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Sample Matching by Inferred Agonal Stress in Gene Expression Analyses of the Brain

ABSTRACT: Gene expression patterns in the brain are strongly influenced by the severity of physiological stress at death. This agonal effect, if not well controlled, can lead to spurious findings in case-control comparisons. While many recent studies match samples by tissue pH and clinically recorded agonal conditions, we found that these commonly used indicators were sometimes at odds with observed stress-related patterns of gene expression, and that matching by these criteria still sometimes results in identifying differences between cases and controls that are primarily driven by residual agonal effects. This problem is analogous to the one in genetic studies, where race and ethnicity are often imprecise proxies for complex environmental and genetic factors. We developed an Agonal Stress Rating (ASR) system that evaluates each sample’s degree of stress based on gene expression data, and used ASRs in post hoc sample matching or covariate analysis. While we found that gene expression patterns are generally correlated across different regions of the same brain, we also found strong region-region differences in empirical ASRs in many subjects that are likely due to inter-individual variabilities in local structure or function, resulting in region-specific vulnerability to agonal stress. Variation of agonal stress from one region of the brain to another differs between individuals, revealing a new level of complexity for gene expression studies of brain tissues. The Agonal Stress Ratings provide a direct assessment of the regulatory responses to agonal stress in individual samples, and allow a strong control of this important confounder. Our strategy is analogous to sample matching by inferred ancestral proportions in genetic association studies to control subtle confounding by ancestry. Keywords: Agonal Stress Rating comparison

ORGANISM(S): Homo sapiens

PROVIDER: GSE6306 | GEO | 2006/11/21

SECONDARY ACCESSION(S): PRJNA100509

REPOSITORIES: GEO

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Project description:Gene expression patterns in the brain are strongly influenced by the severity of physiological stress at death. This agonal effect, if not well controlled, can lead to spurious findings in case-control comparisons. While many recent studies match samples by tissue pH and clinically recorded agonal conditions, we found that these commonly used indicators were sometimes at odds with observed stress-related patterns of gene expression, and that matching by these criteria still sometimes results in identifying differences between cases and controls that are primarily driven by residual agonal effects. This problem is analogous to the one in genetic studies, where race and ethnicity are often imprecise proxies for complex environmental and genetic factors. We developed an Agonal Stress Rating (ASR) system that evaluates each sampleâs degree of stress based on gene expression data, and used ASRs in post hoc sample matching or covariate analysis. While we found that gene expression patterns are generally correlated across different regions of the same brain, we also found strong region-region differences in empirical ASRs in many subjects that are likely due to inter-individual variabilities in local structure or function, resulting in region-specific vulnerability to agonal stress. Variation of agonal stress from one region of the brain to another differs between individuals, revealing a new level of complexity for gene expression studies of brain tissues. The Agonal Stress Ratings provide a direct assessment of the regulatory responses to agonal stress in individual samples, and allow a strong control of this important confounder. Our strategy is analogous to sample matching by inferred ancestral proportions in genetic association studies to control subtle confounding by ancestry. Keywords: Agonal Stress Rating comparison We examined the relationship between the Agonal Stress Ratings (ASRs) and conventional pre hoc indicators such as pH and clinically derived Agonal Factor Scores (AFS), compared the stress ratings across six brain regions in up t0 126 samples, and assessed the performance of different sample matching strategies.

Project description:Prolonged stress has adverse consequences for neurons in the hippocampus (HIPP). The amygdala (AMY) is a brain region that is similar to HIPP across many measures, suggesting that chronic stress might modulate AMY and HIPP function in similar ways. However, studies addressing this issue have produced surprising results. For example, a regimen of chronic stress shown to produce atrophy in HIPP neurons caused dendritic branching in AMY neurons. These and other data have revealed that excessive stress induces fundamentally opposing processes in the AMY and HIPP, such that AMY function is facilitated by levels of stress that produce deleterious effects in HIPP. The mechanisms that contribute to the opposing responses of these brain regions to chronic stress are unknown. Such knowledge may suggest novel directions for pharmacological interventions to protect the HIPP from stress-mediated damage.,We will determine gene expression patterns in the hippocampus and amygdala under basal and stressful conditions, with a particular interest in those genes that are differentially regulated across the two regions. ,We hypothesize that genes that are oppositely regulated in the amygdala and hippocampus after stress mediate the opposing functional consequences of chronic stress in these two brain regions. Moreover, genes which are differentially expressed in these regions under basal conditions may underlie the distinct responses of these regions to stress.,Rats will receive either 30sec of handling (Control group; n=15) or 3hr of immobilization stress (Stress group; n=15) each day for 14 consecutive days. This stress paradigm has previously been shown to produce opposing effects on dendritic morphology and region-dependent behaviors for the hippocampus versus amygdala. Twenty-four hours after the last stress or handling session, the rats will be overanaesthetized, and the brain will be removed and placed in ice-cold buffer. Tissue will be rapidly dissected from the basolateral complex of the amygdala and the CA3 region of the hippocampus of both hemispheres, flash frozen in liquid nitrogen, and stored at -80C until further processing. The tissue from each brain region will be pooled across groups, yielding four samples (Control-Hippocampus, Control-Amygdala, Stress-Hippocampus, and Stress-Amygdala). Total RNA will be extracted using standard methods, and sent to the centers. Each sample will be run in triplicate, utilizing a total of 12 Affymetrix Rat Genome U34A gene chips. A triplicate array assay of pooled samples has previously been shown to both substantially increase the sensitivity of detecting genes of interest and reduce the variability associated with individual microarrays. In addition, the large number of rats used in each group will reduce the variability in gene expression associated with individual responses to chronic stress, as well as variability due to slight anatomical differences in the tissue extracted from each rat.

Project description:Prolonged stress has adverse consequences for neurons in the hippocampus (HIPP). The amygdala (AMY) is a brain region that is similar to HIPP across many measures, suggesting that chronic stress might modulate AMY and HIPP function in similar ways. However, studies addressing this issue have produced surprising results. For example, a regimen of chronic stress shown to produce atrophy in HIPP neurons caused dendritic branching in AMY neurons. These and other data have revealed that excessive stress induces fundamentally opposing processes in the AMY and HIPP, such that AMY function is facilitated by levels of stress that produce deleterious effects in HIPP. The mechanisms that contribute to the opposing responses of these brain regions to chronic stress are unknown. Such knowledge may suggest novel directions for pharmacological interventions to protect the HIPP from stress-mediated damage. We will determine gene expression patterns in the hippocampus and amygdala under basal and stressful conditions, with a particular interest in those genes that are differentially regulated across the two regions. We hypothesize that genes that are oppositely regulated in the amygdala and hippocampus after stress mediate the opposing functional consequences of chronic stress in these two brain regions. Moreover, genes which are differentially expressed in these regions under basal conditions may underlie the distinct responses of these regions to stress. Rats will receive either 30sec of handling (Control group; n=15) or 3hr of immobilization stress (Stress group; n=15) each day for 14 consecutive days. This stress paradigm has previously been shown to produce opposing effects on dendritic morphology and region-dependent behaviors for the hippocampus versus amygdala. Twenty-four hours after the last stress or handling session, the rats will be overanaesthetized, and the brain will be removed and placed in ice-cold buffer. Tissue will be rapidly dissected from the basolateral complex of the amygdala and the CA3 region of the hippocampus of both hemispheres, flash frozen in liquid nitrogen, and stored at -80C until further processing. The tissue from each brain region will be pooled across groups, yielding four samples (Control-Hippocampus, Control-Amygdala, Stress-Hippocampus, and Stress-Amygdala). Total RNA will be extracted using standard methods, and sent to the centers. Each sample will be run in triplicate, utilizing a total of 12 Affymetrix Rat Genome U34A gene chips. A triplicate array assay of pooled samples has previously been shown to both substantially increase the sensitivity of detecting genes of interest and reduce the variability associated with individual microarrays. In addition, the large number of rats used in each group will reduce the variability in gene expression associated with individual responses to chronic stress, as well as variability due to slight anatomical differences in the tissue extracted from each rat. Keywords: dose response

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Sample Matching by Inferred Agonal Stress in Gene Expression Analyses of the Brain

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