X-ray, coronal and sagittal images of micro-CT showed that normal appearance of joints was presented in G1. However, joint destruction was observed in G2 and the destruction of bony surface was reduced in G3 (Fig. 1).
Figure 1. X-ray, coronal and sagittal micro-CT image of hind knee joint of rats treated with collagen.
Micro-CT analysis showed that several bone parameters were altered (Table 1). In G2, bone volume was significantly decreased in tibia of animals compared to G1 (P<0.05). However, bone volume in G3 had no significant difference compared to that of G1. Bone surface/bone volume ratio and trabecular thickness of G3 were significantly decreased compared to G1 (P<0.05). Trabecular number of G2 was significantly decreased compared to G1 (P<0.01), however, it showed no significant difference in G3 compared to G1. H&E staining (Fig. 2A, B and C) showed no inflammation or tissue destruction in G1. However, inflammatory cell infiltration, which was characterized by the collection of lymphocytes and macrophages, as well as cartilage and bone erosion and synovial fibroblast activation, was observed in G2. In contrast, these changes were reduced in G3. In SO staining (Fig. 2D, E and F), cartilage structures in G1 were normal (appearing as red color). However, cartilage staining was remarkably reduced in G2. By comparison, in G3, there was moderate cartilage staining in joints of rats.
Group BV (mm3) BV/TV (%) BS/BV (mm−1) Tb.Th (mm) Tb.N (mm−1) Tb.Sp (mm) G1 14.36±3.65 41.60±4.45 28.67±2.16 0.13±0.01 3.21±0.37 0.25±0.04 G2 10.46±4.22* 28.28±13.05 24.51±2.19 0.15±0.01 1.94±0.86** 0.55±0.41 G3 14.42±5.05 39.39±13.86 24.17±2.64* 0.15±0.02* 2.61±0.80 0.32±0.32 *,**Significantly different from G1 (P<0.05, P<0.01, respectively); Values are shown as mean±SD. BV: bone volume; BV/TV: percent bone volume; BS/ BV: bone surface/volume ratio; Tb.Th: trabecular thickness; Tb.N: trabecular number; Tb.Sp: trabecular separation.
Table 1. Micro-CT analysis parameters of tibial trabecular bone in G1, G2 and G3
Figure 2. Hematoxin & eosin (H&E) and safranin O-fast green (SF) staining of hind knee joint of rats.
By scoring histopathological findings, infiltration of inflammatory cells was significantly increased in G2 compared to G1 (P<0.01), however, it showed no significant difference in G3 compared to G1. Cartilage degradation was significantly increased in G2 and G3, compared to G1 (P<0.01 and P<0.05, respectively). Bone destruction was significantly increased in G2 compared to G1 (P<0.01), however, it showed no significant difference in G3 compared to G1. Synovial hyperplasia was significantly increased in G2 and G3, compared to G1 (P<0.001). Degeneration/necrosis was significantly increased in G2 compared to G1 (P<0.01), however, it showed no significant difference in G3 compared to G1 (Fig. 3).
Effects of methotrexate on collagen-induced arthritis in male Wistar rats
- Received Date: 2017-02-28
- Accepted Date: 2017-04-08
Abstract: To evaluate the effect of methotrexate on collagen-induced arthritis, micro-computed tomography (micro-CT) and histopathological analyses were used in male Wistar rats. Rats were divided randomly into three groups. Group 1 was treated with 0.9% saline, and groups 2 and 3 were boosted with type Ⅱ collagen. From day 21 to 42, groups 1 and 2 were orally treated with 0.9% saline and group 3 was orally treated with 1.5 mg/kg methotrexate. All rats were sacrificed at day 42 after the first collagen treatment. Micro-CT analyses showed bony parameters, such as bone volume and trabecular number, were decreased in group 2 compared to group 1, and these parameters were recovered in group 3. Histopathological examination and pathological parameter scoring showed that the knee joints of rats in group 2 had severe joint destruction, showing cartilage and bone erosion, enlarged cavities with inflammatory cell infiltration and activation of synovial fibroblasts. By contrast, these changes were reduced in group 3. Taken together, methotrexate treatment showed therapeutic potential in male rat collagen-induced arthritis model, and micro-CT analysis and histopathological tools could be integrated to assess the quantification/qualification of arthritic lesions.