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Journal of Psychiatric Research 40 (2006) 550567

JOURNAL OF PSYCHIATRIC RESEARCH


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Review

Assessment of HPA-axis function in posttraumatic stress disorder: Pharmacological and non-pharmacological challenge tests, a review
C.S. de Kloet
a

a,b,*

, E. Vermetten a,b, E. Geuze a,b, A. Kavelaars c, C.J. Heijnen c, H.G.M. Westenberg b

Department of Military Psychiatry, Central Military Hospital, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands b Rudolf Magnus Institute of Neurosciences, Division Psychiatry, Utrecht, The Netherlands c Laboratory of Psychoneuroimmunology, University Medical Center Utrecht, Utrecht, The Netherlands Received 3 June 2005; received in revised form 15 July 2005

Abstract Posttraumatic stress disorder (PTSD) is typically accompanied by acute and chronic alterations in the stress response. These alterations have mostly been described in individuals under baseline conditions, but several studies have also used a challenge model to further assess the role of the hypothalamicpituitaryadrenal (HPA) axis in the stress response. This paper reviews common methodology and research ndings on HPA function in PTSD, and discusses the pathophysiological mechanisms underlying these ndings. We reviewed the literature and selected all English-language, human subject, data driven, pharmacological and non-pharmacological challenge studies pertaining to the HPA axis, and in vitro leukocyte glucocorticoid receptor studies in adult PTSD subjects. Studies using a non-pharmacological stress paradigm (cognitive stress, trauma reminders) to stimulate the HPA axis showed an exaggerated cortisol response in PTSD. The most widely used pharmacological challenge with consistent results was the low dose dexamethasone-suppression test (DST). These DST studies showed enhanced cortisol suppression in subjects with PTSD. Dierent hypotheses have been purported to explain the alterations in HPA axis functioning in PTSD. The results of the reviewed challenge tests, however, did not exclusively support one of the hypothesized mechanisms. Further research assessing hormones at all levels of the HPA axis at both baseline and at challenge conditions with a proper stratication of study population, will be necessary for a better understanding of stress-responsivity on the level of the HPA axis in PTSD. 2005 Elsevier Ltd. All rights reserved.
Keywords: PTSD; HPA; Glucocorticoid; Cortisol; DST; Review

Contents 1. 2. 3. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methodology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Non-Pharmacological stress challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. CRH challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Naloxone challenge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. ACTH challenge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551 551 552 552 553 553 553

Corresponding author. Tel.: +3130 2502 590/124; fax: +3130 250 2586. E-mail address: C.S.deKloet@azu.nl (C.S. de Kloet).

0022-3956/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jpsychires.2005.08.002

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3.5. Dexamethasone suppression test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. DexamethasoneCRH challenge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7. Metyrapone challenges. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8. Glucocorticoid receptor studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Hypothesized mechanism for altered HPA-axis function in PTSD . . . . . . . . . . . . . . . . . . . . 4.2. Other factors involved in HPA-axis regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Confounding factors and limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Recommendations for future research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Posttraumatic stress disorder (PTSD) is a chronic psychiatric disorder that can occur in subjects who have been exposed to or have witnessed a traumatic experience of an extreme nature (APA, 1994). Since PTSD develops after exposure to a stressor and symptoms include hyperarousal, it seems likely that PTSD is accompanied by or will lead to alterations in the biological stress response. The stress response refers to the bodys immediate and long-term reaction to a physical or psychological stressor that endangers homeostasis. Preclinical studies have given insight in the relative contribution of the sympathetic-adrenal-medullary- and the hypothalamicpituitaryadrenal (HPA) axis to the regulation of the stress response. The work in animal models of stress has shown that coordinated functional interactions between the HPA axis, and sympathetic-adrenal-medullary system are critical in promoting adaptive responses to stress, anxiety, or fear (McEwen, 1979; Sapolsky et al., 1990). For review (Bremner et al., 1996 and Vermetten and Bremner, 2002). The ndings in preclinical literature has led to a rapid expansion of studies pertaining to these systems in patients with PTSD in the past decade. HPA axis reactivity especially has been thoroughly investigated. Baseline studies in adult PTSD patients report high levels of corticotrophin releasing hormone (CRH) in cerebrospinal uid (CSF) (Baker et al., 1999; Bremner et al., 1997), while diurnal plasma cortisol levels on average are decreased. (Bremner et al., 2003a; Yehuda et al., 1994). Single point plasma and 24 h urine cortisol measurements, however, reveal mixed results. Lower plasma and 24 h urine cortisol levels have been reported in some (Mason et al., 1986; Yehuda et al., 1990; Yehuda et al., 1995a,b; Kanter et al., 2001; Glover and Poland, 2002; Rohleder et al., 2004) but not in other studies (Pitman and Orr, 1990; Lemieux and Coe, 1995; Maes et al., 1998; Thaller et al., 1999). The combination of high CRH in CSF and lower baseline or diurnal plasma cortisol levels have often been attributed to an enhanced HPA-axis feedback reg-

ulation in PTSD. Other mechanisms that might be involved are an altered perception of stressors, alterations in activity of the central nervous system (CNS), alterations in hormone bioavailability, and hormone receptor function. To further investigate the stress response in PTSD patients, several challenge paradigms, targeting at different levels of the axis, have been applied. In this paper, an overview of the ndings on non-pharmacological challenges and dierent pharmacological challenges, including the dexamethasone suppression test (DST), ACTH-, CRH-, and naloxone challenges, dexamethasone CRH test and metyrapone challenge designs is provided. A special paragraph is devoted to leukocyte glucocorticoid receptor studies, since in vitro leukocyte GR studies have been used as a surrogate measure for central HPA axis feedback regulation, and might thereby contribute to an understanding of the ndings of the reviewed challenge studies in this review. In the discussion the reported alterations will be discussed in view of clinical and preclinical ndings.

2. Methodology We performed a Medline Indexed search with the keywords PTSD in combination with HPA axis, dexamethasone, cortisol, ACTH, CRH, metyrapone, or glucocorticoid receptor. From this database English-language, human subject, data driven papers were selected. From these studies, we selected all pharmacological and non-pharmacological challenge studies pertaining to the HPA-axis, and glucocorticoid receptors in adult PTSD populations. As the outcome of studies in PTSD assessing baseline levels of hormones involved in HPA axis regulation have already been reviewed, studies that exclusively assessed baseline levels were excluded from this review. Thirty-four studies performed in PTSD patients have been reviewed (only papers published before April 2005 were included). Although in some of the studies results of other patient

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groups are also described, this review is conned to the results pertaining to the changes in HPA axis reactivity in PTSD patients.

3. Results In our database of 34 papers, we divided the studies based on the methodology used. In PTSD populations three studies used a non-pharmacological paradigm, including one cognitive challenge test and two studies using traumatic reminders to assess HPA axis function. Pharmacological challenges included three CRH challenges, one naloxone challenge, one ACTH challenge, ve studies using a 1.0 mg dexamethasone challenge, seven studies using a 0.5 mg dexamethasone challenge and one study using a 0.25 mg dexamethasone challenge. Four studies used a design with administration of metyrapone and eight in vitro studies assessing lymphocyte GR function or intracellular GR density were included. In addition one study using a dexamethasone/CRH test is described in patients with a borderline personality disorder with or without a comorbid PTSD. 3.1. Non-Pharmacological stress challenges Researchers have been especially interested in the way the HPA axis is involved in the management of stress in stress related disorders, thus paradigms have been designed to provoke a stress response. Three studies assessing HPA axis responsivity to non-pharmacological stress challenges in patients with PTSD have been reported. Dierent outcome measures have been assessed including cortisol in plasma or saliva, plasma
Table 1 Non-pharmacological challenge paradigms Reference Liberzon et al. (1999) Population Combat related PTSD (n = 17), combat controls (n = 11), healthy controls (n = 14) Challenge paradigm Exposure to nonspecic arousing stimuli compared with exposure to trauma-related stimuli. Arithmetic cognitive tasks with negative feedback.

ACTH, heart rate, and blood pressure. As noradrenergic neurons stimulate CRH release and preclinical and clinical studies show an increased noradrenergic activity and noradrenergic stress responsivity in PTSD patients, an increased ACTH and cortisol response to stressful challenges was hypothesized in all studies. Liberzon et al. (1999) used trauma related acoustic stimuli in combat veterans with and without PTSD and in healthy controls. Subjects were studied in two sessions in randomized order. One session consisted of exposure to non-specic arousing stimuli (white noise) and the other of exposure to trauma-related stimuli (combat sounds). PTSD patients had elevated plasma cortisol levels in comparison to controls at baseline and in both noise conditions. Although there was an enhanced autonomic and adrenergic response in PTSD patients compared to controls there was no dierence in plasma ACTH or cortisol response to combat sounds between groups (see Table 1). Bremner et al. (2003b) used an arithmetic task with negative feedback to elicit a stress reaction. In a mixed gender group, PTSD patients demonstrated signicant more elevation of salivary cortisol levels in anticipation of and during the test as compared to controls. Cortisol remained signicantly elevated for one hour after the challenge in PTSD patients but not in controls. Elzinga et al. (2003) assessed cortisol responsivity to traumatic reminders using a personalized traumatic script in abused women with and without current PTSD. Patients with PTSD had higher salivary cortisol levels in the period leading up to the script exposure, during exposure to the script, and throughout the recovery phase. In these three studies, a stressfull challenge was able to invoke a sympathetic response. In two of the three

Time of challenge 9 a.m.

Outcome measures Subjective distress, skin conductance, heartrate, and blood sampling before and after challenge for plasma catecholamines, ACTH, and cortisol. Heartrate, blood pressure, salivary cortisol at several time points before and after challenge.

Conclusions and remarks PTSD displayed exaggerated response in skin conductance, heartrate, plasma epinephrine and norepinephrine, but not ACTH and cortisol. Cortisol elevated in anticipation phase and in response to challenge in both groups, PTSD displayed elevated cortisol levels in anticipation phase and during challenge. PTSD patients displayed elevated cortisol levels in anticipation phase, during and after challenge.

Bremner et al. (2003)

Civilian PTSD (n = 23), healthy controls (n = 18)

2 p.m.

Elzinga et al. (2003)

Abuse related PTSD (n = 12), trauma controls (n = 12)

Personalized traumatic script.

2 p.m.

Salivary cortisol at several time points before and after challenge, memory for neutral and emotional material.

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PTSD studies an exaggerated salivary cortisol response to stressful or trauma related stimuli was found. In the study by Liberzon et al. no group dierence was found in ACTH and cortisol response, which could be due to the fact that samples were taken immediately after the stressor. Except for the male subjects in the study by Bremner, baseline cortisol levels were higher in PTSD subjects compared to controls. As in all three studies baseline measurements were within 1 h prior to the test, it cannot be excluded that these ndings are due to anticipation stress. The main nding of this paradigm is the presence of augmented salivary cortisol levels in response to a stressful test in PTSD patients. 3.2. CRH challenges Intravenous CRH administration is commonly used to assess the responsivity of the pituitary to CRH, by measuring ACTH levels. In this challenge test 1 lg/kg or 100 lg of CRH is given intravenously as a bolus injection. Blood samples for ACTH and cortisol assays are taken at regular time points during 1 or 2 h after administration of the bolus. In healthy individuals, an increase in ACTH can be observed between 5 and 15 min after CRH administration, with a maximum response between 10 and 15 min. In healthy individuals, the peak cortisol response typically occurs at 30 60 min after administration. Alterations in pituitary responsivity to CRH, may be due to a number of factors, such as alterations in pituitary function, pituitary CRH receptor number and binding anity, alterations in vasopressin levels, CRH binding peptides or other regulatory peptides such as atrial natriuretic peptide (ANP), as well as alterations in feedback inhibition by cortisol. Assessment of the ratio between ACTH, and cortisol response in response to CRH administration can provide more information about adrenal responsivity. To date three CRH challenge studies in PTSD patients have been reported. Since baseline studies have shown higher levels of CRH in CSF of PTSD patients, a down regulation of CRH receptors resulting in a blunted ACTH response was hypothesized in all studies. The rst CRH challenge in PTSD patients performed in 1989, described plasma ACTH and cortisol following administration of 1 lg/kg of CRH in inpatient combat veterans with PTSD and healthy controls (Smith et al., 1989). The total group of PTSD patients (with and without comorbid MDD) exhibited a signicantly smaller ACTH response to CRH than the healthy control group. However, there was no signicant dierence between PTSD patients without comorbid MDD and the healthy control group. Rasmusson et al. (2001) used a similar approach in pre-menopausal women with PTSD and healthy controls (Rasmusson et al., 2001). Plasma ACTH increased

signicantly after CRH administration in both groups. The two groups did not dier in mean baseline ACTH level, but there was a trend for an increased ACTH response in PTSD patients. The CRH-induced increase in plasma cortisol was signicantly higher in PTSD subjects than in controls. Moreover, there was a delay in onset of the cortisol response in the PTSD group. Kellner et al. (2003) investigated ACTH, cortisol and atrial natriuretic peptide (ANP) levels after CRH stimulation in PTSD patients and healthy controls. ANP has been hypothesized to play a regulatory role in the ACTH response and it is known to decrease basal and stimulated pituitaryadrenal activity. Comparing patients with PTSD and healthy controls they found significantly lower basal ANP levels in PTSD patients and reported no signicant dierences in the area under the curve (AUC) of the cortisol and ACTH values between PTSD patients and controls. The three described studies show contrasting results making it impossible to draw any conclusion on pituitary or adrenal reactivity in PTSD. The contrasting results may be due to factors in the realm of methodology and population factors as the design in these studies diers with respect to the time of CRH infusion, the number of blood samples taken, and the time at which blood sampling was performed. Dierences in population factors include trauma type, duration of the disorder, and age when traumatized. 3.3. Naloxone challenge Since it is known that centrally mediated opioid response to traumatic stimuli is an important feature in PTSD and that re-exposure to a traumatic stressor will precipitate opioid-mediated stress induced analgesia in people with PTSD, some studies have been conducted that apply naloxone. Naloxone is an opioid receptor antagonist which ultimately leads to increases in endogenous CRH release. Hockings et al. (1993) administered naloxone in three dosages in patients with combat related PTSD and in healthy volunteers. Plasma cortisol and ACTH levels were measured at baseline and after each dosage. No dierence was found between PTSD patients and controls for all of the measurements. However, in a subgroup of six PTSD patients the ACTH response to naloxone in the lowest dosage was signicantly higher than in the other PTSD patients and controls. This larger response could not be explained by other comorbid psychiatric disorders or medication. In a later study by Vythilingam et al. (2000) no CRH response was found after naloxone administration in normal volunteers. 3.4. ACTH challenge The rapid ACTH stimulation test (RST) was developed to measure the acute adrenal response to ACTH.

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Clinically, this test is used to test adrenocortical function in patients with Cushing disease during and after steroid hormone replacement therapy. In this test, 0.25 mg of synthetic ACTH (cosyntropin) is delivered intravenously within 30 s, which causes a rapid increase of cortisol within 30 min. In the standard procedure, blood samples for cortisol measurements are drawn just before and 30 and 60 min after ACTH infusion. This test measures the reactivity at the level of the adrenal glands, and thus provides information about adrenal responsivity. Alterations in response may be due to adrenal functioning, glucocorticoid receptor number and anity, or feedback inhibition by cortisol. The RST has been applied in one published study in PTSD patients. A blunted cortisol response was hypothesized based on the outcome of a previously performed CRH challenge and ndings of low baseline cortisol in patients with PTSD. The study was performed in women with PTSD and in healthy controls (Rasmusson et al., 2001). Both groups were premenopausal at the time of measurement. The PTSD group varied in age of rst trauma and type of trauma. The challenge revealed a signicantly larger increase of plasma cortisol after ACTH administration in PTSD patients compared to controls (see Table 2). A confounding factor in this study was that the test was not conducted at a xed time. 3.5. Dexamethasone suppression test The dexamethasone suppression test (DST) was originally developed as a diagnostic screening instrument for Cushing disease. It measures the feedback inhibition of dexamethasone on the HPA-axis. In psychiatric patients, the DST was rst used in patients with MDD. In this population, the amount of cortisol suppression after administration of 1.0 mg cortisol was measured (1.0 mg DST). Using this procedure, 40% of the MDD patients were reported to have an abnormal HPA feedback inhibition with an early escape from the dexamethasone suppression (Carroll and Curtis, 1976; Carroll et al., 1980). The notion that suppression of cortisol production seemed to be enhanced in PTSD patients led to the introduction of the 0.5 mg DST in PTSD. This test induces a more modest suppression enabling dierentiation between normal and augmented suppression (Yehuda et al., 1993). The 0.5 mg DST has frequently been used in patients with PTSD and uses the same administration and blood or saliva collection time as the 1 mg test. To date no exact cortisol value has been described as cut o value for enhanced cortisol suppression. Recently studies assessing both ACTH and cortisol in response to a 0.5 mg DST have been published. With this design more information on the role of enhanced feedback inhibition and reduced adrenal output in the pathophysiology of PTSD might be provided.

The DST in PTSD patients originally was conducted with 1 mg of dexamethasone. Due to the clinical overlap of MDD in PTSD populations, the focus in these studies was to test the hypothesis of non-suppression or early escape in PTSD. The rst 1 mg DST performed in PTSD patients did not reveal a signicant dierence in plasma cortisol levels after administration of dexamethasone (Kudler et al., 1987). However, the frequency of non-suppression was substantially lower in PTSD patients without comorbid MDD compared to PTSD patients with comorbid MDD (0% compared to 50%). Two other studies reported a signicant dierence in cortisol suppression between PTSD and MDD patients, but no signicant dierences in cortisol suppression between PTSD patients and controls (Halbreich et al., 1989; Kosten et al., 1990). Thaller et al. (1999) performed a 1 mg DST in Croatian subjects who recently served in the war in Yugoslavia and were diagnosed with PTSD without comorbid psychiatric disorders and found higher cortisol levels in PTSD patients compared to controls after dexamethasone suppression. In another study higher baseline cortisol levels and a signicantly higher rate of non-suppression was found in PTSD patients compared to healthy controls (Atmaca et al., 2002). Reist et al. (1995) measured the eect of treatment on the DST outcome in 21 hospitalized patients with PTSD before and after four weeks of desipramine treatment. Cortisol levels after administration of dexamethasone were not different before and after treatment. The results of the 1 mg DST studies are inconclusive, although the design was comparable. This could be due to dierences in study population and inclusion and exclusion criteria. In all reported studies dexamethasone was administered at 11 p.m., while cortisol levels were assessed at 4 p.m. the following day in four studies (Kudler et al., 1987; Halbreich et al., 1989; Kosten et al., 1990; Atmaca et al., 2002) and at 8 a.m. the next day in one study (Thaller et al., 1999). The study populations diered in in- or exclusion of comorbid disorders, the use of medication, and the presence or absence of a control group (see Table 3). Dierences in outcome could not be explained by the presence of comorbid MDD or medication use. Yehuda et al. (2004c) argued that cortisol suppression is positively correlated with the amount of time passed since the occurrence of initial trauma. Unfortunately, the time passed since traumatization was not mentioned in these studies. Yehuda et al. (1993) hypothesized more suppression of cortisol in response to dexamethasone in patients with PTSD compared to controls and suggested using a lower dose of dexamethasone. Research conrmed this hypothesis because enhanced suppression was found in virtually all studies following 0.5 mg or even 0.25 mg of dexamethasone in PTSD patients compared to controls (Yehuda et al., 1995a,b; Goenjian et al., 1996; Yeh-

Table 2 ACTH and CRH challenges Reference Type of trauma PTSD assessment Medication Diet Durham 1 week medication free No diet CAPS medication free Fasting and no nicotine for 4 hrs. PDS 2 weeks medication free Sober after 1 p.m. CAPS Medication free Fasting and no nicotine for 4 hrs. Method / time of challenge Time of baseline sample(s) Time of post challenge sample(s) CRH 1 lg/kg iv at 8 p.m. 7.45 p.m., 8 p.m. (B) 8.15, 8.30, 8.45, 9.00, 9.30, 10 p.m. CRH 1 lg/kg iv at 8 p.m. 7.45 p.m. (B) 8.15, 8.30, 8.45, 9.00, 9.30, 10 p.m. CRH 100 lg iv at 3 p.m. 2.30, 2.45 p.m. (B) 3, 3.15, 3.30, 3.45, 4, 4.30, 5 p.m. Cosyntropin 250 lg at dierent times. Just before challenge. (B) 30 and 60 min after challenge. PTSD Cortisol (lg/dl) Cortisol (lg/dl) PTSD (n = 8) 7.1 1.7 (B) Values in graph PTSD (n = 12) 4.38 0.70 (B) 19.1 2.0 (PC) PTSD (n = 17) 11.10 1.10 (B) Only AUC values mentioned PTSD (n = 10) 12.6 0.9 (B) Values in graph Controls Cortisol (lg/dl) Cortisol (lg/dl) Controls (n = 11) 4.8 0.5 (B) Values in graph Controls (n = 11) 3.84 1.02 (B) 14.1 1.2 (PC) Controls (n = 17) 10.81 1.38 (B) Only AUC values mentioned Controls (n = 7) 9.1 1.6 (B) Values in graph PTSD ACTH (pg/ml) ACTH (pg/ml) PTSD (n = 8) 5.8 1.8 (B) Values in graph PTSD (n = 12) 7.0 1.6 (B) 40.3 26.0 (PC) PTSD (n = 17) 16.8 2.1 (B) Only AUC values mentioned Controls ACTH (pg/ml) ACTH (pg/ml) Controls (n = 11) 7.6 2.6 (B) Values in graph Controls (n = 11) 7.0 2.3 (B) 21.6 11.4 (PC) Controls (n = 17) 19.4 6.3 (B) Only AUC values mentioned Conclusions/ Remarks

C.S. de Kloet et al. / Journal of Psychiatric Research 40 (2006) 550567

Smith et al. (1989) Combat

ACTH response in PTSD versus controls

Rasmusson et al. (2001) Various trauma

Cortisol (PC) and ACTH (PC) in PTSD No sign. dierence in cortisol (AUC) and ACTH (AUC) after CRH challenge Cortisol increase after cosyntropin in PTSD

Kellner et al. (2003) Various trauma

Rasmusson et al. (2001) Various trauma

AUC, area under the curve; B, baseline value; CSA, childhood sexual abuse; CAPS, clinician administered PTSD scale; PDS, posttraumatic diagnostic scale; PC, peakchange.

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Table 3 Dexamethasone suppression tests C.S. de Kloet et al. / Journal of Psychiatric Research 40 (2006) 550567 Reference Type of trauma Kudler et al. (1987) Combat PTSD assessment Medication Relevant exclusion criteria Structural PTSD interview 1 week medication free Current alcohol abuse DSM IIIR criteriaa 2 weeks medication freeb Method & time of challenge Time of baseline sample(s) Time of post DEX sample I/II Dexamethasone 1 mg, 11 p.m. No baseline 4 p.m. (post DEX) Dexamethasone 1 mg, 11 p.m. 14 p.m. (baseline) 4 p.m. (post DEX) Dexamethasone 1 mg, 11 p.m. No baseline 4 p.m. (post DEX) Dexamethasone 1 mg, 11 p.m. 8 a.m. (baseline) 5 p.m. (baseline) 8 a.m. (post DEX) Dexamethasone 1 mg, 11 p.m. 8 a.m. (baseline) 4 p.m. (post DEX) Dexamethasone 0.5 mg, 11 p.m. 8 a.m. (baseline) 8 a.m. (post DEX) 4 p.m. (post DEX) Dexamethasone 0.5 mg, 11 p.m. 8 a.m. (baseline) 8 a.m. (post DEX) 4 p.m. (post DEX) PTSD Cortisol (lg/dl) Cortisol (lg/dl) PTSD without MDD (n = 18) No baseline 1.86 PTSD with MDD (n = 13) 7.69 1.34 0.96 0.69 PTSDc (n = 11) PTSD (n = 18) No baseline 1.8 1.0 PTSD (n = 34) 21.5 2.07 8.77 0.83 7.14 1.78 PTSD (n = 14) 12.91 3.78 4.76 1.56 PTSD (n = 21) 14.29 3.70 1.78 1.45 1.89 1.36 PTSD (n = 14) 12.68 4.20 1.09 0.62 1.89 1.89 Combat controls (n = 12) 15.40 3.41 10.00 3.59 5.11 2.50 Other Cortisol (lg/dl) Cortisol (lg/dl) PTSD with MDD (n = 10) No baseline 3.83 MDD (n = 23) 12.31 3.43 3.72 3.73 MDD without (n = 28) No baseline 4.1 1.8 Psoriasis (n = 19) 14.64 5.00 6.78 2.90 0.83 0.18 Controls (n = 21) 7.32 17.0 1.37 0.95 Controls No baseline 1.9 0.6 Controls (n = 17) 21.38 1.16 14.57 1.12 <3.12 Controls (n = 14) 10.76 2.22 2.19 1.04 Controls (n = 12) 15.09 4.17 4.78 2.93 4.51 2.28 Controls (n = 14) 14.97 3.19 3.91 2.90 3.91 2.68 5 p.m. baseline cortisollevels in PTSD versus controls, Post DEX cortisol in PTSD versus controls. Percentage of nonsuppressors in PTSD than in controls. More cortisol suppression after correction for dexamethasone plasma level in PTSD. Baseline cortisol and more cortisol suppression in PTSD compared to controls (corrected for baseline cortisol and dexamethasone levels). Controls Cortisol (lg/dl) Cortisol (lg/dl) Conclusions and remarks

No signicant dierence in cortisol suppression between PTSD with and without MDD. Baseline and post DEX cortisollevels in PTSD.

Halbreich et al. (1989) Combat

Kosten et al. (1990) Combat

DSM IIIR criteria Various medication Current substance abuse DSM IV criteria 24 h medication free Alcohol

Post DEX cortisol in PTSD versus MDD.

Thaller et al. (1999) Combat

Atmaca et al. (2002)

CAPS 2 weeks medication free Other psychiatric disorders CMPS medication free 1 month alcohol-/ drugs free

Yehuda et al. (1993) Combat

Yehuda et al. (1995) Combat

Figley PTSD scale medication free for 1 month Alcohol and drugs, MDD

Line missing

Yehuda et al. (1995) Combat

Figley PTSD scale medication free for 1 month Alcohol and drugs, MDD

Dexamethasone 0.25 mg, 11 p.m. 8 a.m. (baseline) 8 a.m. (post DEX) 4 p.m. (post DEX)

PTSD (n = 14) 11.49 3.12 4.49 2.50 4.49 2.21 CSA with PTSD (n = 12) 13.30 3.81 1.45 1.45

Combat controls (n = 12) 15.40 3.41 10.00 3.59 5.11 2.50 CSA (n = 19) 14.21 4.49 1.67 1.38

Controls (n = 14) 14.32 2.79 8.99 3.70 5.58 3.41 Controls (n = 21) 16.31 5.80 3.23 3.33

Baseline cortisol and more cortisol suppression in PTSD compared to controls (corrected for baseline cortisol and dexamethasone levels). Post DEX cortisol in PTSD versus controls, post DEX cortisol in CSA versus controls. CSA with and without PTSD were not compared. C.S. de Kloet et al. / Journal of Psychiatric Research 40 (2006) 550567 % Cortisol suppression in PTSD compared to trauma exposed non PTSD, and HC. Baseline cortisol in PTSD. No sign.dierence in post DEX cortisol. Baseline and post DEX cortisol in PTSD versus controls. No dierence in post DEX cortisol between trauma exposed and non exposed controls. More cortisol and ACTH suppression in PTSD compared to both controlgroups. No dierence in ACTH / cortisol ratio.

Stein et al. (1997) CSA

CAPS Various medications

Dexamethasone 0.5 mg, 11 p.m. 8 a.m. (baseline) 8 a.m. (post DEX)

Yehuda et al. (2002) Holocaust/combat

CAPS 2 months medication free Alcohol/drugs CAPS

Dexamethasone 0.5 mg, 11 p.m. 8 a.m. (baseline) 8 a.m. (post DEX) Dexamethasone 0.5 mg, 10 p.m. 8 a.m., 4 p.m., 10 p.m. (saliva) 8 a.m., 4 p.m., 10 p.m. (saliva) Dexamethasone 0.5 mg, 11 p.m. 8 a.m. (baseline) 8 a.m. (post DEX) Cortisol suppression

PTSD (n = 28) 9.91 2.84 1.81 1.99

Trauma no PTSD no MDD (n = 9) 11.60 4.11 2.00 1.02

Controls (n = 10) 10.94 3.63 2.81 1.92

Lindley et al. (2004) Various trauma Yehuda et al. (2004) Various trauma

PTSD (n = 17) Controls (n = 17) only 8 a.m. baseline values mentioned in article PTSD (n = 15) 12.2 4.7 1.7 1.8 86% Trauma no PTSD no MDD (n = 11) 12.67 4.60 2.68 2.42 79% Controls (n = 10) 17.00 6.90 5.00 3.90 70%

CAPS Various medications Alcohol/drugs

Newport et al. (2004) Childhood abuse

CAPS

Dexamethasone 0.5 mg, 11 p.m. 8 a.m. cortisol (baseline) 8 a.m ACTH (baseline) 8 a.m. cortisol (post DEX)) 8 a.m ACTH (post DEX)) 4 p.m. cortisol (post DEX) 4 p.m. ACTH (post DEX) Dexamethasone 0.5 mg, 11 p.m. 8 a.m. cortisol (baseline) 8 a.m ACTH (baseline) 8 a.m. cortisol (post DEX)) 8 a.m ACTH (post DEX))

ELS with PTSD (n = 20) 15.5 12.9 25.4 12.9 1.3 0.6 5.6 3.9 2.4 2.6 10.4 7.6 PTSD (n = 19) 12.52 3.29 25.47 19.40 1.97 1.28 4.19 3.30

ELS without PTSD (n = 15) 14.5 4.0 33.6 16.9 4.9 6.6 15.4 18.1 2.9 3.2 12.0 9.2

Controls (n = 19) 20.7 8.1 26.8 14.9 4.6 3.7 8.8 5.5 4.5 3.4 14.5 10.0 Controls (n = 19) 11.46 6.58 24.33 16.01 3.14 2.12 8.87 8.38

Yehuda et al. (2004)

CAPS

More ACTH and cortisol suppression in PTSD. No dierence in ACTH / cortisol ratio before and after DEX.

CMPS, Civilian Mississippi PTSD Scale; CSA, childhood sexual abuse; MDD, major depressive disorder; ELS, early life stress; DEX, dexamethasone; HC, healthy control. a Patients with comorbid alcoholabuse/dependence were included in this study. b Five patients used TCA during the study. c Six PTSD patients were diagnosed with a comorbid MDD.

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uda et al., 2002) (see Table 3). Also in adult women with childhood sexual abuse of whom some also met criteria for PTSD, signicantly more suppression of the post DST cortisol level was found compared to the nonabused group (Stein et al., 1997). However, there was no signicant dierence in the abused women with and without PTSD. Two studies also assessed ACTH before and after administration of dexamethasone. In the rst study both a 0.5 and 1 mg DST was used in women with childhood abuse (among which were patients with a diagnosis of PTSD, MDD, PTSD and MDD) and healthy controls (Newport et al., 2004). PTSD patients were compared with the other groups in their cortisol- and ACTH response on the 0.5 mg DST. Patients with early childhood abuse and PTSD revealed signicantly more suppression of ACTH at 8 a.m. and 4 p.m. compared to healthy controls, despite no signicant dierences in baseline ACTH values. Patients with early childhood abuse and PTSD had signicantly lower baseline plasma cortisol values and revealed signicantly more suppression of cortisol at 8 a.m. The second study conrmed increased suppression of ACTH and cortisol at 8 a.m. in patients with PTSD (Yehuda et al., 2004a). The ndings of enhanced ACTH suppression after dexamethasone administration support the hypothesis of inhibition at the level of the pituitary. Correlation analysis showed a signicant correlation between post dexamethasone cortisol and post dexamethasone ACTH values and symptom severity, and a signicant correlation between cortisol and ACTH suppression and symptom severity. A factor that has not been taken into account in most studies is the amount of time passed since the initial trauma. However, Yehuda et al. reported a signicant association between the numbers of years passed since the most recent trauma occurrence and the log transformed plasma cortisol ratio (predexamethasone cortisol level postdexamethasone cortisol level/ predexamethasone cortisol level), showing signicantly more suppression of cortisol when more years had passed (Yehuda et al., 2002; Yehuda et al., 2004c). In a recent study by Yehuda et al. the eect of trauma exposure on the HPA-axis was also measured. A nontraumatized group was compared with a traumatized group without PTSD or MDD. No eect on cortisol levels was found between non-trauma exposed and trauma exposed non-PTSD/non-MDD patients, and no eect of prior traumatization on the extent of cortisol suppression was found in patients with PTSD. However, PTSD patients showed signicantly more reduction of cortisol after administration of 0.5 mg of dexamethasone compared to patients without PTSD. In contrast to all other 0.5 mg DST studies, a study assessing salivary cortisol levels showed no signicant dierences in baseline salivary cortisol and cortisol suppression after dexamethasone administration between patients and controls

(Lindley et al., 2004). The main dierence in the study design used by Lindley et al. is the use of saliva rather than plasma, assessment at home, and the use of more time points before and after dexamethasone administration. In saliva the amount of free cortisol is assessed (biological active), whereas in plasma total cortisol is assessed (biological active and inactive). The dierent outcome might be due to a higher fraction of bound cortisol in PTSD. Another explanation might be the assessment in a non-clinical environment instead of a clinical environment. Although assessment in a non-clinical environment reduces stress caused by a hospital visit, there is little control on the sampling time or the time of dexamethasone administration. In all studies except the latter study, PTSD populations show enhanced suppression of cortisol compared to controls. Baseline cortisol values in PTSD subjects were slightly lower in PTSD patients (between 9.91 and 14.92 (lg/dl)) than in controls (between 10.94 and 20.7 (lg/dl)). Mean 8 a.m. post dexamethasone (0.5 mg) levels were lower in PTSD patients (between 1.09 and 1.97 (lg/dl)) than in controls (between 2.81 and 4.78 (lg/dl)). A correlation between cortisol suppression and symptom severity has only be reported in two studies (Yehuda et al., 1995a,b) (Yehuda et al., 2004a), making the clinical relevance of the observed enhanced cortisol suppression on 0.5 mg DST an issue for further discussion. 3.6. DexamethasoneCRH challenge The dexamethasoneCRH (DEXCRH) challenge test was developed by Holsboer et al. as a rened DST procedure. By combining dexamethasone administration with CRH infusion the neuroendocrine feedback regulation of the HPA-axis system in psychiatric disorders was further elucidated (Holsboer et al., 1987). This combined DEXCRH test requires administration of 1.5 mg of dexamethasone orally at 11 pm the night before testing. The advantage of this procedure is that at the moment of CRH challenge in both patients and controls the HPA-axis is downregulated due to feedback inhibition by dexamethasone. On the day of testing, 100 lg human CRH is administered intravenously as a bolus at 3 p.m. Blood samples are drawn every 15 min between 2 and 6 p.m. (original procedure) or at 5 time points between 3 and 4:15 p.m. (rened procedure). In a population with MDD, it is purported to be the best tool for assessing HPA-axis alterations. It reveals less cortisol suppression after dexamethasone pre-treatment, and a larger cortisol response after CRH administration. Based on baseline CRH measurements, showing high CRH in CSF, a down regulation of CRH receptors in a PTSD population would be expected resulting in a blunted ACTH and cortisol response. Feedback inhibition should not interfere with this outcome as the high

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dose of dexamethasone administrated before CRH infusion will give the same amount of cortisol suppression in patients and controls, thereby blocking feedback inhibition by cortisol completely. Thus far only one study reports on the eect of this paradigm in a traumatized psychiatric population (Rinne et al., 2002). Female patients with borderline personality disorder (BPD) with and without a history of sustained childhood abuse were studied using the rened procedure of the DEXCRH challenge. After DEX CRH a larger mean AUC for both the ACTH and cortisol response in chronically abused BPD patients compared to none or mildly abused BPD patients was found. BPD patients with comorbid PTSD showed a signicantly lower ACTH AUC than BPD patients without PTSD, but a higher response than controls. Patients with PTSD without childhood abuse had the lowest ACTH AUC. The outcome suggests that early childhood abuse is related to an exaggerated ACTH response on stimulation with CRH, whereas PTSD decreases the exaggerated ACTH response in abused BPD patients.

3.7. Metyrapone challenges The metyrapone test was introduced in 1959 for the diagnosis of adrenal insuciency. Metyrapone inhibits 11-beta-hydroxylase, the enzyme that catalyzes the hydroxylation of steroids and is necessary for the conversion of deoxycortisol to cortisol. Inhibition of this enzyme results among other things in decreased cortisol synthesis with lower cortisol levels and higher deoxycortisol levels. The decrease in cortisol levels will result in less negative feedback inhibition on the pituitary and subsequent higher plasma levels of ACTH. In an improved version of this test, metyrapone is administered at midnight and ACTH, deoxycortisol, and cortisol is measured in plasma at 8 a.m. the next day. The authors of the rst metyrapone challenge performed in PTSD patients hypothesized that the enhanced feedback inhibition on the level of the pituitary in PTSD will result in more central stimulation of the HPA-axis in PTSD. A decrease in cortisol levels due to metyrapone administration will block the hypothesized enhanced feedback inhibition in PTSD patients. This would result in a relatively greater increase of ACTH release and an augmented accumulation of 11-deoxycortisol in patients with PTSD (Yehuda et al., 1996). Blood samples for cortisol, 11-deoxycortisol, and ACTH measurements were drawn at baseline, and on the day of testing at 4 time points between 10 a.m. and 3 p.m. On the day of testing, 2500 mg of metyrapone was administered orally at 10 a.m. After metyrapone challenge cortisol was almost completely suppressed in both groups, no group dier-

ence in cortisol suppression was reported. There was a signicantly greater increase in the levels of 11-deoxycortisol and ACTH in the PTSD group compared to the controls. The combination of higher 11-deoxycortisol levels, suggesting enhanced stimulation of the adrenal gland, and higher ACTH levels in this study supported the hypothesized enhanced feedback inhibition at the level of the pituitary. In order to examine negative feedback sensitivity directly cortisol was infused after prior treatment with metyrapone in PTSD patients and controls (Kanter et al., 2001). This procedure enables the measurements of the inhibition of ACTH release in the pituitary by exogenous cortisol. They hypothesized a more rapid decrease of plasma ACTH after the endogenous cortisol administration. Metyrapone was administered in two doses of 750 mg each at 6 and 9 a.m. Blood samples were drawn every 15 min from baseline until 1 p.m. Metyrapone substantially reduced the initial plasma cortisol concentrations, but a signicant smaller reduce in plasma cortisol was reported in PTSD patients. Metyrapone induced an increase in ACTH and 11deoxycortisol concentrations in both groups. There was no signicant dierence in ACTH response between groups, but a signicantly smaller increase in 11-deoxycortisol levels in PTSD patients compared to controls was reported. Plasma levels of 11-deoxycortisol were signicantly lower in the PTSD population throughout the whole study. As the decrease in cortisol levels and subsequent blockage of feedback inhibition did not lead to a more pronounced stimulation of the HPA-axis in PTSD and cortisol infusion did not show more inhibition on ACTH release in the PTSD group compared to controls as well, this study did not support the hypothesized enhanced feedback sensitivity at the pituitary level. However, the lower level of 11-deoxycortisol suggests a decreased adrenocortical responsiveness. Another study assessed the eect of metyrapone on sleep in PTSD. Four doses of 750 mg of metyrapone were administered every 4 h in the morning, and blood for the assessment of cortisol, 11-deoxycortisol and ACTH was collected before and after administration of metyrapone. This study also could not support the hypothesis of enhanced feedback sensitivity; after metyrapone administration, plasma levels of ACTH were signicantly lower in PTSD subjects compared to controls, but cortisol levels revealed no group dierences (Neylan et al., 2003). Recently, a study design with metyrapone was introduced, in which placebo at 12 p.m. (day1), metyrapone at 12 p.m. (day2), or 0.5 mg dexamethasone at 11 p.m. and metyrapone at 12 p.m. (day3) was administered on three consecutive days (Kellner et al., 2004). The doses of metyrapone used were proportional to the bodyweight, between 2000 (<70 kg) and 3000 mg (>90 kg). With this design, the suppressive eect of cortisol and

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dexamethasone was determined. According to the theory of enhanced feedback inhibition in PTSD patients, a larger increase of ACTH and 11-deoxycortisol in response to metyrapone alone and a larger decrease of ACTH and 11-deoxycortisol level on metyrapone and dexamethasone administration in PTSD patients were expected. In both groups metyrapone administration induced a significant increase in ACTH concentrations which rapidly declined after dexamethasone administration. However, no signicant dierences were found between the groups on any of the other parameters. So far studies using a design with metyrapone to inhibit the regulatory eects of cortisol revealed mixed results. Only one study showed the hypothesized increase of ACTH and 11-deoxycortisol after metyrapone administration.

3.8. Glucocorticoid receptor studies Since leukocyte glucocorticoid receptor (GR) studies have been applied to the arena of endocrine studies in PTSD and might provide more information on central GR functioning, these studies will be reviewed as well. Glucocorticoids passively diuse through the cellular membrane and bind to intracellular receptors, which promotes the translocation of GRs to the nucleus, resulting in up or down regulation in the expression of various genes. GRs are expressed everywhere in the human body, but the most accessible cells to study these receptors are leukocytes. Assessment of GR number and function in mononuclear leukocytes has been used by dierent groups as a surrogate model for central GR functioning. However, we have to bear in mind that GR number and anity is also inuenced by the activation state of the immune system, which might complicate the interpretation. In PTSD patients binding studies have been used to assess leukocyte GR density. The rst binding study in PTSD patients showed a signicantly higher density of GRs in leukocytes of PTSD patients, which correlated signicantly with PTSD symptoms (Yehuda et al., 1991). The latter data were conrmed in a second study (Yehuda et al., 1993). A binding study, using 3 H-RU 28362, assessing both baseline and post dexamethasone GR density in combat exposed veterans with PTSD, combat exposed veterans without PTSD, and healthy controls showed increased baseline GR density in both combat related PTSD patients and combat exposed controls compared to healthy controls (Yehuda et al., 1995a,b). After dexamethasone administration only combat veterans with PTSD showed a decrease in leukocyte GR number. This same protocol was used in women with severe childhood sexual abuse and women with no history of abuse (Stein et al., 1997). There was a signicantly higher mean GR density in patients with

childhood sexual abuse compared to subjects without abuse. Subjects who were diagnosed with PTSD had a higher mean leukocyte GR density than subjects who had not been sexually abused (Table 4). In a replication of these studies, in holocaust survivors and combat veterans with PTSD (Yehuda et al., 2002), no dierence in baseline and post DST leukocyte GR density between PTSD patients and controls was reported. However, older PTSD subjects demonstrated signicantly more suppression of leukocyte GR numbers in response to dexamethasone. The amount of suppression was smaller compared to the previously described studies. A study in Croatian combat veterans with PTSD (Gotovac et al., 2003) revealed signicantly higher baseline cortisol levels in PTSD patients compared to healthy controls, while leukocyte GR expression was lower in each of the examined leukocyte populations in PTSD patients. GR function can be assessed by measuring the inhibitory eect of dexamethasone on the immune system in vitro. More inhibition of the immune system reects more functional GR. Rohleder et al. (2004) measured the GR sensitivity, by measuring dexamethasone inhibition of lipopolysaccharide-induced interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-a) production in whole blood. In this study in Bosnian war refugees with PTSD and healthy controls, PTSD patients had a signicantly higher sensitivity to dexamethasone on the level of IL-6 and TNF-a production by leukocytes compared to healthy controls. In a recent study Yehuda et al., 2004b studied GR sensitivity by measuring the inhibition of lysozyme activity by dexamethasone, and GR number using a radioligand binding assay, in mononuclear leukocytes. It was hypothesized that PTSD subjects would show increased glucocorticoid responsiveness as evidenced by an inhibition of 50% (IC50) on a lower dose of dexamethasone in PTSD subjects compared to controls. The mean doseresponse curve indeed showed that a signicantly lower dose of dexamethasone leads to 50% reduction of lysozyme activity in PTSD subjects compared to the total group of controls including both trauma exposed and non-trauma exposed controls non-PTSD subjects. There was no signicant dierence between PTSD subjects and trauma exposed non-PTSD subjects. In this study, there was no group dierence in GR density and plasma cortisol levels at 8 a.m. between PTSD subjects and non-PTSD subjects. With respect to the age of traumatization, IC50 levels and basal GR density correlated with the age of exposure to the rst trauma in PTSD subjects, showing higher leukocyte GR numbers in patients who had been traumatized early in life. Although the rst studies repeatedly showed a higher baseline leukocyte GR density in PTSD patients, this has not been conrmed by the last two studies. However, several studies applying various study designs

Table 4 Leukocyte glucocorticoid receptor studies Reference Yehuda et al. (1991) Population Combat PTSD (n = 15) Controls (n = 11) PTSD (n = 8), MDD (n = 10) Other psych. disorders (n = 22) Combat PTSD (n = 14) Combat controls (n = 12) Controls (n = 14) GR receptors Mononuclear leukocytes Mononuclear leukocytes Method Blood sampling at 8 a.m. and 4 p.m., single point 3 H-RU 28362 binding assay. Blood sampling at 8 a.m., 3 H-RU 28362 binding assay. GR density (rece/cell)/IC50 (nM) PTSD: 3702 Controls: not reported PTSD: 9075 1437 MDD: 2677 386 Conclusions and remarks GR density in PTSD 8 a.m. GR density correlated with PTSD symptoms. GR density in PTSD

Yehuda et al. (1993)

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Yehuda et al. (1995)

Mononuclear leukocytes

Blood sampling 8 a.m. before and after 0.5/0.25 mg DEX, 3 H-RU 28362 or 3H-DEX binding assay.

Baseline: PTSD: 8761 2987 Combat controls: 7783 3048 Controls: 4480 2326 After DEX PTSD: 5034 2462 Combat controls: 6535 3019 Controls: 4431 2804 PTSD: 4638 1515 nCSA: 3475 1634

Baseline GR density in combat exposed PTSD and non-PTSD compared to controls. After DEX more suppression of GR density in PTSD (44%) compared to controls (14%). GR density in PTSD compared to controls (trend).

Stein et al. (1997)

CSA with PTSD (n = 12) CSA without PTSD (n = 7) Controls (n = 21) Holocaust/combat PTSD (n = 28), TC (n = 9) Controls (n = 10)

Mononuclear leukocytes

Blood sampling at 8 a.m. 3 H-RU 28362 binding assay.

Yehuda et al. (2002)

Mononuclear leukocytes

Blood sampling before and after 0.5 mg DEX, 3 H-DEX binding assay.

Baseline: PTSD: 2538 1211 TC: 2603 590 Controls: 2346 506 After DEX: PTSD: 2235 1253 TC: 2417 792 Controls: 2411 1194 PTSD: 2735 940 TC: 3190 1703 Controls: 2912 1340 IC50: PTSD: 4.9 2.1 IC50: TC: 5.7 2.4 IC50: Controls: 8.6 3.8

No main eect of diagnosis in pre- and post DEX GR density.

Yehuda et al. (2004)

PTSD (n = 26) TC (n = 8) Controls (n = 10)

Mononuclear leukocytes

Bloodsampling at 8 a.m. 3H-dex binding assay. Lysozyme activity after 3 days in increasing doses of DEX.

No sign. dierence in GR density. IC-50 dex in PTSD versus controls, consistent with GC-sensitivity. No sign. dierence in IC-50 dex between PTSD and TC.

Gotovac et al. (2003)

Combat PTSD (n = 25), Controls (n = 35) PTSD (n = 12), Controls (n = 13)

Lymphocytes (T, B, NK cells) Whole blood cultures

Blood sampling at 8 a.m., GR antibody staining and FACS. Blood sampling between 9 a.m. and 1 p.m. LPS-induced IL-6 & TNF-a inhibited by 1-1000 nM DEX in vitro. IL-6: IC50: PTSD: 18 IL-6: IC50: Controls: 27 TNF-a: IC50: PTSD: 10 TNF-a: IC50: Controls: 16

GR expression in PTSD, no correlation between cortisol and GR expression. More inhibition through DEX of IL-6 and TNF-a release, consistent with increased GC sensitivity.

Rohleder et al. (2004)

CSA, childhood sexual abuse; TC, traumacontrol; DEX, dexamethasone; GR, glucocorticoid receptor. 561

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repeatedly reported an enhanced sensitivity of GR to dexamethasone in cells of the immune system.

4. Discussion This paper reviewed 26 challenge studies, using dierent methods to asses HPA-axis functioning, and eight in vitro GR studies. Key ndings of the reviewed studies are enhanced salivary cortisol levels in response to cognitive challenge, as well as enhanced plasma cortisol suppression after administration of 0.5 mg of dexamethasone. In addition, in leukocyte GR function studies an enhanced GR sensitivity was reported in PTSD patients. The results of other pharmacological and cognitive challenge paradigms as well as GR density studies on leukocytes are still inconclusive. 4.1. Hypothesized mechanism for altered HPA-axis function in PTSD The combination of baseline high levels of CRH in CSF and low plasma cortisol levels in patients with PTSD contrasts with results seen in MDD patients. Several explanations have been purported to explain this phenomenon, including (a) enhanced feedback sensitivity, (b) pituitary insuciency or reduced pituitary sensitivity, (c) adrenal insuciency or reduced adrenal sensitivity, (d) enhanced bioavailability of dexamethasone, and (e) alterations in vasopressin release. Enhanced feedback sensitivity to cortisol, hypothesizes that a greater availability of GR on pituitary cells leads to enhanced binding of cortisol, resulting in a negative feedback signal and subsequently a decrease in cortisol production (Yehuda et al., 1995a,b). This hypothesis was supported by the rst lymphocyte GR study, used as a model for central GR functioning, which showed enhanced suppression of GR number after administration of a low dose dexamethasone (Yehuda et al., 1995a,b; Yehuda et al., 2002) and increased sensitivity for glucocorticoids (Yehuda et al., 2004b; Rohleder et al., 2004). The nding of both decreased ACTH and cortisol levels after 0.5 mg DST in PTSD patients compared to controls also supports the hypothesized enhanced feedback sensitivity at the level of the pituitary (Newport et al., 2004; Yehuda et al., 2004a). If low cortisol levels are due to enhanced feedback sensitivity, enhanced ACTH and 11-deoxycortisol levels would be expected in response to metyrapone administration. However, the outcome of metyrapone studies is still inconclusive. A confounding factor in these studies could be the interaction of metyrapone with other systems. Metyrapone is known to cause severe nausea, which is a powerful stimulator of the HPA-axis (Kanter et al., 2001). The infusion of cortisol after administration of metyrapone, and the combination of metyrapone

administration with dexamethasone to directly measure the inhibitory eect of glucocorticoids, did not support the hypothesized enhanced feedback sensitivity as well (Kellner et al., 2004; Kanter et al., 2001). The combination of baseline high levels of CRH in CSF and low plasma cortisol levels in patients with PTSD and enhanced cortisol suppression in 0.5 mg DST studies could also be explained by pituitary insuciency or reduced pituitary sensitivity (Yehuda et al., 1996). Administration of dexamethasone leads to low plasma cortisol levels, due to feedback inhibition. Low plasma cortisol levels stimulate the release of CRH and vasopressin in an eort to normalize cortisol levels. Enhanced cortisol suppression in PTSD therefore could be caused by an insucient pituitary response to central stimulation in response to low cortisol levels. For the same reason adrenal insuciency or reduced sensitivity for ACTH could result in enhanced cortisol suppression (Yehuda et al., 1996). This hypothesis is supported by the outcome of the metyrapone study by Kanter et al. (2001) showing lower levels of 11 deoxycortisol, suggesting a decreased adrenocortical responsiveness in PTSD subjects. However, the outcome of the cognitive stress challenge paradigm shows that during stress an enhanced release of cortisol is possible, so pituitary or adrenal insuciency as the main cause for low cortisol levels in PTSD is unlikely. Similarly, if this hypothesis would be true, the expected outcome after ACTH challenge would be a reduced cortisol response, whereas the ACTH challenge showed an enhanced cortisol response (Rasmusson et al., 2001). Another hypothesized mechanism for the enhanced cortisol suppression on low dose dexamethasone is an enhanced bioavailability of dexamethasone. This could be caused by dierent factors including alterations in the interaction with cortisol binding peptides, abnormalities in hepatic metabolism and medication eects. Some studies indeed found higher dexamethasone levels in PTSD patients (Yehuda et al., 1995a,b; Stein et al., 1997; Yehuda et al., 2004c), however, after correction for dexamethasone level there was still signicantly more cortisol suppression in PTSD subjects (Yehuda et al., 1993). In other studies, dexamethasone levels were not found to be signicant covariates (Yehuda et al., 2002; Yehuda et al., 2004a). Therefore, these results do not fully support the hypothesis of an enhanced dexamethasone bioavailability. The results of the dexamethasoneCRH test in MDD revealed a role for other regulatory mechanisms that could also explain the enhanced cortisol suppression in PTSD. In patients with MDD synergistic action of vasopressin on CRH activity is thought to account for the enhanced cortisol response in the DEXCRH test. Suppression of cortisol by dexamethasone leads to stimulation of the HPA-axis by vasopressin in response to the low cortisol level (Holsboer, 2000). An inadequate

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vasopressin reaction could then account for the enhanced cortisol suppression in patients with PTSD. Decreased vasopressin release could also account for the baseline ndings of high CRH levels and low cortisol levels. This hypothesis predicts a low ACTH response after CRH challenges and in cognitive challenge studies. Although a low ACTH response was found by Smith et al., this nding was not replicated in later studies (Smith et al., 1989; Rasmusson et al., 2001; Kellner et al., 2003). The outcome on non-pharmacological stress challenge studies, however, showed an exaggerated cortisol response which is in contrast with the assumed decrease in ACTH response. The results of the reviewed challenges do not exclusively support one of the hypothesized mechanisms. The higher salivary cortisol levels in response to a cognitive challenge do not seem to support the hypothesis of pituitary-, or adrenal insuciency or an inadequate vasopressin reaction. Although dexamethasone bioavailability has not been found to be a signicant covariate, we should make note of the higher levels of dexamethasone after DST often reported in PTSD patients. The infusion of cortisol after administration of metyrapone, and the combination of metyrapone administration with dexamethasone to directly measure the inhibitory eect of glucocorticoids did not support the hypothesized enhanced feedback sensitivity (Kellner et al., 2004; Kanter et al., 2001), which implies that more studies are necessary before it is possible to accept the most often purported hypothesis: namely, enhanced feedback inhibition in PTSD patients. 4.2. Other factors involved in HPA-axis regulation Other factors involved in altered stress regulation in PTSD that need to be further explored are hormone binding proteins (e.g., CRH binding protein and corticosteroid binding globulin), and the immune factors. CRH binding protein is co-localized with CRH at several sites in the brain. It is known to bind CRH with a higher anity than CRH receptors and in this way antagonizes CRH-induced ACTH release. Binding of CRH to CRH-BP promotes a rapid clearance of the complex. Preclinical studies show increased CRH-BP mRNA in response to restraint stress and a decrease of CRH-BP mRNA after adrenalectomy. This suggests an additional feedback mechanism by an upregulation of CRH-BP in response to stress, leading to a decrease of CRH receptor binding, and inhibition of the ACTH releasing activity (McClennen et al., 1998). Studies in patients with Cushings disease and Addisons disease also suggest that in humans plasma cortisol decreases plasma CRH-BP levels, suggesting more binding of CRH to CRH-BP to inhibit pituitary activation (Suda et al., 1990). CRH-BP therefore might also play a role

in the enhanced feedback inhibition on DST in PTSD. To date no studies assessing CRH-BP in PTSD patients have been published. Corticosteroid binding globulin (CBG) binds cortisol with a high anity and thereby regulates the bioavailability of, biological active, free cortisol to target tissues (Breuner and Orchinik, 2002). When plasma cortisol is assessed total cortisol is measured without an indication of the amount of free (biological active) cortisol. Alterations in plasma cortisol baseline or in challenge paradigm do not necessary imply a suppression of biologically active cortisol. CBG levels have been assessed in only one study and revealed signicantly higher plasma CBG levels in PTSD patients compared to controls (Kanter et al., 2001). There is also evidence for CBG synthesis in tissues outside of the liver, including the pituitary. Cellular synthesis of CBG limits the availability of cortisol to intracellular receptors and has been hypothesized to interfere with the negative feedback of cortisol on ACTH secretion (Berdusco et al., 1995). Future studies assessing CBG in combination with cortisol in plasma or CSF could provide additional information on the availability of biologically active cortisol in PTSD. The immune system interfaces with the HPA-axis in a bidirectional way. On the one hand cortisol decreases the production of pro-inammatoire cytokines and increases the production of anti-inammatory cytokines. On the other hand cytokines inuence the HPA-axis and GR expression, e.g., IL-6 stimulates the production of glucocorticoids, ACTH and CRH, and has been found to upregulate GR binding and GR expression in leukocytes; Il-2 has been found to induce CRH and ACTH release and anti-inammatory Il-10 enhances CRH and ACTH production (for review see Wong, 2002). Although in a PTSD population alterations in immune function have been reported frequently (Miller et al., 2001), the role of these immune alterations and their interaction with the HPAaxis still needs to be evaluated in PTSD populations. Leukocyte GR studies might provide a tool in studying this interaction. Alterations in leukocyte GR function can be hypothesized to play a major role in increased cytokines production in PTSD. A reduction or increase in leukocyte GR function may also provide an explanation for sickness behavior which is present in numerous stress related disorders such as PTSD (Boscarino, 2004), and might serve as an explanation for unexplained medical symptoms after deployment (Kang et al., 2003). Based on the observations of altered cytokine production in PTSD we stress the need for further exploration of the interaction between the immune system and the HPAaxis in PTSD (Besedovsky and del Rey, 2000; del Rey and Besedovsky, 2000; Wong, 2002; Raison and Miller, 2003).

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4.3. Confounding factors and limitations For proper interpretation of the results there are factors we should take notice of; e.g., most pituitary and adrenal hormones have a diurnal rhythm and a pulsatile secretion, complicating interpretation of single point baseline measurements as well as comparisons between challenge studies at dierent timepoints during the day. The latter may have inuenced the outcome on the ACTH challenge and metyrapone challenge designs. Population factors which might have inuenced the outcome in the reviewed study designs include gender and age. Furthermore, the time passed since the most recent trauma has been shown to be signicantly associated with the amount of cortisol suppression after low dose dexamethasone, showing a positive association between the amount of cortisol suppression and the number of years passed since the traumatic event occurred. (Yehuda et al., 2004c). However, in most studies the time passed since the traumatic event has not been assessed. Other factors that can inuence the outcome are the age of the individual at the moment of the traumatic event and the chronicity of the traumatic event. Another problem in the interpretation of the results is whether authors correlated their ndings with existing PTSD symptoms or with the experience of a traumatic eect. In the earlier described study by Yehuda et al. (2004c), however, no dierence in cortisol suppression was found between non-traumatized subjects and traumatized subjects without PTSD or MDD, whereas PTSD patients showed signicantly more cortisol suppression compared to non-PTSD subjects. These results conrm the data reported by Grossman et al. (2003), who reported that in a population with a high incidence (88%) of traumatic experiences (as dened by the A criterion of the PTSD diagnosis) neither childhood nor adult history of trauma had a signicant eect on cortisol suppression after 0.5 mg DST. However, subjects with PTSD revealed signicantly more cortisol suppression. These studies make it plausible that at least the altered DST response is correlated with PTSD and not with trauma. Another important factor is the experienced stress during assessments. The outcome of the non-pharmacological paradigm showed enhanced adrenal responsivity in PTSD subjects. In these studies enhanced cortisol levels in PTSD samples may be caused by more severe conditioning. More stimuli are interpreted as stressful, and mild stressful stimuli may be interpreted as threatening stimuli in patients with PTSD (Engelhard et al., 2001). This emphasizes the complexity of baseline assessments in this population, especially in a clinical environment. These factors may also play a crucial role for the assessment of HPA-axis function using challenge paradigms. The interpretation of the results of challenges in which dexamethasone is used instead of cortisol, should

take note of the fact that dexamethasone may not cross the blood brain barrier. Preclinical studies by De Kloet et al. (1998) have shown that access of the synthetic glucocorticoid dexamethasone to central targets is hampered by MDR1a P-glycoprotein, at the level of the endothelial cells of the bloodbrain barrier. However, earlier studies in depressive populations showed comparable cortisol outcome between designs using hydrocortisone and dexamethasone to induce feedback inhibition (Gispen-de Wied et al., 1993). When leukocyte GR studies are used as a model for central GR function the assumption is that leukocyte GR and brain GR expression are correlated. This concept may be doubted as preclinical studies show tissue specic dierences in GR expression between cells and tissues of the immune system (Miller et al., 1998). However, the correlation between in vivo dexamethasone suppression and GR sensitivity in mononuclear leukocytes in healthy controls supports the idea that GR expression in leukocytes may reect central GR expression (Yehuda et al., 2004b). A confounding factor might be that when measuring ex vivo GR density after administration of dexamethasone in vivo, the outcome might be inuenced by the amount of in vivo bound dexamethasone. Assessment of leukocyte GR density has the disadvantage that an increase in GR density does not necessarily imply enhanced receptor sensitivity. GR sensitivity resembles the number of functional GR receptors as well as the transduction of the signal from the receptor to the intracellular eector system. Faced with all the factors inuencing the outcome of HPA-axis measurements we should not be surprised by the inconclusiveness of most challenge designs. In the reviewed studies, the studied populations varied widely. PTSD symptoms could be caused by early childhood abuse, combat experience, motor vehicle accidents, sexual or physical assault. Thus not only the moment of trauma in lifespan and the time passed since the event diered between and within studies, but also the frequency and intensity of the traumatic event. The design of CRH, ACTH and metyrapone tests in this review differed both in time of challenge, and moment, and number of assessments. 4.4. Recommendations for future research The development of a classied system for psychiatric disorders is necessary to gain insight into etiology, course, and treatment of psychiatric disorders. Similarly, consensus in the design of research paradigms, as has been reached in the dexamethasoneCRH test, is necessary to further develop our understanding of HPA-axis dysfunction in PTSD. To overcome the problem of variation in population in PTSD research it is necessary to use larger study populations, preferably perform multicenter research, to enable proper dierentiation in

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gender, type of trauma, time passed since the trauma, and comorbidity. The assessment of diurnal rhythms of the dierent hormones involved in HPA-axis regulation should have a priority. These baseline assessments would provide more information on HPA-axis functioning and will provide the information necessary to interpret the data in challenge studies. Pharmacological challenge designs combining blockade of feedback inhibition with CRH administration, and the assessment of vasopressin and binding-globulins in dierent challenge designs might give more information to allow acceptance or rejection of the hypothesis of enhanced feedback inhibition due to an up regulation of GR receptors. Moreover, suppression tests using cortisol instead of dexamethasone can provide more information on the central eect of glucocorticoids. Leukocyte GR studies are important, however, to investigate if these studies can serve as a model for central glucocorticoid dysregulation more basic research is necessary. Leukocyte GR studies are an interesting tool to assess the role of cortisol in regulation of the immune system and its eects in stress related disorders. Genetic research is developing at a rapid pace. Glucocorticoid receptor polymorphisms have been identied, providing us with tools to expand research on HPA-axis function and assess genetic predisposition for altered GR functioning in PTSD. Future research on PTSD related polymorphism will undoubtedly oer us new insights into the genetic vulnerability factors underlying the HPA-axis dysfunction in this disorder.

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