document: male wistar rats ( 10 weeks old ) were obtained from sankyo labo service , co. ltd . 
   ( sapporo , japan ) . each animal was housed in an individual cage in a temperature- and humidity - controlled room with unlimited food and water and a 12-h light / dark cycle . all animals ( n = 22 ) were given 0.3-mg / kg lps ( from escherichia coli serotype 055 : b5 ; sigma , st . 
   louis , mo , usa ) intravenously on days 1 and 2 . on days 3 , 4 and 5 , 
   the animals were given 20-mg / kg methylprednisolone intramuscularly to promote development of non - traumatic onfh as described previously ( okazaki et al . 
   all the injections were administered at 7:00 p.m. hindlimb unloading was accomplished as described by wronski and morey - holton ( morey - holton & globus 1998 ) with some modifications ( okazaki et al . , unpublished observation ) . 
   the tail was then varnished with instant adhesive ( aron alpha ; toagosei co. , ltd . , 
   tokyo , japan ) , and a strip of traction tape ( skin - trac , 3  40 in . ; zimmer , warsaw , in , usa ) was then attached to the tail beginning at the base of the tail above the hairline . to facilitate free movement about the cage 
   , the cast was attached to a swivel anchored to the cage that allowed a 360 range of movement . 
   the animals were then suspended in a head - down tilted position . over time , the suspension angle was readjusted as the body size increased to prevent any weight coming to bear on the hindlimbs . in nwb rats ( n = 11 ) , the tail was suspended from day 1 to sacrifice . 
   the femur and humerus were harvested from each rat and fixed with 10% formalin-0.1 m phosphate buffer , ph 7.4 . 
   the tissues were processed for routine haematoxylin and eosin staining to assess the general architecture and osteonecrosis of the femoral and humeral heads . 
   we defined histopathological osteonecrosis as the diffuse presence of empty lacunae or pyknotic osteocyte nuclei in the bone trabeculae , accompanied by surrounding bone marrow cell necrosis ( figure 1 ) ( ichiseki et al . 
   histopathological appearance of proximal femoral epiphysis of weight - bearing ( wb ) rat with haematoxylin and eosin staining . 
   data were compared between wb and nwb groups using the chi - square test . a p - value of < 0.05 was considered to be significant . 
   male wistar rats ( 10 weeks old ) were obtained from sankyo labo service , co. ltd . 
   ( sapporo , japan ) . each animal was housed in an individual cage in a temperature- and humidity - controlled room with unlimited food and water and a 12-h light / dark cycle . all animals ( n = 22 ) were given 0.3-mg / kg lps ( from escherichia coli serotype 055 : b5 ; sigma , st . 
   louis , mo , usa ) intravenously on days 1 and 2 . on days 3 , 4 and 5 , 
   the animals were given 20-mg / kg methylprednisolone intramuscularly to promote development of non - traumatic onfh as described previously ( okazaki et al . 
   all the injections were administered at 7:00 p.m. hindlimb unloading was accomplished as described by wronski and morey - holton ( morey - holton & globus 1998 ) with some modifications ( okazaki et al . , unpublished observation ) . 
   the tail was then varnished with instant adhesive ( aron alpha ; toagosei co. , ltd . , 
   tokyo , japan ) , and a strip of traction tape ( skin - trac , 3  40 in . ; zimmer , warsaw , in , usa ) was then attached to the tail beginning at the base of the tail above the hairline . to facilitate free movement about the cage 
   , the cast was attached to a swivel anchored to the cage that allowed a 360 range of movement . 
   the animals were then suspended in a head - down tilted position . over time , the suspension angle was readjusted as the body size increased to prevent any weight coming to bear on the hindlimbs . in nwb rats ( n = 11 ) , the tail was suspended from day 1 to sacrifice . 
   the femur and humerus were harvested from each rat and fixed with 10% formalin-0.1 m phosphate buffer , ph 7.4 . 
   bone samples were decalcified with kalkitox ( wako pure chemical industries , ltd . , osaka , japan ) and then neutralized with sodium sulphate buffer . 
   the tissues were processed for routine haematoxylin and eosin staining to assess the general architecture and osteonecrosis of the femoral and humeral heads . 
   we defined histopathological osteonecrosis as the diffuse presence of empty lacunae or pyknotic osteocyte nuclei in the bone trabeculae , accompanied by surrounding bone marrow cell necrosis ( figure 1 ) ( ichiseki et al . 
   histopathological appearance of proximal femoral epiphysis of weight - bearing ( wb ) rat with haematoxylin and eosin staining . 
    
   all experiments adhered to the guidelines of the ministry of sports , culture , science and technology of japan and followed protocols approved by the animal ethics committee of the sapporo medical university ( # 09 - 008 ) . 
   femoral head osteonecrosis was observed in seven of 11 ( 63.6% ) wb rats and six of 11 ( 54.5% ) nwb rats , with no significant intergroup difference observed ( p = 0.66 ) . 
   figure 2 shows the histopathological appearance of the femoral head after haematoxylin and eosin staining in typical specimens from the wb ( a , b ) and nwb ( c , d ) groups . in the wb group , 
   empty lacunae were seen at the necrotic bone trabeculae , and the number of hematopoietic cells was decreased in the medullary space at most areas of the femoral head ( figure 2a , b ) . in the nwb group , empty lacunae 
   in addition , the number of haematopoietic cells was again decreased , and necrotic cell debris was accumulated in the medullary space at most areas of the femoral head ( figure 2c , d ) , as observed in the wb group . on the other hand , no humeral head osteonecrosis was observed in any of the wb or nwb rats . 
   typical specimens of weight - bearing ( wb ) ( a , b ) and non - weight - bearing ( nwb ) ( c , d ) rats . both wb and nwb group shows diffuse presence of empty lacunae in the bone trabeculae , accompanied by surrounding bone marrow cell necrosis at most areas of femoral head . 
   figure 3 shows the histopathological appearance of the humeral head in typical specimens from the wb ( a , b ) and nwb ( c , d ) groups . in the wb group , 
   normal trabeculae , as well as haematopoietic and fat cells , were observed ( figure 3a , b ) . 
   normal trabeculae were also observed in the nwb group rats . in comparison with the wb group 
   , a large number of haematopoietic cells , but few fat cells were observed in the nwb group rats ( figure 3c , d ) . 
   typical specimens of weight - bearing ( wb ) ( a , b ) and non - weight - bearing ( nwb ) ( c , d ) rats . 
   osteonecrosis of the femoral head often develops after corticosteroid therapy for inflammatory diseases , such as autoimmune diseases ( fukushima et al . 
   , it has been reported that mega - dose corticosteroid therapy for trauma , such as spinal cord injury , does not induce onfh ( wing et al . 
   therefore , we developed a non - traumatic onfh animal model in which onfh was encouraged by the administration of lps ( a ligand of toll - like receptor 4 , and implicated in inducing inflammatory status ) and methylpredonisolone treatment in the rats . in this animal model 
   , however , rats were allowed to walk and move independently in individual cages , with the hip joints supporting the loading from the lower body . 
   we could not exclude the possible influence of weight bearing on the hip joints on the induction of onfh . 
   therefore , we employed a hindlimb unloading procedure , in which the rat tail was fitted on to a suspension system with the rats in a head - down tilted position to prevent weight bearing on the hindlimbs . in this system , 
   we observed that nwb group rats developed onfh at a comparable rate as did wb group rats . 
   taken together , the results of the present study confirm that weight bearing does not contribute to the development of non - traumatic onfh in rats . to clarify the pathogenesis of onfh , 
   several quadruped animal models of osteonecrosis , including both traumatic and non - traumatic forms , have been reported ( yamamoto et al . 1995 ; norman et al . 
   however , the quadruped animals in these models fail to progress to end stage mechanical collapse similar to that in bipedal animals , such as humans ( conzemius et al . 
   xu et al . ( 2010 ) reported that this is caused by a difference in weight bearing through the hip joint between quadruped and bipedal animals . 
   however , our results showed that weight bearing did not contribute to the initial development of onfh in nwb rats . 
   therefore , it is speculated that the rat model may help us to elucidate the pathogenesis of onfh and to devise a prevention strategy for the condition , but may not be useful for the observation of the mechanism of femoral head collapse . in conclusion , the present study revealed that weight bearing is not related to the development of non - traumatic onfh in the rat model , although the aetiology , pathogenesis and prognosis of non - traumatic onfh remain unclear . 
   
summary: the hip joint is one of the major structures in the human body and the resultant force acting through the hip joint is 300% of body weight . 
   therefore , weight bearing , as a cause of ischaemia , may contribute to the development of non - traumatic osteonecrosis of the femoral head ( onfh ) . 
   however , it remains unclear whether weight bearing is related to the development of non - traumatic onfh . 
   therefore the aim of this study was to clarify the role of weight bearing in the development of non - traumatic onfh . 
   non - weight - bearing ( nwb ) rats were tail suspended to prevent any weight coming to bear on the hindlimbs from day 1 to the time of sacrifice . 
   the weight - bearing ( wb ) group rats were also housed individually , although without tail suspension . 
   all rats were injected with lipopolysaccharide and methylprednisolone to promote the development of non - traumatic onfh . 
   all animals were sacrificed three weeks after the final methylprednisolone injection . 
   histopathological analysis was performed . 
   osteonecrosis of the femoral head was observed not only in the nwb but also in the wb rats ; however , no osteonecrosis of the humeral head was observed in either group . 
   we confirmed that non - traumatic onfh developed in nwb rats , suggesting that weight bearing does not contribute to the development of non - traumatic onfh in rats .