Microarray characterisation of gene expression profiles in caudal dorsal root ganglia after tail amputation in pigs

Aim of Investigations
The aim of this study was to conduct a transcriptome analysis of gene expression patterns in the caudal dorsal root ganglia of pigs subjected to tail amputation at two different stages of development consistent with tail docking age (neonate) and tail biting outbreaks (juvenile) to develop a better understanding of the short to long term consequences of these types of traumatic tail injuries on peripheral nociception and pain signalling pathways in commercial pigs.
Methods
Ninety-six pigs were subjected to two tail treatments either: (1) amputation of 2/3rds of the tail (2) tail left intact (sham controls). Tail amputation was conducted at either 3 days of age (neonate) or at 63 days of age (juvenile). Neonatal piglet tails were amputated by hot docking iron consistent with commercial practice. Juvenile pig tails were surgically amputated from under general anaesthesia. All amputation procedures were ethically approved. Caudal dorsal root ganglia (DRG) were collected post-mortem for mRNA microarray analysis at 1, 8 and 16 weeks after tail amputation in both age groups (8 pigs/ tail treatment/treatment age/time). DRG gene expression analysis was performed using the Affymetrix porcine gene ST 1. 1 array. Microarray data were initially analysed using Partek Genomics Suite (RMA 3-way ANOVA). Network correlation analysis was then conducted using Markov Clustering (MCL) Algorithm (Miru v1. 4). ToppGene online database was used for gene ontology (GO) and gene set enrichment analysis (GSEA).
Results
Tail amputation induced highly significant gene expression changes in caudal DRG neurons (both up and down) compared to DRG from intact controls at both treatment ages (518-2,794 genes, false discovery rate (FDR) < 0. 05) that were still evident 16 weeks after tail amputation. Network correlation analysis using the Markov clustering (MCL) algorithm to define expression modules revealed two highly correlated (PCT r2 ≥0. 75), interrelated transcript expression clusters related to (A) neuronal function (759 genes) and (B) wound healing (273 genes). In cluster A, gene ontology (GO) and pathway enrichment analysis identified genes with significant GO terms for voltage- and ligand-gated ion channel activity linked to regulation of membrane potentials, neurotransmitter levels and synaptic signalling. In cluster B significant gene expression was associated with receptor binding, protein transcription activity and regulation, linked to processes such as response to wounding, regulation of response to wounding, inflammatory response and activation of immune response. Cross-reference against an integrated database of known genes involved in the regulation of inflammatory and neuropathic pain revealed 124 and 61 pain–associated genes in clusters A and B, respectively. Key functional families of ion channels and receptors were significantly down-regulated in cluster A, in particular voltage-gated potassium channels and GABA receptors which are linked to increased neuronal excitability. Up-regulated functional gene families in cluster B were mostly associated with inflammation, macrophage activity, neurohormone and opioid peptide activity.
Conclusions
Caudal DRG gene expression profiles appeared to be associated with sustained tissue inflammation and tissue remodelling (still evident 4 months after tail amputation) and pain perception modulation consistent with adaptive, compensatory responses to injury induced increases in peripheral sensory neuron excitability in the injured tail stump. These data link to earlier findings of on-going traumatic neuroma development and neuronal proliferation¹ and long-term mechanical hypersensitivity in the tail stump² measured as part of the same study. The overall findings of this study suggest that tail amputation in neonate and juvenile pigs causes acute and sustained changes in peripheral somatosensory nerve function involving inflammatory and neuropathic pain pathways which have implications for pig welfare.
References
¹Sandercock DA, Smith SH, Di Giminani P, Edwards SA (2016) Histopathological characterization of tail injury and traumatic neuroma development after tail docking in piglets. Journal of Comparative Pathology 155, 40-49.
²Di Giminiani P, Edwards SA, Malcolm EM, Herskin MS, Sandercock DA (2017) Characterization of short- and long-term mechanical sensitisation following surgical tail amputation in pigs. Nature Scientific Reports 7 4827-4836.
Conflicts of Interest
The authors have no conflicts of interest
Source of Financial Support for the Project
Joint funded by BBSRC (BB/L0/3584/1) and DEFRA (AW0129) as part of an ANIWHA ERA-Net Initiative (FareWellDock: Ending tail docking and tail biting in the EU – Hazard characterization and exposure assessment of a major pig welfare problem.