Rheumatology Advance Access originally published online on November 22, 2005
Rheumatology 2006 45(3):287-290; doi:10.1093/rheumatology/kei149
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Oxidative stress by glutathione depletion induces osteonecrosis in rats
Department of Orthopaedic Surgery and 1 Department of Pathophysiological and Experimental Pathology, Kanazawa Medical University, Ishikawa, Japan.
Correspondence to: T. Ichiseki, Department of Orthopaedic Surgery, Kanazawa Medical University, Daigaku 1-1, Uchinada-machi, Kahoku-gun, Ishikawa 920-0293, Japan. E-mail: tsy-ichi{at}kanazawa-med.ac.jp
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Abstract |
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Objective. We recently implicated in vivo oxidative stress in the development of osteonecrosis in a steroid-induced osteonecrosis model in the domestic rabbit. In the present experiment we devised a new non-traumatic model using the rat to investigate the relationship between oxidative stress and the development of osteonecrosis.
Methods. Seven 24-week-old male Wistar rats were subcutaneously injected with the pro-oxidant buthionine sulphoximine (BSO) 500 mg/kg for 14 consecutive days (group B) and eight rats received injections of vehicle (physiological saline; group N). The rats in both groups were killed after 14 days, and their bilateral femurs were examined histopathologically. Blood levels of reduced glutathione (GSH), total cholesterol (T-cho) and triglycerides (TG) were also determined.
Results. GSH was significantly decreased in group B compared with group N (P<0.01). No significant differences were found in T-cho or TG. Osteonecrosis was not detected in any animal in group N in contrast to five of seven animals in group B (P<0.05).
Conclusion. BSO is an inducer of oxidative stress, in particular interfering with the synthesis of GSH in vivo. In the present study, GSH levels were markedly reduced by BSO, whereas neither T-cho nor TG was significantly changed. The high rate of osteonecrosis noted in group B suggests that oxidative stress alone may be sufficient to promote the development of osteonecrosis at certain sites.
KEY WORDS: Osteonecrosis, Oxidative stress, Rat, Glutathione
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Introduction |
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Various theories have been proposed regarding the developmental mechanisms of steroid-induced osteonecrosis, and although the details remain unclear there is wide agreement that disturbed blood flow within bone is involved [1].
Because the development of animal models is considered important to clarify the developmental mechanisms of osteonecrosis and devise prophylactic strategies [2], various such models have been described, including the domestic rabbit disseminated intravascular coagulation shock model induced by administration of lipopolysaccharide [3] and an osteonecrosis model using spontaneously hypertensive rats (SHR) [4]. With regard to animal models of steroid-induced osteonecrosis, Yamamoto et al. [5] first described the development of osteonecrosis in the metaphysis and diaphysis in domestic rabbits given large doses of steroids. Our group also previously reported a model in which domestic rabbits given a single injection of steroid developed osteonecrosis with good reproducibility [6, 7].
However, as mentioned above, the developmental mechanisms of steroid-induced osteonecrosis are not yet clear, none of the available animals models being ideal. For example, in the rabbit, bone-specific genetic studies are relatively difficult to perform, and the sites of predilection of osteonecrosis differ from those in man, while in the SHR genetic abnormalities occur spontaneously [8–10].
On the other hand, we recently demonstrated in a rabbit model of steroid-induced osteonecrosis that oxidative stress underlies various pathological conditions, including the vascular injury associated with osteonecrosis development [6, 7]. Our aim in the present experiment was to expose the Wistar rat, which has been widely used as an animal experimental model that also lends itself to genetic studies, to oxidative stress so as to determine the relation between it and the development of osteonecrosis.
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Materials and methods |
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Animals
This study was performed in accordance with the guidelines of the Animal Research Committee of Kanazawa Medical University. Female Wistar rats aged 24 weeks were studied at the Animal Center of Kanazawa Medical University and were maintained on a standard laboratory diet and water.
Treatment
Eight rats were injected subcutaneously with the pro-oxidant buthionine sulphoximine (BSO) 500 mg/kg for 14 consecutive days (group B) and eight rats received only vehicle injections of physiological saline as a control (group N). In group B, one of the rats died on the second day after the start of the experiment, and so the study was conducted using the remaining seven rats. The rats in both groups were killed 14 days after the start of the injections, and their bilateral femurs were removed.
Tissue preparation
For light microscopic examination, tissue samples were obtained from the femur at the time of death, and were then fixed for 1 week with 10% formalin–0.1 M phosphate buffer, pH 7.4. The bone samples were decalcified with 25% formic acid for 3 days. The specimens were embedded in paraffin, cut into 4 µm sections, and stained with haematoxylin and eosin (HE).
Blood biochemical examinations
Blood levels of reduced glutathione (GSH), total cholesterol (T-cho) and triglycerides (TG) were determined using samples obtained from the abdominal aorta before the animals were killed and compared between the two groups.
Histopathological study
The presence/absence of osteonecrosis was investigated histopathologically using HE-stained specimens. The evaluation criteria of osteonecrosis were based on the report of Yamamoto et al. [5]; the presence of marked bone marrow haematopoietic cell and fatty cell degeneration and/or osteocyte empty lacunae was considered to indicate osteonecrosis. Also, osteonecrosis was judged to have developed in a given animal when it was found even in a unilateral femur, and the proportion of animals with osteonecrosis relative to the total number was determined.
Statistical analysis
Haematological data are given as mean ± S.D. Student's t-test was used to determine the significance of differences in GSH, T-cho and TG between groups N and B. The 
2 test was performed to determine the significance of differences in the presence/absence of osteonecrosis. Differences were considered significant when the P value was less than 0.05.
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Results |
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Blood biochemical examinations
GSH was significantly decreased in group B compared with N group (P<0.01). No significant differences were found in T-cho or TG (Fig. 1).
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Histopathological study
No findings of osteonecrosis were found in any of the rats of group N (Fig. 2a). In contrast, in group B, empty lacunae of osteocytes were noted in five of seven rats (71.4%), and the surrounding fat cells showed marked degeneration and disappearance of their structure. These findings resembled the histopathological picture of osteonecrosis found in man. The rate of development of osteonecrosis was significantly higher in group B than in group N (P<0.05). Particularly noteworthy is the site of osteonecrosis development in this model, namely the femoral head in three animals (42.9%) (Fig. 2b) and the femoral condyle in two (28.6%) (Fig. 2c), which are also the sites of predilection in man. The osteonecrosis in this model developed in all cases in the epiphysis, no animal showing findings of osteonecrosis in the metaphysis or diaphysis, the sites of predilection in the rabbit.
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Discussion |
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It has been demonstrated recently that oxidative stress develops soon after steroid administration, oxidative injury also occurring within bone [7]. Moreover, with regard to approaches to prevent the development of steroid-induced osteonecrosis, significant inhibition has been reported in animal models with the use of lipid-lowering agents such as lovastatin [11], probucol [12] and antioxidant reduced glutathione [6]. The fact that all of these agents exert an antioxidant effect supports the contention that oxidative stress is implicated in the development of steroid-induced osteonecrosis. Meanwhile, it has also been reported that, soon after steroid administration to the domestic rabbit, blood GSH levels significantly decrease [6]. GSH is an antioxidant enzyme in vivo and has attracted attention as a factor related to various functions, such as maintenance of membrane integrity and cell structure and the metabolism of foreign bodies. When GSH levels decrease, oxidative stress is induced, predisposing the organism to tissue and vascular injury [13–15].
The BSO used here to induce oxidative stress impairs redox function by blocking the synthesis of GSH in the organism, thereby inducing oxidative stress [16]. Also, in the present study, to further clarify the relation between oxidative stress and osteonecrosis development, BSO was administered for 14 consecutive days until the animals were killed.
The blood biochemical study confirmed that in group B, given BSO, GSH levels markedly decreased, whereas T-cho and TG did not change significantly, thus providing an opportunity to observe changes in bone induced by oxidative stress alone.
In the histopathological study, osteonecrosis was found in none of the animals in N group in contrast to five of seven in group B (71.4%). This result suggests that oxidative stress induced by decreased GSH is sufficient to cause osteonecrosis even in the absence of lipid abnormalities.
Another issue with the rabbit osteonecrosis model is that the sites of predilection of osteonecrosis differ from those in man. In the rat model devised here the sites of predilection of osteonecrosis were found to be more similar to those in man, osteonecrosis being noted in about 42.9% of rats in the femoral head and about 28.6% in the femoral condyle, thus more faithfully reproducing the pattern of osteonecrosis development seen in man. Thus, the rat may prove to be particularly useful in studies attempting to clarify the developmental mechanisms of osteonecrosis.
In conclusion, this new non-traumatic experimental model demonstrated that oxidative stress is an important element in the development of osteonecrosis. This model of pharmacologically induced osteonecrosis in the rat may facilitate more detailed studies targeting vascular and tissue injury than the rabbit models available hitherto.
The authors have declared no conflicts of interest.
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