Effectiveness of salivary chromogranin A as a stress index in young children during dental treatment

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1 PEDIATRIC DENTAL JOURNAL 22(2): , 2012 Effectiveness of salivary chromogranin A as a stress index in young children during dental treatment Chieko Mitsuhata 1, *, Yukari Ohara 1, Meiko Tachikake 2, Yuko Iwamoto 1 and Katsuyuki Kozai 1 1 Department of Pediatric Dentistry, Integrated Health Sciences, Institute of Biomedical & Health Sciences, Hiroshima University Kasumi, Minami-ku, Hiroshima , JAPAN 2 Department of Pediatric Dentistry, Hiroshima University Hospital Kasumi, Minami-ku, Hiroshima , JAPAN Abstract It is important to understand the level of stress experienced by children during dental treatment, which can be stressful and even lead to permanent dental phobia. We investigated whether chromogranin A (CgA), a stress marker in adults, is useful for determining stress levels in children. Saliva samples were collected before and after treatment from 5 children (3 5 years) who required treatment more than 5 times in a relatively short period. Their parents completed a questionnaire about their child liking/disliking the clinic visit, how they explained to their children the purpose of the dental visit and the type of treatment sought, and to explain descriptively the difference they noticed in their child s behavior. The CgA levels were significantly higher before than after the treatment in all cases. Pre-treatment CgA values were not always related to dislike for the clinic visit, type of treatment sought, and behavioral reaction to the treatment. The CgA values may have been influenced by the children s previous experience of the treatment. The results suggest that CgA might be appropriate for verifying children s stress levels during dental treatment and stress tendency towards dental treatment. Key words Behavior, Chromogranin A, Dental anxiety, Dental stress, Young children Introduction It is well established that dental treatment involves stress. In severe cases, this stress can lead to dental fear anxiety (DFA) or dental behavior management problems (DBMP) for future dental care. Dental phobia can remain in adulthood. DFA and DBMP have multifaceted origins, and several potential etiological factors have been proposed, including general fear 1,2), temperament 2,3), general behavior, and attention 4). Pain and negative experiences from dental treatment are also considered as major causes of DFA and DBMP 5). The precise level of stress experienced by children during dental procedures is currently unknown. The prevalence of dental * Correspondence to: Chieko Mitsuhata chiekom@hiroshima-u.ac.jp Received on March 16, 2012; Accepted on May 23, 2012 anxiety in children has been reported to be between 5.7% and 6.7% 6 8), with anxiety decreasing with increasing age 6). Many measurement techniques have been used to examine the condition of children during dental procedures, including behavioral ratings, psychometric scales, and physiological measures. The Dental Subscale of Children s Fear Survey (CFSS- DS) 9), Dental Fear Scale (DFS) 10), and Dental Anxiety Scale (DAS) 11) are relatively commonly used as assessments of DFA. The other hand, Frankl scale 12) and the Behavior Profile Scale (BPRS) 13) are used as scales of DBMP. Stress is usually defined as the combination of physiological and psychological reactions that mobilize an organism s defense against external and internal threats (stressors). Stress reaction includes activation of the hypothalamus-pituitary-adrenal 163

2 164 Mitsuhata, C., Ohara, Y., Tachikake, M. et al. (HPA) axis. The classical stress hormones, catecholamine (CA) 14,15) and cortisol 16,17), have been used in previous research as physiological and/or psychological markers of various stresses. Recently, other biological materials have been used as stress markers, in an effort to simplify detection and improve sensitivity. The measurement of salivary chromogranin A (CgA) concentration has been developed as a method to evaluate psychological stress 18). CgA is a 457 amino-acid acidic glycoprotein in adrenal chromaffin cells and adrenergic neurons. CgA is secreted with CA into human blood, and is considered a valuable indicator of sympatho-adrenal activity 19). CgA is present in the submandibular glands ducts, and is secreted into saliva during stress 20). Salivary CgA has been proposed as a marker of sympathetic nervous activity involving the sympathetic-adrenomedullary system 18). It has been reported that salivary CgA might be promising as a sensitive index for psychosomatic stress in adults 18,21 24). Salivary CgA changes more rapidly and is more sensitive to psychological stressors than salivary cortisol. This is because the cortisol levels tend to remain high for quite some time even when the person is free from stress. This has been well proved by Nakane et al. 18) who states in his study that CgA levels responds rapidly. After relief of stress these values goes down immediately and attains a stress free level. Biochemical markers in saliva are useful for objectively assessing physiological and/or psychological stress because the collection of saliva is convenient, noninvasive and relatively non-stressful. As such, it is a useful measure in studies where children are experimental subjects. We verified whether saliva CgA might also be used as an index of stress in children 25). Forty-two children patients ranging from 5 to 9 years old (mean 7.5 years old) in our clinic were investigated. CgA values of 83.8% of children decreased after the dental treatment. There was a tendency that pre-treatment CgA values of kindergarten children were higher than school children. These results suggested that CgA might be used to verify child s stress during dental treatment. Meanwhile, it was reported that an experience of past dental treatment influenced the formation of present dental fear in adults 26). It s said that 4 years old child is brought about much fear by visual and audio stimulus. The purpose of the present study was to investigate whether salivary CgA may also be used as an index of stress in younger children during dental treatment. Our results suggest that CgA might be an appropriate measure for verifying young children s stress levels during dental treatment. Patients and Methods Subjects Our subjects were 5 boys aged between 3 years 3 months and 4 years 8 months who visited Hiroshima University Hospital Pediatric Dental Clinic for treatment of caries in 4 months. Of the 5 children, 3 were undergoing dental treatment for the first time while the other 2 children had dental treatment before. All patients were brought to the clinic complaining of spontaneous toothache. All the patients had between 4 and 16 teeth that required treatment, including pulpectomy or root canal treatment. We started observation and saliva sampling after the second visit, and repeated the sampling 6 or 7 times from the same participant in a random manner. All the children had cavities and required more than 5 visits to the dental office. None of the participants had a systemic illness, prescribed drugs and none had growth or mental development problems. Ethical approval for the study was granted by an independent ethical committee of Hiroshima University (D73-1). The study was performed in accordance with the committee s ethical guidelines. Before the experiment, we explained the purpose of this study to the guardians and children, and obtained written consent from all the guardians. Experimental protocol We randomly chose children who needed more than 5 treatments. For each treatment, patients saliva was collected before and after dental treatment. The parents of the children answered the questionnaire for each trip, and we produced a report on each day of treatment and the child s behavior during dental treatment. Each child was treated by a dentist in attendance at all visits. Saliva sampling and CgA analyses Saliva samples were collected just prior to and after each dental treatment in the waiting room using a cotton swab, which was placed inside the mouth for 2 min, and then deposited in a sample tube (Salivette, Sarstedt, Germany). Each child visited our clinic at about the same time period and we asked guardians to stop eating and drinking in 1 2

3 Salivary CgA as stress index in children 165 Fig. 1 Pre- and post-treatment salivary CgA concentrations in all patients (all samples) The distribution of post-treatment CgA values was significantly lower than that of pre-treatment values (P<0.0001). The crossbar indicates median values. Table 1 Pre- and post-treatment salivary CgA concentrations for each child Case No. No.1 No.2 No.3 No.4 No.5 CgA (pmol/mg protein) median pre-treatment [range] [ <] [ ] [ <] [ ] [ ] P=0.028 P=0.028 P=0.031 P=0.075 P=0.016 median post-treatment [range] [ ] [ ] [ ] [ ] [ ] The pre-treatment CgA values were significantly (P<0.05) higher than the post-treatment values for 4 children. hours before treatments. The tubes were stored on ice until centrifugation for 10 min at 3,000 rpm. The collected saliva was stored at 1307C until analysis. The concentration of saliva CgA was measured by an enzyme-linked immunosorbent assay (ELISA) using the YK070 Human CgA EIA Kit (Yanaihara Institute Inc., Japan). Salivary protein concentration was determined using a protein assay (BioRad, USA). The CgA concentration was corrected using protein concentration, and data are presented as pmol/mg protein 18). Saliva samplings and CgA analyses were performed in faithful accordance with the manufacture protocol. Pre- and post-treatment saliva CgA values were compared using a two-sided Wilcoxon s signed rank test. A Kruskal-Wallis rank test was used within each group. Questionnaire and treatment record A questionnaire was administered to the parents, asking them to report whether their child particularly liked/disliked the clinic visit, how they explained to their children about the purpose of the visit to the dentist and the type of treatment to be sought, and to write freely on what they noticed about their child s behavior during the visit to the dental clinic and dental treatment. The treatment record included treatment content, time spent on the chair, use of local anesthesia, and child s cooperation. We used the Frankl scale to classify the degree of cooperation. Each child s behavior was classified into one of the following categories: (1) definitively negative, (2) negative, (3) positive, and (4) definitively positive. Results Figure 1 shows salivary CgA values for all patients at every clinic visit. The results of a two-sided Wilcoxon s signed rank test showed that posttreatment CgA values were significantly lower than the pre-treatment values (P<0.0001). The median of pre-treatment and post-treatment CgA values was 5.94 pmol/mg protein and 1.58 pmol/mg protein, respectively. This indicates that the patients were

4 166 Mitsuhata, C., Ohara, Y., Tachikake, M. et al. Fig. 2 Dental trip details for each child RCF: root canal filling, RCT: root canal treatment, MC: restoration with preformed crown for the primary teeth, Pect: pulpectomy, IDPC: indirect pulp capping, CR: composite resin restoration, EZ: zinc oxide eugenol cement filling. E _: upper right second deciduous molar, _ E : upper left second deciduous molar, E _: lower right second deciduous molar, _ E : lower left second deciduous molar, D _: upper right second deciduous molar, _ D: upper left second deciduous molar, D _: lower right second deciduous molar, _ D: lower left second deciduous molar, C _: upper right deciduous canine, C _: lower right deciduous canine, B _: lower right deciduous lateral incisor.

5 Salivary CgA as stress index in children 167 under stress before start of the treatment, and were relieved from stress soon after the treatment ended. Each child s CgA values are shown in Table 1 and Fig. 2. Pre-treatment CgA values varied widely and exceeded the detection limits in 2 of the 5 cases. The level of stress that the children experienced differed on each visit. Sometimes the same treatment given to the same child on subsequent visits resulted in different pre-treatment CgA values. Similar pretreatment CgA values were observed even in patients who had similar rating for whether they disliked or liked the clinic visit. The distribution of pretreatment CgA values were significantly (P<0.05) higher than the post-treatment distribution for every child except one (Patient No.4), who showed relatively low pre-treatment CgA values and higher values after treatment compared to other participants. Patients CgA values were analyzed separately before and after the treatment using a Kruskal- Wallis rank test. Post-treatment CgA values were significantly different (P=0.0077) among all children, while pre-treatment values were not. The Frankl scale score was found to be between 1 and 3 for all children. There was no correlation between children s cooperation and pre-cga values. We found that there were some children who had low pre-cga value showed less co-operation (Frankl score 1) while some children having high pre-cga value were more cooperative (Frankl score 3). Each child s condition is shown in Fig. 2. Discussion Children often do not sufficiently understand the need for dental treatment, and some children might express fear that appears excessive or unreasonable, similar to dental phobia. In contrast, some children may endure the pain and continue to exhibit good behavior despite their fear. It is widely believed that dental treatment can induce stress in children, but the precise degree of stress experienced remained unclear. In addition, dental stress might contribute to dental phobia, similar to the contribution of stress to Post-traumatic stress disorder: PTSD. The vast majority of dental-care anxiety cases have been reported to stem from aversive dental experiences 27). And some studies have investigated the molecular mechanism involved in cognitive vulnerability stress in vivo and in vitro 28,29). It is possible that patients might have cognitive vulnerability to stress. Some transgenic mice were indicative of high response against stress due to stress vulnerability. Thus, it is important to determine the level of stress experienced by children during dental treatments. It was hypothesized that pre-treatment CgA values indicate the stress associated with dental treatment, and post-treatment CgA values indicate relief from stress after the treatment. The results revealed no significant correlations, but showed that children with a high CFSS-DS score tended to have a higher CgA value 25). We also reported that 83.8% of the children who were targeted between 5 years old and 9 years old on our pediatric dental clinic had lower CgA after treatment and the pre- and post-treatment CgA values of kindergarten boys (5 6 years old) receiving caries treatment differed significantly. In this research, we targeted younger children. The distribution of CgA values as in previous studies was found to decrease significantly after dental treatment (Fig. 1, Table 1). It was a tendency that younger children had higher values and wide variation in pre-treatment CgA and had similar values in post-treatment CgA compared to prior investigation. This result means that CgA values can be used as a stress marker in younger children. Some recent reports have investigated psychological stress caused through multiple stressors, using salivary CgA as an indicator. The stressors examined include psychosomatic stress during computer operation, stress related to academic assessment, and stress related to psychological tension before surgery or anesthesia 18,21 24). Other studies have reported that there were no significant differences in CgA levels between the pre- and posttest saliva samples, because the stressors used were not strong enough to change CgA levels 30). In this study, we found that CgA values across all children were significantly higher in the pretreatment stage, compared with after treatment. This finding suggests that CgA is effective as a biological marker for stress in young children, although CgA values did not correlate with the content of the prearranged dental treatment, and did not always affect the child s behavior during treatment. We predicted that the same dental treatment triggered similar level of stress in the same child if he/she understood the content of the prearranged dental treatment. So we planned to get samples more 5 times from the same child to prove our prediction. Young children tend to experience some degree of stress in any dental treatment. We found that

6 168 Mitsuhata, C., Ohara, Y., Tachikake, M. et al. pre-treatment stress was influenced by experiences that they had on previous treatments. We did not find significant individual differences in pre-treatment CgA values. A possible explanation for the wide variation in CgA values is that in some cases, children who are usually well-behaved suddenly become uncooperative, even for relatively simple dental treatments, due to fear or anxiety. Many factors such as mood swings, differing feelings etc. are involved, and this behavior is not solely related to the dental treatment itself as the CgA readings vary even if the same stress was induced to the same child at a different time point. However, by checking CgA levels several times, it is possible to approximate the level of stress. From each child s median pre-treatment CgA value, it is possible that patients 1, 3 and 4 were more stressed than patient 5. Patient 4 had the highest median post-treatment CgA value. This may be because this patient s steady-state levels are higher than normal or that he needs more time to recover from stress. Each child s median CgA value might indicate the tendency of his/her stress level. It is important to know child s stress levels when planning and conducting dental treatment, in order to prevent dental fear in the future. In particular, when we deal with patients with high pre-treatment CgA values and good cooperation, they need more detailed support best suited them by dentists and dental staff. Our results suggest that salivary CgA might be useful as a stress marker in young children. Dentists, dental staffs, and other practitioners should know patient s stress level objectively using some stress indicators like CgA level estimations while they are visiting dental office or undergoing dental treatment and deal with it appropriately in order to prevent dental phobia in future. Acknowledgments The authors wish to thank the staff of our pediatric dental clinic for their assistance in this study. This study was partly supported by a Grant-in-Aid for Scientific Research (C). Declaration of interest The authors report no conflict of interest. References 1) Brown, D.F., Wright, F.A. and McMurray, N.E.: Psychological and behavioral factors associated with dental anxiety in children. J Behav Med 9: , ) Klingberg, G., Berggren, U., Carlsson, S.G. and Noren, J.G.: Child dental fear: cause-related factors and clinical effects. Eur J Oral Sci 103: , ) Arnrup, K., Broberg, A.G., Berggren, U. and Bodin, L.: Lack of cooperation in pediatric dentistry the role of child personality characteristics. Pediatr Dent 24: , ) Blomqvist, M., Holmberg, K., Fernell, E. and Dahllof, G.: A retrospective study of dental behavior management problems in children with attention and learning problems. Eur J Oral Sci 112: , ) Raadal, M., Strand, G.V., Amarante, E.C. and Kvale, G.: Relationship between caries prevalence at 5 years of age and dental anxiety at 10. Eur J Paediatr Dent 3: 22 26, ) Klingberg, G., Berggren, U. and Noren, J.G.: Dental fear in an urban Swedish child population: prevalence and concomitant factors. Community Dental Health 11: , ) ten Berge, M., Veerkamp, J.S., Hoogstraten, J. and Prins, P.J.: Childhood dental fear in the Netherlands: prevalence and normative data. Community Dental Oral Epidemiol 30: , ) Wogelius, P., Poulsen, S. and Sorensen, H.T.: Prevalence of dental anxiety and behavior management problems among six to eight years old Danish children. Acta Odontol Scand 61: , ) Cuthbert, M.I. and Melamed, B.G.: A screening device: children at risk for dental fears and management problems. ASDC J Dent Child 49: , ) Kleinknecht, R.A., Klepac, R.K. and Alexander, L.D.: Origins and characteristics of fear of dentistry. J Am Dental Assoc 86: , ) Corah, N.L.: Development of a dental anxiety scale. J Dental Res 48: 596, ) Frankl, S.N., Shiere, F.R. and Fogels, H.R.: Should the parent remain with the child in the dental operatory? ASDC J Dent Child 29: , ) Melamed, B.G., Weistein, D., Katin-Borland, M. and Hawes, R.: Reduction of fear-related dental management problems with use of filmed modeling. J Am Dental Assoc 90: , ) Palmer-Bouva, C., Oosting, J., devries, R. and Abraham-Inpijin, L.: Stress in elective dental treatment: epinephrine, norepinephrine, the VAS, and CDAS in four different procedures. Gen Dent 46: , ) Sakuma, N. and Nagasaka, N.: Changes in urinary excretion of catecholamines and their metabolites in pediatric dental patients. ASDC J Dent Child 63: , ) Brand, H.S.: Anxiety and cortisol excretion correlate prior to dental treatment. Int Dent J 49: , 1999.

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