Exposure of young children to general anesthesia (GA) is common in medicine;however, emerging data suggest that early postnatal exposure to GA may be detrimental to brain development, resulting in long- term cognitive impairments. Older literature also suggested that in utero exposure of rodents to GA causes cognitive impairments in the first- as well as the second-generation offspring never exposed to GA. Thus, we must consider the general hypothesis that transient exposure to GA during critical stages of brain development causes epigenetic changes in chromatin with deleterious effects on transcription of target genes crucial for proper synapse formation and cognitive development. My long-term goals are to understand how GA modulates transcriptional machinery of developing neurons and to establish any links between GA-induced epigenetic modulations and long-term memory deficit. The rationale is that this understanding will enable pharmacological targeting of critical steps in gene transcription aimed to prevent GA-induced impairment of synaptogenesis and memory/learning deficits. We will use in vivo and in vitro rat models of GA-induced developmental neurotoxicity to address the specific hypothesis that GA decreases histone acetylase (HAT) activity of cAMP-responsive element binding protein (CREB) Binding Protein (CBP) and thereby reduces transcription of target genes that regulate developmental apoptosis, neuronal morphology, function and long-term memory.
Specific Aim #1 : Determine how GA alters histone acetylation and thereby the activity of a set of transcription factors (CREB, c-FOS) that target genes contributing to developmental neuroapoptosis (e.g. caspase-3,-8 and -9, bcl-XL, BAD) and long-term memory storage and consolidation (e.g. BDNF, NR4A, Cdk-5). Preliminary data suggest that GA significantly decreases CBP content and histone 3 (H3) acetylation, an epigenetic modulation that inhibits transcriptional activation. We will examine GA effects on total HAT and on histone deacetylase (HDAC) activities via ELISA and on specific HAT activity of CBP after immunoprecipitation;and follow acetylation at specific histone sites with antibodies to H3K14, H2BK12 and H4K8. Expression and phosphorylation of cFos and CREB will be assessed, and activation of target genes will be assayed via DNA methylation, targeted assessment of histone acetylation in their promoters, and mRNA production. The dependence of GA-induced changes in histone acetylation will be assessed by determining whether isoform-specific HDAC inhibitors can reverse them.
Specific Aim #2 : Use specific HDAC inhibitors to determine whether GA-induced epigenetic changes in histone acetylation and transcriptional regulation of CREB, c-FOS, and target genes are linked to GA-induced impairment of neuronal survival and morphology, dendritic arborization, synapse formation, synaptic plasticity and memory development. We will attempt to mimic the GA effect in neuronal cultures and slices by using CBP siRNA to impair its HAT and transcriptional activity.

Public Health Relevance

Early exposure to clinical anesthesia can be detrimental to the developing brain resulting in long-term cognitive impairments. Guided by our preliminary findings we propose that transient exposure to anesthesia during critical stages of brain development causes epigenetic changes in neuronal chromatin with deleterious effects on the transcription of target genes crucial for control of developmental apoptosis, proper synapse formation and cognitive development. Hence, we plan to examine the mechanistic aspects and functional consequences of anesthesia-induced epigenetic modulations with ultimate goal being timely prevention of anesthesia-induced impairments of target gene transcription.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Research Scientist Development Award - Research (K02)
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Pediatrics Subcommittee (CHHD)
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Zajicek, Anne
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University of Virginia
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