Nociception related biomolecules in the adult human saliva: A scoping review with additional quantitative focus on cortisol

Nociception related salivary biomolecules can be useful patients who are not able to self-report pain. We present the existing evidence on this topic using the PRISMA-ScR guidelines and a more focused analysis of cortisol change after cold pain induction using the direction of effect analysis combined with risk of bias analysis using ROBINS-I. Five data bases were searched systematically for articles on adults with acute pain secondary to disease, injury, or experimentally induced pain. Forty three articles met the inclusion criteria for the general review and 11 of these were included in the cortisol-cold pain analysis. Salivary melatonin, kallikreins, pro-inflammatory cytokines, soluable TNF-α receptor II, secretory IgA, testosterone, salivary α-amylase (sAA) and, most commonly, cortisol have been studied in relation to acute pain. There is greatest information about cortisol and sAA which both rise after cold pain when compared with other modalities. Where participants have been subjected to both pain and stress, stress is consistently a more reliable predictor of salivary biomarker change than pain. There remain considerable challenges in identifying biomarkers that can be used in clinical practice to guide the measurement of nociception and treatment of pain. Standardization of methodology and researchers’ greater awareness of the factors that affect salivary biomolecule concentrations are needed to improve our understanding of this field towards creating a clinically relevant body of evidence.


AIM
To test the effects of oral hydrocortisone on pain thresholds and explore the sex differences as well as the effects of hydrocortisone on pain related fear METHOD Country: Germany Participants Recruitment: By local advertisement Age: hydrocortisone arm: mean 24.8, placebo arm: mean 25.1 Sex: 50 F, 50 M Total number of participants: 108 Dropouts: 8 Reasons for drop out: medical condition or medication intake (5), technical issues on the study (3) For rectal distension: distention pressure limit precluded measurement of the threshold (8 in hydrocortisone group, 7 in placebo group and in the heat pain 4 excluded (no reliably determined heat thresholds 3 shared with the other 15.Revised sample size: 100 Analgesia intake: None (irregular use of over the counter pain medications permitted) Chronic conditions: Healthy (psychiatric conditions and chronic pain not reported)

Study design:
Measurement of salivary cortisol and alpha-amylase before and after induced noxious visceral and heat stimuli with pain induction taking place before and after hydrocortisone or a placebo control pill, in double blind randomized trial Other measurements: pain intensity Interventions: Combined pressure-controlled rectal distension (

AIM
To assess the effectiveness of thoracic paravertebral regional block for post-operative pain after video-assisted thoracic surgery (VATS) 70 (study group: 37, control: 33) Analgesia intake: post-operative patient-controlled analgesia with oxycodone, no preoperative analgesia Chronic conditions: Participants could have a range of health conditions within American Society of Anesthesiology physical status I-III (psychiatric conditions not specifically excluded, chronic pain part of exclusion criteria) Restrictions: None reported Study design: Salivary cortisol, testosterone, sIgA & alpha-amylase measured before and after surgery in a randomised study comparing thoracic paravertebral block and a control group with no block Other measurement: Pain intensity, blood pressure, heart rate Interventions: VATS Comparison: Change from baseline compared between study (regional block) and control groups Salivary assay: Cortisol & testosterone : Commercial ELISA.sIgA: Commercial ELISA kits.Alpha-amylase: static method with an AMYLAZA kit Saliva type: Mixed stimulated and unstimulated Salivary Collection method: Swab OUTCOMES There was increase in all the biomolecules measured in the study 6 hours and also 24 hours after surgery compared with pre-operatively with the exception of sIgA which fell in the regional block group between at the 6 hour time point.There was no significant difference in this change between the intervention and control groups.Correlation with pain ratings: Alpha-amylase levels were significantly associated with higher one month pain intensity score Sex effects: Not analysed Burns (2004) sIgA AIM To investigate whether differences in the timing of saliva are the explanation for discrepant results in change in salivary IgA after acute stress tasks From CPT: 0. From cognitive stress task: 0. From cardiovascular parameters heart rate variation data analysis: 2. From respiration breathing pattern analysis: 3. From saliva sampling: 0, From pain intensity rating: 1, From voice frequency analysis 3,

OUTCOMES
Change in salivary cortisol after heat pain was not significant before the stress task.In contrast, cortisol levels increased significantly in response to heat pain after the participants had done the stress task.In contrast, cortisol levels increased significantly in response to heat pain after the participants had done the stress task.

NOTES
In participants who were exposed to MIST, cortisol levels increased in men and fell back down during the recovery phase.In women this increase did not reach significance.Among men, temporal summation of pain increased following the MIST but was not predicted by the stress variables.The authors concluded that acute stress manipulation affects stress and pain responses in women and men differently: women exhibited stress-induced anti-nociception and men exhibited stressinduced pro-nociception.

AIM
To investigate the influence of exposure to a cognitive stressor on pain perception and determine the individual characteristics that may be predictors of the pain response We examined pain perception to a mechanical noxious stimulus before and after exposure to a cognitive stressor across a range of pain responses.Mental math was used as the cognitive stressor because it is an established and effective psychosocial technique to induce stress [1].Changes in stress and anxiety were assessed with both self-reported and physiological measures including questionnaires, visual analogue scales (VAS), mean arterial pressure (MAP), heart rate, and salivary cortisol levels.

AIM
To examine the hypothesis that listening to music decreases the pain of vascular access cannulation for haemodialysis

NOTES
Psychological measures of stress such as negative emotion and perceived stress, were stronger predictors of reported pain than physical stress measures such as blood pressure and heart rate Pre-procedure mean cortisol levels were at the high end of the normal range for sex and time of day regardless of which procedure was being done.The authors concluded that anticipatory anxiety leads to cortisol rise that subsides after the event.Author's judgement: High ROB Support for judgement: No control and unclear if steps were taken to minimise participant stress.Unclear whether all potential participants had equitable opportunity to be included.Interactions between participants and experimenters not fully described.No published pre-specified protocol.

NOTES
Healthy participants habituated to the induced pain on the nail bed and were able to subjectively relax.When on the nail bed, signs of both sympathetic and parasympathetic nervous system activity were observed.

AIM
To examine the relationship between trait pain catastrophizing and morning salivary cortisol levels before and after pain induction in pain free and temporomandibular disorder (TMD) participants and whether TMD patients had greater hyperalgesia and hypercortisolism

OUTCOMES
In the group who received TSST placebo (n=43), cortisol levels dropped during the course of the experiment with a significant drop after the first experimental heat pain.There was no change in alpha amylase.There was a rise in both biomolecules in response to TSST induced stress Correlation with pain ratings: No correlation Sex effects: Not applicable (female only study)

NOTES
Experimentally induced social stress did not influence pain in women with or without performance feedback.

AIM
To examine the association between the catechol-O-methyltransferase (COMT) allele and perceived pain, anxiety, cortisol and sAA levels

METHOD
barostat system) & heat pain Comparison: Comparison of biomolecule levels at different experimental time points between hydrocortisone and placebo arms Salivary assay: Cortisol: ELISA.Alpha amylase: Saliva Enzymatic Assay Saliva type: Not recorded Salivary Collection method: swab OUTCOMES No rise in amylase or cortisol after pain stimuli in the placebo arm of the trial Correlation with pain ratings: Not analysed in the control arm Sex effects: Not analysed in the control arm NOTES Heat pain thresholds were not affected by hydrocortisone Hydrocortisone decreased the pain threshold for visceral pain and this was primarily Reason for drop out: Failed to produce sufficient saliva for analysis Revised sample size: 36 Analgesia intake: None Chronic conditions: Healthy (psychiatric conditions and chronic pain not specifically reported) Restrictions: Alcohol, vigorous exercise, food, caffeine Study design: Salivary sIgA measured before and after an induced noxious stimulus (first and second exposures in different arms) Other measurements: Blood pressure and heart rate, pain intensity Intervention: Cold pain induced by CPT Comparison: Change from baseline.Changes compared between first and second exposures.Salivary assay: Radial immunodiffusion (RID) assay (Bind A Rid, The Binding Site Ltd, Winzer 1999) Saliva type: Stimulated saliva Collection method: Swab OUTCOMES sIgA levels fell significantly after first exposure to CPT, but not after second exposure in the same participants' other arm Correlation with pain ratings: No significant difference found Sex effects: No significant difference Advertisement on social media and among undergraduate dental students at Karolinska Institute Age: 23.8 Sex: 13 F, 13 M Total number of participants: 26 Dropouts: 0 Reason for drop out: Not applicable Revised sample size: 26 Analgesia intake: None Chronic conditions: Healthy (psychiatric conditions not specifically reported, chronic pain part of exclusion criteria) Restrictions: All of food, alcohol, smoking, caffeine and precautions to reduce risk of contamination due to bleeding from gums Study design: Salivary AA measured before and after an induced somatic noxious stimulus Other measurements: Depression and anxiety, somatic symptoms, pain intensity Interventions: Hypertonic saline muscle injection Comparison: Change from baseline Salivary assay: Commercially available enzymatic assay kit Saliva type: Stimulated saliva Collection method: time course, duration and magnitude of changes of commonly measured pro-(interleukin [IL]-6, IL-8) and anti-inflammatory (IL-10, IL-4) cytokines in saliva samples and to test for age-related differences METHODS Country: United States of America Participants Recruitment: Details not reported Age: 8 younger participants: mean 21.4; 9 older participants: mean age 68.1 Sex: 8 F, 9 M Total number of participants: 17 Dropouts: 1 Reason for drop out: Vigorous physical activity before arriving for the session Revised sample size: 16 Analgesia intake: None Chronic conditions: Healthy (chronic pain and psychiatric conditions part of exclusion criteria) Restrictions: All of food, alcohol, smoking, caffeineStudy designA panel of salivary cytokines (IL-6, IL-8, IL-10, IL-4) measured before and after an induced noxious stimulus or a non-noxious control in saliva and blood Non-painful task was done on the same participants with and without venepuncture Other measurements: Blood pressure, pain intensity, Intervention: Cold pain induced by CPT with a non-painful thermal water task as control Salivary Assay: MILLIPLEX XMAP human cytokine/chemokine-premixed 13-Plex assay Comparisons: Change from baseline.Changes compared (1) between the CPT and control groups, (2) between the two age groups, (3) with or without venipuncture in control group Saliva type: Unstimulated saliva Salivary collection method: Swab OUTCOMES IL-6, IL-10 & IL-4 concentrations increased from baseline, peaking at 60 minutes after CPT IL-8 peaked at 45 minutes after CPT No significant changes reported in control group Venepunture had no significant effect on the cytokine levels Correlation with pain ratings: Not analysed Sex effects: Not analysedNOTESThe time course of the peak levels of cytokines in the CPT session was nearly identical in saliva and plasma Older adults experienced greater salivary changes in all cytokines during the cold pressor session compared to younger adults in the non-painful sessions 21 Finke (2021) Cortisol AIM To assess how concurrent administration of a cognitive and physical stressor affects stress response patterns on subjective and physiological dimensions METHOD Country: Germany Participants Recruitment: University's email newsletter Age: Mean 23 Sex: 28 F, 28 M Total number of participants: 56 Dropouts: Advertisements posted at the university and internet sites of triathlon Age: Mean 35.9 Gender: Male Total number of participants: 25 Dropouts: 0 Reason for drop out: Not applicable Revised sample size: 25 Analgesia intake: Not reported Chronic conditions: Healthy (psychiatric conditions and chronic pain part of exclusion criteria) Restrictions: Exercise, food, caffeine Study design: Measurement of salivary cortisol before and after noxious heat stimulus twice; before and after the application of the stress manipulation (using the Montreal Imaging Stress Task) Other measurements: Skin conductance, perceived stress, anxiety, heart rate and heart rate variation, pain intensity Interventions: Acute heat pain induced by Peltier-based computerized thermal stimulators Comparison: Change from baseline before and after two noxious heat sessions performed on either side of stress manipulation Salivary assay: ELISA Saliva type: Unstimulated Salivary collection method: SwabOUTCOMESChange in salivary cortisol after heat pain was not significant before the stress task.In contrast, cortisol levels increased significantly in response to heat pain after the participants had done the stress task In contrast, cortisol levels increased significantly in response to heat pain after the participants had done the stress task.Correlation with pain ratings: Not analysed Sex effects: Not analysed Geva (2018) Cortisol AIM To study the effect of acute psychosocial stress manipulation on pain modulation METHOD Country: Israel Participants Recruitment: By advertisements posted around the university campus Age: Mean 34 Gender: Male only Total number of participants: 31 Dropouts: 0 Reason for drop out: Not applicable Revised sample size: 31 Analgesia intake: Not reported Chronic conditions: Healthy (psychiatric conditions and chronic pain part of exclusion criteria) Restrictions: Exercise, food, caffeine Study design: Measurement of salivary cortisol after heat pain induction twice; before and after the application of the stress manipulation (using the Montreal Imaging Stress Task) Other measurements: Skin conductance, anxiety, heart rate and heart rate variation, blood pressure, respiration, heart rate variability, perceived stress, pain intensity Comparison: Change from baseline before and after two testing sessions performed on either side of stress manipulation Interventions: Heat pain using Peltier thermal stimulator Salivary assay: Cortisol: ELISA Saliva type: Unstimulated Salivary collection type: Swab OUTCOME Change in salivary cortisol after heat pain was not significant before the stress task.In contrast, cortisol levels increased significantly in response to heat pain after the participants had done the stress task.Correlation with pain ratings: Not analysed Sex effects: Not analysed 27 Geva (2022) Cortisol AIM To examine the effects of psychosocial stress on pain perception and modulation of women and men METHOD Country: Israel Aim: To examine the effect of psychosocial stress on pain perception and modulation Participants Recruitment: Recruited by advertisements posted around the university campus Age: F: Mean 30.9, M: Mean 28.3 Sex: F 82, M 66 Total number of participants: 148 Dropouts: 0 Reasons for drop out: Not applicable Revised sample size: 148 Analgesia intake: None Chronic conditions: Healthy (psychiatric conditions and chronic pain part of exclusion criteria) Restrictions: Food, caffeine, exercise Study design: Salivary cortisol measured before and after induced noxious heat stimulus followed by MIST (n=133) or sham task (n=15) and then also after a second episode of induced noxious stimulation.Other measurements: Pain intensity, heart rate, heart rate variability, galvanic skin response Interventions: Heat pain using Peltier-based computerized thermal stimulators Comparison: Change in cortisol from baseline Salivary assay: ELISA Saliva type: Stimulated Salivary Collection method: Swab OUTCOMES There was no change in salivary cortisol in the sham group Correlation with pain ratings: Not reported in the sham group Sex effects: No difference in the sham group OUTCOMESsTNFαR-II: Significant reduction immediately after pain induction Cortisol: Significant elevation after pain induction Correlation with pain ratings: Not analysed Sex effects: Not analysed NOTES Researchers were aiming to assess salivary pro-inflammatory cytokines after acute pain induction and chose to measure sTNFαR-II, because it is more stable than TNFα and can be measured more reliably Inclusion in cortisol-cold pain quantitative analysis: No. Reason: Results were not separated for different pain modalities Goodin (2012)-3 Cortisol AIM To investigate the effect of sleep quality on pain intensity and cortisol reactivity METHOD Country: United States of America Participants Recruitment: Recruited from a college campus using posted advertisements Age: Mean 20.2 Sex: 20 F, 20 M Total number of participants: 40 Dropouts: 0 Reason for drop out: Not applicable Revised sample size: 40 Analgesia intake: None Chronic conditions: Healthy (psychiatric conditions and chronic pain part of exclusion criteria) Restrictions: Food, alcohol, smoking, caffeine.Precautions to reduce risk of contamination due to bleeding from gums Study design Salivary cortisol measured before and after an induced noxious stimulus Other measurements: Sleep quality, pain intensity, pain characteristics, affect Intervention: Cold pain induced using CPT Comparison: Change from baseline Salivary assay: High sensitivity immunoassay kits Saliva type: Stimulated Salivary collection method: Swab OUTCOMES Poor sleep quality was significantly associated with greater reports of CPTinduced pain severity and greater cortisol increase from baseline Correlation with pain ratings: Significant positive correlation Sex effects: No significant difference NOTES Inclusion in the cortisol-cold pain quantitative analysis: Yes BIAS Bias Type: Moderate for risks of confounding, selection of participants, and departures from intended exposure and measurement of outcome Author's judgement: Support for judgement: No control but steps taken to minimise participant stress, recruitment from the university community, interactions between participants and experimenter not fully described, no published pre-specified From Luxembourg and the greater region Saar-Lor-Lux Age: Controls: 21.8.Participants with ELA: 22.5 Sex: Healthy participants: 11 F, 11 M. ELA participants: 14 F, 8 M Total number of participants: 44 (22 healthy and 22 ELA) Dropouts: 0 Reason for drop out: Not applicable Revised sample size: 44 Analgesia intake: Yes Chronic conditions: ELA or healthy (psychiatric conditions and chronic pain not specifically recorded in either group) Study design: Salivary cortisol measured before and after an induced noxious cold stimulus combined with a stressful cognitive task in people with ELA and healthy matched controls Other measurements: Stress, arousal, anxiety, pain intensity, heart rate, blood pressure Interventions: Acute cold pain using CPT combined with stress using the Paced Auditory Serial Addition Task (PASAT) Comparison: Change from baseline.Comparison between change in those with ELA and the control group Salivary assay: Time resolved immunoassay with fluorescence detection Saliva type: Unclear Collection method: SwabOUTCOMESIn people with early life adversity (ELA) the cortisol response after CPT combined with PASAT was blunted compared with people who had not experienced ELA (even though there was no difference in reported pain intensity between the groups) Correlation with pain ratings: Not analysed Sex effects: No significant difference NOTES Inclusion in the cortisol-cold pain quantitative analysis: No Reason: Experimental design included stress induced by a cognitive task as well as noxious stimulus for relationship between conditioned pain modulation and SNS and HPA reactivity where pressure pain was studied before and after CPT METHOD Country: Canada Participants Recruitment: university-level participants, purposive recruitment to ensure equal representation of sexes Age: 24.5 Gender: 25 F, 25 M Total number of participants: 50 Dropouts: 0 Reason for drop out: Not applicable Revised sample size: 50 Analgesia intake: None Chronic conditions: Healthy (psychiatric conditions not specifically reported, chronic pain part of exclusion criteria) Restrictions: food, alcohol, exercise Study design: Salivary cortisol measured before and after an induced noxious cold stimulus Other measurements: Pain intensity, galvanic skin response Interventions: Acute cold pain induced by CPT as the noxious conditioning stimulus.Pressure pain detection threshold measured before and after CPT.Comparison: Change from baseline Salivary assay: ELISA Saliva type Unstimulated Salivary Collection method Swab OUTCOMES No significant change in salivary cortisol 30 seconds after exposure to noxious cold Correlation with pain ratings: Not analysed Sex effects: Not analysed NOTES Inclusion in the cortisol-cold pain quantitative analysis: pain inhibition and the cortisol response in chronic fatigue syndrome patients with chronic widespread pain compared with a healthy control group using spatial summation of thermal noxious stimuli METHODS Country: Belgium Participants Recruitment: Study patients: Random selection from the medical files available at the university-based chronic fatigue clinic.Control subjects: From the staff and students of the university physiotherapy department and among friends and family of the researchers (age and gendermatched) Age: Mean: 44.4 Sex: CSF: 21 F, 10 M. Healthy: 21 F, 10 M Total number of participants: 62 (31 CFS-patients with chronic pain, 31 controls) Dropouts: 0 Reason for drop out: Not applicable Revised sample size: 62 Analgesia intake: Not reported Chronic conditions: Healthy control group (psychiatric conditions not specifically recorded, chronic pain excluded in control group) Restrictions: Exertion, caffeine, alcohol, smoking Study design Measurement of salivary cortisol before and after induced noxious heat stimulus in people with chronic fatigue syndrome and widespread pain compared with controls Other measurements: Pain intensity, Interventions: Acute heat pain induced by hot water immersion Comparison: Change from baseline compared in people with chronic fatigue and widespread pain and healthy controls Salivary assay: Radioimmunoassay Saliva type: Unclear Salivary collection method: Swab Restrictions: Exertion, caffeine, alcohol, smoking OUTCOMES No significant change in salivary cortisol either group Correlation with pain ratings: In people with chronic fatigue syndrome (CFS) there was significant negative correlation between change in cortisol and pain intensity Sex effects: Not analysed 55 Muhtz (2013) Cortisol AIM To examine the effects of pain stimuli on cortisol levels in patients with chronic pain and patients with depression METHOD Country: Germany Participants Recruitment: From an outpatient clinic for patients with chronic pain Age: Chronic pain: Mean 44.9.Depression: Mean 36.3.Controls: Mean 33.3 Sex: Chronic pain: 12 F, 8 M. Depression: 6 F, 16M.Controls: 21 F, 12 M Total number of participants: 75 (22 depression, 20 chronic low back pain, 33 controls) Dropouts: 0 Reason for drop out: Not applicable Revised sample size: As above Analgesia intake: None in the healthy control group.Medication for depression and chronic pain in experiment group.Chronic conditions: Controls: psychiatric conditions and chronic pain part of exclusion criteria.Depression group & chronic back pain group: no other psychiatric disorders as part of exclusion criteria Restrictions: None stated Study design: Measurement of cortisol before and after heat pain in 3 groups (1) healthy taking no analgesia, (2) chronic pain (3) depression Other measurements: Pain intensity Interventions: Acute heat pain induced using the peltier device Comparison: Change from baseline compared between the three groups Salivary assay: Radioimmunoassay Saliva type: Unclear Salivary collection method: Swab OUTCOMES No statistically significant change in cortisol levels observed in any of the three groups Correlation with pain ratings: No significant correlation Sex effects: Not analysed Nakajima (2011) Cortisol AIM To examine the extent to which pain perception prior to smoking cessation predicts early relapse METHOD Country: United States of America Participants Recruitment: Newspaper advertisements in the community and flyers in the university and participants completed a phone interview Age: Mean in abstinent group 36.7.Mean in relapsed group 35.4 Sex: 46 F, 45 M Total number of participants: 91 Dropouts: 0 for cortisol-CPT, 20 through the rest of the study Reason for drop out: Follow up non-attendance Revised sample size: 91 (all participants underwent CPT with saliva samples collected at the outset; dropout occurred later in the study) Analgesia intake: None Chronic conditions: Healthy (psychiatric disorders part of exclusion criteria, chronic pain not specifically reported) Restrictions: Alcohol, smoking Study design: Salivary cortisol measured before and after an induced noxious cold stimulus prior to smoking cessation Other measurements: Heart rate, blood pressure, pain intensity, pain characteristics, withdrawal symptoms, mood states, smoking status Interventions: Acute cold pain induced using CPT Comparison: Change from baseline.Changes compared between smokers who remained abstinent and smokers who relapsed Salivary assay: Time-resolved fluorescence immunoassay with a cortisolbiotin conjugate as a tracer Saliva type: Stimulated Saliva collection method: Swab OUTCOMES Increase in salivary cortisol after CPT with no difference between the 2 Correlation with pain ratings: Not analysed Sex effects: Not analysed NOTES Inclusion in the cortisol-cold pain quantitative analysis: Yes BIAS Bias Type: High in confounding.Moderate in selection of participants, departures from intended exposure and selection of reported results.
Details not reported Age: Mean not reported (range: 19-55 years) Sex: 7 F, 11 M Total number of participants: 18 Dropouts: 0 Reason for drop out: Not applicable Revised sample size: 18 Analgesia intake: None Chronic conditions: Healthy (psychiatric conditions not specifically reported, chronic pain part of exclusion criteria) Restrictions: None reported Study design Salivary melatonin measured before and after an induced noxious stimulus Other measurements: Pain intensity Intervention: Acute pain induced by electric stimulation Salivary Assay: Salivary melatonin by direct radioimmunoassay Comparison: Change from baseline Saliva type: Unstimulated saliva Saliva collection method: Not recorded OUTCOMES Melatonin levels changed less than 5 minutes after the pain stimulus with initial decrease followed by a rise and then a reduction until levels similar to those anticipated for the time of day reached Correlation with pain ratings: Not analysed Sex effects: Not analysed Niedbala (2018) Cortisol AIM To determine whether retaliating against a threatening outgroup, enables individuals in a group endure more pain and actually feel less pain intensity METHOD Country: United States of America Participants Recruitment: Introductory psychology students (no further details) Age: Mean 19.32 Sex: 48 F, 26 M Total number of participants: 74 Dropouts: 0 Reason for dropouts: Not applicable Revised sample size: 74 Analgesia intake: Not reported Chronic conditions: Healthy (psychiatric conditions and chronic pain not specifically reported) Restrictions: Smoking, caffeine, mouth rinse before commencement Study design: Salivary cortisol measured before and after an induced noxious cold stimulus compared between retaliation and non-retaliation groups, where completing CPT was a way of subtracting points from the rival (i.e.positive appraisal of pain) Other measurements: Skin conductance, anger, approach motivation, pain intensity Intervention: Acute cold pain induced using CPT Comparison: Change from baseline.Change compared between different behaviour manipulation groups.Salivary assay: Commercially available enzyme immunoassay Saliva type: Stimulated Salivary collection method: Passive drool OUTCOMES The cortisol response was inhibited in participants with positive appraisal of pain compared with controls, even though they did not report less pain.Correlation with pain ratings: Not analysed Sex effects: Not analysed NOTES Inclusion in the cortisol-cold pain quantitative analysis: No Reason: Experimental design included emotional manipulation or a cognitive task as well as CPT 40 Olsson (2011) Cortisol AIM To investigate the differences in physiologic and subjective parameters between lying on a bed of nails compared to a soft bed, and, whether there are any differences between listening to relaxation instructions on a CD versus no CD-instructions METHOD Country: Sweden Participants Recruitment: Internet advertisements and by posters at work places near the study location Age: Mean 39.7 Sex: 20 F, 12 M Total number of participants: 32 Dropouts: 3 Reason for drop out: Saliva samples turned up dry to the lab and could not be analyzed Revised sample size: 29 Analgesia intake: Not reported Chronic conditions: Healthy (psychiatric conditions and chronic pain not specifically recorded) Restrictions: Caffeine, nicotine and any medication 12 hours prior to participation Study design: Salivary cortisol measured before and after a noxious mechanical stimulus compared with a soft stimulus Other measurements: End-tidal carbon dioxide, oxygen saturation, respiration rate, heart rate, heart rate variability, skin conductance level, blood pressure, ECG, pain intensity Intervention: Acute pain induced by mechanical pressure pain (lying on a Shakti-mat), compared with lying on a soft bed, both with and without listening to a relaxing music CD Comparison: Change from baseline, compared in 4 groups: A-lying on nails in silence, B-lying on nails with CD at comfortable volume, C-lying on soft bed in silence, D-lying on a soft bed listening to CD Salivary assay: Spectria [125I]-Coated Tube Radioimmunoassay Saliva type: Stimulated Salivary collection method: Swab OUTCOMES No effects of either bed type or relaxing instructions on saliva cortisol Correlation with pain ratings: No correlation; no rise though participants reporting rapid and significant rise in pain at the start of lying on nail bed Sex effects: Not analysed Country: United States of America Participants Recruitment: TMD patients: from a dental school-based, orofacial pain clinic and media advertisements for a larger prospective study concerning sleep disturbance and TMD pain and function.Healthy controls: from fliers posted at a major teaching hospital and medical school Age: Mean: TMD 33.79 and controls 25.91 Sex: TMD: 32 F, 7 M. Healthy: 21 F, 1 M Total number of participants: 39 TMD, 22 healthy controls Dropouts: 0 Reason for drop out: Not applicable Revised sample size: 61 Analgesia intake: None Chronic conditions: Healthy or TMD (psychiatric conditions part of exclusion criteria, chronic pain excluded in healthy group Restrictions: Smoking, food, caffeine, exercise Study design: Salivary cortisol measured before and after noxious cold, pressure and heat stimuli in TMD and control participants Other measurements: Pain catastrophizing, psychological distress, pain intensity Interventions: Acute pain induced by a combination of pressure, heat and stimuli induced using pressure algometry at defined anatomical sites, peltier stimulator, CPT Comparison: Change from baseline compared between TMD and healthy participants Salivary assays: Commercially available enzyme immunoassay (EIA) Saliva type: Unstimulated Salivary collection method: Swab OUTCOMES No difference in cortisol response from baseline to post-pain between the people with TMD and healthy people.In a separate analysis of the same experiment, in people who had a tendency to catastrophizing there was a reduction in salivary cortisol immediately and 20 minutes after pain compared to baseline Correlation with pain ratings: No significant correlation Sex effects: Not analysed NOTES Inclusion in cortisol-cold analysis: No Reason: Pain induced by a combination of noxious stimuli with no separate analysis of cold effect of psychosocial stress (Trier Social Stress Test, TSST) combined with performance feedback on changes in pain perception and their association with neuroendocrine stress parameters METHOD Country: Germany Participants Recruitment: A web based software for recruiting participants (SONA Systems), postings in a university department of psychology, advertisements in a local on-line newspaper Age: 23.83 Sex: F only Total number of participants: 186 Dropouts: 5 Reason for drop out: Experiment was uncomfortable or did not meet exclusion criteria (5), profound high cortisol levels (2), unaccounted for (2) Revised sample size: 177 Analgesia intake: None Chronic conditions: Healthy (psychiatric disorders and chronic pain part of exclusion criteria) Restrictions: Smoking, food, alcohol, caffeine Study design: Salivary cortisol and alpha-amylase measured before and after induced noxious heat combined with one of: TSST followed by positive feedback (43), negative feedback (46) or no feedback (45) or TSST placebo version (43) Other measurements: pain intensity, anxiety Interventions: Acute phasic heat pain induced by thermal stimulator, tonic heat pain (water bath) Comparison: Change after psychosocial stress induction and differences between the 3 study groups and placebo Salivary assay: Cortisol: chemi-luminescence immunoassay with high sensitivity.Alpha-amylase: enzyme kinetic method Saliva type: Not stated Salivary Collection method: swab

OUTCOMES
IgA: Significant increase one hour after surgery sTNFαR-II: Significant reduction one hour after surgery Cortisol: Rise in the immediate pre-operative period compared to baseline with a further rise 1 hour after surgery sAA: No significant rise in sAA Testosterone: No significant change Correlation with pain ratings: IgA: Significant positive correlation one hour after surgery.sTNFαR-II: No correlation.Cortisol: No correlation Sex effectseffect of Yokucansan (YKS), a traditional Japanese herbal (Kampo) medicine, vs placebo treatment on women undergoing breast cancer surgery without axillary clearance METHOD Country: Japan Participants Recruitment: Not specified Age: range 20-60 Sex: F only Total number of participants: 100 Dropouts: 23 Reason for drop out: Protocol violation (1), refused participation (2), positive sentinel node (12), steroid treatment needed (1), incomplete data (7) Revised sample size: 77 (35 YKS, 42 control) Analgesia intake: None Chronic conditions: Healthy (psychiatric conditions and chronic pain part of exclusion criteria) Restrictions: none reported Study design: Salivary alpha amylase measured before and after breast surgery in women given YKS or placebo in a single blind randomised controlled trial Other measurements: pain intensity, anxiety, depression, quality of life Interventions: Breast surgery Comparison: Change from pre-operative baseline within each group and also compared between the active treatment and placebo group Salivary assay: Handheld monitor (COCORO meter, NIPRO, Osaka, Japan) with disposable test strip-assessment if reaction time of a hydrolyzing reaction Saliva type: Unclear Salivary Collection method: test strip OUTCOMES No change in salivary alpha-amylase in the different time points in the control group.In the YKS group, salivary alpha-amylase scores directly before operation were significantly lower than those on the day before the surgery and one day postoperatively Correlation with pain ratings: Not analysed Sex effects: Reason for drop out: Unavailable pain intensity data (2), took analgesics (2) Revised sample size: 23 Analgesia intake: None Chronic conditions: Healthy (psychiatric conditions and chronic pain not specifically reported) Restrictions: Caffeine, alcohol, food, precautions to avoid blood contamination Study design: Salivary AA measured before and after induced noxious heat stimulus Other measurements: Pain intensity, mood and anxiety Intervention: Acute heat pain induced using Medoc TSA-II thermode Comparison: Change from baseline Salivary assay: Alpha amylase enzyme activity using reagents Saliva type: Unstimulated saliva Collection method: Swab OUTCOME Significant rise in sAA after heat pain Correlation with pain ratings: Positive correlation between sAA activity and pain intensity Sex effects: No correlation NOTES No significant correlation found between the trait anxiety score and the pre-test sAA level After the pain assessment, irrespective of gender, participants felt significantly calmer and their mood was use of salivary amylase activity, as an indicator of pain in people with severe disability who required the daily replacement of gastric and/or bronchial tubes METHOD Country: Japan Participants Recruitment: From a hospital setting, no further details Age: Mean 20.5 Sex: 3 F, 7 M Total number of participants: 10 Dropouts: 0 Reason for drop out: Not applicable Revised sample size: 10 Analgesia intake: Not reported Chronic conditions: Severe motor and cognitive disabilities (psychiatric conditions and chronic pain not specifically reported) Restrictions: None reported Study design: Salivary AA measured before and after a noxious medical procedure stimulus Other measurements: Pain intensity, heart rate Intervention: Acute pain after gastric or bronchial tube exchange Salivary assay: Enzymatic reagent method Saliva type: Stimulated Collection method: Monitor device with test strip under the tongue OUTCOMES Significant rise in sAA Correlation with pain ratings: Significant positive correlation found Sex effects: Not analysed

METHOD Country: Israel Participants Recruitment: Advertisements posted at the university Age: 33 Sex: Male only Total number of participants: 29 Dropouts: 0 Reason for drop out: Not applicable Revised sample size: 29 Analgesia intake: Not reported Chronic conditions: Healthy
Change from baseline before and after two noxious heat stimulus sessions performed on either side of stress manipulation Salivary

number of participants: 46 Dropouts: 0 Reason for drop out: Not applicable Revised sample size: 46 Analgesia intake: None Chronic conditions: Healthy
Cold pain but not heat or ischaemic pain produced significant time-dependent elevation, whereas cortisol significantly decreased for the neutral water task sTNFαRII: The cold pressor, hot water, and ischemic modalities were associated with significant reduction over time, especially 25-35 minutes after pain induction.Response to neutral water initially decreased but returned to approximate baseline.

RISK OF BIAS FOR CORTISOL-COLD PAIN ANALYSIS Bias Type:
Moderate for risks of confounding, selection of participants, and departures from intended exposure and measurement of outcome Author's judgement: Moderate risk of bias Support for judgement: No control but steps taken to reduce stress and anxiety in participants, recruitment limited to university community, interactions between participants and experimenters not fully described and no published pre-specified protocol.

number of participants: 36 Dropouts: 0 Reason for drop out: Not applicable Revised sample size: 36 Analgesia intake: None Chronic conditions:
Healthy (chronic pain and psychiatric conditions part of exclusion criteria) Restrictions: All of food, alcohol, smoking, caffeine

s judgement: High ROB Support for judgement: Method
of participant recruitment not reported.No neutral control but steps taken to minimise participant stress.Missing data excluded from all analysis and not accounted for but no indication of differential loss related to prognostic factors.No published pre-specified protocol.

with pain ratings: Not analysed Sex effects: Not analysed NOTES Pain
ratings increased significantly immediately after CPT but were not affected by COMT polymorphism.The authors concluded that the COMT genotype influences the stress response to painful stimuli.