ReachingBot: An automated and scalable benchtop device for highly parallel Single Pellet Reach-and-Grasp training and assessment in mice.

BACKGROUND: The single pellet reaching and grasp (SPRG) task is a behavioural assay widely used to study motor learning, control and recovery after nervous system injury in animals. The manual training and assessment of the SPRG is labour intensive and time consuming and has led to the development of multiple devices which automate the SPRG task. NEW METHOD: Here, using robotics, computer vision, and machine learning analysis of videos, we describe a device that can be left unattended, presents pellets to mice, and, using two supervised learning algorithms, classifies the outcome of each trial with an accuracy of greater than 94% without the use of graphical processing units (GPUs). Our devices can also be operated using our cross-platform Graphical User Interface (GUI). RESULTS: We show that these devices train and assess mice in parallel. 21 out of 30 mice retrieved > 40% of pellets successfully following the training period. Following ischaemic stroke; some mice showed large persistent deficits whilst others showed only transient deficits. This highlights the heterogeneity in reaching outcomes following stroke. COMPARISON WITH EXISTING METHOD(S): Current state-of-the-art desktop methods either still require supervision, manual classification of trial outcome, or expensive locally-installed hardware such as graphical processing units (GPUs). CONCLUSIONS: ReachingBots successfully automated SPRG training and assessment and revealed the heterogeneity in reaching outcomes following stroke. We conjecture that reach-and-grasp is represented in motor cortex bilaterally but with greater asymmetry in some mice than in others.

Delayed viral vector mediated delivery of neurotrophin-3 improves skilled hindlimb function and stability after thoracic contusion.

Intramuscular injection of an Adeno-associated viral vector serotype 1 (AAV1) encoding Neurotrophin-3 (NT3) into hindlimb muscles 24 h after a severe T9 spinal level contusion in rats has been shown to induce lumbar spinal neuroplasticity, partially restore locomotive function and reduce spasms during swimming. Here we investigate whether a targeted delivery of NT3 to lumbar and thoracic motor neurons 48 h following a severe contusive injury aids locomotive recovery in rats. AAV1-NT3 was injected bilaterally into the tibialis anterior, gastrocnemius and rectus abdominus muscles 48-h following trauma, persistently elevating serum levels of the neurotrophin. NT3 modestly improved trunk stability, accuracy of stepping during skilled locomotion, and alternation of the hindlimbs during swimming, but it had no effect on gross locomotor function in the open field. The number of vGlut1+ boutons, likely arising from proprioceptive afferents, on gastrocnemius α-motor neurons was increased after injury but normalised following NT3 treatment, suggestive of a mechanism in which functional benefits may be mediated through proprioceptive feedback. Ex vivo MRI revealed substantial loss of grey and white matter at the lesion epicentre but no effect of delayed NT3 treatment to induce neuroprotection. Lower body spasms and hyperreflexia of an intrinsic paw muscle were not reliably induced in this severe injury model suggesting a more complex anatomical or physiological cause to their induction. We have shown that delayed intramuscular AAV-NT3 treatment can promote recovery in skilled stepping and coordinated swimming, supporting a role for NT3 as a therapeutic strategy for spinal injuries potentially through modulation of somatosensory feedback.

Harnessing cortical plasticity via gabapentinoid administration promotes recovery after stroke.

Stroke causes devastating sensory-motor deficits and long-term disability due to disruption of descending motor pathways. Restoration of these functions enables independent living and therefore represents a high priority for those afflicted by stroke. Here, we report that daily administration of gabapentin, a clinically approved drug already used to treat various neurological disorders, promotes structural and functional plasticity of the corticospinal pathway after photothrombotic cortical stroke in adult mice. We found that gabapentin administration had no effects on vascular occlusion, haemodynamic changes nor survival of corticospinal neurons within the ipsilateral sensory-motor cortex in the acute stages of stroke. Instead, using a combination of tract tracing, electrical stimulation and functional connectivity mapping, we demonstrated that corticospinal axons originating from the contralateral side of the brain in mice administered gabapentin extend numerous collaterals, form new synaptic contacts and better integrate within spinal circuits that control forelimb muscles. Not only does gabapentin daily administration promote neuroplasticity, but it also dampens maladaptive plasticity by reducing the excitability of spinal motor circuitry. In turn, mice administered gabapentin starting 1 h or 1 day after stroke recovered skilled upper extremity function. Functional recovery persists even after stopping the treatment at 6 weeks following a stroke. Finally, chemogenetic silencing of cortical projections originating from the contralateral side of the brain transiently abrogated recovery in mice administered gabapentin, further supporting the conclusion that gabapentin-dependent reorganization of spared cortical pathways drives functional recovery after stroke. These observations highlight the strong potential for repurposing gabapentinoids as a promising treatment strategy for stroke repair.

Neurotrophin-3 attenuates human peripheral blood T cell and monocyte activation status and cytokine production post stroke.

BACKGROUND AND PURPOSE: Stroke therapy still lacks successful measures to improve post stroke recovery. Neurotrophin-3 (NT-3) is one promising candidate which has proven therapeutic benefit in motor recovery in acute experimental stroke. Post stroke, the immune system has opposing pathophysiological roles: pro-inflammatory cascades and immune cell infiltration into the brain exacerbate cell death while the peripheral immune response has only limited capabilities to fight infections during the acute and subacute phase. With time, anti-inflammatory mechanisms are supposed to support recovery of the ischemic damage within the brain parenchyma. However, interestingly, NT-3 can improve recovery in chronic neurological injury when combined with the pro-inflammatory stimulus lipopolysaccharide (LPS). AIM: We elucidated the impact of NT-3 on human monocyte and T cell activation as well as cytokine production ex vivo after stroke. In addition, we investigated the age-dependent availability of the high affinity NT-3 receptor TrkC upon LPS stimulation. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from acute stroke patients and controls and incubated with different dosages of NT-3 (10 and 100 ng/mL) and with or without LPS or anti-CD3/CD28 for 48 h. Total TrkC expression and cell activation (CD25, CD69 and HLA-DR) were assessed by FACS staining. IFN-γ, TNF-α, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-21 and IL-22 were quantified by cytometric bead array. RESULTS: Most monocytes and only a small proportion of T cells expressed TrkC in blood from humans without stroke. Activation of cells from young humans (without strokes) using anti-CD3/CD28 or LPS partially reduced the proportion of monocytes expressing TrkC whilst they increased the proportion of T cells expressing TrkC. In contrast, activation of cells from elderly humans (without strokes) did not affect the proportion of monocytes expressing TrkC and only anti-CD3/CD28 led to an increase in the proportion of CD4+ T cells expressing TrkC. In blood from stroke patients or controls, NT-3 treatment reduced the percentage of monocytes and CD4+ and CD8+ T cells that were activated and reduced all cytokines investigated besides IL-21. CONCLUSIONS: NT-3 attenuated immune responses in cells from stroke patients and controls. The mechanism whereby human immune cells respond to NT-3 may be via TrkC receptors whose levels are regulated by stimulation. Further work is required to determine whether the induction of sensorimotor recovery in rodents by NT-3 after CNS injury is caused by this attenuation of the immune response.

Peripherally delivered Adeno-associated viral vectors for spinal cord injury repair.

Via the peripheral and autonomic nervous systems, the spinal cord directly or indirectly connects reciprocally with many body systems (muscular, intengumentary, respiratory, immune, digestive, excretory, reproductive, cardiovascular, etc). Accordingly, spinal cord injury (SCI) can result in catastrophe for multiple body systems including muscle paralysis affecting movement and loss of normal sensation, as well as neuropathic pain, spasticity, reduced fertility and autonomic dysreflexia. Treatments and cure for an injured spinal cord will likely require access of therapeutic agents across the blood-CNS (central nervous system) barrier. However, some types of repair within the CNS may be possible by targeting treatment to peripherally located cells or by delivering Adeno-Associated Viral vectors (AAVs) by peripheral routes (e.g., intrathecal, intravenous). This review will consider some future possibilities for SCI repair generated by therapeutic peripheral gene delivery. There are now six gene therapies approved worldwide as safe and effective medicines of which three were created by modification of the apparently nonpathogenic Adeno-Associated Virus. One of these AAVs, Zolgensma, is injected intrathecally for treatment of spinal muscular atrophy in children. One day, delivery of AAVs into peripheral tissues might improve recovery after spinal cord injury in humans; we discuss experiments by us and others delivering transgenes into nerves or muscles for sensorimotor recovery in animal models of SCI or of stroke including human Neurotrophin-3. We also describe ongoing efforts to develop AAVs that are delivered to particular targets within and without the CNS after peripheral administration using capsids with improved tropisms, promoters that are selective for particular cell types, and methods for controlling the dose and duration of expression of a transgene. In conclusion, in the future, minimally invasive administration of AAVs may improve recovery after SCI with minimal side effects.

Refining rodent models of spinal cord injury.

This report was produced by an Expert Working Group (EWG) consisting of UK-based researchers, veterinarians and regulators of animal experiments with specialist knowledge of the use of animal models of spinal cord injury (SCI). It aims to facilitate the implementation of the Three Rs (Replacement, Reduction and Refinement), with an emphasis on refinement. Specific animal welfare issues were identified and discussed, and practical measures proposed, with the aim of reducing animal use and suffering, reducing experimental variability, and increasing translatability within this critically important research field.

Chondroitinase ABC reduces dopaminergic nigral cell death and striatal terminal loss in a 6-hydroxydopamine partial lesion mouse model of Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is characterised by dopaminergic cell loss within the substantia nigra pars compacta (SNc) that leads to reduced striatal dopamine content and resulting motor deficits. Identifying new strategies to protect these cells from degeneration and retain striatal dopaminergic innervation is therefore of great importance. Chondroitin sulphate proteoglycans (CSPGs) are recognised contributors to the inhibitory extracellular milieu known to hinder tissue recovery following CNS damage. Digestion of these molecules by the bacterial lyase chondroitinase ABC (ChABC) has been shown to promote functional recovery in animal models of neurological injury. Although ChABC has been shown to promote sprouting of dopaminergic axons following transection of the nigrostriatal pathway, its ability to protect against nigrostriatal degeneration in a toxin-based module with better construct validity for PD has yet to be explored. Here we examined the neuroprotective efficacy of ChABC treatment in the full and partial 6-hydroxydopamine (6-OHDA) lesion mouse models of PD. RESULTS: In mice bearing a full 6-OHDA lesion, ChABC treatment failed to protect against the loss of either nigral cells or striatal terminals. In contrast, in mice bearing a partial 6-OHDA lesion, ChABC treatment significantly protected cells of the rostral SNc, which remained at more than double the numbers seen in vehicle-treated animals. In the partial lesion model, ChABC treatment also significantly preserved dopaminergic fibres of the rostral dorsal striatum which increased from 15.3 ± 3.5% of the intact hemisphere in saline-treated animals to 36.3 ± 6.5% in the ChABC-treated group. These protective effects of ChABC treatment were not accompanied by improvements in either the cylinder or amphetamine-induced rotations tests of motor function. CONCLUSIONS: ChABC treatment provided significant protection against a partial 6-OHDA lesion of the nigrostriatal tract although the degree of protection was not sufficient to improve motor outcomes. These results support further investigations into the benefits of ChABC treatment for providing neuroprotection in PD.

Cbp-dependent histone acetylation mediates axon regeneration induced by environmental enrichment in rodent spinal cord injury models.

After a spinal cord injury, axons fail to regenerate in the adult mammalian central nervous system, leading to permanent deficits in sensory and motor functions. Increasing neuronal activity after an injury using electrical stimulation or rehabilitation can enhance neuronal plasticity and result in some degree of recovery; however, the underlying mechanisms remain poorly understood. We found that placing mice in an enriched environment before an injury enhanced the activity of proprioceptive dorsal root ganglion neurons, leading to a lasting increase in their regenerative potential. This effect was dependent on Creb-binding protein (Cbp)-mediated histone acetylation, which increased the expression of genes associated with the regenerative program. Intraperitoneal delivery of a small-molecule activator of Cbp at clinically relevant times promoted regeneration and sprouting of sensory and motor axons, as well as recovery of sensory and motor functions in both the mouse and rat model of spinal cord injury. Our findings showed that the increased regenerative capacity induced by enhancing neuronal activity is mediated by epigenetic reprogramming in rodent models of spinal cord injury. Understanding the mechanisms underlying activity-dependent neuronal plasticity led to the identification of potential molecular targets for improving recovery after spinal cord injury.

Stroke Recovery in Rats after 24-Hour-Delayed Intramuscular Neurotrophin-3 Infusion.

OBJECTIVE: Neurotrophin-3 (NT3) plays a key role in the development and function of locomotor circuits including descending serotonergic and corticospinal tract axons and afferents from muscle and skin. We have previously shown that gene therapy delivery of human NT3 into affected forelimb muscles improves sensorimotor recovery after stroke in adult and elderly rats. Here, to move toward the clinic, we tested the hypothesis that intramuscular infusion of NT3 protein could improve sensorimotor recovery after stroke. METHODS: Rats received unilateral ischemic stroke in sensorimotor cortex. To simulate a clinically feasible time to treatment, 24 hours later rats were randomized to receive NT3 or vehicle by infusion into affected triceps brachii for 4 weeks using implanted catheters and minipumps. RESULTS: Radiolabeled NT3 crossed from the bloodstream into the brain and spinal cord in rodents with or without strokes. NT3 increased the accuracy of forelimb placement during walking on a horizontal ladder and increased use of the affected arm for lateral support during rearing. NT3 also reversed sensory impairment of the affected wrist. Functional magnetic resonance imaging during stimulation of the affected wrist showed spontaneous recovery of peri-infarct blood oxygenation level-dependent signal that NT3 did not further enhance. Rather, NT3 induced neuroplasticity of the spared corticospinal and serotonergic pathways. INTERPRETATION: Our results show that delayed, peripheral infusion of NT3 can improve sensorimotor function after ischemic stroke. Phase I and II clinical trials of NT3 (for constipation and neuropathy) have shown that peripheral high doses are safe and well tolerated, which paves the way for NT3 as a therapy for stroke. ANN NEUROL 2019;85:32-46.

RNA sequencing dataset describing transcriptional changes in cervical dorsal root ganglia after bilateral pyramidotomy and forelimb intramuscular gene therapy with an adeno-associated viral vector encoding human neurotrophin-3.

Unilateral or bilateral corticospinal tract injury in the medullary pyramids in adult rats causes anatomical and physiological changes in proprioceptive neurons projecting to the cervical spinal cord accompanied by hyperreflexia and abnormal behavioural movements including spasms. In a previous publication, we showed that "Intramuscular Neurotrophin-3 normalizes low threshold spinal reflexes, reduces spasms and improves mobility after bilateral corticospinal tract injury in rats" (Kathe et al., 2016) [1]. We hypothesize that neurotrophin-3 induces these changes by modifying gene expression in affected cervical dorsal root ganglia (DRG). Therefore in this data article, we analyzed the transcriptomes of cervical DRGs obtained during that previous study from naïve rats and from rats after bilateral pyramidotomy (bPYX) with unilateral intramuscular injections of either AAV1-CMV-NT3 or AAV1-CMV-EGFP applied 24 h after injury (Kathe et al., 2016) [1]. A bioinformatic analysis enabled us to identify genes that are likely to be expressed in TrkC+ neurons after injury and which were regulated by neurotrophin-3 in the direction expected from other datasets involving knockout or overexpression of neurotrophin-3. This dataset will help us and others identify genes in sensory neurons whose expression levels are regulated by neurotrophin-3 treatment. This may help identify novel therapeutic targets to improve sensation and movement after neurological injury. Data has been deposited in the Gene Expression Omnibus (GSE82197), http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=avgpicgcjhknzyv&acc=GSE82197.

The Function of FGFR1 Signalling in the Spinal Cord: Therapeutic Approaches Using FGFR1 Ligands after Spinal Cord Injury.

Extensive research is ongoing that concentrates on finding therapies to enhance CNS regeneration after spinal cord injury (SCI) and to cure paralysis. This review sheds light on the role of the FGFR pathway in the injured spinal cord and discusses various therapies that use FGFR activating ligands to promote regeneration after SCI. We discuss studies that use peripheral nerve grafts or Schwann cell grafts in combination with FGF1 or FGF2 supplementation. Most of these studies show evidence that these therapies successfully enhance axon regeneration into the graft. Further they provide evidence for partial recovery of sensory function shown by electrophysiology and motor activity evidenced by behavioural data. We also present one study that indicates that combination with additional, synergistic factors might further drive the system towards functional regeneration. In essence, this review summarises the potential of nerve and cell grafts combined with FGF1/2 supplementation to improve outcome even after severe spinal cord injury.

Analysis of longitudinal data from animals with missing values using SPSS.

Testing of therapies for disease or injury often involves the analysis of longitudinal data from animals. Modern analytical methods have advantages over conventional methods (particularly when some data are missing), yet they are not used widely by preclinical researchers. Here we provide an easy-to-use protocol for the analysis of longitudinal data from animals, and we present a click-by-click guide for performing suitable analyses using the statistical package IBM SPSS Statistics software (SPSS). We guide readers through the analysis of a real-life data set obtained when testing a therapy for brain injury (stroke) in elderly rats. If a few data points are missing, as in this example data set (for example, because of animal dropout), repeated-measures analysis of covariance may fail to detect a treatment effect. An alternative analysis method, such as the use of linear models (with various covariance structures), and analysis using restricted maximum likelihood estimation (to include all available data) can be used to better detect treatment effects. This protocol takes 2 h to carry out.

The Adaptor Protein CD2AP Is a Coordinator of Neurotrophin Signaling-Mediated Axon Arbor Plasticity.

Growth of intact axons of noninjured neurons, often termed collateral sprouting, contributes to both adaptive and pathological plasticity in the adult nervous system, but the intracellular factors controlling this growth are largely unknown. An automated functional assay of genes regulated in sensory neurons from the rat in vivo spared dermatome model of collateral sprouting identified the adaptor protein CD2-associated protein (CD2AP; human CMS) as a positive regulator of axon growth. In non-neuronal cells, CD2AP, like other adaptor proteins, functions to selectively control the spatial/temporal assembly of multiprotein complexes that transmit intracellular signals. Although CD2AP polymorphisms are associated with increased risk of late-onset Alzheimer's disease, its role in axon growth is unknown. Assessments of neurite arbor structure in vitro revealed CD2AP overexpression, and siRNA-mediated knockdown, modulated (1) neurite length, (2) neurite complexity, and (3) growth cone filopodia number, in accordance with CD2AP expression levels. We show, for the first time, that CD2AP forms a novel multiprotein complex with the NGF receptor TrkA and the PI3K regulatory subunit p85, with the degree of TrkA:p85 association positively regulated by CD2AP levels. CD2AP also regulates NGF signaling through AKT, but not ERK, and regulates long-range signaling though TrkA(+)/RAB5(+) signaling endosomes. CD2AP mRNA and protein levels were increased in neurons during collateral sprouting but decreased following injury, suggesting that, although typically considered together, these two adult axonal growth processes are fundamentally different. These data position CD2AP as a major intracellular signaling molecule coordinating NGF signaling to regulate collateral sprouting and structural plasticity of intact adult axons. SIGNIFICANCE STATEMENT: Growth of noninjured axons in the adult nervous system contributes to adaptive and maladaptive plasticity, and dysfunction of this process may contribute to neurologic pathologies. Functional screening of genes regulated during growth of noninjured axons revealed CD2AP as a positive regulator of axon outgrowth. A novel association of CD2AP with TrkA and p85 suggests a distinct intracellular signaling pathway regulating growth of noninjured axons. This may also represent a novel mechanism of generating specificity in multifunctional NGF signaling. Divergent regulation of CD2AP in different axon growth conditions suggests that separate mechanisms exist for different modes of axon growth. CD2AP is the first signaling molecule associated with adult sensory axonal collateral sprouting, and this association may offer new insights for NGF/TrkA-related Alzheimer's disease mechanisms.

Overexpression of the Fibroblast Growth Factor Receptor 1 (FGFR1) in a Model of Spinal Cord Injury in Rats.

Spinal cord injury (SCI) is a severe condition that affects many people and results in high health care costs. Therefore, it is essential to find new targets for treatment. The fibroblast growth factor receptor 1 (FGFR1) signalling pathway has a history of being explored for SCI treatment. Several groups have examined the effect of high availability of different FGFR1 ligands at the injury site and reported corticospinal tract (CST) regeneration as well as improved motor functions. In this study, we investigated overexpression of the FGFR1 in rat corticospinal neurons in vivo after injury (unilateral pyramidotomy) and in cerebellar granule neurons (CGNs) in vitro. We show that overexpression of FGFR1 using AAV1 intracortical injections did not increase sprouting of the treated corticospinal tract and did not improve dexterity or walking in a rat model of SCI. Furthermore, we show that overexpression of FGFR1 in vitro resulted in decreased neurite outgrowth compared to control. Thus, our results suggest that the FGFR1 is not a suitable therapeutic target after SCI.

Delayed intramuscular human neurotrophin-3 improves recovery in adult and elderly rats after stroke.

There is an urgent need for a therapy that reverses disability after stroke when initiated in a time frame suitable for the majority of new victims. We show here that intramuscular delivery of neurotrophin-3 (NT3, encoded by NTF3) can induce sensorimotor recovery when treatment is initiated 24 h after stroke. Specifically, in two randomized, blinded preclinical trials, we show improved sensory and locomotor function in adult (6 months) and elderly (18 months) rats treated 24 h following cortical ischaemic stroke with human NT3 delivered using a clinically approved serotype of adeno-associated viral vector (AAV1). Importantly, AAV1-hNT3 was given in a clinically-feasible timeframe using a straightforward, targeted route (injections into disabled forelimb muscles). Magnetic resonance imaging and histology showed that recovery was not due to neuroprotection, as expected given the delayed treatment. Rather, treatment caused corticospinal axons from the less affected hemisphere to sprout in the spinal cord. This treatment is the first gene therapy that reverses disability after stroke when administered intramuscularly in an elderly body. Importantly, phase I and II clinical trials by others show that repeated, peripherally administered high doses of recombinant NT3 are safe and well tolerated in humans with other conditions. This paves the way for NT3 as a therapy for stroke.