A prospective double-blinded placebo controlled randomized study was conducted in our institute to compare the efficacy of oral Silodosin, an alpha 1 selective antagonist and Diclofenac in relieving pain after stent removal. All patients with unilateral stent placement following renal and ureteric stone endoscopic surgery were randomized into four groups: group A (Placebo), group B (Diclofenac), group C (Silodosin) and group D (combination of Diclofenac and Silodosin). Visual analogue score (VAS score) and other relevant parameters were assessed during OPD visit. Patients were handed over randomized drug envelope and asked to take the medications one hour before the stent removal. Patients were contacted 24 h after stent removal and relevant parameters were recorded. The mean VAS scores were significantly decreased in Diclofenac (2.9), Silodosin (3.08) and combination group (2.85) when compared to placebo (4.20) (p < 0.001). However there was no statistically significant difference in VAS scores between the treatment groups, i.e., group B, C and D (p > 0.5). Analgesics requirement and severe pain rates were not significantly reduced  in the three treatment groups when compared to placebo. Thus Silodosin and Diclofenac, both are effective in preventing pain after stent removal. Ureteral stents have become an indispensable tool for the urologist but they are often associated with significant morbidity. A significant number of patients often report pain and urinary symptoms with indwelling stents  polyurethane ureteral stent was used. DJ stent was kept for a period of 3 weeks before removal. A total of 272 patients were enrolled in the study from January 2014 to March 2015. Patients with history of peptic ulcer disease, liver impairment, chronic renal failure, coronary artery disease, bleeding diathesis, asthma, urinary tract infections (UTI), chronic painful conditions like arthritis, pregnancy, allergy to medications, significant lower urinary tract symptoms (LUTS) and use of alpha blockers and residual calculus were excluded. Patients with complications during stent removal like hematuria and mucosal injury were also excluded. Out of 272 patients, 240 patients were included in our study after excluding 32 patients not fulfilling the inclusion criteria. The patients were then randomized into four groups: group A (Placebo), group B (Diclofenac), group C (Silodosin) and group D (combination of Diclofenac and Silodosin) using a computer generated model. 60 patients were taken in each group. A vitamin tablet containing folic acid was used as placebo. 50 mg Diclofenac sodium oral tablet and 8 mg Silodosin oral tablet were used in the treatment groups. All medications were placed in numbered envelope as per the computer generated model. All patients and investigators were blinded to the medicine identity and randomistaion design till the end of the study. Patients were handed over the next numbered envelope on their OPD visit and Visual analogue score (VAS score) was also recorded at the same time. Visual analogue score was taken on a scale from zero to ten, zero meaning no pain to 10 meaning excruciating pain. Patients were asked to take the medications 1 h before the procedure. All patients received a single dose of levofloxacin 500 mg before stent removal as per our department protocol. The surgeon removing the stent was also blinded about the grouping. Stent removal was performed under local anesthesia using 2 % xylocaine jelly under vision with 8/9.8 ureteroscope. All patients were contacted after 24 h and VAS score, additional medications requirement and site of pain and any other relevant parameters were recorded. Additional analgesics dose was measured in mgs of i.v morphine equivalents. Patients with severe pain or patient willing for admission were readmitted.Chi square test was used for sex-data analysis. One way Anova, Mann–whitney test and Kruskal–Wallis test were used for analyzing all the other datas. Our sample size calculation of 50 patients per study arm was powered to detect a 20 % decrease in the VAS score with a 95 % confidence level.Out of 240 patients included in the study, 22 patients were further excluded from final analysis Fig. . 10 patiAll the four groups were analysed for baseline characteristics for comparability Table . The resAll the four groups were analysed for post stent removal VAS score. The mean post stent removal VAS score measured at 24 h were 4.2 in placebo group, 2.9 in Diclofenac group, 3.08 in Silodosin group and 2.85 in the combination group . Only 5 % of the patients experienced suprapubic pain in Silodosin group but it was not statistically significant as compared to all other groups (p = 0.15). The mean additional analgesic requirement was least in combination group (3 mg), but did not achieve statistical significance (p = 0.70). The readmission rate also was similar in all four groups. No treatment-related significant side effects were seen in any of the groups.Ureteral stents are associated with significant pain and discomfort . Patients had less severe pain in Silodosin group as compared to placebo group but it was not statistically significant (p = 0.9). The mechanism of pain relief described in previous studies probably explains the pain relief seen with Silodosin in our study. The ureteral dilatory effect as well the decrease in ureteral and detrusor contractility by Silodosin are the likely mechanisms responsible for the pain reduction observed with Silodosin. There was also less incidence of suprapubic pain in Silodosin group but the results were not statistically significant (p = 0.15). This is probably related to Silodosin’s similar effect on lower urinary tract symptoms (LUTS) in BPH patients.There was no difference in pain relief between both Silodosin and Diclofenac groups (p = 0.48). There was higher pain reduction seen with combination of Diclofenac and Silodosin but it did not attain statistical significance . Possible explanation is that there is probably certain overlap of different mechanisms of pain reduction involved between both the drugs. however The incidence of severe pain (VAS > 5) was also least in the combination group (3.4 %) when compared to all the other groups, but contrary to previous study, it was not statistically significant (p = 0.10). Tadros et al. demonstrated significant reduction in analgesic requirement with NSAIDs. However, the additional analgesics requirement in our study was least in combination arm but it was not statistically significant (p = 0.70). However we did not keep any objective criteria for use of additional analgesia and analgesics are used on demand basis. So it is likely that these factors might have influenced on our result.Use of both Diclofenac and Silodosin and their combination did not result in any significant side effects in our study. Like other non-steroidal anti-inflammatory drugs, gastrointestinal side effects are the most commonly reported adverse effects of Diclofenac and seen in about 10 % of patients. These side effects have been found to be dose related (Lehtola and Sipponen Thus our study supports the use of Diclofenac and Silodosin in reducing the pain after stent removal. However, ours is a preliminary study and further research is required in this field to better understand the phenomenon of pain after stent removal and the mechanisms of pain reduction involved.However, there are certain shortcomings to our study. We took the pain score only after 24 h. If we could have taken more frequent pain scores before and even after 24 h, we could have better demonstrated and understood the pain dynamics involved. Moreover, with studies with a larger sample size more clear conclusions could have been derived.We found that use of single oral dose of Diclofenac and Silodosin is very effective in relieving pain after stent removal and the combination was not found to be more effective than the single agent. Thus, we recommend use of oral Diclofenac or Silodosin before stent removal to prevent pain after stent removal.


Our previous study evaluated the effectiveness and safety of radiofrequency thermocoagulation (RFT) of trigeminal gasserian ganglion for idiopathic trigeminal neuralgia (ITN). The aim of this study was to evaluate the optimal radiofrequency temperature of computed tomography (CT)-guided RFT for treatment of ITN.A retrospective study of patients with ITN treated with a single CT-guided RFT procedure between January 2002 and December 2013. Patients were divided into ≤75 °C, 75 °C, and ≥80 °C groups according to the highest radiofrequency temperature used. Pain relief was graded from poor to excellent, and facial numbness/dysesthesia from I (absent) to IV (most severe).A total of 1161 RFT procedures were undertaken in the 1137 patients. The mean follow-up time was 46 ± 31 months. There were no significant differences in the rate of excellent pain relief according to the radiofrequency temperature used. However, more patients experienced with no facial numbness or facial numbness gradually resolved and those patients treated at 75 °C had a lower rate of grade IV facial numbness/dysesthesia than other groups.The optimal radiofrequency temperature to maximize pain relief and minimize facial numbness or dysesthesia may be 75 °C, but this requires confirmation. It should be distinguished from symptomatic trigeminal neuralgia (STN), by the absence of any structural lesion affecting the trigeminal nerve. The pathogeny of ITN is complicated and undefined. Although most favor the vascular compression hypothesis. However, Taha and Tew reported that around 15% of patients with ITN lacked evidence of significant vascular compression; Devor et al considered that compression and demyelination of sensory nerves generally causes numbness or loss of vibration sense, rather than pain.Idiopathic trigeminal neuralgia (ITN) is an uncommon pain disease, mean annual incidence of 12.6 per 100,000 person years.–7 Treatment options include drugs and interventional procedures. Anticonvulsant drugs are generally used first line. There are several neurosurgical procedures available to treat patients if drug treatment is not tolerated or is ineffective. These include ablation technique and microvascular decompression (MVD).Fortunately, whatever the cause, there are effective treatments for ITN, and a number of treatment guidelines have been published.,8 We also found that RFT is a practical treatment option in treating recurrent TN after MVD. Although the RFT has now widespread application in the treatment of TN, different temperatures (55–90 °C) in operation determined by doctor's experience,–12 the optimal temperature to maximize pain relief and minimize complications has no unified standard.Among the ablation techniques, radiofrequency thermocoagulation (RFT) of trigeminal gasserian ganglion is reported to give higher success rates than other ablation techniques, such as glycerol rhizotomy or stereotactic radio suigery. and was introduced to China (by Professor Ni) in 2000. Since 2002, we have performed computed tomography (CT)-guided RFT and have reported promising preliminary results.,14–17 All RFT procedures in our unit are now CT guided. Here we report a retrospective study analyzing the optimal radiofrequency temperature in patients with ITN treated with a single RFT procedure.RFT of the trigeminal ganglion to treat ITN was first reported in 197422.1The study was approved by the institutional review board and local ethics committee. Written informed consent was obtained from each patient before the RFT procedure. Inclusion and exclusion criteria for patients and the methods of follow-up were according to our previous report.2.2,15,16We have previously described our RFT procedure in detail.2.3Demographic information, the duration of symptoms preprocedure, the duration of follow-up postprocedure, the trigeminal nerves and divisions treated, the temperatures used during RFT, and the census point for data collection were obtained from the medical records and follow-up data., respectively; we later allocated the patients into 3 groups for data analysis: ≤75 °C, 75 °C, and ≥80 °C.We initially divided the patients into 5 groups, according to the highest temperature used during the RFT procedure: 65 °C, 70 °C, 75 °C, 80 °C, and 85 °C. However, because there were only 8 and 5 patients treated with temperatures of 65 °C and 85 °C,16Pain intensity evaluation, pain relief , and facial numbness/dysesthesia outcomes classification (grade I–IV) is studied using the methods mentioned in the previous paper.2.4P value of < 0.05 was considered statistically significant.Statistical analysis was performed using the statistical package for the social sciences version 17.0 . Kaplan–Meier curves were used to determine the percentage of patients that were free of pain and free of facial dysesthesia after RFT. The log-rank test was used to compare survival curves. The censor point for data collection was either: the last contact with the patient; the time of any subsequent surgery; or death. For statistical purposes, in patients who died, the duration of pain relief was assessed as the time from the procedure to the time of death. For patients who were lost to follow-up, data obtained at the time of the last contact was used. If a second RFT procedure on the same nerve during the same hospital stay was required, this was considered as a single procedure for statistical purposes. If bilateral RFT procedures were undertaken on the same patient, this was treated as 2 procedures for statistical purposes. A 3A total of 1161 RFT procedures were undertaken in the 1137 patients. The following information is shown in Table 3.1P > 0.05). At follow-up of the census point, the respective NRS scores were: 0.88 ± 2.21, 0.61 ± 1.88, and 0.57 ± 1.47 (P > 0.05), which indicated the mean pain intensity of all patients has been alleviated significantly. Figure P > 0.05), which indicated that excellent pain relief could be obtained as long as the radiofrequency temperature used >65 °C.The mean ± SD NRS scores before RFT in patients treated using temperatures of ≤70 °C, 75 °C, and ≥80 °C were 7.75 ± 1.44, 7.78 ± 1.37, and7.83 ± 1.37, respectively  patients in the ≤70 °C group, 26 (45.6%) patients in the 75°C group, and 14 (24.4%) patients in the ≥89 °C group, never experienced facial numbness which was graded I. A total of 243 patients reported that initial facial numbness gradually resolved and had gone totally after mean ± SD duration of 31.4 ± 14.9 weeks which showed in Table P < 0.01). All patients who experienced facial numbness reported it to lessen with time.The degree of facial numbness in those who continued to experience it varied. It was graded II, III, and IV in 465 (54%), 343 (39.8%), and 53 6.2%) patients, respectively. Grade IV facial numbness was reported by 19 of 247 patients (7.7%), 16 of 790 patients (2%), and 18 of 124 patients (14.5%) who had been treated with RFT using temperatures of ≤70 °C, 75 °C, and ≥80 °C, respectively. We further analyzed using a Kaplan–Meier actuarial curve and setting the event as grade IV facial numbness , Corneitis in 29 patients (2.6%), diplopia in 14 patients (1%); low pressure headache in 2 patients (0.2%), experienced ptosis in 8 patients (0.7%); difficulty with mouth opening in 5 patients (0.4%); and hearing loss in 5 patients (0.4%). These complications maybe related to the operation technique, other than radiofrequency temperature. All these problems tended to improve with time. No other cranial nerve problems occurred, there were no cases of carotid-cavernous fistula, and there was no procedure-related mortality.4The ideal surgical treatment for trigeminal neuralgia should have a high success rate in terms of control of pain, be minimally invasive, have few complications, and be well accepted by patients. No currently available technique meets all of these requirements; each has advantages and disadvantages. RFT satisfies the first 3 of these aims. A relatively high incidence of adverse effects is its principle drawback, limiting its more widespread use. The heat produced by the RPF needle is thought to selectively destroy the Aδ and C pain fibers by thermocoagulation at temperatures above 65 °C. This can prevent both triggering of pain and the intensity of pain, but can also cause numbness and other problems.,19RFT of the trigeminal gasserian ganglion has been popularized since 1974, as an effective treatment which is less invasive than some alternatives.,20 Our previous study reported that complete pain relief was less common in those in whom no facial numbness, or in whom it was transient, indicated that a degree of facial numbness may be inevitable if long-term relief of pain symptoms is to be achieved. It may be more logical to view facial numbness as a predictable side effect of RFT, rather than as an unexpected complication.Postoperative facial numbness is common following RFT. It usually accompanies the relief from pain. Temperatures of >65 °C are known to destroy nerve fibers, with the Aδ and C nociceptive fibers, being destroyed at lower temperatures than are required to destroy the Aα and Aβ tactile fibers. However, to date, selection of radiofrequency temperatures to selectively destroy nociceptive and preserve tactile fibers has not been reported. Generally temperatures of between 55 °C and 85 °C are used during RFT to treat trigeminal neuralgia. In this study, we further explored the outcome, both in terms of pain relief and facial numbness/dysesthesia, of RFT according to the temperature used during the procedure. We found no significant difference in the rate of pain relief according to the temperature used, although more patients experienced with no facial numbness or facial numbness gradually resolved in 75 °C temperatures of 70 °C and below, or of 80 °C and above were more likely to suffer severe facial numbness with dysesthesia than those treated at 75 °C. Facial numbness, particularly dysesthesia, is a serious side effect of RFT that affects the quality of patients’ lives. Given our findings, we recommend that when using RFT to treat ITN, the temperature used should generally be 75 °C, as this seems to optimize pain relief while minimizing the risk of facial numbness and painful dysesthesia.We have analyzed the long-term effective rate of different branches of these 1137 ITN patients after single radiofrequency thermocoagulation in our previous report.,22 We suspect that the choice of which temperature to use during RFT is influenced by the voltage required during motor and sensory stimulations before the thermocoagulation procedure. If >0.1 V produces paresthesia and/or twitching, the operator is likely to use a temperature of ≤70 °C. If 0.1 to 0.3 V is required, 75 °C is likely to be chosen, whereas if >0.3 V is required, a temperature of ≥80 °C is likely to be used. The voltage required to produce effective stimulation reflects the distance of the needle tip from its target nerve tract in the gasserian ganglion; the smaller the distance the less voltage required. This needs to be considered when choosing a voltage which is likely to be effective, while minimizing the risk of painful dysesthesia. We hope our findings will provoke further research into the optimal temperature to use according to different stimulation voltages during RFT.We do not have an explanation why the outcome of RFT is better when using a temperature of 75 °C, rather than with higher or lower temperatures. However, the higher the temperature used, the greater the size of the coagulum.5From our data, the optimal RFT temperature to achieve long-lasting pain relief while minimizing facial numbness and painful dysesthesia may be 75 °C, but this requires further study.


The Japanese Osteoporosis Quality of Life (JOQOL) questionnaire measures quality of life in Japanese patients with osteoporosis. However, several important aspects of the psychometric properties of individual domains, including responsiveness, have not been addressed to enable valid clinical application. This analysis examined the internal and external responsiveness of the JOQOL questionnaire.r) for changes in JOQOL domain scores with Short Form-8 Health Survey and European Quality of Life Instrument scores.This was a post hoc analysis of a 24-week prospective postmarketing study of raloxifene (60 mg/day) administered to postmenopausal Japanese women with osteoporosis (JapicCTI-070465). Internal responsiveness was assessed using Standardized Response Mean (SRM) statistics and changes in JOQOL domain scores. Patients were also stratified into those who did or did not achieve a minimal clinically important change (MCIC) in pain, assessed by a visual analogue scale for pain (VAS pain): comparisons were made between treated patients who achieved VAS pain reduction ≥ 20 mm versus VAS pain reduction < 20 mm. External responsiveness was assessed using Pearson’s correlation coefficient (r ≥ 0.4 to < 0.6) was noted between the domains pain, activities of daily living, and overall health and some Short Form-8 Health Survey subscales and the European Quality of Life total score, suggesting external responsiveness of these domains.Of 506 patients analyzed, 421 had a baseline value for VAS pain; of these, 152 patients (36.1%) had a MCIC, whereas 264 patients (62.7%) did not. The JOQOL domains pain, overall health, and falls/psychological factors had small to moderate SRM values (0.3-0.5) in all patients, but consistently showed significantly larger changes in patients whose pain score changes exceeded the MCIC. Together, these findings suggest some degree of internal responsiveness for these domains. However, activities of daily living domain had a SRM value as low as 0.2, and recreation/social activities and posture/physique domains had SRM values close to 0. Moderate correlation  contains supplementary material, which is available to authorized users. Osteoporosis negatively affects quality of life (QOL) as well as clinical and laboratory indices, such as fractures and bone mineral density . ImproveThe Japanese Osteoporosis Quality of Life (JOQOL) questionnaire was developed to measure QOL in Japanese patients with osteoporosis, with reference to two English language disease-specific QOL questionnaires: the Qualeffo-41 questionnaire and the Osteoporosis Assessment Questionnaire ,5–7. ThePatient-reported outcome (PRO) measures can provide evidence of a treatment benefit from patients’ perspectives . HoweverKumamoto et al. investigated part of the psychometric properties of the overall JOQOL , including several aspects of reliability and validity . HoweverWe previously reported significant improvement in impaired QOL in postmenopausal women receiving raloxifene using two established generic QOL instruments: the Short Form-8 Health Survey (SF-8), and the European Quality of Life Instrument (EQ-5D), as well as a visual analogue scale for pain (VAS pain) and the JOQOL questionnaire in a postmarketing study . The aimThis post hoc analysis examined the psychometric properties of the JOQOL using data from a previous postmarketing study. The 24-week prospective postmarketing study of raloxifene administered in a real-world clinical practice setting was conducted between 1 September 2007 and 28 February 2009 at 60 hospitals in Japan  .The postmarketing study population and inclusion/exclusion criteria have been reported previously . The 24-The postmarketing study was approved by the Institutional Review Board of each participating hospital and conducted in accordance with the Declaration of Helsinki and all applicable local laws and regulations. Informed consent was obtained from the study patients.Pain is the major symptomatic complaint in patients with osteoporosis . Pain inResponsiveness was assessed using two different approaches. Internal responsiveness is the ability of a PRO measure to change over the observation period when treatment-related changes in health status are ‘established’ and is assessed using a distribution-based index such as effect size ,21. Our r) were calculated for changes in JOQOL domains and changes in the external anchors [Summary statistics for patient characteristics and the JOQOL scores were determined as mean ± standard deviation (SD). For internal responsiveness assessment, changes in JOQOL domain scores over the observation period were compared between patients stratified by MCIC and assessed using the Student t-test with a two-sided alpha level of 0.05. The SRM is the mean of the difference of the change in JOQOL domain scores divided by the SD of the difference; SRM values of ≥ 0.2 to < 0.5, ≥ 0.5 to < 0.8, and ≥ 0.8 represented small, moderate, and large internal responsiveness, respectively . For ext anchors . Correla anchors . StatistOf the 536 patients enrolled in the postmarketing study, 506 were eligible for analysis  . In briep < 0.001). These findings confirmed that the predefined MCIC criteria differentiated clearly between Japanese patients with osteoporosis who experienced clinically significant changes in health status related to perceived pain and those without such perceived changes in pain.For the entire treatment group, VAS pain was reduced by 11.7 ± 25.8 mm (n = 416) from a baseline value of 40.2 ± 24.9 mm (n = 421), which corresponded to a moderate SRM value of 0.45. Stratification of patients according to the MCIC criteria showed that 152/421 patients (36.1%) had a VAS pain reduction ≥ 20 mm . These patients had a baseline mean ± SD VAS pain score of 58.4 ± 16.8 mm, a mean ± SD reduction in VAS pain of 38.7 ± 16.2 mm. In contrast, 264/421 patients (62.7%) had a VAS pain reduction < 20 mm . These patients had a mean ± SD baseline VAS pain score of 30.0 ± 22.5 mm and a mean ± SD change in VAS pain of -3.8 ± 15.3 mm, indicating no reduction in pain status and a significant difference with those who achieved the MCIC  and the EQ-5D total score (p < 0.001)  and 174.3 ± 12.0 days (range 120 to 196 days). For the entire treatment group, the JOQOL domains including pain, activities of daily living, overall health, and falls/psychological factors and the total score showed small responsiveness with regard to SRM values , the JOQOL domain overall health and the SF-8 subscale general health (r = 0.43), and the JOQOL domain pain and the SF-8 subscale role physical (r = 0.40). Moderate correlation was also noted for the changes in the JOQOL activities of daily living and changes in the SF-8 physical component summary score (r = 0.43) and the EQ-5D total score (r = 0.52). Overall, weak correlation was noted for changes in the JOQOL domains recreation/social activities, posture/physique, and falls/psychological factors with changes in any of the SF-8 subscales or the EQ-5D total score . Of note, the correlation values of each individual JOQOL domain with the changes in various SF-8 subscales were consistent. This is contrary to the expectation that a domain-specific correlation pattern that reflected the discrete SF-8 constructs and the construct of each JOQOL domain would be observed.Moderate correlation was noted for changes in the JOQOL domains pain, activities of daily living, overall health, and the total score with changes in the external anchors  and the total score showed small internal responsiveness among the treatment population. These were fairly consistent with the significantly larger changes among patients with a VAS pain reduction ≥ 20 mm. In contrast, the JOQOL domains recreation/social activities and posture/physique failed to show adequate internal responsiveness with regard to the SRM values and between-group differences.Assessment of external responsiveness also found moderate correlation between the JOQOL domains pain, activities of daily living, overall health, and the total score and various subscales of the SF-8 and the EQ-5D total score as references. However, homogeneous, non-specific correlation patterns for changes in individual JOQOL domains were observed regardless of the constructs measured by the SF-8 subscales. This non-specific correlation pattern did not support the expectation that a domain-specific correlation pattern between the SF-8 constructs and the JOQOL domains would be observed .The findings of this study partly support the validity of several JOQOL domains in terms of responsiveness for detecting changes over time in QOL and subjective symptoms in patients with osteoporosis. However, the finding that the JOQOL domains recreation/social activities and posture/physique failed to show sufficient internal or external responsiveness requires further investigation. Additionally, the internal responsiveness index for activities of daily living had an SRM value as low as 0.2. Insufficient responsiveness may be because these domains were designed to measure concepts that are not specifically related to subjective pain, although pain was previously shown to change during the course of raloxifene treatment and was assumed to represent an improvement in patient global assessment . Indeed,The internal responsiveness analysis found that pain relief was not associated with improvement in the JOQOL recreation/social activities domain. This is contrary to the fact that the SF-8 social functioning subscale, which is expected to have a similar construct to the JOQOL recreation/social activities, responded significantly in treated patients . FurtherSimilarly, the JOQOL domain posture/physique also appears to have inadequate construct validity as an osteoporosis-specific measure based on its unresponsiveness to treatment. The JOQOL domain posture/physique comprises items related to supposedly incurable symptoms, including shortening of height and hunched back. Further, the lack of correlation of the posture/physique domain with any of the SF-8 subscales/summary score and EQ-5D total score suggests that this domain was not sufficiently sensitive to any treatment-related changes in health-related QOL from the patients’ perspectives.A low SRM value for activities of daily living, representing weak internal responsiveness, appears to be ascribed to a larger SD of the change score. On the contrary, the between-group difference of this domain stratified by the MCIC criteria was large and significant, and moderate or better correlations with external anchors such as the SF-8 general health, social functioning, role emotional and role physical subscales, and the EQ-5D score were noted in the external responsiveness analysis compared with other JOQOL domains. This inconsistency warrants further investigation.The conceptual framework of the JOQOL suggests that the individual JOQOL domains should have correlated in a domain-specific manner with the individual SF-8 subscales . That isThere are several notable strengths and limitations of the present study. In terms of study strengths, the results of internal and external responsiveness were consistent, which confers robustness on the results. Further, we used a sample population that was sufficiently large to determine responsiveness to treatment effects with osteoporosis medications in a real-world setting. Although this confers generalizability to postmenopausal women with osteoporosis, our results can only apply to postmenopausal women with osteoporosis in Japan. This is because treatment responsiveness and MCIC can only be generalized to the population studied . Hence, This is the first report of the validity of individual domains of the JOQOL questionnaire among Japanese women with osteoporosis for the purpose of measuring treatment effects. Pain, overall health, falls/psychological factors, and total score of the JOQOL seem to have limited responsiveness to treatment changes. Activities of daily living, recreation/social activities and posture/physique domains were less responsive based on SRM values than generic QOL measures, such as the SF-8 and EQ-5D. Construct validity of the JOQOL domains also seems to be problematic because all the domains of the JOQOL were correlated with all aspects of health-related QOL covered by SF-8 in an non-specific manner, possibly indicating lack of convergent and discriminant validity. Thus, improvements in the JOQOL are suggested before application to clinical research for osteoporosis. Specifically, scrutiny of the constructs and content , and factor analysis of the question items comprising these domains may be recommended.


Objectives. To compare the mysteriousness scores of the Pain Beliefs and Perceptions Inventory in fibromyalgia. Methods. Two cohorts of patients, one with fibromyalgia (FM) and one with rheumatoid arthritis (RA), completed the Mystery Scale component of the Pain Beliefs and Perceptions Inventory to determine whether subjects in the two diagnostic groups had significantly different scores on the Mystery Scale. Results. A total of 126 subjects  completed all questionnaires. The FM group had a greater percentage of female subjects, more severe pain, more severe anxiety, more severe depression, and a higher perceived injustice score. When the RA and FM group scores for the Mystery Scale were adjusted for age, sex, pain severity, HADS scores, and perceived injustice scores, the FM group still had a higher Mystery Scale score. Discussion. Fibromyalgia is associated with a higher level of perception of mysteriousness in the Pain Beliefs and Perceptions Inventory than is seen with rheumatoid arthritis. This difference appears to be independent of levels of pain, depression, anxiety, and perceived injustice. This sense of mysteriousness may reflect a lack of understanding of pain in fibromyalgia as previously reported and may be an area to be addressed in therapy. It has previously been shown that fibromyalgia patients differ from other widespread pain patients in that fibromyalgia patients are more likely to perceive a much greater degree of difficulty both in understanding the cause of their pain and in explaining the cause of their pain to others . This waOn the other hand, the Pain Beliefs and Perceptions Inventory has had a much more detailed study of its construct , 3. SpecRheumatoid arthritis is useful as a comparator group for fibromyalgia cohorts , 5–7, inThe purpose of this study was therefore to quantify the degree to which fibromyalgia patients perceive mysteriousness in the cause of their pain and to compare this to the perceptions of rheumatoid arthritis patients.The subjects are those described in a previous study that has been part of a larger effort to determine the clinical utility of various questionnaires in the care of fibromyalgia patients . These sThe authors reviewed the charts of all consecutively seen subjects who had a confirmed diagnosis of either rheumatoid arthritis or fibromyalgia. Inclusion criteria also included age over 18. Exclusion criteria included the following: being unable to read English; having lower than grade 8 level education; having suspected or known malignancy, or other serious illness; and having both fibromyalgia and rheumatoid arthritis.From January 2013, the authors had been, as part of clinical care, routinely asking chronic pain patients, including rheumatoid arthritis and fibromyalgia patients to complete various instruments in an effort to determine their utility in practice. As part of a practice audit, the charts of rheumatoid arthritis and fibromyalgia patients were reviewed until May, and data gathered regarding age, gender, and assessment responses (see below). This data was anonymised for the purpose of further analysis. Thus, no data was then available on duration of condition, comorbidities, history of trauma, litigation or compensation status, psychiatric diagnoses, disability status, or treatment.Subjects were asked to specify, on average, how much pain they had because of their condition over the past week. They circled a number between 0 and 10, with 0 being labelled as “no pain” and 10 being labelled as “worst pain imaginable.”The Injustice Experiences Questionnaire (IEQ) was used, as described by Sullivan et al. , to measThe Hospital Anxiety and Depression Scale (HADS) was originally developed by Zigmond and Snaith  to deterThis was assessed using items 1, 4, 8, and 14 from the Pain Beliefs and Perceptions Inventory . These it-tests and after adjusting for potential confounders  using ANCOVA. Adjusted mean scores were determined by estimating marginal means for the diagnosis variable after entering potential confounders into the ANCOVA model. We also examined the adjusted association between diagnostic group and Mystery Scale scores using multivariable linear regression. Our final models were built in a blocked fashion by initially entering diagnostic category into the model and then entering other variables simultaneously using a stepwise strategy . Relevant regression assumptions were examined including normality, linearity, and independence. A 0.05 alpha level was used to judge statistical significance. All analyses were conducted using IBM SPSS 20 .Initially, all data records were reviewed in order to ascertain if any data issues, such as missing data, outliers, or out of range values, existed within the data set. Subjects with missing diagnosis or data were removed from analysis along with subjects diagnosed with both fibromyalgia and rheumatoid arthritis. After data cleaning, the subjects were divided into two groups (RA group and FM group) as the main variable. The mean age, gender distribution, and mean scores of the assessments were calculated for each group. Inferential statistics were then used to determine whether subjects in the two diagnostic groups had significantly different scores on the Mystery Scale items. This was done univariately using We have no previous studies on this topic to assess sample size. However, we had a fixed sample from our practice audit, which limited the sample size. Considering the number of subjects in the study sample previously reported to be 64 rheumatoid arthritis subjects and 62 fibromyalgia subjects, we have calculated that, given a standard deviation in Mystery Scale scores of 5.5 (observed for the two groups combined), we had an 80% power to detect an absolute difference of 2.8 in the total Mystery Scale scores between groups.Initially a total of 147 charts were available for review. Of these, 9 subjects were excluded . This left 138 subjects for analysis. Of these, an additional 12 were excluded from analysis because of significant missing data. Thus, a total of 126 subjects (64 RA and 62 FM) had complete data available for analysis.The gender distribution, mean age, and unadjusted assessment scores are shown in After adjustments by sequentially adding the confounders of age and sex, pain, HADS depression, HADS anxiety, and perceived injustice, a significant difference remains between groups. That is, the adjusted mean for the FM group Mystery Scale score was 1.6 and the RA group had an unadjusted mean Mystery Scale score of −3.4. This indicates again that the FM group supported the notion of having a mysteriousness concerning their pain when compared to the RA group.This study of a referred cohort of subjects with either fibromyalgia or rheumatoid arthritis shows that fibromyalgia patients are much more likely than rheumatoid arthritis patients to endorse the belief that they have a degree of mysteriousness concerning their pain. This remains the case even after adjusting for confounders that included severity of pain, for example. The results of this study are in agreement with a previous study  using noA narrative approach to understanding fibromyalgia patients affirms that both the wide array of symptoms in fibromyalgia and the variability in the illness course make it an illness difficult for patients themselves to understand and even more difficult to explain to others , 11. FibLabelling the condition fibromyalgia, in itself, does not necessarily convey greater meaning or understanding for this suffering. Patients have indicated exactly that while the diagnosis may confer some legitimacy, it does not improve their understanding of their own illness, nor help them explain their illness to others , 33, norThere are a number of limitations to this study. First, the psychometrics of the Mystery Scale score used without the remainder of the Pain Beliefs and Perceptions Inventory are not known. Further study may be required to ascertain the reproducibility, reliability, and validity of using selected items from this inventory in various settings, adding the other constructs of this inventory as confounders in the analysis. The questionnaires we used, however, were part of a practice audit and assessment. They were thus used in a way that, through brevity, would be most applicable in the clinical setting.In terms of selection bias, these subjects were from two clinics. There may be a selection bias in this group that affected their sense of mysteriousness because they were indeed a referral sample, possibly with more severe pain, or seeking more explanation. A primary care sampling of fibromyalgia subjects may have yielded different results. We did not track the scores used to confirm the FM diagnosis via the Modified 2010 ACR criteria, which may have told us how severe these patients were in terms of polysymptomatic distress. Still, we did adjust for illness severity to an extent, in that we adjusted for pain, depression, and anxiety. In addition, we did not measure cognitive dysfunction in the subjects. This could explain why fibromyalgia patients have difficulty understanding their pain, but narrative and other qualitative studies of fibromyalgia patients clearly indicate that fibromyalgia patients are very much capable of logically and earnestly processing their illness and suffering: despite this, they find their illness often inexplicable . CognitiThe question remains as to what to do with the knowledge that fibromyalgia patients perceive a greater perception of mysteriousness to their pain. To the extent that this mysteriousness might contribute to their distress, future studies could examine whether encouraging patients not to seek explanations (but rather solutions) for their pain may help their condition. As far off as such studies may be, the Mystery Scale may be used to inform both clinical practice and research efforts. It may also be that there is no solution to this problem because the research literature is the problem. The mysteriousness of pain perceived by fibromyalgia patients may simply be a reasonable reflection of the polarity reported by researchers holding divergent views on the topic. Fibromyalgia sufferers may be the unfortunate victims of this tug-of-war. To that extent, there may be no mystery at all.


Pain is a prominent feature of acute dengue as well as a clinical criterion in World Health Organization guidelines in diagnosing dengue. We conducted a prospective cohort study to compare levels of pain during acute dengue between different ethnicities and dengue severity.Demographic, clinical and laboratory data were collected. Data on self-reported pain was collected using the 11-point Numerical Rating Scale. Generalized structural equation models were built to predict progression to severe disease.A total of 499 laboratory confirmed dengue patients were recruited in the Prospective Adult Dengue Study at Tan Tock Seng Hospital, Singapore. We found no statistically significant differences between pain score with age, gender, ethnicity or the presence of co-morbidity. Pain score was not predictive of dengue severity but highly correlated to patients’ day of illness. Prevalence of abdominal pain in our cohort was 19%. There was no difference in abdominal pain score between grades of dengue severity.Dengue is a painful disease. Patients suffer more pain at the earlier phase of illness. However, pain score cannot be used to predict a patient’s progression to severe disease. The World Health Organization (WHO) reported that over 40% of the global population is at risk of dengue and an estimated 390 million dengue infections happen worldwide every year.Dengue incidence has increased and its geographic range expanded.Pain is an important component of dengue diagnostic criteria. In WHO 1997 guideline, probable dengue requires the presence of fever with any two of headache, retro-orbital pain, mylagia, arthralgia, rash, positive tourniquet test or or hemarroghic manifestation. Higher prevalence of abdominal pain, backache, headache and myalgia was noted in adult dengue patients.Ethical approval was provided by the Domain Specific Review Board of the National Healthcare Group, Singapore (DSRB/E/2009/432). Written informed consent was obtained from all subjects.Acutely febrile patients above the age of 18 were recruited prospectively from January 2010 to September 2012 at the Communicable Disease Center, Tan Tock Seng Hospital, Singapore, a tertiary referral infectious disease centre. All dengue diagnostic testing was performed by the Enviromental Health Institute, a WHO Collaborating Center for Reference and Research on Arbovirus and its Associated Vectors in Singapore. Patients were classified as having laboratory-confirmed dengue according to WHO standards if they were RT-PCR or NS1 positive using the dengue NS1 antigen (Ag) strip .The Numeric Rating Scale (NRS) is a widely used tool to assess patients’ pain level in chronic and acute illnesses with good responsiveness and sensitivity.The primary outcome was the severity of dengue: 1: Dengue fever, 2: Dengue hemorrhagic fever  Ordinal logistic regression was used to determine how pain score was associated with age, gender, co-morbidity and ethnicity . Generalized structural equation model (GSEM) was applied as a more confirmatory analysis to analyze the data accounting for temporal data structure and the complex dependence among pain score, fever and the outcome.Two sets of analyses were carried out, with the outcome ascertained on day 2 and day 3 of enrolment. In both analyses, the number of fever days reported by patients preceded fever at presentation which in turn preceded days 2 and 3 of enrolment see  and 2.The first GSEM model to predict dengue severity on the second day of enrolment (day 2) involved baseline covariates such as age, gender, ethnicity, Charlson’s co-morbidity scoreEstimated with maximum likelihood, the models were analysed with the multinomial distribution and logit link. Analyzed with Stata MP 13 , all statistical tests were conducted with 95% confidence intervals.Of the 850 patients recruited, 499 tested positive for RT-PCR or NS1 were included in the analysis. There were 111 DHF I–II and 13 DHF III–IV(DSS) cases according to WHO 1997 classification. Twenty were severe dengue cases in accordance with the WHO 2009 classification. The median duration of fever was 6 (3–9) days. One patient died owing to myocarditis. One patient was diagnosed with both DSS and SD. In total, 32 patients fulfilled the severe disease classification.th–95th percentile, 21–51 years). Male patients comprised 79% and 77.6% of the cohort were Chinese. Medical co-morbidity, mainly myocardial infarction, diabetes and cerebrovascular dieseases was present in 1.8%. The majority (76.3%) of patients had fever on the first visit. The median pain score was similar in DF, DHF I–II and severe disease patients . (The median age of the cohort was 34 years (5 = 0.7).  There weFor detailed sample sizes for each day, refer to Pain score on study days 1 and 2, number of fever days, ethnicity and co-morbidity were not significantly associated with dengue severity . The GSESimilarly, pain score on study day 2 was not predictive but age and fever on day 3 of study were predictive of dengue severity. The risk of age on severe disease largely remained the same as the day before. In addition, fever on day 3 increased a patient’s risk of severe disease. Notably, patients who developed severe disease on day 3 were more likely to report higher pain scores .th–95th percentile, 2–9) and 4  for non-severe disease (p = 0.38).Ninety five patients (19%) experienced abdominal pain. Four of them had SD while the other 3 had DSS. Of these 7 cases, 3 of them experienced the worst abdominal pain on the same day as they were diagnosed with severe disease or after progressing on to severe disease. The median abdominal pain score for patients with severe disease was 5 .(DOC)Click here for additional data file.


Cholecystectomy is the therapy of first choice in patients with uncomplicated symptomatic cholecystolithiasis, but it remains unclear which patients truly benefit in terms of health status improvement. Patients generally present with episodic abdominal pain of varying frequency, duration, and intensity. We assessed whether characteristics of abdominal pain episodes are determinants of clinically relevant improvement of health status after cholecystectomy.In a post hoc analysis of a prospective multicenter cohort study, patients of ≥18 years of age with uncomplicated symptomatic cholecystolithiasis subjected to cholecystectomy were included. Preoperatively, patients received a structured interview and a questionnaire consisting of the visual analogue scale  and gastrointestinal quality of life index (GIQLI). At 12 weeks after cholecystectomy, the GIQLI was again administered. Logistic regression analyses were performed to determine significant associations.Questionnaires were sent to 261 and returned by 166 (63.6 %) patients . A total of 131 (78.9 %) patients reported a clinically relevant improvement of health status. The median (interquartile range) frequency, duration, and intensity of abdominal pain episodes were 0.38 (0.18–0.75) a week, 4.00 (2.00–8.00) hours, and 92 (77–99), respectively. None of the characteristics was associated with a clinically relevant improvement of health status at 12 weeks after cholecystectomy.Characteristics of abdominal pain episodes cannot be used to inform patients with symptomatic cholecystolithiasis who are skeptic about the timing of cholecystectomy for optimal benefit. Timing of cholecystectomy should therefore be based on other characteristics and preferences. Cholecystolithiasis represents a clinical spectrum that ranges from asymptomatic gallstone disease to uncomplicated symptomatic gallstone disease to acute cholecystitis. Likewise, therapeutic options may go from conservative treatment to cholecystectomy. Patients with asymptomatic cholecystolithiasis benefit least from cholecystectomy in terms of improvement of health status and should receive conservative care, whereas those with acute cholecystitis benefit most and should receive surgery.–11Uncomplicated symptomatic cholecystolithiasis is frequently characterized by abdominal pain episodes of widely varying frequency. These episodes may last minutes or several hours, and their intensity is variable.6We aimed to assess whether frequency, maximum duration, or intensity of abdominal pain episodes were associated with improvement of health status in order to define which patients with uncomplicated symptomatic cholecystolithiasis may benefit most from cholecystectomy. We also assessed the associations of these episode characteristics with different subscales of health status at 12 weeks after cholecystectomy.We performed a post hoc analysis using the database established during a previous multicenter cohort study conducted in the Netherlands. Details of study design were reported previously.Patients were asked to recall the duration of symptoms, the number of episodes, and longest episode duration. Patients with a history of symptoms for more than 1 year or who reported to have experienced more than five episodes were excluded, because most of these patients could not recall the frequency. Consequently, these data were only sporadically reported in the database. In addition, we excluded those with schizophrenia or other mental disorders that may impair recall. Other exclusion criteria were as follows: a history of complicated symptomatic cholelithiasis ),The questionnaire consisted of the gastrointestinal quality of life index (GIQLI). The GIQLI has been developed in Germany and has been translated and validated in Dutch.The study was approved by the medical ethics committee and reported in accordance with the recommendations in the Strengthening the Reporting of Observational studies in Epidemiology (STROBE) guidelines for reporting observational studies.18The primary outcome was defined as a clinically relevant improvement of overall health status. Secondary outcomes included a clinically relevant improvement of upper and lower gastrointestinal symptoms, or on the emotional, physical, and social subscales, respectively. Based on previous publications, the independent variables included sex,χ2 tests or Fisher’s exact tests for categorical data, Student’s t tests for continuous data, and Mann-Whitney U tests for ordinal data. We determined which variables were associated in univariable analysis with a clinically relevant improvement of health status after surgery using logistic regression analyses. Significant variables in univariable analysis (P < 0.10) were introduced into a backward multivariable regression model to determine whether there were independent predictors of clinically relevant improvement of overall health status or any of the subscales after surgery. Age at operation, sex, center, and baseline GIQLI score were the variables that were retained in the model as co-variables. Results were reported as adjusted odds ratios (ORs) with corresponding 95 % confidence intervals. P < 0.05 was considered statistically significant. All missing values were considered to be completely at random and excluded from analyses. Statistical analyses were performed using SPSS statistical software version 20.0 .We examined whether baseline clinical and abdominal pain characteristics differed between responders and non-responders to the questionnaire, using The database consisted of 870 potentially eligible patients. A total of 261 patients were included. Preoperative and postoperative questionnaires were returned and completed by 166 (63.6 %) patients  (Table P = 0.066).One hundred and thirty-one (78.9 %) patients reported an overall clinically relevant improvement after surgery. Univariable analysis showed maximum intensity of abdominal pain episodes to be associated with clinically relevant improvement of overall health status  patients reported a clinically relevant improvement (Table .3 % patiP = 0.007) and social subscales  in univariable analysis (Table P = 0.025). In multivariable analysis, duration  and frequency  of abdominal pain episodes with clinically relevant improvement of the social subscale remained associated.Duration was associated with clinically relevant improvement of the physical  patients at 12 weeks after cholecystectomy. Episode characteristics of pain were not associated with an overall clinically relevant improvement of health status after surgery, but patients with a higher frequency and a longer duration of abdominal pain episodes were more likely to have a clinically relevant improvement of social functioning after surgery.–The preoperative health status score and improvement of health status were similar to studies using the same patient-reported outcomes.1The main explanation for the discrepant results with literature is the difference in patient-reported outcomes. The most comprehensive patient-reported outcome measure in patients with uncomplicated symptomatic cholecystolithiasis to determine appropriate and efficient utilization of cholecystectomy is still under debate. Postoperative absence of pain, satisfaction, and health status improvement all have been previously used as primary patient-reported outcome measures.Our study has some limitations. First, we cannot fully rule out recall bias, although we limited this type of bias by excluding patients that could not recall abdominal pain frequency. The generalizability of the results may therefore be limited, although the patient characteristics are no different compared with the characteristics of other studies.This study included several strengths as well. First, the database of a prospective observational study was used limiting confounding bias. Second, we used a standardized and validated questionnaire allowing reliable comparisons with other studies using this widely translated and validated questionnaire.Since patients that benefit most in terms of health status improvement cannot be predicted using abdominal pain episode characteristics, future studies should assess which uncomplicated symptomatic cholecystolithiasis patients are at increased risk for complicated cholecystolithiasis. Although the risk of complications because of gallstones in uncomplicated symptomatic cholecystolithiasis patients is estimated to be only 1–3 % a year, these complications can be serious and life threatening as previously reported in this journal.In conclusion, frequency, maximum duration, and intensity of abdominal pain episodes are not associated with a patient-reported clinically relevant improvement of health status at 12 weeks after cholecystectomy. Characteristics of abdominal pain episodes cannot be used to inform patients with symptomatic cholecystolithiasis who are skeptic about the timing of cholecystectomy for optimal benefit. Timing of cholecystectomy for these patients should therefore be based on other characteristics and preferences.


Results from a few studies indicate that intramuscular injections of 5-HT3-antagonists may reduce musculoskeletal pain. The aim of this study was to investigate if repeated intramuscular tender-point injections of the 5-HT3-antagonist granisetron alleviate pain in patients with myofascial temporomandibular disorders (M-TMD).Serotonin (5-HT) mediates pain by peripheral 5-HTwww.randomization.com). 40 patients with a diagnose of M-TMD according to the Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD) were randomized to receive repeated injections, one week apart, with either granisetron  or isotonic saline as control (CTR).This prospective, randomized, controlled, double blind, parallel-arm trial (RCT) was carried out during at two centers in Stockholm, Sweden. The randomization was performed by a researcher who did not participate in data collection with an internet-based application (P < 0.05), but not in the CTR-group . The numbers needed to treat (NNT) were 4 at the 1- and 6-month follow-ups, and 3.3 at the 2-month follow-up in favor of granisetron.The median weekly pain intensities decreased significantly at all follow-ups  in the GRA-group (Friedman test; Repeated intramuscular tender-point injections with granisetron provide a new pharmacological treatment possibility for myofascial pain patients with repeated intramuscular tender-point injections with the serotonin type 3 antagonist granisetron. It showed a clinically relevant pain reducing effect in the temporomandibular region, both in a short- and long-term aspect.NCT02230371.European Clinical Trials Database 2005-006042-41 as well as at Clinical Trials  Musculoskeletal pain is the most common cause of reduced work capacity and sick leave, and as result a significant health problem. Already in 2003, the annual yearly costs in Europe were estimated to be 34 billion Euro . The imp3-receptor to mediate muscle pain and mechanical sensitivity [3A-receptor is highly expressed in human masseter muscles and that more nerve fibers express 5-HT3A-receptors in women with M-TMD compared to healthy women [3-receptors with drugs that block these receptors could be an interesting therapeutic approach for chronic muscle pain.The neurotransmitter serotonin  is an important component of the chemical milieu during inflammation . It is fsitivity –15. In ahy women . Therefo3-antagonists have been tested experimentally and clinically for treatment of muscle pain. Experimentally, systemic administration of granisetron increased the pressure pain threshold (PPT) over healthy muscles [3-antagonists is often associated with bothersome constipation which may affect treatment compliance. For localized pain conditions local administration has therefore been used with positive results [3-antagonists may be effective to use as a new, additional therapeutic approach for both acute and chronic muscle pain. In our knowledge, no randomized controlled study (RCT) has yet investigated the long-term effect of local administration of 5-HT3-antagonists in chronic pain-conditions.Indeed, several specific 5-HT muscles , while l muscles , 18. Sys muscles , 20, whe muscles . However results , 23. EveConsequently, this study was designed to investigate if local treatment with repeated intramuscular tender-point injections of granisetron could be effective in alleviating pain in patients with chronic M-TMD. The hypothesis was that local treatment with granisetron would be superior to isotonic saline (control-substance) regarding the outcome domains recommended for pain trials by the Initiative on Methods, Measurement, and Pain Assessment in Clinical Trials (IMMPACT) , 25.Based on the information given on 1753 referrals for TMD, 437 patients with a main complaint M-TMD pain were subjected to a screening by the principal investigator (N.C.) who enrolled the patients. Forty patients (37 women and 3 men) were found eligible and included in this study; none declined participation  age ≥ 18 years, 2) a diagnosis of myofascial pain according to the Research Diagnostic Criteria for TMD Axis I (RDC/TMD) , 3) duraThe exclusion criteria for the patients were; 1) diagnosed systemic muscular or joint diseases , 2) whiplash associated disorder, 3) neuropathic pain or neurological disorders , 4) history of psychiatric disorders, 5) pain of dental origin, 6) use of muscle relaxants or any medication that might influence the response to pain, 7) pregnancy or lactation, and 8) known hypersensitivity to granisetron.The study was approved by the regional ethical review board in Stockholm, Sweden (2006/192-31/4) and by the Medical Products Agency in Uppsala, Sweden (151:2006/7947), registered in the European Clinical Trials Database (2005-006042-41) as well as at ClinicalTrials.gov (NCT02230371). The study was carried out in compliance with the Declaration of Helsinki as well as the International Conference on Harmonisation Guideline for Good Clinical Practice. All participants received both written and verbal information prior to inclusion and gave their verbal and written consent.This prospective, randomized, controlled, double blind, parallel-arm trial (RCT) follows the consolidated standards of reporting trials (CONSORT) statement , 30 and www.randomization.com). The program generated a numbered list with the substances in random order in blocks of four. The list was kept hidden to the examiners during the entire study and the block size as well as substances not revealed until the last patient had undergone the last follow-up. The study substances have identical appearance, smell and viscosity so both the patients and examiners were blinded to group assignment. A research assistant assigned the patients to treatment substance in consecutive order according to the randomization list, prepared the syringes and marked them with patient number. Three orofacial pain specialists  and two DDS undergoing specialist training in orofacial pain , who were repeatedly calibrated to a gold standard examiner (ME) in the RDC/TMD examination during one day, performed the study.The patients were divided randomly into two groups that received either granisetron or control substance. The randomization was performed by a researcher (ME) who did not participate in data collection with an internet-based application  Screening for study participation and inclusion, V2-V4), Injection of study substances, and V5) Follow-ups at 1-month. Those patients in any group that still reported more than 30 % improvement at the 1-month visit follow-up were scheduled for follow-ups also after 2- (V6) and 6-months (V7), Fig. 3-receptor, with an affinity to the 5-HT3-receptor that is up to 40,000-fold of any other receptor. Granisetron is not dependent on the isoenzyme cytochrome P450 2D6 (CYP2D6), possibly leading to a good efficacy also in ultrarapid metabolizers and has, unlike the other 5-HT3-receptor antagonists, a long half-life [3-receptor antagonist tropisetron used for trigger point injections in similar studies [Granisetron  was used as active treatment (GRA-group) and isotonic saline  was used as control treatment (CTR-group). Granisetron is used to treat chemotherapy-induced and postoperative nausea and vomiting and is among the drugs that exhibit the most selective binding to the 5-HTalf-life –33. It i studies , 26, 34.The injections were made perpendicular to the skin surface over the chosen tender-point with an angle of 90° using a 19-mm long needle (diameter 0.4 mm) from a 2-mL syringe. The solution was administered into each tender-point as a single shot during 10 s. In order to ensure intramuscular administration, during the study, the injections were guided by electromyography  in order to ensure intramuscular administration. The injections were repeated after 1- and 2-weeks in the most painful tender-points at that time.Follow-ups were performed by questionnaires, pain assessment, clinical examination and recordings of PPT. Patients who did not respond to treatment were offered additional treatment at the 1-month follow-up and were excluded from further analyses. They were thus regarded as non-responders in the intention-to-treat-analyses , Fig. The IMMPACT recommends six core domains to be reported in RCTs; participant disposition, pain intensity, physical functioning, emotional functioning, participant ratings of overall improvement and adverse events , 25. TheAdditional outcome measures were changes in PPT, 5-HT platelet poor plasma (P-5-HT), as well as pain distribution.Pain intensity was retrieved from the 1-week pain diary. This was composed of seven 0–100 mm visual analogue scales (VAS) with the endpoints marked “no pain” (0) and “worst experienced pain” (100) for daily assessments of the average orofacial pain intensity the week preceding each visit. For each patient the average pain intensity during that week was calculated . Several repeated assessments are shown to be a more valid measure of pain intensity than a single assessment . The prePhysical functioning was classified using the Graded Chronic Pain Scale (GCPS)  and the JDC was used for assessment of limitation in jaw function. The JDC consists of twelve questions assessing to which extent TMD interferes with activities specifically related to the mandibular function. It has a maximum of 12 points where 0 = no limitation in any mandibular function and 12 = limitations in all mandibular functions.In addition, the pain-free mouth opening and the maximum voluntary mouth opening (MVO), including the vertical overbite, was assessed in millimeters with a ruler.The changes in emotional functioning were assessed using the modified SCL-90-R instrument in the RDC/TMD Axis II questionnaire . This inThe patients overall improvement was assessed on a 6-point rating scale: 0 = symptom-free; 1 = much better; 2 = better; 3 = unchanged; 4 = worse; 5 = much worse. This scale is commonly used and validated , 38.In order to estimate any adverse events the patients were asked to list any adverse event during the week following each injection. If any adverse event occurred they were asked to describe the event and to grad it as mild, moderate or severe.2 probe tip was covered with a 1 mm thick rubber pad to minimize the risk of irritation of the skin. The algometer was held perpendicular to the skin surface over the muscles and the pressure was increased at a standardized rate of 50 kPa per s. The patients were instructed to press a signal button when the sensation of “pressure” changed into “pain”. This was first performed over the soft tissue close to the base of the thumb on the dorsal side of the right hand, in order to accustom the subject to the procedure. Recordings of PPT were made bilaterally over the most prominent points of the masseter muscles as well as over a reference point on the tip of the right index finger. An extra-cranial reference point was chosen to investigate any possible systemic effects by the study treatments. When assessing the PPT the mean value of 3 measurements with an interval of 2 min was used.To assess the PPT an electronic pressure algometer  was used. This device consists of a grip with a pressure-sensitive strain gauge at the tip and a display unit. The 1 cmA blood sample (5 mL) was collected from the decubital vein in EDTA-containing tubes for P-5-HT. The sample was immediately cooled and centrifuged (1700 G) for 30 min. Approximately 200 μL of the supernatant was then collected with a pipette into polystyrene tubes and kept frozen at −80° until the analysis. The plasma samples were analyzed in our laboratory with a commercial EIA-Serotonin kit . The kit has a sensitivity of 0.5 nM, and according to the manufacturer it has an intra-assay coefficient of variation between 8.9 % and 14.5 %, and an inter-assay coefficient variation between 9.9 % and 11.5 %.The patients were asked to avoid tryptophan-rich food  for 24 h before the examination to exclude any risk of interference with the analysis of 5-HT.χ2-test was used. For analyses of group differences of variables on a nominal scale t-test was used, while the Mann–Whitney U-test was used for variables on an ordinal scale. The Friedman’s analysis of variance (ANOVA) on ranks with Dunn’s test as post hoc test was used for analyses of changes between baseline data and follow-up measurements regarding pain variables and emotional functioning, while 2-way repeated measures ANOVA with Holm-Sidak as post hoc test was used for physical functioning and changes in PPT. The Pearson correlation-test with Bonferroni-correction for multiple comparisons was used to correlate the P-5-HT levels with baseline and outcome variables. The significance level was set at P < 0.05.The statistical analyses were performed using SigmaPlot software version 13.0 . Normality of the data was tested with the Shapiro-Wilk test. Descriptive data are reported as frequencies, means and standard deviation (SD) or median and interquartile range (IQR). For analyses of group differences in frequencies The results are presented for both patient centers combined , Stockholm, Sweden) since there were no significant differences for any of the study outcomes between them.There were no differences between the active treatment group (GRA-group) or the control group (CTR-group) regarding demographic data Table .Table 1DFurther, there were no significant differences between the groups regarding baseline pain variables or self-reported clenching/grinding Table . All patThere were no significant differences in mean (SD) P-5-HT levels between the groups at baseline (GRA: 43.5 (66.3); CTR-group: 97.4 (131.4) nmol/L).Finally, no differences in demographic data, pain intensity, physical or emotional functioning and P-5-HT values were found between patients who completed the study and the drop-outs, and there were no differences in baseline variables between responders and non-responders to treatment.P < 0.05), but not in the CTR-group .The median weekly pain intensities are presented in Table At the 1-month follow-up 80 % of the patients in GRA-group and 55 % in CTR-group reported a reduction of 30 % in weekly pain intensity . At the 2-month follow-up these frequencies were 60 % in the GRA-group and 30 % in the CTR-group, and at the 6-month follow-up 35 % and 10 %, respectively. All these frequencies differed significantly between the groups  for a weekly pain reduction of 30 % were 4 at the 1-month follow-up, 3.3 at the 2-month follow-up and 4 at the 6-month follow-up in favor of granisetron . The NNT, for a weekly pain reduction of 50 % were 6.7 at the 2-month follow-up and 5 at the 6-month follow-up. The 1-month NNT could not be calculated because of an equal numbers of responders in both groups.The pain area had decreased significantly with approximately 40 % at the 1- and 2-months follow-ups, and with 55 % at the 6-month follow-up in the GRA-group. There were no significant changes in the CTR-group . There were no significant differences between the groups at any time point  and non-responders to any treatment at baseline. The depression-scores decreased significantly over time, in both groups. The NSPhS-scores decreased significantly at the 2-, and 6-month follow-ups in the GRA-group only  reported an overall improvement of “better”, “much better” or “symptom-free” . At the 2-month follow the corresponding frequencies were 81 % and 62 % respectively, and at the 6-month follow-up 90 % in the GRA-group and 60 % in the CTR-group. The differences between groups were significant at the 2- and 6-month follow-ups Table .Table 4OFour patients in both groups reported mild, short lasting adverse events, such as nausea, constipation, dizziness, hematoma and itching after the first injection of substance. These adverse events did not occur after the second and third injections. In the CTR-group one patient reported hives to the first injection and ended the participation, due to assumed allergic reaction.The PPT over the masseter muscles and the reference point did not differ between groups at baseline and there were no significant differences between the right and left masseter muscles at baseline. Further, PPT did not change over time and there were no differences between substances at any time point Fig. .Fig. 4PrThere were no significant correlations between P-5-HT values and pain or emotional function at baseline. Neither were there any significant correlations between treatment effects regarding pain characteristics or depression- and NSPhS-scores and P-5-HT values at baseline.3 blocker is effective for chronic muscle pain. It was shown that, compared to control, repeated intramuscular tender-point injections with granisetron into jaw muscles of patients with M-TMD had a clinically significant effect on pain intensity and pain area. The effect was still evident 6 months after treatment, when 35 % of the patients in the GRA-group reported a 30 % improvement of pain intensity.To our knowledge this is the first RCT to show that local treatment with a 5-HT3-receptor antagonists are effective for a number of muscular pain conditions [3-receptor antagonists might even be efficacious in more generalized pain conditions, such as fibromyalgia. Although recent studies suggest that peripheral nociceptive inputs, acting as “pain generators”, play an important in maintaining the muscle sensitization in patients with fibromyalgia, indicating that local treatment of these “pain generators” to some extent could decrease pain in patients with fibromyalgia [3-receptor antagonists might have a pain reducing effect in generalized pain conditions such as fibromyalgia.The pain relieving results from this study support previous results that intramuscular injections of 5-HTnditions , 34. Humnditions , 18, 39.nditions , 20, indomyalgia , a previomyalgia . However3-receptors, but also interacts with other substances in the chemical milieu around nociceptors. Thus, blocking of 5-HT3-receptors may inhibit pain by different mechanisms, which may explain the good analgesic effects. For example, tropisetron is reported to inhibit 5-HT-induced PGE2 release from macrophage-like synovial cells in vitro [3-receptor antagonists has a long half-life, and is not dependent on the isoenzyme cytochrome P450 2D6 (CYP2D6), possibly leading to a good efficacy also in ultrarapid metabolizers [5-HT does not only evoke pain directly by 5-HTin vitro  and to hin vitro , 44. Anobolizers –33.As many as 81 % (2-month follow-up) and 90 % (6-month follow-up) of the patients in the GRA-group reported an overall improvement of “better”, “much better” or “symptom-free”, which was significantly higher than the CTR-group with 62 % at the 2-month follow-up and 60 % at the 6-month follow-up, which is in line with previous intervention studies with other treatment modalities in chronic M-TMD –49.The NNT in the GRA-group was 3.3 at the 2-month follow-up which is comparable to commonly used analgesics for acute orofacial pain ranging from NNT 1.8  to NNT 4.5 (aspirin) , or topiPrevious studies in M-TMD patients have reported improved physical functioning after treatment, shown as lower GCPS severity and JDC-scores , 46. ThiTMD-pain is associated with high levels of depression and NSPhS, even higher than the other TMD diagnoses , as was 3-blockers is commonly associated with bothersome side effects such as constipation, headache, and dizziness [In both groups serious adverse events were absent, only a few patients reported mild and short-lasting adverse events after the first injections of test substances. Granisetron is considered efficacious and safe. However, systemic administration of 5-HTizziness , 56. Intizziness , 57, 58.In contrast to several previous experimental studies , 41, 57 Although there was a large difference between the GRA-group and CTR-group in mean P-5-HT levels this difference was not significant. Further, there were no correlations between P-5-HT levels and any of the outcome measures or differences between responders and non-responders to treatment. This contrasts a previous study using systemic ondansetron in 21 patients with fibromyalgia, in which the serum 5-HT levels seem to be lower at baseline in the responders .The strengths with this study are the study design, i.e. a prospective, controlled and double-blind RCT performed in a well-characterized patient sample, that the outcome measures recommended by IMMPACT , 25 to bIn conclusion, the results from the present study show that granisetron has a clinically relevant pain reducing effect, both in a short- and long-term aspect, in patients with chronic M-TMD. Thus, repeated intramuscular injections with granisetron may offer a novel and additional treatment approach for patients with chronic myalgia.


The kynurenine pathway of tryptophan metabolism has been implicated in the pathophysiology of psychiatric disorders, including schizophrenia. We report here that the kynurenine metabolite, xanturenic acid (XA), interacts with, and activates mGlu2 and mGlu3 metabotropic glutamate receptors in heterologous expression systems. However, the molecular nature of this interaction is unknown, and our data cannot exclude that XA acts primarily on other targets, such as the vesicular glutamate transporter, in the CNS. Systemic administration of XA in mice produced antipsychotic-like effects in the MK-801-induced model of hyperactivity. This effect required the presence of mGlu2 receptors and was abrogated by the preferential mGlu2/3 receptor antagonist, LY341495. Because the mGlu2 receptor is a potential drug target in the treatment of schizophrenia, we decided to measure serum levels of XA and other kynurenine metabolites in patients affected by schizophrenia. Serum XA levels were largely reduced in a large cohort of patients affected by schizophrenia, and, in patients with first-episode schizophrenia, levels remained low after 12 months of antipsychotic medication. As opposed to other kynurenine metabolites, XA levels were also significantly reduced in first-degree relatives of patients affected by schizophrenia. We suggest that lowered serum XA levels might represent a novel trait marker for schizophrenia. The first step of the pathway is the conversion of tryptophan into N-formylkynurenine catalyzed by either indolamine 2,3-dioxygenase (IDO) or tryptophan 2,3-dioxygenase (TDO). N-formylkynurenine is converted by formamidase into kynurenine (KYN), which is hydroxylated into 3-hydroxykynurenine (3-HK) by kynurenine monooxygenase (KMO), or, alternatively, transaminated into kynurenic acid (KYNA) by types 1 and 2 kynurenine aminotransferases (KATs), or metabolized into anthranylic acid (ANA) by kynureninase. 3-HK is sequentially transformed into 3-hydroxyanthranylic acid (3-HANA) and quinolinic acid (QUINA)1 (The kynurenine pathway of tryptophan metabolism generates neuroactive compounds that are able to interact with glutamate receptors in the CNS(QUINA)1 . BecauseKYNA and QUINA have been the subject of extensive investigation since they have been shown to interact with ionotropic glutamate receptors. QUINA acts as an orthosteric agonist at the GluN2 subunits of N-methyl-D-aspartate (NMDA) receptors2KMO activity is reduced in the brain of patients affected by schizophrenia, resulting in an increased formation of KYNA at the expenses of 3-HK and its downstream metabolites, including QUINA56791011Recently, two additional metabolites of the kynurenine pathway, cinnabarinic acid and xanthurenic acid (XA) have been suggested to interact with glutamate receptors. Cinnabarinic acid is formed spontaneously by the condensation of two molecules of 3-HANA, whereas XA is the transamination product of 3-HK. Similar to KYNA, brain neosynthesis of XA is catalyzed by KAT-2 in non-neuronal cells2A receptors), thereby restraining the excitatory thalamic input to cortical pyramidal neurons1719in vitro and in vivo models, and we assessed serum levels of XA and other kynurenines in a large cohort of patients affected by schizophrenia, their first-degree relatives, and healthy controls.mGlu2 receptors are considered as drug targets in the treatment of schizophrenia because they negatively regulate the activity of type-2A serotonin receptors . The residual activity of XA in slices from mGlu2−/− mice was antagonized by LY341495  mimicked the action of the mGlu2/3 receptor agonist, LY379268 (1 μM), in inhibiting forskolin-stimulated cAMP formation, and its action was antagonized by LY341495 (1 μM) . In cortLY341495 . Of note3H]inositolmonophosphate (InsP) formation per se, but amplified the stimulation of [3H]InsP formation produced by the mGlu1/5 receptor agonist, DHPG, in adult mouse cortical slices. In contrast, XA (10 and 100 μM) caused a small increase in [3H]InsP formation on its own, but failed to amplify the action of DHPG , and maximal inhibition of about 40% and 60% in mGlu2- and mGlu3-expressing membranes, respectively. Using membranes from mGlu2-expressing cells, we examined whether two orthosteric mGlu2/3 receptor ligands , and one selective allosteric ligand of mGlu2 receptors (LY566332) could displace specifically bound [3H]XA. None of these drugs (tested at concentrations of 1 or 100 μM) significantly inhibited [3H]XA binding  60 min prior to MK-801 (0.32 mg/kg. i.p.), and examined for an overall period of 180 minutes . In mice pretreated with saline, MK-801 caused a sharp increase in locomotor activity, which showed a biphasic kinetic with a first plateau phase from 30 to 75 min, and a subsequent peak at 85–110 min; hyperactivity was still prominent at the end of the observation period . XA had −/− mice. In wild-type mice, we confirmed that 60 mg/kg of XA was able to reduce MK-801-induced hyperactivity (not shown). In contrast, XA was inactive in mGlu2−/− mice . XA-failed to reduce MK-801-induced hyperactivity in the presence of LY341495 , first-degree relatives n = 25), and healthy controls (n = 84) are shown in 5, and hePatients affected by schizophrenia were separated into FES (“first-episode schizophrenia”) and MES (“multi-episode schizophrenia”). The two subgroups showed different demographic and clinical characteristics, with FES patients showing lower BMI and a higher percentage of past alcohol abusers. GAF  scores were similar in the two groups. Scores of PANSS subscales were significantly different between FES and MES patients, with positive symptoms being more prevalent in FES patients, as expected . FourteeLC/MS-MS analysis of kynurenine metabolites showed large and significant reductions in the levels of 3-HK (−59%), XA (−51%), 3-HANA (−48%), and QUINA (−41%) in the serum of the overall population of patients affected by schizophrenia, as compared to healthy controls . Levels Levels of XA and 3-HANA were also reduced in first-degree relatives of patients with respect to healthy controls ; only XA serum levels did not differ between patients and their relatives, but patients had significantly lower 3-HANA levels than relatives. Levels of Trp, KYN and KYNA were higher in relatives of patients than in healthy controls .The subgroup of FES patients also showed a substantial reduction in serum levels of XA (−59%), 3- HK (−83%), 3-HANA (−71%), and QUINA (−41%), and an increase in the levels of ANA (+111%) with respect to healthy controls . In MES One-year treatment with atypical antipsychotics in fourteen FES patients caused large increases in levels of 3-HK (+466%) and 3-HANA (+261%), but no changes in XA or QUINA levels .In the overall population of patients and in the subgroup of FES patients, there were no significant correlations between GAF, CGI, and PANSS  scores and any of the kynurenine metabolites. Weak correlations were found between KYN, KYNA or QUINA levels and PANSS or CGI scores in MES patients see . A few c2+-dependent fashion3H]-XA binding and the evidence that XA stimulates [35S]GTP-γ-S binding in brain membranes1526163H]XA binding could be detected in membranes prepared from cells expressing mGlu2 or mGlu3 receptors, but not in membranes prepared from mock cells or from cells expressing mGlu4 or mGlu7 receptors. How precisely XA interacts with mGlu2 and mGlu3 receptors is unclear. It is unlikely that XA binds to the glutamate recognition site of mGlu2 or mGlu3 receptors because [3H]XA binding was not affected by orthosteric mGlu2/3 receptor ligands, and XA could not displace specifically bound [3H]LY3414953H]XA binding, in membranes prepared from mGlu2-expressing cells. This, however, does not exclude that XA binds to an allosteric site that does not overlap with the LY566332 binding site. Alternatively, XA might interact with a different receptor protein that may signal only in the presence of mGlu2 or mGlu3 receptors. Data obtained in brain tissue also suggest, but do not prove, that XA interacts with mGlu2 and mGlu3 receptors. In cortical slices, XA mimicked the action of LY379268 in reducing cAMP formation, but, unlike LY379268, it failed to amplify mGlu1/5 receptor-mediated PI hydrolysis. One possible explanation for these findings is that XA acts as a biased mGlu2/3 agonistXA is considered as a putative neurotransmitter in the CNS, being stored in synaptic vesicles and released by membrane depolarization in a Ca2A receptors17−/− mice, in line with the evidence that the antipsychotic-like activity of mGlu2/3 receptor agonists is exclusively mediated by mGlu2 receptors30The evidence that mGlu2 receptors negatively regulate responses mediated by 5-HT1; and (ii) enzymes of the kynurenine pathway are segregated in different cell types in the CNS, with KMO and kynureninase being present in microglia, and KATII in astrocytes32We examined blood levels of XA and other kynurenine metabolites in a relatively large cohort of patients affected by schizophrenia, taking into account the following aspects: (i) peripheral KYN and 3-HK enter the brain in large amounts, and fuel the kynurenine pathway in the CNS, whereas brain penetration of KYNA, ANA, 3-HANA, and QUINA, is poorvia KMO-independent reactions, were increased. These changes are consistent with the evidence that KMO activity is reduced and CSF KYNA levels are increased13536Serum levels of kynurenine metabolites that lie downstream of KMO  were largely reduced in patients affected by schizophrenia, whereas levels of KYNA and ANA, which originate from KYN FES patients represent an ideal population for the search of “glutamatergic” biomarkers without the confounding effects of chronic exposure to antipsychotic medication. A growing body of evidence suggests that a glutamatergic dysfunction already occurs in the prodromal phase of schizophrenia and is not affected by antipsychotic treatment3940Interestingly, XA levels were not normalized by one year of treatment with atypical antipsychotic drugs, suggesting that a potential peripheral source of brain mGlu2 receptor activation remains low in spite of the improvement of symptoms in response to medication. This, combined with the evidence that cortical mGlu2 receptors are epigenetically down-regulated by atypical antipsychoticsAs in patients, XA levels were substantially reduced in their first-degree relatives and did not correlate with scores on the clinical scales or neuropsychological tests. Even though other Axis II schizophrenia spectrum disorders were not excluded, this suggests that a reduction in XA levels represents a trait marker, but not a state marker, for schizophrenia.In conclusion, our data demonstrate for the first time that peripheral levels of XA are markedly reduced in patients affected by schizophrenia, regardless of stage of the disorder or medication status. Data obtained in first-degree relatives suggest that peripheral XA levels can be measured as predictive biomarkers in people or family at risk to develop the disorder, but larger studies are needed to verify this hypothesis. In addition, low peripheral XA levels might contribute to a blunted endogenous activation of mGlu2/3 receptors in the CNS, and that subgroup of patients may be more likely to respond to mGlu2/3 receptor agonists or mGlu2 receptor PAMs. Drugs that enhance XA levels might be helpful in the treatment of patients affected by schizophrenia, particularly in the early phases of the disease in which normalization of glutamatergic function represents an unmet therapeutic goal.Clinical samples were collected at the Sant’Andrea Hospital, Psychiatry Unit. All serm samples were collected in accordance with the approved guidelines and relevant regulations. All subjects provided their written informed consent prior to participating in the study. The study design and all experimental procedures were approved by the Ethical Committee at Sant’Andrea Hospital, Sapienza University, Rome, Italy (prot.165/2014). All experiments were performed in accordance with the approved guidelines.in vivo and in vitro experiments were carried out in accordance with the European (86/609/EEC) and Italian (DL.116/92) guidelines of animal care. The experimental protocol was approved by the Italian Ministry of Health (D.M. 209/2011-B).All mGlu2, mGlu3, mGlu4 and mGlu7 receptor cDNAs were kindly provided by J. Blahos , F. Ferraguti , and J.P. Pin , respectively.Human embryonic kidney (HEK) 293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were transfected as described previously43443H]XA binding experiments  or ELISA . Membranes prepared from mGlu2-expressing cells were also used for [ents see .−/− mice and their wild-type counterparts were kindly provided by Prof. Shigetada Nakanishi  and crossed back up to the 17th generation on a C57BL/6J background. These mice were used for behavioural analysis. Mice were housed under standard conditions with a 12 h light/dark cycle and food and water ad libitum. In vivo studies were in accordance with the National Guidelines for Animal Use  and approved by the Italian Ministry of Health (D.M. 209/2011-B).Six to eight week-old male C57BL/6J mice (17–25 g) purchased from Charles River  were used for experiments on cortical slices and for binding studies. mGlu23H]XA and [3H]LY341495 binding was determined in membrane preparations from HEK293 cells or mouse cerebral cortex. Inhibition of cAMP formation was assessed as described above  method for the assay of all kynurenine metabolites. The method allowed a reliable detection of KYN, KYNA, ANA, 3-HANA, 3-HK, XA, and QUINA. Levels of tryptophan (Trp) and 5-hydroxyindolacetic acid (5-HIAA) were also detected. Details on sample preparation, reagents, standard solutions, chromatographic conditions, mass spectrometry conditions, and validation parameters are reported in the The study design was approved by the Ethical Committee at Sant’Andrea Hospital, University Sapienza, Rome, Italy (prot.165/2014). All subjects included in the study signed a free informed consent. M.C., V.C., and A.C. performed the diagnostic assessment at the Psychiatry Unit of Sant’Andrea Hospital. We recruited 60 patients with a history of multi-episode schizophrenia (MES) from the outpatient clinic and 30 patients with first-episode schizophrenia (FES) from the inpatient clinic, all meeting the diagnostic criteria of DSM-IV-TR. Exclusion criteria were: (i) the presence of psychiatric co-morbidities, systemic inflammatory disorders, endocrine disorders, neurological disorders, and mental retardation; (ii) lifetime history of an Axis I disorder ; and (iii) the use of any neurotropic drug or drug of abuse in the last 3 months (except cigarette smoking). In FES patients, the initial diagnosis was confirmed after a 6-month follow-up interview. Fourteen FES patients were re-examined after 12 months of treatment with antipsychotic drugs. Twenty-five first-degree relatives of patients affected by schizophrenia and 84 healthy volunteers also participated to the study. Healthy volunteers were recruited among the hospital personnel and were all unrelated. Only one of the first-degree relatives (i.e. parents or siblings) for each patient, FES and MES, was included in the study. Blood was collected from all subjects for measurements of serum levels of XA and other kynurenine metabolites. The following scales were administered to all participants: PANSS, CGI, and GAF. Neuropsychological assessment was carried out according to the “Measurement and Treatment Research to Improve Cognition in Schizophrenia”plus Fisher’s LSD was applied to the analysis of data obtained in cell cultures, cortical slices, and mice challenged with MK-801.Mann-Whitney, Kruskal-Wallis or Chi-square tests were used for the analysis of the demographic and clinical variables among the groups of patients and control subjects. Bivariate correlations of serum kynurenine metabolites levels with demographic and clinical variables were assessed using the nonparametric Spearman correlation coefficient (rho). The Wilcoxon signed rank test was applied to the analysis of FES patients after 1 year of treatment with antipsychotic drugs. Two-tailed probability of p ≤ 0.05 was considered statistically significant. The STATA software version 11  was used for statistical analyses.One-way ANOVA How to cite this article: Fazio, F. et al. Xanthurenic Acid Activates mGlu2/3 Metabotropic Glutamate Receptors and is a Potential Trait Marker for Schizophrenia. Sci. Rep. 5, 17799; doi: 10.1038/srep17799 (2015).