Elsevier

Bone

Volume 28, Issue 6, June 2001, Pages 641-649
Bone

Original article
Osteonecrosis induced by a single administration of low-dose lipopolysaccharide in rabbits

https://doi.org/10.1016/S8756-3282(01)00460-4Get rights and content

Abstract

We succeeded in developing a novel rabbit model of nonsteroid and nontraumatic osteonecrosis (ON) by use of a single- and low-dose lipopolysaccharide (LPS) injection. This model is simple and highly reproducible for the frequent development of multifocal and widespread ON lesions. Male adult Japanese white rabbits intravenously injected with a single injection of 10 μg/kg body weight of LPS were histopathologically examined in the early phase (3 [n = 3], 5 [n = 3], and 24 h [n = 3]) and at 4 weeks (n = 22). Seventy-seven percent of the rabbits developed multifocal ON 4 weeks after LPS injection. ON was also observed in the femoral and humeral condyle. The average percentage of necrotic area/total area examined was 86.7 ± 29.1% and 78.8 ± 16.7% in the proximal one third of both the femoral and humeral bones, respectively. Organized thrombi in the intraosseous small-sized arteries and arterioles were frequently seen in and around the necrotic tissues. In the early phase, LPS treatment prominently induced thrombocytopenia, hyperlipidemia, and increased plasma levels of plasminogen activator inhibitor-1 (PAI-1). The plasma level of PAI-1 was significantly higher in the rabbits with ON than in those without ON (p < 0.01). The immunohistochemical expression of tissue factor was exaggerated in monocytes/macrophages and adipocytes in both the femoral and humeral bones of the LPS-treated rabbits. Histologically, marrow necrosis and fibrin thrombi could be observed at 24 h. In addition, pretreatment with an anticoagulant, warfarin potassium, significantly decreased the incidence of LPS-induced ON (33%, n = 9, p < 0.05) associated with elongation of prothrombin time. The results of our study show that a single administration of low-dose lipopolysaccharide induces multifocal and widespread ON characterized by the pathophysiological participation of hypercoagulability in ON development. Therefore, this model would be useful for elucidating the pathogenesis of nonsteroid ON in humans especially inflammatory hypercoagulability-induced as well as for developing preventive and therapeutic strategies.

Introduction

There has been much clinical progress in the therapeutic and diagnostic aspects of nontraumatic osteonecrosis (ON) of the femoral head (ONFH). In particular, use and applications of magnetic resonance imaging (MRI) for ONFH have now made possible early detection of lesions and accurate classication of clinical stage of disease.31 Although several theories regarding the pathogenesis of ONFH, including increased bone marrow pressure at the femoral head,18, 29 enlargement of fat cell size,43 accumulation of lipid within osteocytes,22 fat embolism,7, 20, 43 obstruction of venous outflow,15 intravascular coagulation20, 41 and altered fibrinolysis10, 41 have been proposed, the pathogenesis of ONFH remains unclear. In fact, ONFH has often been known to develop following such various morbid conditions as the use of corticosteroids, especially pulse therapy or high-dose therapy for systemic lupus erythematosus1, 35 and renal transplantation,25 alcohol abuse,11, 19, 28 sickle-cell anemia,3, 14, 44 Gaucher disease,21 antiphospholipid antibody syndrome,5, 30 and others.31

The major problem in studying the pathogenesis of human ONFH results from the lack of any clinicopathological information during its earliest events, because it is very difficult to make a clinical diagnosis immediately after the occurrence of ONFH. Secondary bone deformity has normally already occurred by the time patients complain of such clinical symptoms as hip pain, gait disturbance, or limitations in the range of motion of the hip. Therefore, it is necessary to develop useful animal models for human ONFH of various etiologies. Some experimental animal models for nontraumatic ONFH have been reported and proved beneficial in pathophysiological studies of ONFH. Matsui et al.27 reported steroid-induced ON in rabbits with Arthus reaction that was provoked by repeated intravenous injections of horse serum. We previously developed a rabbit model by combining the Shwartzman reaction and corticosteroid injections and thus found steroid treatment to enhance the incidence of ON not only in the metaphysis and diaphysis but also in the epiphysis.41 We also recently reported that a single injection of high-dose corticosteroid could induce multifocal ON in rabbits, which were also associated with thrombocytopenia, hypofibrinogenemia, and hyperlipemia.42

Although steroid-independent ON is known to represent half of all patients with ONFH,2 to our knowledge, only a few animal models have been developed for nontraumatic and nonsteroid ON.34 We therefore attempted to generate a new, highly reproducible rabbit ON model using a single intravenous injection of low-dose LPS, but without steroid treatment. We herein report the histopathological characteristics of ON lesions and refer to the pathogenesis based on the coagulation/fibrinolysis system and lipid metabolism in this animal model.

Section snippets

Animals

Adult male Japanese white rabbits (Kyudo, Tosu, Japan), weighing 3.0–4.0 kg 30–34 weeks old, when growth plates of the femoral and humeral bones had already been closed, were used as experimental animals. All animals tested were kept under routine laboratory conditions at the Animal Center of Kyushu University. They received a standard laboratory diet and had free access to food and water in their cages. All experiments were reviewed by the ethics committee on animal experiments at the Graduate

Body weight

The body weight in the rabbits without ON showed the lowest value at 48 h and recovered to the basal level at 4 weeks, whereas the rabbits with ON continued their reduction in weight up to 2 weeks, with an average weight loss of 0.79 ± 0.28 kg (22.0% of prior body weight at 2 weeks), and 0.60 ± 0.34 kg (16.7% of prior body weight) at 4 weeks. Weight loss was more prominent in rabbits with ON than in those without ON from 72 h through 4 weeks (p < 0.01). The control group showed no remarkable

Discussion

LPS is a constituent of the cell wall of Gram-negative bacilli and has been well known to possess various biological activities, including activation of the immune system and the induction of inflammation-circulatory disturbance, and thus LPS participates intimately in the development of such morbid conditions as intravascular coagulation24 with or without LPS shock, fat embolism,13 multiple-organ dysfunction,40 hyperlipidemia,8 and others.36 In particular, LPS activates vascular endothelial

Acknowledgements

The authors thank Hiroshi Fujii, Shizuko Yugawa, and Yuko Fukai for their very helpful technical assistance and Dr. Brian Quinn for his review of the manuscript. This work was supported in part by a research grant for intractable diseases from the Ministry of Health and Welfare of Japan.

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