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. 2000 Oct;59(10):781–787. doi: 10.1136/ard.59.10.781

Effect of thrombin inhibition on synovial inflammation in antigen induced arthritis

P A Varisco 1, V Peclat 1, K van Ness 1, A Bischof-Delaloye 1, A So 1, N Busso 1
PMCID: PMC1753002  PMID: 11005778

Abstract

OBJECTIVE—To determine the effect of the thrombin inhibitor, hirudin, on the pathogenesis of murine antigen induced arthritis (AIA).
METHODS—AIA was induced by intra-articular injection of methylated bovine serum albumin in the knee joints of previously immunised mice. Hirudin (injected subcutaneously 3 × 200 µg/mouse/day) was given over 13 days, starting three days before arthritis onset, and its anticoagulant effect monitored by clotting times. Arthritis severity was evaluated by technetium-99m (99mTc) uptake in the knee joints and by histological scoring. In addition, intra-articular fibrin deposition was examined by immunohistochemistry, and synovial cytokine mRNA expression measured by RNase protection.
RESULTS—Joint inflammation, measured by 99mTc uptake, was significantly reduced in hirudin treated mice at days 7 and 10 after arthritis onset. Histologically, synovial thickness was markedly decreased in hirudin treated mice compared with untreated ones. By contrast, no difference in articular cartilage proteoglycan content was found between both groups. Intra-articular fibrin deposition and synovial interleukin 1β mRNA levels, were slightly reduced (~20%) in arthritic joints from hirudin treated mice compared with untreated ones at day 10 of AIA.
CONCLUSION—Hirudin reduces joint inflammation associated with AIA by fibrin-dependent and independent mechanisms.



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Figure 1  .

Figure 1  

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Anti-methylated bovine serum albumin (anti-mBSA) antibody levels in control and hirudin treated mice with antigen induced arthritis (AIA). The results were expressed as a percentage of the anti-mBSA antibody content in the plasma of untreated arthritic mice. There was no significant difference between immunisations in untreated (n=11) and treated (n=12) arthritic mice. Non-immunised (n.i.), non-arthritic mice (n=5), were used as negative controls. Results are expressed as means (SEM). Statistical significance was tested by Student's t test. *p<0.05 was considered significant.

Figure 2  .

Figure 2  

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Time course of knee joint inflammation in hirudin treated mice with antigen induced arthritis. Joint inflammation was measured by external gamma counting of 99mTc uptake on days 3, 7, and 10 after antigen challenge into the right knee. Results are expressed as the ratio of 99mTc uptake in the right (R) arthritic knee joint over the left (L) non-inflamed contralateral knee joint, a value higher than 1.1 indicating joint inflammation. For each time point the mean (SEM) of the ratios is shown. Control (n=11), hirudin day 3 (n=12), days 7 and 10 (n=11). Statistical significance was tested by Student's t test. The p values were 0.108 (day 3); 0.028 (day 7); 0.035 (day 10). *p<0.05 was considered significant.

Figure 3  .

Figure 3  

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Histologies and immunohistologies of whole knee joint sections of control and of hirudin treated mice with antigen induced arthritis. Figures A and C show safranin O stained sections of arthritic knee joints at day 10 after arthritis induction. Note the difference of thickness of synovial membrane (S) which is thicker in the control arthritic mice than in the hirudin treated ones. Figures B and C show fibrin(ogen) immunostaining (indicated by (F) on Figure 3B) of adjacent sections.

Figure 4  .

Figure 4  

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Histological grading and fibrin scoring of arthritic knee joints. Synovial thickness (A) and cartilage damage (B) were scored histologically using an arbitrary scale from 0 to 3. Fibrin deposition (C) was scored in the synovial membrane using an arbitrary scale from 0 to 6 and expressed as a percentage of fibrin deposition (control = 100%). Results are expressed as means (SEM), control and hirudin treated mice, n=11 per group. Statistical significance was tested by Wilcoxon/Kruskal-Wallis tests (rank sums). *p<0.05 was considered significant.

Figure 5  .

Figure 5  

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Synovial cytokine mRNA levels in knee joints from untreated or hirudin treated mice. Total RNA was prepared from non-arthritic (left knee) or from arthritic (right knee) joints of untreated or hirudin treated mice at day 10 after induction of arthritis. Expression of different cytokine mRNAs was analysed by RNase protection assay using a multiprobe set which included interleukin 1β (IL1β), IL1 receptor antagonist (IL1Ra), and macrophage migration inhibitory factor (MIF).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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