Study Of The Molecular Mechanisms Of Pain Relief By Low Level Laser Irradiation During Experimental Tooth Movement

Pain is the biggest side effects of orthodontic treatment. It is necessary andmeaningful to reduce orthodontic pain and improve the life quality of patientsduring treatment. To achieve this goal, the orthodontist came up with manymethods, such as non-steroidal anti-inflammatory drugs, and transcutaneouselectrical nerve stimulation, anesthetic gel, chewing gum. However, thesemethods have a variety of side effects or inconvenience.Low energy laser irradiation has been widely used clinically to relievepain, such as rheumatoid arthritis, neuropathic pain and joint pain.And it hasbeen proved to be very effective. The laser treatment has also been successfullyused to relieve pain due to tooth movement caused by the orthodontic treatmentprocess, and this method is effective, simple and no side effects. Themechanism of LLLT remains unclear,and basic research reported in this respectis also very rare. However, its biological mechanism should be clarified beforethe clinical application. Therefore, the mechanisms of pain relief by low levellaser irradiation must be elucidated.Orthodontic load causes the local periodontal tissue injury, which lead toaseptic inflammation, and a variety of important inflammatory mediators werereleased to periodontal tissue, such as the interleukin. These inflammatorycytokines have been shown to cause pain or hyperalgesia. Orthodontic toothmovement may also start the pain by stimulate the nerve endings to releaseneurotransmitter, such as neuropeptides. During experimental tooth movement,the expression of calcitonin gene-related peptide (CGRP), substance P (SP) andgalanin(GAL) was increased in periodontal membrane and trigeminal ganglia,suggesting that these neuropeptides involved in the process of orthodontic pain. In addition to inflammatory mediators and neurotransmitters, in recent yearsthe ion channel (such as P2X3receptor and tetrodotoxin-resistant (TTX-R)Nav1.8sodium channel) was also found important in nociceptive informationtransmission. In addition, the trigeminal ganglion is the primary afferent centralfor the transmission of orofacial pain, and the small and medium-sized neurons(receptors) in trigeminal ganglion are pain sensors involved in the transmissionof nociceptive signals.Therefore, the possible ways to mitigate orthodontic pain includeinhibiting the release of local inflammatory factors, promoting angiogenesis,improving the blood supply, inhibiting the release of the pain-inducedneurotransmitters, promoting the release of endogenous analgesicneurotransmitter, and affecting ion channels. The previous experiments haveshowed that low energy laser irradiation can significantly promote thevascularization of periodontal tissue in experimental tooth movement, so thisstudy will focus on the effect of LLLT on inflammation mediator in periodontalligament, neurotransmitter and ion channels in the trigeminal ganglion duringexperimental tooth movement. Provide objective evidence for low energy laserirradiation to reduce orthodontic pain, and to reveal its mechanisms at themolecular level.1. The effect of low level laser irradiation on local inflammatory mediatorsin peri-odontal tissues during experimental tooth movement.Objective: To detect IL-1β expression in periodontal tissue at each timepoint of experimental tooth movement, and to explore the impact of low energylaser irradiation on the synthesis and/or release of inflammatory mediators inthe periodontal tissues during experimental tooth movement.Methods: Male Wistar rats weighing (200±10) g were randomly dividedinto control group and experimental group, and the latter include loading6h,12h,1d,2d,3d,5d, and7d group. The left side of the experimental group received no irradiation, and the right side received the low energy laserirradiation. Nickel titanium screw spring was set between maxillary first molarand incisor of rat, and exert a force about50g.The right side of theexperimental group received low energy laser irradiation in the first three days,and with a Ga-Al-As semiconductor laser,808nm,100mW, and the buccal andlingual were irradiated for3min, once a day. Each group of experimentalanimals at corresponding time points was dislocated executed to take theperiodontal tissue of the first maxillary molar and IL-1β concentration of eachsample was measured by radioimmunoassay.Results: The expression of IL-1βin the periodontal tissue of thenon-irradiated side of the experimental group was significantly increased after6hours, peaked at day3, and then gradually decreased, but the7d group is stillhigher than control. The expression of IL-1β in irradiation side of thecorresponding time points was significantly lower than non-irradiated side.Conclusion: IL-1β was involved in experimental tooth movementpain.LLLT can inhibit inflammatory mediators synthesis and/or release in theperiodontal tissues induced by experimental tooth movement.2. The effect of low energy laser irradiation on neuropeptides in primaryafferent centr-al (trigeminal ganglion) during experimental toothmovement.Objective: To observe the expression of CGRP and GAL in the trigeminalganglion of experimental groups, and explore the impact of low energy laserirradiation on the synthesis and/or release of neurotransmitter in experimentaltooth movement.Methods: The animal model and the laser irradiation was similar toexperiment1. Five animals of each experimental group were killed at thecorresponding time point, and fresh trigeminal ganglia were taken out.CGRPand GAL gene expression in the trigeminal ganglion was detected by RT-PCR assay, and protein expression was detected by Western blot. Another fiveanimals of experimental groups were killed by cardiac perfusion with4%paraformaldehyde,and the trigeminal ganglions were taken out, expression anddistribution of CGRP and GAL the trigeminal ganglion were observed byimmunohistochemical methods.Results: In control group, only a small number of ganglion cellsexpressed CGRP. Trends over time of CGRP expression in the irradiated side issimilar to the unirradiated side.In6h group, the number of positive cells intrigeminal ganglia has no significant change or even reduce, and the increase ofthe number of positive cells were observed in12h group. The increase becomemore significant after1day, and peaked on the3rd day. A declination wasfound in5d group, the immunoreactive expression in the7d group wassignificantly weakened. It is noteworthy that the number of positive cells inirradiation side were less than the non-irradiated side after1day and thestaining intensity was weaker than the non-irradiated side.trigeminal ganglion CGRP mRNA expression was increased in theprocess of orthodontic force on afterburner, non-irradiated side of theafterburner6h significantly increased expression12h group decreased slightly,1d began to increase, reached the peak at3d,5d,7dstarted to decrease, but stillhigher than normal. Irradiated side of the6h group,12h and control groups nosignificant difference in the12h group and the6h group than there aresignificant differences in expression began to increase and reached its peak in2d, but3d group started to decrease,7d expression at least, but still high thecontrol group. The irradiated side and the non-irradiated side, in addition toboth sides of the12h group, no significant difference in the rest of theirradiated side of the experimental group, CGRP mRNA expression was alwayslower than the non-irradiated side.The Western blot test results showed that the changes trend of CGRP protein expressed in both the unirradiated and irradiated side of are similar:Significant increase was not found between6h group and control group, butwas found between the rest of the group and the control group. The expressionlevel of CGRP protein gradually increased and reached a peak on3d, thengradually decreased.But the expression in7d group is still higher than that inthe control group. From12h to5d, protein expression on were all lower thanthe unirradiated side, P <0.05.After6hours treated by50g force, the number of GAL-immunoreactiveganglion cells in trigeminal ganglia was not increased significantly comparedwith the control side, and increased in a minor extent after12hours and1day.Then significantly increase was found in3d,5d and7d group.And from12hto7d group, GAL-immunoreactive ganglion cells in trigeminal ganglia of theirradiated side was more than the unirradiated side,and the difference wasstatistically significant.Expression of galanin mRNA in the trigeminal ganglion increased in theprocess of orthodontic force application. The expression in the non-irradiatedside did not change significantly at6h, began to increased at12h group, andreached the peak on3d. The side reached the peak on5d and7d reducedsignificantly. Whatever the trends, from12h to5d expression of galanin mRNAin irradiated side was always higher than the non-irradiated side, but on7d theirradiated side was lower than the non-irradiated side.Western blot result showed that expression trend of galanin duringexperimental tooth movement was similar to CGRP, but its peak appered in the5d group. The protein expression were higher in the irradiated side than in theunirradiated side from12h to7d group.Conclusion: CGRP and GAL participated in the experimental toothmovement; During the experimental tooth movement, LLLT inhibitedsynthesis and expression of the pain-caused neuropeptide CGRP in TG, and promote synthesis and expression of analgesic neuropeptide GAL.3. The effect of low energy laser irradiation on P2X3receptor and Nav1.8sodium chan-nel in primary afferent central TG in the experimentaltooth movement.Objective: To explore the influence of LLLT on gene expression of P2X3receptor and Nav1.8ion channel in TG during experimental tooth movement.Methods: The animal model and low level laser irradiation method wassimilar with experiment one. Animals of each experimental group were killed atthe corresponding time point, and fresh trigeminal ganglia were taken out,P2X3receptor and Nav1.8ion channel gene expression were detected byReal-time quantitative PCR.Results:P2X3gene in both the unirradiated side and the irradiated side began toincrease after12hours treated by50g force, peaked on3day and thengradually decreased until7days and its expression is still higher than that in thecontrol group. Side of the irradiated side and unirradiated, From the12h to7dgroup, the gene expression of P2X3receptor in irradiated side were lower thanthe unirradiated side, and the difference is significant(12h,1d,2d,5d and7dgroup, P <0.05;3d group, P <0.01)Gene expression of Nav1.8in non-irradiated side began to increasefrom6h group, reached a peak in3d group, and then began to decline, but thegene expression in7d group is still higher than that in the control group. Geneexpression of Nav1.8in the irradiated side began to increase from12h group,reached the peak in3d group, and then started to decrease. From the1d to5dgroup, the gene expression of Nav1.8ion channel in irradiated side were lowerthan the unirradiated side, and the difference is significant.No significantdifference was found in7d groups.Conclusion: Both P2X3receptor and Nav1.8ion channel were involved in experimental tooth movement pain; LLLT can inhibit geneexpression of P2X3, Nav1.8in TG during experimental tooth movement.In summary, we believe that the mechanisms of low energy laserirradiation to reduce orthodontic pain may be the result of multiple roles,including inhibition of the release of local inflammatory factors, promotingalterations of blood vessels, improving blood supply, inhibiting the release ofpain-caused neurotransmitter, promoting endogenous analgesicneurotransmitter to release, affecting ion channels and so on. Our experimentprovide objective evidence for low energy laser irradiation to reduceorthodontic pain, and provide a theoretical basis for its clinical application.