ARTHRITIS & RHEUMATISM Vol. 63, No. 7, July 2011, pp 2138–2141 DOI 10.1002/art.30365 © 2011, American College of Rheumatology
BRIEF REPORT
Osteonecrosis Development in a Novel Rat Model Characterized by a Single Application of Oxidative Stress Toru Ichiseki, Ayumi Kaneuji, Yoshimichi Ueda, Shintaro Nakagawa, Tomoaki Mikami, Kiyokazu Fukui, and Tadami Matsumoto Objective. To investigate the relationship between transient oxidative stress and the development of osteonecrosis in a rat model. Methods. A total of 160 male Wistar rats (24 weeks old) were injected only once with the pro-oxidant DLbuthionine-(S,R)-sulfoximine (BSO) (500 mg/kg given intraperitoneally) and were killed 12 hours (group A), 1 day (group B), 3 days (group C), 4 days (group D), 5 days (group E), 7 days (group F), or 14 days (group G) after administration (n ⴝ 20 per group). Twenty untreated rats were used as a control group (group N). Femurs were examined histopathologically for the presence of osteonecrosis, and reduced glutathione (GSH) in liver tissue was measured as an index of oxidative stress. Results. GSH decreased rapidly after BSO administration. Significant decreases were noted in groups A and B as compared to group N (P < 0.0001 and P ⴝ 0.0007, respectively), confirming the development of transient extreme oxidative stress soon after BSO administration. The histopathologic study revealed osteonecrosis in 10% of the rats in group E, 35% of the rats in group F, and 40% of the rats in group G. Conclusion. Transient extreme oxidative stress was confirmed to induce osteonecrosis in this model. Since preparation of this model is extremely simple and because rats are well suited to genetic studies, this model may be of use in elucidating the pathophysiology of femoral head osteonecrosis in future studies.
Although it has been shown that oxidative stress is related to osteonecrosis (1–3), the details of its pathogenesis remain unclear. Animal models are needed to clarify the pathophysiology of osteonecrosis and to establish prophylactic treatment strategies. Although previously rabbit models have been most commonly used for the study of osteonecrosis (1–5), we recently hypothesized that models of induced osteonecrosis in rats, which are particularly well suited for genetic studies, would be of greater use. We have previously demonstrated that subcutaneous injection of DL-buthionine-(S,R)-sulfoximine (BSO) in rats on consecutive days was by itself sufficient to induce osteonecrosis (6). However, when BSO is administered to rats for 14 consecutive days, oxidative stress persists for 14 days, making it unclear whether, as in steroid-induced osteonecrosis, osteonecrosis occurs as a single event or whether it results from cytotoxicity associated with chronic oxidation. The mechanisms underlying the process from oxidation induction to the development of osteonecrosis continue to be obscure, and we consider this type of model inadequate to explore them. Steroid-induced osteonecrosis can occur after a single exposure to these agents, and in a model using rabbits administered steroids, oxidation has been shown to also develop soon after a single exposure (2,3). This suggests to us the need for a rat model in which the development of osteonecrosis after a single application of oxidative stress can be reliably observed, to clarify the mechanisms underlying the development of steroidinduced osteonecrosis. We thus undertook the present study, in which we investigated the influence of a single application of extreme oxidative stress on bone and the development of osteonecrosis in rats.
Dr. Ichiseki’s work was supported by the Japanese Ministry of Education, Culture, Sports, Science and Technology (grant 21591963). Toru Ichiseki, MD, PhD, Ayumi Kaneuji, MD, PhD, Yoshimichi Ueda, MD, PhD, Shintaro Nakagawa, MD, PhD, Tomoaki Mikami, MD, PhD, Kiyokazu Fukui, MD, PhD, Tadami Matsumoto, MD, PhD: Kanazawa Medical University, Uchinada, Japan. Address correspondence to Toru Ichiseki, MD, PhD, Department of Orthopaedic Surgery, Kanazawa Medical University, Daigaku 1-1, Uchinada-machi, Kahoku-gun, Ishikawa 920-0293, Japan. E-mail:
[email protected]. Submitted for publication July 11, 2010; accepted in revised form March 17, 2011.
MATERIALS AND METHODS Animals. One hundred sixty male Wistar rats age 24 weeks (body weight 400–450 gm) were studied at the Animal Center of Kanazawa Medical University. All rats were housed 2138
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under standard laboratory conditions (temperature 24°C, 12hour light/dark cycle) and were given food and water ad libitum. They were given a single intraperitoneal injection of BSO (500 mg/kg) and were killed 12 hours (group A), 1 day (group B), 3 days (group C), 4 days (group D), 5 days (group E), 7 days (group F), or 14 days (group G) after administration. Untreated rats were compared as a control group (group N). Each group contained 20 rats. This study was conducted in accordance with the guidelines of the Animal Research Committee of Kanazawa Medical University. Tissue preparation. All rats were killed using an overdose of intraperitoneally injected sodium pentobarbital. The bilateral femurs were harvested 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–0.1M 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 3-m sections using a microtome (Sakura Seiki), and stained with hematoxylin and eosin (H&E). Glutathione (GSH) levels in liver tissue. GSH in liver tissue was measured as an index of in vivo oxidative stress. Two milliliters of 0.1M phosphate buffer (pH 7.0) was added to 100 mg frozen liver tissue and homogenized. Then, 1 ml of the homogenate and an equal volume of 0.6N HClO4–1 mM EDTA were placed in ice water for 10 minutes. After centrifugation for 10 minutes (4°C, 3,000 revolutions per minute), 1.5 ml supernatant was obtained, to which 3.0M K2CO3 was added, and it was left in ice water for 30 minutes. GSH in the supernatant was then quantified using the 5,5⬘-dithiobis(2nitrobenzoic acid)–glutathione reductase recycling method (7). Histopathologic study. H&E-stained specimens were prepared, and the presence or absence of osteonecrosis in the femoral head was investigated histopathologically (5,6). Osteonecrosis was determined as present when there were empty
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lacunae or condensed nuclei in osteocytes, or if degeneration of bone marrow cells was evident, even if limited to a unilateral femur. Finally, the proportion of animals with osteonecrosis relative to the total number was calculated. Statistical analysis. Liver GSH levels were expressed as the mean ⫾ SD. Statistical analysis was performed using analysis of variance with Dunnett’s multiple comparison test. By using this test we were able to determine the point in time after administration of BSO that GSH showed a significant decline, as compared with the values in the control group. P values less than 0.05 were considered significant. Statistical analysis was performed using StatView J-5.0 software (SAS Institute).
RESULTS Levels of GSH in liver tissue. GSH levels in liver tissue were significantly reduced in groups A and B as compared to group N (P ⬍ 0.0001 and P ⫽ 0.0007, respectively). In groups of mice killed 3 or more days after BSO administration, GSH levels tended to gradually recover, with no significant difference found, compared with group N (Figure 1). Histopathology. Group N, as well as groups A, B, C, and D, showed no development of osteonecrosis. In 2 of 20 animals (10%) in group E, 7 of 20 (35%) in group F, and 8 of 20 (40%) in group G, bone cell empty lacunae associated with necrosis of bone marrow hematopoietic cells and fat cells in the femoral head (which are findings typical of osteonecrosis) were confirmed (Figures 2C and D).
Figure 1. Glutathione (GSH) values in liver tissue. Significant decreases were found at 12 and 24 hours after administration of DL-buthionine-(S,R)-sulfoximine, after which levels tended to recover. Values are the mean ⫾ SD. ⴱ ⫽ P ⬍ 0.05 versus controls (group N).
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Figure 2. Histopathologic study of femoral head. In the control group (group N), normal bone tissue was present with no findings of osteonecrosis (A and B). In group F, in addition to marked degeneration of bone marrow hematopoietic cells and fat tissue, empty lacunae were found, representing typical osteonecrosis (C and D). Original magnification ⫻ 40 in A and C; ⫻ 200 in B and D.
DISCUSSION We have recently studied decreased GSH levels and oxidative stress as they are related to osteonecrosis (2,3); the decrease of GSH to ⬃30% of normal levels was shown in a model of osteonecrosis in rabbits (2). Furthermore, induction of osteonecrosis by oxidative stress alone was demonstrated in a model using rats; BSO was subcutaneously injected for 14 consecutive days, which disabled the oxidative reduction function of GSH by interfering with its synthesis (8,9). In order to further clarify the involvement of reduced GSH levels and oxidative stress in osteonecrosis, we attempted in the present study to create a model of transient extreme oxidation in rats by using a single dose of BSO (500 mg/kg), which decreases GSH levels in tissue to ⬍30% of preadministration levels in a few hours (10,11). The fact that all of the groups in our study showed significant decreases in levels of GSH at 12 and 24 hours after administration of BSO, followed by a tendency for levels to recover, confirmed that extreme oxidation was induced with a single application. Also, in the histopathologic study, osteonecrosis was confirmed
on day 5 after administration of BSO in 10% of the rats (group E) and on days 7 and 14 in 35% and 40% (groups F and G), respectively. These findings demonstrate that potent oxidative stress develops soon after a single dose of BSO, and that this is sufficient to induce osteonecrosis in this model. Sato et al reported that, in a rat model, apoptosis in osteocytes began 12 hours after the development of ischemia, and osteonecrosis appeared after 96 hours, while other investigators have noted a similar disease course of osteonecrosis developing 4–5 days after ischemia (12,13). Accordingly, in the present model, by counting back from the time osteonecrosis was confirmed 5–7 days after the BSO injection, we conclude that the intended induction of osteonecrosis (including ischemia) completed by 1–3 days after BSO administration. The results of the current study thus suggest that investigations of the underlying mechanisms of and potential prophylactic treatment for osteonecrosis of the femoral head should focus mainly on a period of up to 3 days from the administration of BSO. In conclusion, we have described a reliable and simple model of osteonecrosis, based on a single admin-
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istration of the pro-oxidant BSO to rats, which are well suited for genetic studies. Moreover, the period from drug administration to desired result is short, making this an extremely useful model for investigating the mechanisms underlying femoral head osteonecrosis and potential treatments for the disease. AUTHOR CONTRIBUTIONS All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Ichiseki had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Ichiseki, Kaneuji, Ueda, Nakagawa, Mikami, Fukui, Matsumoto. Acquisition of data. Ichiseki, Kaneuji, Ueda, Nakagawa, Mikami, Fukui, Matsumoto. Analysis and interpretation of data. Ichiseki, Kaneuji, Fukui.
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