Transcriptional responses of the nerve agent-sensitive brain regions amygdala, hippocampus, piriform cortex, septum, and thalamus following exposure to the organophosphonate anticholinesterase sarin

Abstract

Background: Although the acute toxicity of organophosphorus nerve agents is known to result from acetylcholinesterase inhibition, the molecular mechanisms involved in the development of neuropathology following nerve agent-induced seizure are not well understood. To help determine these pathways, we previously used microarray analysis to identify gene expression changes in the rat piriform cortex, a region of the rat brain sensitive to nerve agent exposure, over a 24-h time period following sarin-induced seizure. We found significant differences in gene expression profiles and identified secondary responses that potentially lead to brain injury and cell death. To advance our understanding of the molecular mechanisms involved in sarin-induced toxicity, we analyzed gene expression changes in four other areas of the rat brain known to be affected by nerve agent-induced seizure (amygdala, hippocampus, septum, and thalamus).

Methods: We compared the transcriptional response of these four brain regions to sarin-induced seizure with the response previously characterized in the piriform cortex. In this study, rats were challenged with 1.0 × LD₅₀ sarin and subsequently treated with atropine sulfate, 2-pyridine aldoxime methylchloride, and diazepam. The four brain regions were collected at 0.25, 1, 3, 6, and 24 h after seizure onset, and total RNA was processed for microarray analysis.

Results: Principal component analysis identified brain region and time following seizure onset as major sources of variability within the dataset. Analysis of variance identified genes significantly changed following sarin-induced seizure, and gene ontology analysis identified biological pathways, functions, and networks of genes significantly affected by sarin-induced seizure over the 24-h time course. Many of the molecular functions and pathways identified as being most significant across all of the brain regions were indicative of an inflammatory response. There were also a number of molecular responses that were unique for each brain region, with the thalamus having the most distinct response to nerve agent-induced seizure.

Conclusions: Identifying the molecular mechanisms involved in sarin-induced neurotoxicity in these sensitive brain regions will facilitate the development of novel therapeutics that can potentially provide broad-spectrum protection in five areas of the central nervous system known to be damaged by nerve agent-induced seizure.

Figure 1

Principal component analysis reveals sample partitioning based on brain region and time following seizure onset. Rat amygdala, hippocampus, piriform cortex, septum, and thalamus were collected at the specified times after seizure onset and processed for oligonucleotide microarray analysis. The raw signal intensities were normalized using the RMA algorithm and visualized using PCA to identify major sources of variability in the data. Each point on the PCA represents the gene expression profile of an individual animal. Point shape corresponds to exposure condition, point color corresponds to the time after seizure onset at which the tissue was collected, and point size indicates absence or occurrence of sarin-induced seizure. Ellipsoids highlight partitioning of samples based on brain region (A) and time point following seizure onset at which the tissues were collected (C). The principal components in the three-dimensional graph represent the variability in gene expression levels seen within the dataset. Principal component 1 (PC#1, x-axis) accounts for 17.10% of the variability in the data; PC#2 (y-axis) represents 12.00% of the variability; and PC#3 (z-axis) represents 9.35% of the variability in gene expression levels seen within the dataset.

Figure 2

Canonical pathways significantly altered in all five brain regions of sarin-exposed seizing animals. A one-way ANOVA was performed to identify genes significantly changed in each brain region at each time point based on exposure (sarin vs. saline). The p-value and geometric fold change for each probeset ID were imported into IPA to identify the biological functions and canonical pathways most significantly affected by sarin-induced seizure at each time point. The top 800 genes that met the p-value cutoff (≤ 0.05) and were associated with a canonical pathway in the IPA Knowledge Base were considered for the analysis. The significance of the association between the dataset and the canonical pathway was calculated using Fisher's exact test. The -log of the p-value is graphed for each time point, with a threshold of 0.05 (or 1.3 when expressed as -log(p-value)) marked by an asterisk. The range of the y-axis was formatted the same to facilitate comparison across all the graphs in the figure.

Figure 3

Sarin-induced seizure up-regulates the expression of TNF-α in all five brain regions examined. Brain tissues were collected at 0.25, 1, 3, 6, and 24 h after seizure onset and processed for oligonucleotide microarray analysis. TNF-α expression was induced as early as 0.25 h following seizure onset, peaked at 3 h, and returned to near control levels by 24 h after seizure onset.

Figure 4

Sarin-induced seizure up-regulates the expression of IL-6 in all five brain regions examined. Tissues were collected at 0.25, 1, 3, 6, and 24 h after seizure onset and processed for oligonucleotide microarray analysis. IL-6 expression peaked at 3 h and decreased at 6 h in all brain regions. Expression then increased at 24 h after seizure onset in the amygdala, hippocampus, piriform cortex, and septum. IL-6 expression appeared to level off after 6 h in the thalamus.

Figure 5

Sarin-induced seizure up-regulates the expression of IL-1β in all five brain regions examined. Tissues were collected at 0.25, 1, 3, 6, and 24 h after seizure onset and processed for oligonucleotide microarray analysis. IL-1β expression peaked at 1 h in the amygdala, hippocampus, and thalamus, while it peaked at 3 h in the septum and at 24 h in the piriform cortex. The expression levels decreased nearer to control level in the hippocampus, septum, and thalamus, while it remained approximately the same in the amygdala. However, IL-1β transcript level was increasing in the piriform cortex at our latest time point of 24 h.

Figure 6

Canonical pathways significantly altered across all examined brain regions of sarin-exposed seizing animals. The dataset was filtered on brain region (amygdala, hippocampus, piriform cortex, septum, or thalamus), and a two-way interaction ANOVA was used to identify genes most significantly altered in each brain region based on exposure (saline or sarin) and time after seizure onset. The significant genes from each ANOVA (p-value ≤ 0.05) were compared using a Venn diagram, and the top 800 overlapping genes that mapped to canonical pathways in the IPA Knowledge Base were analyzed. The pathways that were significantly affected across all five brain regions are shown (p < 0.05, Fisher's exact test), with a threshold of 0.05 (or 1.3 when expressed as -log(p-value)) marked by an asterisk.

Figure 7

Seizure-induced alteration of inflammatory response, cellular movement, and hematological system development and function gene network. The dataset was filtered on brain region (amygdala, hippocampus, piriform cortex, septum, or thalamus), and a two-way interaction ANOVA was used to identify the genes most significantly altered in each region based on exposure (saline or sarin) and time after seizure onset. The significant genes from each ANOVA (p-value ≤ 0.05) were compared using a Venn diagram, and the top 800 overlapping genes that mapped to canonical pathways in the IPA Knowledge Base were overlaid onto a global molecular network developed from information within the IPA Knowledge Base. The networks were then algorithmically generated based on their connectivity. Genes are represented as nodes of various shapes to represent the functional class of the gene product, and the biological relationship between two nodes is represented as a line. The intensity of the node color indicates the degree of differential expression.

Figure 8

Seizure-induced alteration of cell-to-cell signalling/interaction, hematological system development/function, and immune cell trafficking gene network.

Figure 9

Seizure-induced alteration of cell morphology, cell death, and organismal survival gene network.

Figure 10

Seizure-induced alteration of cellular movement, inflammatory response, and cell-to-cell signalling/interaction gene network.

Figure 11

Seizure-induced alteration of lipid metabolism, small molecule biochemistry, and vitamin/mineral metabolism gene network.

Figure 12

Seizure-induced alteration of behavior, nervous system development/function, and cellular growth/proliferation gene network.