ES and Androgens in Bone Loss after SCI: Synergistic Effects and Mechanisms

描述（由申请人提供）： 脊髓损伤（SCI）导致严重的骨丢失，这可能导致病理性骨折，并因此增加发病率和医疗保健费用。到目前为止，最有效的战略，保留或恢复骨脊髓损伤后利用电刺激（ES）诱导的肌肉骨骼负荷。一个关键的问题是ES的有益作用是否反映了骨吸收率的降低，新骨形成的增加，或两者兼而有之。这些问题的答案很可能指导未来策略的发展，以减少SCI后骨丢失，通过确定那些骨代谢的组成部分，没有最佳刺激ES，因此可能是其他类型的干预目标。我们的初步数据表明，一个星期后，通过ES的SCI骨重新加载减少骨吸收活性通过抑制破骨细胞的生成和破骨细胞的活性在大鼠SCI模型。重要的是，ES逆转了SCI诱导的Wnt抑制剂（DKK 1，sFRP 2和SOST）在成骨细胞中表达的上调，并增加了Wnt反应基因骨保护素（OPG）在这些细胞中的表达，OPG是破骨细胞分化和活性的抑制剂。 脊髓损伤相关的骨丢失可能会因睾酮循环水平的降低而恶化，因为这种类固醇激素对骨具有合成代谢作用，并且其水平通常在脊髓损伤后降低。最近，我们观察到诺龙，一种合成代谢类固醇，减少了脊髓损伤后的骨丢失。有趣的是，诺龙增加了脊髓损伤大鼠骨髓来源的成骨细胞中OPG，Runx 2和LRP 5的表达。LRP 5是与控制骨量密切相关的Wnt辅助受体，Runx 2是另一个Wnt应答基因，并参与成骨细胞分化。这些发现表明，诺龙在未加载骨中的一个作用是刺激成骨细胞中的Wnt信号传导，并且Wnt信号传导至少解释了诺龙在SCI后对骨的一些保护作用。 我们的研究结果提出的中心假设是：1）ES对骨的影响是由于骨吸收率降低和新骨形成增加; 2）当与ES联合应用时，合成代谢类固醇将为SCI相关的骨丢失提供额外或协同益处。如果是这样的话，这些发现将很容易转化为临床病例，并将为SCI后明显的骨丢失提供一种创新的治疗策略。因此，提出了以下具体目标：具体目标1。确定ES如何改变骨形成和吸收速率以及骨细胞的性质，并表征ES对骨量的影响。我们将确定ES的强度有多长或多高（即，3个月，每天1小时或1个月，每天3小时）改变BMD和BMC，以及骨吸收和骨形成的代谢和组织形态测量。我们还将测试更长时间或更高强度的ES如何改变骨髓细胞培养物中破骨细胞和成骨细胞的数量，并检查这些细胞中基因表达的变化，以确定其分化和活性。 具体目标2。确定ES和雄激素的组合是否比ES或雄激素单独使用更大程度地减少SCI相关的骨丢失。诺龙将与或不与ES同时给药。我们将比较诺龙与ES组合对骨和骨细胞的作用与观察到的ES或诺龙单独的作用。将使用DNA微阵列分析来研究ES单独或诺龙单独或组合减少SCI相关骨丢失的其他细胞和分子机制的可能作用。 公共卫生相关性： 脊髓损伤（SCI）影响了超过40，000名退伍军人。SCI后骨量和结构完整性的急剧丧失是自发性骨折和发病的重要原因。SCI患者的骨骼变弱可能无法在截肢期间支撑体重，从而限制了神经修复的潜在益处。目前，临床上还没有实际的治疗方法来延迟或预防急性SCI后的骨丢失，或促进慢性SCI患者的骨重建。因此，任何在急性SCI后保留骨骼或在慢性SCI中重建骨骼的干预措施，将具有巨大的长期潜力，以促进承重，而生物或工程干预措施将使截肢成为现实，从而改善生活质量。目前的建议将大大提高我们的 了解如何改善SCI后的骨骼健康。

DESCRIPTION (provided by applicant): Spinal cord injury (SCI) causes severe bone loss which may result in pathologic fractures and, consequently, increased morbidity and health care costs. To date, the most effective strategies for preserving or restoring bone after SCI have utilized electrical stimulation (ES)-induced musculoskeletal loading. One critical question is whether the beneficial effects of ES reflect a decrease in the elevated rate of bone resorption, increased formation of new bone, or both. Answers to these questions are likely to guide the development of future strategies to minimize bone loss after SCI by identifying those components of bone metabolism that are not optimally stimulated by ES and might therefore be targets for additional types of intervention. Our preliminary data indicate that one week of reloading of bone after SCI by ES reduced bone resorptive activity through the inhibition of osteoclastogenesis and osteoclast activity in a rat model of SCI. Importantly, ES reversed SCI-induced upregulation of the expression in osteoblasts of the Wnt inhibitors (DKK1, sFRP2 and SOST) and increased expression in these cells of the Wnt-responsive gene osteoprotegerin (OPG), an inhibitor of the differentiation and activity of osteoclasts. SCI related-bone loss may be worsened by reduced circulating levels of testosterone because this steroid hormone has anabolic actions on bone and its levels are commonly reduced after SCI. Recently, we observed that nandrolone, an anabolic steroid, reduced bone loss after paralysis due to SCI. Interestingly, nandrolone increased the expression of OPG, Runx2 and LRP5 in bone marrow-derived osteoblasts from SCI rats. LRP5 is a Wnt co-receptor closely linked to the control of bone mass, and Runx2 is another Wnt-responsive gene, and is involved in osteoblast differentiation. These findings suggest that one effect of nandrolone in unloaded bone is to stimulate Wnt signaling in osteoblasts and that Wnt signaling explains at least some of the protective effects of nandrolone on bone after SCI. The central hypotheses suggested by our findings are: 1) ES effects on bone are due to a decrease in the elevated rate of bone resorption and increased formation of new bone; 2) anabolic steroids will provide additional or synergistic benefits to SCI-related bone loss when applied in conjunction with ES. If this is the case, these findings would be readily translated to clinical cae, and would provide an innovative therapeutic strategy for the marked loss of bone following SCI. As such, the following specific aims are proposed: Specific Aim 1. To determine how ES alters the rates of bone formation and resorption and the properties of bone cells, and to characterize the effects of ES on bone mass. We will determine how longer or higher intensity of ES (i.e., 3 months for one hour a day or 1 month for 3 hours a day) alters BMD and BMC, and both metabolic and histomorphometric measures of bone resorption and bone formation. We will also test how longer periods or higher intensity of ES alter numbers of osteoclasts and osteoblasts in cultures of bone marrow cells and examine changes in expression in these cells of genes for their differentiation and activity. Specific Aim 2. To determine whether a combination of ES and an androgen will reduce SCI- related bone loss to a greater extent than either ES or androgen alone. Nandrolone will be administered with or without concurrent ES. We will compare the effects on bone and bone cells of nandrolone combined with ES with the effects observed for ES or nandrolone alone. The possible role for other cellular and molecular mechanisms by which SCI-related bone loss is reduced by ES alone or nandrolone alone or in combination will be studied using DNA microarry analysis. PUBLIC HEALTH RELEVANCE: Spinal cord injury (SCI) affects more than 40,000 veterans. The dramatic loss of bone mass and structural integrity that follows SCI is a significant cause of spontaneous fractures and morbidity The weakened bones of persons with SCI may not support the body's weight during ambulation, thus limiting the potential benefit of neurorepair. At present, there is no practical treatment tha is clinically available to delay or prevent bone loss after acute SCI, or to promote rebuilding of bone in individuals with chronic SCI. Therefore, any intervention that preserves bone after acute SCI, or rebuilds bone in chronic SCI, would have a tremendous long-term potential to facilitate weight bearing when biological or engineering interventions which permit ambulation to become a reality, and thus to improve quality of life. The current proposal will significantly enhance our knowledge as to how to improve bone health after SCI.