve resection 14 . On the other hand, central processes of TG cells exhibiting CGRPimmunoreactivities almost completely terminate at the Vc 21 , which means that the released CGRP in Vc derives from the nerve extending from trigeminal neurons 26 . Based on this, we hypothesized that following experimental tooth movement, CGRP synthesized in TG might be bidirectionally transported to both periodontal tissues and Vc (Figure 7). Furthermore, we suggest that smalldose capsaicin was likely to stimulate CGRP release from TG more peripherally than centrally, thereby leading to the difference in CGRP expression levels between TG and Vc.

CONCLUSION
Therefore, CGRP expression levels were elevated following experimental tooth movement both in TG and Vc, suggesting their involvement in the transmission of nociceptive information in tooth movement. Both small-dose and large-dose capsaicin could reduce CGRP expression levels both in TG and Vc following experimental tooth movement in rats at 1 d and 3 d. CGRP synthesized in TG neurons could be bidirectionally transported to peripheral tissues and Vc. Taken together, we suggest that capsaicin could regulate CGRP expression in TG and Vc following experimental tooth movement in rats. However, the underlying mechanism whereby capsaicin regulates CGRP expressions and the effect of capsaicin on orthodontic pain should be further elucidated.