this may cause inconveniences with regard to activities of daily 
 living ; thus physiotherapy is often used to treat problems related to joint movement 
 limitation . in previous studies , we have reported the histopathological changes that occur 
 in the rat knee joint components during immobilization ; however , the changes have not been 
 fully clarified . joint immobilization induces muscle atrophy , articular cartilage loss , and 
 proliferation of connective tissue within the joint space1 , 7 . immobilization of joints 
 surrounded by edematous soft tissue often produces joint stiffness more quickly and more 
 severely than immobilization of nonedematous extremities8 . after two weeks of joint immobilization , the number of synoviocytes 
 in the posterior synovial intima was increased compared with a control group9 . watanabe reported atrophy of fat cells , 
 proliferation of fibroblasts , and narrowing of the spaces between collagen fibers in the rat 
 knee joint after immobilization10 . some 
 studies reported changes in joint components caused by remobilization after 
 immobilization8 , 11,12,13,14,15 . the range of motion of rat knees immobilized for 8 weeks remained 
 substantially reduced after a 4-week period of unassisted remobilization16 . but histopathological findings of the 
 joint components were not described in that study . in the precedent studies , 
 histopathological changes in joint components after remobilization and/or reloading during 
 joint contracture were observed . a few 
 studies have reported the effect of exercise during an immobilization period17 , 18 . tatsuta related that exercise during immobilization periods did not 
 improve range of motion but also that granulation tissue did not infiltrate into the 
 articular cavity19 . in this study , we 
 used external fixation in order to immobilize the joint and subjected the rats to exercise 
 during the immobilization period . the purpose of this study was to clarify the effects of the rom 
 exercise on joint components according to histopathological analysis . the protocol for these experiments were approved by the animal care committee and 
 institutional ethics committee of kanazawa university , and all procedures for animal care 
 and treatment were performed in accordance with the guidelines for the care and use of 
 laboratory animals at kanazawa university . in total , twenty - six 9-week - old adult male wistar 
 rats ( weighing 250275 g ) were used in this study . the rats were randomly divided into three 
 groups , the immobilization group ( n=10 ) , exercise group ( n=10 ) , and control group ( n=6 ) . the 
 animals were kept under normal conditions for one week before the start of experiments in 
 order to acclimatize them to the environment . they were housed , 1 per cage , in a room 
 maintained under a 12-hour light - dark cycle . the immobilization group and exercise group were anaesthetized ( pentobarbital sodium , 
 40  mg / kg bw , ip ) and operated on under sterile conditions . the right knee joints of the rats 
 in the immobilization group and exercise group were immobilized with external fixation at 
 120 degrees of flexion . a 2-mm longitudinal incision was made in the right hind thigh skin ; 
 the right femur and tibia were pierced by kirschner wire with a diameter of 0.8  mm , and the 
 wire was then bent ( fig . the right femur and tibia were pierced by kirschner wire with a diameter of 0.8  mm , 
 and the wire was then bent ( x - ray ) . ) . kirschner wires were fixed outside the wounds in a knee flexion posture with screw 
 ( 4  mm diameter ) and nuts . the instruments for external fixation weighed 6 to 7  g. the rat s 
 knee flexion angle ( 120 degrees ) was determined by the method described in our previous 
 studys13 , 21 , 22 . this immobilization 
 method is low cost , and immobilization can be reversed to perform exercise and then 
 reestablished easily . although the right hindlimb knee joint was immobilized , the rats were 
 able to move freely without restriction in their respective cages and had free access to 
 food and water . the right femur and tibia were pierced by kirschner wire with a diameter of 0.8  mm , 
 and the wire was then bent ( x - ray ) . range of motion exercise ( rom ex ) was started from the day after immobilization . rom ex was 
 performed in the exercise group once a day for 3 minutes , 6 days a week , for 2 weeks . the mean torque 
 necessary for the rat to passively extend the knee joint was 1 n , and this value was adopted 
 as the strength during the rom ex . the exercise consisted in 18 cycles of 10 seconds of 
 exercise ( a total of 3 minutes ) . the exercise time was determinate according to the results 
 of a preliminary experiment . in the first 5 seconds of the exercise cycle , the rat s right 
 hind limb was maintained in 120 degrees of hip flexion , and in the next 5 seconds , the limb 
 was pulled in the caudal direction with 1 n force applied by a manual force gauge . the rom 
 exercise intensity was controlled using a force gauge continuously during the 3 minutes . after each exercise , the rat knee was re - immobilized by external fixation at 120 degrees of 
 knee flexion . the control group did not undergo surgery and was not subjected to 
 exercises . after 2 weeks of intervention , the roms of all rat knees ( extension limitation ) were 
 measured under anaesthesia . in the rats in the immobilization and exercise groups , the 
 external fixation was removed , and shortly after the tight hind limbs of the rats were later 
 pulled with 1 n force in the caudal direction . after angle measurement , the knees of the rats were 
 re - immobilized at 120 degrees of flexion by external fixation . later , the rats were 
 sacrificed by intraperitoneal injection of an overdose of pentobarbital sodium . immediately 
 after euthanasia , their right hind limbs the right knees of the rats were fixed in 10% buffered 
 formalin and decalcified . after decalcification , they knees were cut in the sagittal plane 
 at the level of the anterior and posterior cruciate ligament . following neutralization with 
 5% sodium sulfate solution , the specimens were fixed , decalcified , and neutralized at 4  c 
 for 72 hours . the specimens were embedded in paraffin , sectioned at 3 m , and mounted on 
 microscope slides . all sections were stained with hematoxylin and eosin and toluidine blue 
 stains and used for observation in a histopathological examination . histopathological 
 analysis was performed by examining the synovial membrane , the joint capsule , and the joint 
 cartilage . knee limitation data were statistically analyzed using ibm spss statistics for windows 
 ( version 19.0.1 , ibm corp . rom was analyzed using one - factor analysis of 
 variance ( anova ) with bonferroni post - hoc multiple comparisons . the histopathological analysis showed no 
 swelling or signs of infection in the knees as a result of the intervention . the mean values of knee rom ( extension limitation angle ) were 19.33.0 degrees , 77.37.4 
 degrees , and 51.04.9 degrees in the control group , immobilization group , and exercise 
 group , respectively , and each groups showed a statistically significant difference 
 ( p<0.05 ) . the mean body weights were 326.08.8  g , 310.616.6  g , and 304.57.7  g in the 
 control group , immobilization group , and exercise group , respectively . there were 
 statistically significant differences ( p<0.05 ) between the control group and other 
 groups . in the control group , the joint capsule was typically composed of coarse and relatively 
 loose fibrous connective tissues . the joint capsule in the immobilization group and exercise 
 group showed narrowing of the collagen bundles in interstitial spaces but was less dense in 
 the control group ( fig . the joint capsule in the control group was typically composed of coarse and 
 relatively loose fibrous connective tissues , but those in the immobilization group and 
 exercise group showed narrowing of the collagen bundles of interstitial spaces ; the 
 joint capsule was less dense in the control group . a , control group ; b , immobilization 
 group ; c , exercise group . the surface of the articular cartilage in animals of the control group was smooth , 
 and the hyaline cartilage was directly exposed to the articular cavity ( fig . the surface of the 
 articular cartilage was smooth , and the hyaline cartilage was directly exposed to the 
 articular cavity ( a ) ; the cartilage matrix of the hyaline cartilage was stained with 
 toluidine blue ( b ) . f , femur ; t , tibia ;  m , meniscus . he stain 200 ) . the cartilage 
 matrix of the hyaline cartilage was stained with toluidine blue . in the immobilized group , the 
 articular cartilages were covered with the proliferating tissue composed of fibroblast - like 
 spindle - shaped cells ( fig . 4 . hyperplastic tissues infiltrated into the articular cavity and adhered to the 
 surface of the articular cartilage ( white arrow head ) , and infiltration of vessels 
 ( black arrows ) was observed . conversely , in the exercise group , hyperplasia of membrane - like tissue was localized 
 to the synovial membrane , and the infiltration of fibroblast - like spindle - shaped cells was 
 localized to the articular cavity ( fig . 5 . hyperplasia of tissue was localized to the meniscus ( black arrows ) , and the surface 
 of the articular cartilage was smooth ( a ) . the cartilage matrix of the 
 hyaline cartilage was stained with toluidine blue ( b ) . notably , the presence of erythrocytes that had leaked into the articular cavity was 
 observed in some exercise group animals ( n=6 ) , suggesting the presence of hemorrhage after 
 rom ex .   the joint capsule in the control group was typically composed of coarse and 
 relatively loose fibrous connective tissues , but those in the immobilization group and 
 exercise group showed narrowing of the collagen bundles of interstitial spaces ; the 
 joint capsule was less dense in the control group . a , control group ; b , immobilization 
 group ; c , exercise group . the surface of the 
 articular cartilage was smooth , and the hyaline cartilage was directly exposed to the 
 articular cavity ( a ) ; the cartilage matrix of the hyaline cartilage was stained with 
 toluidine blue ( b ) . he stain 200   hyperplastic tissues infiltrated into the articular cavity and adhered to the 
 surface of the articular cartilage ( white arrow head ) , and infiltration of vessels 
 ( black arrows ) was observed . he stain ( a ) , toluidine blue stain ( b ) 200   hyperplasia of tissue was localized to the meniscus ( black arrows ) , and the surface 
 of the articular cartilage was smooth ( a ) . the cartilage matrix of the 
 hyaline cartilage was stained with toluidine blue ( b ) . proliferation of intracapsular connective tissue and the formation of 
 adhesions are primary responses to limitation of motion5 . in this study , we observed the presence of granulation tissue - like 
 organization and infiltration in the joint cavity after two weeks of knee immobilization in 
 rats . the cartilage appeared to be more or less confluent with the overlying connective 
 tissue20 . these findings are consistent 
 with those reported in our previous study21 , 22 . during the first two weeks of 
 immobilization , rom limitation caused damage in the myogenic component , and after two weeks , 
 the arthrogenic component constituted more than 80% of the total restriction in rom20 . this suggests that in our contracture 
 model , the myogenic factor was stronger than the arthrogenic factor for rom limitation . rom 
 limitation was significantly decreased by exercise , presumably due to its property that 
 contributes to the maintenance of muscle extensibility . however , changes were found not only 
 in the muscular component but also in the joint component . proliferation of intracapsular 
 connective tissue and the formation of adhesions are primary responses to limitation of 
 motion20 . in the present study , extensive 
 hyperplasia of fibroblasts was observed in all animals in the immobilization group , but it 
 was less severe and was focally distributed in the animals in the exercise group . this 
 finding may suggest that rom exercise induces some change within the joint components and 
 tissue metabolism . connective - tissue proliferation was present in all three of the knees 
 immobilized for fifteen days , and it was well established at thirty days20 . the changes in the joint capsule were 
 observed from the early period , and there were no important differences between the 
 immobilized group and exercised group . in our model , we did not observe a significant 
 influence of exercise on the joint capsule ; however we can not rule out that some changes may 
 be observe in response to other exercise methods . we observed slight intra - articular 
 hemorrhage , which indicates that the exercises used in this study might cause laceration of 
 granulation tissue and adhesion . in this study , it was clarified that rom ex maintains range 
 of motion and reduces the changes in the joint components . therefore , it is necessary to examine several kinds of 
 frequency and the exercise strength in a future study . a number of studies have performed 
 remobilization following a joint immobilization period , and some have subjected animals to 
 exercise during an immobilization period .
[ purpose ] the aim of this study was to clarify the effects of the rom exercise on joint 
 components according to histopathological analysis . [ subjects and methods ] in total , 
 twenty - six 9-week - old adult male wistar rats were used in this study . 
 the rats were 
 randomly divided into three groups , the immobilization group ( n=10 ) , exercise group 
 ( n=10 ) , and control group ( n=6 ) . 
 the immobilization group and exercise group were 
 anaesthetized and operated on under sterile conditions . 
 the right knee joints in the 
 immobilization group and exercise group were immobilized with external fixation at 120 
 degrees of flexion . 
 range of motion exercise was started from the day after 
 immobilization . 
 rom exercise was performed in the exercise group once a day for 3 minutes , 
 6 days a week , for 2 weeks . 
 [ result ] the joint capsule in the immobilization group and 
 exercise group showed narrowing of the collagen bundles in interstitial spaces but was 
 less dense in the control group . in the immobilized group , a hyperplastic reaction was 
 associated with infiltration into the articular cavity and adhesion to the surface of the 
 articular cartilage . 
 conversely , in the exercise group , hyperplasia of tissue was 
 localized to the synovial membrane . 
 [ conclusion ] this finding may suggest that rom 
 exercise induces some changes within the joint components and tissue metabolism .