Ó 2014 Eur J Oral Sci
Eur J Oral Sci 2014; 122: 121–124 DOI: 10.1111/eos.12110 Printed in Singapore. All rights reserved
European Journal of Oral Sciences
Evaluation of pain in rats through facial expression following experimental tooth movement
Lina Liao1, Hu Long1, Li Zhang2, Helin Chen2, Yang Zhou1, Niansong Ye1, Wenli Lai1 1
State Key Laboratory of Oral Diseases, Department of Orthodontics, West China Hospital of Stomatology, Sichuan University, Chengdu; 2State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China
Liao L, Long H, Zhang L, Chen H, Zhou Y, Ye N, Lai W. Evaluation of pain in rats through facial expression following experimental tooth movement. Eur J Oral Sci 2014; 122: 121–124. © 2014 Eur J Oral Sci This study was carried out to evaluate pain in rats by monitoring their facial expressions following experimental tooth movement. Male Sprague-Dawley rats were divided into the following five groups based on the magnitude of orthodontic force applied and administration of analgesics: control; 20 g; 40 g; 80 g; and morphine + 40 g. Closed-coil springs were used to mimic orthodontic forces. The facial expressions of each rat were videotaped, and the resulting rat grimace scale (RGS) coding was employed for pain quantification. The RGS score increased on day 1 but showed no significant change thereafter in the control and 20-g groups. In the 40and 80-g groups, the RGS scores increased on day 1, peaked on day 3, and started to decrease on day 5. At 14 d, the RGS scores were similar in control and 20-, 40-, and 80-g groups and did not return to baseline. The RGS scores in the morphine + 40-g group were significantly lower than those in the control group. Our results reveal that coding of facial expression is a valid method for evaluation of pain in rats following experimental tooth movement. Inactivated springs (no force) still cause discomfort and result in an increase in the RGS. The threshold force magnitude required to evoke orthodontic pain in rats is between 20 and 40 g.
Orthodontic pain is one of the most troublesome problems encountered in clinical practice and is frequently reported by patients undergoing orthodontic treatment (1, 2). Its incidence has been reported to be 87–100% (2–4). Although many animal studies have been conducted to elucidate the mechanisms underlying orthodontic pain (5–7), its exact mechanisms are still largely unknown. This can be partly attributed to the difficulty in measuring pain levels in animals following experimental tooth movement. Facial expression analysis has gained wide popularity in humans unable to communicate verbally (e.g. infants and patients with dementia) (8–11). Moreover, it has been recognized as the most sensitive and specific nonverbal cue for pain in humans (12, 13). Likewise, facial expression analysis could be applied in experimental animals that are unable to express verbally. LANGFORD et al. (14) developed a novel method to code facial expressions of mice experiencing pain, and SOTOCINAL et al. (15) modified this method and developed a software package to evaluate pain in rats. Both of these studies demonstrated that facial expression coding is a reliable method for evaluating pain in rodents. However, to date, the facial expressions of rats following experimental tooth movement are still unknown and the pain levels assessed through facial expressions in rats are poorly understood. In this study, we aimed
Wenli Lai, State Key Laboratory of Oral Diseases, Department of Orthodontics, West China Hospital of Stomatology, Sichuan University, No. 14, Section 3, Ren Min Nan Road, Chengdu, China E-mail: [email protected] Key words: behavior assessment; orthodontic pain; orthodontic tooth movement Accepted for publication November 2013
to evaluate pain in rats by monitoring their facial expressions following experimental tooth movement.
Material and methods Animals A total of 40 male Sprague-Dawley rats, weighing 250–300 g, were used in this study. They were supplied by the Animal Experimental Center, Sichuan University. The study was reviewed and approved by the Ethics Committee of the State Key Laboratory of Oral Diseases and also met the standards described in the European Convention. The rats were housed in a temperature-regulated room at 25 2°C with a 12-h light/12-h dark cycle (lights on at 06.00 h and off at 18.00 h). Sample size calculation was performed using STATSDIRECT 2.7.9 (StatsDirect, Cheshire, UK), which gave a minimum sample size of eight rats for each group. Accordingly, eight rats were placed in one of each of the following five groups for treatment with difference magnitudes of orthodontic force (g) and an analgesic: control group (no orthodontic force); 20-g group; 40-g group; 80-g group; and morphine + 40-g group. For each group, closed-coiled springs were ligated between the upper incisors and the first molars to mimic orthodontic forces (16). For rats in the control group, the closed-coil springs were not activated and thus no force was applied. For the morphine + 40-g group, rats received a force magnitude of
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A
B
C
D
E
F
Fig. 1. Images captured from videotapes. (A) No orbital tightening was observed (black arrow). The whiskers did not move forward and showed no tendency to bunch (red arrow). (B) The whisker pad was rounded (black arrow) and a bulge was present on the top of the nose. (C) The ear had a rounded shape (black arrow). (D) The rat displayed an eye squeeze (black arrow). (E) The ears of the rat were folded and angled outwards (black arrow). The bridge of the rat nose was flattened and elongated (red arrow). (F) The whiskers tended to bunch (black arrow).
40 g and were injected with 3 mg kg-1 of morphine 15 min before each videotaping session. Videotaping facial expressions Before the placement of closed-coil springs, rats were videotaped to obtain their baseline RGS scores. In brief, the rats were placed individually in a cubicle (21.0 9 10.5 9 9.0 cm3), without a bottom, on a metal ventilated shelf. The walls of the cubicle were made of transparent glass. Two Sony digital video cameras (Tokyo, Japan) were placed on either side of the cubicle. Fifteen minutes after placement in the cubicle, the rats were videotaped for 30 min continuously. This procedure was repeated at each study time point (i.e. 1, 3, 5, 7, and 14 d) after placement of closedcoil springs and treatment. Videotaping was conducted in a room with 45 dB of background noise and between 21.00 and 24.00 h. Image capture (RFF), the software developed by SOet al. to automate images of rat facial expression extracted from videotapes, was used to capture images for scoring (15). Being able to detect rodent eyes and ears in videos, RFF can capture three clear images in each of the 3-min study time and the most clear image should be chosen for scoring. In addition, on some occasions, manual extraction of images was performed when RFF could not capture images. In total, 10 images should be captured from each videotaping session. The selected image files were copied into Microsoft PowerPoint slides randomly, with one image per slide. Identification of the images was hidden to ensure blind coding and the images were presented on a computer monitor for scoring (Fig. 1). RODENT FACE FINDER
TOCINAL
RGS: orbital tightening; nose/cheek flattening; ear changes; and whisker changes. For orbital tightening, a narrowing of the orbital area is displayed in rats when in pain, presenting as eye closure or eye squeezing. For nose/cheek flattening, rats in pain display the lack of a bulge on the top of the nose. The bridge of the nose flattens and elongates in rat experiencing pain, which causes the whisker pads to flatten, and the crease between the pads and the cheek disappear. For ear changes, the ears of rats in pain tend to fold, curl, and angle forwards or outwards. The space between the ears may broaden and ears display a pointed shape. For whisker changes, the whisker pad of rats is contracted when pain is experienced, and the whiskers move forward and tend to bunch. All four RGS action units were scored with values of 0, 1, or 2 for each image. A score of ‘0’ indicates that the scorer has high confidence of the absence of the action. A score of ‘1’ indicates high confidence of a moderate performance of the action units or equivocation over its presence or absence. A score of ‘2’ indicates that pronounced performance of the action units was observed. The RGS scores were coded in duplicate and independently by two researchers and any disagreement was solved by discussion. An RGS score of each photograph was calculated by averaging the scores of the four RGS action units. Then, a mean RGS score for each video was obtained from 10 photographs and this mean RGS score reflected the level of pain. The mean RGS score of each rat, before the placement of springs, indicated ‘no pain’ and was used as the baseline value. An RGS score difference was obtained by subtracting the baseline RGS score from the RGS scores after placement of springs. This RGS score difference was used as the RGS score in all the analyses. Statistical analyses
RGS coding
analyses were employed to compare RGS scores among different groups and the chronological changes of RGS scores. All statistical analyses were performed in SPSS
ANOVA
The RGS coding was conducted according to SOTOCINAL et al. (15). Specifically, there were four action units of
Evaluation of orthodontic pain
Fig. 2. Changes of rat grimace scale (RGS) score following initiation of experimental tooth movement. The RGS score increased at day 1 and did not change significantly thereafter in the control and 20-g groups. In the 40-g group, the RGS score increased at day 1, peaked at day 3, and had started to decrease at day 5. Similarly, in the 80-g group, the RGS score increased at day 1, remained at that level at day 3, and decreased thereafter. The RGS score in the morphine + 40-g group increased at day 3 and thereafter decreased to a level similar to, or below, baseline. The RGS score in each of the control and 20-, 40-, and 80-g groups did not return to baseline but was similar among the four groups at day 14. Significant differences among groups on the same day are indicated by an asterisk.
16.0 (SPSS, Chicago, IL, USA) and P < 0.05 was considered as statistically significant.
Results Chronological changes of RGS score
As shown in Fig. 2, in the control group, compared with baseline (RGS = 0), the RGS score increased significantly at day 1, peaked at day 3 and was maintained at this increased level on days 5, 7, and 14, and did not return to the baseline level. For the 20-g group, the RGS score had increased significantly at day 1 compared with baseline (RGS=0) and remained at this level at days 3, 5, 7, and 14 (all P > 0.05) (Fig. 2). In the 40-g group, the RGS score had increased significantly at days 1 and 3, compared with baseline (RGS=0). Thereafter, the RGS score decreased at days 5, 7, and 14 (Fig. 2). In the 80-g group, the RGS score increased significantly at days 1 and 3 compared with baseline. Thereafter, the RGS decreased at days 5, 7, and 14 (Fig. 2). For the morphine + 40-g group the RGS score started to increase at day 1, peaked at day 3, and decreased to baseline levels or below at days 5, 7, and 14 (Fig. 2). Differences in RGS scores between different force groups
As shown in Fig. 2, at day 1 the differences in RGS scores were not significantly different between the
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control group and the 20-g group (P > 0.05). In contrast, the RGS scores were significantly higher in the 40-g group (P = 0.003) and the 80-g group (P < 0.001) than in the control group. In addition, the RGS score was significantly higher in the 80-g group than in the 40-g group (P = 0.001). Moreover, RGS scores were significantly lower in the morphine + 40-g group than in the control group (P < 0.001). At day 3, the RGS scores were similar among the control group, the 20-g group, and the morphine + 40g group (P > 0.05) and were significantly higher in the 40-g group (P < 0.001) and the 80-g group (P < 0.02). However, the RGS scores did not differ significantly between the 40-g group and the 80-g group (P > 0.05). At days 5, 7, and 14, the differences in RGS scores were statistically nonsignificant among the control, 20-, 40-, and 80-g groups (P > 0.05). In addition, the RGS scores were significantly lower in the morphine + 40-g group than in the other four groups (P < 0.001).
Discussion In this study, we found that the RGS scores increased at days 1 and 3, and then decreased at days 5, 7, and 14, in the control, 20-, 40-, and 80-g groups. However, the RGS score did not return to baseline at day 14. Particularly for the control group, as no force was applied, it is conceivable that the pain levels should not change compared with baseline (17). However, the RGS score increased at day 1 and peaked at day 3, which we attribute to the discomfort caused by the intra-oral springs. Moreover, the pain levels were similar among all the groups at 14 d, but all did not return to baseline values. We can attribute this also to the discomfort caused by the intra-oral springs. In this study, the general trend of pain levels with time is consistent with that of a previous study (5), in which pain levels were assessed through directed facegrooming activity (i.e. the time spent on mouth wiping). This study revealed that pain levels, as evaluated through face-grooming, started to increase at day 1, decreased thereafter, and had returned to baseline at 14 d. In contrast, in the present study, we found that pain levels, assessed using the RGS, did not return to the baseline level at day 14. Considering that RGS scores increased in the control group, we suggest that the bulky intra-oral springs (even if inactivated) could cause discomfort and hence an increase in the RGS score. Therefore, we suggest that the discrepancy in pain levels between this and the previous study may be attributed to different sensitivities of the two modalities in assessing pain. We found that pain levels, as assessed using the RGS, were significantly correlated with the levels of expression of the P2X3 receptor (reported previously) (5) in trigeminal ganglia following experimental tooth movement (P < 0.05). Moreover, it has been reported that the expression of c-Fos was significantly elevated in the trigeminal nucleus, 1 and 3 d following experimental tooth movement (18), which is consistent with
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the results of the present study (data were unavailable at 5, 7, or 14 d; thus, the correlation test was not performed). These findings suggest that RGS coding would be a valid pain-assessment tool for experimental tooth movement. Moreover, the RGS scores increased only on day 3 and were similar to, or below, the baseline level in the morphine + 40-g group, suggesting that morphine could greatly attenuate pain sensations produced by experimental tooth movement. On the other hand, given that morphine is a well-documented analgesic agent, this decrease of RGS score by morphine would further support the validity of pain assessment through RGS coding. In clinical practice, most orthodontic patients experience the most severe pain 1 d after force application, and the pain levels decrease thereafter (1). However, in this study, we found that the pain levels were similar between days 1 and 3 after force application, or were higher at 3 d than at 1 d. This difference between species may be a result of the discomfort caused by bulky orthodontic appliances: orthodontic appliances are relatively larger in rats’ mouths. Hence, the discomfort led to the continued increase of pain levels at 3 d. Our results revealed that the RGS scores were similar between the control group and the 20-g group at all time points, which indicates that the pain levels were similar between the two groups. This finding suggests that the force magnitude of 20 g was not large enough to evoke pain sensations. Considering that pain was evoked in the 40-g group compared with the control group at days 1 and 3, we suggest that the threshold force magnitude to evoke orthodontic pain lies between 20 and 40 g. Acknowledgements – This work was supported by the National Natural Science Foundation of China (NSFC), nos 81070858 and 81100778. Conflicts of interest – The authors report no conflicts of interest.
References 1. JONES M, CHAN C. The pain and discomfort experienced during orthodontic treatment: a randomized controlled clinical trial of two initial aligning arch wires. Am J Orthod Dentofacial Orthop 1992; 102: 373–381. 2. BERGIUS M, BERGGREN U, KILIARIDIS S. Experience of pain during an orthodontic procedure. Eur J Oral Sci 2002; 110: 92–98. 3. ERDINC AM, DINCER B. Perception of pain during orthodontic treatment with fixed appliances. Eur J Orthod 2004; 26: 79–85.
4. KHATTAB TZ, FARAH H, AL-SABBAGH R, HAJEER MY, HAJ-HAMED Y. Speech performance and oral impairments with lingual and labial orthodontic appliances in the first stage of fixed treatment. Angle Orthod 2013; 83: 519–526. 5. YANG Z, CAO Y, WANG Y, LUO W, HUA X, LU Y, LIAO Z, LAI W, ZHAO Z. Behavioural responses and expression of P2X3 receptor in trigeminal ganglion after experimental tooth movement in rats. Arch Oral Biol 2009; 54: 63–70. 6. LIAO L, HUA X, LONG H, YE N, ZHOU Y, WANG S, LAI W. Expression patterns of nociceptin in rats following experimental tooth movement. Angle Orthod 2013; 83: 1022–1026. 7. YANG Z, WANG Y, LUO W, HUA X, WAMALWA P, WANG J, ZHAO Z, LU Y, LIAO Z, LAI W. Trigeminal expression of Nmethyl-D-aspartate receptor subunit 1 and behavior responses to experimental tooth movement in rats. Angle Orthod 2009; 79: 951–957. 8. SHEU E, VERSLOOT J, NADER R, KERR D, CRAIG KD. Pain in the elderly: validity of facial expression components of observational measures. Clin J Pain 2011; 27: 593–601. 9. GRUNAU RV, CRAIG KD. Pain expression in neonates: facial action and cry. Pain 1987; 28: 395–410. 10. HADJISTAVROPOULOS T, HERR K, TURK DC, FINE PG, DWORKIN RH, HELME R, JACKSON K, PARMELEE PA, RUDY TE, LYNN BEATTIE B, CHIBNALL JT, CRAIG KD, FERRELL B, FERRELL B, FILLINGIM RB, GAGLIESE L, GALLAGHER R, GIBSON SJ, HARRISON EL, KATZ B, KEEFE FJ, LIEBER SJ, LUSSIER D, SCHMADER KE, TAIT RC, WEINER DK, WILLIAMS J. An interdisciplinary expert consensus statement on assessment of pain in older persons. Clin J Pain 2007; 23(Suppl): S1–S43. 11. GRUNAU RV, JOHNSTON CC, CRAIG KD. Neonatal facial and cry responses to invasive and non-invasive procedures. Pain 1990; 42: 295–305. 12. ANAND KJ, CRAIG KD. New perspectives on the definition of pain. Pain 1996; 67: 3–6; discussion 209–211. 13. KENNETH M, PRKACHIN KDC. Expressing pain: The communication and interpretation of facial pain signals. J Nonverbal Behav 1995; 19: 191–205. 14. LANGFORD DJ, BAILEY AL, CHANDA ML, CLARKE SE, DRUMMOND TE, ECHOLS S, GLICK S, INGRAO J, KLASSEN-ROSS T, LACROIX-FRALISH ML, MATSUMIYA L, SORGE RE, SOTOCINAL SG, TABAKA JM, WONG D, VAN DEN MAAGDENBERG AM, FERRARI MD, CRAIG KD, MOGIL JS. Coding of facial expressions of pain in the laboratory mouse. Nat Methods 2010; 7: 447–449. 15. SOTOCINAL SG, SORGE RE, ZALOUM A, TUTTLE AH, MARTIN LJ, WIESKOPF JS, MAPPLEBECK JC, WEI P, ZHAN S, ZHANG S, MCDOUGALL JJ, KING OD, MOGIL JS. The Rat Grimace Scale: a partially automated method for quantifying pain in the laboratory rat via facial expressions. Mol Pain 2011; 7: 55. 16. SHIRAZI M, KHOSROWSHAHI M, DEHPOUR AR. The effect of chronic renal insufficiency on orthodontic tooth movement in rats. Angle Orthod 2001; 71: 494–498. 17. REN Y, MALTHA JC, KUIJPERS-JAGTMAN AM. Optimum force magnitude for orthodontic tooth movement: a systematic literature review. Angle Orthod 2003; 73: 86–92. 18. FUJIYOSHI Y, YAMASHIRO T, DEGUCHI T, SUGIMOTO T, TAKANO-YAMAMOTO T. The difference in temporal distribution of c-Fos immunoreactive neurons between the medullary dorsal horn and the trigeminal subnucleus oralis in the rat following experimental tooth movement. Neurosci Lett 2000; 283: 205–208.
Eur J Oral Sci 2014; 122: 121–124 DOI: 10.1111/eos.12110 Printed in Singapore. All rights reserved
European Journal of Oral Sciences
Evaluation of pain in rats through facial expression following experimental tooth movement
Lina Liao1, Hu Long1, Li Zhang2, Helin Chen2, Yang Zhou1, Niansong Ye1, Wenli Lai1 1
State Key Laboratory of Oral Diseases, Department of Orthodontics, West China Hospital of Stomatology, Sichuan University, Chengdu; 2State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China
Liao L, Long H, Zhang L, Chen H, Zhou Y, Ye N, Lai W. Evaluation of pain in rats through facial expression following experimental tooth movement. Eur J Oral Sci 2014; 122: 121–124. © 2014 Eur J Oral Sci This study was carried out to evaluate pain in rats by monitoring their facial expressions following experimental tooth movement. Male Sprague-Dawley rats were divided into the following five groups based on the magnitude of orthodontic force applied and administration of analgesics: control; 20 g; 40 g; 80 g; and morphine + 40 g. Closed-coil springs were used to mimic orthodontic forces. The facial expressions of each rat were videotaped, and the resulting rat grimace scale (RGS) coding was employed for pain quantification. The RGS score increased on day 1 but showed no significant change thereafter in the control and 20-g groups. In the 40and 80-g groups, the RGS scores increased on day 1, peaked on day 3, and started to decrease on day 5. At 14 d, the RGS scores were similar in control and 20-, 40-, and 80-g groups and did not return to baseline. The RGS scores in the morphine + 40-g group were significantly lower than those in the control group. Our results reveal that coding of facial expression is a valid method for evaluation of pain in rats following experimental tooth movement. Inactivated springs (no force) still cause discomfort and result in an increase in the RGS. The threshold force magnitude required to evoke orthodontic pain in rats is between 20 and 40 g.
Orthodontic pain is one of the most troublesome problems encountered in clinical practice and is frequently reported by patients undergoing orthodontic treatment (1, 2). Its incidence has been reported to be 87–100% (2–4). Although many animal studies have been conducted to elucidate the mechanisms underlying orthodontic pain (5–7), its exact mechanisms are still largely unknown. This can be partly attributed to the difficulty in measuring pain levels in animals following experimental tooth movement. Facial expression analysis has gained wide popularity in humans unable to communicate verbally (e.g. infants and patients with dementia) (8–11). Moreover, it has been recognized as the most sensitive and specific nonverbal cue for pain in humans (12, 13). Likewise, facial expression analysis could be applied in experimental animals that are unable to express verbally. LANGFORD et al. (14) developed a novel method to code facial expressions of mice experiencing pain, and SOTOCINAL et al. (15) modified this method and developed a software package to evaluate pain in rats. Both of these studies demonstrated that facial expression coding is a reliable method for evaluating pain in rodents. However, to date, the facial expressions of rats following experimental tooth movement are still unknown and the pain levels assessed through facial expressions in rats are poorly understood. In this study, we aimed
Wenli Lai, State Key Laboratory of Oral Diseases, Department of Orthodontics, West China Hospital of Stomatology, Sichuan University, No. 14, Section 3, Ren Min Nan Road, Chengdu, China E-mail: [email protected] Key words: behavior assessment; orthodontic pain; orthodontic tooth movement Accepted for publication November 2013
to evaluate pain in rats by monitoring their facial expressions following experimental tooth movement.
Material and methods Animals A total of 40 male Sprague-Dawley rats, weighing 250–300 g, were used in this study. They were supplied by the Animal Experimental Center, Sichuan University. The study was reviewed and approved by the Ethics Committee of the State Key Laboratory of Oral Diseases and also met the standards described in the European Convention. The rats were housed in a temperature-regulated room at 25 2°C with a 12-h light/12-h dark cycle (lights on at 06.00 h and off at 18.00 h). Sample size calculation was performed using STATSDIRECT 2.7.9 (StatsDirect, Cheshire, UK), which gave a minimum sample size of eight rats for each group. Accordingly, eight rats were placed in one of each of the following five groups for treatment with difference magnitudes of orthodontic force (g) and an analgesic: control group (no orthodontic force); 20-g group; 40-g group; 80-g group; and morphine + 40-g group. For each group, closed-coiled springs were ligated between the upper incisors and the first molars to mimic orthodontic forces (16). For rats in the control group, the closed-coil springs were not activated and thus no force was applied. For the morphine + 40-g group, rats received a force magnitude of
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A
B
C
D
E
F
Fig. 1. Images captured from videotapes. (A) No orbital tightening was observed (black arrow). The whiskers did not move forward and showed no tendency to bunch (red arrow). (B) The whisker pad was rounded (black arrow) and a bulge was present on the top of the nose. (C) The ear had a rounded shape (black arrow). (D) The rat displayed an eye squeeze (black arrow). (E) The ears of the rat were folded and angled outwards (black arrow). The bridge of the rat nose was flattened and elongated (red arrow). (F) The whiskers tended to bunch (black arrow).
40 g and were injected with 3 mg kg-1 of morphine 15 min before each videotaping session. Videotaping facial expressions Before the placement of closed-coil springs, rats were videotaped to obtain their baseline RGS scores. In brief, the rats were placed individually in a cubicle (21.0 9 10.5 9 9.0 cm3), without a bottom, on a metal ventilated shelf. The walls of the cubicle were made of transparent glass. Two Sony digital video cameras (Tokyo, Japan) were placed on either side of the cubicle. Fifteen minutes after placement in the cubicle, the rats were videotaped for 30 min continuously. This procedure was repeated at each study time point (i.e. 1, 3, 5, 7, and 14 d) after placement of closedcoil springs and treatment. Videotaping was conducted in a room with 45 dB of background noise and between 21.00 and 24.00 h. Image capture (RFF), the software developed by SOet al. to automate images of rat facial expression extracted from videotapes, was used to capture images for scoring (15). Being able to detect rodent eyes and ears in videos, RFF can capture three clear images in each of the 3-min study time and the most clear image should be chosen for scoring. In addition, on some occasions, manual extraction of images was performed when RFF could not capture images. In total, 10 images should be captured from each videotaping session. The selected image files were copied into Microsoft PowerPoint slides randomly, with one image per slide. Identification of the images was hidden to ensure blind coding and the images were presented on a computer monitor for scoring (Fig. 1). RODENT FACE FINDER
TOCINAL
RGS: orbital tightening; nose/cheek flattening; ear changes; and whisker changes. For orbital tightening, a narrowing of the orbital area is displayed in rats when in pain, presenting as eye closure or eye squeezing. For nose/cheek flattening, rats in pain display the lack of a bulge on the top of the nose. The bridge of the nose flattens and elongates in rat experiencing pain, which causes the whisker pads to flatten, and the crease between the pads and the cheek disappear. For ear changes, the ears of rats in pain tend to fold, curl, and angle forwards or outwards. The space between the ears may broaden and ears display a pointed shape. For whisker changes, the whisker pad of rats is contracted when pain is experienced, and the whiskers move forward and tend to bunch. All four RGS action units were scored with values of 0, 1, or 2 for each image. A score of ‘0’ indicates that the scorer has high confidence of the absence of the action. A score of ‘1’ indicates high confidence of a moderate performance of the action units or equivocation over its presence or absence. A score of ‘2’ indicates that pronounced performance of the action units was observed. The RGS scores were coded in duplicate and independently by two researchers and any disagreement was solved by discussion. An RGS score of each photograph was calculated by averaging the scores of the four RGS action units. Then, a mean RGS score for each video was obtained from 10 photographs and this mean RGS score reflected the level of pain. The mean RGS score of each rat, before the placement of springs, indicated ‘no pain’ and was used as the baseline value. An RGS score difference was obtained by subtracting the baseline RGS score from the RGS scores after placement of springs. This RGS score difference was used as the RGS score in all the analyses. Statistical analyses
RGS coding
analyses were employed to compare RGS scores among different groups and the chronological changes of RGS scores. All statistical analyses were performed in SPSS
ANOVA
The RGS coding was conducted according to SOTOCINAL et al. (15). Specifically, there were four action units of
Evaluation of orthodontic pain
Fig. 2. Changes of rat grimace scale (RGS) score following initiation of experimental tooth movement. The RGS score increased at day 1 and did not change significantly thereafter in the control and 20-g groups. In the 40-g group, the RGS score increased at day 1, peaked at day 3, and had started to decrease at day 5. Similarly, in the 80-g group, the RGS score increased at day 1, remained at that level at day 3, and decreased thereafter. The RGS score in the morphine + 40-g group increased at day 3 and thereafter decreased to a level similar to, or below, baseline. The RGS score in each of the control and 20-, 40-, and 80-g groups did not return to baseline but was similar among the four groups at day 14. Significant differences among groups on the same day are indicated by an asterisk.
16.0 (SPSS, Chicago, IL, USA) and P < 0.05 was considered as statistically significant.
Results Chronological changes of RGS score
As shown in Fig. 2, in the control group, compared with baseline (RGS = 0), the RGS score increased significantly at day 1, peaked at day 3 and was maintained at this increased level on days 5, 7, and 14, and did not return to the baseline level. For the 20-g group, the RGS score had increased significantly at day 1 compared with baseline (RGS=0) and remained at this level at days 3, 5, 7, and 14 (all P > 0.05) (Fig. 2). In the 40-g group, the RGS score had increased significantly at days 1 and 3, compared with baseline (RGS=0). Thereafter, the RGS score decreased at days 5, 7, and 14 (Fig. 2). In the 80-g group, the RGS score increased significantly at days 1 and 3 compared with baseline. Thereafter, the RGS decreased at days 5, 7, and 14 (Fig. 2). For the morphine + 40-g group the RGS score started to increase at day 1, peaked at day 3, and decreased to baseline levels or below at days 5, 7, and 14 (Fig. 2). Differences in RGS scores between different force groups
As shown in Fig. 2, at day 1 the differences in RGS scores were not significantly different between the
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control group and the 20-g group (P > 0.05). In contrast, the RGS scores were significantly higher in the 40-g group (P = 0.003) and the 80-g group (P < 0.001) than in the control group. In addition, the RGS score was significantly higher in the 80-g group than in the 40-g group (P = 0.001). Moreover, RGS scores were significantly lower in the morphine + 40-g group than in the control group (P < 0.001). At day 3, the RGS scores were similar among the control group, the 20-g group, and the morphine + 40g group (P > 0.05) and were significantly higher in the 40-g group (P < 0.001) and the 80-g group (P < 0.02). However, the RGS scores did not differ significantly between the 40-g group and the 80-g group (P > 0.05). At days 5, 7, and 14, the differences in RGS scores were statistically nonsignificant among the control, 20-, 40-, and 80-g groups (P > 0.05). In addition, the RGS scores were significantly lower in the morphine + 40-g group than in the other four groups (P < 0.001).
Discussion In this study, we found that the RGS scores increased at days 1 and 3, and then decreased at days 5, 7, and 14, in the control, 20-, 40-, and 80-g groups. However, the RGS score did not return to baseline at day 14. Particularly for the control group, as no force was applied, it is conceivable that the pain levels should not change compared with baseline (17). However, the RGS score increased at day 1 and peaked at day 3, which we attribute to the discomfort caused by the intra-oral springs. Moreover, the pain levels were similar among all the groups at 14 d, but all did not return to baseline values. We can attribute this also to the discomfort caused by the intra-oral springs. In this study, the general trend of pain levels with time is consistent with that of a previous study (5), in which pain levels were assessed through directed facegrooming activity (i.e. the time spent on mouth wiping). This study revealed that pain levels, as evaluated through face-grooming, started to increase at day 1, decreased thereafter, and had returned to baseline at 14 d. In contrast, in the present study, we found that pain levels, assessed using the RGS, did not return to the baseline level at day 14. Considering that RGS scores increased in the control group, we suggest that the bulky intra-oral springs (even if inactivated) could cause discomfort and hence an increase in the RGS score. Therefore, we suggest that the discrepancy in pain levels between this and the previous study may be attributed to different sensitivities of the two modalities in assessing pain. We found that pain levels, as assessed using the RGS, were significantly correlated with the levels of expression of the P2X3 receptor (reported previously) (5) in trigeminal ganglia following experimental tooth movement (P < 0.05). Moreover, it has been reported that the expression of c-Fos was significantly elevated in the trigeminal nucleus, 1 and 3 d following experimental tooth movement (18), which is consistent with
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the results of the present study (data were unavailable at 5, 7, or 14 d; thus, the correlation test was not performed). These findings suggest that RGS coding would be a valid pain-assessment tool for experimental tooth movement. Moreover, the RGS scores increased only on day 3 and were similar to, or below, the baseline level in the morphine + 40-g group, suggesting that morphine could greatly attenuate pain sensations produced by experimental tooth movement. On the other hand, given that morphine is a well-documented analgesic agent, this decrease of RGS score by morphine would further support the validity of pain assessment through RGS coding. In clinical practice, most orthodontic patients experience the most severe pain 1 d after force application, and the pain levels decrease thereafter (1). However, in this study, we found that the pain levels were similar between days 1 and 3 after force application, or were higher at 3 d than at 1 d. This difference between species may be a result of the discomfort caused by bulky orthodontic appliances: orthodontic appliances are relatively larger in rats’ mouths. Hence, the discomfort led to the continued increase of pain levels at 3 d. Our results revealed that the RGS scores were similar between the control group and the 20-g group at all time points, which indicates that the pain levels were similar between the two groups. This finding suggests that the force magnitude of 20 g was not large enough to evoke pain sensations. Considering that pain was evoked in the 40-g group compared with the control group at days 1 and 3, we suggest that the threshold force magnitude to evoke orthodontic pain lies between 20 and 40 g. Acknowledgements – This work was supported by the National Natural Science Foundation of China (NSFC), nos 81070858 and 81100778. Conflicts of interest – The authors report no conflicts of interest.
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