About 1 in 4 infants comes forward with prolonged crying, agitation, or infant colic (IC) during the first three months of life and is referred for medical evaluation. The pathogenesis remains poorly understood, as do its implications for future health. The aim of this narrative review was to critically examine and discuss the available literature on long-term consequences of excessive crying and/or colic. Most studies display an association between IC and the onset of functional gastrointestinal disorders (FGIDs) years later, probably related to the presence of common etiopathogenetic factors . Although allergic disease in first-degree relatives may be a risk factor for IC, the latter does not appear to be a risk factor for subsequent atopic disease in the individual. Overall, there seems to be a relationship between IC and subsequent headaches, of the migraine type. Similarly, behavioral problems in children with a history of IC appear to be associated with higher parental stress scores. However, the current evidence is based on associations, and currently, a causal relationship between excessive crying and IC and long-term consequences remains not documented. Infant colic (IC) is commonly observed in the first three months of childhood and affects 2% to 73% of newborns worldwide, depending on the diagnostic criteria used ,2. IC geIn this context, it is interesting to note that several sequelae seem to be associated with the presence of IC early in life, such as gastrointestinal disorders, psychological and allergic disorders, such as atopic eczema and food allergy, migraine, and behavioral and developmental disorders ,13,14,15If IC is an early manifestation of these disorders, this would have significant implications for their treatment and prevention.The purpose of this narrative review was to critically explore and discuss the existing published literature on long-term consequences of excessive crying and/or colic. In addition, based on the evidence from the relevant available literature, it is intended to establish whether there is an association between excessive crying time and/or colic in early childhood and the long-term health implications for infants.Two independent authors (VD and FI) searched the PubMed, Embase, and Web of Science databases for articles published from inception through 01 August 2022 on the long-term consequences of excessive crying and/or colic.The following search keywords were used in the databases: IC, functional gastrointestinal disorders (FGIDs), migraine, headache, allergic disorders, asthma, atopy, behavior, sleep mood, hyperactivity, attention-deficit/hyperactivity disorder (ADHD), autism spectrum disorder (ASD), cognitive development, colic symptoms, daily crying, as well as their combinations.Observational studies  and randomized clinical trials that discussed the relationship between excessive crying and/or colic and their long-term consequences were included. The literature search was limited to articles published in English in peer-reviewed journals in order to select studies that underwent a rigorous international peer-review process.Among the articles included there were 5 articles concerning the correlation between IC and onset of FGIDs; 4 articles on allergy-based disorders; 3 on the risk of migraine onset; and 10 on alterations in behavior, sleep mood, hyperactivity, ADHD, ASD, and cognitive development.FGIDs have been defined as a variable combination of chronic or recurrent gastrointestinal symptoms unrelated to organic pathology. A 2015 study estimated the worldwide prevalence of FGIDs-related abdominal pain to be 13.5%, and the most common disorder was found to be irritable bowel syndrome .FGIDs compromise the quality of life of children as they are associated with significant morbidity and require a high cost to health care.Their etiopathogenesis has not yet been clarified, and there is no diagnostic test that leads to a definitive diagnosis. However, some etiological factors have been identified, such as visceral hypersensitivity, infections, genetic factors, stress, and alterations in the gut microbiota. Evidence suggests that there is a constant bidirectional dialogue between brain and gut, which seems to be the mechanism linking emotional state with gastrointestinal dysfunction. Finally, recent preclinical studies indicate that changes in the gut microbiota may influence brain signaling systems related to pain and its associated emotional behavior. Traditionally, they have been considered self-limited diseases; however, altered microbiota and trans-mucosal passage of antigens across the immature intestinal barrier may play a crucial role in the pathogenesis of these functional disorders . FurtherBased on these hypotheses, several researchers have attempted to study the long-term impact of FGIDs in infants not only to identify its pathogenesis but also to seek an effective treatment or preventive approach.p < 0.001, odds ratio (OR) 2.81; p < 0.001, OR 2.30; and p < 0.001, OR 3.24).Indrio et al. conducted a large retrospective study of patients with recurrent abdominal pain that were referred to a pediatric gastroenterology outpatient clinic from January 2002 to December 2009 . The diaDespite the limitations of the study, based on these results obtained from a large population, it can be hypothesized how the early occurrence of bowel discomfort may be a risk factor for the development of recurrent abdominal pain.These hypotheses were subsequently confirmed and extended by further studies.p = 0.037), while complaining about aches “in other parts of body” was somewhat more frequent in healthy subjects, although there was no statistically significant difference (p = 0.08). Similarly soiling and constipation were more common in the Control Group but not statistically significant .A prospective survey with follow-up at 4 years of age on FGIDs conducted on 50 formerly colicky children and 102 healthy controls was conducted in Sweden . Formerlp = 0.001; RR 10.7). No significant difference between the two groups of infants was found for the other gastrointestinal disorders examined. The results of this study show an association between IC and recurrent abdominal pain, in agreement with what has been shown in previous studies listed above.Similarly, Savino et al. conducted a prospective study of 96 infants who were enrolled from January 1991 to December 1993, including 48 infants who had been examined for severe IC and 48 controls . At age Lacticaseibacillus rhamnosus GG  or placebo) to assess the risk of occurrence of FGIDs later in life . This association was mediated by maternal burden of infant care (change in odds’ ratio 1–17 %) and maternal aggressive behavior (change in odds’ ratio 4–10 %) .p < 0.001), externalizing (p < 0.001), and total (p < 0.001) behavioral problems in children with a history of IC were associated with higher parenting stress scores [Finally, in 2022, a prospective cohort study of Dutch infants with IC  had increased odds of scoring in the clinical range of the emotionally reactive . Internalizing (s scores .A 2004 US prospective cohort study with a 5-year follow-up found that children with prolonged excessive crying (n = 15) had an increased risk of lower IQ scores compared with the control group (n = 264) , hyperactivity, and discipline problems. No such problems were found in those with IC only (n = 48). IC had no effect on cognitive development . The smaTo our knowledge, the long-term outcomes of IC remain a very unexplored field, and more evidence is needed to draw strong conclusions.Data obtained from the various studies seem to confirm the multifactorial etiology in IC since a high percentage of children with IC show allergic and psychological disorders later in childhood, so these factors then may be playing an important role in the etiopathogenesis. However, the current evidence is based on associations, and currently, a causal relationship between excessive crying and IC and long-term consequences remains undocumented.Most studies show an association between IC and the occurrence of FGIDs, probably as a consequence of common etiopathogenetic factors  and symptoms  as well. Although allergic disease in first-degree relatives may be a risk factor for IC, IC does not appear to be a risk factor for subsequent atopic disease in the individual. However, these results could be biased by the fact that more breastfed children were included. In addition, although somewhat contradictory regarding the association between IC and type of migraine or headache, overall, there seems to be a relationship between IC and the subsequent headache of the migraine type. Similarly, behavioral problems in children with a history of IC appear to be associated with higher parenting stress scores.Finally, we emphasize the lack of universal definitions of excessive crying and/or IC in all included studies. Limitations of some studies include a rather small study population, a considerable dropout rate, heterogeneity of study designs, outcomes assessed and assessment methods, and the presence of significant independent confounding effects of genetic factors such as parental IQ or mode and duration of breastfeeding. New assessments such as children’s and parents’ quality of life, sleep time, and the impact of the condition on parents could probably be useful.The collection of reliable data in prospective long-term follow-up studies according to agreed, more homogeneous, and well-defined criteria is needed to obtain more accurate estimations of long-term sequelae of these common childhood symptoms, to confirm these correlations, but also to find effective preventive approaches and new treatment options.


We also identified a significant regression model between the body mass index (BMI) and the FQRST (p < 0.001). Conclusions: The physical activity regime of young adults significantly influences the concordance between ventricular depolarization and repolarization, reflected in the FQRST’s width. Also, we found a regression model between FQRST and BMI with statistical significance.Background: The heart’s electrical activity has been the subject of numerous research concerning various physiological parameters. The frontal QRS-T angle (FQRST) is an advanced ECG variable with clinical epidemiological utility. This study aimed to determine the relationship between FQRST and physical activity exposure among young adults. Methods: We recorded the ECG with 12 leads of 124 participants . Next, we measured their physical activity level (PAL) with the International Physical Activity Questionnaire—Short Form (IPAQ), which categorizes activity into three classes: low, moderate, or high. Results: An inferential analysis, based on the Kruskal-Wallis H test and Mann–Whitney U test, revealed a statistically significant difference in FQRST between the three groups of subjects, classified by their PAL ( The heart’s electrical activity has been the subject of numerous research concerning various physiological parameters. The frontal QRS-T angle (FQRST) is an advanced ECG variable with clinical epidemiological utility, especially for long-time prognosis of cardiac mortality and risk in the general population ,2. The dThe FQRST has been proposed in clinical practice for the prediction of many other variables, but there are still controversies regarding the interpretation of research results . This paSeveral authors reported differences in FQRST depending on sex, with women having a smaller angle at baseline than men . HoweverECG changes in the context of regular physical training are common and usually reflect a physiological adaptation of the heart. The experts have systematized the main changes in the heart’s electrical activity induced by exercise. Thus, the common training-related  ECG markers are as follows: increased QRS amplitude (left or right ventricular hypertrophy), ST-segment elevation, incomplete right bundle branch block, sinus bradycardia, physiological arrhythmias, and early repolarization/ST-segment elevation, ectopic atrial or junctional rhythm ,18.The regular and long-term intense training regimes (minimum of 4 h per week) determine particular ECG changes as a consequence of electrical and morphological adaptations secondary to repeated sessions of exercise ,19. GeneAs borderline ECG changes in athletes, we note the left or right axis deviation, voltage criteria for left or right atrial enlargement, and the complete right bundle branch block ,20. The Regarding the FQRST angle, only a few studies have analyzed its changes correlated with exercise. For example, in elite skiers, better athletic performance is associated with a narrower QRS-T angle . In contHowever, it is less established whether these changes occur during physical exercise or are only residual in the post-exercise period as chronic adaptations. Also, in healthy people, the spectrum of FQRST changes, in correlation with the amount of physical activity performed, is not well defined. Considering the inconsistency of the data presented, our research focused on testing the difference in FQRST between groups of individuals with different physical activity levels (PAL).The main objective of the research was to identify the influence of PAL in FQRST in healthy young adults. The secondary objectives were to measure and describe the relationship between FQRST and some variables  as an explanatory approach.n = 124, mean age 20.28 ± 2.23 years, 71 men and 53 women, age range 18–27 years). Before the data gathering, we obtained written informed consent from each subject to participate in the study, according to the ethical principle of research with humans. The study received approval from the Research Ethics Committee of the Research Center for Promoting Excellence in Professional Training, University of Pitesti (reference number 146/7 March 2022).We conducted an observational cross-sectional study on a sample of healthy young adults (Eligible participants were recruited based on the following criteria: young adults (age range 18–27 years), good health status confirmed by a medical professional, acceptance for medical data recording, and completing a physical activity assessment questionnaire. The subjects’ exclusion criteria were: sports performance practice, history of recent acute or chronic diseases, medication intake during the last week, and pregnancy for women. Sports performance practice was defined as the regular practice of a sport, at the elite level, during the last year. This criterion was used to avoid the interference of left ventricular hypertrophy, specific for sports performance, in the FQRST analysis.The assessments were carried out between 8 am and 12 am, based on the same protocol for each participant: measuring the height (H), determining the body weight (W), recording the ECG at rest, and then administering a questionnaire to evaluate the physical activity of the last seven days. Starting from W (in kg) and H (in m), we calculated the body mass index (BMI):The Cardimax FX-7402 device, Fukuda Denshi Co. Ltd. , with 12 derivations, was used for ECG recording. We recorded the ECG signals for all participants in the supine position, for a minimum of 30 s, with a speed of 25 mm/s and a sensitivity of 5 mm/mV. Then, we manually determined the heart rate (HR), measured the QT interval, and calculated the FQRSA, FTA, and FQRST. For the measurement of the QT interval, we used the tangent method in lead II :(2)QTc =For QTc interpretation, according to most experts, the normal values taken into consideration were QTc < 440 ms for adult males and <460 ms for adult females .For the FQRSA and FTA manual measurements, we used the leads I and aVF. On the hexaxial reference system, we determined the areas of positive and negative deflections of the QRS complex and T wave . AccordiThe formula expresses the angle between the mean QRS and T wave vectors, determined in leads I and aVF. For interpretation of FQRST, an angle below 90° was considered normal, and values ≥ 100° abnormal ,13.Another objective for the subjects’ assessment concerned their PAL. For this purpose, we used the International Physical Activity Questionnaire—Short Form (IPAQ). IPAQ short form is an instrument with fair to moderate validity, designed primarily for population surveillance of PAL among adults (age range of 15–69 years) . IPAQ asFrom IPAQ, we obtained categorical and continuous indicators of physical activity. The IPAQ scoring protocol allows us to compute IPAQ scores expressed in MET-minutes/week based on weighting each type of activity by its energy requirements. Computation of the IPAQ scores requires the summation of the duration (in minutes) and frequency (days) of the mentioned activities. The collected data with IPAQ, as a self-report measure, categorize individuals into three classes of physical activity: 1 = low PAL, 2 = moderate PAL, and 3 = high PAL .https://www.bioinformatics.com.cn/en (accessed on 19 January 2023), a free online platform for data analysis and visualization [p < 0.05), as assessed with the Shapiro-Wilk test. The FQRST variable is further described in relation to the PAL level using raincloud plots. Normality hypothesis was not rejected for W, QT, QTc, and FTA, for a level of significance for rejecting the null of 0.05. We applied nonparametric tests for inferential statistical analysis: the Kruskal-Wallis H test and the Mann–Whitney U-test  for independent large samples (n > 20) to check the statistical significance of the differences between groups. The Kruskal-Wallis test was used to address the main objective of the study, with the null hypothesis stating that the medians of the independent samples are equal, at a significance level of 0.05. We conducted the Mann-Whitney test as a post-hoc analysis and corrected the considered p-values of statistical significance of 0.05 using the Bonferroni correction method. Groups were initially defined according to PAL and then to sex. Another analysis considered the average heart rate in the groups of subjects with different PAL. The same methodology was utilized to examine differences in FQRST based on sex levels and to investigate differences in heart rate based on PAL levels.This study provided the following variables: age, sex, W, H, BMI, IPAQ score (MET-minutes/week), category of PAL, HR, QT and QTc intervals, FQRSA, FTA, and FQRST. Data analysis was carried out using the software IBM SPSS 20.0  . The railization . DescripWe also realized a correlational analysis of data based the Spearman’s rank-order correlation. The variables considered for the correlational analysis were age, W, H, BMI, HR, QT, QTc, FQRSA, FTA, and FQRST. Initially, we tested the bivariate normality between the analyzed variables with a normal distribution  required for parametric correlational analysis, but we rejected it. Consecutively, we applied the Spearman analysis for all pairs of variables. Our goal was to understand the relationship between all the recorded variables, including FQRST, as part of a comprehensive analysis. Finally, a linear regression analysis was run to determine the relationship between pair variables that are significantly correlated and are appropriate to the objective of the research .The main results are reported in From the baseline characteristics of the subjects, regarding their nutritional status, we noticed an average healthy weight range (mean BMI = 22.04 ± 2.87), according to the NIH/WHO guidelines for BMI . In the From the ECG records, all subjects had a normal sinus rhythm without significant conduction or repolarization abnormalities. The QT interval varied from 280 to 442 ms (mean QT = 362.28 ± 30.47 ms), for the corresponding HR at rest ranging from 48 to 120 beats/min (mean HR = 72.52 ± 13.67 bpm). The corrected QTc fluctuated in the group of participants between 309 and 465 ms (mean QTc = 394.89 ± 32.40 ms). The QTc intervals were within normal limits for 94.4% of the subjects and prolonged for 5.6% of them, according to the sex reference values .Analysis of FQRSA data revealed normal values for 94.4% of the subjects and a right deviation for 5.6% of cases. Instead, the FTA was in the normal range for each participant. Finally, the FQRST, as a composite variable, was normal in the participants’ group (below 100°).Another investigated parameter was the PAL of the subjects. The analysis of the IPAQ scores showed that 28.2% of the participants had a low PAL, 41.1% had a moderate PAL, and 39.7% had a high PAL.p < 0.001, with a mean rank of FQRST 100.56 for low PAL, 61.19 for moderate PAL and 29.21 for high PAL.We started the inferential statistical analysis with a rank-based nonparametric test  to compare the differences between data series with a nonnormal distribution (FQRST). Thus, we applied the mentioned test to understand whether FQRST differed in the sample group based on PAL. The dependent variable was the FQRST, and the independent variable was the PAL, with three independent groups: subjects with low, medium, and high PAL. The results showed a statistically significant difference in FQRST between the different PAL, χ2(2) = 71.92, Next, we ran a Mann-Whitney test to compare differences between pairs of two independent groups with the continuous dependent variable FQRST. Independent groups were defined based on their PAL: group 1 with low PAL, group II with moderate PAL, and group 3 with high PAL. The results of this inferential analysis are presented in p < 0.001) when comparing groups 1 and 2, 2 and 3, and, respectively, 1 and 3. In our statistical analysis, we used the value of the Bonferroni-corrected threshold of statistical significance of 0.016. The magnitude of the differences between the data series indicated a large effect size (r > 0.5), according to the r classification system [The analysis of the differences between groups revealed statistically significant results for FQRST .Our data were also analyzed using bivariate Spearman’s correlation rariables . In SpeaAfter the correlation analysis, a linear regression was the next step to link the correlated variables of interest for our research purpose. We chose the BMI for the linear regression analysis with FQRST since BMI is a composite variable that reflects the nutritional status and has intensively been studied and associated with FQRST. While FTA is also correlated with FQRST, it is a variable that is used to calculate FQRST through vector geometry, making the relationship between these variables more complex and less relevant to our objective. We run a simple linear regression analysis to determine the effect of BMI (the independent and predictor variable) on the FQRST (the dependent outcome variable). According to the normal P-P plot of regression, the standardized residuals of data considered were approximately normally distributed, and the required assumption to run a linear regression was assured .For the mentioned series of data, we didn’t find significant outliers. The assumption of independence of residuals was checked using the Durbin-Watson test for autocorrelation in the residuals, which assesses the serial dependence of data . The obtp < 0.001). Therefore, a part of the variability of the FQRST (0.09%) is explained by the nutritional status in terms of BMI.We found a regression model between FQRST and BMI  with staThe following regression equation reports how well the analysis fits the data:p < 0.001). Based on the mean rank differences between groups, our results suggest that the widening of FQRST is present in sedentary young adults, even if the angle values do not exceed the limit of normality. At the same time, the narrowing of this angle is characteristic of individuals with an active lifestyle. It should be noticed that, overall, the study participants had no pathological changes on the ECG recordings, except the presence of a prolonged QTc interval in 5.6% of them and a right deviation of FQRSA for 5.6% of them.This analysis found a statistically significant difference in FQRST between the three groups of subjects, classified by their PAL in low, moderate, and high categories of physical activity (p < 0.001). The relationship between these two variables assumes a moderate correlation .Our findings indicate that the interpretation of the FQRST should consider the PAL of the participants since it offers a new perspective for ECG-data stratification. This study also analyzed the relation between BMI and FQRST and found that nutritional status had a predictable influence on the difference in orientation between ventricular depolarization and repolarization , but the lower values were specific to those with high PAL. As a novelty of our study, we demonstrated that PAL influences the FQRST values, even if they were in the normal range. Thus, optimal values of FQRST appear in physically active young adults, while in sedentary individuals the angle tends to enlarge without reaching the abnormal level.As a result, we can estimate that early intervention to increase the PAL in individuals may protect future body dynamics in terms of FQRST values. This explanation is plausible since FQRST reflects the disjunction between ventricular depolarization and repolarization and has a direct relationship with the risk of developing cardiovascular impairments, such as sudden death, cardiac ischemia, or ventricular arrhythmia .Another discussion concerns the consequences of widening the QRS-T angle on hemodynamics. Thus, a widened spatial QRS-T angle may represent poor ventricular stretch . A negatThe final result of our study highlighted the regression model that demonstrated a significant and positive relationship between FQRST and BMI. Previous research suggested a positive weak correlation of FQRST with BMI . Some auOur results are also in accord with those of other authors who investigated the FQRST by referring to the World Health Organization classification for BMI . ThroughIt is interesting that among all of the constitutional variables analyzed in association with FQRST , the highest correlation was achieved with BMI (R = 0.30) and then with H (R = −0.21). Another research stated that H is the most significant constitutional determinant of ECG since an increase in H determines the augmentation of most QRS and ST-T parameters .To conclude, the use of advanced ECG parameters, such as the FQRST, gets new values for the interpretation of the complex factors that interfere with the individual health status. Beyond the controversies in the literature, the data presented here confirm the applicability of FQRST for understanding the body’s adaptations to exercise in healthy young adults. Taking into account the constitutional parameters in various clinical situations, the ECG diagnosis and the stratification of risk factors for health can be improved. Additional research on large samples and with interventional design is needed to refine these findings.This study has several limitations. Firstly, the validity of the IPAQ collected data depends on the honesty and subjectivity of the respondents. Then, the statistical analysis was carried out through non-parametric tests of the ranks, which may lack power compared to parametric tests. For the results’ interpretation, we did not consider other categorical variables and concomitant cardiovascular risk factors of the subjects, such as genetic predisposition, smoking, alcohol consumption, type of diet, etc. Also, for the analysis of the ECG data, we eliminated the cases of left ventricular hypertrophy, which could influence the analyzed variables. However, we tried to minimize this inconvenience by excluding performance athletes from among the subjects, who usually present these ECG changes. Another limitation of the study is that we did not conduct a statistical power analysis to determine the sample size. While we considered other factors such as similar studies in the literature to expect having an appropriate sample size, it remains possible that our study lacked of statistical power for the analyses we conducted.The physical activity regime of young adults significantly influences the concordance between ventricular depolarization and repolarization, reflected in the FQRST’s width. A narrow FQRST is associated with a high PAL in healthy young adults, while in sedentary subjects, the angle tends to increase without exceeding the normal upper limit. We also identified a significant regression model between the BMI and the FQRST.


The chronic pain of patients with knee osteoarthritis (KOA) seriously affects their quality of life and leads to heavy social and economic burden. As a nondrug therapy in Traditional Chinese Medicine (TCM), Tuina is generally recognised as safe and effective for reducing the chronic pain of KOA. However, the underlying central mechanisms of Tuina for improving the pain of KOA are not fully understood.P<0.05 will be the statistical significance level.This study will be a randomised controlled trial with a parallel-group design. A total of 60 eligible participants will be assigned to the Tuina group or healthcare education group (Education group) at 1:1 ratio using stratified randomisation with gender and age as factors. The interventions of both groups will last for 30 min per session and be conducted twice each week for 12 weeks. This study will primarily focus on pain evaluation assessed by detecting the changes in brain grey matter (GM) structure, white matter (WM) structure, and the cerebral functional connectivity (FC) elicited by Tuina treatment, e.g., thalamus, hippocampus, anterior cingulate gyrus, S1, insula, and periaqueductal grey subregions (PAG). The two groups of patients will be evaluated by clinical assessments and multimodal magnetic resonance imaging (MRI) to observe the alterations in the GM, WM, and FC of participants at the baseline and the end of 6 and 12 weeks’ treatment and still be evaluated by clinical assessments but not MRI for 48 weeks of follow-up. The visual analogue scale of current pain is the primary outcome. The Short-Form McGill Pain Questionnaire, Western Ontario and McMaster Universities Osteoarthritis Index, 36-Item Short Form Health Survey, Hamilton Depression Scale, and Hamilton Anxiety Scale will be used to evaluate the pain intensity, pain feeling, pain emotion, clinical symptoms, and quality of life, respectively. MRI assessments, clinical data evaluators, data managers, and statisticians will be blinded to the group allocation in the outcome evaluation procedure and data analysis to reduce the risk of bias. The repeated measures analysis of variance (2 groups × 6 time points ANOVA) will be used to analyse numerical variables of the clinical and neuroimaging data obtained in the study. The results of this randomised controlled trial with clinical assessments and multimodal MRI will help reveal the influence of Tuina treatment on the potential morphological changes in cortical and subcortical brain structures, the white matter integrity, and the functional activities and connectivity of brain regions of patients with KOA, which may provide scientific evidence for the clinical application of Tuina in the management of KOA.Chinese Clinical Trial Registry ChiCTR2000037966. Registered on Sep. 8, 2020.The results will be published in peer-reviewed journals and disseminated through the study’s website, and conferences. Knee osteoarthritis (KOA) is an important cause of knee pain and disability, causing serious personal and social burden all over the world , 2. The The International Society for the Study of Pain redefines pain as an unpleasant sensory and emotional experience associated with, or resembling that associated with, actual or potential tissue damage . KOA paiChronic pain of KOA is complex . The braHuangdi Neijing, which plays an important role in the health of Chinese people for thousands of years. It takes advantage of the hands of the therapist to manipulate the surface of the patient’s body. The relaxation techniques in Tuina such as pressing and kneading exert significant analgaesic effect, improving mood, improving patients’ confidence in prevention and treatment, promoting rehabilitation, and reducing the use of health services [Tuina is recorded in the classic work of ancient Chinese medicine services , 25. Tuiservices , regulatservices . Moreoveservices –34.An education programme such as a self-management programme can provide knowledge and skills that will come in handy in helping patients improve the management of their condition. Self-management skills are essential for treatment and are strongly recommended in the guideline for osteoarthritis management . For mosTuina is a promising treatment option for KOA . It is bHowever, studies on the longitudinal effect and central analgaesic mechanism of Tuina in patients with KOA chronic pain are few , 48–50. All patients with KOA will be recruited from the Tuina department of Yueyang Hospital of Integrated Traditional Chinese and Western medicine, Shanghai University of TCM. Recruitment methods will include posters, online advertisements, and leaflets. Written informed consents will be collected taken from all patients. A total of 60 patients with KOA chronic pain will be randomly assigned into a Tuina group and an Education group at 1:1 ratio.This study’s protocol has been approved by the Ethics Committee of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of TCM. The trial was also registered to the Chinese Clinical Trial Registry.This is a single-centre and parallel randomised controlled trial. A total of 60 participants diagnosed with KOA based on the American College of Rheumatology (ACR) criteria (2019 revised version)  will be Outcome measurements and MRI scans will be assessed at the baseline and at 6 and 12 weeks after treatment, followed by 48 weeks of clinical follow-up. The changes in clinical variables and cerebral activity of each group will be analysed after data collection.Evaluators and statisticians will be blinded to the group allocation during the procedure of outcome evaluation and data analysis , 53. TheMeet the diagnostic criteria for KOA set by the ACR in 2019 Are aged between 40 and 60 years, male or female, right-handed, with left knee painHave I–II degree knee joint radiological change on the Kellgren–Lawrence scaleHave an average knee pain score on a visual analogue scale (VAS) ≥ 3 (range from 0 to 10) in the past 3 monthsVolunteer to take part in the study and sign the informed consent formCan understand and follow the protocol.Mainly to ensure the feasibility of the clinical study and the better baseline balance of the two groups, and because the incidence rate of KOA patients aged 40–60 is relatively high , so it iAre taking analgaesics or anaesthetics in the past 1 month that may influence brain-imaging outcomesHave received any other treatment in the past 1 month, such as exercising therapy, pharmacological intervention, or operationAre pregnant or lactating womenAre suffering from mental disease, neurological disease, infectious disease, gastrointestinal disease, cardiovascular and cerebrovascular disease, immunologic disease, respiratory disease or kidney disease, any other chronic pain symptoms, or have a history of brain injury with loss of consciousnessAre diagnosed as cancer, tuberculosis, rheumatism or rheumatoid arthritis, gout, or joint traumaHave MRI contraindications such as claustrophobia, cardiac pacemaker, defibrillator, heart stent, or intrauterine deviceHave skin lesion around the knee jointAre unsuitable to continue to participate in the study owing to adverse events or serious adverse events during the trialAre unsuitable to continue to participate in the study because of serious deterioration of disease or some complications and special physiological changesUses concomitant medication during the 12-week treatment period is a suspension criteria. Non-compliance to concomitant interventions will be monitored by daily dairies and WeChat queries.Withdrew from the studyLoss of contactσ of the two groups is equal, i.e., σ=2.1. If we set α=0.05 as the type I error, β=0.10 as the type II error, and 1−β=0.90 as the power, the final result is that we need at least 25 samples in each group. For the primary outcome, VAS will be assessed at baseline and at the end of 6 and 12 weeks of treatment, followed by 12, 36, and 48 weeks of follow-up. The statistical significance is defined as P < 0.05, and the 95% confidence interval will be reported.Sample size is required to compare two group mean values. The calculation method of bilateral equality of two samples is adopted  for two Many MRI studies have reported statistical significance with 12 to 21 patients per group , 58–60. A total of 60 eligible participants will be assigned to the Tuina or Education groups at 1:1 ratio using stratified randomisation with gender and age as factors in this research. Stratified randomisation is achieved by performing a separate randomisation procedure within each of two subsets of participants (gender and age). First, participants will be grouped by gender. Then, male and female participants will be grouped by age. After grouping, there will be four subgroups: (1) men aged < 50, (2) men aged ≥ 50, (3) women aged < 50, and (4) women aged ≥ 50. In these four subgroups, the subjects will be randomly divided into the Tuina group and Education group. Finally, all Tuina groups and Education groups are merged to form a new Tuina group and Education group. This step ensures good balance amongst participant characteristics in each group and close balance amongst the numbers of participants receiving each intervention within each stratum. The created random number lists will be generated by a random number generator  and will be sent to the therapist with sequentially numbered, opaque, and sealed envelopes by an independent assistant. The therapist will open random-allocation envelopes and allocate the participants accordingly to the Tuina or Education group.Participants and therapists will not be blinded to treatment allocation, because of the features of the Tuina treatment and the education intervention. For participants, their subjective consciousness will affect the outcomes, which is unavoidable. Nevertheless, the evaluators, data managers, and statisticians will be blinded to the group allocation in the outcome evaluation procedure and data analysis throughout the entire procedure to reduce the risk of bias. The data will not be accessible to the evaluators until the collection is finished.The participants will receive a total of 24 treatments in 12 weeks. The interventionists, who hold a PhD, must have at least 10 years of experience with Tuina treatment and be skillful in healthcare education for the participants. They are also required to have passed a clinical test to ensure consistency of the trial. The clinical test primarily includes the time of Tuina, the positioning of the treatment site, and the application of specific Tuina manipulation types. The standard operating procedures primarily include Tuina’s time, treatment site, and manipulation category, as well as the time and content of health education. In the process of Tuina or Education intervention, the two groups will not allow any concomitant medications or other interventions. However, concomitant medications or other interventions can be used during subsequent follow-up, and the treatment will be recorded in detail.The selected acupoints for the Tuina treatment include EX-LE02 (heding), EX-LE04 (neixiyan), EX-LE05 (xiyan), GB34 (yanglingquan), SP10 (xuehai), ST34 (liangqiu), ST32 (futu), BL40 (weizhong), BL57 (chengshan), GB31 (fengshi), and Ashi acupoints around the knee joint. An ashi point is a temporary acupoint where sensation of acid, distension, and pain exist, which can appear anywhere in knee joints .Referring to previous studies , 62–64, The Osteoarthritis Research Society International (OARSI) and the European Society for Clinical and Economic Aspects of Osteoporosis, Osteoarthritis and Musculoskeletal Diseases (ESCEO) updated the guidelines on KOA in 2019 . In the The education intervention will be given to the participants with two sessions per week. The first health education session is a group meeting using a PowerPoint presentation, which will last 30 min. An educational booklet and a video will be provided to the patients to accomplish the home-based online e-learning module. The later sessions will be conducted as one-to-one communication focused on personal needs to guide patients to implement the key education in their lives. The programme includes two broad components: (1) introduce key concepts of pain biology, and (2) present specification of recovery.Each patient will be given an introduction about the underlying mechanisms, predisposing factors, and prognosis of KOA. Pain will be explained as the conscious part of the response, which can be influenced by many factors as a protective output . IntervePatients with KOA will be instructed not to take any caffeine , as well as other medications, weight-loss techniques, or physical activity to avoid confounding interference for the analgaesic effect of the intervention in the two groups. In cases of severe knee pain, ibuprofen (300 mg per capsule with sustained release) will be allowed as rescue medication and should be recorded on a knee-pain diary. After the end of the study, patients will be taught weight-loss techniques and physical activity. Patients are also asked to record the name, dose, date, frequency, and exact time of the medications used, as well as to report to the researcher if they take any medications during the study. Patient adherence will be maintained and monitored by good communication between the therapist and patient and by constant reminders.The participants’ conditions will be carefully monitored by therapists during the process. If adverse events occur, participants with adverse events will stop participating in the trial. Immediate medical care will be provided to any participant experiencing adverse reactions that can lead to hospitalisation, for example fracture or coma. The details will be recorded in the case report forms (CRFs). The ethics committee will conduct an interim analysis and make a decision, which will be determined according to the severity of the adverse events of the participants. For example, conditions that lead to hospitalisation or even life-threatening are severer, whereas conditions such as muscle pain that can heal on their own are milder. Reports will be made to the ethics committee with statements and detailed reasons for trial suspension. The efficacy and safety of the intervention will be evaluated after 6 and 12 weeks of intervention and 48 weeks of subsequent follow-up. Evaluators can also be reformed with the clinical symptoms and AEs whenever the patients meet something important. After an adverse event is determined to be related to the trial plan, the research team will provide treatment costs and corresponding financial compensation for the damage inflicted by the trial plan.MRI data will be acquired using a 3.0-T magnetic resonance scanner  with a 32-channel phase-array head coil at the Medical Imaging Department of Yueyang Hospital of Traditional Chinese and Western Medicine, Shanghai University of TCM.On the pre-MRI evaluation procedure, the scanner will instruct the subject to rest for 10 min, relax, and keep calm. Throughout the entire scan, participants are asked to close their eyes, wear earplugs, keep their heads still throughout the scan, stay relaxed and awake, and not think of anything in particular.2, number of slices = 160, and slice thickness = 1 mm.Prior to the blood-oxygen-level-independent (BOLD) resting-state functional images, a high-resolution T1 image for each subject will be acquired with spin echo (SE) sequence, transverse sagittal scan, flip angle = 9°, pulse repetition time (TR) = 1900 ms, echo time (TE) = 2.93 ms, field of view (FOV) = 256×256 mm2, B-value2 = 1000 s/mm2, and 30 directions.DTI will be measured with the axial DTI mapping sequences, TR = 10,000 ms, TE = 89 ms, matrix = 240 × 240, slice thickness = 2 mm, B-value1 = 0 s/mm2, voxel size = 3.4 × 3.4 × 4.0 mm3, FA=90°, scanning time = 8 min 8 s, 240 volumes in total.The BOLD resting-state functional images will be obtained with an echo-planar imaging sequence as follows: coronal axial scan, 33 slices, 4 mm thickness, TE = 30 ms, TR = 2000 ms, FOV = 220 × 220 mmOn the post-MRI evaluation procedure, the subjects will be instructed to rest for 5 min. They may leave if there is no uncomfortable feeling.The MRI outcomes include the following: structure–GM density, cortical thickness, subcortical nuclei volumes; diffusion–FA and MD of WM integrity; fMRI–functional connectivity (FC).http://surfer.nmr.mgh.harvard.edu). DTI data-processing pipeline will follow the diffusion toolbox in FSL to obtain FA and MD images, and voxelwise tract-based spatial statistics method will be applied to examine FA/MD changes. For the resting-state fMRI data, preprocessing and functional connectivity analyses will be performed with SPM12 software  by using MATLAB 2013b .The 3D T1 structure data analysis will be performed with FSL tools (FMRIB Software Library) . SIENAX The clinical outcome will be measured by six self-report questionnaires at baseline and the end of 6 and 12 weeks of treatment, followed by 12, 36, and 48 weeks of follow-up. The responses can reflect the degree of pain, sensation, emotion, and quality of life.The primary outcome measurement is VAS . VAS is The secondary outcomes are Short-Form McGill Pain Questionnaire (SF-MPQ) , WesternSF-MPQ is used to evaluate the pain, emotion, and psychological status of patients with KOA. It primarily comprises a 15-descriptor (11 sensory and 4 affective) rating on an intensity scale as follows: 0 = none, 1 = mild, 2 = moderate, and 3 = severe. A higher total score indicates more severe pain. The WOMAC is a self-administered questionnaire comprising 24 items divided into three subscales: pain (5 items), stiffness (2 items), and physical function (17 items). These measurements will be used to evaluate the symptom and quality of life improvement. A repeated longitudinal analysis will be used to evaluate the SF-MPQ mean scores.Furthermore, to investigate the influence of emotional state on the brain activity, HAMD  and HAMAMost items of the HAMD adopt the 5-level scoring method of 0–4 points. The standard of each level as follows: 0 = none; 1 = mild; 2 = moderate; 3 = severe; 4 = extremely severe. A few items adopt the 3-level scoring method of 0–2 points, and the grading standard as follows: 0 = none; 1 = mild to moderate; 2 = severe. The total score of HAMD is 78. A total score less than 8 means normal. A total score of 8–20 means possible mild depression. A total score of 20–35 means possible depression. A total score greater than 35 means severe depression. A repeated longitudinal analysis will be used to evaluate the HAMD mean scores.The total score of HAMA can better reflect the severity of anxiety symptoms. The total score can be used to evaluate the severity of anxiety symptoms and the effect of various drugs and psychological intervention in patients with anxiety and depression. According to the data provided by the China scale collaboration group, a total score ≥29 means possible serious anxiety, ≥21 means possible obvious anxiety, ≥14 means anxiety, ≥7 indicates possible anxiety, and <7 means no anxiety symptoms. A repeated longitudinal analysis will also be used to evaluate the HAMA mean scores.The screeners will collect data on the baseline characteristics when the patients are recruited. CRFs include observation and scanning time points, outcome measures, adverse events, and safety evaluations. Doctors are blinded to evaluate various clinical pain indicators and fill-in relevant information timely and accurately according to the CRF requirement. Only outcome assessors can access the CRFs and perform double-data entry. Then, two data administrators, who are beyond the research team and blinded to group allocation, will independently receive the completed CRF and enter them into an Excel database . They are required to have completed rigorous training for the data monitoring. Then, they will enter the real-time data into the Chinese Clinical Trial Registration Center, in which the electronic data-management system will be used to track and monitor the test data in real-time in the Department of Science and Technology in Yueyang Hospital. During the test, data administrators can access and manage data. After the test, all researchers will have access to the data, and non-researchers will not have access.Clinical data will be analysed through the intention-to-treat assumption. For all outcomes, we use longitudinal models where participant outcomes and treatments are collected at a multiple follow-up times. Moreover, participants who meet the suspension criteria will be removed from the trial, and the modified intention-to-treat analysis will be adopted. For the missing data, several sensitivity analyses will be conducted to evaluate their effects on the results of the experiment. All analyses were performed with SPSS 21.0 statistical software  by statisticians who are blinded to the group allocation.P < 0.05, and the 95% confidence interval will be reported.The baseline characteristics will be expressed with descriptive statistics for the two groups, which are reported as the mean ± standard deviation. A Kolmogorov–Smirnov test with Lilliefors correction will be used to analyse all quantitative variables to determine whether they follow a normal distribution. Parametric statistics (Tukey test) or nonparametric statistics (Wilcoxon rank-sum test) will be used for the within- and between-group analyses in accordance with the results of the homogeneity and normality analyses. When the initial homogeneity and normality of data distribution are found, repeated-measure ANOVA will be implemented, or the Friedman test and Kruskal–Wallis test will be used when initial homogeneity but non-normality of data distribution is found, or only linear mixed model will be adjusted for age and gender if the initial homogeneity is not found. Adverse events in each group will be documented as percentage for safety assessments by using the chi-square test or Fisher’s exact test. Statistical significance is defined as During the processing of the trial, quality control will be conducted with the management of the steering committee. To ensure the consistency of methods, professional trial method and regular monitoring technique should be trained before the researchers participate in the trial. If the study protocol is modified or corrected, the steering committee and ethics committee should be informed. The steering committee and ethics committee will ensure that the trial process works as planned, such as monitoring recruitment, potential harms, Tuina intervention, Education intervention, and data quality.Chronic pain in KOA often seriously affects the patients’ quality of life. Alleviating joint pain is an important part of KOA treatment recommendations in the international OARSI guidelines 2014)  and the 4  and thTuina belongs to the key noninvasive and nondrug therapies of TCM with distinctive characteristics , 85, whiMultimodal MRI is a cutting-edge technology in the study of pain brain-network mapping and analgaesia mechanism. It can explore the mechanism of pain and evaluate and predict pain. It is extensively used in brain science and pain research. Brain MRI is one of the most commonly used neuroimaging technologies in the study of central mechanism given its advantages of its high-quality spatial resolution, no radiation, rapid imaging velocity, non-invasion, and affordable price –89. The This clinical trial will study the multidimensional evaluation of KOA pain and evaluate the physical function, quality of life, mental status (the level of anxiety and depression), and influence of emotional state on brain activity. We will use a combination of clinical behaviour and multimodal MRI indicators such as brain-function indicators, brain-structure indicators , and brain-network properties to perform confirmatory and interactive Tuina analgaesia research, which can fill the knowledge gaps in brain pivot analgaesia research in Tuina discipline in this field.Furthermore, owing to the great difference between humans and animals, as well as the particularity of Tuina manipulation, verifying the central regulatory mechanism of Tuina intervention on KOA analgaesia in animal experiments is difficult because of too many uncontrollable factors. The application of multimodal MRI to Tuina discipline can clearly explore the brain central characteristics of KOA pain only in clinical trial, as well as comprehensively and deeply analyse the central analgaesic regulation mechanism of KOA patients with chronic pain after Tuina, which is a new trend in the future research of Tuina. We can also provide a reference experimental paradigm for the cross-study on the analgaesic effect of TCM nondrug therapy.This research has an inevitable limitation associated with the difficulty in controlling the methodology of blinding during the Tuina and healthcare education intervention, which cannot be blinded for the participants and therapists. Patients can know their group and anticipate the effect of the treatment in advance, which may affect their outcomes. For a study involving complex, multicomponent mind–body therapy, identifying a feasible, useful, and valid sham comparison group remains challenging, with no well-accepted solution.This trial is recruiting patients now. Participant recruitment started in Sep. 2020 and is expected to end in Dec. 2022. The version of this protocol is the 1st version and the completion time is 8 Sep. 2020.


The relationship between physical activity (PA) and physical fitness (PF) has been well established among college students. However, the impact of this relationship after 1 year of COVID-19 pandemic lockdown measures is unclear. This study aimed to test the relationship between PA and PF, exploring the trend across four years, the different components of PF related to PA, and their determinants, by analyzing specific items. A total of 1506 university students  in years 1–4 at two comprehensive universities in Shanghai were recruited after one year of COVID restrictions and asked to complete the PF measurements and International Physical Activity Questionnaire (IPAQ Chinese Short version). The PA level is categorized into three types of intensity (low-moderate-high), and the level of PF is represented by the total test score of each item. Results show that PA was significantly positively correlated with PF; PA levels significantly predicted 1000m-run, 50m-sprint, and standing-long-jump in males, and 800m-sprint and sit-ups in females. Males predominantly had high-intensity PA, whereas females maintained moderate-intensity PA over four academic years. Meanwhile, PA and PF both trended downward as academic years increased in males and females, which could be attributed to a decline in high-intensity PA. The following two recommendations were obtained from the study: first, college students should engage in high-intensity PA activities after the end of the epidemic. Second, colleges offer physical education classes for four academic years of college students to promote PA and PF. The COVID-19 pandemic has led to several impairments in physical and mental health [Two studies reviewed have shown that the direct impact of lockdown measures resulted in a decrease in PA ,3, whichPrevious studies have confirmed that PA and PF are closely related to college students, and they both tend to decrease with the academic year. For example, a study with 17-year-old girls in Lithuania showed a close association between PA and PF . A surveAfter prolonged COVID-19 restrictions, college students returned to campus, and their PA comes primarily from daily life activities on campus, including physical education classes and extracurricular sports activities. During the restriction period, the PA of college students was mainly from daily life at home. On the other hand, to date, most of the studies on the effects of the related COVID-19 pandemic on PA and PF have focused on the onset of the epidemic, but the relationship between PA and PF after one year of lockdown remains unclear. Thus, the objectives of this study were as follows: (1) to explore the relationship between PA and PF of students in four years of college after one year of lockdown restriction, and (2) to determine the effect of PA levels on PF and PF components. By elaborating on the relationship between PA and PF in this context, we expect the results of this study to provide a reliable reference for college PE class settings.This is a cross-sectional study with a random sample. We recruited participants from the following two universities: Shanghai Jiao Tong University and Tongji University. They are public, comprehensive universities located in Shanghai, and students come from all over the country, making the samples more universal and representative.During the weekend of November-December 2020, the Universities conducted the National Student Physical Fitness Test. During the test, students were free to choose the date and time to take the test. We recruited participants from all college students who came to the test. The inclusion criteria for the college students included (1) over the age of 18; (2) healthy ; (3) those who return to school to participate in normal learning activities after experiencing the restrictions of the mainland COVID19 in 2020; (4) availability to complete the National Student Physical Fitness Test (NSFT) and the International Physical Activity Questionnaire (IPAQ).Online sample size calculator was used to obtain sufficient statistical power . AccordiParticipants were required to complete the National Student Physical Fitness Test and IPAQ. The National Student Physical Fitness Test was divided into an indoor test and an outdoor test. After participants completed the questionnaire, they started completing the indoor items and then completed the outdoor items the next day. These two tests were conducted between October 2020 and December 2020, 1 year after the pandemic lockdown.A professional online questionnaire survey tool named “Questionnaire Star” was used for data collection. Prior to the testing, a comprehensive verbal description of the nature and purpose of the test and its experimental risks was elaborated to each participant. At the same time, participants were informed that they could withdraw freely without any reason. Students who indicated their willingness to participate were instructed to scan the code with their cell phones. The QR code was generated on “Questionnaire Star”. After the code was scanned, the content of the participant’s informed consent was displayed on the cell phone screen. If participants read it and selected “I agree”, they were considered voluntarily enrolled and well informed of the purpose and risks of the test, as well as they agreed with the use of the test data for the research.At the end of each week’s testing, we eliminated invalid samples, including unreasonable filling, regularity, continuity in answering, etc. The procedure was repeated during the testing period until the estimated sample size was reached. Finally, 1506 participants (19.48 ± 1.35 years old) were from grades one to four, including 840 males (19.47 ± 1.40) and 666 females (19.49 ± 1.29).Physical fitness measurements were based on the National Student Physical Fitness Test (NSPFT). NSPFT is a battery of tests issued by the Ministry of Education of the People’s Republic of China. It is currently a government-designated test used to evaluate the physical fitness level of university students in China . The tesTo measure the individual’s PA, we used the International Physical Activity Questionnaire (IPAQ), which has both a short and a long form. The IPAQ is recommended for use in adult PA surveys in several countries, which is considered a reliable and effective way to measure PA .In this study, we adopted the short version to measure the PA level of the participants. We asked participants to fill different levels of physical activity and how much time they spent each week performing specific activities, such as heavy lifting, digging, aerobic exercise, or fast cycling. Craig divided the IPAQ results into three levels  by integrating data and calculating the number of PA types , Table 3® Statistical Package for Social Sciences version 25.0  software. The significance level was considered at p < 0.05.Data were stratified by gender and academic year. Descriptive statistics showed the characteristics of participants using means, standard deviations (±SD), and percentages. The normal distributions of variables were verified through histograms and by the Kolmogorov–Smirnoff test. Two-tailed independent sample t-tests were used for normally distributed variables and Mann–Whitney’s U for non-normally distributed variables. Correlations between variables were determined using Pearson correlation analysis. A one-way analysis of variance (ANOVA) was used to compare the differences in PA (MET) and PF scores between the four academic years. A stepwise method of multiple linear regression was performed to test the influence factors between PA levels and PF scores. The univariate linear regression analysis was performed to identify the predictive relationship of PA levels on PF components. The collected data were double entered and analyzed using the IBMp < 0.05). Overall, the PA or PF was different for students in each of the four academic years.p = 0.004) and females  was negative. Both males and females had predominantly moderate PA or high PA in all four years. The percentages of moderate and high PA for males reached 45.8% and 52.5%, and 57.6% and 38.9% for females, respectively, while the percentages of low PA for males and females were only 1.8% and 3.6%.p < 0.001) and females  was significantly negative. Both freshmen and sophomores had significant differences with juniors in total PF scores in both sexes.p < 0.001) and females . Therefore, the higher the PA level, the higher the PF scores for both sexes. The multiple linear regression results showed that the regression equation was significant , where both gender  and grade  affected the total PF score. Age did not affect the total PF score . These variables explained 27.3% of the variance in the total PF score.p < 0.001), 50m sprint , and standing-long-jump  could be significantly predicted by PA levels. For females, it was the 800m run  and sit-up . No other components were significantly predicted.This study aimed to verify the relationship between PA and PF within academic years and determine whether different PA levels could cause differences in PF and PF components after 1 year of restriction. The results revealed a significant linear correlation between PA and PF in both sexes, and both PA and PF decreased each academic year. This result is consistent with other studies ,23,30,31In our study, females consistently had the highest percentage of moderate PA in the four years, and males had the biggest percentage of high PA in all but the junior year. Meanwhile, the percentage of low PA is the lowest each year for both sexes but increases with each academic year. The PA scale used in the study can be repetitive, structured, planned , leisurely , sports-focused , work-related , or transportation-related  . We are Another point worth noting is that the transition from sophomore to junior may become an important demarcation for PA and PF levels. Chinese college students are regarded as a special group on this issue. The study on students at Tsing Hua University also discovered a decline in both PA and PF of Chinese college students with an increase in years, and the total scores of PF increased from year one to grade two, then gradually slowed from year three to year four ,36. In COur results revealed that there is a significant linear correlation between PA and PF levels in both sexes. The sums of the percentages of moderate and high PA for males and females were 98.2% and 96.4%, respectively. Meanwhile, the percentage of low and moderate PA increased with the academic year, while the percentage of high PA decreased from year to year. Therefore, the decline in PF for both sexes is due to the decrease in high PA. It can be seen that the effect of high PA on PF is important. A study found that 17-year-old girls with high PA levels perform better in the standing-long-jump and have a lower BMI than others . LeppäneThe results showed that PA also affected specific PF components. According to the correlation results of PA and PF component scores, the 1000m-run, 50m-sprint, and standing-long-jump for males, and 800m-run and sit-up for females were significant. In PF measurements, these items were adopted to test the speed, endurance, and strength of individuals, including trunk strength and explosive strength. This finding gives us a new understanding of the specific items of PF affected by PA in university students. Murphy et al. identified that using PA to improve aerobic fitness appears to be the most effective at maintaining healthy body composition in college-age women . A studyThis study has the following limitations: (1) This study uses a cross-sectional research design, so the results could not infer the causal relationship between variables. (2) PA was measured using a self-reported questionnaire. Therefore, it is susceptible to potential recall bias, unreliable estimates, and misinterpretation of questions. (3) We only examined the effects of gender, grade, and age on the relationship between PA and PF. In reality, their relationship is likely to be influenced by other factors as well, so the findings may be more complex than the present study shows. A follow-up study design could be used in the future to validate the findings of this study.Despite these limitations, the study has obvious practical significance. To our knowledge, this is the first study exploring the relationship between PA and PF after one year of COVID-19 restriction. The results indicate that PA and PF still tend to decline with the increased academic year and that increasing high PA to improve speed, endurance, and strength becomes a goal after 1 year of lockdown. This contributes to targeted policies or recommendations facilitated for college students after the pandemic outbreak.This study demonstrated a significant correlation between PA levels and PF in college students. Both male and female students showed a decreasing trend in PA and PF with the increasing academic year. In addition, speed, endurance, and strength were strongly correlated with PA levels, and the decline in high PA led to a decline in PA and PF following 1 year of COVID-19 restrictions.


Iron, as an essential microelement, is involved in cell proliferation, metabolism, and differentiation. It also modulates the fate and function of macrophages in hematopoiesis and macrophage-mediated inflammatory responses. On the other hand, anesthetics can affect the inflammatory process by modulating the response to stress or the functions of immune cells. The aim of this paper is to understand how excessive iron intake alters physiological, functional characteristics of peripheral tissues and whether different anesthetics can alter cell metabolism regarding oxidative stress (OS) and inflammation through regulation of macrophage polarization. Y59 rats were injected intraperitoneally with iron dextran solution at a dose of 50 mg/kg or were exposed to inhaled anesthetics sevoflurane and isoflurane and their combination for 28 days every other day. The results show that the use of anesthetics reduces the rat’s organ weight and increases OS in peripheral tissues, leading to M1 macrophage polarization. Excessive iron intake leads to increased OS, inflammation, and an increased ratio of IL-12/IL-10 cytokines to the M1 macrophage phenotype. Iron, in combination with sevoflurane, has a protective effect in tissues showing the M2 phenotype of macrophages. The combination of iron dextran and isoflurane in rats leads to an increase in the erythropoiesis process made possible through the induction of hypoxia. General anesthesia is one of the greatest achievements of medicine; it allows safe surgeries to be performed safely, even in very young or old patients. However, it is not without risk, and this is a fact that is accepted daily by a large number of patients . InhaledAdditional side effects and toxicity include nephrotoxicity, hepatotoxicity, cardiac arrhythmias, postoperative nausea and vomiting, respiratory depression and irritation, malignant hyperthermia, and post anesthesia agitation . InhaledThere is growing evidence to suggest the role of oxidative and nitrative stress in a potential adverse reaction to anesthetics that stimulate reactive oxygen species (ROS) production. Evidence suggests that oxidative stress is the primary cause of the pathogenesis of inflammation, partial ischemia, and metabolic diseases . The maiBesides inflammatory effects, the anti-inflammatory effects of isoflurane and sevoflurane, the most widely used volatile anesthetics in clinical practices, have also been observed in humans. An increasing number of studies indicate the safety of isoflurane and sevoflurane, given oxidative stress and inflammatory response, in patients undergoing minor surgery . HoweverOn the other hand, environmental pollution and excessive metal intake endanger the health of humans and animals, leading to pathophysiological diseases associated with disruption of homeostasis of the whole organism. With the growth of industrialization, increasing environmental pollution, and the use of various products, human exposure to heavy metals has increased dramatically over the past 50 years. Although essential for the normal functioning of metabolism, metals such as iron, zinc, and copper can have negative effects if they are outside the normal range in the body ,12. IncrGiven the lack of data on exposure to inhaled anesthetics in the presence of heavy metals, it is assumed that such exposure will lead to different prooxidative/antioxidative effects on certain tissues and organs, chronic inflammation and metabolic disorders, immune system disorders, endocrine and paracrine interaction leading to long-term tissue damage caused by inflammation, ROS, proinflammatory cytokines, cell death, cell necrosis or autophagy, and the occurrence of autoimmune and chronic disorders. Therefore, the aim of this study is to (a) investigate the effect of long-term use of inhaled anesthetics alone and/or combined with iron dextran on the antioxidant/prooxidative effect of peripheral tissues and organs by monitoring changes in oxido-reduction and functional status in liver, spleen, kidneys, and lungs; (b) investigate how different levels of ROS caused by constant intake of iron, and inhaled anesthetics, lead to immune system disorders connected with macrophages polarization as a heterogeneous cell population, their adaptation, and their response to various stimuli caused by disturbed metal homeostasis.4H3F7O; Mr = 200.005 g/mol) and isoflurane  from Baxter, Deerfield, IL, USA were used as inhaled anesthetics. Iron dextran  from Santa Cruz Biotechnology, Dallas, TX, USA was used in this study as heavy metal. Additionally, from other chemicals saline solution or 0.9% sodium chloride manufactured by B. Braun Adria d.o.o., Zagreb, Croatia was used in the experimental treatment of animals.Sevoflurane (C2O4S in a subchronic dose of 50 mg/kg in purified water (aqua pro), manufactured by B. Braun Adria d.o.o., Zagreb, Croatia.The iron dextran solution was prepared by dissolving FeHad libitum, and housing conditions were standard .Both sexes of inbred Y59 rats, 3 months old, obtained from the Department of Animal Physiology, Faculty of Science, were used in the study. The study was performed on a total of 60 Y59 rats divided into 6 groups of 10 animals each according to the treatment. Animals were fed with standard diet for laboratory animals  with the availability of food and water The research was approved by the Ethical committee of Faculty of Science, University of Zagreb  and was performed in accordance with ethical and legal principles valid in the Republic of Croatia  before the start of the experiment, as well as during the experiment, and were therefore classified into groups with approximately similar body weight (±10 g). Based on the mean body weight of the animals per cage for each group, the number of individual preparations given during the experiment was determined. Animals were weighed at the beginning of the experiment, every seven days during the experiment, and on the day of sacrifice.ip) injection of iron dextran solution in a subchronic dose of 50 mg/kg and/or by exposing rats to inhaled anesthetic—Sevoflurane (2.4 vol %) and Isoflurane (1.3 vol %) [ip iron dextran solution. The fifth group of animals received 2.4 vol % inhaled anesthetic Sevoflurane two hours after ip injection of iron dextran solution, while the sixth group of animals received 1.3 vol % Isoflurane inhaled anesthetic two hours after ip injection of iron dextran solution during the treatment.Pathophysiological changes were induced by intraperitoneal (3 vol %)  in a conip with a combination of ketamine  at a dose of 75 mg/kg and xylazine  at a dose of 10 mg/kg, and sacrificed to collect the blood, tissues, and organs for further analysis.By exposing the animals to 28 days of treatment, the final or total dose of iron dextran was 600 mg/kg. The dose was selected based on the literature . ImmediaA. abdominalis) according to the guidelines of the Clinical Laboratory Standards Institute [After adequate anesthesia of the animals, blood samples were collected from the abdominal aorta  from Abaxis, York, UK was used for analysis, and biochemical parameters were analyzed on a VetScan® VS2 device . From serum (100 μL), the following biochemical parameters were determined: albumin (ALB), alkaline phosphatase (ALP), alanine aminotransferase (ALT), amylase (AMY), total protein (TP), globulin (GLOB), total bilirubin (TBIL), blood urea nitrogen (BUN), phosphorus (PHOS), creatinine (CRE), glucose, sodium, potassium, and calcium.For biochemical analysis, blood was collected in vacutainer without anticoagulants. To assess biochemical parameters, serum was used for further analysis. A Comprehensive Diagnostic Profile reagent rotor , the number of leukocytes (Lkc), differential blood count (DKS), haematocrit (Hct), haemoglobin (Hb), average erythrocyte volume (MCV), average content of haemoglobin in erythrocytes (MCH), average haemoglobin concentration in erythrocytes (MCHC), and erythrocyte size distribution (RDW) were determined. Haematological parameters were determined by the recommended analytical methods on the Horiba ABX169 electronic counter .After sacrifice, the liver, kidneys, spleen, and lungs were isolated and weighed on a digital scale . The obtained values of organ mass and animal mass were used to calculate the animal’s weight change and relative organ weight. The animal’s weight change and the relative organ weights were calculated according to the following formulas:In order to determine the level of 12 cytokines and chemokines after administration of inhaled anesthetics, and iron dextran, the Rat Inflmmatory Cytokines Multi-Analyte ELISArray Kit  was used and performed according to the manufacturer’s instructions. The absorbance values were read at 450 nm on Ao Absorbance Microplate Reader  with a wavelength correction at 540 nm.2− in the samples, expressed as μM/μL, was calculated via the slope line.The Griess reagent system  kit was used to measure nitric oxide. Nitric (II) oxide concentration was measured on homogenized samples of liver, kidney, and spleen, and 0.1 M sodium nitrite dissolved in water in concentrations from 0 to 100 μM was used as a standard. From the standard curve of the dependence of the absorbance on the nitrite concentration, the slope line was determined, and the concentration of NOArginase activity assay kit MAK112  was used to measure the level of arginase 1 activity from a kidney and spleen and was performed according to the manufacturer’s instructions. Arginase is an enzyme that catalyzes the conversion of L-arginine to L-ornithine and urea, whereby the produced urea reacts specifically with the substrate to produce a color proportional to the activity of arginase, and the absorbance was measured at 430 nm . One unit of arginase represents the amount of enzyme sufficient to convert 1 μM L-arginine to ornithine and urea per minute at pH 9.5 and 37 °C.Liver, kidney, spleen, and lung tissue samples were used to measure oxido-reduction status and were prepared as described in our previous paper .Protein concentration in liver, kidney, spleen, and lungs tissue samples was measured by the method of Lowry et al. . Protein2 whose concentration was measured spectrophotometrically  at a wavelength of 532 nm. Total MDA concentration was calculated using the extinction coefficient for MDA (ε = 1.56 × 105/M cm).To determine the concentration of malondialdehyde (MDA), the method of derivatization of MDA with 2-thiobarbiturate acid (TBA) was used to form a red fluorescent complex MDA-(TBA)2O2 amount, and catalase activity was measured spectrophotometrically  at a wavelength of 240 nm for one minute and was calculated using the extinction coefficient of H2O2 (ε = 39.4 m/M cm).The activity of catalase was determined by the Aebium spectrophotometric method . Catalasv/v) ratio with DTNB). To determine SOD activity, the percentage of inhibition of XOD activity (optimized reaction ratio Δ A/min ~0.025) was first calculated, while SOD activity was calculated from % inhibition and expressed as U/mg proteins.Total glutathione and SOD activity were determined according to paper . Regardip < 0.05. Further analysis of the differences between the groups was made by multiple comparison of the mean values of all groups. Some data are represented in a scatter dot plot using GraphPad Prism 7 software .The data were presented as mean ± standard error (mean ± SE). Statistical analysis was performed using the STATISTICA 14 program . Using the Kruskal–Wallis nonparametric test, the data were analyzed, and data were considered significant at the level of p < 0.001) and iron dextran + isoflurane (p < 0.05), compared to the control group  on the body weight change and relative organ weight for 28 days of treatment (chronic effect). The results of the body weight change and relative organ weight of the liver, lungs, kidney, and spleen are shown in ol group .p < 0.01) and iron dextran + isoflurane (p < 0.05)  a.p < 0.001) and iron dextran + isoflurane (p < 0.01), compared to the control group (p < 0.05). The relative weight of kidney was statistically significantly higher in the group treated with iron dextran compared to the groups treated with isoflurane and iron dextran + isoflurane. In the group exposed to isoflurane, a statistically significant decrease in the relative spleen weight was seen compared to the groups treated with iron dextran (p < 0.05) and iron dextran + sevoflurane (p < 0.001)  b.The increase in liver, spleen, and kidneys weight in the iron-dextran- and iron-dextran + sevoflurane-treated groups suggest that iron accumulation in these organs may induce tissue damage and inflammation with respect to iron toxicity, while a decrease in tissue weight in the isoflurane- and iron-dextran + isoflurane-treated groups leads to their accelerated decay.Confirmation of the inflammatory process was further assessed by measuring blood cell counts and biochemical parameters as indicators of tissue and organ damage. Tissue and organ damage were analysed by measuring the amount of circulating enzymes, proteins, metabolites, and substrates.The results of enzyme and protein analysis are shown in p < 0.05) and iron dextran + isoflurane , compared to the group treated with iron dextran (164.33 ± 9.12). The concentration of ALT was statistically significantly decreased in the groups treated with iron dextran, and iron dextran + sevoflurane, compared to the control group, and in the group exposed to isoflurane. The concentration of albumin and total proteins was statistically significantly lower in the groups treated with sevoflurane and iron dextran + isoflurane, compared to the group exposed to isoflurane, while the albumin concentration was also reduced and compared to the control group. Furthermore, the concentration of amylase was statistically significantly reduced in the group treated with iron dextran + sevoflurane, compared to the control group, and the groups exposed to sevoflurane and isoflurane, while the concentration of globulin was increased compared to the control group  and potassium  was statistically significantly increased (p < 0.05), while potassium concentration was also significantly increased and compared to the iron dextran-treated group (p < 0.001).ncreased . Sodium Total bilirubin was not statistically significantly altered in any of the treated groups during the treatment and therefore is not shown in the Macrophages, as key parameters in the inflammatory process and iron homeostasis, recycle iron obtained by spent red blood cell (RBC) catabolism, thus maintaining a normal level of erythropoiesis and preventing anemia. Changes in haematological parameters are shown in Differential blood count includes the number of neutrophils, lymphocytes, monocytes, and eosinophils. The total number of neutrophils was increased in the groups treated with iron dextran, iron dextran + sevoflurane, and iron dextran + isoflurane, compared to the control group, while in the group treated with iron dextran + sevoflurane it was increased and compared to the group exposed to sevoflurane a. Regardp < 0.05) and the group exposed to sevoflurane .The results of other haematological parameters are shown in Summarizing the obtained results, it can be seen that long administration of isoflurane and iron dextran + isoflurane can induce hypotoxic microenvironment.Macrophages, as heterogeneous cells, are fully involved in resolving inflammation at different levels of the inflammatory process. They may be initiators of the inflammatory response and participate in its resolution and maintenance of homeostasis in the second step, through regulation of self-polarization after endogenous or exogenous stimulation or during different environmental conditions, and through reprogramming and continuous plasticity . In normp < 0.001) and iron dextran + sevoflurane (p < 0.05) and compared to the group treated with iron dextran.The results of nitric oxide (NO) activity in animals treated with inhalation anesthetics and iron dextran, and their combinations, are shown in From the results of Additionally, in the spleen tissue of animals treated for 28 days, a statistically significant increase in NO concentration was seen in the iron dextran + sevoflurane and iron dextran + isoflurane treated groups in relation to the groups treated with sevoflurane, isoflurane, and iron dextran c.Macrophage biology is basically driven by the phenotype of macrophage arginine metabolism that is prevalent in ongoing immune response. Additionally, changes in nitric oxide and cytokines levels (Th2) are associated with L-arginine metabolism, which is essential for healing and maintaining healthy states, for example, by activating the immune system (macrophages), and these changes may disturb the L-arginine metabolism, usually by up- or downregulating arginase expression and activity, which may lead to chronic inflammations or other diseases. Thus, arginase generally acts as a dual factor that may cause different outcomes depending on the surrounding biochemical context and can cause pathological processes or a response mechanism to achieve homeostasis; therefore, we measured arginase activity in the kidney and spleen to assess their function.p < 0.05) and the group treated with iron dextran (p < 0.05) (The results of arginase activity in kidney tissue indicate its increase by 11.89% in the group exposed to isoflurane compared to the control group ( < 0.05) a. In the < 0.05) b.Cytokines are secreted by cells of the immune system, and their primary role is mediation and regulation among other participants in the immune system. In addition, they participate in the body’s defences by causing protective local inflammation that can have a systematic impact at the acute level. A total of 12 inflammatory cytokines from rat serum treated with inhaled anesthetics, iron dextran, and their combinations were analysed, and the results are shown in The greatest changes in cytokine levels were found in the group treated with sevoflurane, isoflurane, and iron dextran a,b. In tIncreased amounts of iron dextran and large amounts of inflammatory mediators caused by chronic inflammation can lead to oxidative stress and reactive oxygen species (ROS), so this paper also monitors the parameters of oxidative stress in the liver, kidneys, lungs, and spleen.p < 0.05) and iron dextran + sevoflurane , compared to the control group  and iron dextran + isoflurane , compared to the the control group (p < 0.05)  (p < 0.01)  d. In thectively) c, while  < 0.01) d.p < 0.01), CAT , and SOD  levels was seen in the iron dextran + sevoflurane group compared to the control group, while the SOD level was also increased by 11.62× compared to the group exposed to sevoflurane . In animals exposed only to volatile anesthetics, an increase in MDA level  was seen in the lungs of animals exposed to sevoflurane, while an increase in CAT  and SOD  levels was seen in animals exposed to isoflurane. In the lungs of animals treated with iron dextran + isoflurane, there was a decrease in GSH levels compared to the groups treated with sevoflurane, iron dextran, and iron dextran + sevoflurane  and control group (p < 0.001) (p < 0.01)  a, while  < 0.01) b. In the < 0.01) c and GSH < 0.01) d, compar2−, and H2O2 and contributing to lipid peroxidation in many tissues and organs [None of the currently available halogen anesthetics are ideally adapted to their function, and their negative effects and dose-dependent toxicity have often been reported . Many std organs ,30. Howed organs ,32. Howed organs .In addition, excess iron accumulates in the body, disrupting the normal function of mitochondria and numerous enzymes, worsening the body’s response to inflammation, and causing oxidative stress . AdditioTherefore, the aim of this study was to investigate the effect of inhaled anesthetics, isoflurane, and sevoflurane individually and in the presence of iron on the prooxidative and/or antioxidant effects of peripheral tissues and organs in chronic inflammation. Additionally, it was to find out if anesthetics can alter cell metabolism with respect to the transition metal as an initiator of ROS and inflammation, as well as of macrophage polarization.A good indicator of inflammation, toxicity, and metabolic changes is the change in animal body weight and relative organ weights. The results indicate a loss of body weight, which was observed in all treated groups and was most pronounced in the group treated with sevoflurane (−3%), compared to the control group (+13%) . Weight By injecting iron intraperitoneally, peritoneal macrophages are the first to come into contact with iron and phagocytose it . Specifi2 in the blood, i.e., a reduced ventilation response to hypoxia. At prolonged use of anesthetics, respiratory depression is further promoted by airway atelectasis. Although all anesthetics to some extent compensate for the resulting hypoxia by bronchodilation, isoflurane-induced airway irritation increases the degree of hypoxia compared to the sevoflurane [Inhaled anesthetics significantly affect the respiratory system by reducing respiratory volume and increasing respiratory rate leading to rapid shallow breathing, decreased alveolar ventilation, and consequent systemic hypoxia. Respiratory depression is further stimulated by a reduced ventilation response to elevated levels of COoflurane . In addioflurane  demonstrAdditionally, the analysis of haematological parameters confirms the presence of hypoxia and consequent increase in total erythrocytes in all groups, especially in groups treated with isoflurane and iron dextran + isoflurane a where aAccording to Li et al. , multiplThe presence of oxidative stress was confirmed by the analysis of biochemical parameters that indicate damage of the liver and other organs and increased levels of enzymes, proteins, metabolites, and substrates, as well as pathophysiological consequences of anesthetics and iron dextran. Such low ALP levels in the sevoflurane and iron dextran + isoflurane-treated groups  may indiFurthermore, the kidney and liver damage result from impaired homeostasis between the antioxidant system and increased oxidative stress in cells induced by anesthetics and iron dextran. While higher activities of antioxidant enzymes  indicate the presence of ROS, their low values are an indicator of depletion of the antioxidant system and are associated with damage and toxic effects of ROS. The release of GSH from cells has been observed in healthy hepatocytes and macrophages due to the role of maintaining antioxidant protection in the extracellular space, but its passive release and lower levels were observed immediately before cell death . The greMacrophages play a key role in immunity and tissue homeostasis, protecting the organism from infection, as well as maintaining essential tissue-specific functions . MacrophM1 macrophages metabolize L-arginine to NO and citrulline via nitric oxide synthase, while M2 macrophages metabolize L-arginine to ornithine and urea via the enzyme arginase. The balance between L-arginine consumption by arginase and nitric oxide synthase determines the outcome of an inflammatory reaction in which higher NO concentrations indicate an increase in the inflammatory response, and higher Arg-1 activity indicates a delayed inflammatory response and tissue healing and reparation . In anesThe ratio between IL-12 and IL-10 best indicates the polarization of macrophages. The highest level of inflammatory cytokines in serum  is visible in the group exposed to sevoflurane and less in the group exposed to isoflurane, which indicates M1 polarization a. IFN-γ Escherichia coli, where the authors suggest that sevoflurane may inhibit NF-κB, but the second signaling pathway may activate a direct increase in NO, thereby promoting phagocytosis and bactericidal properties of peritoneal macrophages [A study by Agoro et al.  points orophages . Furtherrophages  show thaSummarizing the results, it is clear that there is multiple regulation and that more signaling pathways are involved in response to increased levels of anesthetics and iron dextran, and that deeper insight at the molecular level is needed to monitor expression of key genes in the regulation process of the liver cells as well as environmental conditions associated with macrophage polarization. In addition, the results clearly indicate that excessive levels of iron and anesthetics lead to increased tissue and organ damage and that the anesthesiologist should assess the risk, select an anesthetic, and adjust techniques for optimizing surgery to reduce multiple anesthetic applications. Furthermore, interprofessional teamwork is needed to reduce the repeated use of anesthetics, especially in children and elderly patients undergoing different surgical procedures that require the use of anesthetics. These different procedures should be combined to ensure that single anesthetic encounter is performed and to reduce toxicity as well as costs for both the hospital and families. Additionally, according to our results, it seems important to monitor the dose of iron before and after surgery, given its toxicity classified as corrosive and cellular, and anesthesiologists should screen patients who are at high-risk for excess iron. Investigations into the possible therapeutic use of antimetabolite and antioxidant drugs with chelating ability would seem to be of interest to reduce the toxic effects of iron and volatile anesthetics and may be used in the management of patients with excess iron as well as improve outcomes of patients with transfusion-dependent anemia. Moreover, we suggest substituting volatile anesthetics for other anesthetic routes  where possible, and searching for new and non-metabolizable anesthetic agents and to establish an international threshold limits of exposure, because this approach would reduce overexposure of medical workers to anesthetics and waste anesthetic gases.