Polyneuropathy (PNP) is a term to describe diseases of the peripheral nervous system, 50% of which present with neuropathic pain. In some types of PNP, pain is restricted to the skin distally in the leg, suggesting a local regulatory process leading to pain. In this study, we proposed a pro-inflammatory pathway mediated by NF-κB that might be involved in the development of pain in patients with painful PNP. To test this hypothesis, we have collected nerve and skin samples from patients with different etiologies and levels of pain. We performed RT-qPCR to analyze the gene expression of the proposed inflammatory pathway components in sural nerve and in distal and proximal skin samples. In sural nerve, we showed a correlation of TLR4 and TNFα to neuropathic pain, and an upregulation of TNFα in patients with severe pain. Patients with an inflammatory PNP also presented a lower expression of TRPV1 and SIRT1. In distal skin, we found a reduced expression of TLR4 and miR-146-5p, in comparison to proximal skin. Our findings thus support our hypothesis of local inflammatory processes involved in pain in PNP, and further show disturbed anti-inflammatory pathways involving TRPV1 and SIRT1 in inflammatory PNP. Polyneuropathy (PNP) is a term to describe diseases of the peripheral nervous system. This can include dysfunction of the motor, sensory, or autonomic system, leading to symptoms such as weakness, numbness, paresthesia, and pain . While aMany neuropathies are accompanied by inflammation, even if they do not belong to the classic immune mediated disorders ,6. ImmunIn PNP, several clinical studies point towards an enhanced systemic inflammation associated with the development of the neuropathy. These studies, nevertheless, have shown a high variability of results, suggesting that the analysis of local inflammatory components might be more valuable ,12,13,14This local inflammatory process might be triggered by the activation of toll-like receptor (TLR)-mediated pathways. In particular, stimulation of TLR4 results in the release of pro-inflammatory cytokines, such as TNFα, through the upregulation of an NF-κB-mediated cascade . In turnFurthermore, small fragments of RNA, or microRNAs (miR), can regulate the expression of different inflammatory mediators. For instance, miR-146a-5p, miR-132-3p, and miR-155-5p are able to modulate the NF-κB mediated cascade and have been found altered in patients with neuropathic pain ,25.PNP symptoms are usually length-dependent and more severe in the extremities . A localHere, we hypothesized that a local pro-inflammatory environment is involved in the severity of the disease and the development of neuropathic pain in PNP. To test our hypothesis, we studied the gene expression of TLR4, TRPV1, TNFα, SIRT1, miR-146a-5p, miR-132-3p, and miR-155-5p, in sural nerve, and in proximal and distal skin from patients with PNP. We correlated their expression to different neuropathy scores, disease severity, and levels of pain. Our results highlight the importance of the immune balance in the development of neuropathic symptoms.Diagnostic subgroups and clinical characteristics are summarized in Patients were further divided according to their levels of pain on the numerical rating scale (NRS): No pain , mild pain , and severe pain . At the time of inclusion, 10 patients had been treated for their neuropathy with intravenous immunoglobulin therapy (IVIG) within the last 6 months, while 56 had not received treatment.While all patients received a sural nerve biopsy, in 33 out of 66 patients, a biopsy of skin from the lower leg  and the upper thigh  was also collected. The analysis of the intraepidermal nerve fiber density (IENFD) showed a median of 2.1 fibers/mm (range 0.0–12.3) in the distal and 7.0 fibers/mm (0.0–18.0) in the proximal skin.p < 0.001). Patients with this treatment also presented a downregulation of miR-155-5p (p < 0.01). The effects of IVIG treatment on each of these markers were confirmed with the 95% confidence intervals  or mild pain (p < 0.01) (p < 0.001) (p < 0.05) (p < 0.05)  and between patients with an inflammatory and non-inflammatory neuropathy showed no differences in the expression of TLR4 A, and th < 0.01) B. Althou< 0.001) F. Furthe < 0.05) C and SIR < 0.05) D in compThirty-three PNP patients out of the sixty-six included in the study received a skin punch biopsy at a proximal (upper thigh) and distal (lower leg) region, where the same inflammatory markers were analyzed. In this case, patients with IVIG treatment did not show any differential expression of the studied components in comparison to those without treatment B,C. Therp < 0.05) . In proximal skin, an increased expression of miR-146-5p was observed in patients with severe pain, in comparison to those with mild pain ( < 0.05) E, while  < 0.05) A, TRPV1  < 0.05) C, TNFα ( < 0.05) B, SIRT1  < 0.05) D, miR-13 < 0.05) E.p < 0.05) and miR-146-5p (p < 0.05) in distal skin in comparison to proximal, specifically in those patients with mild or severe pain  and painless  regions within the same patient. Our results showed a downregulation in the expression of TLR4  (p < 0.01) (Comparison between patients with an inflammatory and non-inflammatory neuropathy did not show any differences in the expression of the studied inflammatory markers . Paired  < 0.01) E, in all < 0.01) A.p < 0.05)  B.p < 0.05) and SIRT1 (p < 0.0001) correlated between both regions, suggesting a systemic modulation of their expression.Moreover, the correlation between distal and proximal expression of each inflammatory component was analyzed to determine the systemic aspect of their role in PNP. Our analysis showed that the expression of TNFα (p < 0.05) and TNFα (p < 0.001). Furthermore, miR-132-3p expression correlated with TCNS (p < 0.01) and MRC (p < 0.05), and negatively with NRS (p < 0.01) , the overall disability sum score (ODSS), and the Medical Research Council (MRC)-sumscore, while pain was specifically evaluated with NRS. In order to determine the involvement of the inflammatory markers in the development of the neuropathy and of neuropathic pain, these neuropathy scales were correlated with the analyzed markers in sural nerve and distal and proximal skin. Our analysis of sural nerve showed that NRS correlated positively with both TLR4 ( < 0.01) A.p < 0.05) (p < 0.05) and miR-155-5p (p < 0.05)  B, while  < 0.05) C. None oIn this study, we analyzed the gene expression of different inflammatory components of the NF-κB pathway in sural nerve and proximal and distal skin from patients with PNP. With this analysis, we aimed to elucidate whether a local pro-inflammatory environment mediated by NF-κB may determine the severity of the disease and the presence of neuropathic pain.The NF-κB-mediated pathway can be activated by the stimulation of TLR4 with a damage-associated molecular pattern (DAMP), resulting in the release of pro-inflammatory cytokines such as TNFα ,27,28,29Since TLRs are expressed in most cell types, fibroblasts and Schwann cells that are supporting the nerve fibers might be involved in the upregulation of this cascade. However, fibroblasts and Schwann cells express low levels of TLR4  and TNFα . Myeloid cells on the other hand, such as monocytes, macrophages, or T-cells, express higher levels of TLR4  and TNFα  in order to respond against stimuli ,31,32. OThese results also confirm the previous literature that describes TNFα as closely related to many neuropathies and, in particular, to neuropathic pain. Not only has TNFα been reported upregulated in the peripheral and central nervous system in animal pain models, but its administration has also reproduced pain hypersensitivity. The mechanisms in which TNFα may induce neuropathic pain are plenty and very variable. One of the proposed mechanisms is through the regulation of cation channels, leading to sensitization of primary afferents, neuronal excitability, and repetitive firing. Another suggested mechanism would be through the activation of NF-κB, p38 Mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinases (JNK) pathways via TNF receptor 1 (TNFR1), promoting pro-inflammatory loops and initiating apoptotic processes that contribute to nerve degeneration and pain ,33,34.In addition, we found a close relation between TRPV1 and SIRT1, both being downregulated in sural nerve from patients with inflammatory PNP. SIRT1 is able to deacetylate NF-κB, and its downregulation would imply an upregulation of the NF-κB pathway, explaining the high expression of TNFα . While tSince PNP symptoms are usually length dependent and more severe in the extremities, the analysis of regions of skin distally located can provide clarity on the inflammatory components associated with more severe and painful neuropathic symptoms . Our resFurthermore, we found that the expression of SIRT1, as well as TNFα, correlated positively between distal and proximal regions of skin, suggesting a systemic regulation of these markers, and highlighting their involvement in systemic inflammation. Moreover, the current literature has reported involvement of SIRT1 in systemic processes such as ageing, metabolism, diet, glucose tolerance, or immune disorders ,53,54,55While our study includes some of the main inflammatory components of the NF-κB pathway, a broader analysis might help elucidate other pathways and mediators that might be involved in PNP. Furthermore, though sural nerve cannot be obtained from healthy individuals, skin biopsies could be performed to include a control group. This may clarify the role of the studied inflammatory components in the onset and worsening of the neuropathy. In addition, the analysis of the protein levels of these inflammatory mediators might shed light on the post-translational regulations that they might encounter and the inter-modulatory activities affecting the NF-κB pathway. Lastly, our results are limited by the well characterized but low number of recruited patients and the variance in etiologies. A bigger cohort might yield clearer results.At the moment, first-line treatments of neuropathic pain are based on the use of antidepressant and antiepileptic drugs, while opioids are recommended as second- and third-line treatment. These therapies are ineffective in many patients or inappropriate for specific painful neuropathies. Many current efforts are being made into identifying new targets to develop novel pharmaceutical agents ,5. Our sWe conclude that stimulation of TLR4 by endogenous ligands in large nerve trunks may result in the upregulation of TNFα, via the activation of the NF-κB pathway, leading to the development of severe neuropathic pain in patients with PNP. Furthermore, we propose that the activation of the NF-κB pathway in large nerve fibers, through the downregulation of SIRT1 and TRPV1, might be involved in the pathophysiology of inflammatory neuropathies. From our results in skin, we cannot resolve the involvement of the NF-κB pathway in the degeneration of nerve fiber endings and the development of localized neuropathic pain. Further analysis of this and other inflammatory pathways may help elucidate the role of inflammation in the skin of patients with PNP. We believe that the study of the NF-κB pathway in large nerve fibers can be an additional indicator of the severity of PNP and improve patient stratification, while a follow-up study may promote the discovery of predictive biomarkers.Between 2017 and 2018, 66 patients with PNP that came to the Department of Neurology, University of Würzburg, Germany for diagnostic work-up, were recruited for this study. Patients were informed about the aim in which their tissue was going to be used and gave written informed consent prior to inclusion. This project was approved by the Würzburg Medical Faculty Ethics Committee (# 230/15).Patients were diagnosed based on neurological examinations, laboratory studies, and nerve conduction examinations. The severity of the neuropathy was determined using standardized scales, including the modified Toronto clinical neuropathy score (mTCNS), the overall disability sum score (ODSS), and the Medical Research Council-sumscore (MRC-sumscore). Pain was evaluated by a numerical rating scale (NRS) from 0 (no pain) to 10 (worst pain).From each patient, a biopsy of sural nerve was collected and embedded in RNA-later over night at 4 °C for gene expression analysis. The RNA-later was removed the next day and the samples were preserved at −80 °C until further analysis. From 33 out of 66 patients, a biopsy of skin from the lower leg and the upper thigh was collected following the same protocol.g at 4 °C in order to separate the aqueous phase (containing the RNA), the organic phase (containing proteins and lipids), and the interphase formed by DNA. The upper phase was collected in a column tube and RNA was cleansed through several centrifugation steps, upon addition of ethanol (EtOH), RWT buffer, and RPE buffer, while discarding the supernatant in every step. Lastly, RNA was retrieved by pipetting 33 µL of RNase-free water onto the membrane of the column and centrifuging the tubes for 1 min at 8000× g. Samples were stored at −80 °C after the RNA quality and quantity was assessed with a NanoDrop™ One .Purification of total RNA including miRNA was performed using the miRNeasy mini kit , following the manufacturer’s protocol. In summary, samples were homogenized with 700 µL of Qiazol lysis reagent in a homogenizer disperser. After incubation with 140 µL of chloroform, samples were centrifuged for 15 min at 12,000× 2, 20 μL deoxyribonucleoside triphosphate, 6.25 μL multiscribe reverse transcriptase, and 2 μL RNase inhibitor was added per sample. Lastly, reaction was performed under these conditions: annealing , reverse transcription , and enzyme inactivation .The cDNA synthesis from mRNA was carried out with TaqMan Reverse Transcription reagents . First, 250 ng mRNA of each sample was pre-incubated with 5 µL random hexamer at 85 °C for 3 min. Next, 10 μL 10× PCR buffer, 22 μL MgClFor miRNA, reverse transcription was performed with the miRCURY LNA RT Kit . For each sample, 10 ng of RNA was mixed with 2 μL of 5x reaction buffer, 5 μL of nuclease free water, and 1 μL of enzyme mix. Reaction was performed using the following program: reverse transcription  and enzyme deactivation .Reactions were carried out on a PRISM 7700 Cycler  and transcribed cDNA was stored at −20 °C until further analysis.Real-time qPCR of mRNA and miRNA targets was performed to analyze gene expression using the StepOnePlus Real-Time PCR System . For mRNA, RT-qPCR was carried out with TaqMan qRT-PCR reagents  and pre-designed assays. For target normalization, experience from our group showed that the ribosomal protein L13a (RPL13A) could act as a good housekeeping gene. Based on its comparability and standard deviation across groups and samples, our tests showed that RPL13A could be selected as suitable endogenous control. For each reaction, 3.5 µL cDNA was mixed with 0.5 µL nuclease free water, 5 µL Fast Advanced Mastermix, and 0.5 µL TBP primer and 0.5 µL target primer  and pre-designed miRCURY LNA miR PCR assays  were used. Based on previous experience from our group ,12, the Each mRNA and miRNA was amplified in triplicates and relative quantitation (RQ) values were obtained by the StepOnePlus™ Software v2.3  using interplate calibrators through the ∆∆Ct method.t-test was used for comparison between two groups, and One-Way ANOVA for three groups. In non-parametric data, the Mann–Whitney U Test was applied for comparison of two groups, and Kruskal–Wallis for three groups. The Spearman test was used for correlations between data groups. Comparisons between distal and proximal skin were analyzed through dependent-samples Wilcoxon signed-rank test. Data results were plotted in GraphPad Prism 9  for visualization.Statistical analysis was performed in SPSS 27 , where the Shapiro–Wilk test was used to determine the normal distribution of the data. For parametric data, a


In assessing and managing pain, when obtaining a self-report is impossible, therapeutic decision-making becomes more challenging. This study aimed to investigate whether monocytes and some membrane monocyte proteins, identified as a cluster of differentiation (CD), could be potential non-invasive peripheral biomarkers in identifying and characterizing pain in patients with severe dementia. We used 53 blood samples from non-oncological palliative patients, 44 patients with pain (38 of whom had dementia) and 0 without pain or dementia (controls). We evaluated the levels of monocytes and their subtypes, including classic, intermediate, and non-classic, and characterized the levels of specific phenotypic markers, namely CD11c, CD86, CD163, and CD206. We found that the relative concentrations of monocytes, particularly the percentage of classic monocytes, may be a helpful pain biomarker. Furthermore, the CD11c expression levels were significantly higher in patients with mixed pain, while CD163 and CD206 expression levels were significantly higher in patients with nociceptive pain. These findings suggest that the levels of monocytes, particularly the classic subtype, and their phenotype markers CD11c, CD163, and CD206 could serve as pain biomarkers in patients with severe dementia. Pain is a multidimensional experience that can significantly impair a patient’s quality of life. It can be classified according to the underlying cause, including nociceptive pain resulting from tissue damage, neuropathic pain caused by nerve injury, and nociplastic pain resulting from altered pain modulation . As multPatient self-reporting is regularly used to characterize pain. However, it is subject to individual interpretation, which can be a problem for proper pain characterization. To overcome this subjectivity, multidimensional hetero-assessment scales have been developed, particularly for patients with dementia or others unable to characterize their pain ,4,5. HowRoutine peripheral blood parameters, including white blood cell counts and mean platelet volume (MPV), have been identified as potential diagnostic, prognostic, and predictive response markers in inflammatory and central nervous system diseases, such as cerebral hemorrhage and dementia in elderly patients . FurtherMonocytes are a type of white blood cell crucial in promoting and resolving inflammation and are implicated in several inflammatory diseases, such as cardiovascular diseases, diabetes, obesity, and metabolic syndrome ,10. TherThis study aims to assess the levels of monocyte subsets  ,16 and tWe initially selected 95 patients. However, the legal representatives of five patients refused to participate in this study. Seventeen patients were excluded from blood sample collection due to their fragile condition, hypovolemia, and difficult venous access. Also, 20 patients were in their last days of life, and we decided to avoid blood collection in this clinical condition.We obtained blood samples from 53 patients with an average age of 74.8 years old, a minimum of 29, and a maximum of 98 years old. Most of the patients were female [n = 39 (73.6%)]. Nine patients had no pain, and forty-four suffered from chronic pain. We had no patients diagnosed with autoimmune diseases such as inflammatory bowel disease, multiple sclerosis, psoriasis, or other conditions.Of the 44 patients with chronic pain, 38 also had severe dementia, making assessing their pain intensity and type difficult. As a result, we relied on the PAINAD scale to identify patients who likely had uncontrolled pain. Patients with PAINAD ≥ 5 (present in 32 patients) are those who probably have uncontrolled pain . Among tWe had six patients with pain without dementia and nine patients without pain and without dementia that were considered controls, as shown in p = 0.025), in particular in the classic monocytes , and we saw a decrease in the intermediate  and non-classic monocytes  compared with patients without pain . On the other hand, we detected, in these patients, a decrease in the expression levels of CD206 , CD163 , and in the ratio of CD163/CD206  in monocytes .When we evaluated, in each monocyte subset, the expression of CD11c, CD163, and CD206, we did not find any statistical differences in these markers in intermediate and non-classic monocytes between patients with and without pain. However, in total and classic monocytes, we found statistically significant differences in CD206 and CD163, when comparing these biomarkers with the presence of pain, as shown in onocytes . In clasNo differences between controlled and uncontrolled pain patients were observed in the studied biomarkers.p = 0.026 and 28.37 vs. 20.1 MFI, p = 0.047).When comparing the biomarkers and the gender of patients, we found significant differences between the percentage of cells expressing CD86 and the ratio CD11c/CD86, which have higher values in men  than in patients with mixed pain.As presented in However, we found statistically significant differences when comparing types of pain and CD11c, CD163, and CD206.The percentage of monocytes expressing CD11c is significantly higher in patients with mixed pain, while in patients with nociceptive pain, we observed an increase in the expression levels of CD163 and CD206 and the ratio CD163/CD206.Concerning the monocyte subpopulations, there is an increase in the expression levels of CD11c in all subpopulations of monocytes in patients with mixed pain compared with those with nociceptive pain, in which the percentage of monocyte expression of this receptor is only changed in classic and intermediate monocytes . Moreovep = 0.016; intermediate—575.94 vs. 517.74 MFI, p = 0.041), while CD163 is only changed in classic monocytes.In patients with nociceptive pain, the expression levels of CD206 are significantly higher in classical and intermediate monocytes compared with patients with mixed pain , with the area under the curve being statistically significant, as is shown in We also analyzed if the treatment with opioids and paracetamol interfered with the monocyte biomarkers. Consequently, we conducted a comparison of monocyte biomarker levels between patients who were receiving treatment with opioids and paracetamol and those who were not. Our findings indicated that CD163 was the sole biomarker with statistical significance. In patients receiving opioid therapy, the percentage of monocytes expressing CD163 was lower than those not receiving opioids , as shown in In order to correlate the phenotypic markers in monocytes with dementia, we analyzed the levels of different monocyte subsets and CD in patients with and without dementia of different subtypes.p = 0.037). Notably, patients with vascular dementia exhibited the lowest levels of relative concentrations of monocytes.As shown in p = 0.032; CD206—562.85 vs. 445.58 MFI, p = 0.001; CD163/CD206—9.2 vs. 4.1, p = 0.01). Moreover, the classic and intermediate monocytes of patients with dementia show a significant decrease in the percentage of monocytes expressing CD163, while the decrease in the expression levels of CD206 is mainly observed in classic and non-classic monocytes, when compared with patients without dementia   .p = 0.028 and 465.70 vs. MFI 419.85, p = 0.007, respectively).When comparing biomarkers with types of dementia, there are statistically significant differences, particularly in the relative concentration of monocytes, as previously mentioned, and in the expression levels of CD206, which are highest in patients with vascular dementia  in the expression of CD206 in non-classical monocytes among patients with vascular dementia (MFI 561.1), whereas the lowest expression was observed in patients with Alzheimer’s disease (MFI 482.0). Our findings showed a significant decrease (p = 0.049) in the expression levels of CD86 (MFI 579.3) in classical monocytes of Alzheimer’s disease patients compared to those with other dementias (MFI 597.1). Moreover, the relative percentage of CD163 was significantly higher in Alzheimer’s patients than in those patients with vascular dementia .When we analyzed the different subsets of monocytes in patients with different types of dementia, in intermediate monocytes, we did not observe significant differences among Alzheimer’s disease, vascular, and other types of dementia. Conversely, we observed a significant increase  and the Faculty of Medicine (FMUP) Ethics Committee accepted the research procedures. The research was conducted following the principles of the Declaration of Helsinki. In compliance with ethical guidelines on confidentiality, data anonymity, and voluntary withdrawal, the participants or their representatives provided informed consent before study enrollment.We collected individual and clinical data by reviewing the records in the patient’s clinical files, which were then saved in a secure Excel sheet . Each paWe also obtained information regarding the diagnosis and type of dementia. In cases where patients had severe dementia, we utilized the Pain Assessment in Advanced Dementia Scale (PAINAD)  to diffeTo analyze the expression of monocyte biomarkers, we selected CD11c , CD86 2, CD163  and CD20We collected peripheral blood samples in EDTA tubes to analyze the monocytes. Monocytes were classified into three subsets based on the expression levels of CD14 and CD16. These subsets are the classical monocytes (CD14+CD16−), non-classical monocytes (CD14−CD16+), and intermediate monocytes (CD14+CD16+) . FollowiThe monocyte analysis was performed in peripheral blood samples collected in EDTA tubes to identify the monocyte subsets and characterize the transmembrane protein receptors related to its recognized functions.+CD16−), non-classical (CD14−CD16+), and intermediate (CD14+CD16+) monocytes. In each monocyte subset, the expressions of CD11c, CD163, and anti-CD206 were determined.We stained 200 µL of whole blood with the following monoclonal antibodies (mAbs): anti-CD14 BD Pharmingen™ (APC), anti-CD16 BD Horizon™ (BV500) mAbs; anti-CD11c BD Pharmingen™ (FITC), anti-CD163 BD Pharmingen™ (PE), anti-CD86 BD Pharmingen™ (PerCP-Cy5.5), and anti-CD206 BD Horizon™ (BV421). Following a 15 min incubation in the dark at room temperature, erythrocytes were lysed using a BD. Pharm Lyse™ reagent according to the manufacturer’s protocol. Cells were run through a FACS Canto II flow cytometer (BD Biosystems), and at least 100,000 events were collected using FACS DIVA software (BD Biosystems). Data were studied with the Kaluza Software (Beckman Coulter), and the results were expressed in a percentage of positive cells for each marker and respective mean fluorescence intensity (MFI). Each marker was analyzed in the total monocyte population (CD14+). Additionally, a sub-analysis was performed based on monocyte classification, using CD14 and CD16 expression, into classical  for statistical analysis. Descriptive statistics measures such as absolute and relative frequencies, means, and standard deviations were employed for data analysis. Additionally, inferential statistics were also utilized. We set the level of significance (α) at ≤0.05 to reject the null hypothesis. The Student’s We incorporated several variables into the analysis, including sex, age, type and intensity of pain, opioid type and dosage, and other analgesics like nonsteroidal anti-inflammatory drugs (NSAIDs) and paracetamol, as well as absolute (MFI) and relative concentration levels of CD11c, CD86, CD163, and CD206. In our analysis, we factored in the type of dementia, since it was prevalent among most patients, and controlled pain time for patients presenting with chronic pain but whose pain was not present during blood collection. In cases where patients had severe dementia, we relied on the Pain Assessment in Advanced Dementia Scale (PAINAD)  to identDue to a lack of evidence in published clinical data regarding this issue, it is not possible to firmly assume the size of the changes . We assuOur study involved patients followed by a specialized palliative care team in the northern region of Portugal with non-oncological illnesses. As previously stated, we only included patients who provided informed consent directly or through their legal representatives.It is difficult to assess pain in patients receiving palliative or end-of-life care or those with severe dementia, cognitive impairment, or speech disorders, as we cannot solely rely on self-reporting ,4.Physical symptoms such as pain, fatigue, and sleep disturbances and psychosocial factors such as stress, coping style, and the availability of emotional support interact in a bidirectional manner and are present at varying levels. These dynamics create a highly personalized disease experience that calls for an individualized, multimodal therapeutic approach ,33. ConsSeveral preclinical and clinical studies have investigated the hypothesis that biomarkers can be utilized to identify and quantify pain. A preclinical study shows that inflammatory and neuropathic pain have different biomarkers . FurtherPreviously, many routine peripheral blood parameters have been used as peripheral novel diagnostic, prognostic, and predictive response markers in several diseases ,9,35,36,This study assesses whether monocytes, their subtypes, and membrane proteins, such as CD11c, CD86, CD163, and CD206, could serve as non-invasive peripheral biomarkers for identifying and characterizing pain in patients with severe dementia. Our findings suggest that the relative concentrations of monocytes, specifically the percentage of classic monocytes, may serve as valuable biomarkers for pain, irrespective of sex, the presence of dementia, and the type of pain. Moreover, the most evident alterations were found in the levels of CD11c, CD163, and CD206 in classic monocytes, which may contribute to characterizing the pain subtype. We did not detect any changes in CD86 expression related to pain and dementia.CD11c is a complement receptor often exploited as a single marker to track murine dendritic cells . CD86 isThis study demonstrated that individuals experiencing pain had higher levels of CD206 and classical monocytes and a lower CD163/CD206 ratio, suggesting the potential use of these parameters as biomarkers for pain. They are independent of gender, dementia, and pain treatment. In fact, in patients with pain, we found a significant increase in the percentage of classic monocytes and the percentage of these cells expressing CD206 and a decrease in the ratio of CD163/CD206 compared to patients without pain. None of these parameters varied with sex, dementia, or treatment with paracetamol and opioids. If these findings are validated in future studies involving a larger number of patients, they should be incorporated into the pain identification process in the peripheral blood of palliative patients with dementia. For patients with well-controlled pain, in comparison with those with non-controlled pain, we did not find statistically significant differences, including in patients with well-controlled pain for seven or more days. These data are relevant because we could expect a reduction in monocyte activation with pain control ,35,37. HDespite the limited sample size, which precluded the assessment of pain types other than nociceptive and mixed pain, we could identify monocyte biomarkers associated with certain types of pain. The levels of CD11c are significantly higher in patients with mixed pain, while the expression levels of CD163 and CD206 are significantly higher in patients with nociceptive pain, with the latter being independent of dementia, as these patients have lower levels of these markers.Another interesting finding is that the percentage of CD163 in intermediate monocytes showed less expression in patients receiving opioid treatment. Freshly isolated peripheral blood monocytes express a relatively low level of CD163, but this expression increases with the differentiation of such cells into macrophages , a role Monocytes can be used as predictors of dementia, as the relative concentration of total monocytes is higher in patients with dementia, particularly in Alzheimer’s disease and vascular dementia compared with non-demented controls. These findings are in agreement with others ,44, and Additionally, we did not observe any differences in the levels of monocyte subtypes, contrasting with the findings reported by other authors . HoweverFurthermore, the decreased expression levels of CD86 and CD206 could be potential markers of Alzheimer’s disease and vascular dementia, respectively . ConversTaken together, these findings suggest that the levels of specific monocyte subtypes, namely the classic subtype, along with their associated CD11c, CD163, and CD206 phenotypes may hold value as objective pain biomarkers for palliative patients with dementia. This is a significant advantage as these biomarkers can be quantified without requiring active patient involvement.The main limitation of our cross-sectional study is the small sample size, which limits the formation of even groups. In this sense, a confirmatory study in a larger cohort of patients should be performed in order to confirm our findings. Additionally, the fact that the pain observed in severe dementia patients can result from different causes and mechanisms, and the fact that patients with pain do not respond similarly to the same treatment may also increase data dispersion. Also, a subtype of dementia may be partially linked to a specific mechanism of neuroinflammation, and therefore this could influence the results ,45,52,53To our knowledge, this is one of the most extensive pain biomarker studies conducted to date and the only study to compare patients with non-oncological pain using specific monocyte biomarkers. Nevertheless, given the small sample size, further studies are warranted to confirm the viability of these markers as indicators of pain and dementia.For vulnerable and dependent patients who cannot provide a self-report of symptoms, identifying pain biomarkers, such as the ones presented in this study, can aid in adjusting therapeutic strategies in line with the WHO ladder , contrib


Sensory profiles of 1090 patients reveal loss of small thermosensory fibre function to be associated with paradoxical heat sensation. Paradoxical heat sensation (PHS) is the perception of warmth when the skin is cooled. Paradoxical heat sensation rarely occurs in healthy individuals but more frequently in patients suffering from lesions or disease of the peripheral or central nervous system. To further understand mechanisms and epidemiology of PHS, we evaluated the occurrence of PHS in relation to disease aetiology, pain levels, quantitative sensory testing parameters, and Neuropathic Pain Symptom Inventory (NPSI) items in patients with nervous system lesions. Data of 1090 patients, including NPSI scores from 404 patients, were included in the analysis. We tested 11 quantitative sensory testing parameters for thermal and mechanical detection and pain thresholds, and 10 NPSI items in a multivariate generalised linear model with PHS, aetiology, and pain (yes or no) as fixed effects. In total, 30% of the neuropathic patients reported PHS in contrast to 2% of healthy individuals. The frequency of PHS was not linked to the presence or intensity of pain. Paradoxical heat sensation was more frequent in patients living with polyneuropathy compared with central or unilateral peripheral nerve lesions. Patients who reported PHS demonstrated significantly lower sensitivity to thermal perception, with lower sensitivity to normally painful heat and cold stimuli. Neuropathic Pain Symptom Inventory scores were lower for burning and electric shock–like pain quality for patients with PHS. Our findings suggest that PHS is associated with loss of small thermosensory fibre function normally involved in cold and warm perception. Clinically, presence of PHS could help screening for loss of small fibre function as it is straightforward to measure or self-reported by patients. Typically, single cold stimuli are perceived accurately, whereas PHS is experienced during temporal alternation of warming and cooling on the same area of the skin. Paradoxical heat sensation is rare in healthy individuals but can be provoked by preheating to noxious temperatures,39 These results suggest that the paradoxical sensation of heat and burning elicited by cold stimuli is mediated by unmyelinated fibres  that are not affected by the compression. This interpretation is consistent with conduction velocity findings demonstrating that PHS is peripherally transmitted through C fibres.30 Mechanistic hypotheses have suggested that PHS is mediated through a specific class of C fibres, known as C2, normally responsive to cooling, heating, and menthol in humans6 or by disinhibition of the heat–pinch–cold (HPC) pathway in the peripheral or central nervous system.14 The HPC disinhibition model is a common explanation of perceptual changes with high similarity to PHS, such as the thermal grill illusion or A-fibre block.39Altered sensation such as PHS can be evoked in healthy individuals by applying nerve compression blocks. Classic studies showed that when the conduction of myelinated fibres is blocked by ischaemic or nerve compression, pain induced by single cold stimuli is perceived at higher temperatures and is typically described as burning hot.19However, these human experimental models are generally linked to a painful, burning heat, rather than painless warmth as mostly reported for PHS as perceived by patients. In this article, we analysed PHS in relation to aetiology (the specific injury or disease of the somatosensory system), presence or absence of pain, somatosensory profiles, descriptors of spontaneous pain, and abnormal sensations in 1090 patients with painful or painless polyneuropathy, unilateral peripheral nerve damage, or central lesions. Patients were part of the prospectively collected (N) European database and published previously in part.The objective of this study was to gain a better understanding of the clinical relevance of PHS in neuropathy by evaluating the relationship between paradoxical heat sensations, aetiology, presence or absence of pain, somatosensory profile, and pain symptoms. Furthermore, we aimed to explore putative mechanisms of this paradoxical phenomenon.11 Finally, we analysed whether PHS could be related to specific neuropathic pain symptoms as captured by the Neuropathic Pain Symptom Inventory.First, we compared the frequency of PHS across patient populations. Second, we tested whether an increase in PHS is associated with changes in the somatosensory profile, especially regarding sensory loss of cold and warm detection, combined with sensory gain of cold pain thresholds, equating PHS to the paradoxical sensations induced by an experimental A-fibre block.The study was approved by the local ethics committees at each participating centre. All patients have given written informed consent for their data to be collected and analysed in the database. All procedures were conducted in accordance with the Declaration of Helsinki.26 during which presence or absence of paradoxical heat sensation is recorded. Patients filled in the questionnaire before assessment. Fifteen pain facilities across Europe participated in gathering data in 3 consortia (German Research Network on Neuropathic Pain (DFNS), IMI (Innovative Medicines Initiative) EUROPAIN, and NEUROPAIN). All centres underwent a strict quality assessment.37 Data were collected in a central database in Bochum, Germany. An analysis of heterogeneity between the participating centres showed a high degree of homogeneity between the different centres, making it possible to analyse the database as a homogenous group.33 Subsets of this database as well as inclusion and exclusion have been published previously.36All patients were tested in a single setting, using the QST protocol of the German Research Network on Neuropathic Pain (DFNS),31 due to polyneuropathy or unilateral peripheral lesions ; patients with central neuropathic pain due to spinal cord injury, syringomyelia, or stroke; and patients with similar painless conditions.19 Exclusion criteria were age < 18 years, insufficient language skills or other communication problems, pain treatment by topical local anaesthetics for ≥7 days in the last 4 months or by topical capsaicin in the last 6 months, comorbidities treated by anticonvulsants or antidepressants, other pain locations with pain intensities ≥6/10 on ≥15 days/month, spinal canal stenosis, peripheral vascular disease (Fontaine stage II or higher), pending litigation, and major cognitive or psychiatric disorders. Patients who were unable to detect any temperature change during thermal sensory limen (TSL) due to extensive sensory loss to thermal stimuli were excluded because PHS could not be determined (see below). We also excluded patients with unilateral syndromes who were also affected by contralateral neuropathy or painful conditions of the contralateral limb, as well as patients with incomplete records .We included patients with confirmed peripheral neuropathic pain26 Patients suffering from polyneuropathy were tested on the dorsum of the foot, whereas patients suffering from other aetiologies were tested on the most affected area. In addition to PHS, QST according to the DFNS protocol assesses 12 other parameters: cold and warm detection thresholds (CDT and WDT), thermal sensory limen, cold and heat pain thresholds (CPT and HPT), mechanical pain threshold and sensitivity (MPT and MPS), dynamic mechanical allodynia (DMA), pressure pain threshold (PPT), wind-up ratio (WUR), tactile  detection threshold (MDT), and vibration detection threshold.25 Thermal detection and pain thresholds were measured using either a TSA 2001-II  or a MSA  thermode at a rate of temperature change of 1°C/second.25 The number of PHS (if any) was recorded during assessment of the TSL. In this task, patients were instructed that the testing device would either cool or warm their skin several times, and they should press a stop button every time they felt a new temperature change. Furthermore, patients were instructed to describe the quality of the temperature change  after each button press. This procedure consisted of alternated warming and cooling of the skin for a total of 6 consecutive temperature changes. The TSL procedure always started with warming of the skin, but no preheating to noxious temperatures was applied.13 The number of PHSs  varied between 0 and 3. According to the DFNS normative data, if patients were female and older than 50 years or male and older than 40 years and they were tested on the feet, abnormal frequency of PHS is defined as at least 2 PHSs during the 3 cooling temperature changes. In younger ages, any number of PHSs was considered abnormal. Henceforth, we will use the term “no PHS” to refer to “no elevated reports of PHS compared with normative data.” Patients who were unable to detect any temperature change during TSL were excluded because PHS could not be determined.We used the QST protocol established by the DFNS.25 Quantitative sensory testing raw or log-transformed results were normalised using a z-transformation, based on normative material defined for 5 different age decades, sex, and 4 testing sites . If the patient's most prominent pain was in an upper limb, the values were z-transformed using the reference data for hands; if the most painful area was at a lower limb, the values were z-transformed using the reference data for feet. Assessments at the torso were z-transformed using the reference data for trunk, whereas all assessments at the face were z-transformed using the reference values for the cheek. Vibration detection threshold was compared with normative data assessed at the ulnar styloid for hand and arm, malleolus for foot and leg, costal arch for trunk, and zygomatic process for face. As a consequence of normalization, all measures had a mean = 0 and a standard deviation = 1 and were comparable between patients irrespective of their demographics or affected body area . Abnormal values were defined as values beyond the 95% confidence interval, corresponding to z values <−1.96 or >1.96.An initial assessment of data from 180 healthy individuals in the DFNS reference database revealed that all parameters, except PHS and DMA, were normally distributed or could be transformed to a standard normal distribution using a log transformation.5 It comprises 10 items, including 5 descriptors of ongoing pain quality , 3 descriptors of evoked pain , and 2 reports of tingling and pins and needles. For each of the 10 items, patients rated the intensity on an 11-point rating scale, ranging from “zero or no [...]” to “10 or worst [...] imaginable.”The NPSI was developed to identify different dimensions of neuropathic pain.t-tests  to investigate whether PHS could discriminate differences between the groups of patients with different aetiologies or with and without pain. Using multivariate analyses, we tested 11 normally distributed QST parameters and 10 NPSI items, with PHS , aetiology , duration of disease , and presence or absence of pain as fixed main effects, and PHS × aetiology, PHS × duration, and PHS × pain as interaction effects. To avoid overinterpretation of false positive results due to alpha-cumulation and multiple testing, we applied a Benjamini–Hochberg correction to control the false discovery rate and only discuss effects that are significant models on a corrected P < 0.05 level.We applied unpaired All data and materials are in the database hosted by the German Research Network of Neuropathic Pain (DFNS) and accessible to the first author (J.V.). Data access for purpose of replication can be requested through submitting an informal data request to the DFNS and will be granted by the DFNS board after review.P < 0.001), within patients with unilateral peripheral lesion more likely to appear after a radicular lesion, and within patients with central lesion less likely to appear after a stroke. There were no differences between painful and nonpainful conditions and neither in sex, duration of disease, or aetiology of polyneuropathy.A total of 1090 patients from 15 European centres were included in the analysis . The NPSI was not collected in all centres, and only for patients with painful conditions, and therefore was available from 404 patients only . Patients' demographics are presented in Table 25 which found that only 2% of healthy participants reported PHS in lower limbs of older participants (P < 0.001). Paradoxical heat sensation was more frequent in polyneuropathy (42%) in comparison to unilateral peripheral nervous lesions (20%) and central nervous lesions (24%) (P < 0.001), whereas there was no difference between unilateral peripheral and central nervous lesions (P = 0.33). Regarding painful vs painless conditions, we found no differences in the frequency of PHS, neither overall (P = 0.67) nor within aetiologies .Abnormal PHS was reported by 30% of all included patients. Notably, although the percentage was higher in patients with loss of thermal detection (35%) compared with those with preserved thermal detection (29%), in absolute numbers, most patients with PHS had preserved thermal detection (n = 248) compared with those with loss of thermal detection (n = 82). This result is significantly different from the frequency of PHS in the DFNS reference data for healthy participants,Figure Neuropathic Pain Symptom Inventory profiles of all patient etiologies can be found in Figure In this large clinical data set of patients experiencing peripheral and central nervous system diseases with and without neuropathic pain, abnormal PHS was frequently reported by all patient groups irrespective of whether they suffered from painful or painless lesion or disease of the somatosensory system (30% each). We found that PHS was much more frequent in patients with polyneuropathy (39%-42%) compared with unilateral peripheral or central lesions. Paradoxical heat sensation was associated with reduced thermosensitive small fibre function. The presence of PHS was not related to mechanical detection or pain thresholds, suggesting that PHS is not a sign of a general, but rather a thermosensory-specific small fibre dysfunction. Finally, we found an association between the presence of abnormal PHS and lower NPSI scores for electric shock–like and burning pain quality.40 and patients with chemotherapy-induced neuropathy experiencing PHS had less warm sensitivity.32 These findings were not reproduced in our population, which covers polyneuropathy of diabetic, HIV, idiopathic, chemotherapy-induced, but not uremic aetiology. A previous study on diabetic children also did not find an association of PHS with sensory loss to cooling.4 These differences across studies may be related to symptoms severity. In our data, all patients with polyneuropathy already had major thermosensory deficits, suggesting potentially more severe polyneuropathy than in the patient populations investigated in other studies.Paradoxical heat sensation was significantly more frequent in polyneuropathy compared with other conditions with peripheral  or central lesions of the somatosensory system, with 42% of patients having abnormal increased frequency of PHS compared with healthy individuals. The QST profiles were different for patients with polyneuropathy compared with patients with other neuropathies. Patients with polyneuropathy showed on average a general sensory loss on all sensory parameters, whereas the other groups also exhibited sensory gain, for example, lower pressure pain thresholds. Interestingly, we found no differences between QST profiles of patients with polyneuropathy with and without PHS. This result is in contrast to previous findings indicating that patients with uremic polyneuropathy experiencing PHS also exhibited less cold sensitivity,39 that selective loss of cold detection is linked to PHS is difficult to assess in this study. However, our finding that PHS is related to functional losses in thermal, but not mechanical, modalities suggests that PHS reflects a specific deficit in the function of small fibres involved in thermosensation, rather than a general sensory deficit.In patients with unilateral nerve damage or central lesions, PHS was similarly frequent across groups and associated with thermal detection deficits. This result suggests that PHS reflects loss of both C-fibre and Aδ-fibre function or their associated pathways in the central nervous system, although the relative importance of individual pathways cannot be determined from this study because the nervous lesions in these patients are rarely selective for single thermal pathways. Thus, the prediction from experimental A-fibre block studies in healthy individuals17 and to be a result of a disinhibition of warm detection neurons caused by rarefication of cold-related fibres.7 In our analysis, spontaneous burning sensation reported by patients was lower for patients reporting PHS. Furthermore, PHS was associated with lower intensity of electric shock–like pain, which is considered to reflect small, myelinated fibre-related pain. These data suggest that impaired or unimpaired function of Aδ fibres and C fibres is relevant.In the NPSI ratings, the presence of burning pain was similar in frequency in patients with and without PHS. Burning sensations are believed to be associated with loss of small fibre function27 However, other methods to assess PHS have been described previously. Yosipovitch and colleagues applied a series of 8 alternating warming and cooling stimuli and considered patients reporting PHS as those individuals who reported at least 3 or 4 warming sensations during cooling of the skin.40 By contrast, in our protocol, patients reporting PHS were patients who experienced 1 or 2 of 3 warm sensations during cooling.In this study, PHS was assessed during 6 alternating warming and cooling stimuli, and in most cases, a single warm report during cooling of the skin was sufficient to classify the patient as reporting PHS. This has been a standard approach in the field in the past 20 years.1 because the battery of thermal and mechanical measures included in the QST according to the DFNS protocol only captures short-term effects , rather than long-term effects, which may be more closely related to spontaneous pain symptoms.Another limitation was the selection of the body site where QST was performed. This selection was not standardised for most patients' aetiologies, except for polyneuropathy. The body region for QST assessment was selected based on where the patient reported the most prominent pain or strongest somatosensory dysfunction in case of painless conditions. Consequently, while the tested body region could have been anywhere on the body, the reference values are only defined for hand, feet, trunk, and face. For each patient, we used reference values that were the closest approximation to the tested area. Finally, temporal and spatial summation effects may be underestimatedWe found that PHS was common not only in patients with polyneuropathy but also in patients with central lesions or unilateral peripheral injury. In patients with central or unilateral peripheral lesions, the presence of PHS was associated with reduced thermosensitive small fibre function, most likely including thinly myelinated Aδ fibres and unmyelinated C fibres. Our findings suggest that PHS is not a general sign of nerve damage, but rather a thermosensory-specific small fibre dysfunction, the mechanisms of which include a disinhibition phenomenon. In healthy subjects, the thermoreceptive and nociceptive circuitries include a pathway by which mild cooling can induce a percept of warmth or heat, and this circuitry is normally suppressed by thermosensory small fibre pathways. By contrast, our patient data indicate that the association between the presence of elevated PHS and lower NPSI scores for electric shock–like and burning pain quality plays an important role for Aδ than C fibres. Mechanistically, the inhibitory interactions between different small fibre pathways deserve more experimental studies; our findings suggest that there likely are additional inhibitory mechanisms distinct from the known gate control or the brainstem descending modulatory system.15 although at face value it seems to be a positive phenomenon. It may be an easy way to clinically screen for loss of small fibre function with a dichotomous outcome (PHS yes or no) and more sensitive than assessing abnormal thermal detection thresholds using DFNS reference values.Paradoxical heat sensation is an interesting sensory phenomenon that can be easily detected and is reported spontaneously by patients. This makes it an interesting parameter for clinical assessment of neuropathy. We argue that PHS should be included in the list of sensory signs of small fibre functional sensory loss,The authors declare that they have no competing interests.


Inguinal-related groin pain (IRGP) in athletes is a multifactorial condition, posing a therapeutic challenge. If conservative treatment fails, totally extraperitoneal (TEP) repair is effective in pain relief. Because there are only few long-term follow-up results available, this study was designed to evaluate effectiveness of TEP repair in IRGP-patients years after the initial procedure.Patients enrolled in the original, prospective cohort study (TEP-ID-study) were subjected to two telephone questionnaires. The TEP-ID-study demonstrated favorable outcomes after TEP repair for IRGP-patients after a median follow-up of 19 months. The questionnaires in the current study assessed different aspects, including, but not limited to pain, recurrence, new groin-related symptoms and physical functioning measured by the Copenhagen Hip and Groin Outcome Score (HAGOS). The primary outcome was pain during exercise on the numeric rating scale (NRS) at very long-term follow-up.p < 0.001). At 83 months follow-up, a median NRS of 0 was observed during exercise (IQR 0–2), which was significantly lower compared to earlier scores (p <0.01). Ten patients (36%) mentioned subjective recurrence of complaints, however, physical functioning improved on all HAGOS subscales (p <0.05).Out of 32 male participants in the TEP-ID-study, 28 patients (88%) were available with a median follow-up of 83 months (range: 69–95). Seventy-five percent of athletes were pain free during exercise (This study demonstrates the safety and effectivity of TEP repair in a prospective cohort of IRGP-athletes, for whom conservative treatment had failed, with a follow-up period of over 80 months. Long-standing groin pain in athletes is a common condition, with an estimated prevalence of 5–10% . DiffereMultidisciplinary evaluation and treatment are highly recommended in athletes with IRGP. Treatment generally starts with conservative measures, which consist of rest, analgesics and physical therapy , 6. In o®) in the preperitoneal space, a low incidence of chronic postoperative pain (CPIP) has been demonstrated in inguinal hernia surgery [As patients that suffer from IRGP are typically young, active and healthy, some controversy exists concerning the implantation of a permanent mesh in regard to chronic pain and foreign body feeling. When positioning a heavyweight polypropylene mesh  were approached by a written letter for additional follow-up , 10. TheInformed consent was newly acquired since the original study protocol did not include very long-term follow-up. The present study protocol was approved by the Medical Research Ethics Committees United (MEC-U) and approval was given by the local ethics board. This manuscript adheres to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines .Relevant patient data were collected retrospectively from the electronic patient file to acquire baseline characteristics, consisting of age, side of operated groin, recurrence, re-operation and other relevant postoperative sequelae directly related to the TEP repair. Patients who consented to participate in the present study were subjected to a single telephone conversation, during which two questionnaires were filled out. The first questionnaire was designed specifically for this study. The questions are illustrated in Table The primary outcome of the present study was pain during exercise, measured on the Numeric Rating Scale (NRS). This score was compared to the patients’ pain scores before treatment, 3- and 19 months after treatment, which were extracted from the TEP-ID-study. Additionally, scores were classified into four NRS score strata .Secondary outcomes were recurrence of initial complaints, re-operation, occurrence of new symptoms, the intensity of sport, time to return to the original level of sport and physical functioning. Recurrence of initial complaints was defined as patient-reported recurrence of inguinal-related groin pain or discomfort of the operated groin. Re-operation was considered any type of inguinal hernia surgery on the ipsilateral side of the original procedure due to recurrence of complaints or new symptoms. New symptoms were defined as any new pain or discomfort in the operated groin area, as reported by patients. The intensity of sport currently practiced was compared to the preoperative situation and expressed as a percentage. Time to return to the original level of sport was the amount of days it took to fully recover after TEP repair and achieve their preoperative level of sport. Physical functioning was determined using the HAGOS. This is a patient-reported questionnaire, in which six different domains are addressed: pain, symptoms, activities of daily living (ADL), sport/ recreation, participation, and quality of life (QoL). These domains are scored from 0 to 100, where 0 is the worst possible score and 100 is the best possible score in that specific domain. No additional outpatient appointments were made and no physical examination was performed.Statistical analyses were performed with SPSS, version 26 . Demographics and baseline characteristics are reported descriptively. Descriptive statistics include median and interquartile range (IQR) or mean and standard deviation (SD) for continuous variables, and frequency counts with percentages for categorical variables. Relevant differences between pre- and postoperative parameters are presented as median with IQR and were analyzed with the Wilcoxon signed rank test for continuous data. For discrete data, McNemar’s test (binary) and the Wilcoxon signed rank test  were used. For between-group comparisons the Mann–Whitney U test was utilized. Patients lost to follow-up were excluded from the final analyses. The same applies for missing data, due to pairwise analyses.From October 11th 2022 until December 12th 2022, patients received a phone call answering two questionnaires. Out of 32 male participants in the TEP-ID-study, 28 patients (88%) were available for follow-up with a median age of 30 years (IQR 27–43). Four patients remained unresponsive despite multiple attempts to get in contact by phone or e-mail . Median p < 0.001). A significant NRS score reduction was also observed between 3- and 83 months after treatment (p = 0.014). However, no significant difference was found between 19- and 83 months follow-up (p = 0.718).The median preoperative pain score during exercise was 8 (IQR 7–8) and this decreased to 0 (IQR 0–2) at 83 months follow-up (p < 0.001) and none reported severe pain (NRS 8–10) during exercise. Ten patients (36%) mentioned subjective recurrence of initial complaints. However, when observing the outcomes of their HAGOS, these recurrences were not perceived as painful or limitative. Six patients described new symptoms and no patients received additional surgical therapy. Mild pain (NRS 1–3) significantly reduced (p = 0.021) between 3- and 83 months postoperatively. Similarly, severe pain reduced significantly at 83 months follow-up (p < 0.001) compared to the preoperative situation. For all other categories, comparing scores between 83 months follow-up and earlier timepoints, no significant differences were objectified , which was higher in the group whom reported recurrence of complaints. For patients experiencing new, different symptoms there was no relationship with NRS score.A significant correlation was observed between recurrence and NRS score . Three patients (11%) did not return to their original level of sport. The median intensity of sport was 80% (IQR 43–80) and 46% of the athletes were able to exercise at 100% intensity.p < 0.05).Improvement on all subscales of the HAGOS was shown at 83 months postoperatively, with high scores on all domains (>80.0) The highest score was observed for ADL (96.3) and the lowest for participation (80.4), which is similar to the TEP-ID-study  were pain free and the two treatment options were assumed to be equally effective [In accordance with the Doha agreement, we selected patients based on the criteria described for IRGP . An RCT ffective . These effective . One majffective . Sadly, n = 55) were included, this definition lacks precision. For retrospective studies it is difficult to clearly specify diagnostic criteria since data are collected retrospectively. Edelman and Le et al. used perioperative characteristics and treatment codes to identify eligible patients [Many other studies report non-specific diagnostic criteria. Also, there is a lack of uniformity in reporting these criteria. Paajanen et al. included patients based on patient history, physical examination and imaging studies in a multidisciplinary manner . On the patients , 27.n = 15) [n = 28) recorded a pain reduction of 100% at 3 months postoperatively (mean follow-up: 15 months) [n = 41) and mesh was again placed bilaterally, with 5% of patients receiving additional tenotomy. In addition, there was no clear definition of the primary outcome or record of their telephone questionnaire assessing the latter. Even though all these studies applied different selection protocols, TEP repair was repeatedly proven to be effective for athletes with IRGP. A possible explanation for these positive outcomes is the significant correlation Serner et al. reported between higher treatment success and lower quality studies [Studies with longer follow-up are of low quality, mainly due to their design. Srinivasan et al. prospectively reported no recurrence of symptoms at 46 months follow-up, yet diagnosis was executed poorly and the sample size was small (n = 15) . In our  months) . Althoug studies .Comparing the results of the present study to NRS scores at 19 months follow-up from the TEP-ID-study in this patient cohort, no significant difference was present. This would imply that the favorable outcome does not change over time and TEP repair remains effective in this specific population . Sport iThe lack of a control group poses a substantial weakness of this and the previous study. However, it would have been ethically debatable to perform an RCT on this uncommon condition with a watchful waiting group, since these patients were urgently in need of pain relief. Another limitation of our study is the possibility of recall bias concerning the questionnaires. When asking the number of days it took to return to their original level of sport after surgery, which happened around seven years ago, it is understandable this is merely an estimation of the exact number of days. Furthermore, follow-up was conducted solely by telephone, so there was no chance to perform physical examination. It could have been interesting to ask patients with recurrent complaints or new symptoms to visit the outpatient clinic and objectively assess these.Finally, this study demonstrates the safety and effectivity of TEP repair in a prospective cohort of athletes with IRGP, for whom conservative treatment had failed, with a follow-up period of over 80 months. We emphasize the importance of a multidisciplinary, systematic work-up for adequate patient selection to gain high success rates of surgical treatment in this specific patient group and to enable generalizability of the current results. To decrease heterogeneity and aid interpreting results between studies, there is a need for more high quality research conducted in larger cohorts with the use of the definitions and criteria of the Doha agreement.


BackgroundLow back pain (LBP) is common and considerably impacts daily lives across all age groups. MRI is not frequently used as a first-line investigation for patients presenting with LBP, except in the presence of red-flag symptoms. This study aimed to use pain severity and its impact as a predictor for MRI findings to help physicians decide whether a patient needs an MRI.MethodsThis cross-sectional study was conducted at the outpatient clinic of the neurosurgery department. The questionnaire included demographic data of the patients, red-flag symptoms, and the Dallas Pain Questionnaire (DPQ). The primary physician then determines whether the patient should have an MRI appointment.ResultsThe study included 100 patients with LBP, of which 71 had chronic LBP (CLBP). Out of these 71, an MRI was requested for 62, but only 26 had findings related to LBP. Regarding the impact of CLBP on daily activities as measured by the DPQ, there was a significant association between those whose CLBP affected their daily activities and the decision to request an MRI. However, no significant statistical association was found between the three other parameters of the DPQ and the primary physician's decision to request an MRI.ConclusionConcerning the use of the DPQ questionnaire to predict MRI findings in patients with CLBP, the study indicates that significant pain impact on the DPQ does not necessarily correlate with MRI findings related to LBP. This suggests that the DPQ evaluation tool has no advantage over a physician's clinical judgment. Low back pain (LBP) is a common condition that considerably impacts daily life activities in all ages . In 2015Study design and selection of patientsThis study is a descriptive cross-sectional design. The study used a convenience sampling technique to include patients with LBP who presented to the neurosurgical outpatients’ department (OPD), Taif, Saudi Arabia, between July 2021 to August 2021 (two months). Ethical approval was granted from the institutional review board (IRB) of the Directorate of Health Affairs - Taif . The total number of neurosurgical clinics was two and managed by four neurosurgeon consultants. Exclusion criteria were patients below adolescent age (12 years) and patients who failed to attend their MRI appointments.Data collection and assessment criteriaA printed, self-reported questionnaire was set up before the clinic appointment in the waiting area for data collection, which was gathered by nine independent data collectors. The collected parameters for all patients included age, sex, nationality, weight, height, comorbidities (hypertension and diabetes mellitus), duration of pain , detailed history of the presenting illness, associated symptoms, and general red flags of LBP (extremes of age (<20 or >55 years), cauda equina syndrome, sudden bladder dysfunction, progressive weakness, gait disturbance, sciatica, saddle  numbness, fecal incontinence, widespread sensory deficit in the lower limbs, pain radiating to the lower limbs). There were also questions regarding malignancy history , fractures, and infection history , and whether an MRI was requested by the clinic physician  for the patient, independent of the questionnaire. The MRI findings of the patients were collected through the hospital imaging system, obtaining T1- and T2-weighted sagittal and axial images. An assigned expert radiologist interpreted all images to evaluate the significance of each image. Significant positive findings included cases with a disc bulge, herniation, or protrusion causing nerve compression or spinal stenosis.For patients with chronic LBP, the Dallas Pain Questionnaire (DPQ) was used to assess the impact of pain . The tooStatistical analysisAfter obtaining data from eligible patients for the study, data were cleaned, coded, and entered into the statistical software IBM SPSS version 23.0 for analysis. Data were analyzed using means ± SDs for quantitative variables, while frequencies and percentages were used for qualitative variables. To compare qualitative variables, a Chi-square test was performed. Means were compared using the ANOVA test. A p-value ≤ 0.05 was regarded as statistically significant. The duration of LBP was classified as acute pain (<6 weeks), sub-chronic pain 6-12 weeks), and chronic pain (>12 weeks) .To inte weeks, a General characteristics of the included participantsThe current study included 100 patients with LBP. Table The impact of pain among patients with CLBP was assessed using the DPQ Figure  in all fFeatures of MRI status and findings among patients with LBPAlmost all of the patients who presented to the clinic with LBP had an MRI spine requested by the physician (86 patients). From those whom an MRI was requested for, only 26 patients had findings related to LBP, such as bulging, herniation, protrusion, or degeneration that causes nerve compression or spinal stenosis. In comparison, 60 patients had no findings related to LBP. Additional information is shown in the concept map , followed by moderate impact 44%) with a statical significance association of 0.03. Additional information on the impact on other domains is presented in Table 4% with aTable The total number of red flags concerning DPQ scores is shown in Table This study attempted to use the DPQ questionnaire to assist physicians in assessing a patient's disability without MRI indications to determine the need for an MRI, especially in the working-age group. However, the results did not demonstrate the superiority of the DPQ evaluation tool over physician judgment. Since recent guidelines recommend against routine MRI imaging in the absence of red flags, further studies with larger sample sizes are needed to assess the DPQ tool as a predictor for MRI findings in patients with CLBP ,8. The rThe study was not limited to a specific age group. The mean age of the participants was 46.5 ± 15.9 years, with a female predominance. A systematic review study conducted in 2020 included five cross-sectional studies that investigated the prevalence and characteristics of LBP in Saudi Arabia. These studies showed that all targeted the working-age group with a mean age of 33.9-38 years .A meta-Strength and limitationsThis study is the first to attempt and utilize the DPQ as an assessment tool to enhance the physician's judgment in making a clearer MRI decision. Nevertheless, there are multiple limitations in the study, starting with the small number of participants (100 participants), with 71 having chronic back pain. The small sample size is because of the short window for data collection (two months), which was further complicated by restrictions related to the COVID-19 pandemic. Furthermore, the study was conducted in only one center in the region. Finally, a major limitation of the study was the funding and the inability to order lumbar MRI for patients determined by the primary physician as non-MRI patients; this can directly affect the results.Regarding using the DPQ questionnaire to predict MRI findings in patients with CLBP, the study indicates that a significant impact of pain in the DPQ tool does not necessarily correspond to findings in MRI imaging related to LBP. This suggests that the DPQ evaluation tool does not hold superiority over a physician's clinical judgment. Current guidelines advise against routine MRI imaging in the absence of red flags. Therefore, further research with larger sample sizes is recommended. Additionally, while the DPQ tool was employed to assess the impact of LBP in this study, exploring other tools that might predict the need for an MRI is suggested.


At the 12-month follow-up, the ASH group showed significant improvements in functional limitation, psychological discomfort, psychological disability, and handicap . ASH treatment did not reduce pain or improve mandibular range of motion more than physical therapy in patients with symptomatic DDwoR. However, ASH could be preferable given its positive long-term effects on patients’ quality of life. The clinician’s main objective is to prioritize the treatment plan order with a focus on the patient’s quality of life. Accordingly, healthcare professionals should consider ASH as a treatment option for patients with symptomatic DDwoR who desire long-term improvement in their quality of life.The aim of this study was to compare the effectiveness of arthrocentesis followed by hyaluronic acid infiltration treatment (ASH) and mandibular exercise therapy (MET) in patients with symptomatic disc displacement without reduction (DDwoR) by examining pain intensity (VAS), mandibular range of motion (MO), and quality of life (QoL). Fifty-two patients were randomly allocated into two groups, MET (N = 26) and ASH (N = 26), and therapy was applied at the baseline and one month after. Patients were followed up at 1 and 12 months after the baseline assessment. Clinical and patient-reported outcomes were compared at the baseline, 1-month follow-up, and 12-month follow-up. The study found no significant differences in VAS and MO between the ASH and MET groups at the baseline. However, while not significant, it was noted that the ASH group showed higher values for MO. Regarding OHIP-14 at 1 month of follow-up, the ASH group showed significant improvements in physical pain ( Intra-articular disorders of the temporomandibular joint (TM) are defined as an abnormal positional relationship between the disc and the condyle, articular eminence, and/or articular fossa . The claDisc displacement with reduction is the most prevalent diagnosis among the intra-articular disorders of the TMJ, corresponding to 41% of TMD clinical diagnoses ,8. On thClinically, the main symptoms of DDwoR are characterized by TMJ pain and limited mouth opening (“closed lock”), which result from the overload of the bilaminar zone by direct contact with the mandible’s head and the mechanical blocking of the translation movement of the mandible’s head due to the displacement of the disc anteriorly, respectively ,16. AddiVarious interventions have been suggested for DDwoR, but, to date, the most effective approach is still unclear. Primary treatment options include conservative, non-surgical treatment modalities, such as mandibular manipulation technique, education and counseling, splints, pharmacotherapy, and physical therapy ,21. ExerMinimally invasive therapies, which include arthrocentesis (AT), are the second treatment option when conservative therapies are not effective in reducing signs and symptoms of DDwoR ,23. In TThus, we conducted a randomized clinical trial to assess the efficacy of AT compared with physical therapy in patients with DDwoR with osteoarthritis, aiming to test the hypothesis that AT is more effective than physical therapy. This was a double-arm randomized controlled trial approved by the institutional review board at the SAMS Hospital (ID-012018) and registered in ISRCTN (number: 48852162) , conductPatients with DDwoR with osteoarthritis treated at or referred to the orofacial pain clinic of Centro Clinico SAMS Serviços de Assistência Medico Social were recruited.Inclusion criteria were age between 18 and 70 years, uni- or bilateral DDwoR diagnosis according to the Portuguese version of the Diagnostic Criteria for Temporomandibular Disorders (DC/TMD)  by one cAfter applying the inclusion criteria, computerized tomography and magnetic resonance imaging was performed on the included patients to confirm the presence of DDwoR and osteoarthritis.n = 26) or arthrocentesis and saline hyaluronate . On the day of treatment administration (visit 2), participants were consecutively allocated (1:1) into treatment groups using a randomization tool  prior to procedures administered by a technician who was not involved in any other procedures in the study. Opaque envelopes were used to achieve allocation concealment. The envelopes were opened by the clinician immediately prior to treatment administration. The order of the envelopes was determined independently prior to the start of the study by an investigator who was not involved in the study. Patients were assessed for pain and functional measures immediately prior to the interventions, and treatment was administered thereafter. Follow-up visits were scheduled at 1 month (visit 3) and 12 months (visit 4). At the beginning of each follow-up visit, patients were assessed for pain and function using the same protocol as at the baseline. Interim phone calls were made at 15 days and 10 months post-treatment to improve retention and to monitor for adverse events. Patients were seen on 4 occasions. During the first visit, patients were screened for inclusion in the study. Patients were then informed of the treatments and assessment tools used in the study. Patients were told that they could receive mandibular exercise therapy  opening and closing the mouth with the tongue on the palate (10 repetitions); (b) moving the mandible right and left laterally with the index finger placed between the incisor teeth, which serve as a guidance platform (10 repetitions); and (c) blowing through the lips, causing the cheeks to vibrate during exhalation (10 repetitions). The exercises were always taught to the participants by the same physiotherapist. Patients were required to perform the set of mandibular exercises five times throughout the day for 1 month, always in the same sequence. In order to dispel doubts about the exercises and to ask if they were being carried out, patients were contacted by telephone fortnightly by the physiotherapist. ® —1.7 kDa) was injected into the upper TMJ compartment. Then, a mandibular maneuver physiotherapy session was conducted by the same physiotherapist. The following intraoral mobilization techniques were applied in the same sequence for 10 min: condylar distraction (mandibular distraction), bilateral (15 repetitions on each side); condylar laterality, bilateral (15 repetitions on each side); and anterior mandible translation (anterior translation mobilization), bilateral (15 repetitions on each side). After this mobilization sequence, participants were asked to perform five maximum mouth openings. The described procedures were performed at the baseline and at the 1-month follow-up.First, a straight line was drawn with a marker pen along the skin from the middle portion of the auricular tragus to the lateral corner of the eyeball. A single point located 10 mm from the tragus and 2 mm below the tragal–canthal line was marked for the insertion of the needle. Next, antisepsis of the whole face was performed with 2% chlorhexidine solution, with an emphasis on the pre-auricular region and the ear. Then, both auriculotemporal nerve block and masseteric and posterior deep temporal nerve block were conducted with lidocaine. A 19 G needle connected to a 5 mL syringe was inserted into the marked point. When in place, 5 mL of saline solution at 0.9% was administered 5 times  in order to distend the joint space, and patients were instructed to open and close their mouths during this procedure to promote wash out of the cavity . At the Variables were assessed at the baseline and again at 1 and 12 months after treatment by a calibrated researcher who was not involved in any other procedure of the study.Patients rated their pain intensity in the TMJ using a 100 mm visual analog scale (VAS) with endpoints “no pain” and “worst pain”. Participants were instructed to make a mark on the VAS indicating their level of pain at each visit . ChangesTo assess OHRQoL, patients completed the validated Portuguese version of the Oral Health Impact Profile 14 (OHIP-14) . Specifi®, Horby, Sweden). Pain-free mouth opening and maximum unassisted opening were measured with patients seated in a dental chair, in a room with adequate lighting, following the instructions of the DC/TMD-Axis I  using a Considering a standard deviation of 1.0 for a 0.89 mean difference  and an 8The statistical approach was based on the site as a unit. Data analysis was performed using R for Macintosh (v4.0.1). An explicit comparison of mean values was performed using the Kruskal–Wallis test, with Benjamini–Hochberg adjustment used for multiple comparisons, as data assumptions for the application of the test did not meet normality and homoscedasticity. A chi-square test was used for comparisons of categorical variables across the groups. The level of statistical significance was set at 5% in all inferential analyses. This trial was initially based on three different arms; however, the third intervention arm (viscosupplementation without arthrocentesis) was deemed impossible to carry out due to lack of available materials and funding.Overall, 83 patients were screened; 27 patients were excluded for not meeting the inclusion criteria, and 56 eligible patients were enrolled in this study (mean age ± 49.4). Included patients were divided in 2 groups, each with 28 patients; however, 2 patients from each group did not complete the study due to discontinued interventions .There were no differences in demographic characteristics regarding age, sex, education level, and DC/TMD diagnoses between treatment groups . In the p > 0.05) in pain intensity (VAS) in post-treatment follow-ups , psychological disability (p > 0.029), and handicap domains (p > 0.033) for the ASH group at the 1-month follow-up (p < 0.008), psychological discomfort and disability , and handicap (p < 0.005)  .p < 0.05) or in treatment follow-ups , all of the assessed domains related to the patients’ quality of life were significantly improved in the AT group.When we consider the pain intensity results, our study showed that there was a decrease in subjective pain in both groups throughout the study, with no significant differences between them. It was very interesting to note that, despite the fact that our studied population was a refractory chronic sample, physical therapy, which is a conservative treatment and generally the first-line treatment, was as effective as AT. Similarly, mandibular opening increased in both groups in all follow-ups, with no differences between groups. These results align with a previous study demonstrating that both treatments are efficacious in diminishing subjective pain and improving mouth opening in patients with symptomatic DDwoR . To imprThe fact that during AT there is a lavage and aspiration of inflammatory fluid mainly composed of proinflammatory cytokines, a joint space expansion, and breaking of joint adhesions could explain its positive effects in the outcomes assessed in this study . In anotIt is well known that TMD patients, independent of the diagnosis, present psychosocial impairment and a reduction in well-being  ,39. It iLimitations of the present study include the potential regression to the mean and the natural disease course of DDwoR, as well as the lack of blinding of patients and researchers and the restricted studied population, with no gender pairing. Randomized controlled clinical trials including groups with only SH, arthrocentesis, and conservative therapies, assessing the efficacy and effects of this treatment on somatosensory variables of symptomatic DDwoR patients, are encouraged in the future. In view of the results and limitations of this study, it can be concluded that ASH treatment is not superior to physical therapy with regard to diminishing pain intensity and improving mandibular range of motion in patients with symptomatic DDwoR. However, ASH could be the preferred treatment for this diagnosis due to its long-term positive effects on patients’ quality of life.