joint contracture occurs when joint movements are reduced , such as by immobilization of a 
 joint with a plaster cast , orthosis , or after prolonged bed rest .
several conditions limit 
 the range of motion ( rom ) of joints , including cerebrovascular diseases , neuromuscular 
 diseases , bone and joint diseases , and various other diseases .
contracture may be classified into three groups : arthrogenic , myogenic , and soft 
 tissue1 .
arthrogenic contracture is 
 caused by immobility caused by damage and inflammation of cartilage , synovial tissue , and 
 joint capsule .
soft 
 tissue contracture occurs because of pathological shortening of connective tissue , including 
 the skin , subcutaneous tissue , tendons , and ligaments .
examples of intrinsic causes include 
 trauma , inflammation , degenerative changes , and ischemia .
examples of extrinsic causes 
 include spasticity , flaccid paralysis , malpositioning , and immobilization .
many investigators have studied the pathogenesis of contracture in dogs , monkeys , rats , and 
 other experimental animals .
these studies provided evidence of a relationship between the 
 period of immobilization and rom limitation factors , and changes in joint components and 
 soft tissue , including muscle and skin . in addition , decreases in mobility and plasticity of 
 the nervous system are considered to cause rom limitation2 .
nervous system mobilization is used as a physical therapeutic 
 approach to improve such conditions .
it uses palpation or joint movements to initiate 
 physical or physiological changes in blood flow or the axonal flow of nerves , aiming to 
 improve the plasticity of the nerve3 . in 
 addition
, nervous system mobilization has been reported to be effective at improving rom 
 limitation and pain , and induces the largest longitudinal excursion of the nerve relative to 
 its surrounding structures without being associated with a potentially detrimental increase 
 in nerve strain4,5,6 . on the other hand ,
there is 
 no evidence that mobilization of neural tissue , independent of other anatomical structures , 
 would be feasible in living people , or that nervous system mobilization is effective in the 
 treatment of musculoskeletal dysfunction .
therefore , it is still unclear whether nervous 
 system mobilization is effective7,8,9,10 . as stated above , nervous system mobilization is used to improve rom limitation and other 
 conditions , but the changes that it causes in the nerves and the periphery in contracture 
 arising from secondary disabilities or prolonged bed rest remain unclear .
the present study was 
 performed to determine changes in the tissue located in the periphery of the sciatic nerve 
 during contracture in an animal model .
  immobilization model male wistar rats ( 9 weeks old , n = 29 , weight 240  280  g ) were divided randomly into a 
 control group ( c group , n = 7 ) and immobilized group ( i group , n = 22 ) , and were kept in 
 plastic cages for 2 weeks .
the rats had unlimited activity in the cage and free access to 
 food and water .
the animal room was maintained at a constant temperature of 22  c , and the 
 animals were maintained under a 12-h light / dark cycle to avoid effects arising from the 
 biological rhythm of the rats .
this experiment was performed in accordance with the 
 guidelines for the care and use of laboratory animals of kanazawa university and the 
 protocol for this experiment was approved by the animal research committee of kanazawa 
 university .
rats in i group had the left knee joint immobilized for 2 weeks in the posture of maximum 
 flexion with a plaster cast of our own making and aluminum wire netting .
the casts were 
 applied under deep anesthesia induced by intraperitoneal injection of pentobarbital sodium 
 ( 40  mg / kg ) ( fig .
care was taken to avoid 
 disturbance of blood flow to the hip and ankle joints due to pressure from the cast . during 
 the immobilization period
, wounds and edema were prevented and if the plaster cast came off 
 immobilization was repeated again as soon as possible .
animals in c group were given food ad 
 libitum , and had no restrictions placed on their mobility .
at the end of the immobilization period , the rats were euthanized with diethyl ether and 
 both hip joints were disarticulated immediately .
the joint tissue was fixed with 10% 
 formalin neutral buffer solution for 72  h decalcified in edta at 4  c for 72  h. after 
 decalcification , the specimens were cut vertically at the center of the femur and 
 neutralized with 5% sodium sulfate for 72 h , and then embedded in paraffin .
the embedded 
 paraffin blocks were cut into sections at a thickness of 3 m .
we examined the periphery of the sciatic nerve under an optical microscope ( bx51 ; olympus ) . 

the software 
 program jmp7 ( sas institute japan inc . ) was used for statistical analyses , and values of p 
 < 0.05 were accepted as statistically significant .
the perineural space ( black arrows ) can be seen between the 
 bundle of nerve fibers and the perineurium ( dotted arrows ) .
adherence between the bundle of nerve fibers and the 
 perineurium ( black arrows ) , and thickening of the perineurium ( dotted arrows ) were 
 observed .
scale bar : c : 500 m , d : 200 m . in c group ,
the bundle of nerve fibers tended to separate from the perineurium , and a space 
 was clearly observed between the bundle of nerve fibers and the perineurium ( fig . 2 and table 
 1 ) .
conversely , in the rats of i group , strong adherence between the bundle of 
 nerve fibers and perineurium was observed in 19 of 22 animals ( fig .
3 and table 1 ) , and 
 thickening of the perineurium was detected in 20 of 22 of these rats ( fig . 3 and table 2 ) . 

generally , peripheral nerves consist of several bundles of nerve fibers . in these bundles 
 of nerve fibers , each nerve fiber , including the schwann cells enveloping it , is surrounded 
 by endoneurium that consists of areolar connective tissues containing blood vessels . each 
 bundle of nerve fibers is covered by perineurium , which is a dense layer composed of 
 collagenous connective tissue enveloped by 7 or 8 layers of squamous epithelial - like 
 cells11 .
there is a space between the 
 perineurium and the bundle of nerve fibers , but there have been no previous anatomical 
 descriptions of this space .
peripheral nerves consist of more than one bundle of nerve 
 fibers and the epineurium consisting of areolar connective tissues .
the bundles of nerve 
 fibers are combined with each other and form a nerve trunk , with a cylindrical strong 
 connective tissue sheath12 .
the sciatic nerve is the largest nerve in humans and represents most of the nerve fibers 
 forming the sacral plexus .
this nerve descends into the posterior compartment of the thigh from the gluteal region 
 through the greater sciatic foramen .
proximal to the knee , and sometimes within the pelvis , 
 the sciatic nerve divides into its two terminal branches , the tibial nerve and the common 
 fibular nerve .
it innervates all muscles in the posterior compartment of the thigh , lower 
 leg , and foot , as well as most of the skin of the lower leg and foot13,14,15 .
the spinal nerves in the rat consist of 34 pairs of 
 nerves : 8 cervical , 13 thoracic , 6 lumbar , 4 sacral , and 3 caudal .
the sacral plexus in the rat is 
 more limited in the extent of its origin than the human one .
it is formed by the fifth , and 
 the adjacent parts of the fourth and sixth lumbar nerves .
the sciatic nerve divides into its 
 two terminal components , the common fibular nerve and the tibial nerve , which in their 
 course through the thigh cross the obturator externus , quadratus femoris , and adductor 
 magnus , lying between these muscles and biceps femoris , until they reach the popliteal fossa 
 where they separate .
as described above , the sciatic nerve tract in the rat is almost the 
 same as in humans16 .
the nervous system adapts to lengthening in two ways ,  movement  and  elongation2 , 3 . 

movement may be considered as gross movement or movement occurring intraneurally between the 
 connective tissues and the neural tissues .
gross movements refer to movement of the system 
 as a whole in relation to the connective tissue interface .
a peripheral nerve sliding 
 through a tunnel , such as the median nerve , is a clear example of this type of gross 
 movement .
intraneural movement refers to movement of the neural tissue elements in relation 
 to the connective tissue interfaces .
the brain or the spinal cord can move in relation to 
 the dura mater and a fascicle can slide in relation to another fascicle in peripheral 
 nerves . on the other hand , elongation is like elastic gum and the nervous system adapts to 
 joint movement by increasing intraneural pressure or intradural pressure .
the looseness of 
 nerve fibers , epineurium , and perineurium decrease as the nerve is elongated .
the epineurium 
 is only slightly involved in resisting elongation of the nerve , and resistance is mainly 
 offered by the perineurium .
reductions in these adaptations induce limitation of rom , pain , 
 and neurological symptoms .
generally , nervous system mobilization can increase the mobility 
 of the nerve by improving the motility and extensibility of the nervous system when movement 
 and elongation are reduced due to the influence of various factors , but there is no 
 histological evidence of such an effect . in this study , a space was observed between the bundle of nerve fibers and the perineurium 
 of the sciatic nerve in the c group .
it is possible that this space is an artificial 
 histopathological image caused by differences in the rate of contraction between the bundle 
 of nerve fibers and perineurium tissue in the process of specimen preparation .
however , in i 
 group this space was not observed , so , we considered it unlikely that this was an artificial 
 image appearing only in i group .
there have been no anatomical descriptions of this space and it has not been the focus of 
 prior study .
however , in pancreatic cancer , gastric cancer , and prostate cancer , the 
 invasion of cancer tissue into this space ( perineural invasion ) has been seen ; therefore , 
 this space has attracted attention and is known as the  perineural space . 
watanabe17 reported that cancer cells do not invade 
 bundles of nerve fibers , but scatter along loose connective tissue in the perineurium .
the 
 results of experimental and clinical studies indicate that the perineural space is 
 independent of the lymphatic system and there is no traffic between the two18 , 19 .
in addition , miyazaki20 reported that perineural invasion shows continuous tumor spread , 
 unlike lymphatic invasion and vein invasion , and there is no flow in the perineural space , 
 unlike lymphatic and blood vessels .
as mentioned above , it is clear that there is a space 
 between the bundle of nerve fibers and the perineurium of autonomic nerves .
however , there 
 have been no previous reports of its presence in somatic nerves and its physiological 
 significance remains unknown .
further studies and descriptions of the perineural space have 
 not been reported to date . in c group ,
the bundle of nerve fibers separated from the perineurium physiologically , and 
 there was a perineural space in the periphery of not only the autonomic nerve , but also the 
 somatic nerve . on the other hand
, there was no perineural space in i group : the bundle of 
 nerve fibers tended to make contact with the perineurium , and there was the possibility of 
 adhesion between the two .
with regard to this adhesion , if the perineurium functions as a 
 buffer between the bundle of nerve fibers and the peripheral tissue of the nerve , it is 
 possible that this adhesion decreased the mobility and plasticity of the bundle of nerve 
 fibers .
thomas21 reported that the 
 perineurium acts as a mechanical barrier to external forces , and sunderland22 reported that it was the structure most 
 resistant to tensile forces .
we suggest that thickening disturbs these functions of the 
 perineurium , causing a decrease in the buffer function and the mobility and plasticity of 
 the nerve .
this is the first study to examine the changes in the periphery of nerve tissue 
 after joint immobilization with histopathological observations . in conclusion
, abnormal histopathological images were observed in not only the joint 
 components and soft tissue , but also in the peripheral tissue of nerves in the rat knee 
 joint contracture model indicating joint immobilization may affect the peripheral tissue of 
 nerves .
   [ purpose ] this study was performed to investigate the histological changes that occur in 
 the periphery of the sciatic nerve in rats undergoing knee immobilization . 
 [ subjects and 
 methods ] 29 male 9-week - old wistar rats were divided randomly into a control group ( c 
 group , n = 7 ) and an immobilized group ( i group , n = 22 ) . 
 the animals in the i group had 
 the left knee joint immobilized in maximal flexion with plaster casts for two weeks . 
 after 
 the experimental period , we obtained cross - sections of tissues from the center of the left 
 thigh , and the periphery of the sciatic nerve was observed under an optical microscope 
 after hematoxylin - eosin staining . 
 [ results ] in contrast to the rats of c group , the rats 
 in i group showed adherence between the bundle of nerve fibers and perineurium , as well as 
 thickening of the perineurium . 
 these histological changes were statistically significant . 
 
 [ conclusions ] immobilization of the knee joints of rats resulted in characteristic 
 histological changes in the connective tissue around the sciatic nerve . 
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION