RNA Sequencing of Trigeminal Ganglia in <i>Rattus Norvegicus</i> after Glyceryl Trinitrate Infusion with Relevance to Migraine

<div><p>Introduction</p><p>Infusion of glyceryl trinitrate (GTN), a donor of nitric oxide, induces immediate headache in humans that in migraineurs is followed by a delayed migraine attack. In order to achieve increased knowledge of mechanisms activated during GTN-infusion this present study aims to investigate transcriptional responses to GTN-infusion in the rat trigeminal ganglia.</p><p>Methods</p><p>Rats were infused with GTN or vehicle and trigeminal ganglia were isolated either 30 or 90 minutes post infusion. RNA sequencing was used to investigate transcriptomic changes in response to the treatment. Furthermore, we developed a novel method for Gene Set Analysis Of Variance (GSANOVA) to identify gene sets associated with transcriptional changes across time.</p><p>Results</p><p>15 genes displayed significant changes in transcription levels in response to GTN-infusion. Ten of these genes showed either sustained up- or down-regulation in the 90-minute period after infusion. The GSANOVA analysis demonstrate enrichment of pathways pointing towards an increase in immune response, signal transduction, and neuroplasticity in response to GTN-infusion. Future functional in-depth studies of these mechanisms are expected to increase our understanding of migraine pathogenesis.</p></div

Trigeminal ganglia gene expression dynamics in response to GTN treatment.

<p>Differential gene expression analysis was conducted in DESeq2 using likelihood-ratio tests with correction for multiple testing using the False Discovery Rate method. 15 genes exhibited statistically significant changes in gene expression at a false discovery rate cutoff of 5%. In the central plot, dots represent estimated log2(fold-changes) with the X- and Y-dimensions representing the responses at 30 and 90 minutes, respectively. The horizontal and vertical lines show the standard error of the log2(fold changes) after 30 and 90 minutes, respectively. The four lateral line-plots provide an alternative visualization of the gene expression trajectories for the four most significantly regulated genes (RT1-A3, Rps10, RT1-A2 and Rgs7bp).</p

qPCR validation of four differentially expressed genes.

<p>Reverse Transcriptase Semi-quantitative PCR (qPCR) analysis on the set of samples used for RNA-seq analysis for four selected differentially expressed genes (A) <i>RT1-a3</i> (B) <i>Per1</i>, (C) <i>Tapbp</i> and, (D) <i>Rgs7bp</i>. Upper panels in each subfigure shows the mean and standard error of the relative copy numbers measured by qPCR normalized to <i>Hprt1</i> (vehicle n = 2, GTN-30 n = 4, and GTN-90 n = 3). Lower panels show the corresponding log-fold changes estimated using RNA-seq as also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155039#pone.0155039.g001" target="_blank">Fig 1</a>. Plots with normalized RNA-seq counts for all significant genes are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155039#pone.0155039.s003" target="_blank">S2 Fig</a>.</p

GSANOVA analysis.

<p>Enriched gene-sets in the trigeminal ganglia at two time points (30 and 90 minutes) after GTN infusion as determined by GSANOVA analysis. The solid black line shows the q-value for each gene-set (upper axis); gene-sets with q<0.25 (dotted line) were considered significantly enriched. The horizontal bars show the size of each gene-set (lower axis) with the upper and lower bars for each gene-set representing the 30 and 90 minutes time points, respectively. Within each bar, the gene expression change of every gene in the gene-set is shown as a colored vertical line with genes sorted in increasing order of change (blue and red colors represent down- and up-regulation, respectively). Gene expression values were further square root transformed to render the data more uniformly distributed on the color-scale.</p