THE EFFECTS OF ANTICONVULSANTS ON NEUROLOGICAL RECOVERY AFTER SPINAL CORD INJURY

by

Freda Warner

B.Sc., University of Guelph, 2011 M.P.H., Simon Fraser University, 2015

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

in

The Faculty of Graduate and Postdoctoral Studies (Kinesiology)

THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)

September 2019

© Freda Warner, 2019

The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled:

The effects of anticonvulsants on neurological recovery after spinal cord injury

Submitted by Freda Warner in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Kinesiology

Examining Committee:

John Kramer, Kinesiology Supervisor

Tania Lam, Kinesiology Supervisory Committee Member

Supervisory Committee Member

Roanne Preston, Anesthesiology, Pharmacology and Therapeutics University Examiner

Thomas Oxland, Mechanical Engineering University Examiner

Additional Supervisory Committee Members:

Supervisory Committee Member

Supervisory Committee Member

ii Abstract

Spinal cord injury is a devastating neurological condition that results in varying degrees of sensory and motor loss, along with other health complications. Neurological recovery after spinal cord injury is generally thought to be limited to the 6-9 month period after injury, and there are currently no approved pharmacological interventions to improve this recovery. Overlapping with a proposed “window of opportunity” for interventions, neuropathic pain can occur early after injury and necessitate pharmacological management. Among the management options, anticonvulsants are routinely administered.

Utilizing longitudinal observational human spinal cord injury data, this thesis explored the effects of anticonvulsants on neurological recovery after spinal cord injury using mixed effects regression, and conduct a meta-analysis on the acute progression of neuropathic pain.

The research studies within this thesis are bookended by an introduction and methodology section (Chapters 1 and 2) and the discussion (Chapter 7). In Chapter 3, I examined the effect of anticonvulsants and found a beneficial association with motor recovery contingent on administration at 4 weeks. A review of patient records revealed that the majority of anticonvulsants being administered were gabapentinoids (i.e. pregabalin and gabapentin). To further examine whether this effect was specific to gabapentinoids or obtained by all anticonvulsants, Chapter 4 examined a unique spinal cord injured population administered non-gabapentinoid anticonvulsants and found no statistically significant associations with neurological recovery. Chapter 5 included a chart review to examine the effect of gabapentinoid-specific administration, and found a continued beneficial association with motor score, as well as the sensory outcome of light touch. Further, this chapter identified that very early administration (e.g. within 5 days) was necessary to achieve the largest benefit. Finally, Chapter 6 produced a longitudinal framework of neuropathic pain progression in clinical trials.

In short, this thesis presents novel findings regarding the administration of anticonvulsants after spinal cord injury, and the beneficial association of gabapentinoid- specific anticonvulsants on motor recovery. Further, it provides an advance in our

iii understanding of neuropathic pain progression after injury and a framework to guide future clinical trials.

iv Lay summary

Spinal cord injuries can result in a loss of sensation and movement. After injury there is usually an initial period of neurological recovery (i.e. recovery of movement and sensation), after which these impairments are permanent. There are currently no approved drugs that can help maximize this neurological recovery after injury. Anticonvulsants, including gabapentinoids, are drugs that are commonly administered after spinal cord injury for complications including neuropathic pain and seizures. Using human spinal cord injury cohort and registry data, I examined the effects of anticonvulsants on neurological recovery after spinal cord injury. Through secondary data analysis I identified that the early administration of gabapentinoid-specific anticonvulsants (and not anticonvulsants in general) was associated with an improvement in recovery. I also describe the progression of neuropathic pain after injury, with the goal of informing future clinical trials.

v Preface

A version of Chapter 1 is a published book chapter [Warner FM, Cragg J, Steeves JC, Kramer JLK. “Clinical trials and spinal cord injury: Challenges and therapeutic interventions.” In Neurological Aspects of Spinal Cord Injury. Springer, 2017]. I was the lead investigator, responsible for concept formation and all major manuscript composition. Cragg J, Steeves JC, and Kramer JLK were also responsible for concept formation and manuscript edits. Kramer JLK was the supervisory author on this project.

A version of Chapter 3 has been published in Cell Reports [Warner FM, Cragg JJ, Jutzeler CR, Röhrich F, Weidner N, Saur M, Maier DD, Schuld C, EMSCI Study Group, Curt A, Kramer JK. Early administration of gabapentinoids improves motor recovery after human spinal cord injury. Cell Reports 2017; 18:1614-1618]. Cragg JJ, Jutzeler CR, Röhrich F, Weidner N, Saur M, Maier DD, Schuld C, and Curt A contributed to concept formation and manuscript edits. EMSCI Study Group was responsible for data collection. Kramer JK was the supervisory author on this project and was involved in concept formation and manuscript edits. I was involved in concept formation and responsible for all analytical methods, data analysis, and manuscript composition.

A version of Chapter 4 has been published in CNS Drugs [Warner FM, Jutzeler CR, Cragg JJ, Tong B, Grassner L, Bradke F, Geisler F, Kramer JK. The effect of non- gabapentinoid anitconvulsants on sensorimotor recovery after spinal cord injury. CNS Drugs 2019; 33(5):503-511]. Jutzeler CR and Cragg JJ contributed to concept formation and manuscript edits. Geisler F was responsible for the data collection and manuscript edits. Tong B, Grassner L, and Bradke F were responsible for content and manuscript edits. Kramer JK was the supervisory author on this project and was involved in concept formation and manuscript edits. I was involved in concept formation and responsible for all analytical methods, data analysis, and manuscript composition.

In Chapter 5, Cragg JJ, Jutzeler CR, Maier D, and Curt A contributed to concept formation and chapter edits. Grassner L, Mach O, Curt A and the EMSCI Study Group were responsible for data collection and chapter edits. Kramer JK was the supervisory author on this project and was involved in concept formation and chapter edits. I was involved in concept formation and responsible for all analytical methods, data analysis, and chapter composition.

A version of Chapter 6 has been published in the Journal of Neurotrauma [Warner FM, Cragg JJ, Jutzeler CR, Finnerup NB, Weargen L, Weidner N, Maier D, Kalke Y-B, Curt A, Kramer JK. Progression of neuropathic pain after acute spinal cord injury: a meta- analysis and framework for clinical trials. Journal of Neurotrauma 2019; 1-8]. Jutzeler CR, and Cragg JJ contributed to concept formation and manuscript edits. Finnerup NB, Weargen L, Weidner N, Maier D, Kalke Y-B, Curt A and the EMSCI sites were responsible for data collection and manuscript edits. Kramer JK was the supervisory author on this project and was involved in concept formation and manuscript edits. I was involved in concept formation and responsible for all analytical methods, data analysis, and manuscript composition.

Ethics approvals for the studies contained in this thesis were obtained by the University of British Columbia’s Research Ethics board (certificates #H16-01119 and H16-03143).

vi Table of contents

Abstract ...... iii Lay summary ...... v Preface ...... vi Table of contents ...... vii List of tables ...... x List of figures...... xii List of abbreviations ...... xiii Acknowledgements ...... xiv Chapter 1 Introduction ...... 1 1.1 Overview: the search for acute interventions to improve neurological outcomes for individuals with spinal cord injuries ...... 1 1.2 Challenges of spinal cord injury trials ...... 1 1.2.1 A “moving target”: effect of the natural progression of spinal cord injury ...... 2 1.2.2 Low numbers becoming lower ...... 2 1.2.3 Enrolment and assessment into an acute clinical trial: very early interventions may be necessary ...... 3 1.2.4 Neurological outcome measures: what is the best outcome to assess efficacy?...... 3 1.2.5 Ethical issues: informed consent and potential risks ...... 4 1.2.6 Translation from animal models to humans with spinal cord injury: what is the right model? ...... 5 1.3 Past and current interventions in spinal cord injury ...... 6 1.3.1 Literature review protocol ...... 6 1.3.2 Non-pharmacological neuroprotection ...... 13 1.3.3 Pharmacological interventions ...... 14 1.3.4 Regeneration and repair ...... 20 1.4 New directions: drug repurposing in spinal cord injury ...... 20 1.5 Spinal cord injury, pain, and pain management ...... 22

vii 1.6 Anticonvulsants and spinal cord injury ...... 24 1.7 Objectives ...... 25 Chapter 2 Methods ...... 27 2.1 Data ...... 27 2.1.1 The European multicenter study about spinal cord injury ...... 27 2.1.2 Sygen ...... 29 2.1.3 Chart review: trauma center Murnau...... 31 2.1.4 Swedish/Danish cohort ...... 31 2.2 Statistical methods ...... 32 2.2.1 Variables ...... 32 2.2.2 Longitudinal mixed effects regression ...... 34 Chapter 3 The effect of anticonvulsants on neurological recovery after spinal cord injury in EMSCI ...... 37 3.1 Introduction ...... 37 3.2 Methods ...... 38 3.2.1 Data ...... 38 3.2.2 Variables ...... 38 3.2.3 Statistics ...... 39 3.3 Results ...... 39 3.4 Discussion ...... 48 Chapter 4 The effect of anticonvulsants on neurological recovery after spinal cord injury in Sygen ...... 51 4.1 Introduction ...... 51 4.2 Methods ...... 52 4.2.1 Data ...... 52 4.2.2 Variables ...... 53 4.2.3 Statistics ...... 53 4.3 Results ...... 54 4.4 Discussion ...... 65 Chapter 5 The effect of gabapentinoids on neurological recovery after spinal cord injury ...... 68

viii 5.1 Introduction ...... 68 5.2 Methods ...... 69 5.2.1 Data ...... 69 5.2.2 Variables ...... 69 5.2.3 Statistics ...... 70 5.3 Results ...... 71 5.4 Discussion ...... 85 Chapter 6 The progression of neuropathic pain after acute spinal cord injury .... 89 6.1 Introduction ...... 89 6.2 Methods ...... 90 6.2.1 Data ...... 90 6.2.2 Variables ...... 90 6.2.3 Statistics ...... 91 6.3 Results ...... 93 6.3.1 Progression ...... 94 6.3.2 Associations with neuropathic pain in the EMSCI ...... 98 6.4 Discussion ...... 102 Chapter 7 General discussion ...... 106 7.1 Key findings ...... 106 7.2 General strengths and limitations ...... 106 7.2.1 Strengths ...... 106 7.2.2 Limitations ...... 107 7.3 Implications and future directions ...... 107 7.3.1 Spinal cord injury as a model for drug repurposing ...... 108 7.3.2 Spinal cord injury data sets ...... 111 7.3.3 Implications for clinical research ...... 112 7.3.4 Implications for observational research ...... 114 7.4 Conclusion ...... 116 Bibliography ...... 117 Appendix ...... 130

ix List of tables

Table 1.1 Characteristics of the trials included in the literature review ...... 8 Table 2.1 An overview of the data sources and methodologies used within each chapter ...... 27 Table 2.2 A comparison of injury types included in the NASCIS vs. Sygen studies ...... 30 Table 2.3 The ASIA grades and modified Benzel classification ...... 34 Table 3.1 Cohort description ...... 41 Table 3.2 Anticonvulsant group descriptions ...... 42 Table 3.3 Linear mixed effects regression model outputs ...... 43 Table 3.4 Examining the addition of confounder to LMER model fit using ANOVA ...... 45 Table 4.1 The characteristics of all early anticonvulsant users vs. non-users and late users ...... 55 Table 4.2 Early anticonvulsants administered in Sygen ...... 56 Table 4.3 The characteristics of all early sodium channel blocker users vs. non- users and late users ...... 57 Table 4.4 Linear mixed effects regression models for total motor score from 4- weeks for all anticonvulsants ...... 58 Table 4.5 Linear mixed effects regression models for total motor score from 4- weeks for sodium channel blockers ...... 60 Table 4.6 Linear mixed effects regression models for total motor score from 4- weeks for sodium channel blockers in a propensity score matched cohort . 62 Table 4.7 Logistic regression models for marked recovery at 26-weeks ...... 64 Table 5.1 Cohort demographics ...... 71 Table 5.2 Anticonvulsants administered ...... 72 Table 5.3 Gabapentinoid user demographics ...... 73 Table 5.4 Duration of use for early vs. late administration ...... 74 Table 5.5 Longitudinal model of motor scores from 4 weeks ...... 74 Table 5.6 Longitudinal model of total light touch scores from 4 weeks ...... 76 Table 5.7 Longitudinal model of total pin prick scores from 4 weeks ...... 77 Table 5.8 Longitudinal model of total SCIM scores from 4 weeks...... 78 Table 5.9 Longitudinal model of total motor scores from 4 weeks in a propensity score matched cohort ...... 80 Table 5.10 Comparison of drug*time term in longitudinal models of total motor scores from 4 weeks using varying timing of exposure ...... 85 Table 6.1 Demographics of the cohorts at first visit ...... 93 Table 6.2 The aggregate estimates of overall, at-level, and below-level neuropathic pain from 1 to 6 months after spinal cord injury...... 94 Table 6.3 The aggregate estimates of overall, at-level, and below-level neuropathic pain from 1 to 12 months after spinal cord injury...... 95 Table 6.4 The results of the logistic regression models for at-level neuropathic pain ...... 96

x Table 6.5 The results of the logistic regression models for below-level neuropathic pain ...... 99

xi List of figures

Figure 1.1 Flow chart of the selection process for the literature review of clinical trials ...... 7 Figure 1.2 Medications administered to an acute spinal cord injury patient in the first 3 months after injury (Sygen) Created by Cheryl Niamath ...... 22 Figure 1.3 The conceptual model for this thesis...... 26 Figure 2.1 International standards for neurological classification of spinal cord injury © 2019 American Spinal Injury Association. Reprinted with permission...... 33 Figure 2.2 The motor score trajectories of 25 randomly selected individuals from the Sygen data in Chapter 4 ...... 36 Figure 3.1 Participants included from the EMSCI dataset ...... 40 Figure 3.2 The effects of early anticonvulsant use on total motor recovery using boxplots ...... 46 Figure 3.3 The modeled effects of anticonvulsant use on motor recovery following spinal cord injury...... 47 Figure 4.1 Boxplots of motor scores over time for sodium channel blockers ...... 63 Figure 5.1 Trajectories and changes in total motor scores of early and late/never gabapentinoid users ...... 81 Figure 5.2 Trajectories and changes in total light touch in early and late/never gabapentinoid users ...... 82 Figure 5.3 Summary of model outputs ...... 84 Figure 6.1 The longitudinal progression of overall neuropathic pain estimates pooled from EMSCI and the Swedish/Danish study ...... 98 Figure 6.2 The and light touch-pinprick differences for developing neuropathic pain in the EMSCI cohort ...... 101 Figure 7.1 Patterns of disease progression ...... 110

xii List of abbreviations

AIS American Spinal Cord Injury Association Impairment Scale ANOVA Analysis of Variance ASIA American Spinal Cord Injury Association BMC Bone Marrow Cells CDE Common Data Elements CI Confidence Interval CNS Central Nervous System EMSCI European Multicenter Study about Spinal Cord Injury EPO Erythropoietin GABA Gamma-Aminobutyric Acid GM-1 Monosialotetrahexosylganglioside ISNCSCI International Standards for Neurological Classification of Spinal Cord Injury LMER Linear Mixed Effects Regression MP Methylprednisolone NASCIS National Acute Spinal Cord Injury Study NINDS National Institute of Neurological Disorders and Stroke NSAID Non-Steroidal Anti-Inflammatory Drug RCT Randomized Controlled Trial ROM Range of Motion SCI Spinal Cord Injury SCIM Spinal Cord Injury Independence Measure SD Standard Deviation SSEP Somatosensory Evoked Potential TRH Thyrotropin-Releasing Hormone

xiii Acknowledgements

I would like to thank the members of my supervisory committee, Drs. Tania Lam, David Whitehurst, and Helen Tremlett for their continued guidance and suggestions. I would especially like to thank my supervisor Dr. Kip Kramer for his support over these many years. Thank you for making sure I always had an array of opportunities and projects to work on and providing valuable feedback on every one. May you continue to use track changes, because with it your comments have helped me to become a better writer.

I would also like to thank those who have helped support me financially over the years including UBC and the CIHR DSECT program. Equally as important, this thesis would not have been possible without the hard work and generosity required in the collection and sharing of data. Chapters 3 and 5 were made possible by the work conducted at the EMSCI study centers. I would like to thank each of them, and in particular Dr. Armin Curt, Dr. Lukas Grassner, Orpheus Mach, and Dr. Doris Maier. Additional thanks are also due to Dr. Fred Geisler for allowing us to examine the Sygen data in Chapter 4, and Dr. Nanna Finnerup for allowing us to include her data in Chapter 6. Finally, I am very thankful to all the participants who agreed to take part in these studies and made this type of research possible.

To my labmates- Dr. Catherine Jutzeler helped guide me through this process from the very first day. Bobo Tong provided continuous help in data cleaning, technically difficulties, and emergency snacks. Jessica Archibald helped to talk me down before presentations. And I can genuinely say that every member that has come through our lab (Eitan, Erin, Anh, Navid, Jessie, and more) has helped make my time here more enjoyable. Thank you to everyone at ICORD, and especially to Matt, Cheryl and Lowell for their constant patience and help! And thank you so much to Dr. Jacquelyn Cragg, who brought me to ICORD, encouraged me to do a PhD, helped me throughout the process, and continues to support and help anyone who is lucky enough to work with her.

I also owe a big thanks to my non-ICORDian friends near and far who helped keep me sane and grounded in the world beyond just research. You made sure I ventured into the real world through beers, hikes, books, shows, trips, dinners, celebrations, and everything in between.

And finally thank you to my family. My parents, for their confidence in me and their constant support as I continued my education (again). My siblings Hannah, Maud and Greg, and their wonderful partners and progeny- thank you as well for your support and putting up with my rants these past four years. And finally thank you to Hugo, who has been next to me since I started and listened tirelessly to every step, frustration, and tiny detail (even when they were incredibly boring). Thank you for your support and unwavering belief in me, I hope I can be half as good as you already think I am.

xiv Chapter 1 Introduction

1.1 Overview: the search for acute interventions to improve neurological outcomes for individuals with spinal cord injuries

In an attempt to overcome the limited capacity for regeneration in the central nervous system (CNS), acute therapeutic interventions after spinal cord injury (SCI) generally fall into one of two categories: neuroprotection and neural regeneration. Following primary damage to the spinal cord, a cascade of biochemical events triggers secondary injury to spared white and gray matter. A window of opportunity for neuroprotection in preventing the cascade of secondary injury is thought to emerge in the initial hours to days after injury.1 While preventing secondary injury represents a reasonable target for improving neurological outcomes, full restoration of sensory and motor function requires regeneration in the spinal cord.2 Emerging regenerative therapies are focused on promoting functional recovery by inhibiting factors that prevent endogenous repair or biochemicals that promote axonal sprouting (e.g., short-distance growth).3 The goal of this introduction is to begin by reviewing the challenges posed in clinical trials in spinal cord injury and provide literature review of the acute randomized controlled trials (RCTs) that have been conducted until now. These largely traditional approaches to RCTs (e.g. bench-to-bedside) will then be compared with the idea of observational research techniques and drug repurposing research. Finally, the use of anticonvulsants as a specific target for drug repurposing will be explored, and my overall thesis objectives stated.

1.2 Challenges of spinal cord injury trials

Before reviewing interventions conducted in the field of spinal cord injury, it is important to briefly describe the challenges facing patients, researchers, and clinicians performing clinical trials. While not necessarily unique to spinal cord injury, these challenges represent a significant barrier to detecting therapeutic benefits of any intervention, and linking neurological improvements with long-term functional benefits.

1 1.2.1 A “moving target”: effect of the natural progression of spinal cord injury

Spinal cord injury is a devastating neurological condition, often characterized by severe and life-long impairments. Similar to other traumatic neurological conditions (e.g. stroke), spinal cord injury is associated with some degree of neurological and functional recovery. Neurological recovery is most evident in the initial days to weeks post-injury and is characterized by a rapid increase in muscle strength for up to 6-9 months, which generally plateaus by 12 months.4–7 Neurological recovery is best predicted by the initial severity of damage in the spinal cord (i.e. degree of impairment), as individuals with less severe injuries are (generally) capable of greater recovery than individuals with more severe injuries.4,8 However, even within a defined injury severity or level of injury, neurological baselines and recovery patterns can vary greatly with some individuals experiencing a great deal of neurological recovery, and others very little.8,9 Neurological recovery is fundamental to the return of functional independence, such as self-care and ambulation. In terms of designing clinical trials, the benefits of a therapeutic intervention must be distinguishable from naturally occurring recovery of function, which is difficult due to the variable recovery patterns, and ideally requires large sample sizes to account for this. At a minimum, it requires careful consideration in terms of study design, ensuring that injury severities are similarly distributed between treatment arms of a clinical trial.10 This can be problematic and is related to our second challenge: low numbers of suitable participants.

1.2.2 Low numbers becoming lower

Spinal cord injury has a relatively low incidence, however, this in turn can make recruiting for a clinical trial very challenging. The heterogeneity in injury severity and neurological recovery profiles further exacerbate this problem.10 Furthermore, most trials apply exclusion criteria regarding timing of intervention (discussed further below), injury level or severity, age, previous treatments, comorbidities, and additional injuries (which are particularly common after trauma). Applying these factors to the already low numbers of spinal cord injuries often prevents individuals from being eligible to participate in trials and creates a ‘funnel-effect’ in which a large number of potentially

2 eligible participants are significantly reduced by the inclusion/exclusion criteria. As an example, 1816 participants were pre-screened for a phase 2 multi-centre study examining the acute neurological effects of autologous cellular therapy in individuals with spinal cord injuries.11 After excluding subjects for being more than 14 days post-injury (a relatively long window for acute trials), having the incorrect injury characteristics and/or failing to meet MRI criteria, the final recruitment number was 50. Of these 50 individuals, only 32 completed the follow-up assessment at 12-months.11

1.2.3 Enrolment and assessment into an acute clinical trial: very early interventions may be necessary

Low subject numbers for clinical trials is particularly problematic for neuroprotective strategies, as they are believed to require very early intervention (i.e. initial hours after spinal cord injury).12,13 Several practical constraints limit recruitment, including patient transportation to a participating centre, acquiring informed consent, and confirming a participant meets all inclusion criteria. Substantial barriers exist for each of these requirements, including ethical debates as to whether an individual with an acute spinal cord injury can reasonably be expected to provide informed consent (see Ethical Issues below). Of paramount importance, detecting efficacy of an acute intervention is contingent on the validity and reliability of the acute neurological assessment. This is important both in terms of enrolling the correct subjects, and establishing baseline measurements that will serve as anchors from which to measure change. In practice, an accurate (i.e. reliable) neurological exam may be very difficult to perform in participants with very acute spinal cord injuries (<72 hours), related to spinal shock, concomitant injuries, ventilator use, or the influence of drugs or alcohol in the very early stages.14

1.2.4 Neurological outcome measures: what is the best outcome to assess efficacy?

As already described, an accurate neurological examination soon after injury is integral for assessing subsequent neurological recovery, and can be difficult to ascertain very early after injury. Equally important, neurological outcomes need to be sensitive to subtle changes, yet representative of important changes in function. The selection of different

3 neurological outcomes may vary depending on the phase of study, and range from neurophysiological (e.g., somatosensory evoked potentials [SSEPs]) to a more conventional examination of muscle strength and sensory function (i.e., International Standards for the Neurological Classification of SCI [ISNCSCI]).10 Neurophysiology may play an important role in early phases of study, supporting a potential mechanism and substrate for neurological repair and regeneration (e.g., decreased latencies of SSEPs as an indication of remyelination). Several outcomes can be derived from the ISNCSCI and have been used to measure efficacy of acute therapeutics, including changes in total motor score (i.e., upper and lower extremities), and American Spinal Injury Association (ASIA) Impairment Scale (AIS) grade conversion.10 Changes in motor levels also have been proposed as a viable clinical trial endpoint among individuals with tetraplegia. Motor level recovery may be particularly important among individuals with cervical sensorimotor complete injuries, where recovery of segmental muscle strength near the injury site may precipitate significant improvement in hand function.5 Several prominent issues with the ISNCSCI remain for applications in clinical trials. For example, it is difficult to ascertain the extent of neurological improvement that is considered a clinically “minimally important difference”, however, attempts have been made to estimate this using ISNCSCI motor scores.15,16 For any intervention, this complex issue requires delicate weighting of potential benefits of neurological improvements with functional outcomes (e.g. the Spinal Cord Independence Measure [SCIM]), as well as an analysis of the potential cost to the individual (i.e. side-effects).

1.2.5 Ethical issues: informed consent and potential risks

There is an ongoing debate as to whether an individual sustaining an acute spinal cord injury can provide informed consent to participate in a clinical trial.17 This is a barrier to recruitment, and worthy of serious consideration. Ethical issues around this topic stem from the capacity of acutely injured individuals to make decisions and process the relevant information regarding potential risks, rewards, and long-term implications (e.g. the potential to be excluded from other clinical trials), and therefore knowledgably consent or decline participation.17 In addition to one’s ability to grant consent, questions have been raised regarding what should be included in the informed consent. Of note is the high termination rate of clinical trials, and whether participants should be informed of

4 this additional risk.18,19 Other ethical issues arise for invasive treatments in spinal cord injury (e.g. first “in-human” therapies), and trials where a true placebo arm might be unethical and/or introduce unnecessary risk or harm.11,14 There is particular concern regarding the possibility that some cellular therapies may lead to tumour growth, and that strategies promoting plasticity may lead to the development of neuropathic pain. 20,21

1.2.6 Translation from animal models to humans with spinal cord injury: what is the right model?

Prior to application in human clinical trials, a pharmacological intervention is required to demonstrate a history of safety and efficacy in animal models of spinal cord injury. Animal models of spinal cord injury have included a variety of experimental injuries (e.g. contusion or transection) in mice, rats, cats, dogs, swine, and non-human primates.2 Despite substantial preclinical evidence of neurological improvements from neuroprotective or reparative therapeutic interventions, no treatment has transitioned successfully into humans (note: more on the specifics of failed clinical trials below). Further, challenges also emerge within animal studies, particularly with regards to the replication of preclinical results. This can be due to reasons reasons such as differences or alterations in animal types or suppliers of the animals (even differences within a single supplier), in batches and lots of reagents, in surgical procedures, in animal housing or post-operative care, or in the injury models.22 Difficulty in replication can even arise within the same species and/or similar model conditions for a variety of experimental and biological reasons.23,24 The assessment of functional recovery within and across species can vary widely, and even commonly accepted measures (such as the Basso, Beattie, Bresnahan scale for hind limb recovery in rat models) may be improperly conducted or subjectively interpreted.25 Other sources of variation across studies include lesion severity, strains of animal, and behavioural influences (e.g. a stressful environment).25 These failures have led some investigators to suggest that animal models do not accurately predict whether a therapy will be effective in humans. As such, the translational path to clinical studies has not been smooth. Among suspected translation problems are: the human condition being more heterogeneous than experimental spinal cord injury (i.e. single uniform injury mechanisms in animal models vs the heterogeneity of injuries in the human population),22 differences in the natural history of recovery, as

5 well as the appropriate design of study protocols and outcome measures.26

1.3 Past and current interventions in spinal cord injury

Despite the numerous challenges, researchers and clinicians have embarked on several acute randomized clinical trials to improve long-term neurological outcomes after spinal cord injury. In order to provide an idea of how neuroprotective or regenerative interventions in human spinal cord injury have progressed thus far, I have conducted a literature review. The objective was to examine the results of randomized clinical trials in acute spinal cord injury participants in terms of long-term neurological recovery.

1.3.1 Literature review protocol

For the literature review, clinical trials included had to (1) be conducted in humans, (2) be limited to those with a spinal cord injury (i.e. not include other diseases), (3) include traumatic spinal cord injury (i.e. not only non-traumatic), (4) have prospectively randomized the participants into 2+ treatment groups, (5) have delivered the intervention to participants within 3 months of injury, (6) have assessed the outcomes measures at least 6 months after injury (as is recommended for neuroprotective and regenerative trials once the majority of recovery has occurred),27 and (7) have included specific baseline and follow-up sensorimotor neurological outcomes. These outcomes had to be derived from the American Spinal Injury Association (ASIA)’s ISNCSCI neurological assessment, which included ASIA Impairment Scale (AIS), the motor evaluation (0-100) and the sensory evaluation (light touch and pinprick, each 0-112).27 As these standards were only developed in 1982,28 near-equivalent measures that were used prior to this were also accepted,29 which included the Frankel Scale upon which AIS was based, or the National Acute Spinal Cord Injury Study system which contained similar motor and sensory measures.30

6

Figure 1.1 Flow chart of the selection process for the literature review of clinical trials

7 I searched the electronic database PubMed using the search terms “spinal cord injury”, and “clinical trial” with the Boolean logic operator “AND” on March 21 2019. This resulted in 3286 studies, and after removing duplicates, 3281 remained. Screening study titles and abstracts reduced this to 144 studies, and after examining these studies in detail a total of 19 clinical trials met the aforementioned criteria (Figure 1.1). I did not assess the risk of bias within individual studies, or across studies. I have reviewed the methodology and outcomes of these studies in terms of neurological outcomes below (Table 1.1). Limitations and potential biases of this search include the use of a single search engine (PubMed), and a single reviewer as opposed to 2 independent reviewers.

Table 1.1 Characteristics of the trials included in the literature review

Timing of Neurological Authors & Length of Intervention Administration first outcome Year follow-up intervention measures Bracken et High dose and 100g initial IV dose Within 48 6 months Measures of al., 198431 low-dose followed by 25g hours motor function, methylpredni- every 6 hours for pin prick, and solone 10 days light touch

1000mg initial IV dose followed by 250g every 6 hours for 10 days Bracken et Methylpredni- 30mg/kg initial MP Within 12 6 months Measures of al., 199032 solone and IV dose followed by hours motor function, naloxone 5.4 mg/kg/hour for pin prick, and 23 hours light touch

5.4mg/kg initial naloxone hydrochloride IV dose followed by 4.0 mg/kg/hour for

8 Timing of Neurological Authors & Length of Intervention Administration first outcome Year follow-up intervention measures 23 hours. Geisler et al., GM-1 100mg daily IV Within 72 6 and 12 Frankel grades 199133 ganglioside dose of GM-1 for hours months and ASIA motor 18-32 doses scores Pitts et al., Thyrotropin- 0.3mg/kg initial IV Within 12 12 months Measures of 199534 releasing dose followed by hours motor function, hormone 0.2mg/kg/hour for 6 pin prick, and hours light touch Vaccaro et Early and late Surgery earlier Within 72 Approxima- Frankel grades al., 199735 surgery than 72 hours post- hours tely 12 and ASIA motor injury months scores

Surgery later than 5 days post-injury Bracken et Methylpredni- 30 mg/kg initial MP Within 8 6 months Measures of al., 199736 solone and IV dose followed by hours motor function, tirilazad 5.4 mg/kg/hour for pin prick, and mesylate 24 hours light touch

30 mg/kg initial MP IV dose followed by 2.5mg/kg tirilizad mesylate every 6 hours for 48 hours

30 mg/kg initial MP IV dose followed by 5.4 mg/kg/hour for 48 hours Pointillart et Methylpredni- 30 mg/kg initial MP Within 8 12 months ASIA motor, light

9 Timing of Neurological Authors & Length of Intervention Administration first outcome Year follow-up intervention measures al., 200037 solone and IV dose followed by hours touch, and pin nimodipine 5.4 mg/kg/hour for prick scores 23 hours

0.15 mg/kg/hour of nimodipine IV for 2 hours followed by 0.03 mg/kg/h for 7 days Geisler et al., High dose and 600mg initial GM-1 Within 8 6 and 12 Modified Benzel 200138 low dose GM- IV dose, followed hours of months Classification and 1 ganglioside by 200 mg/day for injury ASIA motor, light 56 days touch, and pin pick scores 300mg initial GM-1 IV dose, followed by 100 mg/day for 56 days Brodke et al., Anterior and Anterior surgery At admission Minimum 6 Frankel grades 200339 posterior months and ASIA motor surgery Posterior surgery scores Cengiz et al., Early and late Surgery within 8 Within 8 12-20 ASIA grades and 200840 surgery hours post-injury hours months motor scores

Surgery within 3-15 days post-injury Kwon et al., Cerebrospinal Intrathecal lumbar Within 48 6 months ASIA motor 200941 fluid drainage catheter inserted hours scores for 72 hours to allow cerebrospinal

10 Timing of Neurological Authors & Length of Intervention Administration first outcome Year follow-up intervention measures fluid drainage to lower intrathecal pressure to a minimum level of 10mm Hg Lammertse Autologous 6 20μl injections of Within 14 6, 9, and 12 ASIA grades and et al., 201242 incubated 250 000 days months motor, light macrophages macrophages touch, and pin injected with pick scores syringe Casha et al., High dose and 200mg minocycline Within 12 6 and 12 ASIA motor, light 201243 low dose IV dose twice daily hours months touch, and pin minocycline until 7 days post- prick scores injury

800mg initial minocycline IV dose, tapered by 100mg at each 12 hour administration until 400mg followed by 400mg twice daily continued until 7 days post-injury Alibai et al., Erythropoietin 500 unit/kg initial Within 6 6 months ASIA grades 201344 erythropoietin IV hours dose followed by 500 unit/kg dose 24 hours later

11 Timing of Neurological Authors & Length of Intervention Administration first outcome Year follow-up intervention measures Rahimi- Early and late Surgery less than Within 24 6 and 12 ASIA grades, and Movaghar et surgery 24 hours post- hours months motor, light al., 201445 injury touch, and pin prick scores Surgery within 24- 72 hours post- injury Alibai et al., Erythropoietin 500 unit/kg initial Within 8 6 and 12 ASIA motor, light 201546 erythropoietin IV hours months touch, and pin dose followed by prick scores 500 unit/kg dose 24 hours later Chhabra et Autologous 6 300μl injections From 10-14 6 and 12 ASIA grades and al., 201647 bone marrow into spinal cord for days months motor, light cell total of 2 x 108 touch, and pin bone marrow cells prick scores

Intrathecal dose of 2 x 108 bone marrow cells in 1.8ml Aminman- Progesterone 0.5mg/kg Within 8 6 months ASIA grades, and sour et al., and vitamin D intramuscular hours motor and pin 201648 injection of prick scores progesterone injection twice daily for 5 days and 5μg/kg oral vitamin D3 twice daily for 5 days

12 Timing of Neurological Authors & Length of Intervention Administration first outcome Year follow-up intervention measures Meshkini et Riluzole 50mg orally every At admission 6 months Frankel grades al., 201849 12 hours for 8 weeks IV: intravenous; g: gram; mg: milligram; kg: kilogram; μl: microliter; μg: microgram

1.3.2 Non-pharmacological neuroprotection

Surgical decompression

Surgical decompression and/or stabilization following spinal cord injury is performed to reduce pressure on the spinal cord and can involve the removal of bone, as well as instrumentation of the vertebrae to stabilize the spinal column and prevent further injury.1,50 Although successful in animal models, surgical decompression in humans has generated more variable outcomes. In terms of neurological recovery, retrospective studies have largely confirmed the safety of surgical decompression, but provide limited evidence of efficacy.51 As such, the only randomized trials conducted on the topic examine the importance of timing of surgery after injury. The first single-centre American study examined the outcomes of early (<72 hours of injury) vs. late (> five days after injury) decompressive surgery. 35 After randomizing 62 participants with cervical traumatic spinal cord injuries with cord compression from 1992-1995, they found no statistically significant effect on changes in Frankel grades from admission to hospital to discharge from rehabilitation, nor in motor scores. The authors proposed that perhaps 72 hours was too long to constitute an “early” surgery. Another RCT began recruiting 27 thoracolumbar spinal cord injury participants in 2004 to compare even earlier decompressive surgery (<8 hours) to very late (3-15 days) surgery.40 They found that the changes in ASIA scores were statistically significantly better in the early surgery group vs. the late surgery group. A single-centre RCT that began recruitment in 2010 randomized 35 traumatic thoracolumbar spinal cord injury participants to early (<24 hours) or late (>24 hours) decompressive surgery.45 Although appropriate statistical analyses were described in the methodology, no risk estimates or p-values were reported, and it appears that there were no statistically significant differences between

13 the groups with regards to changes in AIS grades or motor scores. Finally, a study examining the technique of an anterior vs. posterior stabilization surgery in 52 cervical spinal cord injury participants (who were determined not to benefit from either approach) found no statistically significant differences in Frankel grade or ASIA motor score changes at 6 months post-surgery.39

Cerebrospinal fluid drainage

Systemic hypotension after acute spinal cord injury is believed to contribute to secondary injury via cord ischemia.41,52 In the interest of maintaining sufficient perfusion to the spinal cord, decreases in the spinal cord perfusion pressure (the difference between the mean arterial blood pressure and the intrathecal pressure) must be limited. To achieve this, cerebral spinal fluid can be drained to lower the intrathecal pressure. Beginning recruitment in 2006, 24 acute spinal cord injury participants within 48 hours of injury were randomized to cerebrospinal fluid drainage down to 10 mm Hg for 72 hours, or no drainage at all.41 This study recorded ASIA motor scores at baseline and 6 months post- injury, and found no statistically significant differences in changes between the groups.

1.3.3 Pharmacological interventions

Methylprednisolone

The application of corticosteroids to reduce secondary inflammation is an extensively tested neuroprotective intervention for human spinal cord injury. Based on animal studies corticosteroids (methylprednisolone, [MP]) became widely used in humans to improve neurological outcomes after spinal cord injury. In the first multicentre double- blind randomized controlled trial initiated in 1979, 330 participants with acute spinal cord injury were randomized to receive a low dose (100mg loading dose followed by 25mg every 6 hours for 10 days) or a high dose (1000mg loading dose followed by 250mg every 6 hours for 10 days) intravenous (IV) treatment of MP.31 The neurological outcomes (motor function, light touch, and pin prick scores) were measured at 6 months after injury, and revealed no statistically significant neurological benefits. A 1-year follow- up study confirmed a sustained lack of benefit. This study has became known as the first

14 National Acute Spinal Cord Injury Study (NASCIS).53

Based on new data from animal models indicating a requirement for even higher dosages to have a beneficial neurological effect, a second larger, placebo-controlled RCT was initiated in 1985 (NASCIS II). This protocol randomized 487 participants within 12 hours of injury to the IV treatment of MP (30 mg/kg initial dose then 5.4mg/kg/hour for 23 hours), naloxone hydrochloride (5.4 mg/kg initial dose then 4.0mg/kg/hour for 23 hours), or a placebo control. The same neurological change scores were compared at 6 months. The beneficial effects of MP were driven by those who received the treatment within 8 hours of injury: these participants demonstrated improved neurological function (i.e., sensory and motor scores), whereas administration after 8h or administration of naloxone had no statistically significant benefit compared to placebo.32 Similar findings were reported in a 1-year follow up study,54 and the timing and dosages from this study were then adopted as the “standard of care” and included in all participants in many of the subsequent trials.2,55,56

The NASCIS III double-blind, multicentre trial (n=499) began recruitment in 1991, and was conducted without a placebo in order to refine the ideal duration of MP treatment (i.e. 24 vs. 48 hours), and compare with tirilazad mesylate.36 All participants received an initial dose of 20-40 mg/kg of MP prior to randomization, and then received a 24 or 48 hour IV MP protocol (5.4mg/kg/hour), or a 48 hour protocol of tirilazad mesylate (25mg/kg every 6 hours). The NASCIS studies used motor and sensory index scores as primary endpoints, and NASCIS III also incorporated a functional outcome measure (i.e., Functional Independence Measure).32,36,53 They found that the administration of MP for 48h significantly improved motor function at 6 months, though it was no longer statistically significant at a 1-year follow-up.57 This beneficial effect was not seen in treatment initiated within 3 hours after injury, but only treatment within 3-8 hours after injury. The 48-hour MP treatment group also had statistically significant increased rates of pneumonia.

These studies spurred other trials examining the effects of MP, which produced uncertain or mixed results. This included a study conducted in France which began in 1990 and randomized 106 acute spinal cord injury participants to IV MP (30 mg/kg for 1 hour, then 5.4 mg/kg/h for 23h), nimodipine (0.15mg/kg/h for 2 hours, then 0.03 mg/kg/h

15 for 7 days), or both treatments, or neither.37 Using ASIA sensory and motor scores, this study found no statistically significant differences between the study groups at 1 year after injury. In addition to this, criticisms regarding the claims and methodologies (e.g. inadequate or misleading reporting, skewed groups, lack of surgical information, post- hoc analysis) of the original NASCIS studies began to grow.58,59 Also of major concern were the documented adverse effects of MP treatment, including immunological compromise, sepsis, pneumonia, gastrointestinal and pulmonary complications, and myopathy.2,55,56 As a result of these concerns, as well as a growing lack of evidence supporting efficacy, MP use in spinal cord injury began to fade.1,56 Interestingly, for a time, MP was widely adopted as a treatment even though it was never approved by a regulatory agency as a treatment for acute spinal cord injury.

GM-1 Ganglioside (Sygen)

Monosialotetrahexosylganglioside (GM-1) is a naturally occurring ganglioside found in human cell membranes, believed to have neuroprotective and regenerative properties.2,38 In 1986, a small pilot RCT began recruiting and randomizing 37 participants to an IV GM-1 treatment, or placebo.33 Participants received 100mg daily dose of GM-1 or placebo daily for 18-32 doses, with the first dose occurring within 72 hours of injury. This study reported that the GM-1 treatment group had statistically significant better mean improvements in ASIA motor scores and Frankel grades with no associated adverse effects.33

The safety and early success of this pilot study propelled a much larger phase III multicentre trial (Sygen), which began in 1992 and compared an IV high dose GM-1 (600mg loading dose, then 200mg/day for 56 days) vs. low-dose GM-1 (300mg loading dose then 100mg/day for 56 days) vs. placebo control. A priori, a two-grade improvement from baseline in the Modified Benzel Scale (a scale similar to the AIS grades, but with additional grades to more clearly identify improvements within the mildest severity of spinal cord injury) was chosen as the primary outcome measure. The primary trial endpoint was 6 months after injury.38 Despite the encouraging evidence from the pilot study, this larger RCT showed no statistically significant differences between those treated with GM-1 and the placebo control group. Although the results were disappointing, the study introduced many important criteria to improve the rigor of

16 human spinal cord injury studies, including a defined a priori end point, improved outcome definitions, and ongoing assessment for the training and reliability of outcome examinations.56

Thyrotropin-releasing hormone (TRH)

In the same year that the initial GM-1 study was recruiting, a trial for thyrotropin- releasing hormone (TRH) vs. placebo was recruiting and randomized 20 spinal cord injury participants. At the time, TRH was believed to provide neuroprotective effects by antagonizing substances involved in secondary injury mechanisms, including endogenous opioids.34,60 TRH was administered intravenously within 12 hours of injury; with a 0.2mg/kg initial dose and then 0.2mg/kg/hour over 6 hours. Outcomes were in line with the NASCIS studies and included motor and sensory exams at 4 months and 12 months.34 Randomization was stratified by severity of injury, and although there were promising results at 4 months after injury, there was insufficient follow-up for the authors to consider the 12-month analysis informative.

Autologous activated macrophages

The inflammatory and immune response by the damaged CNS includes a prominent mobilization of endogenous microglia and exogenous macrophages to the spinal cord injury site, which has been suggested to be a significant source of secondary damage and cell death. However, some experimental studies in animals have shown that autologous activated macrophages promote growth and healing, as opposed to harmful inflammation.2,56 A promising phase I study was completed,61 and these results stimulated a multicentre phase III RCT in 2003.42 Fifty participants were randomized to a macrophage injection group or standard care, though financial constraints cut both recruitment and follow-up early. Outcomes included AIS grade improvement and ISNCSCI motor and sensory scores at 6 months and 1 year. Autologous activated macrophages failed to show statistically significant differences in the primary outcome measure (AIS grade conversion) at 6 and 12-months, and only light touch scores showed significantly different change scores at 6 months, benefitting the control group.42 Although disappointing, this study represented a milestone: the first time a cell-based

17 therapy had been administered after acute spinal cord injury.56

Minocycline

Minocycline is a broad-spectrum antibiotic that has been investigated for its potential to decrease inflammatory reactions following spinal cord injury.2,50 Although laboratory results have shown conflicting results with regards to its efficacy in animal models, a phase II placebo-controlled clinical trial was recently completed in Canada, administering minocycline within 12 hours of injury.2,43 This study began in 2004 and randomized 52 individuals with spinal cord injuries to an IV minocycline treatment (generally 800mg loading dose which tapered by 100mg at every 12-hour administration until 400mg, and continued at 400mg/dose) for 7 days and compared it to a placebo treatment. Outcomes included the ASIA motor and sensory scores. This study confirmed the safety of minocycline use in humans, but did not show any statistically significant neurological improvement compared with the placebo group. Although motor improvement in the treatment arm was greater than that of the placebo group, it did not reach statistical significance. The authors suggested that the low numbers in each treatment arm and the heterogeneity of the study groups warranted further research into the efficacy of minocycline after acute spinal cord injury.62

Riluzole

Increased activation of voltage-gated sodium ion channels is thought to play a pivotal role in secondary cell death after spinal cord injury through a variety of secondary injury mechanisms (e.g. swelling, acidosis, and glutaminergic excitotoxicity).63 Based on this information, riluzole, a sodium channel blocker, has been proposed as a pharmacological intervention to modulate concentrations of glutamate, thereby protecting the spinal cord from secondary damage.1,2 Currently administered for management of amyotrophic lateral sclerosis (ALS), riluzole is in the early stages of clinical trials in spinal cord injury.2 A centre in Iran began recruiting acute spinal cord injury participants to an RCT in 2014, and randomized 60 participants.49 The participants in the treatment group received 50mg of riluzole orally every 12 hours for 8 weeks, although the timing of admission from surgery is unclear and is simply defined as “acutely upon admission”. The distribution of the Frankel grades between treatment and

18 control were similar at earlier timepoints, but at 6 months there were statistically significant differences in that the treatment group had higher proportions of less severe neurological deficits.

Erythropoietin

Erythropoietin (EPO) is a hematopoietic growth factor believed to provide neuroprotective effects after injuries such as brain injuries, stroke, or spinal cord injury.64–66 It was purported at the time that its unclear mechanisms included inhibiting glutamate release,67 preserving vascular integrity, protecting from hypoxia,65 or reducing inflammation. Promising preclinical studies lead to an RCT that randomized 30 acute spinal cord injury participants within 6 hours of trauma to receive IV erythropoietin or a placebo.44 Participants in the treatment groups received 500 unit/kg of alpha- recombinant human EPO initially, then 500 unit/kg 24 hours after the first dose. Six months post-injury the treatment and control groups had statistically significant differences AIS grades, such that the treatment group had a high proportion of ASIA E participants. Following this study, a second Iranian RCT for EPO used the same protocol in 27 acute traumatic cervical spinal cord injury participants who presented within 8 hours of injury. This study found no statistically significant differences in ASIA sensory or motor scores at 6 or 12 months post-injury.46

Progesterone and vitamin D

Progesterone and vitamin D both have preclinical evidence to suggest they can provide neuroprotective benefits after spinal cord injury, such as protection from oxidative stress and reactive gliosis.68–70 Furthermore, a combination of these treatments has shown potential synergistic effects in studies of traumatic brain injury.71,72 In 2012 a 2-centre study recruited 64 participants for randomization within 8 hours of spinal cord injury.48 Participants in the treatment group received 0.5mg/kg injection of progesterone and a 5μg/kg oral dose of vitamin D, both twice daily for 5 days. Using ASIA neurological measures, the study found that the treatment group had significantly higher motor scores at 6-months when compared to the placebo for all extremities (right and left upper and lower), and higher sensory scores for three of the four extremities. The differences in AIS grades at 6 months between the two groups were not statistically significantly different.

19 1.3.4 Regeneration and repair

Cell transplants

Cell transplantation after spinal cord injury has become increasingly popular as a potential therapeutic agent in recent years. Although multiple cell types have been transplanted after spinal cord injury with the hopes of promoting regeneration, the vast majority of these studies were excluded as they focused on safety and feasibility, had participants that were not randomized to treatment, or were conducted in chronic spinal cord injury.73–76 Bone marrow cells (BMCs) are believed to be able to induce neuroregeneration or repair after injury by generating neural cells or myelin producing cells, or by guiding axon growth.47,77 Recruitment began in 2011 for a study which randomized traumatic spinal cord injury participants with thoracic, AIS A injuries from 10- 14 days after injury.47 Twenty-one participants were randomized to one of three treatment groups: BMCs injected into the injured spinal cord during durotomy (2x 108 cells), BMCs injected intrathecally, or a control group. There were no statistically significant differences between the groups in motor or sensory scores at 6 or 12 month follow-ups.

1.4 New directions: drug repurposing in spinal cord injury

Thus far, there are currently no approved pharmacological interventions to improve recovery for acute human spinal cord injury.1 This may be in large part due to the challenges faced by human spinal cord injury trials, as previously discussed. These challenges not only include those of conducting a trial (e.g. recruitment, outcomes), but also in deciding which therapies should be chosen for translation from experimental models to humans trials. Therapies are required to show evidence of effectiveness in animal studies prior to human trials, and despite the strong evidence shown in the past there have been no successful translations. While suggestions to improve preclinical evidence are paramount to the future success of translations (e.g. types of animals and injuries, replication of findings),78 observational studies should not be overlooked as an additional tier of evidence of effectiveness whenever possible.

20 The possibility of identifying interventions beyond the traditional bench-to-bedside approach should be explored. In contrast to the trajectory of traditional pharmacological RCTS (e.g. animal studies to human translation), the process of dug repurposing research commonly starts with a compound that has been tested in humans, demonstrated an acceptable safety and tolerability profile, and has already received regulatory approval for one indication being repurposed for another. This strategy has several distinct advantages over traditional translation.79 Above all else, the time from discovery to applications in humans can be substantially reduced. This is achievable because these medications are already being administered and represent a relatively “low-risk” intervention. Furthermore, repurposing strategies may help to identify therapies which animal models could not identify, or may have missed. In this thesis I propose to complement this traditional search for pharmaceutical interventions, and focus on the potential of repurposing existing treatments in spinal cord injury.

Drug repurposing has shown promise in other neurological disorders (e.g. Parkinson’s disease, Alzheimer’s disease), and there has been increasing interest in pharmacoepidemiological approaches to this strategy.79,80 Many sources of clinical data may be useful for these pharmacoepidemiological approaches, including administrative records, patient charts, and completed clinical trials. Clinical trials often collect more data than the investigators are able to analyze, and these data can be used to document interesting results that may have gone unnoticed. Specifically, these data can be used to examine therapeutic agents administered after spinal cord injury and their associations with recovery.

Anecdotally, it is well known that nearly every individual sustaining a spinal cord injury receives multiple types and classes of medications to manage a litany of problems associated with the traumatic injury (Figure 1.2). These range from medications to manage blood pressure, to analgesics for concomitant traumatic injuries, to anticholinergics for spasms.81 Less obvious psychological problems also require pharmacological management, like anxiety and problems sleeping.81 Somewhat surprisingly, very little is known to what degree medications administered for the management of acute spinal cord injury have downstream and unintended effects that can be beneficial or detrimental for neurological recovery. Therefore, depending on management strategies aimed at treating secondary complications, neurological

21 recovery could be attenuated or facilitated by different medications.

Figure 1.2 Medications administered to an acute spinal cord injury patient in the first 3 months after injury (Sygen) Created by Cheryl Niamath

1.5 Spinal cord injury, pain, and pain management

In addition to sensorimotor loss, pain is one of the major complications necessitating medications after injury, as well as negatively impacting quality of life among individuals with spinal cord injuries. Two types of pain are commonly associated with spinal cord injury, and each type often results in a unique management strategy. First, concomitant injuries suffered in the course of a traumatic injury (e.g. broken bones) can result in immediate nociceptive pain. Nociceptive pain, which is not specific to spinal cord injury, is best managed by common analgesic medications (e.g. NSAIDs and opioids) early after injury. Nociceptive pain tends to diminish somewhat in the first year, but can remain highly prevalent into the chronic stages (e.g. shoulder pain due to overuse).82 Nociceptive pain after spinal cord injury tends to be located in areas of preserved sensation, be described as “dull” or “aching”, and responds to NSAID or opioid treatments.83

The other type of pain associated with spinal cord injury is neuropathic pain, which

22 arises from damage to the nervous system and can present in the days to weeks post- injury.84 Despite its appearance after injury, there is limited information about the acute progression of neuropathic pain after spinal cord injury. Neuropathic pain after spinal cord injury is classified relative to the level of injury as occurring at-level (at the level of injury or within 3 levels below), or below-level (occurring more than 3 levels below the level of injury).85 Neuropathic pain is often described as “hot/burning”, “tingling”, “pricking”, “pins and needles”, “sharp”, “shooting”, “squeezing”, “cold” or “electric shock”, as well as allodynia or hyperalgesia.85 The mechanisms underlying the development and maintenance of neuropathic pain are likely complex and due to multiple changes occurring in the CNS after injury (i.e. neuroplasticity). These changes may include glial activation, up-regulation of chemokines and receptors, changes in ion channel expressions and proteins, neuroinflammation, and signalling of neurotrophic factors.83 Neuropathic pain is generally refractory to nociceptive pain treatments, and the current front-line treatments for neuropathic pain include gabapentinoid anticonvulsants (i.e. pregabalin and gabapentin), and tricyclic antidepressants (i.e. amitriptyline). 83

Overlapping with the proposed “window of opportunity” for spontaneous neurological recovery following spinal cord injury, nociceptive and neuropathic pain can present early after injury. However, the potential impact that pain medications may be having on neurological outcomes after injury remains largely unknown. Pain itself has been proposed to limit plasticity, driving maladaptive changes that limit the recovery of locomotor function.86 Similarly, opioids have been shown to have detrimental effects on recovery and increase the size of the lesion in the spinal cord.87,88 In contrast to the detrimental effects of pain and opioids, non-steroidal anti-inflammatories drugs (NSAIDs) have been proposed as neuroprotective.89,90 This notion that some medications may give rise to unintended benefits supports the exciting concept of repurposing pain medications in the field of spinal cord injury. Repurposing (sometimes also called rescuing) has demonstrated increased interest in other fields (e.g. multiple sclerosis) to improve outcomes in the absence of novel therapies79,91,92. Drug repurposing has demonstrated success in the past, including the early development of galanthamine for polio which was subsequently approved as a treatment for Alzheimer’s disease.93 Many of the drugs used in pain management drugs, such as anticonvulsants, present as viable options for drug repurposing: they are administered early after injury (during the “window of opportunity”), they are centrally acting (can cross the blood-brain barrier), and they

23 modulate activity in the central nervous system.94

1.6 Anticonvulsants and spinal cord injury Seizures are believed to be caused by multiple mechanisms resulting in the excessive firing of neurons, and anticonvulsant medications are targeted to increase inhibition or decrease excitation in the nervous system.95 Anticonvulsants with these targets are also believed to exert effects in the CNS related to the neuronal excitability of neuropathic pain (e.g. modulating sodium and calcium channels).96

In line with modulating neuronal excitability, anticonvulsants aiming to increase the effects of the inhibitory neurotransmitter GABA may also provide relief for neuropathic pain.95 Clonazepam increases receptor affinity for GABA, increasing its inhibitory response.97 Although originally thought to be a GABA analogue, gabapentinoids bind to the alpha2delta subunit of voltage gated calcium channels in the CNS, and inhibit presynaptic calcium channel activation. 83,96 They are believed to relieve neuropathic pain by decreasing excitatory pronociceptive transmitter release.83 Anticonvulsants that block sodium channels (e.g. carbamazepine, phenytoin, mexiletine, lignocaine), and therefore limit the repetitive firing of neurons, are also of interest in neuropathic pain.95 Lamotrigine, another sodium channel blocker, is currently a second-line treatment for neuropathic pain.

In addition to their effects on neuropathic pain, anticonvulsants have been associated within neurological recovery after spinal cord injury. Valproic acid has demonstrated neuroprotective effects after injury via inhibition of histone deacetylase, resulting in improved functional outcomes in rats.98,99 Pregabalin has also shown neuroprotective effects in a study on rats, exhibiting anti-apoptotic and anti-inflammatory effects in additional to improved functional recovery.100 However, pregabalin has also recently demonstrated effects on axon regeneration. Using transcriptome sequencing, Tedeschi et al. identified the gene encoding the alpha2delta2 calcium channel subunit as a developmental switch to limit axon growth and regeneration. They then used pregabalin as a pharmacological blockage, and found that it enhanced axon regeneration after spinal cord injury.101

24 As previously highlighted in this chapter, promising animal and phase I safety results102– 104 gave rise to a riluzole (sodium channel blocker) clinical trial in Iran, which showed some benefit to neurological recovery.49 In addition to these findings, a North American trial is currently recruiting acute spinal cord injury participants to another placebo- controlled RCT of riluzole, with ISNCSCI motor scores as the primary outcome (ClinicalTrials.goc identifier: NCT01597518). Finally, a recent observational cohort study examined the effects of multiple broad-classes of drugs administered to manage pain on neurological recovery.105 The categories of drugs included were antidepressants, anticonvulsants, NSAIDs, and opioids. The only category of drug that had a statistically significant effect on the rate of motor recovery after spinal cord injury were anticonvulsants.105 It is clear that anticonvulsants have preclinical evidence of improving recovery after spinal cord injury, and are valid candidates for drug repurposing research.

1.7 Objectives Animal studies have previously reported on the potential neuroprotective and regenerative effects of anticonvulsants after spinal cord injury.100,106,107 Furthermore, a beneficial association between anticonvulsants and neurological recovery after spinal cord injury was reported in an observational human study.105 In line with this seminal evidence in humans and animal evidence of the neuroprotective and neuroregenerative potential of anticonvulsants (Figure 1.3), the overarching objectives of this thesis were to:

1. Determine the effect of acute anticonvulsants on neurological recovery after spinal cord injury  Primary aim: Determine the effect on ISNCSCI motor scores  Secondary aims: Determine the effects on functional measures (SCIM) and ISNCSCI sensory scores (light touch and pin prick) 2. Examine the longitudinal progression of neuropathic pain after spinal cord injury  Primary aim: describe the progression of neuropathic pain  Secondary aim: identify predictors of neuropathic pain progression

25

Figure 1.3 The conceptual model for this thesis

Note: - - - - - indicates a relationship that is sometimes true

26 Chapter 2 Methods

2.1 Data In this thesis I utilized 4 data sources: (1) The European Multicenter Study About Spinal Cord Injury (EMSCI), (2) The Sygen clinical trial, (3) A chart review from a trauma centre in Germany, and (4) a cohort of patients from Sweden/Denmark. Aspects of EMSCI were used in multiple chapters, and the inclusion/exclusion criteria for each dataset used in each chapter is defined in that chapter’s methods accordingly. In Table 2.1 I have provided an overview of the data sources and methodologies included within this thesis. The measurements discussed in the Data section are described in more detail below, under Variables.

Table 2.1 An overview of the data sources and methodologies used within each chapter

Analytical Chapter Data Source N Exposure/Outcome Methods Anticonvulsant exposure on -Mixed effects Chapter 3 EMSCI 550 neurological regression recovery -Mixed effects Anticonvulsant regression exposure on Chapter 4 Sygen 570 neurological -Propensity recovery score matching -Mixed effects Gabapentinoid EMSCI regression anticonvulsant

Chapter 5 201 exposure on -Propensity neurological Chart Review score recovery matching EMSCI 251/144 Progression of Chapter 6 -Meta-analysis Swedish/Danish neuropathic pain 77/87 cohort

2.1.1 The European multicenter study about spinal cord injury

Data for several chapters came from EMSCI database (https://www.emsci.org, ClinicalTrials.gov Identifier: NCT01571531). The EMSCI is a network of centres in

27 Europe that prospectively collects detailed neurological, neurophysiological, and functional outcomes from individuals with spinal cord injuries during the first year after injury for research purposes. The EMSCI currently includes information from 19 centres in Spain, Germany, Italy, the Netherlands, the Czech Republic, Switzerland, and an additional centre in India, with the coordinating centre located in Zurich. These assessments were performed at fixed time points after injury: very acute (0-15 days), 4 weeks (16-40 days), 12 weeks (70-98 days), 24 weeks (150-186 days), and 48 weeks (>=300 days). Individuals were included if: they experienced a single event traumatic or ischemic spinal cord injury, their first EMSCI assessment was possible within 6 weeks post-injury, and the patient was able and willing to provide written informed consent. Individuals were excluded if their spinal cord injury is caused by a non-traumatic event (excluding single event ischemia), if they had previous dementia or severely reduced capabilities of cooperation or giving consent, if they had peripheral nerve lesions above the level of injury, if they had a previous polyneuropathy, or if they had a severe craniocerebral injury. EMSCI has been approved at each site by local research ethics boards, with individuals consenting to have their data entered into the EMSCI database.

The neurological outcomes recorded in EMSCI included sensory scores, motor score, and classification of injury severity according to the International Standards for the Neurological Classification of Spinal Cord Injury (ISNCSCI, described in more detail below).108 The EMSCI also measured independence using the Spinal Cord Independence Measure (SCIM). Additional assessments included functional, neurophysiological, hand function, and urodynamic measures.

Beginning in 2007, and updated in 2011, a subset of EMSCI individuals completed an additional pain measure via a questionnaire.105,109 Although the questionnaires differed slightly, they included many identical or near-identical questions about pain. In both cases, a trained interviewer in a structured interview ascertained pain outcomes. The questionnaires allowed up to three pain sites to be reported. Each pain site was characterized by the interviewer according to key descriptors (e.g. hot-burning), location (i.e. relative to lesion level), alleviating and aggravating factors, frequency, and intensity (0-10 numeric rating scale, with 0 indicating no pain and 10 indicating the strongest pain imaginable). Pain was classified according to the International Association for the Study of Pain guidelines (IASP) as nociceptive (musculoskeletal or visceral) or neuropathic (at-

28 level or below-level). Four published studies have utilized the EMSCI pain questionnaire.105,109–111 Self-reported pain medications (i.e. NSAIDs, anticonvulsants, antidepressants, opioids, spasmolytics, hypnotics, and drug/alcohol abuse) were also tracked at each time point in the questionnaire.

2.1.2 Sygen

Briefly discussed in Chapter 1, the Sygen trial was a multicentre trial (28 North American neurotrauma centres) conducted in acute spinal cord injury with the treatment of monosialotetrahexosylganglioside sodium salt, or GM-1. Unlike some previous clinical trials (e.g. the NASCIS studies), Sygen was more exclusive in its inclusion of spinal cord injuries (Table 2.2).112 The Sygen trial investigators wanted to recruit participants with a “pure” spinal cord injury and a higher potential for recovery. As such, they included spinal cord injuries rostral to T10 with at least one lower extremity ASIA motor score less than 15 points (of a possible 25 points). Patients were further excluded if their trauma was caused by a penetrating injury; if it was a traumatic spinal cord anatomic transection; if there was the presence of cauda equina damage, major brachial injury, major lumbar plexus injury, significant head trauma, other injury that the physician deemed sufficient to interfere with the assessment of spinal cord function or compromise the validity of the data; if an existing systemic disease or active malignancy or other disease could alter the distribution/accumulation/metabolism/ excretion of the study medication, cause a neurological deficit, or cause an expected life expectancy of less than 2 years; if any pre-existing polyneuropathy, focal or multifocal neuropathy, myelopathy, or radiculopathy could be expected to interfere with the study assessments; if the presence of any medical condition could be expected to cause the patient to have unwarranted risk from participating in the study or cause significant deterioration in their clinical course; if there was a history of Guillain-Barré syndrome; if they had a psychoactive substance abuse disorder in the previous 6 months; if they had a history of a psychotic disorder; if they were a pregnant or nursing woman; if they had a history of life-threatening allergic or immune-mediated reaction; if they were not likely to be available for follow-up evaluations; if they were unable to communicate effectively with the examiner; or if they had a previous use of any ganglioside preparation.112

29 Table 2.2 A comparison of injury types included in the NASCIS vs. Sygen studies

Category of spinal cord injury Included in Included in Sygen NASCIS studies study Anatomic Transection Yes No AIS A Yes Yes AIS B Yes Yes AIS C Yes Yes AIS D Yes Yes Minor motor deficit, not disabled, Yes No functional walking Thoracic/lumbar injuries with Yes No conus/cauda equina injury Sensory only with no motor deficit Yes No Hysterical Yes No Bony or ligament injury only No No Muscle sprain No No

In total, 3165 participants were screened and 797 were randomized to treatment between 1992-1997. Due to the protocol at the time, all participants of the Sygen trial received the NASCIS II regiment of MP, and then received their dose for the study within 72 hours after injury.112

Participants in Sygen had an emergency room evaluation, and a detailed baseline evaluation prior to receiving their first dose of GM-1 (i.e. before, then 72 hours after injury).113 Neurological recovery measures included the ASIA motor and sensory scores, as described in greater detail below. Functional measures in Sygen included the Benzel and ASIA impairment scales. These were examined by using the AIS grade at baseline and the modified Benzel Classification at week 26, and used the binary outcome of “success vs. failure” as defined by “marked recovery”. Marked recovery is defined as a 2-point improvement in classification, such that a baseline AIS grade of A, B, C or D would convert to a 26-week Benzel Classification of III, IV, V, and VI (or higher), respectively.113 Modified Benzel Classification and ASIA examinations were conducted at 4, 8, 16, 26, and 52 weeks after injury.38 The treatment did not have a statistically

30 significant effect on marked recovery at week 26.

2.1.3 Chart review: trauma center Murnau A review of patient charts was performed at the participating EMSCI centre Trauma Center Murnau in Germany. Information from these charts was then retrospectively linked with neurological outcomes from the original EMSCI database (as previously described), to create the observational cohort examined in Chapter 5. Information gathered from the EMSCI data included ISNCSCI measures, AIS grades, levels of injury, and SCIM scores.

The extraction of data from patient charts was conducted by two research assistants and supervised and approved by Dr. Grassner on site. The data extracted included anticonvulsant use (Y/N), type of anticonvulsant, first date of administration, and duration of use.

2.1.4 Swedish/Danish cohort

The original aims of the study that prospectively gathered the Swedish/Danish cohort information were to identify pain phenotypes in the first year after a traumatic spinal cord injury, and to determine if sensory hypersensitivity predicted the development of neuropathic pain at 1 year after injury.114 Ninety participants with a traumatic spinal cord injury who were 18 years or older were recruited from 2 centres in Denmark and Sweden from 2007-2010. Participants were excluded if they had alcohol or substance abuse, if they had a psychiatric disease, or if they were unable to participate (e.g. brain damage, language problems etc.). The first examination occurred within 1 month of injury if possible, and otherwise within a maximum of 3 months of injury. Follow-up visits occurred at 6 and 12 months after injury. If a visit was not possible, telephone interviews were performed instead.

At the first examination participants were asked about their chronic pain preceding the spinal cord injury (e.g. pain at least once a week at least 3 months prior to their injury). At each visit, injuries were classified according to ISNCSCI. Patients were interviewed regarding the presence of paresthesia and dyesthesia classified by adjectives to describe the sensations, and any pain that had presented within the previous 7 days.

31 Pain was the classified according the to the International Spinal Cord Injury Pain Classification (e.g. nociceptive vs. neuropathic, neuropathic at-level vs. below-level pain).85 For each pain type reported by a participant, they completed the short-form McGill Pain Questionnaire, and the examiner completed the International Spinal Cord Injury Pain Basic Data Set. For overall pain, participants completed the Brief Pain Inventory and the Pain Interference scale. Pain treatment was recorded from medical records and by participant self-report of pain treatments.114

At each visit, sensory testing was performed above, at, and below injury level. Testing using the numeric rating scale (0-10) included pain and unpleasantness to brush, single pinprick, repetitive pinprick, and cold and warm stimuli. The study found that hypersensitivity at 1 month was predictive of below-level neuropathic pain at 12 months. From this study the authors provided us with data on participants’ injuries (AIS grade and level), presence of each pain at each timepoint, and the results of the McGill Pain Questionnaire.114

2.2 Statistical methods The statistical methods and variables utilized in each chapter are briefly described within the chapter’s methodology, but a more in-depth description is provided below.

2.2.1 Variables The data in this thesis include sensory and motor scores as developed by ISNCSCI. The ISNCSCI is developed and maintained by the American Spinal Injury Association (ASIA). The worksheet for the ISNCSCI exam is shown in Figure 2.1. The motor scores are derived from assessments of muscle strength in key upper and lower extremity myotomes (C5-T1 and L2-S1, respectively), in addition to the examination of voluntary anal contraction. Each measurement is graded from 0-5, with 0 being total paralysis, 1 being palpable or visible contraction, 2 being active movement with full range of motion (ROM) with gravity eliminated, 3 being active movement, full ROM against gravity, 4 being active movement and full ROM against gravity and moderate resistance in a muscle specific position, and 5 being (normal) active movement and full ROM against gravity and full resistance in a functional muscle position expected from an otherwise unimpaired person. In testing 5 myotomes in each extremity, there is a total motor score ranging from 0-100. 108

32 Then sensory scores are similarly tested in 28 distinct dermatomes, from C2-S4-5 and contain upper, lower, and left and right subscores. Each dermatome is graded from 0-2, with 0 being absent sensation, 1 being altered sensation (either decreased/impaired sensation or hypersensitivity), and 2 being normal sensation. The upper and lower scores of each sensory examination can have a maximum score of 56, results in total sensory scores that range from 0-112. The sensory examination includes light touch, in which a tapered wisp of cotton is stroked across the area, and pin prick, in which sharp/dull discrimination is tested using the pointed and rounded end of a safety pin.108

INTERNATIONAL STANDARDS FOR NEUROLOGICAL Patient Name Date/Time of Exam CLASSIFICATION OF SPINAL CORD INJURY Examiner Name Signature (ISNCSCI)

MOTOR SENSORY MOTOR KEY MUSCLES KEY SENSORY POINTS KEY MUSCLES RIGHT Light Touch (LTR) Pin Prick (PPR) Light Touch (LTL) Pin Prick (PPL) LEFT C2 C2 C2 C3 C3 C4 C2 C4 C3 Elbow fleor s C5 C3 C5 Elbow fleor s UER Wrist extensors C6 C4 C4 C6 Wrist extensors UEL T2 (Upper Extremity Right) Elbow extenxsors C7 T3 C7 Elbow extenxsors (Upper Extremity Left) Finger fleor s T4 Finger fleor s C8 T5 C8 Finger axbductors (little finer ) T1 T6 T1 Finger axbductors (little finer ) T7 8 T2 C T2 6 Comments (Non-key Muscle? Reason for NT? Pain? C T8 7 MOTOR C Non-SCI condition?): T3 T9 T3 (SCORING ON REVERSE SIDE) Dorsum T4 T10 T4 0 = Total paralysis T5 T11 T5 1 = Palpable or visible contraction T12 2 = Active movement, gravity eliminated T6 L1 T6 3 = Active movement, against gravity 4 = Active movement, against some resistance Palm T7 T7 5 = Active movement, against full resistance T8 T8 NT = Not testable S3 0*, 1*, 2*, 3*, 4*, NT* = Non-SCI condition present T9 Key Sensory T9 L2 Points T10 S4-5 T10 SENSORY (SCORING ON REVERSE SIDE) T11 L T11 2 0 = Absent NT = Not testable 1 = Altered 0*, 1*, NT* = Non-SCI T12 L L3 T12 L1 S2 3 L1 2 = Normal condition present Hip fleor s L2 L2 Hip fleor s Knee extensorsx L3 L3 Knee extensorsx LER L4 LEL (Lower Extremity Right) Ankle dorsifleor s L4 L4 Ankle dorsifleor s (Lower Exxtremity Left) L L5 Long toe extensorsx L5 4 L5 Long toe extensorsx Ankle plantar fleor s S1 S1 S1 Ankle planxtar fleor s L5 S2 S2 S3 S3 (VAC) Voluntary Anal Contraction (DAP) Deep Anal Pressure (Yes/No) S4-5 S4-5 (Yes/No) RIGHT TOTALS LEFT TOTALS (MAXIMUM) (50) (56) (56) (56) (56) (50) (MAXIMUM) MOTOR SUBSCORES SENSORY SUBSCORES UER +UEL = UEMS TOTAL LER + LEL = LEMS TOTAL LTR + LTL = LT TOTAL PPR + PPL = PP TOTAL MAX (25) (25) (50) MAX (25) (25) (50) MAX (56) (56) (112) MAX (56) (56) (112)

NEUROLOGICAL R L 4. COMPLETE OR INCOMPLETE? (In injuries with absent motor OR sensory function in S4-5 only) R L 3. NEUROLOGICAL 6. ZONE OF PARTIAL LEVELS 1. SENSORY LEVEL OF INJURY Incomplete = Any sensory or motor function in S4-5 SENSORY Steps 1- 6 for classifict ion PRESERVATION MOTOR as on reverse 2. MOTOR (NLI) 5. ASIA IMPAIRMENT SCALE (AIS) Most caudal levels with any ginnervation

Page 1/2 This form may be copied afreely but should not be altered without permission from the American Spinal Injury Association. REV 04/19

Figure 2.1 International standards for neurological classification of spinal cord injury © 2019 American Spinal Injury Association. Reprinted with permission.

33

Using the sensory and motor score, ASIA grades are assigned to indicate injury severity as "complete" injuries, or "incomplete" injuries. The criteria for the ASIA grades are defined in Table 2.3, alongside the Modified Benzel Classifications.

Table 2.3 The ASIA grades and modified Benzel classification

AIS Grade Definition MBC Grades A Complete. No sensory or motor function is preserved in I the sacral segments S4-S5. B Incomplete. Sensory but not motor function is preserved II below the neurological level and includes the sacral segments S4-S5, AND no motor function is preserved more than three levels below the motor level on either side of the body. C Incomplete. Motor function is preserved below the III neurological level, and more than half of key muscles below the neurological level have a muscle grade less than 3. D Incomplete. Motor function is preserved below the IV, V, VI neurological level, and at least half of key muscles below the neurological level have a muscle grade greater than or equal to 3. E Normal. Sensory and motor functions are normal. VII MBC= Modified Benzel Classification

The EMSCI also includes the Spinal Cord Independence Measure (Version 3 in Chapter 5). The SCIM 3 was developed for individuals with spinal cord injuries to assess the daily assistance required to score their independence. It is comprised of 3 main subsections: self-care (scored 0-20), respiration and sphincter management (scored 0-40), and mobility (scored 0-40), for a possible total score of 100.

2.2.2 Longitudinal mixed effects regression To effectively measure how anticonvulsant use may change in the progression of

34 neurological recovery after spinal cord injury, this thesis contains varying forms of longitudinal data (i.e. repeating measurements for an individual across multiple time points). Longitudinal data is complex in that individuals produce multiple measurements across time, thus violating the assumption of independence intrinsic to linear regression. To analyze this longitudinal (i.e. clustered) data, I used linear mixed effect models. This allowed me to model trajectories for each individual, as well as incorporate individual intercepts and slopes, thus creating models with random effects. Linear mixed effect models are advantageous for this data because they can accommodate irregular time intervals between observations or participants with missing observations (and not exclude them), non-linear trends, increased variability over time, and capture participant- level variability.115,116

The analysis of longitudinal data begins with examining the empirical growth plots, or trellis plots, of a group of individuals to see how their motor score changes over time. In doing this we can identify that the trend after spinal cord injury is for motor scores to increase initially, and then plateau around 9-12 months after injury. This indicates that a statistical model would benefit from the inclusion of a quadratic time variable so accommodate the curve. The motor scores from a random subset of participants from the Sygen data are plotted below in Figure 2.2, and the fitted lines from a mixed-effects model (with random slope and intercept) including a quadratic time variable are superimposed to demonstrate the fit for each individual. These models are described as multilevel because they see to examine how individuals change over time, but also how these changes vary across individuals.115 They do not assume all participants have the same recovery trajectory, but allow each to have a personal intercept and slope (as clearly depicted to be the case in Figure 2.2). Following this basic model outline, additional confounders can be added to the multilevel model.

35

Figure 2.2 The motor score trajectories of 25 randomly selected individuals from the Sygen data in Chapter 4

36 Chapter 3 The effect of anticonvulsants on neurological recovery after spinal cord injury in EMSCI

3.1 Introduction

In line with addressing the effect of pain and pain medications on neurological recovery, Cragg et al. conducted a seminal analysis of the initial EMSCI cohort linked to the pain questionnaire (2007-2011).105 For the first time, this analysis identified that human participants who were administered any anticonvulsants at 4 weeks post-injury achieved greater neurological recovery compared to participants who did not receive anticonvulsants. Neuropathic pain intensity was also significantly reduced by anticonvulsants administered at 4 weeks post-injury.105 This first analysis had not specifically set out to address the effects of anticonvulsants on neurological recovery. Medications that did not confer a statistically significant benefit or harm included antidepressants, NSAIDs, and opioids.

However, critical information was missing from this investigation (e.g., the type of anticonvulsant that had been administered), and a biological mechanism was unknown. Prior to this observational analysis, Tedeschi et al. demonstrated that the anticonvulsant pregabalin promoted neural regeneration in the injured mouse spinal cord. 101 In short, this research applied transcriptome sequencing and bioinformatics to identify the Alpha2delta2 subunit of voltage-gated calcium channels as a developmental switch to limit axon regeneration, and found that the pharmacological blockade of this subunit using pregabalin enhanced axon regeneration after spinal cord injury.101 These findings provided a novel potential mechanism explaining the beneficial effects of anticonvulsant administration at 4 weeks post injury in humans: gabapentinoid-induced axonal regeneration.101

Following these promising clinical and preclinical findings, I aimed to explore the potential effects of anticonvulsants further. Specifically, I wanted to answer some of the limitations from the previous analysis regarding type of anticonvulsants associated with recovery, if timing of administration altered the effect, and if pain-related variables were responsible for the effect in an expanded EMSCI dataset.

37 3.2 Methods

3.2.1 Data

Our observational cohort study analyzed prospectively gathered data from EMSCI. The cohort previously utilized by Cragg et al. (2007-2011) was combined with an updated cohort from 2011-2015. From all EMSCI participants, we only included individuals with a defined level of injury within the cervical and thoracic cord (C1-T12), injury severity measures with a degree of impairment (AIS grades A-D), and a valid pain assessment at 4 weeks post-injury. Further, in order to be categorized as late vs. non-user, participants who did not receive anticonvulsants in the first month were required to have a minimum of one other valid pain measure (Figure 3.1).

3.2.2 Variables

Pain characteristics, descriptors, classifications, and medications were tracked post- injury via a questionnaire by trained examiners. Specific data extracted included self- reported pain medication class, self-reported pain intensity (numeric rating scale, representing the average intensity in the last week prior), and pain classification (nociceptive or neuropathic) at each time assessment. For timing, three anticonvulsant groups were defined: ‘non-users’ (i.e., never administered anticonvulsants), ‘late users’ (i.e., administered anticonvulsants after week 4), and ‘early users’ (i.e., administered anticonvulsants at the 4 week time point). As a proxy for frequency of use, we examined the number of times anticonvulsant administration was recorded (i.e., recorded as being taken at how many of the four time points: 0, 1, 2, or 3+ times). Finally, we also examined a model in which anticonvulsant was a time-varying covariate (i.e. yes/no at each time point). In a subset of participants, we extracted what type of anticonvulsant was being administered.

As anticonvulsants after spinal cord injury are often administered for pain relief, we also examined the effects of pain (e.g. presence, severity, relief) itself on motor recovery. The questionnaire permitted subjects to report up to three different types of pain at each time point. Pain intensity described the average intensity (the highest of the three reported pains) experienced in the previous week, using a Numeric Rating Scale. Regarding type of pain, we examined the presence of nociceptive or neuropathic pain at 4 weeks. As

38 pain intensity varied over the four assessments, we examined pain intensity at each time point, the average pain intensity across the 4 time points, and changes in pain intensity scores over the 4 time points. Specifically, we examined the change in pain intensity from 1-3 months, change in intensity from 1-6 months, and change in intensity from 1-12 months.

3.2.3 Statistics

Potential confounding variables examined included: neurological level of injury and injury severity according to the AIS grades. To account for the longitudinal data and potential confounders, multivariable analyses were performed using linear mixed effects regression (LMER) models (R package: lme4). To assess differences between groups, we examined covariate-by-time interactions for each group. Specifically, the original longitudinal model included anticonvulsant exposure, time (weeks after injury), 4 week AIS grade, 4 week level of injury, a quadratic time variable, and the primary variable of interest: an interaction term of anticonvulsant exposure and time. However, subsequent analyses (Chapter 4) highlighted the need for an AIS Grade * Time interaction, and this too was added to the final model in this chapter. As each confounder was added to the model, its improvement on model fit was assessed using ANOVA to confirm statistically significant improvements. The primary models of interest were as follows:

Outcome ~ Drug Exposure + Drug Exposure*Time + AIS Grade +AIS Grade*Time + Injury Level + Time + Time2 + (Time|ID)

In addition, the use of anti-spasmodics was examined as a potential confounder. The primary outcome variable was total motor score as defined by ISNCSCI.108 Secondary outcomes included light touch and pin prick sensory scores. Neurological measures were recorded at 4, 12, 24, and 46 weeks post injury and were modeled as a continuous, longitudinal outcome. RStudio statistical software Version 0.99.484 was used for all analyses. 117

3.3 Results

There were 70 ‘early users’ of anticonvulsants, 64 ‘late users’, and 416 ‘non-users’, for a

39 total of 550 individuals with a valid categorization (Table 3.1; Table 3.2; Figure 3.1). Those who were excluded were not statistically significantly different with regards to age (p=0.31), or sex (p=0.23), but they had higher baseline motor scores (p<0.00001) and were more likely to have incomplete (p<0.00001), thoracic (p<0.00001) injuries compared to those who were included. In a larger sample than the aforementioned study (i.e. a cohort of 225 with 40 early users),105 longitudinal analysis confirmed that anticonvulsants administered at 4 weeks post-injury significantly improved motor recovery (estimate= 0.09, p= 0.03 for the drug by time interaction term) after adjusting for 4 week injury-related characteristics.

Figure 3.1 Participants included from the EMSCI dataset

40

Table 3.1 Cohort description

Characteristics N (%)

Total 550 (100.00)

Sex

Male 445 (80.90)

Female 105 (19.09)

Age at injury

Mean (SD) 48.47 (19.11)

AIS at 4-weeks

A 227 (41.27)

B 54 (9.81)

C 78 (14.18)

D 191 (34.73)

Level of injury at 4-weeks

Upper cervical 180 (32.73)

Lower cervical 140 (25.45)

Thoracic 230 (41.82)

Total motor score at 4-weeks

Mean (SD) 51.43 (26.05)

41 Table 3.2 Anticonvulsant group descriptions

Characteristics Non-users (%) Late users (%) Early users (%)

Total 416 (100.00) 64 (100.00) 70 (100.00)

Sex

Male 336 (80.77) 54 (84.38) 55 (78.57)

Female 80 (19.23) 10 (15.63) 15 (21.43)

Age at injury

Mean (SD) 47.94 (19.33) 52.06 (17.45) 48.34 (19.19)

AIS at 4-weeks

A 184 (44.23) 24 (37.50) 19 (27.14)

B 43 (10.34) 6 (9.38) 5 (7.14)

C 53 (12.74) 13 (20.31) 12 (17.14)

D 136 (32.69) 21 (32.81) 34 (48.57)

Level of injury at 4-weeks

Upper cervical 124 (29.81) 24 (37.50) 32 (45.71)

Lower cervical 110 (26.44) 15 (23.44) 15 (21.43)

Thoracic 182 (43.75) 25 (39.06) 23 (32.86)

Total motor score at 4- weeks

Mean (SD) 51.04 (25.98) 47.85 (25.36) 56.97 (26.61)

The frequency of use measured by number of times a participant was recorded as receiving anticonvulsants was not statistically significant (estimate= 0.04, p=0.14). Our next analysis addressed the effects of timing of initial administration. Unlike administration at 4 weeks, late administration at 12, 24, or 48 weeks had no statistically

42 significant effect on recovery. Further, including anticonvulsant use as a time-varying covariate in the model was not statistically significant (estimate= 0.73, p= 0.37). Given the results regarding timing of anticonvulsant administration, we concluded that early anticonvulsant use improved motor recovery when compared with later (or no) administration (Figure 3.2; Figure 3.3). The initial Model 1 revealed similar results to the previous study (8.84 points improvements in the first year, p=0.001), and further adjustment found that early use conferred a benefit of 5.17 more motor points on average over the course of 1 year compared to non-users (p=0.02; Table 3.3). Early users recovered 3.82 more motor points more than late users. Including anticonvulsant use (as an interaction term) also statistically significantly improved the baseline statistical model (Table 3.4).

Table 3.3 Linear mixed effects regression model outputs

Model 1 Model 2 Characteristics Estimate P-value Estimate P-value (95% CI) (95% CI) Intercept 12.94 <0.00001 14.03 <0.00001 (10.46-15.43) (11.48-16.57) Time (weeks) 0.80 <0.00001 0.63 <0.00001 (0.70-0.89) (0.53-0.73) Quadratic time -0.01 <0.00001 -0.01 <0.00001 variable (-0.21- -0.15) (-0.21- -0.15) 4-week AIS A ref - ref - B 0.31 0.87 -1.15 0.54 (-3.35-3.97) (-4.84-2.53) C 17.23 <0.00001 13.97 <0.00001 (13.64-20.83) (10.74-17.21) D 49.90 <0.00001 48.61 <0.00001 (47.37-52.44) (46.05-51.16) 4-week level of injury Upper cervical ref - ref - Lower cervical 13.62 <0.00001 13.59 <0.00001

43 Model 1 Model 2 Characteristics Estimate P-value Estimate P-value (95% CI) (95% CI) (10.89-16.34) (10.87-16.32) Thoracic 30.55 <0.00001 30.36 <0.00001 (27.97-33.13) (27.79-32.94) Anticonvulsant use Never ref - ref - Late -1.46 0.38 -1.41 0.39 (-4.70-1.77) (-4.64-1.81) Early 1.94 0.24 2.39 0.14 (-1.25-5.12) (-0.78-5.57) Anticonvulsant use * time (interaction term) Never ref - ref - Late 0.05 0.36 0.03 0.52 (-0.05-0.14) (-0.05-0.11) Early 0.17 0.001 0.10 0.02 (0.07-0.27) (0.02-0.18) 4-week AIS * time (interaction term) A - - ref - B - - 0.93 0.00002 (0.13-0.34) C - - 2.24 <0.00001 (0.48-0.64) D - - 0.81 <0.00001 (0.13-0.27)

44 Table 3.4 Examining the addition of confounder to LMER model fit using ANOVA

Variables included Variable added ANOVA model comparison Model 1 Time, random effects - - Model 2 Time, Time2, random effects Time2 P<0.00001 Model 3 Time, Time2, AIS, random effects 4-week AIS grade P<0.00001 Model 4 Time, Time2, AIS, Level, random 4-week level of P<0.00001 effects injury Model 5 Time, Time2, AIS, Level, Motor 4-week AIS * time P<0.00001 Score, random effects Model 6 Time, Time2, AIS, Level, Motor Anticonvulsant use P=0.04 Score, Anticonvulsant use, (early vs. late/none), Anticonvulsant use*Time, random Anticonvulsant effects use*Time Note: * indicated an interaction term, 2 indicates a quadratic term

The inclusion of various pain measures, and the administration of anti-spasmodics had no statistically significant effects on motor recovery.

A retrospective chart review on 40 participants from the original analysis who were administered anticonvulsants within the 4 week timepoint revealed that n=33 (83%) received gabapentinoids (n=24 pregabalin, n=9 gabapentin). When comparing only early gabapentinoid users versus non-users using the same longitudinal modeling approach, we found that the beneficial effect remained significant (n=225, P<0.05 for greater recovery in motor points over the first year) for this group.

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Figure 3.2 The effects of early anticonvulsant use on total motor recovery using boxplots

(A) The effects of anticonvulsant use (non vs. late vs. early) on motor recovery following spinal cord injury at 4, 12, 24, and 48 weeks. Boxplots show raw data at each time point, with horizontal lines indicating the first quartile, median, and third quartile. (B) Proportional recovery indicates the proportion of ‘available’ recovery (for a total motor score of 100) achieved at 48 weeks. For example, if an individual had a one-month motor score of 40, their potential recovery would be 60 points (100-40).

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Figure 3.3 The modeled effects of anticonvulsant use on motor recovery following spinal cord injury.

The modeled effects of anticonvulsant use on motor recovery following spinal cord injury. This model was derived from linear mixed effects methods including early users (n= 70), late users (n= 64) and non-users (n= 416). There was a statistically significant drug-by- time interaction (i.e., greater slope or recovery) in early users compared with non-users,

47 even after adjusting for injury characteristics. The unadjusted fitted curve is based on the unadjusted mixed effects model. 3.4 Discussion

In a large sample of acute spinal cord injury patients, our findings confirm that anticonvulsants, administered at therapeutic doses for the management of neuropathic pain, enhance motor recovery after injury. Timing of initial administration was integral for enhanced recovery, with early (i.e. at 4 weeks) but not late (i.e. at 12, 24, or 48 weeks) benefiting the recovery of muscle strength. Our new analysis also ruled out one potential mechanism (i.e. reductions in pain), and identified a common class of anticonvulsants as the potential driving this result (i.e. pregabalin and gabapentin).

Experimental studies in rodent models have clearly demonstrated a “window of opportunity” for pharmacological interventions to repair the injured spinal cord.118,119 The observation of a time dependent effect in humans (i.e., at 4 weeks but not later) suggests that anticonvulsants may be directly, through their biological activity in the CNS, benefiting motor outcomes after spinal cord injury. This is bolstered by the observation that changes in pain had no effect, discounting the theory that anticonvulsants are impacting motor outcomes indirectly through pain relief.

Both regeneration and neuroprotection may be important mechanisms to consider underlying the direct effects of anticonvulsants. First, a recent study reported that the administration of pregabalin 1 hour post-injury resulted in an increased number of regenerating axons rostral to the lesion site. 101 Further, delaying pregabalin treatment for weeks showed anatomical regeneration of axons, but to a lesser extent, with the rationale that axons may be set too late in their growth state (when the glial scar is already formed).101 This beneficial effect was mediated via blocking the α2δ2 subunit, for which both pregabalin and gabapentin have a high affinity and selectively bind.120,121 Second, the neuroprotective effects of gabapentinoids have also been widely demonstrated across a number of animal models of neurological conditions, including spinal cord injury.100,106,107 Neuroprotection has been attributed to various other biological actions of gabapentinoids in the central nervous system (e.g. changes in glutamate metabolism). 100

48 That gabapentinoids have the potential to improve function via multiple pathways (i.e. neuroregeneration and neuroprotection) may make them suitable candidates for translation into humans. First, a regenerative window could mean a longer opportunity for delivery outside the boundary of conventional neuroprotective interventions (e.g. minutes to hours post injury), though the exact timing of initial administration remains vague. This has important clinical implications, increasing the number of patients that can be treated based on later admission times to acute care facilities. The translational potential of gabapentinoids is also enhanced by the fact that they have an established safety profile in the acute stage of spinal cord injury. A clinical trial to assess the efficacy of gabapentinoids to improve motor outcomes after spinal cord injury could administer gabapentin and pregabalin in routine clinical dosages as related to the management of pain without changing practice guidelines for neuropathic pain. Time from discovery to translation is long and arduous, and means that even existing preclinical therapies currently being tested in animal models are years away from applications in humans.1 Gabapentinoids offer a rare and exciting opportunity to repurpose an already in use medication, which in turn circumvents many of the difficulties of performing early phase clinical trials in the field of spinal cord injury (e.g. expensive and time consuming).

It is well known that nearly every individual sustaining a spinal cord injury receives multiple types and classes of medications to manage a litany of problems associated with traumatic injury. Somewhat surprisingly, very little is known to what degree these acute medications have downstream and unintended effects that could be beneficial or detrimental on neurological recovery. This is all the more surprising in light of the fact that many common medications coincidentally administered in early phases of spinal cord injury have been tested in experimental models.122–124 As an example, phenytoin (tradename Dilantin), a potent sodium channel blocker and anticonvulsant administered for neuropathic pain in the 90’s (i.e. predating gabapentinoids), has demonstrated comparable benefits to other neuroprotective treatments currently in clinical a trial (e.g. riluzole).125 Some medications have demonstrated detrimental effects. This includes opioids, which have been shown to limit the recovery of locomotor function in animal studies.87,126,127 This should be considered a major concern, as opioids are ubiquitously administered for pain management in humans sustaining an acute traumatic spinal cord injury. That these important preclinical observations have not yet been examined in the context of human spinal cord injury points to a failure in translation and potentially

49 missed opportunities to maximize neurological recovery.

A limitation of our study is that we do not have information on indication, exact timing, or dosage of anticonvulsant administration at 4 weeks. However, anticonvulsants, specifically gabapentin and pregabalin, are currently the front-line treatments for neuropathic pain after spinal cord injury. Both drugs are administered at a base dose, with flexible dosing increases dependent on effectiveness and tolerance. 128 Very little information is known about neuropathic pain in the very acute stages of injury. This makes speculation of when anticonvulsant administration may have been initiated more difficult. Additionally, anticonvulsant use in EMSCI is self-reported, which may introduce inaccuracies or biases. Moreover, outside the scope of the current study, an important and remaining issue is whether anticonvulsant-induced motor recovery results in improved functional outcomes (e.g. ambulation and use of the hands) 15. At this point, the observed 5-point improvement in muscle strength based on uncertain dosages and frequency should be interpreted as evidence of a modest change. The next step, which may only be achievable in a clinical trial, would be to determine if optimizing dosages and timing (within the 4 week timeframe) could enhance this effect and, in turn, lead to improvements in function (i.e. Functional Independence Measure, Spinal Cord Independence Measure).

In summary, we have provided corresponding evidence in humans that anticonvulsants have beneficial effects on motor recovery after an acute spinal cord injury. In line with the study goals we have concluded that these effects are time dependent and primarily related to the application of gabapentinoids. Future studies may be warranted to assess the efficacy of anticonvulsants as a repurposed therapy to enhance motor outcomes after acute spinal cord injury.

50 Chapter 4 The effect of anticonvulsants on neurological recovery after spinal cord injury in Sygen

4.1 Introduction

In chapter 3 I identified that anticonvulsant administration was associated with an improved motor recovery after spinal cord injury. These findings were likely related to gabapentinoid administration, as they comprised the majority of anticonvulsants in a subset of patients. The focus of this chapter is to confirm whether this beneficial effect was gabapentinoid-specific, or whether it was related more broadly to the administration of anticonvulsants in general.

Damage to the spinal cord disrupts neural signals to and from the brain, resulting in varying degrees of sensory and motor impairments. Partial neurological recovery can occur in the first year after injury, presenting a theoretical “window of opportunity” for repair or protection.14,24 During this time, other complex medical conditions arise and necessitate the administration of a large number of medications. Among these, anticonvulsants have been routinely administered over the past three decades for the management of neuropathic pain. 96,129

The administration of anticonvulsants post-injury is notable because they cross the blood-brain barrier and alter key physiological processes involved in secondary injury mechanisms (e.g. calcium influx).130 In addition to neuroprotection, a recent preclinical study found that gabapentinoid anticonvulsants, i.e. pregabalin and gabapentin, increased the regenerative capacity of damaged central nervous system axons.101 This was achieved by binding to the Alpha2delta2 subunit of voltage-gated calcium channels. Corresponding with observations in rodent models, we found in a series of observational studies in humans that exposure to anticonvulsants was associated with greater motor recovery after injury,105 and that this benefit was driven by the administration of gabapentinoid anticonvulsants in the analysis of Chapter 3.109

To this point, it is not known whether other anticonvulsants are similarly beneficial. Therefore, the aim of the current study was to examine the effect of non-gabapentinoid

51 anticonvulsants on neurological recovery after spinal cord injury, compared to the benefit previously observed in Chapter 3.

4.2 Methods

4.2.1 Data

An observational study was planned with data from the Sygen (monosialotetrahexosylganglioside GM-1 sodium salt) clinical trial. The Sygen trial was mostly completed before gabapentinoids came to market and introduced in the field of spinal cord injury, as the FDA only approved them for the treatment of seizures in 1993. The trial incorporated modern standards of clinical evaluation, including ISNCSCI measures.131 In brief, Sygen is the largest acute spinal cord injury trial.112 Patients were recruited from 28 neurotrauma centres across North America between 1992 and 1997. Efficacy was determined using sensory and motor scores according to ISNCSCI.131 Despite accelerated recovery (i.e., at 8 and 16 weeks), treated and placebo groups did not statistically significantly differ with respect to sensory and motor scores at 26 and 52 weeks post injury. Due to the treatment guidelines at the time, all patients were administered methylprednisolone based on the second National Acute Spinal Cord Injury study (NASCIS II) regimen.54

The study recruited spinal cord injury patients with an initial AIS grade of A, B, C, or D.112 However, patients were additionally required to have at least one lower extremity motor score less than 15 (of a maximum of 25) in order to maximize the potential for recovery, thereby excluding some AIS grade D patients.112 Exclusion criteria included: spinal cord injury caused by a traumatic penetrating injury or an anatomic transection; injury including cauda equina damage; other injuries that could interfere with collection or interpretation of data; history of Guillain-Barré syndrome; recent psychoactive substance use; history of a mental health disorder; history of high risk allergic or immune-mediated reaction; previous use of any ganglioside preparation; pregnant or nursing women; unlikely to be available for follow-up; unable to communicate effectively; presence of other significant systemic diseases that could alter the effectiveness of the study medication or put the patient at risk. Of the initial 3165 patients screened, 2368 were excluded, and 797 were randomized for treatment.112 We applied additional inclusion

52 criteria to the 797 patients randomized in the Sygen trial: we required a valid contemporary AIS grade at 4 weeks (described in more detail below), level of injury at baseline, and total motor score at 4 weeks, with at least one follow-up total motor score at 16, 26, or 52 weeks.

4.2.2 Variables

Sensorimotor examinations were conducted at baseline (within 72 hours, after the initial trauma evaluation), and 4, 8, 16, 26, and 52 weeks after injury. The primary outcome was total motor score according to ISNCSCI,131 which ranges from 0-100. The secondary outcomes were sensory scores: light touch and pinprick scores, which both range from 0-112,131 as well as ‘marked recovery’. Described in greater detail elsewhere,113 marked recovery as measured using the original AIS grades was achieved by improving 2 levels on the modified Benzel Classification between baseline and 26 weeks (range: 1-7).

The original Sygen trial investigators recorded all concomitant medications and related information, including the name of the medication, indication, start and stop dates, doses, and frequency and route of administration. The World Health Organization Collaborating Centre for Drug Statistics Methodology (https://www.whocc.no/) Anatomical Therapeutic Chemical (ATC) classification system was used to determine anticonvulsant exposure (ATC code: N03) which were recorded and assessed with regards to number of users, timing of injury, and reason for administration. DrugBank (https://www.drugbank.ca/) was then used to classify anticonvulsant medications according to mechanism of action.132 For timing of initial administration, we defined three groups: early users (recorded use within 30 days post injury, as described in a previous study),109 late users (recorded use within study period but after 30 days), and never users. Additionally, we examined anticonvulsant use as a time-varying covariate such that any recorded use in the 4-weeks prior to a neurological exam was considered as yes/no exposed.

4.2.3 Statistics

RStudio statistical software Version 0.99.484 was used for all analyses.117 Multivariable

53 analyses were performed using linear mixed effects regression (LMER) models (R package: lme4) for continuous outcomes and logistic regression for binary outcomes. Differences in rates of recovery between exposure groups were examined with a drug- by-time interaction term. Potential confounding was addressed by adjustment for 4-week contemporary AIS grades and baseline neurological level (i.e. cervical vs. thoracic). Due to the evolving nature of the AIS grades since their acceptance in 1982, we recalculated the AIS grades according to contemporary measures using the raw sensory and motor examinations.133 A quadratic time variable was also included to better fit the acute trajectory of motor recovery. The initial analysis was for all anticonvulsants combined as a group, then separately for identified subclasses based on mechanism of action. For any models with a statistically significant drug-by-time interaction, additional analyses were performed. Namely, to further control for potential confounding, we created a matched cohort using propensity scores from variables AIS grade and neurological level of injury. Briefly, this involves creating propensity scores (i.e. the conditional probability of being assigned to a group based on selected covariates) for each individual in order to create a cohort that is as similar as possible based on these values.134 The cohort was matched using a 2:1 ratio (late/non users: early users), and the optimal matching algorithm (R package: MatchIt). The matched set was then examined longitudinally using a LMER model.

4.3 Results

Of the 797 patients from the original Sygen trial, 570 remained after applying our exclusion criteria. The cohort consisted primarily of males with complete, cervical injuries (Table 4.1). Those who were excluded were not statistically significantly different with regards to sex (p= 0.39), 4-week motor scores (p= 0.46), or level of injury (p= 0.84), when available. However, those with less severe injuries (AIS D) were more likely to be excluded (p= 0.002) and older in age (p=0.02).

54 Table 4.1 The characteristics of all early anticonvulsant users vs. non-users and late users

Characteristic Non-Users and Early Users P-value§ Late Users n (%) n (%)

Total 536 (100.00) 34 (100.00)

Sex 0.12

Female 113 (21.08) 3 (8.82)

Male 423 (78.92) 31 (91.18)

Age at Injury 0.02

Mean (SD) 31.46 (12.83) 38.44 (15.73)

4-week AIS 0.001

A 330 (61.57) 11 (32.35)

B 61 (11.38) 3 (8.82)

C 85 (15.86) 11 (32.35)

D 60 (11.19) 9 (26.47)

Baseline level of injury 0.04

Cervical 405 (75.56) 31 (91.18)

Thoracic 131 (24.44) 3 (8.82)

4-week motor score 0.01

Mean (SD) 30.07 (22.93) 42.27 (25.81)

§P-values derived from t-test for continuous variables, and Fisher’s exact test for categorical variables

55 A total of five non-gabapentinoid anticonvulsants were administered as concomitant medications: carbamazepine, phenytoin, clonazepam, phenobarbital, and valproic acid, for a variety of different indications (Table 4.2). There were a total of 49 users of one or more anticonvulsants, 34 of whom were early users. Of the anticonvulsants categorized as sodium channel blockers (phenytoin and carbamazepine), 26 were early users. In early users of all anticonvulsants (Table 4.1), there was a lower proportion of complete injuries and females, and a higher baseline motor score and age, as well as a higher proportion of cervical injuries compared to the late and non-users. The sodium channel blockers were the largest subclass, and were examined in separate analyses. The early sodium channel users presented with similar characteristics as early users of all anticonvulsants (Table 4.3).

Table 4.2 Early anticonvulsants administered in Sygen

Number Median days Reasons for Generic name Target of “early after injury of administration users” first use carbamazepine Sodium 20 10 Pain, seizures, spasms channel phenytoin Sodium 8 12 Seizures channel clonazepam GABA 6 10 Spasms, depression, anxiety, sedation, seizures phenobarbital GABA 4 6.5 Agitation, seizures valproic acid Histone 1 15 Seizures deacetylase

56 Table 4.3 The characteristics of all early sodium channel blocker users vs. non- users and late users

Characteristic Non-users and Early users P-value§ late users [n (%)] [n (%)]

Total 544 (100.00) 26 (100.00)

Sex 0.13

Female 114 (20.96) 2 (7.69)

Male 430 (79.04) 24 (92.30)

Age at Injury 0.10

Mean (SD) 30.10 (22.93) 45.77 (25.72)

4-week AIS 0.001

A 334 (61.40) 7 (26.92)

B 61 (11.21) 3 (11.54)

C 88 (16.18) 8 (30.77)

D 61 (11.21) 8 (30.77)

Baseline level of injury 0.06

Cervical 412 (75.74) 24 (92.31)

Thoracic 132 (24.26) 2 (7.69)

4-week motor score 0.005

Mean (SD) 31.64 (12.96) 36.85 (15.34)

§P-values derived from t-test for continuous variables, and Fisher’s exact test for categorical variables

The results from the linear mixed effects regression models are shown in Tables 4.4 and 4.5. In brief, there was no statistically significant interaction effect between time and

57 anticonvulsant administration (early versus late and never) on motor scores (effect= 0.10, p-value= 0.10) (Table 4.4). Further, including anticonvulsant use as a time-varying covariate was not statistically significant (effect= -1.85, p-value= 0.06). This suggests that exposure to any anticonvulsant drugs does not modify the course of neurological recovery. Conversely, there was a statistically significant drug-by-time interaction effect among the sodium channel blocker subclass. Specifically, those exposed to sodium channel blockers within the first 30 days after injury recovered, on average, 7.8 additional motor points (Table 4.5; see Figure 4.1 panel A for raw data). However, the introduction of an injury severity (AIS) by time interaction term eliminated this association: the estimated effect size decreased from approximately 8 to 1 motor point (p= 0.67; Table 4.5). This contrasts with the results of Chapter 3, in which the inclusion of the interaction term decreased the effect size but maintained statistical significance. A power analysis revealed that an effect size of 7.8 motor points for sodium channel blockers, or all anticonvulsants, would be detected with a power of 87.5% and 91.5% respectively. All 78 individuals (26 early users of sodium channel blockers were matched with 52 late/non-users) were included in the propensity scores analysis due to strong overlap. Similarly, propensity-score matching confirmed no statistically significant association between the use of sodium channel blockers and motor recovery (Table 4.6; Figure 4.1 panel B for raw data). The inclusion of the treatment with the original Sygen study drug (GM-1) did not alter the effect sizes in any of the models.

Table 4.4 Linear mixed effects regression models for total motor score from 4- weeks for all anticonvulsants

Model 1 Model 2

Variable Coefficient P-value Coefficient P-value (95% CI) (95% CI)

Intercept 9.54 (7.94-11.13) <0.00001 9.88 (8.31-11.45) <0.00001

4-week AIS

A ref - ref -

B 5.52 (2.14-8.89) 0.001 5.11 (1.83-8.40) 0.003

C 21.29 (17.75-24.83) <0.00001 20.04 (17.26-22.83) <0.00001

58 Model 1 Model 2

Variable Coefficient P-value Coefficient P-value (95% CI) (95% CI)

D 57.84 (54.53-61.15) <0.00001 57.28 (54.11-60.46) <0.00001

Baseline level of injury

Cervical ref - ref -

Thoracic 31.36 (28.90-33.83) <0.00001 31.25 (28.78-33.71) <0.00001

Time (weeks) 0.88 (0.82-0.94) <0.00001 0.73 (0.66-0.79) <0.00001

Quadratic time -0.01 (-0.01- -0.01) <0.00001 -0.01 (-0.01- -0.01) <0.00001 variable

All anticonvulsants

Never ref - ref -

Late -4.37 (-10.67-1.92) 0.18 -4.36 (-10.67-1.93) 0.18

Early 7.39 (3.13-11.64) <0.00001 7.67 (3.45-11.90) 0.0004

Time * all anticonvulsants (interaction term)

Never ref - ref -

Late -0.004 (-0.19-1.18) 0.96 -0.04 (-0.18-0.10) 0.58

Early 0.10 (-0.02-0.21) 0.10 -0.04 (-0.13-0.05) 0.38

Time * 4-week AIS (interaction

59 Model 1 Model 2

Variable Coefficient P-value Coefficient P-value (95% CI) (95% CI) term)

A - ref -

B - 0.23 (0.16-0.30) <0.00001

C - 0.58 (0.53-0.64) <0.00001

D - 0.27 (0.21-0.34) <0.00001

Table 4.5 Linear mixed effects regression models for total motor score from 4- weeks for sodium channel blockers

Model 1 Model 2

Variable Coefficient P-value Coefficient P-value (95% CI) (95% CI)

Intercept 9.52 (7.93-11.11) <0.00001 9.89 (8.33-11.44) <0.00001

4-week AIS

A ref - ref -

B 5.36 (1.20-8.72) 0.001 4.93 (1.65-8.21) 0.003

C 21.38 (17.87-24.91) <0.00001 20.07 (17.30-22.84) <0.00001

D 57.73 (54.43-61.03) <0.00001 57.14 (53.97-60.32) <0.00001

Baseline level of injury

Cervical ref - ref -

Thoracic 31.49 (28.95-33.85) <0.00001 31.28 (28.82-33.74) <0.00001

Time 0.87 (0.81-0.94) <0.00001 0.73 (0.66-0.79) <0.00001

60 Model 1 Model 2

Variable Coefficient P-value Coefficient P-value (95% CI) (95% CI)

Quadratic time -0.01 (-0.01- -0.01) <0.00001 -0.01 (-0.01- -0.01) <0.00001 variable

Sodium blockers

Never ref - ref -

Late -5.45 (-13.49-2.60) 0.19 -5.46 (-13.51-2.60) 0.19

Early 9.44 (4.63-14.24) 0.0001 9.75 (4.97-14.53) 0.00008

Time * sodium blockers (interaction term)

Never ref - ref -

Late 0.10 (-0.13-0.33) 0.39 0.073 (-0.11-0.24) 0.47

Early 0.15 (0.02-0.28) 0.022 0.01 (-0.09-0.11) 0.83

Time * 4-week AIS (interaction term)

A - - ref -

B - - 0.22 (0.15-0.29) <0.00001

C - - 0.58 (0.52-0.64) <0.00001

D - - 0.27 (0.20-0.34) <0.00001

61 Table 4.6 Linear mixed effects regression models for total motor score from 4- weeks for sodium channel blockers in a propensity score matched cohort

Variable Coefficient P-value (95% CI)

Intercept 34.67 (26.75-42.58) <0.00001

4-week AIS

Time 1.48 (1.28-1.67) <0.00001

Quadratic time variable -0.02 (-0.02- -0.02) <0.00001

Sodium blockers

Late/Never ref -

Early 8.19 (-5.32-21.70) 0.24

Time * sodium blockers (interaction term)

Late/Never ref -

Early -0.00 (-0.17-0.16) 0.96

62 A 100

75

50

25 e

r 0

o

c

s

r

o B t

o 100

m

l

a

t o

T 75

50

25

0 04 08 16 26 52 Weeks after injury

Late/never users Early users

Figure 4.1 Boxplots of motor scores over time for sodium channel blockers

(A) entire cohort and (B) propensity matched cohort

63 With regards to the secondary outcomes, analysis of sensory outcomes (light touch and pin prick scores) yielded no statistically significant associations (data not shown). Further, there was no statistically significant association between the use of any anticonvulsants (Table 4.7 Model 1, n=547, n=32 early users), or specifically sodium channel blockers (Table 4.7 Model 2, n=547, n=25 early users), with marked recovery at 26 weeks post injury.

Table 4.7 Logistic regression models for marked recovery at 26-weeks

Model 1 Model 2

Variable Odds Ratio P-value Odds Ratio P-value (95% CI) (95% CI)

Intercept 0.15 (0.10-0.21) <0.00001 0.14 (0.10-0.21) <0.00001

4-week AIS

A ref - ref -

B 6.26 (3.67-10.77) <0.00001 6.19 (3.62-10.67) <0.00001

C 60.26 (29.22-139.31) <0.00001 60.25 (29.16-138.63) <0.00001

D 44.97 (11.76-295.73) <0.00001 44.35 (11.58-291.93) <0.00001

Baseline level of injury

Cervical ref - ref -

Thoracic 0.10 0.59 (0.30-1.09) 0.10

All 1.39 (0.51-3.86) 0.52 - - anticonvulsants

Sodium channel - - 1.57 (0.52-4.97) 0.43 blockers

64 4.4 Discussion

Non-gabapentinoid anticonvulsants were administered in the acute phase of spinal cord injury, often within the first 30 days post injury. The most prevalent were carbamazepine and phenytoin (i.e. sodium channel blockers), which were primarily administered for pain and seizures, respectively (Table 4.2). In contrast to our previous analysis of gabapentinoids109, there was no significant association between the included non- gabapentinoid anticonvulsants and long-term neurological recovery when adjusted for potential confounders (Table 4.4, Table 4.5). These findings suggest that non- gabapentinoid anticonvulsants do not impact neurological recovery when administered early for the acute management of spinal cord injury.

A necessary first step in our study was to identify a data source where non- gabapentinoids had been administered in the early stages of spinal cord injury, which could be linked to neurological outcomes. The Sygen trial was optimal for this purpose because it: i) ran before the widespread use of gabapentinoids, ii) tracked neurological outcomes according to the International Standards from 24-72 hours to 52 weeks post injury, and iii) captured concomitant medication information administered alongside the delivery of the study medication (i.e. GM-1 gangliosides). Our review revealed a number of anticonvulsants administered within the first 30 days of injury, a time period in which anticonvulsants had previously demonstrated an effect on recovery of muscle strength.109

In our initial analysis of the sodium channel blockers phenytoin and carbamazepine, early exposure was associated with an over 8-point greater recovery in muscle strength greater relative to late or no exposure (Table 4.5). This observation was similar to the initial results reported for the Model 1 of gabapentinoids in Chapter 3 (8.84 points). However, unlike the analysis of EMSCI data in Chapter 3, the addition of the AIS-by-time interaction term eliminated the large effect size and statistical significance of the results. Furthermore, propensity score matching demonstrated that sodium channel blockers had no statistically signifcant effect on motor recovery. These analyses are crucial because injury severity is known to impact neurological recovery over the first-year post injury, such that more severe injuries (e.g. AIS A) recover to a lesser extent 135. The

65 cancellation of a significant effect suggests that the motor recovery initially observed in patients exposed to sodium channel blockers was largely attributable to differences in baseline injury characteristics (i.e. less severe injuries prone to exposure earlier).

Preclinical studies have demonstrated that sodium channel blockers enhance recovery in experimental models of spinal cord injury.136–138 This knowledge has directly contributed to initiation of a clinical trial in humans (i.e. riluzole, which has a similar molecular target as carbamazepine and phenytoin).104 Interventions targeting sodium channels are typically aimed at limiting secondary injury mechanisms, such as oxidative stress. Secondary injury exacerbates the extent of primary damage in the spinal cord in the immediate aftermath of traumatic injury, and thus represents a target to improve long-term neurological function.103,136 Very early timing is critical to the success of neuroprotective interventions and delays beyond 12 to 24 hours may render otherwise promising therapies less effective, or ineffective. This could explain why sodium channel blockers administered during the routine management of acute spinal cord injury, as in our study, had no impact on neurological recovery. Comparatively, gabapentinoids’ effect on neurological recovery has been ascribed to “regenerative” mechanisms associated with the inhibition of calcium signaling.101 In the latter case, the window of opportunity may be longer, extending the period in which normally administered medications (i.e. pregabalin) impact neurological recovery.

A limited number of studies have adopted a pharmacoepidemiological approach to examine existing medications in spinal cord injury. This is somewhat surprising in light of: 1) disease onset that is extremely well defined by a single traumatic event and 2) the pharmacological management of complications within a defined period of time. Pharmacoepidemiological approaches to examine disease-modifying effects of medications applied in other neurological conditions are often difficult to conduct due to the time between disease onset and administration of medications at varying time-points throughout disease. Although non-gabapentinoid anticonvulsants had no effect on neurological outcomes after acute spinal cord injury, future studies should consider other medications, or combinations of medications, using a pharmacoepidemiological approach.

A limitation of the current study, as in all observationally designed studies, is that

66 exposure was not randomized. As a result, other confounding variables may have influenced the results (e.g. social or financial status, frailty indices). Nevertheless, well- designed and carefully controlled observational studies tend to yield similar effects as conventionally designed clinical trials.139 The exposure group has a low sample size (n= 34 administered non-gabapentinoids anticonvulsants in the first 30 days post injury). While comparable to our previous analysis,105 we may have had a limited ability to detect small and statistically significant effects, though further power analyses revealed high power for an effect of approximately 8 motor points. Finally, this landmark trial is nearly 20 years old and the medical management of spinal cord injury has changed since that time (e.g. use of methylprednisolone). To this point, studies have demonstrated remarkable similarities between recovery in the Sygen and more contemporary datasets, suggesting very little has changed in terms of neurological recovery profiles with the evolution of management protocols.6

In summary, non-gabapentinoid medications were administered in the acute phase of spinal cord injury (≤ 30 days). Among them were sodium channel blockers, administered for pain and seizures, which have been demonstrated as neuroprotective in preclinical models. Our analysis in humans revealed that non-gabapentinoid anticonvulsants (i.e. carbamazepine, phenytoin, clonazepam, phenobarbital, and valproic acid) do not modify neurological recovery after acute spinal cord injury.

67 Chapter 5 The effect of gabapentinoids on neurological recovery after spinal cord injury

5.1 Introduction

Gabapentinoid anticonvulsants (i.e. pregabalin and gabapentin) represent the front line interventions for the management of neuropathic pain.83 In cases arising from damage to the spinal cord, administration often occurs early after injury (e.g. within 4 weeks).105,109 Within this acute time frame, these medications have the potential to modify the protection or repair of the spinal cord by antagonizing voltage-gated calcium channels in the central nervous system. 100,101,140

In support of these modifying effects, Chapters 3 revealed an exciting association between the early administration of gabapentinoids (but not other anticonvulsants; Chapter 4), and enhanced neurological recovery.109,141 However, a notable limitation of Chapter 3 was that anticonvulsant use was self-reported. This is problematic, particularly in acute care settings, as self-reported use of medications can be inaccurate.142 Chapter 3 was also limited in its ability to discern the exact timing of initial drug exposure. Specifically, gabapentinoids administration was only reported at fixed time-points (i.e. at 4 weeks). This is problematic with regards to accurately defining exposure, in that a patient administered gabapentinoids early after injury (e.g. at 2 weeks) who discontinued use at a later time-point (e.g. at 4 weeks) may be incorrectly classified as a “non-user” early after injury.

To address these limitations, we performed an observational study, tracking patients’ neurological recovery after spinal cord injuries alongside chart-reviewed medication usage from a single centre. The fundamental goal was to further elucidate the effect of gabapentinoids on neurological recovery after spinal cord injury using well-defined criteria on usage, and timing of initial administration.

68 5.2 Methods 5.2.1 Data

Patients were identified from their participation in EMSCI between 2007 and 2016. The chart review was performed at a participating centre (i.e., Trauma Center Murnau, Germany). This chart review provided information on specific anticonvulsant use (the generic name), the first date of administration, and the duration. Charts were then retrospectively linked with neurological outcomes from the original EMSCI database to create an observational cohort. The EMSCI database was queried for the following individual patient information for each patient: date of injury, sensory and motor scores from the ISNCSCI (i.e. motor, light touch, and pinprick scores),131 SCIM 3 scores, AIS injury severity grades, and level of injury (including upper cervical [C1-C4], lower cervical [C5-C8], thoracic [T1-T12], and lumbar [L1-L5]). EMSCI sites obtained approval by local research ethics boards, and individuals consented to have their data entered into the database.

The EMSCI included patients with spinal cord injury caused by a traumatic event (including single event ischemia). They were excluded if they had prior dementia or reduced learning disabilities, if they had peripheral nerve lesions above the level of injuries or a prior polyneuropathy, or if they had a severe craniocerebral injury. For the purpose of our study, additional inclusion/exclusion criteria were applied. We calculated “baseline” measures for injury severity (AIS grade) and level of injury by using 4-week recordings if available, and 1-week recordings if 4-week recordings were missing. Patients were then excluded if they were missing either their baseline level of injury or their baseline AIS grade. Patients were also excluded if they presented with baseline injury severity of AIS E (normal sensory and motor function), if they had fewer than 2 motor scores, or if they presented with an injury level below T12 (e.g. lumbar or sacral injuries).

5.2.2 Variables

Neurological outcomes included motor and sensory scores as defined by ISNCSCI performed at 1, 4, 12, 24, and 48 weeks.131 The primary outcome was total motor score (0-100). Secondary outcomes included the sensory measures pin prick score and light

69 touch score (each 0-112), and the functional score from the of SCIM 3 (0-100).143 Outcomes were modeled using available scores from 4 weeks to 52 weeks after injury (i.e. after exposure), as was done previously.109

The extraction of anticonvulsant use through a review of patient charts was conducted by two research assistants and supervised and approved on-site by Dr. Grassner. Anticonvulsant classification was then verified using the World Health Organization Collaborating Centre for Drug Statistics Methodology’s (https://www.whocc.no/) Anatomic Therapeutic Chemical (ATC) classification system (code: NO3). Administration of anticonvulsants was then classified as having occurred “early” (within 30 days after injury), or “late/never”, as previously examined.109 Gabapentinoid use was also examined as a time-varying covariate, such that any use in the 4 weeks prior to a neurological exam time point would be considered “exposed” at that measure.

5.2.3 Statistics

The longitudinal progression of total motor score was first visually examined using Trellis plots. A multivariable longitudinal analysis was then conducted using a linear mixed effects regression model (R package: lme4). The model was adjusted for baseline level of injury and AIS grade, as well as an AIS-by-time interaction term to account for potential differences in recovery profiles between injury severities across time.8 A quadratic term for the time variable was included to account for the non-linear profile of neurological recovery after spinal cord injury. A time-by-drug interaction term was included to examine the effects of gabapentinoids on neurological recovery (when not time-varying), and an analysis of variance (ANOVA) was used to statistically compare model fit after the addition of each variable. These analyses were conducted for the primary outcome measure (total motor score), and then repeated for sensory and functional outcomes. In addition to this, we created a matched cohort using propensity scores using variables AIS grade and level of injury. The cohort was matched using a 1:1 ratio (late/non users: early users), and the optimal matching algorithm (R package: MatchIt). Motor scores were then examined longitudinally using a mixed effects regression model in the matched cohort.

Following our initial models, we took a closer look at the timing of initial administration.

70 Specifically, initial gabapentinoid exposure was redefined relative to time of injury, and each of these new exposures were run in respective longitudinal models. The exposures run in these models were gabapentinoids that had been initially administered 0-5, 6-15, 16-30, 31-60, and 61-120 days after injury. P <0.05 was considered statistically significant. All analyses were performed using RStudio statistical software, version 1.1.453.144

5.3 Results

The chart review included 251 patients who sustained a spinal cord injury. After applying our study-specific exclusion criteria, a total of 201 patients remained. Those who were excluded were not statistically significantly different with regards to their 4-week motor scores (p=0.80), age (p=0.74), sex (p=0.18), baseline AIS (p=0.12), or level of injury (p=0.13) compared to those who were included. The cohort was primarily male with a high proportion of motor and sensory complete injuries (AIS A) (Table 5.1).

Table 5.1 Cohort demographics

Characteristics N (%)

Total 201 (100.00)

Sex

Female 30 (14.93)

Male 171 (85.07)

Age at Injury

Mean (SD) 45.23 (18.16)

Baseline AIS

A 101 (50.25)

B 16 (7.96)

C 25 (12.44)

D 59 (29.35)

71 Characteristics N (%)

Baseline level of injury

Upper cervical 57 (28.36)

Lower cervical 56 (27.86)

Thoracic 88 (43.78)

The anticonvulsants administered included gabapentin, pregabalin, and carbamazepine (Table 5.2). Of the 201 patients, 70 (35%) received gabapentinoids within the first 30 days after injury (i.e. “early” administration). Those who received “early” administration did not significantly differ from the late/non-users with regards with age at injury, sex, or baseline AIS grades, but were different with regards to baseline level of injury. Late/non- users tended to have a lower proportion of upper cervical injuries, and a higher proportion of thoracic injuries (Table 5.3). The duration of drug use of those receiving early vs. late administration was similar (Table 5.4).

Table 5.2 Anticonvulsants administered

Generic name Target Users “Early users”

Gabapentin Calcium channel 26 12 Pregabalin Calcium channel 89 59 Carbamazepine Sodium channel 5 1 Gabapentinoids Calcium channel 102 70

72 Table 5.3 Gabapentinoid user demographics

Characteristics Non-Users and Early Users (%) P-value§ Late Users (%)

Total 131 (100.00) 70 (100.00)

Sex 1.00

Female 20 (15.27) 10 (14.29)

Male 111 (84.73) 60 (85.71)

Age at Injury 0.92

Mean (SD) 45.15 (18.51) 45.41 (17.61)

Baseline AIS 0.66

A 69 (52.67) 32 (45.71)

B 11 (8.40) 5 (7.14)

C 14 (10.69) 11 (15.71)

D 37 (28.24) 22 (31.43)

Baseline level of injury 0.007

Upper cervical 29 (22.14) 28 (40.00)

Lower cervical 35 (26.72) 21 (30.00)

Thoracic 67 (51.15) 21 (30.00)

§P-values derived from t-test for continuous variables, and Pearson’s chi-squared or Fisher’s exact for categorical variables

73 Table 5.4 Duration of use for early vs. late administration

Duration Early users (n=70) Late users (n=32) Pregabalin Gabapentin Pregabalin Gabapentin (n=59) (n=12) (n=30) (n=14) Range 6-252 2-126 1-223 4-204 Mean (SD) 111.20 (64.78) 54.92 (37.90) 107.23 (69.00) 59.79 (51.46) *Note: some users were administered both pregabalin and gabapentin

The early administration of gabapentinoids was associated with improved motor recovery. Figures 5.1 and 5.2 depict the improved recovery profiles of the early gabapentinoid group (also stratified by AIS), and the change in scores from 4 to 48 weeks. In the longitudinal analysis, the drug-by-time interaction term was statistically significant (Table 5.5). The early administration of gabapentinoids had an overall benefit of approximately 4.36 motor points in the first year after injury. The fitted recovery trajectories from the unadjusted model (including only time and drug variables and interactions) are depicted in Figure 5.3 panel i. Further, ANOVA confirmed that including the drug*time interaction term statistically significantly improved the model fit. Gabapentinoid use as a time varying covariate was not statistically significant in the mode (estimate= -0.59, p-value= 0.55).

Of the secondary outcomes, early gabapentinoid administration was statistically significantly associated with greater recovery of light touch scores (Table 5.6, 5.7). Early administration had no statistically significant effect on SCIM scores (Table 5.8). For the propensity score matched cohort, all 140 individuals (70 early users of sodium channel blockers were matched with 70 late/non-users) were included. Similar results were found in the longitudinal model of the propensity score matched cohort (n=140, 70 “early users”), in which a large effect size was reported for the drug-by-time interaction term that neared statistical significance (p=0.06) (Table 5.9).

Table 5.5 Longitudinal model of motor scores from 4 weeks

Characteristics Estimate P-value (95% CI)

Intercept 12.71 (8.49-16.92) <0.00001

74 Characteristics Estimate P-value (95% CI)

Time (weeks) 0.55 (0.39-0.71) <0.00001

Quadratic time variable -0.01 (-0.01- -0.01) <0.00001

Baseline AIS

A ref -

B -1.49 (-8.04-5.07) 0.66

C 18.92 (13.37-24.48) <0.00001

D 48.64 (44.27-53.01) <0.00001

Baseline level of injury

Upper cervical ref -

Lower cervical 17.30 (12.64-21.97) <0.00001

Thoracic 31.01 (26.85-35.18) <0.00001

Gabapentinoid use

Late/Never ref -

Early 0.16 (-3.56-3.88) 0.93

Baseline AIS * Time (interaction term)

A ref -

B 0.16 (0.02-0.30) 0.03

C 0.62 (0.51-0.74) <0.00001

D 0.15 (0.07-0.23) 0.0005

Gabapentinoid use * Time (interaction term)

75 Characteristics Estimate P-value (95% CI)

Late/Never ref -

Early 0.08 (0.01-0.16) 0.03

Table 5.6 Longitudinal model of total light touch scores from 4 weeks

Characteristics Estimate P-value (95% CI)

Intercepts 28.30 (22.60-34.05) <0.00001

Time (weeks) 0.33 (0.13-0.54) 0.001

Quadratic time variable -0.01 (-0.01- -0.00) 0.004

Baseline AIS

A ref -

B 14.64 (5.40-23.84) 0.002

C 43.98 (36.20-51.73) <0.00001

D 51.79 (45.77-57.78) <0.00001

Baseline level of injury

Upper cervical ref -

Lower cervical 15.01 (8.86-21.16) 0.00004

Thoracic 25.47 (19.84-31.07) <0.00001

Gabapentinoid use

Late/Never ref -

Early -1.91 (-7.11-3.28) 0.48

76 Characteristics Estimate P-value (95% CI)

Baseline AIS * Time (interaction term)

A ref -

B -0.02 (-0.24-0.22) 0.89

C -0.06 (-0.24-0.12) 0.52

D -0.11 (00.24-0.03) 0.12

Gabapentinoid use * Time 0.16 (0.03-0.28) 0.01 (interaction term)

Table 5.7 Longitudinal model of total pin prick scores from 4 weeks

Characteristics Estimate P-value

Intercepts 18.45 (12.05-24.91) <0.00001

Time (weeks) 0.62 (0.34-0.90) 0.00002

Quadratic time variable -0.01 (-0.02- -0.01) 0.0001

Baseline AIS

A ref -

B 6.49 (-3.82-16.83) 0.23

C 25.09 (16.41-33.76) <0.00001

D 45.51 (38.79-52.28) <0.00001

Baseline level of injury

Upper cervical ref -

Lower cervical 18.83 (12.03-25.59) <0.00001

77 Characteristics Estimate P-value

Thoracic 32.32 (26.23-38.40) <0.00001

Gabapentinoid use

Late/Never ref -

Early -0.60 (-6.43-5.22) 0.84

Baseline AIS * Time (interaction term)

A ref -

B 0.08 (-0.20-0.36) 0.58

C 0.18 (-0.04-0.39) 0.11

D -0.02 (-0.18-0.13) 0.78

Gabapentinoid use * Time 0.06 (-0.09-0.20) 0.45 (interaction term)

Table 5.8 Longitudinal model of total SCIM scores from 4 weeks

Characteristics Estimate P-value (95% CI)

Intercepts -10.06 (-16.49- -3.65) 0.002

Time (weeks) 2.39 (2.10-2.68) <0.00001

Quadratic time variable -0.03 (00.04- -0.03) <0.00001

Baseline AIS

A ref -

B 4.66 (-5.82-15.08) 0.39

C 11.65 (2.94-20.38) 0.01

78 Characteristics Estimate P-value (95% CI)

D 27.04 (20.83-33.80) <0.00001

Baseline level of injury

Upper cervical ref -

Lower cervical 15.06 (8.35-21.81) 0.00002

Thoracic 27.84 (21.83-33.84) <0.00001

Gabapentinoid use

Late/Never ref -

Early -1.95 (-7.79-3.87) 0.52

Baseline AIS * Time (interaction term)

A ref -

B -0.16 (-0.47-0.16) 0.33

C 0.19 (-0.03-0.41) 0.09

D 0.32 (0.16-0.49) 0.0002

Gabapentinoid use * Time 0.01 (-0.14-0.17) 0.87 (interaction term)

79

Table 5.9 Longitudinal model of total motor scores from 4 weeks in a propensity score matched cohort

Characteristics Estimate P-value

Intercept 37.89 (31.01-44.75) <0.00001

Time (weeks) 0.93 (0.71-1.15) <0.00001

Quadratic time variable -0.01 (-0.02- -0.01) <0.00001

Gabapentinoid use

Late/Never ref -

Early 3.84 (-5.74-13.43) 0.43

Gabapentinoid use * Time (interaction term)

Late/Never ref -

Early 0.14 (-0.00-0.29) 0.06

80 i

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82 Longitudinal models of the motor scores were then conducted using the redefined exposures for gabapentinoids. The time categories for initial administration included 0-5 (n=14 users), 6-15 (n=32), 16-30 (n=24), 31-60 (n=17), and 61-120 (n=13) days after injury. Though the numbers were low within each model and none reached statistical significance, the effect sizes decreased as administration occurred later after injury, and the p-values increased accordingly (Table 5.10, Figure 5.2B). For example, the exposure interaction term from 0-5 days after injury indicated the greatest improvement in total motor score in the first year after injury (5.53 points, p= 0.11).

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Figure 5.3 Summary of model outputs

(A) The fitted motor scores of early and late/never users of gabapentinoids from an unadjusted longitudinal model of gabapentinoid administration within 30 days after injury. (B) The effect sizes for the longitudinal models using exclusive time points of administration of gabapentinoids (0-5, 6-15, 16-30, 31-60, and 61-120 days after injury).

84 Table 5.10 Comparison of drug*time term in longitudinal models of total motor scores from 4 weeks using varying timing of exposure

Drug initiation Number of Estimate from P-value (days after injury) exposed drug*time term Model 1 0-5 14 0.11 0.11 Model 2 6-15 32 0.06 0.26 Model 3 16-30 24 0.03 0.65 Model 4 31-60 17 0.03 0.61 Model 5 60-120 13 0.006 0.94

It was then examined if there was a bias in exclusion between our original analysis of early users (within 30 days), and late/never users. For example, if more severe injuries were more prone to exclusion in the “early users”. Of the 50 participants excluded, 19 were early users and 31 were late/never users. These two groups did not statistically significantly differ with regards to age, sex, level of injury, 4-week motor scores, or AIS grade (when available).

5.4 Discussion

In a cohort of patients with acute spinal cord injury, early exposure to gabapentinoids was associated with a beneficial effect on the recovery of motor and sensory function. The current analysis was based on medication details extracted directly from patient charts which, compared to self-report in Chapter 3, revealed higher rates of gabapentinoid exposure.109 In addition to the confirmed beneficial association between gabapentinoids and neurological recovery, further analyses indicated the largest effect occurred when initiated in the first 5 days of injury. This is consistent with concept of a “window of opportunity” to promote protection and/or repair in the injured spinal cord. Despite evidence of neurological benefit, early exposure had no long-term benefit on pin prick scores, or a functional independence measure. According to the Oxford Centre of Evidence-Based Medicine, this study has provided level 3 evidence of their benefit on neurological function after acute spinal cord injury.145

As in previous studies, there was a beneficial effect of administering gabapentinoids within the 30 days after injury on the recovery of muscle strength, which appeared to have been driven by very early exposure (e.g. within 5 days after injury). Our results indicated an overall benefit of 4.36 motor points when gabapentinoids were administered

85 within 30 days, which increased marginally to 5.54 points if administered within 5 days of injury. These results are in line with previous findings utilizing multicentre observational cohorts.105,109 A strength of multicentre cohort studies (as is often used in large-scale clinical trials and previous Chapters) is that they have the benefit of including larger sample sizes, and thus have a greater likelihood of detecting an effect. Additionally, by including a more diverse sample of the spinal cord injury population at large they can increase the generalizability of their findings. However, single-centre studies, as used here, offer their own advantages. Collecting detailed data such as drug administration is more feasible when dealing with a single centre. Further, in utilizing single-centre data there is a decreased likelihood of variable methodologies or failures to comply with a given protocol across centres (thus reducing across-centre errors).

In contrast to previous studies (e.g. Chapter 3), this analysis identified a beneficial effect on sensory (light touch) scores. It has been previously noted that that total motor scores have the highest level of inter-rater reliability, followed by light touch and then pin prick scores.146,147 These novel findings may indicate that using single-centre data decreased the variability and was therefore able to elucidate the effect on light touch scores, though not pin prick. However, they may also indicate chance findings from a single centre. The lack of statistically significant effects in the functional (SCIM) measure thus far could indicate that this effect is very small, or that the changes in neurological recovery did not result in clinically meaningful results. If so, it may be that an improved protocol (i.e. one in which a large number of patients receiving gabapentinoids within 5 days after injury) could identify these effects. It may also be the case that in the search for a treatment after spinal cord injury, multiple therapies providing marginal changes must be combined in order to obtain an overall clinical benefit.

Gabapentinoids provide an exciting opportunity for drug repurposing in spinal cord injury because they are already administered to spinal cord injury patients, and thus have an established safety profile. For this reason, repurposing could avoid many of the costly and time-consuming hurdles of early-phase clinical trials. Unlike our previous chapters which examined the general administration of anticonvulsants,105,109 this study focused solely on the effects of gabapentinoids. In addition to this, Chapter 3 included self- reported drug use at a specific time point (i.e. 4 weeks after injury), whereas in conducting a chart review this study may have more accurately identified of all

86 participants who received anticonvulsants within 30 days of injury. Furthermore, this study was able to look further into the more exact initiation of administration (e.g. multiple time points), and highlight its importance.

The increase in effect size closer to time of injury (i.e. within 5 days) strengthens the time-dependent effect of gabapentinoids, and is important for several reasons. Early administration is believed to be necessary for both neuroprotective and regenerative strategies after spinal cord injury, and is clearly demonstrated in gabapentinoids.140 Indeed, the 30-day window defining “early” administration is somewhat longer than hypothesized for either neuroprotection (hours to days) or neuroregeneration (days to weeks). Furthermore, many previous clinical trials have emphasized the need to administer pharmacological interventions very early, within hours of injury.32,43,63 This in turn greatly reduces the already limited number of candidates that may be included in a study’s protocol. However, the 5-day window is longer, and may be taken important practical consideration for future trials. A window of opportunity of 5 days after injury not only increases the likelihood of recruiting participants, but also of gathering accurate baseline neurological evaluations (which are generally recommended at around 72 hours) in clinical trials.148

Although this chapter provides benefits over previously conducted studies, it still presents with some limitations. Firstly, as in all observational cohorts, the individuals receiving gabapentinoids were not randomized to their treatment, and thus some unknown or unavailable confounder may have caused the effect. Specifically, there could be an indication bias, in that the beneficial effects of gabapentinoids were linked to the underlying reason for administration (i.e. neuropathic pain) and not the drug itself. Information regarding neuropathic pain status was unavailable in this cohort, however, Chapter 3 examined the presence and progression of neuropathic pain as a potential confounder.109 Further, Chapter 4 found that early (within 30 days) use of non- gabapentinoid anticonvulsants was not associated with any benefit to neurological recovery. These results indicate the specificity of gabapentinoids for a benefit, but also highlight the lack of effect for drugs of similar indications.141 Secondly, while specific initiation of administration was available, we did not have information on overall dosages for each patient, and thus cannot surmise a dose-response effect. Finally, the information regarding very early administration (i.e. within 5 days) is based on very low

87 numbers (n= 14), as it is a short time frame for administration to occur. These limitations may be further explored within a clinical trial, where these factors may be better controlled. As such, the overall benefit of gabapentinoids on total motor score recovery has persisted in several studies, is unique to gabapentinoid anticonvulsants, and has strengthened with improved methodology.105,109,141 From these results it is clear that gabapentinoids are an exciting new candidate for drug repurposing in spinal cord injury and a progression to further research is warranted.

88 Chapter 6 The progression of neuropathic pain after acute spinal cord injury

6.1 Introduction

Gabapentinoids are currently the front-line treatment for neuropathic pain after spinal cord injury. As such, any clinical trials to investigate the effects of gabapentinoid anticonvulsants for recovery would run into the ethical issue of administering gabapentinoid anticonvulsants to those not in the treatment group, but experiencing neuropathic pain. It would be helpful, in this scenario, to estimate the patterns of progression of neuropathic pain after acute spinal cord injury. In addition, there is growing interest in the application of acute pharmacological interventions to prevent the development of pain.149–152 Furthermore, many other therapeutic interventions have been explored in animal models with the goal of improving neurological outcomes after spinal cord injury.153 A chief concern among those translated to humans154–156 is that promoting axonal growth and repair in the central nervous system could, inadvertently, drive pathophysiological mechanisms associated with the development of neuropathic pain.154,157,158 This concern exists in early trials that lack a control group, or those that are underpowered to detect potentially detrimental secondary outcomes (e.g. cell based interventions).159 To this end, historical control data will serve an important role in establishing safety guidelines.

Of particular value is a clear understanding of the natural history of neuropathic pain progression after injury. This includes knowledge of the probability that individuals with neuropathic pain will have their pain resolve, as well as the probability that individuals without neuropathic pain will develop symptoms. To date, two large studies have prospectively described the progression of neuropathic pain after acute traumatic spinal cord injury.114,160 Due to variability in methodology and reporting, estimates to characterize the temporal changes of neuropathic pain are difficult to ascertain.

The aim of this study was to develop a framework to evaluate the probability that a therapeutic intervention for spinal cord injury modifies the progression of neuropathic pain in acute trials. In a two step process, data was analyzed from the European

89 Multicenter Study about Spinal Cord Injury (EMSCI) and meta-analyzed with outcomes from a previously published study.114 A secondary analysis explored the association of neuropathic pain progression with demographic, pain, and injury characteristics.

6.2 Methods

6.2.1 Data

Data from the EMSCI and a published observational cohort study performed in Denmark and Sweden114 were included in our analysis. EMSCI has been approved at each site by local research ethics boards, and individuals consent to have their data entered into the EMSCI database. Individuals were excluded if their spinal cord injury was caused by a non-traumatic event (excluding single event ischemia), if they had previous dementia/reduced learning capabilities, if they had peripheral nerve lesions above the level of injury, if they had a previous polyneuropathy, or if they had a severe craniocerebral injury. We further excluded individuals who did not have baseline injury characteristics (level and severity of injury), and those who did not have the pain questionnaires at the necessary time points.

The Swedish/Danish cohort data has been previously published. The objectives of the prospective study were to identify pain phenotypes after traumatic spinal cord injury and determine if sensory hypersensitivity predicted the development of neuropathic pain. The inclusion criteria and methods for this study, as well as the evaluation of pain, has been previously described.114

6.2.2 Variables

The EMSCI prospectively collects detailed neurological, neurophysiological, and functional outcomes from individuals with traumatic or ischemic spinal cord injuries during the first year after injury for research purposes. This includes sensory and motor scores according to the ISNCSCI.108 Motor scores are calculated from select muscle groups, each muscle scored from 0-5 (0= total paralysis, 5= normal), for a maximum score of 25 for each extremity and a maximum total motor score of 100.108 Sensory scores are composed of light touch scores (sensation with a cotton wisp) and pinprick scores (sharp/dull discrimination with a safety pin) scored from 0-1 (0= absent, 1= altered, 2= normal). Select dermatomes are tested with comparison to the same

90 sensation on the cheek, such that each sensory measure has a maximum total score of 112 points.108 These assessments are performed at fixed time points after injury: very acute (0-15 days), 4 weeks (16-40 days), 12 weeks (70-98 days), 24 weeks (150-186 days), and 48 weeks (>=300 days).

Beginning in 2007 (and updated in 2011), a subset of EMSCI individuals completed an additional pain questionnaire.105,109 Pain outcomes were ascertained by a trained interviewer. The questionnaire allowed up to three pain sites to be reported, with the categorization of musculoskeletal, visceral, or neuropathic pain. Each pain was characterized by the interviewer according to descriptors, location (i.e. relative to lesion level), alleviating and aggravating factors, frequency, and intensity (0-10 numeric rating scale). In agreement with the International Spinal Cord Injury Pain Classification,84 neuropathic pain status was determined according to its location (i.e., in an area of complete/partial sensory loss), key descriptors (e.g., hot-burning), lack of relationship with movement, and the presence of allodynia and paresthesia. Neuropathic pain was then further classified based on proximity to the level of injury (i.e., at-/below level).84 Three published studies have utilized the EMSCI pain questionnaire.105,109,110 For our analysis, only individuals with a valid 4-week (1-month) examination for the American Spinal Injury Association Impairment Scale (AIS) grade (grade of A, B, C or D) and level of injury (including upper cervical [C1-C4], lower cervical [C5-C8], thoracic [T1-T12], and lumbar [L1-L5]), and a complete pain questionnaire were included.

The evaluation of pain conducted in the Swedish/Danish cohort has been previously described.114

6.2.3 Statistics

The primary analysis addressed the progression of neuropathic pain by dichotomizing pain status (yes/no) at each time point. The inclusion of three time points (i.e., 1, 6, and 12 months, common to both EMSCI and Swedish/Danish study) and two pain states (yes/no) resulted in two progression timelines for the progression of acute neuropathic pain: 1-6 months and 1-12 months. Individuals who had complete pain information at the required time points (e.g. 1 and 6 months or 1 and 12 months) were included in these estimates, creating 2 overlapping cohorts. These frameworks were constructed for at- level, below-level, and overall (at-level and below-level) neuropathic pain. Meta-analyses

91 were performed using logit transformations to fit random-effects logistic models (inverse variance methods, such that the included values are weighted inversely proportionally to their variance, R function: rma.glmm). The pooled estimates were then calculated using the R package metaphor. All analyses were conducted using open access R Studio version 1.0.153.

As a secondary analysis, we explored the associations between demographics, pain characteristics, and injury characteristics with neuropathic pain using bivariable logistic regression in the EMSCI dataset only (previously performed in Swedish/Danish cohort).114 To assess these associations, individuals were grouped into two categories: “developed pain” (i.e., no neuropathic pain present at 1 month, with symptoms occurring for the first time at 6 or 12 months) and 6 month “persistent pain” (neuropathic pain present at 1 month, with symptoms persisting into 6 months). The former definition required individuals with a valid pain examination at 6 or 12 months as the pooled outcome, as pain appearing at either time would be classified as “developing” later after injury. The reference group was comprised of those with no recorded neuropathic pain at 1 month or at the later time points. Six month persistent pain was defined as neuropathic pain at 1 month AND at 6 months, with a reference group of those with neuropathic pain at 1 month and no neuropathic pain at 6 months (i.e., those who had pain that did not persist over time). The persistent pain was limited to pain persisting at 6-months, as extending this definition to 12 months decreased the numbers available for analysis.

Demographic variables included age at injury and sex. Injury characteristics were measured at 1 month post-injury and included level of injury, total motor score, total light touch score, and total pinprick score.108 We further examined sensory subscores, as previously described in a published study.161 This included at-level light touch and pinprick scores (sum of scores from 3 dermatomes of the neurological level of injury excluding injuries at L3/L4/L5) and low-level light touch and pinprick scores (sum of scores from dermatomes L3-L5). Additionally, the difference between light touch and pinprick (light touch – pinprick scores) for total, at-level, and low-level scores were included.161 Pain characteristics included the presence of musculoskeletal pain, and the presence of at-level neuropathic pain (for the evaluation of below-level pain progression) or below-level neuropathic pain (for the evaluation of at-level pain progression). In the 6- month persistent pain group, we additionally examined associations with 1-month pain

92 severity.

6.3 Results

A total of 1005 participants completed the EMSCI pain questionnaire. After excluding those with baseline injury characteristics and a valid 1-month pain questionnaire, 728 remained. From this cohort the two overlapping cohorts of those with an additional valid 6-month and a valid 12-month pain questionnaire were taken. A total of 251 participants were included in the 1-6 month analysis and 144 in the 1-12 month analysis from a total of 8 participating centres. From the original 90 individuals in the published Swedish/Danish cohort,114 one participant with an AIS E injury was excluded. Seventy- seven participants had a valid neuropathic pain status for 1-6 months, and 87 for 1-12 months after injury. The demographics for the EMSCI and Swedish/Danish cohorts are summarized in Table 6.1.

Table 6.1 Demographics of the cohorts at first visit

EMSCI cohorts (%) Swedish/Danish cohorts (%)

1-6 month 1-12 month 1-6 month 1-12 month

Total 251 (100.00) 144 (100.00) 77 (100.00) 87 (100.00)

Mean age (SD) 47.88 (18.53) 47.09 (19.02) 48.0 (15.5) 47.2 (16.1)

Sex

Males 208 (82.87) 127 (88.19) 68 (88.31) 77 (88.51)

Females 43 (17.13) 17 (11.81) 9 (11.69) 10 (11.49)

Level of injury

Upper cervical 74 (29.48) 39 (27.08) 22 (28.57) 23 (26.44)

Lower cervical 55 (21.91) 27 (18.75) 22 (28.57) 26 (29.89)

Thoracic 97 (38.64) 54 (37.50) 25 (32.47) 28 (32.18)

93 Lumbar 25 (9.96) 24 (16.67) 8 (10.39) 10 (11.49)

AIS Grade

A 107 (42.63) 51 (35.42) 31 (40.26) 37 (42.52)

B 26 (10.36) 11 (7.64) 8 (10.39) 8 (9.20)

C 43 (17.13) 18 (12.50) 19 (24.67) 19 (21.84)

D 75 (29.88) 64 (44.44) 19 (24.67) 23 (26.44)

6.3.1 Progression

The combined EMSCI and Swedish/Danish aggregate estimates from the meta-analysis for the progression of overall neuropathic pain are shown in Figure 6.1. The estimates for overall, at-, and below-level neuropathic pain are summarized (from 1-6 months and 1-12 months) and reported in Tables 6.2 and 6.3, respectively. The respective estimates from each of the EMSCI versus the Swedish/Danish cohort are available for comparison in Tables A.1 and A.2. These aggregate estimates highlight the number incident cases, that is, the number of new cases that develop within first year after injury, in addition to the rates of neuropathic pain relief or persistence, given the initial pain state and timeline of a cohort.

Table 6.2 The aggregate estimates of overall, at-level, and below-level neuropathic pain from 1 to 6 months after spinal cord injury

Estimates from 1 to 6 months Neuropathic pain At 1 month N At 6 months % (95% CI) Overall Yes 89 Yes 72% (62, 80) No 28% (20, 38) No 239 Yes 23% (14, 37) No 77% (63, 86) At-level Yes 60 Yes 67% (54, 77)

94 Estimates from 1 to 6 months Neuropathic pain At 1 month N At 6 months % (95% CI) No 33% (23, 46) No 268 Yes 14% (7, 25) No 86% (75, 93) Below-level Yes 44 Yes 55% (40, 68) No 45% (32, 60) No 284 Yes 14% (11, 19) No 86% (81, 89)

Table 6.3 The aggregate estimates of overall, at-level, and below-level neuropathic pain from 1 to 12 months after spinal cord injury

Estimates from 1 to 12 months Neuropathic pain At 1 month N At 12 months % (95% CI) Overall Yes 66 Yes 69% (53, 81) No 31% (19, 47) No 165 Yes 36% (23, 52) No 64% (48, 77) At-level Yes 45 Yes 47% (33, 61) No 53% (39, 67) No 186 Yes 19% (13, 26) No 81% (74, 87) Below-level Yes 29 Yes 52% (34, 69) No 48% (31, 66) No 202 Yes 23% (17, 29) No 77% (71, 83)

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Table 6.4 The results of the logistic regression models for at-level neuropathic pain

Outcome (yes/no) Characteristic Odds ratio (95% CI) P-value

Developed at-level neuropathic pain N=107 (35 with pain) Age at injury 1.03 (1.00-1.05) 0.02 Sex Female Ref - Male 0.65 (0.22-1.94) 0.42 Level of injury Upper cervical Ref - Lower cervical 0.93 (0.27-3.13) 0.90 Thoracic 0.66 (0.24-1.79) 0.41 Lumbar 0.31 (0.06-1.24) 0.12 Below-level neuropathic pain 1.66 (0.50-5.20) 0.39 Musculoskeletal pain 1.00 (0.45-2.26) 1.00 Motor score 0.99 (0.98-1.01) 0.50 Light touch 1.00 (0.98-1.01) 0.79 Pinprick 0.99 (0.98-1.00) 0.19 Light touch – pinprick 1.04 (1.01-1.08) 0.03 Low-level light touch 1.00 (0.91, 1.09) 0.98 Low-level pinprick 0.96 (0.87, 1.06) 0.45 Low-level light touch-pinprick 1.15 (0.95, 1.40) 0.14 N=106 (34 with pain) At-level light touch 1.00 (0.85, 1.18) 0.95 At-level pinprick 0.95 (0.82, 1.11) 0.54 At-level light touch- pinprick 1.12 (0.88, 1.43) 0.38 Six month persistent at-level neuropathic pain N=41 (26 with pain) Age at injury 0.93 (0.87-0.97) 0.008 Sex Female Ref -

96 Outcome (yes/no) Characteristic Odds ratio (95% CI) P-value Male 2.00 (0.40-10.01) 0.39 Level of injury** Upper cervical Ref - Lower cervical - - Thoracic - - Lumbar - - Pain severity 1.10 (0.73-1.73) 0.66 Below-level neuropathic pain 2.36 (0.62-10.32) 0.22 Musculoskeletal pain 0.61 (0.16-2.23) 0.45 Motor score 1.00 (0.97-1.03) 0.91 Light touch 1.01 (0.98-1.03) 0.69 Pinprick 1.01 (0.98-1.03) 0.59 Light touch – pinprick 0.99 (0.95-1.03) 0.64 Low-level light touch 1.08 (0.94, 1.27) 0.30 Low-level pinprick 1.18 (0.99, 1.47) 0.09 Low-level light touch-pinprick 0.89 (0.69, 1.13) 0.33 At-level light touch 1.37 (1.05, 1.91) 0.04 At-level pinprick 1.42 (1.11, 1.94) 0.01 At-level light touch- pinprick 0.85 (0.63, 1.13) 0.27 **Level of injury could not be included due to insufficient numbers

97

Figure 6.1 The longitudinal progression of overall neuropathic pain estimates pooled from EMSCI and the Swedish/Danish study

(A) Evaluated from 1-6 months, and (B) 1-12 months.

The rates of neuropathic pain persistence and development were quite similar between the two cohorts, with statistically significant differences only occurring in the proportions of developing pain from 1-6 months for at-level and overall neuropathic pain (a higher proportion was reported in the Swedish/Danish cohort).

From 1-6 months, the aggregate estimates indicate that at-level pain was more likely to persist than below-level (67% vs. 55%), and was equally likely to develop (14% vs. 14%) (Table 6.2). From 1-12 months post-injury, at-level and below-level neuropathic pain had similar rates of persistence (47% and 52%, respectfully), and development (19% and 23%, respectfully) (Table 6.2). Higher rates of pain persistence and development were found for overall neuropathic pain from 1-6 months (72% and 23%, respectively), and 1- 12 months (69% and 36%, respectively) (Tables 6.2 and 6.3).

6.3.2 Associations with neuropathic pain in the EMSCI

The bivariable logistical regression analysis revealed that the odds of developing at-level neuropathic pain (i.e. first appeared at 6 or 12 months) was statistically significantly

98 higher in older individuals and those with a greater total light touch and pinprick score difference (Figure 6.2, Table 6.4). The odds of developing below-level neuropathic pain symptoms were similarly increased in older individuals, as well with higher motor scores (i.e., less severe injuries), and higher low-level light touch and at-level light touch scores (Table 6.5). Developing overall neuropathic pain was only statistically significantly associated with increased age (Table A.3).

Table 6.5 The results of the logistic regression models for below-level neuropathic pain

Outcome (yes/no) Characteristic Odds Ratio (95% CI) P-value

Developed below- level neuropathic pain N=112 (45 with pain) Age at injury 1.03 (1.01-1.06) 0.005 Sex Female Ref - Male 0.44 (0.12-1.47) 0.18 Level of injury Upper cervical Ref - Lower cervical 0.89 (0.29-2.66) 0.83 Thoracic 0.52 (0.20-1.31) 0.17 Lumbar 0.67 (0.17-2.46) 0.55 At-level neuropathic pain 2.45 (0.86-7.30) 0.10 Musculoskeletal pain 1.41 (0.66-3.03) 0.37 Motor score 1.02 (1.00, 1.04) 0.03 Light touch 1.01 (1.00, 1.02) 0.18 Pinprick 1.00 (0.99, 1.02) 0.60 Light touch – pinprick 1.02 (0.99-1.05) 0.14 Low-level light touch 1.10 (1.01, 1.19) 0.03 Low-level pinprick low 1.07 (0.98, 1.16) 0.12 Low-level light touch-pinprick 1.13 (0.95, 1.35) 0.19 N=111 (44 with pain) At-level light touch 1.25 (1.05, 1.52) 0.02

99 Outcome (yes/no) Characteristic Odds Ratio (95% CI) P-value At-level pinprick 1.09 (0.08, 1.11) 0.27 At-level light touch- pinprick 1.20 (0.97, 1.50) 0.10 Six month persistent below- level neuropathic pain N=36 (19 with pain) Age at injury 0.98 (0.94-1.01) 0.21 Sex Female Ref - Male 2.22 (0.45-12.63) 0.33 Level of injury Upper cervical Ref - Lower cervical 3.75 (0.35-53.50) 0.29 Thoracic 3.93 (0.65-33.21) 0.16 Lumbar 2.50 (0.26-29.56) 0.43 Pain severity 1.11 (0.75-1.70) 0.60 At-level neuropathic pain 3.30 (0.86-14.13) 0.09 Musculoskeletal pain 1.01 (0.27-3.81) 0.99 Motor score 0.98 (0.94-1.01) 0.20 Light touch 0.97 (0.94-1.00) 0.09 Pinprick 0.98 (0.95-1.01) 0.21 Light touch – pinprick 0.98 (0.93-1.03) 0.54 Low-level light touch low 0.90 (0.75, 1.08) 0.27 Low-level pinprick low 0.92 (0.74, 1.13) 0.44 Low-level light touch-pinprick At-level light touch 1.06 (0.82, 1.39) 0.63 At-level pinprick 0.96 (0.76, 1.19) 0.69 At-level light touch- pinprick 1.31 (0.90, 2.09) 0.19

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- Did not develop pain - Developed pain Figure 6.2 The and light touch-pinprick differences for developing neuropathic pain in the EMSCI cohort

(A) For age at injury, there as a statistically significant difference between groups for at- level neuropathic pain (p=0.02) and below-level neuropathic pain (p=0.005). (B) For light

101 touch – pinprick difference, there was a statistically significant difference between groups for at-level neuropathic pain (p=0.03), and not for below-level neuropathic pain (p=0.14).

In contrast to developed pain, the odds of 6-month persistent at-level neuropathic pain (i.e., present at 1 and 6 months) were associated with younger age at injury, and higher at-level light touch and pinprick scores (Table 6.4). Persistent below-level neuropathic pain was not statistically significantly associated with any of the available characteristics (Table 6.5). Persistent overall neuropathic pain demonstrated similar trends to at-level pain, and was significantly associated with younger age and greater at-level pinprick scores (Table A.3).

6.4 Discussion

The present analysis utilized two large, independent, observational studies to generate aggregate probability estimates for the progression of acute neuropathic pain after spinal cord injury given current standards of treatment. Neuropathic pain status changed considerably over the course of the first year. This included individuals with no neuropathic pain at 1 month developing symptoms at later time-points, as well as individuals whose initial neuropathic pain symptoms resolved at follow-up. The estimates could be incorporated as benchmarks to evaluate the safety of interventions being assessed in early phase clinical trials in patients with acute spinal cord injury.

The extent to which safety concerns are likely to occur in acute spinal cord injury clinical trials is currently unknown.153 However, there is already precedence for neuropathic pain to emerge as a consequence of cell-based intervention intended to enhance motor outcomes (e.g. autologous bone marrow mesenchymal stem cells).162,163 Unfortunately, neuropathic pain is arguably among the worst possible outcomes for an acute intervention aimed at regenerating or enhancing plasticity in the central nervous system.158,164 This is particularly true if signs and symptoms of neuropathic pain are unaccompanied by other neurological improvements (e.g. increased muscle strength) – a type of “unmitigated failure”.165 Given that pain measures in animal models do not exactly reflect clinical signs of neuropathic pain in humans, sensitive benchmarks are needed for human clinical trials.166

102 This study highlights the likelihood of developing or resolving neuropathic pain over the course of the first year following spinal cord injury. Trajectories of neuropathic pain have been presented in time periods relevant to acute clinical trials (i.e. beginning at 1 month, with follow-up at 6/12 months). As is evident by our observations (and not entirely surprising), the probability of neuropathic pain developing at a later time point is dependent on earlier pain status. Unlike the report of prevalence, measures of pain progression (e.g. incidence during a trial) should be considered as a safety benchmark for clinical trials.

As an example, consider a single-arm trial where all the individuals are recruited without neuropathic pain, but at a 6-month follow-up, 50% report symptoms. This estimate may NOT be initially concerning when compared to previously published estimates of overall prevalence.167 However, our benchmarks suggest only 23% (95% CI: 14-37) of an initially pain-free population is expected to develop symptoms (Figure 6.1). A less obvious example of an adverse outcome is a treatment that reduces the likelihood of pain relief. Without incorporated benchmarks, such a subtle change could easily go overlooked.

The proportions outlined in Figure 6.1 (and in Appendix) should, however, be interpreted cautiously. Recruitment for acute spinal cord injury clinical trials is difficult, and many early phases will only include a small number of patients.168 As a result, the proportion developing signs and symptoms of neuropathic pain will be highly variable. To avoid erroneously penalizing trials tracking neuropathic pain, sample size needs to be considered. For example, our aggregate estimates indicated that 23% of initially pain- free individuals are expected to present with symptoms at 6 months (Figure 6.1). Compared to this benchmark, 40% developing symptoms of neuropathic pain in a trial of 46 subjects may represent cause for concern (i.e. a statistically significant increase), whereas 30% would not (alpha = 0.05, power = 0.8, binomial tests).169

A similar process could also be applied to determine the effectiveness of an acute intervention to prevent neuropathic pain (i.e. a beneficial outcome). With some success, the concept of prophylactically managing chronic pain has emerged in other health conditions (e.g. post-surgical).170 To our knowledge, carbamazepine, a first-generation anticonvulsant, is the only medication trialed so far to prevent the onset of neuropathic

103 pain symptoms after spinal cord injury.171 While unsuccessful in the long-term (i.e. pain emerged when carbamazepine was withdrawn), newer generation medications such as gabapentin and pregabalin may be more effective in this regard.172 To plan future trials aimed at preventing neuropathic pain after spinal cord injury, an understanding of progression is important.

For prognosis and treatment planning purposes, predictors of neuropathic pain early after injury are of high clinical value. Understanding factors associated with developing neuropathic pain symptoms are also important in terms of gaining novel insights into mechanisms. In our exploratory analysis, age was the most consistent subject-level predictor of neuropathic pain. The logistical regression analyses revealed an association between developed neuropathic pain and older age at injury (both at- and below-level). An age effect has been reported elsewhere167,173 and is not entirely surprising given that older individuals are at increased odds of other forms of neuropathic pain.174 Although younger age at injury was statistically significantly protective against developed pain, the odds of persistent at-level pain were higher. This is difficult to explain in light of the fact that other neuropathic pain conditions with persistent symptoms are associated with older age.174,175 Furthermore, the small effect sizes presented here may fall below clinical significance, and the lack of consistent or further statistically significant predictors may be due to the small sample sizes.

Individuals with preserved motor sparing were at increased odds of developing below- level neuropathic pain. This suggests that sparing at the lesion site acts as an anatomical substrate for neuropathic pain to develop, protecting those with complete injuries (i.e. AIS-A). Based on a previous study,161 sensory scores and their subscores and differences were also considered in the logistic regression models. In line with earlier observations, a larger difference between total light touch and pinprick scores at 1 month, such that pinprick demonstrating greater deficits than light touch, was associated with a higher likelihood of developing at-level neuropathic pain. This association may be attributable to preferential sparing in the dorsal columns relative to the spinothalamic tract, which has been postulated as a mechanism of neuropathic pain.161 The association between greater light touch scores and the development of below-level neuropathic pain follows a similar pattern. Persistent at-level pain, however, was associated with both increased at-level light touch and pinprick scores, indicating more

104 general sparing at the lesion site (which was not the case for persistent below-level pain).

A strength of our analysis is that both datasets are from multicentre studies. This is important because the design of acute clinical trials will almost certainly involve multiple centres.32,176,177 However, the diagnosis of neuropathic pain presents with some risk of misclassification. Overall there was high correspondence between the two independently collected datasets, which illustrates the robustness of neuropathic pain progression after spinal cord injury. Nonetheless, the Swedish/Danish cohort tended towards a higher incidence of neuropathic pain with statistically significantly higher proportions of developed at-level and overall neuropathic pain (likely drive by the at-level proportions) than the EMSCI cohort. This could be attributed to differences in methodology, including, for example, a bedside examination to assess evoked neuropathic pain sensations (e.g., allodynia) in the Swedish/Danish cohort but not EMSCI.114 Another difference exists in the timing of the initial pain measurement: the initial EMSCI assessment was administered within the first 6 weeks following injury, and the initial assessment in the Swedish/Danish occurred between 1-3 months after injury. This may have introduced some variability regarding the earlier reported rates of pain. Furthermore, individuals in the EMSCI cohort were limited to reporting their top three sources of pain, which may have caused some underreporting. Another limitation exists in the elusive nature of reporting neuropathic pain: questions about pain were specified at the given examination, and these timelines may have missed pain that developed and relieved within a period of time. It has also been reported that neuropathic pain at one time point may progress into a non-painful sensations (e.g., dyesthesia) at another time point, and be classified as “no pain” when the symptoms have not actually resolved.178 Other discrepancies may be the result of individual and study related differences, including drop-out. Indeed, the dropout rate in the EMSCI was markedly higher than the Swedish/Danish cohort.

This study comprehensively explored the progression of distinct forms of acute neuropathic pain after spinal cord injury. Furthermore, we have identified factors associated with developing and persisting neuropathic pain. In summary, we have provided an initial framework by which to assess the risks and benefits of future acute therapeutic interventions administered in spinal cord injury.

105 Chapter 7 General discussion

7.1 Key findings The key objectives of this thesis were to examine the effect of acute anticonvulsants on neurological recovery after spinal cord injury, and the longitudinal progression of neuropathic pain after spinal cord injury. These objectives were met in four original research studies using secondary data analyses. The key findings are the following:

1. Anticonvulsants used to treat complications after spinal cord injury are administered early after injury during what may be an important period of neurological recovery (Chapters 3-5) 2. Gabapentinoid anticonvulsants are consistently associated with improved motor recovery in the first year after spinal cord injury (Chapters 3 and 5) 3. The beneficial effect of gabapentinoid anticonvulsants is dependent on timing of initial administration, and appears to benefit from administration very early after injury (Chapters 3 and 5) 4. Non-gabapentinoid anticonvulsants (carbamazepine, phenytoin, clonazepam, phenobarbital and valproic acid) are not associated with an effect on neurological recovery after spinal cord injury, suggesting that this benefit is unique to gabapentinoid anticonvulsants (Chapter 4) 5. Neuropathic pain in the first year after spinal cord injury changes considerably, persisting and remitting, and benchmarks for progression should be examined in future clinical trials as a measure of efficacy, or adverse events (Chapter 6)

7.2 General strengths and limitations Chapters 3-6 each contain a more comprehensive description of their respective strengths and limitations, and these will not be restated in this section. Below is a description of the broader strength and limitations from the overall findings of this thesis.

7.2.1 Strengths Analyses conducted across Chapters 3-6 make use of relatively larger dataset from a registry and previously run clinical trials (within the field of spinal cord injury) and represent a broad range of participants with spinal cord injuries. Furthermore, by using longitudinal data (Chapters 3-6) and longitudinal models (i.e. mixed effects regression in

106 Chapters 3-5), the trajectories of motor recovery and neuropathic pain are modeled after true individual change, and not population averages. This type of analysis avoids the common pitfall of ecological fallacy. Longitudinal analyses also permit the use of all participant data, even if they are missing time points. This limits the bias that could arise from further excluded participants with incomplete data. Finally, observing neurological recovery after spinal cord injury allowed for the use of ISNCSCI scores to classify of motor and sensory in Chapters 3-5. This uniformity in outcome measures is important when contrasting the results of different data sources or comparing the consistency of results in this thesis.

7.2.2 Limitations A consistent limitation throughout this thesis (Chapters 3-5) is that in using observational studies the subjects were not randomized to the treatment (anticonvulsants). For this reason, there is the risk that the results were influenced by unmeasured or unknown confounders. However, there is evidence to suggest that effect estimates between RCTs and observational studies are usually not statistically significantly different, even in pharmacological-specific comparisons.139 As with any examination of observational drug repurposing, indication bias of anticonvulsant administration is also a concern. For example, indication bias could occur if the reason for administration of anticonvulsants (e.g. neuropathic pain) were responsible for the improvement in recovery, and not the drug itself. While the reasons for administration were not available for analysis in Chapters 3 and 5, it is know that gabapentinoids are the primary treatment for neuropathic pain after spinal cord injury and can also be used to treat seizures. The study in Chapter 3 attempted to adjust for the presence, severity, and progression of neuropathic pain as a potential bias, and Chapter 4 examined anticonvulsants that likely presented with many similar indications and did not demonstrate an effect. Finally, within the chapters examining a beneficial effect of anticonvulsants (Chapters 3 and 5), we did not have exact information on dosing, and therefore could not ascertain an ideal dosage or a dose-response relationship. However, knowing the primary indications for gabapentinoids (e.g. neuropathic pain), we can determine that the effects observed within this thesis were obtained using clinically-relevant dosing for these conditions.179

7.3 Implications and future directions The research in this thesis raises several further questions regarding data gathering, clinical practice, and future observational and clinical research. These questions are

107 summarized below, along with considerations and recommendations.

7.3.1 Spinal cord injury as a model for drug repurposing

Drug “repurposing” or “repositioning” are examples of different terms that are being used with varying definitions and distinctions across studies, with little overall consensus.180 However, a broad and widely accepted umbrella definition is “the process of finding new uses outside the scope of the original medical indication for existing drugs”.79 This is indeed the case in this thesis, in which new uses (neurological recovery) are being sought from existing drugs (anticonvulsants) for a different indication (neuropathic pain or seizures after spinal cord injury). In fact, many drug repurposing methodologies focus on the translation of a drug from one disease to other (e.g. galanthamine from polio to Alzheimer’s disease),79,93 whereas the approach taken in this thesis is simplified by focussing on a drug already administered in the spinal cord injured population. Pharmaceutical companies are currently investing more money into the discovery of new drugs, but creating proportionally fewer products, which had increased interest in the untapped potential of repurposing existing drugs on the market.79 Drug repurposing can also offer numerous benefits over de novo drug discovery, including faster development times, reduced development costs, and reduced risks when a drug is already administered in the population of interest (e.g. anticonvulsants after spinal cord injury).79

As touched upon in several chapters of this thesis, spinal cord injury presents as a unique disease model that, in many ways, is ideal for drug repurposing research. Contrasting spinal cord injury with a different neurological disorder, such as multiple sclerosis, can further emphasize these differences. Although both diseases present with numerous and overlapping complications (e.g. pain, depression, spasticity), and therefore overlapping drug candidates, they also present with several key differences. Firstly, in order to define and compare the rates of progression of a disease it is necessary to quantify time since the disease started, and therefore the time of disease onset. Disease onset after traumatic spinal cord injury is caused by an easily identifiable single event, making time since injury simple to quantify. In multiple sclerosis, however, disease onset is much more difficult to ascertain as underlying disease processes might begin weeks, months, or even years before clinical symptoms.181 Because of this, modeling of the disease can rely on the time of first symptom or first diagnosis, which may vary largely between individuals and populations depending on factors such as

108 symptom presentation, accessibility to healthcare, or clinical practice.

Secondly, disability (e.g. neurological impairment) after spinal cord injury usually improves somewhat in the first 6-9 months after injury, after which it plateaus and generally won’t show further improvement (Figure 7.1 panel A). In contrast, multiple sclerosis is a neurological disorder in which disability (e.g. the Expanded Disability Status Scale) continues to progress over time.182 Multiple sclerosis is first indicated by a clinically isolated syndrome, which includes the first clinical presentation of multiple sclerosis without formally meeting the disease criteria. Primary progressive multiple sclerosis is an uncommon form of the disease (approximately 10-15% of the population) and is characterized by a steady decline in neurological function over time. Relapse- remitting multiple sclerosis is the most common form (approximately 85%) in which neurological dysfunction presents, but then the patient appears to recover. This pattern repeats until remission slowly declines and the majority of patients develop secondary progressive multiple sclerosis. Secondary progressive multiple sclerosis is the development of a steady decline after the initial relapsing disease course (Figure 7.1 panel B).183,184 As these descriptions indicate, multiple sclerosis is progressive disease, which can in turn make the overall or isolated effects of a given treatment more difficult to ascertain. Ideally the neurological recovery that occurs after a spinal cord injury could continue indefinitely past the 1-year time point; however, this finite time period of recovery permits the examination of the total recovery that will occur (i.e. the total treatment effect), without the influence of further progression. The continued progression of multiple sclerosis also complicates the examination of several drug candidates because many of the complications requiring treatment (e.g. spasticity, pain) are linked to the progression of the disease itself. For that reason, drugs administered may have an indication that is intrinsically linked to the worsening of the disease, potentially making the drug appear to worsen disease progression. The finite treatment and progression window of spinal cord injury limit this confounding aspect of neurologically progressive diseases.

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(A) spinal cord injury and (B) multiple sclerosis

110

Finally, related to the finite progression of spinal cord injury, there is believed to be a window of opportunity early after injury (i.e. hours/days/weeks) during which neuroprotective and neuroregenerative treatments should be administered. While it would be beneficial if treatments could be administered within a broader time frame after injury, this treatment window provides a finite time period in which we can examine drugs of interest, potentially limiting the options of a multitude of different drugs administered over many years. Together, these factors describe a disease that has a distinct onset, a finite recovery period, and a specific window in which to examine potential therapies, creating a unique model well designed for observational drug repurposing research.

7.3.2 Spinal cord injury data sets

Spinal cord injury may be a suitable model for observational research; however, this type of research is only possible with the appropriate data. In this thesis we have highlighted 3 data sources used for drug repurposing: a prospectively gathered longitudinal database for research purposes, a completed clinical trial, and a patient chart-review. As discussed in Chapter 1, clinical trials in spinal cord injury are met with numerous obstacles limiting their success (e.g. heterogeneity of injuries and recovery, low numbers for recruitment). If data sources like this were widely available, secondary analyses could help maximize the information gained and accelerate new discoveries. EMSCI provides an example of how we can utilize such a resource, with numerous studies having been conducted.5,6,9,111 The sharing of clinical trial data has successfully begun in other neurological diseases (e.g. traumatic brain injury, ALS, Parkinson’s disease), but it has yet to expand into spinal cord injury.185 Such platforms are necessary in order to gather sufficient participant information to inform research in areas such as drug repurposing, drug safety and effectiveness, or creating aggregate estimates to inform disease progression. Indeed, aggregate estimate (as highlighted in Chapter 6) can be used to more accurately predict recovery after injury, and have been used for this purpose.186

While the data sources used in this thesis each provided valuable information, they also presented with their own unique challenges related to data formatting (e.g. long vs. wide formats), data recording (e.g. inconsistencies), and data availability (e.g. missing or incomplete variables). The National Institute of Neurological Disorders and Stroke (NINDS) in the United States aims to increase knowledge that can reduce the burden of

111 neurological disease (https://www.ninds.nih.gov/). As part of this mission, they created data standards for specific neurological disorders, including common data elements for spinal cord injury created in 2014. These elements include the ISNCSCI exam as a core outcome measure (i.e. strongly recommended for all studies to collect), and the availability of these measures across datasets has proven to be a benefit throughout this thesis. The drug elements, however, are not core elements, and are categorized as supplementary or exploratory (i.e. they should be collected in clinical trials, or when they are deemed to be appropriate for a particular study). Although potentially time consuming, difficult, and ethically problematic, the consistent collection of drug data that could be incorporated into open-access datasets may help to expedite research in the field, particularly among disorders as uncommon as spinal cord injury. NINDS highlights the importance of recording concomitant mediations, and describes their use in terms of eligibility, treatment interactions, and adverse events. This thesis also highlights their potential in secondary data analysis of drug safety, effectiveness, or repurposing. Indeed, having consistent recordings of drug data in addition to outcomes measure would strengthen our ability to compare and combine the potential effects across data sets. The exploration of multiple data sets has highlighted the specific data challenges in this area of research, and barriers included the limitations of self-reported drug data, the manner in which drugs are reported (e.g. brand names, generic names, broad drug categories), drug data only pertaining to set time points (e.g. cross-sectional), missing data, a lack of detailed dosages or routes of administration, inconsistent formatting, and errors when recording drug data. Careful pre-trial consideration of how and when concomitant medications are recorded could help to minimize these obstacles and propel advances in future observational research (discussed further below).

7.3.3 Implications for clinical research

In addition to the potential for observational research, clinical trials are warranted to further elucidate the effects of preclinical and observational findings. While this thesis has provided evidence for the benefit of gabapentinoid anticonvulsants, further questions remain. Observational research offers multiple benefits, including maximizing the usage of existing data and minimizing the cost, time, and prospectively collecting data. However, in observational research participants are not randomized to a treatment and placebo group, which frequently raises the issue of unmeasured confounders (such as indication bias), and a lack of comparison to a placebo or active control group. These

112 issues could be addressed using the gold standard of research: a randomized controlled trial. However, these avenues of research have cost an enormous amount of spinal cord injury-dedicated time and money. Like the administration of drugs, conducting a clinical trial requires a careful weighting of the potential risks and benefits. Is there enough evidence to justify the trial? What are the risks of the treatment? Is it ethical to withhold the treatment from the control group? What are the opportunity costs? How great is the potential benefit?

The current argument for the benefits of gabapentinoid anticonvulsants after spinal cord injury is fairly strong, as there exists both preclinical and observational human evidence. Furthermore, the risks of its administration are small because of the observational research supporting its administration, and because it is already being administered to the population of interest for a different indication. Together these factors arguably eliminate the need for any Phase I or Phase II trials in spinal cord injury and would support moving directly to a Phase III clinical trial. As highlighted in Chapter 1, the recruitment of spinal cord injury participants for clinical trials already presents with numerous challenges: the low disease incidence (particularly when recruiting for acute injuries), the high heterogeneity of injuries (or alternatively excluding certain injuries and limiting candidates), and the natural progression of recovery. These factors alone make it difficult to recruit a sufficient number of participants for a trial, and to recruit enough participants to be able to randomize them to a placebo and a treatment group requires a great deal of time and money. Alternatives to the traditional RCT include, for example, an open-label uncontrolled trial. This option would remove the necessary indication for the drug (i.e. participants would receive the treatment regardless of neuropathic pain status), and results could be compared with a historical control. Alternatively (or sequentially), randomization could focus on clarifying the ideal timing and dosing of gabapentinoids after spinal cord injury, which have not been clearly defined from observational research methods alone. Indeed, if there is very little risk from the administration of gabapentinoid anticonvulsants and evidence of a potential gain, is it ethical to randomize patients not to receive it? The current use of gabapentinoids as the primary treatment of neuropathic pain presents with a unique challenge: it is expected that, to some extent, individuals in both groups would develop neuropathic pain (as demonstrated in Chapter 6).111 Given this knowledge, how would we ethically treat those individuals not assigned to receive gabapentinoids (i.e. a control or low-dose group)? It

113 would be inappropriate to deny treatment, but treating them could potentially diminish the expected effect, or require an even greater sample size to overcome it. There are options beyond the gold standard of placebo-controlled randomized trials, and these may indeed be the next logical step in the exploration of gabapentinoid anticonvulsants.

This is not to say that an alternative trial would not present with its own set of challenges. Regardless of what kind of trial is conducted, it would still be time-consuming and costly. Furthermore, the effect of gabapentinoids on total motor recovery reported in this thesis is modest (e.g. 4.36-5.54 points). Included in the rationale for conducting a clinical trial is a desired effect - that is to say, an effect of a treatment that can be defined as meaningfully or clinically significant to the population of interest. Although there is no clear and accepted definition of what improvement in motor scores is required to justify a trial (and it would be expected to vary across injury types), approximately 5-points has been calculated and discussed as a small clinically relevant improvement.14,16 While this is in line with the findings in this thesis, a trial looking to identify a small or modest effect would require a greater number of participants in order to detect the effect. Treatments that restore small improvements are still ranked as highly desirable by individuals with spinal cord injury,187 however the challenges facing their translation into clinical trials are many.

7.3.4 Implications for observational research

While this thesis focuses on a specific class of drug, it also raises potential avenues of research beyond anticonvulsants. In highlighting the potential for observational drug repurposing studies after spinal cord injury, this thesis should propel future studies to examine drugs beyond anticonvulsants, or those used for pain management. As previously described, individuals with spinal cord injury are at increased risk for a wide range of complications after injury (e.g. pain, spasticity, pressure sores, urinary tract infections, depression, cardiovascular disease), and thus often receive a multitude of medications. Many of these medications are also administered early after injury, during the aforementioned “window of opportunity” for protection and repair. There is very little research regarding how the numerous drugs administered after injury may affect neurological recovery, though progress is being made.188 This thesis has provided evidence of drugs that may be beneficial to neurological recovery (gabapentinoid anticonvulsants), as well as those that have no effect (non-gabapentinoid

114 anticonvulsants). Whether these drugs are providing beneficial, detrimental, or null effects is of the utmost importance. For example, if a drug is detrimental, this could provide evidence informing clinicians to limit its administration early after injury, thus optimizing recovery. The prescription of drugs by clinicians is based upon a careful weighting of the known potential risks and benefits as well as the preferences and decisions of the patients. For this reason, any risk to recovery after injury must be known in order to inform clinical guidelines as to how to best manage complications, particularly early after injury. Although there is still no pharmacological treatment specifically approved to improve or maximize neurological recovery, identifying drugs with a beneficial effect on recovery (or those with a detrimental effect) could alter clinical protocols to maximize the recovery process after spinal cord injury. Although work in this area has started to progress (e.g. baclofen),188,189 much remains unknown and should be reconsidered as a priority area of research in spinal cord injury.

Related to the many potential candidates for drug repurposing after spinal cord injury is the issue of polypharmacy. Though studied extensively in geriatric populations, polypharmacy presents with varying definitions in the literature, and can broadly be defined as taking multiple medications.190 Like the geriatric populations, individuals with spinal cord injuries can present with many co-existing health conditions and each may require their own treatments. In addition to the risks introduced by these conditions and their respective treatments, polypharmacy itself is associated with increased risks of adverse drug events, falls, mortality, impaired cognition or functional status, and extended hospitalization or readmission.191 Therefore, while weighing the risks and benefits of each individual drug, clinicians and patients must also weight the risks of administering multiple drugs. As a disease with a relatively small population (estimated Canadian prevalence of 85,556 individuals, 51% of which were traumatic),192 research into polypharmacy in spinal cord injury is still relatively recent.193 As research into the effects of drugs on neurological recovery continues to expand it will need to incorporate polypharmacy into the equation. Unfortunately, large datasets and longitudinal outcomes are needed to explore such complex interactions and relationships, and these are still lacking in spinal cord injury. Furthermore, there may be another degree of complexity in the interaction of gabapentinoid administration and neuropathic pain. In pain research, including neuropathic pain research, there is increasing interest in the use of prophylactically administering medications to prevent the development of pain. Animal

115 models of prophylactic drug use preventing the development of neuropathic pain have shown promising results (including the use of gabapentinoids).149,150,194 Research in humans thus far has not extended to gabapentinoids and spinal cord injury-specific neuropathic pain, though the results for post-operative pain have been mixed.151,152 This area of research lends an additional potential benefit to the administration of gabapentinoids after spinal cord injury (i.e. preventing neuropathic pain), and another potential confounder in analyzing its affect on neurological recovery.

7.4 Conclusion This thesis presents novel findings regarding the administration of anticonvulsants after spinal cord injury, and the beneficial association of gabapentinoid-specific anticonvulsant on motor recovery. It has provided key evidence in the search for a pharmaceutical treatment to improve neurological recovery after spinal cord injury and demonstrated the potential for human observational studies to work in conjunction with preclinical research with the hope of improving the successful translation into human trials. It highlighted the potential of using secondary spinal cord injury data, the benefits of uniform outcome measures, and the improvements that can be made in future gathering and sharing of drug data. This work may have long-term implications for the use of anticonvulsants after spinal cord injury, but also in the exploration of drug repositioning.

116 Bibliography 1. Ramer LM, Ramer MS, Bradbury EJ. Restoring function after spinal cord injury: Towards clinical translation of experimental strategies. Lancet Neurol. 2014;13:1241–1256. 2. Silva NA, Sousa N, Reis RL, Salgado AJ. From basics to clinical: A comprehensive review on spinal cord injury. Prog. Neurobiol. 2014;114: 25–57. 3. Filli L, Schwab ME. Structural and functional reorganization of propriospinal connections promotes functional recovery after spinal cord injury. Neural Regen Res. 2015;10:509–513. 4. Curt A, Van Hedel HJ a, Klaus D, Dietz V. Recovery from a spinal cord injury: significance of compensation, neural plasticity, and repair. J Neurotrauma. 2008;25:677–685. 5. Kramer JLK, Lammertse DP, Schubert M, Curt A, Steeves JD. Relationship between motor recovery and independence after sensorimotor-complete cervical spinal cord injury. Neurorehabil Neural Repair. 2012;26:1064– 1071. 6. Steeves JD, Kramer JK, Fawcett JW, et al. Extent of spontaneous motor recovery after traumatic cervical sensorimotor complete spinal cord injury. Spinal Cord. 2011;49:257–265. 7. Zariffa J, Kramer JLK, Jones LAT, Lammertse DP, Curt A, Steeves JD. Sacral sparing in SCI: Beyond the S4-S5 and anorectal examination. Spine J. 2012;12:389–400. 8. Khorasanizadeh M, Yousefifard M, Eskian M, et al. Neurological recovery following traumatic spinal cord injury: a systematic review and meta- analysis. J Neurosurg Spine. 2019;15:1-17. 9. Zariffa J, Kramer JLK, Fawcett JW, et al. Characterization of neurological recovery following traumatic sensorimotor complete thoracic spinal cord injury. Spinal Cord. 2011;49:463-471. 10. Steeves JD. Bench to bedside: Challenges of clinical translation. Prog. Brain Res. 2015;218:227–239. 11. Jones LAT, Lammertse DP, Charlifue SB, et al. A phase 2 autologous cellular therapy trial in patients with acute, complete spinal cord injury: pragmatics, recruitment, and demographics. Spinal Cord [online serial]. Nature Publishing Group; 2010;48:798–807. 12. Yoon DH, Kim YS, Young W. Therapeutic time window for methylprednisolone in spinal cord injured rat. Yonsei Med J. Epub 1999. 13. Gavira M, Privat A, d’Arbigny P, Kamenka J-M, Haton H, Ohanna F. Neuroprotective Effects of Gacyclidine After Experimental Photochemical Spinal Cord Lesion in Adult Rats: Dose-Window and Time-Window Effects. J Neurotrauma. 200;17:19-30. 14. Fawcett JW, Curt A, Steeves JD, et al. Guidelines for the conduct of clinical trials for spinal cord injury as developed by the ICCP panel: spontaneous recovery after spinal cord injury and statistical power needed for therapeutic clinical trials. Spinal Cord. 2007;45:190–205.

117 15. Wu X, Liu J, Tanadini LG, et al. Challenges for defining minimal clinically important difference (MCID) after spinal cord injury. Spinal Cord [online serial]. 2015;53:84–91. 16. Scivoletto G, Tamburella F, Laurenza L, Molinari M. Distribution-based estimates of clinically significant changes in the International Standards for Neurological Classification of Spinal Cord Injury motor and sensory scores. Eur J Phys Rehabil Med. 2013;49:373-384. 17. Eijkholt M, Kwon BK, Mizgalewicz A, Illes J. Decision-making in stem cell trials for spinal cord injury: the role of networks and peers. Regen Med. 2012;7:513–522. 18. Curt A, Levi AD, Schwab JM. Challenges to Translation and the Hippocratic Oath by Premature Termination of Spinal Cord Stem Cell– Based Trials. JAMA Neurol. 2017;74:635-636. 19. Scott CT, Magnus D. Wrongful Termination: Lessons From the Geron Clinical Trial. Stem Cells Transl Med. 2014;3:1398-1401. 20. Garraway SM, Huie JR. Spinal Plasticity and Behavior: BDNF-Induced Neuromodulation in Uninjured and Injured Spinal Cord. Neural Plast. Epub 2016:9857201. 21. Boadas-Vaello P, Castany S, Homs J, Álvarez-Pérez B, Deulofeu M, Verdú E. Neuroplasticity of ascending and descending pathways after somatosensory system injury: Reviewing knowledge to identify neuropathic pain therapeutic targets. Spinal Cord. 2016;54:330-340. 22. Steward O, Popovich PG, Dietrich WD, Kleitman N. Replication and reproducibility in spinal cord injury research. Exp Neurol. 2012;233:597- 605. 23. Popovich PG, Tovar CA, Lemeshow S, Yin Q, Jakeman LB. Independent evaluation of the anatomical and behavioral effects of Taxol in rat models of spinal cord injury. Exp Neurol. 2014;261:97–108. 24. Reier PJ, Lane MA, Hall ED, Teng YD, Howland DR. Translational spinal cord injury research: Preclinical guidelines and challenges. Handb Clin Neurol. 2012;109:411–433. 25. Fouad K, Hurd C, Magnuson DSK. Functional testing in animal models of spinal cord injury: not as straight forward as one would think. Front Integr Neuros. 2013;7:85. 26. Dietz V, Fouad K. Restoration of sensorimotor functions after spinal cord injury. Brain. 2014;137:654–667. 27. Tator CH. Review of treatment trials in human spinal cord injury: issues, difficulties, and recommendations. Neurosurgery. 2006;59:957-982. 28. Kirshblum SC, Waring W, Biering-Sorensen F, et al. Reference for the 2011 revision of the international standards for neurological classification of spinal cord injury. J Spinal Cord Med. 2011;34:547-554. 29. Bracken MB, Webb SB, Wagner FC. Classification of the severity of acute spinal cord injury: Implications for management. Paraplegia. 1978;15:319– 326. 30. Frankel HL, Hancock DO, Hyslop G, et al. The value of postural reduction in the initial management of closed injuries of the spine with paraplegia and

118 tetraplegia. Paraplegia. 1969;7:179-192. 31. Bracken MB, Collins WF, Freeman DF, et al. Efficacy of methylprednisolone in acute spinal cord injury. JAMA. 1984;251:45–52. 32. Bracken MB, Shepard MJ, Collins WF, et al. A randomized, controlled trial of methylprednisolone or naloxone in the treatment of acute spinal-cord injury. Results of the Second National Acute Spinal Cord Injury Study. N Engl J Med [online serial]. 1990;322:1405–1411. 33. Geisler FH, Dorsey FC, Coleman WP. Recovery of motor function after spinal-cord injury--a randomized, placebo-controlled trial with GM-1 ganglioside. N Engl J Med. 1991;324:1829–1838. 34. Pitts LH, Ross A, Chase GA, Faden AI. Treatment With Thyrotropin- Releasing Hormone (TRH) in Patients With Traumatic Spinal Cord Injuries. J Neurotrauma. 1995;12:235–243. 35. Vaccaro AR, Daugherty RJ, Sheehan TP, et al. Neurologic outcome of early versus late surgery for cervical spinal cord injury. Spine (Phila Pa 1976). 1997;22:2609-2613. 36. Bracken MB, Shepard MJ, Holford TR, et al. Administration of methylprednisolone for 24 or 48 hours or tirilazad mesylate for 48 hours in the treatment of acute spinal cord injury. JAMA. 1997;277:1597–1604. 37. Pointillart V, Petitjean M, Wiart L, et al. Pharmacological therapy of spinal cord injury during the acute phase. Spinal Cord. 2000;38:71-76. 38. Geisler FH, Coleman WP, Grieco G, Poonian D, Group SS. The Sygen multicenter acute spinal cord injury study. Spine (Phila Pa 1976). 2001;26:S87-98. 39. Brodke DS, Anderson PA, Newell DW, Grady MS, Chapman JR. Comparison of anterior and posterior approaches in cervical spinal cord injuries. J Spinal Disord Tech. 2003;16:229-235. 40. Cengiz ŞL, Kalkan E, Bayir A, Ilik K, Basefer A. Timing of thoracolomber spine stabilization in trauma patients; impact on neurological outcome and clinical course. A real prospective (rct) randomized controlled study. Arch Orthop Trauma Surg. 2008;128:959–966. 41. Kwon BK, Curt A, Belanger LM, et al. Intrathecal pressure monitoring and cerebrospinal fluid drainage in acute spinal cord injury: a prospective randomized trial. J Neurosurg Spine. 2009;10:181-193. 42. Lammertse DP, Jones LA, Charlifue SB, et al. Autologous incubated macrophage therapy in acute, complete spinal cord injury: results of the phase 2 randomized controlled multicenter trial. Spinal Cord. 2012;50:661– 671. 43. Casha S, Zygun D, Mcgowan MD, Bains I, Yong VW, Hurlbert RJ. Results of a phase II placebo-controlled randomized trial of minocycline in acute spinal cord injury. Brain. 2012;135:1224–1236.. 44. Alibai E, Zand F, Rahimi A, Rezaianzadeh A. Erythropoietin plus methylprednisolone or methylprednisolone in the treatment of acute spinal cord injury: A preliminary report. Acta Med Iran. 2013;52:275-279. 45. Rahimi-Movaghar V, Niakan A, Haghnegahdar A, Shahlaee A, Saadat S, Barzideh E. Early versus late surgical decompression for traumatic

119 thoracic/thoracolumbar (T1-L1) spinal cord injured patients. Primary results of a randomized controlled trial at one year follow-up. Neurosciences (Riyadh). 2014;19:183–191. 46. Alibai EA, Baghban F, Farrokhi MR, Mohebali N, Ashraf MH. Effects of Human Erythropoietin on Functional Outcome of Patients with Traumatic Cervical Cord Injury; A Pilot Randomized Clinical Trial. Bull Emerg Trauma. 2015;3:79-85. 47. Chhabra HS, Sarda K, Arora M, et al. Autologous bone marrow cell transplantation in acute spinal cord injury - An Indian pilot study. Spinal Cord. 2016;54:57-64. 48. Aminmansour B, Asnaashari A, Rezvani M, et al. Effects of progesterone and vitamin D on outcome of patients with acute traumatic spinal cord injury; a randomized, double-blind, placebo controlled study. J Spinal Cord Med. 2016;39:272-280. 49. Meshkini A, Salehpour F, Aghazadeh J, Mirzaei F, Naseri Alavi S. Riluzole can improve sensory and motor function in patients with acute spinal cord injury. Asian J Neurosurg. 2018;13:656–659. 50. Witiw CD, Fehlings MG. Acute Spinal Cord Injury. J Spinal Disord Tech [online serial]. 2015;28:202–210. 51. Fehlings MG, Perrin RG. The timing of surgical intervention in the treatment of spinal cord injury: A systematic review of recent clinical evidence. Spine (Phila. Pa. 1976). 2006;15:S28-S35. 52. Harrop JS, Sharan AD, Vaccaro AR, Przybylski GJ. The cause of neurologic deterioration after acute cervical spinal cord injury. Spine (Phila Pa 1976). 2001;26:340-346. 53. Bracken MB, Shepard MJ, Hellenbrand KG, et al. Methylprednisolone and neurological function 1 year after spinal cord injury. Results of the National Acute Spinal Cord Injury Study. J Neurosurg. 1985;63:704–713. 54. Bracken MB, Shepard MJ, Collins WF, et al. Methylprednisolone or naloxone treatment after acute spinal cord injury: 1-year follow-up data. Results of the second National Acute Spinal Cord Injury Study. J Neurosurg. 1992;76:23–31. 55. Bydon M, Lin J, Macki M, Gokaslan ZL, Bydon A. The current role of steroids in acute spinal cord injury. World Neurosurg. 2014;82:848–854. 56. Lammertse DP. Clinical trials in spinal cord injury: lessons learned on the path to translation. The 2011 International Spinal Cord Society Sir Ludwig Guttmann Lecture. Spinal Cord. 2013;51:2–9. 57. Bracken MB, Shepard MJ, Holford TR, et al. Methylprednisolone or tirilazad mesylate administration after acute spinal cord injury: 1-year follow up. Results of the third National Acute Spinal Cord Injury randomized controlled trial. J Neurosurg. 1998;89:699-706. 58. Hanigan WC, Anderson RJ. Commentary on NASCIS-2. J Spinal Disord. 1992;5:125-131. 59. Sayer FT, Kronvall E, Nilsson OG. Methylprednisolone treatment in acute spinal cord injury: the myth challenged through a structured analysis of published literature. Spine J. 2006.

120 60. Faden AI, Jacobs TP, Holaday JW. Thyrotropin-Releasing Hormone Improves Neurologic Recovery After Spinal Trauma in Cats. N Engl J Med. 1981;305:1063–1067. 61. Knoller N, Auerbach G, Fulga V, et al. Clinical experience using incubated autologous macrophages as a treatment for complete spinal cord injury: phase I study results. J Neurosurg Spine. 2005;3:173–181. 62. Kramer JLK, Curt A. When is the time right for a phase III clinical study in spinal cord injury (P = 0.05)? Brain. 2012;135:e221–e221. 63. Fehlings MG, Nakashima H, Nagoshi N, Chow DSL, Grossman RG, Kopjar B. Rationale, design and critical end points for the Riluzole in Acute Spinal Cord Injury Study (RISCIS): a randomized, double-blinded, placebo- controlled parallel multi-center trial. Spinal Cord. 2016;54:8-15. 64. Celik M, Gokmen N, Erbayraktar S, et al. Erythropoietin prevents motor neuron apoptosis and neurologic disability in experimental spinal cord ischemic injury. Proc Natl Acad Sci. 2002;99:258-2263. 65. Brines ML, Ghezzi P, Keenan S, et al. Erythropoietin crosses the blood- brain barrier to protect against experimental brain injury. Proc Natl Acad Sci. 2000;97:10526-10531. 66. Ehrenreich H, Hasselblatt M, Dembowski C, et al. Erythropoietin therapy for acute stroke is both safe and beneficial. Mol Med. 2002;8:495-505. 67. Kawakami M, Sekiguchi M, Sato K, Kozaki S, Takahashi M. Erythropoietin Receptor-mediated Inhibition of Exocytotic Glutamate Release Confers Neuroprotection during Chemical Ischemia. J Biol Chem. 2001;276:39469- 39475. 68. Labombarda F, González S, Lima A, et al. Progesterone attenuates astro- and microgliosis and enhances oligodendrocyte differentiation following spinal cord injury. Exp Neurol. 2011;231:135-146. 69. Vandenberk N, Unluer EE, Gokmen N, et al. Neuroprotective effects of progesterone in spinal cord ischemia in rabbits. Am J Emerg Med. 2013;31:581-584. 70. Zhou KL, Chen DH, Jin HM, et al. Effects of calcitriol on experimental spinal cord injury in rats. Spinal Cord. 2016;54:510-516. 71. Aminmansour B, Nikbakht H, Ghorbani A, et al. Comparison of the administration of progesterone versus progesterone and vitamin D in improvement of outcomes in patients with traumatic brain injury: A randomized clinical trial with placebo group. Adv Biomed Res. 2012;1:58. 72. Tang H, Hua F, Wang J, et al. Progesterone and Vitamin D combination therapy modulates inflammatory response after traumatic brain injury. Brain Inj. 2015;29:1165-1174. 73. Vaquero J, Zurita M, Rico MA, et al. Intrathecal administration of autologous mesenchymal stromal cells for spinal cord injury: Safety and efficacy of the 100/3 guideline. Cytotherapy. 2018;20:806-819. 74. Ghobrial GM, Anderson KD, Dididze M, et al. Human Neural Stem Cell Transplantation in Chronic Cervical Spinal Cord Injury: Functional Outcomes at 12 Months in a Phase II Clinical Trial. Clin Neurosurg. 2017;64:87-91.

121 75. Oh SK, Choi KH, Yoo JY, Kim DY, Kim SJ, Jeon SR. A Phase III Clinical Trial Showing Limited Efficacy of Autologous Mesenchymal Stem Cell Therapy for Spinal Cord Injury. Neurosurgery. 2016;78:436-447. 76. Shin JC, Kim KN, Yoo J, et al. Clinical Trial of Human Fetal Brain-Derived Neural Stem/Progenitor Cell Transplantation in Patients with Traumatic Cervical Spinal Cord Injury. Neural Plast. Epub 2015. 77. Karamouzian S, Nematollahi-Mahani SN, Nakhaee N, Eskandary H. Clinical safety and primary efficacy of bone marrow mesenchymal cell transplantation in subacute spinal cord injured patients. Clin Neurol Neurosurg. 2012;114:935-939. 78. Hug A, Weidner N. From Bench to Beside to Cure Spinal Cord Injury: Lost in Translation? Int Rev Neurobiol. 2012;106:153–172. 79. Ashburn TT, Thor KB. Drug repositioning: identifying and developing new uses for existing drugs. Nat Rev Drug Discov. 2004;3:673–683. 80. Corbett A, Pickett J, Burns A, et al. Drug repositioning for Alzheimer’s disease. Nat. Rev. Drug Discov. 2012;7:184-188. 81. Hwang M, Zebracki K, Vogel LC. Medication profile and polypharmacy in adults with pediatric-onset spinal cord injury. Spinal Cord. 2015;53:673– 678. 82. Siddall PJ, McClelland JM, Rutkowski SB, Cousins MJ. A longitudinal study of the prevalence and characteristics of pain in the first 5 years following spinal cord injury. Pain. 2003;103:249–257. 83. Widerström-Noga E. Neuropathic Pain and Spinal Cord Injury: Phenotypes and Pharmacological Management. Drugs. 2017;77:967-984. 84. Siddall PJ, Taylor DA, Cousins MJ. Classification of pain following spinal cord injury. Spinal Cord. 1997;35:69–75. 85. Bryce TN, Biering-Sørensen F, Finnerup NB, et al. International Spinal Cord Injury Pain Classification: Part I. Background and description. Spinal Cord. 2012; 50:413-417. 86. Ferguson AR, Russell Huie J, Crown ED, et al. Maladaptive spinal plasticity opposes spinal learning and recovery in spinal cord injury. Front Physiol. 2012;3:399. 87. Hook MA, Moreno G, Woller S, et al. Intrathecal Morphine Attenuates Recovery of Function after a Spinal Cord Injury. J Neurotrauma. 2009;26:741–752. 88. Woller SA, Hook MA. Opioid administration following spinal cord injury: Implications for pain and locomotor recovery. Exp. Neurol. 2013. p. 328– 341. 89. Sharp KG, Yee KM, Stiles TL, Aguilar RM, Steward O. A re-assessment of the effects of treatment with a non-steroidal anti-inflammatory (ibuprofen) on promoting axon regeneration via RhoA inhibition after spinal cord injury. Exp Neurol. 2013;248:321–337. 90. Xing B, Li H, Wang H, et al. RhoA-inhibiting NSAIDs promote axonal myelination after spinal cord injury. Exp Neurol. 2011;231:247–260. 91. Marino F, Cosentino M. Multiple sclerosis: Repurposing dopaminergic drugs for MS — the evidence mounts. Nat Publ Gr. Nature Publishing

122 Group; 2016;12:191–192. 92. Vesterinen HM, Connick P, Irvine CMJ, et al. Drug repurposing: A systematic approach to evaluate candidate oral neuroprotective interventions for secondary progressive multiple sclerosis. PLoS One. 2015;10:1–18. 93. Heinrich M, Teoh HL. Galanthamine from snowdrop - The development of a modern drug against Alzheimer’s disease from local Caucasian knowledge. J. Ethnopharmacol. 2004;92:147-162. 94. Finnerup NB, Gottrup H, Jensen TS. Anticonvulsants in central pain. Expert Opin Pharmacother. 2002;3:1411-1420. 95. Dickenson AH, Matthews EA, Suzuki R. Neurobiology of neuropathic pain: Mode of action of anticonvulsants. Eur J Pain. 2002;6:A51-A60. 96. Guy S, Mehta S, Leff L, Teasell R, Loh E. Anticonvulsant medication use for the management of pain following spinal cord injury: systematic review and effectiveness analysis. Spinal Cord. 2014;52:89-96. 97. Corrigan R, Derry S, Wiffen PJ, Moore RA. Clonazepam for neuropathic pain and fibromyalgia in adults. Cochrane Database Syst. Rev. 2012;16:CD009486. 98. Lee JY, Kim HS, Choi HY, Oh TH, Ju BG, Yune TY. Valproic acid attenuates blood-spinal cord barrier disruption by inhibiting matrix metalloprotease-9 activity and improves functional recovery after spinal cord injury. J Neurochem. 2012;121:818-829. 99. Lv L, Sun Y, Han X, Xu CC, Tang YP, Dong Q. Valproic acid improves outcome after rodent spinal cord injury: Potential roles of histone deacetylase inhibition. Brain Res. 2011;1396:60-68. 100. Ha K-Y, Kim Y-H, Rhyu K-W, Kwon S-E. Pregabalin as a neuroprotector after spinal cord injury in rats. Eur Spine J. 2008;17:864–872. 101. Tedeschi A, Dupraz S, Laskowski CJ, et al. The Calcium Channel Subunit Alpha2delta2 Suppresses Axon Regeneration in the Adult CNS. Neuron. 2016;92:416–434. 102. Nagoshi N, Nakashima H, Fehlings MG. Riluzole as a neuroprotective drug for spinal cord injury: From bench to bedside. Molecules. 2015;20:7775– 7789. 103. Fehlings MG, Wilson JR, Frankowski RF, et al. Riluzole for the treatment of acute traumatic spinal cord injury: rationale for and design of the NACTN Phase I clinical trial. J Neurosurg Spine. 2012;17:151–156. 104. Grossman RG, Fehlings MG, Frankowski RF, et al. A prospective, multicenter, Phase i matched-comparison group trial of safety, pharmacokinetics, and preliminary efficacy of riluzole in patients with traumatic spinal cord injury. J Neurotrauma. 2014;31:239–255. 105. Cragg JJ, Haefeli J, Jutzeler CR, et al. Effects of Pain and Pain Management on Motor Recovery of Spinal Cord-Injured Patients: A Longitudinal Study. Neurorehabil Neural Repair. 2016;30:753–761. 106. Emmez H, Börcek AÖ, Kaymaz M, et al. Neuroprotective effects of gabapentin in experimental spinal cord injury. World Neurosurg. 2010;73:729–734.

123 107. Kale A, Börcek AÖ, Emmez H, et al. Neuroprotective effects of gabapentin on spinal cord ischemia-reperfusion injury in rabbits. J Neurosurg Spine. 2011;15:228–237. 108. Kirshblum SC, Burns SP, Biering-Sorensen F, et al. International standards for neurological classification of spinal cord injury (Revised 2011). J Spinal Cord Med. 2011;34:535–546. 109. Warner FM, Cragg JJ, Jutzeler CR, et al. Early Administration of Gabapentinoids Improves Motor Recovery after Human Spinal Cord Injury. Cell Rep. 2017;18:1614–1618. 110. Hassanpour K, Hotz-Boendermaker S, Dokladal P, Curt A. Low depressive symptoms in acute spinal cord injury compared to other neurological disorders. J Neurol. 2012;259:1142–1150. 111. Warner FM, Cragg JJ, Jutzeler CR, et al. Progression of Neuropathic Pain after Acute Spinal Cord Injury: A Meta-Analysis and Framework for Clinical Trials. J Neurotrauma. 2019;36:1461-1468. 112. Geisler FH, Coleman WP, Grieco G, Poonian D. Recruitment and early treatment in a multicenter study of acute spinal cord injury. Spine (Phila Pa 1976). 2001;26:S58–S67. 113. Geisler FH, Coleman WP, Grieco G, Poonian D. Measurements and recovery patterns in a multicenter study of acute spinal cord injury. Spine (Phila Pa 1976). 2001;26:S68-S86. 114. Finnerup NB, Norrbrink C, Trok K, et al. Phenotypes and predictors of pain following traumatic spinal cord injury: a prospective study. J Pain. 2014;15:40–48. 115. Singer JD, Willett JB. Applied Longitudinal Data Analysis. 1st ed. Oxford University Press; 2003. 116. Boisgontier MP, Cheval B. The anova to mixed model transition. Neurosci. Biobehav. Rev. 2016;68:1004-1005. 117. R Core Team. R: A Language and Environment for Statistical Computing [online]. Vienna, Austria: R Foundation for Statistical Computing; 2015. Accessed at: https://www.r-project.org/. 118. Elkabes S, Nicot AB. Sex steroids and neuroprotection in spinal cord injury: a review of preclinical investigations. Exp. Neurol. 2014;259:28–37. 119. Wu Y, Satkunendrarajah K, Teng Y, Chow DS-L, Buttigieg J, Fehlings MG. Delayed post-injury administration of riluzole is neuroprotective in a preclinical rodent model of cervical spinal cord injury. J Neurotrauma. 2013;30:441–452. 120. Gee NS, Brown JP, Dissanayake VUK, Offord J, Thurlow R, Woodruff GN. The novel anticonvulsant drug, gabapentin (neurontin), binds to the alpha2delta subunit of a calcium channel. J Biol Chem. 1996;271:5768–5776. 121. Gong HC, Hang J, Kohler W, Li L, Su TZ. Tissue-specific expression and gabapentin-binding properties of calcium channel alpha2delta subunit subtypes. J. Membr. Biol. 2001;184:35–43. 122. Hirsch EC, Hunot S. Neuroinflammation in Parkinson’s disease: a target for neuroprotection? Lancet Neurol. 2009;8:382–397.

124 123. Wang Q, Liu Y, Zhou J. Neuroinflammation in Parkinson’s disease and its potential as therapeutic target. Transl Neurodegener. 2015;4:19. 124. Melzer N, Meuth SG, Torres-Salazar D, et al. A beta-lactam antibiotic dampens excitotoxic inflammatory CNS damage in a mouse model of multiple sclerosis. PLoS One. 2008;3:e3149. 125. Schwartz G, Fehlings MG. Evaluation of the neuroprotective effects of sodium channel blockers after spinal cord injury: improved behavioral and neuroanatomical recovery with riluzole. J Neurosurg. 2001;94:245–256. 126. Hook MA, Washburn SN, Moreno G, et al. An IL-1 receptor antagonist blocks a morphine-induced attenuation of locomotor recovery after spinal cord injury. Brain Behav Immun. 2011;25:349–359. 127. Woller SA, Moreno GL, Hart N, Wellman PJ, Grau JW, Hook MA. Analgesia or addiction?: implications for morphine use after spinal cord injury. J Neurotrauma. 2012;29:1650–1662. 128. Guy SD, Mehta S, Casalino A, et al. The CanPain SCI Clinical Practice Guidelines for Rehabilitation Management of Neuropathic Pain after Spinal Cord: Recommendations for treatment. Spinal Cord. 2016;54:S14-23. 129. Kitzman P, Cecil D, Kolpek JH. The risks of polypharmacy following spinal cord injury. J Spinal Cord Med. 2014;0:1–7. 130. Sitges M, Chiu LM, Reed RC. Effects of Levetiracetam, Carbamazepine, Phenytoin, Valproate, Lamotrigine, Oxcarbazepine, Topiramate, Vinpocetine and Sertraline on Presynaptic Hippocampal Na+ and Ca2+ Channels Permeability. Neurochem Res. 2016;41:758-769. 131. Kirshblum S, Waring W. Updates for the international standards for neurological classification of Spinal Cord Injury. Phys. Med. Rehabil. Clin. N. Am. 2014;25:505–517. 132. Wishart DS, Feunang YD, Guo AC, et al. DrugBank 5.0: A major update to the DrugBank database for 2018. Nucleic Acids Res. 2018;46:D1074- D1082. 133. Van Middendorp JJ, Hosman AJF, Pouw MH, Van De Meent H. Is determination between complete and incomplete traumatic spinal cord injury clinically relevant Validation of the ASIA sacral sparing criteria in a prospective cohort of 432 patients. Spinal Cord. 2009;47:809-816. 134. Garrido MM, Kelley AS, Paris J, et al. Methods for constructing and assessing propensity scores. Health Serv Res. 2014;49:1701-1720. 135. Dvorak MF, Noonan VK, Fallah N, et al. Minimizing Errors in Acute Traumatic Spinal Cord Injury Trials by Acknowledging the Heterogeneity of Spinal Cord Anatomy and Injury Severity: An Observational Canadian Cohort Analysis. J Neurotrauma. 2014;31:1540-1547. 136. Hains BC, Saab CY, Lo AC, Waxman SG. Sodium channel blockade with phenytoin protects spinal cord axons, enhances axonal conduction, and improves functional motor recovery after contusion SCI. Exp Neurol. 2004;188:365–377. 137. Ates O, Cayli SR, Gurses I, et al. Comparative neuroprotective effect of sodium channel blockers after experimental spinal cord injury. J Clin Neurosci. 2007;14:658–665.

125 138. Schwartz G, Fehlings MG. Evaluation of the neuroprotective effects of sodium channel blockers after spinal cord injury: improved behavioral and neuroanatomical recovery with riluzole. J Neurosurg Spine. 2001;94:245– 256. 139. Anglemyer A, Horvath HT, Bero L. Healthcare outcomes assessed with observational study designs compared with those assessed in randomized trials. Cochrane Database Syst. Rev. 2014;4:MR000034. 140. Kabu S, Gao Y, Kwon BK, Labhasetwar V. Drug delivery, cell-based therapies, and tissue engineering approaches for spinal cord injury. J Control Release. 2015;219:141–154. 141. Warner FM, Jutzeler CR, Cragg JJ, et al. The effect of non-gabapentinoid anticonvulsants on sensorimotor recovery after human spinal cord injury. CNS Drugs. 2019;33:503-511. 142. Wai A, Salib M, Aran S, Edwards J, Patanwala AE. Accuracy of patient self-administered medication history forms in the emergency department. Am J Emerg Med. Epub 2019. 143. Itzkovich M, Gelernter I, Biering-Sorensen F, et al. The Spinal Cord Independence Measure (SCIM) version III: Reliability and validity in a multi- center international study. Disabil Rehabil. 2007;29:1926-1933. 144. Team RDC, R Development Core Team R. R: A Language and Environment for Statistical Computing. R Found Stat Comput. Epub 2016. 145. Howick J, Chalmers I, Glasziou P, Greenhalgh P, Heneghan C, Liberati A. The Oxford 2011 Levels of Evidence. Oxford Cent. Evidence-Based Med. 2011. 146. Marino RJ, Jones L, Kirshblum S, Tal J, Dasgupta A. Reliability and repeatability of the motor and sensory examination of the international standards for neurological classification of spinal cord injury. J Spinal Cord Med. 2008;31:166-170. 147. Jonsson M, Tollbäck A, Gonzales H, Borg J. Inter-rater reliability of the 1992 international standards for neurological and functional classification of incomplete spinal cord injury. Spinal Cord. 2000;38:675-679. 148. Fawcett JW, Curt A, Steeves JD, et al. Guidelines for the conduct of clinical trials for spinal cord injury as developed by the ICCP panel: Spontaneous recovery after spinal cord injury and statistical power needed for therapeutic clinical trials. Spinal Cord. 2007;45:190-205. 149. Arcos M, Palanca JM, Montes F, Barrios C. Antioxidants and gabapentin prevent heat hypersensitivity in a neuropathic pain model. J Investig Surg. 2013;26:109-117. 150. Wang RR, Lou GD, Yu J, et al. Oral Administration of Pregabalin in Rats before or after Nerve Injury Partially Prevents Spontaneous Neuropathic Pain and Long Outlasts the Treatment Period. Pharmacology. 2016;97:251-258. 151. Homma T, Doki Y, Yamamoto Y, et al. Efficacy of 50 mg pregabalin for prevention of postoperative neuropathic pain after video-assisted thoracoscopic surgery and thoracotomy: A 3-month prospective randomized controlled trial. J Thorac Dis. 2019;11:694-701.

126 152. Martinez V, Pichard X, Fletcher D. Perioperative pregabalin administration does not prevent chronic postoperative pain: Systematic review with a meta-analysis of randomized trials. Pain 2017. 153. Cote DJ, Bredenoord AL, Smith TR, et al. Ethical clinical translation of stem cell interventions for neurologic disease. Neurology. 2017;88:322–328. 154. Anderson KD, Guest J, Dietrich WD, et al. Safety of Autologous Human Schwann Cell Transplantation in Subacute Thoracic Spinal Cord Injury. J. Neurotrauma 2017. 155. Gomes-Osman J, Cortes M, Guest J, Pascual-Leone A. A Systematic Review of Experimental Strategies Aimed at Improving Motor Function after Acute and Chronic Spinal Cord Injury. J Neurotrauma. 2016;33:425– 438. 156. Assinck P, Duncan GJ, Hilton BJ, Plemel JR, Tetzlaff W. Cell transplantation therapy for spinal cord injury. Nat Neurosci. 2017;20:637– 647. 157. Fan C, Zheng Y, Cheng X, et al. Transplantation of D15A-expressing glial- restricted-precursor-derived astrocytes improves anatomical and locomotor recovery after spinal cord injury. Int J Biol Sci. 2012;9:78–93. 158. Hofstetter CP, Holmström N a V, Lilja J a, et al. Allodynia limits the usefulness of intraspinal neural stem cell grafts; directed differentiation improves outcome. Nat Neurosci. 2005;8:346–353. 159. Levi AD, Okonkwo DO, Park P, et al. Emerging Safety of Intramedullary Transplantation of Human Neural Stem Cells in Chronic Cervical and Thoracic Spinal Cord Injury. Neurosurgery. 2018;82:562-575. 160. Siddall PJ, Taylor D a, McClelland JM, Rutkowski SB, Cousins MJ. Pain report and the relationship of pain to physical factors in the first 6 months following spinal cord injury. Pain. 1999;81:187–197. 161. Levitan Y, Zeilig G, Bondi M, Ringler E, Defrin R. Predicting the Risk for Central Pain Using the Sensory Components of the International Standards for Neurological Classification of Spinal Cord Injury. J Neurotrauma. 2015;32:1684–1692. 162. Kishk NA, Gabr H, Hamdy S, et al. Case Control Series of Intrathecal Autologous Bone Marrow Mesenchymal Stem Cell Therapy for Chronic Spinal Cord Injury. Neurorehabil Neural Repair. 2010;24:702–708. 163. Oraee-Yazdani S, Hafizi M, Atashi A, et al. Co-transplantation of autologous bone marrow mesenchymal stem cells and Schwann cells through cerebral spinal fluid for the treatment of patients with chronic spinal cord injury: Safety and possible outcome. Spinal Cord. 2016;54:102–109. 164. Macias MY, Syring MB, Pizzi MA, Crowe MJ, Alexanian AR, Kurpad SN. Pain with no gain: Allodynia following neural stem cell transplantation in spinal cord injury. Exp Neurol. 2006;201:335–348. 165. Schulzer M, Mancini GBJ. “Unqualified success” and “unmitigated failure”: Number-needed-to-treat-related concepts for assessing treatment efficacy in the presence of treatment-induced adverse events. Int J Epidemiol. 1996;25:704–712. 166. Kramer JLK, Minhas NK, Jutzeler CR, Erskine ELKS, Liu LJW, Ramer MS.

127 Neuropathic pain following traumatic spinal cord injury: Models, measurement, and mechanisms. J Neurosci Res. 2016;95:1295-1306. 167. Burke D, Fullen BM, Stokes D, Lennon O. Neuropathic pain prevalence following spinal cord injury: A systematic review and meta-analysis. Eur J Pain. 2016;21:29-44. 168. Thibault-Halman G, Rivers CS, Bailey CS, et al. Predicting Recruitment Feasibility for Acute Spinal Cord Injury Clinical Trials in Canada Using National Registry Data. J Neurotrauma. 2017;34:599–606. 169. Faul F, Erdfelder E, Lang A-G, Buchner A. G*Power 3: A flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav Res Methods. 2007;39:175–191. 170. De Oliveira GS, Almeida MD, Benzon HT, McCarthy RJ. Perioperative single dose systemic dexamethasone for postoperative pain: A meta- analysis of randomized controlled trials. Anesthesiology. 2011;115:575– 588. 171. Salinas FA, Lugo LH, García HI. Efficacy of Early Treatment with Carbamazepine in Prevention of Neuropathic Pain in Patients with Spinal Cord Injury. Am J Phys Med Rehabil. 2012;91:1020–1027. 172. Cardenas D., Nieshoff EC, Suda K, et al. A randomized trial of pregabalin in patients with neuropathic pain due to spinal cord injury. Neurology. 2013;80:533–539. 173. Margot-Duclot A, Tournebise H, Ventura M, Fattal C. What are the risk factors of occurence and chronicity of neuropathic pain in spinal cord injury patients? Ann Phys Rehabil Med. 2009;52:111–123. 174. Boogaard S, Heymans MW, de Vet HCW, et al. Predictors of Persistent Neuropathic Pain--A Systematic Review. Pain Physician. 2015;18:433– 457. 175. Jung BF, Johnson RW, Griffin DRJ, Dworkin RH. Risk factors for postherpetic neuralgia in patients with herpes zoster. Neurology. 2004;62:1545–1551. 176. Inada T, Takahashi H, Yamazaki M, et al. Multicenter prospective nonrandomized controlled clinical trial to prove neurotherapeutic effects of granulocyte colony-stimulating factor for acute spinal cord injury: Analyses of follow-up cases after at least 1 Year. Spine (Phila Pa 1976). 2014;39:213–219. 177. Fehlings MG, Theodore N, Harrop J, et al. A phase I/IIa clinical trial of a recombinant Rho protein antagonist in acute spinal cord injury. J Neurotrauma. 2011;28:787–796. 178. Finnerup NB, Jensen MP, Norrbrink C, et al. A prospective study of pain and psychological functioning following traumatic spinal cord injury. Spinal Cord. 2016;54:816–821. 179. Tzellos TG, Papazisis G, Amaniti E, Kouvelas D. Efficacy of pregabalin and gabapentin for neuropathic pain in spinal-cord injury: An evidence-based evaluation of the literature. Eur. J. Clin. Pharmacol. 2008;64:851-858. 180. Langedijk J, Mantel-Teeuwisse AK, Slijkerman DS, Schutjens MHDB. Drug repositioning and repurposing: terminology and definitions in literature.

128 Drug Discov. 2015;20:1027-1034. 181. Wijnands JMA, Kingwell E, Zhu F, et al. Health-care use before a first demyelinating event suggestive of a multiple sclerosis prodrome: a matched cohort study. Lancet Neurol. 2017;16:445-451. 182. Kurtzke JF. Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS). Neurology. 1983;33:1444-1452. 183. Lublin FD, Reingold SC, Cohen JA, et al. Defining the clinical course of multiple sclerosis: The 2013 revisions. Neurology. 2014;83:278-286. 184. Howard J, Trevick S, Younger DS. Epidemiology of Multiple Sclerosis. Neurol. Clin. 2016;34:919-939. 185. Kramer JLK, Geisler F, Ramer L, Plunet W, Cragg JJ. Open Access Platforms in Spinal Cord Injury: Existing Clinical Trial Data to Predict and Improve Outcomes. Neurorehabil Neural Repair. 2017;31:399-401. 186. Aimetti AA, Kirshblum S, Curt A, Mobley J, Grossman RG, Guest JD. Natural history of neurological improvement following complete (AIS A) thoracic spinal cord injury across three registries to guide acute clinical trial design and interpretation. Spinal Cord. 2019;57:753-762. 187. North American Spinal Cord Injury Consortium (NASCIC). NASCIC Report of SCI 2020 Panel and Consumer Survey. 2019. p. 1–11. 188. Cragg JJ, Tong B, Jutzeler CR, et al. A Longitudinal Study of the Neurologic Safety of Acute Baclofen Use After Spinal Cord Injury. Neurotherapeutics. 2019;16:858-867. 189. Kohout RK, Saunders LL, Krause JS. The relationship between prescription medication use and ability to ambulate distances after spinal cord injury. Arch Phys Med Rehabil. 2011;92:1246-1249. 190. Masnoon N, Shakib S, Kalisch-Ellett L, Caughey GE. What is polypharmacy? A systematic review of definitions. BMC Geriatr. 2017. 191. Fried TR, O’Leary J, Towle V, Goldstein MK, Trentalange M, Martin DK. Health outcomes associated with polypharmacy in community-dwelling older adults: A systematic review. J. Am. Geriatr. Soc. 2014;62:2261-2272. 192. Noonan VK, Fingas M, Farry A, et al. Incidence and prevalence of spinal cord injury in Canada: A national perspective. Neuroepidemiology 2012;38:219–226. 193. Hand BN, Krause JS, Simpson KN. Polypharmacy and adverse drug events among propensity score matched privately insured persons with and without spinal cord injury. Spinal Cord. 2018;56:591-597. 194. Rashid H, Ueda H. Pre-injury administration of morphine prevents development of neuropathic hyperalgesia through activation of descending monoaminergic mechanisms in the spinal cord in mice. Mol Pain. 2005;1:19.

129 Appendix

Table A.1 Overall, at-level, and below-level neuropathic pain estimates from 1 to 6 months after spinal cord injury in the EMSCI and Swedish/Danish cohort

Estimates from 1 to 6 months EMSCI cohort Swedish/Danish cohort Neuropathic At 1 month At 6 months At 1 month At 6 months P-value pain (n) (%) (n) (%) Overall Yes (67) Yes (82) 0.15 Yes (61) Yes (28) No (33) No (18) Yes (17) Yes (35) 0.01* No (190) No (49) No (83) No (65) At-level Yes (63) Yes (74) 0.41 Yes (41) Yes (19) No (37) No (26) Yes (10) Yes (22) 0.02* No (210) No (58) No (90) No (78) Below-level Yes (53) Yes (63) 0.61 Yes (36) Yes (8) No (47) No (38) Yes (13) Yes (17) 0.41 No (215) No (69) No (87) No (83)

Table A.2 Overall, at-level, and below-level neuropathic pain estimates from 1 to 12 months after spinal cord injury in the EMSCI and Swedish/Danish cohort

Estimates from 1 to 12 months EMSCI cohort Swedish/Danish cohort At 12 At 12 Neuropathic At 1 month At 1 month months months P-value pain (n) (n) (%) (%) Overall Yes (59) Yes (79) 0.09 Yes (37) Yes (29) No (41) No (21)

130 Yes (27) Yes (48) 0.01* No (107) No (58) No (73) No (52) At-level Yes (42) Yes (53) 0.47 Yes (26) Yes (19) No (58) No (47) Yes (15) Yes (25) 0.09 No (118) No (68) No (85) No (75) Below-level Yes (45) Yes (67) 0.28 Yes (20) Yes (9) No (55) No (33) Yes (19) Yes (28) 0.14 No (124) No (78) No (81) No (72)

Table A.3 The results of the logistic regression models for overall neuropathic pain

Outcome (yes/no) Characteristic Odds Ratio (95% CI) P-value

Developed overall neuropathic pain N=102 (50 with pain) Age at injury 1.03 (1.01-1.06) 0.01* Sex Female Ref - Male 0.44 (0.11-1.49) 0.20 Level of injury Upper cervical Ref Lower cervical 0.55 (0.15-1.90) 0.35 Thoracic 0.69 (0.26-1.83) 0.46 Lumbar 0.92 (0.24-3.61) 0.90 Musculoskeletal pain 1.61 (0.74-3.56) 0.23 Motor score 1.00 (0.99-1.02) 0.81 Light touch 1.00 (0.99-1.02) 0.62 Pinprick 1.00 (0.99-1.01) 0.91 Light touch – pinprick 1.03 (0.99-1.07) 0.16

131 Outcome (yes/no) Characteristic Odds Ratio (95% CI) P-value Low-level light touch low 1.03 (0.94, 1.12) 0.52 Low-level pinprick low 1.01 (0.93, 1.11) 0.75 Low-level light touch- 1.10 (0.88, 1.40) 0.39 pinprick N=101 (49 with pain) At-level light touch 1.19 (1.01, 1.43) 0.05 At-level pinprick 1.07 (0.92, 1.26) 0.35 At-level light touch- 1.18 (0.94, 1.51) 0.17 pinprick Six month persistent overall neuropathic pain N=61 (41 with pain) Age at injury 0.97 (0.94-1.00) 0.04* Sex Female Ref - Male 2.62 (0.76-9.14) 0.13 Level of injury Upper cervical Ref - Lower cervical 7.50 (1.00-157.53) 0.09 Thoracic 1.50 (0.39-5.72) 0.55 Lumbar 0.94 (0.17-5.28) 0.94 Pain severity 1.09 (0.82-1.50) 0.55 Musculoskeletal pain 0.52 (0.17-1.54) 0.24 Motor score 1.00 (0.98-1.03) 0.89 Light touch 1.00 (0.98-1.02) 0.94 Pinprick 1.01 (0.99-1.03) 0.50 Light touch – pinprick 0.97 (0.94-1.01) 0.22 Low-level light touch low 1.08 (0.95, 1.24) 0.22 Low-level pinprick low 1.15 (0.99, 1.38) 0.08 Low-level light touch- 0.94 (0.78, 1.14) 0.54 pinprick

132 Outcome (yes/no) Characteristic Odds Ratio (95% CI) P-value At-level light touch 1.12 (0.91, 1.38) 0.27 At-level pinprick 1.22 (1.03, 1.48) 0.03* At-level light touch- 0.78 (0.59, 1.00) 0.06 pinprick

Table A.4 Pain medication used in EMSCI cohort for the total number of individuals available at each time point

EMSCI cohort Neuropathic pain (%) No neuropathic pain (%) 1 month Total 121 (100) 418 (100) NSAIDs 97 (80.2) 197 (47.1) Antidepressants 32 (26.7) 55 (13.2) Anticonvulsants 29 (24.0) 43 (10.3) Opioids 41 (33.9) 68 (16.3) All drugs 106 (87.6) 237 (56.7) 6 months Total 73 (100) 178 (100) NSAIDs 27 (37.0) 47 (36.7) Antidepressants 16 (21.9) 38 (21.3) Anticonvulsants 33 (45.2) 26 (14.6) Opioids 15 (20.5) 28 (15.7) All drugs 44 (60.3) 68 (38.2) 12 months Total 51 (100) 93 (100) NSAIDs 20 (39.2) 21 (22.6) Antidepressants 10 (19.6) 11 (11.8) Anticonvulsants 18 (35.3) 9 (9.7) Opioids 8 (15.7) 4 (4.3) All drugs 27 (52.9) 27 (29.0)

133 Table A.5 Pain medication in the Swedish/Danish cohort for the total number of individuals available at each time point

Swedish/Danish cohort Neuropathic pain (%) No neuropathic pain (%) 1 month Total 31 (100) 58 (100) NSAIDs 4 (12.9) 16 (27.6) Antidepressants 3 (9.7) 4 (6.9) Anticonvulsants 10 (32.3) 3 (5.2) Opioids 19 (61.3) 32 (55.2) All drugs 21 (67.7) 36 (62.1) 6 months Total 40 (100) 37 (100) NSAIDs 31 (77.5) 34 (91.9) Antidepressants 14 (35.0) 5 (13.5) Anticonvulsants 13 (32.5) 4 (10.8) Opioids 10 (25.0) 3 (8.1) All drugs 23 (57.5) 9 (24.3) 12 months Total 51 (100) 36 (100) NSAIDs 41 (80.4) 33 (91.7) Antidepressants 13 (25.5) 1 (2.8) Anticonvulsants 17 (33.3) 4 (11.1) Opioids 10 (19.6) 2 (5.6) All drugs 23 (45.1) 5 (13.9)

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