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Demonstration of the correlation between salivary cortisol concentration and post-operative healing times following nail avulsion
The Foot 16 (2006) 76–81
Demonstration of the correlation between salivary cortisol concentration and post-operative healing times following nail avulsion K.J. Davies ∗ Podiatry Surgical Services, Saint Andrew’s Hospital, Bromley-by-Bow, London, United Kingdom.
Abstract Twenty-eight subjects complaining of nail disorders were randomly selected over a period of three months. Each subject underwent either partial or a total nail avulsion with phenolisation under local anaesthesia. Subjects underwent weekly follow-up appointments to debride any eschar or callosity from the wound site. Following debridement post-operative healing was assessed. Post-operative healing was recorded as the period when there was no further discharge evident and there was normal healthy epidermis at the site of the wound. Saliva samples were collected from each subject. Salivary cortisol concentration was determined by radioimmunoassay of the samples. Three samples were collected for each subject, the first at the initial assessment, the second immediately prior to the administration of the local anaesthesia and the third 20 min after the second sample. Salivary cortisol concentration was compared with post-operative healing times. It was found that as the mean salivary cortisol concentration increased, there was an associated increase in the post-operative healing times following the nail surgery, r = 0.84. Cortisol in excess has been found to suppress immunological and anti-inflammatory responses. Increases in salivary cortisol concentrations may indicate a greater inflammatory response. It is likely that this response suppresses the post-operative healing times following nail surgery. © 2006 Elsevier Ltd. All rights reserved. Keywords: Salivary cortisol; Nail avulsion; Post-operative healing times; Phenolisation
1. Introduction
1.1. Physiology
The measurement of cortisol in saliva provides the scientist with a reliable tool for investigations of hypothalamic pituitary–adrenal axis (HPA) activity [1]. Cortisol is a corticosteroid [2]. It is a 21-carbon glucocorticoid secreted by the adrenal cortex [3]. It is not principally controlled by direct innervation but by the action of other hormones [2] in response to various biochemical agents and psychosocial stimuli [1]. Changes in the plasma concentration of adrenocorticotrophic hormone (ACTH), a peptide hormone released from the anterior pituitary gland, regulate the release of cortisol [2,4]. Once released into the circulation it has widespread effects on immune functioning and is a powerful anti-inflammatory. The aims of this study are to determine whether levels of cortisol produced may affect wound healing following nail surgery.
ACTH is regulated by the effect of a hypothalamic peptide, corticotrophin-releasing factor on the pituitary [4]. This system is referred to as the pituitary–adrenal cortex (PAC) axis [2,4]. Decreased output of cortisol results from negative feedback [3] as increased levels of cortisol inhibit the release of ACTH [4,5]. The HPA axis can respond sensitively to external stimulation. A variety of agents and treatments known as stressors are able to over-ride feedback systems [1]. Levels of glucocorticoid can vary considerably with psychological variables being among the most potent stimuli to release this hormone from the adrenal cortex [6]. Stress is a state of arousal usually brought about by exposure to stressors [4]. 1.2. Stress and stressors
∗
Correspondence to: 13, Locksley Close, Walderslade, Chatham, Kent ME5 9BT, United Kingdom. Tel. +44 7981 687 633. E-mail address: [email protected]. 0958-2592/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.foot.2005.12.005
There appears to be a bi-directional relationship between central nervous system, immune system and endocrine
K.J. Davies / The Foot 16 (2006) 76–81
system [7]. Primarily psychological coping with stressors induces emotions. The perception and expression of anxiety or experienced fear constitutes psychological stress [5]. Stress is associated with pituitary–adrenal cortical arousal [8]. The adrenocortical hormones maintain peripheral homeostasis and regulate cardiovascular, metabolic and immune functions during stress reactions. This is known as the ‘fight or flight’ response [5]. This response helps to increase readiness and performance of behaviour, which will ultimately augment the probability of survival. This is known as positive stress [5]. Cortisol levels are consistently elevated in acute situations, which are considered stressful [2]. 1.3. Factors affecting cortisol Environmental changes can either elevate or suppress the secretion of cortisol [9]. Blood cortisol is generally raised in human subjects exposed to unfamiliar situations that evoke uncertainty and anxiety [10]. Studies as early as 1960 found links between anxiety, fear and apprehension associated with dental surgery and stress [11]. Studies measuring cortisol levels in patients undergoing dental treatment found that anticipation of oral surgery produced a significant increase in cortisol levels compared to a control group [3]. This showed that apprehension could be a stimulus to adrenocortical secretion [3]. It is believed that emotional processing is the major trigger of cortisol secretion in response to stressful psychological stimulation [1]. The more novel uncontrollable or unpredictable the situation, the greater the hormone output [1]. Pituitary–adrenal activation can be shown to be associated with negative feelings of distress, and the adrenal cortisol suppression can be characterised by high controllability and predictability [9]. Trait anxiety, agitation and depression showed significant correlations with elevated cortisol [12]. Alternatively the cortisol response may be associated with levels of any kind of emotional arousal positive or negative [4]. 1.4. Physiological effects of cortisol levels Cortisol is often regarded as a stress hormone and may participate in the mechanism linking stress to disease [13]. Cortisol in excess has been found to suppress both immunological and inflammatory response [4]. Cortisol is known to impair protein synthesis on a molecular basis thus making it both immunosuppressive and anti-inflammatory [14]. Measurement of cortisol is considered an objective cross-culturally valid method to test the amount of stress experienced by individuals [4]. 1.5. Stress, cortisol and the immune system Stress may have an important role as a risk factor for some diseases or physiological processes [13]. If stress becomes chronic, harm can come to the individual, with increased
77
risk of psychosomatic disorders and can eventually lead to organic disease. This process is referred to as negative stress [5]. It is widely believed that chronically elevated levels of cortisol will lead to suppression of the immune system and therefore increased susceptibility to disease [2]. Psychological stressors can have stimulating effects on the immune system, such as increasing the number of T-cells, altering IgM concentration and the component C3. These effects on the immune system lead to immunosuppression [8]. Studies have shown that anxiety and depression can be associated with small but statistically significant cortisol elevation [12]. Therefore, even minor daily events and fluctuations in mood states may have an effect on health and well being [12]. 1.6. The advantages of salivary cortisol Salivary cortisol is a marker of psycho-physiological stress reactions [8]. Previously measurement of plasma cortisol required a number of venepuntures, which is a stressful event [15] and could change the cortisol levels being measured [16]. Investigations into cortisol levels in blood donors 10, 20 and 30 min after insertion of the catheter, found that those subjects who had donated blood for the first time showed the highest salivary cortisol levels [1]. This indicates that venepuncture has no true impact on the HPA axis but the anticipation of threatening or noxious events associated with the venopuncture is responsible for the psychoendocrine reaction observed. This may imply that with repeated exposure to the same psychological stimulation may gradually habituate the saliva cortisol response [1]. Measuring salivary cortisol is non-invasive and stress-free, so that samples can be taken as often as 10 min intervals, with little effect on the cortisol levels [17]. Using this technique cortisol levels were found to peak at 20 min after the stress of an injection [18]. Studies of cortisol responses to any treatment require an appropriate baseline value, which allow for conclusions as to what extent the subjects show increased adrenal activity following stimulation. Baseline control samples should be obtained one to three weeks before the stressful period [6].
2. Methodology Subjects were selected from those complaining of nail disorders within a three-month period. Subjects underwent a full assessment to determine their medical history, surgical history and drug history. Subjects excluded from the study were those taking any steroid-based medications, hormone medications, i.e., contraceptive pill and HRT, and those with Cushing’s syndrome. These may have affected the salivary cortisol levels. Any subjects with a disorder known to affect wound healing, such as diabetes, were also excluded. Subjects underwent a pre-surgical vascular assessment including Doppler indices as vascular insufficiency diminishes the rate
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of nail growth significantly [19]. Subjects with an anklebrachial index of less than one were excluded. 2.1. Demographic details Twenty-eight subjects, 13 males and 15 females, were selected for the purpose of the study. A total of 37 nails underwent a surgical procedure. The age range was 12–63 years with a mean age of 28 years. The age range for males was 12–36 years, mean age 13.6 years and for females it was 12–63 years, mean age 32.7 years. One subject failed to attend their surgical appointment and one subject was lost to follow-up appointments. One subject who had surgery on a total of four nails had one re-grow.
appointment was made for the following morning where the dressing was changed to a melolin and tube-gauze dressing. Subjects were instructed to bathe their feet daily in saline for a period of 5 min then to apply a melolin and tube-gauze dressing. Subjects were reviewed on a weekly basis to monitor healing and assess for any post-operative infection. In every second visit, callous and eschar were debrided from the nail bed as necessary using a surgical blade. Healing was defined as the time following the reduction of callous or eschar, where there was no further discharge from the toe and a normal healthy epidermis was noted over the nail bed. This was recorded as number of weeks post-operatively.
3. Results 2.2. Saliva collection Saliva was collected and stored using a ‘salivette’ collection tube, which consists of a small cotton swab and two plastic tubes. Saliva was obtained by instructing the subject to chew on the cotton swab for a period of 2 min. This was timed using a standard stop clock. The subject then removed the cotton swab from their mouth and placed it back in the ‘salivette.’ The samples were then frozen immediately. A total of three samples were obtained. The first sample was collected at the initial assessment in order to establish a baseline salivary cortisol level. The second sample was obtained immediately prior to surgery and the third sample 20 min after the second. The third sample was always taken post-surgery. Once all saliva samples were collected the salivettes were defrosted then centrifuged to extract the saliva from the swabs. Cortisol levels were determined by using radioimmunoassay kit. All samples were processed on the same day by the same operator. The results were not released from the laboratory until all the nails had been assessed for total healing time to avoid operator bias. 2.3. Surgical methodology All subjects underwent one of the two surgical procedures, partial or total nail avulsion with phenolisation. All procedures were performed under local anaesthesia. Anaesthesia was administered using Citanest (Prilocaine) 4% solution to perform a digital block to the affected digit. All anaesthetics and surgery were performed by the same practitioner to eliminate inter-practitioner errors. A digital ‘tournicot’ type tourniquet was applied to create a bloodless field. This prevents neutralisation of the phenol by blood or serum [20]. The tourniquet time did not exceed 6 min. The phenolisation time for all the digits was 3 min. The nail bed was dried to remove excess phenol but the phenol was not neutralised. 2.4. Post-operative care and advice Post-operatively the subject was instructed to rest and elevate the foot and to keep the dressing in situ. A return
Of the 28 subjects selected for this project, 26 (92.9%) underwent surgery and attended regular follow-up appointments to enable healing time to be assessed. Surgery was performed on a total of 37 nails. Fifty-nine percent of the procedures were performed on the right foot and 41% on the left foot. Of the 37 nails, 22 (59.5%) had total nail avulsion and 15 (40.5%) had partial nail avulsion. 3.1. Salivary cortisol levels Three salivary cortisol measurements were obtained for each subject. These were used to calculate the mean cortisol, the highest recorded cortisol and the cortisol peak. Cortisol peak was the difference between the highest and the lowest recorded cortisol measurements. Sample one was collected at the initial assessment clinic. Sample two was collected immediately prior to surgery. Sample three was collected 20 min after sample two. It was hypothesised that salivary cortisol concentrations for sample one would be the lowest as this was the baseline measurement. It was also hypothesised that sample three would be the highest as the salivary cortisol concentrations peak 20 min after the stressful event [1], in this case the surgery. To determine whether the data obtained supported this hypothesis, both one- and two-tailed t-tests were performed for related samples. It was observed that there were no significant differences in salivary cortisol concentrations between samples. The hypothesis was therefore rejected. 3.2. Incidence of salivary cortisol concentrations Mean salivary cortisol concentrations were divided into five sub-groups; very low, values of 0–5 nmol/l, values of 6–10 nmol/l, medium, 11–15 nmol/l, high, 16–20 nmol/l and very high, 21–25 nmol/l. Values were plotted against frequency as shown on Graph 1. Fourteen subjects (53.8%) had low or very low cortisol levels, over half of the population. The frequency decreases as salivary cortisol concentrations
K.J. Davies / The Foot 16 (2006) 76–81
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4. Discussion 4.1. Salivary cortisol measurement
Graph 1. The incidence of salivary cortisol levels.
Graph 2. The frequency of healing times.
increase. Mean cortisol concentration 10.8 nmol/l, standard deviation 4.4 nmol/l. 3.3. Post-operative healing times Healing times were assessed as the number of weeks postoperatively that healing took to occur. Post-operative healing times varied from 2 to 8 weeks, mean 4.8 weeks, standard deviation 1.8 weeks. The frequency of healing times is shown on Graph 2. 3.4. Salivary cortisol and healing times The hypothesis of this study was that there was a direct correlation between salivary cortisol concentration and postoperative healing times. Regression values were obtained for healing times against mean cortisol, cortisol peak and against the highest recorded cortisol value for each subject. There was found to be a significant correlation between mean salivary cortisol concentration and healing time r = 0.84 (see Graph 3). There was no significant correlation between cortisol peak and post-operative healing times.
Graph 3. Mean salivary cortisol and healing time.
When the design of the study was outlined, it was hypothesised that collecting the salivary cortisol samples at the initial assessment would provide suitable baseline cortisol concentrations for each subject. The surgical procedure was considered the most stressful event so cortisol levels were expected to rise prior to surgery and to peak 20 min after the maximal point of stress [1]. The maximum point of stress was taken to be the administration of local anaesthetic. However, approximately 30% of the salivary cortisol concentrations were found to be highest at initial assessment, 22% were high immediately prior to surgery and only 48% were highest 20 min after the anaesthetic. The standard deviation was found to be the largest at the initial assessment indicating that this was when the greatest variance of salivary cortisol concentrations occurred. This may be partially explained as some subjects were observed to be apprehensive at the initial assessment. Some were expecting to undergo surgery at that appointment. Many were unsure of the new situation that they faced. It can be concluded that the initial assessment clinic may be considered a stressful situation for some subjects and it is, therefore, not an appropriate time to collect baseline salivary cortisol concentration measurements. To obtain appropriate baseline cortisol, saliva samples would be taken at each of the follow-up redressing appointments and a mean baseline cortisol could be calculated. 4.2. Salivary cortisol in direct relation to healing There was found to be a significant correlation between mean salivary cortisol concentrations and post-operative healing times, r = 0.84. As post-operative healing times were significantly higher for surgery to the hallux in comparison to surgeries to the lesser toes, P = 0.02, it was hypothesised that the correlation between post-operative healing times and salivary cortisol concentration would be stronger if the lesser toe surgeries were excluded. However, this was not found to be the case. There were also no significant differences for gender. There was no significant correlation between cortisol peak and post-operative healing times. This was probably due to the inappropriate timing of the baseline samples. If this study was repeated using mean baseline cortisol concentrations measured at appropriate times, for example, at the followup redressing appointments, then a more appropriate cortisol peak could be calculated. There was also no significant correlation between the highest recorded salivary cortisol concentration and postoperative healing times but there was evidence of a trend. However, as these results are relying on one single cortisol measurement it cannot be considered a very reliable method of representing the highest cortisol measurement. Salivary
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cortisol samples are very easy to obtain. As salivary cortisol concentration is not dependent on the flow rate (with minimal trauma to the subject) [10,17], saliva samples could be collected at 5 min intervals pre- and post-operatively. These figures could be plotted to enable accurate determination of the highest cortisol concentration levels. This may also lead to a more accurate determination of post-operative peak salivary cortisol concentrations especially in association with appropriate baseline measurements. 4.3. Perception of stress An individual’s perception of a stressful situation is detrimental to the trigger of cortisol secretion in response to stressful psychological stimulation [1]. Blood cortisol is generally raised in human subjects exposed to unfamiliar situations that evoke uncertainty and anxiety [10]. However, despite subjects perceiving psychological stress and presenting activation of HPA axis activity, Canals et al. [13] found that there was no significant rise in cortisol levels in relation to perceived stress. This was found to be true in the clinical setting of this study. Subjects undergoing nail surgery that showed excessive stress pre- and peri-operatively were noted. It was noted that cortisol levels were not significantly higher than nonagitated subjects who appeared to be exhibiting less signs of stress. A personality questionnaire could be devised to assess personality type and perceived stress. This could be used to determine whether there is a true correlation between perceived stress and cortisol levels. Actual stressful events are associated with increased cortisol secretion, the magnitude of the effect depending on whether the event is still ongoing and how frequently a similar event may have occurred [12]. A study by Kirschbaum and Hellhammer [1] found that venopuncture has no HPA axis impact. It was personality traits like anxiety or sensitisation that may lead to the reactive endocrine responses. Anticipation of threatening or noxious events associated with venopuncture is responsible for the psychoendocrine reaction observed. After termination of the stress exposure, cortisol levels typically return to baseline levels within 1–2 h [12]. 4.4. Habituation Several of the subjects reported having experiences of past surgery for similar nail pathologies. Some experiences were associated with positive emotions and others negative emotions. Some subjects had experiences of multiple exposures to venopuncture or the administration of local anaesthesia and had varying psychological responses to the surgery. With repeated exposure to the same psychological stimuli initial cortisol response will gradually habituate [1]. Recurrent events were found to have effects on cortisol production. The more novel the event the higher the cortisol response suggesting that habituation can occur [12]. Habituation to recurrent stressors is less likely in individuals with high perceived stress, trait anxiety or depression [12]. Habituation
may, therefore, bias real stress responses and lead to misinterpretation of results [22]. 5. Conclusion Glucocorticoids have an inhibitory effect on the healing and production of tissues [21]. Cortisol in excess has been found to suppress both immunological and inflammatory responses [2]. Cortisol is known to impair protein synthesis on a molecular basis making it immunosuppressive and antiinflammatory [14]. If elevated cortisol concentrations can be significantly related to an increase in post-operative healing times, then they must also be related to other systemic problems related to the inflammatory response. Salivary cortisol concentration was found to be correlated with post-operative healing times. This correlation was not affected by the age or the gender of the subject. As salivary cortisol concentrations increased the post-operative healing time was also found to increase. Cortisol is often regarded as a stress hormone. For the purpose of this study, one acute episode of stress, marked by the surgical procedure, was presumed to be linked to an acute rise in salivary cortisol concentration in response to stress. But, as baseline cortisol concentrations were not appropriately established, it may be that these elevated cortisol concentrations were linked to chronic stress. The high cortisol concentrations found in some subjects may be normal for these subjects. It may be that these levels were associated with chronic stress. Chronic stress would lead to more constantly elevated levels of salivary cortisol. These subjects may have such high baseline cortisol concentrations that they only exhibit a small rise in salivary cortisol concentration in response to the surgical stressor. Even if the increase in cortisol concentrations was associated with acute stress, subjects who lead a stressful lifestyle may regularly reach such elevated cortisol levels on a daily basis. If subjects regularly demonstrate such chronically elevated levels of cortisol then it may not be just the postoperative healing times, which are affected. There may also be an affect on any other systemic disease, which is linked to the inflammatory response. If this is the case then chronically elevated cortisol concentrations could be an indicator of more serious long-term health problems. If further research is carried out on the implications of elevated cortisol concentration levels, healing times and the effect on health and well-being, then it may be possible to predict the onset of certain diseases. If this is the case then salivary cortisol may be used as a screening device. It is, therefore, essential that long-term research be carried out on the chronic effects of elevated salivary cortisol concentrations and disease. Acknowledgements Ethical approval was obtained through East London and the City Health Authority, Research Ethics Committee.
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References [1] Kirschbaum C, Hellhammer DH. Salivary cortisol in psychological research: an overview. Neuropsychology 1989;22:150–69. [2] Pollard TM. Physiological consequences of everyday psychological stress. Coll Anthropol 1997;21:17–28. [3] Miller CS, Dembo JB, Falace DA, Kaplan AL, Ky L. Salivary cortisol responses to dental treatment of varying stress. Oral Surg Oral Med Oral Pathol 1995;79(4):436–41. [4] Pollard TM. Use of cortisol as a stress marker: practical and theoretical problems. Am J Human Biol 1995;7:265–74. [5] De La Torre B. Psychoendocrinologic mechanisms of life stress. Stress Med 1994;10:107–14. [6] Kirschbaum C, Strasburger CJ, Jammers W, Hellhammer DH. Cortisol and behaviour: 1. Adaptation of a radioimmunoassay kit for reliable and inexpensive salivary cortisol determination. Pharmacol Biochem Behav 1989;34:747–51. [7] Henning J, Netter P. Local immunocompetence and salivary cortisol in confinement. Adv Space Biol Med 1996;5:115–32. [8] Zeier H, Brauchli P, Joller-Jemelka HI. Effects of work demands on immunoglobulin A and cortisol in air traffic controllers. Biol Psychol 1996;42:413–23. [9] Lunberg U, Frankenhaeuser M. Pituitary–adrenal and sympathetic– adrenal correlates of distress and effort. J Psychosom Res 1980;24:124–30. [10] Burton RF, Hinton JW, Neilson E, Beastall G. Concentrations of sodium, potassium and cortisol in saliva, and self reported chronic work stress factors. Biol Psychol 1996;42:425–38. [11] Ship II, White CL. Physiologic response to dental stress. Oral Surg Oral Med Oral Pathol 1960;13(3):363–76.
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[12] Eck MV, Berkhof H, Nicolson N, Sulon J. The effects of perceived stress, traits, mood states and daily stressful events on salivary cortisol. Psychosom Med 1996;58:447–58. [13] Canals J, Colomina MT, Gallart I, Domingo JL. Stressful events and salivary cortisol. Psychol Rep 1997;80:305–6. [14] Weicker H, Werle E. Internation between hormones and the immune system. Int J Sports Med 1991;12:S30–7. [15] Kahn JP, Rubinow DR, Davis CL, Kling M, Post RM. Salivary cortisol: a practical method for evaluation of adrenal function. Biol Psychiatry 1998;23:335–49. [16] Woolston JL, Gianfreidi S, Gertner JM, Paugus JA, Mason JW. Salivary cortisol: a non-traumatic sampling technique for assaying cortisol dynamics. J Am Acad Child Psychiatry 1983;22(5): 474–6. [17] Riad-Fahmy D, Read GF, Walker RF, Griffiths K. Steroids and saliva for assessing endocrine function. Endocr Rev 1982;3(4):367– 95. [18] Hiramatsu R. Direct assay of cortisol in human saliva by solid phase radioimmunoassay and its clinical applications. Clin Chim Acta 1981;117:239–49. [19] Bean WB. Nail growth: 30 years of observation. Arch Intern Med 1974;134:497–501. [20] Byrne DS, Caldwell D. Phenol cauterisation for ingrowing toenails: a review of five years experience. Br J Surg 1989;76:598–9. [21] Woolfe N. Cells, tissue and diseases: the basis of pathology. 2nd ed. Balliere Tindall; 1986. [22] Beerda A, Schilder MBH, Janssen NSCRM, Mol JA. The use of saliva cortisol, urinary cortisol and catecholamine measurement for non-invasive assessment of stress responses in dogs. Horm Behav 1996;30:272–9.
Demonstration of the correlation between salivary cortisol concentration and post-operative healing times following nail avulsion K.J. Davies ∗ Podiatry Surgical Services, Saint Andrew’s Hospital, Bromley-by-Bow, London, United Kingdom.
Abstract Twenty-eight subjects complaining of nail disorders were randomly selected over a period of three months. Each subject underwent either partial or a total nail avulsion with phenolisation under local anaesthesia. Subjects underwent weekly follow-up appointments to debride any eschar or callosity from the wound site. Following debridement post-operative healing was assessed. Post-operative healing was recorded as the period when there was no further discharge evident and there was normal healthy epidermis at the site of the wound. Saliva samples were collected from each subject. Salivary cortisol concentration was determined by radioimmunoassay of the samples. Three samples were collected for each subject, the first at the initial assessment, the second immediately prior to the administration of the local anaesthesia and the third 20 min after the second sample. Salivary cortisol concentration was compared with post-operative healing times. It was found that as the mean salivary cortisol concentration increased, there was an associated increase in the post-operative healing times following the nail surgery, r = 0.84. Cortisol in excess has been found to suppress immunological and anti-inflammatory responses. Increases in salivary cortisol concentrations may indicate a greater inflammatory response. It is likely that this response suppresses the post-operative healing times following nail surgery. © 2006 Elsevier Ltd. All rights reserved. Keywords: Salivary cortisol; Nail avulsion; Post-operative healing times; Phenolisation
1. Introduction
1.1. Physiology
The measurement of cortisol in saliva provides the scientist with a reliable tool for investigations of hypothalamic pituitary–adrenal axis (HPA) activity [1]. Cortisol is a corticosteroid [2]. It is a 21-carbon glucocorticoid secreted by the adrenal cortex [3]. It is not principally controlled by direct innervation but by the action of other hormones [2] in response to various biochemical agents and psychosocial stimuli [1]. Changes in the plasma concentration of adrenocorticotrophic hormone (ACTH), a peptide hormone released from the anterior pituitary gland, regulate the release of cortisol [2,4]. Once released into the circulation it has widespread effects on immune functioning and is a powerful anti-inflammatory. The aims of this study are to determine whether levels of cortisol produced may affect wound healing following nail surgery.
ACTH is regulated by the effect of a hypothalamic peptide, corticotrophin-releasing factor on the pituitary [4]. This system is referred to as the pituitary–adrenal cortex (PAC) axis [2,4]. Decreased output of cortisol results from negative feedback [3] as increased levels of cortisol inhibit the release of ACTH [4,5]. The HPA axis can respond sensitively to external stimulation. A variety of agents and treatments known as stressors are able to over-ride feedback systems [1]. Levels of glucocorticoid can vary considerably with psychological variables being among the most potent stimuli to release this hormone from the adrenal cortex [6]. Stress is a state of arousal usually brought about by exposure to stressors [4]. 1.2. Stress and stressors
∗
Correspondence to: 13, Locksley Close, Walderslade, Chatham, Kent ME5 9BT, United Kingdom. Tel. +44 7981 687 633. E-mail address: [email protected]. 0958-2592/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.foot.2005.12.005
There appears to be a bi-directional relationship between central nervous system, immune system and endocrine
K.J. Davies / The Foot 16 (2006) 76–81
system [7]. Primarily psychological coping with stressors induces emotions. The perception and expression of anxiety or experienced fear constitutes psychological stress [5]. Stress is associated with pituitary–adrenal cortical arousal [8]. The adrenocortical hormones maintain peripheral homeostasis and regulate cardiovascular, metabolic and immune functions during stress reactions. This is known as the ‘fight or flight’ response [5]. This response helps to increase readiness and performance of behaviour, which will ultimately augment the probability of survival. This is known as positive stress [5]. Cortisol levels are consistently elevated in acute situations, which are considered stressful [2]. 1.3. Factors affecting cortisol Environmental changes can either elevate or suppress the secretion of cortisol [9]. Blood cortisol is generally raised in human subjects exposed to unfamiliar situations that evoke uncertainty and anxiety [10]. Studies as early as 1960 found links between anxiety, fear and apprehension associated with dental surgery and stress [11]. Studies measuring cortisol levels in patients undergoing dental treatment found that anticipation of oral surgery produced a significant increase in cortisol levels compared to a control group [3]. This showed that apprehension could be a stimulus to adrenocortical secretion [3]. It is believed that emotional processing is the major trigger of cortisol secretion in response to stressful psychological stimulation [1]. The more novel uncontrollable or unpredictable the situation, the greater the hormone output [1]. Pituitary–adrenal activation can be shown to be associated with negative feelings of distress, and the adrenal cortisol suppression can be characterised by high controllability and predictability [9]. Trait anxiety, agitation and depression showed significant correlations with elevated cortisol [12]. Alternatively the cortisol response may be associated with levels of any kind of emotional arousal positive or negative [4]. 1.4. Physiological effects of cortisol levels Cortisol is often regarded as a stress hormone and may participate in the mechanism linking stress to disease [13]. Cortisol in excess has been found to suppress both immunological and inflammatory response [4]. Cortisol is known to impair protein synthesis on a molecular basis thus making it both immunosuppressive and anti-inflammatory [14]. Measurement of cortisol is considered an objective cross-culturally valid method to test the amount of stress experienced by individuals [4]. 1.5. Stress, cortisol and the immune system Stress may have an important role as a risk factor for some diseases or physiological processes [13]. If stress becomes chronic, harm can come to the individual, with increased
77
risk of psychosomatic disorders and can eventually lead to organic disease. This process is referred to as negative stress [5]. It is widely believed that chronically elevated levels of cortisol will lead to suppression of the immune system and therefore increased susceptibility to disease [2]. Psychological stressors can have stimulating effects on the immune system, such as increasing the number of T-cells, altering IgM concentration and the component C3. These effects on the immune system lead to immunosuppression [8]. Studies have shown that anxiety and depression can be associated with small but statistically significant cortisol elevation [12]. Therefore, even minor daily events and fluctuations in mood states may have an effect on health and well being [12]. 1.6. The advantages of salivary cortisol Salivary cortisol is a marker of psycho-physiological stress reactions [8]. Previously measurement of plasma cortisol required a number of venepuntures, which is a stressful event [15] and could change the cortisol levels being measured [16]. Investigations into cortisol levels in blood donors 10, 20 and 30 min after insertion of the catheter, found that those subjects who had donated blood for the first time showed the highest salivary cortisol levels [1]. This indicates that venepuncture has no true impact on the HPA axis but the anticipation of threatening or noxious events associated with the venopuncture is responsible for the psychoendocrine reaction observed. This may imply that with repeated exposure to the same psychological stimulation may gradually habituate the saliva cortisol response [1]. Measuring salivary cortisol is non-invasive and stress-free, so that samples can be taken as often as 10 min intervals, with little effect on the cortisol levels [17]. Using this technique cortisol levels were found to peak at 20 min after the stress of an injection [18]. Studies of cortisol responses to any treatment require an appropriate baseline value, which allow for conclusions as to what extent the subjects show increased adrenal activity following stimulation. Baseline control samples should be obtained one to three weeks before the stressful period [6].
2. Methodology Subjects were selected from those complaining of nail disorders within a three-month period. Subjects underwent a full assessment to determine their medical history, surgical history and drug history. Subjects excluded from the study were those taking any steroid-based medications, hormone medications, i.e., contraceptive pill and HRT, and those with Cushing’s syndrome. These may have affected the salivary cortisol levels. Any subjects with a disorder known to affect wound healing, such as diabetes, were also excluded. Subjects underwent a pre-surgical vascular assessment including Doppler indices as vascular insufficiency diminishes the rate
78
K.J. Davies / The Foot 16 (2006) 76–81
of nail growth significantly [19]. Subjects with an anklebrachial index of less than one were excluded. 2.1. Demographic details Twenty-eight subjects, 13 males and 15 females, were selected for the purpose of the study. A total of 37 nails underwent a surgical procedure. The age range was 12–63 years with a mean age of 28 years. The age range for males was 12–36 years, mean age 13.6 years and for females it was 12–63 years, mean age 32.7 years. One subject failed to attend their surgical appointment and one subject was lost to follow-up appointments. One subject who had surgery on a total of four nails had one re-grow.
appointment was made for the following morning where the dressing was changed to a melolin and tube-gauze dressing. Subjects were instructed to bathe their feet daily in saline for a period of 5 min then to apply a melolin and tube-gauze dressing. Subjects were reviewed on a weekly basis to monitor healing and assess for any post-operative infection. In every second visit, callous and eschar were debrided from the nail bed as necessary using a surgical blade. Healing was defined as the time following the reduction of callous or eschar, where there was no further discharge from the toe and a normal healthy epidermis was noted over the nail bed. This was recorded as number of weeks post-operatively.
3. Results 2.2. Saliva collection Saliva was collected and stored using a ‘salivette’ collection tube, which consists of a small cotton swab and two plastic tubes. Saliva was obtained by instructing the subject to chew on the cotton swab for a period of 2 min. This was timed using a standard stop clock. The subject then removed the cotton swab from their mouth and placed it back in the ‘salivette.’ The samples were then frozen immediately. A total of three samples were obtained. The first sample was collected at the initial assessment in order to establish a baseline salivary cortisol level. The second sample was obtained immediately prior to surgery and the third sample 20 min after the second. The third sample was always taken post-surgery. Once all saliva samples were collected the salivettes were defrosted then centrifuged to extract the saliva from the swabs. Cortisol levels were determined by using radioimmunoassay kit. All samples were processed on the same day by the same operator. The results were not released from the laboratory until all the nails had been assessed for total healing time to avoid operator bias. 2.3. Surgical methodology All subjects underwent one of the two surgical procedures, partial or total nail avulsion with phenolisation. All procedures were performed under local anaesthesia. Anaesthesia was administered using Citanest (Prilocaine) 4% solution to perform a digital block to the affected digit. All anaesthetics and surgery were performed by the same practitioner to eliminate inter-practitioner errors. A digital ‘tournicot’ type tourniquet was applied to create a bloodless field. This prevents neutralisation of the phenol by blood or serum [20]. The tourniquet time did not exceed 6 min. The phenolisation time for all the digits was 3 min. The nail bed was dried to remove excess phenol but the phenol was not neutralised. 2.4. Post-operative care and advice Post-operatively the subject was instructed to rest and elevate the foot and to keep the dressing in situ. A return
Of the 28 subjects selected for this project, 26 (92.9%) underwent surgery and attended regular follow-up appointments to enable healing time to be assessed. Surgery was performed on a total of 37 nails. Fifty-nine percent of the procedures were performed on the right foot and 41% on the left foot. Of the 37 nails, 22 (59.5%) had total nail avulsion and 15 (40.5%) had partial nail avulsion. 3.1. Salivary cortisol levels Three salivary cortisol measurements were obtained for each subject. These were used to calculate the mean cortisol, the highest recorded cortisol and the cortisol peak. Cortisol peak was the difference between the highest and the lowest recorded cortisol measurements. Sample one was collected at the initial assessment clinic. Sample two was collected immediately prior to surgery. Sample three was collected 20 min after sample two. It was hypothesised that salivary cortisol concentrations for sample one would be the lowest as this was the baseline measurement. It was also hypothesised that sample three would be the highest as the salivary cortisol concentrations peak 20 min after the stressful event [1], in this case the surgery. To determine whether the data obtained supported this hypothesis, both one- and two-tailed t-tests were performed for related samples. It was observed that there were no significant differences in salivary cortisol concentrations between samples. The hypothesis was therefore rejected. 3.2. Incidence of salivary cortisol concentrations Mean salivary cortisol concentrations were divided into five sub-groups; very low, values of 0–5 nmol/l, values of 6–10 nmol/l, medium, 11–15 nmol/l, high, 16–20 nmol/l and very high, 21–25 nmol/l. Values were plotted against frequency as shown on Graph 1. Fourteen subjects (53.8%) had low or very low cortisol levels, over half of the population. The frequency decreases as salivary cortisol concentrations
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4. Discussion 4.1. Salivary cortisol measurement
Graph 1. The incidence of salivary cortisol levels.
Graph 2. The frequency of healing times.
increase. Mean cortisol concentration 10.8 nmol/l, standard deviation 4.4 nmol/l. 3.3. Post-operative healing times Healing times were assessed as the number of weeks postoperatively that healing took to occur. Post-operative healing times varied from 2 to 8 weeks, mean 4.8 weeks, standard deviation 1.8 weeks. The frequency of healing times is shown on Graph 2. 3.4. Salivary cortisol and healing times The hypothesis of this study was that there was a direct correlation between salivary cortisol concentration and postoperative healing times. Regression values were obtained for healing times against mean cortisol, cortisol peak and against the highest recorded cortisol value for each subject. There was found to be a significant correlation between mean salivary cortisol concentration and healing time r = 0.84 (see Graph 3). There was no significant correlation between cortisol peak and post-operative healing times.
Graph 3. Mean salivary cortisol and healing time.
When the design of the study was outlined, it was hypothesised that collecting the salivary cortisol samples at the initial assessment would provide suitable baseline cortisol concentrations for each subject. The surgical procedure was considered the most stressful event so cortisol levels were expected to rise prior to surgery and to peak 20 min after the maximal point of stress [1]. The maximum point of stress was taken to be the administration of local anaesthetic. However, approximately 30% of the salivary cortisol concentrations were found to be highest at initial assessment, 22% were high immediately prior to surgery and only 48% were highest 20 min after the anaesthetic. The standard deviation was found to be the largest at the initial assessment indicating that this was when the greatest variance of salivary cortisol concentrations occurred. This may be partially explained as some subjects were observed to be apprehensive at the initial assessment. Some were expecting to undergo surgery at that appointment. Many were unsure of the new situation that they faced. It can be concluded that the initial assessment clinic may be considered a stressful situation for some subjects and it is, therefore, not an appropriate time to collect baseline salivary cortisol concentration measurements. To obtain appropriate baseline cortisol, saliva samples would be taken at each of the follow-up redressing appointments and a mean baseline cortisol could be calculated. 4.2. Salivary cortisol in direct relation to healing There was found to be a significant correlation between mean salivary cortisol concentrations and post-operative healing times, r = 0.84. As post-operative healing times were significantly higher for surgery to the hallux in comparison to surgeries to the lesser toes, P = 0.02, it was hypothesised that the correlation between post-operative healing times and salivary cortisol concentration would be stronger if the lesser toe surgeries were excluded. However, this was not found to be the case. There were also no significant differences for gender. There was no significant correlation between cortisol peak and post-operative healing times. This was probably due to the inappropriate timing of the baseline samples. If this study was repeated using mean baseline cortisol concentrations measured at appropriate times, for example, at the followup redressing appointments, then a more appropriate cortisol peak could be calculated. There was also no significant correlation between the highest recorded salivary cortisol concentration and postoperative healing times but there was evidence of a trend. However, as these results are relying on one single cortisol measurement it cannot be considered a very reliable method of representing the highest cortisol measurement. Salivary
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cortisol samples are very easy to obtain. As salivary cortisol concentration is not dependent on the flow rate (with minimal trauma to the subject) [10,17], saliva samples could be collected at 5 min intervals pre- and post-operatively. These figures could be plotted to enable accurate determination of the highest cortisol concentration levels. This may also lead to a more accurate determination of post-operative peak salivary cortisol concentrations especially in association with appropriate baseline measurements. 4.3. Perception of stress An individual’s perception of a stressful situation is detrimental to the trigger of cortisol secretion in response to stressful psychological stimulation [1]. Blood cortisol is generally raised in human subjects exposed to unfamiliar situations that evoke uncertainty and anxiety [10]. However, despite subjects perceiving psychological stress and presenting activation of HPA axis activity, Canals et al. [13] found that there was no significant rise in cortisol levels in relation to perceived stress. This was found to be true in the clinical setting of this study. Subjects undergoing nail surgery that showed excessive stress pre- and peri-operatively were noted. It was noted that cortisol levels were not significantly higher than nonagitated subjects who appeared to be exhibiting less signs of stress. A personality questionnaire could be devised to assess personality type and perceived stress. This could be used to determine whether there is a true correlation between perceived stress and cortisol levels. Actual stressful events are associated with increased cortisol secretion, the magnitude of the effect depending on whether the event is still ongoing and how frequently a similar event may have occurred [12]. A study by Kirschbaum and Hellhammer [1] found that venopuncture has no HPA axis impact. It was personality traits like anxiety or sensitisation that may lead to the reactive endocrine responses. Anticipation of threatening or noxious events associated with venopuncture is responsible for the psychoendocrine reaction observed. After termination of the stress exposure, cortisol levels typically return to baseline levels within 1–2 h [12]. 4.4. Habituation Several of the subjects reported having experiences of past surgery for similar nail pathologies. Some experiences were associated with positive emotions and others negative emotions. Some subjects had experiences of multiple exposures to venopuncture or the administration of local anaesthesia and had varying psychological responses to the surgery. With repeated exposure to the same psychological stimuli initial cortisol response will gradually habituate [1]. Recurrent events were found to have effects on cortisol production. The more novel the event the higher the cortisol response suggesting that habituation can occur [12]. Habituation to recurrent stressors is less likely in individuals with high perceived stress, trait anxiety or depression [12]. Habituation
may, therefore, bias real stress responses and lead to misinterpretation of results [22]. 5. Conclusion Glucocorticoids have an inhibitory effect on the healing and production of tissues [21]. Cortisol in excess has been found to suppress both immunological and inflammatory responses [2]. Cortisol is known to impair protein synthesis on a molecular basis making it immunosuppressive and antiinflammatory [14]. If elevated cortisol concentrations can be significantly related to an increase in post-operative healing times, then they must also be related to other systemic problems related to the inflammatory response. Salivary cortisol concentration was found to be correlated with post-operative healing times. This correlation was not affected by the age or the gender of the subject. As salivary cortisol concentrations increased the post-operative healing time was also found to increase. Cortisol is often regarded as a stress hormone. For the purpose of this study, one acute episode of stress, marked by the surgical procedure, was presumed to be linked to an acute rise in salivary cortisol concentration in response to stress. But, as baseline cortisol concentrations were not appropriately established, it may be that these elevated cortisol concentrations were linked to chronic stress. The high cortisol concentrations found in some subjects may be normal for these subjects. It may be that these levels were associated with chronic stress. Chronic stress would lead to more constantly elevated levels of salivary cortisol. These subjects may have such high baseline cortisol concentrations that they only exhibit a small rise in salivary cortisol concentration in response to the surgical stressor. Even if the increase in cortisol concentrations was associated with acute stress, subjects who lead a stressful lifestyle may regularly reach such elevated cortisol levels on a daily basis. If subjects regularly demonstrate such chronically elevated levels of cortisol then it may not be just the postoperative healing times, which are affected. There may also be an affect on any other systemic disease, which is linked to the inflammatory response. If this is the case then chronically elevated cortisol concentrations could be an indicator of more serious long-term health problems. If further research is carried out on the implications of elevated cortisol concentration levels, healing times and the effect on health and well-being, then it may be possible to predict the onset of certain diseases. If this is the case then salivary cortisol may be used as a screening device. It is, therefore, essential that long-term research be carried out on the chronic effects of elevated salivary cortisol concentrations and disease. Acknowledgements Ethical approval was obtained through East London and the City Health Authority, Research Ethics Committee.
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