Scand J Med Sci Sports 2015: 25: e11–e19 doi: 10.1111/sms.12195
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Interaction between gastrocnemius muscle weakness and moderate exercise deteriorates joint integrity in rat knee J. Ozawa1, A. Kaneguchi2, R. Tanaka1, S. Kawamata3, T. Kurose3, H. Moriyama4, N. Kito1, N. Kawaguchi5, N. Matsuura5 Department of Rehabilitation, Faculty of Rehabilitation, Hiroshima International University, Hiroshima, Japan, 2Graduate School of Medical Technology and Health Welfare Sciences, Hiroshima International University, Hiroshima, Japan, 3Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan, 4Graduate School of Health Sciences, Kobe University, Hyogo, Japan, 5Graduate School of Medicine, Osaka University, Osaka, Japan Corresponding author: Junya Ozawa, Department of Rehabilitation, Faculty of Rehabilitation, Hiroshima International University, Kurose-Gakuendai 555-36, Higashi-Hiroshima, Hiroshima 739-2695, Japan. Tel: +81 823 70 4547, Fax: +81 823 70 4542, E-mail: [email protected]
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Accepted for publication 17 January 2014
The aim of this study was to determine the effect on the knee joint of the interaction between ankle muscle weakness and moderate exercise. Gastrocnemius muscle weakness was induced by intramuscular injection of botulinum toxin type A (BTX) in rats. Low-speed treadmill running (12 m/min for 60 min) was applied for 6 weeks in rats with and without BTX. Untreated animals were used as controls. After BTX injection, the gastrocnemius muscle weakness was confirmed by 3-D motion analysis in kinematic features of the hindlimb during locomotion as an increased maximal dorsiflexion angle during the stance phase. Serum biomarker analysis by
enzyme-linked immunosorbent assay revealed that lowspeed running decreased the catabolic effect on type II collagen. However, the inhibition of catabolism induced by running exercise was significantly counteracted by BTX injection. In addition, thinning of the cartilage layer and a reduction in the chondrocyte density was also found in the tibial plateau of the knee in the BTX-injected rats after running for 6 weeks. These data suggest that moderate exercise have a positive effect on joint homeostasis. However, ankle muscle weakness may alter the mechanical environment of the knee and impair the integrity of joint cartilage with moderate exercise.
Joint injury, obesity, genetics, aging, sex, and anatomical and mechanical alterations are considered to result in osteoarthritis (OA) (Yuan et al., 2003; Moriyama et al., 2008; Abramson & Attur, 2009). Nonsurgical, conservative treatments for OA, such as provision of information, drugs, and physical therapies including walking exercise are provided for the purpose of symptomatic relief (Zhang et al., 2008), but do not inhibit progressive changes in OA joints. As joint cartilage has limited capacity to repair or regenerate, it is important to retain the integrity of the cartilage extracellular matrix (ECM) to prevent damage. Many investigators have reported effects of exercise on the knee joint pathology. In these studies, harmful effects were predominantly focused on changes such as proteoglycan decrease (Tang et al., 2008; Sekiya et al., 2009; Ni et al., 2011), decrease of keratan sulfate (a component of proteoglycan), discontinuity of the articular surface, suppression of type II collagen expression (Tang et al., 2008), cell cloning (Ni et al., 2011), and cyst formation (Beckett et al., 2012) in rat knee joints after involuntary treadmill running. On the other hand,
adverse effects were not documented in the knees of OA patients after walking exercise (Bautch et al., 2000), nor in experimentally injured joints in dogs after intensified loading (Vos et al., 2009). Rather, positive effects have been reported in OA patients after moderate exercise, both on metabolism-related biomarkers (Helmark et al., 2011) and glycosaminoglycan (GAG) content (Roos & Dahlberg, 2005). Therefore, appropriate joint loading is essential for the integrity of ECM in cartilage, although excessive mechanical loading may contribute to harmful effects. Skeletal muscles surrounding the joint are considered a key factor in the regulation of mechanical stress on the joint. Several reports have focused on the relationship between quadriceps muscle strength and knee OA in humans and animals (Mikesky et al., 2000; Herzog & Longino, 2007; Rehan Youssef et al., 2009; Hunt et al., 2010). Quadriceps muscle weakness may alter the kinetic and kinematic features in gait, and consequently, change impact loading and stability of the knee joint. Interestingly, it has been indicated that quadriceps muscle weakness contributes not only to joint instability and
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Ozawa et al. locomotion difficulty, but also to the development and progression of knee OA(Herzog & Longino, 2007; Rehan Youssef et al., 2009). Like the quadriceps muscle, the muscles of the lower leg that cross the ankle joint also acts as a shock absorber when walking. In humans, eccentric dorsiflexion of the tibialis anterior muscle reduces impact from the ground during the heel contact phase. In contrast, in the quadruped animal hindlimb, the forefoot rather than the heel comes into contact with the ground during the initial phase in walking. Therefore, it is predicted that the rat gastrocnemius – one of the antagonists of the dorsiflexor muscle – plays an important role in the shock absorption of impact loading during walking. Misiaszek and Pearson (2002) previously investigated the effects of BTX injection into the cat gastrocnemius muscle on locomotion. However, the effects of muscle weakness on joint cartilage metabolism and structure have not been investigated, except for those of knee extensor muscle weakness (Herzog & Longino, 2007). The aims of this study were to determine the effects of gastrocnemius muscle weakness on knee joint cartilage in low-speed running rats using biomarkers and histological studies. We hypothesized that moderate running exercise contributes to the maintenance or induction of anabolic effects on joint cartilage metabolism, and that ankle planter-flexor muscle weakness interferes with such anabolic changes. In addition, we considered the relationship between morphological changes in knee joint cartilage and walking disturbance as a result of BTX-induced muscle weakness, from the kinematic viewpoint, using 3-D locomotion analysis. Methods Skeletally mature, 14-week-old male Wistar rats weighing 352 ± 13 g (mean ± standard deviation) were used in this study. A previous radiographic study reported that the growth of the rat hindlimb bone and epiphyses in these rats stopped at 60 days after birth (Hughes & Tanner, 1970). Therefore, we considered rats that were 14 weeks (98 days) old as skeletally mature. Thirty-five and 15 rats were used for biomarker analysis and histological studies, respectively. The animals were housed in standard cages, given ad libitum access to food and water, and were maintained in a thermoneutral environment (22–25 °C), with a constant humidity of 55 ± 5% and a 12-h light–dark cycle. All procedures for animal care and treatment were approved by the Committee of Research Facilities for Laboratory Animal Sciences, Hiroshima International University. Rats were divided as follows: untreated control (Cont) groups (5 rats for the biomarker study and 5 rats for the histological study were sacrificed at 14 and 20 weeks old, respectively); RUN groups (1 week running, 1wRUN, n = 5; 3 weeks running, 3wRUN, n = 5; and 6 weeks running, 6wRUN, n = 10); BTX + RUN groups (1 week running with BTX injection, BTX + 1wRUN, n = 5, 3 weeks running with BTX injection, BTX + 3wRUN, n = 5, and 6 weeks running with BTX injection, BTX + 6wRUN, n = 10).
Intramuscular injection of BTX BTX injection was used to reduce the strength of the gastrocnemius muscles. BTX is a neuromuscular blocking agent that inac-
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tivates muscle fibers via inhibition of acetylcholine release from motor nerve terminals (Brin, 1997). By referring to a previous report (Pickett et al., 2008), the appropriate dosage of BTX required to induce muscle force reduction for 6 weeks was determined. The lyophilized Clostridium botulinum toxin type A neurotoxin complex (BOTOX, Allergan, Inc., Ireland) was diluted with saline to a final concentration of 5 units/mL. In BTX + RUN groups, rats were anesthetized by intraperitoneal administration of pentobarbital sodium (50 mg/kg). The skin covering the lower leg was incised and the calf muscle on the right hindlimb was exposed. The gastrocnemius muscle was injected with 10 units/mL of BTX solution including 1% Indian ink, at the total dose of 2 units/kg body weight, at two sites of each of the medial and lateral heads. After BTX injection, rats were returned to their home cage and allowed to recover for 3 days before starting the exercise regime.
Treadmill running exercise Previously, excessive running conditions such as 1 h at 20 m/min with 5° incline for 6 weeks (Tang et al., 2008; Sekiya et al., 2009) and 25 m/min with 12° incline for 6 weeks (Ni et al., 2011), induced degeneration in the rat knee joint. In this study, therefore, relatively low-speed running was applied to animals for up to 6 weeks in the RUN and BTX + RUN groups. A motor-driven rodent treadmill machine (Rat Runner, Agawa, Ltd., Shimane, Japan) was used for the running exercise. Initially, rats in the RUN and BTX + RUN groups became accustomed to using the treadmill by running for 10 min at a speed of 8 m/min without incline, everyday for 3 days. After acclimation, they were forced to run at a speed of 12 m/min with a 5° of incline for a total of 60 min, 5 days a week. The 60-min running consisted of six sessions of 10-min running and 1-min rest between successive running sessions. At 1, 3, and 6 weeks after the start of the running regime, rats were sacrificed with an overdose of diethyl ether inhalation.
Biomarker analysis Blood samples were collected from rats in all groups. In RUN and BTX + RUN groups, blood was collected within 15 min of the end of the last running session. In the Cont group, blood samples were collected just before sacrifice. All samples were drawn from the left ventricle. Samples were centrifuged at 2000 g for 15 min to obtain serum and stored at −80 °C until analysis. Three markers of cartilage metabolism were used in this study. Procollagen type II C-propeptide (CPII) and aggrecan chondroitin sulfate 846 epitope (CS846) were used as cartilage synthesis markers. Type II collagen cleavage (CIIC) was used as a marker of cartilage degradation. The concentration of each biomarker was evaluated using enzyme-linked immunosorbent assay (ELISA) kits, according to the manufacturer’s instruction (all ELISA kits were purchased from IBEX Technologies, Montreal, Canada). Absorbance was measured using a Multiscan FC Microplate reader (Thermo Scientific, Kanagawa, Japan).
Histological examination Gastrocnemius muscles and knee joint tissues were removed from the right hindlimbs of the 6wRUN group and from both hindlimbs of the BTX + 6wRUN group. To compare the joint cartilage thickness among age-matched groups, the right knee joints from 20-week-old, untreated rats (n = 5) were also collected. Gastrocnemius muscles were weighed and snap frozen in isopentane cooled by liquid nitrogen and stored at −80 °C. Transverse cryosections (10 μm) were cut and stained with hematoxylin and eosin. Knee joint tissues were dissected and immersion-fixed in 0.1 M phosphate-buffered 4% paraformaldehyde (pH 7.4) for 2 days at 4 °C. During fixation, the knee joint angle was kept at 90°
Ankle muscle alters knee joint integrity flexion. Samples were decalcified and cut into two pieces in the frontal plane, at the central level of the tibia, and embedded in paraffin wax. Transverse sections (5 μm) were cut and stained with Safranin-O for histological observation. In previous studies, running-induced knee joint cartilage degeneration was more pronounced in the lateral compartment than the medial one (Tang et al., 2008), and joint cartilage was more sensitive to altered loading conditions in the tibial plateau than in the femoral condyle (Liphardt et al., 2009). In addition, the loading site of the femoral condyle may be altered by changes in the knee joint angle, which is due to the fact that the gastrocnemius muscle weakens during the stance phase, and thus, in this study, we focused on joint cartilage in the lateral tibial plateau of the knee.
Histometrical analysis To evaluate cartilage metabolism in the knee joint, articular cartilage thickness was measured. Cartilage thickness, including uncalcified and calcified layers, was defined as the distance from the cartilage surface to the osteochondral junction. Accordingly, mean values of cartilage thickness were calculated by dividing the area of cartilage matrix by the length of the cartilage surface. The central portion of articular cartilage from each section was photographed at a magnification of ×10. Images were processed digitally, and cartilage matrix areas and surface length were measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA). To evaluate chondrocyte number, central portions of articular cartilage in the lateral plateau of each section were photographed at a magnification of ×20. The area of the cartilage matrix and the number of chondrocytes within this area were measured by ImageJ software and manually, respectively. Chondrocyte density was expressed as number per mm2.
3-D locomotion analysis To evaluate the effects of gastrocnemius muscle weakness, kinematic features of quadrupedal locomotion were analyzed both preand post-BTX injection at 1, 3, and 6 weeks in BTX + 6wRUN rats (n = 4). Hindlimb movement was assessed during treadmill locomotion, using a KinemaTracer system (Kissei Comtec, Nagano, Japan) and a treadmill machine (MK680S; Muromachi, Ltd., Tokyo, Japan). In this system, 3-D video recordings (60 Hz) were made with four digital cameras (Tokina, Tokyo, Japan), two of which were placed on the right side and two on the left side of the treadmill, to trace and analyze hindlimb joint movement during locomotion. Color markers corresponding to the lateral cleft between articulations (knee), lateral malleolus (ankle), the and fifth metatarsophalangeal joint were placed during each test day (Fig. 1). Each marker was traced using KinemaTracer software. The movement of the colored markers was recorded by video during locomotion at a speed of 10 m/min on a treadmill. The markers were automatically and manually traced, and the KinemaTracer software used to analyze the coordinates was calibrated by recording a cube of known size [20 × 10 × 5 (x × y × z)]. The position of each marker in the xyz plane was automatically calculated by the system, allowing it to measure the angle between ankle dorsal flexion and plantar flexion. We obtained values for the maximal ankle dorsiflexion angle in stance phase during locomotion. Excessive dorsiflexion range of motion (ROM) in the stance phase was considered as gastrocnemius muscle weakness induced by BTX. Four to eight consecutive steps were analyzed in each trial, and average values for the maximal dorsiflexion ROM in ankle joints were calculated using KinemaTracer software.
Statistics Statistical analysis was performed using Dr. Statistical Package for the Social Sciences (SPSS) II for Windows (SPSS Japan,
Fig. 1. Schematic presentation of marker positions for kinematic data collection from botulinum toxin type A (BTX)injected rats. In total, six reflective markers (2-mm diameter) were used to identify two segments of both hindlimbs. Ankle dorsiflexion range of motion (ROM) was defined through the following equation: angle = θ–90°. Inc., Tokyo, Japan) and modified R (The R Foundation for Statistical Computing, Perugia, Italy). Differences in maximal ankle ROM between right and left sides were evaluated using MannWhitney’s U-test at each time point. One-way analysis of variance (ANOVA) was used to statistically evaluate the differences between Cont and RUN groups, and between Cont and BTX + RUN groups, in muscle wet weight, ankle ROM (of each side of the animal), all biomarkers, and chondrocyte density. Tukey’s honestly significant difference post-hoc test was performed to clarify significant effects. Two-way repeated measures ANOVA was also conducted to examine the relationship between muscle weakness (with or without BTX) and running period (1, 3, and 6 weeks). For each ANOVA model with a significant direct or interaction effect, Bonferroni tests were performed posthoc to localize the effects. Cartilage thickness was evaluated by Kruskal–Wallis test and Steel–Dwass post-hoc tests were conducted. For all tests, statistical significance was accepted as P < 0.05.
Results Behavior All animals could run at experimental settings 3 days post-BTX injection. However, ankle plantarflexor movement during treadmill running was compromised on the experimental side in BTX + RUN groups. Importantly, the heel was in contact with the floor during the stance phases (in a normal rat, unlike a human, heel contact does not occur during the stance phase). The foot on the BTX-injected side was oriented outward when running. Such trends were prominent until 3–4 weeks post-BTX injection, and slightly reduced thereafter. Ankle joint ROM during locomotion Representative changes in ankle joint ROM during locomotion in BTX-injected rats are shown in Fig. 2(a). Temporal changes in maximum dorsiflexion ROM
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Ozawa et al. during the stance phase are shown in Fig. 2(b). The right (BTX-injected) side vs the left (contralateral) side were 37 ± 4° vs 33 ± 11°, respectively (right/left = 110%) before BTX injection, and 51 ± 8° vs 29 ± 4° (175%, P = 0.003), 47 ± 7° vs 33 ± 11° (168%, P = 0.003), and 35 ± 9° vs 24 ± 11° (142%, P = 0.13) at 1, 3, and 6 weeks post-BTX injection, respectively. Compared with the baseline (pre-injection), the right maximum dorsiflexion angles at 1, 3, and 6 weeks post-injection were 163% (P = 0.017), 148% (P = 0.080), and 114% (P = 0.971), respectively. On the left side, there was no statistically significant difference at any time point (109%, 99%, and 81% at 1, 3, and 6 weeks postinjection, respectively). These results suggest that compensatory changes in the contralateral side did not appear, at least in ankle ROM during the stance phase. Gastrocnemius muscle wet weight and histology
Fig. 2. Locomotion analysis data on ankle joint range of motion (ROM) during treadmill walking. (a) Representative locomotion patterns at 1w after botulinum toxin type A (BTX) injection and (b) mean values of maximal dorsiflexion angle during the stance phase at 1, 3, and 6 weeks pre- and post-injection. Values are means ± standard deviation. *indicates significant difference from contralateral side during the same running period (P < 0.05). ** indicates significant difference from the ipsilateral side at pre-injection (P < 0.05).
Table 1 shows the wet weight of gastrocnemius muscles in all groups. As compared with Cont, muscle wet weight was not affected in 1wRUN (95%, P = 0.638), but was significantly increased in 3wRUN (117%, P = 0.02) and in 6wRUN (117%, P = 0.002). In contrast, muscle wet weight was progressively decreased in BTX + 1wRUN (83%, P < 0.001), in BTX + 3wRUN (66%, P < 0.001), and in BTX + 6wRUN (49%, P < 0.001) despite prolonged exposure to exercise. Comparison between RUN groups and BTX + RUN groups over the same period revealed that wet weights of BTX-injected muscles were significantly decreased at 1 (87%, P = 0.001), 3 (56%, P < 0.001), and 6 weeks (42%, P < 0.001). Comparison between the ipsi- and contra-lateral sides of gastrocnemius muscles in BTX + RUN groups revealed that BTXinjected muscles decreased over time, which parallels the comparison between RUN groups and BTX + RUN groups. Comparing the RUN groups and the contralateral side of the BTX + RUN groups, which reflects the contralateral effects of BTX on gastrocnemius muscles, there were no differences in muscle wet weight at 1 (104%, P = 0.60) and 6 weeks (105%, P = 0.50), but muscle weight in the BTX + RUN group was slightly decreased at 3 weeks (87%, P < 0.05).
Fig. 3. Histological photographs of gastrocnemius muscles in botulinum toxin type A (BTX) + 6wRUN group. (a), Contralateral (left) side and (b), BTX-injected (right) side. Severely atrophied muscle fibers were observed at the BTX-injected site. Hematoxylin and eosin staining. Arrows = Indian ink included in injection. Scale bar: 100 μm.
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Ankle muscle alters knee joint integrity Table 1. Wet weight of the gastrocnemius muscle
Running period, week (s)
0 1 3 6
Cont, mg
1805 ± 61
(a) RUN, mg
1722 ± 95 2114 ± 93* 2112 ± 162*
BTX + RUN (b) Ipsilateral side, mg
(c) Contralateral side, mg
1505 ± 34* 1185 ± 117* 883 ± 115*
1710 ± 147 1875 ± 165 2020 ± 77*
(b)/(a), %
(b)/(c), %
88 ± 5† 51 ± 5† 42 ± 4†
88 ± 7‡ 58 ± 8‡ 42 ± 4‡
*Indicates significant difference from Cont (P < 0.05). † Indicates significant difference between BTX + RUN group and RUN group at the same running periods (P < 0.05). ‡ Indicates significant difference between Rt side and Lt side in BTX + RUN group at the same running periods (P < 0.05). Values are means ± SD.
Fig. 4. Concentrations of cartilage metabolism-related biomarkers in serum. (a) Type II collagen cleavage (CIIC), (b) Procollagen type II C-propeptide (CPII), (c) aggrecan chondroitin sulfate 846 epitope (CS846), and (d) CPII/CIIC ratio in Cont and groups with or without botulinum toxin type A (BTX) injection after 1, 3, and 6 weeks running. Data represent the means ± standard deviation (Cont and RUN groups, □; BTX + RUN groups, ■). *indicates simple main effect between groups with or without BTX at the same running period (P < 0.05).**indicates significant difference from the ipsilateral side at pre-injection (P < 0.05).
Histological observation also revealed that areas of gastrocnemius muscle exposed to the injectate were severely atrophied [Fig 3(b) ] compared with those in the contralateral (un-injected) side [Fig. 3(a)] of the BTX + 6wRUN group.
Cartilage metabolism biomarkers CIIC concentrations in Cont, 1wRUN, 3wRUN, and 6wRUN groups were 148 ± 16 ng/mL, 139 ± 28 ng/mL (94% of Cont), 127 ± 19 ng/mL (86% of Cont), and
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Fig. 5. Representative histological photographs of changes in the rat knee joint. Frontal section of the lateral tibial plateau were stained with Safranin O for articular cartilage matrix proteoglycan. (a) Age-matched control (Cont), (b) 6wRUN, (c) BTX + 6wRUN [botulinum toxin type A (BTX)-injected side], (d) BTX + 6wRUN (contralateral side). Arrow indicates cyst in cartilage layer. Scale bar: 100 μm.
104 ± 24 ng/mL (70% of Cont), respectively [Fig. 4(a)]. In RUN groups, CIIC tended to decreased over time, but had not reached significance even at 6 weeks. In BTX + RUN groups, CIIC decreased in BTX + 1wRUN (107 ± 20 ng/mL, 73%), and then recovered in BTX + 3wRUN (122 ± 42 ng/mL, 84%) and BTX + 6wRUN (149 ± 29 ng/mL, 100%). Interaction effect was detected between running and muscle weakness (P = 0.035), and the simple main effect was also detected between running and muscle weakness at 6 weeks (P = 0.033). CPII concentrations were 1876 ± 279 ng/mL, 2084 ± 119 ng/mL (111%), 2061 ± 276 ng/mL (110%), and 1950 ± 206 ng/mL (104%) in Cont, 1wRUN, 3Wrun, and 6wRUN groups, respectively [Fig 4(b) ]. In BTX + RUN groups, CIIC concentrations were 2000 ± 195 ng/mL (107%), 1941 ± 236 ng/mL (104%), and 1986 ± 176 ng/mL (106%) in BTX + 1wRUN, BTX + 3wRUN, and BTX + 6wRUN group, respectively. There was no significant difference between Cont and RUN groups, or Cont and BTX + RUN groups. No interaction effect between running and muscle weakness was detected. CS846 concentrations were 43.6 ± 3.0 ng/mL, 39.5 ± 6.3 ng/mL (91%), 39.9 ± 6.8 ng/mL (92%), and 41.0 ± 8.1 ng/mL (94%) in Cont, 1wRUN, 3wRUN, and 6wRUN groups, respectively [Fig 4(c)]. In the BTX + RUN groups, CS846 concentrations were 42.2 ± 8.3 ng/mL (87%), 39.3 ± 8.9 ng/mL (90%), and 35.8 ± 7.1 ng/mL (82%) in BTX + 1wRUN, BTX + 3wRUN, and BTX + 6wRUN, respectively. There was no significant difference between Cont and RUN groups, or Cont and BTX + RUN groups. No interaction effect between running and muscle weakness was detected.
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CPII/CIIC ratios, which represent total change of type II collagen, were increased in 1wRUN and 3wRUN groups (120% and 128%, respectively), and reached significance at 150% (P = 0.012) in the 6wRUN group [Fig. 4(d) ]. In BTX + RUN groups, CPII/CIIC tended to increase in BTX + 1wRUN (148%, P = 0.10), and then tended to decrease in BTX + 3wRUN and BTX + 6wRUN groups (135% and 107%, respectively). Interaction effect was detected between running and muscle weakness (P = 0.022), and a simple main effect was also detected at 6 weeks after running (P = 0.020). Histological analysis In Cont, dense staining with Safranin O, indicating rich GAG content, and arranged chondrocytes were observed at the lateral tibial plateau of the knee joint cartilage[Fig. 5(a)]. In 6wRUN, GAG content and cartilage layer thickness were well preserved (103%), and chondrocyte density (cell number per mm2) was not changed (86%, P = 0.17) [Figs 5(b), 6(a,b)]. Interestingly, single or multiple cyst formations were observed in the deeper zone of the calcified and/or uncalcified layer in three (60%) of the five subjects. In the experimental side of BTX + 6wRUN animals, although degenerative changes were not so severe, weakened staining with Safranin O in the cartilage matrix and partial disappearance of the tidemark, were occasionally observed [Fig. 5(c)]. Importantly, the cartilage layer was significantly thinner than in Cont (77%, P = 0.045) and in 6wRUN (75%, P = 0.045), and the chondrocyte density was also decreased compared with the Cont group (78%,
Ankle muscle alters knee joint integrity
Fig. 6. Histometrical scores in joint cartilage from the lateral plateau of rat knee joint. (a) Cartilage thickness and (b) the chondrocyte density. *indicates significant difference from Cont group (P < 0.05). **indicates significant difference from 6wRUN group (P < 0.05).
P = 0.021) [Fig. 6(b)]. In the contralateral side of BTX + 6wRUN group, a slight reduction of Safranin O staining and discontinuities of the tidemark were sometimes detected, but less than those in the experimental side. Chondrocyte density and cartilage thickness were not statistically different as compared with the Cont group (84% and 88%, respectively) [Figs 5(d), 6(a,b)]. With regard to the contralateral effects of BTX on the knee, there were no significant differences in cartilage thickness and chondrocyte density between the 6wRUN and BTX + 6wRUN groups. In the medial tibial plateau, we could not distinguish marked differences among all groups, in accordance with previous studies (Tang et al., 2008; Liphardt et al., 2009). Discussion The major finding of our study is that the inhibition of cartilage catabolism induced by low-speed treadmill
running is counteracted by gastrocnemius muscle weakness elicited by BTX injection. In addition, a marked decrease in chondrocyte density and cartilage thinning, which are known features of OA, are observed in the lateral plateau of the knee joint in BTX + 6wRUN, but not in the 6wRUN group. These results suggest that moderate running, combined with weakened ankle plantarflexor muscle strength has a harmful effect on the maintenance of cartilage integrity, although the intensity of running was not enough to exert cartilage degradation. The association between muscle weakness and the onset and progression of knee joint degradation has not been elucidated except by reports from Herzog and coworkers (Herzog & Longino, 2007; Rehan Youssef et al., 2009) focusing on quadriceps muscle weakness. Both increased and decreased loading conditions resulted in a reduction in joint cartilage thickness and cartilage degradation (Eckstein et al., 2006; Leong et al., 2011). Accordingly, we speculate that the adverse effects on joint cartilage might be concerned with changes in the mechanical environment of the surface of the knee joint, induced by ankle muscle weakness. Under motion analysis, maximal dorsiflexion ROM during the stance phase was larger in the BTX-injected side than in the contralateral side, at both 1 and 3 weeks after BTX injection. Forward progression of the tibia is controlled by the gastrocnemius muscle during the foot-flat to heel-off phases in humans (Dugan & Bhat, 2005). Thus, it was suggested that weakened plantarflexor muscles could not sufficiently resist the dorsiflexion generated by frontal moment in the stance phase. In addition, heel contact was observed during the stance phase, during locomotion after BTX injection. Unlike in humans, initial contact arises not from the heel but from the forefoot in rats, and heel contact does not appear during the stance phase in normal rat locomotion. Therefore, heel contact may reflect dysfunction in the ability to absorb the impact of ground reaction forces because of eccentric contraction of ankle plantarflexor muscles. Excessive impact loading on the hindlimb as a result of receiving ground reaction forces directly may give rise to the collapse of cartilage matrix homeostasis in the knee joint. In fact, excessive joint loading by strenuous running (Tang et al., 2008; Sekiya et al., 2009; Ni et al., 2011; Beckett et al., 2012) induced reduction in type II collagen expression, thinning of joint cartilage, and worsened histopathological score. However, a possible contradicting factor of disruption to cartilage homeostasis is decreased loading. Rehan Youssef et al. (2009) reported that quadriceps muscle weakness induced by BTX injection resulted in knee joint degeneration in rabbits. They noted that this degeneration might be attributed to muscle weakness, which interferes with normal loading of the knee. Weakness of the quadriceps muscle decreased vertical ground reaction forces in the push-off phase of hopping in BTX-injected animals (Longino et al., 2005). Likewise,
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Ozawa et al. force-transmission to the forefoot at heel rise was decreased in response to reduction in gastrocnemius muscle force (Chen et al., 2012). In this study, we could not determine whether the effects on knee joint cartilage induced by gastrocnemius muscle weakness depended on excessive or decreased loading. Reduction in chondrocyte density in the BTX + 6wRUN group was thought to be a result of increased cell death because there is little or no cell division or cell death in normal adult cartilage (Heijink et al., 2012). Increased cell death in joint cartilage was also observed in aging rats (Moriyama et al., 2012), in human OA (Harmand et al., 1982), and after electrical stimulation of the quadriceps muscle, which mimics joint loading in athletes or those performing heavy labor who have an increased rate of knee OA (Horisberger et al., 2012). Our findings indicate that ankle muscle weakness sufficient to induce walking disturbance might have negative effects on the maintenance of cartilage integrity in the knee joint, even after moderate exercise. Normal joint kinematics is important to the integrity of the knee joint cartilage. Breakdown of segmental regulation in motion, which manifests as walking disturbances, can shift the loading patterns to the knee joint cartilage in the stance phase. In addition, uncontrolled segmental motion, such as an increase in peak knee adduction moment and excessive tibiofemoral rotation, are assumed to increase mechanical stresses on the knee joint surface, contributing to the onset and progression of OA (Vincent et al., 2012). Our results show that our experimental regime had a protective effect of catabolism on the matrix of joint cartilage. Several studies also documented the positive effects of moderate exercise on knee joint cartilageboth morphologically and/or biochemically in animals (Otterness et al., 1998) and in humans (Bautch et al., 2000; Roos & Dahlberg, 2005; Helmark et al., 2011). However, both degradation and synthesis biomarkers of type II collagen were up-regulated in OA-affected horses (Frisbie et al., 2008) and dogs (Chu et al., 2002). These results indicate that anabolic effects on biomarker levels were not necessarily representative of positive effects on joint integrity. In addition, we observed cyst formation in 60% of 6wRUN subjects. Cyst formation is a possible sign of adverse effects on normal homeostasis of the knee joint cartilage (Beckett et al., 2012). Further, longterm study (> 6 weeks under our running conditions) is needed to evaluate the utility of moderate running exercise on knee joint cartilage. Various kinds of therapeutic exercises (i.e., quadriceps muscle strengthening, aerobic exercise, and
walking, etc.) have been recommended for symptomatic relief both in knee OA patients in systematic reviews (Bennell & Hinman, 2005; Fransen & McConnell, 2009) and clinical guidelines (Zhang et al., 2008). However, our results suggest that ankle muscle weakness is a possible risk factor for knee joint cartilage degradation, even under moderate running exercise. Therefore, it is crucial to pay attention to ankle muscle weakness during moderate intensity exercise therapy for knee OA patients. The current study has some limitations. First, notable differences in muscle function exist between rats and humans during walking and other locomotion patterns as described earlier. In addition, we did not undertake a histological study of the patellofemoral joint. It was reported that the quadriceps muscle weakness resulted in joint degeneration in retro-patellar cartilage rather than tibia or femur cartilage in rabbits (Rehan Youssef et al., 2009).
Perspectives In conclusion, using serum biomarker data, our study has shown that low-speed running exercise inhibited catabolic changes in rat knee joint cartilage. However, these effects were counteracted by gastrocnemius muscle weakness. Histological analysis showed thinning of the cartilage layer and reduction of chondrocyte density in BTX-injected knee joints. Using 3-D locomotion analysis, dysfunction of ankle plantarflexion was confirmed during the stance phase. These data suggest that the ankle muscle weakness might impair the homeostasis and integrity of the knee joint cartilage, albeit with a moderate exercise, which is proposed to be positive on the joint homeostasis. Therefore, patients with lower limb weakness should be considered for exercise programs designed for those with metabolic disorders of the joint cartilage such as knee OA. Key words: Articular cartilage, type II collagen, botulinum toxin type A, the gastrocnemius muscle, treadmill running.
Acknowledgements This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sport, Science, and Technology of Japan. We thank Mr Tomoaki Nishino for his technical assistance.
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Interaction between gastrocnemius muscle weakness and moderate exercise deteriorates joint integrity in rat knee J. Ozawa1, A. Kaneguchi2, R. Tanaka1, S. Kawamata3, T. Kurose3, H. Moriyama4, N. Kito1, N. Kawaguchi5, N. Matsuura5 Department of Rehabilitation, Faculty of Rehabilitation, Hiroshima International University, Hiroshima, Japan, 2Graduate School of Medical Technology and Health Welfare Sciences, Hiroshima International University, Hiroshima, Japan, 3Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan, 4Graduate School of Health Sciences, Kobe University, Hyogo, Japan, 5Graduate School of Medicine, Osaka University, Osaka, Japan Corresponding author: Junya Ozawa, Department of Rehabilitation, Faculty of Rehabilitation, Hiroshima International University, Kurose-Gakuendai 555-36, Higashi-Hiroshima, Hiroshima 739-2695, Japan. Tel: +81 823 70 4547, Fax: +81 823 70 4542, E-mail: [email protected]
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Accepted for publication 17 January 2014
The aim of this study was to determine the effect on the knee joint of the interaction between ankle muscle weakness and moderate exercise. Gastrocnemius muscle weakness was induced by intramuscular injection of botulinum toxin type A (BTX) in rats. Low-speed treadmill running (12 m/min for 60 min) was applied for 6 weeks in rats with and without BTX. Untreated animals were used as controls. After BTX injection, the gastrocnemius muscle weakness was confirmed by 3-D motion analysis in kinematic features of the hindlimb during locomotion as an increased maximal dorsiflexion angle during the stance phase. Serum biomarker analysis by
enzyme-linked immunosorbent assay revealed that lowspeed running decreased the catabolic effect on type II collagen. However, the inhibition of catabolism induced by running exercise was significantly counteracted by BTX injection. In addition, thinning of the cartilage layer and a reduction in the chondrocyte density was also found in the tibial plateau of the knee in the BTX-injected rats after running for 6 weeks. These data suggest that moderate exercise have a positive effect on joint homeostasis. However, ankle muscle weakness may alter the mechanical environment of the knee and impair the integrity of joint cartilage with moderate exercise.
Joint injury, obesity, genetics, aging, sex, and anatomical and mechanical alterations are considered to result in osteoarthritis (OA) (Yuan et al., 2003; Moriyama et al., 2008; Abramson & Attur, 2009). Nonsurgical, conservative treatments for OA, such as provision of information, drugs, and physical therapies including walking exercise are provided for the purpose of symptomatic relief (Zhang et al., 2008), but do not inhibit progressive changes in OA joints. As joint cartilage has limited capacity to repair or regenerate, it is important to retain the integrity of the cartilage extracellular matrix (ECM) to prevent damage. Many investigators have reported effects of exercise on the knee joint pathology. In these studies, harmful effects were predominantly focused on changes such as proteoglycan decrease (Tang et al., 2008; Sekiya et al., 2009; Ni et al., 2011), decrease of keratan sulfate (a component of proteoglycan), discontinuity of the articular surface, suppression of type II collagen expression (Tang et al., 2008), cell cloning (Ni et al., 2011), and cyst formation (Beckett et al., 2012) in rat knee joints after involuntary treadmill running. On the other hand,
adverse effects were not documented in the knees of OA patients after walking exercise (Bautch et al., 2000), nor in experimentally injured joints in dogs after intensified loading (Vos et al., 2009). Rather, positive effects have been reported in OA patients after moderate exercise, both on metabolism-related biomarkers (Helmark et al., 2011) and glycosaminoglycan (GAG) content (Roos & Dahlberg, 2005). Therefore, appropriate joint loading is essential for the integrity of ECM in cartilage, although excessive mechanical loading may contribute to harmful effects. Skeletal muscles surrounding the joint are considered a key factor in the regulation of mechanical stress on the joint. Several reports have focused on the relationship between quadriceps muscle strength and knee OA in humans and animals (Mikesky et al., 2000; Herzog & Longino, 2007; Rehan Youssef et al., 2009; Hunt et al., 2010). Quadriceps muscle weakness may alter the kinetic and kinematic features in gait, and consequently, change impact loading and stability of the knee joint. Interestingly, it has been indicated that quadriceps muscle weakness contributes not only to joint instability and
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Ozawa et al. locomotion difficulty, but also to the development and progression of knee OA(Herzog & Longino, 2007; Rehan Youssef et al., 2009). Like the quadriceps muscle, the muscles of the lower leg that cross the ankle joint also acts as a shock absorber when walking. In humans, eccentric dorsiflexion of the tibialis anterior muscle reduces impact from the ground during the heel contact phase. In contrast, in the quadruped animal hindlimb, the forefoot rather than the heel comes into contact with the ground during the initial phase in walking. Therefore, it is predicted that the rat gastrocnemius – one of the antagonists of the dorsiflexor muscle – plays an important role in the shock absorption of impact loading during walking. Misiaszek and Pearson (2002) previously investigated the effects of BTX injection into the cat gastrocnemius muscle on locomotion. However, the effects of muscle weakness on joint cartilage metabolism and structure have not been investigated, except for those of knee extensor muscle weakness (Herzog & Longino, 2007). The aims of this study were to determine the effects of gastrocnemius muscle weakness on knee joint cartilage in low-speed running rats using biomarkers and histological studies. We hypothesized that moderate running exercise contributes to the maintenance or induction of anabolic effects on joint cartilage metabolism, and that ankle planter-flexor muscle weakness interferes with such anabolic changes. In addition, we considered the relationship between morphological changes in knee joint cartilage and walking disturbance as a result of BTX-induced muscle weakness, from the kinematic viewpoint, using 3-D locomotion analysis. Methods Skeletally mature, 14-week-old male Wistar rats weighing 352 ± 13 g (mean ± standard deviation) were used in this study. A previous radiographic study reported that the growth of the rat hindlimb bone and epiphyses in these rats stopped at 60 days after birth (Hughes & Tanner, 1970). Therefore, we considered rats that were 14 weeks (98 days) old as skeletally mature. Thirty-five and 15 rats were used for biomarker analysis and histological studies, respectively. The animals were housed in standard cages, given ad libitum access to food and water, and were maintained in a thermoneutral environment (22–25 °C), with a constant humidity of 55 ± 5% and a 12-h light–dark cycle. All procedures for animal care and treatment were approved by the Committee of Research Facilities for Laboratory Animal Sciences, Hiroshima International University. Rats were divided as follows: untreated control (Cont) groups (5 rats for the biomarker study and 5 rats for the histological study were sacrificed at 14 and 20 weeks old, respectively); RUN groups (1 week running, 1wRUN, n = 5; 3 weeks running, 3wRUN, n = 5; and 6 weeks running, 6wRUN, n = 10); BTX + RUN groups (1 week running with BTX injection, BTX + 1wRUN, n = 5, 3 weeks running with BTX injection, BTX + 3wRUN, n = 5, and 6 weeks running with BTX injection, BTX + 6wRUN, n = 10).
Intramuscular injection of BTX BTX injection was used to reduce the strength of the gastrocnemius muscles. BTX is a neuromuscular blocking agent that inac-
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tivates muscle fibers via inhibition of acetylcholine release from motor nerve terminals (Brin, 1997). By referring to a previous report (Pickett et al., 2008), the appropriate dosage of BTX required to induce muscle force reduction for 6 weeks was determined. The lyophilized Clostridium botulinum toxin type A neurotoxin complex (BOTOX, Allergan, Inc., Ireland) was diluted with saline to a final concentration of 5 units/mL. In BTX + RUN groups, rats were anesthetized by intraperitoneal administration of pentobarbital sodium (50 mg/kg). The skin covering the lower leg was incised and the calf muscle on the right hindlimb was exposed. The gastrocnemius muscle was injected with 10 units/mL of BTX solution including 1% Indian ink, at the total dose of 2 units/kg body weight, at two sites of each of the medial and lateral heads. After BTX injection, rats were returned to their home cage and allowed to recover for 3 days before starting the exercise regime.
Treadmill running exercise Previously, excessive running conditions such as 1 h at 20 m/min with 5° incline for 6 weeks (Tang et al., 2008; Sekiya et al., 2009) and 25 m/min with 12° incline for 6 weeks (Ni et al., 2011), induced degeneration in the rat knee joint. In this study, therefore, relatively low-speed running was applied to animals for up to 6 weeks in the RUN and BTX + RUN groups. A motor-driven rodent treadmill machine (Rat Runner, Agawa, Ltd., Shimane, Japan) was used for the running exercise. Initially, rats in the RUN and BTX + RUN groups became accustomed to using the treadmill by running for 10 min at a speed of 8 m/min without incline, everyday for 3 days. After acclimation, they were forced to run at a speed of 12 m/min with a 5° of incline for a total of 60 min, 5 days a week. The 60-min running consisted of six sessions of 10-min running and 1-min rest between successive running sessions. At 1, 3, and 6 weeks after the start of the running regime, rats were sacrificed with an overdose of diethyl ether inhalation.
Biomarker analysis Blood samples were collected from rats in all groups. In RUN and BTX + RUN groups, blood was collected within 15 min of the end of the last running session. In the Cont group, blood samples were collected just before sacrifice. All samples were drawn from the left ventricle. Samples were centrifuged at 2000 g for 15 min to obtain serum and stored at −80 °C until analysis. Three markers of cartilage metabolism were used in this study. Procollagen type II C-propeptide (CPII) and aggrecan chondroitin sulfate 846 epitope (CS846) were used as cartilage synthesis markers. Type II collagen cleavage (CIIC) was used as a marker of cartilage degradation. The concentration of each biomarker was evaluated using enzyme-linked immunosorbent assay (ELISA) kits, according to the manufacturer’s instruction (all ELISA kits were purchased from IBEX Technologies, Montreal, Canada). Absorbance was measured using a Multiscan FC Microplate reader (Thermo Scientific, Kanagawa, Japan).
Histological examination Gastrocnemius muscles and knee joint tissues were removed from the right hindlimbs of the 6wRUN group and from both hindlimbs of the BTX + 6wRUN group. To compare the joint cartilage thickness among age-matched groups, the right knee joints from 20-week-old, untreated rats (n = 5) were also collected. Gastrocnemius muscles were weighed and snap frozen in isopentane cooled by liquid nitrogen and stored at −80 °C. Transverse cryosections (10 μm) were cut and stained with hematoxylin and eosin. Knee joint tissues were dissected and immersion-fixed in 0.1 M phosphate-buffered 4% paraformaldehyde (pH 7.4) for 2 days at 4 °C. During fixation, the knee joint angle was kept at 90°
Ankle muscle alters knee joint integrity flexion. Samples were decalcified and cut into two pieces in the frontal plane, at the central level of the tibia, and embedded in paraffin wax. Transverse sections (5 μm) were cut and stained with Safranin-O for histological observation. In previous studies, running-induced knee joint cartilage degeneration was more pronounced in the lateral compartment than the medial one (Tang et al., 2008), and joint cartilage was more sensitive to altered loading conditions in the tibial plateau than in the femoral condyle (Liphardt et al., 2009). In addition, the loading site of the femoral condyle may be altered by changes in the knee joint angle, which is due to the fact that the gastrocnemius muscle weakens during the stance phase, and thus, in this study, we focused on joint cartilage in the lateral tibial plateau of the knee.
Histometrical analysis To evaluate cartilage metabolism in the knee joint, articular cartilage thickness was measured. Cartilage thickness, including uncalcified and calcified layers, was defined as the distance from the cartilage surface to the osteochondral junction. Accordingly, mean values of cartilage thickness were calculated by dividing the area of cartilage matrix by the length of the cartilage surface. The central portion of articular cartilage from each section was photographed at a magnification of ×10. Images were processed digitally, and cartilage matrix areas and surface length were measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA). To evaluate chondrocyte number, central portions of articular cartilage in the lateral plateau of each section were photographed at a magnification of ×20. The area of the cartilage matrix and the number of chondrocytes within this area were measured by ImageJ software and manually, respectively. Chondrocyte density was expressed as number per mm2.
3-D locomotion analysis To evaluate the effects of gastrocnemius muscle weakness, kinematic features of quadrupedal locomotion were analyzed both preand post-BTX injection at 1, 3, and 6 weeks in BTX + 6wRUN rats (n = 4). Hindlimb movement was assessed during treadmill locomotion, using a KinemaTracer system (Kissei Comtec, Nagano, Japan) and a treadmill machine (MK680S; Muromachi, Ltd., Tokyo, Japan). In this system, 3-D video recordings (60 Hz) were made with four digital cameras (Tokina, Tokyo, Japan), two of which were placed on the right side and two on the left side of the treadmill, to trace and analyze hindlimb joint movement during locomotion. Color markers corresponding to the lateral cleft between articulations (knee), lateral malleolus (ankle), the and fifth metatarsophalangeal joint were placed during each test day (Fig. 1). Each marker was traced using KinemaTracer software. The movement of the colored markers was recorded by video during locomotion at a speed of 10 m/min on a treadmill. The markers were automatically and manually traced, and the KinemaTracer software used to analyze the coordinates was calibrated by recording a cube of known size [20 × 10 × 5 (x × y × z)]. The position of each marker in the xyz plane was automatically calculated by the system, allowing it to measure the angle between ankle dorsal flexion and plantar flexion. We obtained values for the maximal ankle dorsiflexion angle in stance phase during locomotion. Excessive dorsiflexion range of motion (ROM) in the stance phase was considered as gastrocnemius muscle weakness induced by BTX. Four to eight consecutive steps were analyzed in each trial, and average values for the maximal dorsiflexion ROM in ankle joints were calculated using KinemaTracer software.
Statistics Statistical analysis was performed using Dr. Statistical Package for the Social Sciences (SPSS) II for Windows (SPSS Japan,
Fig. 1. Schematic presentation of marker positions for kinematic data collection from botulinum toxin type A (BTX)injected rats. In total, six reflective markers (2-mm diameter) were used to identify two segments of both hindlimbs. Ankle dorsiflexion range of motion (ROM) was defined through the following equation: angle = θ–90°. Inc., Tokyo, Japan) and modified R (The R Foundation for Statistical Computing, Perugia, Italy). Differences in maximal ankle ROM between right and left sides were evaluated using MannWhitney’s U-test at each time point. One-way analysis of variance (ANOVA) was used to statistically evaluate the differences between Cont and RUN groups, and between Cont and BTX + RUN groups, in muscle wet weight, ankle ROM (of each side of the animal), all biomarkers, and chondrocyte density. Tukey’s honestly significant difference post-hoc test was performed to clarify significant effects. Two-way repeated measures ANOVA was also conducted to examine the relationship between muscle weakness (with or without BTX) and running period (1, 3, and 6 weeks). For each ANOVA model with a significant direct or interaction effect, Bonferroni tests were performed posthoc to localize the effects. Cartilage thickness was evaluated by Kruskal–Wallis test and Steel–Dwass post-hoc tests were conducted. For all tests, statistical significance was accepted as P < 0.05.
Results Behavior All animals could run at experimental settings 3 days post-BTX injection. However, ankle plantarflexor movement during treadmill running was compromised on the experimental side in BTX + RUN groups. Importantly, the heel was in contact with the floor during the stance phases (in a normal rat, unlike a human, heel contact does not occur during the stance phase). The foot on the BTX-injected side was oriented outward when running. Such trends were prominent until 3–4 weeks post-BTX injection, and slightly reduced thereafter. Ankle joint ROM during locomotion Representative changes in ankle joint ROM during locomotion in BTX-injected rats are shown in Fig. 2(a). Temporal changes in maximum dorsiflexion ROM
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Ozawa et al. during the stance phase are shown in Fig. 2(b). The right (BTX-injected) side vs the left (contralateral) side were 37 ± 4° vs 33 ± 11°, respectively (right/left = 110%) before BTX injection, and 51 ± 8° vs 29 ± 4° (175%, P = 0.003), 47 ± 7° vs 33 ± 11° (168%, P = 0.003), and 35 ± 9° vs 24 ± 11° (142%, P = 0.13) at 1, 3, and 6 weeks post-BTX injection, respectively. Compared with the baseline (pre-injection), the right maximum dorsiflexion angles at 1, 3, and 6 weeks post-injection were 163% (P = 0.017), 148% (P = 0.080), and 114% (P = 0.971), respectively. On the left side, there was no statistically significant difference at any time point (109%, 99%, and 81% at 1, 3, and 6 weeks postinjection, respectively). These results suggest that compensatory changes in the contralateral side did not appear, at least in ankle ROM during the stance phase. Gastrocnemius muscle wet weight and histology
Fig. 2. Locomotion analysis data on ankle joint range of motion (ROM) during treadmill walking. (a) Representative locomotion patterns at 1w after botulinum toxin type A (BTX) injection and (b) mean values of maximal dorsiflexion angle during the stance phase at 1, 3, and 6 weeks pre- and post-injection. Values are means ± standard deviation. *indicates significant difference from contralateral side during the same running period (P < 0.05). ** indicates significant difference from the ipsilateral side at pre-injection (P < 0.05).
Table 1 shows the wet weight of gastrocnemius muscles in all groups. As compared with Cont, muscle wet weight was not affected in 1wRUN (95%, P = 0.638), but was significantly increased in 3wRUN (117%, P = 0.02) and in 6wRUN (117%, P = 0.002). In contrast, muscle wet weight was progressively decreased in BTX + 1wRUN (83%, P < 0.001), in BTX + 3wRUN (66%, P < 0.001), and in BTX + 6wRUN (49%, P < 0.001) despite prolonged exposure to exercise. Comparison between RUN groups and BTX + RUN groups over the same period revealed that wet weights of BTX-injected muscles were significantly decreased at 1 (87%, P = 0.001), 3 (56%, P < 0.001), and 6 weeks (42%, P < 0.001). Comparison between the ipsi- and contra-lateral sides of gastrocnemius muscles in BTX + RUN groups revealed that BTXinjected muscles decreased over time, which parallels the comparison between RUN groups and BTX + RUN groups. Comparing the RUN groups and the contralateral side of the BTX + RUN groups, which reflects the contralateral effects of BTX on gastrocnemius muscles, there were no differences in muscle wet weight at 1 (104%, P = 0.60) and 6 weeks (105%, P = 0.50), but muscle weight in the BTX + RUN group was slightly decreased at 3 weeks (87%, P < 0.05).
Fig. 3. Histological photographs of gastrocnemius muscles in botulinum toxin type A (BTX) + 6wRUN group. (a), Contralateral (left) side and (b), BTX-injected (right) side. Severely atrophied muscle fibers were observed at the BTX-injected site. Hematoxylin and eosin staining. Arrows = Indian ink included in injection. Scale bar: 100 μm.
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Ankle muscle alters knee joint integrity Table 1. Wet weight of the gastrocnemius muscle
Running period, week (s)
0 1 3 6
Cont, mg
1805 ± 61
(a) RUN, mg
1722 ± 95 2114 ± 93* 2112 ± 162*
BTX + RUN (b) Ipsilateral side, mg
(c) Contralateral side, mg
1505 ± 34* 1185 ± 117* 883 ± 115*
1710 ± 147 1875 ± 165 2020 ± 77*
(b)/(a), %
(b)/(c), %
88 ± 5† 51 ± 5† 42 ± 4†
88 ± 7‡ 58 ± 8‡ 42 ± 4‡
*Indicates significant difference from Cont (P < 0.05). † Indicates significant difference between BTX + RUN group and RUN group at the same running periods (P < 0.05). ‡ Indicates significant difference between Rt side and Lt side in BTX + RUN group at the same running periods (P < 0.05). Values are means ± SD.
Fig. 4. Concentrations of cartilage metabolism-related biomarkers in serum. (a) Type II collagen cleavage (CIIC), (b) Procollagen type II C-propeptide (CPII), (c) aggrecan chondroitin sulfate 846 epitope (CS846), and (d) CPII/CIIC ratio in Cont and groups with or without botulinum toxin type A (BTX) injection after 1, 3, and 6 weeks running. Data represent the means ± standard deviation (Cont and RUN groups, □; BTX + RUN groups, ■). *indicates simple main effect between groups with or without BTX at the same running period (P < 0.05).**indicates significant difference from the ipsilateral side at pre-injection (P < 0.05).
Histological observation also revealed that areas of gastrocnemius muscle exposed to the injectate were severely atrophied [Fig 3(b) ] compared with those in the contralateral (un-injected) side [Fig. 3(a)] of the BTX + 6wRUN group.
Cartilage metabolism biomarkers CIIC concentrations in Cont, 1wRUN, 3wRUN, and 6wRUN groups were 148 ± 16 ng/mL, 139 ± 28 ng/mL (94% of Cont), 127 ± 19 ng/mL (86% of Cont), and
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Fig. 5. Representative histological photographs of changes in the rat knee joint. Frontal section of the lateral tibial plateau were stained with Safranin O for articular cartilage matrix proteoglycan. (a) Age-matched control (Cont), (b) 6wRUN, (c) BTX + 6wRUN [botulinum toxin type A (BTX)-injected side], (d) BTX + 6wRUN (contralateral side). Arrow indicates cyst in cartilage layer. Scale bar: 100 μm.
104 ± 24 ng/mL (70% of Cont), respectively [Fig. 4(a)]. In RUN groups, CIIC tended to decreased over time, but had not reached significance even at 6 weeks. In BTX + RUN groups, CIIC decreased in BTX + 1wRUN (107 ± 20 ng/mL, 73%), and then recovered in BTX + 3wRUN (122 ± 42 ng/mL, 84%) and BTX + 6wRUN (149 ± 29 ng/mL, 100%). Interaction effect was detected between running and muscle weakness (P = 0.035), and the simple main effect was also detected between running and muscle weakness at 6 weeks (P = 0.033). CPII concentrations were 1876 ± 279 ng/mL, 2084 ± 119 ng/mL (111%), 2061 ± 276 ng/mL (110%), and 1950 ± 206 ng/mL (104%) in Cont, 1wRUN, 3Wrun, and 6wRUN groups, respectively [Fig 4(b) ]. In BTX + RUN groups, CIIC concentrations were 2000 ± 195 ng/mL (107%), 1941 ± 236 ng/mL (104%), and 1986 ± 176 ng/mL (106%) in BTX + 1wRUN, BTX + 3wRUN, and BTX + 6wRUN group, respectively. There was no significant difference between Cont and RUN groups, or Cont and BTX + RUN groups. No interaction effect between running and muscle weakness was detected. CS846 concentrations were 43.6 ± 3.0 ng/mL, 39.5 ± 6.3 ng/mL (91%), 39.9 ± 6.8 ng/mL (92%), and 41.0 ± 8.1 ng/mL (94%) in Cont, 1wRUN, 3wRUN, and 6wRUN groups, respectively [Fig 4(c)]. In the BTX + RUN groups, CS846 concentrations were 42.2 ± 8.3 ng/mL (87%), 39.3 ± 8.9 ng/mL (90%), and 35.8 ± 7.1 ng/mL (82%) in BTX + 1wRUN, BTX + 3wRUN, and BTX + 6wRUN, respectively. There was no significant difference between Cont and RUN groups, or Cont and BTX + RUN groups. No interaction effect between running and muscle weakness was detected.
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CPII/CIIC ratios, which represent total change of type II collagen, were increased in 1wRUN and 3wRUN groups (120% and 128%, respectively), and reached significance at 150% (P = 0.012) in the 6wRUN group [Fig. 4(d) ]. In BTX + RUN groups, CPII/CIIC tended to increase in BTX + 1wRUN (148%, P = 0.10), and then tended to decrease in BTX + 3wRUN and BTX + 6wRUN groups (135% and 107%, respectively). Interaction effect was detected between running and muscle weakness (P = 0.022), and a simple main effect was also detected at 6 weeks after running (P = 0.020). Histological analysis In Cont, dense staining with Safranin O, indicating rich GAG content, and arranged chondrocytes were observed at the lateral tibial plateau of the knee joint cartilage[Fig. 5(a)]. In 6wRUN, GAG content and cartilage layer thickness were well preserved (103%), and chondrocyte density (cell number per mm2) was not changed (86%, P = 0.17) [Figs 5(b), 6(a,b)]. Interestingly, single or multiple cyst formations were observed in the deeper zone of the calcified and/or uncalcified layer in three (60%) of the five subjects. In the experimental side of BTX + 6wRUN animals, although degenerative changes were not so severe, weakened staining with Safranin O in the cartilage matrix and partial disappearance of the tidemark, were occasionally observed [Fig. 5(c)]. Importantly, the cartilage layer was significantly thinner than in Cont (77%, P = 0.045) and in 6wRUN (75%, P = 0.045), and the chondrocyte density was also decreased compared with the Cont group (78%,
Ankle muscle alters knee joint integrity
Fig. 6. Histometrical scores in joint cartilage from the lateral plateau of rat knee joint. (a) Cartilage thickness and (b) the chondrocyte density. *indicates significant difference from Cont group (P < 0.05). **indicates significant difference from 6wRUN group (P < 0.05).
P = 0.021) [Fig. 6(b)]. In the contralateral side of BTX + 6wRUN group, a slight reduction of Safranin O staining and discontinuities of the tidemark were sometimes detected, but less than those in the experimental side. Chondrocyte density and cartilage thickness were not statistically different as compared with the Cont group (84% and 88%, respectively) [Figs 5(d), 6(a,b)]. With regard to the contralateral effects of BTX on the knee, there were no significant differences in cartilage thickness and chondrocyte density between the 6wRUN and BTX + 6wRUN groups. In the medial tibial plateau, we could not distinguish marked differences among all groups, in accordance with previous studies (Tang et al., 2008; Liphardt et al., 2009). Discussion The major finding of our study is that the inhibition of cartilage catabolism induced by low-speed treadmill
running is counteracted by gastrocnemius muscle weakness elicited by BTX injection. In addition, a marked decrease in chondrocyte density and cartilage thinning, which are known features of OA, are observed in the lateral plateau of the knee joint in BTX + 6wRUN, but not in the 6wRUN group. These results suggest that moderate running, combined with weakened ankle plantarflexor muscle strength has a harmful effect on the maintenance of cartilage integrity, although the intensity of running was not enough to exert cartilage degradation. The association between muscle weakness and the onset and progression of knee joint degradation has not been elucidated except by reports from Herzog and coworkers (Herzog & Longino, 2007; Rehan Youssef et al., 2009) focusing on quadriceps muscle weakness. Both increased and decreased loading conditions resulted in a reduction in joint cartilage thickness and cartilage degradation (Eckstein et al., 2006; Leong et al., 2011). Accordingly, we speculate that the adverse effects on joint cartilage might be concerned with changes in the mechanical environment of the surface of the knee joint, induced by ankle muscle weakness. Under motion analysis, maximal dorsiflexion ROM during the stance phase was larger in the BTX-injected side than in the contralateral side, at both 1 and 3 weeks after BTX injection. Forward progression of the tibia is controlled by the gastrocnemius muscle during the foot-flat to heel-off phases in humans (Dugan & Bhat, 2005). Thus, it was suggested that weakened plantarflexor muscles could not sufficiently resist the dorsiflexion generated by frontal moment in the stance phase. In addition, heel contact was observed during the stance phase, during locomotion after BTX injection. Unlike in humans, initial contact arises not from the heel but from the forefoot in rats, and heel contact does not appear during the stance phase in normal rat locomotion. Therefore, heel contact may reflect dysfunction in the ability to absorb the impact of ground reaction forces because of eccentric contraction of ankle plantarflexor muscles. Excessive impact loading on the hindlimb as a result of receiving ground reaction forces directly may give rise to the collapse of cartilage matrix homeostasis in the knee joint. In fact, excessive joint loading by strenuous running (Tang et al., 2008; Sekiya et al., 2009; Ni et al., 2011; Beckett et al., 2012) induced reduction in type II collagen expression, thinning of joint cartilage, and worsened histopathological score. However, a possible contradicting factor of disruption to cartilage homeostasis is decreased loading. Rehan Youssef et al. (2009) reported that quadriceps muscle weakness induced by BTX injection resulted in knee joint degeneration in rabbits. They noted that this degeneration might be attributed to muscle weakness, which interferes with normal loading of the knee. Weakness of the quadriceps muscle decreased vertical ground reaction forces in the push-off phase of hopping in BTX-injected animals (Longino et al., 2005). Likewise,
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Ozawa et al. force-transmission to the forefoot at heel rise was decreased in response to reduction in gastrocnemius muscle force (Chen et al., 2012). In this study, we could not determine whether the effects on knee joint cartilage induced by gastrocnemius muscle weakness depended on excessive or decreased loading. Reduction in chondrocyte density in the BTX + 6wRUN group was thought to be a result of increased cell death because there is little or no cell division or cell death in normal adult cartilage (Heijink et al., 2012). Increased cell death in joint cartilage was also observed in aging rats (Moriyama et al., 2012), in human OA (Harmand et al., 1982), and after electrical stimulation of the quadriceps muscle, which mimics joint loading in athletes or those performing heavy labor who have an increased rate of knee OA (Horisberger et al., 2012). Our findings indicate that ankle muscle weakness sufficient to induce walking disturbance might have negative effects on the maintenance of cartilage integrity in the knee joint, even after moderate exercise. Normal joint kinematics is important to the integrity of the knee joint cartilage. Breakdown of segmental regulation in motion, which manifests as walking disturbances, can shift the loading patterns to the knee joint cartilage in the stance phase. In addition, uncontrolled segmental motion, such as an increase in peak knee adduction moment and excessive tibiofemoral rotation, are assumed to increase mechanical stresses on the knee joint surface, contributing to the onset and progression of OA (Vincent et al., 2012). Our results show that our experimental regime had a protective effect of catabolism on the matrix of joint cartilage. Several studies also documented the positive effects of moderate exercise on knee joint cartilageboth morphologically and/or biochemically in animals (Otterness et al., 1998) and in humans (Bautch et al., 2000; Roos & Dahlberg, 2005; Helmark et al., 2011). However, both degradation and synthesis biomarkers of type II collagen were up-regulated in OA-affected horses (Frisbie et al., 2008) and dogs (Chu et al., 2002). These results indicate that anabolic effects on biomarker levels were not necessarily representative of positive effects on joint integrity. In addition, we observed cyst formation in 60% of 6wRUN subjects. Cyst formation is a possible sign of adverse effects on normal homeostasis of the knee joint cartilage (Beckett et al., 2012). Further, longterm study (> 6 weeks under our running conditions) is needed to evaluate the utility of moderate running exercise on knee joint cartilage. Various kinds of therapeutic exercises (i.e., quadriceps muscle strengthening, aerobic exercise, and
walking, etc.) have been recommended for symptomatic relief both in knee OA patients in systematic reviews (Bennell & Hinman, 2005; Fransen & McConnell, 2009) and clinical guidelines (Zhang et al., 2008). However, our results suggest that ankle muscle weakness is a possible risk factor for knee joint cartilage degradation, even under moderate running exercise. Therefore, it is crucial to pay attention to ankle muscle weakness during moderate intensity exercise therapy for knee OA patients. The current study has some limitations. First, notable differences in muscle function exist between rats and humans during walking and other locomotion patterns as described earlier. In addition, we did not undertake a histological study of the patellofemoral joint. It was reported that the quadriceps muscle weakness resulted in joint degeneration in retro-patellar cartilage rather than tibia or femur cartilage in rabbits (Rehan Youssef et al., 2009).
Perspectives In conclusion, using serum biomarker data, our study has shown that low-speed running exercise inhibited catabolic changes in rat knee joint cartilage. However, these effects were counteracted by gastrocnemius muscle weakness. Histological analysis showed thinning of the cartilage layer and reduction of chondrocyte density in BTX-injected knee joints. Using 3-D locomotion analysis, dysfunction of ankle plantarflexion was confirmed during the stance phase. These data suggest that the ankle muscle weakness might impair the homeostasis and integrity of the knee joint cartilage, albeit with a moderate exercise, which is proposed to be positive on the joint homeostasis. Therefore, patients with lower limb weakness should be considered for exercise programs designed for those with metabolic disorders of the joint cartilage such as knee OA. Key words: Articular cartilage, type II collagen, botulinum toxin type A, the gastrocnemius muscle, treadmill running.
Acknowledgements This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sport, Science, and Technology of Japan. We thank Mr Tomoaki Nishino for his technical assistance.
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