Smoking Associated T-Cell Imbalance in Patients With Chronic Pain.

INTRODUCTION: Smoking is associated with several diseases and affects the immune system. Recently, published data demonstrate an involvement of T helper 17 cells (Th17) and regulatory T cells (Tregs) in the pathogenesis of chronic pain and pain intensity. The role of these T-cell subsets in smoking patients with chronic pain is nebulous so far. We therefore analyzed Th17 cells and Tregs in smokers and nonsmokers with chronic pain. METHODS: Analyses of T-cell subsets, mRNA expression and T-cell related cytokine profiles were done in 44 patients with chronic pain. Twenty-two of these patients were smokers. Numbers of T-cell subsets were quantified by flow cytometry. mRNA expression of the Th17- (RAR-related orphan receptor gamma) and Treg (forkhead box protein P3)-specific transcription factors was determined by quantitative real-time PCR, and levels of cytokines were measured by Human Cytokine Multiplex Immunoassay. RESULTS: Compared to nonsmokers, smokers showed significantly enhanced pain levels. On cellular basis, the number of pro-inflammatory Th17 cells (smokers: 2.2 ± 2.5% vs. nonsmokers: 0.5 ± 0.4%; p = .04) was increased, whereas the number of anti-inflammatory Tregs (smokers: 2.5 ± 0.9% vs. nonsmokers: 3.1 ± 1.1%; p = .02) was significantly decreased, resulting in an altered Th17/Treg ratio (Th17/Treg ratio: 0.9 ± 1.0 in smokers vs. 0.2 ± 0.1 in nonsmokers; p < .01). These findings were confirmed by quantitative real-time PCR. Analyses of cytokines revealed only marginal changes. CONCLUSIONS: In patients with chronic pain, smoking is associated with enhanced pain levels together with an imbalance of the Th17/Treg ratio. The shift of the Th17/Treg ratio toward inflammation may explain in part the increased pain intensity in these patients. IMPLICATIONS: Smoking is associated with increased pain levels and a pro-inflammatory Th17/Treg shift. The altered Th17/Treg ratio in smoking patients with chronic pain may partly explain their increased pain intensity. GERMAN CLINICAL TRIAL REGISTER (DRKS): Registration Trial DRKS00005954.

Altered regulation of the T-cell system in patients with CRPS.

The aim of this study was to investigate T-cell subsets and immunomodulatory factors in patients with complex regional pain syndrome (CRPS). We found decreased numbers of pro-inflammatory Th17 cells in patients with CRPS as compared to healthy volunteers. The expression of Th17 related RORγT mRNA was also significantly decreased. Patients with CRPS showed an increased proportion of CD39+ Tregs. CD39 is a known inhibitor of Th17 cell differentiation. Systemic cytokine levels were almost unchanged in patients with CRPS. These findings suggest that the decrease in Th17 cells in CRPS is regulated by an increase in CD39+ Tregs and that this anti-inflammatory T-cell shift may be a mechanism to control inflammation in CRPS. GERMAN CLINICAL TRIAL REGISTER: Registration Trial DRKS00005954.

miR-124a and miR-155 enhance differentiation of regulatory T cells in patients with neuropathic pain.

BACKGROUND: Accumulating evidence indicates that neuropathic pain is a neuro-immune disorder with enhanced activation of the immune system. Recent data provided proof that neuropathic pain patients exhibit increased numbers of immunosuppressive regulatory T cells (Tregs), which may represent an endogenous attempt to limit inflammation and to reduce pain levels. We here investigate the molecular mechanisms underlying these alterations. METHODS: Our experimental approach includes functional analyses of primary human T cells, 3'-UTR reporter assays, and expression analyses of neuropathic pain patients' samples. RESULTS: We demonstrate that microRNAs (miRNAs) are involved in the differentiation of Tregs in neuropathic pain. We identify miR-124a and miR-155 as direct repressors of the histone deacetylase sirtuin1 (SIRT1) in primary human CD4(+) cells. Targeting of SIRT1 by either specific siRNA or by these two miRNAs results in an increase of Foxp3 expression and, consecutively, of anti-inflammatory Tregs (siRNA: 1.7 ± 0.4; miR-124a: 1.5 ± 0.4; miR-155: 1.6 ± 0.4; p < 0.01). As compared to healthy volunteers, neuropathic pain patients exhibited an increased expression of miR-124a (2.5 ± 0.7, p < 0.05) and miR-155 (1.3 ± 0.3; p < 0.05) as well as a reduced expression of SIRT1 (0.5 ± 0.2; p < 0.01). Moreover, the expression of these two miRNAs was inversely correlated with SIRT1 transcript levels. CONCLUSIONS: Our findings suggest that in neuropathic pain, enhanced targeting of SIRT1 by miR-124a and miR-155 induces a bias of CD4(+) T cell differentiation towards Tregs, thereby limiting pain-evoking inflammation. Deciphering miRNA-target interactions that influence inflammatory pathways in neuropathic pain may contribute to the discovery of new roads towards pain amelioration. TRIAL REGISTRATION: German Clinical Trial Register DRKS00005954.

Differential expression of P2X7 receptor and IL-1ß in nociceptive and neuropathic pain.

BACKGROUND: Despite substantial progress, pathogenesis and therapy of chronic pain are still the focus of many investigations. The ATP-gated P2X7 receptor (P2X7R) has previously been shown to play a central role in animal models of nociceptive inflammatory and neuropathic pain. Recently, we found that the adaptive immune system is involved in the pathophysiology of chronic nociceptive and neuropathic pain in humans. So far, data regarding P2X7R expression patterns on cells of the adaptive immune system of pain patients are scarce. We therefore analyzed the P2X7R expression on peripheral blood lymphocytes and monocytes, as well as serum levels of IL-1ß in patients suffering from chronic nociceptive and neuropathic pain in comparison to healthy volunteers in order to identify individuals who might benefit from a P2X7R modulating therapy. METHODS: P2X7R messenger RNA (mRNA) and protein expression were determined in patients with either chronic nociceptive low back pain (CLBP) or neuropathic pain (NeP), and in healthy volunteers by quantitative real-time PCR (qPCR) and by fluorescence-assisted cell-sorting (FACS), respectively. IL-1ß serum levels were measured with a multiplex cytokine assay. RESULTS: Compared to healthy volunteers, P2X7R mRNA (1.6-fold, p = 0.038) and protein levels were significantly increased on monocytes (NeP: 24.6 ± 6.2, healthy volunteers: 17.0 ± 5.4; p = 0.002) and lymphocytes (NeP: 21.8 ± 6.5, healthy volunteers: 15.6 ± 5.2; p = 0.009) of patients with NeP, but not in patients with CLBP. Similarly, IL-1ß serum concentrations were significantly elevated only in NeP patients (1.4-fold, p = 0.04). CONCLUSIONS: A significant upregulation of P2X7R and increased IL-1ß release seems to be a particular phenomenon in patients with NeP. P2X7R inhibitors may therefore represent a potential option for the treatment of this frequently intractable type of pain. German Clinical Trial Register (DRKS): Registration Trial DRKS00005954.

Pharmacology of peripheral opioid receptors.

PURPOSE OF REVIEW: Since the detection of morphine by the pharmacologist Friedrich Sertürner in 1806, opioids have been used as potent centrally acting analgesics. In addition to the central site of action, peripheral endogenous opioid analgesic systems have been extensively studied, especially in the past two decades. This review is not only mentioned to give a brief summary in this well investigated field of peripheral opioid receptors, but also to highlight the role of peripheral opioid receptors in other physiological and pathophysiological conditions. RECENT FINDINGS: A number of studies, which initially focused on nociception, also revealed an important role of the peripheral opioid receptor system in opioid-induced bowel dysfunction and pruritus, as well as in wound healing, cardioprotection, and the analgesic effects of celecoxib. SUMMARY: Efforts continue to develop opioid analgesics unable to cross the blood-brain barrier, which act only peripherally in low doses, thus providing adequate analgesia without central and systemic side-effects.The awareness of the influence of peripheral opioid receptors beyond nociception may also have therapeutic ramifications on the other fields mentioned above. For example, the treatment of opioid-induced bowel dysfunction by methylnaltrexone is one of the major findings in the previous years.

Implications of the putamen in pain and motor deficits in complex regional pain syndrome.

Complex regional pain syndrome (CRPS) develops after-limb injury, with persistent pain and deficits in movement frequently co-occurring. The striatum is critical for mediating multiple mechanisms that are often aberrant in CRPS, which includes sensory and pain processing, motor function, and goal-directed behaviors associated with movement. Yet, much remains unknown with regards to the morphological and functional properties of the striatum and its subregions in this disease. Thus, we investigated 20 patients (15 female, age 58 ± 9 years, right-handed) diagnosed with chronic (6+ months of pain duration) CRPS in the right hand and 20 matched, healthy controls with anatomical and resting-state, functional magnetic resonance imaging. In addition, a comprehensive clinical and behavioral evaluation was performed, where each participant's pain, motor function, and medical history were assessed. Complex regional pain syndrome patients harbored significant abnormalities in hand coordination, dexterity, and strength. These clinical pain- and movement-related findings in CRPS patients were concomitant with bilateral decreases in gray matter density in the putamen as well as functional connectivity increases and decreases among the putamen and pre-/postcentral gyri and cerebellum, respectively. Importantly, higher levels of clinical pain and motor impairment were associated with increased putamen-pre-/postcentral gyri functional connectivity strengths. Collectively, these findings suggest that putaminal alterations, specifically the functional interactions with sensorimotor structures, may underpin clinical pain and motor impairment in chronic CRPS patients.

Can self-reported pain characteristics and bedside test be used for the assessment of pain mechanisms? An analysis of results of neuropathic pain questionnaires and quantitative sensory testing.

Hyperalgesia and allodynia are frequent in neuropathic pain. Some pain questionnaires such as the Leeds Assessment of Neuropathic Symptoms and Signs (LANSS) and the Neuropathic Pain Scale (NPS) include self-assessment or bedside testing of hyperalgesia/allodynia. The aim of this study was to determine to what extent LANSS and NPS data are congruent with findings on quantitative sensory testing (QST). Self-reported presence of dynamic mechanical allodynia (DMA) and descriptors of hot, cold, or deep ongoing pain (the NPS and LANSS) as well as bedside findings of mechanical allodynia (LANSS) were compared with signs of DMA and thermal hyperalgesia on QST in 617 patients with neuropathic pain. Self-reported abnormal skin sensitivity (LANSS) showed a moderate concordance with DMA during bedside test (67.9%, κ = 0.391) or QST (52.8%, κ = 0.165). Receiver operating curve analysis for self-reported DMA yielded similar area-under-the-curve values for the LANSS (0.65, confidence interval: 0.59%-0.97%) and NPS (0.71, confidence interval: 0.66%-0.75%) with high sensitivity but low specificity. Self-reported deep pain intensity was higher in patients with blunt pressure hyperalgesia, but not in patients with DMA or thermal hyperalgesia. No correlations were observed between self-reported hot or cold pain quality and thermal hyperalgesia on QST. Self-reported abnormal skin sensitivity has a high sensitivity to identify patients with DMA, but its low specificity indicates that many patients mean something other than DMA when reporting this symptom. Self-reported deep pain is related to deep-tissue hypersensitivity, but thermal qualities of ongoing pain are not related to thermal hyperalgesia. Questionnaires mostly evaluate the ongoing pain experience, whereas QST mirrors sensory functions. Therefore, both methods are complementary for pain assessment.

Cannabinoid receptor type 1 located on presynaptic terminals of principal neurons in the forebrain controls glutamatergic synaptic transmission.

It is widely accepted that cannabinoids regulate GABA release by activation of cannabinoid receptor type 1 (CB1). Results obtained from a variety of brain regions consistently indicate that cannabinoid agonists can also reduce glutamatergic synaptic transmission. However, there are still conflicting data concerning the role of CB1 in cannabinoid-induced inhibition of glutamatergic transmission in cortical areas. Here, we provide direct evidence that activation of CB1 on terminals of principal neurons controls excitatory synaptic responses in the forebrain. In slices of the basolateral amygdala, the CA1 region of the hippocampus, and the primary somatosensory cortex of wild-type mice, application of the CB1 agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55,212-2; WIN) (5 mum) reduced evoked excitatory postsynaptic responses. In contrast, in slices obtained from conditional mouse mutants lacking CB1 in all principal forebrain neurons but not in GABAergic interneurons (CB1(f/f;CaMKIIalphaCre)), WIN no longer affected glutamatergic synaptic transmission in any of the brain regions tested. Compatible with a presynaptic mechanism, WIN did not change the sensitivity to focally uncaged l-glutamate. WIN reduced glutamatergic responses in slices obtained from mice lacking CB1 exclusively in GABAergic neurons (CB1(f/f;Dlx5/6-Cre)), thus excluding the involvement of CB1 expressed on GABAergic neurons in this effect of the drug. The present data strongly indicate that excitatory synaptic transmission in forebrain areas is directly modulated by CB1 expressed on presynaptic axon terminals originating from glutamatergic neurons.

Circuitry for associative plasticity in the amygdala involves endocannabinoid signaling.

Endocannabinoids are crucial for the extinction of aversive memories, a process that considerably involves the amygdala. Here, we show that low-frequency stimulation of afferents in the lateral amygdala with 100 pulses at 1 Hz releases endocannabinoids postsynaptically from neurons of the basolateral amygdala of mice in vitro and thereby induces a long-term depression of inhibitory GABAergic synaptic transmission (LTDi) via a presynaptic mechanism. Lowering inhibitory synaptic transmission significantly increases the amplitude of excitatory synaptic currents in principal neurons of the central nucleus, which is the main output site of the amygdala. LTDi involves a selective mGluR1 (metabotropic glutamate receptor 1)-mediated calcium-independent mechanism and the activation of the adenylyl cyclase-protein kinase A pathway. LTDi is abolished by the cannabinoid type 1 (CB1) receptor antagonist SR141716A and cannot be evoked in CB1 receptor-deficient animals. LTDi is significantly enhanced in mice lacking the anandamide-degrading enzyme fatty acid amide hydrolase. The present findings show for the first time that mGluR activation induces a retrograde endocannabinoid signaling via activation of the adenylyl cyclase-protein kinase A pathway and the release of anandamide. Furthermore, the results indicate that anandamide decreases the activity of inhibitory interneurons in the amygdala. This disinhibition increases the activity of common output neurons and could provide a prerequisite for extinction by formation of new memory.

The major brain endocannabinoid 2-AG controls neuropathic pain and mechanical hyperalgesia in patients with neuromyelitis optica.

Recurrent myelitis is one of the predominant characteristics in patients with neuromyelitis optica (NMO). While paresis, visual loss, sensory deficits, and bladder dysfunction are well known symptoms in NMO patients, pain has been recognized only recently as another key symptom of the disease. Although spinal cord inflammation is a defining aspect of neuromyelitis, there is an almost complete lack of data on altered somatosensory function, including pain. Therefore, eleven consecutive patients with NMO were investigated regarding the presence and clinical characteristics of pain. All patients were examined clinically as well as by Quantitative Sensory Testing (QST) following the protocol of the German Research Network on Neuropathic Pain (DFNS). Additionally, plasma endocannabinoid levels and signs of chronic stress and depression were determined. Almost all patients (10/11) suffered from NMO-associated neuropathic pain for the last three months, and 8 out of 11 patients indicated relevant pain at the time of examination. Symptoms of neuropathic pain were reported in the vast majority of patients with NMO. Psychological testing revealed signs of marked depression. Compared to age and gender-matched healthy controls, QST revealed pronounced mechanical and thermal sensory loss, strongly correlated to ongoing pain suggesting the presence of deafferentation-induced neuropathic pain. Thermal hyperalgesia correlated to MRI-verified signs of spinal cord lesion. Heat hyperalgesia was highly correlated to the time since last relapse of NMO. Patients with NMO exhibited significant mechanical and thermal dysesthesia, namely dynamic mechanical allodynia and paradoxical heat sensation. Moreover, they presented frequently with either abnormal mechanical hypoalgesia or hyperalgesia, which depended significantly on plasma levels of the endogenous cannabinoid 2-arachidonoylglycerole (2-AG). These data emphasize the high prevalence of neuropathic pain and hyperalgesia in patients with NMO. The degree of mechanical hyperalgesia reflecting central sensitization of nociceptive pathways seems to be controlled by the major brain endocannabinoid 2-AG.

Effects of low-dose intranasal (S)-ketamine in patients with neuropathic pain.

BACKGROUND: NMDA receptors are involved in the development and maintenance of neuropathic pain. We evaluated the efficacy and safety of intranasal (S)-ketamine, one of the most potent clinically available NMDA receptor antagonists. METHODS: Sixteen patients with neuropathic pain of various origins were randomized into two treatment groups: (S)-ketamine 0.2mg/kg (group 1); (S)-ketamine 0.4mg/kg (group 2). Plasma concentrations of (S)-ketamine and (S)-norketamine were measured over 6h by High Performance Liquid Chromatography combined with mass spectrometry. Quantitative sensory testing (QST) was conducted before, during and after treatment. Side effects and amount of pain reduction were recorded. RESULTS: Intranasal (S)-ketamine administration lead to peak plasma concentrations of 27.7+/-5.9ng/ml at 10+/-6.3min (group 1) and 34.3+/-22.2ng/ml at 13.8+/-4.8min after application (group 2). Maximal plasma concentrations of (S)-norketamine were 18.3+/-14.9ng/ml at 81+/-59min (group 1) and 34.3+/-5.5ng/ml at 75+/-40min (group 2). Pain scores decreased significantly in both groups with minimal pain at 60min after drug administration (70+/-10% and 61+/-13% of initial pain in groups 1 and 2). The time course of pain decrease was significantly correlated with plasma concentrations of (S)-ketamine and (S)-norketamine (partial correlations: (S)-norketamine: -0.90 and -0.86; (S)-ketamine: -0.72 and -0.71 for group 1 and group 2, respectively). Higher dosing elicited significantly more side effects. Intranasal (S)-ketamine had no significant impact on thermal or mechanical detection and pain thresholds in normal or symptomatic skin areas. CONCLUSIONS: Intranasal administration of low dose (S)-ketamine rapidly induces adequate plasma concentrations of (S)-ketamine and subsequently of its metabolite (S)-norketamine. The time course of analgesia correlated with plasma concentrations.

Activation of kappa opioid receptors decreases synaptic transmission and inhibits long-term potentiation in the basolateral amygdala of the mouse.

BACKGROUND: The amygdala plays an important role in the processing of chronic pain and pain memory formation. Particularly, it is involved in the emotional and affective components of the pain circuitry. The role of kappa opioid receptors in these pain conditions is only partly known. The present study investigates the effect of kappa receptor activation on synaptic transmission and synaptic plasticity in the amygdala. METHODS: Electrophysiological in vitro experiments were carried out in brain slices of male C57BL/6JOlaHsd mice. The effect of the kappa opioid receptor agonist U50,488H (5 microM) and the selective kappa opioid receptor antagonist nor-BNI (3 microM) on field potential (FP) amplitude and the induction of long-term potentiation (LTP) in the basolateral amygdala (BLA) was examined. RESULTS: High frequency stimulation (HFS) of afferents in the lateral amygdala with two trains of 100 pulses at 50 Hz increased the FP amplitudes to 119+/-2% (mean+/-SEM; n=6) in the BLA. U50,488H decreased synaptic transmission (baseline: 100+/-0.5%; U50,488H: 86.3+/-2.4%; n=6) and blocked the induction of LTP (U50,488H: 100+/-4.1%; HFS: 102.6+/-7%; n=6). The effect on synaptic transmission and on LTP was completely reversed or prevented by application of nor-BNI, which itself had no effect on synaptic transmission or the induction of LTP. CONCLUSION: Kappa opioid receptor activation decreases synaptic transmission and inhibits the induction of LTP in the BLA of the mouse. These findings may be associated with the effects of kappa opioid agonists in chronic pain and pain memory formation.

Impact of a functional restoration program on pain and health-related quality of life in patients with chronic low back pain.

OBJECTIVE: Functional restoration programs for chronic low back pain (CLBP) have been shown to be successful in improving function and, to a lesser extent, in reducing pain. The Munich Functional Restoration Program (MFRP) is a 4-week outpatient program designed to reduce pain and to improve health-related quality of life in patients with a long history of CLBP. DESIGN: In a retrospective matched concurrent-controls therapeutic study, 44 patients with CLBP, who had either undergone MFRP or received an outpatient standard treatment (control) after initial evaluation at the pain center, completed questionnaires 1 year after the respective therapy (t1). The following parameters were assessed: health-related quality of life with Short Form-36 (SF-36), Pain Disability Index (PDI), Numeric Rating Scale (NRS) for pain, depression with the Center for Epidemiological Studies Depression Test (CES-D), and occupational situation. These data were compared with baseline values assessed by a questionnaire completed before starting the respective treatment (baseline, t0). RESULTS: Compared with control, NRS and PDI were significantly better in patients completing the MFRP. Patients of the MFRP group showed also a significant reduction in CES-D as well as an improvement in three of eight SF-36 subscales. No changes were detected in the control group receiving standard treatment. CONCLUSIONS: Compared with standard treatment, a functional restoration program for CLBP significantly improves some aspects of health-related quality of life. It results in a decrease of pain and pain-related disability even in patients with a long history of CLBP.

The endogenous cannabinoid system controls extinction of aversive memories.

Acquisition and storage of aversive memories is one of the basic principles of central nervous systems throughout the animal kingdom. In the absence of reinforcement, the resulting behavioural response will gradually diminish to be finally extinct. Despite the importance of extinction, its cellular mechanisms are largely unknown. The cannabinoid receptor 1 (CB1) and endocannabinoids are present in memory-related brain areas and modulate memory. Here we show that the endogenous cannabinoid system has a central function in extinction of aversive memories. CB1-deficient mice showed strongly impaired short-term and long-term extinction in auditory fear-conditioning tests, with unaffected memory acquisition and consolidation. Treatment of wild-type mice with the CB1 antagonist SR141716A mimicked the phenotype of CB1-deficient mice, revealing that CB1 is required at the moment of memory extinction. Consistently, tone presentation during extinction trials resulted in elevated levels of endocannabinoids in the basolateral amygdala complex, a region known to control extinction of aversive memories. In the basolateral amygdala, endocannabinoids and CB1 were crucially involved in long-term depression of GABA (gamma-aminobutyric acid)-mediated inhibitory currents. We propose that endocannabinoids facilitate extinction of aversive memories through their selective inhibitory effects on local inhibitory networks in the amygdala.

CB1 cannabinoid receptors and on-demand defense against excitotoxicity.

Abnormally high spiking activity can damage neurons. Signaling systems to protect neurons from the consequences of abnormal discharge activity have been postulated. We generated conditional mutant mice that lack expression of the cannabinoid receptor type 1 in principal forebrain neurons but not in adjacent inhibitory interneurons. In mutant mice,the excitotoxin kainic acid (KA) induced excessive seizures in vivo. The threshold to KA-induced neuronal excitation in vitro was severely reduced in hippocampal pyramidal neurons of mutants. KA administration rapidly raised hippocampal levels of anandamide and induced protective mechanisms in wild-type principal hippocampal neurons. These protective mechanisms could not be triggered in mutant mice. The endogenous cannabinoid system thus provides on-demand protection against acute excitotoxicity in central nervous system neurons.