Endophenotypes or intermediate phenotypes are of great interest in neuropsychiatric genetics because of their potential for facilitating gene discovery. We evaluated response inhibition, latency and variability measures derived from the stop task as endophenotypes of ADHD by testing whether they were related to ADHD traits in the general population, heritable and shared genetic risk with ADHD traits. Participants were 16,099 children and adolescents, ages 6 to 18 years who visited a local science center. We measured ADHD traits using the Strengths and Weaknesses of ADHD-symptoms and Normal-Behavior (SWAN) rating scale and performance on the stop signal task (SST)—response inhibition (SSRT), response latency (GoRT), and response variability (GoRTSD). Regression analysis was used to assess the relationship of cognitive measures and ADHD traits while controlling for family, age, sex, ethnicity, socioeconomic status and treatment status. Heritability of ADHD and cognitive traits was estimated using SOLAR in 7,483 siblings from 3,507 families that included multiple siblings. Bivariate relationships between pairs of variables were examined. Individuals with greater ADHD trait scores had worse response inhibition, slower response latency, and greater variability. Younger participants and girls had inferior performance although the gender effects were minimal and evident in youngest participants. Inhibition, latency, variability, total ADHD traits, inattention and hyperactivity-impulsivity scores were significantly heritable. ADHD traits and inhibition, but not latency or variability were coheritable. In the largest study in the general population, we found support for the validity of response inhibition as an endophenotype of ADHD.The online version of this article (doi:10.1007/s10802-012-9693-9) contains supplementary material, which is available to authorized users. Attention deficit hyperactivity disorder (ADHD) is a complex disorder characterized by extremes of inattention, hyperactivity and impulsivity, by clinical and genetic heterogeneity and by high heritability whether it is measured as a discrete diagnostic entity or as a continuous trait in the general population . The OSC has about one million visitors each year of all ages, social backgrounds and ethnic groups and is a leading developer of interactive exhibitions for science centres around the world. Participants were tested at workstations with dividers in a specifically designed exhibition on genes, brain and behaviour. Visitors that showed interest in the display were invited to participate. Typically, there are several such participatory research studies going on at the Science Centre at any one time as part of the mission of the Centre. Participants were individually supervised during testing by undergraduate university students.We evaluated 16,099 individuals, 6 to 18 years of age, at the Ontario Science Centre (OSC) in Toronto, Canada  were excluded if they performed the task in an invalid manner . As an added check, we recalculated all models on participants who inhibited more than 0.40 or less than 0.60 . We present models for the entire sample because results were not affected by this additional set of exclusion criteria. Participants (147) were also excluded if they interrupted the task before completion because of time constraints on their family visit to the Science Centre or pushed the wrong buttons on the game pad device used to collect responses (180).Regression analysis was conducted to identify the best combination of variables making a unique contribution to SSRT, GoRT and GoRTSD while considering family as a random effect Singer . In ordeIn order to evaluate the effect of age, gender, and ADHD trait on the stop task values, we calculated an approximation of Cohen’s effect size using the equationTo determine if extremely low SWAN scores might also be maladaptive, we compared participants with high, medium and low SWAN scores on rates of parent-reported ADHD, anxiety, depression, learning disability and other disorders.We estimated SSRT for 414 (2.6 %) participants who reported taking medication for ADHD within 48 h of testing by adjusting SSRT by 0.75 effect size based on the results of published studies of the effect of a moderate dose of stimulant medication on SST performance : the ratio of the genetic variance component to the residual  phenotypic variance. We calculated confidence intervals for heritability estimates under the asymptotic normality assumption of the Maximum Likelihood estimator where sample size is ~ 7,000 using the formulaHeritability of each SST variable and of ADHD traits with age and sex as covariates was estimated using SOLAR  is indicated by a genetic correlation significantly different from zero . We estimated heritability of SST variables with and without SWAN ADHD traits as covariates in order to control for the influences of ADHD traits on cognitive performance. Heritability estimates were essentially unchanged and are presented with ADHD controlled.Bivariate analyses were conducted on pairs of neuropsychological measures that demonstrated significant univariate heritability to partition the phenotypic correlation between two traits (RhoP) into genetic (Rhog) and environmental (Rhoe) correlations according to the equationTable There was a bias toward more economically privileged participants. Approximately 16 % of study participants were from the poorest neighbourhoods compared with 37 % in the surrounding metropolitan area, whereas 25 % of participants were from the richest neighbourhoods compared with 16 % in the surrounding area. Neither ethnicity nor socioeconomic status had a significant effect on the ADHD traits or SST variables or interactions and are not considered further. Rates of reported psychiatric disorder were lowest for individuals with lowest ADHD trait scores and increased with increasing ADHD trait scores suggesting that extremely low scores were not maladaptive (data available).Supplementary Figures p = 0.0002), ADHD traits (p < 0.0001), age (p < 0.0001), age*gender (p = 0.02) and age*age (p < 0.0001) were significantly associated with SSRT scores. Children with higher ADHD trait scores had longer SSRT representing inferior inhibition, as did girls, and younger individuals. There was a significant gender effect on SSRT across all levels of ADHD traits at age 6, with the largest difference (36 ms) seen at an ADHD trait score of 54 , ADHD traits (p < 0.0001), age (p < 0.0001), age*age (p < 0.0001), age*gender (p < 0.0001) and age*age*gender (p = 0.0005) were significantly associated with GoRT. On average, boys, older children and those with lower ADHD trait scores had faster response latencies. ADHD traits (p < 0.0001), gender (p < 0.0001), age (p < 0.0001), age*age (p < 0.0001) and age*gender (p < 0.0001) significantly predicted GoRTSD. Models of GoRTSD that included GoRT did not change estimates of these effects. On average, individuals with lower ADHD trait scores, boys and older individuals were less variable than girls, younger participants and those with more ADHD traits. The largest difference in GoRT between the lowest and highest ADHD trait score of 30 ms (ES = 0.35) was seen among females at age 6; however, differences were similar across other genders and age combinations. The difference of 51 ms seen on the GoRTSD between an ADHD trait score of −54 and 54 for females at age 6 corresponded to an effect size of 1.4. As with SSRT, the gender effects for GoRT and GoRTSD were greater at age 6 with a difference of 43 ms (ES = 0.50) on the GoRT and a difference of 21 ms (ES = 0.60) on the GoRTSD , p = 6.3 × 10−23], GoRT  and GoRTSD . Total ADHD trait scores , hyperactivity-impulsivity  and inattention  were also heritable. Age and sex accounted for differing proportions of phenotypic variance, but had little effect on estimates of heritability. Exclusion of ADHD trait scores in the heritability estimates had no impact on heritability estimates.Univariate heritability  adjusted for age and sex were significant for SSRT [hTable In the largest general population study reported to date we found support for the validity of response inhibition as an endophenotype of ADHD. Response inhibition, latency and variability were significantly correlated with ADHD traits even after controlling for age and gender. Analysis of the family data indicated that inhibition, latency and variability were heritable; however, of these three measures only inhibition shared genetic risk with ADHD traits. Current results have implications for understanding the genetic contributions to ADHD traits and to higher-order executive functions as well as for the design of genetic studies.Participants with greater ADHD traits had longer SSRTs indicating inferior inhibition, slower response latencies and greater response variability than did those with fewer ADHD traits. The relationship of ADHD traits and inhibition was strong. Participants with highest ADHD trait scores had SSRTs that were substantially slower indicating less efficient inhibition than among those with the lowest trait scores across age and sex. The magnitude of these differences was substantial for both girls  and boys . These effects are comparable to those found in a meta-analysis of studies comparing ADHD clinic cases and healthy controls  are comparable to heritability estimates from prior studies of executive functions, specifically those on SSRT  and high ADHD trait scores whereas there was little evidence for shared genetic risk for ADHD traits and either response latency (GoRT) or variability (GoRTSD)  approach using endophenotypes is a particularly powerful and cost-effective design for detecting genetic association Below is the link to the electronic supplementary material.


Bmax) of [3H] Quinuclidinyl benzilate (3H-QNB) to mAChRs was decreased by almost 50 % in the children with ADHD  in comparison with controls . The decreased Bmax indicates a reduced cholinergic receptor density, which might constitute a biomarker for ADHD. However, these preliminary findings need to be replicated in larger ADHD and comparison cohorts.Monoaminergic dysregulation is implicated in attention-deficit/hyperactivity disorder (ADHD), and methylphenidate and amphetamines are the most frequently prescribed pharmacological agents for treating ADHD. However, it has recently been proposed that the core symptoms of the disorder might be due to an imbalance between monoaminergic and cholinergic systems. In this study, we used fibroblast cell homogenates from boys with and without ADHD as an extraneural cell model to examine the cholinergic receptor density, that is, muscarinic acetylcholine receptors (mAChRs). We found that the binding capacity ( Attention-deficit/hyperactivity disorder (ADHD) is the most common neurodevelopmental disorder, with a worldwide prevalence of about 5–7 % in childhood , implicating a monoaminergic–muscarinic receptor interaction , one being that after several passages in culture, fibroblasts are free from state-related changes and effects of medications, drugs and hormones, and thus patients do not have to be unmedicated at the time of the biopsy collection  in fibroblasts from children with ADHD, and in a comparison group, in an in vitro receptor binding assay.The study was approved by the Regional Ethics Committee in Gothenburg, Sweden. A written and signed consent was obtained from the parent/s and their child before performing the studies.The index group consisted of fibroblast cell lines from 11 boys with ADHD of the combined type (inattention combined with hyperactivity and impulsivity) according to the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) APA . They we75 tissue culture flasks  containing minimal essential medium , l-glutamine (2 mM/L), penicillin (100 mg/mL), streptomycin (100 mg/mL) and Amino-Max™ (0.06 % vol/vol). All growth media, antibiotics and FBS were obtained from Invitrogen, Life Technologies Europe BV, Sweden. The cells were incubated at a temperature of 37 °C in the presence of 5 % CO2 and high humidity. When the cells reached confluence, they were harvested by trypsinization, washed twice with phosphate-buffered saline  and diluted in 1.5 mL of sterile Milli Q water. Protein concentrations for each cell homogenate were determined by the Bradford method, using bovine serum albumin as a standard (approximately 1 mg/mL of cell protein per cell line was used in the receptor binding assay). The cell homogenates were stored at −20 °C until used in the receptor binding assay. Cell lines between 7th and 15th passages (i.e. the number of splitting) were used in the experiments.In order to prepare fibroblast cell homogenates to be used in the receptor binding assays, each cell line was seeded in T3H] Quinuclidinyl benzilate  into plastic tubes, containing 200 μL sodium phosphate buffer (pH 7.4). Half of the tubes contained unlabelled atropine  to correct for non-specific binding. Fibroblast cell homogenates  were added to the tubes, making a total volume of 250 μL. Tubes were incubated at 22 °C for 60 min and filtered rapidly through Whatman GF/C glass fibre filters  presoaked for 50 min in 0.05 % polyethyleneimine solution. The filters were washed twice under vacuum with ice-cold sodium phosphate buffer. The filters were then placed in scintillation vials to which scintillation liquid was added  and the disintegrations per minute (DPM) was counted by a scintillation counter .The receptor binding assay in fibroblast cell homogenate was done according to the modified procedure of Vestling et al. . This wa3H-QNB was calculated by subtracting the values for non-specific binding in the presence of unlabelled atropine from those of total binding in the absence of atropine. All incubations were performed in duplicates and mean values were used for the calculation of binding characteristics. The binding capacity (Bmax) and the equilibrium dissociation constant (KD) of mAChR antagonist QNB were determined by nonlinear regression analysis.The specific binding of KD of ADHD patients, but not for KD of controls or for Bmax of ADHD patients and controls . Therefore, the nonparametric Mann–Whitney U test was used to compare the Bmax and KD values between patients and controls. However, three outliers were identified by the scaled Median Absolute Deviation (MADE) method and by Tukey’s method; one in the comparison group and two in the ADHD group. If excluding these outliers, assumptions about parametric methods were met and Student’s unpaired t test was used to compare the Bmax and KD values between patients and controls. For all statistical analyses, a significant level of 5 % was accepted and all calculations and analyses were performed using GraphPad Prism version 5 for Windows.Assumptions about parametric methods were met for Bmax) of the mAChR ligand QNB in comparison with controls (p ≤ 0.01) when excluding the three outliers identified by the MADE method. The KD did not differ significantly between the two groups. These results suggest that the children with ADHD had a reduced density (~50 %) of mAChRs in fibroblasts, but that the affinity of 3H-QNB binding to the cholinergic receptors was not affected  and/or post-transcriptional (e.g. mRNA stability) factors. To our knowledge, similar findings have not been reported previously. However, three outliers were identified and we can only speculate about the reason for these extreme values, as they were not caused by experimental errors. The two ADHD outliers may represent different subgroups of ADHD and at least one of these boys had no evidence of hereditary origin. Since ADHD is a very heterogeneous disorder with regard to molecular genetics and phenotypic diversity  was found to be lower (determined in lymphocytes) in children with ADHD (Coccini et al. We are aware of the limitations of our study, such as a small sample size, a small comparison group and the index group consisting of only boys. The identification of outliers would possibly be explained by the large heterogeneity and complexity of molecular factors underlying ADHD. Therefore, the results should be considered as preliminary. Future studies have to include larger sample size consisting of both boys and girls with hereditary and non-hereditary ADHD.We conclude that fibroblasts derived from patients with ADHD offer a useful model for exploring neurological aspects in vitro, thus allowing new hypothesis and drugs to be tested. The indicated reduced density of mAChRs in fibroblasts from children with ADHD may constitute a biomarker for ADHD; however, these preliminary findings need to be replicated.


Objective: Attention Deficit Hyperactivity Disorder (ADHD) is one of the most prevalent psychiatric disorders in children. According to concern regarding the growth of these children, this study was carried out to compare height and weight between 5-6-year-old boys with ADHD and those without ADHD in Ahvaz, IranMethods: In this cross-sectional study, 32 5-6-year-old ADHD boys with the Conners’ rating scale (CRS) of ≥ 15 were compared to 32 non-ADHD same-age boys with CRS of < 15. Exclusion criteria were some special disease with negative effect on growth and psychostimulant treatment. Centers for Disease Control and Prevention (CDC) curves were used to determine the growth status.Results: Comparison between ADHD and non-ADHD boys regarding mean height (111.95 ± 6.12 vs. 110.77 ± 6.22 cm), weight (19.39 ± 3.65 vs. 19.19 ± 3.75), and body mass index (15.44 ± 1.82 vs. 15.54 ± 1.67) showed no statistically significant difference (P>0.05).Conclusion: Our study does not support an association between problems in growth outcomes and ADHD in 5-6 years old boys Attention Deficit Hyperactivity Disorder (ADHD) is one of the most common childhood psychiatric disorders, affecting 5-12% of children worldwide  and amphPrenatal and postnatal growth rates are independent risk factors for later mental health outcomes and poor nutrition early in life can lead to adult mental illness . Also loAccording to over mentioned subjects and prevalence of nutritional disorder, short stature and underweight in preschool children , and difIn this cross-sectional study, 32 5-6-year-old boys with the diagnosis of ADHD and 32 same-age boys without ADHD were selected by cluster sampling from nine kindergartens of Ahvaz, Iran. Inclusion criteria consisted of boys aged 5-6 years and the Conners’ rating scale (parent or teacher) was ≥ 15 (ADHD group) or < 15 (non-ADHD group). The children with positive history of diseases that can compromise growth, a history of growth failure due to medical conditions and those who were under treatment with psychostimulants were excluded. 2)  . For determining nutritional status indicators, anthropometric indices were assessed by comparing of BMI and height of each child with same age and same sex growth curves. The height under 5th percentile age was defined as short stature. BMI under 5th percentile age was defined as underweight and over 95th percentile age was defined as overweight. BMI between 85-95th percentile ages was defined as at risk of overweight . A highly abbreviated form of the Connors Rating Scale was developed to be used for parents and teachers by Connors in 1973. It consists of ten items that assess both hyperactivity and inattention . Statistical analysesThe Statistical Package for the Social Sciences (SPSS) Windows version 15.0 and Excel 2003 were used to perform the statistical analyses. A probability of P < 0.05 was considered statistically significant. For descriptive analyses, mean, standard deviation (SD) and incidence were used. Z test was used to compare quantitative variables. The Fisher exact test and Chi-square test were used to retest results. Informed consent was obtained from the parents and teachers. Institutional review board of Ahvaz Jundishapur University of Medical Sciences (AJUMS) approved the study. Results of Z test showed no significant differences between ADHD and non-ADHD groups in terms of mean weight, height and BMI . Also thOur results found no association between ADHD and growth failure in 5-6-year-old males. This is consistent with Amani et al. study in school-aged children . Their sAlso Spencer et al.  in a croOur results should be considered in the light of some methodological limitations. Psychiatric assessment and diagnosis of ADHD relied on the Conners Rating Scale and we could not conduct interview with parents. The current result does not support an association between problems in growth outcomes in height and weight and ADHD. These findings support other literature on this topic and provide information for patients, clinicians, and parents about the growth of children with ADHD. Suggestions for future studies:Future studies should be carried out about relationship between existence of depression and growth outcomes in preschool children with ADHD. Because the majority of studies are carried out in male subjects , future AT conceived and designed the evaluation, drafted the manuscript and re-analyzed statistical data. KR participated in designing the evaluation. RA collected the clinical data. AHA performed the statistical analysis. All authors read and approved the final manuscript.


Within the last 2 years the bulk of information on evidence based treatments in ADHD was reviewed quite intensively and new empirical studies could be added. This update reports comprehensively about actual and essential facts in the field related to brain development and sensitive periods, predictors of treatment, safety of medication, value of naturalistic studies, new drugs and complementary medicine, behavioral interventions including neurofeedback and psychosocial treatment, treatment of comorbidity, and ethical considerations including preventive aspects. The updated combination of well selected evidence based treatments  seems to be clinically and ethically recommended as also suggested by the European and American guidelines on ADHD. Attention deficit hyperactivity disorder (ADHD) is one of the most common  ; and debFrom the year 2011 to 2012, several reviews and meta-analyses covered the field of treatment options in ADHD and their degree of empirical evidence  of the child’s brain. Within the last few years we learned from experiments of ADHD on animal models that stimulants may bring the increased density of dopamine transporters back to a normal range . MoreoveHowever, some recent clinical investigations give hope. For example, Hoekzema et al.  assessedIn summary, pharmacological as well as non-pharmacological interventions in ADHD seem to support developmental normalization and/or compensation of brain alterations in these patients. The chance may be highest during sensitive phases of neuronal plasticity. Thus there might be a potential for secondary prevention in case of early detection of ADHD.A very early start of treatment should qualify for a safe use. Unfortunately, clear predictors for this and successful symptom reduction are lacking. At the moment pharmacogenetic approaches are en vogue, but before the field can offer data to be used for personalized ADHD treatment, pharmacogenetic studies with larger samples and range of outcomes related to efficacy, effectiveness, and safety are needed to determine the utility of genomic information ). Their Concerning non-genetic predictors, probably the prepotent response inhibition reaction time in a neuropsychological Stop-Signal-Task seems a good candidate. Children with lower levels of inhibition showed worse outcome after 10 weeks of MPH treatment, independent of dose .Finally, success of neurofeedback training (NF) is the better the higher the pre-NF-training amplitude of the contingent negative variation (CNV); the latter is an event-related electrophysiological brain potential generated mainly by anterior cingulate cortex [ACC]) .Hence, preparation for and inhibition of a goal directed behavior is related to the dopaminergic genes  and thusTreatment options for ADHD are always lively debated in light of probable side effects, especially if medication with stimulants is considered. Fortunately, guidelines help clinicians to provide high-quality care to their patients by delivering evidence-based treatment programs.The European ADHD Guidelines Group (EAGG) reviewed the published literature on adverse effects of the drugs that are licensed in Europe in order to treat ADHD ••  are the gold standard in order to clarify the efficacy of an intervention. Post-RCT observational studies are required to ensure well-monitored, up-to-date treatment management regarding effectiveness (“is it of use?”), and efficiency (“how much benefit at what cost?”), especially for a long-term perspective . Today, Concerning pharmacological treatment of ADHD observational studies contributed a great deal to the improvement of daily clinical practice. Although it is not yet clear from the naturalistic part of the MTA-study, what treatment contributed what in the long run , 28 someFinally, van der Oord et al.  pointed There is no break-through for treatment of ADHD expected within the short-term, but the new recommendations of the American Academy of Pediatrics •• are heIn clinical practice, the different drugs in use are sometimes administered adjunctively, without having a controlled evidence base for this. For the combined application of guanfacine XR plus stimulants, Wilens et al.  showed aFor the practitioner it would be important to know from the beginning, what stimulant or noradrenergic drug may be the best one to support the patient. This predictive approach is limited since, so far, individual predictors (like CNV and certain gene-profiles) only predict treatment outcome in general, but not for a specific drug or intervention. Also, recent comparisons of Medikinet-R vs Concerta  and betwFinally, co-existing sleep problems in ADHD may complicate family interaction at home and increase sleepiness during the day. So far, melatonin is often used off-label in order to reorganize the disturbed circadian rhythm. A first RCT on the effect of l-theanine on objective sleep quality (using actigraphy) in 98 ADHD boys aged 8–12 years demonstrated that 400 mg daily of l-theanine might improve some aspects of sleep quality but more rigorous studies are needed.Complementary medicine in ADHD  is again in the focus of ADHD treatment options.While some news about zinc and iron , 36• neeHence, adherence problems are foreseeable even if indication is given only in selected patients, contradicting Pelsser et al. • who “thn = 699 patients) showed a small but significant constant effect size (SMD = 0.31). However with the better investigated, more effective, and approved psychopharmacological agents like stimulants and atomoxetine at hand, only an augmentation seems reasonable. Because of poor quality and weak blinding in earlier clinical studies, further trials each involving at least 330 children with ADHD are needed to demonstrate efficacy with an effect size of about 0.3.When talking about complementary medicine in ADHD, omega-3 fatty acid supplementation is always a favorite •, 36•. TIn their meta-analytic review on non-pharmacological treatments for ADHD Hodgson et al. • reporteConsidering the overall effect on the range of the reported 20 outcome measures examined, . Hodgson et al. • favoredHowever, the main purpose of behavioral interventions is to modify behavior and self-regulation. From this point of view Hodgson et al. • assume,Two recent narrative reviews confirm the recommendation of neurofeedback for ADHD on the basis of the increasing evidence for its short-term clinical efficacy and stability of treatment effects on ADHD symptoms for at least 6 months ••, 40.Working memory training is another neurotraining but without controlling for brain activity. A meta-analytic review on 23 studies concluded that these programs produce short-term improvement in working memory skills, which do not generalize to other neuropsychological functions or clearly to ADHD symptom-reduction. Therefore, this review cast doubt on its clinical relevance in ADHD [Since there exist already a multitude of reviews and meta-analyses on psychosocial treatments for ADHD, the purpose of the systematic appraisal of the evidence by Watson et al. •• was toTwo Cochrane reviews, one on parent training • and anoFurther studies which try to test combined parent and teacher/child psychosocial treatment programs are ongoing with some preliminary success mainly on children’s externalizing behavior , 44). WiIn ADHD, comorbidity is the rule (60 %–80 %) and, psychosocially, the most impairing disorder needs to be treated first, but sometimes the co-occurring problems need to be treated in parallel . The guiIn summary, studies on efficacy, effectiveness, and safety of poly-pharmacy in ADHD comorbidity are needed to provide a better empirical guideline for the widespread use of multiple medications, often in combination with psychotherapy.There exists an ongoing ethical discussion about how the physician should handle treatment when sharing and selling of prescribed stimulants may play a role. Especially, when these drugs are used to treat patients with ADHD plus a substance use disorder (SUD). Diversion of stimulants varies across adolescent populations and shows the highest prevalence among ADHD patients using other illicit drugs . FortunaAnother point of on-going ethical controversy is a probable pharmacological neuroenhancement (doping for performance) in ADHD. Neuroenhancement (and probably related to this faking ADHD) is merely a problem of adults –58, espeThis discussion includes different views on ADHD concerning, eg, inclusion/exclusion criteria, the question of dimensionality vs categorization, validity, and reliability of diagnostics in clinical practice and, thus, the sound empirical and practical basis for clear treatment decisions in contrast to hidden neuroenhancement within a grey zone of diagnostics .Ethical problems and controversies could be minimized if a normalization of Quality of Life in patients with ADHD and their families  would beADHD can be a lifelong threat starting early in life with long-term treatments of different kind. Unfortunately, medication nonadherence seems to be common in childhood/adolescent ADHD ; thus, iClose to this process is the ethical impetus to increase shared decision-making in ADHD •, 64•. SThe handling of children’s willingness to use ADHD treatments, medication refusal, and their developmentally adequate self-management of medication are also important issues for ethical considerations, since they are closely related to compliance and adherence, ie, the success of treatment with good psychosocial functioning giving the youngster a better chance for his/her future. This seems to be ethically important with regard to prevention of severe symptoms, adverse events concerning high dosages of medication, and difficulties in social life.Summarizing the following empirical publications of Bussing et al. , BrinkmaWithin the last 2 years, the bulk of studies on ADHD treatment could be reviewed to get their essential content and usefulness for further clinical practice including prevention. A broad variety of evidence-based treatments is available like behavior therapy, neurofeedback, family coaching, and psychopharmacology. As the combination of treatments seems to be clinically and ethically recommended, studies of combined treatment are needed to clarify which profile of multimodal treatment fits best the needs of families with an ADHD patient.


This study presents an evaluation of the bidirectional correlation between attention deficit hyperactivity disorder (ADHD) and epilepsy using 2 cohorts from the same population database.We used data from the Taiwan National Health Insurance Research Database to establish 2 separate cohort studies with participants <19 years old. We subdivided Cohort 1 in 2 groups: (1) 2468 patients initially diagnosed with epilepsy during the period 1999–2008, and (2) 9810 randomly selected sex- and age-matched non-epileptic controls. We subdivided Cohort 2 into 2 groups: (1) 3664 patients with newly diagnosed ADHD and (2) 14 522 sex- and age-matched non-ADHD patients. We evaluated the risk of subsequent ADHD in relationship to epilepsy and vice versa in the 2 cohorts at the end of 2008.The ADHD incidence in Cohort 1 was 7.76 in patients with epilepsy and 3.22 in those without epilepsy (per 1000 person-years) after a median follow-up of 7–7.5 years. The adjusted hazard ratio (HR) for ADHD was 2.54 (95% CI 2.02–3.18) in the epilepsy group compared to the non-epilepsy group. In Cohort 2, the incidence of epilepsy was 3.24 in patients with ADHD and 0.78 in those without ADHD (per 1000 person-years) after a median follow-up of 3–3.5 years and an HR of 3.94 (95% CI 2.58–6.03).This study shows a bidirectional association between ADHD and epilepsy in the 2 cohort studies. Causative factors may be common between these 2 disorders, leading to a cascade of transcriptional changes in the brain that alter behavior or cognition prior to seizures. Epilepsy and attention deficit hyperactivity disorder (ADHD) have significant effects on the social and behavioral development of children Previous research has indicated that ADHD is the most common disorder in preschool and school-aged children with epilepsy Whether ADHD is a non-specific symptom caused by anti-epileptic drugs (AEDs), non-convulsive epileptiform discharges, or negative chronic seizure effects remains unknown. Whether ADHD is associated with epilepsy because of overlapping pathophysiologic mechanisms is also unclear. Comorbidity patterns that show shared neurobiological mechanisms involved in multiple disorders may provide useful insights.Researchers previously conducted a two-way population-based retrospective cohort study to determine the possibility of developing schizophrenia in patients with epilepsy, and vice versa The Taiwan National Health Insurance Research Database (NHIRD), an electronic claims database of the Taiwan National Health Insurance (NHI) program, covers 99% of the 23 million population of Taiwan and contracts with more than 90% of national health care facilities We conducted 2 cohort studies to assess whether epilepsy  is associated with the subsequent incidence of ADHD  (Cohort 1) or vice versa (Cohort 2) by using the same procedure to select the study participants . The stuA diagnosis of outpatient and inpatient claims revealed the onset of ADHD and epilepsy for Cohorts 1 and 2, respectively, during the follow-up period. This period started from the index date and terminated on the date of cancellation of the insurance coverage or December 31, 2008 (whichever came first).P< .05.We performed all statistical analyses using SAS . In Cohort 1, we compared the differences in demographic characteristics, including age, sex, and urbanization level of the residential area, between patients with epilepsy and the comparison group by using chi-square and Wilcoxon two-sample tests for categorical and continuous variables, respectively. We used Cox proportional-hazards regression models to estimate hazard ratios (HRs) and their 95% confidence intervals (95% CI) for ADHD incidence in patients with epilepsy in a relationship to the comparison group. Accessibility to health care varies among geographic regions in Taiwan, requiring the adjustment of the urbanization level in the multivariate models. We repeated Cox regression analyses in subgroups stratified by age and sex. We used the same statistical analysis in Cohort 2. All significance levels were set as a two-sided Cohort 1 included 2468 epileptic and 9810 non-epileptic children  matched During the median follow-up of 7.0 years for the epilepsy group and 7.5 years for the comparison group, the incidence of ADHD (per 1000 person-years) was 7.76 and 3.22, respectively . EpilepsP < .0001).Cohort 2 included 3664 ADHD and 14 522 non-ADHD children . More thDuring the median follow-up period of 3.3 years for the ADHD group and 3.5 years for the comparison group, the incidence of epilepsy (per 1000 person-years) was 3.24 and 0.78, respectively . ChildreThe prevalence rates of ADHD and epilepsy in the general pediatric population are 3%–7% and 0.05%–1%, respectively The complex relationship between epilepsy and ADHD remains unclear. Researchers have proposed several hypotheses on the possible pathophysiology of their comorbidity in the context of brain development, including the effects of chronic seizures, EEG epileptiform discharges, and AEDs The pathogenesis of the relationship between epilepsy and ADHD remains unknown. A frontostriatal network dysfunction is related to ADHD without epilepsy The results of this study show that common neurobiological mechanisms may be present in epilepsy and ADHD. Identifying these mechanisms is an important consideration in the clinical management of epilepsy. Further studies should investigate genetic abnormalities that, through variable penetrance, can lead to epilepsy alone, epilepsy with ADHD, or ADHD alone. The effects of epileptiform activity in different neural network functions also require better understanding.


STS gene (encoding steroid sulfatase) have been observed in subjects with neurodevelopmental disorders, including attention deficit hyperactivity disorder (ADHD). Recently, two single nucleotide polymorphisms (SNPs) within STS (rs12861247 and rs17268988) have been reported to be associated with ADHD risk and inattentive symptoms in ADHD, respectively. Using a UK sample of ADHD subjects (aged 5–18 years), we tested the hypothesis that rs12861247 is associated with ADHD risk using a case–control approach . Using a subset of males from the ADHD sample, we also examined whether variation within STS is associated with symptomatology/cognitive function in ADHD. We then tested whether SNPs associated with cognitive function in ADHD were also associated with cognitive function in healthy male subjects using a German sample , and whether STS was expressed in brain regions pertinent to ADHD pathology during development. We did not replicate the previously identified association with rs12861247. However, in ADHD males, variation at rs17268988 was associated with inattentive symptoms, while variation within STS was significantly associated with performance on three cognitive measures. Three SNPs associated with cognitive function in ADHD males were not associated with cognitive function in healthy males. STS was highly expressed in the developing cerebellar neuroepithelium, basal ganglia, thalamus, pituitary gland, hypothalamus and choroid plexus. These data suggest that genetic variants affecting STS expression and/or activity could influence the function of brain regions perturbed in ADHD.Deletions encompassing the X-linked  STS) gene have been noted in cases of neurodevelopmental disorders associated with abnormal cognition, including attention deficit hyperactivity disorder (ADHD) ], thereby modulating their neural function and activity , are at significantly increased risk of developing ADHD (STS (rs2270112 and rs12861247) have been reported to be associated with ADHD, although only the latter survived correction for multiple testing; in the same study SNP rs17268988 was associated with inattentive symptoms (STS gene in mice (or inhibition of the associated enzyme) is associated with attention deficits, with alterations in impulsivity, and with elevated aggression  has been strengthened by four observations: first, subjects with deletions of STS gene might influence symptomatology and general cognitive function in ADHD subjects; with regard to effects on symptomatology, we focussed on possible association with inattentive, impulsive and aggressive symptoms given the previous findings in mouse and man, (3) to ascertain whether variants associated with symptomatology and/or cognitive function in an ADHD sample were also associated with effects on cognition in healthy volunteers and (4) to identify the pattern of expression of STS in the developing human brain with a view to understanding how STS activity might influence normal neurodevelopment, and thereby to understand whether there are plausible biological underpinnings to any observed genetic associations with cognitive endophenotypes.The aims of the present study were fourfold: (1) to try and replicate the most robust association finding previously identified with rs12861247 using a case–control protocol, (2) to investigate how variation across the STS in UK and Irish ADHD samples  between the ages of 5 and 18 years who had recently been diagnosed with ADHD was recruited from Child and Adolescent Psychiatrists and Paediatricians in the Greater Manchester, South Wales and Avon areas of the United Kingdom. The presence of symptoms and research diagnoses of Diagnostic and Statistical Manual of Mental Disorders, Version IV (DSM-IV ) ADHD and comorbid conditions were obtained from parent reports using the Child and Adolescent Psychiatric Assessment (CAPA) , with pea priori to give a power of >80% to detect an odds ratio of >1.52, and a power of 100% to detect an odds ratio of >2.00, assuming a disorder prevalence of 6% (The control sample genotyped at rs12861247 for the case–control study (hereafter referred to as the control group) consisted of 360 males from the Wellcome Trust Case Control Consortium 1958 Birth Cohort sample, used in previous association studies in Cardiff . These cSTS variation on symptomatology and cognition. Importantly, given the possible long- and short-term effects of medication on behavior and cognition, these subjects had been on medication for <1 year, and had been medication-free for at least 24 h prior to assessment. The mean age of this subset of subjects was 111.5 ± 1.5 months.Of the overall ADHD sample described above, a total of 266 male subjects were used to assay the effects of Using the CAPA, the number of parent-reported DSM-IV inattentive symptoms (maximum nine) and impulsive symptoms (maximum four) was ascertained . The presence of DSM-IV aggressive conduct disorder symptoms, again ascertained using the CAPA, was also investigated. This subgroup of conduct disorder symptoms consists of seven measures: (1) often bullies, threatens or intimidates others, (2) often initiates physical fights, (3) has used a weapon that can cause serious physical harm to others, (4) has been physically cruel to people, (5) has been physically cruel to animals, (6) has stolen while confronting a victim and (7) has forced someone into sexual activity. The final measure was not assessed in this sample, resulting in a maximum of six possible aggressive symptoms being present.Cognitive performance in this sample was tested using the Wechsler Intelligence Scales for Children Version III  were drawn from a sample of 2337 healthy volunteers of German descent who were randomly selected from the general population of Munich, Germany, and contacted by mail. To exclude subjects with central neurological diseases and psychotic disorders or subjects who had first-degree relatives with psychotic disorders, several screenings were conducted before the volunteers were enrolled in our study. First, subjects who responded were initially screened by telephone for the absence of neuropsychiatric disorders. Second, detailed medical and psychiatric histories were assessed for both themselves and their first-degree relatives by using a semistructured interview. Third, if no exclusion criteria were fulfilled, the subjects were invited to a comprehensive interview including the Structured Clinical for DSM-IV Axis I Disorders – Patient Edition .STS gene (aploview Tagger (r2 = 0.8 in order to achieve widespread coverage of the gene locus (tagging ∼68% of the STS gene). Six of these SNPs (with the exceptions of rs12861247 and rs5978405) were genotyped in 233 ADHD males using a Sequenom MassARRAY system and Sequenom iPLEX GOLD chemistry according to the manufacturer's instructions. Assays were designed using Assay Design software version 3.1  and genotyping was performed using Typer software version 4.0 (Sequenom Inc.). All thermocycling reactions were performed using an MJ Thermocycler. Specific details of reaction conditions and primers are available on request. Quality control assessment of genotyping assays after optimization involved analysis in the CEU individuals genotyped as part of the HapMap project prior to genotyping in the association sample (http://www.hapmap.org). Only assays with 100% concordance with HapMap genotypes were analyzed further. All association sample plates contained cases, controls, blanks and CEU samples. Genotypes were called in duplicate blind to sample identity and blind to the other rater. Single-nucleotide polymorphism rs12861247 was genotyped as described above. rs5978405 was genotyped using TaqMan SNP genotyping assay C_30005846_10 according to the manufacturer's instructions (Applied Biosystems). Linkage disequilibrium analysis was performed using Haploview (Subjects for which symptomatology and cognitive data are presented were genotyped for eight Tag-SNPs covering the promoter and coding regions of the STS gene . Single-http://www.hapmap.org). Only assays with 100% concordance with HapMap genotypes were analyzed further.Three SNPs, rs5978405, rs5933863 and rs17268988, which showed evidence for association (replicated in the case of rs17268988) with symptomatology/cognitive measures in the ADHD sample, were successfully genotyped in healthy volunteer subjects using the Sequenom MassARRAY system; rs12861247, which also showed strong evidence for association, could not be typed using this panel. Genomic DNA was extracted from whole blood samples according to routine procedures. One nanogram of DNA was assayed using the iPLEX assay on the MassARRAY MALDI-TOF mass spectrometer . DNA concentration was adjusted using the PicoGreen quantitation reagent . Quality control assessment of genotyping assays involved analysis in the CEU individuals genotyped as part of the HapMap project  were regarded as significant , and transcribed using T7 or Sp6 polymerases to generate antisense or sense ribonucleotide probes, respectively. Digoxigenin-uridine triphosphate was incorporated into riboprobes during in vitro transcription using DIG RNA labeling mix  according to the manufacturer's instructions. Labeled probes were then hybridized to human embryonic or fetal tissue obtained from the Human Developmental Biology Resource  according to standard protocols (A polymerase chain reaction (PCR) product corresponding to NM_000351.3 (bp 2570–3249) was subcloned into pGEM-T Easy vector . The vector was linearized using rotocols .P = 0.762, odds ratio = 1.08, 95% confidence interval 0.667–1.737. When the analysis was limited to males only, again there was no significant difference between cases and controls . Data from the females genotyped indicated that there was no significant deviation from Hardy–Weinberg equilibrium at this SNP .For our case–control study, a total of 327 ADHD cases  and 358 male control subjects were successfully genotyped for rs12861247. The frequency of the minor allele (A) at this SNP did not differ significantly between the two groups: 0.107 in cases  and 0.101 in control subjects, Our genotyping success rate across all eight SNPs analyzed in the ADHD sample for which behavioral/cognitive data are presented was 90.5 ± 2.6%. The minor allele frequencies we observed were comparable with those seen previously in British and Irish ADHD samples . The TagOur genotyping success rate across the three SNPs analyzed in the healthy volunteer sample was 96.3 ± 1.3%. Minor allele frequencies within the healthy volunteer sample are shown in STS is a non-expressed pseudogene  being associated with the number of DSM-IV inattentive symptoms, with males possessing a G allele (n = 51) showing more inattentive symptoms than those possessing the more common C allele (n = 163)  and subjects ≥109 months of age ; this resulted in two evenly sized groups . We found no effect of allelic variation on inattentive symptoms in the younger subgroup , but a significant effect in the older subgroup, with males possessing a G allele showing, on average, 0.8 more inattentive symptoms than males possessing a C allele  (As the Y-linked homolog of = 0.005) . None of= 0.005) .STS variants was initially tested in males only  , with a second SNP rs4403552 showing suggestive evidence for association with this subtest  . Significant associations were also noted between rs5978405 and verbal IQ  and between rs12861247 and comprehension subtest performance , indicated by corrected P values <0.05 in each case. These positive results cannot be attributed to age-dependent effects  data could not be obtained for particular subtests. As with the symptomatology analysis, association with les only . One SNP effects .STS variants at SNPs rs5978405, rs5933863 and rs17268988 was initially tested in hemizygous young, male healthy volunteer subjects; cognitive data were obtained for all of these subjects. Data from measures analogous to those in the WISC-III were examined . We detected no evidence for association between the SNPs tested and any of the cognitive measures examined within our healthy volunteer sample , d plexus . High led plexus . The gend plexus . At Carnd plexus . At 9 wed plexus . High exd plexus .STS gene, encoding the enzyme steroid sulfatase, were associated with vulnerability to ADHD and with cognitive function in ADHD and healthy subjects. A secondary aim was to map the expression of STS during development, with a view to understanding how its altered expression/function might influence ADHD endophenotypes.The primary aim of this study was to examine whether genetic variants within the X-linked STS that influence ADHD risk, and that only complete loss of function of the gene (as in the mutant mouse and XLI studies) results in ADHD endophenotypes. It is also important to be aware of two possible limitations of our control sample (from the Wellcome Trust Case Control Consortium): first, it was not specifically matched for IQ to our ADHD sample, although we did exclude ADHD subjects with evidence of learning disability (IQ < 70) from our study. Second, no formal assessment of childhood ADHD symptoms was made in this sample; it is plausible that the presence of subjects with undiagnosed ADHD in the control group may have slightly biased our study toward producing a type II error, although given the incidence of ADHD, this would be expected to have little impact on power  ] ] .An important potential limitation with studies such as this is the possibility of type I errors. The fact that the results above remain significant upon correction for stringent multiple testing, that the association between rs5933863 and picture completion is replicated in a small sample of ADHD females whereby homozygotes for the G allele significantly outperform heterozygotes and homozygotes for the A allele (data not shown), and that the only subject from previous publications (of whom we are aware) with STS deficiency and ADHD tested using the WISC  has been reported to show an uneven cognitive profile, with relative strength in picture completion and weakness in comprehension (We could find no evidence that the three SNPs genotyped in healthy male volunteers were associated with cognitive function. This may be because the associations are only manifest in the context of an abnormally developing brain, because the associations vary with age, because of subtle methodological differences between the WISC-III and the WAIS-R, because any effects within this homogeneous population would be too small to detect with our limited sample size or because the findings from the ADHD sample were indeed false positives. However, as we have argued above, we believe that the latter possibility is unlikely.STS was found to be most highly expressed in various epithelia during development, including the olfactory, thalamic and cerebellar neuroepithelia. High STS expression was also observed in the choroid plexus, the hypothalamus and pituitary gland, the thalamus and the basal ganglia. Expression was noted throughout much of the rest of the brain at lower levels, including the developing neocortex. These findings are broadly consistent with previous limited data examining the localization of STS protein/enzyme activity in specific adult brain regions in man (STS expression in the thalamus, basal ganglia and cerebellar neuroepithelium (which gives rise to GABAergic and glutamatergic neurons of the cerebellum; STS are consistent with the associated protein having discrete effects on attention and other aspects of cognition.Recent data from monkeys have shown that the thalamus is a key mediator of visual attention . AlthougSTS might be associated with altered brain development and affect the severity of inattention symptoms in ADHD. It is possible that there is an association with cognitive function in ADHD, but some caution is required due to testing of multiple phenotypes.Several of the analyses presented herein are exploratory and, as such, should be confirmed in alternative samples. However, when taken together, these data suggest the possibility that variation within


XbaI) vector followed by insertion of the NT4-6His-PR39 gene segment and, thus, the recombinant plasmid pSS-HRE-CMV-NT4-6His-PR39-PolyA-AAV was successfully constructed. The expression of 6xHis in CRL-1730 cells under the regulation of HRE was assayed using the immunohistochemical method and results showed that the expression was positive in the experimental group. Myocardium perfusion MRI displayed that the infracted area significantly decreased under the action of pSS-HRE-CMV-NT4-PR39-PolyA-AAV. The artificial minimal HRE in CRL-1730 cells effectively and rapidly regulates the expression of the downstream gene NT4-TAT-His-PR39 of the CMV promoter. Recombinant pSS-HRE-CMV-NT4-PR39-Poly-AAAV promotes neoangiogenesis in the ischemic area, reduces the area of infarction and improves heart function.The objective of this study was to investigate the effect of the PR39 recombinant adeno-associated virus (AAV) controlled by the hypoxia-responsive element (HRE) on gene therapy of ischemic heart disease. The minimal HRE was artificially synthesized and the AAV vector controlled by HRE was introduced with NT4-TAT-His-PR39 to investigate the expression of AAV-PR39 in hypoxic vascular endothelial cells (VEC) of human umbilical vein (CRL-1730 cell line) and the angiogenesis-promoting effect in pigs with acute myocardial infraction (AMI). The minimal HRE/CMV was designed and artificially synthesized using the PCR method and cloned with the T vector cloning method. The pSS-HRE-CMV-NT4-6His-PR39-PolyA-AAV plasmid was constructed. Using the calcium phosphate precipitation method, HEK-293 cells were co-transfected with three plasmids to produce the recombinant virus. An equal volume of pSS-HRE-CMV-NT4-6His-PR39-PolyAAAV and enterovirus  was transfected into CRL-1730 cell lines, respectively. The immunohistochemical method was used to assay the expression of 6xHis in CRL-1730 cell lines and the expression of PR39 under hypoxia. Eighteen AMI miniature pigs were randomized into the experimental group (HRE-AAV-PR39 group), control group 1  and control group 2 (EV group). The area of ischemia was assessed with conventional MRI and myocardium perfusion MRI. Pigs were sacrificed at preset time-points to obtain samples of ischemic myocardium. Morphological and pathological data were collected. According to data in the literature and databases, the minimal HRE was designed and synthesized with the PCR method. A large number of HREs were connected to modified pSSHGAAV (pSSV9int-/ Data from over 70 genes suggest that endogenous HREs are composite regulatory elements comprising the conserved HIF-binding site (HBS) with an A/GCGTG core sequence and a highly variable flanking sequence. A single HBS is essential but not sufficient for activation by hypoxia. Binding sites of transcription factors provided by the flanking sequence are not necessary for hypoxia regulation but are required to amplify the hypoxic response or make the HRE tissue-specific (The hypoxia-responsive element (HRE) is the minimal indirect specific . In contEscherichia coli TOPIO, T/T AT-His vector, pBV220/NT4 vector, AAV vector (pSSHG-CMV), helper virus pAAV/Ad, helper packaging plasmid PFG140 and biological enzyme were purchased from the Xi’an Sino-American Biotechnology Co., Ltd. Hypoxic (1% O2) incubator was provided by the Department of Pathology, School of Basic Medical Sciences, Fourth Military Medical University. CRL-1730 cells were provided by the Department of Pharmacology, Xian Jiaotong University. CoCl2 was provided by the Department of Molecular Genetics, Fourth Military Medical University. Recombinant virus pSS-HRE-CMV-NT4-TAT-6His-PR39-PolyA-AAV was packed. Experimental miniature pigs were purchased from the Animal Center, Fourth Military Medical University . Pentobarbital was purchased from the Reagent Center, Fourth Military Medical University. Suminaxin injection was manufactured by the Jilin Huamu Animal Health Product Co., Ltd. The experimental procedures in the present study were approved by the local ethics committee and the animals were treated according to guidelines set by our University’s Animal Laboratory .Design and synthesis of primers. According to data in the literature and databases, the minimal HRE sequence was CTGCACGTA CTGCACGTA CTGCACGTA CTGCACGTA and 35 bp sequences spacing in the TA Box. Based on nucleotide sequences in GenBank, XbaI-recognition sequence TCTAGA (cohesive end) was added to the 5′ and 3′ ends and the protective base G was introduced to the 5′ end of forward and reverse primers. Using the DNAsis software, forward and reverse primers were designed as follows and were synthesized by the Shanghai Generay Biotech Co., Ltd.: F1, ‘-g 5′-TCTAGA CTGCACGTA CTGCACGTA CTGCACGTA CTGCACGTA atcaattacg gggtcattag-3′; ‘-g 5′-TCTAGA acgcgttaag atacattgat gag-3′. The complete pGEM-T-HRE-CMV-MCS-PolyA was obtained by PCR amplification and this sequence was completely consistent with that of gene expression box designed, as proved by DNAsis software.Construction of pSS-HRE-CMV-MCS-PolyA-AAV vector. pSS-HRE-CMV was digested with both E. co 721 and BamHI. The digested HRE-CMV segment was connected with the human pBV220/NT4-TAT-His-PR39 vector with the method aforementioned. The recombinant T/TAT-His-PR39 was harvested and digested by both NaeI and BamHI. The digested segment was connected with the human pBV220/NT4 vector. The recombinant pBV220/NT4-TAT-His-PR39 was harvested and sent to Sangon Biotech (Shanghai) Co., let for gene sequencing.Package of recombinant virus. Using the calcium phosphate precipitation method, HEK-293 cells were co-transfected with plasmid pssHG-9H-CMV-NT4-PR39 helper packaging plasmid pAAV/Ad with cloning Rep and Cap genes, and adenoviral plasmid PFG140 to produce the recombinant AAV-9H-CMV-NT4-PR39.Construction of myocardial infarction in miniature pigs. Eighteen miniature pigs were randomized into the experimental group , control group 1  and 2 . Every pig was fasted for 8 h and then injected intramuscularly with 2 ml of Suxinmian injection and 18 ml of 5% pentobarbital (1 ml/kg). Under electrocardiographic (ECG) monitoring, left ventriculography was performed by introducing the guide wire and 6F arterial sheath; subsequently, 6F Amplatz guide catheter was exchanged to conduct coronary angiography. The occluded site of the left anterior descending artery (LAD) was identified. Next, a PTCA balloon catheter was introduced into the distal LAD artery with the aid of a microcatheter and microguide wire. The balloon (2.0 or 2.5) was placed 0.5–1.0 cm below the first diagonal branch of LAD and inflated with 4–6 times of atmospheric pressure to occlude LAD for 90 min. If LAD was completely occluded, the AMI model was successfully constructed. One milliliter of AAV/HRE-PR39 or 1 ml of physical saline/control virus was injected through the microcatheter. After that, the catheter and sheath were removed. Magnetic resonance imaging (MRI) was performed preoperatively and 24 h, 1, 2 and 3 weeks postoperatively.MRI scanning post AMI. In this study, a magnetom triotim 3.0-T magnetic resonance scanner  was used to perform MRI scanning under both the conventional MR sequence and a novel sequence sensitive to myocardium edema (T2-weighted TrueFISP imaging of myocardium). Additionally, myocardium delayed perfusion scanning was also performed under the TrueFISP-PSIR sequence. Changes in MR images of MI area over time were observed. Bright-blood sequence: scanning was performed using the novel sequence sensitive to myocardium edema (T2-weighted TrueFISP imaging of myocardium). Scanning parameters were as follows: TR/TE, 281.95/1.09 msec; section thickness, 6 mm; gap, 0; matrix, 256x256; FOV, 360 mm; RFOV, 75%; acquisition window, 715 msec; 2 trigger pulse, 2; trigger delay, 433 msec and Flip, 90°C. For the TrueFISP-PSIR sequence to performed myocardium delayed perfusion scanning, 2D TrueFISP-PSIR sequence was used. The contrast agent was injected at a rate of 0.1 mmol/kg. Scanning was started 10 min later. Scanning parameters were as follows: TR/TE, 82.24/1.09 msec; section thickness, 6 mm; gap, 0; matrix, 256x256; FOV, 360 mm; RFOV, 75%; acquisition window, 750 msec; 2 trigger pulse, 2; trigger delay, 300 msec and Flip, 90°C.A pig of every group was sacrificed respectively 1, 2 and 3 weeks postoperatively. The fresh heart was harvested for immunohistochemical examination to measure expression of HIF-1α at the area of ischemia. The myocardium tissue was fixed with paraform, dehydrated with 60–100% alcohol solution, and was embedded in soft, neutral and hard paraffin wax. Sections were made followed by dewaxing in 170–100%. Ischemia myocardium samples of each pig were collected for SABC immunohistochemical staining to observe expression of HIF-1α around the area of infarction and compare expression levels between the experimental and control groups.The OD of the 6xHis staining area was calculated with the IPP Image Processing software, which represented the level of PR39 protein. In the AAV/HRE-PR39 group, massive brown area was present in and out of the cytoplasm under hypoxia but only in the cytoplasm under common oxygen; in non-viral cells, a small quantity of brown area was observed in the cytoplasm . The average OD value for positive 6xHis was 132.3±8.5 in control group 2, 23.3±5.4 in the experimental group and 48.5±7.3 in control group 1 , illustrating that HRE triggers the expression of NT4-TAT-His-PR39 of hypoxic CRL-1730 cells .Delayed perfusion MR images in different stages of AMI models could significantly distinguish normal and infarcted myocardium. The area of infarction was displayed clearly. Significant differences in myocardium changes over time post AMI were noted between the experimental and control group. The area of infarction in the control group changed slightly over time (no inflammatory edema period was taken into account). In the experimental group, the area of infarction decreased over time and the difference was statistically significant, as measured by the eFilm software. The left ventricular ejection fraction post AMI also differed significantly between the two groups.Pathological examinations showed that the cardiac apex, the anterior wall of the left ventricle, and the anterior septal were infarcted. The infarcted myocardium was crisp and white and significantly decreased over time in the experimental group. The white myocardium was collected for paraffin sectioning. Expression of HIF-1α in myocardial cells was assayed using immunohistochemistry, as described previously. Digital microphotographs showed that the average OD value for positive HIF-1α was 5469±395.7 in the experimental group and 1686±423.2 in the control group (P<0.001), respectively, suggesting that rAAV expressing PR39 could significantly increase the level of HIF-1α proteins in hypoxic myocardial cells .cis-activation , inducing excessive expression of VEGF protein. VEGF will accumulate in the systemic circulation, causing hemangioma ,12. In tAccording to previous literature, the minimal HRE is planned to insert to the site of 35 bp in the upstream of CMV. However, there is no digestion site in the upstream of the CMV promoter . TherefoIn this study, AAV was used as the viral vector carrying PR39, which could stably express PR39 in the host cell during a long period. Recovery of AMI is time enduring. Hence, AAV-PR39 is superior to the conventional method for treating AMI and also valuable in the prevention and management of AMI. It is promising for thr gene therapy of IHD.The HRE-CMV-MCS-PolyA gene segment is a hypoxia-regulatory promoter requiring increasing research for hypoxia-regulation. This promoter has more potent hypoxia-inducible ability and is more minor than current hypoxia-regulatory promoters. It offers a minor promoter with the regulating ability for constructing thr rAAV vector, i.e. it offers a relatively large remaining space to insert the objective gene for the 4-kb space which can be inserted in the endogenous gene. We believe that HRE/CMV can be valuable in future gene therapy.To better investigate the effect of HRE-regulated PR39 on IHD, AMI was modeled. Since miniature pigs have a coronary artery similar to that of humans with less collateral circulation , it is used for modeling. We constructed AMI models by introducing a balloon to block the coronary artery and verified the model by monitoring ECG and measuring troponin I and creatine kinase isoenzymes at different stages.Delayed perfusion MR images at different stages of AMI models significantly distinguish normal and infracted myocardium. The infarcted area was displayed clearly. Significant differences in changes of myocardium over time post AMI were noted between the experimental and control group. The area of infarction in the control group changed slightly over time (no inflammatory edema period was taken into account). In the experimental group, the area of infarction decreased over time and the difference was statistically significant, as measured by the EFilm software. The left ventricular ejection fraction post AMI was also significantly different between the two groups.Myocardial cells of the AMI miniature pigs receiving recombinant viruses and physical saline were analyzed using immunohistochemistry and the results showed that rAAV secreting PR39 could significantly increase the level of HIF-1α protein in hypoxic myocardial cells, thereby upregulating the expression of VEGF and promoting angiogenesis.These animal experiments utilized only imaging data to show the ability of the recombinant virus to reduce the infarcted myocardial area and increase the HIF-1α protein in hypoxic myocardial cells. In this study, PR39 was administered immediately after AMI modeling and only pathological and immunohistochemical examinations were performed. However, in clinical practice, most AMI patients are not treated in a timely manner but are treated several hours post onset. Therefore, quantitative studies of the effects of the recombinant virus on IHD gene therapy should be performed to investigate the efficacy of the recombinant virus in this study following longer periods of AMI.


The Strengths and Difficulties Questionnaire (SDQ) is widely used as an international standardised instrument measuring child behaviour. The primary aim of our study was to examine whether behavioral symptoms measured by SDQ were elevated among children with autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD) relative to the rest of the population, and to examine the predictive value of the SDQ for outcome of parent-reported clinical diagnosis of ASD/ADHD. A secondary aim was to examine the extent of overlap in symptoms between children diagnosed with these two disorders, as measured by the SDQ subscales. A cross-sectional secondary analysis of data from the Millennium Birth Cohort , was conducted. Data were weighted to be representative of the UK population as a whole. ADHD or ASD identified by a medical doctor or health professional were reported by parents in 2008 and this was the case definition of diagnosis; . Study children's ages ranged from 6.3–8.2 years; (mean 7.2 years). Logistic regression was used to examine the association between the parent-reported clinical diagnosis of ASD/ADHD and teacher and parent-reported SDQ subscales. All SDQ subscales were strongly associated with both ASD and ADHD. There was substantial co-occurrence of behavioral difficulties between children diagnosed with ASD and those diagnosed with ADHD. After adjustment for other subscales, the final model for ADHD, contained hyperactivity/inattention and impact symptoms only and had a sensitivity of 91% and specificity of 90%; (AUC) = 0.94 . The final model for ASD was composed of all subscales except the ‘peer problems’ scales, indicating of the complexity of behavioural difficulties that may accompany ASD. A threshold of 0.03 produced model sensitivity and specificity of 79% and 93% respectively; AUC = 0.90 . The results support changes to DSM-5 removing exclusivity clauses. The Strengths and Difficulties Questionnaire (SDQ) is a brief dimensional measure of psychopathology among children aged 4–16 that has been widely adopted in both research and in clinical practice The SDQ has been used in in clinical practice as a screening and/or assessment tool by both school psychologists Woerner and colleagues Various studies have examined the utility of the SDQ as a screening device in predicting childhood psychiatric cases Goodman and colleagues According to the International Classification of Diseases (ICD-10), for a diagnosis of ASD to be made, children must display impairments in social interactions and communication, as well as restricted interests and repetitive behaviour Given the wide and on-going use of the SDQ in research on developmental disorders, we sought to clarify the predictive power of the SDQ subscales in the identification of parent-reported clinical diagnosis of two specific disorders: ADHD and ASD. The primary aim of our study was to examine whether all behavioral symptoms measured by SDQ were elevated in children with ASD and ADHD relative to the rest of the population, and the utility of the SDQ as an indicator of these disorders. A secondary aim was to examine the extent to which symptoms co-occurred in children diagnosed with ASD or ADHD. We hypothesised hyperactivity/inattention symptoms would predict clinical diagnosis of ADHD, and prosocial and peer relationship problems would predict ASD. This study therefore assesses the utility of the SDQ in identifying these disorders.Information was gathered from the sample, the first Millennium Cohort Study (MCS) survey when children were 9 months old, and three, five and seven years of age: four sweeps of data collection Our study sought to clarify the predictive power of the SDQ subscales in the identification of parent-reported diagnosis of ASD and ADHD using logistic regression models. This was compared to the predictive power of the PHD algorithm already in existence st September 2000 and 11th January 2002 and listed on the Child Benefit Records were eligible for the study. Child Benefit was a financial benefit payable to all parents of UK children at this time, with near universal take up. Data were first collected when children were 9 months old (1st wave), further data were recorded concerning the children's health and development when the children were 3 years old (2nd wave), 5 years old (3rd wave) and 7 years old (4th wave). Within the total MCS cohort of 19, 519 children, the current study outcomes, ASD and ADHD status, were recorded for 14, 043 children at wave 4 (over 70%). The MCS provides appropriate standardised weightings to adjust for the effect of attrition and oversampling, making these results representative of the UK population as a whole. Details of sampling design and weighting calculations are documented in detail elsewhere The Millennium Cohort Study (MCS) is a UK-representative birth cohort study that used a disproportionate stratified cluster sampling design The case definition of the two conditions was based on responses to an MCS question duplicated from the US National Health Interview Survey questionnaire reported in previous studies Has a doctor or health professional ever told you that (sample child) had attention deficit hyperactivity disorder (ADHD)Has a doctor or health professional ever told you that (sample child) had autism, Asperger's syndrome or autistic spectrum disorder?n = 30 ASD, n = 44 ADHD, of these, n = 17 refused/don't know in both categories). We took this measure to represent a clinical diagnosis of disorder in line with other studies Families at wave 4 whose study children were seven years old, who responded with positive or negative answers to the above questions, were included. Families who answered ‘don’t know' or refused to answer were excluded from the analysis (www.sdqinfo.org).The SDQ is composed of 25 items that ask about behavioural attributes of the child and are combined to form five subscales (composed of 5 items each). The emotional symptoms subscale contains items that ask about fears, worries, misery, nerves and somatic symptoms, the conduct problems subscale inquires about tantrums, obedience, fighting, lying and stealing, and the hyperactivity/inattention subscale covers restlessness, fidgeting, concentration, distractibility and impulsivity. The peer relationships subscale items include questions about popularity, victimization, isolation, friendship and ability to relate to children as compared to adults, and the prosocial subscale covers consideration of others, ability to share, kindness to younger children, and helpfulness when other children are distressed and willingness to volunteer to comfort. For all the subscales except the prosocial subscale, high scores indicate difficulties. As the prosocial items ask about the presence of prosocial behaviour, the subscale measures the strengths of the child in this area, and increasing scores represent increasingly prosocial behaviour, unlike the other sub-scales where increasing score represents increasing impairment. In all cases, answer options for each item are: ‘Not true’ ‘Somewhat true’ or ‘Certainly true’, and these are scored 0, 1 or 2, giving a total score out of a possible 10 for each subscale. A further ‘impact’ subscale measures the impact of any difficulties on carers and the children themselves in terms of chronicity, distress, social impairment, and burden to others. This is again scored 0–10 with increasing impact producing a higher score. More details about the SDQ, the probable hyperactivity disorder (PHD) algorithm, normative data, background research and how the subscales are scored are available at the SDQ website . Several studies have stressed the need for information from multiple informants when rating symptoms of a child psychiatric disorder The ASD, ADHD and general population were compared on SDQ subscale scores. Box plots were provided for teacher and parent report of behaviour separately to illustrate how the three groups  differed in SDQ scores. Children reported as having both diagnoses (n = 44) were included in both ASD and ADHD groups.th wave when the study outcomes were measured, making the sample representative of the UK population Logistic regression (LR) established the odds of diagnosis of ASD/ADHD using SDQ subscales as independent variables. Parent and teacher ratings of behaviour were treated as separate covariates. The odds ratios (OR) from the analyses indicate that the relative increase in odds of being identified with ASD/ADHD corresponded to a one-point increase in the SDQ subscales. All the sub-scales bar the prosocial scale measure impairment, therefore the reciprocal of the odds ratios for the prosocial scores was used to fit conceptually with the rest of the model. This means that for all SDQ subscales, an odds ratio greater than 1 represents greater prediction of diagnosis as children's difficulties increase. Unadjusted logistic regression models were fitted in which just one predictor at a time was included. Multivariable (adjusted) logistic regression models were then fitted in which predictors significant at the 10% level in the unadjusted analyses were included as covariates. Estimates from LR were weighted to take account of the disproportionate stratified sample of electoral wards and attrition/non-response by the 4For 96.7% of families participating, the main respondent on the outcome measure of ASD or ADHD was the child's mother. At the birth of the child, mothers had a mean age of 28 years (range 13 to 48 years), and over 99% were resident at home with the study child all of the time. The mean child age when outcome measures were taken was 7.2 years . As LR was also used to explore the predictive value of the subscales for ASD .Again all the SDQ subscales were significant in unadjusted analysis. After adjustment for interdependencies between subscales, several still remained significantly associated with the outcome of ASD at 10% levels. These were the impact and hyperactivity subscales from both raters, and the prosocial and emotional symptoms scores rated by parents. The measures with the largest effect were the parent-rated subscales of the prosocial behaviour and impact subscales. Peer problems from either rater did not appear in the final model. A threshold of 0.03 produced the optimal values for model sensitivity and specificity of 79% and 93% respectively; AUC = 0.90, . The PPV was again low at 18%. Examples of scores that exceed the threshold for this ASD model using optimal values are given in the supporting information in The prevalence of ASD and ADHD was not the focus of this paper: we have written about this elsewhere Despite the exclusivity clause in the current ICD-10 diagnostic classification systems, there was a high proportion of dual diagnosis in the two conditions: 23% of children with ADHD had a diagnosis of ASD, and 21% with ASD had identified ADHD. Several other recent studies After adjustment for other subscales in multivariable models, the final model for ADHD was composed of the hyperactivity/inattention and impact symptoms only. This finding is highly predictable and as initially hypothesised, although the selection biases inherent in obtaining a clinical diagnosis may have clouded the relationship. Although the findings suggest that ADHD symptoms are also relatively common in children with ASD and vice-versa, in line with findings from other studies In our study LR models, after statistical adjustment for interdependencies between different types of behavioral problems, a distinctive symptom profile emerged for ADHD based on hyperactivity and impact sub-scales, but not for ASD. The finding provides evidence to support the assertion of Nicalsen et al. The second main difference is that cut-offs (e.g. for identifying 91% of children with disorder) were not fixed as are those in the PHD model. This is consistent with the findings of Ullebo and colleagues Goodman and Mullick The resulting LR model for autism shows that many types of difficulties may complicate the picture for a child with ASD. This is to be expected, as there is not a specific “autism spectrum” subscale that focuses on the core difficulties as there is with ADHD. Prosocial behaviour emerged as the strongest predictor of ASD, which again is not surprisingly as social impairments are core deficits. Furthermore, ASD diagnosis has been associated with the low scores on the prosocial subscale in other UK cohorts The current study used parent-report of clinical identification of ASD and ADHD by a doctor or another professional. This means that parents are likely to be well aware of symptoms of these conditions and may therefore be more likely to report them than a parent of a child with similar difficulties that have not been clinically highlighted. Furthermore, parents may have been over-inclusive in their interpretation of the question: inferring a positive answer in cases where ASD or ADHD was suggested by a health worker but not confirmed by further assessment. Clinician diagnoses themselves can be inaccurate if unguided by structured assessment In addition, the sample will contain other children with ASD and ADHD, and other disorders, as yet unrecognised Other limitations relate to the ADHD group. First, we did not have access to pharmaceutical data, but evidence suggests treatment with methylphenidate may have improved symptoms of hyperactivity and may have led parents and teachers to under-report difficulties in any children with diagnoses who were taking medication Despite these limitations our results provide further evidence to suggest that the SDQ algorithm is a useful tool as an indicator of ADHD symptoms for research purposes. As the SDQ instrument is widely used in research studies already The study is part of a large and growing literature that demonstrates that ADHD symptoms are also relatively common in children and adults diagnosed with ASD and vice-versa Table S1Examples of scores over the threshold for ADHD model. There are 1331 combinations of 3 SDQ scales (0–10) of which 928 combinations would produce a value over the threshold. The most frequent combinations in MCS are included for illustrative purposes.(DOCX)Click here for additional data file.Table S2Examples of scores over the threshold for ASD model. In MCS data there are 671 combinations which produce a value over the threshold. They are all unique. (DOCX)Click here for additional data file.


Isotonic saline has been proposed as a safer alternative to traditional hypotonic solutions for intravenous (IV) maintenance fluids to prevent hyponatremia. However, the optimal tonicity of maintenance intravenous fluids in hospitalized children has not been determined. The objective of this study was to estimate and compare the rates of change in serum sodium ([Na]) for patients administered either hypotonic or isotonic IV fluids for maintenance needs.This was a masked controlled trial. Randomization was stratified by admission type: medical patients and post-operative surgical patients, aged 3 months to 18 years, who required IV fluids for at least 8 hours. Patients were randomized to receive either 0.45% or 0.9% saline in 5.0% dextrose. Treating physicians used the study fluid for maintenance; infusion rate and the use of additional fluids were left to their discretion.Sixteen children were randomized to 0.9% saline and 21 to 0.45% saline. Baseline characteristics, duration (average of 12 hours) and rate of study fluid infusion, and the volume of additional isotonic fluids given were similar for the two groups. [Na] increased significantly in the 0.9% group  and increased, but not significantly, in the 0.45% group . The rate of change and absolute change in serum [Na] did not differ significantly between groups.When administered at the appropriate maintenance rate and accompanied by adequate volume expansion with isotonic fluids, 0.45% saline did not result in a drop in serum sodium during the first 12 hours of fluid therapy in children without severe baseline hyponatremia. Confirmation in a larger study is strongly recommended.NCT00457873  For almost half a century, pediatricians have ordered "maintenance" intravenous (IV) fluids for children according to the guidelines set out by Holliday and Segar: 100 cc/kg/day for the first 10 kg, plus 50 cc/kg/day for the next 10 kg, plus 20 cc/kg/day for each remaining kilogram  would fall among children receiving 0.45% saline and remain stable or increase among children receiving isotonic fluids. The primary aim of this study was to estimate the rate of change in [Na] for patients administered each of hypotonic  and isotonic  IV fluids for maintenance needs, and to compare these rates. Rate of change in [Na] was selected as the primary outcome to allow 'standardization' of changes in [Na] to a fixed time interval . The absolute change in serum [Na], and the proportions with a decrease in [Na], hyponatremia ([Na] < 136 mmol/L), hypernatremia ([Na] > 145 mmol/L), hypertension, and adverse events related to hyponatremia were also considered.This prospective, double-blind, randomized controlled trial was conducted at the Montreal Children's Hospital, a tertiary care pediatric hospital. Two populations of children between 3 months and 18 years of age were recruited: children with medical illnesses admitted via the emergency department , and children admitted following elective surgery . Only those requiring at least 8 hours of IV fluids were eligible. Exclusion criteria included a baseline serum [Na] of less than 133 or greater than 145 mmol/L, renal disease, cardiac dysfunction, pre-existing hypertension, diuretic use, edema, known adrenal dysfunction, and acute or severe chronic neurological illness. Children with neurological illnesses were excluded for safety reasons .A retrospective chart review of hospitalized children similar to those included in this trial indicated that the standard deviation for the rate of change in [Na] among patients administered hypotonic IV fluids was 0.29 mmol/L/h. This estimate was based, of necessity, on the change in [Na] observed among children in whom [Na] was measured at baseline and repeated at least once during IV fluid administration; both baseline and follow-up [Na] were obtained in only 20% of children receiving IV fluids. We planned to enroll 25 subjects per group, giving us 80% power to detect a difference of 0.21 mmol/L/h between groups with a one-sided test, setting alpha at 0.05. A one-sided test was used since it was felt to be physiologically very unlikely that the 0.9% saline group would experience a larger drop  in [Na] than the 0.45% saline group. This number also gave us 80% power to detect a change of at least 0.17 mmol/L/hr in each group, with a two-sided test.Eligible children were invited to participate if they were expected to receive maintenance IV fluids for at least 8 hours. Participants were randomized to receive either 0.9% saline in 5.0% dextrose or 0.45% saline in 5.0% dextrose. Solution bags were covered with an opaque plastic covering by the pharmacist. Participants, the treating team and the research team were blinded to the contents of the solution bags.Randomization was stratified by admission type  and carried out in blocks of six using a computerized random number generator. Baseline serum [Na] was drawn prior to starting study fluid. In medical patients, this was done when the IV catheter was inserted; surgical patients had their blood drawn upon completion of the surgical procedure. The rate of infusion of study fluid, use of fluid boluses or additional IV fluids, and addition of potassium to solutions were left to the discretion of the treating physicians. Treating physicians were informed that the study fluid was either 0.9% or 0.45% saline in 5% dextrose, and advised to prescribe it as they would normally prescribe maintenance fluids. Oral fluids were not restricted and were considered to be composed of free water. Serum [Na] was repeated approximately 12 hours  after the beginning of the infusion, at which point the study ended. Serum [Na] was measured using indirect potentiometry using the Beckman Coulter DxC analyzer, which has a range of [Na] detection between 100-200 mmol/L and a coefficient of variation of 1%.All participants received routine in-patient nursing care, with vital signs including blood pressure monitored at least every 4 hours. Nurses also monitored for possible adverse events including symptoms potentially associated with hyponatremia  or volume overload . All fluid intake during the study interval, including oral, was recorded.The rate of change in serum [Na] was calculated as (exit [Na] - baseline [Na]) divided by the number of hours between baseline and exit blood draws. The rate of maintenance fluid administration was expressed as a percentage of the standard recommended rate . The volBecause the rate of change in [Na], and absolute change in [Na], were not normally distributed, the medians were compared using the Wilcoxon rank sum test, and difference from zero was tested using the Wilcoxon signed rank test. Categorical variables were compared with chi-square. A P-value < 0.05 was considered significant. All statistical analyses were performed using Stata/SE 10.0 .The study was approved by the institutional Research Ethics Board. Written informed consent was obtained from parents or guardians for all participants and assent was obtained from children over 7 years of age if appropriate.As illustrated in Figure Characteristics of children receiving 0.9% saline n = 16) were similar to those receiving 0.45% saline (n = 21) with respect to age, sex, diagnosis, and baseline [Na]  years), more likely to be male (60%), and more likely to have a medical, rather than surgical, condition (57%).The duration and rate of study fluid administration, as well as the volume of other isotonic fluids received during the study period were similar for the two treatment groups Table .The majority of participants received supplemental isotonic fluids in addition to the study fluid. Most of this supplemental fluid was administered as boluses, but some surgical patients received infusions of isotonic fluid in the interval between the baseline blood draw and start of study fluid.On average, [Na] increased in both treatment groups Table . HoweverAs illustrated in Figure There were no adverse events. No participant developed hypertension. A 6 year-old boy with gastroenteritis in the 0.9% saline group developed mild hypernatremia, with an exit [Na] of 147 mmol/L.Fluid management differed between medical and surgical patients. Therefore, although numbers were small, an exploratory analysis, stratified by admission type, was also conducted. Medical patients received the study fluid at 142%  of the traditional maintenance rate, and 19.7 ml/kg  of additional isotonic fluids, mostly in the form of boluses. In contrast, surgical patients received study fluid at 101%  of the traditional maintenance rate, and received only 3.4 ml/kg  of additional isotonic fluids. The duration and rate of study fluid administration, and volume of additional isotonic fluids were similar for the 0.9% and 0.45% saline groups overall. The changes in [Na] observed when medical and surgical patients were analyzed separately mirrored those in the unstratified analysis.Contrary to our expectations, we observed increases in serum sodium in both the 0.45% and 0.9% groups, although this change was only statistically significant in the 0.9% group. Furthermore, similar proportions of individuals in both treatment arms developed hyponatremia.composition was dictated by study protocol; physicians were advised that study fluid was to be used for maintenance needs, and allowed the freedom to choose the rate of administration and any additional fluids according to their usual practice. This allowed us to isolate the impact of fluid composition on changes in serum [Na] in a 'real world' clinical setting in which physicians determined the rate of fluid administration.After decades of using hypotonic fluids, the best choice of solution for maintenance IV fluid therapy has recently become a topic of intense debate. Six randomized trials favored isotonic over hypotonic fluids for the prevention of hyponatremia ,27-31. TMaintenance fluids are one of the three key components of an IV fluid prescription, along with deficit replacement, and replacement of ongoing losses . The goaRecommendations for replacement of a volume deficit vary. For rapid volume expansion, only isotonic fluids are considered safe and effective . AlthougRegardless of the recommendation to consider deficit replacement separately from maintenance fluid needs, it has been common practice to simply increase the rate of infusion of the maintenance fluid solution to "1.5 times or 2 times maintenance" in an effort to replace volume deficits that remain after bolus isotonic fluids have been given for rapid intravascular volume expansion. This practice was evident among the medical patients participating in the present study, who received study fluid at an average of 142% of the traditional maintenance rate. Our study design isolated the impact of differing maintenance fluid composition on [Na] in the context of current prescription practices.As expected, we observed a significant increase in serum [Na] among patients in the 0.9% saline group. A smaller and slower, though not statistically significant, increase in [Na] was observed in the 0.45% saline group. This was not anticipated. Rather, we had hypothesized that patients randomized to receive hypotonic fluids would experience a drop in serum [Na]. The rationale for this hypothesis was that the patients enrolled in the study were at risk for high ADH secretion, stimulated by either volume depletion (appropriately) or the syndrome of inappropriate ADH secretion . Because free water excretion is impaired in the presence of ADH, the administration of hypotonic solutions to patients secreting ADH will inevitably lead to a fall in serum [Na] .The fact that most children who received 0.45% saline did not experience a drop in [Na] suggests that most did not have an ongoing stimulus for ADH secretion. Adequate volume repletion with isotonic fluids prior to and during IV maintenance fluid administration likely protected patients against hyponatremia despite hypotonic maintenance fluids. Those who did experience a drop in [Na] can be assumed either to have not been adequately volume expanded (and therefore have ongoing physiologic volume-related stimulus for ADH secretion) or to have had inappropriate ADH secretion . Children in the 0.9% saline group who experienced a decrease in [Na] were very likely to have had inappropriate ADH secretion; unless they developed new onset adrenal insufficiency, hypothyroidism, or renal salt wasting, inappropriate ADH secretion is the only reasonable explanation for a drop in [Na] in this group ,36.The average rate of study fluid administration in both treatment groups was only slightly greater than the traditionally recommended maintenance rate. This likely also played a role in the relative stability in serum [Na]. Hypotonic fluids prescribed at high rates have been implicated in many of the reported deaths due to iatrogenic hyponatremia .Two features of the study design may have resulted in bias toward finding no difference between treatment groups. First, the exclusion for safety reasons of children with baseline [Na] < 133 mmol/L or serious neurological disease may have eliminated those children at the highest risk for progressive hyponatremia. Second, this study focused on the first 12 hours of maintenance fluids. This was done in part for practical reasons, but also because we hypothesized that more pain and a greater degree of volume depletion would make this early period the period of highest risk for non-osmotic stimuli for ADH secretion. However, the syndrome of inappropriate ADH secretion may occur at any time, and may be more likely to occur later in the hospitalization, particularly in surgical patients ,27. If tThis was a small trial. The small sample limited our ability to detect small differences between the two treatment groups. It is possible that both the observed difference between treatment groups and the change in [Na] in the 0.45% saline group would have been statistically significant with a larger sample. The relatively large proportion of patients who failed to complete the study was an additional limitation. There were some differences in the characteristics of patients who completed the study compared with those who did not. This may limit generalizability of findings.It is also important to recognize that this study was designed to examine intermediate outcomes  rather than a clinically important outcome (complications associated with choice of fluid). This was done for power reasons: clinical complications are rare and would require a very large sample. Complications of hyponatremia are believed to depend on both the magnitude of the serum [Na] and the rate of change; therefore, these were considered relevant outcomes. However, much larger trials, examining clinically important outcomes, are required to establish the safety of different maintenance fluids.The most important finding of this study was that there was no decrease in [Na] in the 0.45% saline group; this can likely be attributed to judicious use of volume expansion with isotonic fluids and to the fact that, in most patients, study fluids were prescribed at rates generally not exceeding traditional maintenance rates. This result highlights the importance of considering maintenance fluids separately from deficit replacement.It is important to recognize that there is no IV fluid strategy that will completely eliminate the risk of iatrogenic hyponatremia. Hypotonic fluids in the setting of subtle, unrecognized volume depletion will result in a drop in [Na]. In the setting of the syndrome of inappropriate ADH secretion, [Na] may fall even with isotonic fluids - although larger drops will occur with hypotonic fluids. The use of isotonic fluids may reduce the risk of important hyponatremia, but if tragic consequences of IV fluids are to be avoided, monitoring of serum [Na] is important.Firm conclusions regarding the best solution for maintenance IV fluids cannot be drawn from this small study. Larger studies, powered to detect differences in the incidence of clinically important adverse events such as symptomatic hyponatremia or hypernatremia and hypertension, in addition to differences in change in [Na], are needed. In addition, studies of longer duration would be useful. This study suggests that 0.45% saline does not result in a drop in [Na] during the first 12 hours of fluid therapy among children with a baseline [Na] > 133 mmol/L and < 145 mmol/L, if appropriate care is taken to restore volume deficits with isotonic fluids, and excessive fluid administration rates are avoided. However, confirmation of these results in a larger study is strongly recommended.The following abbreviations appear: ADH: antidiuretic hormone; IQR: interquartile range; IV: intravenous; Na: sodium.The authors declare that they have no competing interests.TS was involved in patient recruitment, data collection, data synthesis, statistical analysis and drafted the manuscript; JF helped in the chart review and data entry; DL and FH recruited patients; BF conceived of the study, oversaw the entire project, and edited the manuscript. All authors have read and approved this manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2431/11/82/prepub