The role of prostaglandin E2 in intervertebral disc degeneration in dogs
Bie, M.H. de
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Abstract Introduction: Intervertebral disc (IVD) degeneration is a common problem in dogs and humans, and has significant effects on spinal biomechanics. Present-day therapies are either conservative or surgical and do not solve the problem of the degenerative process. In the search for targeted therapies and more clarification towards the process, prostaglandin E2 (PGE2) is proposed to have impact on the disc matrix. Multiple in vitro studies have revealed increased levels PGE2 in degenerative discs, but thus far in vivo studies are lacking. The beagle dog breed has been shown to be a valuable translational model to study spontaneous IVD degeneration in humans. Methods: In an on-going study from N.Willems (PhDc) two types of hydrogels and microspheres were loaded with a COX-2 selective inhibitor (Celecoxib, CXB) and injected intradiscally in Beagles to accomplish a slow release of and subsequently a reduction in PGE2 in the IVD. To determine the PGE2 contents, three types of ELISAs of two different manufacturers (R&D and ENZO) were tested. As part of the overarching study, this research project was designed to establish the most reliable ELISA. In order to preserve valuable canine test material bovine material was included. IVDs from two cowtails were collected from the slaughterhouse. From the IVDs transversal and sagittal cuts and subsequently cryoslides were performed and the resulting sample material was gathered in Ambion® lysis buffer. Nucleus pulposus (NP) material was separately collected, and after Ambion® lysis, the resulting NP supernatant was applied in the available ELISAs. Dilution outcomes, standard deviations (SD), covariance rates (CV), spike-and-recovery experiments and intra-assay precisions (IAPs) were compared, and PGE2 levels were relativized to quantified protein concentrations by using the Thermo Scientific Pierce® BCA protein Assay. All results were calculated by Microplate Manager® (MPM) 6 Software. Results: Particularly the IAP, spike, and dilution results demonstrated inconsistency in the R&D, with especially high SDs in the lower ranges. The outcomes of both ENZO ELISAs were more reliable, but PGE2 concentrations in the canine and bovine IVDs appeared to be less than expected. In the bovine ENZO-HS the PGE2 amounts of sample dilutions were immeasurable low, but in the canine material, compared to the ENZO, the ENZO-HS was steadier and better able to quantify the PGE2 levels in these lower ranges. Conclusion: As a result, the ENZO-HS was considered to be the most appropriate test for the further research in the Beagle experiment.