RNA‐sequence analysis reveals unique changes in the dorsal root ganglia transcriptome of rats with femoral artery occlusion

Femoral artery occlusion elicits exaggerated increases in blood pressure during treadmill running in rats that resemble responses in peripheral artery disease patients. These augmented responses may be caused in part by an enhanced exercise pressor reflex (EPR) originating from skeletal muscle afferents that project to the brain stem via the dorsal root ganglia (DRG) and dorsal horn of the spinal cord. Femoral artery occlusion has been shown to increase the expression of several ion channels and receptors (ASIC3, TRPA1, P2X3) in DRG neurons which may contribute to the exaggerated EPR. However, this work has been limited to short duration occlusion (24–72 h) and traditional hypothesis‐driven approaches that typically focus on a single molecular pathway. To gain insight into genome‐wide expression changes and potentially identify novel mechanistic pathways underlying the exaggerated EPR, we performed a pilot study using RNA‐sequence analysis (RNA‐seq) to characterize the DRG transcriptome of rats following prolonged bilateral femoral artery occlusion. We isolated RNA from L4–L6 DRG of rats with freely perfused (n = 3; 309 ± 11 g) or occluded femoral arteries (n = 3; 311 ± 19 g; 57 ± 7 days post‐occlusion) and performed RNA‐seq using an Illumina HiSeq 2500 platform. A total of 14,872 protein‐coding, annotated transcripts were identified including 109 that were differentially expressed in occluded vs freely perfused rats (p<0.05; fold‐change ≥ ±2). Gene ontology (GO) functional annotation analysis indicated that a large number of these genes encode signaling molecules (17%), receptors (13%), and defense/immunity proteins (9%). Differentially expressed genes with putative roles in neuronal signaling and neuropathic pain were identified, including Crh (corticotropin‐releasing hormone), Reg3b (regenerating islet‐derived 3 beta), Kcnj4 and Kcnj16 (inward rectifier K + channels), Slc2a8 and Slc6a2 (K + and norepinephrine transporters), and Arhgap9 and Rgs18 (GTPase activating proteins). To determine if any of these changes were unique to the femoral artery occlusion model, we compared our findings to a survey of 8 published neuropathic pain studies that used microarray or RNA‐seq to map gene expression changes in the DRG of rats subjected to sciatic nerve ligation or transection. A total of 114 genes were differentially expressed in 3 or more of the pain studies. Surprisingly, only one gene, Reg3b , was commonly regulated between the occlusion and pain models. GO enrichment analysis identified 45 biological processes that were over‐represented in the occlusion model; 19 of these were shared with the pain models, including many that were immune system related (p<0.05). However, we observed differential changes in gene expression within these shared processes; genes showing upregulation in the pain models were often downregulated in the occlusion model. In summary, prolonged femoral artery occlusion produces a unique DRG transcriptome distinct from that of neuropathic pain models. Whether these novel changes in gene expression contribute to the exaggerated EPR in the femoral artery occlusion model remains to be determined.