Deconstructing Spasticity after Spinal Cord Injury

本研究的目的是研究痉挛的细胞和分子机制，以及 为今后的临床翻译研究奠定基础。大多数患有脊髓损伤的美国退伍军人 经历临床上显著的痉挛，这可能会扰乱康复并对生活质量产生负面影响， 例如，机动性、个人卫生、亲密关系(Holtz等人，2017；Skold等人，1999；Walter等人， 2002年)。目前的痉挛治疗策略是姑息性的，无法解决根本原因。 现有的治疗方案也存在因非特定作用或长期使用而产生不良反应的高风险。 (Adams等人，2005年；Kheder等人，2012年)。开发更好的脊髓损伤治疗方法面临的一个主要障碍- 诱发痉挛是对损伤如何导致残疾缺乏机械性的洞察力。 为了推进循证调查以改善痉挛治疗，我们将开展 有两个目标的实验： 在目标1中，我们将实施有条件的基因敲除研究，以了解 脊髓损伤后运动神经元和星形胶质细胞中的rac1与痉挛有关。我们之前的工作表明 药物抑制物可阻断rac1调节的运动神经元树突棘重塑并减少 痉挛(Bandaru等人，2015；赵等人，2016)。然而，到目前为止，我们的研究依赖于使用 药理的rac1-抑制剂，NSC23766，这使得我们无法确定该药物对 神经元。也不清楚为什么NSC23766只部分恢复了正常的反射输出，以及剂量- 限制副作用阻碍了长期治疗。因此，为了阐明rac1信号在体内的作用 神经元和星形胶质细胞，我们将使用病毒介导的Cre-Lox系统来敲除Rac1的表达 运动神经元，以及2)在星形胶质细胞中特异性缺失rac1的转基因小鼠。星形胶质细胞是 突触可塑性和维持神经元的超兴奋性，但尚未在 脊髓损伤后的痉挛。我们将使用电生理和行为测试来测量诱发的H反射 兴奋性和痉挛。为了控制运动功能的其他变化，我们还将监测粗大运动 功能。为了评估与痉挛和其他解剖学变化相关的树突棘发育不全，我们 将在“清除”的脊髓组织中进行图像分析。 在目标2中，为了为临床翻译奠定基础，我们还将评估两种方法的可行性 针对rac1途径的翻译相关方法。具体地说，首先我们将评估 以病毒为基础的基因治疗平台，敲除rac1的表达，缓解痉挛。我们有 以前使用的定制shRNA构建物的病毒传递有效地靶向错误表达的蛋白质 并在受伤或疾病后改变神经病理性疼痛(Samad等人，2013年；Tan等人，2015年)。在第二个 方法，我们将确定“再用途”罗米地辛的潜在效用，一种临床上可用的药物 破坏PAK1，一种连接rac1和树突棘突重组的下游效应器(Hayashi等人，2007年)。 综上所述，这项研究的发现不仅可以提高对 痉挛和推动该领域的临床应用，但也潜在地延伸到脊髓损伤，以 多发性硬化症、脑外伤、中风等在美国退伍军人中广泛流行的疾病。

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.