Deconstructing Spasticity after Spinal Cord Injury

本研究的目的是探讨痉挛的细胞和分子机制， 为将来临床上的转化研究奠定基础。大多数美国退伍军人SCI 经历临床上显著的痉挛，这可能会破坏康复并对生活质量产生负面影响， 例如，在一个实施例中，移动性、个人卫生、亲密关系（Holtz等人，2017; Skold等人，1999; Walter等人， 2002年）。目前的痉挛管理策略是姑息性的，并未能解决根本原因。 由于非特异性作用或长期使用，现有的治疗方案也存在不良反应的高风险 （亚当斯等人，2005; Kheder等人，2012年）。开发更好的SCI治疗方法面临的一个主要障碍是- 诱发性痉挛是对伤害如何导致残疾缺乏机械性洞察。 为了推进以证据为基础的调查，以改善痉挛管理，我们将开展 实验有两个目标： 在目标1中，我们将实施条件性敲除研究，以了解 Rac1在运动神经元和星形胶质细胞中特异性表达，与脊髓损伤后的痉挛状态有关。我们以前的工作表明， 药理学抑制剂可以阻断Rac1调节的运动神经元树突棘重塑， 痉挛状态（Bandaru等人，2015; Zhao等人，2016）。然而，到目前为止，我们的研究依赖于使用 药理学Rac1-抑制剂，NSC23766，这排除了我们确定药物对 神经元还不清楚为什么NSC23766仅使正常反射输出部分恢复，并且剂量- 有限的副作用阻碍了长期治疗。因此，为了阐明Rac1信号传导在细胞内的作用， 神经元和星形胶质细胞，我们将使用1）病毒介导的Cre-Lox系统敲除Rac1表达， 运动神经元，和2）转基因小鼠缺乏Rac 1特异性星形胶质细胞。星形胶质细胞是 突触可塑性和维持神经元的过度兴奋，但尚未在 SCI后痉挛我们将使用电生理和行为测试来测量诱发H反射 兴奋性和痉挛性。为了控制运动功能的其他变化，我们还将监测总体运动 功能为了评估与痉挛相关的树突棘发育不全和其他解剖学变化，我们 将在"清除的"脊髓组织中进行图像分析。 在目标2中，为了建立临床翻译的基础，我们还将评估两种翻译的可行性。 靶向Rac1途径的预防相关方法。具体来说，首先，我们将评估 基于病毒的基因治疗"平台"，以敲低Rac1表达和缓解痉挛。我们有 以前使用的定制shRNA构建体的病毒递送，以有效靶向错误表达的蛋白质， 并在损伤或疾病后改变神经性疼痛（Samad等，2013; Tan等人，2015年）。在第二 方法，我们将确定"重新利用"罗米地辛的潜在效用，罗米地辛是一种临床可用的药物， 破坏PAK 1，其是连接Rac 1与树突棘重组的下游效应物（Hayashi等，2007年）。 总之，这项研究的发现不仅可以提高对 痉挛，并推动该领域的临床应用，但也有可能超越SCI， 条件，如MS，TBI，中风，是广泛流行的美国退伍军人。

The goal of this study is to investigate the cellular and molecular mechanisms underlying spasticity, and establish the groundwork for future translational studies in the clinic. A majority of US Veterans with SCI experience clinically significant spasticity, which can disrupt rehabilitation and negatively impact quality-of-life, e.g., mobility, personal hygiene, intimate relationships (Holtz et al., 2017; Skold et al., 1999; Walter et al., 2002). Current spasticity management strategies are palliative, and fail to address the underlying cause. Available treatment options also carry high risk for adverse effects due to non-specific action or long-term use (Adams et al., 2005; Kheder et al., 2012). A major hurdle facing the development of better treatments for SCI- induced spasticity is a lack of mechanistic insight into how injury leads to disability. To advance an evidence-based investigation toward improving spasticity management, we will carry out experiments with two objectives: In Objective 1, we will implement conditional knockout studies to understand the mechanistic contribution of Rac1 specifically in motor neurons and astrocytes to spasticity after SCI. Our previous work demonstrates that pharmacological inhibitors can block Rac1-regulated dendritic spine remodeling in motor neurons and reduce spasticity (Bandaru et al., 2015; Zhao et al., 2016). However, our studies thus far have relied upon the use of a pharmacological Rac1-inhibitor, NSC23766, which precluded our ability to determine the drug’s direct action on neurons. It is also unclear why NSC23766 rendered only partial restoration of normal reflex output, and dose- limiting side effects have prevented longer-term treatment. Thus, to clarify the contribution of Rac1 signaling in neurons and astrocytes, we will use a 1) virally-mediated Cre-Lox system to knockout Rac1 expression in motor neurons, and 2) transgenic mice lacking Rac1 specifically in astrocytes. Astrocytes are integral to synaptic plasticity and maintain neuronal hyperexcitability, but have not been studied within the context of spasticity after SCI. We will use electrophysiological and behavioral tests to measure evoked H-reflex excitability and spasticity. To control for other changes in motor function, we will also monitor gross locomotor function. To assess dendritic spine dysgenesis associated with spasticity, and other anatomical changes, we will perform image analyses in “cleared” spinal cord tissue. In Objective 2, to establish the groundwork for clinical translation, we will also assess the feasibility of two translationally-relevant approaches targeting the Rac1-pathway. Specifically, first we will assess the utility of a viral-based gene therapy “platform” to knockdown Rac1 expression and alleviate spasticity. We have previously used viral-delivery of custom-made shRNA constructs to effectively target misexpressed proteins and modify neuropathic pain after injury or disease (Samad et al., 2013; Tan et al., 2015). In the second approach, we will determine the potential utility of “repurposing” romidepsin, a clinically available drug to disrupt PAK1, a downstream effector linking Rac1 to dendritic spine reorganization (Hayashi et al., 2007). In summary, findings from this study could be expected to not only improve the mechanistic understanding of spasticity and advance the field toward clinical application, but also potentially extend beyond SCI, to conditions such as MS, TBI, stroke that are widely prevalent among US Veterans.