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The salivary-α-amylase level after stroop test in anxious patients can predict the severity of anxiety
Journal Pre-proof The Salivary-␣-amylase level after stroop test in anxious patients can predict the severity of anxiety Liu Yuting, Yin Han, Ma Huan, Yu Xueju, Liu Guihao, Guo Lan, Geng Qingshan
PII:
S0304-3940(19)30716-5
DOI:
https://doi.org/10.1016/j.neulet.2019.134613
Reference:
NSL 134613
To appear in:
Neuroscience Letters
Received Date:
10 April 2019
Revised Date:
1 November 2019
Accepted Date:
4 November 2019
Please cite this article as: Liu Y, Yin H, Ma H, Yu X, Liu G, Guo L, Geng Q, The Salivary-␣-amylase level after stroop test in anxious patients can predict the severity of anxiety, Neuroscience Letters (2019), doi: https://doi.org/10.1016/j.neulet.2019.134613
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The Salivary-α-amylase level after stroop test in anxious patients can predict the severity of anxiety LIU Yutinga,Yin Hana,MA Huanb,YU Xuejua,LIU Guihaoc,GUO Lanb,GENG Qingshanb1 1
Corresponding author：GENG Qingshan
email:[email protected]
(a.School of Medicine,South China University of Technology,Guangdong
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General Hospital,School of Medicine,South China University of Technology,Guangzhou 510080,China；b.Guangdong General Hospital,School of Medicine,South China University of
Technology,Guangdong Academy of Medical Sciences,Guangdong
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Cardiovascular Institute,Guangzhou 510080,China；c.Department of
Epidemiology,Guangdong Cardiovascular Institute,Guangdong General
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Hospital,Guangdong Academy of Medical Sciences,Guangzhou,China.)
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Highlight:
Found a positive correlation between baseline sAA level and anxiety severity；
2.Found a negative correlation between anxiety level and sAA changes before and
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after mental stress；
the basic function of sympathetic - adrenal medulla system was hyperfunctional in
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GAD patients while theirs reactivity was limited； The change of sAA after stroop test in GAD patients can predict the severity of
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anxiety,but subjective psychological perception can not.
Abstract Individuals with anxiety disorder often exhibit as imbalance in response to stressors.We sought to explore the relationship between physiological as well as psychological responses under acute mental stress and the severity of the disease.20 Generalized 1
Anxiety Disorder(GAD) patients (14 males,mean age 46±10 years) were confronted with the stroop test,during which salivary-α-amylase(sAA),salivary cortisol,and heart rate variability (HRV) were assessed.The results showed that stroop test as a stressor induced autonomic nervous response in GAD patients,which was mainly manifested as the increase in HRV representing sympathetic nervous system and the decrease in HRV representing vagal nerve activity.Moreover,the basic function of sympathetic-adrenal medulla system was hyperfunctional in GAD patients while theirs reactivity was limited,which showed the more serious the lesion was,the higher the baseline value of sAA
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was,and the less sAA secretion increase would be,or even decrease after mental stress.The change of sAA after stroop test in GAD patients can predict the severity of anxiety,but subjective psychological perception can not.
Key Words:Anxiety Disorder;heart rate variability;Sympathetic-adrenal medulla;salivary
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alpha amylase
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Introduction
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Generalized anxiety disorder(GAD) is characterized by excessive,uncontrollable and often irrational worry about events or activities,often accompanied by somatic anxiety symptoms and significant autonomic nervous system dysfunction symptoms.Up to
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now,GAD’s neurobiological characteristics were equivocal,including the serotonergic system,noradrenergic
system
and
adrenocortical
system[1-4].For
example,while
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sympathetic was hyperactive,increased catecholamine secretion may induce sudden increase in blood pressure,arterial spasm,chest tightness and palpitation,and the risk of
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late progression to coronary heart disease is also increased.In fact,the incidence of cardiovascular disease,Cardiovascular mortality and all-cause mortality in anxiety patients are higher than that in the normal population[5],however,the precise mechanisms underlying greater cardiovascular risk among GAD patients remain uncertain.Therefore,it is of great clinical significance to strengthen the understanding of anxiety disorder. Non-anxious individuals show rapid adaptation to stressors and respond strongly to threats,but can also quickly return to baseline once the threat is removed.Persistent 2
overstimulation caused by chronic stress,such as anxiety disorder,may lead to allostatic load,manifested as an imbalance in response to stressors,or a dysfunction to return to baseline[6-8].Prior findings suggested that GAD was characterized by hyperactive in sympathetic system at resting state,in which the sympathetic-adrenal medulla (SAM) system is more active than normals[4].And it was also found that patients with GAD were lesser variability in response to stressors,and was related to the basic expression level of SAM system[9].However,no relevant studies have been found to explore the relationship between the severity of the disease and the pathological response of the GAD
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sympathetic system. In order to simulate the mental stress(MS) people face in daily life,researchers artificially give subjects a certain amount of mental stress under laboratory conditions,while detecting the changes of physiological indicators at the same time,which is called “mental
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stress-induced test”.It has been found that mental stress mainly affects the function of
various systems through autonomic nerves system and the SAM system.In the SAM
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system,sympathetic activation causes catecholamines such as epinephrine and norepinephrine to be released into the blood.Salivary-α-amylase (sAA) is one of the main
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salivary enzymes in human beings which is secreted from the salivary glands under sympathetic stimulation.Chatterton et al.[10] has reported that there was a good correlation
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between sAA concentration and serum catecholamine level.Therefore,the determination of salivary enzyme is an effective tool for evaluating SAM system. Heart rate variability (HRV) is heartbeats interval fluctuation controlled by the activity of
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the autonomic nervous system,so it can be used as a noninvasive index for detecting autonomic nervous function.Low HRV variability could reflect the inflexibility of the
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autonomic nervous system and was a marker of less favorable health[11].In healthy subjects,reduced HRV associated with changes in cardiac autonomic regulation was associated with an increased risk of cardiac events,even after adjusted for known risk factors[11,12].In anxiety disorders,including GAD,post-traumatic stress disorder,social anxiety disorder and panic disorder,there were also reports of decrease in HRV which represents parasympathetic[13-16],but higher LF/HF which reflecting the balance of parasympathetic and sympathetic nervous systems[17],indicating GAD is associated with an 3
altered sympathovagal balance with reduced vagal modulation.These were consistent with the symptoms of GAD,for example,attentional biases like hypervigilance to threat. In most previous studies,HRV has been evaluated in a resting state.In the study of autonomic function,it is necessary to analyze not only the resting,but also the activated state. Stress response is a complex phenomenon involving multiple response systems.It not only causes physiological reaction,but also causes individual's subjective psychological reaction.Subjective psychological response reflects individual's feeling and evaluation of stress,which is crucial for individual's survival[18].And it is also closely related to the
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individual's basic mental health.Previous studies have found that compared with healthy individuals,people with social phobia were more stressful and less content with their
performance after mental stress[19].Therefore,investigating both physiological and
psychological responses simultaneously is conducive to a more comprehensive
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understanding of the functional status of stress system in anxiety individuals.
In present study,patients with GAD were selected as subjects,sAA was used as
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indicator of neuroendocrine response and HRV represented autonomic nervous response.The aim of our study was therefore to provide further insight into the changes
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of sAA and HRV under mental stress in GAD and evaluate the relationship between physiological as well as subjective psychological responses under mental stress and the
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severity of anxiety.
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Materials and methods 2.1 Participants
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Patients with GAD admitted to the psychiatric department of Guangdong Provincial People's Hospital were eligible for participation if they were order than 18 years of age.The diagnosis was made by an psychiatrist using DSM-IV[20].Subjects combined with other serious mental disorders(e.g.,obsessive-compulsive disorder,schizophrenia,etc),could have been pregnant,had a current substance abuse disorder (e.g.,drug or alcohol),or demonstrated an apparent cognitive or language deficit such that they would have been unable to reliably complete the study were excluded.Patients were also excluded if they 4
were regular(daily) users of antipsychotics.Totally 20 patients were enrolled from April to June,2018 (mean age:46±10 years;14 males and 6 females).The aims of the present study and the procedures involved were explained to the subjects before.The written informed consenting to participate in this study and use their data was obtained from all subjects.They were also ensured that all of their personal and medical information would remain confidential.The study was approved by the Institutional Review Board of Guangdong Provincial People's Hospital. 2.2 Study procedures
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Experiment took place in the afternoon (between 14:00 and 17:00).Before the beginning,the Hamilton Anxiety Scale(HAMA) score was assessed by a psychiatrist.At the beginning of the experimental session,subjects rest for 10 min in a quiet room,while hrv machine was collecting data the whole time for analyzing short-term hrv in every 5 min.At
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the end of the baseline period,subjects provided the first saliva sample.After the sample collection,subjects need to wear a head-mounted virtual reality (VR) device to perform the
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stroop test (see below).Saliva samples were collected immediately after,10 min after and 20 min after the Stroop test.Self-perceived stress level and Self-performance level were
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obtained by using visual analogue scale (VAS) after the experiment was done.The subjects were instructed to abstain from eating,smoking,drinking any beverages except water and
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exercising 2 h before the experiment.The study procedure is shown in Fig.1.
（baseline）
（MS）
（MS+5min）
（MS+10min）
（MS+15min）
Enrollment HAMA Evaluation
Baseline period Rest for 10min
MS period MS for 5min
Recovery period Post-MS 5min
Recovery period 5 Post-MS 10min
Recovery period Post-MS 15min
sAA、HRV
sAA、HRV
HRV
sAA、HRV
HRV
Fig. 1 Study Procedure. HAMA= Hamilton Anxiety Rating Scale for Anxiety; sAA= salivary-αamylase; HRV= heart rate variability;MS=mental stress. In baseline period,subject rest for at least 10
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min,in which baseline sAA and HRV were collected(Labeled as baseline).In MS period,subject underwent 5 min mental stress test,in which MS-sAA and HRV were collected(Labeled as MS).In recovery period,post-mental stress test 5min,10min,15min,20min HRV were collected
respectively(Labeled as MS+5min,MS+10min,MS+15min and MS+20min). And post-mental stress
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test10min and 20min sAA were also collected (Labeled as MS+10min and MS+20min).
2.3 Psychological assessment
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Subjects were evaluated on the following scales:(1) Hamilton Anxiety Rating Scale for Anxiety (HAMA):It is used to evaluate the severity of Anxiety,with a rate of 5 (0-4).(2) Self-
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perceived stress Visual analogue scale (VAS):For the assessment of self-perceived stress level during the stroop test,subjects chose how stressed they felt on the stress level
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expressed as 1 to 10,with 1 representing "no pressure at all" and 10 representing "maximum pressure".(3) Self-performance VAS:For the assessment of self-performance level during the stroop test.The degree of self-performance was expressed as 1 to 10,with
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1 representing "extremely bad performance" and 10 representing "extremely good performance".
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2.4 Measurement
(1) HRV measurement:5-minute short-term electrocardiogram analysis was performed
on the subjects.The electrocardiogram was recorded by single lead dynamic electrocardiograph(Zhongruiqi electronic technology co.LTD,Shenzhen,China),and the data was analyzed automatically by computer soft ware.The commonly used timedomain and frequency-domain indexes are measured in resting period,mental stress
6
period and recovery period respectively:①mean R-R interval:the mean R-R interval length measures the sum of parasympathetic and sympathetic influences; ②Square root of difference mean square (rMSSD):rMSSD is considered an index of parasympathetic neuromod ulation changes of heart rate[21];③Very Low frequency (VLF) in range 0.00330.04 Hz; ④Low frequency (LF) in range 0.04-0.15hz; ⑤High frequency (HF) in range 0.15-0.4hz; ⑥LF / HF ratio.LF is affected by both parasympathetic and sympathetic
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nervous systems,reflecting the overall activity of the autonomic nervous system[22].Its standardized ratio (LFnu=100%×LF / TP-VLF）was used to represent sympathetic
activity[23].HF was used as a marker reflecting vagus nerve activity,and the LF / HF ratio reflected the balance of parasympathetic and sympathetic nervous systems[24]. Alpha-Amylase
Activity:Saliva
samples
were
collected
using
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(2)Salivary
Salivette(Sarstedt,Rommelsdorft,Germany).Gargling was ordered before the test to
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reduce the effects of oral residues on saliva collection.For salivary collection,subjects were asked to chew on the swab for 1 min.During the experiment,four saliva samples were
min
after
mental
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collected,reflecting the sAA levels at baseline (baseline),the end of mental stress (MS),10 stress
(MS+10min),and
20
min
after
mental
stress
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(MS+20min),respectively.The Salivette tubes were centrifuged at 4 ℃ and 3000 rpm for 10 min,and the supernatant was stored in a refrigerator at-80 ℃ until tested.The sAA was
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determined by enzyme-linked immunosorbent assay(ELISA)(Human saliva amylase ELISA kit,Shanghai Keshun Biotechnology Co.,Ltd.,China).The detection range of sAA
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was 25-800U/mL.Due to the symptom of somatic anxiety or the response to stress leading to dry mouth,2 subjects lacked a complete set of saliva collection.sAA in 18 subjects were finally included in the analysis. 2.5 Mental stress-induced test Stroop test has been widely used in psychophysiology.In present study,the test was applied as an acute mental stressor based on previous studies showing the test can induce mental stress,manifested as increased cortisol and catecholamine levels and 7
increased heart rate[25,26].The test lasted for 5min,during which subjects would see a Chinese character representing color(RED or GREEN or BLUE or YELLOW) on the VR screen while the character were printed in incongruent colors (e.g.word RED printed in green color).The subject was asked to indicate the color and to ignore the meaning of the character.The four Chinese character s displayed at the bottom of the screen,and subjects need to select the correct color option quickly and accurately as much as possible(i.e.,select " GREEN ").During the test,the device automatically gave the requirements of "faster" and "more accurate" every 30 s.The computerized stroop test
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provided each subject with a standardized level of stimulation. 2.6 Statistical Analyses
Analyses were performed using SPSS 20.0.Data were described as the means ±
standard error (se).All data were tested for normality of distribution by Shapiro-Wilk’s
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test.Due to the positive skewness of some HRV data,natural logarithms (ln)
transformations were computed for VLF,LF and LF/HF ratio.All other data passed the
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normality test.The sAA and HRV levels of different time were analyzed by repeated measures ANOVA(with Greenhouse—Geisser correction in the case of violation of the
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sphericity assumption).Post-hoc comparisons were performed using Bonferroni adjustments.The Pearson’s correlation coefficient(r) was determined for the relationship
Results
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between all variables and HAMA score.P ≤ 0.05 was considered significant.
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3.1 Salivary alpha-amylase responses There was no significant difference in the levels of sAA at baseline,at the end of mental
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stress,at 10 min and at 20 min after mental stress(440.64±185.12U/mL VS 449.44±156.37U/mL VS 445.39±151.55U/mL VS 472.41±140.48U/mL,F(3,27)=0.556,P=0.648,ηp2=0.058).
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(a)
Fig.2
(b)
(c)
Variation trend of HRV. rMSSD=square root of difference mean square; HF=high frequency;LFnu=
standardized ratio of low frequency; MS=mental stress. MS+5min,10min,15min,20min=post-mental stress test 5min,10min,15min,20min; (a)rMSSD was significantly lower during MS than that at 10 min and 20 min after(P<0.05). (b)HF was significantly lower during MS than that at 5 min,10 min,15 min and 20 min after(P<0.05,MS VS MS+5min,MS+10min,MS+20min;P=0.001,MS VS MS+15min). (c)LFnu during MS was significantly higher than that at 20 min after(P<0.05).
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Data are expressed as mean ± S.D. * P<0.05,***P=0.001.
3.2 HRV responses
The R-R interval at baseline was 754.35±95.47ms,and it was significantly lower during mental stress and then recover after stress（707.55±92.82 ms VS 748.95±
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96.65 ms VS 765.60±102.97 ms VS 768.90±102.60 ms VS 774.60±107.40 ms,P＜ 0.01).
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The rMSSD at baseline was 19.50±11.80ms,and it was significantly lower during mental stress than that at 10 min and 20 min after（16.34±7.09ms VS 19.38±9.43ms VS
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19.71±8.70ms,P＜0.05）(see Fig.2a).
The HF at baseline was 176.89±138.70ms2.It was significantly lower during mental
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stress than that at 5 min,10 min,15 min and 20 min after（97.54±77.26ms2 VS 170.44±113.16ms2 VS 174.15±131.10ms2 VS 169.87±102.96ms2 VS 171.14±117.01ms2,P=0.017、0.026、0.001、0.021）(see Fig.2b).
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The LFnu at baseline was 51.53±20.13.LFnu during mental stress was significantly higher than that at 20 min after（58.88±11.25 VS 49.46±20.26,P=0.023）(see Fig.2c).
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There was no significant difference in lnLF,lnVLF or lnLF/HF at baseline,mental
stress,5min,10min,15min and 20min after stress（lnLF:0.08±0.95 ms2 VS 0.37±0.49 ms2 VS 0.32±0.83 ms2 VS 0.03±1.00 ms2 VS 0.10±0.57 ms2 VS -0.42±0.91 ms2,P=NS ;lnVLF :5.79±1.12 ms2 VS 5.38±0.95 ms2 VS 5.79±0.96 ms2 VS 5.58±0.86 ms2 VS 5.82±0.94 ms2 VS 5.44±0.90 ms2,P=NS; LF/HF :1.73±2.22 VS 1.61±0.75 VS 1.96±1.88 VS 1.63±1.83 VS 1.27±0.64 VS 1.37±1.13,P=NS）； 3.3 Correlation between anxiety severity and baseline levels of sAA and HRV 9
It showed that there was a significant positive correlation between HAMA score and sAA levels both at baseline and during mental stress（P<0.01,r=0.602； P<0.05,r=0.505;see Fig.3a&b）.The levels of HRV were not correlated with HAMA score at each time point. 3.4 Correlation between anxiety severity and changes of sAA and HRV The correlation between HAMA score and the changes of sAA and HRV before and after mental stress found that there was a significant negative correlation between HAMA score and the changes values of sAA after 20 min of mental stress（P<0.05,r=-0.483；
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see Fig.3c）.In contrast to that of sAA,the change value of HRV did not statistically
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correlate with HAMA score.
(b)
(a) Fig.3
(c)
Correlation between HAMA score and sAA levels. HAMA= Hamilton Anxiety Rating Scale for
Anxiety; sAA= salivary-α-amylase; MS=mental stress; 20min-R= the differences between sAA at post-MS
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20min and baseline; (a)Correlation between the baseline sAA level and HAMA score is highly significant(P<0.01,r=0.602). (b)Correlation between the sAA level during MS and HAMA score is highly significant(P<0.05,r=0.505). (c) Correlation between the changes values of sAA after 20 min of MS and
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HAMA score is highly significant(P<0.05,r=-0.483).
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3.5 Correlation between anxiety severity and psychological response The correlation coefficient between self-perceived stress level and HAMA score was
r=0.080 (P>0.05),while the correlation coefficient between self-performance level and HAMA score was r=0.294 (P>0.05).No significant correlation was found.
Discussion 10
4.1 Physiological responses to mental stress The present study found that HRV,rMSSD and HF,which represented parasympathetic response,decreased
significantly
while
LFnu,which
reflected
sympathetic
response,increased significantly during mental stress.The results showed that the stroop test as a stressor induced autonomic nervous response in patients with GAD,which is mainly manifested as increased sympathetic nervous activity and decreased vagus nerve activity.Previous studies have found that rMSSD and HF decreased[27,28],LF did not change[29] or RR interval increased during mental stress in healthy subjects[30].There are
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very few studies have been done on anxiety population.Similar to our results,Olga V.Crowley et al.[31] found a significant decrease in rMSSD after mental stress among 905 subjects with anxiety traits.
The analysis of SAM system responses showed that the differences were not
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significant between each time point,but the means of sAA level were consistently
increased after mental stress.Although the sympathetic system of patients with GAD is
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pathologically hyperactive in resting state[4],there is no unified conclusion on the changes of the system in the face of acute stress.Few studies have been conducted on patients
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with anxiety.Similar to the results of our study,the levels of epinephrine and norepinephrine in patients with panic disorder increased after mental stress,but there was
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not significant[32].The reason for the present study showed that there was no significant increase in sAA compared with the baseline value,may be GAD patients had a high initial secretion level,as the "ceiling effect",which makes it difficult to the further activation of
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norepinephrine system[32].
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4.2 Relationship between anxiety severity and physiological responses GAD patients had hyperfunction of adrenal medulla system,as their catecholamine level
in resting state was higher than that of normal subjects,and could be improved after the intervention of norepinephrine system[33,34].But the relationship between anxiety and disease severity has not been seen before.The present findings showed that there was a positive correlation between baseline sAA level and anxiety severity,which means that the more serious the disease is,the more hyperactive the baseline level of SAM system 11
function may be.This is similar to the results of previous studies.Compared with those with low anxiety levels,the levels of norepinephrine in patients with high anxiety levels were higher at rest and during tension[35].Studies in both animals and humans have shown that long-term mental stress can alter the response capacity of the central-peripheral regulatory systems[36-38],and these changes may have different effects on different pathways.For example,it may lead to an increase or decrease in the output of the target-specific sympathetic or vagus nerve,or an up-regulation or down-regulation of the HPA axis[39].Altered responsiveness may be less efficient and less adaptive,and therefore may
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be detrimental to health on a long-term basis. We found a blunted sympathetic response to acute stress in the GAD patients.This finding is supported by a significant negative correlation of anxiety scores with the absolute
sAA activity during the mental stress test, suggesting that the more severe the anxiety was,
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the lower the activation degree of SAM system would be. Similarly, Fisher et al.[9,40] found that the level of basic sympathetic neurotransmitter was correlated with the responsiveness
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of the sympathetic nervous system.The higher level of sAA at baseline,predicting the less change of sAA in patients with GAD after mental stress.
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A healthy organism is characterized by its flexibility to adapt to environmental requirements[8,41].Stress should be harmless,as long as the organism can respond
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appropriately.The fast neuroendocrine response to stress is largely mediated by norepinephrine (NE) and corticotropin releasing hormone (CRH),followed by a slower and more sustained release of corticosterone[42]. Acute stress quickly activates the locus
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coeruleus(LC)-noradrenergic system,which is the main source of NE in the center[43-45]. Central catecholaminergic activation is followed by activation of the peripheral SAM
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system,which triggers release of epinephrine from the adrenal medulla.Epinephrine further increases NE release through ascending vagal projections to the nucleus of the solitary tract. Ceiling effect for both neuroendocrine and sympathetic stress related activity may be one possible interpretation of the blunted sAA activity response in our GAD patients.Since the sAA level reflects the same of peripheral catecholaminergic level, the increased peripheral level of baseline in GAD patients may be a negative feedback to restrain the stress activity. Furthermore,this noradrenergic projection from the LC widely 12
throughout the most brain regions,including the brainstem,the cerebellum,the diencephalon and the paleo- and neocortex,and are known to be involved in a range of stress-related diseases such as anxiety,depression,and posttraumatic stress[46,-48].The anxiety projection from LC may infect the stress response in some or how, which underlying mechanism remains to unknow. According to the above,it may not be surprised for us to find out disease severity dependent response to acute stress in SAM systems in GADs. Based on the results of present study, it reflected the hyperfunction of SAM system in patients with GAD,the more serious the lesion,the higher the baseline value of
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sAA.However,its reactivity was reduced,which showed that the more serious the lesion,the less change of sAA after stress.
Evidence suggested that the physiological characteristics of GAD was limiting or
inhibiting the body's normal response to environment,which known as diminished
indicators
such
as
heart
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physiological flexibility[8,49].It referred to the lack of expected variability of physiological rate,electrocardiogram,skin
conduction
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reaction,catecholamine,and cortisol,which is not only reflected in the decreased physiological response to stress,but also in the decreased ability to recover after
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stress.Consistent with previous studies[49-51],the GAD patients showed a diminished physiological flexibility,which we believe reflects the weaker adaptability when facing
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stress.Lack of flexibility in regulation may be problematic because it was associated with mortality and morbidity[12,52,53].Perhaps this is the potential mechanism for the development
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or progression of many psychosomatic and physical diseases.
4.3 Psychological response to mental stress
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In present study,it was found that the HAMA score was not correlated with the degree
of self-perceived stress and self-performance,indicating that the severity of anxiety disorder may not related to stress perception in the face of acute mental stress.Nor does it affect the individual's evaluation of his or her self-coping ability.Previous studies have also shown that the degree of lesion is not completely consistent with the individual's own psychological response.Crowley OV et al.[31]found that among the individuals with higher trait anxiety,reductions levels in perceived stress were positively associated with recovery 13
in ln rMSSD after the challenge.In contrast,among the individuals with lower trait anxiety,changes in perceived stress had no impact on ln rMSSD recovery.This indicates that the relationship between perceived stress and recovery of cardiac vagal was only found in individuals with high degree of trait anxiety.A similar finding was also found in another study.Subjects who were actually felt stressful due to the mental stress-induced test but showed only weak physiological response,and subjects who were only slightly stressed but showed a marked response[29].The present study did not obtain consistent results with social phobia patients[19],suggesting that for GAD subjects,there is an
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inconsistency between the psychological response of self-perception and the degree of disease lesion,so individual subjective psychological perception may not be objective as a prediction of disease severity.
One possible weakness of the current study is that for the GAD subjects enrolled,the
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effect of early intake of antianxiety drugs on the adrenal medulla system cannot be
excluded.Additionally,the present sample size is limited.Expanding the sample size and
Conclusions
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generalizability of the present findings.
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trying to enroll subjects with first diagnosed GAD would further strengthen the
In present study,by measuring the indicators of different stress response systems,it was
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found that stroop test did induce obvious cardiac autonomic nervous responses in patients with GAD,and confirmed the activation function of SAM system in GAD patients was
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impaired and correlated with anxiety severity.Also,ours study examined the subjective psychological response of individuals,and found that there was no consistency between
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the anxiety severity and the psychological response of self-perception.Proving the diminished physiological flexibility of GAD patients may be of great enlightening significance to explore the potential mechanism for the development or progression of many psychosomatic and physical diseases,which needs further research.
Funding：This research did not receive any specific grant from funding agencies in the public,commercial,or not-for-profit sectors. 14
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PII:
S0304-3940(19)30716-5
DOI:
https://doi.org/10.1016/j.neulet.2019.134613
Reference:
NSL 134613
To appear in:
Neuroscience Letters
Received Date:
10 April 2019
Revised Date:
1 November 2019
Accepted Date:
4 November 2019
Please cite this article as: Liu Y, Yin H, Ma H, Yu X, Liu G, Guo L, Geng Q, The Salivary-␣-amylase level after stroop test in anxious patients can predict the severity of anxiety, Neuroscience Letters (2019), doi: https://doi.org/10.1016/j.neulet.2019.134613
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The Salivary-α-amylase level after stroop test in anxious patients can predict the severity of anxiety LIU Yutinga,Yin Hana,MA Huanb,YU Xuejua,LIU Guihaoc,GUO Lanb,GENG Qingshanb1 1
Corresponding author：GENG Qingshan
email:[email protected]
(a.School of Medicine,South China University of Technology,Guangdong
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General Hospital,School of Medicine,South China University of Technology,Guangzhou 510080,China；b.Guangdong General Hospital,School of Medicine,South China University of
Technology,Guangdong Academy of Medical Sciences,Guangdong
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Cardiovascular Institute,Guangzhou 510080,China；c.Department of
Epidemiology,Guangdong Cardiovascular Institute,Guangdong General
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Hospital,Guangdong Academy of Medical Sciences,Guangzhou,China.)
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Highlight:
Found a positive correlation between baseline sAA level and anxiety severity；
2.Found a negative correlation between anxiety level and sAA changes before and
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after mental stress；
the basic function of sympathetic - adrenal medulla system was hyperfunctional in
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GAD patients while theirs reactivity was limited； The change of sAA after stroop test in GAD patients can predict the severity of
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anxiety,but subjective psychological perception can not.
Abstract Individuals with anxiety disorder often exhibit as imbalance in response to stressors.We sought to explore the relationship between physiological as well as psychological responses under acute mental stress and the severity of the disease.20 Generalized 1
Anxiety Disorder(GAD) patients (14 males,mean age 46±10 years) were confronted with the stroop test,during which salivary-α-amylase(sAA),salivary cortisol,and heart rate variability (HRV) were assessed.The results showed that stroop test as a stressor induced autonomic nervous response in GAD patients,which was mainly manifested as the increase in HRV representing sympathetic nervous system and the decrease in HRV representing vagal nerve activity.Moreover,the basic function of sympathetic-adrenal medulla system was hyperfunctional in GAD patients while theirs reactivity was limited,which showed the more serious the lesion was,the higher the baseline value of sAA
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was,and the less sAA secretion increase would be,or even decrease after mental stress.The change of sAA after stroop test in GAD patients can predict the severity of anxiety,but subjective psychological perception can not.
Key Words:Anxiety Disorder;heart rate variability;Sympathetic-adrenal medulla;salivary
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alpha amylase
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Introduction
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Generalized anxiety disorder(GAD) is characterized by excessive,uncontrollable and often irrational worry about events or activities,often accompanied by somatic anxiety symptoms and significant autonomic nervous system dysfunction symptoms.Up to
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now,GAD’s neurobiological characteristics were equivocal,including the serotonergic system,noradrenergic
system
and
adrenocortical
system[1-4].For
example,while
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sympathetic was hyperactive,increased catecholamine secretion may induce sudden increase in blood pressure,arterial spasm,chest tightness and palpitation,and the risk of
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late progression to coronary heart disease is also increased.In fact,the incidence of cardiovascular disease,Cardiovascular mortality and all-cause mortality in anxiety patients are higher than that in the normal population[5],however,the precise mechanisms underlying greater cardiovascular risk among GAD patients remain uncertain.Therefore,it is of great clinical significance to strengthen the understanding of anxiety disorder. Non-anxious individuals show rapid adaptation to stressors and respond strongly to threats,but can also quickly return to baseline once the threat is removed.Persistent 2
overstimulation caused by chronic stress,such as anxiety disorder,may lead to allostatic load,manifested as an imbalance in response to stressors,or a dysfunction to return to baseline[6-8].Prior findings suggested that GAD was characterized by hyperactive in sympathetic system at resting state,in which the sympathetic-adrenal medulla (SAM) system is more active than normals[4].And it was also found that patients with GAD were lesser variability in response to stressors,and was related to the basic expression level of SAM system[9].However,no relevant studies have been found to explore the relationship between the severity of the disease and the pathological response of the GAD
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sympathetic system. In order to simulate the mental stress(MS) people face in daily life,researchers artificially give subjects a certain amount of mental stress under laboratory conditions,while detecting the changes of physiological indicators at the same time,which is called “mental
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stress-induced test”.It has been found that mental stress mainly affects the function of
various systems through autonomic nerves system and the SAM system.In the SAM
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system,sympathetic activation causes catecholamines such as epinephrine and norepinephrine to be released into the blood.Salivary-α-amylase (sAA) is one of the main
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salivary enzymes in human beings which is secreted from the salivary glands under sympathetic stimulation.Chatterton et al.[10] has reported that there was a good correlation
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between sAA concentration and serum catecholamine level.Therefore,the determination of salivary enzyme is an effective tool for evaluating SAM system. Heart rate variability (HRV) is heartbeats interval fluctuation controlled by the activity of
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the autonomic nervous system,so it can be used as a noninvasive index for detecting autonomic nervous function.Low HRV variability could reflect the inflexibility of the
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autonomic nervous system and was a marker of less favorable health[11].In healthy subjects,reduced HRV associated with changes in cardiac autonomic regulation was associated with an increased risk of cardiac events,even after adjusted for known risk factors[11,12].In anxiety disorders,including GAD,post-traumatic stress disorder,social anxiety disorder and panic disorder,there were also reports of decrease in HRV which represents parasympathetic[13-16],but higher LF/HF which reflecting the balance of parasympathetic and sympathetic nervous systems[17],indicating GAD is associated with an 3
altered sympathovagal balance with reduced vagal modulation.These were consistent with the symptoms of GAD,for example,attentional biases like hypervigilance to threat. In most previous studies,HRV has been evaluated in a resting state.In the study of autonomic function,it is necessary to analyze not only the resting,but also the activated state. Stress response is a complex phenomenon involving multiple response systems.It not only causes physiological reaction,but also causes individual's subjective psychological reaction.Subjective psychological response reflects individual's feeling and evaluation of stress,which is crucial for individual's survival[18].And it is also closely related to the
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individual's basic mental health.Previous studies have found that compared with healthy individuals,people with social phobia were more stressful and less content with their
performance after mental stress[19].Therefore,investigating both physiological and
psychological responses simultaneously is conducive to a more comprehensive
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understanding of the functional status of stress system in anxiety individuals.
In present study,patients with GAD were selected as subjects,sAA was used as
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indicator of neuroendocrine response and HRV represented autonomic nervous response.The aim of our study was therefore to provide further insight into the changes
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of sAA and HRV under mental stress in GAD and evaluate the relationship between physiological as well as subjective psychological responses under mental stress and the
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severity of anxiety.
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Materials and methods 2.1 Participants
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Patients with GAD admitted to the psychiatric department of Guangdong Provincial People's Hospital were eligible for participation if they were order than 18 years of age.The diagnosis was made by an psychiatrist using DSM-IV[20].Subjects combined with other serious mental disorders(e.g.,obsessive-compulsive disorder,schizophrenia,etc),could have been pregnant,had a current substance abuse disorder (e.g.,drug or alcohol),or demonstrated an apparent cognitive or language deficit such that they would have been unable to reliably complete the study were excluded.Patients were also excluded if they 4
were regular(daily) users of antipsychotics.Totally 20 patients were enrolled from April to June,2018 (mean age:46±10 years;14 males and 6 females).The aims of the present study and the procedures involved were explained to the subjects before.The written informed consenting to participate in this study and use their data was obtained from all subjects.They were also ensured that all of their personal and medical information would remain confidential.The study was approved by the Institutional Review Board of Guangdong Provincial People's Hospital. 2.2 Study procedures
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Experiment took place in the afternoon (between 14:00 and 17:00).Before the beginning,the Hamilton Anxiety Scale(HAMA) score was assessed by a psychiatrist.At the beginning of the experimental session,subjects rest for 10 min in a quiet room,while hrv machine was collecting data the whole time for analyzing short-term hrv in every 5 min.At
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the end of the baseline period,subjects provided the first saliva sample.After the sample collection,subjects need to wear a head-mounted virtual reality (VR) device to perform the
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stroop test (see below).Saliva samples were collected immediately after,10 min after and 20 min after the Stroop test.Self-perceived stress level and Self-performance level were
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obtained by using visual analogue scale (VAS) after the experiment was done.The subjects were instructed to abstain from eating,smoking,drinking any beverages except water and
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exercising 2 h before the experiment.The study procedure is shown in Fig.1.
（baseline）
（MS）
（MS+5min）
（MS+10min）
（MS+15min）
Enrollment HAMA Evaluation
Baseline period Rest for 10min
MS period MS for 5min
Recovery period Post-MS 5min
Recovery period 5 Post-MS 10min
Recovery period Post-MS 15min
sAA、HRV
sAA、HRV
HRV
sAA、HRV
HRV
Fig. 1 Study Procedure. HAMA= Hamilton Anxiety Rating Scale for Anxiety; sAA= salivary-αamylase; HRV= heart rate variability;MS=mental stress. In baseline period,subject rest for at least 10
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min,in which baseline sAA and HRV were collected(Labeled as baseline).In MS period,subject underwent 5 min mental stress test,in which MS-sAA and HRV were collected(Labeled as MS).In recovery period,post-mental stress test 5min,10min,15min,20min HRV were collected
respectively(Labeled as MS+5min,MS+10min,MS+15min and MS+20min). And post-mental stress
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test10min and 20min sAA were also collected (Labeled as MS+10min and MS+20min).
2.3 Psychological assessment
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Subjects were evaluated on the following scales:(1) Hamilton Anxiety Rating Scale for Anxiety (HAMA):It is used to evaluate the severity of Anxiety,with a rate of 5 (0-4).(2) Self-
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perceived stress Visual analogue scale (VAS):For the assessment of self-perceived stress level during the stroop test,subjects chose how stressed they felt on the stress level
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expressed as 1 to 10,with 1 representing "no pressure at all" and 10 representing "maximum pressure".(3) Self-performance VAS:For the assessment of self-performance level during the stroop test.The degree of self-performance was expressed as 1 to 10,with
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1 representing "extremely bad performance" and 10 representing "extremely good performance".
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2.4 Measurement
(1) HRV measurement:5-minute short-term electrocardiogram analysis was performed
on the subjects.The electrocardiogram was recorded by single lead dynamic electrocardiograph(Zhongruiqi electronic technology co.LTD,Shenzhen,China),and the data was analyzed automatically by computer soft ware.The commonly used timedomain and frequency-domain indexes are measured in resting period,mental stress
6
period and recovery period respectively:①mean R-R interval:the mean R-R interval length measures the sum of parasympathetic and sympathetic influences; ②Square root of difference mean square (rMSSD):rMSSD is considered an index of parasympathetic neuromod ulation changes of heart rate[21];③Very Low frequency (VLF) in range 0.00330.04 Hz; ④Low frequency (LF) in range 0.04-0.15hz; ⑤High frequency (HF) in range 0.15-0.4hz; ⑥LF / HF ratio.LF is affected by both parasympathetic and sympathetic
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nervous systems,reflecting the overall activity of the autonomic nervous system[22].Its standardized ratio (LFnu=100%×LF / TP-VLF）was used to represent sympathetic
activity[23].HF was used as a marker reflecting vagus nerve activity,and the LF / HF ratio reflected the balance of parasympathetic and sympathetic nervous systems[24]. Alpha-Amylase
Activity:Saliva
samples
were
collected
using
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(2)Salivary
Salivette(Sarstedt,Rommelsdorft,Germany).Gargling was ordered before the test to
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reduce the effects of oral residues on saliva collection.For salivary collection,subjects were asked to chew on the swab for 1 min.During the experiment,four saliva samples were
min
after
mental
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collected,reflecting the sAA levels at baseline (baseline),the end of mental stress (MS),10 stress
(MS+10min),and
20
min
after
mental
stress
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(MS+20min),respectively.The Salivette tubes were centrifuged at 4 ℃ and 3000 rpm for 10 min,and the supernatant was stored in a refrigerator at-80 ℃ until tested.The sAA was
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determined by enzyme-linked immunosorbent assay(ELISA)(Human saliva amylase ELISA kit,Shanghai Keshun Biotechnology Co.,Ltd.,China).The detection range of sAA
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was 25-800U/mL.Due to the symptom of somatic anxiety or the response to stress leading to dry mouth,2 subjects lacked a complete set of saliva collection.sAA in 18 subjects were finally included in the analysis. 2.5 Mental stress-induced test Stroop test has been widely used in psychophysiology.In present study,the test was applied as an acute mental stressor based on previous studies showing the test can induce mental stress,manifested as increased cortisol and catecholamine levels and 7
increased heart rate[25,26].The test lasted for 5min,during which subjects would see a Chinese character representing color(RED or GREEN or BLUE or YELLOW) on the VR screen while the character were printed in incongruent colors (e.g.word RED printed in green color).The subject was asked to indicate the color and to ignore the meaning of the character.The four Chinese character s displayed at the bottom of the screen,and subjects need to select the correct color option quickly and accurately as much as possible(i.e.,select " GREEN ").During the test,the device automatically gave the requirements of "faster" and "more accurate" every 30 s.The computerized stroop test
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provided each subject with a standardized level of stimulation. 2.6 Statistical Analyses
Analyses were performed using SPSS 20.0.Data were described as the means ±
standard error (se).All data were tested for normality of distribution by Shapiro-Wilk’s
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test.Due to the positive skewness of some HRV data,natural logarithms (ln)
transformations were computed for VLF,LF and LF/HF ratio.All other data passed the
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normality test.The sAA and HRV levels of different time were analyzed by repeated measures ANOVA(with Greenhouse—Geisser correction in the case of violation of the
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sphericity assumption).Post-hoc comparisons were performed using Bonferroni adjustments.The Pearson’s correlation coefficient(r) was determined for the relationship
Results
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between all variables and HAMA score.P ≤ 0.05 was considered significant.
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3.1 Salivary alpha-amylase responses There was no significant difference in the levels of sAA at baseline,at the end of mental
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stress,at 10 min and at 20 min after mental stress(440.64±185.12U/mL VS 449.44±156.37U/mL VS 445.39±151.55U/mL VS 472.41±140.48U/mL,F(3,27)=0.556,P=0.648,ηp2=0.058).
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(a)
Fig.2
(b)
(c)
Variation trend of HRV. rMSSD=square root of difference mean square; HF=high frequency;LFnu=
standardized ratio of low frequency; MS=mental stress. MS+5min,10min,15min,20min=post-mental stress test 5min,10min,15min,20min; (a)rMSSD was significantly lower during MS than that at 10 min and 20 min after(P<0.05). (b)HF was significantly lower during MS than that at 5 min,10 min,15 min and 20 min after(P<0.05,MS VS MS+5min,MS+10min,MS+20min;P=0.001,MS VS MS+15min). (c)LFnu during MS was significantly higher than that at 20 min after(P<0.05).
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Data are expressed as mean ± S.D. * P<0.05,***P=0.001.
3.2 HRV responses
The R-R interval at baseline was 754.35±95.47ms,and it was significantly lower during mental stress and then recover after stress（707.55±92.82 ms VS 748.95±
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96.65 ms VS 765.60±102.97 ms VS 768.90±102.60 ms VS 774.60±107.40 ms,P＜ 0.01).
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The rMSSD at baseline was 19.50±11.80ms,and it was significantly lower during mental stress than that at 10 min and 20 min after（16.34±7.09ms VS 19.38±9.43ms VS
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19.71±8.70ms,P＜0.05）(see Fig.2a).
The HF at baseline was 176.89±138.70ms2.It was significantly lower during mental
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stress than that at 5 min,10 min,15 min and 20 min after（97.54±77.26ms2 VS 170.44±113.16ms2 VS 174.15±131.10ms2 VS 169.87±102.96ms2 VS 171.14±117.01ms2,P=0.017、0.026、0.001、0.021）(see Fig.2b).
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The LFnu at baseline was 51.53±20.13.LFnu during mental stress was significantly higher than that at 20 min after（58.88±11.25 VS 49.46±20.26,P=0.023）(see Fig.2c).
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There was no significant difference in lnLF,lnVLF or lnLF/HF at baseline,mental
stress,5min,10min,15min and 20min after stress（lnLF:0.08±0.95 ms2 VS 0.37±0.49 ms2 VS 0.32±0.83 ms2 VS 0.03±1.00 ms2 VS 0.10±0.57 ms2 VS -0.42±0.91 ms2,P=NS ;lnVLF :5.79±1.12 ms2 VS 5.38±0.95 ms2 VS 5.79±0.96 ms2 VS 5.58±0.86 ms2 VS 5.82±0.94 ms2 VS 5.44±0.90 ms2,P=NS; LF/HF :1.73±2.22 VS 1.61±0.75 VS 1.96±1.88 VS 1.63±1.83 VS 1.27±0.64 VS 1.37±1.13,P=NS）； 3.3 Correlation between anxiety severity and baseline levels of sAA and HRV 9
It showed that there was a significant positive correlation between HAMA score and sAA levels both at baseline and during mental stress（P<0.01,r=0.602； P<0.05,r=0.505;see Fig.3a&b）.The levels of HRV were not correlated with HAMA score at each time point. 3.4 Correlation between anxiety severity and changes of sAA and HRV The correlation between HAMA score and the changes of sAA and HRV before and after mental stress found that there was a significant negative correlation between HAMA score and the changes values of sAA after 20 min of mental stress（P<0.05,r=-0.483；
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see Fig.3c）.In contrast to that of sAA,the change value of HRV did not statistically
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correlate with HAMA score.
(b)
(a) Fig.3
(c)
Correlation between HAMA score and sAA levels. HAMA= Hamilton Anxiety Rating Scale for
Anxiety; sAA= salivary-α-amylase; MS=mental stress; 20min-R= the differences between sAA at post-MS
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20min and baseline; (a)Correlation between the baseline sAA level and HAMA score is highly significant(P<0.01,r=0.602). (b)Correlation between the sAA level during MS and HAMA score is highly significant(P<0.05,r=0.505). (c) Correlation between the changes values of sAA after 20 min of MS and
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HAMA score is highly significant(P<0.05,r=-0.483).
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3.5 Correlation between anxiety severity and psychological response The correlation coefficient between self-perceived stress level and HAMA score was
r=0.080 (P>0.05),while the correlation coefficient between self-performance level and HAMA score was r=0.294 (P>0.05).No significant correlation was found.
Discussion 10
4.1 Physiological responses to mental stress The present study found that HRV,rMSSD and HF,which represented parasympathetic response,decreased
significantly
while
LFnu,which
reflected
sympathetic
response,increased significantly during mental stress.The results showed that the stroop test as a stressor induced autonomic nervous response in patients with GAD,which is mainly manifested as increased sympathetic nervous activity and decreased vagus nerve activity.Previous studies have found that rMSSD and HF decreased[27,28],LF did not change[29] or RR interval increased during mental stress in healthy subjects[30].There are
ro of
very few studies have been done on anxiety population.Similar to our results,Olga V.Crowley et al.[31] found a significant decrease in rMSSD after mental stress among 905 subjects with anxiety traits.
The analysis of SAM system responses showed that the differences were not
-p
significant between each time point,but the means of sAA level were consistently
increased after mental stress.Although the sympathetic system of patients with GAD is
re
pathologically hyperactive in resting state[4],there is no unified conclusion on the changes of the system in the face of acute stress.Few studies have been conducted on patients
lP
with anxiety.Similar to the results of our study,the levels of epinephrine and norepinephrine in patients with panic disorder increased after mental stress,but there was
na
not significant[32].The reason for the present study showed that there was no significant increase in sAA compared with the baseline value,may be GAD patients had a high initial secretion level,as the "ceiling effect",which makes it difficult to the further activation of
ur
norepinephrine system[32].
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4.2 Relationship between anxiety severity and physiological responses GAD patients had hyperfunction of adrenal medulla system,as their catecholamine level
in resting state was higher than that of normal subjects,and could be improved after the intervention of norepinephrine system[33,34].But the relationship between anxiety and disease severity has not been seen before.The present findings showed that there was a positive correlation between baseline sAA level and anxiety severity,which means that the more serious the disease is,the more hyperactive the baseline level of SAM system 11
function may be.This is similar to the results of previous studies.Compared with those with low anxiety levels,the levels of norepinephrine in patients with high anxiety levels were higher at rest and during tension[35].Studies in both animals and humans have shown that long-term mental stress can alter the response capacity of the central-peripheral regulatory systems[36-38],and these changes may have different effects on different pathways.For example,it may lead to an increase or decrease in the output of the target-specific sympathetic or vagus nerve,or an up-regulation or down-regulation of the HPA axis[39].Altered responsiveness may be less efficient and less adaptive,and therefore may
ro of
be detrimental to health on a long-term basis. We found a blunted sympathetic response to acute stress in the GAD patients.This finding is supported by a significant negative correlation of anxiety scores with the absolute
sAA activity during the mental stress test, suggesting that the more severe the anxiety was,
-p
the lower the activation degree of SAM system would be. Similarly, Fisher et al.[9,40] found that the level of basic sympathetic neurotransmitter was correlated with the responsiveness
re
of the sympathetic nervous system.The higher level of sAA at baseline,predicting the less change of sAA in patients with GAD after mental stress.
lP
A healthy organism is characterized by its flexibility to adapt to environmental requirements[8,41].Stress should be harmless,as long as the organism can respond
na
appropriately.The fast neuroendocrine response to stress is largely mediated by norepinephrine (NE) and corticotropin releasing hormone (CRH),followed by a slower and more sustained release of corticosterone[42]. Acute stress quickly activates the locus
ur
coeruleus(LC)-noradrenergic system,which is the main source of NE in the center[43-45]. Central catecholaminergic activation is followed by activation of the peripheral SAM
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system,which triggers release of epinephrine from the adrenal medulla.Epinephrine further increases NE release through ascending vagal projections to the nucleus of the solitary tract. Ceiling effect for both neuroendocrine and sympathetic stress related activity may be one possible interpretation of the blunted sAA activity response in our GAD patients.Since the sAA level reflects the same of peripheral catecholaminergic level, the increased peripheral level of baseline in GAD patients may be a negative feedback to restrain the stress activity. Furthermore,this noradrenergic projection from the LC widely 12
throughout the most brain regions,including the brainstem,the cerebellum,the diencephalon and the paleo- and neocortex,and are known to be involved in a range of stress-related diseases such as anxiety,depression,and posttraumatic stress[46,-48].The anxiety projection from LC may infect the stress response in some or how, which underlying mechanism remains to unknow. According to the above,it may not be surprised for us to find out disease severity dependent response to acute stress in SAM systems in GADs. Based on the results of present study, it reflected the hyperfunction of SAM system in patients with GAD,the more serious the lesion,the higher the baseline value of
ro of
sAA.However,its reactivity was reduced,which showed that the more serious the lesion,the less change of sAA after stress.
Evidence suggested that the physiological characteristics of GAD was limiting or
inhibiting the body's normal response to environment,which known as diminished
indicators
such
as
heart
-p
physiological flexibility[8,49].It referred to the lack of expected variability of physiological rate,electrocardiogram,skin
conduction
re
reaction,catecholamine,and cortisol,which is not only reflected in the decreased physiological response to stress,but also in the decreased ability to recover after
lP
stress.Consistent with previous studies[49-51],the GAD patients showed a diminished physiological flexibility,which we believe reflects the weaker adaptability when facing
na
stress.Lack of flexibility in regulation may be problematic because it was associated with mortality and morbidity[12,52,53].Perhaps this is the potential mechanism for the development
ur
or progression of many psychosomatic and physical diseases.
4.3 Psychological response to mental stress
Jo
In present study,it was found that the HAMA score was not correlated with the degree
of self-perceived stress and self-performance,indicating that the severity of anxiety disorder may not related to stress perception in the face of acute mental stress.Nor does it affect the individual's evaluation of his or her self-coping ability.Previous studies have also shown that the degree of lesion is not completely consistent with the individual's own psychological response.Crowley OV et al.[31]found that among the individuals with higher trait anxiety,reductions levels in perceived stress were positively associated with recovery 13
in ln rMSSD after the challenge.In contrast,among the individuals with lower trait anxiety,changes in perceived stress had no impact on ln rMSSD recovery.This indicates that the relationship between perceived stress and recovery of cardiac vagal was only found in individuals with high degree of trait anxiety.A similar finding was also found in another study.Subjects who were actually felt stressful due to the mental stress-induced test but showed only weak physiological response,and subjects who were only slightly stressed but showed a marked response[29].The present study did not obtain consistent results with social phobia patients[19],suggesting that for GAD subjects,there is an
ro of
inconsistency between the psychological response of self-perception and the degree of disease lesion,so individual subjective psychological perception may not be objective as a prediction of disease severity.
One possible weakness of the current study is that for the GAD subjects enrolled,the
-p
effect of early intake of antianxiety drugs on the adrenal medulla system cannot be
excluded.Additionally,the present sample size is limited.Expanding the sample size and
Conclusions
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generalizability of the present findings.
re
trying to enroll subjects with first diagnosed GAD would further strengthen the
In present study,by measuring the indicators of different stress response systems,it was
na
found that stroop test did induce obvious cardiac autonomic nervous responses in patients with GAD,and confirmed the activation function of SAM system in GAD patients was
ur
impaired and correlated with anxiety severity.Also,ours study examined the subjective psychological response of individuals,and found that there was no consistency between
Jo
the anxiety severity and the psychological response of self-perception.Proving the diminished physiological flexibility of GAD patients may be of great enlightening significance to explore the potential mechanism for the development or progression of many psychosomatic and physical diseases,which needs further research.
Funding：This research did not receive any specific grant from funding agencies in the public,commercial,or not-for-profit sectors. 14
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