Delayed, subcutaneous neurotrophin-3 infusion improves recovery after stroke: mechanisms of recovery, automation of testing and markerless kinematics

背景：当血栓限制血流或血管破裂时，中风就会发生在大脑中。没有氧气和能量，脑细胞会迅速死亡，身体的另一侧往往会失去能力，一只手往往会变得虚弱或受损。目前还没有针对中风的完全恢复性疗法。数以百万计的人受到影响。神经营养素-3(NT3)是一种生长因子，通常在人类和其他哺乳动物的婴儿期由肌肉产生。它对神经系统从肌肉到脊髓的连接是必不可少的，而这些回路对正常的运动很重要。NT3水平随年龄增长而下降。我们开始确定在临床可行的时间范围内补充NT3蛋白是否可以改善卒中后的预后。创新：我们的工作是新颖的，因为我们发现，皮下注射NT3可以使控制手部肌肉的脊髓回路正常化，并提高中风后老年大鼠的灵活性和行走能力。临床意义：这些发现令人兴奋，原因有三：1)我们在中枢神经系统损伤后24小时开始治疗。大多数新的中风幸存者可以在这个时间框架内得到治疗(不像现有的中风血栓溶解疗法需要在几个小时内进行治疗)。2)我们通过临床简单的途径提供治疗，不需要侵入性神经外科手术，原则上可以在康复期间使用现有的小型可穿戴输液泵进行治疗。3)临床试验表明，在200多人中，重复皮下大剂量NT3的耐受性和安全性都很好。这为NT3治疗中风铺平了道路。知识差距：目前尚不清楚神经营养素-3是如何促进中风后的康复的。也许令人惊讶的是，我们的证据表明，NT3通过在血液中移动，并通过与位于中枢神经系统外的外周感觉神经元上的“受体”(分子靶标)结合来促进康复，这些感觉神经元将有关身体位置的信息从肌肉传递到脊髓。这项拨款旨在了解皮下注射NT3如何促进康复，因为这一信息可能有助于未来NT3在中风幸存者中的临床试验。目标1：我们希望评估NT3改善小鼠手臂和手功能的机制。NT3于卒中后24小时皮下注射，疗程1个月。灵巧度和灵活性将在12周内每周进行一次测量。我们的“鼠标机器人”是一种新颖的笼内自动化设备，可以全天候自动评估群养小鼠的抓握情况(使我们从重复的手动测试中解脱出来)。神经刺激和记录将每两周进行一次，以评估参与运动的脊髓回路的恢复情况。为了确认NT3治疗后恢复灵巧性的关键神经通路的身份，我们将使用可诱导的遗传和药理学方法来沉默来自大脑的通路。目标2：我们将发现NT3在哪里起作用。我们预测，中风后转基因小鼠外周感觉神经元(而不是运动皮质神经元)中TrkC受体的缺失将阻止NT3诱导的康复。如果是真的，这将证明这种疗法不需要进入大脑或脊髓就能促进康复，并将促进临床试验。目标3：我们将通过开发软件来自动化我们的所有行为分析，该软件使用人工神经网络跟踪手指、鼻子和小球(在伸手和抓取期间)和肢体运动(在行走期间)，而不需要在关节上安装反光标记。这将使我们和世界各地的其他用户能够加快对中风和其他影响肢体运动的疾病的研究。这些实验将帮助我们使这种潜在的治疗方法离临床试验更近一步。

Background: Stroke occurs in the brain when a clot restricts blood flow or when a blood vessel breaks. Without oxygen and energy, brain cells die rapidly and the opposite side of the body is often disabled, with a hand often weak or impaired. There are no fully restorative therapies for stroke. Millions of people are affected.Neurotrophin-3 (NT3) is a growth factor normally produced by muscles during infancy in humans and other mammals. It is essential for wiring up the nervous system from muscles to the spinal cord and these circuits are important for normal movement. Levels of NT3 decline with age. We set out to determine whether supplementary NT3 protein can improve outcome after stroke when it is administered in a clinically-feasible time frame. Innovation: Our work is novel because we have discovered that infusion of NT3 subcutaneously (under the skin) normalises a spinal circuit that controls a hand muscle, and improves dexterity and walking in elderly rats after stroke. Clinical relevance: These discoveries are exciting for three reasons: 1) We start treatment 24 hours after CNS injury. Most new stroke survivors could be treated within this time frame (unlike the existing clot-busting therapies for stroke which need to be given within a few hours). 2) We deliver the treatment by a clinically-straightforward route that does not require invasive neurosurgery and could, in principle, be delivered during rehabilitation using existing small, wearable infusion pumps. 3) Clinical trials have shown that repeated, subcutaneous, high doses of NT3 are well-tolerated and safe in more than 200 humans. This paves the way for NT3 as a therapy for stroke.Gaps in knowledge: It is not yet known how Neurotrophin-3 promotes recovery after stroke.Perhaps surprisingly, our evidence indicates that NT3 promotes recovery by travelling in the bloodstream and by binding to "receptors" (molecular targets) that are located outside the central nervous system on peripheral sensory neurons that convey information about body position from muscle to the spinal cord. This grant seeks to understand how subcutaneously infused NT3 promotes recovery because this information could help a future clinical trial of NT3 in stroke survivors.Aim 1: We wish to assess the mechanisms whereby NT3 improves arm and hand function in mice. NT3 will be infused subcutaneously 24 hours after stroke for one month. Dexterity and mobility will be measured weekly for 12 weeks. Our "MouseBots" are novel in-cage automated devices which automatically assess grasping in group-housed mice 24/7 (freeing us from repetitive manual testing). Neural stimulation and recording will be performed fortnightly to assess recovery of spinal circuits involved in movement. To confirm the identity of the critically-important neural pathways that restore dexterity after NT3 treatment, we will silence pathways from the brain using inducible genetic and pharmacological methods.Aim 2: We will discover where NT3 acts. We predict that deletion of the TrkC receptors in peripheral sensory neurons (and not neurons in motor cortex) of transgenic mice after stroke will prevent recovery induced by NT3. If true, this would prove that this therapy does not need to enter the brain or spinal cord for it to promote recovery, and would facilitate clinical trials.Aim 3: We will automate all our behavioural analyses by developing software which uses artificial neural networks to track digits, snout and pellets (during reaching and grasping) and limb movements (during walking) without the need to attach reflective markers to joints. This will enable us and other users worldwide to accelerate research into stroke and other diseases affecting limb movements. These experiments will help us take this potential therapy one step closer to clinical trials.