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

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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.