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

背景：脑卒中发生在血栓限制血流或血管破裂时。如果没有氧气和能量，脑细胞会迅速死亡，身体的另一侧通常会残疾，手通常会虚弱或受损。中风没有完全的恢复疗法。神经营养素-3（NT3）是一种生长因子，通常由人类和其他哺乳动物在婴儿期的肌肉产生。它是连接从肌肉到脊髓的神经系统所必需的，这些回路对正常运动很重要。NT3的水平随着年龄的增长而下降。我们着手确定是否补充NT3蛋白可以改善中风后的结果时，它是在临床上可行的时间范围内管理。创新性：我们的工作是新颖的，因为我们发现皮下（皮肤下）输注NT3使控制手部肌肉的脊髓回路正常化，并改善中风后老年大鼠的灵活性和行走。临床相关性：这些发现令人兴奋，原因有三：1）我们在CNS损伤后24小时开始治疗。大多数新的中风幸存者可以在这一时间范围内得到治疗（不像现有的中风血栓破坏疗法需要在几个小时内给予）。2)我们通过临床上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，重复皮下注射高剂量的NT 3具有良好的耐受性和安全性。这为NT3作为中风治疗方法铺平了道路。目前尚不清楚神经营养素-3如何促进中风后的恢复。也许令人惊讶的是，我们的证据表明NT3通过在血液中流动和与"受体"结合来促进恢复。（分子靶点）位于中枢神经系统外的外周感觉神经元上，这些神经元将有关身体位置的信息从肌肉传递到脊髓线.这项资助旨在了解皮下注射NT3如何促进恢复，因为这些信息可以帮助未来的临床试验NT3中风survivor.Aim 1：我们希望评估机制，使NT3改善小鼠手臂和手的功能。NT3将在卒中后24小时皮下输注1个月。每周测量一次灵活性和活动性，持续12周。我们的"MouseBots"是一种新型笼内自动化设备，可全天候自动评估分组饲养小鼠的抓握能力（使我们免于重复的手动测试）。每两周进行一次神经刺激和记录，以评估参与运动的脊髓回路的恢复情况。为了确认NT3治疗后恢复灵活性的至关重要的神经通路的身份，我们将使用诱导遗传和药理学方法沉默来自大脑的通路。目的2：我们将发现NT3的作用。我们预测，转基因小鼠中风后外周感觉神经元（而不是运动皮层神经元）中TrkC受体的缺失将阻止NT3诱导的恢复。如果这是真的，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所有的行为分析，该软件使用人工神经网络来跟踪手指，鼻子和小球（在伸手和抓握过程中）和肢体运动（在行走过程中），而不需要在关节上附加反射标记。这将使我们和世界各地的其他用户能够加速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将这种潜在的疗法更接近临床试验。

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.