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Local intra-articular injection of rapamycin delays articular cartilage degeneration in a murine model of osteoarthritis.

Takayama K, Kawakami Y, Kobayashi M, Greco N, Cummins JH, Matsushita T, Kuroda R, Kurosaka M, Fu FH, Huard J - Arthritis Res. Ther. (2014)

Bottom Line: VEGF, COL10A1, and MMP13 expressions were further examined via quantitative RT-PCR (qPCR).A reduction in mTOR expression and the activation of LC3 (an autophagy marker) in the chondrocytes was observed in the rapamycin treated mice.Rapamycin treatment also reduced VEGF, COL10A1, and MMP13 expressions at 8 and 12 weeks after DMM surgery.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: Recent studies have revealed that rapamycin activates autophagy in human chondrocytes preventing the development of osteoarthritis (OA) like changes in vitro, while the systemic injection of rapamycin reduces the severity of experimental osteoarthritis in a murine model of OA in vivo. Since the systemic use of rapamycin is associated with numerous side effects, the goal of the current study was to examine the beneficial effect of local intra-articular injection of rapamycin in a murine model of OA and to elucidate the mechanism of action of rapamycin on articular cartilage.

Methods: Destabilization of the medial meniscus (DMM) was performed on 10-week-old male mice to induce OA. Intra-articular injections of 10 μl of rapamycin (10 μM) were administered twice weekly for 8 weeks. Articular cartilage damage was analyzed by histology using a semi-quantitative scoring system at 8 and 12 weeks after surgery. Mammalian target of rapamycin (mTOR), light chain 3 (LC3), vascular endothelial growth factor (VEGF), collagen, type X alpha 1 (COL10A1), and matrix metallopeptidase 13 (MMP13) expressions were analyzed by immunohistochemistry. VEGF, COL10A1, and MMP13 expressions were further examined via quantitative RT-PCR (qPCR).

Results: Intra-articular injection of rapamycin significantly reduced the severity of articular cartilage degradation at 8 and 12 weeks after DMM surgery. A reduction in mTOR expression and the activation of LC3 (an autophagy marker) in the chondrocytes was observed in the rapamycin treated mice. Rapamycin treatment also reduced VEGF, COL10A1, and MMP13 expressions at 8 and 12 weeks after DMM surgery.

Conclusion: These results demonstrate that the intra-articular injection of rapamycin could reduce mTOR expression, leading to a delay in articular cartilage degradation in our OA murine model. Our observations suggest that local intra-articular injection of rapamycin could represent a potential therapeutic approach to prevent OA.

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Histological evaluation of osteoarthritis (OA). (A, B) Representative images of toluidine blue staining from dimethyl sulphoxide (DMSO) (A) and rapamycin (B) treated mice at 8 and 12 weeks after destabilization of the medial meniscus (DMM) or sham surgery. Boxed areas in the toluidine blue stained image at the left side (x4) indicate the regions shown in the enlarged toluidine blue stained area at the right side (x20). Scale bar = 500 μm (x4) and 100 μm (x10). Arrows indicate the osteoarthritic change after DMM surgery. (C) Graph indicating the summed OA scores. Summed OA scores were calculated from all four quadrants and eight sections from each knee. ***P <0.001, **P <0.01.
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Fig1: Histological evaluation of osteoarthritis (OA). (A, B) Representative images of toluidine blue staining from dimethyl sulphoxide (DMSO) (A) and rapamycin (B) treated mice at 8 and 12 weeks after destabilization of the medial meniscus (DMM) or sham surgery. Boxed areas in the toluidine blue stained image at the left side (x4) indicate the regions shown in the enlarged toluidine blue stained area at the right side (x20). Scale bar = 500 μm (x4) and 100 μm (x10). Arrows indicate the osteoarthritic change after DMM surgery. (C) Graph indicating the summed OA scores. Summed OA scores were calculated from all four quadrants and eight sections from each knee. ***P <0.001, **P <0.01.

Mentions: There were no structural changes at the anterior cruciate ligament or meniscus identified in the sham knees that were treated with rapamycin or DMSO. Side effects such as weight loss, skin rashes, delayed wound healing, or diarrhea were not observed after local intra-articular injection of rapamycin. Histological sections demonstrated significantly less articular cartilage degeneration in the experimental group treated with local intra-articular injections of rapamycin at 8 and 12 weeks after induction of OA with DMM surgery compared to the OA-induced mice treated with DMSO. Histological grading showed that the DMSO-treated mice had a loss of proteoglycan staining with articular fibrillation at 8 weeks and losses of hyaline cartilage, proteoglycan staining, and lesions extending into the calcified cartilage at 12 weeks after surgery (Figure 1A). In contrast, rapamycin-treated mice showed a focal loss of proteoglycan staining without severe articular cartilage loss at 8 weeks and lesions with a loss of proteoglycan staining slightly increased 12 weeks after surgery; however, hyaline cartilage was preserved (Figure 1B). The extent of OA was evaluated by scoring specific parameters of OA, and is presented as summed scores (the higher the score the greater the articular cartilage degeneration) (Figure 1C, Table 2). Using the summed OA score, DMSO-treated mice developed OA in a time-dependent manner and had a significantly higher score than the rapamycin-treated mice at 8 and 12 weeks following DMM surgery (Figure 1C, P = 0.001 at 8 weeks and P <0.001 at 12 weeks). The summed OA score in the rapamycin-treated mice at 12 weeks was increased compared to the score at 8 weeks (Figure 1C, P <0.001). These results suggested that the intra-articular injection of rapamycin did not completely prevent, but delayed articular cartilage degradation in the presence of meniscus injury after DMM surgery.Figure 1


Local intra-articular injection of rapamycin delays articular cartilage degeneration in a murine model of osteoarthritis.

Takayama K, Kawakami Y, Kobayashi M, Greco N, Cummins JH, Matsushita T, Kuroda R, Kurosaka M, Fu FH, Huard J - Arthritis Res. Ther. (2014)

Histological evaluation of osteoarthritis (OA). (A, B) Representative images of toluidine blue staining from dimethyl sulphoxide (DMSO) (A) and rapamycin (B) treated mice at 8 and 12 weeks after destabilization of the medial meniscus (DMM) or sham surgery. Boxed areas in the toluidine blue stained image at the left side (x4) indicate the regions shown in the enlarged toluidine blue stained area at the right side (x20). Scale bar = 500 μm (x4) and 100 μm (x10). Arrows indicate the osteoarthritic change after DMM surgery. (C) Graph indicating the summed OA scores. Summed OA scores were calculated from all four quadrants and eight sections from each knee. ***P <0.001, **P <0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig1: Histological evaluation of osteoarthritis (OA). (A, B) Representative images of toluidine blue staining from dimethyl sulphoxide (DMSO) (A) and rapamycin (B) treated mice at 8 and 12 weeks after destabilization of the medial meniscus (DMM) or sham surgery. Boxed areas in the toluidine blue stained image at the left side (x4) indicate the regions shown in the enlarged toluidine blue stained area at the right side (x20). Scale bar = 500 μm (x4) and 100 μm (x10). Arrows indicate the osteoarthritic change after DMM surgery. (C) Graph indicating the summed OA scores. Summed OA scores were calculated from all four quadrants and eight sections from each knee. ***P <0.001, **P <0.01.
Mentions: There were no structural changes at the anterior cruciate ligament or meniscus identified in the sham knees that were treated with rapamycin or DMSO. Side effects such as weight loss, skin rashes, delayed wound healing, or diarrhea were not observed after local intra-articular injection of rapamycin. Histological sections demonstrated significantly less articular cartilage degeneration in the experimental group treated with local intra-articular injections of rapamycin at 8 and 12 weeks after induction of OA with DMM surgery compared to the OA-induced mice treated with DMSO. Histological grading showed that the DMSO-treated mice had a loss of proteoglycan staining with articular fibrillation at 8 weeks and losses of hyaline cartilage, proteoglycan staining, and lesions extending into the calcified cartilage at 12 weeks after surgery (Figure 1A). In contrast, rapamycin-treated mice showed a focal loss of proteoglycan staining without severe articular cartilage loss at 8 weeks and lesions with a loss of proteoglycan staining slightly increased 12 weeks after surgery; however, hyaline cartilage was preserved (Figure 1B). The extent of OA was evaluated by scoring specific parameters of OA, and is presented as summed scores (the higher the score the greater the articular cartilage degeneration) (Figure 1C, Table 2). Using the summed OA score, DMSO-treated mice developed OA in a time-dependent manner and had a significantly higher score than the rapamycin-treated mice at 8 and 12 weeks following DMM surgery (Figure 1C, P = 0.001 at 8 weeks and P <0.001 at 12 weeks). The summed OA score in the rapamycin-treated mice at 12 weeks was increased compared to the score at 8 weeks (Figure 1C, P <0.001). These results suggested that the intra-articular injection of rapamycin did not completely prevent, but delayed articular cartilage degradation in the presence of meniscus injury after DMM surgery.Figure 1

Bottom Line: VEGF, COL10A1, and MMP13 expressions were further examined via quantitative RT-PCR (qPCR).A reduction in mTOR expression and the activation of LC3 (an autophagy marker) in the chondrocytes was observed in the rapamycin treated mice.Rapamycin treatment also reduced VEGF, COL10A1, and MMP13 expressions at 8 and 12 weeks after DMM surgery.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: Recent studies have revealed that rapamycin activates autophagy in human chondrocytes preventing the development of osteoarthritis (OA) like changes in vitro, while the systemic injection of rapamycin reduces the severity of experimental osteoarthritis in a murine model of OA in vivo. Since the systemic use of rapamycin is associated with numerous side effects, the goal of the current study was to examine the beneficial effect of local intra-articular injection of rapamycin in a murine model of OA and to elucidate the mechanism of action of rapamycin on articular cartilage.

Methods: Destabilization of the medial meniscus (DMM) was performed on 10-week-old male mice to induce OA. Intra-articular injections of 10 μl of rapamycin (10 μM) were administered twice weekly for 8 weeks. Articular cartilage damage was analyzed by histology using a semi-quantitative scoring system at 8 and 12 weeks after surgery. Mammalian target of rapamycin (mTOR), light chain 3 (LC3), vascular endothelial growth factor (VEGF), collagen, type X alpha 1 (COL10A1), and matrix metallopeptidase 13 (MMP13) expressions were analyzed by immunohistochemistry. VEGF, COL10A1, and MMP13 expressions were further examined via quantitative RT-PCR (qPCR).

Results: Intra-articular injection of rapamycin significantly reduced the severity of articular cartilage degradation at 8 and 12 weeks after DMM surgery. A reduction in mTOR expression and the activation of LC3 (an autophagy marker) in the chondrocytes was observed in the rapamycin treated mice. Rapamycin treatment also reduced VEGF, COL10A1, and MMP13 expressions at 8 and 12 weeks after DMM surgery.

Conclusion: These results demonstrate that the intra-articular injection of rapamycin could reduce mTOR expression, leading to a delay in articular cartilage degradation in our OA murine model. Our observations suggest that local intra-articular injection of rapamycin could represent a potential therapeutic approach to prevent OA.

Show MeSH
Related in: MedlinePlus