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，可能导致病理性骨折，从而增加发病率和医疗费用。到目前为止，在脊髓损伤后保存或恢复骨骼的最有效的策略是使用电刺激(ES)诱导的肌肉骨骼负荷。一个关键的问题是ES的有益效果是否反映了骨吸收升高的速度的降低、新骨形成的增加，或者两者兼而有之。这些问题的答案可能会指导未来的策略的发展，以减少脊髓损伤后的骨丢失，通过识别那些不是ES最佳刺激的骨代谢成分，因此可能成为其他类型干预的靶点。我们的初步数据表明，在脊髓损伤大鼠模型中，ES对脊髓损伤后一周的骨重新加载通过抑制破骨细胞的生成和破骨细胞的活性来降低骨吸收活性。重要的是，ES逆转了脊髓损伤诱导的成骨细胞中Wnt抑制物(Dkk1、SFRP2和SOST)的表达上调，并增加了Wnt反应基因OPG的表达，OPG是一种抑制破骨细胞分化和活性的基因。由于这种类固醇激素对骨骼有合成代谢作用，在脊髓损伤后其水平通常会降低，因此降低循环中的睾酮水平可能会加剧与脊髓损伤相关的骨丢失。最近，我们观察到一种合成类固醇能减少脊髓损伤后瘫痪后的骨丢失。有趣的是，诺得龙增加了脊髓损伤大鼠骨髓来源的成骨细胞中OPG、Runx2和LRP5的表达。LRP5是一个与骨量控制密切相关的Wnt共同受体，Runx2是另一个Wnt反应基因，参与成骨细胞的分化。这些发现表明，去负荷骨中的一种作用是刺激成骨细胞中的Wnt信号，而Wnt信号至少解释了Nandroone对脊髓损伤后骨的部分保护作用。我们的发现提出的中心假设是：1)ES对骨的影响是由于骨吸收增加和新骨形成增加的减少；2)合成类固醇与ES联合应用将对脊髓损伤相关的骨丢失提供额外或协同的好处。如果是这样的话，这些发现将很容易转化为临床CAE，并将为脊髓损伤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如何改变骨形成和骨吸收的速率以及骨细胞的特性，并表征ES对骨量的影响。我们将确定更长或更高强度的ES(即3个月每天1小时或1个月每天3小时)如何改变BMD和BMC，以及骨吸收和骨形成的代谢和组织形态计量学指标。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中的破骨细胞和成骨细胞的数量，并检测这些细胞中分化和活性基因表达的变化。明确目标2.确定ES和雄激素联合使用是否能比单独使用ES或雄激素更大程度地减少脊髓损伤相关的骨丢失。使用或不使用并发ES均可给药。我们将与单独使用ES或单独使用ES对骨和骨细胞的影响进行比较。其他细胞和分子机制的可能作用将通过DNA微阵列分析来研究，通过这些机制，ES单独或单独或联合应用能减少脊髓损伤相关的骨丢失。 公共卫生相关性： 脊髓损伤(SCI)影响着40,000多名退伍军人。脊髓损伤后骨量和结构完整性的急剧丧失是自发性骨折和发病率的重要原因。脊髓损伤患者虚弱的骨骼在行走时可能无法支撑身体的重量，因此限制了神经修复的潜在好处。目前，临床上尚无有效的治疗方法来延缓或预防急性脊髓损伤后的骨丢失，或促进慢性脊髓损伤患者的骨重建。因此，任何在急性脊髓损伤后保留骨骼或在慢性脊髓损伤中重建骨骼的干预措施，都将具有巨大的长期潜力，当允许步行成为现实的生物或工程干预时，将促进负重，从而提高生活质量。目前的建议将大大加强我们的 关于如何改善脊髓损伤后骨骼健康的知识。

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.