Spaceflight and hind limb unloading induces an arthritic phenotype in knee articular cartilage and menisci of rodents

Abstract

Reduced knee weight-bearing from prescription or sedentary lifestyles are associated with cartilage degradation; effects on the meniscus are unclear. Rodents exposed to spaceflight or hind limb unloading (HLU) represent unique opportunities to evaluate this question. This study evaluated arthritic changes in the medial knee compartment that bears the highest loads across the knee after actual and simulated spaceflight, and recovery with subsequent full weight-bearing. Cartilage and meniscal degradation in mice were measured via microCT, histology, and proteomics and/or biochemically after: (1) ~ 35 days on the International Space Station (ISS); (2) 13-days aboard the Space Shuttle Atlantis; or (3) 30 days of HLU, followed by a 49-day weight-bearing readaptation with/without exercise. Cartilage degradation post-ISS and HLU occurred at similar spatial locations, the tibial-femoral cartilage-cartilage contact point, with meniscal volume decline. Cartilage and meniscal glycosaminoglycan content were decreased in unloaded mice, with elevated catabolic enzymes (e.g., matrix metalloproteinases), and elevated oxidative stress and catabolic molecular pathway responses in menisci. After the 13-day Shuttle flight, meniscal degradation was observed. During readaptation, recovery of cartilage volume and thickness occurred with exercise. Reduced weight-bearing from either spaceflight or HLU induced an arthritic phenotype in cartilage and menisci, and exercise promoted recovery.

Figure 1

Decreased articular cartilage thickness at the tibial-femoral cartilage-cartilage contact point (indicated at approximately the white circle in the coronal view, and center of the white cross in the axial view, and at the end of the arrow from the upper right plot) and volume lining the medial tibial plateau occurred after spaceflight (FLIGHT) vs Ground Control (GC), Vivarium (VIV), and Baseline. (a) Coronal image indicating the contact point. (b) Cartilage thickness was lower from FLIGHT compared to controls only at the tibial-femoral cartilage contact point (upper right), but not other regions (n = 10/group). (c) Cartilage volume was lower after FLIGHT compared to controls (n = 10/group). **p < 0.01, ***p < 0.001.

Figure 2

Meniscal volume and sulfated glycosaminoglycan (GAG) content is reduced after spaceflight. (a) Representative 3D reconstructions of medial menisci used for biometric analysis, with arrows identifying the respective anterior and posterior horns. (b) Meniscal volume measurements were lower in FLIGHT compared to controls (n = 10/group). (c) GAG content determined via a dimethylmethylene blue assay was not significantly lower in RR9 FLIGHT vs controls (n = 10/group). (d) GAG content from menisci harvested from the right and left knees (side identified in each plot) of mice flown on STS-135 were significantly (Left, n = 12–14/group) or marginally (Right, n = 8/group) lower than Ground Control. *p < 0.05, **p < 0.01, ****p < 0.0001. Scale bar = 100 µm.

Figure 3

Sulfated glycosaminoglycans (GAG) are lower and catabolic enzymes are higher within the articular cartilage lining the medial tibial plateau and the medial meniscus from mice after spaceflight (FLIGHT) vs Ground Control (GC) and Vivarium Control (VIV). (a) Representative safranin-O stained sections (n = 5–7/group). (b) Sulfated GAGs in the articular cartilage are lower in FLIGHT vs GC, and vs GC and VIV in meniscus (n = 5–7/group). (c) Representative MMP-13 stained sections (n = 5–7/group). (d) MMP-13 staining was higher in the articular cartilage and menisci in FLIGHT vs GC and VIV (n = 5–7/group). (e) Representative ADAMTS5 stained sections (n = 5–7/group). (f) ADAMTS5 was higher in FLIGHT vs both GC and VIV in articular cartilage (n = 5–7/group). *p < 0.05, **p < 0.01. Scale bar = 100 µm.

Figure 4

Ingenuity Pathway Analysis (IPA) identifying altered canonical pathways, and supportive Western blot analyses (a) Canonical pathways comparisons of FLIGHT vs GC (top) and FLIGHT vs VIV (bottom) indicate involvement of catabolic response and increased oxidative stress via p70S6K, mitochondrial dysfunction, mTOR signaling and NRF2-mediated oxidative stress; color coding indicates directional change in pathway activity (n = 10/group). (b) p70S6 Kinase was reduced vs controls post-FLIGHT (n = 3/group); pAKT (s473) was marginally lower post flight (n = 3/group); Cleaved Caspase-3 was increased post-flight (n = 3/group). *p < 0.05, **p < 0.01.

Figure 5

Exercise after a 30 day period of HLU results in recovery of cartilage and meniscal thickness and volume (n = 7–10/group). Arrows point to the approximate location of where measures were performed using 3D reconstructions of cartilage overlying the bone (a) Cartilage thickness at the tibial-femoral cartilage-cartilage contact point was lower in HLU compared to GROUND after 30 days, but not at other regions of the tibial plateau. (b) Thinning of cartilage at the tibial-femoral cartilage contact point remained in mice that had previously been HLU but returned to full weight-bearing from Days 31–80 but performed No Exercise. However, thickness of cartilage at the tibial-femoral contact point was significantly greater in previously HLU mice with both Climbing and Running from Days 31–80 than HLU-No Exercise. (c) Loss of cartilage volume observed throughout the medial tibial plateau was observed after Day 30 of HLU. For the mice that were previously HLU, recovery of cartilage volume was observed in HLU-Climbing and HLU-Running by Day 80. (d) Meniscal volume was marginally lower in HLU vs GROUND on DAY 30. Readaptation to full weight bearing from Days 31–80 in all HLU groups (HLU-No exercise, HLU-Climbing, and HLU-Running) resulted in recovery vs HLU on Day 30. *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 6

Sulfated glycosaminoglycan (GAG) abundance was lower and catabolic enzymes elevated within the articular cartilage lining the medial tibial plateau and the medial menisci from mice after HLU. (a) Sulfated GAG abundance was lower in both the articular cartilage and menisci after 30 Days of HLU (n = 7–8/group). (b) MMP-13 was significantly increased in the articular cartilage after HLU, and was marginally greater in the meniscus (n = 7–8/group). (c) ADAMTS5 was similar between both GROUND and HLU on Day 30 (n = 7–8/group). **p < 0.01.