Previous studies have demonstrated that cytokines, transforming growth factor (TGF-β1), and brain-derived neurotrophic factor (BDNF) can impact the intensity of pain in rodents. However, the roles of cytokines, TGF-β1 and BDNF in humans with chronic pain in osteoarthritis remains unclear, and no comparison between plasma and central cerebral spinal fluid (CSF) has been conducted.Patients with osteoarthritis who were scheduled to receive spinal anesthesia were enrolled. The intensity of pain was evaluated with a visual analogue scale (VAS). In addition, patients with genitourinary system (GU) diseases and without obvious pain (VAS 0–1) were included as a comparison (control) group. The levels of TGF-β1, BDNF, tumor necrosis factor-α (TNF-α), and interleukin (IL)-8 within the CSF and plasma were collected and evaluated before surgery.The plasma and CSF TGF-β1 levels were significantly lower in the osteoarthritis patients with pain (VAS ≥ 3) than in the GU control patients. Downregulation of plasma BDNF was also found in osteoarthritis patients with pain. The Spearman correlation analysis showed that the VAS pain scores were significantly negatively correlated with the levels of TGF-β1 in the CSF of patients with osteoarthritis. However, there was no significant correlations between the pain scores and the levels of BDNF, TNF-α, and IL-8 in either the CSF or plasma.TGF-β1 but not BDNF, TNF-α, or IL-8 may be an important biological indicator in the CSF of osteoarthritis patients with chronic pain. Pain is an unpleasant stressful sensation that is caused by an acute injury or chronic disease . PersistAlthough the mechanisms of chronic pain in patients with OA is not well understood , the infOn the other hand, brain-derived neurotrophic factor (BDNF) can increase the intensity of pain in rodent inflammatory and neuropathic pain models , 18. BDNTherefore, in this study TGF-β1, TNF-α, IL-8 and BDNF were simultaneously collected from the cerebrospinal fluid (CSF) and plasma samples taken from patients with OA with (VAS score ≥ 3) or without (VAS score ≤ 2) significant clinical pain. The relationship between pain perceptions and molecular expression in CSF and plasma was evaluated and compared. We hypothesized that cytokines, TGF-β1 and the neurotrophic factors in CSF and plasma are correlated to the intensity of pain and that these molecules in subjects with chronic OA experiencing pain may serve as pain indicators of chronic pain in OA.The trial was registered prior to patient enrollment at clinicaltrials.gov . This study was approved by the University’s Institutional Review Board (IRB #B-ER-104-070) for the Protection of Human Subjects at National Cheng Kung University Hospital and Tainan Hospital of Ministry of Health and Welfare, Taiwan. The study procedures were performed according to the rules of the IRB and fully explained to the participants. After the study had been completely described to the participants, informed consent was obtained from all patients in the study. This study was performed in accordance with the standards of ethics outlined in the Declaration of Helsinki.To evaluate the changes in the biological molecules in patients at different levels of pain, patients with or without chronic pain who had received spinal anesthesia for lower body surgery were enrolled in this study. The patients’ pain intensity was evaluated based on the pain score prior to surgery. The levels of TNF-α, IL-8, TGF-β1, and BDNF both in CSF and plasma were also evaluated.We collected patients with a pain history from 07/2015 to 02/2017 before they received spinal anesthesia for knee/hip OA surgery. Moreover, patients with genitourinary system (GU) diseases without obvious current pain at rest served as a comparison (control) group. None of the patients were pregnant; they had an American Society of Anesthesiologists physical status of 1 to 3 and were scheduled for lower body surgery. The inclusion criteria included 1) patients undergoing spinal anesthesia who were over 20 years of age, 2) patients who were able to understand the purpose of the study and had provided written informed consent. The exclusion criteria were 1) patients with contraindications for spinal anesthesia  or refusal to participate in the study, 2) patients with pre-existing neurological diseases, 3) patients using immunomodulatory drugs, and 4) patients over 70 years old or with cancer. All patient medical records were reviewed.We evaluated the intensity of pain before surgery. All patients were asked to evaluate their current pain intensity using the Visual Analogue Scale , based on their native language Mandarin or Taiwanese a day before the operation and biosample collection. The pain intensity was recorded by experienced nurses and was believed to be their current pain intensity as impacted by their disease. To confirm the situation of chronic pain, the duration of pain (days) was collected. Patients were asked to recall how long the pain or discomfort caused by the disease has persisted. Complete blood count, liver and renal function, chest X-ray, and electrocardiogram were also routinely checked to exclude other medical conditions. The patients typically received their surgery and CFS sampling with the next few days. According to the patients’ statements and their medical records, they were then grouped into 3 different groups: GU without obvious pain , OA with very mild pain (VAS ≤ 2), and OA with pain (VAS ≥ 3) based on their pain level a day before the surgery. The analgesic drugs used by the patients were also recorded based on their medical records within one month prior to sampling.g at 4°C for 10 min), and the CSF and plasma was isolated. The CSF and plasma were immediately stored at −80°C.All patients were sampled in the operating room when they received spinal anesthesia for their scheduled surgery. All patients were collected in the daytime (8:30~16:30) and had no food and water intake for at least 8 hours. In the operating room, patients were in the lateral decubitus knee-chest position. After sterilizing and dressing the patient’s lower back, we introduced a 25G spinal needle into the spinal canal and CSF (1 ml) was drained, collected, and then replaced with 0.5% bupivacaine (2–3 ml) as the spinal anesthesia. We also collected whole blood simultaneously before surgery began via venipuncture. Whole blood (10 ml) was drawn into a test tube that contained the anticoagulant ethylenediaminetetraacetic acid. The blood and CSF samples were ice bathed and then transported to a laboratory for a biological molecule analysis. The CSF and whole blood samples were then centrifuged   as before . These k2) tests, a one-way ANOVA with a Bonferroni post-hoc multiple comparison the demographic data. A multivariate analysis of covariance (MANCOVA)  was used for the statistical evaluations. For the biological molecules are often non-normality, a non-parametric statistical test  was also chosen to analysis the data. To determine the correlation between the VAS pain scores and the plasma and CSF molecule, the Spearman correlations were calculated. SPSS 22.0 for Windows was used for the statistical computations. Significance was set at p < 0.05.Chi-square  when they received spinal anesthesia for total knee or hip replacement surgery. Our data demonstrated that some patients with chronic OA do not feel obvious pain when resting . Therefore, in order to understand the cause of different expressions of pain between patients, the OA patients were then divided into groups according to their VAS score as very mild pain  and pain  groups. Moreover, 14 patients with GU diseases without obvious pain (VAS score 0–1 at rest) were included as a control group .p < 0.0001) respectively, which indicated the different degrees of pain intensity in these patients at rest. Moreover, to better understand the situation of chronic pain, the duration of pain (days) were recorded according to the recollection of the patients. The significant different duration of pain experience caused by the disease were found among GU control patients (mean duration of pain is 75.1 days), OA patients with very mild pain , and OA patients with pain  (p < 0.0001).There were no significant between-group differences in age, body weight, or body mass index (BMI) . There wp < 0.05).A multivariate analysis of covariance adjusted for the covariates  was used. And because of the non-normal distribution of these biological molecules, a non-parametric method  was used to analyze the data. OA patients with pain (VAS ≥ 3), but not those with VAS ≤ 2, had plasma TGF-β1 and BDNF levels that were significantly lower compared to the GU control patients (p < 0.05) was significantly down regulated in the OA patients with pain compared to the GU control group. The levels of BDNF, TNF-α or IL-8 in CSF did not change in these patients with chronic OA compared to the GU control group  was used. And a non-parametric method  was also used to analyze the data. We demonstrated that the level of TGF-β1 (ol group .p < 0.05). However, there were no significant correlations between plasma and CSF BDNF, TNF-α and IL-8 levels and the VAS scores . In addition, patients with end-stage OA who experience very mild pain (VAS ≤ 2) at rest had a higher CSF TGF-β1 than those patients who experience pain at rest. Thus, we hypothesize that the levels of TGF-β1 in CSF in humans may modulate pain in OA subjects. Although the mechanism of TGF-β1 down-regulation in OA patients is still unclear, the findings of our study indicate that in patients with end stage of osteoarthritis chronic pain, the downregulation of TGF-β1 in CSF may be a significant indicator of the pain intensity associated with osteoarthritis.With the exception of the downregulation of plasma and CSF TGF-β1, we also found a significant decrease in the level of plasma BDNF in patients with chronic pain. However, we did not find a significant downregulation of CSF BDNF in those experiencing chronic pain. In the CNS, BDNF is produced from neurons and the glia cells , 44 and There are some limitations in this study. First of all, there was no CSF from the healthy subjects. We collected it from a disease reference group without obvious pain (VAS score 0–1). We did not exclude the molecular differences in circulation or CSF between unhealthy and healthy control subjects. Second, the use of analgesics (acetaminophen and NSAIDs) may impact the expression of cytokines. Although we attempted to exclude their impact through the use of a partial correlation with adjusted the used of pain-relieving drugs, further studies still need to be conducted to confirm the effects of analgesics on cytokines in CSF and blood in patients with pain. Third, although the ELISA kits were validated to measure these molecules in human plasma and supernatants of cell culture. And the recombined human IL-8, TNF-α, BDNF and TGF-β1 diluted in buffer were used as a calibration. However, this is the first time ELISA kits is used to detect these molecules in CSF, and the interpretation of CSF data should be more cautious.Our findings revealed that the levels of TGF-β1 in plasma and CSF may be biological indicators in patients with end stage osteoarthritis experiencing chronic pain. Therapeutics that promote TGF-β1 expression may serve as a therapy for pain control in patients with osteoarthritis. The levels of BDNF, TNF-α, or IL8, were not shown to be suitable biological indicators of chronic pain. 22 Jun 2021PONE-D-20-38558TGF-β1 in plasma and cerebrospinal fluid can be used as a biological indicator of chronic pain sensation in patients with osteoarthritisPLOS ONEDear Dr. Chen,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by Aug 06 2021 11:59PM. 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If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: NoReviewer #2: No**********  4. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: NoReviewer #2: No**********  5. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: This interesting manuscript presents data on cytokine-immunoreactivity in paired plasma and CSF from people with OA and urological controls. The findings are of interest, not least because they are not what one might expect. As such, the manuscript raises more questions than it answers, although much of this might be addressed by more detailed presentation of methods and data.Specific points:What was the lead question and recall timeframe for the pain VAS?Please refer to molecule detection by ELISA to indicate that what was measured was immunoreactivity (e.g. BDNF-LI), or otherwise provide evidence of assay validation with this sample type that confirms molecular identity with e.g. BDNF.Please specify diagnoses in control group in more detail, as, in the absence of a healthy matched control group, it might be that controls are abnormal, rather than the patients with OA. How do the authors explain that 5 in the control group were using pain-relieving medications if they did not have pain?The mean VAS for those with OA pain (3.4) is very low (Table 1) considering that all these people had at least VAS=3. What was the range/distribution of VAS scores in the groups?Were cytokine levels and VAS scores normally distributed? Cytokine levels will usually be positively skewed, requiring logarithmic transformation prior to parametric analysis. It seems that the range of VAS scores is very low (mild pain). Descriptives (table 1) should normally be presented with SD or interquartile range rather than SEM, to enable the reader to gain an impression of the distribution of data, rather than just the precision of the mean estimate. Additionally, the regression analyses might be clearer if presented through scatterplots, to demonstrate that the significant Pearson correlation coefficients are not driven by a small number of extreme values.Please include diagnostic group as a covariate (ie control or OA) in regression analyses in case observed differences are attributable to other aspects of diagnosis rather than pain.The origin of TGF beta in the current population is unclear, however, differences in plasma levels as well as CSF levels might well point to a peripheral source. TGF beta has been associated with OA pathology and it would be important to relate the current findings to previous data on circulating TGF beta in OA. Furthermore, please describe the `stage’ of OA in more detail (mentioned in discussion as end stage). Given that knee structural changes in OA are also associated with pain, might TGF beta be a marker for structural severity rather than pain severity?The authors should be commended for not emphasising conclusions from their negative findings, which might have resulted from power limitations.Pain is, by definition, a sensation. Please avoid the term `pain sensation’ which is easily misread for `pain sensitisation’ which is not studied here.Reviewer #2: PLos ONETGF-β1 in plasma and cerebrospinal fluid can be used as a biological indicator of chronic pain sensation in patients with osteoarthritis.Yen-Chin Liu, Hung-Tsung Hsiao, Jeffrey Chi-Fei Wang, Tzu-Cheng Wen, Shiou-Lan Chen.Cytokines can affect pain in osteoarthritis (OA), as well as in many other conditions, in various ways e.g. by enhancing or depressing inflammatory, and various other tissue changes, but also by direct effects on the nervous system. The authors present their data on the plasma and CSF levels of 4 cytokines in patients with OA of the hip, and/or knee. The cytokines they studied were TGF-β1, BDNF, TNF-α, and IL-8. Plasma and CSF samples were obtained from patients scheduled to have spinal anesthesia for surgery. Plasma and CSF samples were also obtained from patients with genitourinary system disease (GU), scheduled for surgery under spinal anesthesia. The samples were assessed for levels of the above 4 cytokines. The authors compared the levels of each of the 4 cytokines between the group of OA patients with little or no pain, and OA patients with more severe pain. The authors also compared cytokine levels between OA patients and GU patients. Their findings showed a significant difference in TGF-β1 levels in OA patients with severe pain and OA patients with mild or no pain; CSF and plasma TGF-β1 levels were significantly lower in the OA patients with severe pain compared to those levels in OA patients with no or mild pain. Similarly, the OA patients with severe pain also had significantly lower TGF-β1 levels (CSF and plasma) than the GU patients. BDNF plasma levels were lower in patients with OA associated with severe pain, but there was no significant lowering of BDNF levels in CSF. The lower plasma BDNF levels represented an unexpected finding for which there does not appear to be a good explanation. There were no significant differences between the patients with severe OA, mild OA, and GU with respect to IL-8 and TNF-α levels in either plasma or CSF. In OA patients with severe pain there was a significant negative correlation between pain severity (assessed by VAS) and TGF-β1 levels (both plasma and CSF).Assessment.Major Points1.These results are interesting, but it is uncertain whether they are generalizable because of some questions with respect to choices in the patient populations and their clinical assessments.*The authors included 36 patients with OA; they excluded 5 for “a chronic pain history with acute pain surgery”. What does this mean?*Two more were excluded because they had neural disease “when they received spinal anesthesia for the osteoarthritis of knee or hip surgery” What does this mean?*It is not clear what surgery the other 29 OA patients had. I assume that it would have been either knee or hip arthroplasties but this has to be specified.*If the above is correct, it appears that such OA patients differ from the usual population selected for arthroplasties in Europe or North America where such operations are performed in a somewhat older population with a male predominance.*Eight out of the 26 patients with OA had a VAS of 0-2. Patients with OA of hips or knees proceedingto an arthroplasty have severe pain. A VAS of 0-2 would be unusual in such patients.2. The VAS was assessed at the time the patients entered the hospital. I am concerned about the validity of such an assessment. An average VAS for the preceding week would be more informative.3. The GU comparison group was selected as “having no pain”. Yet in Table 1 it appears that 5/14 of these patients were taking “pain relief drugs”. Somewhat surprisingly, only 7/21 OA patients with VAS scores of > were taking such medications.4. The low levels of TGF-β1 in patients with more severe pain are an interesting finding. The authors should discuss the possible reasons for this. Could this be due to inadequate production or excessive consumption? What do animal experiments suggest?Minor Points1. The paper requires extensive corrections with respect to the English language. Examples : Page 2 (Introduction) line 2 : “The persisted chronic pain has been considered”; Page 11, lines 240-241 ( first and second from the top) in the “Discussion” part : “Our data suggest that then discrepancy of cytokines expression in different disease may provide the multiple aspect of observation in pain sensation”.2. Each table should be presented on a separate page.**********  6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  5 Aug 2021Reviewers' comments:Reviewer #1: This interesting manuscript presents data on cytokine-immunoreactivity in paired plasma and CSF from people with OA and urological controls. The findings are of interest, not least because they are not what one might expect. As such, the manuscript raises more questions than it answers, although much of this might be addressed by more detailed presentation of methods and data.Response: We thank the reviewer for the positive words. As recommended by the reviewer, we have added the revision in the manuscript.Specific points:What was the lead question and recall timeframe for the pain VAS? Response: This is a cross-sectional study of CSF and plasma molecules in human subjects with chronic pain. We evaluated the intensity of pain before surgery. So even the GU patients were evaluated. About the OA patients, all patients fit the OA X ray criteria (including joint space loss and osteophyte formation) and passed another objective orthpedicst's check, their chronic OA diagnosis was confirmed. About patients' VAS recall timeframe, we asked patient recall their VAS when admission. We also added the duration of pain to confirm their chronic pain situation. The related descriptions were revised in method , Table 1 and results .Please refer to molecule detection by ELISA to indicate that what was measured was immunoreactivity (e.g. BDNF-LI), or otherwise provide evidence of assay validation with this sample type that confirms molecular identity with e.g. BDNF.   1 and mature form of BDNF .Response: As we mentioned in method, the commercial ELISA kit  were used for the measurement of protein levels of TNF-α, IL-8, TGF-�Please specify diagnoses in control group in more detail, as, in the absence of a healthy matched control group, it might be that controls are abnormal, rather than the patients with OA. How do the authors explain that 5 in the control group were using pain-relieving medications if they did not have pain? Response: We agree with the comment, thus this point was described in the limitation of this study . We also revised our Table 1 to present more details of each groups. We used the GU patients with no pain as a control group and presented no significant differences of age, gender, body weight, or body mass index (BMI) between-groups. However, the significant difference of VAS scores and pain duration were found between groups (Table 1). About the 5 GU patients that using pain-relieving medications that we founded from their outpatient clinic record, we think it is the ethic issue that doctors prescribed the pain -relieving medication because of the complain of pain of patients in the outpatient clinic. Thus, only weak pain killers were used in this group. And as we revised in Table 1 that 3 of those GU patients used acetaminophen and 2 of those GU patients used NSAIDs  which were adjusted as covariates in Table 2 and figure 1. We also described this point in the limitation of this study .The mean VAS for those with OA pain (3.4) is very low (Table 1) considering that all these people had at least VAS=3. What was the range/distribution of VAS scores in the groups? Response: In this study, we found that OA patients with very mild pain (n=8), their VAS score are 0 to 2. And in OA subjects with chronic pain (n=21), the range of VAS scores are 3 to 6. Were cytokine levels and VAS scores normally distributed? Cytokine levels will usually be positively skewed, requiring logarithmic transformation prior to parametric analysis. It seems that the range of VAS scores is very low (mild pain). Descriptives (table 1) should normally be presented with SD or interquartile range rather than SEM, to enable the reader to gain an impression of the distribution of data, rather than just the precision of the mean estimate. Additionally, the regression analyses might be clearer if presented through scatterplots, to demonstrate that the significant Pearson correlation coefficients are not driven by a small number of extreme values.Response: We had revised the Table 1 as the suggestion. And we thanks the reviewer's comments that the distribution of cytokine levels are indeed positively skewed. Thus, we also transformed the levels of cytokines logarithmically prior to the analysis of Pearson correlation in Table 3 (Page 16). The scatterplots of VAS with log CSF TGF-β1 and log plasma TGF-β1 are also shown in Fig 2 (Page 17).Please include diagnostic group as a covariate (ie control or OA) in regression analyses in case observed differences are attributable to other aspects of diagnosis rather than pain.Response: In Table 3 (Page 16), we only included the patients with same diagnosis . And we transformed the levels of cytokines logarithmically prior to the analysis of Pearson correlation. We also added the scatterplots of VAS with log CSF TGF-β1 and log plasma TGF-β1 are also shown in Fig 2 (Page 17).The origin of TGF beta in the current population is unclear, however, differences in plasma levels as well as CSF levels might well point to a peripheral source. TGF beta has been associated with OA pathology and it would be important to relate the current findings to previous data on circulating TGF beta in OA. Response: We thank the reviewer for the excellent comments. We had added the description of origin of TGF beta in blood and brain. And the possible relationship of TGF-β1 in OA Furthermore, please describe the `stage’ of OA in more detail (mentioned in discussion as end stage). Given that knee structural changes in OA are also associated with pain, might TGF beta be a marker for structural severity rather than pain severity? Response: Thanks the comments. We added the describe in result  that the definition of end-stage OA was that X-ray presented articular cartilage wears away almost completely and bone on bone contact occurs and passed another objective orthpedicst’s check. We also added the duration of pain of each groups in Table 1.In addition, we agree with the comments of reviewer, that the levels of plasma TGF-β1 may as a marker for structural severity of OA. However, in this study we collect the CSF TGF-β1 that may explain the intensity of central pain in OA subjects. However, the expression of TGF-β1 in different stage of OA need more study in the future. The authors should be commended for not emphasising conclusions from their negative findings, which might have resulted from power limitations.Response: Thank you for this comment.Pain is, by definition, a sensation. Please avoid the term `pain sensation’ which is easily misread for `pain sensitisation’ which is not studied here. Response: Thank you for this comment. In this revision, we revised VAS as the measurement of pain intensity. We revised most of the “pain sensation” description as “pain intensity” or “the intensity of pain”. Reviewer #2: PLos ONECytokines can affect pain in osteoarthritis (OA), as well as in many other conditions, in various ways e.g. by enhancing or depressing inflammatory, and various other tissue changes, but also by direct effects on the nervous system. The authors present their data on the plasma and CSF levels of 4 cytokines in patients with OA of the hip, and/or knee. The cytokines they studied were TGF-β1, BDNF, TNF-α, and IL-8. Plasma and CSF samples were obtained from patients scheduled to have spinal anesthesia for surgery. Plasma and CSF samples were also obtained from patients with genitourinary system disease (GU), scheduled for surgery under spinal anesthesia. The samples were assessed for levels of the above 4 cytokines. The authors compared the levels of each of the 4 cytokines between the group of OA patients with little or no pain, and OA patients with more severe pain. The authors also compared cytokine levels between OA patients and GU patients. Their findings showed a significant difference in TGF-β1 levels in OA patients with severe pain and OA patients with mild or no pain; CSF and plasma TGF-β1 levels were significantly lower in the OA patients with severe pain compared to those levels in OA patients with no or mild pain. Similarly, the OA patients with severe pain also had significantly lower TGF-β1 levels (CSF and plasma) than the GU patients. BDNF plasma levels were lower in patients with OA associated with severe pain, but there was no significant lowering of BDNF levels in CSF. The lower plasma BDNF levels represented an unexpected finding for which there does not appear to be a good explanation. There were no significant differences between the patients with severe OA, mild OA, and GU with respect to IL-8 and TNF-α levels in either plasma or CSF. In OA patients with severe pain there was a significant negative correlation between pain severity (assessed by VAS) and TGF-β1 levels (both plasma and CSF).Assessment.Major Points1.These results are interesting, but it is uncertain whether they are generalizable because of some questions with respect to choices in the patient populations and their clinical assessments.*The authors included 36 patients with OA; they excluded 5 for “a chronic pain history with acute pain surgery”. What does this mean? Response: Sorry for the misunderstanding. Actually, we collected 5 OA patients with low-leg trauma and with acute pain. Thus, these patients with chronic and acute pain were excluded. We deleted this sentence (Page 8) for possible misunderstanding.*Two more were excluded because they had neural disease “when they received spinal anesthesia for the osteoarthritis of knee or hip surgery” What does this mean? Response: We deleted this sentence (Page 8). Actually, we had mentioned in the exclusion criteria 2) we excluded those patients having pre-existing neurological diseases. The pre-existing neurological disease may have influence on CSF data. *It is not clear what surgery the other 29 OA patients had. I assume that it would have been either knee or hip arthroplasties but this has to be specified. Response: Thank you for your comment. We revised the Table 1 and presented the surgery type which was OA hip , OA knee  and knee arthroscopy. *If the above is correct, it appears that such OA patients differ from the usual population selected for arthroplasties in Europe or North America where such operations are performed in a somewhat older population with a male predominance. Response: Thank you for your comment. We agree that generally, male may receive more OA surgery than female. However, some data still claim that OA is more common in women than in men. Although we have the limitation in the selection of case, however, the comparison of VAS scores with CSF and plasma molecules may provide a reference in the observation of OA with chronic pain.*Eight out of the 26 patients with OA had a VAS of 0-2. Patients with OA of hips or knees proceeding to an arthroplasty have severe pain. A VAS of 0-2 would be unusual in such patients. Response: Thank you for your comment. We agree that OA surgery caused a lot of pain. However, we had described clearly that patients’ VAS was recorded when they administered to the hospital before surgery, not after surgery. And patients were asked to present their VAS before operation. It is also reasonable that OA patients have low VAS once they reduced activity or taking drugs. We thought their low VAS may present some clinical meanings and separated as another groups 2. The VAS was assessed at the time the patients entered the hospital. I am concerned about the validity of such an assessment. An average VAS for the preceding week would be more informative. Response: Thank you for your comment. We agreed that the VAS validity is an important issue. However, we also concerned the recall validity if we asked patient recall the preceding week. Furthermore, there is no data showed that recalling preceding week VAS is a better presentation than VAS at administration. So we used the VAS at administration as grouping category and we also noticed that those with low VAS at administration also used less pain drug than those with high VAS at administration which also proved that our grouping using VAS at administration is reasonable.3. The GU comparison group was selected as “having no pain”. Yet in Table 1 it appears that 5/14 of these patients were taking “pain relief drugs”. Somewhat surprisingly, only 7/21 OA patients with VAS scores of > were taking such medications. Response: Thank you for your comment. About the 5 GU patients that using pain killers, that we founded from their outpatient clinic record, we think it is the ethic issue that doctors prescribed the pain killer because of the complain of pain of patients in the outpatient clinic. Thus, only weak pain killers were used in this group. And as we presented in the revised Table 1 that 3 of those GU patients used acetaminophen and 2 of those GU patients used NSAIDs  which were adjusted as covariates in Table 2 and figure 1. We also described this point in the limitation of this study For OA patients, we still find that less pain group patient used less pain drugs (1/8 vs.7/21) We also think that, in our hospital, many patients receive surgery because they don’t want taking pain drugs because of many reasons . So even the surgeon prescribed less drugs if patients claimed they don’t want take medicine. The surgeon just arrange operation as soon as possible. So there are less ratio for patients receiving drugs.4. The low levels of TGF-β1 in patients with more severe pain are an interesting finding. The authors should discuss the possible reasons for this. Could this be due to inadequate production or excessive consumption? What do animal experiments suggest?Response: This is a first study that compared the levels of TGF-β1, BDNF and cytokines in CSF and plasma of human subjects with no pain, very mild pain and chronic pain condition. As we discussed in page 19-20 that TGF-β1 may involve in the articular cartilage homeostasis and inhibit pain sensation in rodents. Thus we hypothesize that the levels of TGF-β1 in CSF/plasma in humans may associated with the progression of disease and modulate pain. Although the mechanism of TGF-β1 down-regulation in OA patients is still unclear. However, our study represented that patients with end stage of osteoarthritis and with chronic pain, the downregulation of TGF-β1 in CSF may be a significant indicator of the pain intensity associated with osteoarthritis. The related description of TGF-β1 with OA progression and chronic pain were added in Page 19 last paragraph and Page 20 lines 299-315.Minor Points1. The paper requires extensive corrections with respect to the English language. Examples : Page 2 (Introduction) line 2 : “The persisted chronic pain has been considered”; Page 11, lines 240-241 ( first and second from the top) in the “Discussion” part : “Our data suggest that then discrepancy of cytokines expression in different disease may provide the multiple aspect of observation in pain sensation”.Response: Thank you for your comment. We revised this newest version again and make it easy to read. We also corrected the grammar errors with the native speaker's help.2. Each table should be presented on a separate page.Response: We had presented each table on a separate page.Attachment2021-0805-PONE-reply to the reviewers comments.docxSubmitted filename: Click here for additional data file. 13 Sep 2021
 PONE-D-20-38558R1

 TGF-β1 in plasma and cerebrospinal fluid can be used as a biological indicator of chronic pain in patients with osteoarthritis

 PLOS ONEDear Dr. Chen,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by Oct 28 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at
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For instructions see: We look forward to receiving your revised manuscript.Kind regards,Firas H Kobeissy, PhDAcademic EditorPLOS ONEJournal Requirements:Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author  1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: (No Response)Reviewer #2: All comments have been addressed**********  2. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: Yes**********  3. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: NoReviewer #2: Yes**********  4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: NoReviewer #2: Yes**********  5. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: NoReviewer #2: Yes**********  6. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: I appreciate the authors’ efforts in revising their manuscript, which goes some way to address my original concerns. I suspect that my original comments (and those of the second reviewer where similar) might have been incompletely understood by the authors and I attempt to explain my residual concerns more clearly below.What was the lead question and recall timeframe for the pain VAS? (?p6) I note also that this point was raised by the second reviewer, who made additional pertinent points. Having read the authors’ response, I think that I might understand the source of apparent confusion and discrepancies, but this is not yet clear to the general reader of the manuscript. Am I correct to think that participants were recruited on the basis of clinical evidence of chronic pain (undergoing arthroplasty), but that the pain VAS used in analyses asked about `recent pain’. As in my original comments, please give the exact text and anchors for the VAS used. I expect this was not in English language, and so the exact text in the original language, plus a verbatim translation might suffice. It makes a very big difference what time-frame defined `recent’ if the original text used the word `recent’. Does `recent’ mean since admission, over the past few minutes while you have not been standing, or over the past few months? I suspect from the results that participants rated their `current’ pain whilst not mobilising or standing. Our experience also is that patients listed for arthroplasty might not have severe pain at rest, but as soon as they try to do something, then their pain becomes severe. Chronic pain  typically refers to pain over the past week; either average pain, or worst pain, thereby reflecting the intermittent nature often of OA pain. On reflection, I can understand that `current pain’ might be the most appropriate index here, because molecules in CSF might change over short time periods, and the authors might wish to measure the pain experienced at a time point closest to biosample collection. However, if this is the case, they might wish to speculate as to how relevant this might be to clinical pain. At least, they should explain the apparent inconsistency commented on by both reviewers that people with very low pain scores were undergoing joint surgery. Also I note that reviewer 2, like myself, commented on the GU comparison group was selected as “having no pain”; so am I correct to understand that they had no `current pain on admission to hospital’ but that they might have had chronic pain prior to admission? This again should be clarified, and might be part of the same explanation as the clarification of what the VAS score represents.The authors have chosen to not `indicate that what was measured was immunoreactivity (e.g. BDNF-LI)’. I do not feels strongly about this, but the fact that ELISA measurements, especially competitive ELISAs, might measure factors other than the specific molecular species targeted, particularly when they are applied to biofluids in which the manufacturer and investigator have not validated them. I would still suggest that the authors provide data on validation in plasma and CSF, rathe than assume that these kits do what the manufacturers say they do when they sell them for profit.Please provide statistical evidence that log transformed data are normally distributed. If, as stated in the authors’ response, untransformed data were significantly positively skewed, then please use log transformed  data for all parametric analyses, including between group comparisons. If log transformation converts non-normal data to normal data, then please only present analyses on log transformed data (e.g. in Table 2). In other words, only present the statistically appropriate analyses.I’m afraid that I don’t understand the data in the statement; ` The mean duration of pain was 75.1, 1,665, and 1,060 days with no pain, OA patients with very mild pain, and OA patients experiencing pain according to the recall of the patients, respectively (p < 0.0001).’ Are the authors really stating that participants were asked how long they had had pain to the nearest 0.1 days??? Given my above comments about variability of pain in OA, this seems meaningless if I am interpreting correctly. What is the method for this?Reviewer #2: My concerns have been answered. The authors' findings are interesting although their significance is uncertain in view of conflicting evidence in thre literature on the roles of TGFbeta1 and BDNF.**********  7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Yes: David Andrew WalshReviewer #1: Reviewer #2: Nohttps://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.
 While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  26 Oct 2021Reviewers' comments:Response: We thank the reviewers for the positive words. As recommended by the reviewers, we have added the revision in the manuscript.Reviewer #1: I appreciate the authors’ efforts in revising their manuscript, which goes some way to address my original concerns. I suspect that my original comments (and those of the second reviewer where similar) might have been incompletely understood by the authors and I attempt to explain my residual concerns more clearly below.What was the lead question and recall timeframe for the pain VAS? (?p6) I note also that this point was raised by the second reviewer, who made additional pertinent points. Having read the authors’ response, I think that I might understand the source of apparent confusion and discrepancies, but this is not yet clear to the general reader of the manuscript. Am I correct to think that participants were recruited on the basis of clinical evidence of chronic pain (undergoing arthroplasty), but that the pain VAS used in analyses asked about `recent pain’. As in my original comments, please give the exact text and anchors for the VAS used. I expect this was not in English language, and so the exact text in the original language, plus a verbatim translation might suffice. It makes a very big difference what time-frame defined `recent’ if the original text used the word `recent’. Does `recent’ mean since admission, over the past few minutes while you have not been standing, or over the past few months? I suspect from the results that participants rated their `current’ pain whilst not mobilising or standing. Our experience also is that patients listed for arthroplasty might not have severe pain at rest, but as soon as they try to do something, then their pain becomes severe. Chronic pain  typically refers to pain over the past week; either average pain, or worst pain, thereby reflecting the intermittent nature often of OA pain. On reflection, I can understand that `current pain’ might be the most appropriate index here, because molecules in CSF might change over short time periods, and the authors might wish to measure the pain experienced at a time point closest to biosample collection. Response: Thank you for your comments. Correctly, all patients were asked to evaluate their current pain intensity using the Visual Analogue Scale , based on their native language Mandarin or Taiwanese a day before the operation and biosample collection. All of these were revised in method  and results .However, if this is the case, they might wish to speculate as to how relevant this might be to clinical pain. At least, they should explain the apparent inconsistency commented on by both reviewers that people with very low pain scores were undergoing joint surgery. Response: Thank you for your comments. And, as you recognized, our data support that patients with end-stage OA experienced with chronic pain . In addition, our data also revealed that some patients with chronic OA do not feel obvious pain when resting . And this patients (OA with very mild pain) at rest had higher CSF TGF-β1 than those OA patients with pain. Thus, we hypothesize that the levels of TGF-β1 in CSF in humans may modulate pain in OA subjects. All of these were revised in result  and discussion . Also I note that reviewer 2, like myself, commented on the GU comparison group was selected as “having no pain”; so am I correct to understand that they had no `current pain on admission to hospital’ but that they might have had chronic pain prior to admission? This again should be clarified, and might be part of the same explanation as the clarification of what the VAS score represents. Response: Thank you for your comments. We apology that we did not described the definition and condition of the GU-control group clearly. In this study, patients with GU diseases without obvious current pain at rest  before surgery were included as a comparison (control) group. Furthermore, to access that they might have had chronic pain, we also checked the experience of pain of GU patients according to their recollection . All of these were revised in method  and result (Page 9).The authors have chosen to not `indicate that what was measured was immunoreactivity (e.g. BDNF-LI)’. I do not feels strongly about this, but the fact that ELISA measurements, especially competitive ELISAs, might measure factors other than the specific molecular species targeted, particularly when they are applied to biofluids in which the manufacturer and investigator have not validated them. I would still suggest that the authors provide data on validation in plasma and CSF, rathe than assume that these kits do what the manufacturers say they do when they sell them for profit. Response: We agree that although the ELISA kit were validated to measure these molecules in human plasma and supernatants of cell culture. And the recombined human IL-8, TNF-α, BDNF and TGF-β1 diluted in buffer were used as a calibration. However, this is the first time that using of ELISA kits to detect these molecules in CSF, and the interpretation of CSF data should be more cautious. We have added and revised such description in method  and limitations (Page 21).Please provide statistical evidence that log transformed data are normally distributed. If, as stated in the authors’ response, untransformed data were significantly positively skewed, then please use log transformed  data for all parametric analyses, including between group comparisons. If log transformation converts non-normal data to normal data, then please only present analyses on log transformed data (e.g. in Table 2). In other words, only present the statistically appropriate analyses. Response: Thanks for the suggestion of statistics. Because of the log transformed data are still not normally distributed. For the biological molecules are often non-normality, thus, a non-parametric statistical test  was added to analysis the original data (Table 2). To determine the correlation between the VAS pain scores and the plasma and CSF molecule, the Spearman correlations with control of age, gender, body mass index and pain drug were performed (Table 3). All of these were added in method , Table 2, Table 3 and results .I’m afraid that I don’t understand the data in the statement; ` The mean duration of pain was 75.1, 1,665, and 1,060 days with no pain, OA patients with very mild pain, and OA patients experiencing pain according to the recall of the patients, respectively (p < 0.0001).’ Are the authors really stating that participants were asked how long they had had pain to the nearest 0.1 days??? Given my above comments about variability of pain in OA, this seems meaningless if I am interpreting correctly. What is the method for this? Response: We apology that we did not clearly described the definition of this data. In this study, to confirm the situation of chronic pain, the duration of pain (days) was collected. Patients were asked to recall how long the pain or discomfort caused by the disease has persisted. All of these were added and revised in Table 1, method  and in result .Reviewer #2: My concerns have been answered. The authors' findings are interesting although their significance is uncertain in view of conflicting evidence in thre literature on the roles of TGFbeta1 and BDNF.Response: Thank you for you insightful feedback and comments.Attachment2021-1026-Reply to the Reviewers comment.docxSubmitted filename: Click here for additional data file. 19 Dec 2021TGF-β1 in plasma and cerebrospinal fluid can be used as a biological indicator of chronic pain in patients with osteoarthritisPONE-D-20-38558R2Dear Dr. Chen,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Firas H Kobeissy, PhDAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author  1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #2: All comments have been addressed**********  2. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #2: Yes**********  3. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #2: Yes**********  4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #2: Yes**********  5. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #2: Yes**********  6. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #2: The authors have addressed my concerns. The patient population of osteoarthritis patients is somewhat atypical, but I am willing to accept that. The results with respect to the low levels of TGF-beta are very interesting.**********  7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Yes: ManfredHarthReviewer #2:  22 Dec 2021PONE-D-20-38558R2 TGF-β1 in plasma and cerebrospinal fluid can be used as a biological indicator of chronic pain in patients with osteoarthritis Dear Dr. Chen:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Firas H Kobeissy Academic EditorPLOS ONE


Background: Multiple sclerosis is a chronic neurological disease with increasing incidence and prevalence worldwide being the main cause of non-traumatic disability in young adults. Both acute and chronic pain have been mentioned as the most common symptoms among those patients.Objective: This study was designed to evaluate the pain experience among patients with multiple sclerosis by describing its prevalence, characteristics, analgesic treatment and its efficacy, and also the impact of pain on quality of life.Methods: A cross-sectional observation survey was carried out on patients with multiple sclerosis followed in a tertiary hospital. Data were collected between December 2019 and March 2021 from a structured telephone inquiry, applying two questionnaires, the Brief Pain Inventory and the McGill Pain Questionnaire (MPQ), to evaluate the prevalence of pain and its impact on quality of life (QoL). Clinical records were also consulted to obtain data on disease duration, year of diagnosis, MS type, Expanded Disability Status Scale (EDSS) score.Results: Our sample included 305 patients in a universe of 1500, mainly women, with mean age of 44.27 years, and most of them presented with an outbreak-remission subtype of disease. One hundred twenty-four patients experienced pain which corresponds to 41% of the patients. Considering the patients who experienced pain, 67.7% were under treatment and of these, 64.3% with only one painkiller. Pain significantly interfered with general activity, mood, and regular work.Conclusion: Pain was an important symptom in this group of patients with MS and significantly interfered with mood, general activity, and regular work. The maximum intensity of pain felt by patients was significant and only 67.7% of patients were under analgesic treatment with mean pain relief of 54. NSAIDs were the most used drugs followed by gabapentinoids and acetaminophen for the management of pain. Medical community must continue to study this population in order to improve the approach to pain in these patients and improve quality of life. Multiple sclerosis (MS) is an autoimmune disorder of the central nervous system (CNS) that is characterized by patchy inflammation, gliosis, and demyelination within the central nervous system. Its increasing incidence and prevalence worldwide make it the main cause of non-traumatic disability in young adults . It is uThe severity of the disease depends on its form, duration, location, and size of lesions in the CNS, which are highly variable. Several forms of the disease can be described like relapsing-remitting MS (RRMS), secondary progressive MS (PSMS), and primary progressive MS (PPMS). Common symptoms include sensory impairment, extremity weakness, urinary dysfunction, visual impairment, fatigue, spasticity, incoordination, and pain ,4.Pain is a frequent complaint among individuals with MS. The prevalence of pain in MS differs in the literature, ranging from 28% to 92% ,6. This There is only one Portuguese study with a small sample size that shows data on prevalence of pain . This stThis cross-sectional observational study took place in an MS clinic at Centro Hospitalar Universitário de Lisboa Central (CHULC), a tertiary hospital in Lisbon. All patients older than 18 years old were considered eligible. Exclusion criteria were inability to read or write or to consent to voluntary participation.Data was collected between December 2019 and March 2021 from a structured telephone inquiry, applying two questionnaires, the Brief Pain Inventory and The McGill Pain Questionnaire (MPQ), to evaluate the prevalence of pain and its impact on quality of life (QoL). Clinical records were also consulted to obtain data on disease duration, year of diagnosis, MS type, Expanded Disability Status Scale (EDSS) score. The project was approved by the CHULC Ethics Committee with the approval number AGFC/79/2019 and all patients gave informed consent.Pain evaluation and questionnairesBrief Pain InventoryThe BPI is a simple and short questionnaire composed of 15 items aiming to assess two scales: pain intensity and pain interference . The paiLong-Version of the McGill Pain QuestionnaireThe McGill Pain Questionnaire (MPQ) measures the sensory, affective, evaluative, and miscellaneous components of pain . It was Statistical analysisThe information gathered was entered into an excel sheet. An exploratory analysis was carried out for all variables. Categorical data were expressed as frequencies and percentages, and continuous variables as mean or median and SD. Univariable analysis was carried out using Student’s t-test and nonparametric chi-square test as appropriate. Pearson’s correlation was used to investigate association between pain intensity and disease duration. Results were considered significant when p was less than 0.05. Confidence intervals are presented when appropriate (95% CI). Statistical analysis was carried out using the IBM SPSS Statistics, version 27.0 .Description of the sampleFour hundred phone calls were made, and only 305 patients answered the questionnaire and consented to data collection. Of the remaining 95 patients, 40 patients did not consent to participate in the study, 45 patients did not attend, and 10 were dependent, so only the caregiver could provide data and consent. These last 10 patients were also excluded from the sample. The patient’s demographic data and disease characteristics are presented in Table Concerning multiple sclerosis, the meantime since initial diagnosis was 9.72 years (SD: 7.63). The mean EDSS score was 2.18 (SD: 1.99). Two hundred and seventy patients (88.5%) presented with relapsing-remitting subtype of disease, 21 (6.9%) with primary progressive, and 14 (4.6%) with secondary progressive subtype.Pain prevalence and characteristicsThe prevalence of pain in this population was 40.65%. One hundred eighty-one did not experience any pain. One hundred twenty-four experienced pain a week before and answered the Brief Pain Inventory (BPI) questionnaire and the Portuguese version of McGill pain questionnaire (MPQ).Regarding pain location, only 54 (43.5%) patients experienced pain in a single point, against 49 (39.5%), 14 (11.3%), and seven (5.6%) that experienced pain at two, three, or four locations, respectively. The most frequent sites were lower limb pain (68.5%), back pain (40.3%), upper limb (36.3%), and head pain (16.1%) Table .Using BPI, pain severity was calculated through mean value and standard deviation from a 10-point scale. Pain severity calculated was 6.25 (SD: 2.02) for maximum, 1.94 (SD: 2.04) for minimum, 4.47 (SD: 2.71) for mean, and 3.29 (SD: 2.74) for current pain intensity. At MPQ, almost 97% of the patients described their pain with words of the four dimensions, proposed by the questionnaire. One hundred twenty-four patients used at least one word of the sensitive category, 112 a word of the affective category, 104 a word of the evaluative category, and 111 one word of the miscellaneous group. Table The most selected descriptors were tender (45.2%), boring (44.4%), tingling (41.9%), miserable (37.1%), and nagging (35.5%). The calculated pain index (standard deviation) for the sensitive dimension was 17.4 (7.34), while for the affective dimension a value of 4.24 (3.04) was obtained, and the total pain index was 29.03 (5.92). Considering current pain, and a 0 to 6 points scale, the mean intensity was 1.81 (0.9), below the middle point, and so non-significant. No association between pain intensity and disease duration was statistically significant (p=0.21). Concerning pain patterns, 46.8% of the patients defined it as rhythmic, periodic, or intermittent, while 36.3% recognized their pain as being continuous, steady, or constant.Pain and interference with daily functionPain interference with daily function is scored 4.77 (SD: 3.10). The mean effect of pain over general activity was 5.83 (SD: 2.728), humor 5.31 (SD: 2.948), walking 4.72 (SD: 3.360), regular work 5.65 (SD: 2.981), relationships 4.11 (SD: 3.323), sleep 4.11 (SD: 3.323), and pleasure of life 3.68 (SD: 3.070).Pain managementEighty-four patients (67.7%) who experienced pain were under analgesic treatment. Among patients who take one drug (64.3%), NSAIDs were the most used followed by gabapentinoids and acetaminophen. Seventeen patients (20.2%) were under treatment with two drugs, eleven (13%) with three drugs, and four (4.7%) with more than three drugs. The mean pain relief analgesic treatment was 54%. No association between a specific type of treatment and pain relief was found.Comparisons of persons with and persons without painRegarding both cohorts, patients without pain and with pain, no statistically significant differences were found respect to gender , EDSS score , or mean time of disease . However, the difference of ages between samples is statistically significant , with older patients in the pain group. This information is detailed in Tables According to 2016 data, it was estimated that there were between 56,000 and 8000 patients with MS in Portugal . This crThe socio-demographic data of our sample are similar to other studies, with the majority of patients being female and suffering from the clinical subtype of outbreak-remission of the disease ,6. The hAs previously mentioned, 40.65% of patients experienced pain during the week prior to the questionnaire, which demonstrates a prevalence of pain similar to other populations studied ,2,7,12. Regarding pain location, our studies are consistent with others ,19. RegaRegarding the management of pain, a greater number of patients should be under treatment. Perhaps the effectiveness of AINE's is related to nociceptive component of pain, which is not negligible, taking into account the McGill Pain Questionnaire results ,21. PainFinally, the greatest difficulty found in the studies is the lack of a standardized questionnaire to assess the prevalence of pain and distinguish the different types of pain in this population. It would be very interesting to develop new questionnaires aimed at this population that would allow the prevalence of pain to be measured in a more uniform way and a valid distinction between neuropathic pain and nociceptive pain .With our cross-sectional survey using two validated questionnaires, we found that pain is an important symptom in patients with MS with several implications for patient satisfaction and quality of life. The maximum intensity of pain felt by patients is significant and only 67.7% of patients are under analgesic treatment with mean pain relief of 54. NSAIDs were the most drug used followed by gabapentinoids and acetaminophen for management of pain. Since pain remains a frequent and often overlooked problem, medical community must continue to study this population in order to improve the approach to pain in these patients and improve quality of life.


Postendodontic pain is one of the problems of root canal therapy. This clinical study aimed to evaluate the effect of infiltration injection of dexamethasone and methylprednisolone on postendodontic pain in patients with necrotic pulp. n = 40). After the administration of local anesthesia and before root canal therapy, in group 1, an infiltration injection of 1 ml of dexamethasone was done and in group 2, an infiltration injection of 1 ml of methylprednisolone was done in the buccal vestibule of each tooth. Patients' pain was reported using a visual analogue scale at pretreatment and 6, 12, 24, and 48 hours after treatment.  A total of 80 volunteers with necrotic pulp teeth were included and assigned to two groups ( There was no significant difference between the two groups receiving dexamethasone and methylprednisolone at pretreatment and 6, 12, 24, and 48 hours after endodontic treatment.  Infiltration injection of dexamethasone and methylprednisolone had a significant effect in reducing pain after the endodontic treatment in necrotic pulp teeth, but between 6 and 12 hours, methylprednisolone had significantly more effect on pain relief than dexamethasone. Overall, the use of any of these drugs to reduce postendodontic pain is recommended. Despite the advances in root canal treatment, endodontic postoperative pain is common and can occur between 3% and 58% of patients depending on the variables: host-related factors, such as patient's age, gender, tooth type, psychological and immunological factors; the iatrogenic factors, such as over instrumentation and chemical or obturating material; and the microbial factors. Proper anesthesia techniques reduce pain during treatment, but postendodontic pain is still a problem .Clinical signs and symptoms and the radiological findings of the treated tooth determine the success or failure of endodontic treatment. The success symptoms are the absence of pain, inflammation, and fistulas and the maintenance of the functional tooth . AlthougIn necrotic teeth, the passage of infected debris into the periapical area causes postoperative endodontic pain .Several strategies are suggested to manage or prevent postoperative endodontic pain. Several pharmacological  and nonpharmacological methods  have been used to treat and relieve postendodontic pain. One of the suggested methods to control pain is to prescribe medications before endodontic treatment (premedication) .Postoperative endodontic pain is contributed to inflammatory mediators such as prostaglandins that activate sensitive nociceptors and have a key function in the pathogenesis of periradicular and pulpal diseases .Nonsteroidal anti-inflammatory agents and steroids have been used to interfere with inflammation and to prevent or stop endodontic pain .In some severe and uncontrollable postoperative endodontic pain, it seems that the prescription of steroidal anti-inflammatory drugs is a suitable treatment .Corticosteroids are the most effective anti-inflammatory therapy and they suppress inflammation .Dexamethasone is a long-acting glucocorticoid that has more anti-inflammatory activity than other steroids. For example, the relative potency of dexamethasone is 25 times larger than that of hydrocortisone and 5 times larger than that of methylprednisolone  which haSome studies were done to evaluate the effect of corticosteroid on postendodontic pain in teeth with irreversible pulpitis. Studies by Yavari et al. , Aksoy aThe study by Moradi et al. on the efficacy of infiltration injection of dexamethasone on postendodontic pain of necrotic teeth showed infiltration administration of dexamethasone did not reduce postoperative pain severity in the first 12 hours after endodontic treatment . Becausen = 80): (1) patients with good general health , (2) patients who were willing to participate, (3) patients who have not received anti-inflammatory or analgesic drugs in the last 8 hours, (4) patients who have not taken any drugs, (5) patients who have not an allergy to dexamethasone and methylprednisolone and (6) aged between 18 and 60 years, and (7) with necrotic tooth without periapical lesions [After taking approval from the ethical committee (IR.YUMS.REC.1396.136), this randomized clinical study was conducted in the endodontic department of dental school. Inclusion criteria were as follows (Exclusion criteria were as follows: (1) patients who have systematic complexity , (2) patients who were unwilling to cooperate; (3) patients who have taken drugs, and (4) patients whose treatment was more than one session.After taking informed consent, 80 patients of both genders with good general health were selected for this study using a convenient sampling technique and were randomly divided into two equal groups with the help of a scientific random number table. All participants who fit the criteria volunteered, received verbal and written descriptions of the study design, and signed informed consent forms.A (no report of pain in patients receiving dexamethasone) = 60%; population proportion B = 30% difference in pain relief in treatment with methylprednisolone (dexamethasone is stronger than methylprednisolone).The sample size was based on the study of Jalalzadeh et al. . With a n = 40). In two groups, pulpal anesthesia of mandibular molars was done with the alveolar nerve block technique by injections of 1.8 mL  of 2% lidocaine with 1/100000 epinephrine , and for maxillary molars, 1 cartridge of 2% lidocaine with 1/100000 epinephrine was injected by infiltration.Demographic information, medical and dental history, relevant clinical examination, and periapical radiographs were taken, and all data  were recorded on the patient chart. The study procedure was explained to each volunteer, and they were divided into two groups, randomly  with similar conditions and devices of infiltration anesthesia.In group 2, infiltration injection was performed using 0.2 mL of methylprednisolone  with similar conditions and devices of infiltration anesthesia. The reason for not injecting a placebo in this study was ethical considerations.As this study was accomplished in a double-blinded manner, the drugs were coded A and B.After 15 minutes in mandibular teeth and 7 minutes in maxillary teeth, root canal treatment was initiated under rubber dam isolation. Working length within 2 mm shorter from radiographic apex was taken with apex locator. The root canals were instrumented with S1-S2-F1-F2 ProTaper rotary files . The canals were irrigated with 2.5% sodium hypochlorite (NaOCl), dried with paper point and obturated with laterally condensed gutta-percha  and sealer . The access cavity was restored with temporary restoration , and to prevent future pain, the occlusion and proximal contact were checked. All procedure was completed by one root canal specialist use of single-visit endodontics treatment .The patients were also asked to record the type and dosage of analgesic consumption if required to relieve their postoperative pain. The pain intensity was measured by a nonnumeric VAS ruler. According to this scale, the level of pain was recorded by the operator in the range of 0–170 . A scoreT-test, and ANOVA test were used to evaluate the study data. The level of significance was set at 0.05.Statistical analysis was performed using SPSS version 16. Mean, standard deviation, and frequency distribution were measured for each group. Chi-square test, independent-samples P = 0.0001)  .P value <0.05).There were no reports of pain at 24 and 48 hours after the intervention. The frequency distribution of pain duration in the two groups was not statistically significant. In each group, the pain score decreased significantly over time. In the methylprednisolone group, unlike the dexamethasone group, the pain was significantly reduced 12 hours after the intervention compared with 6 hours after the intervention  are greatly expressed, and the patient requires urgent treatment sooner than the scheduled appointment. The etiology of the postendodontic flare-up involves mechanical, chemical, and microbial factors .Despite modern root canal treatment methods and new rotary systems, flare-up remains a major problem for all dentists .It occurs particularly for the patients exhibiting preoperative pain, and up to 80% of these patients may experience it. Flare-up rarely lasts longer than 72 h and usually is not so severe that it cannot be managed by NSAID agents .Various categories of drugs have been prescribed for the management of this condition, including corticosteroids. Corticosteroids include dexamethasone and methylprednisolone, which reduce pain after endodontic treatment. Steroids bind to the glucocorticoid receptor, inhibiting proinflammatory signals and promoting anti-inflammatory signals . DexametIn the present study, VAS score data showed that infiltration injection of dexamethasone and methylprednisolone had no statistically significant effect at 6 hours after the intervention in the two groups and this result is consistent with some studies , 25. At Sharma et al. in their study suggested that preoperative administration of SAID resulted in significantly less postoperative endodontic pain at all time intervals and preoperative oral administration of dexamethasone reduces pain postoperatively .Another finding of this study is that, in each group, the pain score decreased considerably over time. Some studies are in agreement with this result , 29, 30.In the present study, the mean pain score before the intervention and 6 hours after the intervention in each group was reduced. This result is consistent with some studies , 32.A comparison of pain scores between 6 and 12 hours showed that the percent of patients that recorded no pain in the methylprednisolone group is more than that in the dexamethasone group. It could be because of its shorter half-life when compared with dexamethasone. Methylprednisolone requires less time to diffuse across cell membranes and alter gene transcription. Such an effect could not be observed at other time points. This could be explained by the fact that the intensity of postoperative endodontic pain decreased with time .This study also has some limitations, such as sample size and lack of placebo group. Future randomized clinical trials with placebo group are required to obtain more accurate results.There was no significant difference between infiltrated injections of dexamethasone and methylprednisolone in reducing pain after the endodontic treatment in necrotic teeth. Therefore, the use of any of these drugs to reduce pain after endodontic treatment is recommended.