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AB0061 35sulphate incorporation assay as a new tool for measuring early cartilage degradation following blood exposure in vitro and in vivo in f8 ko rats
  1. AE Pulles1,2,
  2. KR Christensen3,4,
  3. K Coeleveld1,
  4. M Kjelgaard-Hansen4,
  5. RE Schutgens2,
  6. FP Lafeber1,
  7. K Roepstorff4,
  8. SC Mastbergen1
  1. 1Department of Rheumatology & Clinical Immunology
  2. 2Van Creveldkliniek, University Medical Center Utrecht, Utrecht, Netherlands
  3. 3Veterinary Disease Biology, University of Copenhagen, Copenhagen
  4. 4Global research, Novo Nordisk A/S, Maaloev, Denmark

Abstract

Background Joint damage upon bleeding causes significant morbidity in patients with hemophilia, and adds to joint degeneration after trauma or major joint surgery. To study the pathophysiology, rodent models with histology as primary outcome measure are often used. However, histological changes take time to develop and are subject to interpretation. Determining proteoglycan synthesis rate (PSR) in cartilage, by incorporation of radioactive 35sulphate, is a sensitive method previously applied in human tissue and larger animal models to detect early cartilage changes. Isolating cartilage of small animals can be challenging, so a technique to shave off rat cartilage was developed.

Objectives To study cartilage degradation following blood exposure, applying the 35sulphate incorporation assay on rat tibial cartilage.

Methods A total of 13 factor VIII knock out (F8KO) rats were bred and housed at Novo Nordisk A/S, Maaloev, Denmark. After euthanasia, the legs were removed and transported to UMC Utrecht, The Netherlands.

In vitro: within 24 hours after euthanasia the cartilage of 6 healthy F8KO rats was obtained by shaving off cartilage pieces of the tibia plateau by use of a scalpel. All cartilage explants were cultured for four days; in addition to culture medium, half of the cartilage samples were cultured with 50% v/v whole blood. After four days PSR was determined by adding 4uCi Na235SO4 to the cultures for four hours. 35SO42– is incorporated into newly synthesized proteoglycans. After digesting the cartilage pieces and precipitating the proteoglycans with cetylpyridinium chloride, the amount of radioactivity was measured by liquid scintillation analysis and normalized to the specific activity of the pulse medium, labeling time and wet cartilage weight.

In vivo: in 7 F8KO rats a unilateral joint bleed was induced by needle puncture and in the following four days until euthanasia, the animals received analgesia. At UMCU, the tibial cartilage was removed and PSR determined as described above.

All animal experiments were performed in accordance with and approved by the Danish Animal Experiments Council, Ministry of Food, Agriculture and Fisheries, Denmark.

Results On average, a total of 1.6mg (0.8–3.1mg) cartilage per tibia could be obtained. The PSR of healthy cartilage determined after four days of culturing in vitro was on average 49.5 nmol/h.g. In vitro blood exposure resulted in a diminished synthesis: 7.7 nmol/h.g (p=0.0191), corresponding to an 84% decrease comparable to previously published experiments using human tissue [1]. In vivo, an induced joint bleed led to a 44% decrease in PSR (13.5 vs 7.5 nmol/h.g, p=0.0151).

Conclusions This study demonstrates for the first time that PSR, by use of the 35sulphate incorporation assay, can be determined in rat tibial cartilage and is affected by blood exposure in vitro and in vivo. As such, this assay can be a valuable tool to detect cartilage changes using joint degenerative rat models.

References

  1. L. van Vulpen, R. Schutgens, K. Coeleveld, E. Alsema, G. Roosendaal, S. Mastbergen, F. Lafeber. IL-1beta: in contrast to TNFalpha, is pivotal in blood-induced cartilage damage and is a potential target for therapy. Blood, 2015;126(19):2015, pp.2239–2246.

References

Disclosure of Interest None declared

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