As previously indicated an association may exist between early sleep problems in infants and toddlers, and a diagnosis of attention deficit hyperactivity disorder (ADHD). The aim of this study was to study if this association could be replicated in a complete nationwide cohort of children. Prospective cohort study using national registries. All children born in Norway from January 2004 to December 2010 were included . Information on hypnotic drugs dispensed to children 0–3 years of age outside of institutions was collected from the Norwegian Prescription Database and used as a proxy for sleep problems. The outcome ADHD (ICD-10), as diagnosed by specialists in the Child Mental Health Service, was obtained from the Norwegian Patient Registry. Data were analysed using weighted estimation in Cox regression. The unadjusted weighted hazard ratio (wHR) for a later diagnosis of ADHD in children dispensed two or more prescriptions for any hypnotic drug, compared to zero prescriptions, was 2.30 [95% confidence interval (CI) 1.63–3.23] for girls and 1.75 (95% CI 1.48–2.07) for boys. For the sedative antihistamine trimeprazine the corresponding wHR was 3.71 (95% CI 1.83–7.52) for girls and 2.78 (95% CI 2.04–3.80) for boys. After adjusting for parental ADHD and parental education the wHR for trimeprazine users was 2.81 (95% CI 1.34–5.88) for girls and 2.33 (95% CI 1.70–3.20) for boys. Infants and toddlers who were dispensed hypnotics had an increased risk of ADHD at school age. This association was most pronounced with the use of trimeprazine, a drug traditionally prescribed to toddlers for sleep problems in Norway. After adjusting for parental ADHD and educational level the risk for ADHD among the trimeprazine users was still more than twice the risk among controls. Regulation problems such as sleep–wake regulation, crying and transition to solid foods are important challenges for all human babies in the first years of life , and proPharmacological treatment of childhood sleep problems is frequent in many countries , 16. An Norway has several national health registries with compulsory and automatically collected information. Several registries can be linked on an individual level, making it possible to conduct large cohort studies, with information from different sources and follow up over time. In such national registries, only emigrants are lost to follow up. We wanted to utilize these registries to create a large-scale cohort and study if previous findings on a relationship between early sleep problems and later development of ADHD could be replicated. As ICD-10 diagnosis for sleep disturbances are rarely used in the paediatric population, dispensed hypnotic medications for children was used as the exposure. We studied the association between dispensed hypnotics before age of 3 years, and diagnosed ADHD between 5 and 11 years of age, in all children born in Norway between 2004 and 2010. We further wanted to explore to what extent adjusting for parental ADHD and parental educational level affected the association.In this cohort study, we linked information from the Norwegian Prescription Database (NorPD), the Norwegian Patient Registry (NPR) and Statistics Norway, and used data spanning an 12 year time period (2004–2015). The unique person identifier held by all Norwegian inhabitants enabled us to link the different registries at an individual level..All pharmacies have, since 2004, been obliged to register electronically all dispensed prescribed drugs to individuals and send the information to the NorPD . Each prThe NPR is an administrative database containing activity data from all hospitals, private specialists and outpatient clinics in Norway. Reporting to the NPR is mandatory and linked to the governmental reimbursement system for funding of health services. Every admission to a hospital or appointment with a specialist or outpatient clinic is registered with information such as date, diagnosis and patient data. All Norwegian residents are covered by the public health care system through a national insurance scheme. Health care is free of charge for children under 16 years. This implies that our data should capture all children diagnosed with ADHD in Norway. All diagnoses are reported according to the International Classification of Diseases, 10th Revision, ICD-10 . IndividStatistics Norway provided the link between parents and children and information on the highest achieved educational level in 2014. Education is reported in nine categories according to the Norwegian Standard Classification of Education . Dates oData from the NorPD, the NPR, Statistics Norway and the National Registry were linked using the encrypted personal identifier. All children born in Norway in the years 2004–2010 were initially included in our study population, in total 429,158 children. 18,603 Children who died or emigrated before the age of 5 years were excluded. Our final selection consisted of 410,555 children.From the NorPD, we collected information about all prescriptions for children under 3 years, for drugs known to be used in Norway as hypnotic drugs. In addition to the traditional anxiolytic and hypnotic drugs we also chose to include first generation antihistamines classified in ATC under respiratory system—antihistamines for systemic use (R06), but known to be sedating. There is a clinical tradition for using sedating antihistamines for sleep induction in small children in Norway , 31. TheAs we only had access to birth month and birth year, we assigned a birth date in order to use the age of the children in the analysis. We chose to set the birth date to the 15th of the month for all children. This over- or under-estimated the actual birthdate by no more than 16 days. We wanted to investigate use of medication in infancy and toddler age, which is commonly considered to be from birth until 36 months of age. From the estimated birth date, a 36 months “exposure window” was calculated and we collected all dispensed prescriptions in this period. The medications were analysed separately and grouped together as the variable “all hypnotics”. A negligible number of toddlers received more than one hypnotic drugs during the exposure window. Also, investigating the effect of combined medication use was beyond the scope of this study. We stratified the exposure variable into those receiving no prescriptions, one prescription and two or more prescriptions. From before we know that around 70% of children who receive trimeprazine before 3 years of age only receive one prescription .For outcome, we identified all children who had at least two registered visits in specialised health care, resulting in an ICD-10 F90 (hyperkinetic disorder/ADHD) diagnosis from around school age of 5 years and onwards, either as main or secondary diagnosis in the NPR in the years 2008–2015. Time of outcome was for these children defined as time of the first diagnosis. Although the ICD-10-diagnosis F90 hyperkinetic disorder is slightly narrower than the DSM-IV diagnosis ADHD it is considered equivalent in this study. The children were censored at death, at emigration or as of December 31st 2015, whichever occurred first.For confounding factors, parental education was included to control for socioeconomic status of the family. Education was stratified into two levels; “low” up to high school diploma and “high” including college and university education. The level of education for one or both parents was registered for 407,608 children. All parents who had ever (2008–2015) been diagnosed with F90 hyperkinetic disorder registered in NPR or had ever (2004–2015) been dispensed a prescription for a stimulant medication  in NorPD were considered to have ADHD. Treatment with stimulant medication is regulated by the Norwegian authorities and must be initiated by a specialist in psychiatry, neurology or paediatrics.2 testing and weighted Cox regression.Two types of statistical analyses were performed; χ2 test was performed for the association of children’s ADHD with use of (i) any hypnotic drug, (ii) dexchlorpheniramine  and (iii) trimeprazine . We performed the same analysis for (iv) parental ADHD and (v) parental educational level. Cramér’s bias corrected V was computed using the “cramerV” function in the R-package “rcompanion”.A bivariate analysis with χSince follow-up time varied from 1 day to 7 years, Cox regression analyses were performed to estimate hazard rate ratios (HRs) associated with use of (i) any hypnotic drug, (ii) dexchlorpheniramine and (iii) trimeprazine. Time of outcome was the time of the first of two ADHD diagnoses, and exposure was a 3-level categorical variable: 0 (reference), 1, or ≥ 2 prescriptions before age 3. The Cox regression was adjusted with inclusion of parental ADHD as a two- level categorical variable; no parental ADHD (reference)/at least one parent with ADHD, and maternal and paternal education as two two-level categorical variables; high school or less/university or college education (reference). We present Kaplan–Meier survival plots, and we tested the proportional hazards assumption using the “cox.zph” function in R. In some cases there was a significant deviance from proportionality at a 5% level, and we therefore performed a weighted Cox regressions  using thThe analyses were performed using IBM SPSS statistics version 22 and R version 3.5.2.Before the age of 3 years, 10.4% of the girls and 12.3% of the boys had been dispensed at least one prescription of a hypnotic drug. The two most prevalent drugs were dexchlorpheniramine (girls 6.7% and boys 7.9%) and trimeprazine (2.8% and 3.6%). The prevalence of the other hypnotics was as follows: diazepam , melatonin (0.1% and 0.2%), nitrazepam (0.05% and 0.06%), midazolam (0.02% and 0.03%), clobazam (0.02% and 0.02%), oxazepam, promethazine, zopiclone, zolpidem  (data not shown in table).Use of hypnotic drugs at age 0–3 was more prevalent among children who later received an ADHD diagnosis than among other children . This can also be seen from the Kaplan–Meier survival plots . The weighted Cox regression did not reveal any statistically significant interactions, but we have chosen to still present the results separately for boys and girls.The unadjusted wHR for receiving a later diagnosis of ADHD when dispensed one prescription for any hypnotics from birth to 3 years of age, as compared to no prescriptions, was 1.23 (95% CI 1.02–1.49) for girls and 1.32 for boys (1.18–1.47) (Table Being dispensed any hypnotic drug twice or more before the age of 3 years, doubled the risk of a later ADHD diagnosis for girls and increased the risk by 60% for boys even after controlling for known risk factors such as parental ADHD and parental educational level. The risk after being dispensed the sedative antihistamine trimeprazine was even higher. The increased risk of ADHD diagnosis when receiving two or more prescriptions as opposed to one prescription indicates a dose–response relationship where more severe sleep problems further increased the risk of later receiving a diagnosis of ADHD.From birth to the age of 3 years, a little over 10% of both girls and boys received prescriptions for sleep inducing drugs. The most frequent drugs, dexchlorpheniramine and trimeprazine, are both first generation antihistamines, not traditional hypnotics. But unlike newer antihistamines these substances cross the blood–brain barrier and have sedating effects. In Norway there is a long-standing clinical tradition for prescribing the sedative antihistamine trimeprazine on a large scale for infants and toddlers as a sleep inducer . DexchloGirls who received sleep medication were more likely to be diagnosed with ADHD than boys who received sleep medication. Several studies find that toddler boys sleep less that girls , 40. PerThe association between dispensed hypnotic drugs and later ADHD could have several explanations. Although we believe that hypnotic use serves as a proxy for an underlying sleep problem, it could be argued that a direct drug effect on the brain development explains the association. The aetiology of ADHD is multifaceted, but even if drugs like trimeprazine and dexchlorpheniramine influence a number of receptors in the brain , such a Problems with sleep are frequent in both adults and children with ADHD , 46. A mThere could be several explanations for the association between sleep problems and later ADHD. Sleep problems could be an early symptom of ADHD preceding diagnosis. Other studies have shown modest to strong stability of ADHD-like symptoms from 18 months and throughout preschool years . AlternaThe association between dispensed sleep medication and later ADHD diagnosis could be biased by family and social background. Family adversities have been shown to be a more important explanation for the development of hyperkinetic traits than infant regulation problems . The assThe ADHD prevalence rates in the present study were low compared to what is found internationally , but simWe do unfortunately not have information on sleep diagnosis in the age 0–3 years as the Norwegian prescription database does not contain diagnosis unless medications are reimbursed and hypnotics are not reimbursed for insomnia or sleep disturbances in general . Sleep dICD-10 diagnosis of hyperkinetic disorder is narrower than the DSM IV diagnosis of ADHD, as the inattentive type ADHD is not formally included in ICD-10. This subgroup was thus not included in our study. This could introduce an unknown bias, but overall, the exposed group in this study probably includes children with more severe sleep problems and possibly more severe forms of ADHD, possibly leading to a slight overestimation of the association between sleep problems and ADHD.The proportional hazards assumption of the Cox regression was not fulfilled for all of the regressions, and we therefore present average hazard ratios from weighted estimation in Cox regression. These were quite close to the estimates from standard Cox regression although the confidence intervals were roughly twice as large.This study investigates a complete cohort of 410,555 children born in Norway in the years 2004–2010. We have captured dispensed hypnotic drugs from birth to 3 years of age and all diagnoses for ADHD from 5 years of age and up to the age of 11 years set by specialists in Child and Adolescent Psychiatry. It has previously been shown that infant regulation problems, including sleep problems, is associated with ADHD diagnosis. We replicate this finding in large cohort, where continuous data collection ensures no recall bias and no loss to follow up. We find that the use of hypnotic drugs before 3 years of age signifying substantial sleeping problems increases the risk of a later ADHD diagnosis. This was especially true for the antihistaminergic drug, trimeprazine.


Left Cornu Ammonis 3 (CA3) volume was a positive predictor of peer victimization . Left CA3 volume was a positive predictor of WM and left Cornu Ammonis 4 (CA4) volume negatively predicted WM . A cluster analysis revealed that children displaying symptoms of hyperactivity-impulsivity are the most at risk for peer victimization. Interventions focusing on minimizing peer victimization may aid in mitigating adverse downstream effects, and assist in promoting brain health and cognitive function.The symptoms of hyperactivity-impulsivity and inattention displayed by children with ADHD put them at risk of experiencing peer victimization. Hippocampal maturation, may reduce a child’s vulnerability to the experience of peer victimization, as it has been associated with decreased ADHD symptomatology. Working memory is an important executive function in the formation and maintenance of social relationships, which is often impaired in ADHD. We aimed to evaluate the relationship between problem behaviours, peer victimization, hippocampal morphology, and working memory in children with and without ADHD. 218 typically-developing participants  and 232 participants diagnosed with ADHD  were recruited. The ADHD group was subdivided into inattentive (ADHD-I) or combined (ADHD-C) types. The Child Behavior Checklist measured problem behaviours and peer victimization. Children underwent Magnetic Resonance Imaging (MRI). Hippocampal subfield volumes were obtained using FreeSurfer. The Wechsler Intelligence Scale for Children-fifth edition measured working memory (WM). The ADHD-C group displayed significantly higher levels of problem behaviours and peer victimization (all,  ADHD is further characterized into three diagnostic subtypes, including ADHD hyperactive-impulsive type (ADHD-H), ADHD inattentive type (ADHD-I), and ADHD combined type (ADHD-C)3. ADHD-H is characterized by clinically elevated symptoms of hyperactivity-impulsivity1. ADHD-I is categorized by clinically elevated symptoms of inattention and children with ADHD-C display both clinically elevated hyperactivity-impulsivity and inattention symptoms1.Attention Deficit Hyperactivity Disorder (ADHD) affects approximately 5% of the child and youth population worldwide and is characterized by symptoms of hyperactivity-impulsivity and inattention5. These social difficulties make children with ADHD susceptible to peer victimization and research has suggested that children with ADHD experience the highest rates of peer victimization when compared to children with other disabilities such as Autism Spectrum Disorder (ASD), Oppositional Defiant Disorder (ODD), intellectual disabilities, learning disabilities, and physical disabilities6. However, recent research has demonstrated that the levels of peer victimization experienced by children with ADHD vary according to their diagnosis subtype, with symptoms of hyperactivity-impulsivity centrally implicated as a factor that increases risk of experiencing higher levels of peer victimization7.Children with more severe hyperactivity-impulsivity symptoms also tend to exhibit more problem behaviours, such as rule breaking and externalized aggression, which can negatively influence the ability to form healthy peer relationships5. This suggests that children and youth with ADHD-H and ADHD-C are more susceptible to experiencing peer victimization than those with ADHD-I. Peer relationships play an important role in child development, and victimization by peers, including verbal, emotional, and physical abuse, can be a significant and potentially traumatizing stressor in a young person’s life9.Children with ADHD have deficits in social skills and often act inappropriately in social situations, such as being disruptive and intrusive, which make maintaining healthy peer relationships difficult10. However, for many brain regions, including the hippocampus, how the structure differs between ADHD and TD children is unclear15. In some studies, enlarged hippocampal volumes are reported in children with ADHD compared to TD children12, some studies report decreased hippocampal volumes in children with ADHD compared to TD children14, and others report no differences15. However, few studies have taken diagnosis subtype into account when assessing hippocampal volume in ADHD. In a recent study with a large group of 880 children, Al-Amin et al. (2018)16 found that those diagnosed with ADHD-C had significantly smaller hippocampal volumes compared to a TD group, however hippocampal volumes were not significantly different between TD children and those with ADHD-I. In addition, they found that that larger hippocampal volumes negatively correlated with levels of ADHD symptomatology, a finding consistent with Plessen et al. (2006)12. As such, it has been suggested that hippocampal growth acts as a compensatory mechanism in response to the presence of ADHD symptoms12. Therefore, we hypothesized that in children with ADHD, those with larger hippocampal volumes might be at a decreased risk of experiencing the adverse effects of peer victimization and those that present with smaller hippocampal volumes might be more vulnerable, as they most likely display higher levels of ADHD symptomatology that more severely impact their ability to form peer relationships7.Children with ADHD differ in their brain structure compared to their typically-developing (TD) counterparts17. Further, the association between peer victimization and hippocampal volume in the ADHD population and how this differs between ADHD subtypes has not been studied.The research on peer victimization and its association with hippocampal volume is scarce. In some studies, smaller hippocampal volumes were associated with higher levels of reported victimization in TD children, but further research is required to obtain a better understanding of this association19. Some studies have found that, in children, WM ability is  associated with social functioning20, and one study specifically observed WM deficits were related to poorer social functioningin children with ADHD21. The hippocampus is a key structure in WM and other executive functions, however the relationship between hippocampal volume and performance on tasks of executive function in children is not well defined24. In adult and aging populations, larger hippocampal volumes are associated with better performance on tasks of executive functioning, including WM24. However, this same relationship is not consistently observed in children, with some evidence suggesting that children with larger hippocampal volumes perform worse on tasks of memory23. The relationship between hippocampal volume and WM ability in the ADHD population remains largely unexplored.In addition to being at an increased risk of peer victimization due to their symptomatology, children with ADHD also display deficits in working memory (WM)In the current work, we examined the association amongst problem behaviours, peer victimization, hippocampal volume and WM function in a large heterogenous sample of children with ADHD and TD children. We addressed three main research questions: 1) Are problem behaviours and peer victimization predicted by ADHD diagnostic category? 2) Do hippocampal subfield volumes predict problem behaviours and peer victimization levels? 3) Do hippocampal subfield volumes predict WM ability? Our central hypothesis is that participants with ADHD will report higher levels of problem behaviours and peer victimization compared to TD children, and that hippocampal subregion volumes will significantly predict levels of peer victimization as well as WM ability.25. TD participants and those with a clinical diagnosis of ADHD were tested at three different sites: Rutgers University (RU), Citigroup Biomedical Imaging Center (CBIC), and Staten Island Diagnostic Research Center (SI). The research ethics boards at all respective institutions approved the study. Written informed consent was obtained from the participants’ parents and written assent was obtained from the participants. A breakdown of participant demographics can be found in Table Participants in this study ranged in age from 6.0 to 17.7 years. Data were collected by researchers at the Child Mind Institute and participants were recruited as part of the Healthy Brain Network initiative26. Participants diagnosed with ADHD were further divided into subgroups, those diagnosed with ADHD-I and those diagnosed with ADHD-C. Participants with an ADHD-H diagnosed were excluded from this study due to a small sample size of only 15 participants having received this subtype diagnosis. All participants in the study were fluent in English, had a parent that was able to complete informant questionnaires, had an Intelligence Quotient (IQ) score of over 66, and were free of brain injury or disease. Participants were excluded if they presented with schizophrenia or bipolar disorder, psychosis, substance dependence, acute intoxication, a neurodegenerative disorder, or any other neurodevelopmental disorder. The study was conducted in accordance with the Declaration of Helsinki.In combination with clinical judgment, the computerized version of the Kiddie Schedule for Affective Disorders and Schizophrenia (K-SADS-COMP) was administered under the supervision of a licensed clinician to make diagnoses of ADHD, and to confirm the absence of ADHD diagnoses in TD participantsThe data for this study was collected in 4 study visits. In the first visit, the WISC-V was administered by a trained psychometrist. The second visit consisted of the MRI protocol, including the T1-weighted sequence conducted by a trained MRI technician. During the scan participants watched two cartoon movies, the first movie was a short-film titled ‘The Present’ and the second was a 10-min clip from the full-length film ‘Despicable Me.’ Parent questionnaires were completed during the third visit and the K-SADS-COMP was completed during the fourth and final visit.27. The BSMSS assesses level of education and occupation for both of a child’s parents, or from one parent if the child is from a single-parent household27. The scores were converted into a total score between 8 and 66 for each child, with a higher score indicative of a higher SES27.The Barratt Simplified Measure of Social Status (BSMSS) was used as a measure of socioeconomic status (SES) and was completed by a parent28. To measure levels of problem behaviours in the participants, the Child Behaviour Checklist (CBCL) was administered to parents29. The subscales used from the CBCL in this study included the aggressive behaviour, rule breaking behaviour, social problems and withdrawn scales. In addition, peer victimization was measured using the CBCL subscale developed by McCloskey and Stuewig30. The peer victimization subscale contained four questions: “doesn’t get along with other kids”, “gets in many fights”, “gets teased a lot”, and “not liked by other children.” The Wechsler Intelligence Scale for Children-fifth edition (WISC-V) was used to obtain a Working Memory Index (WMI) in all participants, which included a digit span and a picture span task31.To assess symptoms of hyperactivity-impulsivity and inattention, the Strengths and Weaknesses Assessment of ADHD and Normal Behaviour (SWAN) was completed by a parent25. At the Staten Island Diagnostic Research Center, participants were scanned on a 1.5 T Siemens Avanto scanner and at Citigroup Biomedical Imaging Center participants were scanned on a Siemens 3 T Prisma25. To adjust for the differences in scanners, statistical models included Magnetic Resonance Imaging (MRI) site as a covariate.A Siemens 3 T Trio scanner was used at the Rutgers University siteHigh-resolution anatomical images were acquired using a 3D-MPRAGE pulse sequence with 192 T1-weighted, straight sagittal slices (1 mm thickness).http://surfer.nmr.mgh.harvard.edu) version 6.0. was used to segment and isolate hippocampal structures at high-resolution32. The hippocampus was segmented, bilaterally, into the hippocampal tail, subiculum, CA1, hippocampal fissure, presubiculum, parasubiculum, molecular layer, granule cell layers of the dentate gyrus (GCMLDG), CA3, CA4, fimbria, and the hippocampus-amygdala-transition-area (HATA). The total cerebral volumes were extracted using the Freesurfer pipeline.To obtain hippocampal subfield volumes from the structural images, FreeSurfer .Our first aim was to examine problem behaviour and peer victimization in children with TD, ADHD-I, and ADHD-C. In five general linear models, we examined parent-reported problem behaviours and peer victimization  related to diagnostic group . Age, sex, SES (using the BSMSS), and study site were adjusted for in each model.. In four general linear models we examined peer victimization , aggressive behaviour , rule breaking behaviour , and social problems  in relation to hippocampal subfield volumes , while adjusting for age, sex, SES , diagnostic group, total cerebral volume (TCV), and MRI site.Our second aim was to examine problem behaviours and peer victimization and their relationships to hippocampal subfield volume. A general linear model was used to examine the relationship between the left and right hippocampal subfield volumes , and WM . We adjusted for age, sex, SES , diagnostic group, TCV, and MRI site.Our third aim was to examine the association of hippocampal subfield volumes and working memory abilityA total of 218 TD participants  and 232 participants diagnosed with ADHD  were recruited. Of the 232 participants diagnosed with ADHD, 108 participants had the ADHD-C subtype  and 124 participants had the ADHD-I subtype . Participant characteristics can be found in Table In our first aim, we examined the relationship between problem behaviours from the CBCL, including social problems, rule breaking behaviour, aggressive behaviour, and withdrawn, and diagnostic group . We also examined how the experience of peer victimization differs by diagnostic group . Age, sex, SES, and study site were adjusted for in all the models. In the problem behaviours models 433 participants were included in analysis, 204 TD children, 123 children with ADHD-I, and 106 children with ADHD-C. In the peer victimization model, a total of 431 participants were included in the analysis, 203 TD children, 123 children with ADHD-I, and 105 children with ADHD-C. p < 0.001) and ADHD-I  groups had significantly higher scores on the aggressive behaviour subscale. ADHD-C participants also had significantly higher scores than the TD participants on the rule breaking behaviour  and social problems  subscales. No significant differences were found between the TD and ADHD-C participants on the withdrawn subscale . Nor were significant differences found between the TD and ADHD-I participants on the rule breaking behaviour , social problems , and withdrawn  subscales. In comparing children with ADHD-C to children with ADHD-I, we found that those with ADHD-C had significantly higher scores on aggressive behaviour, rule breaking behaviour, and social problems .Compared to the TD group, children from both the ADHD-C , but there was no significant difference found for peer victimization between the ADHD-I and TD groups . In addition, the children with ADHD-C had significantly higher peer victimization scores than the children with ADHD-I (p < 0.001). Refer to Fig. When examining the peer victimization subscale, children with ADHD-C had significantly higher levels of peer victimization compared TD children . Left CA3 volume showed a positive but not-significant association with rule breaking behaviour , aggressive behaviour , and social problems .Left CA3 volume was positively associated with peer victimization , children with ADHD-C , and children with ADHD-I .We subsequently examined the interaction between left CA3 volume and diagnostic group (left CA3 volume x diagnostic group) in the peer victimization model. We found that left CA3 volume was significantly and positively associated with peer victimization in in TD children , but the ADHD-C group did not significantly differ from the TD group .This analysis included a total of 243 participants, including 109 TD children, 76 children with ADHD-I, and 58 children with ADHD-C. When adjusting for age, sex, SES, MRI site, TCV, and hippocampal subfield volumes the ADHD-I group had significantly poorer WMI scores than the TD group . Statistical interactions were tested in the WMI model between left CA3 volume and diagnostic group (left CA3 x diagnostic group). Left CA3 volume was significantly and positively associated with WMI in TD children , children with ADHD-C , and children with ADHD-I .We examined the relationship between hippocampal subfield volumes and WMI. Left CA3 volume was found to be significantly and positively associated with WMI . Statistical interactions were also tested in the WMI model between left CA4 and diagnostic group (left CA4 x diagnostic group). Left CA4 volume was significantly and negatively associated with WM in TD children , children with ADHD-C , and children with ADHD-I .Left CA4 volume was found to be significantly and negatively associated with WMI . In this post-hoc model, the associations between WMI and left CA3  and left CA4  remained significant.As anxiety and depression may also be important predictors of hippocampal volumes in children with ADHD, we further examined the association amongst WMI and brain development in a post-hoc analysis using the anxious/depressed subscale of the CBCL as a covariate. Scores on the anxious/depressed scale were not significantly related to WMI , peer victimization score (p < 0.001), WMI (p < 0.001), and the hyperactivity-impulsivity and inattention subscales from the SWAN (both p < 0.001). Refer to Table To examine the associations amongst diagnosis, hippocampal volumes, WM as well as ADHD symptomatology, a K-means cluster analysis was performed with 245 participants: 111 TD, 58 ADHD-C, and 76 ADHD-I. The analysis included variables of brain morphology, behaviour, and cognition (Z-scored). The model included, left CA3 volume  was grouped into cluster 1, 17% were grouped into cluster 2, and 47% were grouped into cluster 3. Of the 58 total ADHD-C participants, 41% were grouped into cluster 2, and 59% were grouped into cluster 3. Of the 76 total ADHD-I participants, 1% belonged to cluster 1, 14% were grouped into cluster 2, and 84% were grouped into cluster 3. Refer to Fig. p = 0.018), WMI  = 15.44, p < 0.001), peer victimization  = 364.86, p < 0.001), hyperactivity-impulsivity  = 103.63, p < 0.001), and inattention  = 112.24, p < 0.001). Post hoc multiple comparison using Bonferroni correction revealed the differences between the clusters. Significant differences were found between Cluster 1 and Cluster 2 on WMI, peer victimization, hyperactivity-impulsivity, and inattention . Significant differences were found between Cluster 1 and 3 on Left CA3 volume (p = 0.015), WMI, hyperactivity-impulsivity, and inattention . Significant differences were found between Cluster 2 and 3 on peer victimization (p < 0.001) and hyperactivity-impulsivity (p = 0.001).A one-way ANOVA was conducted to examine the differences between the clusters on all the variables. Significant between-group differences were found for all the variables: left CA3 volume  = 4.07, The presence of problem behaviours, levels of peer victimization, hippocampal subfield morphology, and working memory were assessed in a large heterogenous sample of children further divided by diagnostic group: TD, ADHD-C and ADHD-I. We found that problem behaviours and levels of peer victimization differed between TD, ADHD-C, and ADHD-I groups. We also report that hippocampal subfield volumes predict the presence of peer victimization levels in children with ADHD and TD children. Lastly, hippocampal subfield volumes were associated with working memory ability in children with ADHD and TD children. Peer victimization was highest in children that displayed elevated levels of hyperactivity-impulsivity. Findings suggest that children with ADHD-C who display elevated levels of hyperactivity-impulsivity may be at greater risk for peer victimization.As hypothesized, children in the ADHD diagnostic groups had elevated levels problem behaviours and peer victimization compared to TD children. Parents of children with ADHD-C reported higher levels of aggressive behaviour, rule breaking behaviour, social problems, and higher levels of experiencing peer victimization in their children compared to parental reports for TD children and those with ADHD-I. The children with ADHD-I only differed from the TD children when comparing levels of aggressive behaviour; with significantly higher reported levels in the ADHD-I group. No significant differences were found in levels of withdrawal between any of the diagnostic groups.5. Children with ADHD who display more severe levels of externalizing behaviours have been observed to be the most at-risk pediatric population to experience peer victimization6. Investigation into how peer victimization differs amongst the diagnostic subtypes of ADHD is sparse. Recent findings into the symptomatology of ADHD in relation to peer victimization suggest that more severe symptoms of hyperactivity-impulsivity are associated with higher rates of peer victimization among children7. Not only are hyperactive-impulsive symptoms related to experiencing more victimization, it has also been demonstrated that individuals with these symptoms are also more likely to be perpetrators of bullying themselves7. This notion is consistent with the findings from the present study. Although the peer victimization measure in the present study only accounted for experiencing victimization, the significantly elevated aggressive behaviour, rule breaking behaviour, and social problems scores in the ADHD-C group suggest that this group might also be perpetrators of victimization and fit the profile of a bully-victim7. As the ADHD-I group has levels of hyperactivity-impulsivity that are comparable to the TD group, it follows that they are victimized by their peers at rates similar to the TD group. Although the children with ADHD-I also had significantly elevated aggression scores compared to TD children, the lack of hyperactivity-impulsivity symptoms combined with low rates of peer victimization suggest that this group might fit into a different category than the ADHD-C group; that is, one of aggressive children and adolescents that are accepted by their peers33.Children with ADHD are typically not well-liked by peers and can often experience social rejection and victimization35. In the present study, the children in the ADHD-C group were younger than the group diagnosed with ADHD-I; however all models were adjusted for age. Age was not significantly associated with peer victimization, aggressive behaviour or rule breaking behaviour. However, younger age was associated greater social problems, and older age was associated with withdrawn behaviours. In the present study, we did not examine the associations of problem behaviours in younger versus older children. It would be a worthwhile avenue of future exploration to investigate problem behaviours and peer victimization between the ADHD subtypes in  specific age groups when these behaviours may be more likely to occur.Although significant differences were found between the diagnostic subtypes in peer victimization and problem behaviours, some research has highlighted instability and unreliability in the ADHD diagnostic subtypes, especially as children age36. It is crucial that future research focuses on examining the impact of different types of victimization on child and adolescent psychological health. Determining how different types of victimization effect children can inform intervention strategies and can be useful for informing school-based interventions.Overall, experiencing peer victimization may be traumatizing, with a tremendous impact on later psychological development12. However, contrary to our hypothesis, in the current study larger left CA3 volumes were associated with higher parent-reported levels of peer victimization in children with ADHD-C, ADHD-I and TD children. Our peer victimization scale measured victimization in the past 6 months, however it is possible, and perhaps likely that the children who experienced peer victimization in the past 6 months, had been experiencing victimization over a longer period of time. The CA3 region is a particularly stress-sensitive area and has previously been associated with other forms of childhood adversity37. Typically, smaller hippocampal volumes have been associated with a greater incidence of trauma and adversity in TD children39. However, this association has been found to be different in children with mental health and neurodevelopmental disorders42. Some models suggest that chronically elevated levels of stress during childhood may cause reduced stress responsivity, which in turn may mitigate the neurotoxic effects glucocorticoids (GCs) typically have in atrophying the neurons of the hippocampus43. This cascade of events results in larger hippocampal volumes, as found in our study.Research suggests children with smaller hippocampal volumes are usually observed to display more severe ADHD symptomatology, and as a result may be more vulnerable to the adverse effects of peer victimization42. A recent study found larger subiculum, presubiculum, and CA1 volumes to be associated with childhood trauma in bipolar children41. Children with ASD have also been found to have enlarged hippocampal volumes compared to TD children40 and larger hippocampal volumes in children with ADHD have also been observed12.In addition, research conducted with pediatric post-traumatic stress disorder (PTSD) populations has provided evidence that hippocampal volumes are enlarged in PTSD when compared to  matched non-traumatized groups, suggesting that anxiety and stress may be associated with increased growth of the hippocampusFuture research in this area could focus on examining if and how the relationship between hippocampal volume and peer victimization differs according to cortisol levels. In addition, age and gender should be directly examined. To maintain the large sample size in the current study we were not able to split the data into subgroups; however, these factors were used as covariates. Age was not a significant covariate in the model relating hippocampal volumes and peer victimization. In addition, future research is warranted to elucidate the relationships between ADHD subtype, symptomatology and peer victimization to hippocampal volume, specifically including the ADHD-H subtype.19. However, there is a lack of consensus on how the different ADHD subtypes differ in respect to WM ability, and cognitive ability in general. Some studies report no differences between high and low inattention symptoms and WM scores44. Other studies have found inattention to be a significant predictor of WM ability in adolescents with ADHD45. As children with ADHD-C also have clinically elevated symptoms of inattention, in turn it is unclear why the children with ADHD-C did not display WM deficits when compared to the TD children in our study.The children with ADHD-I had significantly lower scores on the WMI when compared to TD children. The ADHD-C group did not significantly differ from the ADHD-I group nor the TD group on WMI scores. WM deficits are often seen in children with an ADHD diagnosis46. Larger volumes predict better cognitive function in adult samples24. Yet, findings in children and adolescents provide inconsistent results to support this view. In a meta-analysis by Van Petten23 multiple studies examining hippocampal volume and memory performance in child and adolescent populations found negative correlations between volume and memory abilities. This suggests that the positive relationship between cognitive ability and hippocampal brain volume may increase with age23. It has been found more recently that as children age, smaller hippocampal volumes are associated with superior memory abilities49. Previous studies examined whole hippocampal volumes. Future studies should investigate the relationship between hippocampal subfield volumes and WM ability in ADHD populations.Larger left CA3 volumes and smaller left CA4 volumes were significantly predictive of higher WMI scores in all of the diagnostic groups. CA3 and CA4 are both key hippocampal subfields that are involved in WM51. However, some studies have suggested that children with ADHD and comorbid disorders do not perform signficiantly differently on tasks of executive functioning, such as WM, from children with an ADHD diagnosis alone50. A post-hoc analysis was conducted including the anxious/depressed subscale as a covariate in the WMI model. We found that the anxious/depressed scale was not significantly related to WMI and that left CA3 and left CA4 remained significant predictors of WMI, however future research should examine this further.Anxiety, depression, and other common disorders that co-occur with ADHD can also impact WM performanceA three-cluster K-means model was used to characterize the present study population. Cluster 1 included 40 TD children and 1 child with ADHD-I who have low levels of inattentive and hyperactive-impulsive symptomatology, good WM ability, and low peer victimization. Cluster 2 included 19 TD children, 24 children with ADHD-C and 11 children with ADHD-I who have significantly elevated levels of peer victimization and hyperactivity-impulsivity when compared to the two other clusters. Cluster 3 included 52 TD children, 34 children with ADHD-C and 64 children with ADHD-I who have significantly enlarged left CA3 volumes and significantly higher hyperactivity-impulsivity and WMI scores compared to children in Cluster 1.12 observed between CA3 volume and symptoms of inattention in children with ADHD. In our model children in cluster 3 have the most prominent enlargement of left CA3 volumes, but milder symptoms of inattention than children in cluster 2. This suggests the possibility that enlarged left CA3 volumes are representative of a compensatory mechanism by which the hippocampus hypertrophies in response to the presence of ADHD symptoms and results in less severe symptomatology12. The relationship between symptoms of inattention in children with ADHD and hippocampal volume, specifically in the CA3 subfield should be further explored. Our results suggest that deep phenotyping of brain morphology, cognition, and behaviour can identify subtle differences in ADHD subtypes.The results of the cluster analysis suggest that ADHD symptomatology, especially hyperactivity-impulsivity, is related to increased levels of peer victimization. Our model also suggests that left CA3 volumes are associated with ADHD symptomatology. In both Clusters 2 and 3, symptoms of hyperactivity-impulsivity and inattention are significantly elevated and, in both groups, left CA3 volumes are enlarged compared to Cluster 1, although only significantly enlarged in cluster 3. Similarities can be seen between our model and the inverse correlation Plessen et al.52. As children age, an ADHD-H diagnosis becomes less common and could be the reason we only had access to the data of very few children with this subtype. Another limitation is that different scanners with different strengths were used at the three MRI sites. However, a majority of the participants were scanned at CBIC and RU, which both had 3 T scanners. The medication status of the children and adolescents in the study sample are unknown and this is a potential confound for behavioural reports, hippocampal volumes and WM performance.A limitation of the current study is our use of a parent-report questionnaire, the CBCL, as a measure of problem behaviour and peer victimization. Children have many experiences without their parents present, such as in school, recreational activities, etc., thus child and teacher reports may be able to provide additional information that cannot be captured by parent-report alone. However, only a limited subset of children had self- and teacher- reports available. This is an important area of future research. Another limitation of the current study was the absence of the ADHD-H. We only had access to data for 15 participants diagnosed with ADHD-H , which was not a large enough sample for valid analyses. In children and adolescents ranging from 3 to 18 years of age, ADHD-I is the most common ADHD subtype with a prevalence of between 2.2 and 5.7%, the prevalence of ADHD-C ranges from 1.1 to 2.4%, and the prevalence of ADHD-H ranges from 1.1 to 4.9%


Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder common from childhood to adulthood, affecting 5% to 12% among the general population in developed countries. Potential etiological factors have been identified, including genetic causes, environmental elements and epigenetic components. Nutrition is currently considered an influencing factor, and several studies have explored the contribution of restriction and dietary supplements in ADHD treatments. Iron is an essential cofactor required for a number of functions, such as transport of oxygen, immune function, cellular respiration, neurotransmitter metabolism (dopamine production), and DNA synthesis. Zinc is also an essential trace element, required for cellular functions related to the metabolism of neurotransmitters, melatonin, and prostaglandins. Epidemiological studies have found that iron and zinc deficiencies are common nutritional deficits worldwide, with important roles on neurologic functions , finicky appetite, and mood changes (sadness and irritability). Altered levels of iron and zinc have been related with the aggravation and progression of ADHD. Objective: This is a systematic review focused on the contribution of iron and zinc in the progression of ADHD among children and adolescents, and how therapies including these elements are tolerated along with its effectiveness (according to PRISMA guidelines). Method: The scientific literature was screened for randomized controlled trials published between January 2000 to July 2021. The databases consulted were Medline, PsycINFO, Web of Science, and Google Scholar. Two independent reviewers screened studies, extracted data, and assessed quality and risk of bias . Conclusion: Nine studies met the eligibility criteria and were selected. Evidence was obtained regarding the contribution of iron-zinc supplementation in the treatment of ADHD among young individuals. The discussion was focused on how the deficits of these elements contribute to affectation on multiple ADHD correlates, and potential mechanisms explaining the mediational pathways. Evidence also suggested that treating ADHD with diet interventions might be particularly useful for specific subgroups of children and adolescents, but further investigations of the effects of these diet interventions are needed. Attention-deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder, usually beginning in early childhood and with a chronic progression to adulthood with several negative consequences, such as low self-esteem, difficulties in interpersonal relationships, and problems in school learning . SubtypeEtiological research outlines that ADHD constitutes a complex condition with multiple interactive factors . SpecifiDiverse treatment plans have demonstrated to improve the symptoms of ADHD, usually combining medication (stimulants and non-stimulants have proved efficacy) with behavioral therapy ,34. EmpiOn the other hand, despite growing pharmacological discoveries, mental disorders (such as ADHD) have shown a gradual rise during the last few decades, and rates are expected to continue increasing in the coming years ,43,44. IDietary models have proved to be relevant to both metabolic performance and individuals’ behavior. Restriction and elimination diets have been tested in ADHD treatments , and supSome studies have analyzed the contribution of zinc and iron on ADHD occurrence, but conflicting findings have been obtained . As a trOn the basis of the controversial results published regarding the role of dietary nutrients with zinc and iron for the treatment of ADHD ,60, we pPreferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) . The next filters were employed: (a) type of publication:  and ; and (b) Date of publication: (From 1 January 2000–31 July 2021).PsycINFO: search = [((zinc OR iron) AND (ADHD)) AND ((treatment or therapy))]. The next filters were defined: (a) Type of publication: [Peer Reviewed Journal]; and (b) Date of publication: [1 January 2000–31 July 2021];Web of Science: search = [(((zinc OR iron OR ferritin) AND (ADHD)) AND ((treatment or therapy))))]. The next filters were defined: (a) Document Types: [Clinical Trial], and (b) Publication years: [1 January 2000–31 July 2021];Google Scholar: search = [(zinc OR iron) AND (supplement) AND (ADHD) AND (treatment or therapy) AND  AND (children or adolescent) AND ].The research was conducted on 20 July 2021. The strategy research (keywords and search sequence) for each database was:Two authors of this systematic review independently analyzed the title and abstract of each record, according to the inclusion/exclusion criteria. Only studies meeting the eligibility criteria were then extracted. Data validation was discussed by the same authors, and disagreements were resolved by discussion and if needed by consulting a third team member until a consensus was reached.The authors also extracted data of the identified screening records to be described and evaluated in the results section. The information considered for applying the eligibility criteria was: the date and location of publication, type of publication, sample size, participants’ sex and age range, study design, type and duration of the treatment, and measures and outcomes .Consolidated Standards of Reporting Trials guidelines were employed (CONSORT-2010) , obtaining the precision of the estimates  and carrying out a cost-effectiveness analysis cost). p = 0.02), but no differences between the groups were found in the other measures for the hyperactivity and impulsivity scales. The lack of differences in the total ADHD score in this study suggested that augmentation with zinc could only partially improve ADHD severity levels. The authors of the study also noticed that the difference between the two groups regarding the gender ratio compromised the generalization capacity, and that the use of the Connors Parent’s Questionnaire as the only measure for ADHD was a significant limitation .The study of Noorazar and colleagues used the next intervention : (a) a dn = 40 pediatric children, and obtained an improvement in ADHD signs associated to the experimental group in the questionnaires answered by teachers (no differences emerged from the parents’ reports) [The study of Zamora and colleagues also valued the effect of zinc supplementation (10 mg/day) as an adjuvant therapy  for ADHD in reports) . These an = 52 ADHD children treated with d-amphetamine (weight-standardized) and zinc complementation (15 mg/day or 30 mg/day) or d-amphetamine and a placebo, a similar improvement in ADHD was observed in all the conditions  [In the research by Arnold et al. with placebo) . InteresThe study carried out by Akhondzadeh et al. aimed to compare two groups of patients with ADHD  and both conditions treated with methylphenidate (1 mg/kg/day), and the experimental group obtained more improvement according to parents’ and teachers’ ratings at 6 weeks of the intervention . Trend an = 400 children with ADHD observed that the group receiving the zinc supplement (approximate dose of 40 mg/day) compared with the control group (receiving placebo) obtained greater improvements in multiple measures  but results were similar for attention deficiency [The study by Bilici and colleagues among a large sample of ficiency . The gren = 23 children who met the criteria for ADHD, which showed that the experimental group reported more improvement than the control group in different treatment outcomes [The study by Konofal and colleagues aimed to examine the contribution of iron supplementation  in a sample of outcomes : ADHD syn = 42 children with ADHD also obtained benefits for the use of ferrous sulfate (5 mg/kg/day) plus methylphenidate (1 mg/kg/day), in the inattentiveness, hyperactivity, and impulsive symptom levels reported by parents at 2 months of treatment [The study of Panahandeh et al. in a sample of reatment . In the reatment ,78.Some randomized clinical studies have also valued the contribution of multimineral-vitamin supplements which include zinc and iron for ADHD treatment in children. The rationale for selecting trails with multi-supplements therapy plans for ADHD was the low number of trails using simultaneously zinc and iron, and those identified used preparations with vitamins, dietary minerals and other nutritional elements. Therefore, considering these studies facilitates generalizability of the results related with the use of zinc–iron supplements. n = 93 patients used Daily Essential Nutrients , which contains a comprehensive range of micronutrients . The participants were instructed to titrate the dose up to over a week, starting with 3 capsules per day and increasing it to up to 12 capsules per day  is consistent with other non-randomized studies that explored the contribution of these micronutrients within clinical- and population-based samples. For example, a longitudinal study with a large sample with p period . A positchildren . The potential explanations about the paths between the zinc–iron levels and the various ADHD manifestations are discussed in a number of studies, which showed that lower levels of these elements in childhood  could be related to a dopaminergic impairment, which could be the origin of the high inattentiveness–hyperactivity levels and the other correlates of the ADHD disease (like the presence of restless leg syndrome and other multiple sleep problems) ,84,85. Nn = 810 monitored children aged 5–12 years old showed that consumption of omega-3 and omega-6 with complementation of zinc improved ADHD symptoms, as well as other emotional problems, and sleeping disease  after 12 weeks [The link between iron and zinc supplementation and the observed decrease in ADHD symptoms in the controlled clinical trials selected for this systematic review also makes sense with regard to the role of zinc and iron as dopamine reuptake inhibitors, which was precisely the core target of the stimulant medications used in the combined treatment plans for the disease. Other non-randomized studies using a combination of polyunsaturated fatty acids with zinc supplementation also showed benefits for the treatment of ADHD. For example, the observational study of Huss et al. in a large sample of 12 weeks . The rat12 weeks ,93.n = 18 children by comparing d-amphetamine with Efamol, assessed the moderate role of zinc [The studies included in this review support the existence of subgroups  that could be particularly benefitted from treatments including dietary zinc and iron supplements, such as the results of Bilici and colleagues : the ass of zinc , categor of zinc  analyzed of zinc . This st of zinc . HoweverBased on the results obtained in this systematic review, the specific role of dietary nutrients with zinc and iron still seems controversial for the treatment of ADHD, being most consistent with the evidence for zinc. Moreover, although the reviewed studies found a relationship between the use of dietary supplements containing these elements with the improvement of ADHD symptoms, neither the mono-causal role of a concrete specific nutritional deficiency among ADHD children nor the role of a concrete dietary nutrient in the management of this disorder were proven (as was reported quite recently ). FutureThe results obtained in this systematic review should be considered for combining these multi-facet interventions with additional precise interventions, specifically focused on the nutritional state and clinical profile of each patient. The ultimate objective of these treatments, including medical, psychological, and diet plans, should be to decrease the levels of ADHD, but also its multiple negative correlates, in order to achieve better performance in vigilance and sustained attention tasks, executive functioning tasks (like planning and organization), set shifting and response monitoring, and complex problem-solving.Finally, this study should be interpreted in consideration of certain limitations. First, a meta-analysis was not conducted. Systematic reviews of treatment plans often include statistical methods to summarize the results of independent research by combining the information of selected studies  with the aim to provide more precise estimates of the effects of health care. However, it must be outlined that not all topics allow a meta-analysis to be conducted, and these concrete systematic reviews use alternative methods of synthesis described as “narrative synthesis”. This was the case of our study: we did not employ meta-analyses because of the clinical heterogeneity of individual studies, with different intervention characteristics , diverse outcome/effects measurement, different research settings, and differences in participants’ features . Since current studies have proven that this heterogeneity could cause significant inaccurate summary effects and associated conclusions, and thus misleading decision-markers , we opte


ObjectiveN = 136) with AUD. Methods: We assessed ADHD symptoms , health-related quality of life , psychiatric comorbidity , and impulsive personality traits . Results: 19% of patients screened positive in the retrospective assessment of childhood ADHD, and 17% for adult ADHD. Participants reported moderate levels of problem severity in the HRQL dimensions, and 65% had ≥1 current psychiatric disorders other than AUD and ADHD. Multiple mediation indicated that there was a significant direct effect of childhood ADHD symptoms on psychiatric comorbidity , and indirect effects of both reacting impetuously when experiencing negative emotions  and the tendency to not finish tasks . Conclusions: The subcomponents of impulsivity to react rashly when experiencing negative emotions and the tendency to not persist in activities seem to contribute to the relationship between ADHD symptoms (particularly those in childhood) and psychiatric comorbidity among patients with severe AUD.Attention-deficit/hyperactivity disorder (ADHD) is an established risk factor for developing alcohol use disorder (AUD), and AUD-ADHD comorbidity is associated with additional psychiatric diagnoses. Several lines of evidence support the role of impulsivity as a pathway of these relationships; however, impulsivity is not a unitary construct. Thus, we sought to explore whether separate aspects of impulsivity may explain the relationship between ADHD symptoms and psychiatric comorbidity among inpatients ( Alcohol use disorder (AUD), characterized by an impaired ability to stop or control alcohol use despite adverse social, occupational, or health consequences, is among the most common mental health problems , a large inpatient clinic for addiction treatment in Vienna, Austria. Participation in the study was voluntary and anonymous. There was a complete discussion of the study with potential participants and written informed consent was obtained after this discussion. The study was conducted in accordance with the Declaration of Helsinki  Diagnostic and Statistical Manual of Mental Disorders 5th edition , aged 22–77 years, took part in this study. No patient who expressed interest in the study had to be excluded based on the criteria listed above. On average, participants endorsed 8.76 symptoms . Participants were compensated for the time spent in the study with €10. Besides socio-demographic information, the following variables were assessed.The DSM-5 checklist for AUD was used to determine the severity of the patients’ primary AUD diagnosis during the past 12 months. Endorsement of 2–3 criteria is indicative of mild AUD, endorsement of 4–5 symptoms is indicative of moderate AUD, and endorsement of six or more symptoms is indicative of severe AUD.The Adult ADHD Self-Report Scale  is a widespread standardized instrument with 25 items rated on a 5-point scale, and was used for the retrospective assessment of childhood ADHD symptoms in adults  represents the effects of an illness upon the physical, mental, and social dimensions of an individual’s well-being. The EuroQol-5 Dimension (EQ-5D) is a short questionnaire that consists of a description and a valuation of HRQOL as a summarized single index score reflecting individual preferences for different health states , a brief structured interview for major Axis I psychiatric disorders in DSM-IV and ICD-10 including mood disorders, anxiety disorders, SUDs, psychotic disorders, and eating disorders, as well as antisocial personality disorder (ASPD). The validity and reliability of the MINI are similar to the Structured Clinical Interview for DSM .Between-group analyses with ADHD screening status  as the independent variable were conducted using analysis of variance (ANOVA). Welch’s statistic was reported if the assumption of homogeneity of variances was not met. In the case of significant ANOVAs, Tukey pairwise comparisons identified specific group differences. Other between-group analyses were conducted using tests that were appropriate for the dependent variable . A multiple mediation model was built to test whether impulsivity  mediated the relationship of ADHD screening status and number of psychiatric comorbidities. In mediation analysis, the total effect  can be broken down into two parts: the direct and indirect effect. The direct effect is the effect of the independent variable (ADHD screening status) on the outcome (number of psychiatric comorbidities) absent the mediators. The indirect pathway is the effect of the independent variable on the outcome that works through the mediators (impulsivity traits).We performed these analyses using Jamovi software, version 1.0.7.0  screened positive in the retrospective assessment of childhood ADHD  but symptoms of ADHD were no longer present . ADHD symptoms persisted in adulthood in 23 patients (16.9% of the sample); i.e., these patients exceeded the cutoff for childhood ADHD (WURS-k) and adult ADHD (ASRS-v1.1). Based on WURS-k and ASRS-v1.1 scores, it was possible to classify participants into three ADHD groups see . FourteeΧ2(2) = 7.30, p = .026. Women were less frequently classified in childhood and adult ADHD groups than men. In addition, there were differences between the three ADHD groups in several aspects of impulsivity. F = 10.48, p < .001, partial η2 = .136. Compared to participants without a history of ADHD, both those with a history of childhood ADHD symptoms  and those with adult ADHD symptoms  had higher negative urgency scores. Similarly, there was a significant association between ADHD group classification and lack of perseverance, F = 12.09, p < .001, partial η2 = .154. However, only the difference between participants without a history of ADHD and those with adult ADHD symptoms reached significance . Moreover, there was an overall difference between the three ADHD groups in sensation seeking, F = 4.35, p = .015, partial η2 = .061. However, post-hoc comparisons between the three groups did not reach significance.There was a significant association between sex and ADHD group classification, Five participants reported that they had been treated for ADHD in the past, and two of those reported that they had received ADHD medication as part of this treatment. Four of those participants screened positive for adult ADHD in the current study, and one for childhood ADHD only.SD = 14.20). Participants who reported at least one problem in the EQ-5D dimensions indicated a significantly worse current overall health  than those who reported no problems , t(133) = 5.23, p < .001, d = 0.99.The separate EQ-5D VAS rating indicated that participants perceived their current overall health as relatively good, with a mean score of 75.77  = 2.23, p = .112, or between ADHD screening status and current overall EQ-5D VAS rating, F = 1.37, p = .258.There was no significant association between ADHD screening status and overall severity of problems regarding HRQL, Overall, 88 participants (64.7% of the sample) screened positive for at least one current psychiatric comorbidity other than ADHD. F = 8.23, p < .001, partial η2 = .110. Participants who had no history of ADHD had significantly fewer comorbidities than those with a history of childhood ADHD symptoms .β = 0.327, 95% CI , p < .001; β = 0.224, 95% CI , p < .038; result not shown). This indicates that impulsivity partially mediated the effect of a history of ADHD symptoms in childhood on psychiatric comorbidity  or direct effect of adult ADHD symptoms on psychiatric comorbidity , there were indirect effects of both negative urgency and lack of perseverance subscales; i.e., 95% bias-corrected confidence intervals (CIs) for indirect effects of mediating variables did not contain zero . In concordance with previously raised concerns Levin, , only fiPrevious studies from Austria, using the same ADHD assessment instruments as the current study, reported a higher frequency of childhood ADHD symptoms but similar adult ADHD symptoms among prisoners with opioid use disorder . Consistent with the literature, the most common psychiatric comorbidities were major depression, manic episodes, anxiety disorders, and suicide risk  perseverance, mediated the relationship between childhood ADHD symptoms and psychiatric comorbidity. These results are in line with findings in individuals with gambling disorder  perseverance indirectly affected the relationship between adult ADHD symptoms and psychiatric comorbidity. Previous research found similar results with regard to adult ADHD symptoms of impulsivity/emotional liability and AUD severity, highlighting the indirect effects of ADHD on AUD severity through unique facets of impulsivity , showed good operating characteristics  and the tendency to not persist in activities (lack of perseverance) contribute to the relationship between ADHD symptoms (particularly those present in childhood) and psychiatric comorbidity among patients with severe AUD. An increasing focus on impulsive personality traits, in addition to diagnosing ADHD, may provide a more precise characterization of SUD patients and help guide individually tailored treatment decisions.


As a selective histone deacetylase (HDAC) inhibitor developed in China, chidamide has been applied for the treatment of refractory peripheral T-cell lymphoma (PTCL) and multiple solid tumors, including lung cancer. However, the underlying mechanisms are not well elucidated. In our present study, we found that chidamide and radiation acted synergistically to suppress cell and xenograft growth of lung squamous cell carcinoma cells by inducing cell apoptosis. Moreover, chidamide alone or a combination of chidamide and radiation treatment inhibited cancer cell stemness. miRNA microarray analysis demonstrated that miR-375 was the highest upregulated microRNA (miRNA) in NCI-2170 and NCI-H226 cells treated with chidamide alone or treated with chidamide plus radiation, compared with normal control. Inhibition of miR-375 attenuated the promoting effect of chidamide alone and chidamide plus radiation-induced NCI-2170 and NCI-H226 cell apoptosis and reverted the suppression of cancer stemness caused by chidamide alone or chidamide plus radiation treatment. Moreover, EIF4G3, a scaffold protein in the translation initiation complex, was found to be a direct target of miR-375 based on the luciferase reporter assay and western blot analysis. Interestingly, both chidamide alone and chidamide plus radiation treatments suppressed the mRNA and protein expression of EIF4G3. Silence of EIF4G3 also induced cell apoptosis and suppressed tumor growth in NCI-2170 and NCI-H226 cells. These data suggest that chidamide shows a synergistic effect with radiation therapy on lung squamous cell carcinomas by modulating the miR-375-EIF4G3 axis, which may afford an effective strategy to overcome the drug resistance of chidamide in clinical cancer therapy. Lung cancer is one of the leading causes of cancer-associated deaths all over the world, with a high metastatic potential . Lung caChidamide, a selective inhibitor of HDAC1, 2, 3, and 10 developed wholly in China, has been entered into clinical trials both in the United States and China. In December 2014, chidamide has been approved by the China Food and Drug Administration (CFDA) as a treatment strategy for peripheral T-cell lymphoma (PTCL) . InteresMicroRNAs (miRNAs) represent a class of noncoding short RNAs with 19–24 nucleotides in length, which was highly conserved in eukaryotes. miRNAs play an important role in regulating multiple physiological and pathological processes by binding to the 3′-untranslated regions (3′-UTRs) of target genes , 13. DysAmong these cancer-related miRNAs, miR-375 was initially identified as a critical regulator of insulin secretion and a novel therapeutic target for diabetes treatment . FurtherIn the present study, we found that a combination of chidamide and radiation treatment promoted synergistic cytotoxicity and suppressed tumor stemness in LSCCs. Importantly, miR-375 was upregulated in NCI-2170 and NCI-H226 cells treated with chidamide alone or with chidamide plus radiation, compared with normal control. In addition, suppression of miR-375 attenuated chidamide alone and chidamide plus radiation-induced NCI-2170 and NCI-H226 cell apoptosis and suppressed tumor growth and stemness. Moreover, EIF4G3 was identified as a direct target of miR-375. Interestingly, both chidamide alone and chidamide plus radiation treatments suppressed the mRNA and protein expression of EIF4G3. Silence of EIF4G3 also induced cell apoptosis and suppressed tumor growth in NCI-2170 and NCI-H226 cells. These data suggest that chidamide shows a synergistic effect with radiation therapy on lung squamous cell carcinomas by modulating the miR-375-EIF4G3 axis.2 incubator. Chidamide  was diluted in dimethyl sulfoxide . Cells were exposed to 300 nM of chidamide for 24 h or/and 6 MV X-ray radiation using a linear accelerator  at single doses of 0, 1, 2, and 4 Gy.Human lung squamous cell carcinoma NCI-H2170 and NCI-H226 cells were obtained from ATCC  and maintained in DMEM  with 10% fetal bovine serum  and 1% penicillin/streptomycin  at 37°C in a 5% COCells were cultured to about 75% confluence before transfection. Control mimic, miR-375 mimic, control siRNA, and EIF4G3 siRNA were transfected into cells using Lipofectamine RNAiMAX Reagent  following the manufacturer's manual. Forty-eight hours after transfection, the cells were applied for the following experiment. The siRNA oligos were synthesized by Santa Cruz .Cell Counting Kit-8 was applied to determine the proliferation rate of NCI-H2170 and NCI-H226 cells with indicated treatment as previously described. Briefly, two thousand treated cells were plated into 96-well plates. Then, CCK-8 solution was added at the harvest time and incubated for an additional 30 min. The absorbance was determined at 450 nm on the microplate reader .μl of FITC-labeled Annexin V and 5 μl of propidium iodide (PI) were added to the suspended cells and gently mixed, following by the incubation at room temperature for 15 min in dark. At last, the samples were detected on the flow cytometer (BD Biosciences).Apoptosis assay was performed by Annexin V/PI double staining using the Annexin V-FITC apoptosis detection kit  following the standard manual. Briefly, treated cells were washed with cold 1x PBS and resuspended in 1x binding buffer. Five miRNA microarray was performed as previously described .Real-time RT-PCR was performed as previously described . The priThe luciferase reporter assay was determined using the psi-CHECK2 dual-luciferase system  following the standard manual. The QuickMutation™ Site-Directed Mutagenesis Kit  was applied for construction of EIF4G3 3′-UTR reporter plasmids with a mutant miR-375 binding site. Primer sequences used for construction of these plasmids were as follows: 5′-GCGCGATCGCAACTTCAAATACACAAAATG-3′ , 5′-GCGTTTAAACCTGTCCAAAGGAGAAGTCAC-3′ ; 5′-AGGCTTGTAAATACATACTTGTTTTATTTAAAAAAAC-3′ , 5′-GTTTTTTTAAATAAAACAAGTATGTATTTACAAGCCT-3′ ; 5′- CACTTTGAAAATATAAACTTGTTTTAAAGACAAAC-3′ , and 5′-GTTTGTCTTTAAAACAAGTTTATATTTTCAAAGTG-3′ .β-actin .Western blot analysis was performed as previously described . The pri+ cell analyses according to the manufacturer's manual. For each sample, one-half of cells was treated with 50 mM of diethylaminobenzaldehyde (DEAB) to define negative gates.The ALDEFLUOR Kit (Stemcell Technologies) was used for ALDHSphere formation assay was determined as previously described .2 each other day after palpable tumors appeared. The study protocol was approved by the Animal Care and Use committee of Harbin Medical University.Treated cells were subcutaneously injected into both sides of flank areas of 6–8-week-old BALB/c nude mice for 42 days. Tumor volumes were measured using the following equation: 0.5 × length × widthImmunohistochemistry assay was performed as previously described .t-test was used to determine the statistical differences, in which p < 0.05 was considered to be significant.All data were expressed as mean ± standard deviation (SD). Statistical analysis was performed using the software GraphPad Prism 5. Student's Initially, to determine the effect of chidamide on cellular proliferation in LSCC cells, we treated NCI-H2170 and NCI-226 cells with 300 nM of chidamide for 24 h or/and 6 mV X-ray radiation using a linear accelerator at single doses of 0, 1, 2, and 4 Gy. The results indicated that chidamide, radiotherapy, and their combinational treatment inhibited cell proliferation in NCI-H2170 and NCI-H226 cells Figures . Next, t+ cells  play a central role in tumor progression, metastasis, recurrence, and chemotherapy resistance. Herein, we also detect the effect of chidamide on lung cancer stemness. Results of sphere formation assay demonstrated that the sizes and number of spheres were suppressed by chidamide alone or a combination treatment of chidamide and radiation Figures . To dete Figures .Next, we sought to elucidate the molecular mechanism underlying antitumor activity of chidamide. As miRNAs play a critical role in tumorigenesis, we hence focused on the expression profile alterations of miRNAs. The Affymetrix miRNA 2.0 Array was applied to identify differentially expressed miRNAs in NCI-H2170 and NCI-H226 cells in response to chidamide, radiotherapy, and their combinational treatment. Among the upregulated miRNAs, miR-375 showed the most remarkable fold . Moreove+ cells suppressed by chidamide alone or chidamide and radiation combinational treatment . Among the numerous targets, eukaryotic translation initiation factor 4 gamma 3 (EIF4G3) was selected as the candidate target gene for miR-375 for further analysis, as its 3′-UTR contains two conserved binding regions of miR-375. The binding sites between miRNA-375 and EIF4G3 are shown in Furthermore, we identified the target genes of miR-375 using TargetScan online software (To determine the role of EIF4G3 in the biological behaviors of LSCC cells, we first detected the expression of EIF4G3 in NCI-H2170 and NCI-H226 cells treated with chidamide or chidamide plus radiation combinational treatment. The results demonstrated that both chidamide and chidamide plus radiation combinational treatment suppressed EIF4G3 expression both at the mRNA and protein levels Figures . Next, wTaken together, our study systematically demonstrates that chidamide and radiation synergistically promote LSCC cell apoptosis and suppressed tumor growth and stemness by modulating the miR-375-EIF4G3 axis.The HDAC inhibitors can be used for various diseases, some of which have entered clinical trials. In cancer, HDAC inhibitors are becoming promising novel tumor therapeutic drugs exerting anticancer function across a wide range of cancers, especially in leukemia. Chidamide, an orally active novel HDAC inhibitor of the benzamide class, selectively inhibits HDAC1, HDAC2, HDAC3, as well as HDAC10. In pancreatic cancers, chidamide augments gemcitabine-induced cell growth arrest and apoptosis by downregulating the antiapoptotic gene MCL-1 . ChidamiAlthough previously published data also demonstrate chidamide induces cell apoptosis, the underlying mechanism is not very clear. Our data showed that both chidamide and chidamide plus radiation combinational treatment induced the expression of endogenous miR-375 in NCI-H2170 and NCI-H226 cells. Moreover, rescue experiments indicated that the apoptosis-promoting function of chidamide depended on the upregulation of miR-375 expression. In addition, the scaffold protein EIF4G3 was identified as a direct target of miR-375 and the suppression of EIF4G3 induced cell apoptosis and inhibited tumor growth in NCI-2170 and NCI-H226 cells. These data conclude that chidamide exhibits a synergistic effect with radiation therapy on LSCC by modulating the miR-375-EIF4G3 axis. Our finding may represent a universal mechanism underlying the synergistic antitumor interactions between HDAC inhibitors and DNA damaging agents in tumorigenesis, which should be confirmed in our future studies.In the study of NCI-H2170 and NCI-H226 cell lines, it has been found that chidamide, radiation, and their combinational treatment could exert an anti-LSCC effect. However, unlike chidamide and chidamide plus radiation combinational treatment, radiation alone could not upregulate the expression of miR-375 and downregulate the expression of EIF4G3. These results suggest the miR-375/EIF4G3 axis may not be involved in the regulation of radiotherapy alone-induced cell apoptosis. On the other hand, acquired drug resistance frequently occurred to destroy effective therapy with chemotherapeutic agents, leading to an unsatisfactory clinical outcome. Therefore, HDAC inhibitor combined with radiation at different dose might have multiple targets and pathways to induce cell apoptosis.In conclusion, our results systematically explored the role of miR-375/EIF4G3 axis in chidamide-induced LSCC apoptosis and tumor growth arrest, which may afford an effective strategy to overcome the drug resistance of chidamide in clinical cancer therapy.