Univ. Assistant, PhD student, Dept. Pharmaceutical Biochemistry, Faculty of Pharmacy, University of Medicine and Pharmacy,

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1 Pediatric dentistry Univ. Assistant, PhD student, Dept. Pharmaceutical Biochemistry, Faculty of Pharmacy, University of Medicine and Pharmacy, 3 Associate Professor, PhD, Dept. Pharmaceutical Biochemistry, Faculty of Pharmacy, University of Medicine and Pharmacy, Professor, PhD, Dept. Morphology of Teeth and Dental Arches; Technology of Dental Prosthesis and Dental Materials, Faculty of Corresponding author: Dental treatment represents a stress factor for most children. The aim of the study was to analyse the variation of salivary alpha-amylase concentration in children after a video viewing on dental treatments. In this study, 7 to 10 year-old school children were evaluated (n=119). Unstimulated whole saliva was collected before and after viewing a 15 min video on dental treatments performed on children. Changes in salivary alpha-amylase levels have been assessed. increase of the alpha-amylase level in the whole sample group. This was noticeable in terms of gender as well as age groups. From the viewpoint of age and gender, girls groups, while this could be observed only in younger boys. In conclusion, analysis of salivary alpha-amylase revealed that the sight of dental treatment represents a salivary alpha-amylase, dental treatment, stress Anxiety and fear are frequently related to dental treatments even in children who did not have such an experience before. To overcome this problem, Bandura [1] introduced a procedure called modelling, assuring attaining of a correct attitude towards dental interventions through the presentation of a behavioural model to be followed. The method is based on the social principles of learning, called modelling and imitation. Generally, people learn more about the surrounding world by observing the behaviour of other people, by following their ways to success and by avoiding their failures. For this reason, it is recommended for both parents and children to attend the treatment of a child already adapted to dental interventions. The other possibility is to watch videos on docile children getting dental treatments [2]. Dental fear and anxiety is a form of psychological stress, which has such impacts on the body as physical challenges do [3]. Activation of the sympatho-adrenomedullary system (SAM) is one of its effects [4]. response to sympathetic stimuli [5-7] and regulated by norepinephrine via the sympathoadrenomedullary system. Its activity is reported to increase during distress and to decrease during comfort [4,8-9], with a good association between the concentration of salivary amylase and blood levels of cathecolamines [8]. Currently, it is considered that measurement of this salivary to clinical settings, as well as a useful tool for evaluating the SAM system [4, 10-12]. The aim of the present study was to analyse the variation of salivary alpha-amylase as a biomarker of stress in children who have viewed a short video on different dental treatments performed on docile children. International Journal of Medical Dentistry 77

2 The subjects of this study were 119 randomly selected children from three primary schools of between the ages of 7 and 10 years were evaluated. The aim of the study and the procedures involved were explained to the subjects and to their parents, who gave their written informed consent. The study protocol was approved by the Ethical Research Committee of the University of the participating schools (43/ ). Unstimulated whole saliva samples were collected twice from each subject, prior to and after viewing a 15-min long video on different dental treatments (professional dental prophylaxis, getting a toothfilling and an injection, a primary tooth extraction) performed on co-operative children. Subjects were instructed on the method of saliva collection, which was absolutely noninvasive and consisted of collecting the unstimulated whole saliva for 5 min into a sterile recipient. After collection, samples were transposed to sterile test tubes and centrifuged for 10 min at 5,000 rpm. After centrifugation, the supernatant and the sediment were stored separately at -20 C prior to samples analysis (Fig. 1). The experimental sessions took place at school in the morning (between 9 and 11 a.m.), to alpha-amylase secretion has a pronounced and distinct diurnal rhythm, with a strong drop in steady increase towards the evening. It is independent on the diurnal rhythm of free Quantitative determination of salivary alphaamylase was done by analysing its activity with Wolhgemuth s starch reduction method. Meanwhile, the total salivary protein level was determined by the Bradford micromethod, so that, in each case, salivary alpha-amylase values For statistical analysis, the independent t-test was used by SPSS Statistics, version 17.0, with The mean age of the subjects was 8.46 (±0.78) years. presents the mean values of salivary alpha-amylase in the evaluated children. Video viewing on dental procedures led to a highly amylase level within the whole sample group Subjects p (n) Total ± ±0.034 p< Female ± ±0.034 p< Male ± ±0.033 p<

3 SALIVARY ALPHA-AMYLASE AS A BIOMARKER OF DENTAL FEAR AND ANXIETY IN CHILDREN According to their age, the subjects were distributed into two groups, a younger one from 7 to 8 years, and an older one from 9 to 10 years, respectively. The activity of salivary alpha-amylase after video viewing presented age, as well (Table 2). Age groups Subjects (n=119) p 7 8 years ± ±0.036 p< years ± ±0.023 p= From the viewpoint of age and gender, girls in both age groups, a situation perceptible only in the case of younger boys (independent t-test, FEMALES 7 8 years ± ±0.036 p= years ± ±0.032 p= MALES 7 8 years ± ±0.037 p= years ± ±0.026 p= Recently, stress stimuli during dental procedures have been audio-visually simulated using audiovisual clips. Several studies have investigated autonomic responses, cerebral activation and subjective ratings using such simulated stimuli [15,16]. Lueken et al. [15] have reported that video stimuli could be a valuable research tool in evaluating sensitivity to changes in symptom intensity. Koda et al. [17] have compared the subjective ratings and autonomic responses to dental and neutral stimulation in children with the ratings and responses of their mothers. Two types of dental stress stimulation were perceived more negatively than neutral stimulation. Furthermore, the subjects felt more disgust, fear and pain International Journal of Medical Dentistry 79

4 during the dental drilling scene than during the vacuum suction scene. In terms of autonomic responses, the dental drilling scene had a activity than that of the vacuum suction and neutral stimulation. They concluded that stimulation using dental videos is useful in assessing emotional stress and subjective perspectives even in children. Melamed et al. [18] have conducted a study on 5 to 9 year-old children, who attended three was made, in the second one they were examined, the third visit, the children were randomly included into two groups, one of which watched and was rewarded with a toy for good behaviour. At the same time, children from the control group had to draw the visit to the dentist. It was different between the two groups, however, during the third visit, those who watched the from the control group [2]. Takai et al. [4] have examined the effects of two videos, one of them as a psychological stressor and the other as a soother, on the salivary cortisol and amylase levels in young adults. They concluded that the salivary amylase level was rapidly than cortisol by psychological stressor, suggesting that it is a better index of stress. In the present study, the increase of salivary alpha-amylase activity after video viewing could be observed in the whole sample group. This means that video viewing on dental treatments was a stress factor for the subjects. Therefore, observing dental treatments, even if only on the screen, represents a real stressor for the children from this sample. With respect to gender, consistently with some other studies from the international literature [19,20], females presented a higher level of stress than males. At the same time, it was stated by fellow researchers that, as children grow older, they develop methods to control fear and anxiety [21,22]. In this study, the age-related tendency of stress reduction was also observed. To sum up our results, the sight of dental among children aged 7 to 10 years. Statistically levels was revealed in the older age groups in boys. Salivary alpha-amylase analysis could be used as a biomarker of dental fear and anxiety in children. This paper was supported by the Sectorial Operational Programme Human Resources Development (SOP HRD), Government, under the contract number POSDRU/159/1.5/S/ Bandura A. (1979) Psychological mechanisms of aggression. Cambridge University Press, Cambridge.. Ed. 3. Sadi H, Finkelman M, Rosenberg M. (2013) Salivary cortisol, salivary alpha amylase and teh Dental Anxiety Scale. Anesth Prog, 60: Takai N, Yamaguchi M, Aragaki T, Eto K, Uchihashi K, Nishikawa Y. (2004) Effect of psychological stress on the salivary cortisol and amylase levels in healthy young adults. Archives of Oral Biology, 49: Gallacher DV, Petersen OH. (1983) Stimulus-secretion coupling in mammalian salivary glands. Int Rev Physiol, 28: Deshpande RR, Panvalkar PS, Kulkarni AA, Gadkari TV. (2011) Age-related Changes of the Human Salivary Secretory Total Protein Complex and Trace Elements in Children between the Age Group of 3-16 years. J Biomed Sci and Res, 3(1): Sivakumar T, Hand AR, Mednieks M. (2009) Secretory proteins in the saliva of children. Journal of Oral Science, 51(4): Chatterton RT, Vogelsong KM, Lu YC, Ellman AB, Hudgens GA. (1996) Salivary alpha-amylase as a measure of endogenous adrenergic activity. Clin Physiol, 16(4): Karibe H, Aoyagi-Naka K, Koda A. (2014) Maternal anxiety and child fear during dental procedures: a preliminary study. Journal of Dentistry for Children, 81(2): Walsh NP, Blannin AK, Clark AM, Cook L, Robson PJ, Gleeson M. (1999) The effects of high-intensity 80

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