Chondrodystrophic Dogs as a Pre-clinical Large Animal Model of Discogenic Back Pain
Background: Intervertebral disc (IVD) degeneration is a major cause of low back pain (LBP) in humans and canines. IVD degeneration affects the structure and function of both the disc and the innervating dorsal root ganglion (DRG) neurons. Pre-clinical animal models are necessary for elucidating the mechanisms of IVD degeneration (IVDD) and the pain signaling pathways involved in discogenic back pain. The chondrodystrophic (CD) dog exhibits similar characteristics to the clinical population affected by IVDD-associated LBP. However, further investigation of the translational tools to study these conditions and the efficacy of novel treatments is needed in this canine model. The objectives of the present study are to: (1) assess the changes in the structure and function of the IVD and DRG, including pain behaviors, in response to injury using a comprehensive set of outcome measures and (2) evaluate the efficacy of potential therapeutics in mitigating these pathologic changes due to injury in the CD canine model. Methods: Retired female research beagles underwent spinal surgery where T11/T12, T12/T13, and T13/L1 IVDs were identified and punctured with a needle containing either a protease-activated receptor 2 antagonist (PAR2A) and cromolyn sodium (CS) solution (N=3) or phosphate-buffered saline (PBS) (N=3). Pain phenotyping and related outcomes were assessed longitudinally or at the 12-week endpoint via RNA-seq on the DRG, von Frey analysis, FitBark activity, and C-reactive protein (CRP) plasma levels. Changes in the structure/function of the IVD were assessed via MRI, mechanics, Dimethylmethylene Blue Assay (DMMB), histological staining using Picrosirius red/ Alcian Blue (PR/AB) and fluoroscopy, and electrophysiology on the DRG neurons. Results: We evaluated a comprehensive series of outcome measures to determine the effects of IVD injury on the structure/function of the canine IVD and DRG, and pain in the in vivo CD dog model of IVDD and back pain. Specifically, we established methods to obtain high-quality messenger RNA from canine DRGs to perform bulk RNA-seq. We demonstrated that injury to the disc resulted in significant upregulation of inflammatory and pain-signaling genes, and downregulation of developmental genes in the adjacent innervating DRG neurons. Additionally, we isolated and cultured viable neurons from canine DRGs and found through whole-cell patch-clamp that that DRGs innervating the injured disc demonstrated altered voltage-gated sodium channel (VGSCs) activity compared to controls. Using T2 weighted MRI, we demonstrated that relaxation time in punctured discs was reduced in four out of the six dogs compared to internal control discs, indicating potential compositional changes in these injured IVDs. No significant effect of PAR2A and CS treatment was observed in this small cohort.Conclusion: This study evaluated a rigorous series of outcome measures to determine the effects of IVD injury on the disc joint and pain in a CD canine in vivo model of back pain. This was the first study to investigate the effects of disc injury on canine DRG transcriptome and whole-cell patch clamping on canine DRG neurons. Results support the CD dog as a clinically relevant translational model for studying IVDD and LBP, and for evaluating the potential efficacy of novel therapeutics in mitigating the changes associated with these conditions. Overall design: DRGs from L4/5 (internal control) and T12/L1 (puncture) were dissected and RNA isolated using the TRIzol Plus RNA Purification Kit (Cat#12183555). RNA-seq was performed at The Ohio State University Genomics Core with library generation using a NEBNext rRNA Depletion and Prep Kit. cDNA libraries were sequenced on Novaseq SP system (Illumina, USA).