Gene transcripts expressed in equine white blood cells are potential biomarkers of extracorporeal shock wave therapy

Abstract

Extracorporeal shockwave therapy (ESWT) is a treatment applied to musculoskeletal injuries in equine athletes to alleviate pain and accelerate healing. ESWT also causes acute tissue damage. Therefore, its ability to act as an analgesic and cause tissue damage potentially increases the risk of a catastrophic event if used shortly before a strenuous competition such as horseracing. While ESWT is prohibited by many racing jurisdictions within 10 days prior to competition, a test to detect whether a horse has received ESWT is needed. ESWT changes the protein levels of inflammatory mediators in blood, and white blood cells (WBC) typically produce these proteins. Changes in gene expression precede changes in protein production; thus, it was hypothesized that WBC gene transcripts might serve as biomarkers of ESWT. To test this hypothesis, six thoroughbred horses received a single administration of ESWT to the distal limb, and WBC RNA was extracted from blood samples collected before (0 h) and after ESWT (2, 4, 6, 24, 48, and 72 h). Targeted and untargeted analyses evaluated the transcriptome using quantitative PCR (qPCR) and microarray. The expression of IL‐1α, IL‐1β, TNF‐α, IL‐1Ra1, IL‐1Ra2 and TGF‐β1, and BMPR1A in circulating WBCs was significantly up‐regulated, while IFN‐γ, ZNF483, TMEM80, CAH6, ENPP, and S8723 were significantly down‐regulated at various time points following ESWT. These data support the hypothesis that changes in WBC gene transcripts could serve as biomarkers for ESWT.

Biomarker analysis

“…The potential for white blood cell (WBC) gene transcripts to serve as biomarkers of ESWT was investigated by targeted and untargeted transcriptomics using qPCR and microarray. Seven biomarkers were found to be upregulated, and six were significantly down‐regulated at various time points following ESWT 20 …”

“…This demonstrated the potential of an intelligencebased approach to detect sub-threshold doping at 3-MT levels greater than 776 ng/ml and 3-MT/Tyr values greater than 5. 20 The potential for white blood cell (WBC) gene transcripts to serve as biomarkers of ESWT was investigated by targeted and untargeted transcriptomics using qPCR and microarray. Seven biomarkers were found to be upregulated, and six were significantly down-regulated at various time points following ESWT.…”

“…Seven biomarkers were found to be upregulated, and six were significantly down-regulated at various time points following ESWT. 20 Finally, a perspective is provided by our group to propose that biomarker research should complement the focus on up-or downregulation of individual target compounds with exploring orthogonal approaches that exploit the use of ratios involving ERCs, which display no change and/or the opposite change. With supporting validation, this can result in greater sensitivity and specificity (i.e.…”

“…This study developed a qPCR screening test to target conserved AAV sequences for detection of recombinant AAV gene therapy products for 2 to 4 days' post‐administration in equine plasma and up to 28 days in synovial fluid. This research group also contributes a transcriptomic study of gene expression to identify extracorporeal shockwave therapy (ESWT) 20 . The potential for white blood cell (WBC) gene transcripts to serve as biomarkers of ESWT was investigated by targeted and untargeted transcriptomics using qPCR and microarray.…”