Stepwise Imperatives for Improving the Protection of Animals in Research and Education in Canada.

In this paper, we review the standard-setting tools of different levels of government in Canada for overseeing the care of animals used in science against a landscape of other international efforts. We find regulatory inconsistencies, argue that the related shortcomings are detrimental to the level of care afforded to animals, and offer suggestions for a centralized and proactive approach that could close the existing gaps. Given the resources, cost, and time it would take to transform the current system into a single cohesive one, the proposed approach is a stepwise one, and begins with the addition of two new Rs-Reflection and Responsiveness-to the existing 3Rs framework: Replacement, Refinement, and Reduction. Reflection emphasizes more continuous and specific attention to progress in the research pathway as it applies to animals than is currently required by institutional review and reporting; Responsiveness speaks to the immediate action that researchers can take responsively to that ongoing evaluation.

Connecting Researchers and Clinicians Before Connecting the Injured Spinal Cord.

Background: Translating research findings from animal models of spinal cord injury (SCI) to humans is a challenging enterprise. It is likely that differences in the use of common terms contribute to this. Objectives: The purpose of this study was to identify how scientists and clinicians define terms used across the research and clinical care continuum. Methods: We utilized the Delphi technique to develop consensus on the opinions of experts (defined as researchers and/or clinicians working in the field of SCI) through a series of structured, iterative surveys. A focus group of stakeholders developed the terms on the initial survey. Results were used to create definitions and formulate questions for a second and third survey. Results: Survey 1 yielded one definition for eight terms and multiple definitions for six terms in addition to three new terms that respondents believed should be defined. In Survey 2, definitions for eight terms reached at least 80% agreement: anatomically complete spinal cord injury, functionally complete spinal cord injury, neuromodulation, physical exercise, physical rehabilitation, plasticity, task specificity, and training intensity. Consensus was not reached for six terms. In Survey 3, definitions for seven additional terms reached at least 80% agreement: recovery, repair, compensation, regeneration, physical function, physiological function, and chronic. There were three terms that did not reach agreement after the three rounds: acute, translational research, and sprouting. Conclusion: We found that different terminology contributes to the gap between preclinical and clinical research and clinical application. This suggests that increased communication among different disciplines could be a way to advance the field.

Locomotor-related propriospinal V3 neurons produce primary afferent depolarization and modulate sensory transmission to motoneurons.

When a muscle is stretched, sensory feedback not only causes reflexes but also leads to a depolarization of sensory afferents throughout the spinal cord (primary afferent depolarization, PAD), readying the whole limb for further disturbances. This sensory-evoked PAD is thought to be mediated by a trisynaptic circuit, where sensory input activates first-order excitatory neurons that activate GABAergic neurons that in turn activate GABAA receptors on afferents to cause PAD, though the identity of these first-order neurons is unclear. Here, we show that these first-order neurons include propriospinal V3 neurons, as they receive extensive sensory input and in turn innervate GABAergic neurons that cause PAD, because optogenetic activation or inhibition of V3 neurons in mice mimics or inhibits sensory-evoked PAD, respectively. Furthermore, persistent inward sodium currents intrinsic to V3 neurons prolong their activity, explaining the prolonged duration of PAD. Also, local optogenetic activation of V3 neurons at one segment causes PAD in other segments, due to the long propriospinal tracts of these neurons, helping to explain the radiating nature of PAD. This in turn facilitates monosynaptic reflex transmission to motoneurons across the spinal cord. In addition, V3 neurons directly innervate proprioceptive afferents (including Ia), causing a glutamate receptor-mediated PAD (glutamate PAD). Finally, increasing the spinal cord excitability with either GABAA receptor blockers or chronic spinal cord injury causes an increase in the glutamate PAD. Overall, we show the V3 neuron has a prominent role in modulating sensory transmission, in addition to its previously described role in locomotion.NEW & NOTEWORTHY Locomotor-related propriospinal neurons depolarize sensory axons throughout the spinal cord by either direct glutamatergic axoaxonic contacts or indirect innervation of GABAergic neurons that themselves form axoaxonic contacts on sensory axons. This depolarization (PAD) increases sensory transmission to motoneurons throughout the spinal cord, readying the sensorimotor system for external disturbances. The glutamate-mediated PAD is particularly adaptable, increasing with either an acute block of GABA receptors or chronic spinal cord injury, suggesting a role in motor recovery.

Challenges in Translating Regenerative Therapies for Spinal Cord Injury.

Regenerating the injured spinal cord is a substantial challenge with many obstacles that need to be overcome to achieve robust functional benefits. This abundance of hurdles can partly explain the limited success when applying regenerative intervention treatments in animal models and/or people. In this article, we elaborate on a few of these obstacles, starting with the applicability of animal models and how they compare to the clinical setting. We then discuss the requirement for combinatorial interventions and the associated problems in experimental design, including the addition of rehabilitative training. The article expands on differences in lesion sizes and locations between humans and common animal models, and how this difference can determine the success or failure of an intervention. An additional and frequently overlooked problem in the translation of interventions that applies beyond the field of neuroregeneration is the reporting bias and the lack of transparency in reporting findings. New data mandates are tackling this problem and will eventually result in a more balanced view of the field. Finally, we will discuss strategies to negotiate the challenging course of successful translation to facilitate successful translation of regeneration promoting interventions.

GABA facilitates spike propagation through branch points of sensory axons in the spinal cord.

Movement and posture depend on sensory feedback that is regulated by specialized GABAergic neurons (GAD2+) that form axo-axonic contacts onto myelinated proprioceptive sensory axons and are thought to be inhibitory. However, we report here that activating GAD2+ neurons directly with optogenetics or indirectly by cutaneous stimulation actually facilitates sensory feedback to motor neurons in rodents and humans. GABAA receptors located at or near nodes of Ranvier of sensory axons cause this facilitation by preventing spike propagation failure at the many axon branch points, which is otherwise common without GABA. In contrast, GABAA receptors are generally lacking from axon terminals and so cannot inhibit transmitter release onto motor neurons, unlike GABAB receptors that cause presynaptic inhibition. GABAergic innervation near nodes and branch points allows individual branches to function autonomously, with GAD2+ neurons regulating which branches conduct, adding a computational layer to the neuronal networks generating movement and likely generalizing to other central nervous system axons.

Metabolomic Fingerprint of Behavioral Changes in Response to Full-Spectrum Cannabis Extracts.

Numerous existing full-spectrum cannabis extract products have been used in clinical trials for the treatment of various diseases. Despite their efficacy, the clinical use of some of these full-spectrum cannabis extracts is limited by behavioral side effects such as cognitive dysfunction and impaired motor skills. To better understand what constitutes cannabis-induced behavioral effects, our objective was to identify a novel panel of blood-based metabolites that are predictive, diagnostic, and/or prognostic of behavioral effects. At 8 weeks of age, male rats were randomly assigned to groups and were gavage fed with full-spectrum cannabis extract (tetrahydrocannabinol/cannabidiol (THC/CBD) along with all other cannabis compounds, 15 mg/kg), broad-spectrum cannabis extract (CBD along with all other cannabis compounds, 15 mg/kg), or vehicle oil. Four hours after being gavage fed, behavioral assessments were determined using the open field test and the elevated plus maze. Following these assessments, serum was collected from all rats and the serum metabolites were identified and quantified by LC-MS/MS and 1H NMR spectroscopy. We found that only rats treated with full-spectrum cannabis extract exhibited behavioral changes. Compared to vehicle-treated and broad-spectrum extract-treated rats, full-spectrum extract-treated rats demonstrated higher serum concentrations of the amino acid phenylalanine and long-chain acylcarnitines, as well as lower serum concentrations of butyric acid and lysophosphatidylcholines. This unique metabolomic fingerprint in response to cannabis extract administration is linked to behavioral effects and may represent a biomarker profile of cannabis-induced behavioral changes. If validated, this work may allow a metabolomics-based decision tree that would aid in the rapid diagnosis of cannabis-induced behavioral changes including cognitive impairment.

Rehabilitative training improves skilled forelimb motor function after cervical unilateral contusion spinal cord injury in rats.

Animal models of cervical spinal cord injury (SCI) have frequently utilized partial transection injuries to evaluate plasticity promoting treatments such as rehabilitation training of skilled reaching and grasping tasks. Though highly useful for studying the effects of cutting specific spinal tracts that are important for skilled forelimb motor function, cervical partial-transection SCI-models underappreciate the extensive spread of most human SCIs, thus offering poor predictability for the clinical setting. Conversely, moderate cervical contusion SCI models targeting the spinal tracts important for skilled reaching and grasping can better replicate the increased size of most human SCIs and are often considered more clinically relevant. However, it is unknown whether animals with moderate cervical contusion SCIs that damage key spinal motor tracts can train in skilled reaching and grasping tasks. In this study, we quantify the impact of injury size and distribution on recovery in a skilled motor task called the single pellet reaching, grasping and retrieval (SPRGR) task in rats with cervical unilateral contusion injuries (UCs), and compare to rats with a partial transection SCIs (i.e., dorsolateral quadrant transection; DLQ). We found that UCs damage key tracts important for performing skilled motor tasks, similar to DLQs, but UCs also produce more extensive grey matter damage and more ventral white matter damage than DLQs. We also compared forelimb functionality at 1, 3, and 5 weeks of rehabilitative motor training between trained and untrained rats and found a more severe drop in SPRGR performance than in DLQ SCIs. Nevertheless, despite more severe injuries and initially low SPRGR performance, rehabilitative training for contusion animals resulted in significant improvements in SPRGR performance and proportionally more recovery than DLQ rats. Our findings show that rehabilitative motor training can facilitate considerable amounts of motor recovery despite extensive spinal cord damage, especially grey matter damage, thus supporting the use of contusion or compression SCI models and showing that ventral grey and white matter damage are not necessarily detrimental to recovery after training.

Promoting FAIR Data Through Community-driven Agile Design: the Open Data Commons for Spinal Cord Injury (odc-sci.org).

The past decade has seen accelerating movement from data protectionism in publishing toward open data sharing to improve reproducibility and translation of biomedical research. Developing data sharing infrastructures to meet these new demands remains a challenge. One model for data sharing involves simply attaching data, irrespective of its type, to publisher websites or general use repositories. However, some argue this creates a 'data dump' that does not promote the goals of making data Findable, Accessible, Interoperable and Reusable (FAIR). Specialized data sharing communities offer an alternative model where data are curated by domain experts to make it both open and FAIR. We report on our experiences developing one such data-sharing ecosystem focusing on 'long-tail' preclinical data, the Open Data Commons for Spinal Cord Injury (odc-sci.org). ODC-SCI was developed with community-based agile design requirements directly pulled from a series of workshops with multiple stakeholders (researchers, consumers, non-profit funders, governmental agencies, journals, and industry members). ODC-SCI focuses on heterogeneous tabular data collected by preclinical researchers including bio-behaviour, histopathology findings and molecular endpoints. This has led to an example of a specialized neurocommons that is well-embraced by the community it aims to serve. In the present paper, we provide a review of the community-based design template and describe the adoption by the community including a high-level review of current data assets, publicly released datasets, and web analytics. Although odc-sci.org is in its late beta stage of development, it represents a successful example of a specialized data commons that may serve as a model for other fields.

Beyond the lesion site: minocycline augments inflammation and anxiety-like behavior following SCI in rats through action on the gut microbiota.

BACKGROUND: Minocycline is a clinically available synthetic tetracycline derivative with anti-inflammatory and antibiotic properties. The majority of studies show that minocycline can reduce tissue damage and improve functional recovery following central nervous system injuries, mainly attributed to the drug's direct anti-inflammatory, anti-oxidative, and neuroprotective properties. Surprisingly the consequences of minocycline's antibiotic (i.e., antibacterial) effects on the gut microbiota and systemic immune response after spinal cord injury have largely been ignored despite their links to changes in mental health and immune suppression. METHODS: Here, we sought to determine minocycline's effect on spinal cord injury-induced changes in the microbiota-immune axis using a cervical contusion injury in female Lewis rats. We investigated a group that received minocycline following spinal cord injury (immediately after injury for 7 days), an untreated spinal cord injury group, an untreated uninjured group, and an uninjured group that received minocycline. Plasma levels of cytokines/chemokines and fecal microbiota composition (using 16s rRNA sequencing) were monitored for 4 weeks following spinal cord injury as measures of the microbiota-immune axis. Additionally, motor recovery and anxiety-like behavior were assessed throughout the study, and microglial activation was analyzed immediately rostral to, caudal to, and at the lesion epicenter. RESULTS: We found that minocycline had a profound acute effect on the microbiota diversity and composition, which was paralleled by the subsequent normalization of spinal cord injury-induced suppression of cytokines/chemokines. Importantly, gut dysbiosis following spinal cord injury has been linked to the development of anxiety-like behavior, which was also decreased by minocycline. Furthermore, although minocycline attenuated spinal cord injury-induced microglial activation, it did not affect the lesion size or promote measurable motor recovery. CONCLUSION: We show that minocycline's microbiota effects precede its long-term effects on systemic cytokines and chemokines following spinal cord injury. These results provide an exciting new target of minocycline as a therapeutic for central nervous system diseases and injuries.

What Makes a Successful Donor? Fecal Transplant from Anxious-Like Rats Does Not Prevent Spinal Cord Injury-Induced Dysbiosis.

Spinal cord injury (SCI) causes gut dysbiosis and an increased prevalence of depression and anxiety. Previous research showed a link between these two consequences of SCI by using a fecal transplant from healthy rats which prevented both SCI-induced microbiota changes and the subsequent development of anxiety-like behaviour. However, whether the physical and mental state of the donor are important factors in the efficacy of FMT therapy after SCI remains unknown. In the present study, rats received a fecal transplant following SCI from uninjured donors with increased baseline levels of anxiety-like behaviour and reduced proportion of Lactobacillus in their stool. This fecal transplant increased intestinal permeability, induced anxiety-like behaviour, and resulted in minor but long-term alterations in the inflammatory state of the recipients compared to vehicle controls. There was no significant effect of the fecal transplant on motor recovery in rehabilitative training, suggesting that anxiety-like behaviour did not affect the motivation to participate in rehabilitative therapy. The results of this study emphasize the importance of considering both the microbiota composition and the mental state of the donor for fecal transplants following spinal cord injury.

New Mechanistic Insights, Novel Treatment Paradigms, and Clinical Progress in Cerebrovascular Diseases.

The past decade has brought tremendous progress in diagnostic and therapeutic options for cerebrovascular diseases as exemplified by the advent of thrombectomy in ischemic stroke, benefitting a steeply increasing number of stroke patients and potentially paving the way for a renaissance of neuroprotectants. Progress in basic science has been equally impressive. Based on a deeper understanding of pathomechanisms underlying cerebrovascular diseases, new therapeutic targets have been identified and novel treatment strategies such as pre- and post-conditioning methods were developed. Moreover, translationally relevant aspects are increasingly recognized in basic science studies, which is believed to increase their predictive value and the relevance of obtained findings for clinical application.This review reports key results from some of the most remarkable and encouraging achievements in neurovascular research that have been reported at the 10th International Symposium on Neuroprotection and Neurorepair. Basic science topics discussed herein focus on aspects such as neuroinflammation, extracellular vesicles, and the role of sex and age on stroke recovery. Translational reports highlighted endovascular techniques and targeted delivery methods, neurorehabilitation, advanced functional testing approaches for experimental studies, pre-and post-conditioning approaches as well as novel imaging and treatment strategies. Beyond ischemic stroke, particular emphasis was given on activities in the fields of traumatic brain injury and cerebral hemorrhage in which promising preclinical and clinical results have been reported. Although the number of neutral outcomes in clinical trials is still remarkably high when targeting cerebrovascular diseases, we begin to evidence stepwise but continuous progress towards novel treatment options. Advances in preclinical and translational research as reported herein are believed to have formed a solid foundation for this progress.

Automation of training and testing motor and related tasks in pre-clinical behavioural and rehabilitative neuroscience.

Testing and training animals in motor and related tasks is a cornerstone of pre-clinical behavioural and rehabilitative neuroscience. Yet manually testing and training animals in these tasks is time consuming and analyses are often subjective. Consequently, there have been many recent advances in automating both the administration and analyses of animal behavioural training and testing. This review is an in-depth appraisal of the history of, and recent developments in, the automation of animal behavioural assays used in neuroscience. We describe the use of common locomotor and non-locomotor tasks used for motor training and testing before and after nervous system injury. This includes a discussion of how these tasks help us to understand the underlying mechanisms of neurological repair and the utility of some tasks for the delivery of rehabilitative training to enhance recovery. We propose two general approaches to automation: automating the physical administration of behavioural tasks (i.e., devices used to facilitate task training, rehabilitative training, and motor testing) and leveraging the use of machine learning in behaviour analysis to generate large volumes of unbiased and comprehensive data. The advantages and disadvantages of automating various motor tasks as well as the limitations of machine learning analyses are examined. In closing, we provide a critical appraisal of the current state of automation in animal behavioural neuroscience and a prospective on some of the advances in machine learning we believe will dramatically enhance the usefulness of these approaches for behavioural neuroscientists.

A TrkB Antibody Agonist Promotes Plasticity after Cervical Spinal Cord Injury in Adult Rats.

After spinal cord injury (SCI) in mammals, there is only limited repair and, consequently, only moderate recovery. One mechanism frequently discussed to be involved in this recovery is plasticity (i.e., adaptations in spared neuronal circuitries). In the current study, we tested the effect of an intrathecal application of the TrkB agonist antibody, 29D7, on plasticity after cervical SCI in adult rats. Treatment with 29D7 for 4 weeks resulted in an ∼50% increase in collateral sprouting of severed corticospinal tract fibers above the lesion compared to the control group and enhanced branching in the gray matter rostral to the injury. Growth-associated protein 43 immunoreactivity in the spinal cord rostral to the level of the injury as well as contralateral to the lesion was also increased. These indications of enhanced plasticity by 29D7 were paralleled by improved performances of the mildly affected paw, as assessed by Montoya and tray reaching tasks. The reaching behaviors of the paw ipsilateral to the side of severe injury to the cortico- and rubrospinal tract were not altered by the treatment. The present study suggests that 29D7 may be a potential candidate to promote plasticity and functional recovery, especially after moderate SCI. Future studies confirming these results, along with a potential combinatory therapy including rehabilitative training, will be needed to evaluate the clinical value of such a treatment.

Self-directed rehabilitation training intensity thresholds for efficient recovery of skilled forelimb function in rats with cervical spinal cord injury.

Task specific rehabilitation training is commonly used to treat motor dysfunction after neurological injures such as spinal cord injury (SCI), yet the use of task specific training in preclinical animal studies of SCI is not common. This is due in part to the difficulty in training animals to perform specific motor tasks, but also due to the lack of knowledge about optimal rehabilitation training parameters to maximize recovery. The single pellet reaching, grasping and retrieval (SPRGR) task (a.k.a. single pellet reaching task or Whishaw task) is a skilled forelimb motor task used to provide rehabilitation training and test motor recovery in rodents with cervical SCI. However, the relationships between the amount, duration, intensity, and timing of training remain poorly understood. In this study, using automated robots that allow rats with cervical SCI ad libitum access to self-directed SPRGR rehabilitation training, we show clear relationships between the total amount of rehabilitation training, the intensity of training (i.e., number of attempts/h), and performance in the task. Specifically, we found that rats naturally segregate into High and Low performance groups based on training strategy and performance in the task. Analysis of the different training strategies showed that more training (i.e., increased number of attempts in the SPRGR task throughout rehabilitation training) at higher intensities (i.e., number of attempts per hour) increased performance in the task, and that improved performance in the SPRGR task was linked to differences in corticospinal tract axon collateral densities in the injured spinal cords. Importantly, however, our data also indicate that rehabilitation training becomes progressively less efficient (i.e., less recovery for each attempt) as both the amount and intensity of rehabilitation training increases. Finally, we found that Low performing animals could increase their training intensity and transition to High performing animals in chronic SCI. These results highlight the rehabilitation training strategies that are most effective to regain skilled forelimb motor function after SCI, which will facilitate pre-clinical rehabilitation studies using animal models and could be beneficial in the development of more efficient clinical rehabilitation training strategies.

Inducing inflammation following subacute spinal cord injury in female rats: A double-edged sword to promote motor recovery.

The inflammatory response following spinal cord injury is associated with increased tissue damage and impaired functional recovery. However, inflammation can also promote plasticity and the secretion of growth-promoting substances. Previously we have shown that inducing inflammation with a systemic injection of lipopolysaccharide in the chronic (8 weeks) stage of spinal cord injury enhances neuronal sprouting and the efficacy of rehabilitative training in rats. Here, we tested whether administration of lipopolysaccharide in female rats in the subacute (10 days) stage of spinal cord injury would have a similar effect. Since the lesioned environment is already in a pro-inflammatory state at this earlier time after injury, we hypothesized that triggering a second immune response may not be beneficial for recovery. Contrary to our hypothesis, we found that eliciting an inflammatory response 10 days after spinal cord injury enhanced the recovery of the ipsilesional forelimb in rehabilitative training. Compared to rats that received rehabilitative training without treatment, rats that received systemic lipopolysaccharide showed restored motor function without the use of compensatory strategies that translated beyond the trained task. Furthermore, lipopolysaccharide treatment paradoxically promoted the resolution of chronic neuroinflammation around the lesion site. Unfortunately, re-triggering a systemic immune response after spinal cord injury also resulted in a long-term increase in anxiety-like behaviour.

The neuroanatomical-functional paradox in spinal cord injury.

Although lesion size is widely considered to be the most reliable predictor of outcome after CNS injury, lesions of comparable size can produce vastly different magnitudes of functional impairment and subsequent recovery. This neuroanatomical-functional paradox is likely to contribute to the many failed attempts to independently replicate findings from animal models of neurotrauma. In humans, the analogous clinical-radiological paradox could explain why individuals with similar injuries can respond differently to rehabilitation. We describe the neuroanatomical-functional paradox in the context of traumatic spinal cord injury (SCI) and discuss the underlying mechanisms of the paradox, including the concepts of lesion-affected and recovery-related networks. We also consider the various secondary complications that further limit the accuracy of outcome prediction in SCI and provide suggestions for how to increase the predictive, translational value of preclinical SCI models.

Electrical Stimulation as a Tool to Promote Plasticity of the Injured Spinal Cord.

Unlike their peripheral nervous system counterparts, the capacity of central nervous system neurons and axons for regeneration after injury is minimal. Although a myriad of therapies (and different combinations thereof) to help promote repair and recovery after spinal cord injury (SCI) have been trialed, few have progressed from bench-top to bedside. One of the few such therapies that has been successfully translated from basic science to clinical applications is electrical stimulation (ES). Although the use and study of ES in peripheral nerve growth dates back nearly a century, only recently has it started to be used in a clinical setting. Since those initial experiments and seminal publications, the application of ES to restore function and promote healing have greatly expanded. In this review, we discuss the progression and use of ES over time as it pertains to promoting axonal outgrowth and functional recovery post-SCI. In doing so, we consider four major uses for the study of ES based on the proposed or documented underlying mechanism: (1) using ES to introduce an electric field at the site of injury to promote axonal outgrowth and plasticity; (2) using spinal cord ES to activate or to increase the excitability of neuronal networks below the injury; (3) using motor cortex ES to promote corticospinal tract axonal outgrowth and plasticity; and (4) leveraging the timing of paired stimuli to produce plasticity. Finally, the use of ES in its current state in the context of human SCI studies is discussed, in addition to ongoing research and current knowledge gaps, to highlight the direction of future studies for this therapeutic modality.

Fecal transplant prevents gut dysbiosis and anxiety-like behaviour after spinal cord injury in rats.

Secondary manifestations of spinal cord injury beyond motor and sensory dysfunction can negatively affect a person's quality of life. Spinal cord injury is associated with an increased incidence of depression and anxiety; however, the mechanisms of this relationship are currently not well understood. Human and animal studies suggest that changes in the composition of the intestinal microbiota (dysbiosis) are associated with mood disorders. The objective of the current study is to establish a model of anxiety following a cervical contusion spinal cord injury in rats and to determine whether the microbiota play a role in the observed behavioural changes. We found that spinal cord injury caused dysbiosis and increased symptoms of anxiety-like behaviour. Treatment with a fecal transplant prevented both spinal cord injury-induced dysbiosis as well as the development of anxiety-like behaviour. These results indicate that an incomplete unilateral cervical spinal cord injury can cause affective disorders and intestinal dysbiosis, and that both can be prevented by treatment with fecal transplant therapy.