Zheng Li-Zhen, Wang Jia-Li, Kong Ling, Huang Le, Tian Li, Pang Qian-Qian, Wang Xin-Luan, Qin Ling
Musculoskeletal Research Laboratory, Department of Orthopaedics & Traumatology, The Chinese University of Hong Kong, Hong Kong Special Administrative Region.
Translational Medicine R&D Center, Institute of Biomedical and Health Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, PR China.
J Orthop Translat. 2018 Feb 6;13:13-24. doi: 10.1016/j.jot.2018.01.003. eCollection 2018 Apr.
Established preclinical disease models are essential for not only studying aetiology and/or pathophysiology of the relevant diseases but more importantly also for testing prevention and/or treatment concept(s). The present study proposed and established a detailed induction and assessment protocol for a unique and cost-effective preclinical steroid-associated osteonecrosis (SAON) in rats with pulsed injections of lipopolysaccharide (LPS) and methylprednisolone (MPS).
Sixteen 24-week-old male Sprague-Dawley rats were used to induce SAON by one intravenous injection of LPS (0.2 mg/kg) and three intraperitoneal injections of MPS (100 mg/kg) with a time interval of 24 hour, and then, MPS (40 mg/kg) was intraperitoneally injected three times a week from week 2 until sacrifice. Additional 12 rats were used as normal controls. Two and six weeks after induction, animals were scanned by metabolic dual energy X-ray absorptiometry for evaluation of tissue composition; serum was collected for bone turnover markers, Microfil perfusion was performed for angiography, the liver was collected for histopathology and bilateral femora and bilateral tibiae were collected for histological examination.
Three rats died after LPS injection, i.e., with 15.8% (3/19) mortality. Histological evaluation showed 100% incidence of SAON at week 2. Dual energy X-ray absorptiometry showed significantly higher fat percent and lower lean mass in SAON group at week 6. Micro-computed tomography (Micro-CT) showed significant bone degradation at proximal tibia 6 weeks after SAON induction. Angiography illustrated significantly less blood vessels in the proximal tibia and significantly more leakage particles in the distal tibia 2 weeks after SAON induction. Serum amino-terminal propeptide of type I collagen and osteocalcin were significantly lower at both 2 and 6 weeks after SAON induction, and serum carboxy-terminal telopeptide was significantly lower at 6 weeks after SAON induction. Histomorphometry revealed significantly lower osteoblast surface and higher marrow fat fraction and oedema area in SAON group. Hepatic oedema appeared 2 weeks after SAON induction, and lipid accumulation appeared in the liver of SAON rats 6 weeks after SAON induction.
The present study successfully induced SAON in rats with pulsed injection of LPS and MPS, which was well simulating the clinical feature and pathology. Apart from available large animal models, such as bipedal emus or quadrupedal rabbits, our current SAON small model in rats could be a cost-effective preclinical experimental model to study body metabolism, molecular mechanism of SAON and potential drugs developed for prevention or treatment of SAON.
The present study successfully induced SAON in a small animal model in rats with pulsed injection of LPS and MPS. The evaluation protocols with typical histopathologic ON features and advanced evaluation approaches to identify the metabolic disorders of SAON could be used in future rat SAON studies. The SAON rat model is a suitable and cost-effective animal model to study molecular mechanism of SAON and potential drugs developed for prevention and treatment of SAON.
建立临床前疾病模型不仅对于研究相关疾病的病因和/或病理生理学至关重要，更重要的是还可用于测试预防和/或治疗方案。本研究提出并建立了一种详细的诱导和评估方案，用于通过脉冲注射脂多糖（LPS）和甲基强的松龙（MPS）在大鼠中建立独特且经济高效的临床前类固醇相关骨坏死（SAON）模型。
选用16只24周龄雄性Sprague-Dawley大鼠，通过静脉注射一次LPS（0.2mg/kg）和腹腔注射三次MPS（100mg/kg），间隔24小时来诱导SAON，然后从第2周开始至处死，每周三次腹腔注射MPS（40mg/kg）。另外12只大鼠用作正常对照。诱导后2周和6周，对动物进行代谢双能X线吸收法扫描以评估组织成分；收集血清检测骨转换标志物，进行微血管灌注造影，收集肝脏进行组织病理学检查，并收集双侧股骨和双侧胫骨进行组织学检查。
LPS注射后有3只大鼠死亡，即死亡率为15.8%（3/19）。组织学评估显示第2周时SAON发病率为100%。双能X线吸收法显示SAON组在第6周时脂肪百分比显著更高，瘦体重更低。微计算机断层扫描（Micro-CT）显示SAON诱导6周后胫骨近端有明显的骨质破坏。血管造影显示SAON诱导2周后胫骨近端血管明显减少，胫骨远端渗漏颗粒明显增多。SAON诱导后2周和6周时，血清I型胶原氨基端前肽和骨钙素均显著降低，SAON诱导后6周时血清羧基端肽显著降低。组织形态计量学显示SAON组成骨细胞表面显著更低，骨髓脂肪分数和水肿面积更高。SAON诱导2周后出现肝水肿，SAON诱导6周后SAON大鼠肝脏出现脂质蓄积。
本研究通过脉冲注射LPS和MPS成功在大鼠中诱导出SAON，很好地模拟了临床特征和病理。除了现有的大型动物模型，如双足鸸鹋或四足兔外，我们目前的大鼠SAON小型模型可能是一种经济高效的临床前实验模型，用于研究SAON的机体代谢、分子机制以及开发用于预防或治疗SAON的潜在药物。
本研究通过脉冲注射LPS和MPS成功在大鼠小型动物模型中诱导出SAON。具有典型组织病理学骨坏死特征的评估方案以及用于识别SAON代谢紊乱的先进评估方法可用于未来的大鼠SAON研究。SAON大鼠模型是研究SAON分子机制以及开发用于预防和治疗SAON的潜在药物的合适且经济高效的动物模型。
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