A novel microassay for the quantitation of the sulfated glycosaminoglycan content of histological sections: its application to determine the effects of Diacerhein on cartilage in an ovine model of osteoarthritis.
OBJECTIVE
A new micro-histological method of assessing the sulfated glycosaminoglycan (S-GAG) content in unstained histological sections of articular cartilage was developed and used to study the effects of orally administered Diacerhein (DIA) on joint cartilage in an ovine model of osteoarthritis (OA).


METHODS
Twenty adult, age-matched Merino wethers were subjected to bilateral lateral meniscectomy, while 10 served as non-operated controls (NOC groups). Half of the operated sheep (N=10) remained untreated (MEN groups), while the other 10 animals were given DIA (25 mg/kg orally) daily for 3 months, then 50 mg/kg daily for a further 6 months (DIA groups). Five animals each of the DIA, MEN and NOC groups, respectively, were sacrificed at 3 months post-operatively, and the remainder 6 months later. For the present study only one knee joint of each animal was used for histological processing. The tissues studied were from the lateral femoral condyles (LFC) and lateral tibial plateaux (LTP). Each of these joint regions was further subdivided into inner (I), middle (M), and outer (O) zones. Unstained histological sections from these AC regions and zones were then analysed for S-GAG content using the following procedure. Images of each section of 6 microm thickness were acquired using a flatbed scanner and the area determined with an image analysis software program. The sections were then transferred to wells of a microtiter plate, digested with papain and the S-GAG content quantitated using a modification of the 1,9-dimethylmethylene blue dye binding assay. The data was represented as microg S-GAG/mm(3)of each tissue section. These data were also compared with toluidine blue stained sections from the same paraffin blocks.


RESULTS
The results obtained showed that the area of histological sections could be very accurately determined by computer assisted image analysis using a 10 mmx10 mm calibration grid. Cartilage sections of areas ranging from 1 mm(2)up to 25 mm(2)were analysed for S-GAG content with this simple technique. There was a linear relationship between section thickness (2-10 microm) and S-GAG content per unit area (R(2)=0.993). Sections of 6 microm thickness were found to be optimal. S-GAG analyses of serial sections from tibial and femoral articular cartilage (I, M and O zones) revealed an average coefficient of variation of 7.0+/-2.3% (range 4.9-10.2%) confirming the accuracy and reproducibility of this assay method. A separate experiment showed that no significant losses of S-GAG occurred during the histological sample processing. The different regions and zones of the knee joint AC in the six experimental groups revealed variable levels of S-GAG which did not necessarily correlate with the histochemical distribution of toluidine blue staining. The major S-GAG changes occurred in the middle (lesion zone) and outer zones (hypertrophic zone) of both the LFC and LTP of the MEN groups. In the lesion (M) zone the S-GAG content was reduced while in the O zone levels were increased at both 3 and 6 months post-surgery. In animals receiving Diacerhein S-GAG levels in the M zone were lower than or equivalent to those of non-drug treated OA or non-operated controls for both joint regions at 3 and 6 months. While the hypertrophic response in the outer zone of the LFC, as assessed by S-GAG content, was enhanced by drug treatment, the cartilage of the outer zones of the LTP was not affected by drug treatment.


CONCLUSIONS
The results of this study have demonstrated that the S-GAG (and therefore proteoglycan [PG]) content in different cartilage zones of OA joints can be readily quantitated by direct biochemical analysis of unstained histological sections. By this means subtle changes in PG distribution in different cartilage zones, which were not evident using traditional histochemical staining methods, could be readily detected.
Burkhardt
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Institute of Bone and Joint Research (University of Sydney), Royal North Shore Hospital, St Leonards, New South Wales, 2065, Australia. dburkhar@mail.usyd.edu.au
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D000818:Animals; D000880:Anthraquinones; D000894:Anti-Inflammatory Agents, Non-Steroidal; D002358:Cartilage, Articular / Q000737:chemistry* / Q000187:drug effects; D016022:Case-Control Studies; D006025:Glycosaminoglycans / Q000032:analysis*; D006614:Hindlimb; D006652:Histological Techniques / Q000379:methods / Q000592:standards; D007090:Image Interpretation, Computer-Assisted / Q000379:methods; D007596:Joints; D015207:Osteoarthritis, Hip / Q000175:diagnosis*; D011509:Proteoglycans / Q000032:analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012756:Sheep
D000880:Anthraquinones; D000894:Anti-Inflammatory Agents, Non-Steroidal; D006025:Glycosaminoglycans; D011509:Proteoglycans; C013786:A73025; C025292:diacerein