The human brain is rapidly growing during the last trimester and there is evidence that premature birth is related to adverse neurological events. Magnetic resonance (MR) imaging of premature neonates and subsequent automated segmentation allows us to quantify subtle disruptions in cortical growth and folding in large population based studies. We use an age-specific automated tissue segmentation technique combined with an age-specific parcellation scheme to study regional patterns of tissue growth in a large study of brain development in premature neonates. We aimed to investigate the additional information provided by regional analysis of MR scans when determining risks for delayed neurocognitive outcomes. Our image analysis framework incorporates a spatiotemporal reference atlas that covers the age range of 27.43 to 46.43 gestational weeks (GW), and consisted of manually segmented brain tissues and 8 hemispheric lobe regions. The tissue atlas was used to automatically segment all 269 MR scans, age range from 27.29 to 47.00 GW, using a hybrid framework that combines an age-specific atlas-based EM segmentation with a patch-based prior. This tissue segmentation was then used to re-align the subject anatomy with an age-specific tissue template and the region parcellation of each subject was carried out by template probability propagation into the subject anatomy from the spatiotemporal parcellation model. Multiple linear regression models of the parcellation volumes, divided into gray matter and white matter tissues, showed a significant correlation between regional volume and Bayley’s Scale of Infant and Toddler Development scores, beyond gestational age at birth. Parietal and occipital lobes were clear markers for language and motor abilities, while the temporal lobe volumes were correlated with cognitive abilities. These findings support a more anatomically-specific analysis of brain structure after premature birth and may provide the possibility of earlier and more specific clinical diagnostics and timely developmental interventions.

Neurological outcomes for premature infants with cerebellar hemorrhages are poor, but the mechanisms of injury are not understood. We hypothesize that the addition of blood to cerebellar slices would decrease external granule layer (EGL) proliferation and impair migration of cells leading to developmental disruption. Our objective is to develop a slice culture model for understanding the effects of cerebellar hemorrhage on cerebellum development. Postnatal day six mice were decapitated and trunk blood obtained using a heparinized syringe. Whole brains were removed and 200 µm thick sagittal cerebellar slices were obtained using a Vibratome. Slices were placed on a cell culture insert with serum containing media. Trunk blood (4µL) was applied directly to treatment slices. Slices are incubated for 48 hours. Media samples were collected for lactate dehydrogenase (LDH) assays. BrdU was added 24 or 2 hours before fixation. Slices were fixed in 4% paraformaldehyde overnight. Immunohistochemistry for calbindin, BrdU, GFAP, PCNA was performed. Slices were processed with LacZ and Nissl staining. Images were taken on a confocal microscope and analyzed for EGL cell counts and thickness. LDH, a marker of cell death, increased in the media of blood-exposed slices than control slices for 4, 24, and 48 hours. Blood-exposed slices have thinner EGL (control 141 versus 120 µm blood-exposed). Bergmann glia were more disorganized after blood exposure and lacked the radial appearance of control slices. Purkinje cell arborization was decreased after blood exposure. Cell proliferation was lower in blood-exposed slices than in control slices (control 77 versus 26 blood-exposed for PCNA; control 80 versus 55 blood-exposed for BrdU). In conclusion, the addition of blood to neonatal mouse cerebellum slice cultures decreased proliferation of cells in the EGL after 48 hours. Additional experiments are underway to better characterize the mechanism of action.

While better social support can lead to positive outcomes for caregivers of youth with chronic disease, less is known about the specific types of social support used, and what may drive caregiver preferences for these types of support. Our pilot study describes the type of social support used in 46 caregivers of youth with type 1 diabetes who were recently diagnosed (diagnosed longer than 6 months but less than 24 months) at Seattle Children’s Hospital. Interested caregivers signed informed consent and completed a social support survey. Categories of support included: in-person support group options, internet-based support, or a friend/family member with diabetes. In addition, they were asked to rank their top 3 sources of support. Of the 46 caregivers enrolled, 9 (20%) reported using in-person support groups. Caregivers who used in-person groups reported an average satisfaction score of 3.6/4.0. Of those who did not use in-person groups, the most frequently cited reasons included: I don’t know of any available groups (41%), too far (35%) and I have too much else going on (22%). Use of the internet-based support groups such as Facebook was reported by 23 (53%). The average satisfaction score for these was 3.5. Use of friends/family with diabetes for support was used by 19 (41%) with a satisfaction score of 3.7. In summary, internet-based support was the most widely used amongst the types of social support while the use of friends/family ranked the highest amongst the types of social support. All types of support seemed to be satisfactory. When asked to rank their types of support used, the top-cited sources of support were: local and national diabetes organization support (54.2%), family (37.0%) and Seattle Children’s (21.7%). Efforts should be made to better publicize sources of support as many reported not knowing about their availability.

Hemophilia, a common genetically inherited blood disorder, is currently treated by infusion of clotting factors. However, protein infusion poses disadvantages due to high cost, pain on patient, and sometimes immune responses against infused proteins. In recent years, gene delivery using viral vectors has been developed as a promising alternative strategy for treating inherited diseases. Among the viral vectors, adeno-associated virus (AAV) is favorably studied due to its non-pathogenicity and persistence; however, the uptake of AAV by host cells can be inhibited by neutralizing antibodies (NAb) against vectors, and heparin, a molecule that competes with AAV for endocytosis. This project explores if sonoporation can help overcome these barriers and enhance AAV-mediated gene delivery. Therapeutic ultrasound and microbubbles can induce the formation of transient pores in the cell membrane to enhance the cellular uptake of gene carrier vectors. Our in vitro transfection experiments in 293T cells revealed that using ultrasound and microbubbles has potential to enhance the transduction efficiency of AAV by overcoming inhibition by NAb and heparin. Additional in vivo experiments will be performed to study the relationship between endocytosis and ultrasound-microbubble induced sonoporation. Ultimately, we hope to apply our understanding of this relationship to develop an ultrasound-facilitated method to achieve non-invasive, localized, and efficient AAV-mediated gene delivery for treating inherited diseases.

Leigh Syndrome (LS) is a common pediatric mitochondrial disease comprised of many biochemically distinct but symptomatically similar disorders, and affects approximately 1 in 40,000 children. We are currently studying what is considered classical Leigh Syndrome by using a mouse model with a homozygous loss of the Ndufs4 gene, which encodes an essential subunit of Complex I in the mitochondrial electron transport chain, and thus results in energetic defects. Animals lacking Ndufs4 (Ndufs4KO mice) display progressive neurodegeneration, ataxia, and respiratory failure that leads to death. Selective inactivation of Ndufs4 in neurons and glia leads to manifestation of a disease phenotype indistinguishable from Ndufs4KO mice. Accordingly, lesions in the brainstem and cerebellum are common in human LS patients. However, the neuronal cell types susceptible to the disease are unknown. Thus, to determine the role of certain neuronal types in disease progression, we used the Cre-Lox system to inactivate Ndufs4 in glutamatergic, cholinergic, and GABAergic neurons. Glutamate, acetylcholine, and GABA are three primary neurotransmitters which define specific subsets of neurons widely present in the brain. So far, it appears that inactivation of Ndufs4 in cholinergic and GABAergic neurons does not yield a disease phenotype comparable to that of a full-body Ndufs4 knockout, as they are indistinguishable from wild-type mice. Inactivation of Ndufs4 in glutamatergic neurons, however, appears to yield a mouse with a significantly shortened lifespan that is statistically indistinguishable from the full-body Ndufs4KO mouse.  

The brainstem's important role in the motor and sensory systems makes it a vital structure for studying pediatric development. However, the developing brain's rapid growth presents significant challenges when studying preterm infants. We investigated the regional differences of the brainstem between healthy and developmental delayed preterm infants. A spatiotemporal atlas was constructed from 28 training subject magnetic resonance (MR) scans and their brainstem manual tissue segmentations (whole brainstem) and parcellations (midbrain, pons, medulla) to capture a wide range of growth over time. The brainstem in all 269 MR scans in our database was automatically segmented using an age-specific template derived from the tissue-atlas training data to initiate an atlas-based Expectation-Maximization framework. We used tissue segmentations to re-register the age-specific tissue maps and derive an accurate non-rigid transformation from the atlas into the space of the subject scan. Template propagation was used to place an age-specific brainstem parcellation from the atlas into the space of the subject MRI. Multiple linear regression analysis was performed to model the relationship between regional brainstem volume calculated from the automated parcellations and specific Bayley Scales of Infant and Toddler development-Third Edition (BSID-III) cognitive, language, and motor composite scores at 18 months of age. We covaried with post-menstrual age at scan, age at birth, gender, and with and without supratentorial volume. Significant association existed for all brainstem volumes except for the pons and all BSID-III scores near birth age (mean=32.7+/-3.2 weeks) while there was nearly no significance for the same models in children near full-term age (mean=40.6+/-2.7 weeks). This supports the hypothesis that smaller total brainstem, midbrain, and medulla volumes measured shortly after preterm birth may be useful as a biomarker to predict poor developmental outcome at 18 months of age, while measurements later (near full term age) may be less useful as brain volume recovers.

Cleft lip with or without cleft palate is a common birth defect with wide etiological and phenotypic variability, stemming from diverse genetic and environmental factors. Many classification systems have been developed to aid in documentation, diagnosis, and management of clefting. Recent efforts under the Ontology of Craniofacial Development and Malformation (OCDM) project, though the National Institute of Dental and Craniofacial Research’s FaceBase Program, have been aimed toward data pooling and integrating these different fields into a unified anatomical ontology to facilitate diverse research into craniofacial disorders. However, researchers, geneticists, clinicians, surgeons, and other specialists often interpret cleft features differently and utilize classification systems best geared toward their areas of focus. In order to facilitate integration of clinical and research studies that use different classification systems, it is first necessary to identify and consolidate inconsistencies among systems. We have comprehensively reviewed existing classification systems and tested their abilities to subclassify clefts of the lip, and have found several discrepancies and contradictions between and among systems. Variations in terminology, anatomical precision, and depth of detail among every system may result in an inability to accurately compare studies using different systems. Although the scope of an individual classification system can be sufficient for a specific goal or research outcome, it is becoming more appreciated that in order to continue advancements in preventing and treating clefts, it is necessary to encourage cross-institutional collaboration. Ultimately, our work is a key first step in creating an integrated, standardized framework to better understand orofacial clefting.

A major public health aim is to effectively intervene with college students who are irresponsibly using alcohol. Defensiveness is a response that is often associated with this kind of behavior change intervention, and evidence suggests that mitigating a student’s defensiveness during this process can help increase the effectiveness of the alcohol intervention. This is a notion that is of great interest to university administrators, however it remains unclear how to best approach such students in order to minimize their defensiveness. In addition, it is also unknown with what accuracy students perceive the intentions of the alcohol intervention process, which is another factor that may contribute to their defensiveness towards an intervention. The purpose of this study was to investigate two factors that may contribute to a student's defensiveness towards the intervention process: the way in which they are approached when they violate alcohol policy on campus, and how accurately they understand alcohol policy and the consequences associated with violating it. Twenty five University of Washington students were interviewed to collect this qualitative data. Participants’ quantity and frequency of alcohol use over 28 days was also collected in order to observe if levels of alcohol consumption was related to different opinions on the topics of interest. Content analysis was used for identifying themes present in participants' interview responses, while participants' alcohol use data was analyzed with descriptive statistics. 

Childhood epileptic encephalopathies (CEE) are a subset of rare epilepsies, largely characterized by seizures and mental disabilities. Recent studies, including those by the Mefford lab, have shown that about 30% of CEE is caused by mutations in specific genes.These mutations are ‘de novo’ – they arise during fertilization or the development of the embryo. While many types of epilepsy have a hereditary basis, de novo mutations are increasingly recognized as significant risk factors in some epilepsies. The mutations also change the amino acids that make up proteins and are mostly in exons – parts of the DNA that code for protein. Few studies focus on synonymous mutations that do not change the amino acids of a protein or mutations in introns –pieces of DNA that have regulatory function but do not code for protein. We have previously sequenced 29 known epilepsy genes in a cohort of 800 CEE patients, but focused primarily on exonic mutations that change the protein sequence. My goal is to understand whether intronic or synonymous mutations that we have already found in these patients are inherited or de novo. We hypothesize that these classes of de novo mutations disrupt the typical formation of RNA transcripts, which in turn results in an absent or mutant protein. I will use molecular inversion probe (MIP) capture to determine the inheritance of intronic variants in patients with CEE. A MIP is a piece of DNA that is designed to target specific regions of DNA which are then amplified and analyzed; the technique enables us to capture a large number of variants at once. Discovering de novo variants in intronic regions of genes will deepen our understanding of the cause of CEE. These variants will inform us of affected biological pathways and contribute information for future CEE treatments.

Sepsis is a medical condition characterized by potentially fatal generalized inflammation, typically resulting from infection. Despite previous attempts to identify mechanistic therapies, no targeted intervention has impacted survival rates. Prior hypotheses focused on immunologic mechanisms, but this project investigates metabolic responses in sepsis using a mouse model. The current target of investigation is a nuclear hormone transcription factor called PPARalpha. PPARalpha regulates the expression of proteins and enzymes associated with inflammation, stress, lipid metabolism and is related to both human and mouse survival in sepsis. We hypothesize that PPARalpha is necessary to maintain energy production sufficient to support organ function in sepsis and that deficient PPARalpha activity results in organ system failure. Because cardiac tissues preferentially use fatty acids as an energy source, we hypothesize that it is the organ that will be most affected in sepsis. Sepsis was surgically induced in mice, tissues were harvested at specific time points and gene expression was quantified using real time qPCR. Seven genes involved in fatty acid metabolism were analyzed at time points 0, 24, 48 and 72 hours. PPARalpha expression was higher in wild-type tissues, but it was down-regulated in both wild type and knockout mice. In addition, wild type mice showed greater expression of fatty acid oxidation genes than PPARalpha-knockout mice at every time point though this group of genes was down-regulated in both cohorts. Knock out mice also showed increased cardiac PPARgamma expression at 24 and 48 hours, which is a marker of reduced survival in other models. Our findings indicate that wild type mice are better able to maintain energy production necessary to preserve tissue function. These results support the hypothesis that the major cause of death in septic mice is cardiac failure due to impaired PPARalpha expression and therefore inability to sufficiently metabolize fatty acids.

Social media in this day and age has become a network of information both relied upon and consumed by the general public. Of these, studies have shown that Youtube.com, a video sharing site, has more than a billion unique visits each month, and that this network is proliferated by decades worth of videos watched and shared every day. Thus, Youtube.com becomes a valuable platform for the inquiry of contemporary public issues such as the legalization of marijuana. The controversial social, economic, political and health consequences of the legalization of marijuana has recently moved into the spotlight due to Washington and Colorado State’s new marijuana legalization laws. By analyzing content of public Youtube videos and their user comments on the topic of “legalization of marijuana” – categorized by key subjects (i.e., political, health, crime, etc.) and attitude towards legalization – information and opinion both relayed and consumed are shown. A maximum number of comments are chosen in order of date posted, and videos are selected in terms of relevance pertaining to the search results of key word: “legalization of marijuana.” Content analysis included are subject and attitude towards marijuana displayed in comments, likes on user generated comments, and video details such as views, orientation towards legalization of marijuana, published date, and length. I anticipate my results will include a comprehensive evaluation of approximately 900 comments generated on at least forty different videos relating to legalization of marijuana to illustrate contemporary issues, concerns, beliefs and attitudes towards marijuana and legalization. This data could prove useful in determining public attitude, knowledge and responses towards critical issues.

“Fetal alcohol spectrum disorders” (FASD) are considered to be the leading preventable cause of mental retardation. Among this spectrum Fetal Alcohol Syndrome (FAS) represents the most extreme consequence of maternal alcohol consumption and as such associated facial dysmorphologies and neurological defects have been heavily investigated. In this project, our lab is interested in assessing the extent craniofacial development is affected due to chronic paternal and pre-conception maternal alcohol consumption using a mouse model. For the paternal exposure project, male mice were first bred with normal females as control and then given a 10% ethanol water solution for six weeks, which covers a full cycle of spermatogenesis in mice, and mated with normal females again as experimental compare. For the pre-conception exposure project, female mice are divided into three groups given different solutions respectively, four days a week for ten weeks: 10% ethanol water exposure, calorie-matched sucrose solution, and water. Upon completion of exposure, they were bred with normal males. When litters are at 60 days of age, the craniofacial phenotype of the specimens will be assessed using high-resolution microCT and their shape differences will be statistically assessed using a set of landmark and geometric morphometric methods (GMM). By the time of the Symposium, we expect to complete the image acquisition of the control group, as well as some of the initial GMM analysis. We will also be able to report differences in litter size, birth weight, and growth rate of litters between groups in both projects. As a whole, we expect to see subtle but significant differences in craniofacial development between mice that are born to fathers or mothers consuming alcohol and their controls. From there, the leading mechanism and degrees of alteration of craniofacial development can be related to the effects of chronic alcohol exposure in humans.