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Normalizing cardiovascular measurements for body size allows for comparison among children of different ages and for distinguishing pathologic changes from normal physiologic growth. Because of growing interest to use height for normalization, the aim of this study was to develop height-based normalization models and compare them to body surface area (BSA)-based normalization for aortic and left ventricular (LV) measurements. The study population consisted of healthy, non-obese children between 2 and 18 years of age enrolled in the Pediatric Heart Network Echo Z-Score Project. The echocardiographic study parameters included proximal aortic diameters at 3 locations, LV end-diastolic volume, and LV mass. Using the statistical methodology described in the original project, Z-scores based on height and BSA were determined for the study parameters and tested for any clinically significant relationships with age, sex, race, ethnicity, and body mass index (BMI). Normalization models based on height versus BSA were compared among underweight, normal weight, and overweight (but not obese) children in the study population. Z-scores based on height and BSA were calculated for the 5 study parameters and revealed no clinically significant relationships with age, sex, race, and ethnicity. Normalization based on height resulted in lower Z-scores in the underweight group compared to the overweight group, whereas normalization based on BSA resulted in higher Z-scores in the underweight group compared to the overweight group. In other words, increasing BMI had an opposite effect on height-based Z-scores compared to BSA-based Z-scores. Allometric normalization based on height and BSA for aortic and LV sizes is feasible. However, height-based normalization results in higher cardiovascular Z-scores in heavier children, and BSA-based normalization results in higher cardiovascular Z-scores in lighter children. Further studies are needed to assess the performance of these approaches in obese children with or without cardiac disease.

Background Growth abnormalities in single-ventricle survivors may reduce quality of life (QoL) and exercise capacity. Methods This multicenter, longitudinal analysis evaluated changes in height and body mass index (BMI) compared to population norms and their relationship to mortality, ventricular morphology, QoL, and exercise capacity in the Pediatric Heart Network Fontan studies. Results Fontan 1 (F1) included 546 participants (12 ± 3.4 years); Fontan 2 (F2), 427 (19 ± 3.4 years); and Fontan 3 (F3), 362 (21 ± 3.5 years), with ~60% male at each time point. Height z-score was −0.67 ± −1.27, −0.60 ± 1.34, and− 0.43 ± 1.14 at F1-F3, lower compared to norms at all time points (P ≤ .001). BMI z-score was similar to population norms. Compared to survivors, participants who died had lower height z-score (P ≤ .001). Participants with dominant right ventricle (n = 112) had lower height z-score (P ≤ .004) compared to dominant left (n = 186) or mixed (n = 64) ventricular morphologies. Higher height z-score was associated with higher Pediatric Quality of Life Inventory for the total score (slope = 2.82 ± 0.52; P ≤ .001). Increase in height z-score (F1 to F3) was associated with increased oxygen consumption (slope = 2.61 ± 1.08; P = .02), whereas, for participants >20 years old, an increase in BMI (F1 to F3) was associated with a decrease in oxygen consumption (slope = −1.25 ± 0.33; P ≤ .001). Conclusions Fontan survivors, especially those with right ventricular morphology, are shorter when compared to the normal population but have similar BMI. Shorter stature was associated with worse survival. An increase in height z-score over the course of the study was associated with better QoL and exercise capacity; an increase in BMI was associated with worse exercise capacity.

Background:The Single Ventricle Reconstruction Trial randomised neonates with hypoplastic left heart syndrome to a shunt strategy but otherwise retained standard of care. We aimed to describe centre-level practice variation at Fontan completion.Methods:Centre-level data are reported as median or median frequency across all centres and range of medians or frequencies across centres. Classification and regression tree analysis assessed the association of centre-level factors with length of stay and percentage of patients with prolonged pleural effusion (>7 days).Results:The median Fontan age (14 centres, 320 patients) was 3.1 years (range from 1.7 to 3.9), and the weight-for-age z-score was −0.56 (−1.35 + 0.44). Extra-cardiac Fontans were performed in 79% (4–100%) of patients at the 13 centres performing this procedure; lateral tunnels were performed in 32% (3–100%) at the 11 centres performing it. Deep hypothermic circulatory arrest (nine centres) ranged from 6 to 100%. Major complications occurred in 17% (7–33%). The length of stay was 9.5 days (9–12); 15% (6–33%) had prolonged pleural effusion. Centres with fewer patients (Conclusions:Fontan perioperative practices varied widely among study centres. Strategies to decrease the duration of pleural effusion and minimise complications may decrease the length of stay. Further research regarding deep hypothermic circulatory arrest is needed to understand its association with prolonged pleural effusion.

Background:Using existing data from clinical registries to support clinical trials and other prospective studies has the potential to improve research efficiency. However, little has been reported about staff experiences and lessons learned from implementation of this method in pediatric cardiology.Objectives:We describe the process of using existing registry data in the Pediatric Heart Network Residual Lesion Score Study, report stakeholders’ perspectives, and provide recommendations to guide future studies using this methodology.Methods:The Residual Lesion Score Study, a 17-site prospective, observational study, piloted the use of existing local surgical registry data (collected for submission to the Society of Thoracic Surgeons-Congenital Heart Surgery Database) to supplement manual data collection. A survey regarding processes and perceptions was administered to study site and data coordinating center staff.Results:Survey response rate was 98% (54/55). Overall, 57% perceived that using registry data saved research staff time in the current study, and 74% perceived that it would save time in future studies; 55% noted significant upfront time in developing a methodology for extracting registry data. Survey recommendations included simplifying data extraction processes and tailoring to the needs of the study, understanding registry characteristics to maximise data quality and security, and involving all stakeholders in design and implementation processes.Conclusions:Use of existing registry data was perceived to save time and promote efficiency. Consideration must be given to the upfront investment of time and resources needed. Ongoing efforts focussed on automating and centralising data management may aid in further optimising this methodology for future studies.

Survival for hypoplastic left heart syndrome (HLHS) has improved dramatically. Little is known about early family function, quality of life (QOL), or well-being/adjustment for parents of infants with HLHS. Parent/family outcomes over time, predictors, and differences in 143 mothers and 72 fathers were examined. Parents reported better family function compared with published norms, but 26% experienced family dysfunction. QOL and well-being were significantly lower than adult norms. QOL scores generally declined over time, whereas self-reported well-being improved. Responses from mothers and fathers showed different trends, with mothers having worse scores on most measures and at most time points. Being a single parent was a risk factor for poorer family function, but not for lower individual QOL or well-being. Family characteristics, stress, and coping skills were predictive of outcomes. Parents’ psychosocial responses to the challenges of life with infants with HLHS change over time. Individually tailored psychosocial support is needed.

Background/Aims: The Pediatric Heart Network Marfan Trial was a randomized trial comparing atenolol versus losartan on aortic root dilation in 608 children and young adults with Marfan syndrome. Barriers to enrollment included a limited pool of eligible participants, restrictive entry criteria, and a diverse age range that required pediatric and adult expertise. Retention was complicated by a 3-year commitment to a complex study and medication regimen. The Network partnered with the Marfan Foundation, bridging the community with the research. The aims of this study are to report protocol and medication adherence and associated predictive factors, and to describe recruitment and retention strategies. Methods: Recruitment, retention, and adherence to protocol activities related to the primary outcome were measured. Retention was measured by percentage of enrolled participants with 3-year outcome data. Protocol adherence was calculated by completion rates of study visits, ambulatory electrocardiography (Holter monitoring), and quarterly calls. Medication adherence was assessed by the number of tablets or the amount of liquid in bottles returned. Centers were ranked according to adherence (high, medium, and low tertiles). Recruitment, retention, and adherence questionnaires were completed by sites. Descriptive statistics summarized recruitment, retention, and adherence, as well as questionnaire results. Regression modeling assessed predictors of adherence. Results: Completion rates for visits, Holter monitors, and quarterly calls were 99%, 94%, and 96%, respectively. Primary outcome data at 3 years were obtained for 88% of participants. The mean percentage of medication taken was estimated at 89%. Site and age were associated with all measures of adherence. Young adult and African American participants had lower levels of adherence. Higher adherence sites employed more strategies; had more staffing resources, less key staff turnover, and more collaboration with referring providers; utilized the Foundation’s resources; and used a greater number of strategies to recruit, retain, and promote protocol and medication adherence. Conclusion: Overall adherence was excellent for this trial conducted within a National Institutes of Health–funded clinical trial network. Strategies specifically targeted to young adults and African Americans may have been beneficial. Many strategies employed by higher adherence sites are ones that any site could easily use, such as greeting families at non-study hospital visits, asking for family feedback, providing calendars for tracking schedules, and recommending apps for medication reminders. Additional key learnings include adherence differences by age, race, and site, the value of collaborative learning, and the importance of partnerships with patient advocacy groups. These lessons could shape recruitment, retention, and adherence to improve the quality of future complex trials involving rare conditions.

Background Different methods have resulted in variable Z scores for echocardiographic measurements. Using the measurements from 3,215 healthy North American children in the Pediatric Heart Network (PHN) echocardiographic Z score database, the authors compared the PHN model with previously published Z score models. Methods Z scores were derived for cardiovascular measurements using four models (PHN, Boston, Italy, and Detroit). Model comparisons were performed by evaluating (1) overlaid graphs of measurement versus body surface area with curves at Z = −2, 0, and +2; (2) scatterplots of PHN versus other Z scores with correlation coefficients; (3) Bland-Altman plots of PHN versus other Z scores; and (4) comparison of median Z scores for each model. Results For most measurements, PHN Z score curves were similar to Boston and Italian curves but diverged from Detroit curves at high body surface areas. Correlation coefficients were high when comparing the PHN model with the others, highest with Boston (mean, 0.99) and lowest with Detroit (mean, 0.90). Scatterplots suggested systematic differences despite high correlations. Bland-Altman plots also revealed poor agreement at both extremes of size and a systematic bias for most when comparing PHN against Italian and Detroit Z scores. There were statistically significant differences when comparing median Z scores between the PHN and other models. Conclusions Z scores from the multicenter PHN model correlated well with previous single-center models, especially the Boston model, which also had a large sample size and similar methodology. The Detroit Z scores diverged from the PHN Z scores at high body surface area, possibly because there were more subjects in this category in the PHN database. Despite excellent correlation, significant differences in Z scores between the PHN model and others were seen for many measurements. This is important when comparing publications using different models and for clinical care, particularly when Z score thresholds are used to guide diagnosis and management.

Background/Aims:Registry-based trials have emerged as a potentially cost-saving study methodology. Early estimates of cost savings, however, conflated the benefits associated with registry utilisation and those associated with other aspects of pragmatic trial designs, which might not all be as broadly applicable. In this study, we sought to build a practical tool that investigators could use across disciplines to estimate the ranges of potential cost differences associated with implementing registry-based trials versus standard clinical trials.Methods:We built simulation Markov models to compare unique costs associated with data acquisition, cleaning, and linkage under a registry-based trial design versus a standard clinical trial. We conducted one-way, two-way, and probabilistic sensitivity analyses, varying study characteristics over broad ranges, to determine thresholds at which investigators might optimally select each trial design.Results:Registry-based trials were more cost effective than standard clinical trials 98.6% of the time. Data-related cost savings ranged from $4300 to $600,000 with variation in study characteristics. Cost differences were most reactive to the number of patients in a study, the number of data elements per patient available in a registry, and the speed with which research coordinators could manually abstract data. Registry incorporation resulted in cost savings when as few as 3768 independent data elements were available and when manual data abstraction took as little as 3.4 seconds per data field.Conclusions:Registries offer important resources for investigators. When available, their broad incorporation may help the scientific community reduce the costs of clinical investigation. We offer here a practical tool for investigators to assess potential costs savings.

ObjectiveThe aim of this study was to evaluate the safety and feasibility of a passive range of motion exercise programme for infants with CHD.Study designThis non-randomised pilot study enrolled 20 neonates following Stage I palliation for single-ventricle physiology. Trained physical therapists administered standardised 15–20-minute passive range of motion protocol, for up to 21 days or until hospital discharge. Safety assessments included vital signs measured before, during, and after the exercise as well as adverse events recorded through the pre-Stage II follow-up. Feasibility was determined by the percent of days that >75% of the passive range of motion protocol was completed.ResultsA total of 20 infants were enrolled (70% males) for the present study. The median age at enrolment was 8 days (with a range from 5 to 23), with a median start of intervention at postoperative day 4 (with a range from 2 to 12). The median hospital length of stay following surgery was 15 days (with a range from 9 to 131), with an average of 13.4 (with a range from 3 to 21) in-hospital days per patient. Completion of >75% of the protocol was achieved on 88% of eligible days. Of 11 adverse events reported in six patients, 10 were expected with one determined to be possibly related to the study intervention. There were no clinically significant changes in vital signs. At pre-Stage II follow-up, weight-for-age z-score (−0.84±1.20) and length-for-age z-score (−0.83±1.31) were higher compared with historical controls from two earlier trials.ConclusionA passive range of motion exercise programme is safe and feasible in infants with single-ventricle physiology. Larger studies are needed to determine the optimal duration of passive range of motion and its effect on somatic growth.

OBJECTIVES: The Residual Lesion Score (RLS) was developed as a novel tool for assessing residual lesions after congenital heart operations based on widely available clinical and echocardiographic characteristics. The RLS ranks postoperative findings as follows: Class 1 (no/trivial residua), Class 2 (minor residua), or Class 3 (major residua or reintervention prior to discharge for residua). The multi-center prospective RLS study aims to analyze the impact of residual lesions on outcomes in common congenital cardiac operations. We hypothesize that RLS will predict postoperative adverse events, resource utilization, mortality and reinterventions by one year postoperatively. METHODS: The study cohort consists of infants ≤12 months of age undergoing definitive surgery for: (1) complete atrioventricular septal defect, (2) tetralogy of Fallot, (3) dextro-transposition of the great arteries with or without intact ventricular septum, (4) single ventricle (Norwood procedure), and (5) coarctation or interrupted/hypoplastic arch with ventricular septal defect. Subjects with major congenital or acquired extra-cardiac anomalies that could independently affect the primary endpoint, which is number of days alive and out of the hospital within 30 days of surgery (60 days for Norwood procedure), were excluded. Secondary outcomes include: ≥1 early major postoperative adverse event; days of intensive care unit and hospital stay, and initial and total ventilator time; mortality/transplant after discharge; unplanned reinterventions after discharge; and cost. All analyses will be performed separately by surgical operation. CONCLUSION: This is the first multi-center prospective validation of a tool for surgical outcome assessment and quality improvement specific to congenital heart surgery.

BACKGROUND: The reliability of LV systolic functional indices calculated from blinded echocardiographic measurements of LV size has not been tested in a large cohort of healthy children. The objective of this study was to estimate interobserver variability in standard measurements of left ventricular (LV) size and systolic function in children with normal cardiac anatomy and qualitatively normal function. METHODS: The Pediatric Heart Network Normal Echocardiogram Database collected normal echocardiograms from healthy children ≤18 years old distributed equally by age, gender, and race. A core lab used 2-dimensional echocardiograms to measure LV dimensions from which a separate data coordinating center calculated LV volumes and systolic functional indices. To evaluate interobserver variability, two independent expert pediatric echocardiographic observers re-measured LV dimensions on a subset of studies, while blinded to calculated volumes and functional indices. RESULTS: Of 3215 subjects with measurable images, 552 (17%) had a calculated LV shortening fraction (SF)

Background Heart size and function in children with single right ventricle (RV) anomalies may be influenced by shunt type at the Norwood procedure. We sought to identify shunt-related differences during early childhood after staged surgical palliations using echocardiography. Methods We compared echocardiographic indices of RV, neoaortic, and tricuspid valve size and function at 14 months, pre-Fontan, and 6 years in 241 subjects randomized to a Norwood procedure using either the modified Blalock-Taussig shunt or RV-to-pulmonary-artery shunt. Results At 6 years, the shunt groups did not differ significantly in any measure except for increased indexed neoaortic area in the modified Blalock-Taussig shunt. RV ejection fraction improved between pre-Fontan and 6 years in the RV-to-pulmonary artery shunt group but was stable in the modified Blalock-Taussig shunt group. For the entire cohort, RV diastolic and systolic size and functional indices were improved at 6 years compared with earlier measurements, and indexed tricuspid and neoaortic annular area decreased from 14 months to 6 years. The prevalence of ≥moderate tricuspid and neoaortic regurgitation was uncommon and did not vary by group or time period. Diminished RV ejection fraction at the 14-month study was predictive of late death/transplant; the hazard of late death/transplant when RV ejection fraction was Conclusions By 6 years after staged palliation, shunt type has not impacted RV size and function, and RV and valvar size and function show beneficial remodeling. Poor RV systolic function at 14 months predicts worse late survival independent of the initial shunt type. Clinical Trial Registration URL: https://www.clinicaltrials.gov . Unique identifier: NCT00115934.

The Fontan operation creates a circulation characterized by elevated central venous pressure and low cardiac output. Over time, these characteristics result in a predictable and persistent decline in exercise performance that is associated with an increase in morbidity and mortality. A medical therapy that targets the abnormalities of the Fontan circulation might, therefore, be associated with improved outcomes. Udenafil, a phosphodiesterase type 5 inhibitor, has undergone phase I/II testing in adolescents who have had the Fontan operation and has been shown to be safe and well tolerated in the short-term. However, there are no data regarding the long-term efficacy of udenafil in this population. The Fontan Udenafil Exercise Longitudinal (FUEL) Trial is a randomized, double blind, placebo controlled phase III clinical trial being conducted by the Pediatric Heart Network in collaboration with Mezzion Pharma Co., Ltd. This trial is designed to test the hypothesis that treatment with udenafil will lead to an improvement in exercise capacity in adolescents who have undergone the Fontan operation. A safety extension trial, the FUEL Open-Label Extension Trial (FUEL OLE), offers the opportunity for all FUEL subjects to obtain open-label udenafil for an additional 12 months following completion of FUEL, and evaluates the long-term safety and tolerability of this medication. This manuscript describes the rationale and study design for FUEL and FUEL OLE. Together, these trials provide an opportunity to better understand the role of medical management in the care of those who have undergone the Fontan operation.

Purpose The purpose of this study was to determine the major subcategories and clinicopathologic features of sudden unexpected death in young children in a large retrospective cohort, and to confirm the association of sudden unexplained death in children (abbreviated by us for unexplained deaths as SUDC) with hippocampal pathology and/or febrile seizures. Methods We undertook analysis of a retrospective cohort of 151 cases, of which 80 % (121/151) were subclassified as SUDC, 11 % (16/151) as explained, 7 % (10/151) as undetermined, and 3 % (4/151) as seizure-related. Results There were no significant differences between SUDC and explained cases in postnatal, gestational, or postconceptional age, frequency of preterm birth, gender, race, or organ weights. In contrast, 96.7 % (117/121) of the SUDC group were discovered during a sleep period compared to 53.3 % (8/15) of the explained group (p

Resins used in dental composites, derived from bisphenol-A (BPA), have been shown to alter immune cells. The objective of this study was to explore children's immune function changes in relation to resin composite treatment.We conducted secondary data analysis of the New England Children's Amalgam Trial immune function substudy (N = 59). Immune function was measured pre-treatment and up to five times post-treatment through 5-year follow-up. Multivariable generalized linear regression models were used to estimate the association between three classes of resin composites (bisphenol-A-diglycidyl-dimethacrylate [BisGMA]-based flowables used for preventive sealants; urethane dimethacrylate [UDMA]-based compomer restorations; bisGMA-based restorations) and changes in immune function markers measured annually.Total white blood cell counts and responsiveness of T cells or neutrophils were not appreciably altered by composite treatment levels. Changes in B cell responsiveness were greater throughout follow-up among children with more bisGMA-based composite restorations, which opposed findings for amalgam treatment levels. Monocyte responsiveness changes were decreased at 6 months with greater treatment, but not over longer follow-up.Results of this analysis showed no overt immune function alterations associated with resin composites. Additional research regarding lymphocyte activation may be warranted given the consistency of results within these analyses and with a prior study showing increased B cell activation.

BackgroundPost-traumatic stress disorder (PTSD) is associated with elevated risk for metabolic syndrome (MetS). However, the direction of this association is not yet established, as most prior studies employed cross-sectional designs. The primary goal of this study was to evaluate bidirectional associations between PTSD and MetS using a longitudinal design.MethodA total of 1355 male and female veterans of the conflicts in Iraq and Afghanistan underwent PTSD diagnostic assessments and their biometric profiles pertaining to MetS were extracted from the electronic medical record at two time points (spanning ~2.5 years, n = 971 at time 2).ResultsThe prevalence of MetS among veterans with PTSD was just under 40% at both time points and was significantly greater than that for veterans without PTSD; the prevalence of MetS among those with PTSD was also elevated relative to age-matched population estimates. Cross-lagged panel models revealed that PTSD severity predicted subsequent increases in MetS severity (β = 0.08, p = 0.002), after controlling for initial MetS severity, but MetS did not predict later PTSD symptoms. Logistic regression results suggested that for every 10 PTSD symptoms endorsed at time 1, the odds of a subsequent MetS diagnosis increased by 56%.ConclusionsResults highlight the substantial cardiometabolic concerns of young veterans with PTSD and raise the possibility that PTSD may predispose individuals to accelerated aging, in part, manifested clinically as MetS. This demonstrates the need to identify those with PTSD at greatest risk for MetS and to develop interventions that improve both conditions.

Resin-based dental restorations may intra-orally release their components and bisphenol A. Gestational bisphenol A exposure has been associated with poorer executive functioning in children.To examine whether exposure to resin-based composite restorations is associated with neuropsychological development in children.Secondary analysis of treatment level data from the New England Children's Amalgam Trial, a 2-group randomized safety trial conducted from 1997 to 2006. Children (N=534) aged 6-10 y with ≥2 posterior tooth caries were randomized to treatment with amalgam or resin-based composites (bisphenol-A-diglycidyl-dimethacrylate-composite for permanent teeth; urethane dimethacrylate-based polyacid-modified compomer for primary teeth). Neuropsychological function at 4- and 5-year follow-up (N=444) was measured by a battery of tests of executive function, intelligence, memory, visual-spatial skills, verbal fluency, and problem-solving. Multivariable generalized linear regression models were used to examine the association between composite exposure levels and changes in neuropsychological test scores from baseline to follow-up. For comparison, data on children randomized to amalgam treatment were similarly analyzed.With greater exposure to either dental composite material, results were generally consistent in the direction of slightly poorer changes in tests of intelligence, achievement or memory, but there were no statistically significant associations. For the four primary measures of executive function, scores were slightly worse with greater total composite exposure, but statistically significant only for the test of Letter Fluency (10-surface-years β=-0.8, SE=0.4, P=0.035), and the subtest of color naming (β=-1.5, SE=0.5, P=0.004) in the Stroop Color-Word Interference Test. Multivariate analysis of variance confirmed that the negative associations between composite level and executive function were not statistically significant (MANOVA, P=0.18). Results for greater amalgam exposure were mostly nonsignificant in the opposite direction of slightly improved scores over follow-up.Dental composite restorations had statistically insignificant associations of small magnitude with impairments in neuropsychological test change scores over 4- or 5-years of follow-up in this trial.

Aim To assess the feasibility of collecting electronic pain data from thalassaemia patients, based on its acceptability and convenience to the participants and study team. Methods Participants in the Thalassemia Clinical Research Network Assessment of Pain Survey Study completed the Brief Pain Inventory (BPI) quarterly by paper or phone interview. Participants in a substudy completed the BPI Short Form daily over three non-consecutive transfusion cycles through an automated telephone system. Results The consent rate for the main study was 93%, with 93% retention. The substudy had 75% retention, with more than 75% of scheduled calls completed. Regular monitoring of enrolment, missed calls, data quality, and the performance of the subcontractor for the automated system was crucial to fulfillment of the study goals. Conclusions Use of electronic data collection for patient-reported outcomes was convenient for both patients and study personnel but required human interactions beyond the automated system to maximise data quantity and quality.

Objective: To assess health-related quality of life (HRQOL) in a large multicenter cohort of children and young adults with Marfan syndrome participating in the Pediatric Heart Network Marfan Trial. Study design: The Pediatric Quality of Life Inventory (PedsQL) 4.0 Generic Core Scales were administered to 321 subjects with Marfan syndrome (5-25 years). PedsQL scores were compared with healthy population norms. The impact of treatment arm (atenolol vs losartan), severity of clinical features, and number of patient-reported symptoms on HRQOL was assessed by general linear models. Results: Mean PedsQL scores in children (5-18 years) with Marfan syndrome were lower than healthy population norms for physical (P ≤.003) and psychosocial (P

Project There is limited literature concerning the effect of urinary flow rate on mercury excretion at low-level exposure. The aim of the present study is to examine the influence of urinary flow rate on mercury excretion in children. Also of interest is the influence of flow rate on creatinine excretion and creatinine-corrected mercury, which arisearises with spot urine samples. Procedure A substudy of the New England Children's Amalgam Trial collected pairs of urine samples from children aged 10–16 years: a timed overnight collection and a spot daytime sample collected the following day. These samples were analyzed for mercury and creatinine concentration. Regression analysis was used to model the effect of urinary flow rate in the timed overnight samples. A paired t -test compared concentrations and creatinine-corrected mercury between overnight and daytime samples. Results Creatinine excretion rate (mg/h) increased significantly with urinary flow rate (mL/h), whereas creatinine concentration (g/L) decreased with flow rate. We found a non-significant increase in mercury excretion rate (ng/h) with flow rate, and mercury concentration decreased with flow rate. Mercury and creatinine concentrations were significantly higher in the overnight compared to daytime samples. For creatinine-corrected mercury, no significant impact of urinary flow rate was found. Conclusions Although the creatinine excretion rate, and probably the mercury excretion rate, increased with urinary flow rate, the mercury/creatinine ratio seemed relatively unaffected by urinary flow rate.

Recently, we reported hippocampal and temporal lobe abnormalities in 5 toddlers with sudden unexplained death in childhood (SUDC). The association of these anomalies with a high incidence (40%) of individual/family histories of simple febrile seizures in the cases raised concern that febrile seizures can be associated with death. In a series of 64 toddlers with sudden death, we tested the hypothesis that an SUDC subset is characterized by hippocampal and temporal lobe maldevelopment and an individual and/or family history of simple familial seizures. Cases of sudden and unexplained death in children aged 1.0 to 5.9 years (median 1.7 years) were divided into groups based upon a history of febrile or nonfebrile seizures, familial febrile seizures, and autopsy classification of cause of death. Forty-nine of the 64 cases (77%) were classified as SUDC, of which 40% had an individual/family history of febrile seizures. Of the 26 SUDC cases with available hippocampal sections, 62% (16/26) had hippocampal and temporal lobe anomalies, including 82% (9/11) of cases with an individual/family history of febrile seizures. Cases with these anomalies were all found dead during a sleep period, typically in the prone (87%) position. We conclude that a potential new entity may account for the majority of SUDC in toddlers, defined by sleep-related death in the prone position, individual/family history of febrile seizures, and hippocampal and temporal lobe anomalies. The mechanism of death appears analogous to sudden death in (temporal lobe) epilepsy, with a putative unwitnessed seizure during sleep leading to airway occlusion and death. This study mandates further research into the potential link between simple febrile seizures and death.

We previously identified immunocytochemical evidence of nitrative and oxidative injury in premyelinating oligodendrocytes in periventricular leukomalacia (PVL). Here, we tested the hypothesis that free radical injury occurs in the overlying cerebral cortex and subplate neurons in PVL. We immunostained for nitrotyrosine, malondialdehyde, and hydroxynonenal adducts and scored neuron staining density in PVL (n = 11) and non-PVL (n = 15) cases (postconceptional ages from 34 to 109 weeks). Analysis of covariance controlled for age. Mean malondialdehyde scores in PVL cases were increased over controls (p = 0.005). Hydroxynonenal scores increased with age only in PVL cases (diagnosis vs age interaction; p = 0.024). Nitrotyrosine scores were not significantly increased. In 11 PVL and 23 control cases between 20 and 183 postconceptional weeks, cells morphologically consistent with subplate and Cajal-Retzius neurons showed qualitatively increased free radical modification in PVL over control cases with statistically significant odds ratios for hydroxynonenal and nitrotyrosine in both subplate neurons and Cajal-Retzius cells. Glial fibrillary acidic protein and CD68 scores for reactive astrocytes and microglia, respectively, were not significantly increased, suggesting a minimal inflammatory response. Thus, oxidative/nitrative damage to cortical and "pioneer" neurons, although mild overall, may contribute to cortical volume loss and cognitive/behavioral impairment in survivors of prematurity.

Children may be at particular risk from toxic effects of mercury (Hg). Previous studies of hair (organic) and urine (inorganic) Hg levels in US children were unable to assess Hg levels while accounting for exposure to amalgam dental restorations. This analysis describes, over a 5-year period, levels and correlates/predictors of scalp hair (H-Hg) and urinary (U-Hg) mercury in 534 New England Children's Amalgam Trial (NECAT) participants, aged 6-10 years and without exposure to dental amalgam at baseline.Mean H-Hg levels were between 0.3 and 0.4 microg/g over 5 years. 17-29% of children had H-Hg levelsor = 0.5 microg/g, and 5.0 to 8.5% of children had levelsor = 1 microg/g, in any given study year. In adjusted models, fish consumption frequency was the most robust predictor of high H-Hg. U-Hg mean levels were between 0.7 and 0.9 microg/g creatinine over two years. The percentage of those with U-Hgor 2.3 microg/g creatinine ranged from 4% to 6%. Number of amalgam restorations had a significant dose-response relationship with U-Hg level. Daily gum chewing in the presence of amalgam was associated with high U-Hg.

The major neuropathological correlate of cerebral palsy in premature infants is periventricular leukomalacia (PVL), a disorder of the immature cerebral white matter. Cerebral ischemia leading to excitotoxicity is thought to be important in the pathogenesis of this disorder, implying a critical role for glutamate transporters, the major determinants of extracellular glutamate concentration. Previously, we found that EAAT2 expression is limited primarily to premyelinating oligodendrocytes early in development and is rarely observed in astrocytes until >40 weeks. In this study, we analyzed the expression of EAAT2 in cerebral white matter from PVL and control cases. Western blot analysis suggested an up-regulation of EAAT2 in PVL compared with control cases. Single- and double-label immunocytochemistry showed a significantly higher percentage of EAAT2-immunopositive astrocytes in PVL (51.8% +/- 5.6%) compared with control white matter (21.4% +/- 5.6%; P = 0.004). Macrophages in the necrotic foci in PVL also expressed EAAT2. Premyelinating oligodendrocytes in both PVL and control cases expressed EAAT2, without qualitative difference in expression. The previously unrecognized up-regulation of EAAT2 in reactive astrocytes and its presence in macrophages in PVL reported here may reflect a response to either hypoxic-ischemic injury or inflammation.

Clear adverse effects of blood lead levelsor=10 microg/dL have been documented in children. Given that the majority of US children have levels below 10 microg/dL, clarification of adverse effects below this cutoff value is needed. Our study evaluated the associations between blood lead levels10 microg/dL and a broad spectrum of children's cognitive abilities. Data were analyzed from 534 children aged 6-10, enrolled in the New England Children's Amalgam Trial (NECAT) from the urban area of Boston, Massachusetts and rural Farmington, Maine. Adjusting for covariates (age, race, socioeconomic status, and primary caregiver IQ), children with 5-10 microg/dL had 5.0 (S.D. 2.3) points lower IQ scores compared to children with blood lead levels of 1-2 microg/dL (p=0.03). Verbal IQ was more negatively affected than performance IQ, with the most prominent decrement occurring in children's vocabulary. Wechsler Individual Achievement Test scores were strongly negatively associated with blood lead levels of 5-10 microg/dL. In adjusted analyses, children with levels of 5-10 microg/dL scored 7.8 (S.D. 2.4) and 6.9 (S.D. 2.2) points lower on reading and math composite scores, respectively, compared to children with levels of 1-2 microg/dL (p0.01). Finally, levels of 5-10 microg/dL were associated with decreased attention and working memory. Other than associations of lead exposure with achievement, which even persisted after adjustment for child IQ, the most pronounced deficits were in the areas of spatial attention and executive function. Overall, our analyses support prior research that children's blood levels10 microg/dL are related to compromised cognition and highlight that these may especially be related to academic achievement.

Bisphenol A-glycidyl methacrylate (bis-GMA)-based dental composite restorations may release bisphenol A (BPA). The authors assessed changes in urinary BPA concentrations over a 6-month follow-up period in children and adolescents who received bis-GMA-based restorations.The authors collected data from 91 study participants aged 3 to 17 years who needed composite restorations. Participants provided urine samples and information on BPA-related exposures before and at approximately 1 day, 14 days, and 6 months after treatment. The authors used multivariable linear regression models to test associations between the number of surface restorations placed and the changes in urinary BPA concentrations.Participants had a mean (standard deviation [SD]) of 1.4 (1.0) for surfaces restored with composite at the first treatment visit and 2.3 (1.6) for surfaces restored during the entire study period. Mean (SD) change in urinary BPA concentrations between pretreatment and day 1 was 1.71 (9.94) nanograms per milliliter overall and 0.87 (5.98) after excluding 1 participant who had 8 surfaces restored at the visit. Overall, the authors observed an association between a greater number of composite surface restorations placed and higher urinary BPA concentrations in the 1-day sample (posterior-occlusal exponentiated coefficients [e(β)] = 1.47; 95% confidence interval [CI], 1.18-1.83; P .001), but the association was attenuated after the authors restricted the sample to the 88 participants who had up to 4 restorations (e(β) = 1.19; 95% CI, 0.86-1.64), and they did not observe any association using 14-day (e(β) = 0.94; 95% CI, 0.75-1.18) or 6-month (e(β) = 0.88; 95% CI, 0.74-1.04) samples.Placement of bis-GMA-based restorations in children and adolescents may produce transient increases in urinary BPA concentrations that are no longer detectable in urine samples taken approximately 14 days or 6 months after treatment. After placement of a few restorations, increases in urinary BPA concentrations may not be detectable, owing to a high level of variation in background BPA exposure.These results suggest that leaching of BPA from newly placed composite restorations ceases to be detectable in urine within 2 weeks after restoration placement. The potential human health impact of such short-term exposure remains uncertain.

This report is the second of a two-part evaluation of developmental differences in alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor (AMPAR) subunit expression in cell populations within white matter and cortex. In part I, we reported that, in rat, developmental expression of Ca2+-permeable (GluR2-lacking) AMPARs correlated at the regional and cellular level with increased susceptibility to hypoxia/ischemia (H/I), suggesting an age-specific role of these receptors in the pathogenesis of brain injury. Part II examines the regional and cellular progression of AMPAR subunits in human white matter and cortex from midgestation through early childhood. Similarly to the case in the rodent, there is a direct correlation between selective vulnerability to H/I and expression of GluR2-lacking AMPARs in human brain. For midgestational cases aged 20-24 postconceptional weeks (PCW) and for premature infants (25-37 PCW), we found that radial glia, premyelinating oligodendrocytes, and subplate neurons transiently expressed GluR2-lacking AMPARs. Notably, prematurity represents a developmental window of selective vulnerability for white matter injury, such as periventricular leukomalacia (PVL). During term (38-42 PCW) and postterm neonatal (43-46 PCW) periods, age windows characterized by increased susceptibility to cortical injury and seizures, GluR2 expression was low in the neocortex, specifically on cortical pyramidal and nonpyramidal neurons. This study indicates that Ca2+-permeable AMPAR blockade may represent an age-specific therapeutic strategy for potential use in humans. Furthermore, these data help to validate specific rodent maturational stages as appropriate models for evaluation of H/I pathophysiology.

Dental sealant materials may intraorally release their components, including bisphenol-A (BPA), but long-term health effects are uncertain. The New England Children's Amalgam Trial (NECAT) found that composite restorations were associated with psychosocial, but not neuropsychological or physical, outcomes. The previous analysis did not consider sealants and preventive resin restorations (PRRs), which were routinely placed. The purpose of this analysis was to examine sealant/PRR exposure in association with psychosocial and other health outcomes.NECAT recruited 534 six- to 10-year-olds and provided dental care during a five-year follow-up. Annually, examiners conducted psychosocial and neuropsychological tests and measured body mass index (BMI) and fat percentage (BF%). Associations between surface years (SY) of sealants/PRRs and outcomes were tested using multivariable models.Cumulative exposure level to sealants and/or PRRs was not associated with psychosocial assessments (eg, total problems: Child Behavior Checklist, 10-SY β=-0.2 ± 0.3, P=.60) or neuropsychological tests (eg, full-scale IQ, 10-SY β=0.1 ± 0.2, P=.60). There were no associations for changes in BMI-for-age z-score (P=.40), BF% (girls 10-SY β=-0.2 ± 0.3; boys 10-SY β=-0.1 ± 0.3), or menarche (10-SY hazard ratio=0.91, 95% confidence interval=0.83-1.01, P=.08).This study showed no associations between exposure level of dental sealants or PRRs and behavioral, neuropsychological, or physical development in children over 5-years.

Thalassemia, a chronic blood disease, necessitates life-long adherence to blood transfusions and chelation therapy to reduce iron overload. We examine stability of health-related quality of life (HRQOL) in thalassemia and adherence to chelation therapy over time, especially after changes in chelator choice.Thalassemia Longitudinal Cohort participants in the USA, UK, and Canada completed the SF-36v2 (ages 14+) and the PF-28 CHQ (parents of children14 years). Chelation adherence was defined as self-reported percent of doses administered in the last 4 weeks.Two hundred and fifty-eight adults/adolescents (mean 29.7 years) and 133 children (mean 8.5 years) completed a mean of 2.8-years follow-up. Children made few chelator changes, whereas a mean of 2.2 changes was observed among the 37% of adults/adolescents who made chelator changes, mainly due to patient preference or medical necessity. Physical HRQOL improved among those with lower iron burden (better health status) at baseline who made a single change in chelator, but declined among participants with multiple changes and/or high iron burden (worse health status). Mental health improved among participants with lower iron burden, but iron overload was negatively associated with social functioning. Adherence did not significantly change over follow-up except for an increase after a change from deferoxamine (DFO) infusion to oral deferasirox (p = 0.03). Predictors of lower adherence for adults/adolescents at follow-up included side effects, smoking, younger age, problems preparing DFO, increased number of days per week DFO prescribed, and lower physical quality of life .Strategies to balance medical needs with family, work, and personal life may assist in adherence.

Rationale, aims, and objectives : Variation in physician decisions may reflect personal styles of decision making, as opposed to singular clinical actions, and these styles may be applied differently depending on patient complexity. The objective of this study is to examine clusters of physician decision making for type 2 diabetes, overall and in the presence of a mental health comorbidity. Method: This randomized balanced factorial experiment presented video vignettes of a “patient” with diagnosed but uncontrolled type 2 diabetes. “Patients” were systematically varied by age, sex, race, and comorbidity (depression, schizophrenia with normal or bizarre affect, eczema as control). 256 primary care physicians, balanced by gender and experience level, completed a structured interview about clinical management. Results : Cluster analysis identified three styles of diabetes management. “Minimalists” (N=84) performed fewer exams or tests compared to “middle of the road” physicians (N=84). “Interventionists” (N=88) suggested more medications and referrals. A second cluster analysis, without control for comorbidities, identified an additional cluster of “information seekers” (N=15) who requested more additional information and referrals. Physicians ranking schizophrenia higher than diabetes on their problem list were more likely “minimalists” and none were “interventionists” or “information seekers”. Conclusions : Variations in clinical management encompass multiple clinical actions, and physicians subtly shift these decision making styles depending on patient comorbidities. Physicians’ practice styles may help explain persistent differences in patient care. Training and continuing education efforts to encourage physicians to implement evidence-based clinical practice should account for general styles of decision making and for how physicians process complicating comorbidities.

To examine whether medical decisions regarding evaluation and management of musculoskeletal pain conditions varied systematically by characteristics of the patient or provider.We conducted a balanced factorial experiment among primary care physicians in the US. Physicians (n = 192) viewed 2 videos of different patients (actors) presenting with pain: undiagnosed sciatica symptoms or diagnosed knee osteoarthritis. Systematic variations in patient gender, socioeconomic status, and race and physician gender and experience (20 versus ≥20 years in practice) permitted estimation of unconfounded effects. Analysis of variance was used to evaluate associations between patient or provider attributes and clinical decisions. Quality of decisions was defined based on the current recommendations of the American College of Rheumatology, American Pain Society, and clinical expert consensus.Despite current recommendations, less than one-third of physicians would provide exercise advice (30.2% for osteoarthritis and 32.8% for sciatica). Physicians with fewer years in practice were more likely to provide advice on lifestyle changes, particularly exercise (P ≤ 0.01), and to prescribe nonsteroidal antiinflammatory drugs for pain relief, both of which were appropriate and consistent with current recommendations for care. Newer physicians ordered fewer tests, particularly basic laboratory investigations or urinalysis. Test ordering decreased as organizational emphasis on business or profits increased. Patient factors and physician gender had no consistent effects on pain evaluation or treatment.Physician education on disease management recommendations regarding exercise and analgesics and implementation of quality measures may be useful, particularly for physicians with more years in practice.

In neonatal rodents, serotonin (5-HT) neurons are critical for successful autoresuscitation. We hypothesized that caffeine, a respiratory stimulant, would hasten the onset of gasping and improve autoresuscitation in 5-HT-deficient, Pet-1(-/-) mice.Using a head-out system and electrocardiogram, we measured respiratory and heart rate (HR) responses of Pet-1(-/-) rodents and their littermates during episodic asphyxia at postnatal days 8-9 (P8-9). After a baseline recording, we injected either vehicle or caffeine (i.p.) at doses of 1, 5, or 10 mg/kg. We then induced 10 brief (~30 s) episodes of asphyxia, each interspersed with 5 min of room air to allow autoresuscitation. In addition to measuring survival, we measured the duration of hypoxic apnea (time to initiate gasping) and time to recover eupnea and HR.Caffeine had a dose-dependent effect of hastening the onset of gasping, recovery of breathing, and restoration of HR in Pet-1(-/-) (but not in wild-type) rodents, thereby improving survival across asphyxic episodes. Increased survival was strongly correlated with hastened onset of gasping.Our data suggest that caffeine reduces mortality relating to asphyxia and 5-HT deficiency. These findings may be relevant for efforts to reduce the incidence of sudden infant death syndrome (SIDS), given that SIDS is associated with failed autoresuscitation and reduced brainstem 5-HT.

OBJECTIVE:To test the hypothesis that the profile of sudden infant death syndrome (SIDS) changed after the Back-to-Sleep (BTS) campaign initiation, document prevalence and patterns of multiple risks, and determine the age profile of risk factors.METHODS:The San Diego SIDS/Sudden Unexplained Death in Childhood Research Project recorded risk factors for 568 SIDS deaths from 1991 to 2008 based upon standardized death scene investigations and autopsies. Risks were divided into intrinsic (eg, male gender) and extrinsic (eg, prone sleep).RESULTS:Between 1991–1993 and 1996–2008, the percentage of SIDS infants found prone decreased from 84.0% to 48.5% (P < .001), bed-sharing increased from 19.2% to 37.9% (P < .001), especially among infants CONCLUSIONS:SIDS infants in the BTS era show more variation in risk factors. There was a consistently high prevalence of both intrinsic and especially extrinsic risks both before and during the Back-to-Sleep era. Risk reduction campaigns emphasizing the importance of avoiding multiple and simultaneous SIDS risks are essential to prevent SIDS, including among infants who may already be vulnerable.

Despite the key role of γ-aminobutyric acid (GABA) neurons in the modulation of cerebral cortical output, little is known about their development in the human cortex. We analyzed several GABAergic parameters in standardized regions of the cerebral cortex and white matter in a total of 38 human fetuses and infants from 19 gestational weeks to 2.7 postnatal years utilizing immunocytochemistry, Western blotting, tissue autoradiography and computer-based cellular quantitation. At least 20% of GABAergic neurons in the white matter migrated toward the cortex over late gestation. After term, migration declined and ended within 6 postnatal months. In parallel, the GABAergic neuronal density increased in the cortex over late gestation, also with a peak at term. From midgestation to infancy, the pattern of GABAA receptor binding changed from uniformly low across all cortical layers to high levels concentrated in the middle laminae; glutamic acid decarboxylase (GAD65 and GAD67) levels differentially increased. Thus, the second half of gestation is a period of rapid development of the cortical GABAergic system that continues into early infancy. This time period corresponds to the peak window of vulnerability to perinatal hypoxia-ischemia in which GABAergic neurons are potentially developmentally susceptible, including in the preterm infant.

Periventricular leukomalacia (PVL) in the premature infant represents the major substrate underlying cognitive deficits and cerebral palsy and is characterized as focal periventricular necrosis and diffuse gliosis in the immature cerebral white matter. We have recently shown a significant decrease in the density of neurons in PVL relative to controls throughout the white matter, including the subventricular, periventricular, and subcortical regions. These neurons are likely to be remnants of the subplate and/or GABAergic neurons in late migration to the cerebral cortex, both of which are important for proper cortical circuitry in development and throughout adulthood. Here, we tested the hypothesis that intrinsic repair occurs in PVL to attempt to compensate for the deficits in white matter neurons. By using doublecortin (DCX) immunopositivity as a marker of postmitotic migrating neurons, we found significantly increased densities (p < 0.05) of DCX-immunopositive cells in PVL cases (n = 9) compared with controls (n = 7) in the subventricular zone (their presumed site of origin), necrotic foci, and subcortical white matter in the perinatal time-window, i.e. 35-42 postconceptional weeks. These data provide the first evidence suggestive of an attempt at neuronal repair or regeneration in human neonatal white matter injury.

Objective: To provide recommendations for design and analysis of studies using urine specimens to evaluate renal function or mercury excretion in children.Methods: An analysis of mercury, albumin, γ-glutamyl transpeptidase (γ-GT) and N-acetyl-β-D-glucosaminidase (NAG) concentrations was carried out.Results: Mercury concentration and creatinine-corrected renal markers were higher in daytime compared with overnight samples. Excretion rates increased with urinary flow rate. γ-GT and NAG concentrations decreased with storage time at −20°C. Differences by age, sex and race were noted.Conclusions: We recommend use of these creatinine-corrected markers and collection of timed overnight urine samples, stored at −70°C, with control for urinary flow rate, age, sex and race in statistical models.

Objective. The objective of this study is to examine the influence of storage time at −20°C on the concentration of albumin, γ-glutamyl transpeptidase (γ-GT), N-acetyl-β-d-glucosaminidase (NAG), α1-microglobulin (A1M) and creatinine in a large sample of healthy children. Material and methods. The New England Children's Amalgam Trial followed 534 children, aged 6–10 at baseline, for 5 years, with annual urine collections. Urine samples were analysed for creatinine, albumin, γ-GT, NAG and A1M concentrations. Repeated measures analysis of covariance was used to model the effect of storage time on these concentrations. Results. The γ-GT concentration decreased significantly with storage time at −20°C. There was also a limited decrease in NAG. Albumin, A1M and creatinine concentrations did not appear to be affected by storage time at −20°C. Conclusions. If it is necessary to interpret results from samples stored for a long time at −20°C, it is advisable to account for storage time in statistical models.

The role of the cerebral cortex in the cognitive deficits in preterm survivors is poorly understood. Periventricular leukomalacia (PVL), the key feature of encephalopathy of prematurity, is characterized by periventricular necrotic foci and diffuse gliosis in the surrounding cerebral white matter. Here, we tested the hypothesis that reductions in the density of layer I neurons and/or pyramidal neurons in layers III and/or V are associated with PVL, indicating cortical pathology potentially associated with cognitive deficits in long-term survivors. In controls (23 gestational weeks to 18 postnatal months) (n = 15), a lack of significant differences in pyramidal density among incipient Brodmann areas suggested that cytoarchitectonic differences across functional areas are not fully mature in the fetal and infant periods. There was a marked reduction (38%) in the density of layer V neurons in all areas sampled in the PVL cases (n = 17) compared to controls (n = 12) adjusted for postconceptional age at or greater than 30 weeks, when the six-layer cortex is visually distinct (P < 0.024). This may reflect a dying-back loss of somata complicating transection of layer V axons projecting through the necrosis in the underlying white matter. This study underscores the potential role of secondary cortical injury in the encephalopathy of prematurity.

Sudden infant death syndrome (SIDS) is the sudden death of an infant younger than 1 year that remains unexplained after a complete autopsy and death scene investigation.1 Typically, an apparently healthy infant is found dead after a sleep period,2 with death presumably occurring during sleep or one of the many transitions between sleep and waking.3,4 The recognition that prone sleep position increases the risk for SIDS led to national campaigns,5 but despite initial success, the overall SIDS rate has plateaued over the last decade.3,4 SIDS remains the leading cause of postneonatal infant mortality in the United States, with an overall rate of 0.54 per 1000 live births.6,7 One model underlying SIDS research is the triple-risk model, which posits that SIDS results from the simultaneous occurrence in the infant of an underlying vulnerability, a critical developmental period, and an exogenous stressor.8 In 3 independent data sets assessing infants with SIDS, our laboratory has consistently reported serotonin (5-hydroxytryptamine [5-HT]) receptor binding abnormalities in regions of the medulla oblongata critical to state-dependent homeostatic regulation,9–11 ie, the medullary 5-HT system.3,12 In the third data set, we also found increased 5-HT neuronal densities as well as decreased 5-HT transporter binding relative to 5-HT neuronal number.11 Thus, we propose that SIDS results from an abnormality of the medullary 5-HT system that causes an inability to restore homeostasis following life-threatening challenges, eg, asphyxia, during a sleep period and leads to sudden death in the critical first year of life, when homeostatic systems are still maturing.12 The question remains as to whether underproduction or overproduction of 5-HT is associated with abnormal 5-HT receptor binding in SIDS. In this study we tested the main hypothesis that SIDS is associated with reductions in tissue levels of 5-HT, its key biosynthetic enzyme (tryptophan hydroxylase [TPH2]), or both, thereby representing a 5-HT deficiency disorder. The 3 other study objectives were (1) to compare infants dying from SIDS with hospitalized infants who had chronic hypoxia-ischemia prior to death to evaluate the putative effects of impaired oxygenation on 5-HT tissue markers, given that some infants with SIDS experience repetitive apnea and agonal impaired gasping prior to death4,12–15; (2) to analyze 5-HT1A receptor binding to verify that this data set displays the same alterations we observed previously9–11; and (3) to examine levels of norepinephrine and dopamine and the metabolite 3,4-dihydroxyphenylacetic acid to address whether medullary abnormalities in SIDS involve the catecholamine system.

Periventricular leukomalacia (PVL) is a lesion of the immature cerebral white matter in the perinatal period and associated predominantly with prematurity and cerebral ischemia/reperfusion as well as inflammation due to maternofetal infection. It consists of focal necrosis in the periventricular region and diffuse gliosis with microglial activation and premyelinating oligodendrocyte (pre-OL) injury in the surrounding white matter. We previously showed nitrotyrosine in pre-OLs in PVL, suggesting involvement of nitrosative stress in this disorder. Here we hypothesize that inducible nitric oxide synthase (iNOS) expression is increased in PVL relative to controls. Using immunocytochemistry in human archival tissue, the density of iNOS-expressing cells was determined in the cerebral white matter of 15 PVL cases [29-51 postconceptional (PC) weeks] and 16 control cases (20-144 PC weeks). Using a standardization score of 0-3, the density of iNOS-positive cells was significantly increased in the diffuse component of PVL (score of 1.8 +/- 0.3) cases compared to controls (score of 0.7 +/- 0.3) (P = 0.01). Intense iNOS expression occurred in reactive astrocytes in acute through chronic stages and in activated microglia primarily in the acute stage, suggesting an early role for microglial iNOS in PVL's pathogenesis. This study supports an important role for iNOS-induced nitrosative stress in the reactive/inflammatory component of PVL.

gamma-Amino butyric (GABA) critically influences serotonergic (5-HT) neurons in the raphe and extra-raphe of the medulla oblongata. In this study we hypothesize that there are marked changes in the developmental profile of markers of the human medullary GABAergic system relative to the 5-HT system in early life. We used single- and double-label immunocytochemistry and tissue receptor autoradiography in 15 human medullae from fetal and infant cases ranging from 15 gestational weeks to 10 postnatal months, and compared our findings with an extensive 5-HT-related database in our laboratory. In the raphe obscurus, we identified two subsets of GABAergic neurons using glutamic acid decarboxylase (GAD65/67) immunostaining: one comprised of small, round neurons; the other, medium, spindle-shaped neurons. In three term medullae cases, positive immunofluorescent neurons for both tryptophan hydroxylase and GAD65/67 were counted within the raphe obscurus. This revealed that approximately 6% of the total neurons counted in this nucleus expressed both GAD65/67 and TPOH suggesting co-production of GABA by a subset of 5-HT neurons. The distribution of GABA(A) binding was ubiquitous across medullary nuclei, with highest binding in the raphe obscurus. GABA(A) receptor subtypes alpha1 and alpha3 were expressed by 5-HT neurons, indicating the site of interaction of GABA with 5-HT neurons. These receptor subtypes and KCC2, a major chloride transporter, were differentially expressed across early development, from midgestation (20 weeks) and thereafter. The developmental profile of GABAergic markers changed dramatically relative to the 5-HT markers. These data provide baseline information for medullary studies of human pediatric disorders, such as sudden infant death syndrome.

The EPA reference dose for methylmercury (MeHg) was established using data from populations with greater exposures than those typical of the US. Few data are available on potential adverse health effects at lower levels. We examined relationships between hair mercury (Hg) levels and neuropsychological outcomes in a population of US children. This study included data from 355 children ages 6-10 enrolled in the New England Children's Amalgam Trial. Data on total hair Hg levels, sociodemographic information and neuropsychological function were collected. We evaluated associations between hair Hg and neuropsychological test scores with linear regression methods and used generalized additive models to determine the shape of associations that departed from linearity. Models controlled for relevant covariates, including the potential beneficial effects of consuming fish. In adjusted models, we observed no significant linear relationships between hair Hg level and any test score. Significant departures from linearity were identified for WIAT Math Reasoning and WRAMVA Visual-Motor Composite scores. The association was positive for hair Hg levels below 0.5 microg/g and negative for levels between 0.5 and 1.0 microg/g. Overall, test scores of children with hair Hg levels 1.0 microg/g appeared to be lower than those of children with levels < 1.0 microg/g, but few children had levels in this upper range and these differences did not reach statistical significance. Hair Hg levels below 1.0 microg/g in US school-age children were not adversely related to neuropsychological function.

High-dose exposures to elemental mercury vapor cause emotional dysfunction, but it is uncertain whether the levels of exposure that result from having dental amalgam restorations do so. As part of the New England Children’s Amalgam Trial, a randomized trial involving 6- to 10-year-old children, we evaluated the hypothesis that restoration of caries using dental amalgam resulted in worse psychosocial outcomes than restoration using mercury-free composite resin. The primary outcome was the parent-completed Child Behavior Checklist. The secondary outcome was children’s self-reports using the Behavior Assessment System for Children. Children’s psychosocial status was evaluated in relation to three indices of mercury exposure: treatment assignment, surface-years of amalgam, and urinary mercury excretion. All significant associations favored the amalgam group. No evidence was found that exposure to mercury from dental amalgams was associated with adverse psychosocial outcomes over the five-year period following initial placement of amalgams.

The cellular basis of myelin deficits detected by neuroimaging in long-term survivors of periventricular leukomalacia (PVL) is poorly understood. We tested the hypothesis that oligodendrocyte lineage (OL) cell density is reduced in PVL, thereby contributing to subsequent myelin deficits. Using computer-based methods, we determined OL cell density in sections from 18 PVL and 18 age-adjusted control cases, immunostained with the OL-lineage marker Olig2. Myelination was assessed with myelin basic protein (MBP) immunostaining. We found no significant difference between PVL and control cases in Olig2 cell density in the periventricular or intragyral white matter. We did find, however, a significant increase in Olig2 cell density at the necrotic foci, compared with distant areas. Although no significant difference was found in the degree of MBP immunostaining, we observed qualitative abnormalities of MBP immunostaining in both the diffuse and necrotic components of PVL. Abnormal MBP immunostaining in PVL despite preserved Olig2 cell density may be secondary to arrested OL maturation, damage to OL processes, and/or impaired axonal-OL signaling. OL migration toward the “core” of injury may occur to replenish OL cell number. This study provides new insight into the cellular basis of the myelin deficits observed in survivors of PVL.

The major brain abnormality underlying cerebral palsy in premature infants is periventricular leukomalacia (PVL), a lesion of the immature cerebral white matter. Oligodendrocyte precursors (pre-OLs; O4+O1−) predominate in human cerebral white matter during the peak time frame for PVL (24–32 gestational weeks) and are vulnerable to excitotoxicity. We hypothesize that PVL reflects, in part, excitotoxicity to pre-OLs resulting from cerebral ischemia/reperfusion. Reversal of glutamate transport in the setting of energy failure is a major source of pathologic accumulation of extracellular glutamate. Here, we identify and localize the glutamate transporters in human cerebral white matter during the age range of PVL. In situ hybridization was performed with digoxigenin-labeled probes directed against the full-length coding regions of EAAT1, EAAT2, and EAAT3. EAAT2 mRNA was abundant in human fetal white matter during the period of peak incidence of PVL and virtually disappeared by 2 postnatal months. Its developmental profile differed significantly from that of both EAAT1 and EAAT3 mRNA. Immunoblotting demonstrated that EAAT2 protein was highly expressed in early development relative to adult values. Double-label immunocytochemistry detected EAAT2 in OLs but not astrocytes or axons in the human fetal white matter. We conclude that transient expression of EAAT2 occurs during the window of peak vulnerability for PVL, suggesting that this developmentally up-regulated transporter may be a major source of extracellular glutamate in ischemic injury to the cerebral white matter of the preterm infant. J. Comp. Neurol. 501:879–890, 2007. © 2007 Wiley-Liss, Inc.

Dysfunction of medullary serotonin (5-HT)-mediated respiratory and autonomic function is postulated to underlie the pathogenesis of the majority of sudden infant death syndrome (SIDS) cases. Several studies have reported an increased frequency of the LL genotype and L allele of the 5-HT transporter (5-HTT) gene promoter polymorphism (5-HTTLPR), which is associated with increased transcriptional activity and 5-HT transport in vitro, in SIDS cases compared with controls. These findings raise the possibility that this polymorphism contributes to or exacerbates existing medullary 5-HT dysfunction in SIDS. In this study, we tested the hypothesis that the frequency of LL genotype and L allele are higher in 179 SIDS cases compared with 139 controls of multiple ethnicities in the San Diego SIDS Dataset. We observed no significant association of genotype or allele with SIDS cases either in the total cohort or on stratification for ethnicity. These observations do not support previous findings that the L allele and/or LL genotype of the 5-HTTLPR are associated with SIDS.