N = 42 CP patients and n = 42 matched controls were analyzed (age range: 33–68 years). Numerical-spatial abilities were investigated by using number line tasks, where participants either estimated the position of a given number (position marking) or the value of a predefined mark (number naming). Pain intensity was assessed using numerical rating (NRS), verbal rating (VRS), and visual analog (VAS) scales. Additional measures included attention and working memory, verbal intelligence, medication and depression. Results revealed that in number naming, patients deviated more from expected (correct) responses than controls, and that VAS scores were significantly higher than both NRS and VRS and correlated with deviations in position making. Changes in number naming were predicted by pain intensity, sex and IQ but not by attention, memory or opioid medication. This article presents new insight on which cognitive mechanisms are influenced by CP with the focus on numerical spatial abilities. It could therefore provide useful knowledge in developing new pain assessment tools specifically for patients suffering from CP.Chronic pain (CP) is linked to changes in cognitive function. However, little is known about its influence on number sense, despite the fact that intact numerical-spatial processing is a prerequisite for valid scale-based pain assessments. This study aimed to elucidate whether number sense is changed in CP, to determine if changes have an impact on pain assessments using pain rating scales and what patient factors might contribute.  A mutual relationship between chronic pain (CP) perception and cognitive impairments, e.g., in attentional mechanisms, memory or executive functioning has repeatedly been reported . TamburiStill, another, albeit under-recognized but potentially clinically important cognitive modality, is the number sense. Number sense refers to the intuitive skill to estimate and compare numerical magnitudes and orders and is thought to be based on the “mental number line,” a spatial representation of numbers in the human brain. As it allows people to manipulate numbers as well as non-numerical quantities in form of rounding and approximations as well as exact calculations and measurements, it is suggested to be a key basic skill in today’s societies . In the Hence, the aims of the present study were (a) to assess whether number sense is changed in CP patients compared to healthy controls; (b) to assess the impact of possible number sense changes on the accuracy of responses of pain assessment tools; and (c) to evaluate whether changes in number sense are associated with performance in attention, intelligence or memory tests.Chronic pain (CP) patients were recruited from the pain clinic of the Klinikum Oldenburg AöR, Germany, between March and August 2016. In addition, healthy controls and further patients were recruited through advertisement in a local newspaper. To ensure that results were not biased by specific demographic characteristics of participants, we matched pain patients and healthy controls for sex and age (±3 years). Inclusion criteria for patients were CP, perceived for at least 12 months (CP group), and for controls the absence of any pain related issues . Because the direction of the mental number line is presumed to depend on cultural background, resulting in a left-to-right oriented representation for persons from cultures with left-to-right reading and writing, and from right-to-left in cultures with right-to-left reading, respectively , we onlyn = 8) and vertically (n = 8) with the anchors shown in a “familiar” or “unfamiliar” order. In “familiar” order “0” was either shown on the left of horizontal or at the bottom of vertical number lines. This was reversed in “unfamiliar” conditions where “0” was shown on the right  or at the top , respectively. Lines were presented in random order and participants were asked to mark each line at the position which they thought would best represent a given number  .For this task, each participant was exposed to a total of 16 23 cm long number lines printed on separate A4 papers. The ends of each line were anchored with “0” and “100,” respectively. Lines were presented horizontally . To evaluate the influence of landmarks on the results obtained in the number naming and position marking tasks, items were ordered according to their distance to the nearest clue . Thus, eight numbers were categorized as “low distance” (within a 13-unit distance from the landmark), and 8 as “high distance” (between 17 and 24 units from a landmark).Similar to position marking, each participant was exposed to a total of 16 23 cm long number lines printed on separate sheets of A4 paper. The lines were again presented either horizontally or vertically and anchored with “0” and “100” at each end in familiar and unfamiliar order, respectively. In contrast to position marking, the lines in this experiment were pre-marked and participants needed to indicate the numbers they thought these marks represented  as described before . In shorFigure 1). Participants were instructed to indicate the middle of each line as accurately as possible. The outcome measure was the mean deviation from the midline in mm.This task served as a control task to rule out possible influences of spatial neglect on the number line tasks . Each paParticipants completed three subtests of the computerized TAP battery 2.3 . All tesThis task measures control of an attentional focus and concentration over a longer time period (15 min). Participants were shown stimuli comprising sequences of symbols in different color, size, and filling. If a stimulus matched the preceding stimulus in either color or shape, the stimulus was considered a target stimulus and the participant was instructed to press a button as quickly as possible. An increase in the number of omissions over time indicates a decline in the ability to concentrate or focus on the task. Outcome measures were the number of omissions across the whole duration of task performance (15 min) and across each of three consecutive time bins with a 5 min duration .This task assesses the ability to shift the focus of visual attention onto the peripheral space without changing the direction of the gaze. Participants were briefly presented a cue (arrow) which pointed either to the left or right-hand side of the screen until it disappeared. This cue was intended to direct the participant’s attention. Then, a target cross was shown either on the side which was indicated by the cue , or on the opposite side . Participants were instructed to respond to the target stimulus by pressing a key as fast as possible, regardless of the validity of the cue. Outcome measures were mean reaction times for valid and invalid cues and for stimuli presented on the left or right side of the fixation point.This task probes the ability to control continuous updating of information flow in working memory. Participants were shown a sequence of 100 one-digit numbers at a constant rate. They were instructed to press a key as soon as possible whenever a stimulus matched the one that was presented two trials before . During the task, 15 critical stimuli were presented. The number of omissions and errors were recorded as outcome measures. Higher omission rates indicate a lack of control over the flow of information whereas an increased number of errors suggest attentional lapses.We used the Wortschatztest which is a standardized German vocabulary test with good correlation to general intelligence and education. In a multiple choice format, participants are required to identify, in each of 42 rows, a word within five non-words . The sumChronic pain (CP) patients were asked to assess the average pain intensity which they had perceived during the last 24 h by using the following instruments: the VAS, which is presented as a 10 cm high pyramid with the top indicating “no pain” and the bottom “worst pain imaginable,” an 11-point NRS, with 0 representing “no pain” and 10 “worst pain imaginable” , and a sAllgemeine Depressionsskala (ADS-K) which is the short form of the German version of the Center for Epidemiological Studies Depression Scale (CES-D) (We used the  (CES-D) . The que (CES-D) .t-tests or repeated measures analyses of variance (ANOVA) were used where appropriate. Within-group analyses were performed within the CP sample using dependent samples t-tests to investigate differences in results of pain rating scales. For correlation analyses we used Pearson correlation to explore the relationship of number sense tasks with pain rating scales.All participants with missing values were removed from the respective analyses: one participant was excluded from analysis of position marking, clinical pain assessment and covert shift attention. Also due to missing data, two patients were excluded from analysis of working memory and three from analysis of sustained attention. One additional participant was excluded from all number sense analyses, because of presenting as an extreme outlier . All analyses were performed with SPSS 24 . To detect differences between groups in cognitive function, number sense and intelligence relating to our first research question either independent samples Table 1) to predict number sense performance within the CP sample.Two stepwise linear regressions with forward selection were used to identify the relevance of demographical and cognitive factors for number sense performance as a dependent variable. We used the MADER for number naming overall as outcome variable based on the observed influence of landmarks in the position marking task. Participant demographics , as well as depression (ADS-K raw score), attentional measures  and working memory (misses) were included as predictors into the model. A second regression analysis used pain intensity and duration, and main pain syndrome  was considered significant.To correct for all measures of the number line tasks, which were the primary measures of the experiment, we used Bonferroni corrected alpha-levels of 0.005, two-sided (0.05/10). These comprised the overall performance, as well as horizontal and vertical lines, and lines in familiar and unfamiliar order of the numerical anchors. Additionally, Cohen’s  effects . For alln = 93 participants (50 controls and 43 patients) were invited to participate, but nine participants had to be excluded due to the following conditions: neurological or psychiatric diseases (n = 3), technical issues (n = 1), the absence of pain for more than 1 month (n = 1), lack of matching pain patients (n = 4). Thus, n = 84  could be included in our final analyses. Demographic and clinical variables of these patients and controls are depicted in Table 1. Compared to controls (C), chronic pain patients (CP) were similar with respect to age and sex. However, verbal IQ  and educational level  were significantly lower in CP patients and patients experienced significantly more depressive symptoms than controls .A total of Table 2).Between-group analyses revealed that pain patients consistently had a significantly higher MADER in the overall condition  and the vertically unfamiliar condition of the number naming task, with large effect sizes. Although no significant differences were observed in the overall position marking performance between pain patients and healthy controls, similar trends were noted here as well, with small to median effect sizes, and a significant difference in the horizontal unfamiliar condition (Table 3).Evaluating the influence of landmarks on results of the number naming task, we found that there were no differences between high- and low-distance stimuli in both, controls and CP patients. In position marking, however, patients were significantly more accurate with low-distance than with high-distance stimuli. A similar but non-significant trend was observed for controls  = 0.531, p = 0.597] and for those anchored with the smaller quantity on the left . As the line anchor type did not seem to influence response accuracy, we pooled data obtained with the small symbolic and non-symbolic quantities on the left (MADERleft), and those obtained with the smaller quantities on the right (MADERright). A 2 × 2-repeated-measures ANOVA was employed with group (chronic pain patients vs. controls) as between subjects and MADER (left vs. right) as within subject factor. Results revealed a non-significant main effect of group , a significant main effect of MADER , but no interaction between group and MADER , suggesting that visual-spatial attention was not different between groups.We first determined whether responses differed depending on the type of stimulus (symbolic: Arabic digits vs. non-symbolic: dot arrays) participants were exposed to. Across all participants, response accuracy for stimulus type was not different for lines anchored with the smaller quantity on the right  and VRS . However, there was no difference between the VRS and NRS .To determine possible differences between pain rating scales, CP patients were included in a repeated measures ANOVA with r = 0.321, p < 0.05), but not in number naming  were associated with higher values in pain ratings employing VAS.To further examine the relationship between numerical tasks and VAS, correlation analyses including all CP patients were employed between overall number naming and VAS scores, and overall position marking and VAS scores. Results showed that only deviations in position marking  = −0.542, p = 0.591] and position marking  compared to patients without opioid medication .Finally, the role of opioid medication on number sense performance was evaluated, suggesting that patients with opioid medication performed equally well on both number naming  in their time to detect left and right targets , as well as in their ability to shift their attention to both sides .Results of the repeated measures ANOVA showed no significant difference between healthy controls and CP patients  as well as for the interaction of group with measurement-interval  showed no significant differences between CP patients and healthy controls (Table 4).Results for the main effect of r = 0.173, p = 0.116).Still, since there was a trend toward higher omissions of patients compared to controls over time, we employed a correlation analysis between the total number of omissions and overall number naming performance (MADER). Results suggest no significant relationship between number of omissions and number naming performance (t(65.572) = −1.591, p = 0.117] and misses  for this task (Table 4).There were no significant differences between healthy controls and CP patients in the number of errors  (Table 5).For the pain-unrelated regression, results showed that of all demographic and cognitive variables, only IQ, group and sex were included into the final model and in combination explained 29.8%  = 9.205, p < 0.01] significantly predicted number naming performance (adjusted R2 = 0.167) (Table 5).For the pain-related regression within the CP sample, results showed that out of pain duration, pain syndrome and pain intensity, only pain intensity [The present study aimed to assess whether number sense is changed in CP patients compared to healthy controls. Using different variants of a number naming and position marking task, we found number naming to be more effective in detecting differences between C and CP participants than position marking. Here, patients deviated more from the expected value compared to healthy controls, with medium to large effect sizes. Although similar but not statistically significant trend was observed in overall position marking, we assume that patients were able to compensate and hence improve their accuracy in this task through the use of landmarks. Our results are well in line with findings of However, number sense assessments are new to the research on cognitive consequences of CP , and theAlthough often thought of as equivalent tools in assessing pain intensity , resultsThere are two major components involved in the processing of numbers: each number can be perceived either in cardinality (its magnitude) or ordinality (its rank or position in a given sequence)  ratings, our finding of an altered number naming performance in patients might bear some clinical relevance. Finally, despite attention and working memory not contributing to the phenomenon, number sense changes in CP might nevertheless be the result of complex processes involving pain intensity judgments, sex, intelligence and possibly the working memory span which was not measured in this study.The anonymized raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.CB and CT designed the study, applied for funding, and the ethical approval. Both were also involved in preparing the manuscript. MS recruited participants, analyzed the data, and prepared the manuscript. KK recruited participants and prepared the manuscript. EB recruited participants and prepared the manuscript. JÖ and PS analyzed the data and prepared the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Dermatophagoides pteronyssinus, D.farinae, Blomia tropicalis) were highest in this population and correlated with atopic dermatitis (p < 0.001). Shrimp and peanut were the most common food allergens. A dominance of house/storage mite sensitization is seen in Brunei which is consistent with other tropical countries.IgE sensitisation in tropical areas is under-reported. A 2 year retrospective cohort study of allergy data specific to aero and food allergens in Brunei demonstrated that specific IgE levels to house dust mite ( Allergy is on the increase, and there are differences in the development of allergic disease dependent on climate, diet and lifestyle. Allergy may present as respiratory symptoms , skin manifestations  or others . House dust mite is a potent allergen and sensitisation can lead to asthma, rhinitis, conjunctivitis and atopic dermatitis . StrategIn Brunei, the number of inpatient and outpatient submissions with asthma and related respiratory symptoms increased from 2538 patients per 100,000 in 2005 to 2935 patients per 100,000 in 2010 equalling to about 15% increment over a 5-year period. In 2017, asthma was the 5th cause of in-patient morbidity  [Sensitisation to food allergens varies according to age, climate, geography and genetic background. Atopic dermatitis is often associated with but not limited to food allergy. It can also be a manifestation of a skin allergy to house dust mite .A retrospective cohort study was performed on all patients subjected to specific IgE blood tests (sIgE) in 2015 and 2016. The data  was collected anonymously from the patients’ electronic record (BruHIMS) with no further sampling or exclusion criteria. All data collection was done with permission of RIPAS Hospital and approved by the Medical and Health Research Ethics Committee, Ministry of Health (MHREC).Specific IgE levels to a panel of defined inhaled allergens were measured in patient serum by fluoroenzymeimmunoassay using the Phadia 100 platform. Serum ≥ 0.10 kUA/L was considered to be positive for specific IgE tests (sIgE) and Serum ≥ 100 kUIgE/L was considered positive for total IgE tests (tIgE).p < 0.05 considered statistically significant. Graphpad Prism v8.1.2 was used to plot data.IBM SPSS Statistics v21 was used to analyse data with Dermatophagoides pteronyssinus  followed by D. farinae  and Blomia tropicalis . Shrimp  was the most common food allergen, followed by peanut  and egg white . Total IgE significantly correlated with sIgE p < 0.001. Figure One hundred and seventy patients had at least one positive sIgE (out of 223 patients tested). The mean age of patients was 27.8 (SD = 18.05 median 25 range 0–80), including 99 (44.4%) males and 124 (55.6%) females. The frequency of positive sIgE was highest in younger patients, peaking in the 16–20 year age group. Most patients were Malay (74.1%) followed by Chinese (19.3%). The rest (4.2%) were indigenous (e.g. Iban), expatriate  or Indian (2.4%). The majority of samples came from Dermatology (73%), followed by Oral Maxillo-Facial Department (16%). Eleven percent came from other departments . The most prevalent aeroallergen was D. pteronyssinus, D2 D. farinae, D201 B. tropicalis, F13 Peanut, F24 Shrimp all p < 0.001). The levels of sIgE to the most common aeroallergens and food allergens in atopic dermatitis patients are illustrated in Fig. p > 0.05).Atopic Dermatitis was associated with sensitisation to all 5 most common allergens . Atopic dermatitis appears to be predominantly a skin manifestation of allergy to house dust mite in Brunei. There was also a statistically significant association with sIgE to shrimp and peanut, both common in the local diet.It is apparent that there is a very high sensitisation to house dust mite species  where a dominant mono-specific sensitization to house dust mite was also linked to the burden of allergic disease , 4 A preD. pteronyssinus, d1 and 37/60 to D. farinae d2 [It was surprising that there were relatively few asthmatic patients referred for sIgE testing. It would be expected that asthmatic patients would be sensitized to house dust mite. An earlier study from Brunei published in 1980 demonstrated that 40/60 asthmatics tested at the same hospital had a positive skin prick test to D. pteronyssinus (d1), D. farinae (d2) and B. tropicalis (d201).Most patients referred allergy testing came from dermatology, 89 were diagnosed with atopic dermatitis and 16 of these patients also had asthma. Education to raise awareness of reducing house dust mite growth could help reduce sensitization and response to environmental allergens, particularly D. pteronyssinus allergens, in particular tropomyosin which is a pan allergen across arthropods including arachnids e.g. mites, insects e.g. cockroach and crustaceans e.g. shrimp [There is limited data on food allergy in tropical countries. Brunei’s profile was dominated by shrimp allergy which matches the international trend. There may be some may be some cross reactivity between shrimp and . shrimp . FurtherSpecific IgE and total IgE are positively associated with each other. Total IgE can be used as a screening tool before following up on requests for sIgE. Future studies should investigate tIgE and sIgE to house dust mite in asthmatic patients, given the increasing prevalence of this disease in Brunei.


Moderate-severe pain after surgical procedures is associated with decreased quality of life and increased costs. This study aimed to identify the incidence and predictive factors of moderate-severe postoperative pain within 48 hours following video-assisted thoracoscopic surgery (VATS) in a tertiary hospital. A retrospective cohort analysis was performed using medical records of adult patients who underwent VATS between January 2015 and December 2016. Logistic regression was performed to identify predictive factors for moderate-severe pain  within 24 hours and within 48 hours postoperatively. Of the 1164 participants, the incidence of moderate-severe pain was 12.7% within the first 24 hours and 15.6% within the first 48 hours after surgery. In multivariable analysis, the independent risk factors related to moderate-severe pain within 24 hours after surgery were younger age, increased body mass index, preoperative pain within 1 month and history of smoking. The risk factors for moderate-severe acute pain within 48 hours were almost the same, except that the number of chest tubes were also included. Moderate-severe postoperative pain following VATS is not rare, and presence of several risk factors deserves more aggressive pain management strategies perioperatively. It is widely accepted that better management of postoperative pain results in better patients’ recovery.Moderate-severe pain after surgical procedures is associated with prolonged hospital stays, readmissions, patient dissatisfaction, development of chronic pain, decreased quality of life and increased costs3. Although potentially less postoperative pain was generally expected as one of the benefits of VATS4, many patients still suffer moderate to severe pain postoperatively7. There are several reasons contributing to this unsatisfactory situation, one of them being that some patients may have certain risk factors for occurrence of more severe postoperative pain8. Early identification of patients at risk may help to allow early application of effective treatment strategies to prevent unexpected suffering in these patients.Video assisted thoracoscopic surgery (VATS), compared to conventional thoracotomy, is notable for less tissue trauma, improved postoperative respiratory function, and increased tolerability for the patients9. However, most of them are cohorts with mixed surgical procedures while VATS is not included. Moreover, various subtypes of VATS  exist currently, and the lack of guideline results in variation of analgesia regimens and effectiveness of analgesia in different medical centers10. To date, the predictive factors of moderate-severe postoperative pain following VATS were not reported, which is important for individual-specific pain management perioperatively.Previously, several studies have focused on the predictors of postoperative pain and demographic, social psychological and clinical factors have been identified contributing to the development of severe postoperative painThis study was therefore conducted to investigate the incidence and risk factors of moderate-severe postoperative pain over the first 48 hours following VATS in a tertiary hospital.A total of 1177 patients underwent VATS during the study period. 13 patients were excluded because of bilateral surgery (n = 3), conversion to thoracotomy (n = 9) and VATS together with inguinal hernia repair (n = 1), leaving 1164 patients for analysis. The average age of the study population was 58 ± 11 years and 48.4% of study participants were male. In this population, 1 (0.1%) patient underwent pneumonectomy, 840 (72.2%) patients underwent lobectomy, 108 (9.3%) patients underwent segmentectomy, 180 (15.5%) patients underwent wedge resection and 35 (3.0%) patients underwent bullectomy. The preoperative and intraoperative characteristics of 1164 participating patients are presented in Table The incidence of moderate-severe pain was 12.7% within the first 24 hours and 15.6% within the first 48 hours postoperatively. The average VAS was 4 (4–5) for patients with moderate-severe pain vs. 2 (1–2) for patients without moderate-severe pain. Patients with moderate-severe pain pressed the PCA/PVB button significantly more frequently , and required more morphine than patients without moderate-severe pain , while PVB combined with systemic analgesia was applied in 69 patients (5.9%) Table . Inadequ.1%, whilIn multivariable analysis, the independent risk factors related to moderate-severe pain within 24 hours after surgery were younger age , increased BMI , preoperative existing pain within 1 month  and smoking history . As shown in Table In regards to early postoperative outcomes, occurrence of moderate-severe pain was not associated with increased incidence of nausea or vomiting. And postoperative hospital days were comparable between patients with or without adequate analgesia. However, moderate-severe acute pain lead to significantly lower patient satisfaction  . This is mainly owing to different patient characteristics, diverse types of surgery performed and distinct follow-up periods.Acute pain following VATS is not rare because of multiple muscle incisions and irritation of the pleura with chest tubes. In this retrospective study of 1164 adult patients undergoing VATS, the prevalence of moderate-severe postoperative pain over 48 hours was 15.7%. This finding differs from the results obtained by Emine 16. Our study was the first to demonstrate that these associations also present in patients undergoing VATS and that patients with high BMI and pre-existing pain deserved additional care. A 1 kg/m2 increase in BMI was associated with an increased risk of postoperative pain by 10% , and the presence of preoperative pain increased the risk of pain by 55% . A possible explanation for the relationship of BMI to moderate-severe acute pain may be that high BMI is associated with development of postoperative complications, such as wound infection, contributing to frequent debridement, change of dressing and increased postoperative pain17. Besides, opioids such as sulfentanil as we used in this study were distributed at least as extensively in the excess body mass as in the lean tissues, therefore the same dose of opioids might be less effective in obese patients19. Additionally, obesity and preoperative pain were both characterized by elevated proinflammatory cytokines and subclinical systemic inflammation, leading to decreased pain threshold22. Further study is needed to elucidate the potential mechanisms behind our observations.Elevated BMI as well as preoperative existing pain have previously been reported to be associated with increased postoperative pain in different types of surgeries other than VATS5, but in contrast to a recent study reporting no age-related differences by limited sample size6. This finding could be explained by the fact that the old had higher pain threshold, more peripheral neuropathy and less scar with less stiffness, contributing to less pain. Besides, elderly patients showed increased sensitivity to perioperative opioids with pharmacokinetic and psychosocial mechanisms24. More smokers underwent VATS compared with non-smokers, and it is therefore important to detect whether the increased risk for postoperative pain was smoking-related actually or if there was other explanations. And in VATS, the association between smoking history and postoperative pain was controversial26. The multiple analysis of this study indicated that presence of previous history of smoking increased the risk of moderate-severe acute pain following VATS by 86% . The potential mechanism was that chronic nicotine exposure was associated with pain intolerance and mechanical hyperalgesia by elevating spinal dynorphin and cytokines in rats27. Chest tube placement contributes to postoperative pain, and early removal of chest tubes was recommended28.Younger age is another consistent risk factor for moderate-severe pain following VATS, similar to a study including robotic-assisted thoracic surgery, VATS and open lobectomyEven though female sex was shown to be related to increased pain and narcotic use in various procedures, this was not seen in our cohort. Diabetes and educational level also did not have any association with pain either. Despite our expectations, there was no association between the analgesic methods and moderate-severe acute pain, which might be confounded by the severe imbalance of group distribution and variability in analgesia modalities of different anesthesiologists. Further studies comparing the efficiency and safety of different analgesia strategies in VATS should be undertaken in the future.We did not find a significant association between moderate-severe pain and postoperative hospital days. This could be explained, at least in part, by the prolonged hospital days (8–9 days) in our institution. In most western studies, the hospital days after VATS are much shorter (1–4 days), and the pain level at days 1–2 may have had more influence on hospital stay if the average length of stay were shorter. Our data showed that occurrence of moderate-severe acute pain following VATS was negatively related to patients’ satisfaction rate . So investigation of risk factors of postoperative pain is appealing. The independent factors in this study were simple and were confirmed in two models (pain within postoperative 24 hours and postoperative 48 hours) to be well correlated with risks of acute pain. Since patients with younger age, increased BMI, preoperative existing pain within 1 month, history of cigarette smoking, and increased number of chest tube placement have an extremely high risk, they deserve extra attention to perioperative pain management. Preoperatively, patient expectations should be set according to the anticipated pain intensity and postoperative analgesia protocol. Epidural analgesia and auxiliary analgesia methods should be considered, and sometimes consultation with a pain physician is necessary.Several limitations are worth mentioning. First is the retrospective nature of this study. It is possible that confounding factors for pain could not obtained in our study, such as psychological variables. Some additional complexities to each case including different surgeons/anesthesiologists, distinctive incisions and variance of postoperative courses contributing differently to pain were not taken into account. And the anesthetics in this trial were not standardized, but followed routine practice. For example, at the end of surgery, three different kinds of analgesics were administered for different patients. Prospective studies controlling for these variables were recommended. Secondly, VAS is subjective and only the maximum score within 24 hours and 48 hours was recorded and analyzed, while the median of pain score was inaccessible during the study period. However, the VAS is used widely across institutions for pain assessment, and the patients were followed up by the same nurse during the study period. Besides, patient demographics and management varies from centers to centers, so our results should be drawn with caution.In conclusion, moderate-severe acute pain occurred in 15.6% of adult patients undergoing VATS and was associated with patients’ dissatisfaction. Younger age, increased BMI, preoperative existing pain within 1 month, history of cigarette smoking, and increased number of chest tube placement were risk factors of postoperative pain. Gender and educational level did not appear to be associated with acute pain in patients undergoing VATS.This single-center retrospective study was approved by institutional review board (IRB) of the Second Affiliated Hospital of Zhejiang University, School of Medicine. All methods were performed in accordance with the guidelines and regulations. The requirement for informed consent was waived by the IRB based on the retrospective nature of the study and the minimum intrusion on participants’ privacy. All adult patients undergoing elective VATS for lung disease between January 2015 and December 2016 in our institution were identified. Excluded criteria was bilateral surgery, conversion to thoracotomy and another procedure performed at the same time.In VATS procedures, rib retractors were not applied. Single-port VATS and multi-port VATS were selectively used decided by the attending surgeon according to patient’s condition and personal preference. The maximum size of the utility incision was 1–4 centimeters routinely in our institution. Disposable drainage tubes with a diameter of 7.33 mm  were used at the end of surgery.11. In addition to standard monitors, central venous catheter and an arterial catheter were placed. Bispectral index was monitored routinely. Postoperatively, patients were transferred to the postanesthesia care unit or intensive care unit as needed where they were continuously monitored. Postoperative care was standardized as per institutional routines.Anesthetic management followed the standard care at our institution. Anesthesia was induced and maintained with intravenous and inhalational anesthetics combined with sulfentanyl/remifentanil/fentanyl. Muscle relaxants were administered as indicated. Intraoperative opioids were converted to morphine so the dosages could be compared−1•h−1 and a bolus dose of 0.02–0.03 mL•kg−1•h−1 with a lock-out of 15 minutes. PCA/PVB pump was attached to the patient immediately after surgery and was stopped after 48 hours. If the analgesia was inadequate  during the postoperative period, patients were recommended to press the PCA/PVB button. And if relief was not obtained, additional analgesics were given intravenously as rescue. All patients received tropisetron at the end of surgery to prevent postoperative nausea and vomiting, additional bolus was supplemented as needed.Patients received either of the two analgesia regimens, continuous paravertebral block (PVB) by the surgeon under video-assisted vision combined with a single bolus of intravenous analgesic , or a single-shot intercostal nerve block (ICNB) by the surgeon combined with patient-controlled analgesia (PCA) with opioids. Continuous PVB protocol was 0.15–0.20% ropivacaine with a continuous dose of 5 mL and a bolus dose of 1 mL with a lock-out of 15 minutes. A single bolus of 15–20 mL ropivacaine were administered to make sure of the appropriate location of the catheter after placement at the end of surgery. ICNB was performed using 0.75% ropivacaine 10 mL under video-assisted vision. The PCA protocol was 0.1 mg of sulfentanyl diluted to 100 mL with a continuous dose of 0.03–0.05 mL•kg12.Patients’ demographic characteristics , comorbidity , cigarette smoking history and American Society of Anesthesiologists (ASA) grade were retrospectively collected. If the patient has been smoking cigarette everyday for more than 1 year, no matter he quit smoking or not perioperatively, he was considered as having smoking history in this study. And preoperative pain experience within 1 month before surgery was collected from medical records. It was evaluated and recorded by the nurses in wards on the first day of admission in our institution, defined as continuous pain feeling lasting for more than 1 day during the past 1 month, no matter of the degree, location and character of the pain. Intraoperative characteristics included subtypes of surgery (single-port VATS or multi-port VATS), volume of fluid resuscitation, morphine consumption, surgery duration, anesthesia duration, pathological diagnosis and total number of chest tube placement. The postoperative days of chest tube in place were also recorded. In our institution, postoperative pain and related variables were followed up every day by a nurse at 24 and 48 hours after surgery. The most severe pain at rest since the last visit evaluated by VAS  and total dose of morphine requirement were recorded. The primary outcome was the incidence of moderate-severe pain (VAS ≥ 4) within 24 hours and within 48 hours postoperatively. Secondary outcomes included incidence of postoperative nausea and vomiting, postoperative hospital days and patient satisfaction. Patient satisfaction rate was defined by the incidence of the “extremely satisfactory” on a 5-point scale  as previously describedData were reported as mean value ± standard deviation or median (interquartile range) for continuous variables, or percentages for categorical variables. Student t or Mann Whitney U test were performed for continuous variables and the Chi square test or Fisher’s test was performed for categorical variables as appropriate. Independent risk factors were identified using a multivariable logistic regression model that include preoperative and intraoperative variables with P < 0.15 in univariable analysis and odds ratio (OR) and 95% confidence interval (CI) were calculated. All tests were performed using SPSS, version 22.0 , with P < 0.05 considered to be statistically significant.


The Pain Disability Index (PDI) is a widely-used instrument to measure pain-related disability. The aim of this study was to assess the responsiveness and interpretation of change score of the PDI in patients with chronic musculoskeletal pain (CMP) at discharge of vocational rehabilitation.Retrospective data of patients with CMP who attended vocational rehabilitation between 2014 and 2017 was used. The anchor-based method was used to assess the responsiveness of the total sample and of PDI baseline quartile groups. A receiver operating characteristic curve was performed, including Area Under the Curve (AUC) and Minimal Important Change (MIC).The PDI showed responsive to detect clinically relevant changes in pain-related disability at discharge of vocational rehabilitation (AUC 0.79). A PDI change score of 13 points (MIC 12.5) can be considered as a real change in pain-related disability for the total study sample, and a PDI change score of 7–20 points can be considered as a real change in pain-related disability for PDI lowest and highest baseline quartile scores.The PDI is responsive in patients with CMP at discharge of vocational rehabilitation. The interpretation of change score depends on PDI baseline score. Patients with a PDI baseline score of ≤27 should decrease minimal 7 points, patients with a baseline score between 28 and 42 should decrease minimal 15 points, and patients with a baseline score ≥ 43 should decrease minimal 20 points. Chronic Musculoskeletal Pain (CMP) negatively affects quality of life, daily activities and social and working lives . A decreHowever, the responsiveness, measurement error, and interpretability of change score of the PDI have scarcely been addressed. Responsiveness is the ability of a questionnaire to detect clinically important changes over time  . An outcThe aforementioned studies on responsiveness and measurement error were performed with patients attending pain rehabilitation in the Netherlands. It is unknown, however, what the responsiveness and interpretation of change score of the PDI is for patients at discharge of vocational rehabilitation (VR). Vocational rehabilitation is a “multi-professional evidence-based approach” that is provided in different settings, services, and activities to working age individuals with health-related impairments, limitations, or restrictions with work functioning, and whose primary aim is to “optimize work participation” . HoweverThe COnsensus-based Standards for the selection of health Measurement INstruments (COSMIN) checklist was applied in the design of the study , 15, 16.The study sample consisted of CMP patients who attended vocational rehabilitation (VR) between November 2014 and July 2017 in the Netherlands. Vocational rehabilitation is a multidisciplinary bio-psychosocial group-based program for workers with CMP and decreased work participation. The VR program is described in detail elsewhere [Data were collected using a core set of standardized web-based patient-reported questionnaires . For thiThe Pain Disability Index (PDI) is a 7-item questionnaire to investigate the magnitude of self-reported pain-related disability, independent from region of pain or pain-related diagnosis. The items of the questionnaire are assessed on a 0–10 numeric rating scale in which 0 means no disability and 10 is maximum disability. The sum of the seven items equals the total score of the PDI, which ranges from 0 to 70, with higher scores reflecting higher interference of pain with daily activities. The PDI measures family / home responsibilities, recreation, social activity, occupation, sexual behavior, self-care and life support activity . MissingA global perceived effect (GPE) item was used as the anchor  in this study. An anchor is a global rating scale in which patients are asked, in a single question at follow-up, to indicate how much their pain has changed since baseline . The pain = 14). We used the improved and unchanged groups to calculate the MIC [Responsiveness in this study was defined as the ability of the PDI to detect clinically relevant changes in pain-related disability at discharge of vocational rehabilitation . To calc the MIC , 20.In a secondary analysis we stratified the analysis on PDI baseline quartile scores, to assess whether the level of pain-related disability on baseline had a modifying effect on the MIC. Based on earlier research , 22 we hFloor or ceiling effects were considered to be present if more than 15% of the respondents achieved the lowest or highest possible score  . We gavep > 0.05). Independent samples T test showed a non-significant change in PDI score when the PDI change score ranged from − 6 to + 6. The ICC of the SEM formula was obtained from a study with a similar study sample [Measurement error was analyzed by calculating the Standard Error of Measurement (SEM = SD√1-ICC) . The SD Interpretability is defined as the degree to which one can assign qualitative meaning to quantitative scores . To enhaAll analyses were performed using SPSS 23 for Windows . The demographic data of the individuals were described by means and standard deviations (SD), or inter-quartile range in the case of no normal distribution. The assumption of normal data distribution was visually verified using histograms and QQ-plots.A total of 341 patients completed the PDI questionnaire on baseline and discharge. Mean age was 46.5 (±10.9) years, and 57% of the patients were woman. Ninety-one percent of the patients were employed and 63% were on sick leave in the preceding month prior to baseline measurement. Patients suffered from 3.4 (±2.4) pain locations, which were located in the back (76%), lower extremities (35%) and upper extremities (29%). Seventy-four percent had pain complaints for longer than 6 months. The average pain score was 5.4 (±2.3), the worst pain score was 6.8 (±2.5) and the PDI mean score was 34.7 (±11.7). Mean duration between baseline questionnaires and the start of VR was 8 ± 4.4 weeks and mean duration between the start of VR and completion of the discharge questionnaires was 15 ± 1.1 weeks. Table The responsiveness parameters  of the total study sample and the baseline quartile scores are presented in Table Floor and ceiling effects were absent in this study. The PDI total baseline score (min-max) was 3–60; 2.6% of the study sample had a total PDI baseline score < 10 and 0.3% (1 person) of the study sample had a total PDI baseline score of 60.The SEM was 1.2, the SDC for group level was 0.3 and the SDC for individuals was 3.4 . A PDI change score of 13 points (MIC 12.5) can be considered as a real change in pain-related disability for the total study sample, and a PDI change score of 7–20 points can be considered as a real change in pain-related disability for PDI lowest and highest baseline quartile scores.The responsiveness of the total study sample is in line with others  who founThe mean change score of the present study (10.5) is somewhat higher compared to a study that found a mean change score in PDI of 9.4 in patients with chronic pain after a multidisciplinary pain program . This isThe interpretation of change score of the PDI can be interpreted as a “real” change in pain-related disability if the mean change score is at or above the MIC and if the SDC for individuals does not exceed the MIC Fig. , Table 2The baseline PDI score of the current study is similar compared to patients with chronic back pain , 8, but The first methodological consideration of this study was the assessment of the MIC. Two common methods can be used to calculate the MIC: the distribution-based method and the anchor-based method . In the Practitioners can use the following cutoff scores to decide if a PDI change score is clinically relevant at discharge of VR: patients with a baseline score of ≤27 should decrease minimal 7 points, patients with a baseline score between 28 and 42 should decrease minimal 15 points, and patients with a baseline score ≥ 43 should decrease minimal 20 points.The PDI is a responsive questionnaire which can detect real change in decrease of pain-related disability in patients with CMP at discharge of vocational rehabilitation. Future research should focus on assessing the SDC and the MIC of the PDI in various patient samples and settings. Also, when using longitudinal cohorts, researchers are encouraged to report the portion of the sample with a change score at or above the MIC since this will enhance comparability and clinical relevance.


Local infiltration analgesia (LIA) supports early mobilization after hip and knee arthroplasty. Inspired by this, we studied the effectiveness of wound infiltration with the long acting local anesthetic ropivacaine in an effort to decrease the need for postoperative opioids after osteosynthesis of extracapsular hip fracture. NCT01119209). The patients received intraoperative infiltration followed by 6 postoperative injections through a wound catheter in eight-hour intervals. 23 patients received ropivacaine and 26 received saline. The intervention period was 2 days, and the observation period was 5 days. In both groups, there were no restrictions on the total daily dose of opioids. Pain was assessed at specific postoperative time points, and the daily opioid usage was registered.  Forty-nine patients undergoing osteosynthesis with a sliding hip screw were randomized into two groups in a double-blind study (ClinicalTrials.gov: Intraoperative infiltration with 200 mg ropivacaine and postoperative repeated infiltration with 100 mg ropivacaine did not result in statistically significant difference between the groups regarding postoperative opioid consumption or pain.  Ropivacaine as single component in postoperative treatment of pain after hip fracture is not effective. In our setup, wound infiltration with ropivacaine is not statistically significantly better than placebo. Sufficient treatment of pain is essential for early postoperative rehabilitation after osteosynthesis of hip fractures. The final outcome after hip fracture may be impaired by undertreated pain , and conα = 0.05 and a type II error of β = 0.2. From a pilot study, we estimated SD to 9. To be conservative and to meet the risk of dropouts in this cohort of fragile patients, we decided to include 74 individuals.Based on a probable clinical relevant difference of 10 mg of oxycodone per day, a sample size of 13 patients in each study group was calculated . We permitted a type I error of 201 patients with extracapsular fractures  were screened on admission to our department  Table 1Table 1.Screening was performed prior to surgery by the first author during a period of 3 years. All included participants scored above a specified cutoff point of seven (out of 28) on the inverted version of the Short Orientation-Memory-Concentration Test (SOMC) .NCT01119209), and the study was conducted in accordance with the Helsinki Declaration and the principles of Good Clinical Practice. Anonymized research data used to support the findings of this study are available for five years after publication from the corresponding author upon request.The study was approved by the regional ethics committee (Region of Southern Denmark), the Danish Medicines Agency , and was reported to the Danish Data Protection Agency. Registration was done at ClinicalTrials.gov (49 patients (ASA I–III) completed the intervention period  after asAll patients underwent osteosynthesis with a sliding hip screw  after repositioning of the fracture. Surgery was performed by 35 different surgeons. Standard anesthesia was short acting spinal anesthesia (35 patients) using hyperbaric bupivacaine 0.5% , but 14 patients received general anesthesia at the discretion of the anesthesia service because of technical and medical complications. For general anesthesia, we used fentanyl  as analgesic component with an expected duration of 30 minutes after intravenous injection. No nerve blocks were performed before or after surgery. Infiltration was done by the surgeon just before skin closure according to an illustrated step by step instruction and supervision by the first author when needed: through the incisional opening and under the guidance of fluoroscopy, 50 mL of the assigned solution was injected into the soft tissues surrounding the fracture and into the anterior and posterior part of the capsule. 25 mL were distributed into the fascia, subcutaneous tissues, and the skin. A multihole ON-Q Soaker Catheter™  was placed along the anterior side of the greater trochanter and connected to a bacterial filter. Wound drains were not used. Dicloxacillin (2 g) was given intravenously before surgery. For thromboprophylaxis, an injection of low molecular weight heparin 5.000 IE was administered subcutaneously for 7 days after surgery. In the case of nausea, metoclopramide 10 mg or ondansetron 4 mg was administered intravenously or orally. All patients were prescribed daily laxative treatment with 10 mg bisacodyl. Treatment of pain was standardized during the whole period of hospitalization. From time of admission to the hospital, all patients received 1 g paracetamol orally 4 times a day supplemented with opioid rescue analgesia as needed. Opioid drugs consisted in immediate-release oxycodone, 5 mg, and there were no restrictions on the frequency or the total daily dose of the opioid drug. During preoperative fast and postoperatively in the recovery ward, rescue medication was provided by the attending nurse as morphine bolus intravenously according to the cutoff values of pain as described in the following section. If patients were unable to rate pain during the recovery phase after general anesthesia, the administration of analgesics were based on the caregivers' subjective estimate of patients' needs.The primary outcome measure was consumption of opioid rescue analgesics. We registered the amount of consumed opioid drugs 5 days postoperatively from wound closure. A conversion factor  was usedhttp://www.epidata.dk) and Stata software . Data were analyzed using the Mann–Whitney U test (presented as medians with interquartile ranges) preceded by the Shapiro–Wilk test and Q-Q plots for normality. A p value of <0.05 was considered statistically significant. To compare the patients' pain at rest, by passive straight leg rise, and during weight-bearing physiotherapy, we analyzed our data by examining the maximum and medium pain ratings reported at rest and by passive straight leg rise. We also analyzed the number of pain scores that exceeded the predefined limits of tolerable pain (VRS > “slight pain”at rest or VRS > “moderate pain” by straight leg rise) as a measure of sufficient treatment of pain. This stratification of the data provides us with information about the quality of pain relief offered to the 2 groups of patients. Boxplots show interquartile ranges [P25–P75] with medians highlighted. Lengths of whiskers cover the interval from (P25 − 1.5 ∗ interquartile range) to (P75 + 1.5 ∗ interquartile range) or the nearest inside value. Outliers from this interval are indicated as circles.Data entry and statistical analyses were performed with EpiData software , (ondansetron P=0.6) during POD 1–5.We did not observe any serious adverse events directly related to the experimental treatment, and there were no statistically significant differences in the occurrence of side effects between the groups . The patTo our knowledge, this is the first randomized clinical trial evaluating the effect of wound infiltration for postoperative pain relief after osteosynthesis of extracapsular hip fractures. In previous studies, we investigated the effect of wound infiltration with ropivacaine in intracapsular hip fractures  and afteTraditional nerve blocks require advanced anesthesiological skills, and they involve potential risks of nerve injury. Not least, they might restrict early mobilization due to motor inhibition. The infiltration method has been reported to be safe and simple to perform, and in the view of the fact that hip fracture patients suffer from substantial postoperative pain and that undertreatment of postoperative pain remains a problem , 16, we There are several possible explanations for the lack of substantial effect. A striking feature is that the pain ratings are generally low if the patients are at rest. This may affect our results since assessment of the impact of our intervention presupposes a certain level of pain which might be restricted by limited mobility alone.P=0.005), which fits with these previous findings. This supports the exclusion of general anesthesia patients when quantifying intake of rescue analgesia , and we did not observe adverse effects directly attributable to the injections. We chose the 75 mL volume to ensure sufficient amount for the extensive perioperative infiltration. Likewise, the 20 mL volumes for catheter injections were chosen to aim for a widespread distribution of the solution. In contrast to other investigators , 14, 18 Pain was rated using the 5-point Verbal Rating Scale which we have found reliable for assessment of pain after hip fracture in a previous study . CognitiThe strength of our study is the randomized design with blinding of surgeons, patients, caregivers, and assessors which limits bias by diminishing conceivable differences in outcome caused by other factors than the intervention.Since our data does not support the practice of wound infiltration with ropivacaine after osteosynthesis of extracapsular hip fracture, we do not use the technique any longer.


Brachial plexus injuries can be debilitating. We have observed that manual reduction of the patients' shoulder subluxation improves their pain and have used this as a second reason to perform the trapezius to deltoid muscle transfer beyond motion. The authors report a series of nine patients who all had significant improvement of pain in the shoulder girdle and a decrease in pain medication use after a trapezius to deltoid muscle transfer. All patients were satisfied with the outcomes and stated that they would undergo the procedure again if offered the option. The rate of major complications was low. The aim is not to describe a new technique, but to elevate a secondary indication to a primary for the trapezius to deltoid transfer beyond improving shoulder function: pain relief from chronic shoulder subluxation. Brachial plexus injuries can be devastating. For optimal recovery, the current recommendations suggest treatment within 6 months from the time of injury.Patients with brachial plexus injuries present with two types of pain: (1) phantom pain from nerve root avulsion or associated nerve pain and 2) chronic subluxation pain in the shoulder. Most of the muscles around the shoulder are involved in the injury, leaving few muscles available for tendon transfer. The trapezius muscle is typically spared after pan-plexopathy because of its innervation by the spinal accessory nerve and contributions of C3 and C4. chronic We have observed that manual reduction of patients' shoulder subluxation often improves their pain. This observation provides a second reason to perform the muscle transfer beyond motion. Despite undergoing brachial plexus reconstruction, some patients request an elective amputation. With such amputation, pain was only improved in five of nine patients (55.6%), as reported by Maldonado et al.Patients were considered candidates for this procedure if there was pain relief on manual reduction of the shoulder subluxation in the office, which would provide a second reason for the surgery beyond motion. Shoulder shrug test was performed to ensure a functioning trapezius muscle, a prerequisite for the tendon transfer surgery. All patients had a brachial plexopathy involving the deltoid muscle. The procedures were timed to take place after the window for neurotization. A total of nine patients underwent a trapezius to deltoid transfer .We are describing our operative technique, which is not meant to be unique from those previously described in the literature. A U-shaped incision with a vertical extension was made surrounding the acromial joint and over the humerus inferiorly . The deWith the aid of fluoroscopy, three 4.5-mm cannulated screws were placed through the acromion process to mobilize it anterolaterally and as far distally as possible on the humerus while the arm was in an abducted and slightly rotated position . In somThe deltoid was then transferred into the trapezius muscle using modified figure-of-eight #0 PDS suture . Local anesthetic for postoperative anesthesia was injected into the surrounding muscle and incision. The patient was placed in a gunslinger splint in maximal shoulder abduction for 6 weeks.Six males and three females underwent the procedure described above. The patients' age ranged from 22 to 63 years (median: 39 years). Body mass index (BMI) of the patients ranged from 19.7 to 33.2 (median BMI: 27.1). The follow-up ranged from 5 to 118 months (median: 27.5 months). All patients (100%) had significant improvement in pain in the shoulder girdle. Fifty percent of patients who were taking pain medication prior surgery no longer required medication. Out of the remaining 50% of patients, two-thirds required less medication. All patients were happy with the outcomes and stated that they would undergo the procedure again if offered the option. Only seven of nine patients had data regarding postoperative shoulder abduction, two were lost to follow-up. Of those seven patients, the range of shoulder abduction was from 45 to 90 degrees (median: 45 degrees) . There In brachial plexus injuries, neurotization or primary nerve reconstruction is the gold standard in restoration of function. Sulaiman et al demonstrated that nerve transfers for reconstruction of brachial plexus injuries could result in excellent recovery of elbow and shoulder function. The accessory nerve is often utilized for the treatment of brachial plexus injuries. Sulaiman et al also reported that shoulder abduction recovery to Medical Research Council grade 3 after spinal accessory to suprascapular and/or thoracodorsal to axillary nerve transfer was 95 and 36%, respectively.We observed that manual reduction of our brachial plexus patients' shoulder subluxation improved their pain. All patients reported significant improvement of pain in the shoulder. The need for pain medication decreased in 33% of patients, while 50% discontinued use of all pain medication. Seven of the nine patients had documented shoulder abduction of at least 45 degrees (range: 45–90 degrees). The shoulder joint is unique in that the surrounding musculature must be functional to maintain reduction in the joint. Paralysis of the surrounding muscles will lead to subluxation, which is different from other joints in the body, i.e., the knee.Limitations to our report include the retrospective design of the study and small sample size heterogeneity in patient population. Most patients also had other procedures performed in the past with limited success and persistent shoulder subluxation. It is unlikely that these additional procedures would have addressed the chronic shoulder subluxation pain.Our results demonstrate that the trapezius-to-deltoid transfer may be considered in patients with shoulder subluxation pain with relief upon reduction as a primary reason for performing the surgery. The benefits of the procedure outweighed the potential complications with significant reduction in pain.Our article elevates an additional primary indication for the trapezius-to-deltoid transfer beyond improving shoulder function: pain relief from chronic shoulder subluxation.


Background: Central neuropathic pain represents one of the most common symptoms in multiple sclerosis (MS) and it seriously affects quality of life. Spinal mechanisms may contribute to the pathogenesis of neuropathic pain in MS. Converging evidence from animal models and neurophysiological and clinical studies in humans suggests a potential effect of transcranial direct current stimulation (tc-DCS) on neuropathic pain. Spinal application of DCS, i.e., transcutaneous spinal DCS (ts-DCS), may modulate nociception through inhibition of spinal reflexes. Therefore, ts-DCS could represents an effective, safe and well-tolerated treatment for neuropathic pain in MS, a largely unexplored topic. This study is a pilot randomized double-blind sham-controlled trial to evaluate the efficacy of ts-DCS on central neuropathic pain in MS patients.Methods: Thirty-three MS patients with central neuropathic pain were enrolled and randomly assigned to two groups in a double-blind sham-controlled design: anodal ts-DCS group  or sham ts-DCS group . The following clinical outcomes were evaluated before ts-DCS treatment (T0), after 10 days of treatment (T1) and 1 month after the end of treatment (T2): neuropathic pain symptoms inventory (NPSI), Ashworth Scale (AS) for spasticity and Fatigue Severity Scale (FSS). A subgroup of patients treated with anodal ts-DCS (n = 12) and sham ts-DCS (n = 11) also underwent a parallel neurophysiological study of the nociceptive withdrawal reflex (NWR) and the NWR temporal summation threshold (TST), two objective markers of pain processing at spinal level.Results: Anodal ts-DCS group showed a significant improvement in NPSI at T1, which persisted at T2, while we did not detect any significant change in AS and FSS. Sham ts-DCS group did not show any significant change in clinical scales. We observed a non-significant trend towards an inhibition of NWR responses in the anodal ts-DCS group at T1 and T2 when compared to baseline.Conclusions: Anodal ts-DCS seems to have an early and persisting  clinical efficacy on central neuropathic pain in MS patients, probably through modulation of spinal nociception.Clinical Trial Registration:www.ClinicalTrials.gov, identifier #NCT02331654. Pain represents one of the most disabling symptoms of multiple sclerosis (MS), in that it adversely affects most aspects of health-related quality of life and is often neglected and undertreated. The overall prevalence of pain syndromes in MS patients is 63%  represents a non-invasive, safe and well tolerated method for selectively modulating cortical excitability in a polarity-dependent way. Tc-DCS was documented to have an impact on a range of motor , may represent a non-invasive, safe, non-pharmacological and potentially self-administered approach to those conditions where pain is generated or becomes chronic through changes in spinal interneurons. In humans, anodal ts-DCS has been proven to inhibit nociceptive specific responses, such as the nociceptive withdrawal reflex  is to evaluate the short and middle-term effect of treatment with anodal ts-DCS on central neuropathic extremity pain in MS, using clinical scales and neurophysiological measures of spinal nociception. We focused on central neuropathic extremity pain, because it is by far the most common type of neuropathic pain in MS  and subsequently approved by the Local Institutional Ethical Committees of the participating centers. Before enrollment, all subjects gave their written informed consent in accordance with the Declaration of Helsinki.Thirty-three subjects  with a definite diagnosis of MS according to McDonald criteria , secondary-progressive (SP) and primary-progressive (PP) MS patients were enrolled in a follow-up procedure that included a general and neurological evaluation scored according to the Expanded Disability Status Scale of Kurtzke and its functional systems Kurtzke, . RR patiFor all participants, exclusion criteria were: (a) other neurological disorders, including primary or secondary headaches; (b) clinical or family history of neurological disorders; (c) any systemic or psychiatric disorder; (d) Beck Depression Inventory (BDI) scale score >9; (e) cognitive impairment ; (f) use of analgesics or steroids in the previous 24 h; (g) clinical or instrumental (including MRI) evidence of any central or peripheral disease/lesion potentially causing sensory impairment, including spinal lesions at lumbar level; (h) fibromyalgia; (i) complex regional pain syndrome; (j) chronic low back pain and other pain conditions not related to MS; and (k) changes in the schedule or dose of Disease Modifying Drugs (DMDs) for MS, antidepressants, antiepileptic drugs, tetrahydrocannabinol/cannabidiol or any other drug that may have a definite or potential effect on pain in the previous 3 months. Patients were excluded from the study if:-any change in the schedule or dose of drugs listed at point (l) above became necessary at any time during the observation period.-they had taken analgesics or steroids in the 24 h before the clinical and neurophysiological evaluations.Patients were randomly assigned to two groups: 19 patients were assigned to the anodal ts-DCS treatment group and 14 patients to the sham ts-DCS group. A subgroup of 12 patients treated with anodal ts-DCS and 11 patients treated with sham ts-DCS also underwent the neurophysiological evaluation of the NWR (see below).2 for both active and reference electrode. Electrodes were fixed inside by elastic customized stripes.Anodal and sham ts-DCS was delivered by a constant direct current electrical stimulator  connected to a pair of electrodes: the anode was placed on the thoracic spinal cord  and the cathode (reference) on the right shoulder in the suprascapular region. Stimulating electrodes consisted in 1-mm thick, rectangular (7 × 5 cm), rubber membranes, enveloped in a saline-soaked sponge. Conducting surface was 35 cm2 and delivered a total charge of 63.9 mC/cm2. These parameters are far below both the threshold for tissue damage and the conscious sensory threshold, apart from transient, and short-lasting tingling sensation below the electrodes at the start of the stimulation.For the real anodal ts-DCS group, we delivered a 2 mA constant direct current for 20 min in each session with a density of 0.071 mA/cmFor the sham ts-DCS group, electrodes were placed in the same spots than real anodal stimulation, but the stimulator was programmed to automatically turn to 0 mA after 10 s.We based the choice of the stimulation site and the related stimulation parameters on the results of our previous study in healthy subjects in which we demonstrated the effectiveness of the tsDCS in modulating the TST of the NWR  and a follow-up period of 4 weeks. Patients and assessing physicians were blind to group allocation; stimulator programming and electrodes applications were made by physicians not involved in the enrollment, clinical and neurophysiological evaluation, and data analysis.Patients were evaluated with clinical scales at baseline (T0), at the end of the 2-week treatment period (T1) and at 1 month from the end of treatment (T2). Characteristics and intensity of pain symptoms were collected at each stage with the Validated Italian Version of Neuropathic Pain Symptoms Inventory , NWR threshold (Rth), area under curve response (Area) and NWR TST and was administered at T0, T1 and T2.The NWR from the lower limbs was investigated according to a previously described and validated method . Electromyographic reflex responses were recorded from the capitis brevis of the biceps femoris muscle via a standard pair of Ag/AgCl surface electrodes. The analysis time was 300 ms, with the sensitivity set at 100 mV, the filter bandpass setting was between 3 Hz and 3 kHz .The sural nerve was stimulated percutaneously In order to evaluate the efficiency of the sensory pathways, the Sth was determined at the start of each experimental session. The previously described pulse train, increased or decreased in 0.1 mA steps were delivered at unpredictable intervals of ±10 s. Subjects were asked to indicate verbally the stimulation levels at which they became aware of sensory sensations. The sensory threshold was defined as the lowest stimulation intensity generating a stable sensory sensation.The staircase method was used to evaluate the Rth, defined as the lowest stimulation intensity generating a clearly detectable nociceptive reflex response exceeding 20 μV for 10 ms or more in the time interval 80–130 ms over five pulse train randomly delivered every 20–40 s and hence the related pain perception. The intensity of stimulation was then fixed at 1.2 Th and randomly delivered every 5–20 s to avoid habituation phenomena. Each response was full-wave rectified and integrated between set points from 80 to 130 ms after the start of the test stimulus. Five reflex responses were recorded and the mean NWR Area was computed by means of a computerized method.To evaluate the NWR TST, the previously described stimulation/recording setting was used. The pulse train of five individual 1-ms pulses delivered at 200 Hz was repeated five times at a frequency of 2 Hz. The stimulus intensity was increased (in 1 mA steps) from 2 mA until detection of temporal summation of the NWR. The NWR TST was defined as the lowest stimulation intensity generating a stable NWR response at the fourth and fifth pulse train of the five-train series and exceeding 20 μV in the time interval between 80 and 130 ms for a period of more than 10 ms. A threshold was accepted when three consecutive recordings resulted in the same threshold. Subjects rated the subjective pain sensation for each stimulus on a 0–11 numerical rating scale, . The mean psychophysical pain sensation at Th (NRS at Th) and at first (NRS1) and fifth (NRS5) stimulus of the five delivered to evoke the TST was calculated.t-test for related samples or, in presence of multiple time measurements, a repeated measures analyses of variance (ANOVA) and post hoc with Bonferroni’s correction. To assess differences between groups at each time point we used a Student’s t-test for unpaired samples. The level of significance (α) was set for convention as p = 0.05 for all the tests, and always corrected where necessary.The Statistical Package for the Social Sciences (SPSS) for Windows, version 21.0, was used for the calculation. For each variable we evaluated skewness and kurtosis to assess normality. Moreover, the data were plotted using a “Q-Q plot” that confirmed normal distribution of all tested variables. For qualitative variables we used cross-tabs analysis, performing statistical significance with chi-square or Fisher exact test. Quantitative variables are presented as mean values ± standard deviation. To assess the intragroup acute effect, we used a Student’s Demographic features and baseline parameters of participating groups are reported in F = 5.175; p = 0.013) at both T1 (Bonferroni’s corrected paired t-test: p = 0.023) and T2 (p = 0.008) when compared to T0  4.194; p = 0.049) and T2  6.637; p = 0.015; The intergroup analysis showed a significant improvement in the NPSI score in the active ts-DCS group compared to the sham ts-DCS group at both T1  in the active ts-DCS group at T1 when compared to T0, suggesting a reduced pain processing. However, no significant difference was found in either active or sham ts-DCS groups when comparing the neurophysiological and related psychophysical parameters recorded at T1 or T2 with T0 values . SimilarThe main result of this study is that a 2-week period of daily anodal ts-DCS at the thoracic level induced a significant and persisting improvement in pain severity in patients with MS and central neuropathic extremity pain. Indeed, the observed improvement in pain symptoms persisted for further 4 weeks after the end of the stimulation period. On the contrary, the sham stimulation did not influence clinical parameters,  in the control group. Neurophysiological parameters, including Rth and TST of the NWR, showed a parallel pattern to the clinical findings when considering the T0 and T1 time point, although the neurophysiological changes did not reach a statistically significant level. Taken as a whole, these findings are in line with previous reports of inhibition of pain reflex responses after anodal ts-DCS in healthy subjects  or lesion load site . Further studies are needed to evaluate the effect of ts-DCS on different types of MS-related pain, either neuropathic or nociceptive. Moreover, studies that stratify patients according to disease type and the presence or spinal lesions could better clarify mechanism of ts-DCS in MS.In conclusion, our results suggest that ts-DCS may improve central neuropathic pain in MS patients for up to 4 weeks. Spinal DCS could be a promising, non-invasive, well-tolerated and potentially self-administered treatment for neuropathic pain in MS patients. This type of DCS may play a role as an add-on treatment in drug-resistant pain or in patients who poorly tolerate pharmacological treatment.The study was designed by RB, GS and CT. Patients selection and enrolment, collection of clinical and instrumental data was performed by EB, RB and CD in IRCCS Mondino Foundation, by AP in IRCCS Neuromed, by MR and GP in IRCCS Santa Maria Nascente, by MG and FM in Santa Lucia Foundation. Data was analyzed by EB and RDI and interpreted by RDI and ST. The manuscript was drafted by EB. AP, MR, RDI, CD, MG, ST, RB, GP, FM, GS and CT revised critically the manuscript. All authors approved the final version of the manuscript, and agreed to be accountable for all aspects of the work submitted.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Evaluate the effectiveness of LLL (Low level laser therapy) in auriculotherapy points for pain reduction following lower third molar extractions.Randomized, controlled, single-blinded study.Eighty-four bilateral, symmetrical third molar surgeries were performed in 42 healthy patients using a split-mouth design. In the immediate postoperative period, each side was randomly treated in a single-blind method with an LLL at the auriculotherapy points or simulation of its use  over a 21-day interval. This protocol was repeated 24 and 48 hours after surgery. All patients used the same analgesic (paracetamol) but only in case of pain. The primary variable was postoperative pain according to the visual analogue scale, and the secondary variables were mouth opening, edema, local temperature, dysphagia, and the presence of infection . These variables were evaluated at baseline and at 24 hours, 48 hours and seven days after surgery. Adverse effects were recorded and reported.There was no difference between the groups in relation to any of the evaluated parameters (p>0.05).For this experimental model, application of a low-intensity laser at auriculotherapy points did not prevent postoperative pain following lower third molar surgery.https://register.clinicaltrials.gov/prs/app/template/EditRecord.vm?epmode=View&listmode=Edit&uid=U0002BEY&ts=11&sid=S0006026&cx=6g4wff).This trial is registered at ClinicalTrials.gov; the registration number is NCT02657174 and the Unique Protocol ID number is 1.100.869. ( The third molar extraction postoperative period is accompanied by pain and edema; the control of which are essential for both the patient and dental surgeon. The intense inflammatory reaction within the three first days of the postoperative period compromises the quality of life for patients ,2,3,4. TAcupuncture has been used for many years in several areas of healthcare to control postoperative pain ,14, and Artemisia vulgaris. This plant appears to have unique features.In auriculotherapy practice, various modalities can be adopted including auricular acupuncture, auricular electroacupuncture, acupressure, moxibustion, injection, and auricular bloodletting therapy ,14,16,18Auriculotherapy can be defined as a system of diagnosis and treatment through stimulation of localized points on the ear ,20,24 inth, 2015  under number 1.100.869 on June 10th, 2015 –S4 Filesth, 2015 , so the th, 2016 (before patient recruitment). The protocol was submitted for publication in Trials [th, 2016. The study started  in June 2016. Recruitment of the participants for this study started in July 2016, which was six months after the study was registered on clinicaltrials.gov. Primary completion of the study occurred in February 2017, and study completion (including follow up) occurred in April 2017. The authors confirm that all ongoing and related trials for this drug/intervention are registered. This study follows CONSORT statement  on January 11n Trials  on Januatatement Surgeries were performed at the Dental Clinic of Nove de Julho University–UNINOVE and at the Military Police Dental Center of São Paulo State in the city of São Paulo, Brazil. In June 2015, when our protocol was approved by the Research Ethics Committee, the plan was to perform 120 surgeries on 60 patients based on the scenario with largest required sample size. At that time, we had not found any similar studies to compare the differences between the mean values in auriculotherapy field and pain. Subsequently, we found a study that evaluated pain after auriculotherapy treatment. Therefore, we decided to re-calculate the sample size for better accuracy. The mean values of the control and treated groups, as well as SD, were obtained from this study  and the Forty-two patients of both genders, aged between 18 and 28 years, underwent 84 surgical removals of bilateral and symmetric third molars. Two surgeries were performed in the same patient with a 21-day interval; thus, this was a split-mouth study. In general, the population is young healthy adults because these are the individuals who typically need to undergo third molar surgery.The following patients were excluded from the study: pregnant or breastfeeding women, smokers, those who had undergone head and neck radiotherapy, those who were allergic to any drugs used in the research (such as paracetamol or 2% chlorhexidine), those with systemic or local infections , those who had used anti-inflammatory drugs in the last 3 months, those who used medications differently than the method in which they were prescribed, and those with lesions or radiolucent images associated with the teeth to be extracted. Patients who presented with complications during surgery  were also excluded because these cases are not expected for third molar surgeries, and only in the aforementioned cases, a centrally acting analgesic was prescribed. These data were not part of the statistical analysis but were described and discussed including trans-surgical complications and possible adverse effects. Patients with teeth in position IIB  with indAn external researcher (I.P.T.) performed the randomization . The randomization was blocked in 45 pairs of numbers (1:1), i.e., 45 AB or BA blocks. Neither the patient nor the surgeon or those assessing the outcomes knew which treatment was applied in each of the two surgeries. The only individual who was aware of the treatment performed was the acupuncturist who applied the laser (single-blinded study). The drawn treatments (A or B) were placed inside opaque envelopes identified with sequential numbers. The envelopes were sealed and remained sealed in the same numerical order in a secure place until the surgery was performed. The study design is shown in Each patient served as his or her own control:G1 -  (n = 42 surgeries)–surgery was performed in a conventional manner, and the patient received LLL treatment at auriculotherapy points to prevent pain and inflammation in the immediate postoperative period and within 24 and 48 hours after intervention .G2 - (control) (n = 42 surgeries)–the procedures were performed in the same way as in Group 1 but with the laser turned off.The use of a verum or placebo laser was randomized for the first surgery. For the second surgery, the opposite treatment was performed , then the second surgery must be B and vice-versa). We did this to prevent the patient from receiving the same treatment twice.® 500 mg every 8 hours was prescribed. All patients were instructed to use the medication only in case of pain. Additionally, in case of unbearable pain, a prescription of paracetamol with codeine phosphate 30 mg Tylex® (Janssen-Cilag) was provided for ethical reasons. The medication data were described in the results analysis. The auriculotherapy points used in this study have been described previously [®) with a wavelength of 660 nm (±10 nm) and power of 100 mW was used. The optical fiber diameter was 600 μm, so the spot (area) was 0.002826 cm2. The energy delivered per point was 1 J over 10 seconds. Six points were irradiated, with a total energy of 6 J. The power density was 35.4 mW/cm2. For accuracy in determining the points, we used the ear locator for auriculopuncture , which is based on the principles of electrical potential least resistance, allowing greater accuracy [One researcher assigned participants to their interventions (HSF). In both groups, postoperative doses were applied 24 h and 48 h after the intervention. For all patients, paracetamol Tylenoleviously . A red dAll variables were evaluated by the same operator (G.N.) at baseline and after 24 hours, 48 hours and 7 days. The primary variable of the study was postoperative pain, which was assessed by visual analogue scale (VAS). The secondary variables included mouth opening, edema, local temperature, dysphagia and presence of infection (systemic temperature or lymphadenopathy).rd molar (including muscular tissue). Difficulty in mouth opening shows more exudate and higher levels of inflammation. The temperature was measured locally because of local inflammation and systemically to detect fever and verify the presence of an infection. Systemic temperature was measured 3 cm above the glabella using a digital thermometer . The local measurement was performed in the jaw angle region 2 cm above the lower border of the mandible and 3 cm paramesial from mandibular branch, both on the operated and opposite sides. Dysphagia was also evaluated to complement the detection of clinical features of inflammation/infection. Dysphagia assessment was performed using a numerical scale as follows: (0) total absence of dysphagia; (1) dysphagia to solid food and (2) dysphagia to any liquid or solid food.Postoperative pain was assessed by applying the visual analog scale (VAS) with a 100-mm line with two ends including "0" meaning no pain and "100" meaning unbearable pain; the VAS was always evaluated by the same operator. Each patient was instructed to mark with a vertical dash the point that best corresponded to his or her pain intensity at the time of evaluation . Edema mWe decided to exclude 4 patients. One of the patients was excluded due to omission of systemic problems during anamnesis. Another patient had a postoperative infection, and two patients withdrew from the study during the postoperative period.The data were analyzed using STATA 15. We chose to use mixed effects models, including random effects at the individual patient level in this analysis, and treated the data as an unbalanced panel, given that not all measurements were recorded for all patients at each time point. This approach is consistent with that described by Wooldridge (2010). All models included the variables sex, use of medication and length of surgery as covariates as these can be considered potential confounding variables. The models were evaluated with regards to the relationship between the primary (or secondary) outcome variables and the variable Group, which was set up as a dummy equal to zero if the patient had received a placebo treatment and equal to one if the patient received LLL treatment. Our null hypothesis was that the proposed protocol for LLL in auriculotherapy points is not effective for pain reduction following lower third molar surgeries. Statistical significance was declared at the 0.05 level. F-tests of joint significance and t-tests of individual significance were conducted and are reported for all models. Additionally, we fitted the predicted regression results against residual plots to evaluate normality, symmetry and independence, although given the use of the mixed methods models, those plots only referred to the fixed effects component of the general modeling equation. The regression results of the modeling performed can be found in the supplementary materials  also theWhen evaluating the presence of lymph nodes as a dependent (secondary) outcome variable, due to lack of variation in the data, we also performed a random effects model analysis, as the fixed effects component resulted in problems of multicolinearity.The demographic data are presented in the tables . ScatterAs well as the inferential analysis, we have compared means and standard deviations at four timepoints: baseline, postoperative, 24 hours, 48 hours and 7 days. The presented graphs compare means and standard deviations of all variables for the two groups in the four timepoints. Furthermore, given that our outcome measures are all time sensitive, we have explored mixed effects models in which measured outcomes in time 12hours, 24hours and 7 days have been used to explain the variation in the outcome measured at baseline. We acknowledge that this further analysis serves a different purpose, i.e. does not explain the variation between groups at any given time point, but it provides some reassuarance about the data collected, as it is expected that a significant relationship between any outcome at baseline and its repeated later measurements.Forty-two patients underwent surgery, but we have excluded some participants from analysis. One of the patients was excluded due to omission of systemic problems during anamnesis. Another patient had a postoperative infection, and two patients withdrew from the study during the postoperative period. Thus, 38 patients and 76 surgeries are included in the statistical analysis.The regression results of the modeling performed can be found in the supplementary materials  also theAlthough they can each be examined individually, the results all point to the same direction and indicate that we cannot reject the null hypothesis as there was no significant difference between the placebo and the LLL treatment groups. It is worth noting that only the secondary outcome variables Edema (I), (II) and (III) and mouth opening passed the F-test of joint significance at p = 0.05.There was no difference between the groups regarding the mean number of anesthetic cartridges (p = 0.19). The mean duration of surgery was 31 min in the laser group and 33 min in the placebo group (p = 0.409). The number of tablets required for postoperative pain management was 1.47± 2.50 in the laser group and 1.57 ± 3.12 in the placebo group.There was no significant difference in postoperative pain between G1 and G2 at baseline (p>0.05), 24 hours (p>0.05) after surgery, 48 hours after surgery (p>0.05) or 7 days after surgery (p>0.05) . There wFor edema Figs , 6 and 7Regarding the variables, we performed the correlation test between them for each timepoint. The only correlation detected was in the 24-hour period between pain and mouth opening . The PeaThe intake of pain medication was described in Regarding temperature, local temperature on the operated side  and oppoThere was also no difference between groups in systemic temperature  and lynpThere were some clinical complications during the study. One of the patients was excluded due to omission of systemic problems during anamnesis. Another patient had a postoperative infection and two others gave up in the postoperative period of the second surgery.Considering the importance of studying postoperative pain after third molar surgery, this study evaluated a new, alternative treatment to minimize the use of analgesics and anti-inflammatory drugs because many adverse effects have been observed with their use ,12. In tAs an alternative to this protocol, various modalities of auriculotherapy could be tested including auricular acupuncture, auricular electroacupuncture, injection, acupressure, moxibustion, and auricular bloodletting therapy . AuriculArtemisia vulgaris can be used for several treatments with moxibustion yielding impressive results [Artemisia vulgaris extract has anti-inflammatory and antioxidant properties [Classical acupuncture is another interesting option to test pain reduction after third molar surgery. Acupuncture can be used in local  and distant body points . Currently,  results . Artemisoperties . This pl2 [2. Since the amount of energy required for this type of treatment is low, we chose to deliver 1 J at each auriculotherapy point with a power density of 35.4 mW/cm2. According to the precepts of Chinese medicine, each individual is a unique being, and an individualized protocol should be developed for each patient. However, to obtain scientific evidence, we had to standardize the interventions to be performed in each patient. Therefore, we sought comprehensive, classically recognized and simple points of application in the daily clinic. The ear points chosen were based on the Chinese school [Regarding the study methodology, rehabilitation of patients was accomplished with a red diode laser at a wavelength of 660 nm (± 10 nm). It has been shown that wavelengths of 650 to 950 nm can penetrate biological tissues up to 3 millimeters . Other a2  using dee school  as suggee school  and weree school , 41–43. e school . The Shee school ,26,41–43e school ,43. The e school . To datee school ,18,25.To ensure better methodological quality, this study followed the recommendations of the Standards for Reporting Interventions in Clinical Trials of Acupuncture (STRICTA), which is a new protocol specific to acupuncture studies . To ensuFinally, although some of the residual plots presented –S11 FigsA systematic review  of 17 raFor this experimental model, low-intensity laser treatment at auriculotherapy points did not prevent postoperative pain in lower third molar surgeries.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(PDF)Click here for additional data file.S5 File(PDF)Click here for additional data file.S6 File(PDF)Click here for additional data file.S7 File(PDF)Click here for additional data file.S8 File(DOCX)Click here for additional data file.S1 FigX axis—residuals, Y axis–predicted values.(JPG)Click here for additional data file.S2 FigX axis—residuals, Y axis–predicted values.(JPG)Click here for additional data file.S3 FigX axis—residuals, Y axis–predicted values.(JPG)Click here for additional data file.S4 FigX axis—residuals, Y axis–predicted values.(JPG)Click here for additional data file.S5 FigX axis—residuals, Y axis–predicted values.(JPG)Click here for additional data file.S6 FigX axis—residuals, Y axis–predicted values.(JPG)Click here for additional data file.S7 FigX axis—residuals, Y axis–predicted values.(JPG)Click here for additional data file.S8 FigX axis—residuals, Y axis–predicted values.(JPG)Click here for additional data file.S9 FigX axis—residuals, Y axis–Fitted values(TIFF)Click here for additional data file.S10 FigX axis—residuals, Y axis–Fitted values.(TIFF)Click here for additional data file.S11 FigX axis—residuals, Y axis–Fitted values.(TIFF)Click here for additional data file.