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A clinical genetic method to identify mechanisms by which pain causes depression and anxiety

A clinical genetic method to identify mechanisms by which pain causes depression and anxiety Background: Pain patients are often depressed and anxious, and benefit less from psychotropic drugs than pain-free patients. We hypothesize that this partial resistance is due to the unique neurochemical contribution to mood by afferent pain projections through the spino-parabrachial-hypothalamic-amygdalar systems and their projections to other mood-mediating systems. New psychotropic drugs for pain patients might target molecules in such brain systems. We propose a method to prioritize molecular targets by studying polymorphic genes in cohorts of patients undergoing surgical procedures associated with a variable pain relief response. We seek molecules that show a significant statistical interaction between (1) the amount of surgical pain relief, and (2) the alleles of the gene, on depression and anxiety during the first postoperative year. Results: We collected DNA from 280 patients with sciatica due to a lumbar disc herniation, 162 treated surgically and 118 non-surgically, who had been followed for 10 years in the Maine Lumbar Spine Study, a large, prospective, observational study. In patients whose pain was reduced >25% by surgery, symptoms of depression and anxiety, assessed with the SF-36 Mental Health Scale, improved briskly at the first postoperative measurement. In patients with little or no surgical pain reduction, mood scores stayed about the same on average. There was large inter-individual variability at each level of residual pain. Polymorphisms in three pre-specified pain-mood candidate genes, catechol-O-methyl transferase (COMT), serotonin transporter, and brain-derived neurotrophic factor (BDNF) were not associated with late postoperative mood or with a pain-gene interaction on mood. Although the sample size did not provide enough power to persuasively search through a larger number of genes, an exploratory survey of 25 other genes provides illustrations of pain-gene interactions on postoperative mood – the mu opioid receptor for short-term effects of acute sciatica on mood, and the galanin-2 receptor for effects of unrelieved post-discectomy pain on mood one year after surgery. Conclusion: Genomic analysis of longitudinal studies of pain, depression, and anxiety in patients undergoing pain-relieving surgery may help to identify molecules through which pain alters mood. Detection of alleles with modest-sized effects will require larger cohorts. Page 1 of 15 (page number not for citation purposes) Molecular Pain 2006, 2:14 http://www.molecularpain.com/content/2/1/14 drugs than those effective in pain-free patients. This Background Decades of cross-sectional surveys have shown that hypothesis is based on the neuroanatomical finding that chronic pain, depression, and anxiety often coexist. How- spinal cord and brainstem pain-signaling neurons project ever, data derived from a single time point is consistent via the parabrachial and solitary nuclei to densely inner- with diverse causal links [1] e.g., that (1) pain causes vate the hypothalamus, amygdala, nucleus accumbens, mood or anxiety disorders; (2) these affective disorders medial orbital cortex, cingulum, and other brain struc- increase pain; (3) a common biological predisposition tures mediating mood [11], and the clinical observation underlies both pain and affective disorders; or (4) pain or that the presence of pain renders depressed patients rela- affective disorder do not directly cause the other but fre- tively resistant to antidepressant drugs [12]. We predict quently associate with a "true" causal variable such as that genetic analysis of inter-individual variability of pain- somatization, occupational or social stress, or ineffective related mood change (Fig. 1) will identify novel therapeu- coping style. tic targets in these neural connections, whose molecular components are just beginning to be defined [13]. Recent reports have more directly examined the direction of causation by assessing pain and mood over time in An economical approach to searching for these molecular thousands of individuals. In primary care practices and mediators is to piggyback on existing longitudinal studies diverse occupational settings, mood or anxiety disorder at of painful diseases caused by definite structural lesions baseline predicts the subsequent onset of any chronic that include serial pain measurements and standard qual- pain syndrome [2]; chronic widespread pain [3]; or ity of life questionnaires. Most such questionnaires assess chronic low back [4], neck [5], abdominal [6] or shoulder, depression and anxiety. Perturbations of affect on these arm, or knee pain [7]. Chronic pain at baseline predicts measures, although not diagnostic of clinical disorder, later anxiety or depressive symptoms [2]. Treatment of provide a convenient assessment of these negative emo- depression in patients with osteoarthritis reduces pain tions. To pursue this approach, we collected DNA from one year later [8]. former participants in a large study of surgical and non- operative treatment of sciatica caused by intervertebral Although these studies have strengthened the evidence for disc herniation [14,15]. Discectomy variably relieves bidirectional causal links between pain and mood, the patient's pain with effects occurring soon after a uniform designs are not suited for inferring physiological mecha- time point, creating a quasi-experimental design for test- nisms. A crucial limitation is that idiopathic "central pain ing the effects of pain on later mood. In this sciatica study amplification" or "multisomatoform" conditions [9] were and others ([16] and RR Edwards et al., in preparation), mixed with conditions in which a measurable structural baseline mood accounts for only a small component of injury dominates the pain phenotype. To optimize mech- the pain relief afforded by discectomy, so one can get a anistically-oriented clinical studies one should collect rel- clearer look at the effect of pain on later mood. atively homogeneous patient samples and assess putative physiological mediators. Predominantly structurally In this paper we present a descriptive analysis of mood determined pains may have a different causal relationship during the year following surgery, and illustrate a method to mood than multisomatoform pains. Cohorts with pain for detecting genetic polymorphisms that predispose to caused by common and measurable structural lesions like pain-influenced mood and or anxiety disorders. This acute surgical wounds or degenerative joint disease would approach adapts a gene-environment interaction model be expected to resemble the general population in preva- that has been proposed to facilitate the detection of genes lence of previous affective disorder. In patients with mul- that predispose to psychiatric disorders in the presence of tisomatoform pains, however, lifetime prevalence of specific quantified stressors, such as the interaction of depressive and anxiety disorders is triple that of the gen- serotonin transporter polymorphisms and life stress to eral population [10]. In these patients, some feature of influence depression [17], and of monoamine oxidase brain physiology may predispose to both pain and mood polymorphism and parental abuse to influence conduct disorders, and it may be more challenging to tease out disorder [18]. In the current paper, we are defining the causal relations between pain and affect. lumbar spine, nerve root, and its pain input to the central nervous system as part of the "environment" in which the We propose a method to investigate the following brain generates an affective state. hypothesis: Depression and anxiety triggered or worsened by pain are mediated by anatomical and neurochemical links that Methods differ in part from those mediating depression and anxiety dis- Patients orders that occur independent of pain. A corollary is that opti- Participants were members of the sciatica group of the mal treatment of the pain patient's mood disorder might Maine Lumbar Spine Study (MLSS [14]), a prospective require different types of antidepressant or anxiolytic cohort study conducted by approximately half of Maine's Page 2 of 15 (page number not for citation purposes) Molecular Pain 2006, 2:14 http://www.molecularpain.com/content/2/1/14 Baseline depression/anxiety Delayed surgery Workers’ compensation Baseline pain Herniated Disc Depression, Pain at 3-6 Discectomy Back + Leg Pain Anxiety at 1 yr months median ~ 3 mos Pain-Mood Genes Mood Genes H Figure 1 ypothesis regarding variability in depression and anxiety observed one year after lumbar discectomy Hypothesis regarding variability in depression and anxiety observed one year after lumbar discectomy. Two types of genetic contribution to one-year mood scores are shown. (1) Mood genes currently studied by biological psychia- trists may contribute to the late mood effects of a stressful surgical illness. Because the diagnosis and treatment are shared by all participants, and one can measure residual pain and surgical delay as "environmental variables" and correct for their effect on moo, this design may enhance the sensitivity to detect gene effects, compared to designs that study affective disorders in patients with widely varying life stressors. These influences would show up in the statistical analysis as main effects on late mood. (2) Pain-mood genes may alter the direct effects of pain upon mood, possibly by effects on signaling molecules in the dense connections between spinal pain afferent inputs and mood-processing brain structures such as hypothalamus, amygdala, nucleus accumbens, medial orbital cortex, and cingulum. These gene effects would vary with the amount of residual chronic pain after surgery; i.e., they would show up as significant interactions between gene and pain levels in their effects upon mood. orthopedists and neurosurgeons who actively treat spine this group was smaller, often lacked confirmatory spine disease [19]. Patients were enrolled between 1990 and imaging studies, and was probably more heterogeneous 1992, and surgical or nonsurgical treatment was deter- with regard to underlying pathology and treatment. More- mined by clinician judgment and patient preference. over, their changes in pain during the early months of the Patients completed questionnaires at study entry, after 3, study were much smaller and more gradual than the sur- 6, and 12 months, and then annually through year 10. gical group [15]. This temporal course does not offer as Individuals who initially embarked on nonsurgical treat- clear an experimental model as the abrupt one-time surgi- ment but elected surgery at or before month 6 were cal perturbation of pain. included in the surgical group because recent data was available to serve as a baseline. Patients who crossed over Pain measure to surgery after month 6 were not included in this study The primary measure of pain for this pain-mood study because the "baseline" may have occurred 6–12 months was the Bodily Pain intensity item on the Short-Form-36 preoperatively. After completion of the 10-year study, the (SF-36) quality of life instrument [20] at baseline, 3, and NIDCR and MLSS investigators developed a collaboration 6 months. Patients responded to the question "How to collect DNA from consenting patients, under a protocol much bodily pain have you had during the past 4 weeks?" approved by the NIDCR Institutional Review Board. Of by choosing from "very severe," "severe," "moderate," the 277 patients treated surgically, 162 contributed DNA. "mild," "very mild," and "none." This has been a standard We also collected DNA from 118 patients treated nonsur- scale used in analgesic clinical trials for more than 50 gically and included them in our analysis of pain and years. As part of the SF-36, the scale has been shown to be mood scores at baseline. We did not include nonsurgical reliable and to be sensitive to changes in pain produced by patients in genetic analyses of late mood change because joint replacement [20]. Page 3 of 15 (page number not for citation purposes) Molecular Pain 2006, 2:14 http://www.molecularpain.com/content/2/1/14 Depression and anxiety measures chemokine receptor 5; purinergic receptor ligand-gated The mood measure was the Mental Health (MH) subscale ion channel P2X4; calcium channel, voltage-dependent, of the SF-36 health survey. This subscale includes three α2/δ subunit 2; and chemokine (C-X-C motif) ligand 5 Likert scale items about the frequency in the previous (ENA-CXCL5). month of depressed vs. happy moods, and two items about the frequency of anxious vs. peaceful moods, each Genotyping methods SNP markers with 6 possible responses ranging from "all of the time" to "none of the time." Because depressive and anxious symp- The physical position and frequency of minor alleles toms usually coexist in medically ill patients, the develop- (>0.05) from a commercial database (Celera Discovery ers of the scale combined the items into a single score, System, CDS) were used to select SNPs spaced at 2–5 kb which correlates closely with DSM-IV psychiatric diag- intervals throughout each gene region plus 4–6 kb noses [21]. upstream and 4–6 kb downstream of each gene. Allele fre- quencies of markers and their locations in the genes Choice of mood candidate genes described in the Results appear in Additional file 1. In order to control the false-positive error rate in this modestly sized sample, prior to data analyses we chose Genomic DNA three high-priority candidate polymorphisms that we pre- Genomic DNA was extracted from lymphoblastoid cell dicted would be associated with pain-related mood dete- lines and diluted to a concentration of 5 ng/µl. Two-µl rioration: aliquots were dried in 384-well plates. (1) The met allele at the val158met polymorphism in the Polymerase Chain Reaction (PCR) amplification catechol-O-methyltransferase gene (COMT) reduces the Genotyping was performed by the 5' nuclease method ability of the enzyme to metabolize catecholamines, and [30] using fluorogenic allele-specific probes. Oligonucle- has been associated with variability in an experimentally otide primer and probe sets were designed based on gene evoked pain threshold and unpleasant pain-related affect sequences from the CDS. Reactions were performed in a 5 [22] and with anxiety disorders [23]. µl volume containing 2.25 µl TE (ABI Assays On Demand) or 2.375 µl TE (ABI Assays By Design), 2.5 µl (2) The short allele in the intron 2 tandem repeat poly- PCR Master Mix (ABI, Foster City, CA), 10 ng genomic morphism of the serotonin transporter gene (SLC6A4) DNA, 900 nM of each forward and reverse primer, and lowers the level of expression of the transporter protein 100 nM of each reporter and quencher probe. DNA was [24] and alters cerebral processing of fear stimuli [25]. incubated at 50°C for 2 min and at 95°C for 10 min, and This allele has been associated with neuroticism and the amplified on an ABI 9700 device for 40 cycles at 92°C risk of lifetime major depression [26]; and with depres- (ABI Assays on Demand) or 95°C (ABI Assays By Design) sion related to stressful life events [18]. for 15 s and 60°C for1 min. Allele-specific signals were distinguished by measuring endpoint 6-FAM or VIC fluo- (3) The met allele of the val66met polymorphism in the rescence intensities at 508 nm and 560 nm, respectively, brain-derived neurotrophic factor gene (BDNF) lowers and genotypes were generated using Sequence Detection activity-induced secretion of this trophic factor. The met System V.1.7 (ABI). Genotyping error rate was directly allele has been associated with geriatric depression [27] determined by re-genotyping 25% of the samples, ran- and anxious temperament [28]. domly chosen, for each locus. The overall error rate was <0.005. Genotype completion rate was 0.96. In addition to the a priori selection of three putative mood Inference of haplotypes genes, we carried out exploratory analyses of polymor- phisms in 25 additional genes that we had previously gen- Haplotype phases – i.e., how the directly measured SNP otyped for pain genetics studies. We recognized that alleles were distributed into two chromosomes in each correction for multiple testing a cohort of several hundred patient – were inferred by the expectation-maximization patients [29] might render these analyses suitable only for (EM) algorithm (SAS/Genetics, Cary, North Carolina, generating hypotheses for future study. The genes were: USA). galanin; galanin receptors 1, 2, and 3; interleukin (IL)-1α and β; IL-1 receptor antagonist; IL-6; IL-10; IL-13; tumor Statistical analysis necrosis factor α; adrenergic receptors 2A, 2B, and 2C; mu We specified as our primary analysis the test of the inter- opioid receptor; glial cell derived neurotrophic factor action between a (1) particular genetic polymorphism (GDNF); tyrosine hydroxylase; kainate-3 glutamate recep- and (2) the mean bodily pain at the 3 and 6 month post- tor; downstream regulatory element antagonistic modula- operative time points in predicting the SF-36 MH at 12 tor (DREAM); bradykinin receptors B1 and B2; months. The secondary analysis was the same analysis car- Page 4 of 15 (page number not for citation purposes) Molecular Pain 2006, 2:14 http://www.molecularpain.com/content/2/1/14 ried out on the baseline SF-36 MH score, examining the (about 13% of variance), receiving workers' compensa- effect of genotype, baseline pain score, and their interac- tion (5% of variance), prolonged pain prior to presenta- tion. This latter analysis included patients who elected tion (3% of variance), and younger age (1% of variance) non-surgical treatment as well. If there is no interaction, were significantly associated with more symptoms of the functions describing bodily pain as a contributor to depression and anxiety over the month before presenta- mood (i.e., regression of mood on bodily pain) are the tion. same across the different SNP genotypes. The initial selec- tion of covariates from the MLSS dataset was based upon Time course of mood after successful or unsuccessful discectomy the chronic pain literature and winnowed by modeling of 153 surgical patients provided baseline and 3 month the data prior to analyzing for genotypes. We pre-specified questionnaires and DNA. We divided these patients into the additive model as most plausible for the effects of 0, four groups according to the percent reduction they 1, or 2 copies of a polymorphism on mood but also car- reported on the 0–5 point SF-36 bodily pain intensity ried out analyses of dominant and recessive models. from pre-surgical baseline to the first postoperative obser- vation at 3 months: 75–100%, 50–75%, 25–50%, and ≤ Results 25%. Fig 3a (top left) shows the time course of bodily pain Relation between pain and mood intensity over 3 years for the four quartiles. Fig 3b (top Baseline pain and mood right) shows the time course of SF-36 MH; and Figs 3c and Fig 2 shows the relation at study baseline between the SF- 3d (bottom) break this down into the items of the SF-36 36 bodily pain intensity question and the SF-36 MH sub- MH relating to depression and anxiety, considered sepa- scale (top panel) and its items assessing depression (mid- rately. dle) and anxiety (bottom) in 276 patients, including those who subsequently underwent surgical or non-surgical The baseline points in Figs 3b–d show little difference in treatment. Consistent with the literature, patients' scores mood between the groups at baseline, suggesting that fac- on anxiety and depression items were closely correlated (r tors other than baseline mood account for most of the = 0.68, p < 0.001). Table 1 shows the results of a regres- large inter-individual differences in surgical relief of pain. sion analysis of contributions to the variance in baseline At the three-month time point, the three groups of symptoms of depression and anxiety. High baseline pain patients with the greatest reduction of pain from baseline Table 1: Contributions of the variables to baseline SF-36 Mental Health scores Correlation (r) with Quantitative variables Mean STD Baseline SF36-MH R p-value Baseline bodily pain 3.67 1.12 -0.36 0.130 <0.0001 Age 41.8 10.6 0.15 0.023 0.0121 Baseline SF36-MH Categorical variables Category n Mean STD Sex Male 166 61.25 19.88 0.002 0.5065 Female 110 62.95 19.35 Workers' compensation Yes 105 56.15 20.53 0.052 0.0001 No 171 65.47 18.27 Length of episode <=6 week 54 68.52 18.99 0.033 0.0109 6–26 weeks 133 61.29 17.95 >26 weeks 89 58.88 21.68 0.006 0.2024 Comorbid illnesses Yes 69 59.36 19.81 No 207 62.78 19.58 R from the model including all above variables 0.227 Note: R was calculated using the model including only one variable. SF-36 MH scores range from 0–100, where 100 means "always happy and calm over past month." 280 (surgical+ non-surgical) patients have DNA, four patients, 3 missing baseline SF36-MH and 1 missing baseline bodily pain, were dropped from analysis In covariate selection, linear regression with backward selection method (p = 0.1) was applied, where baseline Sf36-MH was dependent variables, age, sex, workers' compensation, baseline bodily pain, comorbid illnesses, patient group (nonsurgical vs. surgical), marital status, education, and prior episodes were independent variables, and age, sex and workers' compensation were fixed in the model Page 5 of 15 (page number not for citation purposes) Molecular Pain 2006, 2:14 http://www.molecularpain.com/content/2/1/14 Baseline mood vs. Figure 2 pain in 277 patients with subacute sciatica, regardless of subsequent surgical or nonsurgical treatment Baseline mood vs. pain in 277 patients with subacute sciatica, regardless of subsequent surgical or nonsurgical treatment. Overall intensity of "bodily pain" over the month before seeing a surgeon for sciatica explains about 13% of the variance in depressive and anxious feelings over the same period (p < 0.0001), assessed by the five-item SF-36 Mental Health Subscale (top panel). Higher scores on the y axis correspond to better mood. The other panels show similar relations to pain of the three items of the subscale pertaining to depressed mood (middle) and the two items pertaining to anxiety (bottom). Page 6 of 15 (page number not for citation purposes) Molecular Pain 2006, 2:14 http://www.molecularpain.com/content/2/1/14 Pain and Figure 3mood over time after surgical discectomy Pain and mood over time after surgical discectomy. In each panel, the four curves represent subgroups of 153 surgical patients divided according to the percentage reduction in "bodily pain" from the baseline to the three month rating. Top left: Bodily pain plotted against time over three years postoperatively. Top right: SF-36 Mental Health scores plotted against time. Higher values on the y axis correspond to less depressed or anxious feelings. Note that mood sharply improves at the first postoperative point in the three subgroups of patients with 25–100% reduction in pain, but mood does not worsen in the group with minimal pain relief. Bottom: The items specifically related to depression (left) and anxiety (right) show similar rela- tions to pain reduction. reported an immediate improvement in mood. Surpris- Genetic analysis of relation between pain and mood ingly, the quartile of patients with the least improvement Chi-square tests showed that all SNPs used in the study were in Hardy-Weinberg equilibrium. No polymorphism in pain reported, on average, a small improvement in mood from 6 to 24 months. in any of the three prespecified mood candidates, COMT, BDNF, and 5HTT had a significant main effect on 12- However, patients demonstrated considerable variability month mood, or a significant gene-pain interaction on in the relationship between pain and mood. Fig 4 shows mood. None of the other 25 genes we examined showed that there is a large amount of individual variability in a strong enough association with the mood endpoint to mood change from baseline to one year at each level of remain significant after correction for the multiple candi- acute surgical pain improvement. Table 2 shows the date genes, and where appropriate, for multiple analysis results of a regression analysis of SF-36 MH scores at 12 models, or multiple SNPs within one gene. In order to months. More intense pain at the 3 and 6 month time illustrate the method, however, we show the results for points (about 20% of variance), baseline depression and the galanin-2 receptor. Three of the four SNPs tested in the anxiety (19% of variance), "crossing over" to surgery sev- gene (Fig. 5) showed uncorrected p values of 0.003 to eral months after choosing nonsurgical treatment at base- 0.008 for a recessive model of interaction with 3–6 month line (8% of variance), receiving workers' compensation at pain to explain variance in 12-month mood. An analysis baseline (4% of variance), and more intense pain at base- of a haplotype incorporating these SNPs showed a nomi- line (1% of variance) were associated with more anxiety nally significant interaction term (p = .01). However, a and depression at 12 months. nominal p value < 0.001 would have been necessary to Page 7 of 15 (page number not for citation purposes) Molecular Pain 2006, 2:14 http://www.molecularpain.com/content/2/1/14 Individual variation tion produ Figure 4 ced by surg in improvement in ery mood over first year after discectomy plotted against the percent of bodily pain reduc- Individual variation in improvement in mood over first year after discectomy plotted against the percent of bodily pain reduction produced by surgery. Each point represents one patient. Table 2: Contributions of the variables to 1-year SF-36 Mental Health scores Correlation (r) with Quantitative variables Mean STD 1 year SF36-MH R p-value Mean bodily pain at 3 and 6 month 2.03 1.11 -0.450 0.203 <0.0001 Baseline SF36-MH 61.62 18.15 0.436 0.190 <0.0001 Baseline bodily pain 3.9 0.99 0.118 0.014 0.1642 Age 42.84 10.07 0.042 0.002 0.623 1 year SF36-MH Categorical variables Category n Mean STD Sex Male 86 74.88 20.34 0.001 0.724 Female 55 73.60 21.92 Workers' compensation Yes 42 68.00 25.05 0.040 0.018 No 99 77.09 18.44 Crossover 0 month 120 76.83 18.54 0.079 0.001 3 or 6 month 21 60.38 28.03 R from the model including all above variables 0.435 Note: R was calculated using the model including only one variable. In covariate selection, linear regression with backward selection method (p = 0.1) was applied, where 1 year SF-36 MH was the dependent variable; age, sex, workers' compensation, baseline bodily pain, prior episodes, and the SF-36 General Health, Vitality, Social Function, and Emotional Role subscales were independent variables; and age, sex and workers' compensation were fixed in the model. 141 of 162 surgical patients with both 1 year mood data and all 7 covariates and DNA were used in the 1 year mood analysis. "Crossover" refers to the time point when a questionnaire showed that a patient who initially chose nonsurgical treatment underwent lumbar discectomy. Page 8 of 15 (page number not for citation purposes) Molecular Pain 2006, 2:14 http://www.molecularpain.com/content/2/1/14 Ga Figure 5 lanin-2 receptor gene Galanin-2 receptor gene. Physical locations of the four genotyped single nucleotide polymorphisms (SNPs). Coding exons are shown as solid blocks. SNP locations are from the SNP Browser software and the Panther Classification System public database, February, 2006. P values for the effect on one-year SF-36 Mental Health scores of the SNP x 3–6 month pain interac- tion term are shown above each SNP. correct for the 28 candidate genes and multiple analysis significant after correction for multiple testing. However, models. With that caveat, we suggest that Fig. 6 illustrates three SNPs in the mu opioid receptor gene (Fig. 7) showed a pattern consistent with a neurochemical interaction nominally significant pain-gene interactions on baseline between pain and mood processing. In patients with 3–6 mood, with p values ranging from 0.006 to 0.02. Fig. 8 month pain levels of none to moderate (0–3), GAL2R shows that at baseline levels of pain from 0 – 3 (none- genotype did not appear to affect 12-month mood. In moderate), the relation between genotype and mood is patients with residual pain that was moderate/severe, similar. However, patients who are homozygous for the severe, or very severe (3.5–5), patients with one or two uncommon allele at rs495491 appear more susceptible to copies of the common allele of this SNP had greater emo- late emotional distress at high pain levels than patients tional distress (i.e., lower SF-36 Mental Health scores) with at least one copy of the common allele. than patients homozygous for the uncommon allele. Discussion Baseline mood scores These data illustrate an approach to investigating causal Results of genetic analyses for baseline mood were similar relationships among pain, mood, and genetic polymor- to those for the 12-month mood data. No polymorphism phisms patients who undergo a surgical procedure that in COMT, BDNF, and 5HTT had a significant main effect produces variable degrees of pain relief. The degree of sur- on baseline mood, or a significant gene-pain interaction gical relief of pain at the first two postoperative time on mood. None of the other 25 genes showed a strong points explains 20% of the variance in mood at 1 year (p enough association with the mood endpoint to remain <0.0001), but there is also additional inter-individual var- Page 9 of 15 (page number not for citation purposes) Molecular Pain 2006, 2:14 http://www.molecularpain.com/content/2/1/14 Patte Figure 6 rn of interaction between galanin-2 receptor SNP and residual pain upon 12-month mood score Pattern of interaction between galanin-2 receptor SNP and residual pain upon 12-month mood score. SNP rs8836 showed a significant interaction term in the recessive model (p = 0.003, uncorrected for multiple tests). The 12 month SF-36 MH mood scores are plotted against the mean of bodily pain scores at 3 and 6 mos. Each point is the mean 12-month mood for all of the patients with that bodily pain score and genotype. The curve connecting the triangles represents 33 patients homozygous for the uncommon allele; the curve connecting the filled circles represents 93 patients with one or no copies of the uncommon allele. In the presence of high residual postoperative pain, 2 copies of the uncommon allele appear to be associated with relative protection against symptoms of depression and anxiety, but the small numbers of such patients make this result illustrative, not statistically persuasive. iation in mood (Fig. 4), some of which may result from with sample size. For example, just an eight-fold increase inherited genetic variation. Because surgical relief of pain in N permits a million-fold increase in independent tests, is a large abrupt change occurring at a fixed time point, sufficient to examine the genome in detail [29]. this experimental design is well-suited for studying physi- ological events over time. Another limitation of this study is the lack of clinical diag- nosis of anxiety or depressive disorder. Across cohorts of Although the galanin-2 receptor and mu opioid receptor patients, the SF-36 MH subscale correlates strongly with are plausible candidates to mediate effects of pain on research psychiatric diagnoses and changes with success- mood, and showed graphical patterns consistent with a ful treatment, but cannot provide individual diagnoses pain-gene interaction, we cannot prove these specific [20]. The SF-36 pain measure does not have optimal pre- effects because of our modest sample size. Although this cision either, for estimating the actual pain level over was the largest prospective study of pain from a uniform many months. Bellamy et al. [31] found that a 0–10 point lesion that we could identify at the outset of the project, a numerical scale and 100 mm VAS were more sensitive several hundred patient cohort does not provide sufficient than a 5-category pain intensity scale, while Jensen and power to correct for the tests of dozens of genes and mul- McFarland [32] reported that the average of 7 pain meas- tiple analytical models unless the relative risk conferred urements at different times gives a better estimate of by the polymorphism is more than 2.5, larger than most actual pain than 1–2 measurements. Quality of life common polymorphisms for which a link to medical dis- researchers are currently seeking to improve diagnostic eases have been established [29]. However, major medical precision with computerized adaptive algorithms that centers perform thousands of many types of pain-reliev- choose items to hone in on each subject's response range. ing operations each year, making possible more powerful searches of this type. The number of simultaneous statis- It is possible that some of patients with the most severe tical tests supported by candidate studies increases steeply mood disorder may have refused to return questionnaires Page 10 of 15 (page number not for citation purposes) Molecular Pain 2006, 2:14 http://www.molecularpain.com/content/2/1/14 Figure 7 Mu opioid receptor gene Mu opioid receptor gene. Physical locations of the eight genotyped single nucleotide polymorphisms (SNPs). Coding exons are shown as solid blocks. SNP locations are from SNP Browser software and the Panther Classification System public database, February, 2006. P values for the effect on one-year SF-36 Mental Health scores of the SNP x 3–6 month pain interaction term are shown above each SNP. SNP #1 m tge wekk-known Asn 40 Asp polymorphism, was not associated with mood scores. or contribute DNA, lessening the study's power to exam- humans [37]. The three galanin receptor subtypes, ine this link. Another possible gap is that in the 16 years GALR1, GALR2, and GALR3 [38], are widely distributed in since this study was begun, pain psychologists learned mood-related brain areas such as hypothalamus, central that styles of coping with pain and environmental stres- amygdaloid nucleus, and thalamus [39] and may mediate sors are as important determinants of many pain out- anxiety-associated behavior [40]. No common functional comes as mood. This study did not include detailed polymorphism has yet been identified in the human measurements of pain catastrophizing [33], pain self-effi- GALR2 gene. There is strong linkage disequilibrium (LD) cacy, and stressors in the work and personal environment between all SNP pairs we genotyped, and on the related [34], which one might consider for a new prospective chromosome region (HapMap; http://www.hapmap.org). study of pain and mood. Although the SNPs used in our study are not located within GALR2, they are within 5 kb of the start and end While we cannot exclude a false positive in this study, the sites of the gene (Fig 4), and are within a haplotype block possible mood mediating effects of the galanin-2 and mu encompassing GALR2, its regulatory elements, and neigh- opioid receptor polymorphisms should be studied in boring genes. The nominally significant pain-gene inter- additional cohorts of patients with pain. The neuropep- action on mood of three out of four SNPs in this region tide galanin is widely expressed in the central nervous sys- and the haplotype may reflect the contribution of a func- tem, including areas regulating emotionality [35]. It has tional allele in GALR2 or the genes located nearby. been implicated in a wide range of physiological func- tions including pain control and cognition and in behav- The mu opioid receptor is also a plausible candidate to iors such as anxiety and depression [36]. We recently mediate between pain and mood. The endogenous opioid reported that haplotypes in the galanin gene were associ- system and µ-opioid receptors modulate affective behav- ated with anxiety-associated alcoholic phenotypes in iors [41] as well as affective components of acute pain Page 11 of 15 (page number not for citation purposes) Molecular Pain 2006, 2:14 http://www.molecularpain.com/content/2/1/14 Figure 8 Pattern of interaction between mu opioid receptor SNP and baseline pain upon baseline mood score Pattern of interaction between mu opioid receptor SNP and baseline pain upon baseline mood score. SNP rs495491 was one of the three SNPs that showed a significant interaction term in the recessive model (p = 0.003, uncorrected for multiple tests). The baseline SF-36 MH mood scores are plotted against the baseline bodily pain scores. Each point is the mean baseline mood for all of the patients with that bodily pain score and genotype. The curve connecting the triangles represents 17 patients homozygous for the uncom- mon allele; the curve connecting the diamonds represents the 252 patients with one or no copies of the uncommon allele. In the pres- ence of high baseline pain, 2 copies of the uncommon allele appear to be associated with symptoms of depression and anxiety, but the modest sample size and multiple genes tested make this result illustrative, not statistically persuasive. Data from patients with baseline pain of 0–2 (none, very mild, or mild) were pooled because few patients had such low pain scores at presentation. [22]. Opioid treatment of chronic pain is often accompa- the genes for corticotropin-releasing hormone (CRH) nied by striking improvements in mood [42]. Although [45], the adenosine A(2A) receptor [46], the dopamine the human mu opioid receptor gene (OPRM1) has poly- D4 receptor [47], and tryptophan hydroxylase [48]. morphisms that affect receptor function and are associ- ated with some behavioral phenotypes [43], no It is interesting that among patients in the quartile with associations with depression and anxiety disorders have the least surgical pain relief, mood did not deteriorate, yet been reported [44]. The three OPRM1 SNPs most and actually improved from 6 months on. Within this closely associated with pain-related mood scores in our quartile of patients (Fig. 4), for example, pain levels at 24 study are in high linkage disequilibrium and in the same months after surgery were equivalent to the presurgery haploblock, which also includes the previously reported levels, but mood at 24 months was considerably functional non-synonymous SNP Asn40Asp (rs1799971). improved from baseline. It would be interesting to exam- However, the latter SNP was not associated with mood ine individual differences in resilience factors, including scores. Therefore we assume that the association signal, if genetic resilience factors, that are associated with replicated, could be attributable to another functional improvements in mood in the face of the surgical failure allele that is still unknown. to relieve pain. In this study, we have tested mood effects of genes that we The preceding analysis of gene effects mediating mood had already genotyped based on their involvement in responses to unrelieved pain in surgical patients is a gene chronic pain processing. For future studies on pain-mood × environment (G × E) interaction study. Thus far, the interaction, one might select additional candidate genes most widely cited examples of G × E interactions on reportedly associated with anxiety and depression; e.g., behavioral endpoints [17,18] involve an environmental Page 12 of 15 (page number not for citation purposes) Molecular Pain 2006, 2:14 http://www.molecularpain.com/content/2/1/14 stress. Although our most suggestive results for the genes Authors' contributions we examined pertained to unique pain-gene interactions MBM initiated and supervised the clinical genetics study, on mood, the same analytic method might help to iden- proposed the method, and drafted the manuscript. TW tify other genes that have a general effect on mood, dem- designed the specific statistical method and carried out onstrated by a main effect in the regression. A potential the analyses. AK advised on statistical analyses. SJA was advantage of this study design is that patients are all sub- co-PI of the original clinical study, contributed the clinical ject to a similar stress – several months of a painful condi- data, and advised on the analysis. RBK was co-PI of the tion followed by major surgery – and then experience original clinical study, contributed the clinical data, and variable levels of residual pain, a stressor that can be well- advised on the DNA collection. RRE and JAH advised on quantified and corrected for in the analysis. Without the analyses and interpretation of psychological data. detailed measurements of environmental stress, it may be AFB, HH, CDM, IAO, ENC, JN, DCA, and MAS did the difficult to identify genes for depression or anxiety. For bioinformatics work and genotyping. IB trained and example, although twin studies suggest that the heritabil- supervised the bioinformatics and genotyping team, inter- ity of unipolar major depression disorder is 40–50% [49] preted this data, and wrote the molecular parts of the it has been difficult to replicate candidate gene associa- Methods and Discussion. MP advised on psychiatric tions, possibly because the environmental circumstances aspects of the manuscript. DG provided laboratory facili- triggering depression are so varied [50]. A major surgical ties, trained the authors in bioinformatics and genotyp- procedure may provide a somewhat uniform stressful sit- ing, and advised on the methods and interpretation. All uation. Within this setting, the two factor analysis shown authors read and approved the final manuscript. above may remove a large amount of variance in late mood due to pain, increasing sensitivity to a main effect Additional material of a genetic polymorphism involved in general mood-reg- ulating mechanisms not specific to patients with pain. Additional File 1 Galanin receptor-2 gene polymorphisms and 1 year SF-36-MH It is possible that alternative statistical methods may bet- Click here for file ter detect "mood" or "pain-mood" genes in the types of [http://www.biomedcentral.com/content/supplementary/1744- surgical cohort studies illustrated above. For example, 8069-2-14-S1.doc] patients without any pain might be excluded from the pain-mood analyses, or different time-points might be chosen for the pain and mood variables. The relative mer- its of alternative methods can be compared more persua- Acknowledgements Supported by NIDCR Intramural Grant DE00366 (MBM) and NIAAA Intra- sively once a robust gene effect is identified to use as a mural Grant Z01AA000301 (DG). The Maine Lumbar Spine Study was sup- gold standard. ported by grants from the Agency for Healthcare Research and Quality (HS-06344, HS-08194, and HS-09804). SJA (P60 AR048094) and RRE (K23 Conclusion AR051315) is supported in part by funding from the National Institute of A better understanding of the specific mechanisms linking Arthritis and Musculoskeletal and Skin Diseases. JAH is supported in part pain and mood may open up a set of new interventions to by funding from the National Institute of Neurological Disorders and decrease the morbidity of chronic pain conditions. Basic Stroke (K24 NS02225). We thank Yen A. Wu, MPH for preparing the clin- science studies in animals [13,50] including microarray ical dataset, Ann Scher for suggestions on data presentation, and Suzan search methods are likely to yield a long list of candidates. Khoromi and Beata Buzas for reviewing the manuscript. The funding bodies had no input into the design or publication of the current study. We have demonstrated a simple method to search for genetic polymorphisms that contribute to interindividual References variation in the manner in which pain produces depres- 1. Robinson ME, Riley JL: The role of emotion in pain. In Psychosocial sion or anxiety, using cohorts of patients who have under- Factors in Pain: Critical Perspectives Edited by: Gatchel RJ, Turk DC. gone a surgical procedure that variably relieves pain. New York: Guilford Press; 1999:74-88. 2. Gureje O, Simon GE, Von Korff M: A cross-national study of the Cross-correlation with the results of the types of human course of persistent pain in primary care. 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A clinical genetic method to identify mechanisms by which pain causes depression and anxiety

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Springer Journals
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Copyright © 2006 by Max et al; licensee BioMed Central Ltd.
Subject
Medicine & Public Health; Pain Medicine; Molecular Medicine; Neurobiology
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1744-8069
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1744-8069
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10.1186/1744-8069-2-14
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16623937
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Abstract

Background: Pain patients are often depressed and anxious, and benefit less from psychotropic drugs than pain-free patients. We hypothesize that this partial resistance is due to the unique neurochemical contribution to mood by afferent pain projections through the spino-parabrachial-hypothalamic-amygdalar systems and their projections to other mood-mediating systems. New psychotropic drugs for pain patients might target molecules in such brain systems. We propose a method to prioritize molecular targets by studying polymorphic genes in cohorts of patients undergoing surgical procedures associated with a variable pain relief response. We seek molecules that show a significant statistical interaction between (1) the amount of surgical pain relief, and (2) the alleles of the gene, on depression and anxiety during the first postoperative year. Results: We collected DNA from 280 patients with sciatica due to a lumbar disc herniation, 162 treated surgically and 118 non-surgically, who had been followed for 10 years in the Maine Lumbar Spine Study, a large, prospective, observational study. In patients whose pain was reduced >25% by surgery, symptoms of depression and anxiety, assessed with the SF-36 Mental Health Scale, improved briskly at the first postoperative measurement. In patients with little or no surgical pain reduction, mood scores stayed about the same on average. There was large inter-individual variability at each level of residual pain. Polymorphisms in three pre-specified pain-mood candidate genes, catechol-O-methyl transferase (COMT), serotonin transporter, and brain-derived neurotrophic factor (BDNF) were not associated with late postoperative mood or with a pain-gene interaction on mood. Although the sample size did not provide enough power to persuasively search through a larger number of genes, an exploratory survey of 25 other genes provides illustrations of pain-gene interactions on postoperative mood – the mu opioid receptor for short-term effects of acute sciatica on mood, and the galanin-2 receptor for effects of unrelieved post-discectomy pain on mood one year after surgery. Conclusion: Genomic analysis of longitudinal studies of pain, depression, and anxiety in patients undergoing pain-relieving surgery may help to identify molecules through which pain alters mood. Detection of alleles with modest-sized effects will require larger cohorts. Page 1 of 15 (page number not for citation purposes) Molecular Pain 2006, 2:14 http://www.molecularpain.com/content/2/1/14 drugs than those effective in pain-free patients. This Background Decades of cross-sectional surveys have shown that hypothesis is based on the neuroanatomical finding that chronic pain, depression, and anxiety often coexist. How- spinal cord and brainstem pain-signaling neurons project ever, data derived from a single time point is consistent via the parabrachial and solitary nuclei to densely inner- with diverse causal links [1] e.g., that (1) pain causes vate the hypothalamus, amygdala, nucleus accumbens, mood or anxiety disorders; (2) these affective disorders medial orbital cortex, cingulum, and other brain struc- increase pain; (3) a common biological predisposition tures mediating mood [11], and the clinical observation underlies both pain and affective disorders; or (4) pain or that the presence of pain renders depressed patients rela- affective disorder do not directly cause the other but fre- tively resistant to antidepressant drugs [12]. We predict quently associate with a "true" causal variable such as that genetic analysis of inter-individual variability of pain- somatization, occupational or social stress, or ineffective related mood change (Fig. 1) will identify novel therapeu- coping style. tic targets in these neural connections, whose molecular components are just beginning to be defined [13]. Recent reports have more directly examined the direction of causation by assessing pain and mood over time in An economical approach to searching for these molecular thousands of individuals. In primary care practices and mediators is to piggyback on existing longitudinal studies diverse occupational settings, mood or anxiety disorder at of painful diseases caused by definite structural lesions baseline predicts the subsequent onset of any chronic that include serial pain measurements and standard qual- pain syndrome [2]; chronic widespread pain [3]; or ity of life questionnaires. Most such questionnaires assess chronic low back [4], neck [5], abdominal [6] or shoulder, depression and anxiety. Perturbations of affect on these arm, or knee pain [7]. Chronic pain at baseline predicts measures, although not diagnostic of clinical disorder, later anxiety or depressive symptoms [2]. Treatment of provide a convenient assessment of these negative emo- depression in patients with osteoarthritis reduces pain tions. To pursue this approach, we collected DNA from one year later [8]. former participants in a large study of surgical and non- operative treatment of sciatica caused by intervertebral Although these studies have strengthened the evidence for disc herniation [14,15]. Discectomy variably relieves bidirectional causal links between pain and mood, the patient's pain with effects occurring soon after a uniform designs are not suited for inferring physiological mecha- time point, creating a quasi-experimental design for test- nisms. A crucial limitation is that idiopathic "central pain ing the effects of pain on later mood. In this sciatica study amplification" or "multisomatoform" conditions [9] were and others ([16] and RR Edwards et al., in preparation), mixed with conditions in which a measurable structural baseline mood accounts for only a small component of injury dominates the pain phenotype. To optimize mech- the pain relief afforded by discectomy, so one can get a anistically-oriented clinical studies one should collect rel- clearer look at the effect of pain on later mood. atively homogeneous patient samples and assess putative physiological mediators. Predominantly structurally In this paper we present a descriptive analysis of mood determined pains may have a different causal relationship during the year following surgery, and illustrate a method to mood than multisomatoform pains. Cohorts with pain for detecting genetic polymorphisms that predispose to caused by common and measurable structural lesions like pain-influenced mood and or anxiety disorders. This acute surgical wounds or degenerative joint disease would approach adapts a gene-environment interaction model be expected to resemble the general population in preva- that has been proposed to facilitate the detection of genes lence of previous affective disorder. In patients with mul- that predispose to psychiatric disorders in the presence of tisomatoform pains, however, lifetime prevalence of specific quantified stressors, such as the interaction of depressive and anxiety disorders is triple that of the gen- serotonin transporter polymorphisms and life stress to eral population [10]. In these patients, some feature of influence depression [17], and of monoamine oxidase brain physiology may predispose to both pain and mood polymorphism and parental abuse to influence conduct disorders, and it may be more challenging to tease out disorder [18]. In the current paper, we are defining the causal relations between pain and affect. lumbar spine, nerve root, and its pain input to the central nervous system as part of the "environment" in which the We propose a method to investigate the following brain generates an affective state. hypothesis: Depression and anxiety triggered or worsened by pain are mediated by anatomical and neurochemical links that Methods differ in part from those mediating depression and anxiety dis- Patients orders that occur independent of pain. A corollary is that opti- Participants were members of the sciatica group of the mal treatment of the pain patient's mood disorder might Maine Lumbar Spine Study (MLSS [14]), a prospective require different types of antidepressant or anxiolytic cohort study conducted by approximately half of Maine's Page 2 of 15 (page number not for citation purposes) Molecular Pain 2006, 2:14 http://www.molecularpain.com/content/2/1/14 Baseline depression/anxiety Delayed surgery Workers’ compensation Baseline pain Herniated Disc Depression, Pain at 3-6 Discectomy Back + Leg Pain Anxiety at 1 yr months median ~ 3 mos Pain-Mood Genes Mood Genes H Figure 1 ypothesis regarding variability in depression and anxiety observed one year after lumbar discectomy Hypothesis regarding variability in depression and anxiety observed one year after lumbar discectomy. Two types of genetic contribution to one-year mood scores are shown. (1) Mood genes currently studied by biological psychia- trists may contribute to the late mood effects of a stressful surgical illness. Because the diagnosis and treatment are shared by all participants, and one can measure residual pain and surgical delay as "environmental variables" and correct for their effect on moo, this design may enhance the sensitivity to detect gene effects, compared to designs that study affective disorders in patients with widely varying life stressors. These influences would show up in the statistical analysis as main effects on late mood. (2) Pain-mood genes may alter the direct effects of pain upon mood, possibly by effects on signaling molecules in the dense connections between spinal pain afferent inputs and mood-processing brain structures such as hypothalamus, amygdala, nucleus accumbens, medial orbital cortex, and cingulum. These gene effects would vary with the amount of residual chronic pain after surgery; i.e., they would show up as significant interactions between gene and pain levels in their effects upon mood. orthopedists and neurosurgeons who actively treat spine this group was smaller, often lacked confirmatory spine disease [19]. Patients were enrolled between 1990 and imaging studies, and was probably more heterogeneous 1992, and surgical or nonsurgical treatment was deter- with regard to underlying pathology and treatment. More- mined by clinician judgment and patient preference. over, their changes in pain during the early months of the Patients completed questionnaires at study entry, after 3, study were much smaller and more gradual than the sur- 6, and 12 months, and then annually through year 10. gical group [15]. This temporal course does not offer as Individuals who initially embarked on nonsurgical treat- clear an experimental model as the abrupt one-time surgi- ment but elected surgery at or before month 6 were cal perturbation of pain. included in the surgical group because recent data was available to serve as a baseline. Patients who crossed over Pain measure to surgery after month 6 were not included in this study The primary measure of pain for this pain-mood study because the "baseline" may have occurred 6–12 months was the Bodily Pain intensity item on the Short-Form-36 preoperatively. After completion of the 10-year study, the (SF-36) quality of life instrument [20] at baseline, 3, and NIDCR and MLSS investigators developed a collaboration 6 months. Patients responded to the question "How to collect DNA from consenting patients, under a protocol much bodily pain have you had during the past 4 weeks?" approved by the NIDCR Institutional Review Board. Of by choosing from "very severe," "severe," "moderate," the 277 patients treated surgically, 162 contributed DNA. "mild," "very mild," and "none." This has been a standard We also collected DNA from 118 patients treated nonsur- scale used in analgesic clinical trials for more than 50 gically and included them in our analysis of pain and years. As part of the SF-36, the scale has been shown to be mood scores at baseline. We did not include nonsurgical reliable and to be sensitive to changes in pain produced by patients in genetic analyses of late mood change because joint replacement [20]. Page 3 of 15 (page number not for citation purposes) Molecular Pain 2006, 2:14 http://www.molecularpain.com/content/2/1/14 Depression and anxiety measures chemokine receptor 5; purinergic receptor ligand-gated The mood measure was the Mental Health (MH) subscale ion channel P2X4; calcium channel, voltage-dependent, of the SF-36 health survey. This subscale includes three α2/δ subunit 2; and chemokine (C-X-C motif) ligand 5 Likert scale items about the frequency in the previous (ENA-CXCL5). month of depressed vs. happy moods, and two items about the frequency of anxious vs. peaceful moods, each Genotyping methods SNP markers with 6 possible responses ranging from "all of the time" to "none of the time." Because depressive and anxious symp- The physical position and frequency of minor alleles toms usually coexist in medically ill patients, the develop- (>0.05) from a commercial database (Celera Discovery ers of the scale combined the items into a single score, System, CDS) were used to select SNPs spaced at 2–5 kb which correlates closely with DSM-IV psychiatric diag- intervals throughout each gene region plus 4–6 kb noses [21]. upstream and 4–6 kb downstream of each gene. Allele fre- quencies of markers and their locations in the genes Choice of mood candidate genes described in the Results appear in Additional file 1. In order to control the false-positive error rate in this modestly sized sample, prior to data analyses we chose Genomic DNA three high-priority candidate polymorphisms that we pre- Genomic DNA was extracted from lymphoblastoid cell dicted would be associated with pain-related mood dete- lines and diluted to a concentration of 5 ng/µl. Two-µl rioration: aliquots were dried in 384-well plates. (1) The met allele at the val158met polymorphism in the Polymerase Chain Reaction (PCR) amplification catechol-O-methyltransferase gene (COMT) reduces the Genotyping was performed by the 5' nuclease method ability of the enzyme to metabolize catecholamines, and [30] using fluorogenic allele-specific probes. Oligonucle- has been associated with variability in an experimentally otide primer and probe sets were designed based on gene evoked pain threshold and unpleasant pain-related affect sequences from the CDS. Reactions were performed in a 5 [22] and with anxiety disorders [23]. µl volume containing 2.25 µl TE (ABI Assays On Demand) or 2.375 µl TE (ABI Assays By Design), 2.5 µl (2) The short allele in the intron 2 tandem repeat poly- PCR Master Mix (ABI, Foster City, CA), 10 ng genomic morphism of the serotonin transporter gene (SLC6A4) DNA, 900 nM of each forward and reverse primer, and lowers the level of expression of the transporter protein 100 nM of each reporter and quencher probe. DNA was [24] and alters cerebral processing of fear stimuli [25]. incubated at 50°C for 2 min and at 95°C for 10 min, and This allele has been associated with neuroticism and the amplified on an ABI 9700 device for 40 cycles at 92°C risk of lifetime major depression [26]; and with depres- (ABI Assays on Demand) or 95°C (ABI Assays By Design) sion related to stressful life events [18]. for 15 s and 60°C for1 min. Allele-specific signals were distinguished by measuring endpoint 6-FAM or VIC fluo- (3) The met allele of the val66met polymorphism in the rescence intensities at 508 nm and 560 nm, respectively, brain-derived neurotrophic factor gene (BDNF) lowers and genotypes were generated using Sequence Detection activity-induced secretion of this trophic factor. The met System V.1.7 (ABI). Genotyping error rate was directly allele has been associated with geriatric depression [27] determined by re-genotyping 25% of the samples, ran- and anxious temperament [28]. domly chosen, for each locus. The overall error rate was <0.005. Genotype completion rate was 0.96. In addition to the a priori selection of three putative mood Inference of haplotypes genes, we carried out exploratory analyses of polymor- phisms in 25 additional genes that we had previously gen- Haplotype phases – i.e., how the directly measured SNP otyped for pain genetics studies. We recognized that alleles were distributed into two chromosomes in each correction for multiple testing a cohort of several hundred patient – were inferred by the expectation-maximization patients [29] might render these analyses suitable only for (EM) algorithm (SAS/Genetics, Cary, North Carolina, generating hypotheses for future study. The genes were: USA). galanin; galanin receptors 1, 2, and 3; interleukin (IL)-1α and β; IL-1 receptor antagonist; IL-6; IL-10; IL-13; tumor Statistical analysis necrosis factor α; adrenergic receptors 2A, 2B, and 2C; mu We specified as our primary analysis the test of the inter- opioid receptor; glial cell derived neurotrophic factor action between a (1) particular genetic polymorphism (GDNF); tyrosine hydroxylase; kainate-3 glutamate recep- and (2) the mean bodily pain at the 3 and 6 month post- tor; downstream regulatory element antagonistic modula- operative time points in predicting the SF-36 MH at 12 tor (DREAM); bradykinin receptors B1 and B2; months. The secondary analysis was the same analysis car- Page 4 of 15 (page number not for citation purposes) Molecular Pain 2006, 2:14 http://www.molecularpain.com/content/2/1/14 ried out on the baseline SF-36 MH score, examining the (about 13% of variance), receiving workers' compensa- effect of genotype, baseline pain score, and their interac- tion (5% of variance), prolonged pain prior to presenta- tion. This latter analysis included patients who elected tion (3% of variance), and younger age (1% of variance) non-surgical treatment as well. If there is no interaction, were significantly associated with more symptoms of the functions describing bodily pain as a contributor to depression and anxiety over the month before presenta- mood (i.e., regression of mood on bodily pain) are the tion. same across the different SNP genotypes. The initial selec- tion of covariates from the MLSS dataset was based upon Time course of mood after successful or unsuccessful discectomy the chronic pain literature and winnowed by modeling of 153 surgical patients provided baseline and 3 month the data prior to analyzing for genotypes. We pre-specified questionnaires and DNA. We divided these patients into the additive model as most plausible for the effects of 0, four groups according to the percent reduction they 1, or 2 copies of a polymorphism on mood but also car- reported on the 0–5 point SF-36 bodily pain intensity ried out analyses of dominant and recessive models. from pre-surgical baseline to the first postoperative obser- vation at 3 months: 75–100%, 50–75%, 25–50%, and ≤ Results 25%. Fig 3a (top left) shows the time course of bodily pain Relation between pain and mood intensity over 3 years for the four quartiles. Fig 3b (top Baseline pain and mood right) shows the time course of SF-36 MH; and Figs 3c and Fig 2 shows the relation at study baseline between the SF- 3d (bottom) break this down into the items of the SF-36 36 bodily pain intensity question and the SF-36 MH sub- MH relating to depression and anxiety, considered sepa- scale (top panel) and its items assessing depression (mid- rately. dle) and anxiety (bottom) in 276 patients, including those who subsequently underwent surgical or non-surgical The baseline points in Figs 3b–d show little difference in treatment. Consistent with the literature, patients' scores mood between the groups at baseline, suggesting that fac- on anxiety and depression items were closely correlated (r tors other than baseline mood account for most of the = 0.68, p < 0.001). Table 1 shows the results of a regres- large inter-individual differences in surgical relief of pain. sion analysis of contributions to the variance in baseline At the three-month time point, the three groups of symptoms of depression and anxiety. High baseline pain patients with the greatest reduction of pain from baseline Table 1: Contributions of the variables to baseline SF-36 Mental Health scores Correlation (r) with Quantitative variables Mean STD Baseline SF36-MH R p-value Baseline bodily pain 3.67 1.12 -0.36 0.130 <0.0001 Age 41.8 10.6 0.15 0.023 0.0121 Baseline SF36-MH Categorical variables Category n Mean STD Sex Male 166 61.25 19.88 0.002 0.5065 Female 110 62.95 19.35 Workers' compensation Yes 105 56.15 20.53 0.052 0.0001 No 171 65.47 18.27 Length of episode <=6 week 54 68.52 18.99 0.033 0.0109 6–26 weeks 133 61.29 17.95 >26 weeks 89 58.88 21.68 0.006 0.2024 Comorbid illnesses Yes 69 59.36 19.81 No 207 62.78 19.58 R from the model including all above variables 0.227 Note: R was calculated using the model including only one variable. SF-36 MH scores range from 0–100, where 100 means "always happy and calm over past month." 280 (surgical+ non-surgical) patients have DNA, four patients, 3 missing baseline SF36-MH and 1 missing baseline bodily pain, were dropped from analysis In covariate selection, linear regression with backward selection method (p = 0.1) was applied, where baseline Sf36-MH was dependent variables, age, sex, workers' compensation, baseline bodily pain, comorbid illnesses, patient group (nonsurgical vs. surgical), marital status, education, and prior episodes were independent variables, and age, sex and workers' compensation were fixed in the model Page 5 of 15 (page number not for citation purposes) Molecular Pain 2006, 2:14 http://www.molecularpain.com/content/2/1/14 Baseline mood vs. Figure 2 pain in 277 patients with subacute sciatica, regardless of subsequent surgical or nonsurgical treatment Baseline mood vs. pain in 277 patients with subacute sciatica, regardless of subsequent surgical or nonsurgical treatment. Overall intensity of "bodily pain" over the month before seeing a surgeon for sciatica explains about 13% of the variance in depressive and anxious feelings over the same period (p < 0.0001), assessed by the five-item SF-36 Mental Health Subscale (top panel). Higher scores on the y axis correspond to better mood. The other panels show similar relations to pain of the three items of the subscale pertaining to depressed mood (middle) and the two items pertaining to anxiety (bottom). Page 6 of 15 (page number not for citation purposes) Molecular Pain 2006, 2:14 http://www.molecularpain.com/content/2/1/14 Pain and Figure 3mood over time after surgical discectomy Pain and mood over time after surgical discectomy. In each panel, the four curves represent subgroups of 153 surgical patients divided according to the percentage reduction in "bodily pain" from the baseline to the three month rating. Top left: Bodily pain plotted against time over three years postoperatively. Top right: SF-36 Mental Health scores plotted against time. Higher values on the y axis correspond to less depressed or anxious feelings. Note that mood sharply improves at the first postoperative point in the three subgroups of patients with 25–100% reduction in pain, but mood does not worsen in the group with minimal pain relief. Bottom: The items specifically related to depression (left) and anxiety (right) show similar rela- tions to pain reduction. reported an immediate improvement in mood. Surpris- Genetic analysis of relation between pain and mood ingly, the quartile of patients with the least improvement Chi-square tests showed that all SNPs used in the study were in Hardy-Weinberg equilibrium. No polymorphism in pain reported, on average, a small improvement in mood from 6 to 24 months. in any of the three prespecified mood candidates, COMT, BDNF, and 5HTT had a significant main effect on 12- However, patients demonstrated considerable variability month mood, or a significant gene-pain interaction on in the relationship between pain and mood. Fig 4 shows mood. None of the other 25 genes we examined showed that there is a large amount of individual variability in a strong enough association with the mood endpoint to mood change from baseline to one year at each level of remain significant after correction for the multiple candi- acute surgical pain improvement. Table 2 shows the date genes, and where appropriate, for multiple analysis results of a regression analysis of SF-36 MH scores at 12 models, or multiple SNPs within one gene. In order to months. More intense pain at the 3 and 6 month time illustrate the method, however, we show the results for points (about 20% of variance), baseline depression and the galanin-2 receptor. Three of the four SNPs tested in the anxiety (19% of variance), "crossing over" to surgery sev- gene (Fig. 5) showed uncorrected p values of 0.003 to eral months after choosing nonsurgical treatment at base- 0.008 for a recessive model of interaction with 3–6 month line (8% of variance), receiving workers' compensation at pain to explain variance in 12-month mood. An analysis baseline (4% of variance), and more intense pain at base- of a haplotype incorporating these SNPs showed a nomi- line (1% of variance) were associated with more anxiety nally significant interaction term (p = .01). However, a and depression at 12 months. nominal p value < 0.001 would have been necessary to Page 7 of 15 (page number not for citation purposes) Molecular Pain 2006, 2:14 http://www.molecularpain.com/content/2/1/14 Individual variation tion produ Figure 4 ced by surg in improvement in ery mood over first year after discectomy plotted against the percent of bodily pain reduc- Individual variation in improvement in mood over first year after discectomy plotted against the percent of bodily pain reduction produced by surgery. Each point represents one patient. Table 2: Contributions of the variables to 1-year SF-36 Mental Health scores Correlation (r) with Quantitative variables Mean STD 1 year SF36-MH R p-value Mean bodily pain at 3 and 6 month 2.03 1.11 -0.450 0.203 <0.0001 Baseline SF36-MH 61.62 18.15 0.436 0.190 <0.0001 Baseline bodily pain 3.9 0.99 0.118 0.014 0.1642 Age 42.84 10.07 0.042 0.002 0.623 1 year SF36-MH Categorical variables Category n Mean STD Sex Male 86 74.88 20.34 0.001 0.724 Female 55 73.60 21.92 Workers' compensation Yes 42 68.00 25.05 0.040 0.018 No 99 77.09 18.44 Crossover 0 month 120 76.83 18.54 0.079 0.001 3 or 6 month 21 60.38 28.03 R from the model including all above variables 0.435 Note: R was calculated using the model including only one variable. In covariate selection, linear regression with backward selection method (p = 0.1) was applied, where 1 year SF-36 MH was the dependent variable; age, sex, workers' compensation, baseline bodily pain, prior episodes, and the SF-36 General Health, Vitality, Social Function, and Emotional Role subscales were independent variables; and age, sex and workers' compensation were fixed in the model. 141 of 162 surgical patients with both 1 year mood data and all 7 covariates and DNA were used in the 1 year mood analysis. "Crossover" refers to the time point when a questionnaire showed that a patient who initially chose nonsurgical treatment underwent lumbar discectomy. Page 8 of 15 (page number not for citation purposes) Molecular Pain 2006, 2:14 http://www.molecularpain.com/content/2/1/14 Ga Figure 5 lanin-2 receptor gene Galanin-2 receptor gene. Physical locations of the four genotyped single nucleotide polymorphisms (SNPs). Coding exons are shown as solid blocks. SNP locations are from the SNP Browser software and the Panther Classification System public database, February, 2006. P values for the effect on one-year SF-36 Mental Health scores of the SNP x 3–6 month pain interac- tion term are shown above each SNP. correct for the 28 candidate genes and multiple analysis significant after correction for multiple testing. However, models. With that caveat, we suggest that Fig. 6 illustrates three SNPs in the mu opioid receptor gene (Fig. 7) showed a pattern consistent with a neurochemical interaction nominally significant pain-gene interactions on baseline between pain and mood processing. In patients with 3–6 mood, with p values ranging from 0.006 to 0.02. Fig. 8 month pain levels of none to moderate (0–3), GAL2R shows that at baseline levels of pain from 0 – 3 (none- genotype did not appear to affect 12-month mood. In moderate), the relation between genotype and mood is patients with residual pain that was moderate/severe, similar. However, patients who are homozygous for the severe, or very severe (3.5–5), patients with one or two uncommon allele at rs495491 appear more susceptible to copies of the common allele of this SNP had greater emo- late emotional distress at high pain levels than patients tional distress (i.e., lower SF-36 Mental Health scores) with at least one copy of the common allele. than patients homozygous for the uncommon allele. Discussion Baseline mood scores These data illustrate an approach to investigating causal Results of genetic analyses for baseline mood were similar relationships among pain, mood, and genetic polymor- to those for the 12-month mood data. No polymorphism phisms patients who undergo a surgical procedure that in COMT, BDNF, and 5HTT had a significant main effect produces variable degrees of pain relief. The degree of sur- on baseline mood, or a significant gene-pain interaction gical relief of pain at the first two postoperative time on mood. None of the other 25 genes showed a strong points explains 20% of the variance in mood at 1 year (p enough association with the mood endpoint to remain <0.0001), but there is also additional inter-individual var- Page 9 of 15 (page number not for citation purposes) Molecular Pain 2006, 2:14 http://www.molecularpain.com/content/2/1/14 Patte Figure 6 rn of interaction between galanin-2 receptor SNP and residual pain upon 12-month mood score Pattern of interaction between galanin-2 receptor SNP and residual pain upon 12-month mood score. SNP rs8836 showed a significant interaction term in the recessive model (p = 0.003, uncorrected for multiple tests). The 12 month SF-36 MH mood scores are plotted against the mean of bodily pain scores at 3 and 6 mos. Each point is the mean 12-month mood for all of the patients with that bodily pain score and genotype. The curve connecting the triangles represents 33 patients homozygous for the uncommon allele; the curve connecting the filled circles represents 93 patients with one or no copies of the uncommon allele. In the presence of high residual postoperative pain, 2 copies of the uncommon allele appear to be associated with relative protection against symptoms of depression and anxiety, but the small numbers of such patients make this result illustrative, not statistically persuasive. iation in mood (Fig. 4), some of which may result from with sample size. For example, just an eight-fold increase inherited genetic variation. Because surgical relief of pain in N permits a million-fold increase in independent tests, is a large abrupt change occurring at a fixed time point, sufficient to examine the genome in detail [29]. this experimental design is well-suited for studying physi- ological events over time. Another limitation of this study is the lack of clinical diag- nosis of anxiety or depressive disorder. Across cohorts of Although the galanin-2 receptor and mu opioid receptor patients, the SF-36 MH subscale correlates strongly with are plausible candidates to mediate effects of pain on research psychiatric diagnoses and changes with success- mood, and showed graphical patterns consistent with a ful treatment, but cannot provide individual diagnoses pain-gene interaction, we cannot prove these specific [20]. The SF-36 pain measure does not have optimal pre- effects because of our modest sample size. Although this cision either, for estimating the actual pain level over was the largest prospective study of pain from a uniform many months. Bellamy et al. [31] found that a 0–10 point lesion that we could identify at the outset of the project, a numerical scale and 100 mm VAS were more sensitive several hundred patient cohort does not provide sufficient than a 5-category pain intensity scale, while Jensen and power to correct for the tests of dozens of genes and mul- McFarland [32] reported that the average of 7 pain meas- tiple analytical models unless the relative risk conferred urements at different times gives a better estimate of by the polymorphism is more than 2.5, larger than most actual pain than 1–2 measurements. Quality of life common polymorphisms for which a link to medical dis- researchers are currently seeking to improve diagnostic eases have been established [29]. However, major medical precision with computerized adaptive algorithms that centers perform thousands of many types of pain-reliev- choose items to hone in on each subject's response range. ing operations each year, making possible more powerful searches of this type. The number of simultaneous statis- It is possible that some of patients with the most severe tical tests supported by candidate studies increases steeply mood disorder may have refused to return questionnaires Page 10 of 15 (page number not for citation purposes) Molecular Pain 2006, 2:14 http://www.molecularpain.com/content/2/1/14 Figure 7 Mu opioid receptor gene Mu opioid receptor gene. Physical locations of the eight genotyped single nucleotide polymorphisms (SNPs). Coding exons are shown as solid blocks. SNP locations are from SNP Browser software and the Panther Classification System public database, February, 2006. P values for the effect on one-year SF-36 Mental Health scores of the SNP x 3–6 month pain interaction term are shown above each SNP. SNP #1 m tge wekk-known Asn 40 Asp polymorphism, was not associated with mood scores. or contribute DNA, lessening the study's power to exam- humans [37]. The three galanin receptor subtypes, ine this link. Another possible gap is that in the 16 years GALR1, GALR2, and GALR3 [38], are widely distributed in since this study was begun, pain psychologists learned mood-related brain areas such as hypothalamus, central that styles of coping with pain and environmental stres- amygdaloid nucleus, and thalamus [39] and may mediate sors are as important determinants of many pain out- anxiety-associated behavior [40]. No common functional comes as mood. This study did not include detailed polymorphism has yet been identified in the human measurements of pain catastrophizing [33], pain self-effi- GALR2 gene. There is strong linkage disequilibrium (LD) cacy, and stressors in the work and personal environment between all SNP pairs we genotyped, and on the related [34], which one might consider for a new prospective chromosome region (HapMap; http://www.hapmap.org). study of pain and mood. Although the SNPs used in our study are not located within GALR2, they are within 5 kb of the start and end While we cannot exclude a false positive in this study, the sites of the gene (Fig 4), and are within a haplotype block possible mood mediating effects of the galanin-2 and mu encompassing GALR2, its regulatory elements, and neigh- opioid receptor polymorphisms should be studied in boring genes. The nominally significant pain-gene inter- additional cohorts of patients with pain. The neuropep- action on mood of three out of four SNPs in this region tide galanin is widely expressed in the central nervous sys- and the haplotype may reflect the contribution of a func- tem, including areas regulating emotionality [35]. It has tional allele in GALR2 or the genes located nearby. been implicated in a wide range of physiological func- tions including pain control and cognition and in behav- The mu opioid receptor is also a plausible candidate to iors such as anxiety and depression [36]. We recently mediate between pain and mood. The endogenous opioid reported that haplotypes in the galanin gene were associ- system and µ-opioid receptors modulate affective behav- ated with anxiety-associated alcoholic phenotypes in iors [41] as well as affective components of acute pain Page 11 of 15 (page number not for citation purposes) Molecular Pain 2006, 2:14 http://www.molecularpain.com/content/2/1/14 Figure 8 Pattern of interaction between mu opioid receptor SNP and baseline pain upon baseline mood score Pattern of interaction between mu opioid receptor SNP and baseline pain upon baseline mood score. SNP rs495491 was one of the three SNPs that showed a significant interaction term in the recessive model (p = 0.003, uncorrected for multiple tests). The baseline SF-36 MH mood scores are plotted against the baseline bodily pain scores. Each point is the mean baseline mood for all of the patients with that bodily pain score and genotype. The curve connecting the triangles represents 17 patients homozygous for the uncom- mon allele; the curve connecting the diamonds represents the 252 patients with one or no copies of the uncommon allele. In the pres- ence of high baseline pain, 2 copies of the uncommon allele appear to be associated with symptoms of depression and anxiety, but the modest sample size and multiple genes tested make this result illustrative, not statistically persuasive. Data from patients with baseline pain of 0–2 (none, very mild, or mild) were pooled because few patients had such low pain scores at presentation. [22]. Opioid treatment of chronic pain is often accompa- the genes for corticotropin-releasing hormone (CRH) nied by striking improvements in mood [42]. Although [45], the adenosine A(2A) receptor [46], the dopamine the human mu opioid receptor gene (OPRM1) has poly- D4 receptor [47], and tryptophan hydroxylase [48]. morphisms that affect receptor function and are associ- ated with some behavioral phenotypes [43], no It is interesting that among patients in the quartile with associations with depression and anxiety disorders have the least surgical pain relief, mood did not deteriorate, yet been reported [44]. The three OPRM1 SNPs most and actually improved from 6 months on. Within this closely associated with pain-related mood scores in our quartile of patients (Fig. 4), for example, pain levels at 24 study are in high linkage disequilibrium and in the same months after surgery were equivalent to the presurgery haploblock, which also includes the previously reported levels, but mood at 24 months was considerably functional non-synonymous SNP Asn40Asp (rs1799971). improved from baseline. It would be interesting to exam- However, the latter SNP was not associated with mood ine individual differences in resilience factors, including scores. Therefore we assume that the association signal, if genetic resilience factors, that are associated with replicated, could be attributable to another functional improvements in mood in the face of the surgical failure allele that is still unknown. to relieve pain. In this study, we have tested mood effects of genes that we The preceding analysis of gene effects mediating mood had already genotyped based on their involvement in responses to unrelieved pain in surgical patients is a gene chronic pain processing. For future studies on pain-mood × environment (G × E) interaction study. Thus far, the interaction, one might select additional candidate genes most widely cited examples of G × E interactions on reportedly associated with anxiety and depression; e.g., behavioral endpoints [17,18] involve an environmental Page 12 of 15 (page number not for citation purposes) Molecular Pain 2006, 2:14 http://www.molecularpain.com/content/2/1/14 stress. Although our most suggestive results for the genes Authors' contributions we examined pertained to unique pain-gene interactions MBM initiated and supervised the clinical genetics study, on mood, the same analytic method might help to iden- proposed the method, and drafted the manuscript. TW tify other genes that have a general effect on mood, dem- designed the specific statistical method and carried out onstrated by a main effect in the regression. A potential the analyses. AK advised on statistical analyses. SJA was advantage of this study design is that patients are all sub- co-PI of the original clinical study, contributed the clinical ject to a similar stress – several months of a painful condi- data, and advised on the analysis. RBK was co-PI of the tion followed by major surgery – and then experience original clinical study, contributed the clinical data, and variable levels of residual pain, a stressor that can be well- advised on the DNA collection. RRE and JAH advised on quantified and corrected for in the analysis. Without the analyses and interpretation of psychological data. detailed measurements of environmental stress, it may be AFB, HH, CDM, IAO, ENC, JN, DCA, and MAS did the difficult to identify genes for depression or anxiety. For bioinformatics work and genotyping. IB trained and example, although twin studies suggest that the heritabil- supervised the bioinformatics and genotyping team, inter- ity of unipolar major depression disorder is 40–50% [49] preted this data, and wrote the molecular parts of the it has been difficult to replicate candidate gene associa- Methods and Discussion. MP advised on psychiatric tions, possibly because the environmental circumstances aspects of the manuscript. DG provided laboratory facili- triggering depression are so varied [50]. A major surgical ties, trained the authors in bioinformatics and genotyp- procedure may provide a somewhat uniform stressful sit- ing, and advised on the methods and interpretation. All uation. Within this setting, the two factor analysis shown authors read and approved the final manuscript. above may remove a large amount of variance in late mood due to pain, increasing sensitivity to a main effect Additional material of a genetic polymorphism involved in general mood-reg- ulating mechanisms not specific to patients with pain. Additional File 1 Galanin receptor-2 gene polymorphisms and 1 year SF-36-MH It is possible that alternative statistical methods may bet- Click here for file ter detect "mood" or "pain-mood" genes in the types of [http://www.biomedcentral.com/content/supplementary/1744- surgical cohort studies illustrated above. For example, 8069-2-14-S1.doc] patients without any pain might be excluded from the pain-mood analyses, or different time-points might be chosen for the pain and mood variables. The relative mer- its of alternative methods can be compared more persua- Acknowledgements Supported by NIDCR Intramural Grant DE00366 (MBM) and NIAAA Intra- sively once a robust gene effect is identified to use as a mural Grant Z01AA000301 (DG). The Maine Lumbar Spine Study was sup- gold standard. ported by grants from the Agency for Healthcare Research and Quality (HS-06344, HS-08194, and HS-09804). SJA (P60 AR048094) and RRE (K23 Conclusion AR051315) is supported in part by funding from the National Institute of A better understanding of the specific mechanisms linking Arthritis and Musculoskeletal and Skin Diseases. JAH is supported in part pain and mood may open up a set of new interventions to by funding from the National Institute of Neurological Disorders and decrease the morbidity of chronic pain conditions. Basic Stroke (K24 NS02225). We thank Yen A. Wu, MPH for preparing the clin- science studies in animals [13,50] including microarray ical dataset, Ann Scher for suggestions on data presentation, and Suzan search methods are likely to yield a long list of candidates. Khoromi and Beata Buzas for reviewing the manuscript. The funding bodies had no input into the design or publication of the current study. We have demonstrated a simple method to search for genetic polymorphisms that contribute to interindividual References variation in the manner in which pain produces depres- 1. Robinson ME, Riley JL: The role of emotion in pain. In Psychosocial sion or anxiety, using cohorts of patients who have under- Factors in Pain: Critical Perspectives Edited by: Gatchel RJ, Turk DC. gone a surgical procedure that variably relieves pain. New York: Guilford Press; 1999:74-88. 2. Gureje O, Simon GE, Von Korff M: A cross-national study of the Cross-correlation with the results of the types of human course of persistent pain in primary care. 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Molecular Pain – Springer Journals

Published: Apr 19, 2006

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