Differences in the expression of oxidative stress (OS) markers between female patients with temporomandibular disorders (TMD) and healthy individuals indicate that OS plays a role in the pathogenesis of TMD. Because chronic exposure to stress generates oxidative damage during continuous stimulation of the hypothalamic-pituitary-adrenal axis, we expected that higher levels of cortisol might be associated with higher oxidative damage. Our aim was to test the association between OS markers, stress perception, and salivary cortisol (SC) in chronic, female TMD patients. We tracked changes in OS markers and SC during occlusal splint therapy in order to evaluate the influence of treatment on oxidative status. We hypothesized that the effects of TMD therapy would differ among individuals depending on the source and intensity of pain.Sixteen female patients were recruited, and 12 finished the study. Clinical assessment and saliva sampling were performed at the baseline and follow-up appointments. Repeated measures analysis of variance and Pearson’s correlation were used for analyzing the data.p < 0.05). A significant reduction in afternoon malondialdehyde (MDA) (p = 0.021) and a decrease in afternoon MDA to superoxide dismutase ratios (p = 0.017) were present in high-intensity pain patients. At baseline, higher levels of perceived stress were significantly associated with higher morning cortisol (ρ = 0.67). At the end of the therapy,  reduced  perceived stress was positively correlated with morning SC changes when considering all TMD patients, but the association between perceived stress with OS markers was present only in myofascial pain (MP) group. The effect of treatment on the self-perceived quality of life was more pronounced in female MP patients while the reduction of spontaneous pain was significantly greater in high-intensity pain patients.After 3 months, a significant reduction in afternoon total antioxidant capacity (TAC) was observed (Our data indicate that occlusal splint therapy in female TMD patients contributes to increasing their capacity to remove free radicals. The question remains whether or not TAC decreases in this process as a result of avoiding unnecessary processes, once the increase in antioxidants effectively compensates for OS. The intensity and the source of pain should be considered important factors in future investigations evaluating salivary OS markers and their association with perceived stress and SC in TMD patients.NCT03029494. Registered on 2017-01-19.ClinicalTrials.gov  Temporomandibular disorders (TMD) encompass the most common painful musculoskeletal and neuromuscular orofacial conditions and affect jaw joints, masticatory muscles, and the surrounding structures. These conditions are still primarily diagnosed through medical history and clinical examination due to their multifactorial pathophysiology, which is still not completely clear . SymptomThe main goal of TMD therapy is the reduction of clinical symptoms such as pain and limited lower jaw movement . PhysicaResearch has recently suggested that oxidative stress (OS) can play a part in the pathophysiological processes that occur in TMD. OS occurs as a result of an imbalance between oxidants and antioxidants in favor of oxidants, and it is believed to play a role in various adverse processes in an organism . OS is bStudies have found significant differences in the expression of OS biomarkers and antioxidants between TMD patients and healthy individuals , 18. TheThe connection between OS, cortisol, and psychological stress has been previously discussed . CortisoThe aim of this study was to: 1) test the association between OS biomarkers, stress perception, and salivary cortisol (SC) in chronic TMD patients, and 2) to follow up on changes in OS markers and SC during 3 months of occlusal splint therapy in order to evaluate the influence of treatment on oxidative status. Our hypothesis was that there would be a positive association between SC, perceived stress, and OS biomarkers. We also hypothesized that the effects of TMD therapy on salivary oxidative status would differ between individuals with HIP and LIP as well as between MP and DD patients.This study was performed at the Department of Removable Prosthodontics, School of Dental Medicine, University of Zagreb with the approval of the Ethics Committee  and regulated in accordance with the Helsinki Declaration. All subjects were informed of the procedures involved in the study and provided with written consent before being included in the research.N = 12 (6 per group).A statistical power analysis was performed for sample size estimation based on data from Rodríguez de Sotillo et al.  which coParticipants were diagnosed according to the diagnostic criteria for TMD (DC/TMD) . The incSixteen female patients met the inclusion criteria. During recruitment, 1 of them refused to take part in the study due to travelling complications; thus, 15 patients were enrolled in the study, but 3 patients dropped out before the salivary measurements were performed. Ultimately, 12 patients had all of the required measurements performed at the baseline and follow-up appointments .Next, the patients were grouped according to pain intensity as measured by the characteristic pain intensity (CPI) using the graded chronic pain scale . To calcAll patients received a hard acrylic  occlusal splint (stabilization splint) to wear on the maxillary dental arch. The splint was made using a stone cast in an ARTEX articulator  in the referent position of the centric relation, with a thickness of 1.5 mm at the level of the first molar. The same dental technician made all of the splints. A clinician (IZA) adjusted the splints so that opposing teeth occluded simultaneously with the splint surface. The same clinician adapted the splints as necessary at follow-up appointments.The study followed changes in spontaneous pain as assessed by a visual analogue scale (VAS), maximal comfortable mouth opening (MCO), the Oral Health Impact Profile (OHIP-14), and the level of perceived stress .A 100 mm horizontal VAS scale was used to evaluate spontaneous pain from the temporomandibular joint and the masticatory muscles. The range on the scale went from “no pain” (0 mm) to “worst pain imaginable” (100 mm) . Only suMCO was measured as the distance between the maxillary and mandibular central incisors. A pain-free opening was defined as the maximum distance the participant could open her mouth without experiencing any additional pain or discomfort .The PSS is a standard 10-item questionnaire that assesses subjective perceptions of stress over the previous month. Response options form a 5-point Likert scale: 0 = never, 1 = almost never, 2 = sometimes, 3 = fairly often, 4 = very often. Possible scores ranged from 0 to 40, with higher numbers indicating higher levels of stress. The questionnaire was previously translated into and validated in Croatian by Hudek-Knežević et al. .The short-form OHIP-14 questionnaire , which hA clinician (EV) performed a clinical examination of the patients at the baseline and follow-up appointments at the first and third months after the initiation of treatment; the clinician was blinded as to each patient’s CPI and specific diagnostic subgroup.Five mL whole, unstimulated saliva samples were collected in graduated tubes . Saliva aliquots (1 mL) were stored at − 80 °C until the analysis. Some of the markers show significant diurnal variations; therefore, the saliva was collected in the morning (7 AM) and in the afternoon (5 PM) .Morning sampling was performed at home before tooth brushing. Subjects were instructed to fast before saliva collection in the morning and to not eat or drink anything but water for at least 2 hours before sampling in the afternoon. All saliva samples were collected after rinsing the mouth with water. Brushing teeth before saliva sampling was forbidden to avoid blood contamination. Saliva was collected at follow-up measurements at the first and third months of occlusal splint therapy, following the same procedure. The sample collection and storage principles used in the study are described in detail by Alajbeg et al. .The sample analysis was performed at the Department of Laboratory Diagnostics, University Hospital Center, Zagreb.The following OS biomarkers were analyzed: MDA, TAC, SOD, and UA.MDA was determined using the MDA Adduct Competitive ELISA kit , which measures the MDA-stable hybrid protein adducts (MDA adducts), referred to as lipid peroxidation end products, formed by binding MDA to proteins.TAC and SOD were measured using the commercial colorimetric reagent kits TAS and RANSOD  while saliva UA levels were measured using the enzymatic uricase method using commercially available reagents from Roche Diagnostics . The total protein concentration was determined using Roche Diagnostics’ automated turbidimetric urinary and cerebrospinal fluid total protein assay; this measuring range (0.02–2 g/L) proved to have satisfactory analytical performance for the measurement of total proteins in the saliva samples. Measurements of TAC, SOD, UA, and total proteins were performed on a Cobas c501 automated biochemistry analyzer . All OS markers’ values were normalized to the total protein concentration.For the assays, the analytical performance (including intra- and inter-assay variability) was assessed and is available in our previously published study .Free SC was determined using a commercially available enzyme-linked immunosorbent assay (ELISA)  according to the manufacturers’ recommendations. This assay measures free SC based on the principle of competitive binding of cortisol from samples and a cortisol-enzyme conjugate to a polyclonal antibody that is pre-coated onto the microtiter wells. The resultant binding is measured spectrophotometrically and is inversely proportional to the concentration of free SC in the tested samples. The intra- and inter-assay variabilities of this assay kit are 5.8 and 6.4%, respectively, as stated by the manufacturer.p < 0.05. Data distribution was tested using the Shapiro-Wilk test. Before performing the statistical analyses, a log transformation was used for all data that were non-normally distributed (OS markers and SC).Data analyses were performed using the Statistica 13.4.0 software package (1984–2018 TIBCO Software Inc.) with the alpha set to The Student’s t-test was used to examine differences upon admission between the TMD subgroups. The changes in the means of measured variables were analyzed using repeated measures analysis of variance (ANOVA) with time  as the within factor and pain intensity (HIP and LIP) and source of pain (MP and DD) as the between factors. Bonferroni’s post hoc test was used to show where the differences occurred. Pearson’s correlation was used to evaluate the association between changes in SC, the level of perceived stress and OS markers in the TMD subgroups.The baseline characteristics of the patients are listed in Table p = 0.46) or pain intensity  were found.No significant age differences in the TMD subgroups in terms of pain source . LIP and HIP were equally distributed among the patients diagnosed with MP and with DD. Three MP subjects and 3 DD subjects had LIP . Similarly, 3 MP subjects and 3 DD subjects had HIP . However, no significant differences among the groups were found (p > 0.05). Therefore, patients were pooled into pain intensity subgroups, regardless of the source of pain. Similarly, patients were pooled into pain source subgroups, regardless of the pain intensity.The mean CPI of the MP subjects (48.6 ± 13.36) and DD subjects (53.55 ± 24.12) was not statistically different . but changed significantly over time . A decrease of morning TAC was observed in all TMD subgroups. However, the change was not statistically significant.The overall afternoon TAC did not differ between TMD subgroups . HIP patients showed a significant reduction in afternoon MDA at the first and third month of treatment compared to the baseline  while in LIP patients the levels of afternoon MDA remained unchanged. A greater reduction in afternoon MDA levels during the therapy period was observed in the MP group when compared to DD patients, but the decrease was not statistically significant.When evaluating afternoon MDA levels, the analysis revealed a significant interaction between time and pain intensity  = 6.12, F = 0.38, p = 0.55; source of pain: F = 1.24, p = 0.29)).A greater reduction in morning MDA was observed in HIP patients when compared to LIP patients, as well as in MP patients when compared to DD patients, but the difference was not statistically significant . A significant reduction in afternoon UA in LIP patients was observed in the third month of treatment compared to the baseline. In HIP patients, the decrease in afternoon UA was not statistically significant.The analysis revealed a significant interaction between time and pain intensity in terms of afternoon UA  = 5.11, F = 6.38, p = 0.03) with higher values in MP patients at all-time points. The effect of treatment on afternoon SC differed significantly between high and low intensity pain subgroups  = 4.71, p = 0.039, ηp2 = 0.34). Post hoc tests revealed that afternoon SC decreased significantly in LIP patients and increased in HIP patients.Morning SC did not change significantly over time, but differed significantly between the MP and DD subgroups . Afternoon MDA to SOD ratios decreased significantly in HIP patients at the first and third month of treatment compared to the baseline  while in LIP patients, the MDA to SOD ratios remained unchanged at the end of therapy when compared to baseline levels.When evaluating afternoon MDA to SOD ratios Table , the repF = 1.07; p = 0.032), but changed significantly over time  = 13.5, p = 0.002, ηp2 = 0.63)  and were significantly influenced by time  = 16.48, p = 0.001, ηp2 = 0.68).When considering the source of pain, VAS scores did not differ between TMD subgroups , but did not differ between groups . Significant improvement in OHIP-14 scores during the treatment period was observed both in MP and DD patients  = 8.36, p = 0.008, ηp2 = 0.57). The effect of treatment on OHIP-14 scores was more pronounced in MP patients  = 4.29, p = 0.048, ηp2 = 0.19).  = 7.61, F = 5.01, p = 0.049)  ρ = 0.67, p = 0.016). However, the association between perceived stress and SC with OS markers was not significant when considering all TMD patients. When correlations were tested separately for the TMD subgroups, a significant positive correlation between morning cortisol and OS markers was found in MP patients  when all TMD patients were considered.At the end of the therapy, the percentage change in morning SC showed a significant positive correlation with reduced perceived stress  was observed in MP patients.When correlations were tested separately for the TMD subgroups, a significant positive correlation between reduced perceived stress and the percentage change in morning TAC (When it comes to TAC levels in various diseases, data are inconsistent. Research addressing oxidative status in TMD patients is scarce , 29–31, The higher levels of TAC previously found in TMD patients were explained as a compensatory reaction to a distorted oxidative balance ; therefoResearchers have pointed out wide variability as a problem when assaying the antioxidant biomarker MDA . BecauseUA is often mentioned as a paradoxical biomarker because it can have both antioxidant and oxidant characteristics . Our stuThe reduction in morning SC levels was more prominent in the MP patients when compared to the DD patients. This change in morning SC levels and the correlation between morning SC and perceived stress with OS markers observed in the MP patients implies that stress responses may have a larger impact on female patients with pain of myofascial origin. MP is often related to enhanced stress reactivity, and coping with chronic pain may lead to a larger stress response . MoreoveMCO improved during the treatment period, but the improvement was not statistically significant. We need to take into consideration the clinical importance of these results because an increase of only a few millimeters in MCO can have an enormous impact on a patient’s quality of life. In the current study, the MCO’s tendency to increase was a clear sign of patients’ improvement in TMD symptomatology, and it was naturally followed by an improvement in patients’ oral health-related quality of life. The significant difference observed between MP and DD patients showed that, in terms of the MCO, female MP patients had a better response to occlusal splint therapy. Research shows that there is probably no possibility of restoring the disc to its natural position, especially in chronic DD TMD ; hence, There were a few limitations to this study. One of these was the small sample size, especially when considering the variability of the tested biomarkers. With a larger number of participants, more significant differences would most certainly occur. Second, our subjects were exclusively women; this was because few men came to the clinic with TMD-associated difficulties, which is in accordance with the fact that women are much more likely to be affected . BecauseThe results of this study, conducted among female, chronic TMD patients, show that during 3 months of occlusal splint therapy, the TAC levels in the patients’ saliva significantly decreased. The MDA to SOD ratios decreased, and this reduction was more pronounced in HIP patients. This decrease in the MDA to SOD ratio might indicate that the effect of splint therapy, which may be seen as a reduction of TMD symptoms in female TMD patients, might help in body’s cleansing capacity to remove free radicals. The question remains whether or not TAC decreases in this process as a result of avoiding unnecessary processes, once the increase in antioxidants effectively compensates for OS. When considering the association between SC and perceived stress with OS markers, it is important to emphasize that the positive correlation was present in female MP patients. The effect of treatment on the self-perceived quality of life was more pronounced in female MP patients while the reduction of spontaneous pain, although present in all TMD subgroups, was significantly greater in patients with HIP.Although the study used a small sample size of only women, our results emphasize that both the source and the intensity of pain should be considered in future investigations.


In acute and ongoing pain, the spontaneous oscillatory activity of electroencephalogram (EEG) has been characterized by suppression of alpha band oscillations and enhancement of gamma band oscillations. In pathological chronic pain which is more severe and common in clinic practice, it is of great interest to investigate the oscillatory activity especially at the broad gamma frequency bands. Our present study explored the resting state oscillatory activities of EEG in patients with post-herpetic neuralgia (PHN) over 3 months which is a typical neuropathic pain model in clinical researches. It was found that the PHN patients showed anxiety and depression revealed by Beck Anxiety Inventory (BAI) and Beck Depression Inventory (BDI) examinations. Power spectrum analysis revealed that the power at gamma frequency band (from 40 to 70 Hz) of EEG was significantly higher in the PHN patients, and positively correlated with pain intensity, anxiety, and depression indexes. Further, increased gamma activity derived from the prefrontal cortex and the cerebellum were revealed by cluster-based sensor level and the beamforming source level analyses. These results suggest the enhanced gamma oscillatory activity in the prefrontal cortex and cerebellum is a characteristic marker in chronic neuropathic pain patients. Brain oscillation is rhythmic electrical activity and has distinct functions at different frequencies in various neural processing. It has been shown that oscillatory activities at alpha, beta, and gamma frequency bands are involved in short-lasting acute pain. Decreased alpha power together with increased beta and gamma power of electroencephalogram (EEG) was detected after painful stimulation . The powChronic pain, especially neuropathic pain, is more severe and common in clinic practice. Pathological pain severely impairs the life quality of patients. It is characterized as persistent and long-lasting pain , which iThalamocortical dysrhythmia (TCD) refers to a persistent oscillatory imbalance which manifests increased spectrum power at theta and gamma band . TCD is Post-herpetic neuralgia (PHN) is a common type of neuropathic pain, which lasts beyond the healing of rash caused by herpes zoster . It is cTherefore, in our present study, we investigated the oscillatory activities of EEG, their spatial distribution and source location, and its correlation with pain symptoms in chronic PHN patients.Table 1.Fourteen patients  with PHN were recruited at the outpatient clinic of Peking University People’s Hospital. PHN was diagnosed based on medical history and physical examination according to diagnostic criteria . The incThe healthy controls (HC) group comprised 14 healthy participants matched by age (65.2 ± 7.9) and gender. Both patients and healthy participants gave their written informed consent to use the data for scientific purposes. All subjects were right handed and the education level was above junior high school . Procedures were approved by the Ethics Committee of Peking University People’s Hospital.All subjects filled the Short-Form of McGill Pain Questionnaire (SF-MPQ) . The SF-Net station 5.3 software  was used for the EEG recording. The subjects sit relaxed in a dim room to prevent from disturbing and weared the 128-channel electrode cap suitable for his/her head size. Electrode CPz served as the common reference. All electrodes impedance were below 50 kΩ. The sampling rate was 500 Hz. Twenty minutes of resting EEG were recorded during eyes closed. During the recording, the subject remained awake to avoid sleep.1). Large muscle or eye movement artifacts were removed using independent component analysis (ICA), which showed a large electrooculogram (EOG) channel contribution and a frontal channel distribution (t(26) = 1.26, p > 0.05].The raw EEG data were preprocessed offline using MATLAB-based EEGLAB toolbox  and FieldTrip. Power between 1 and 70 Hz in step of 1 Hz was estimated using the multitaper method . Data wet-statistic at each channel was calculated. The neighbor channels with t-value bigger than the 95th percentile were selected and calculated as the maximum sum of each cluster. Then 500 times permutation test was conducted between two groups. The Monte-Carlo significance probability was the proportion of random partitions resulted in higher cluster-statistics than that of the actual data.Topographical distribution of gamma power was plotted over channels. The non-parametric statistical significance of the difference between the two conditions was evaluated using a cluster-based randomization test . Clustert-test was performed on the result of each grid point to estimate the signal change between groups across subjects. Subsequently, t-values were transformed to Z-scores, MNI-coordinates of peak voxel were transformed into Talairach-coordinates  beamforming method  implemenrdinates  and wereL(n), aH(n)] with low frequency phase time series ϕL(n) and high frequency amplitude time series aH(n). The modulation index was obtained by measuring the degree of asymmetry of the probability density function of the complex-valued signal. The modulation index of the signal was compared with that of 100 surrogate data generated by shifting the amplitude time series to obtain the significance of coupling. The significant PACs with p < 0.05 were kept while those with p > 0.05 were set to zero.Phase–amplitude coupling (PAC) was estimated by the method proposed by t-test was used to compare the difference between the two groups. The Cohen’s d was calculated for estimating the effect size. Spearman correlation was applied to calculate the pain rating correlation with psychological behavior scores. Pearson correlation and linear regression were applied to calculate the gamma power correlation with pain intensity and related symptoms. BAI scores were controlled by logistic regression. The significance level was set at α = 0.05.Student Table 2. As revealed by independent t-test, PHN patients had significantly higher VAS pain ratings and BAI scores compared with healthy Controls  indicating the chronic neuropathic pain and anxiety. Spearman correlation analysis further showed that the BAI score was positively correlated with VAS rating in PHN patients  but not in healthy Controls.The pain rating and psychological behavior scores have been listed in Figure 1A, the power at 1–40 Hz averaged across all channels was similar between Patient group and Control group, while the power at 40–70 Hz was significantly higher in Patient group compared with that in healthy Control group  , indicating that the gamma power was enhanced in PHN patients.We calculated the power spectrum of spontaneous EEG from 1 to 70 Hz for both healthy Control and PHN patients. As shown in p = 0.14), suggesting that the gamma power was more related to anxiety symptoms.Since the BAI scores were different in the Patient group from the Control group, we then compared gamma power after controlling the BAI scores. It showed that the gamma power was not significantly different any more  was higher in several channel locations. In order to find the areas with different gamma between groups, we compared gamma power of each channel between two groups with the t-test. Compared with permutation, a group of neighbor channels with sum bigger 95th t-value was then considered significant. Statistical analysis showed that several channels at right parietal and temporal regions, left temporal and prefrontal regions had higher gamma activities in Patient group than in Control group .We further explored the difference of gamma activity in spatial distribution and plotted the topographic distribution of gamma power in both groups. As shown in Figures 3A,B).Then, we investigated the source location of the increased gamma activities in patients at the source level. By Beamforming source level analysis, it was found that the gamma powers in bilateral dorsolateral cortex, medial prefrontal cortex, median and anterior cingulate gyri, and the cerebellum were significantly higher in Patient group than in Control group (p < 0.01). Linear regression showed that gamma power could predict 48% changes of VAS  . This strong correlation indicates the more increased gamma power, and the more severe pain.We further analyzed the relationship between the increased gamma activity and the pain intensity. It was shown that gamma power was positively correlated with VAS rating in PHN patients  and BAI  scores, and could explain 37 and 64% variability of BDI and BAI, respectively  .Moreover, the gamma power was also strongly correlated with BDI  was free of pain after the treatment. We took the advantage of self-comparison and measured the EEG of this patient again. By analyzing the power spectrum and power topographic distribution, we found that the power of oscillation (15–70 Hz) was reduced in the pain-free state compared with the pain state. Interestingly, the reduced gamma power was over prefrontal and parietal areas where was the similar location of the enhanced gamma power in PHN patients . This result, although from only one case, suggests a reduction of gamma power after pain relief.One of the PHN patients . While, neither the theta–gamma coupling nor delta–gamma coupling in PHN group was significantly different from that in the Control group .As the amplitude of gamma frequency is usually modulated by the phase of low frequency, we measured the phase–amplitude coupling between gamma frequency and other frequencies. It was found that the spontaneous gamma amplitude, especially from 40 to 55 Hz, was modulated by the phase of delta and theta frequency (1–8 Hz) for both groups . Although the narrow band low gamma power (30–48 Hz) exhibited a trend of increase in fibromyalgia patients (Our research explored the changes of oscillatory activity in chronic pain patients with PHN and found enhanced gamma activity (40–70 Hz) . In the current study, this relationship was further evidenced by one patient whose gamma power was reduced when the pain was relieved . Besides, the gamma power was also found to correlate with patient’s BDI and BAI scores. It is reasonable since the pain intensity was positively correlated with both BAI and BDI scores . As the concomitant symptoms of PHN, depression, and anxiety represent the affective aspects of chronic pain. This result is consistent with a previous finding that gamma power at narrow low frequency band in the DLPFC and orbitofrontal cortex was positively correlated with affective pain , median and anterior cingulate and paracingulate cortices and cerebellum. The location information of gamma activity is comparable to that in the ongoing experimental pain  in PHN chronic pain patients. Though the theta phase seems have stronger modulation index, no statistical difference between chronic pain patients and healthy controls was found. It is different from transient acute pain in rats  and chro2+ channels in the thalamus. It then produces disinhibition of cortical GABAergic interneurons that exhibit enhanced gamma band activity in cortical areas received the projection from thalamus  . BecauseIn summary, our results uncover the enhanced gamma oscillatory activity in prefrontal cortex and cerebellum in PHN patients which strongly correlates with pain intensity and pain relief. The altered gamma oscillatory activity in chronic pain provides evidence for TCD theory and a potential therapeutic target for oscillation intervention.This study was carried out in accordance with the recommendations of Measures for the Ethical Review of Biomedical Research Involving Humans, Ethics Committee of Peking University People’s Hospital. The protocol was approved by the Ethics Committee of Peking University People’s Hospital. All subjects gave written informed consent in accordance with the Declaration of Helsinki.RZ, JW, HG, and YW designed the experiments. RZ, JW, and WQ performed the experiments. JW, F-YL, and MY analyzed the data. RZ, JW, and YW wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Massage therapies such as cupping are often applied in patients with chronic neck and back pain with the assumption that they can reduce increased tissue stiffness and, therefore, improve pain. The aim of this study was to clarify whether tissue stiffness is related to pain experience in patients with chronic (>3 months) back and neck pain and whether it can be altered by a cupping massage. n = 20) or lower back pain (n = 20) was measured by a myometer. Exact contralateral side served as an individual control. Side of higher stiffness was then treated with a cupping massage. 5, 10, 15, and 20 minutes as well as 24 hours after treatment, tissue stiffness was measured again. Patients rated their pain on a standardized pain questionnaire (neck pain disability score (NDI) or Oswestry disability index (ODI), respectively) before and 24 hours after treatment.  The tissue stiffness of the point of subjectively felt maximum pain intensity of 40 patients with neck (p=0.827). The tissue stiffness and the side difference between treated and nontreated control sides decreased significantly after cupping  but returned to baseline after 24 hours. NDI and ODI scores significantly decreased 24 hours after cupping .  Compared to the contralateral control side, the more painful side did not exhibit an increased stiffness of myofascial tissue before treatment (DRKS00011281). Tissue stiffness might not be related to pain experience in patients with chronic neck and lower back pain. Trial registration: German Clinical Trial Register ( Chronic neck and back pain are common and relevant health problems  by affecDRKS00011281) and approved by the ethical committee of the University Medical Center of Freiburg, Germany (EK Freiburg 387/16) before onset. The study was performed according to the principles of the declaration of Helsinki and to the guidelines of ICH for a good clinical practice (GCP). All patients gave their written informed consent before participation. All data were handled strictly confidential.The monocentric, individually controlled, experimental trial was performed between November 2016 and January 2017, reported according to the CONSORT guidelines . It was Measurement of all patients was performed by only one skilled investigator (CM) under supervision of another skilled investigator (RH) at University Medical Center Freiburg.Patients were recruited via newspaper announcement. Interested patients were screened for eligibility by phone call and, if eligible, invited for a personal examination the following days. All patients were intensively informed about the aim and the implementation of the study before signing written informed consent. Eligible for the study were patients between 18 and 60 years of age with chronic (>3 months) nonspecific pain of the neck or lower back  and a miTissue stiffness was measured with the myometer MyotonPro®  . The metAfter putting the probe of the device with a size of 1 × 1 mm on the surface of the skin, it applies a mechanical impulse with a prepressure of 0.18 N. The aim of the prepressure is compression of subcutaneous tissue. It is followed by a quick mechanical impulse with a pressure of 0.40 N. Consequence is a natural oscillation of the fascia, which is recorded by the accelerometer of the device. The device automatically calculates arithmetic means of 5 single measures of dynamic stiffness, oscillation frequency, and logarithmic decrement of the tissue [During the whole trial, the point of measurement was the place of subjectively felt maximum pain intensity on the neck or back before treatment, which was marked with a skin-friendly and water-proofed pen. The place of subjectively felt maximum pain intensity was chosen due to the hypothesis that it might be the place of highest stiffness. An additional mark was set on the exactly contralateral side of the neck or back. Tissue stiffness, oscillation frequency, and logarithmic decrement were measured at both sides. Measurement was performed before intervention and 5, 10, 15, and 20 minutes as well as 24 hours after intervention.Due to the hypotheses that higher tissue stiffness correlates with more pain and cupping would reduce tissue stiffness, the side with the higher stiffness was chosen for intervention. All patients were seated putting their head stress-free on a prepared shelf . After aAll patients had to answer the NDI or ODI as standardized and validated questionnaires for neck pain and back pain, respectively, before and 24 hours after cupping.The NDI as well as the ODI captures 10 dimensions of neck-specific disability . Each dimension is assessed with 1 item, measured on a scale ranging from “no disability” (=0 points) to “full disability” (=5 points) , 25. A mPrimary outcome was the proportion of concordance between subjectively felt maximum pain and higher myometrically measured tissue stiffness in this area compared to tissue stiffness of the contralateral area of less pain. Secondary targets were the course of tissue stiffness 5, 10, 15, and 20 min as well as 24 hours after cupping in comparison to the tissue stiffness of the nontreated side. NDI and ODI after 24 hours in comparison to baseline and in relation to tissue stiffness were further secondary targets.d = 0.66), it was calculated that 40 patients (20 patients with neck pain and 20 patients with lower back pain) were needed to detect a statistical difference of p < 0.05 for correlation of stiffness and pain intensity within the groups.The study was planned as an explorative trial. Considering a statistical power of 80% and an expected medium effect size  was used for analysis. 2. They suffered from neck or back pain for almost 9 years (mean 107 ± 141 months). 20 patients had chronic nonspecific neck pain  and 20 patients had chronic nonspecific pain of the lower back . All patients were able to indicate a side (left or right) with dominant pain.40 patients were included in the study and analyzed ; there wn = 9) of the patients with neck pain and 50% (n = 10) of the patients with lower back pain located their subjectively felt maximum of pain intensity on the side with higher tissue stiffness. 55% of the neck pain patients and 50% of the patients with lower back pain had higher tissue stiffness on the control side which was less painful. There was no correlation between the side of maximum pain and tissue stiffness .Only 45% (p=0.002). Tissue stiffness returned to baseline values after 24 hours . The mean baseline score of both subgroups is shown in p=0.012, d = 0.62). Patients with lower back pain had a mean ODI score of 16 (range 14–18) before and 14 (range 12–17) after treatment .The mean score of NDI/ODI decreased significantly from 17 (range 15–27) to 15 (range 2–26) points 24 hours after cupping  and the interventions (defined by measuring the pressure and surface area). A limitation is the lack of blinding, which was not possible due to the nature of the intervention. The study focused on the myometrically measurable tissue stiffness at one single spinal level. Therefore, nothing is known about the pain of other spinal levels as well as the relation between pain and other types of stiffness, which are not measurable by a myometer. Furthermore, the most painful single point, which was chosen for measurement, might not be the point of pathophysiology as it is known that pain and dysfunction can occur far from tissue irritation. A parallel group design or a crossover trial would have been other options to analyze the effect of cupping on tissue stiffness, but we expected high variations when measuring at different days or when comparing different individuals and, therefore, decided to prefer an intraindividual comparison of two laterally reversed sides. Finally, 40 study participants, especially with the numerous exclusion criteria, do not necessarily represent the many different types and variations of conditions that can cause neck and back pain. It is still possible that some persons exhibit tissue stiffness changes that could be measured using this method and that this study failed to recruit enough (or any) to demonstrate it or that using different sites (not based on most painful region) could have yielded different results.Our study gives new insights into the relation between the myometrically measurable tissue stiffness and pain. This study found no relation between the most painful site and tissue stiffness as measured with a myometer in 40 participants with chronic neck and back pain. It also found that tissue stiffness as measured with a myometer did not change following cupping massage applied at the most painful site. The results of the study should lead to further, confirmatory research.


Half of the patients with high degree of pain at inclusion reported decreased pain at follow-up, median change (IQR) 45 (35 to 65), p = 0.021; fatigue, 8 (8 to 17), p = 0.018; anxiety, 4 (1 to 4), p = 0.035; and depression, 4 (2 to 5), p = 0.018 and improvements in most dimensions of SF-36. The remaining half of the patients reported no changes regarding pain and PROs except for a worsening in vitality in SF-36, 20 (15 to 35), p = 0.0018. All patients with remaining high level of pain indicated long-standing widespread pain. After 7 years, a subgroup of patients with SLE reported remaining high level of SLE-related pain and a high symptom burden, including long-standing widespread pain. Such patients require more observant attention to receive appropriate treatment.We have previously shown that most patients with systemic lupus erythematosus (SLE) reported low degree of SLE-related pain. However, 24% of the patients reported high degree of SLE-related pain, more fatigue, anxiety and depression, and worse health-related quality of life (HRQoL). To explore SLE-related pain, the presence of long-standing widespread pain, and patient-reported outcomes (PROs) after 7 years. Sixty-four out of 84 patients participated in a 7-year follow-up of the original survey and completed the same questionnaires answered at inclusion: pain (VAS 100 mm), fatigue (MAF), HRQoL (SF-36), anxiety and depression (HADS), and, if appropriate, a pain-drawing. Differences between inclusion and follow-up (change) were calculated. The patients with a low degree of SLE-related pain at inclusion reported no changes at follow-up in pain and PROs except for worsening in physical function in SF-36, median change (IQR) 0 (− 10 to 5),  Living with the chronic disease systemic lupus erythematosus (SLE) may imply many difficulties. Beyond organ-specific symptoms and unpredictable courses , patientPain is a frequent self-reported symptom in SLE , 5 and iWe have previously shown that nearly one quarter of a cohort with patients with SLE reported a high degree of pain on a visual analogue scale (VAS) ≥ 40 mm, and these patients also reported more fatigue, reduced health-related quality of life, and more signs of anxiety and depression , 17.Considering the chronic nature of SLE and the great symptom burden that disease-related pain and associated symptoms can mean for the patients, it seems important to investigate the long-term course of SLE-related pain.The aim of the present study was to explore self-reported SLE-related pain after 7 years in the previously studied cohort, as well as fatigue, health-related quality of life, and mental health. In addition, characteristics and disease activity were captured.The studied cohort comprised 84 patients with SLE who were recruited consecutively in the period from 2006 to 2008 from one rheumatology centre. Also, 91 age- and sex-matched population controls were recruited. Characteristics of the cohort and the recruitment process are described in our previous studies –18.n = 64), and those who reported pain ≥ 40 mm on VAS were classified as the high pain group in our study (n = 20).In the inclusion study, the average of SLE-related pain intensity that patients experienced in the last week was measured using VAS 100 mm. The distribution of reported pain identified a clear cutoff value of 40 mm on the VAS . The patn = 4), migration (n = 3), unable to participate due to illness (n = 2), and unwilling to participate (n = 11) and for the controls: migration (n = 1), unable to participate due to illness (n = 5) and unwilling to participate (n = 17). There was no significant difference in age (years) between those patients who participated median (IQR) 52 (38.5 to 64) and those who did not 54.3 (45 to 64.6) (p = 0.289 for between group difference) and those controls who participated 56 (46.5 to 66) and those who did not 49.7 (38.2 to 67.5) (p = 0.146), and there was no significant difference in disease duration (years) among the patients who participated 16 (11.5 to 22.5) and who did not participate 16 (13 to 25) (p = 0.74) in this follow-up study.Data for the present 7-year follow-up study was collected between 2013 and 2015. In all, it was possible to follow 64 out of 84 patients and 68 out of 91 controls from the original inclusion study. Reasons for not participating in the follow-up study for the patients were death  were asked to complete the questionnaires at the follow-up assessment by a rheumatologist and a research nurse (year 7) Fig. .Fig. 1ChDisease activity was measured with the Systemic Lupus Activity Measure (SLAM)  and the Pain intensity was measured by VAS (100 mm) , 21 and To measure the clinical pain quantitatively, the total index and numbers of pain descriptors in the short form of the McGill Pain Questionnaire (SF-MPQ)  were useThe distribution of pain was captured by a pain drawing with predefined body regions .The global fatigue index (GFI) of the multidimensional assessment of fatigue (MAF) –26 was uThe Hospital Anxiety and Depression Scale (HADS) , 28 was The Medical Outcomes Survey-Short Form 36 (SF-36), standard Swedish version 1.0, was used to measure health-related quality of life (HRQoL) , 30. Thip value less than 0.05 was considered statistically significant.The change between year 0 and year 7 in VAS pain and index for fatigue, anxiety, and depression were measured by calculating the change between the variables at year 0 and at year 7 (year 0 minus year 7). For all variables, the change represented an improvement if it was positive, and a worsening if negative. The change for health-related quality of life (SF-36) was calculated as a measure at year 7 minus year 0 because a higher value of SF-36 means better health-related quality of life. Descriptive results are presented as median and interquartile range (IQR) and nonparametric statistical tests were used for comparative analysis due to the non-normally distributed data and different sizes of the groups compared. A The statistical analyses were conducted using the software STATISTICA 12 .The study was approved by the Stockholm Regional Ethical Review Board and all participants gave their written informed consent.Before characterisation of the participants, sub-grouping was performed based on reported SLE-related pain Fig. .n = 64), no statistical differences were found between year 0 and year 7 in respect to reported overall pain and SLE-related pain (Table In the whole patient group (in Table .Table 1Pn = 50) reported statistically similar levels of overall pain as well as SLE-related pain in the last week at year 0 and at year 7 (Table n = 14) reported lower levels of overall pain at year 7, VAS median (IQR) 49 (14 to 70) compared to year 0, 70 (49 to 79) (p = 0.050) as well as SLE-related pain at year 7, VAS median 43 (15 to 66) and 69 (50 to 72) (p = 0.035), respectively. The change of SLE-related pain at year 7 was greater in the high pain group, 32 (− 13 to 48), while it was 0 (− 6 to 2) in the low pain group.When dividing the patients into the groups by pain intensity at inclusion, the low pain group ( 7 Table . Howevern = 7) reported a lower level of SLE-related pain at year 7 and the other half of the patients in the high pain group (n = 7) reported similar levels of SLE-related pain at year 0 and at year 7  reported pain lasting more than 3 months and the pain drawing indicated chronic widespread pain according to the definition by Wolf [All the patients with remaining pain ( by Wolf  Table 1n = 7 repp = 0.032 and p = < 0.001 respectively), the controls reported lower overall pain at year 7.There was no significant difference in overall pain between the controls and the patients with low pain and those with decreased pain Table  at year p = 0.006), the patients with low pain (p = 0.002), and the controls (p = <0.001).The patients with remaining pain at year 7 reported the greatest number of body regions with pain outlined on the pain drawing Table , and sigCharacteristics of the patients and the controls are presented in Table There were no significant differences in age or disease duration at year 7 between the patients by pain groups in this study Table .p = 0.017) as well as the patients with low pain (p = 0.006). Also, the physician’s estimated global disease activity on VAS/SLAM at year 7 was highest for the patients with remaining pain (Table p = 0.007).At year 7, the disease activity index SLAM had decreased for the patients with low pain and for the patients with decreased pain. The patients with decreased pain had the greatest improvement between year 0 and year 7 regarding SLAM and self-reported disease activity Table . Howeverin Table  and signp = < 0.001), the patients with decreased pain (p = 0.043), and the patients with remaining pain (p = 0.028). The difference between the physician’s and the patient’s estimated global disease activity was greatest for the patients with remaining pain  and to the patients with remaining pain (p = 0.073). Yet, there was no significant difference in SLEDAI at year 0 between the patients with low pain and those with remaining pain (p = 0.99).No statistical differences in SLEDAI at year 7 were found between the pain sub-groups. SLEDAI at year 7 decreased significantly only for the patients with decreased pain Table . HoweverThe damage index SLICC at year 7 was highest in the patients with decreased pain Table  but was The use of glucocorticoids and disease modifying antirheumatic drugs (DMARDs) as well as the current dose of glucocorticoids decreased for all patients at year 7 Table . The greAccording to the SF-McGill pain questionnaire, the patients with decreased pain reported a significantly lower total index as well as fewer words to describe pain and pain intensity at year 7 Fig. a, b. ForOnly the patients with decreased pain reported a significantly lower general fatigue index at year 7 compared to year 0 Fig. a.Fig. 3ap = 0.017) and the patients with decreased pain (p = 0.017), as well as the controls (p = 0.001)  Fig. a. There As assessed by the HADS, no symptoms of clinically significant anxiety or depression were found in any of the groups at year 7. Only the patients with decreased pain reported a significantly lower index for anxiety and depression at year 7 compared to year 0 Fig. b, c. Thep = 0.011), the patients with low pain (p = 0.011) and to the controls (p = 0.004)  Fig. c.For the patients with decreased pain, there were improvements in all the dimensions of SF-36 at year 7 compared to year 0 Fig. b.Fig. 4aIn contrast, the patients with remaining pain did not report any changes in SF-36 dimensions at year 7 except for worsening in the dimension of vitality, median change (IQR), − 20 (− 35 to − 15) Fig. c.p = < 0.001–0.012).At year 7, the patients with remaining pain, compared with the patients in the decreased and low pain groups, had significantly poorer HRQoL in all dimensions of SF-36 at year 7, but not for the dimension mental health (There were no differences in any dimension of SF-36 at year 7 between the patients in the decreased pain and low pain groups (data not shown).physical function, median change (IQR) 0 (− 10 to − 5), and bodily pain median change 0 (IQR) (− 21 to 1)  Fig. a.p = 0.005 to < 0.001). Also, the controls reported better health status than the patients with low pain in the dimensions of physical function (p = 0.016), general health , and bodily pain (p = 0.053). At year 7, no differences were found between the controls and the patients with decreased pain.Among the controls, there were no significant differences in any of the dimensions in SF-36 between year 0 and year 7 Fig. d. At yeaIn general, previous studies have shown impaired HRQoL and mental health as well as a high degree of fatigue in patients with SLE . In addiEven if the patients with remaining pain reported higher indices in HADS and were thereby distinguished from the other participants, SLE and pain did not seem to affect the mental dimension of health as much as the physical dimension among the patients in this study. Mental health was the only dimension of SF-36 in which none of the groups differed from each other after 7 years. These results conflict with those of a previous study where SLE generally is reported to be associated with poor mental health, i.e. depression and anxiety . Yet in The disparity between disease activity indices as well as the discrepancy between estimations of disease activity of the physician and the patients, especially in the patients with remaining pain, assumes a risk of communication barriers. The discrepancies found in this study are in accordance with results from previous studies in which patients with SLE associated their assessment of disease activity with more subjective experiences, including the impact of the disease on life as a whole. Healthcare providers, on the other hand, base their assessments on more objective measures such as laboratory findings and physical examinations , 35, 36.Higher SLEDAI at year 0 in the patients with decreased pain, in combination with a higher damage index SLICC at year 7, may suggest a higher degree of inflammatory activity at year 0 for the patients with decreased pain. In this group, after treatment and the natural course of the disease, the inflammatory activity had decreased at follow-up, yet patients had permanent damage. Despite permanent disease damage, the patients with decreased pain reported less pain and other subjective symptoms. For the patients with remaining pain, this discrepancy may illustrate the patient’s perception that pain is related to SLE and not to comorbidity, for example chronic widespread pain. For patients, it may be difficult to distinguish between inflammatory pain and other types of pain. For physicians, the symptoms of long-standing pain may be confused with SLE-related symptoms. Different types of pain require different treatments, and it is thus a major challenge for health care providers to distinguish SLE-related pain from pain of non-inflammatory origin , 39. LikOne limitation of this study is the relatively low number of participants in the sub-groups. However, the objective of the study was to investigate the variation over time of pain and important patient-reported outcomes in the original cohort, and thus recruitment of other patients with SLE outside this cohort was not appropriate. A strength of the study is that it had a sufficiently long follow-up to assess variation in pain and quality of life measures, and that it involved a comprehensive survey of both patients and controls, consistent with the measures used at inclusion of participants in the original study.Taken together, the results from this 7-year follow-up study showed consistently that decreased disease-related pain is related to decreased fatigue, anxiety, and depression as well as improved HRQoL. Vice versa, the patients with remaining high levels of pain at follow-up maintained a high degree of fatigue and poor health-related quality of life. Following the principle of shared decision-making with the patient as an active participant of care, our results stress the importance of accurate monitoring of pain using self-report questionnaires incorporated in the assessment of disease burden in patients with SLE.


Extraintestinal manifestations (EIM) contribute significantly to the burden of disease in inflammatory bowel disease (IBD). Pain is a leading symptom in IBD and could be seen as an EIM itself. Treatment of IBD associated pain is challenging and insufficiently studied. A better knowledge on the association of pain and IBD specific treatment is warranted to improve the management of IBD patients.All patients of the Swiss IBD Cohort Study (SIBDCS) (n = 2152) received a questionnaire regarding pain localization, pain character, and the use of IBD specific medication.1263 completed questionnaires were received. Twenty-one out of 184 patients (10%) receiving anti-TNF treatment compared to 142 out of 678 patients (21%) not receiving anti-TNF medication reported elbow pain (p = 0.002) while 28 out of 198 patients (14%) receiving steroid treatment significantly more often reported elbow pain compared to 59 from 696 patients (8%) not receiving steroids (p = 0.021). Furthermore, we found significantly more female patients under anti-TNF treatment to report knee/ lower leg pain and ankle/ foot pain compared to their male counterparts . The frequency of knee, lower leg, ankle and foot pain was especially low in male patients under anti-TNF treatment, indicating a high benefit of male patients from anti-TNF therapy regarding EIM.The frequency of elbow pain was lower in IBD patients treated with anti-TNF but higher in patients treated with steroids. Pain is a common symptom in patients with inflammatory bowel disease (IBD) , 2. In aTreatment of both, IBD and IBD associated pain is challenging. The mainstay of IBD treatment includes systemic immunosuppressive medications, such as corticosteroids, anti-tumor necrosis factor (TNF) antibodies or immunomodulators. Furthermore, the management of an acute flare differs from the strategies for maintenance of remission , 23. MorFurthermore, non-steroidal anti-inflammatory drugs (NSAIDs) can very effectively mediate pain relieve due to their analgetic and also anti-inflammatory effects. However, due to the risk of disease exacerbation and induction of flares their use in IBD is limited –30.Here, we used the well-characterized patient collective of the Swiss IBD Cohort Study (SIBDCS) to study the association of pain and IBD treatment with a focus on anti-TNF treatment.Ethics approval was obtained from the regional Swiss Ethics Committees in which cohort participants were enrolled . Written, informed consent was obtained from each patient included in the study. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the institution's human research committee.Patients of the nationwide SIBDCS have been prospectively included since 2006 with a yearly follow-up. The cohort goals und methodology of SIBDC have been described elsewhere.A questionnaire addressing various aspects of pain including pain duration localization and frequency was mailed to 2152 SIBDC patients, representing the entire cohort. The questionnaire also inquired about the use of pain specific medication in detail. Our questionnaire contained several questions from a validated German pain questionnaire . The queDescriptive statistical analyses were performed: Categorical variables were summarized as frequencies and percentages, whereas quantitative variables as median and range. To assess differences in categorical data distribution between groups of different sizes, Fisher’s exact test was used.The statistical analysis was performed using GraphPad Prism 7 for MacOS. A p-value of <0.05 was considered statistically significant.The patients’ characteristics shown in When comparing the use of IBD specific medication and ten different pain localizations, we found several significant differences. Regarding elbow pain, only 21 patients (10%) receiving anti-TNF treatment compared to 142 patients (21%) not receiving anti-TNF were affected (p = 0.002). Other pain localizations did not reveal significant differences regarding anti-TNF treatment .Comparing other IBD specific therapy and the different pain localization, we found patients not receiving steroid treatment significantly less often to be suffering from elbow pain compared to patients receiving steroids . For the evaluation of other pain localizations and other IBD specific therapy  no significant differences were observed  Tables.Duration of pain did not differ between patients on anti-TNF treatment versus those not on anti-TNF treatment . The durThe frequency of pain in patients with and without anti-TNF treatment did not significantly differ . When coFurther, there was no association between the pain character and the use of IBD specific medication. 36 patients (20%) on anti-TNF treatment described their pain to be constant with slight fluctuations compared to 115 patients (19%) without anti-TNF treatment (p>0.999). Constant pain with strong fluctuations was reported by 15 patients (8%) using anti-TNF treatment and by 64 patients (11%) not receiving anti-TNF treatment (p = 0.331). 115 patients (61%) with TNF treatment and 349 (58%) without anti-TNF treatment experienced pain attacks with pain free intervals (p = 0.554). Pain attacks with constant pain were reported by 30 patients (16%) receiving anti-TNF treatment compared to 75 (12%) not receiving anti-TNF (p = 0.268). When comparing the pain character of patients receiving other IBD specific medication , no significant differences across treatment groups were seen  Tables.Moreover, the duration of pain attacks was not influenced by IBD specific medication , neitherFrom a total of 894 patients, a similar fraction of male and female patients (24% for both) received anti-TNF therapy .When comparing the pain localizations of male and female patients receiving anti-TNF treatment significantly fewer male patients with anti-TNF treatment suffered from knee/ lower leg pain compared to female patients receiving anti-TNF therapy . Also, significantly fewer male patients receiving anti-TNF treatment reported ankle/ foot pain compared to female patients with anti-TNF treatment . For the other pain localizations, no differences regarding gender were seen .We did not observe any differences in pain localizations in patients with versus without anti-TNF-therapy, neither in male nor female patients Tables  and 9.In our study population 5-aminosalycylicacid (5-ASA) (37%) was the most frequently used IBD specific medication, followed by immunomodulators (35%) and anti-TNF antibodies (24%). As for anti-TNF, Vavricka et al. showed that in more than 40% of the cases, this therapy regime is initiated to treat EIM rather than bowel inflammation and over 70% showed a clinical response of EIM to anti-TNF therapy . Our stuRegarding gender specific differences in treatment of EIM/ pain in IBD patients, data is not consistent. Concerning IBD treatment, Lopetusa et al. found no general influence of the gender on the therapy of ulcerative colitis (UC) with anti-TNF (infliximab) . HoweverOne strength of our study is the size of the cohort with 1263 completed questionnaires. Together with our former study evaluating pain in the SIBDCS [However, our study also has limitations. Due to the study design and the lack of control regarding unreturned questionnaires, a reporting bias cannot be excluded. Patients who actually suffer from pain due to IBD therefore might be overrepresented compared to patients without pain, since the former might be more motivated to return the questionnaire. The patients not responding to the survey might have represented a different phenotype regarding our topic of interest. The existing data of the SIBDCS doesn’t include any information about pain, preventing us from compairing pain specific parameters between responders and non-responders. Furthermore, regarding the use of IBD specific therapy and pain localizations, we do not have information on the reason to initiate medical therapy  and how high the prevalence of pain has been before treatment initiation. Our statistical evaluation of the data represents another limitation. We have performed a mostly derscriptive analysis of the dataset. To remain a high response rate and not no overstrain the goodwill of the patients, we intended to keep our questionnaire on a simplistic level. We further see the sizes of our subgroups as a potential limitation. We are aware that small subgroups may be linked to random positive findings. In comparison to other studies about pain in IBD, the subgroups examined here are not considerably small. Furthermore, we aimed to include as many pain localizations as possible to thoroughly analyse the distribution of pain. Additionally, our findings, particularly regarding Anti-TNF, match the clinical observations, depicting a genuine outcome.In summary, we could show that the frequency of elbow pain was lower in patients treated with anti-TNF but higher under steroid treatment. There were no significant differences regarding the use IBD specific therapy and the character, duration and frequency of pain. Furthermore, our data point towards a higher treatment benefit of anti-TNF with regards to EIM in male patients which should be followed up in future studies.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S7 Table(PDF)Click here for additional data file.S8 Table(PDF)Click here for additional data file.S9 Table(PDF)Click here for additional data file.S10 Table(PDF)Click here for additional data file.S11 Table(PDF)Click here for additional data file.S12 Table(PDF)Click here for additional data file.S13 Table(PDF)Click here for additional data file.S14 Table(PDF)Click here for additional data file.S15 Table(PDF)Click here for additional data file.S16 Table(PDF)Click here for additional data file.S17 Table(PDF)Click here for additional data file.S18 Table(PDF)Click here for additional data file.S19 Table(PDF)Click here for additional data file.S20 Table(PDF)Click here for additional data file.S21 Table(PDF)Click here for additional data file.S22 Table(PDF)Click here for additional data file.S23 Table(PDF)Click here for additional data file.S24 Table(PDF)Click here for additional data file.S25 Table(PDF)Click here for additional data file.S26 Table(PDF)Click here for additional data file.


Background: Postoperative pain control in craniotomies poses multiple challenges. Pain must be addressed, but the use of medications must be weighed against risks. Craniotomies risk neurologic injury and so postoperative examinations are critical. Medications used to address pain can alter the neurological examination or cause bleeding leading to misdiagnosis of complications.Objective: Determine if there is a significant difference in postoperative pain from emergent craniotomies vs. non-emergent craniotomiesMethods: A retrospective review included 102 cases performed from 2010-2016; pain scores were compared on post-operative days one, two, and three between emergent and non-emergent craniotomies.Results: Pain scores for emergent cases on post-operative days one through three were 5.1 (standard deviation (SD)=2.9), 5.9 (SD=2.1), 4.7 (SD=3.0) respectively. Pain scores for non-emergent cases on post-operative days one through three were 5.7 (SD=2.6), 4.8 (SD=2.8), and 4.6 (SD=3.0) respectively. A one-way analysis of variance (ANOVA) was conducted to compare pain scores between groups for each post-operative day. On post-operative day, one there was no significant difference between the groups . On post-operative day two, there was no significant difference between the groups . On post-operative day three, there was no significant difference between the groups .Conclusion: There is no significant difference in the level of pain on postoperative days one through three between emergent and non-emergent craniotomy patients. Postoperative pain control in craniotomies has been a challenge for years. Research has been directed at trying to identify improved analgesic regimens -9. TheseA retrospective study at a single institution of all patients undergoing craniotomy from January 2010 to December 2016 were assessed for postoperative pain as recorded by nursing staff during medication administration for the first three postoperative days using the visual analog scale. Scores were averaged over each day for post-operative days one, two, and three. Inclusion criteria included craniotomy performed at the hospital of study and age greater than 18 years old. Exclusion criteria included the inability to communicate pain score verbally either through hand gestures or through writing, previous craniotomy or craniectomy, pregnancy, prison inmate, or incomplete records (for example missing recorded pain scores).After a review of 939 potential records, 102 were included in the study after inclusion and exclusion criteria were applied. The average age of patients was 53 and approximately 53% of study participants were male (n=54) with 47% female (n=48). Reasons for surgery include tumor resection 70% (n=71), intracranial hemorrhage including epidural, subdural, or intraparenchymal 21% (n=21), arteriovascular malformation (AVM) or aneurysm 6% (n=6), or other 4% (n=4). Cases that satisfied criteria were largely non-emergent at 87% (n=89) and 13% emergent (n=13). Demographic information is shown in Table Aggregate pain scores of both emergent and non-emergent groups from post-operative days one to three scored 5.6 (standard deviation (SD)=2.7), 4.9 (SD+2.7), and 4.6 (SD=3.0), respectively. Pain scores for emergent cases on post-operative days one through three were 5.1 (SD=2.9), 5.9 (SD=2.1), 4.7 (SD=3.0), respectively. Pain scores for non-emergent cases on post-operative days one through three were 5.7 (SD=2.6), 4.8 (SD=2.8), and 4.6 (SD=3.0), respectively. Average pain scores are shown in Table A one-way analysis of variance (ANOVA) was conducted to compare pain scores in patients who underwent emergent craniotomy versus non-emergent craniotomy for each post-operative day. On post-operative day one, there was no significant difference between the groups . On post-operative day two, there was no significant difference between the groups . On post-operative day three, there was no significant difference between the groups . Post-operative day three has fewer data points as one patient was intubated and another taken back to the operating room for recurrent subdural hematoma. Regarding opioids, the majority of patients 95% (n=97) received some form of opioid for pain control with only 1% (n=1) of all patients returning to the operating room for recurrent subdural hematoma. ANOVA scores are shown in Table Pain scores in emergent versus non-emergent craniotomy cases did not show a statistically significant difference which could suggest that post-operative pain is dominated by surgical disruption of anatomy as opposed to the mechanism of injury. However, this study includes a disproportionate amount of non-emergent cases since emergent cases are likely left intubated or are unable to communicate pain effectively post operatively. Therefore, this study population likely represents less severe mechanisms of injury since they were able to be extubated post operatively. Scalp nerve blocks were inconsistently used in non-emergent cases with infiltration typically only taking place along the planned site of incision primarily to aid with hemostasis. Therefore, this could be a potential confounder in post operative pain score comparison. Trauma patients included in the study population may also have had additional injuries such as fractured bones which would confound the reported pain scores. Also, the perception of pain by each individual is different which will lead to significant variability. The averages of all patients for each group were computed and the variability is partially reflected in the standard deviation. Emergent craniotomies represent 13% of the study population and non-emergent craniotomies representing 87% which also makes comparison difficult due to the disproportionate group sizes.In this review, only one patient out of 102 patients receiving opioid analgesics post operatively had to return to the operating room. Concerns with opioids typically include obscuring the clinical exam to the point of missing a catastrophic event which could lead to further morbidity or mortality ,11,15-17No statistical difference was found in pain scores in post-operative days one through three of patients who underwent craniotomy for emergent (within 24 hours of diagnosis) and non-emergent reasons when they were able to communicate a pain score either verbally or in writing. In our series, pain was reported verbally postoperatively, therefore, emergent cases represented in this patient population likely only included minor mechanisms of injury since they were able to be extubated postoperatively and there was a disproportionate amount of non-emergent craniotomies compared to emergent craniotomies for data analysis limiting the generalizability of findings.


Which specific targets of voltage-gated potassium channel–complex antibodies are found in acquired neuromyotonia, and are these antibodies associated with additional clinical features of tumors, pain, or autonomic or central disorders?This cohort study combined with a patient-led survey found that antibodies to the extracellular aspects of leucine-rich glioma inactivated protein, contactin-associated protein 2, and contactin 2 were variably present in 45% of patients with neuromyotonia. Paresthesia and various pain manifestations were common in neuromyotonia, and the type and severity of pain were found to exert a substantial influence on quality of life.Antibodies to voltage-gated potassium channel–complex proteins are not found in all patients with neuromyotonia and do not individually relate to specific clinical features, but the presence of pain and its effects on quality of life need greater recognition. Although acquired autoimmune neuromyotonia (NMT) is associated with voltage-gated potassium channel (VGKC)–complex antibodies, to date there has been no systematic study of autoantibodies to the specific antigens leucine-rich glioma inactivated protein 1 (LGI1), contactin-associated protein 2 (CASPR2), and contactin 2 together with the full clinical syndrome, particularly pain and autonomic and central nervous system involvement.To study the full spectrum of clinical features and serum autoantibodies in patients with NMT, including the effects of pain on quality of life.A cohort study of clinical features and serologic testing in 38 patients with electrophysiologically-confirmed NMT, reviewed clinically between February 2007 and August 2009, in the Universities of Sydney and Kagoshima and followed up across 2 to 4 years. Association of NMT with quality of life was researched in an independent, patient-led, online pain survey conducted from April 2012 to May 2012. Serologic analyses were performed in 2012, and final data analysis was performed in 2016.Clinical data and scores on the modified Rankin Scale (mRS), which measures disability on a range of 0 to 6, with 0 indicating normal and 6 indicating death, before and after treatments were combined with CASPR2, LGI1, and contactin 2 antibody status.Among the 38-person NMT cohort, 25 (65.8%) were male and the median (range) age was 55 (12-85) years. Twenty-three (60.5%) were Japanese and 15 (39.5%) were of white race/ethnicity. Symptomatic treatments (mainly antiepileptic drugs) were used in most patients with mild disease (12 patients with mRS <3), whereas immunotherapies were successful in most patients with mRS scores greater than 2. Autoantibodies to VGKC-complex antigens (17 patients [45%]), bound to CASPR2 (5 [13%]), contactin 2 , LGI1 (2 [5%]), or both LGI1 and CASPR2 (6 [16%]). The last group of 6 patients had high mRS scores , thymoma (4 patients), pain (5 patients), autonomic (6 patients) and sleep (5 patients) disturbance, suggesting Morvan syndrome. The 56 responders to the independent patient-led survey reported pain that could be severe, anatomically widespread, and that often resulted in unemployment, domestic problems, and poor quality of life.The cohort study detailed underrecognized aspects of the clinical and serologic spectrum of NMT. The heterogeneity of clinical features and of specific antibodies limit associations, but the common existence of thymoma, pain, and autonomic and central nervous system features, often with both LGI1 and CASPR2 antibodies, should be better recognized to more completely address the range of comorbidities and consequences of the disease regarding quality of life. This combined clinical cohort and survey study examines associations between neuromyotonia and voltage-gated potassium channel–complex–specific antibodies and the consequences of neuromyotonia for quality of life. Acquired neuromyotonia (NMT) was first associated with antibodies that immunoprecipitated voltage-gated potassium channels (VGKCs).16 Other studies have included only a few patients with NMT.17 The full spectrum of clinical features , the specific antigenic targets, and whether the clinical features relate to antibody specificity have not been determined in NMT.The reported frequency of VGKC-complex antibodies in patients with NMT, usually at relatively low titers (100-400pM), or in those with the less severe variant, cramp fasciculation syndrome, has varied between 2% and 40%.18We describe detailed clinical and serologic characteristics of patients with NMT from Japan and Australia combined with the results of a novel, independent, patient-led pain questionnaire sent to individuals registered on an online forum for persons with Isaacs syndrome. The study is particularly timely given recent evidence that injection of patient-derived CASPR2 antibodies or genetic deletion of CASPR2 causes afferent nerve hyperexcitability and mechanical allodynia in mice.19 after VGKC-complex antibody screening, and Kagoshima, Japan, between February 2007 and August 2009 for routine testing at the time of patient review and were studied in detail in 2012. Final data analysis was performed in 2016. The diagnosis was reached according to established criteria, with symptoms or signs of muscle twitching or muscle cramps affecting at least 2 regions of skeletal muscles.14 All patients demonstrated the characteristic electromyographic (EMG) discharges consisting of doublet, triplet, or multiplet single–motor unit discharges with a high intraburst frequency of between 40 and 400 per second.21 Eleven patients with similar symptoms and serologic results, but without confirmation or testing by EMG, were excluded. Ethical approval was granted by the South Eastern Sydney Local Health District , the University of Sydney (Sydney), and the University of Kagoshima  human research ethics committees, and patients gave written informed consent for their data to be used. The serologic study was performed under the authority of the Oxford Regional Ethics Committee.Thirty-eight serum samples from patients with clinical and neurophysiologic features consistent with a diagnosis of NMT were collected in Sydney, Australia,8 Antibodies to LGI1, CASPR2, and contactin 2 were detected by live cell–based assays. For these assays, DNA encoding the different proteins was transfected into human embryonic kidney 293 cells cultured on glass coverslips and left overnight at 37°C. The cells were gently washed and left in medium for 1 additional day before each coverslip was placed in a 20-well microtiter plate and incubated in 200 μL of medium containing 10 μL (1:20 for LGI1 or contactin 2) or 2 μL (1:100 for CASPR2) for 2 hours. Binding of IgG to the cells was detected with Alexa fluor anti-human IgG (Thermo Fisher Scientific) after fixation.8 The binding was scored visually on a scale from 0 (negative), 1 (low positive), and 2 to 4 (increasing strength of binding).Serum samples were originally tested for binding to VGKC-complex antibodies by radioimmunoprecipitation, as previously described.The clinical data recorded shown in  covered http://isaacsyndrome.proboards.com/ (now https://www.facebook.com/groups/isaacs.pnh/) and asked whether they had pain. One hundred seventy-six patients responded, of whom 165 reported pain and were sent the questionnaire  through t tests were used to compare scores before and after treatments, and a 2-sided P < .05 was considered significant.Prism software, version 7 (GraphPad Software) was used to create graphs and perform statistical analysis. Two-tailed paired Of the 38 patients, 25 (66%) were male and the median (range) age was 55 (12-85) years. Twenty-three (60.5%) were Japanese and 15 (39.5%) were of white race/ethnicity. The cohort was defined by typical history and EMG findings of peripheral nerve hyperexcitability. Nerve conduction was normal in 10 of 12 patients examined; 2 patients had evidence of neuropathy .Initial presenting symptoms on referral comprised cramps (16 patients [42%]), fasciculations (7 [18%]), and, less commonly, symptoms of myokymia, stiffness, or spasms . SensoryThe symptoms recorded in response to specific questions at the first clinic visit were more widespread, including cramps (32 [84%]) and muscle twitching (30 [79%]), as well as sweating (12 [32%]), weakness (13 [34%]), and stiffness (12 [32%]) . In addiThe most striking additional complaint reported was sensory disturbance. Specifically, 20 patients (53%) complained of paresthesia (5 [13%]), pain (8 [21%]), or both (7 [18%]). Pain and paresthesia were typically reported in the legs or arms and sometimes in all limbs. Pain was described as burning or throbbing in 7 patients (18%). One patient specifically reported pain from muscle cramps.With respect to central nervous system symptoms, sleep disturbance, particularly insomnia, was present in 13 patients (34%), anxiety and agitation in 10 (26%), and depression in 5 (13%). Two male patients (5%) attempted suicide. One patient had new-onset seizures.Additional investigations included cerebrospinal fluid analysis in 8 patients, which had normal results with the exception of 2 patients (25%) who had a small increase in lymphocyte counts. Electroencephalogram results were normal in 8 of 9 patients (89%) , and normal findings on magnetic resonance imaging in 18 of 20 others (90%) .Eight of the 38 patients (21%) had recent tumors, 5 thymomas , and 3 prostate tumors; 1 had a previously treated non–small cell cancer; and 1 had acute lymphatic leukemia after bone marrow transplantation. Acetylcholine receptor antibodies were positive in 3 patients with thymoma and Hu antibodies in another patient with thymoma.Overall, autoimmunity or other comorbidities (eTable 3 in the P = .001) and had received a range of immunotherapies only or in combination with drugs for symptoms, with clear benefits in most (P = .004) but also responded well to treatments . Eleven of the serum samples (29%) (9 from men and 2 from women)were VGKC-complex antibody positive. However, results of the live cell–based assays were positive in 17 of the 38 patients (45%), including CASPR2 antibodies in 11 (29%), LGI1 antibodies in 8 (21%), and contactin 2 antibodies in 5 (13%) A. Result11 although 1 had an additional central nervous system feature (seizures). As a group, they had higher mean pretreatment mRS scores  compared with all other patients (2.71 [1.08]), but both groups had similar posttreatment scores  had thymoma and 1 had developed prostate cancer; 1 had mild disease that responded to carbamazepine alone. Each of these patients had autonomic symptoms as well as typical NMT, 5 complained of pain, and 4 had neuropsychiatric features. Five had sleep disturbance and 3 fulfilled the criteria for Morvan syndrome ,Serum samples were also tested simultaneously using a widely available commercial assay. Positivity for LGI1 and CASPR2 antibodies was confirmed except for 1 CASPR2 antibody that was not detected with the commercial assay (data not shown).Because of the reports of pain in 20 patients (53%) that were often unrelated to muscle cramps, an independent survey was conducted via an Isaacs syndrome website, and questionnaires were sent to 165 individuals who reported pain. The patients were asked to describe the level of pain at best and at worst (0 representing no pain to 10 representing very bad or incapacitating pain), describe the nature of the pain, and detail where the pain was, what factors made it better or worse, and the extent to which it had responded to treatments. To assess the effects on quality of life, patients were asked the extent to which pain affected sleep, relationships, work, exercise, mood, and general activities .How bad is the pain at best ?How bad is the pain at worst ?How would you describe the types of pain(s) you feel, and where on the body they occur? Please also add how bad is/are the pain(s) in each area.What if anything make the pain(s) worse ?When do your pain symptoms occur ?How effective have your prescriptions been at helping with pain(s) ?For the following questions, please select between 1 and 10 as appropriate .How much would you say pain affects your enjoyment of life?How much would you say the pain(s) affects sleep?How much would you say pain affects your relations at /home/with loved ones/friends?How much would you say pain affects your ability to work (includes work around the home)?How much would you say pain affects your walking ability?How much would you say pain affects your mood?How much would you say pain affects overall day-to-day living and general activities?Finally, if you wish, comment further about the pain(s) you suffer and how they affect you.Abbreviations: CFS, cramp fasciculation syndrome; NMT, neuromyotonia.aThis is a simplified version of the form sent to patients who responded to the internet survey as having pain. The full form, which includes example answers for each question, is available in eTable 1 in the Supplement20 and none have studied in detail the associated clinical features, serologic characterization, and treatment responses. The results of this cohort study of 38 patients with EMG-defined NMT found 8 (21%) with concurrent tumors ; when asked, a high proportion reported additional features of autonomic disturbance, pain, and central nervous system dysfunction. Antibodies to the now well-recognized VGKC-complex antigens were present in 17 of the 38 patients (45%), but these included 6 (16%) patients with both LGI1 and CASPR2 antibodies, and these patients had more severe disease, thymoma (in 50%), and features of Morvan syndrome. Contactin 2 antibodies were relatively common (5 patients [13%]) compared with their incidence in other VGKC-complex antibody disorders.8 After receiving treatment, all but 4 patients with follow-up data improved by at least 1 mRS score. Therapies to treat symptoms were moderately effective in the few patients with relatively mild disease; however, in others, improvement was related to immunotherapy with or without symptom treatments.There have been few detailed descriptions of NMT,23 and may previously have been ascribed mainly to muscle cramps. However, the study patients with NMT reported a variety of painful symptoms as reflected in the detailed responses reported via the independent, patient-led online questionnaire that included fuller descriptions of the pain, its distribution and effects on aspects of daily living, such as relationships and work. A systematic, prospective study of pain in NMT needs to be undertaken.The high frequency of pain among the patients with EMG-defined NMT (20 patients [53%]) exceeded that reported in previous studies, and the nature of the pain in NMT has only occasionally been described13 but direct questioning resulted in almost 50% of both male and female patients reporting diverse symptoms. In addition, sleep disturbance, including insomnia, were frequent. Morvan syndrome, which is defined by NMT, autonomic disturbance, and insomnia with encephalopathy, is thought to be a rare disease, with fewer than 100 cases reported in the literature.24 One typical patient with Morvan syndrome and high-titer CASPR2 antibodies was not included here because the diagnosis had already been given and serum was no longer available. Nevertheless, 3 of the 6 patients (50%) with both CASPR2 and LGI1 antibodies had many features of Morvan syndrome, including thymoma; however, none had developed encephalopathy, and the diagnosis remained NMT. Thus, NMT can be seen as a forme fruste of Morvan syndrome.Another feature of this study was the number of patients who reported autonomic features. These have always been recognized in NMT,8 in patients with NMT, the titers originally reported13 were mostly lower (<400pM), and in the present study, 47% of patients were negative for all antibody tests . In fact, the clinical relevance of many lower titers, often without evidence of antibodies to LGI1, CASPR2, or contactin 2, is unclear.26 By contrast, LGI1 antibodies modulate VGKC function on brain slices and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid currents in hippocampal neurons28 and internalize the LGI1–disintegrin and metalloproteinase 22 receptor complex in transfected human embryonic kidney cells.29 CASPR2 antibodies did not internalize CASPR2 on hippocampal neurons in one study,30 but in vivo transfer of CASPR2-antibodies to mice produced afferent nerve hyperexcitability and mechanical allodynia with loss of surface CASPR2 and VGKC Kv1 subunits in the dorsal root ganglia; these and additional dorsal horn changes were found in CASPR2−/− mice.18 These findings are typical of neuropathic pain models and consistent with a contribution of CASPR2 antibodies to producing pain via an effect at the levels of the dorsal root in NMT. Autoimmunity in patients with pain is an emerging field of research.31In this study, 3 of the patients had VGKC-complex antibodies without evidence of positivity for CASPR2, LGI1, or contactin 2. Although VGKC-complex antibodies can be at high titer (>400pM)23 suggest that CASPR2, LGI1, and contactin 2 antibodies are more relevant than VGKC-complex antibodies in patients with suspected NMT. Nevertheless, fewer than half of the patients with suspected acquired autoimmune NMT were positive for the antibodies tested here, and other targets may need to be identified. At present, neurophysiologic assessment remains the criterion standard for diagnosis combined with clinical phenotyping.The data here and from other studiesTreatment protocols for NMT have not been formalized. Most patients will be given antiepileptic therapies or muscle relaxants as a first-line treatment because these can be sufficient; in the present series, these therapies were most often prescribed for patients with less severe disability. The remaining patients were given these treatments plus corticosteroids, immunotherapies, or both. Such therapies were effective and most patients reported benefit, but some patients noted little benefit. In patients with symptoms of NMT refractory to immunotherapies, the possibility of nonimmunologic disorders should be considered.Neuromyotonia is a rare disorder and consequently study numbers tend not to be large. Furthermore, as a study driven by patients and their clinical needs rather than through an established protocol, data were typically collected at follow-up reviews. As such, it was not always possible to obtain mRS scores from all patients before and after treatment. Serologic studies of LGI1, CASPR2, and contactin-2 antibodies were performed on archived samples that had been sent to the Oxford reference laboratory as part of routine diagnostic screening from Sydney and Kagoshima and were then retested for this study. Separately, although there are limitations with relying on a patient-initiated questionnaire—including a lack of response in those patients who did not report pain—it is increasingly recognized that patient-reported outcome measures may better reflect real-world experiences, which in this case have provided novel insight into the variable frequency and characteristics of pain in patients diagnosed with NMT and on the effects on their daily activities.Although cramps, muscle fasciculations, and pain are common symptoms, neuromyotonia remains a relatively rare diagnosis, and it is likely that less severe cases are often unrecognized. Nevertheless, comorbidities such as thymoma or myasthenia and sometimes associated symptoms of autonomic and central nervous system disturbance, and pain, a potentially disabling feature that is not always appreciated in clinical practice, mean that the disease deserves to be investigated for underlying triggers while also treated appropriately. Serologic studies are not positive in all patients, and neurophysiology remains a key diagnostic tool that also serves as a means of phenotyping patients. Moreover, although heterogeneity of clinical features and related antibodies may limit interpretation of significant correlations, the coexistence of LGI1 and CASPR2 antibodies may suggest the presence of thymoma, often accompanied by autonomic and central nervous system involvement.