It is known that loading force to bone is one of the key factors influencing bone metabolism. Unloading, such as long-term bed rest,6,13,14,28,32,37,42 space flight,3,4,18,32 leg fixation after orthopaedic surgery,22 and bone fracture,7 induces bone loss. Animal experimental models are useful for investigating the underlying mechanism of osteopenia under reduced loading, and for developing a method of its prevention or treatment or both. To mimic this type of osteopenia, several animal models have been proposed, including tail suspension,17,19,20 back suspension,39 limb taping,15,16 nerve resection,26,41 tenotomy,35 confinement to restricted caging,12 spaceflight,19,36 amputation,30 and limb casting.10,11,34 There are few experimental data concerning unloading a murine model.26,31 The cellular responses in bone tissue after unloading, such as osteoblast surface and osteoclast number, have not been well documented.
All the above models have their advantages and disadvantages. The tail suspension model seems to be good for simulating spaceflight-induced osteopenia because it reproduces the fluid shifts under microgravity in space.24 However, it causes hypertension resulting from increased cephalic arterial pressure,38 and this model also induces selective skeletal muscle atrophy.8 Moreover, the unloading causes adrenal hypertrophy,6 which is an indicator for stress in experimental animals. In the limb-tapping model,15 the cephalic fluid and cerebral blood flow seem to be normal; however, animals are stressed because of unbalance of body dimension and overloading to anterior limbs. We think that it still is useful to develop an animal model for osteopenia caused by reduced loading if the new model is simple and accurately mimics disuse osteopenia in human patients.
Therefore, the purpose of this study was to develop a simple osteopenia model under reduced loading using mice, plastic tubes, and wires, and to characterize this model in histomorphometric analysis.
MATERIALS AND METHODS
Thirty male ICR mice (8 weeks old; average weight, 39 g) were used. Twenty mice were divided into four experimental groups for immobilization periods of 3, 7, 10, or 14 days. Ten mice were used as controls. The baseline control and 14-day control animals were sacrificed at Day 0 and Day 14, respectively. Five mice in each group were housed in a conventional plastic cage for small animals (inside length x width x height of 22x17x12 cm), floored with small wood chips. Five pellets (weight, approximately 3 g each) of standard laboratory food containing 1.25% calcium and 1.06% phosphate (CE-2 pellet type for mice, rats, and hamsters; Japan Clea, Tokyo, Japan) per day were placed directly in the cage for pair-feeding as described elsewhere.1 A limited amount of food, approximately 3 g/day, was given to each animal. Water was allowed ad libitum. The protocol for our study was approved by our institution’s animal experiment committee and all the experimental processes followed the institutional guidelines for the care and manipulation of laboratory animals.
Leg Immobilization Procedure
The leg immobilization procedure is shown in Figure 1. Conventional Eppendorf-type 1.5-mL plastic tubes were used, with the bottom ⅓ of the tube cut off at an angle. The cap also was removed and two small holes were drilled as close to the top as possible and another hole was drilled close to the cut edge. Under Nembutal (pentobarbital; Dainippon Pharmaceutical Co, Osaka, Japan) anesthesia, a thick wire (0.9 mm in diameter, 15 cm in length) was looped around the abdominal region like a belt. Both hind legs of the animals in the experimental group were inserted into these tubes; the tubes then were fixed to the thick wire (0.9 mm in diameter) belt with two thin wires (0.3 mm in diameter, 6 cm in length). Both tubes again were fixed with medium wire (0.6 mm in diameter, 10 cm in length) positioned at the back of the animal to hold the animal’s legs open during immobilization. The medium wire was fixed to the thick wire with another thin wire.
All mice except those in the Day 0 and Day 3 groups were injected intraperitoneally with 5 mg/kg of body weight of calcein (CL; Sigma Chemical Co, St Louis, MO) and 10 mg/kg of body weight of tetracycline HCl (TC; Lederle Laboratories, Pearl River, NY) for histomorphometric analysis 7 days and 1 day before sacrifice, respectively. At 3, 7, 10, and 14 days after immobilization, the mice in each group were sacrificed under chloroform anesthesia. The femurs and tibias were dissected out and then fixed in 10% neutralized formalin (10% formalin in phosphate buffered saline) for 2 days at 4°C.
Bone Mineral Density (BMD) Measurement and Histomorphometric Analyses
After washing the dissected samples with deionized water overnight, total BMD and BMD of 20-fragmented regions of femurs and tibias were measured with dual-energy xray absorptiometry (DEXA) for small animals (DCS-600R, Aloka Co, Tokyo, Japan). For histomorphometric analysis, the left femurs and tibiae were embedded in methylmethacrylate as described elsewhere,2 and undecalcified 10-μm thick frontal sections were prepared using a Reichert Jung microtome (Leica Microsystems, Heidelberg, Germany). One part of these sections was stained with Von Kossa stain. Under a fluorescence microscope (Axiophoto, Carl Zeiss, Eching, Germany), the samples were subjected to measurement for interlabeling fluorescence and double labeling distance. Right femurs and tibias, however, were first decalcified in 10% EDTA and embedded in glycol methacrylate (JB4 Plus Embedding Kit, Polysciences, Warrington, PA). The 1.5-μm thick frontal sections were prepared and stained for tartrate-resistant acid phosphatase (TRAP) and counterstained with toluidine blue.29 Standard histomorphometric measurements23 were done in the area of the secondary spongiosa of the distal femur in a 0.4-mm2 area (defined as the area between 1 and 3 mm proximal to the distal growth plate-metaphyseal junction of the femur) and in the same area of the proximal tibia (defined as the area between 0.5 and 2 mm distal to the proximal growth plate-metaphyseal junction of the tibia) under x20 magnification using an image-analyzing system (KS 400, Carl Zeiss, Eching, Germany). Static and dynamic bone histomorphometric parameters were obtained from decalcified and undecalcified sections, respectively.
All measurements are presented as mean ± one standard deviation (SD). The Kruskal-Wallis test followed by the Mann-Whitney U test in post hoc analysis were done to compare the effects of immobilization at each experimental time by using Statview Statistical Analysis Software v5.0 (SAS Institute Inc, Cary, NC). Values of p < 0.05 were regarded as statistically significant.
We did not observe any physical problems in any of the mice during the experiment. Under pair-feeding conditions, body weights of the control mice decreased (Table 1): 39.4 ± 2.6 g at the beginning and 36.0 ± 1.2 g on Day 14 (8% decrease of their initial weight). However, this change was not statistically significant (p = 0.06). Body weight in the experimental group decreased from 37.2 ± 2.4 g on Day 0–33.2 ± 3.4 g on Day 7 (10% decrease of their initial weight) in this study but subsequently remained unchanged from Day 7 (33.2 ± 3.4 g) to Day 14 (33.3 ± 1.7 g). Although we tried to maintain pair-feeding conditions, a significant difference between the control and immobilized groups was observed at Day 14 (36.0 ± 1.2 g versus 33.3 ± 1.7 g; p < 0.05). There was no significant difference in the length of femurs between the control and immobilized mice (data not shown), suggesting that this model does not affect bone growth in young growing mice.
The decrease in total femoral BMD in the experimental group compared with the control group was observed in DEXA analysis (Table 2). This change was statistically significant (p < 0.05). Total BMD of the tibia of the immobilized animals was not affected. Fragmented regional analysis of BMD measurement revealed a marked decrease in BMD at the trabecular bone-rich region (fragments 1–5 and 15–19) of the immobilized femurs, whereas the BMDs of the cortical-bone-rich midshaft were less affected (Fig 2A). In the tibia (Fig 2B), the fragmented regional analysis of BMD measurement showed a slight decrease in BMD, but only in the proximal trabecular bone-rich region (fragments 2–3). Figure 3 shows the histologic findings of distal femurs of the control and immobilized groups. The trabecular bone in the immobilized femurs decreased markedly compared with those of the control femurs at Days 10 and 14, and trabecular number in the immobilized femurs was lower than in the femurs of the control animals. In the tibias, however, the degree of trabecular bone loss was not remarkable (data not shown).
Histomorphometric analysis of distal femurs (approximately fragment 18 of Figure 2A) and proximal tibias (approximately fragment 3 of Figure 2B) revealed deterioration of trabecular bone, which started from Day 7 and continued until Day 14 (Tables 3, 4). In the immobilized animals, although trabecular thickness was not affected, trabecular numbers of the femur at Day 7, Day 10, and Day 14 dramatically decreased by 23.4%, 38.4%, and 42.6%, respectively, and bone volumes of the femur at Day 7, Day 10, and Day 14 also decreased by 26.8%, 36.5%, and 46.7%, respectively. These alterations were statistically significant compared with the controls (p < 0.05). In contrast, only the trabecular number of the tibia at Day 10 decreased by 30.2%, which was significantly different compared with those of the controls (p < 0.05). These histomorphometric changes in the trabecular bone support the results of the slice analysis of BMD measurement of DEXA. Osteoblast surface of femurs and tibias together with dynamic histomorphometric parameters, such as mineral apposition rate and bone formation rate had decreased in the immobilized animals at Days 7, 10, and 14. At Day 14, the osteoblast surface of the femurs and tibias decreased by 51.4% and 49.7%, and bone formation of the femurs and tibias decreased by 48.4% and 18.5%, respectively. The osteoclastic parameters (osteoclast surface, osteoclast number) increased at Day 7 in the femurs and the tibias of the immobilized animals and were elevated until Day 14. The osteoclast number of femurs and tibias at Day 7 increased to 182% and 134%, respectively.
We developed an apparatus for immobilizing mouse hind legs and showed that leg immobilization using this apparatus induced bone loss. The decrease in trabecular bone in the femurs was the most characteristic feature of this experimental model. Total tibial BMD, however, was not affected in our immobilized model. The possible reason for this is that during the experiment the mouse was able to partially move the lower part of the leg, below the knee while wearing the apparatus. Because long bones comprise mainly cortical bone, it would mask the change in BMD in the trabecular bone. Loading force to bone is one of the key factors that controls bone metabolism, and it is accepted that a decrease in the loading force induces osteopenia.35,39
Several osteopenia models10–12,15–17,19,26,30,34–36,39,41 already have been proposed to mimic osteopenia under reduced loading. Among them, the tail suspension model is frequently used. However, numbers of special cages, similar to the number of animals in the experiment,20 are necessary in this model. Furthermore, keeping the animals in a suspended condition for a long period requires extensive care. It is possible that bone loss in the tail-suspension model is not only caused by removal of loading but also is attributable to mental distress because of nonphysiologic posture during tail traction. Furthermore, the blood distribution in the suspended animal is obviously not physiologic. In addition, the head tilt-down osteopenia model has been shown to induce a marked shift in cephalic fluid,24,38 cardiovascular deconditioning such as an increase in mean arterial pressure and heart rate,21 adrenal hypertrophy,5 and altered renal function.33 None of these problems is likely to occur in our immobilization model. The other model, involving leg immobilization by taping or casting, is similar to our model, although one hind limb, not both, usually is immobilized in these models; it also is technically difficult. In our model, because only the experimental site is immobilized, the animal can ambulate, therefore the above problems seem to be avoided. The key advantage of our model is its simplicity and ease of application. Because the immobilized mice still could ambulate, they all tolerated the treatment and showed fewer physical problems. Moreover, compared with nerve resection and the tenotomy osteopenia model, which induce irreversible osteopenia, our immobilized animals are able to recover from the immobilization so we are able to study the recovery process. Therefore, our model could induce reversible localized osteopenia, which mimics the surgically fixed leg of the patient.
One limitation of our immobilized model is that the femurs are immobilized in extension and the animal cannot flex the hind legs. In our preliminary study, erosive skin lesions under the immobilized apparatus were not observed through 14 days; however, they did appear in some of the animals by 21 days. Therefore, the period of immobilization is limited and our immobilized model remains to be improved.
Simske et al31 reported that immobilized mice consume less food than control animals. Generally, if the animals had free access to food, the weight of the control animals might have exceeded that of the immobilized animals. Differences in food intake and body weight affect bone metabolism.40 Pair-feeding conditions are needed in immobilized models. In our immobilized animals, a decrease in body weight from Day 0 to Day 3 was inevitable. Although we tried pair feeding, at Day 14 the body weights of the experimental group mice were lower than those of the control group. In our model, achieving accurate pair-feeding conditions is a difficult issue. In the current study, however, the BMD of femurs in the experimental groups decreased because of the effects of the conditions of reduced loading, not because of decreased body weight, because the total BMD of the tibias in the experimental group was not affected, even with loss of body weight, although the tibias might experience sufficient weightbearing to prevent bone loss during immobilization.
Bone is a remodeled tissue, and bone volume is a result of the balance between bone formation and resorption. Bone remodeling is a coupled process between bone formation and bone resorption; however, absence of loading force results in reduced bone formation and leaves bone resorption activity temporarily unopposed, as has been shown in previous immobilization models.26,27 In our study, histomorphometric analysis showed the characteristic features of osteopenia in the cancellous region of the femur, as previously observed in other osteopenia models with less mechanical stress.12,15–17,25,33–35,37 At Day 7 after immobilization, femoral bone formation parameters (osteoblast surface, mineral apposition rate, and bone formation rate) decreased, whereas bone resorption parameters (osteoclast surface and osteoclast number) increased. Similar findings in static and dynamic parameters alterations were observed by Rantakokko et al25 who used the tail suspension model; however, the degree of alteration was different. After 14 days of immobilization, the osteoblast surface decreased to ⅓ and osteoclast number increased 1.7-fold compared with those of the control whereas Rantokokko et al reported that after 10 days of immobilization the osteoblast number reduced to 70% and the osteoclast number increased threefold. In our study, increased osteoclast population occurred before the decrease in osteoblast population. However, the mineral apposition rate and bone formation rate, which indicate osteoblast activity, were significantly reduced as early as the osteoclast number increased at Day 7. Therefore, it is possible that osteoclast and osteoblast activities initially affect the process of osteopenia. The balance between bone formation and resorption was disturbed in our immobilized model and consequently the trabecular bone in the femur decreased, supporting the results of previous reports.25,27 This lack of balance peaked at Day 7 and continued until Day 14. Similarly, in previous osteopenia models17 for reduced loading force, decreased bone formation together with increased bone resorption is evident, although the degree and time of this uncoupling is different for each model. Similar results were obtained in tibias. It is conceivable that the proximal tibia might be subjected to decreased load, which may in part be attributable to the extension of the immobilizing apparatus to cover the proximal tibia. Our dynamic histomorphometric parameters showed that our model, as in the other models,15,25,27,35,41 induced bone resorption and suppressed bone formation.
Systemic stresses are factors that burden the animal and influence bone metabolism. Morey-Holton and Globus20 proposed the indicators for stress included thymus atrophy, adrenal hypertrophy, and the increase of plasma glucocorticoid such as corticosterone. Corticosterone level in the plasma has not been examined in detail in murine osteopenia models of reduced loading. It is controversial whether the hind-limb unloading models cause excessive stress. Deavers et al5 reported adrenal hypertrophy in rat models at the end of a 7-day experiment, whereas Halloran et al9 showed that plasma corticosterone do not change in normally loaded and unloaded rats at all times. We did not examine the plasma corticosterone level in the current study. However, we think that our model results in less stress because the animal can ambulate to some extent. The reduction in body weight possibly resulted from the pair-feeding procedure and not from systemic stress of our apparatus. Although blood flow may affect bone remodeling, we did not observe any muscular morphologic or dimensional changes that might affect blood flow. The precise relationship between blood flow and bone remodeling under osteopenic conditions still must be found.
We developed a simple immobilized osteopenia model using mice, plastic tubes, and wires. Loading force to bone is a key factor affecting bone remodeling; however, the mechanism by which loading force affects bone remodeling is not clear. Application of our immobilization model to transgenic or knockout mice is one way to clarify this mechanism. Furthermore, our immobilization model has potential for use in development of therapeutic drugs for osteopenia.
We thank Dr. Tatsuya Shibata, Department of Dental Pharmacology, Faculty of Dentistry, Tsurumi University, Japan, for extensive technical support.
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