Clinical Assessment of Anti-inflammatory Activity of 940 Nanometer Low Level laser Therapy on Carrageenan Induced Arthritis in Temporomandibular Joint in Wistar Albino Rats

The aim of this study was to evaluate the possible anti-inflammatory effect of 940nm diode laser in treating carrageenan induced arthritis in temporomandibular joint in Wistar albino rats, using different energy density of LLLT, by clinical assessment of the inflammation. it was concluded that 74J/cm2 LLLT had very efficient in edema reduction and restore function with good anti-inflammatory effect, on the other hand 171J/cm2 had appear to be in or near the borderline between non-thermal and thermal lasers eradiation in considering to Westar rat skin.


iNtRODUCtiON
Temporomandibular joint disorders (TMDs) are considered as the most common nondental-related orofacial pain conditions which are usually presents with an inflammatory component 1,2 many types of non-surgical treatments are usually used to treat TMDs to improve and enhance function and to reduce or cure symptoms 3 such as physical therapy 4 , drug therapy 2,5 , various types of occlusal splints 4,6 , acupuncture7 and low level laser therapy 3 which is very promising method to treat TMDs 2, 3,8 . Carrageenan is an extract of seaweeds; Chondrus spp. and Gigartinaspp which is commonly known as Irish moss, chemically it is a sulphatedmucopolysaccharide 9 , for many years it had been used to induce inflammation in rat joints as in footpad , paw 10 and temporomandibular joint 2,5 , carrageenan induced arthritis is the most common and reliable method to test new anti-inflammatory drugs effectiveness and assessment of new strategies to treat inflammatory arthritis 2,11 . Low level laser therapy (LLLT) is a light-source therapeutic method that utilize light of a single wavelength, and acts through photochemical non-thermal reactions in the cells 5,12 , LLLT is a good option to treat arthritis in the temporomandibular joint due to its known analgesic, anti-inflammatory and regenerative effects 13 , another important advantage of LLLT is patient acceptance and reduce or stop using drugs thus get rid of its side effects 2,14 such as gastric ulcer and nephrotoxicity 15 .Previous studies have shown very good effects of LLLT of different wavelength such as 780, 850, 660 on carrageenan induced arthritis in rat TMJ 2,5,[15][16][17][18] . The aim of this study was to clinical evaluation of the effectiveness of 940nm LLLT on carrageenan induced arthritis in Wistar albino rats TMJ, using two different energy density (171J/cm 2 and 74J/cm 2 ).

MAteRiAls AND DisCUsiON Subjects
Following approval by the Committee of the college of Dentistry of the University of Baghdad, sixty male Wistar rats (150-200g) obtained from the animal house of the higher institute of infertility diagnosis and research assisting production of the University of Al-Nahrain were used in this study. Animals kept in acrylic cages lined with wood chips and maintained at 24°C in a day/night light cycle with limitless access to food and water. Procedure was carried out in the animal house during light period of the day from 8:30 a.m. to 12:30 p.m.

Preparation and calibration of laser device
Epic X diode laser device and deep tissue handpiece were used in this experiment, the handpiece was covered with tin foil diaphragm with central 10mm radius circular opening, the tin foil diaphragm was used to cover and protect eyes and ears from laser beam. Laser power was calibrated using Laser power and energy meter (Mastero, Gentec, Quebec, Canada), in put power was set to 4 watts as manufacturer recommendation to pain therapy and the output power measured by power meter was around 1.48 watts, then the input power set at 1.6 watts and the output power were measured by the power meter which was around 0.650 watts.

Formation of the experimental groups
Animals were randomly assigned to four experimental groups as following: Negative control group: injected by normal saline into TMJ area and applying deep tissue handpiece with 0 J/cm 2 .
Positive control group: injected with carrageenan solution into TMJ area with and applying deep tissue handpiece with 0 J/cm 2 .
LLLT with 171 J/cm 2 group: injected with carrageenan solution into TMJ area with and applying deep tissue handpiece with 171 J/cm 2 .
LLLT with 74 J/cm 2 group: injected with carrageenan solution into TMJ area with and applying deep tissue handpiece with 171 J/cm 2 . 2 Injection of saline and applying 0 J/cm 2 were made to ensure that all animals undergo the same circumstances and stressful procedures.

Carrageenan injection
Freshly prepared 1% carrageenan solution was made by dissolving carrageenan powder (Modernist, USA) in normal saline (Pioneer Co. IRAQ). The animals were anesthetized by inhalation of Diethyl ether (stab/ BHT, Netherlands) in close plastic container then hair on TMJ area were removed using hair removal cream and plastic spatula. thepostero-inferior border of the left zygomatic arch was palpated and a needle of an insulin syringe was inserted immediately below this point and then advanced in the anterior direction until reaching the postero-lateral aspect of the condyle. Then, 50 µL of the carrageenan solution was injected into the left TMJ 2,5,19,20 . lllt procedure Low level laser therapy procedure was made transcutaneously in one contact point facing the central position of left TMJ, immediately after carrageenan injection while the animal is under anesthesia, using diode laser device ( Biolase epic X, USA) and deep tissue handpiece ( Biolase epic, USA), using wavelength 940 nm, spot radius 10mm, irradiation time 90 second and energy density for first experimental group was 171J/cm 2 and 74 J/cm 2 for the second experimental group, the procedure were repeated every 48 hours for the next seven days (four session of irradiation).

Clinical assessment
Each animal was weighted before the induction of arthritis and every next day till the end of experiment (eight times) to evaluate weight changes 21,22 , and percentage of weight changes were recorded for each animal.Circumferences of animal head were measured using measuring tape in a circle around the head just anteriorly to the ears in line over TMJs in both sides before the induction of arthritis and every next day till the end of experiment (eight times) to evaluate circumferences changes which represent edema in TMJ 23 and percentage of circumferences changes were recorded for each animal.

Statistical analysis
Data were analyzed using SigmaStat for Windows version 4.0 build 4.0.0.37, Systat, USA. and the figures were made using Microsoft office 2016. Comparison between study groups were made using one-way ANOVA and multiple post hoc comparisons were performed using the Tukey test. A probability level of less than 0.05 was considered to indicate statistical significance

ResUlts AND DisCUsiON
Clinical assessment was done by evaluation of weight changes and edema in TMJ area caused by injection of carrageenan solution into TMJ between study groups, 128 Westar albino rats were used in this experiment divided into four study groups; negative control, positive control, 171J/cm 2 LLLT and 74J/cm 2 LLLT, 30 animals were randomly assigned in each experimental group and eight were died during or briefly after induction of arthritis.

Weight changes
Each animal was weighted by digital weight meter after light anesthesia early in the morning from 8-9 am from day of arthritis induction by carrageenan (day zero) and for the next seven days, statistically there was highly significant increase of weight in the negative control group (p=<0.001) and non-significant decrease in weight in positive control and 171J/ cm 2 laser therapy group (P = 0.903 and 0.426 respectively) and there was non-significant increase in weight in 74J/cm 2 laser therapy group (table 1), a non-significant increase in weight was reported from day one to day four in LLLT group treated by 171J/cm 2 laser radiation then a nonsignificant decrease of weight was reported till the end of the experiment (Fig. 1). Comparison of weight changes between study groups on each day of the experiment (table 2) showed a nonsignificant differences between them in the first four days of the experiment, but a highly significant differences were reported from day 4-7 so Pairwise Multiple Comparison Procedures using Holm-Sidak method were done to clarify the particular differences in every study group for the day 4-7. Analysis of day four (Table 3) results reveals highly significant differences in weight between negative and positive study groups (p=0.002) and nonsignificant differences between all other groups. In day five of the experiment; mean of weight of the positive control group was highly-significantly lower than that of negative control group (P= <0.001) and there was statistically significant decrease in weight of 171J/cm 2 group in comparing to negative control ( P=0.029), regarding 74 J/cm 2 groupa non-significant differences were recorded (  table 4). Day six results; weight mean of positive control group continued to be highly significantly lower than that of negative control one with P= <0.001, 171 J/cm 2 group also continued to be highly significantly lower than that of negative control group with P=0.005, regarding 74 J/cm 2 there were non-significant differences in comparing to negative control, but there was significant increase in weight when comparing 74 J/cm 2 to the positive control group with P= 0.035 as shown in table (5).in the last day of the experiment, day seven; weight differences were continued to increase between groups, regarding positive control group and 171 J/cm 2 group the mean of weight in both were statistically highly significantly lower than that of negative control(P=<0.001), regarding 74 J/cm 2 group there was non-significantly different from mean of negative control group, but significant increase in weight were obvious in comparing to positive and 171 J/cm 2 croup and the P value were 0.021 and 0.038 respectively as shown below in table (6). edema assessment Injection of carrageenan solution into TMJ area caused marked edema (figure 2) was obvious with naked eye, to assess the validity of LLLT treatment the circumference of rat head was measured before and every successive day to the end of the experiment (figure 3), one-way ANOVA to compare between means of head circumferences with negative control group which was injected with normal saline solution instead of carrageenan solution. The analysis of one-way ANOVA of head circumferences in day zero showed non-significant differences between study groups (P=0.648) which indicate no edema present at this moment, 24  hours later highly significant differences with P value <0.001, for the next two days till day three the same results were reported with the exact P value, then in day four and day five the differences begun to decrease but the differences still statistically significant with P value 0.017, 0.027 respectively, in the last two days of the experiment ( day 6 and 7) the decrease in differences continued to reach a non-significant level as shown in table (7). Post hoc analysis were done to compare all pair of study groups in each day of the experiment, in day zero the analysis revealed as it was expected a non-significant differences between study groups (table 8). Twenty-four hours later the numbers dramatically changed with highly significant increase of head circumferences (P=<0.001) in positive control, 171J/cm2 and 74J/cm2 groups in comparing to negative control group (table 9) which were due to the massive edema caused by carrageenan solution injected in TMJ in these groups, non-significant differences were recorded between all three group which were injected with carrageenan solution which revealed that the swelling was almost identical at this stage between them. In day two the head circumferences were still highly significantly higher in positive control and 171J/cm2 groups when compared with negative control group thus the edema was still obvious in these groups 48 hours after the initial induction of inflammation by carrageenan solution, but things were different in 74J/cm2 LLLT group, a non-significant difference with P value = 0.197 was reported between 74J/cm2 LLLT group and negative control group and highly significant differences between 74J/cm2 LLLT group and  positive control group with P value =0.007 which indicate that the edema begun to subside only 48 hours after the initial induction of inflammation, also there were a non-significant difference with P value = 0.407 between 171J/cm2 and positive control which mean that this parameter are still not effective in this stage (table 10). Day three results analysis with post hoc showed that the edema was still present in positive control group and the highly significant increase in head circumference compared to negative control group with P value =<0.001, both LLLT groups (171J/cm2 and 74J/cm2) showed non-significant differences compared to negative control and significant / highly significant lower head circumferences in comparing to positive control group indicating the edema begun to subside in both groups as shown in table (11).Post hoc analysis of day four showed significant increase in head circumference in positive control group compared to negative control group with P value =0.014, other pairs comparison results were non-significantly different compared to each other as shown below in table (12).Day five results almost the same as day before with significant increase in head diameter in positive group comparing to negative group (p= 0.015), all other pairs comparing were non-significantly different (table 13).In day six there was non-significant increase in head diameter of the positive control group in comparing to negative control group (p= 0.059) which indicated that inflammation in positive control group started to subside. All other pairs were non significantly different when compared to each other (table 14).
Comparison of head circumferences of all pairs in the last day of the experiment revealed non statistically significant differences between all pairs, indicating that the inflammation end in all groups (table 15). Induction of arthritis was made using the most common method used to evaluate the effects of different types of treatment protocols on inflammatory arthritis which is induction of arthritis using carrageenan solution injected directly into TMJ, carrageenan is polysaccharide obtained from sea weed, it have potent pro-inflammatory effect in animal joint when injected into the joint space 11 , previous studies had proof the carrageenan effectiveness in arthritis induction in TMJ of rats 19,20,24,25 . Pilot study revealed massive edema into TMJ area and decrease amount of food intake by rats suffering from arthritis comparing to normal rats and apparently that decrease in food consumption caused these rats look thinner, thus we made an  31 , others relied on volume changes in paws to assess i n d u c e d a r t h r i t i s u s i n g d i s p l a c e m e n t plethysmometry 29,36,37 , apparently this method can precisely measure edema progression and regression but unfortunately cannot applied in our study due to possible rat suffocation in water during the procedure hence it was anesthetized, thus our study relied on circumference measurement as parameter to assess edema in area TMJ. Before discussion inflammatory results it's important to mention An unexpected observation noticed during this experiment in the study group treated with 171J/cm 2 which is developing of red lesion of burns in the area of TMJ in 21 of the 30 rats exposed to two or more session of laser radiation(figure 4) noticed in the day after the session, none of these lesions were reported in other groups (P = <0.001), thus it was concluded that 171J/cm 2 was in or near the borderline between non-thermal and thermal lasers eradiation in considering to Westar rat skin. Another observation was the slight hair growth noticed in 74J/cm2 LLLT and in rats which didn't developed burns in 171J/cm 2 LLLT group at last day of the experiment, no such growth was observed in other rats, which can be explained by biostimulation of hair follicles by LLLT 38,40 . The results of this study showed that irradiation of the TMJ area of rats with 940 nm diode laser with 74 J/cm 2 lead to faster resolution of the inflammation regarding swelling size and normal weight gain of the rat. This laser dose didn't cause burning or negative impact to skin overlying the TMJ area. Typically, edema is caused by intensification in vascular permeability, fluid retention, extravasation of protein and other cellular component at the site of inflammation 41 , LLLT with suitable dosimetry can revert these effects by increasing blood supply, clearing inflammatory agent and downgrading inflammatory response 3,42,44 .Slowing the process of weight gain is caused by difficulty in eating due to induced arthritis followed by limited mouth opening 33 . Using the lower laser setting of 74 J/ cm 2 had led to nearly normal weight gain which was close to the negative control group. This finding further support the results of rat head circumference (edema suppression). This means that the reduction in the head circumference passing the TMJ area has led to normal mouth opening and normal feeding process 45 . on the other hand, LLLT using 171J/cm 2 caused initial good result when edema decreased and body weight was somewhat identical to 74J/cm 2 till day four when result begun to change and weight started to decrease reaching similar level of positive control group, these finding can be due to burnings in TMJ area occurred in some of the rats which was an indication that this dose represents the threshold settings. This finding accomplices with Arndt-Schulz law which states that biostimulation falls in a window area and doses over this window will have no or even adverse effect to the biological tissues 46 .

CONClUsiON
The results of this study demonstrate that LLLT using 74J/cm 2 have a very powerful antiinflammatory effect on inflamed TMJ.