Background and Objective: Thrombosis due to inherited hypercoagulability is an issue that has been raised in microvascular flap surgery previously. We analyzed the association of a single nucleotide polymorphism (SNP) in rs2066865 in the fibrinogen gamma chain (FGG) gene, alteration in plasma fibrinogen concentration, and presence of microvascular flap thrombosis. Materials and Methods: A total of 104 adult patients with microvascular flap surgery were subjected to an analysis of the presence of SNP rs2066865 in the FGG gene. Alterations in plasma fibrinogen concentration according to genotype were determined as a primary outcome, and flap thrombosis was defined as a secondary outcome. Results: Flap thrombosis was detected in 11.5% of patients (n = 12). Successful revision of anastomosis was performed in four patients, resulting in a microvascular flap survival rate of 92.3%. We observed an increase in plasma fibrinogen concentration in genotype G/A and A/A carriers ; G/A, 4.28 (IQR 5.38-3.18); A/A, 6.87 (IQR 8.25-5.49) . Within group differences in microvascular flap thrombosis incidence rates were observed—G/G 6/79 (7.59%); G/A 5/22 (22.7%); A/A 1/3 (33.3%) , p = 0.016; RR 3.2—when G/G versus G/A and A/A were analyzed respectively. Conclusions: A/A and G/A genotype carriers of a single nucleotide polymorphism in rs2066865 in the fibrinogen gamma chain gene had a higher plasma fibrinogen concentration, and this might be associated with an increased microvascular flap thrombosis incidence rate. Determined polymorphism could be considered as a genetic marker associated with microvascular flap thrombosis development. To confirm the results of this study, the data should be replicated in a greater sample size. Flap thrombosis is still the leading cause of flap loss, resulting in patient and surgeon disaffection as well as increased hospital stay and costs. Overall, thrombotic events are attributable to 3% to 12% of flap complications. Although technical factors are of constant concern, inherited thrombophilia has been mentioned as a potential risk factor. Factor V Leiden, prothrombin gene mutation, protein C and protein S deficiencies, antithrombin deficiency, and mutation of the Thrombosis is a heterogeneous disorder . Microcifibrinogen alpha (FGA), fibrinogen beta (FGB), and fibrinogen gamma (FGG), clustered in a region of approximately 50 kb on chromosome 4q31.3 [FGG gene is described as a reason for alterations in the coagulation system. Particularly, fibrinogen gamma H2 haplotype-tagging SNP (FGG 10034C > T) has been demonstrated as a risk factor for deep venous thrombosis [Fibrinogen is a plasma glycoprotein with a molecular weight of 340 kDa and is synthesized by hepatocytes. Fibrinogen molecules are elongated structures 45 nm long, which are comprised of two sets of three polypeptide chains—A alpha; B beta; and gamma . The thre 4q31.3 . Polymorrombosis . Haplotyrombosis . FGG gene alters the level of plasma fibrinogen concentration and could associate with an increase in the microvascular flap thrombosis rate.Therefore, we tested the hypothesis that SNP rs2066865 in the In this observational case series, we included a total of 104 patients who underwent microvascular flap transfer in The Centre of Plastic and Reconstructive Microsurgery of Latvia from 2016–2018.We enrolled all adult patients undergoing microvascular flap surgery during the study period. The protocol and the informed consent form, including the request to donate genetic material, were approved by the Latvian Central Ethics Committee (Nr.1/28-11-16). All patients provided written, informed consent.The exclusion criteria were: pregnancy, peripartum period; transfusion of allogeneic blood components, and/or coagulation factors within 72 h perioperatively; proven left ventricular failure; allogenic bone marrow transplantation; liver failure, liver transplantation; and end-stage kidney disease.For patients on direct oral anticoagulants, medication was stopped 72 h prior to sample collection to avoid any inaccuracy in testing. For patients taking a vitamin K antagonist, medication was stopped, according to the patient’s international normalized ratio (INR) level, 4 to 5 d prior to sample collection. Patients with a high thrombosis risk were switched to low-molecular-weight heparin (LMWH) and discontinued use 12 h prior to the surgery. FGG gene and collected laboratory data, including total plasma fibrinogen concentration, platelets, white blood count, and C-reactive protein.We determined SNP rs2066865 (G > A) in the An interview was performed to register patient histories, particularly with interest in any previous thrombotic event, use of antithrombotics, regular medication , family history of thrombotic events, and any previously diagnosed inherited thrombophilias  We registered all tissue injury causative factors and defined 30 d as a recent trauma period in case of trauma or polytrauma etiology.In regards to demographics, history, and family history collection, patient interviews were performed by the same clinician. A positive history of thrombosis was defined as any thrombotic event with either arterial or venous origin. Group malignancy consisted of patients with orofacial tumors in different stages  after treatment with actinotherapy and palliation.t 1°C was considered as the optimal difference between core and peripheral temperature. Infusion of warm fluids and a warming blanket were used to prevent a drop in the core temperature. During the study, a total of 104 microvascular flap transfers were performed by highly trained specialists. The venue of thrombosis  was assured by direct visualization during the revision of anastomosis.All patients received standardized general anesthesia. According to our local guidelines, administration of 10 mL/kg of Dextran 40  intravenously, at the time the anastomoses were assured, was performed. Patient temperatures were monitored during the surgery. We measured axillar and nasopharyngeal temperatures, and ∆Blood samples were drawn on the day of surgery prior to the induction of anesthesia and any crystalloid infusion. All tests were processed within an hour. 9/L) was measured using the Sysmex XP-300 . C-reactive protein was measured in serum with radial immunodiffusion with fixed-point immune rate methodology . Total plasma fibrinogen concentration was measured by the Clauss method    on a Viit-tests by using the SPSS 23 Statistics software . The Kolmogorov–Smirnov test was used to check whether the variables followed a normal distribution. Normally distributed, continuous variables were presented as mean ± standard deviation (M ± SD) and categorical variables as percentages (%). In case values did not follow a normal distribution, the medians and interquartile ranges (IQRs) were presented. Odds ratios and 95% confidence intervals were calculated to evaluate factor impacts between groups. Comparisons between genotype groups were performed with Kruskal–Wallis H tests for nonparametric variables and with ANOVA for parametric variables. Pearson’s x2 correlation coefficient and p values were calculated, and Spearman’s rank correlation coefficient was used where applicable. Statistical significance was assumed as two-tailed p < 0.05.We compared variables with independent-sample (unpaired) FGG-carrying genotype: G/G, n = 79; G/A, n = 22; and A/A, n = 3. The genotype results of rs2066865 in the FGG A/G polymorphism were all in Hardy–Weinberg equilibrium. The characteristics of the studied group are listed in FGG. In total, 104 consecutive patients scheduled for microvascular flap surgery were subjected to analysis after inclusion and exclusion criteria were met. We analyzed 86 males and 18 females classified according to rs2066865 in the n = 12). After unsuccessful salvage, re-anastomosis total flap necrosis eventuated in 7.7% (n = 8) of patients, and partial flap necrosis occurred in 4.8% (n = 5) of patients, resulting in a microvascular flap survival rate of 92.3%  respectively , white blood count, C-reactive protein, and platelet count parameters did not differ between G/G, G/A, and A/A genotype carriers. A/A carriers showed a tendency to have higher plasma C-reactive protein levels without reaching significance (G/G 18.39 vs. G/A 17.62 vs. A/A 31.00 (mg/L); p = 0.372).Although we observed a positive association between C-reactive protein and increased plasma fibrinogen concentrations (FGG gene carriers of A/A and G/A genotypes had higher levels of plasma fibrinogen concentrations (G/G 3.9 (IQR 4.76–3.04); G/A 4.28 (IQR 5.38–3.18); A/A 6.87 (IQR 8.25–5.49); A/A vs. G/A, p = 0.003; and A/A vs. G/G, p = 0.001), as shown in Patients with SNP rs2066865 in FGG gene, alteration in plasma total fibrinogen concentration, and thrombotic events in microvascular flap surgery.We evaluated the association between a single nucleotide polymorphism rs2066865 in the In our studied group, patients with a variety of injury-causing factors underwent microvascular flap surgery. Overall, our results are within the range of microvascular flap survival rates reported in literature. Shechter et al. analyzed two groups of patients with breast cancer and radiation therapy who underwent postmastectomy breast reconstruction with a deep inferior epigastric artery perforator (DIEP) flap. They reported a total of 8.3% flap loss and vascular anastomosis failure in 5.6% of patients with additional boost radiation compared to zero percent for standard post mastectomy radiation therapy. They concluded that added radiation therapy potentially increased the risk for surgical complication . Bendon An increased plasma fibrinogen concentration has been proposed to reflect the inflammatory state , and infFGG gene. None of the haplotypes was associated with alterations in the total fibrinogen level, although one of the haplotypes, H2, was strongly associated with reduced fibrinogen γ’ levels and a reduced γ’/γ ratio, and both markers were associated with increased risk for venous thrombosis. This indicates that only one FGG H2 haplotype increases the risk of venous thrombosis by reducing fibrinogen γ’ levels [Interestingly, the reduced levels of fibrinogen γ’/γ and reduction in the γ’/γ ratio were strongly associated with an increase in venous thrombosis. In a study by Uitte de Willige et al., five haplotypes were identified in the ’ levels . In our Rosendaal et al. found correlation between increased age and changes in fibrinogen levels and an increased risk for thrombosis mainly of venous origin . We founThe study by Hollenbeck et al. presented data about the role of preoperative platelet count as a predictor of free flap thrombosis. They evaluated a total of 565 acute trauma patients who underwent lower extremity free tissue transfer, and they concluded that acute trauma patients with elevated preoperative platelet counts were at an increased risk for lower extremity free flap thrombotic complications . In contThere were a few limitations in this study. First, as this is a clinical, observational case series study, our sample size was rather small for genetic study. In the Centre of Plastic and Reconstructive Microsurgery of Latvia, around forty microvascular flap surgeries are performed per annum; therefore, we were not able to strengthen our finding by means of genotype intergroup analysis, demonstrating that A/A carriers were associated with an increased risk of microvascular flap thrombosis. In addition, development of thrombosis is multifactorial, including a patient’s related factors and other gene mutations, such as factor V Leiden and prothrombin gene mutation in the heterozygous state, which both present the most common hereditary thrombophilias but possess a relatively low risk for venous thrombosis. Risk increases in the presence of other risk factors . Deficiencies in antithrombin, protein C, protein S, factor V Leiden, and prothrombin gene in the homozygous state hold greater risk for venous thrombosis, but these mutations are considerably rarer. The observed patients and their family members had a positive history of thrombotic events. As we were focusing on a particular SNP, we were not able to exclude the role of other gene mutations. To add, many patients harbor one or more hereditary or biological risk factors that are not recognized with available methods, and venous thrombosis due to biological causes is found in up to 16.93% of unselected patients . It woulNature (1968) [Secondly, our local perioperative guideline suggests administration of Dextran 40 because of its rheological properties in all patients at the time anastomosis is assured. Thus, its contribution to outcomes in our studied group should be kept in mind. The multiplicity of Dextran 40 effects is governed either by the ability to expand the volume of plasma and, therefore, reduce the hematocrit, which in turn lowers blood viscosity, and, on the other hand, by inhibiting the formation of erythrocyte aggregates as reported by Rosenblum in e (1968) . In the e (1968) , Dextrane (1968) .Another limitation of the study is the multifactorial nature of the thrombotic event and the difficulty in analyzing the variables separately. Patient-related factors for both arterial and venous thrombosis are described in arterial and venous risk assessment scores, CHAD2S2-VASc and Caprini scores, respectively. In microvascular flap thrombosis, both arterial and venous pools play a role; therefore, we were not able to define an appropriate score for patient thrombotic risk assessment. However, it has been mentioned that the 2005 Caprini Risk Assessment Model could also be applied to patients undergoing microvascular flap surgery, although its ability to predict microvascular thrombosis has not been evaluated .And finally, the patients were localized in the trauma department, and during the postoperative period, a lack of critical clinical monitoring of flap perfusion provoked late readmission to the operating theater, resulting in a 40% salvage rate after revision of anastomosis.FGG gene have higher plasma fibrinogen concentrations, and this might be associated with an increased free flap thrombosis incidence rate.The results demonstrate that A/A and G/A genotype carriers of a single nucleotide polymorphism in rs2066865 in the The determined polymorphism could be considered as a genetic marker associated with microvascular flap thrombosis development. Patients that have the determined polymorphism could benefit from perioperative thromboprophylaxis. To confirm the results of this study, the data should be replicated in a greater sample size.


Clinical depression is characterized by high levels of negative affect (NA) and attenuated positive affect (PA). Psychological and pharmacological treatments have been shown to reduce NA and to enhance PA in depressed patients. Following dual-process models, two types of affect can be distinguished: explicit (or self-reported) affect, which is formed by conscious reflections, and implicit affect, which relates to automatic affective reactions. The present study was conducted to examine, for the first time, both implicit and explicit affectivity in patients suffering from acute depression. Moreover, changes in patients’ implicit and explicit affectivity were investigated over the course of inpatient treatment.Thirty-nine patients suffering from major depression and 39 healthy individuals participated in the study. Implicit affectivity was assessed using the Implicit Positive and Negative Affect Test. The explicit state and trait affectivity were measured by the Positive and Negative Affect Schedule. The level of depressive symptoms was assessed with the Beck Depression Inventory. Tests were administered to patients after admission and after 7 weeks of therapy, whereas healthy controls were investigated only once. We examined whether either comorbidity or antidepressant medication has an effect on affectivity.Patients with acute depression had lower implicit and explicit PA scores and higher implicit and explicit NA scores than the healthy controls. After treatment, patients’ level of depression decreased significantly. At posttreatment, patients exhibited heightened implicit and explicit PA and diminished explicit trait NA. Independent of antidepressant medication and comorbidity, no significant change in implicit NA was observed over the course of treatment. Implicit NA was correlated with explicit NA in acute depression but not during recovery.Acute depression appears to be characterized by decreased implicit and explicit PA and increased implicit and explicit NA. After 7 weeks of treatment, depressed patients’ implicit and explicit PA increased, and explicit trait NA decreased. No decrease in implicit NA and explicit state NA occurred over the course of treatment. Finally, it seems that in the state of acute depression, the interplay between the automatic and reflective systems could be increased for negative affectivity. The essential features of major depressive disorder are a prolonged depressed mood and diminished interest or pleasure in response to situations or stimuli that previously generated positive emotions. Watson and Tellegen  proposedThere is growing evidence that the impaired ratio between PA and NA in depression can be altered by psychological and pharmacological treatments. Oren-Yagoda et al.  demonstrexplicit affect. Explicit affect is formed by conscious reflections and comparisons between affective episodes ).ch other . The IPAch other . Since ich other . Thus, bch other . The invThe IPANAT has been shown to be a valuable predictor of spontaneous behavioral and psychophysiological reactions to emotion stimuli and stressors in healthy individuals. Implicit NA, as assessed by the IPANAT, is associated with unintentionally occurring processes of attention allocation to dysphoric stimuli . These fThe aim of the present study was to examine, for the first time, both implicit and explicit affectivity in patients suffering from acute clinical depression. We hypothesized that depressed patients differ from healthy control subjects concerning positive and negative affect. Specifically, on the basis of previous findings on explicit affectivity, it was expected that depressed patients would manifest lower positive state and trait affect scores and higher negative state and trait affect scores than healthy controls. Similarly, it was hypothesized that depressed patients exhibit heightened negative implicit affect and reduced positive implicit affect compared to healthy individuals. A second aim of our study was to investigate changes in the implicit (and explicit) affectivity of depressed patients over the course of a naturalistic inpatient treatment program. Therefore, patients were reexamined after 7 weeks. We expected that depressed patients’ implicit and explicit positive affectivity increases, and that implicit and explicit negative affectivity decreases over the treatment program. Whether posttreatment patients show a prevalence of PA versus NA, as typically observed in healthy individuals, was explored. Finally, we examined the relationships between implicit and explicit affectivity in acute and partially remitted depressed patients compared to healthy individuals.The patient group included 39 inpatients (26 women and 13 men) with an acute major depression episode according to the Structured Clinical Interview for DSM-IV Axis I Disorders (SCID-I ). The ext(58.81)p > .05. However, the patients reported, on average, a lower education level compared to the controls  without a history of psychiatric diseases. Diagnoses of current or past psychiatric disorders were determined by the Mini International Neuropsychiatric Interview . The MIM = 2.23 weeks, SD = 0.81 weeks) and, on average, after 7 weeks of therapy . The SCID-I [This study was approved by the local ethics committee of the Medical School of the University. After a detailed explanation of the study, written informed consent was obtained from all participants. All subjects received financial compensation after completion of the tasks. Healthy control subjects served as the reference group and completed the questionnaires only once. Depressed patients were initially tested approximately 2 weeks after admission to the clinic . The BDductions . There iductions  for an ot-tests revealed significant group differences in all affective measures. Compared to healthy controls, depressed patients demonstrated higher scores in depressive symptoms (BDI-II: t(54.75) = 15.92; p < .001), explicit NA (PANAS-NA trait: t(52.05) = 10.27; p < .001; state: t(42.55) = 7.40; p < .001), and implicit NA (IPANAT-NA: t(76) = 2.12; p < .05). Moreover, patients scored lower in explicit PA (PANAS-PA trait: t(75) = 10.45; p < .001; state: t(76) = 7.23; p < .001) and implicit PA (IPANAT-PA: t(76) = 4.34; p < .001).The questionnaire characteristics of healthy controls and depressed patients at baseline are presented in Table t(37) = 21.84; p < .001; PANAS state: t(38) = 19.40; p < .001; IPANAT: t(38) = 9.11; p < .001), indicating a prevalence of positive mood at explicit and implicit levels. In depressed patients, dependent t-tests revealed no significant differences between mean PA and NA scores (PANAS state: t(38) = 0.44; p > .05; IPANAT: t(38) = 1.42; p > .05), except for higher scores in explicit trait NA compared to PA (PANAS trait: t(38) = 2.71; p < .05).Within the healthy group, individuals scored significantly higher in all PA scales when compared to the mean scores of the NA scales (PANAS trait: n = 12) had higher implicit PA scores at baseline (2.25 (SD: 0.32) vs. 1.94 (SD: 0.43); t(37) = 2.25; p < .05) and reported more explicit positive state affect (25.92 (SD: 7.95) vs. 19.96 (SD: 5.68); t(37) = 2.69; p < .05)) than medicated patients (n = 27). No other group differences in explicit and implicit affectivity measures were revealed between medicated and unmedicated depressed patients.Unmedicated patients (n = 13) did not differ from depressed patients without comorbid disorders (n = 26) in explicit and implicit affectivity measures with one exception. Patients with comorbidity had higher implicit PA scores at baseline than patients without comorbidity (2.14 (SD: 0.40) vs. 1.84 (SD: 0.39); t(37) = 2.21; p < .05).Depressed patients with comorbid disorders , we applied a Bonferroni correction for multiple comparisons to reduce the likelihood of type I error. Specifically, we divided the threshold of p < 0.05 by the number of critical tests , resulting in a corrected threshold of p < 0.0071 (two-tailed). Depressive symptoms and explicit trait NA scores (but not explicit state NA) were significantly reduced at posttreatment (see Table Table p < 0.05 was maintained. Patients with and without comorbidity both showed significant increases in implicit PA over time (2.14 (SD: 0.40) vs. 2.40 (SD: 0.42); t(24) = 2.73; p < .05 and 1.84 (SD: 0.39) vs. 2.28 (SD: 0.64); t(12) = 2.85; p < .05), but for both groups, no significant changes in implicit NA were observed. Moreover, both medicated and unmedicated depressed patients exhibited significant increases in implicit PA over time (1.94 (SD: 0.43) vs. 2.32 (SD: 0.56); t(26) = 3.45; p < .01 and 2.26 (SD: 0.33) vs. 2.44 (SD: 0.29); t(11) = 2.36; p < .05); for both groups, no significant changes in implicit NA were found.In the following explorative comparisons between patients with and without comorbidity and between medicated and unmedicated patients, a conventional statistical significance threshold of t(37) = 3.76; p < .001; PANAS trait: t(37) = 3.07; p < .01; IPANAT: t(37) = 5.24; p < .001). These findings suggest a restored prevalence of positive mood, as could be observed in healthy controls.At posttreatment, depressed patients had higher mean values in all explicit and implicit PA scales than in NA scales , but implicit NA was not significantly related to explicit negative state or trait affect.In the control subjects, implicit PA was correlated with explicit positive state and trait affect  and of implicit NA with explicit negative state and trait affect  in the patient sample. At posttreatment, implicit PA was again correlated with explicit positive state and trait affect , but implicit NA was not found to be correlated with explicit negative state or trait affect.At baseline, there were significant correlations of implicit PA with explicit positive state and trait affect , a decrease in explicit state NA (but not in implicit NA) from pretreatment to posttreatment would have been revealed.Our data confirm the hypotheses that after 7 weeks of inpatient treatment, depressed patients’ implicit and explicit PA increased, and their explicit trait NA decreased. Explicit state NA was not found to decrease over time. These results are consistent with the findings of intervention studies, demonstrating increases in PA and decreases in NA in depressed patients after psychotherapy or psychopharmacological treatment –14. In tTo our knowledge, our investigation is the first to examine the alterations of implicit affectivity in depression using an indirect psychometric test. However, no evidence was found in our study for a significant decrease in implicit NA over the course of treatment. It appears that NA may improve first on the explicit level and only later on the implicit level. Thus, changes in the automatically elicited negative affectivity of depressed patients may occur later and require more time to develop. Interestingly, this pattern of findings seems to not depend on antidepressant medication or comorbidity. Medicated and unmedicated patients as well as patients with and without comorbid disorders showed improvements in implicit PA but no changes in implicit NA over the course of treatment.However, it has yet to be shown that implicit NA normalizes in the long term in depressed patients. It appears possible that, at least in some patients, heightened implicit NA may persist, which could be interpreted as vulnerability to developing NA. Implicit NA is thought to be linked to the automatic activation of the cognitive representation of negative affective experiences . It has It has been recognized that deficiency in PA represents a core mechanism underlying depression . The earThe IPANAT, as an indirect measure of affectivity, could also represent a useful supplement to classic self-report measures in depression research, as it appears less amenable to distortions caused by reflections about one’s state, self-presentation or appellative tendencies . Some paspecific, discrete affect. It has been argued that a certain number of basic emotions, such as joy, fear, sadness, or anger, universally exist across all human cultures [Future research should investigate the alterations of affective experience in acute depression and during recovery on the level of cultures . To assecultures ) could bcultures . SpecifiAccording to our findings, patients exhibited a prevalence of PA versus NA at posttreatment on both an implicit and explicit level. It appears that although no significant changes were observed in implicit NA over the course of treatment, the ratio between PA and NA tended to normalize. At posttreatment, depressed individuals showed a preponderance of implicit and explicit PA, which is characteristic of healthy individuals. Finally, the relationship between implicit and explicit affectivity was examined in acutely ill and partially remitted depressed patients in comparison to healthy individuals. Interestingly, implicit PA was related to explicit positive state and trait affect in healthy controls as well as in depressed patients at both assessment points. Moreover, for healthy controls and depressed patients in recovery, implicit NA was not found to be correlated with explicit negative state or trait affect, but in the state of acute depression, patients manifested correlations between implicit NA and explicit negative state and trait affect. Thus, in the state of acute depression, the interplay between the automatic and reflective systems could be increased for negative affectivity. This observation may be interpreted within the context of neuroimaging findings, suggesting that in clinical depression, the functional balance between the amygdala and prefrontal structures is impaired. The amygdala is hyperresponsive to negative stimuli in depression and biases perception and higher-order cognition, while prefrontal executive control is decreased . During Some limitations of the present study should be acknowledged. This limitation of our study is that the healthy control group was tested only once. Moreover, our sample consisted mainly of female patients. Thus, it would be important to examine, in future studies, the course of implicit and explicit affect, as a function of gender, with a focus on male patients. Since personality disorders may influence the experience and reporting of affectivity, the presence of personality disorders should be explicitly assessed and controlled using standardized diagnostic interviews in future research on the subject.It has been demonstrated that clinical depression is associated with a substantially increased risk for coronary heart disease and myocardial infarction . The undIn the present study, implicit and explicit affectivity were investigated in patients suffering from acute clinical depression and during remission. According to our results, acute depression is characterized by decreased implicit and explicit PA as well as by increased implicit and explicit NA. At posttreatment, after 7 weeks of inpatient treatment, depressed patients’ implicit and explicit PA increased, and explicit trait NA decreased. However, no evidence was found for a decrease in implicit NA over the course of treatment. Changes in automatically elicited negative affectivity may occur later in depressed patients and need more time to develop compared to affectivity on the explicit processing level. It appears also possible that, at least in some patients, heightened implicit NA may persist as a vulnerability to developing NA. It appears that in the state of acute depression, the interplay between the automatic and reflective systems could be increased for negative affectivity.


The main factor characteristic for low-grade systemic inflammation typical for obesity is oxidative stress (OS). Reactive oxygen species (ROS) production is higher and more increased in time in the obese patients than in lean subjects. fa).  To assess the effect of ileal transposition (IT) and sham types of bariatric procedures on the antioxidative systems in the liver tissue of Zucker rats (Crl:ZUC Leprn = 7), sham group (n = 7), and IT group (n = 7). Sham and IT animals underwent selected surgery. The concentration of total antioxidant capacity (TAC), total antioxidant status (TOS), and activity of glutathione reductase , catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), and total superoxide dismutase activity (SOD) were assessed in liver tissue 3 months after surgery.  21 animals were divided into the experimental groups: control group ( IT procedure significantly increased TAC when compared to sham and the control group. Animals after IT showed higher levels of TOS when compared to sham procedure. The total amount of TOS was similar in IT and control groups. GPx activity was increased in the groups submitted to the sham and IT surgery in relation to control. GR and CAT activities were significantly higher after IT in comparison to control and sham procedures. Total SOD and MnSOD were significantly higher in sham-operated animals in comparison to IT intervention and control groups.  IT procedure had a positive impact on the diminishing of oxidative stress measured by TAC and TOS markers. The dynamic, adaptive, and protective mechanisms of enzymatic antioxidant systems were observed after the IT but not sham procedure. Nevertheless, 3 months after surgery, the midterm effect of bariatric surgery was observed, which might not fully balance the antioxidative response. One of the main factors characteristic for low-grade systemic inflammation and an immune activation characteristic for obesity is OS. ROS generation and inflammation increase more in time in obese patients than in lean subjects .fa, ZF) as an animal model of obesity, phenotypically close to human obesity [Bariatric surgery is, so far, the most efficient procedure for achieving significant and long-term body mass reduction in morbidly obese subjects . It has  obesity .The antioxidant defence under the conditions of OS caused by obesity considers the cumulative action of enzymatic and nonenzymatic antioxidant systems present in liver tissue . The dynfa, Charles River Breeding Laboratories, Wilmington, Mass, USA). The animals were kept under controlled conditions on a 12/12-hour light and dark cycle, in humidity 70 ± 1%, with unlimited access to water and rat food . Food contained 24% of protein, 4.9% of fat, 7% of crude ashes, 4.7% of crude fibre, lysine (13.6 g/kg), calcium (12 g/kg), methionine (4.5 g/kg), and phosphorus (8.3 g/kg).The study was conducted according to the Guide for the Care and Use of Laboratory Animals (Directive 2010/63/EU) and the Ethics Committee of the Medical University of Silesia in Katowice, Poland. 21 male, obese12-week-old Zucker rats (Crl:ZUC Leprn = 7), sham group (n = 7), and IT group (n = 7). The sham and IT groups underwent two different types of surgery: sham, which is a control type of surgery, and IT  was homogenized in 1 ml of a homogenising buffer. All samples were snap frozen in liquid nitrogen and stored at −80°C until further analysis. All experimental procedures were approved by the Ethical Committee for Animal Experimentation of the Medical University of Silesia (58/2014).The total antioxidant capacity (TAC), total antioxidant status (TOS), and the activity of the following antioxidant enzymes: glutathione peroxidase (GPx), glutathione reductase , catalase (CAT), glutathione S-transferase (GST), total superoxide dismutase activity (SOD) in the liver tissue of the control, IT and Sham operated rats were determined.TAC was measured using a commercial kit . The 2.2′azino-di-(3-ethylbenzothiazoline sulphonate) (ABTS) was incubated with a peroxidase (metmyoglobin) and hydrogen peroxide to produce the radical cation ABTS+, which has a relatively stable blue-green colour and was measured at 600 nm. The suppression of the colour was compared to the standard for TAC measurement assays (Trolox). The assay results are expressed as a Trolox equivalent (mmol/l). The inter- and intra-assay coefficients of variations (CV) were 1.1% and 3.8%, respectively.2O2 as the standard. Values are expressed in μmol/l.The method according to Erel  uses theGR enzymatic activity in the liver tissue was evaluated by a decrease in the concentration of NADPH in the samples using a GR buffer  and kinetic reading was performed at a wavelength of 340 nm for 10 minutes [μl of tissue. After 10 seconds, the absorbance was read at λ = 240 nm, every 30 seconds for 2 minutes. The enzymatic activity was expressed in IU/mg protein [The catalase activity in the liver tissue was measured using Aebi methods. Briefly, a 50 mM TRIS/HCl buffer, pH 7.4, and perhydrol were mixed with 50  protein .To measure the activity of glutathione peroxidase, 40 mM sodium azide, GSH (diluted in 5% metaphosphoric acid), GR (GPx diluted in the buffer), NADPH (diluted with sodium bicarbonate 5%), and 0.5 mM tert-butyl were incubated in the liver tissue with a GPx buffer (100 mM potassium phosphate with 1 mM EDTA pH 7.7). The decay of NADPH concentration was evaluated for 10 minutes in a spectrophotometer, at 340 nm .Transferase activity of glutathione S-transferase in the liver tissue was estimated by the kinetic method, previously described by Habig and Jakoby . 1-ChlorSOD isoenzymes' activity was determined with the use of the spectrophotometric method by Oyanagui . KCN wasProtein concentration was determined by Lowry methods using bovine serum albumin as the standard .p value below 0.05 was accepted as statistically significant. All tests were two tailed. Interval data were expressed as a mean value ± standard deviation in the case of a normal distribution or as median/lower-upper quartile range in the case of data with skewed or nonnormal distribution. Distribution of variables was evaluated by the Shapiro-Wilk test, and the quantile-quantile plot. The homogeneity of variances was assessed by the Levene test. The two-way parametric ANOVA with post hoc contrast analysis, nonparametric Kruskal-Wallis test, or Mann-Whitney U test were used in order to compare the data. In the case of skewed data distribution, logarithmic transformation was done before analysis.Statistical analysis was completed using STATISTICA 12.5 PL . A p < 0.001) and control groups (p < 0.01). The lowest level of this parameter was observed in the sham-operated group . After IT surgery, the CAT activity was twofold higher than in other studied animals  while oxidising glutathione (GSH) [Hne (GSH) . Oxidisene (GSH) . Changesne (GSH) . The incne (GSH) . Other hne (GSH) , 33. Thene (GSH) . The conne (GSH) .Under the conditions of long persisting obesity, the pool of antioxidant sources can be diminished, further affecting the activity of enzymes such as superoxide dismutase (SOD). In human subjects, the activity of SOD in obese individuals was significantly reduced in comparison to that in healthy subjects, intensifying the development of obesity-related health problems . ObesityWe conclude that the IT procedure had a positive impact on the reduction of oxidative stress, measured by TAC and TOS parameters in the liver tissue of obese rats. The dynamic, adaptive, and protective mechanisms of enzymatic antioxidant systems were observed after the IT but not sham procedure. Nevertheless, 3 months after surgery, the midterm effect of bariatric surgery was observed, which might not fully balance the antioxidative response.


The Chikungunya (CHIK) virus is epidemic in Brazil, with 170,000 cases in the first half of 2016. More than 60% of patients present relapsing and remitting chronic arthralgia with debilitating pain lasting years. There are no specific therapeutic agents to treat and rehabilitee infected persons with CHIK. Persistent pain can lead to incapacitation, requiring long-term pharmacological treatment. Advances in non-pharmacological treatments are necessary to promote pain relief without side effects and to restore functionality. Clinical trials indicate transcranial direct current stimulation (tDCS) can treat a broad range of chronic pain disorders, including diffuse neuromuscular pain and arthralgia. Here, we demonstrate that the tDCS across the primary motor cortex significantly reduces pain in the chronic phase of CHIK. High-resolution computational model was created to analyze the cortical electric field generated during tDCS and a diffuse and clustered brain current flow including M1 ipsilateral and contralateral, left DLPFC, nucleus accumbens, and cingulate was found. Our findings suggest tDCS could be an effective, inexpensive and deployable therapy to areas lacking resources with a significant number of patients with chronic CHIK persistent pain. CHIK transmission occurs through the bite of an infected mosquito, Aedes aegypti or Aedes albopictu, from mother to fetus in the intrauterine perinatal period, through blood transfusion and sexual intercourse2. Mainly transmitted in urban areas, CHIK constitutes a significant public health problem in many countries, including in the Brazilian Northeast3. The burden of CHIK in Latin America is especially severe in Brazil with 170,000 cases in the first half of 2016, accounting for 94% of confirmed CHIK cases in the Americas4.The Chikungunya (CHIK) virus is a mosquito-transmitted alphavirus, first isolated in 1953 during an epidemic in Tanzania1. CHIK virus induced disease shares many similarities with illnesses caused by other arthritogenic alphaviruses1. Most patients develop severe and often debilitating polyarthralgia that is usually bilateral and symmetric, most commonly in ankles, wrists, and phalanges1. Although complete recovery of these symptoms occurs in some cases within four weeks, several individuals evolve into chronic disability pain for up to 6 years6. According to Silva, a large number of patients experience chronic musculoskeletal disorders and usually respond to some extent to analgesics, anti-inflammatory treatments, and physiotherapy1. In an attempt to reduce the physical symptoms caused by CHIK virus, several medications have been tested, such as chloroquine and meloxicam with limited effectiveness7.Patients infected with CHIK virus will develop persistent rheumatologic and general disabling symptoms such as joint pain, fever, asthenia, headache, retro-orbital pain, photosensitivity, muscular pains, back pain and tenosynovitis9. However, approximately 5% of patients meet the criteria for chronic inflammatory rheumatism, which may be destructive and deforming with severe pain symptoms and disability1. The current challenge for rehabilitation medicine is to provide an optimal treatment for CHIK rheumatic disorders and effectively disrupt the development course of CHIK symptoms.The acute or chronic painful process generated by CHIK virus appears to be associated with the expression of specific metabolites in the human body influencing its severity after infection10. Clinical trials indicate tDCS can treat a broad range of chronic pain disorders, including diffuse neuromuscular pain13. Studies with anodal stimulation of the primary motor cortex (M1) for the treatment of chronic pain syndromes including chronic back pain, fibromyalgia, trigeminal neuralgia, arthrosis and polyneuropathy demonstrated the effectiveness of tDCS on pain and mood symptoms15. Chronic pain may reflect not only local inflammation and peripheral sensitization, by having origins in the central nervous system reflecting maladaptive changes in brain excitability16. Active stimulation was found to be effective for reducing chronic rheumatic symptoms, with no adverse events and good tolerability16.Transcranial Direct Current Stimulation (tDCS) is a battery-powered non-invasive neuromodulation technique in which low amplitude direct current is conducted to the cortex18. tDCS thus offers promise as a novel neuromodulation approach for pain-related networks to alleviate CHIK rheumatic chronic symptoms, such as muscular pain and arthralgia. Moreover, tDCS is a low cost, safe, and mobile intervention that can be deployed on a large scale across locations such as health posts, clinics and in home-visit rehabilitation19.Anodal M1 and cathodal contra lateral supraorbital right (M1-SO) montages of tDCS may treat chronic pain by influencing pain matrix includes motor cortex and deep brain regions producing long-lasting changes in cortical connectivity or excitabilityThe present study aim to investigate the hypothesis that tDCS would improve pain and functionality in subjects with chronicle CHIK arthralgia.20. The study was registered in clinicaltrials.gov on December 5, 2016 with the identifier NCT02993952. All participants provided written informed consent.The study was conducted in the Faculty of Health Science of Trairí, Federal University of Rio Grande do Norte. It was approved by the local institutional Ethical Committee  and conducted according to Declaration of Helsinki (1964), resolution No. 466/12 of the National Health Council, and the CONSORT recommendations22, and with significance of 0.05 and power of 0.80. Fregni suggested the mean reduction of 3 points in Visual Analogue Scale (VAS) for the experimental group was expected in contrast to no improvement in the sham group in rheumatic disease such as fibromialgia22. Thus, the sample size resulted in two groups of 10 participants.The sample size was estimated based on previous studies of tDCS effect on rheumatic diseasesAll participants were included with previous serologic confirmation of Chikungunya (CHIK) virus infection based on CHIK virus IgG and IgM detected by direct ELISA/IgM/Euroimmun, according to the Central Laboratory . Paticipants from local communities of the Northeast of Brazil were recruited through advertisements in the electronic media and by health professionals from the communities. The inclusion criteria were positive laboratory tests for the CHIK virus for at least 6 months (chronic phase), preserved intellectual capacity determined by the Mini Mental State Examination (MMSE), physical capacity to do physical evaluation, and between 18 and 65 years old. The exclusion criteria were pain clearly related to any other etiology, such as dengue, zika, rheumatoid arthritis, gout, lupus, neurologic and muscular diseases, psychiatric illness, and history of drug abuse, signs or history of dizziness or epileptic disease, pregnancy, signs of severity and/or indication of hospitalization and metal implants in the head. Participants were not taking analgesic or other pain relieve medication during this study. All participants were women and only one men was included in Sham-tDCS.The flowchart illustrating the process of the study is shown in Fig. www.randomization.com) to assign each participant to either the active-tDCS group or the sham-tDCS group by an independent researcher not involved with either stimulation or assessments. Participants and researchers were blind to group allocation throughout the trial.Participants were evaluated 1 week before the intervention (baseline), 1 day after the first intervention (day 1), after the last day of intervention (day 5), and 1 week after the last day of intervention. A random numerical sequence was generated (Participants were seated in a comfortable chair with back and arm support. tDCS was carried out with anode electrode on the left primary motor cortex (C3) and the cathode electrode on the right supraorbital region (Fp2) according to the international 10–20 EEG system (“M1-SO” assembly). Electrodes were encased in electrode sponges (7 × 5 cm) soaked in saline solution (150 mMols of NaCl diluted in water Milli-Q), and supported by a rubber band. Active tDCS was performed on 5 consecutive days of constant current of 2 mA for 20 min. A gradual current ramp-up and ramp-down of 30 s was used. Sham-tDCS was performed on 5 consecutive days with electrodes placed on the same position, and a constant current of 2 mA was delivered only for 30 s (10-s ramp-up) of the 20 min. Electrodes (anode and cathode) were connected to a battery (9 V) powered constant current stimulator with current verified by a precision digital multimeter .Finite element models were created to analyze the cortical electric field generated during tDCS. High-resolution MRIs were segmented into seven tissue/material masks of varying conductivities through a combination of automated and manual tools. Computer generated models of electrodes, gel, and/or sponge pads were incorporated into the segmentation. Volume meshes were generated, boundary conditions were applied, and the Laplace equation Socio-demographic data were collected, such as age, disease duration, pain intensity, pain location, marital status, and education. The measures of the outcomes were collected one week before the first simulation (baseline), after the first stimulation (day 1), after the last stimulation (day 5) and one week after the last stimulation (follow-up). Pain levels were evaluated with the self-reported Visual Analogue Scale (VAS), McGill Pain Questionnaire and Brief Pain Inventory (BPI) (short form).23. VAS was asked by a researcher after the first interview (one week before the first simulation - baseline), after day 1 and day 5 of tDCS and one week follow-up. In addition, participants recorded their daily pain intensity using VAS every night before sleeping. This pain diary completed 21 days of measurement. To analyze the homogeneity between groups, initial VAS pain levels were calculated for each patient by using the average of 1 week of pain diary reports.VAS consisted of a gradient color-coded scale ranging from 0  to 10  in intervals of 1 cm23. The BPI (short form) was used to assess pain severity and impact in daily living activities. It presents 15 items, including 2 multi-item scales to measure pain and its impact on functionality and well-being24.McGill Pain Questionnaire was used to characterize participants’ pain. It presents 20 items 10 sensorial, 5 affective, 1 evaluative and 4 miscellaneous. Each of these items displays from 2 to 5 options, and only one or none must be chosen® model SH5001) seated in a chair with armrests, shoulders at 0° of adduction, abduction, flexion, extension, internal and external rotation; elbow at 90° of flexion and wrist between 0° and 30° of extension. They were instructed to squeeze three times as hard as possible for 3 seconds and rest for 30 seconds between repetitions to have the arithmetic mean25. Strength of the lower limbs was evaluated by the 30-second chair stand test. A chair with 43 cm high, with backrest, without armrest and a stopwatch was used. Participants were instructed to keep their arms crossed on their chest, to stand and to sit with their backs resting on the backrest as fast as possible in 30 seconds. The score corresponds to the number of times performed to repeat the movement in 30 seconds26.Participants performed a hand grip test (hydraulic hand dynamometer Saehan27.Upper limb flexion strength (dominant or more painful) was evaluated by the 30-second arm curl test. The test consisted of requiring the participants to hold a 2 kg weight and to perform full flexions and extensions as quickly as possible within 30 seconds. Participants could discontinue the test at any time, if necessary27.To assess the flexibility of the lower limbs (posterior thigh muscles), a chair sit and reach test was performed. A chair with 43 cm high and 50 cm of backrest was used. Participants were instructed to sit on the edge of the chair with flat feet on the floor, knees and ankles at 90° flexion; then the dominant or painful leg was stretched (hip and knee) with the calcaneus supported on the floor and with the ankle flexed at 90°. With overlapping hands and with tips of the middle fingers even, participants tried to reach their toes and hold the reach for two seconds in three repetitions to get the arithmetic mean. A negative score was recorded if the middle fingers did not reach the toes, and a score was positive if the middle fingers were beyond the toes27.Flexibility of the upper limbs was performed using the scratch flexibility test. The participants were instructed to pass one hand (dominant or more painful) over the shoulder to assess flexibility of the shoulder in flexion, abduction and external rotation and try to reach the other hand to assess extension, adduction and internal rotation on the center of the back. Three repetitions were made to obtain the arithmetic mean of the results. The scores were considered negative if there was any distance between the middle fingers, and positive if the middle fingers overlapped28 to evaluate quality of life in the last four weeks. It consists of a 36 item questionnaire divided into 8 domains: functional capacity, limitation by physical aspects, pain, general health, vitality, social and emotional aspects and mental health. These domains have between 2 and 6 response options.A short form health survey (SF-36) was usedAnalyses were performed using the SPSS software  and Graph Pad Prism 5. Quantitative variables were expressed as means and standard deviations (SD). The Shapiro-Wilk and Levene’s test were applied to assess the normality of the distribution and homogeneity of variance of the data, respectively. Mauchly’s test of sphericity was used to validate the correlation of the repeated measures, and if the assumption of sphericity was violated, the Greenhouse-Geisser correction was applied. To compare age, time with CHIKV and VAS baseline between groups, an unpaired t-test or a Mann-Whitney test were used. The effects of stimulation on VAS were calculated using a mixed ANOVA model, in which the dependent variable was the level of pain, and the independent fixed variables were the treatment , the group of stimulation (active and sham) and the interaction term group vs. time. To determine the difference between groups at each category of time and vice versa it was performed three separate between-subjects ANOVAs. When appropriate, post-hoc comparisons were carried out using Bonferroni correction for multiple comparisons.2 were calculated as measures of effect size in the ANOVA results (main effects and interaction effects). Partial η2 was used to calculate the effect size, where η2 = 0.01 was considered small, η2 = 0.06 moderate and η2 = 0.14 large effect. Statistical significance was set at p ≤ 0.05.For other outcome variables , a non-parametric Friedman test was used. Missing data were treated by intention-to-treat analysis, taking into account the method of the last observation carried forward. Partial ηTwenty patients in the chronic phase of CHIK were randomized into two groups receiving active-tDCS or sham-tDCS with a “M1-SO” assembly. Nineteen individuals completed the intervention period and there was one withdrawal in active-tDCS group. No differences in age (p = 0.969), time with CHIKV (p = 0.806) and VAS baseline (p = 0.257) were found between groups. There were no socio-demographic differences between groups (Table Active-tDCS simulation of brain current flow (M1-SO) is predicted to produce electric fields across the brain pain neuromatrix regions, including motor cortex, DLPFC, nucleus accumbens and cingulate Fig. .Figure 22 = 0.175. Similarly, there were significant main effects of time, F(3.54) = 17.712, p = 0.001, partial η2 = 0.956. It was performed the simple main effect for group and time for testing differences in VAS between groups at each category of the within-subjects factor using three separate one-way ANOVAs. There was no statistically significant simple main effect difference in VAS between groups at the baseline, F = 1.894, p = 0.18, partial η2 = 0.09; day 1 F = 3.200, p = 0.48, partial η2 = 0.02; day 5 F = 1.250, p = 0.65, partial η2 = 0.01 and follow-up F = 12.800, p = 0.21, partial η2 = 0.08. There was a statistically significant simple main effect of time on VAS for active-group F = 7.300, p = 0.008, partial η2 = 0.44. No significant effect of time was showed for sham-group F = 2.019, p = 0.135, partial η2 = 0.18 . A mixed ANOVA model indicated significant interaction between the group and time on pain evaluated using VAS, F(3.54) = 3.815, p = 0.01, partial η.18 Fig. .Figure 3McGill’s pain questionnaire showed significant effect of Active-tDCS during the intervention, χ2(4) = 18.113, p = 0.001 Fig. , while t29.Hand grip strength test showed a trend to improve in Active-tDCS group (χ2(3) = 6.704, p = 0.08; Sham-tDCS: χ2(3) = 2.526, p = 0.47). Chair stand, Arm-curl, Chair-sit and reach showed no difference between time and intervention: Active-tDCS (χ2(3) = 4.225, p = 0.23); Sham-tDCS (χ2(3) = 6.148, p = 0.10); Active-tDCS (χ2(3) = 4.014, p = 0.26); Sham-tDCS (χ2(3) = 1.479, p = 0.68); Active-tDCS: (χ2(3) = 6.750, p = 0.08); Sham-tDCS: (χ2(3) = 0.273, p = 0.96), respectively. Back scratch test showed significant difference in the Active-tDCS group (χ2(3) = 15.289, p = 0.002). Baseline back scratch indicated significant difference with day 5 (p = 0.01) and follow-up (p = 0.001). However, Sham-tDCS was also significant (χ2(3) = 11.762, p = 0.008). SF-36 questionnaire showed non-significant difference in both Active-tDCS groups (χ2(3) = 5.055, p = 0.16) and Sham-tDCS (χ2(3) = 3.935, p = 0.26). These tests aimed to evaluate the functionality as function of deficits in resistance, power, mobility and flexibility. These physical fitness tests are simple, quick and economical evaluation, especially in primary care settings and rheumatologic diseases31.Here we showed significant benefits of tDCS on pain sensation following five consecutively days of M1-SO tDCS at 2 mA for 20 min. Our results suggest tDCS could be a critical cost-effective approach for a non-pharmacological strategy to pain relief in a large number of people afflicted by Brazilian CHIK virus epidemic. tDCS has not been previously evaluated to treat CHIK, and the ineffectiveness of the rehabilitation methods to treat the chronic phase of CHIK inspired us to conduct the first clinical trial with tDCS in this population. This preliminary study indicates that several aspects of pain were already ameliorated when M1-SO tDCS was applied for only 5 consecutive days. These results encourage further clinical trials, including long term tDCS treatment (e.g. 10 or 20 sessions) to enhance efficacy. Indeed, CHIK very often results in severe chronic arthralgia and/or arthritis, which can last months to years following the initial infection33. Thus, these areas in the pain neuromatrix  may exhibit abnormal functional and structural changes33. Therefore tDCS can modulate the neuromatrix and provide a significant reduction of pain in patients with chronic pain conditions14. One physiological explanation would be that the tDCS may change maladaptive plasticity both in originally impacted and secondary brain regions in chronic pain syndromes34. Consistent with this hypothesis, the pain treatment related to CHIK virus in this trial was performed during the chronic phase (after 6 months) and significant improvements were shown for pain and its impacts on daily activities according to BPI. Nonetheless, tDCS effect must be investigated during acute stage, where the maladaptive plasticity might be more susceptible to change, and to increase the benefit. We hypothesize that the association between chronic pain in the CHIK virus and brain reorganization could also exist in others chronic pain syndromes, and tDCS may show similar beneficial results. Mechanism of tDCS action should prevent or revert the ongoing maladaptive plasticity within the pain matrix35.Several studies suggest that chronic pain conditions, such as primary dysmenorrhea, irritable bowel syndrome, interstitial cystitis, fibromyalgia and low back pain, may develop an adaptive neuroplasticity and functional reorganization in pain-related areas38. These evidences support the use of neuromodulation for analgesic effects in chikungunya. While, the M1/SO is the most common tDCS electrode montage for pain14, Several meta-analysis suggest an evidence for the effectiveness of anodal M1 stimulation over pain relief34, and others tDCS montage such as dorsolateral prefrontal cortex (DLPFC/SO), primary sensory cortex (S1/SO) or cerebellar (cerebellum/Shoulder) could be used for pain relief39.Moreover, repetitive transcranial magnetic stimulation (rTMS) on M1 was associated with improvement in quality of life and pain in fibromyalgia, chronic pelvic pain and neuropathic pain14, and several adult population, including rheumatic diseases40. Our results showed a reduction over a 30% in VAS, which can be clinically relevant according to Klein at el41. The absence of differences in physical outcomes between the active and sham group could be related to: (1) fitness tests to discriminate specific physical function for CHIK; (2) number of participants ; (3) need to further optimized dose including more sessions. M1-SO tDCS for fibromyalgia showed reduction on pain scores, anxiety, depression and improvement in sleep quality and cognitive performance, but no difference in physical performance14. Therefore, there remains the need of specific physical performance test for CHIK in chronic phase, especially in arthritic symptoms.VAS is the most common method of pain evaluation in clinical trials with tDCS and TMS42. This may reflect the limited number of session and/or the period for participants’ evaluation was insufficient to induce changes in normal routine. In addition, quality of life aspects in CHIK are multifocal and include not only joint pain, but also mental health, emotional role and social functioning5. It can be suggested that future intervention studies with neuromodulation and CHIK should observe not only rheumatic symptoms, but also the social and emotional context of the participants. BPI could be used to evaluate intensity, location and pain effect on the quality of life in patients with CHIK according to Andrade et al.40. Moreover, BPI showed significant improvement in normal work and general activities, suggesting that tDCS could improve the functional status of daily activities.According to SF-36 no significant improvement of quality of life was found, and similar studies using the same intervention, but for chronic abdominal pain also showed the same resultset al. showed a significant self-reported over morbidity and impaired quality of life persisting in CHIK patients for 6 years after infection5, while in our study the average was 10 months of CHIK infection. Furthermore, chronic CHIK arthralgia has a large psychological impact, frequent depressive moods and social disabilities5. These clinical findings influence quality of life and functionality. The M1-SO tDCS used in this trial is note the best assemble to treat depression and others montages should be tested.Marimoutou 5. Understanding new rehabilitation approaches will greatly contribute to future decision-making on appropriate allocation of resources for public health care and research.In countries that have already suffered large CHIK outbreaks, attention is focused on the long-term burden of persistent arthralgiaOur results provide evidence that tDCS reduces the pain levels with clinically significant changes for patients with the CHIK virus. Future investigations involving additional numbers of sessions of tDCS in CHIK patients could prove more effective not only in pain relief, but also in cognitive function, physical activity assessment, quality of life and general functionality. The tDCS treatment could be useful, inexpensive and attractive to areas lacking resources and with a high number of patients with CHIK symptoms. This study brings a novel approach to the non-pharmacological treatment for CHIK fever with tDCS.


Univariate and multivariate Cox regression models were used to assess the hazard for composite outcome at the endpoint (death or cardiovascular event) for each liver pathology. In 111 COPD patients, 75% exhibited liver damage with a prevalence of steatosis, NASH and fibrosis of 41%, 37% and 61%, respectively. During 5-year follow-up, 31 experienced at least one cardiovascular event and 7 died. In univariate analysis, patients with liver fibrosis had more cardiovascular events and higher mortality  than those with no fibrosis; this remained significant in multivariate analysis . We also found that steatosis and NASH were not associated with increased cardiovascular events or mortality. To conclude, early assessment of liver damage might participate to improve cardiovascular outcomes in COPD patients.Chronic Obstructive Pulmonary Disease (COPD) and Non-Alcoholic Fatty Liver Disease (NAFLD) both independently increase cardiovascular risk. We hypothesized that NAFLD might increase the incidence of cardiovascular disease and death in COPD patients. The relationship between NAFLD, incident cardiovascular events, and death was assessed in a prospective cohort of COPD patients with 5-year follow-up. Noninvasive algorithms combining biological parameters (FibroMax Recent literature and the Global initiative for chronic Obstructive Lung Disease (GOLD) recommendations emphasize the importance of an integrated approach to COPD with a careful assessment and treatment of comorbidities4. While CV comorbidities5 and type 2 diabetes6 have been extensively described in COPD patients, there is an emerging interest regarding liver damage and Non-alcoholic Fatty Liver Disease (NAFLD) in COPD patients7.Chronic obstructive pulmonary disease (COPD) is a growing public health concern, causing considerable health-related costs and increased premature mortality. Although diagnosis is mainly based on chronic airflow limitation, as assessed by post-bronchodilator spirometry, COPD is nowadays considered a complex, heterogeneous and multi-organ condition. It is increasingly recognized that the presence of comorbidities such as obesity and/or cardiovascular (CV) and metabolic diseases substantially contributes to the recurrence of hospitalization for exacerbation and significantly impacts prognosis and the incidence of late CV events8. Some patients have an additional inflammatory infiltration ) and can eventually develop liver fibrosis leading to an increased risk of hepatocellular carcinoma and/or cirrhosis10.NAFLD is characterized by the accumulation of hepatic triglycerides (steatosis) in patients without significant alcohol consumption or viral hepatitis11. We recently showed that the prevalence of NAFLD is elevated in COPD patients, with 41% of liver steatosis, 37% of NASH and 61% of liver fibrosis7. COPD severity is also recognized as an independent risk factor of NAFLD7. Compelling evidence over the past several years has substantiated a significant link between NAFLD and CV diseases22. We hypothesized that NAFLD might increase the incidence of CV diseases and death in COPD patients. Therefore, the relationship between NAFLD, incident cardiovascular events, and death was assessed in a prospective cohort of COPD patients with 5-year follow-up.COPD and NAFLD share common risk factors, such as reduced physical activity, and pathophysiological mechanisms including oxidative stress, low grade inflammation, and metabolic syndrome1/FVC) <70% were eligible. Patients with active pulmonary infection, chronic heart failure, left ventricular ejection fraction <45%, active smoking >10 cigarettes per day, neoplasia, antioxidant treatment , and pregnant women were excluded. Also those with a daily consumption of alcohol ≥20g for women and ≥30 g for men and those with viral hepatitis were excluded as these are confounding factors for liver diseases. All patients have given their informed consent.A prospective COPD cohort with associated biobanking was initiated in 2007 in Grenoble Alps University Hospital having been approved by an independent ethics committee . All methods were performed in accordance with the relevant guidelines and regulations. Patients aged 18 years or over, with documented COPD or Forced Expiratory Volume in one second/Forced Vital Capacity At study enrolment, fasting serum samples were collected from all patients, frozen and stored at −80 °C until analyses were conducted.Dimension Vista (Siemens©) and CE-IVD kits.Ten biochemical markers, including liver enzymes, lipid profile, fasting glycaemia and insulin were measured in order to characterize metabolic comorbidities and calculate FibroMax® scores: α2-macroglobulin, apolipoprotein A1, haptoglobin, γ-glutamyltransferase (GGT), total bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), fasting blood glucose, triglycerides and total cholesterol. Measurements were performed with −1)24.Anonymized data were sent to the calculation centre  to obtain FibroMax® scores blinded to the severity of COPD and comorbidities. Insulin resistance was assessed using the homeostatic model assessment for insulin resistance (HOMA-IR), calculated using the following formula: insulinaemia × glucose/22.5 (glucose units mmol.L7. It combines three tests: FibroTest (FT), SteatoTest (ST) and the NashTest (NT)26. FT includes α2-macroglobulin, apolipoprotein A1, haptoglobin, GGT and total bilirubin values, age and sex, and provides a quantitative estimate ranging from 0.00 to 1.00, with higher values corresponding to a greater probability of lesions. Scores correspond to the METAVIR stages27 as follows: F0 (0.00–0.21); F0–F1 (0.22–0.27); F1 (0.28–0.31); F1–F2 (0.32–0.48); F2 (0.49–0.58); F3 (0.59–0.72); F3–F4 (0.73–0.74) and F4 (0.75–1.0) [19]. ST combines FT parameters with height and weight plus ALT, fasting serum glucose, triglycerides and cholesterol. ST scores range from 0.00 to 1.00 used to attribute four steatosis stages from S0 to S4, as follows, S0 (0.00–0.37): no steatosis; S1 (0.38–0.56): minimal steatosis <5%; S2 (0.57–0.68): moderate steatosis 6–32%; and S3–S4 (0.69–1.0): severe steatosis >32%. Finally, the NT score is calculated from weight, height, AST, fasting serum glucose, triglycerides and cholesterol in addition to age, sex and FT components. The NT distinguishes three NASH categories: N0 (0.00–0.25) for absence of NASH; N1 (>0.25–0.50) for borderline NASH; and N2 (>0.50–0.75) for NASH . FT ≥ F0-F1, ST ≥ S2 and NT ≥ N1 were considered as positives, as previously applied by Minville et al. and Viglino et al.28.The Fibromax® algorithm was used to noninvasively evaluate liver damage as previously describedPatients included in the cohort were systematically followed-up at 1, 2, 3 and 5 years. At each visit, patients were questioned regarding the occurrence of incident CV events and AECOPD since the last visit. Incident CV events were defined by the new occurrence of a cardiac event during follow-up: acute myocardial infarction; stroke; new diagnosis of peripheral arterial disease or acute limb ischemia; venous thromboembolic disease and/or pulmonary embolism and new onset arrhythmias. When necessary complementary information was obtained through electronic medical records from Grenoble Alps University Hospital and the patient’s general practitioner was phoned to collect additional information. Cardiovascular events were defined as myocardial infarction, stroke, peripheral arterial disease, pulmonary embolism, atrial fibrillation, and acute pulmonary edema and were validated by the two first authors.Qualitative variables are presented in numbers and percentages and quantitative variables in median and interquartile range. Comparison of the variables was performed separately for NASH, fibrosis and steatosis using a Mann-Whitney test for quantitative variables and a Chi-square test or an exact Fisher test for qualitative variables.First a univariate survival analysis was performed using the Kaplan-Meier estimator and the Log-Rank test to assess the impact of the presence of a liver disease on the composite outcomes. The Kaplan-Meier analysis was also performed to assess separately the impact of each liver disease on the composite outcome. The results were reported by using the Kaplan-Meier curves and the Log-rank p-value.In a second time, to identify the factors associated with the 5-year composite outcome, a univariate and a multivariate Cox regression model were performed. Univariate Cox models were performed to select variables associated with the 5-year composite outcome with a p-value threshold of 0.20. Selected variables were introduced into the multivariable Cox model including age and sex as confounding factors. For all Cox models, the proportionality hazard assumption was tested and verified, and log-linearity of continuous variables was also tested. Statistical analyses were performed using SAS v9.4 . A p-value <0.05 was considered as significant.th; 75th percentiles]), with a body mass index (BMI) of 26  kg/m2. At inclusion, 28 patients (25%) had no liver disease and 83 (75%) had at least one type of liver disease  had at least one exacerbation, 31 28%) experienced at least one late CV event and 7 (6%) patients died  than for those with no liver disease Fig. . In patiAfter adjustment on age, BMI, gender, inhaled corticosteroids, dyslipidemia and diabetes, fibrosis remained a significant risk factor for CV events and death  is associated with an increased probability of fatal and non-fatal incident CV events 31. This risk appears to further increase with NAFLD severity; as in this meta-analysis fibrosis appears to have a more severe impact on the risk of CV events and death than other NAFLD subtypes . Our results in a COPD population are fully consistent with these data, showing an even higher risk  and supporting an elevated risk in cases with combined COPD-NAFLD. Our results are in line with a longitudinal multicenter prospective study of 859 patients with NAFLD, in which liver fibrosis was independently associated with long-term overall mortality  whereas other histologic features of steatohepatitis were not17. Another cohort study found that liver fibrosis predicted overall and disease-specific mortality32.Over the past decade it has been shown that NAFLD is strongly associated with an increased risk of CV diseases, which are the leading cause of mortality among NAFLD patients36, eosinophilic patients and those with asthma-COPD overlap syndrome38, and patients with severe airflow limitation or patients with milder airflow limitation but with obesity and CV comorbidities39. The identification of such phenotypes may have an impact both on long term integrated care and exacerbations41. The identification of comorbidities is a key step in characterizing these phenotypes1. Interestingly, the NAFLD patients in our cohort were older, they were more often men, and they had more other CV risk factors, although their FEV1 at baseline was not different from that of COPD patients without NAFLD. These NAFLD patients have characteristics very similar to those of COPD patients with an increased risk of myocardial infarction43.Previous studies have tried to identify different phenotypes of COPD patients: frequent exacerbatorsThis study had some limitations. Indeed, although it is a well phenotyped prospective cohort, there was no information about time-dependent additional confounders which might have impact the outcomes. Moreover, the relatively limited sample size might under-powered the assessment of liver diseases outcomes. No definite relationship between observed deaths and NAFLD severity can be established owing to the lack of power for this specific type of events. However, we included deaths in the analysis of composite “cardiovascular events and death” as is usually the case in studies addressing hard outcomes in the cardiovascular field. According to these limitations, our results should be confirmed by external validation. However, we strongly suggest that NAFLD should be systematically investigated in COPD patients as a significant contributing factor to CV morbi-mortality. We acknowledge that the ideal design to study the potential cumulative effects of NAFLD and COPD would have been to provide information in 4 conditions: COPD and healthy patients, patients presenting with NAFLD and not in each of these groups. However, it is nearly impossible to achieve an appropriate matching for confounders in these populations. The effect of liver damage in COPD is a very novel area of research and we think that our data really represent an original dataset interesting to share with the scientific community. Our current objective was to demonstrate the association in COPD population with NAFLD and cardiovascular disease. The second step will be intervention studies.45.The diagnosis of NAFLD in COPD could have therapeutic implications. Specific and non-specific treatments for NAFLD are currently being evaluated and subgroup analyses of COPD patients may be of interest in order to explore the possibilities for reducing the risk of CV eventsThis longitudinal prospective study was the first attempt to evaluate the impact of NAFLD on the incidence of cardiovascular events and death in COPD patients. COPD patients suffering from liver fibrosis exhibited a higher risk of incident CV events. This risk appears greater than in the general hepatic fibrosis population. These results suggest that liver disease should be considered when planning the integrative care of COPD patients.Supplementary materials