5-6 September 2014

The 16th

Spinal Research Network Meeting ABSTRACTS

ABSTRACTS Speakers’ abstracts appear in presentation order, followed by poster abstracts in alphabetical order POSTER PRESENTATIONS Poster session is scheduled from 5pm at the end of the first day, immediately after the main meeting, on Friday, 5th September. The posters are also available to view during the coffee and lunch breaks on Friday and Saturday. SCIENTIFIC ORGANIZING COMMITTEE Professor Susan Barnett BSc PhD FSB University of Glasgow Professor James Fawcett PhD FRCP University of Cambridge Professor Stephen B. McMahon PhD, FMedSci King's College, London Professor Wolfram Tetzlaff MD DrMed PhD University of British Columbia Professor Joost Verhaagen PhD Netherlands Institute for Neuroscience

Table of Contents

Presentation Abstracts

Friday ………………………………………………………………………… 3

Saturday………………………………………………………………….13

Poster Abstracts…………………………………………………………...26

Delegate list………………………………………………………………..67

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Table of Contents – Presentation Abstracts

5th September 2014

Mechanisms of intermittent hypoxia-induced functional recovery after spinal cord injury

Gordon S. Mitchell ........................................................................................................................................ 3

Diaphragm pacing: early utilization to decrease invasive mechanical ventilation and optimize functional

respiratory recovery after spinal cord injury

Raymond P. Onders ...................................................................................................................................... 4

Enhancing respiratory plasticity following cervical SCI

Michael Lane ................................................................................................................................................. 5

Restoring bladder function

Graham Creasey ............................................................................................................................................ 6

Promoting regeneration with electrical stimulation

Thomas M. Brushart ..................................................................................................................................... 7

Autonomic control and sport performance in Paralympic athletes with spinal cord injury: What we

learned from London 2012 Games?

Andrei Krassioukov ....................................................................................................................................... 8

Pharmacological management of autonomic dysreflexia: Effects on intraspinal plasticity and

inflammation after complete spinal cord injury

Alexander G. Rabchevsky .............................................................................................................................. 9

Regulation of autonomic control of bladder voiding after a complete spinal cord injury

Parag Gad .................................................................................................................................................... 10

Infections as ‘outcome modifying riskfactor’ after spinal cord injury (SCI) – phenotype and underlying

mechanisms: a bed-side to bench approach

Jan M. Schwab ............................................................................................................................................ 11

Promoting neuroplasticity after spinal cord injury by over-expressing polysialic acid

Louise Adams .............................................................................................................................................. 12

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6th September 2014

Recent progress with a high-performance brain-computer interface

Andrew Schwartz ........................................................................................................................................ 13

Improvements in nerve-electrode integration for closed-loop control of neuroprostheses

Daniel Chew ................................................................................................................................................ 14

Brain machine interface controlled functional electrical stimulation therapy

Milos R. Popovic .......................................................................................................................................... 15

Systemic administration of Epothilone B promotes axon regeneration and functional recovery after

spinal cord injury

Frank Bradke ............................................................................................................................................... 16

Successful spinal cord regeneration in the zebrafish

Catherina G. Becker .................................................................................................................................... 17

Visualization of neuronal networks in the mouse brain, spinal cord and mouse embryos by

ultramicroscopy

Hans-Ulrich Dodt ......................................................................................................................................... 18

A transgenic approach to permanently labeling stressed or damaged neurons

Matt S. Ramer ............................................................................................................................................. 19

Transplantation of corticospinal motor neurons derived from human iPS to repair spinal cord cervical

injuries

Giles W. Plant .............................................................................................................................................. 20

Intravenous multipotent adult progenitor cell treatment for acute spinal cord injury: promoting

recovery through immune modulation

Sarah A. Busch ............................................................................................................................................. 21

Role of endogenous neural stem cells in spinal cord injury

Moa Stenudd ............................................................................................................................................... 22

Inclusive SCI clinical trials: Predicting homogeneous trial participants and modeling outcome measures

for incomplete SCI participants

John D. Steeves ........................................................................................................................................... 23

The SCIentinel study - prospective multicenter study to define the spinal cord injury-induced immune

depression syndrome (SCI-IDS)': aiming at protection of the endogenous recovery potential after SCI

Marcel A. Kopp ............................................................................................................................................ 24

Cell transplantations for SCI – will we really need them?

Sue Barnett , Frank Bradke, Armin Blesch, Simone Di Giovanni, Karim Fouad, James Guest,

Dana McTigue, Adina Michael-Titus, Giles Plant , Phil Popovich, Jerry Silver, Wolfram Tetzlaff ........... 25

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Day 1 Friday, 5th September 2014

Session I: RESPIRATION AND SCI Chair: Professor Joost Verhaagen

Mechanisms of intermittent hypoxia-induced functional recovery after spinal cord injury Gordon S. Mitchell Department of Comparative Biosciences, University of Wisconsin - Madison, Madison, USA After December, 2014: McNight Brain Institute and Physical Therapy, University of Florida, Gainesville, Florida, USA

Mitchell@vetmed.wisc.edu

Spinal plasticity is essential for even limited spontaneous functional recovery of respiratory and non-respiratory motor function after spinal cord injury. Strategies that induce spinal motor plasticity have considerable potential to further improve function after incomplete spinal injuries. An important model of spinal motor plasticity that has guided us in our development of new strategies to treat chronic spinal injury is phrenic long-term facilitation, a long-lasting increase in phrenic motor induced by exposure to modest protocols of acute intermittent hypoxia (AIH). Preconditioning with repetitive AIH enhances subsequent expression of AIH-induced phrenic long-term facilitation, demonstrating that meta-plasticity amplifies its functional effect (and that repetitive AIH may amplify therapeutic benefits). We have identified multiple, distinct cellular cascades, each capable of giving rise to similar long-lasting phrenic motor facilitation in different circumstances. Two important pathways are initiated and orchestrated by spinal G-protein coupled metabotropic receptors activated during hypoxic episodes, including serotonin 2 and adenosine 2A receptors; these receptors, in turn, differentially up-regulate BDNF or TrkB function in the phrenic motor nucleus, leading to amplification of phrenic motor output. AIH-induced respiratory motor plasticity can be harnessed to improve respiratory function in rodent models of clinical disorders that cause severe respiratory insufficiency, including cervical spinal injury and motor neuron disease (ALS). However, we have also come to realize that AIH and AIH pre-conditioning elicit similar mechanisms in non-respiratory motor nuclei, improving limb/leg function after incomplete chronic spinal injuries in both rodent models and humans with chronic, incomplete spinal injuries. Intermittent hypoxia induced spinal motor plasticity may be a general feature of motor systems, reflecting an evolutionary coupling of hypoxia, breathing and movement (swimming) in aquatic vertebrates. We continue progress towards an understanding of cellular mechanisms giving rise to intermittent hypoxia induced motor plasticity, factors that amplify or constrain this plasticity, its biological significance, and its clinical application. Although research on intermittent hypoxia induced motor plasticity is still in its infancy, progress has been rapid, and there is considerable promise that it will lead to novel, safe and effective therapeutic approaches to treat devastating clinical disorders that compromise respiratory and non-respiratory motor function, particularly chronic, incomplete spinal injury. References Dale, E.A., F. Ben Mabrouk and G.S. Mitchell (2014). Unexpected benefits of intermittent hypoxia: enhanced respiratory and non-respiratory motor function. Physiology 29: 39-48. Devinney, M.J., A.G. Huxtable, N.L.Nichols and G.S. Mitchell (2013). Hypoxia-induced phrenic long-term facilitation: emergent properties. Ann. N.Y. Acad. Sci. 1279:143-53. Lovett-Barr, M.R.*, I. Satriotomo*, G. Muir*, J.E.R. Wilkerson, M.S. Hoffman and G.S. Mitchell (2012). Repetitive intermittent hypoxia induces respiratory and somatic motor recovery following chronic cervical spinal injury. J. Neuroscience. 32: 3591-3600. Nichols, N.L., G. Gowing, I. Satriotomo, L.J. Nashold, E.A. Dale, M. Suzuki, P. Avalos, P. Mulcrone, J. McHugh, C.N. Svendsen and G.S. Mitchell (2013). Intermittent hypoxia and stem cell implants preserve breathing capacity in a rat model of ALS. Am. J. Resp. Crit. Care Med. 187(5): 535-42. Hayes, H.B., A. Jayaraman, A., M. Herrmann, G.S. Mitchell, W.Z. Rymer and R.D. Trumbower (2014). Daily intermittent hypoxia enhances walking after chronic spinal cord injury: a randomized trial. Neurology 82: 104-13. Supported by NHLBI R3769064, 080209 and 111598; and DoD CDMRP SC090355, SC120226 and SC130298.

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Diaphragm pacing: early utilization to decrease invasive mechanical ventilation and optimize functional respiratory recovery after spinal cord injury Raymond P. Onders Department of Surgery, University Hospitals Case Medical Center, Cleveland, Ohio, USA

Raymond.onders@uhhospitals.org

High cervical tetraplegics with intact phrenic nerves can be removed from mechanical ventilation with diaphragm pacing (DP). Being removed from tracheostomy mechanical ventilation has been shown to decrease pneumonia rates, decrease secretions, decrease cost of care and improve multiple aspects of quality of life. The long term benefits of DP have shown that it is durable and can be used to maintain natural negative pressure ventilation. There are no significant adverse effects of early implantation or long term use of DP. Recent reports have shown that early use of DP, within days and weeks, after the initial trauma, will allow more rapid weaning from ventilators. Up to 36% of patients implanted early can have recovery of respiration and removal of the implanted electrodes. Control of respiration is incompletely understood, but utilizing the therapeutically implanted electrodes to analyze diaphragm electromyographic activity(dEMG) has allowed a greater understanding of what is occurring to the respiratory system after SCI injury in humans.

Functional electrical stimulation has been shown to have neuroplasticity effects. There is growing evidence in compensatory plasticity of the respiratory system. Recent reports of DP in amyotrophic lateral sclerosis have shown not only improvement in maintaining ventilation but improvement in the central control of the phrenic motor neurons. Mechanical ventilation rapidly causes atrophy of the diaphragm converting Type I muscle fibers to the less functional glycolytic fast fatigable Type IIb muscle fibers. DP converts the entire diaphragm to Type I muscle fibers with subsequent changes to the involved motor neurons. So not only will early use of DP after SCI decrease atrophy of the diaphragm muscle and decrease pneumonia, but it will help in recovery of respiration. This presentation will outline how DP can have the maximum effect with early use in SCI and should become a primary intervention in the intensive care units treating these patients acutely. References Posluszny JA, Onders R, Kerwin AJ, Weinstein MS, Stein DM, Knight J, Lottenberg L, Cheatham ML, Khansarinia S, Dayal S, Byeno PM. Multicenter Review of Diaphragm Pacing in Spinal Cord Injury: Successful not only in weaning from ventilators but also in bridging to independent respiration. J Trauma Acute Care Surg 2014;76:303-310. Onders R, Elmo MJ, Kaplan C, Katirji B, Schilz R. Extended Use of Diaphragm Pacing in Patients with Unilateral of Bilateral Diaphragm Dysfunction: A New Therapeutic Option. Accepted for Surgery 2014 Onders R, Elmo MJ, Kaplan C, Katirji B, Schilz R. Identification of Unexpected Respiratory Abnormalities in Patients with Amyotrophic Lateral Sclerosis through Electromyographic Analysis Using Intramuscular Electrodes Implanted for Therapeutic Diaphragmatic Pacing. Accepted for American Journal Onders RP. Functional Electrical Stimulation: Restoration of Respiratory Function. Handbook Clinical Neurol. 2012;109:275-82

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Enhancing respiratory plasticity following cervical SCI

Michael Lane

Department of Neurobiology, Drexel University College of Medicine, PA, USA

mlane.neuro@gmail.com

Impaired breathing is a devastating consequence of cervical spinal cord injury (SCI), representing a significant burden to injured people and increasing the risk of mortality. While there is mounting evidence for spontaneous improvements in respiration, the extent of recovery – or functional plasticity – remains limited. Elucidating the mechanisms of respiratory plasticity and enhancing functional recovery after cervical SCI is becoming an important experimental and clinical goal. Many of the anatomical and functional changes contributing to post-injury respiratory plasticity remain a subject of ongoing investigation. The results from experimental studies by our research team and others, however, suggests that spinal interneurons may contribute to spontaneous diaphragm recovery and represent a therapeutic target for enhancing functional improvement following SCI.

Our ongoing research aims to improve our understanding of spinal and supraspinal changes that may influence respiration following cervical SCI, and assess whether therapeutic strategies can harness ongoing neuroplastic changes to improve function post-injury. The primary goal of the present work is to test whether transplantation of neural precursor cells can restore anatomical continuity, contribute to formation of novel interneuronal relays, and enhance diaphragm recovery. Adult, female Sprague Dawley rats receive lateralized C3/4 contusion injuries were allowed to recover for 1 week. At that time, the injury site is re-exposed and allogeneic donor tissues obtained from fetal spinal cord (E13.5) were transplanted directly into the lesion epicenter. Ventilatory patterns were assessed using whole-body plethysmography weekly pre- and post-injury for 4 weeks. Transneuronal tracing with pseudorabies virus was then used to examine the extent of synaptic integration between host and donor neurons, and between transplanted cells and host phrenic circuitry. One month post-transplantation, terminal neurophysiological studies were used to assess diaphragm activity or phrenic motor output, and record multiunit activity from transplanted neurons. These experiments have revealed that transplanted neural precursor cells survive, proliferate and become integrated with host phrenic circuitry ipsilateral to injury. Host neurons also become integrated with donor cells. Furthermore, terminal electrophysiology has shown improvement in diaphragm function in transplant recipients. Multiunit recordings made from within transplanted tissue have revealed phasic patterns of activity consistent with inspiration. These results suggest that transplantation of neural progenitor tissue from the developing spinal cord may contribute to an interneuronal relay capable of improving diaphragm recovery following cervical SCI.

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Session II: ELECTRICAL STIMULATION AND SCI Chair: Professor Robin Franklin

Restoring bladder function Graham Creasey Department of Neurosurgery, Stanford University, California

gcreasey@stanford.edu

The essential functions of the bladder are storage of urine at safe pressures for convenient intervals, and nearly complete emptying of urine at safe pressures when desired. The majority of methods of managing the bladder after spinal cord injury restore one but not both of these functions. Restoring these functions fully requires methods of dealing with both paralysis and hyper-reflexia of the bladder and sphincter muscles.

Voiding of the paralyzed bladder can be restored by intermittent electrical stimulation of the sacral preganglionic parasympathetic neurons, provided these are still intact, to produce bladder contraction. Although this stimulation by the Finetech-Brindley Bladder Controller also directly causes intermittent contraction of the sphincter, urine can be effectively voided in a pattern of post-stimulus voiding.

Implantation of this stimulator has usually been combined with cutting the sacral sensory roots to reduce hyper-reflexia of the bladder and restore storage of urine at safe pressures. This posterior rhizotomy has the added benefits of reducing hyper-reflexia of the sphincter, which improves voiding, and reducing autonomic dysreflexia originating from the bladder and bowel. However, it also abolishes reflex erection and reflex ejaculation, and although erection and ejaculation can be restored by other methods this has limited acceptance of the technique.

It is possible that neuromodulation may be able to inhibit hyper-reflexia of the bladder sufficiently to restore storage of urine at safe pressures for convenient intervals. It is also possible that electrical block may be able to reduce hyper-reflexia of the sphincter sufficiently to restore voiding of urine at safe pressures when desired. It has been shown that conduction of action potentials can be blocked rapidly and reversibly, and probably safely, by application of high frequency stimuli to nerves. This has been used to prevent sphincter contraction during electrically stimulated voiding in animals with chronic spinal cord injury. We are now starting a clinical trial to test the feasibility and efficacy in human subjects of restoring both continence and voiding by electrical stimulation without posterior rhizotomy.

References Neuromodulation through sacral nerve roots 2 to 4 with a Finetech-Brindley sacral posterior and anterior root stimulator. Kirkham AP, Knight SL, Craggs MD, Casey AT, Shah PJ. Spinal Cord 40(6):272-81, 2002 High frequency sacral root nerve block allows bladder voiding. Boger AS, Bhadra N, Gustafson KJ. Neurourol Urodyn. 31(5):677-82, 2012 Reversible nerve conduction block using kilohertz frequency alternating current. Kilgore KL, Bhadra N. Neuromodulation 17(3):242-54, 2014

Supported by US Department of Veterans Affairs, US Department of Defense and National Institutes of Health

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Promoting regeneration with electrical stimulation Thomas M. Brushart Department of Orthopaedics, Johns Hopkins University, Baltimore Maryland, USA

tbrusha@jhmi.edu

Electrical stimulation has been used for over a century in attempts to promote neural regeneration. In 2000 the Brushart and Gordon laboratories began a systematic evaluation of electrical stimulation at the time of peripheral nerve repair. Experiments were performed in the rat femoral nerve model. Proximally, at the site of transection and repair, sensory and motor axons intermingle. Distally, the nerve bifurcates into the saphenous (cutaneous) nerve and the motor branch to the quadriceps muscle. We found that one hour of 20 Hz stimulation at the time of nerve repair nearly doubled the number of motoneurons projecting axons correctly to the muscle branch 3 weeks later. Subsequent investigations revealed that the stimulation effect was mediated by the neuron and not the peripheral pathway, and that it involved enhanced promotion of motor axons across the repair site rather than increased regeneration speed in the distal pathway. Stimulation without axotomy did not serve as a conditioning lesion as determined by radiotracer transport after nerve crush or retrograde labeling after subsequent nerve transection and repair. Examination of the neuronal consequences of stimulation revealed accelerated and enhanced

upregulation of BDNF and its receptor TrkB, GAP-43, and T1 tubulin. Expression of the motor-specific HNK-1 carbohydrate epitope was enhanced in the femoral muscle branch after stimulation; this could be a secondary consequence of promoting motor axons into the motor pathway rather than a direct effect of stimulation on Schwann cells. Clinically, electrical stimulation at the time of carpal tunnel decompression was found to enhance the electrophysiologic parameters of regeneration without affecting outcome. Potential challenges to the clinical use of stimulation as an adjunct to nerve repair include the interval between injury and repair/stimulation and the dramatic difference in regeneration distance between the rodent and man. References Brushart, TM. Nerve Repair. Oxford University Press, 2012 Brushart lab supported by NIH RO1 NS034484

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Session III: REHAB AND AUTONOMIC FUNCTION Chair: Professor Stephen McMahon

Autonomic control and sport performance in Paralympic athletes with spinal cord injury: What we learned from London 2012 Games? Andrei Krassioukov ICORD; University of British Columbia; Vancouver, BC, Canada

krassioukov@icord.org

The field of autonomic function and exercise performance in athletes with spinal cord injury (SCI) is still in its infancy. For individuals with SCI who partake in competitive sport, the cardiovascular and autonomic consequences of SCI, such as blood pressure, heart rate, and temperature dysregulation are of critical importance for athletic performance[4]. For example, it is well known that cervical or high-thoracic SCI is associated with life-long abnormalities in systemic arterial pressure control, whereby resting arterial pressure is lower than that of individuals with mid-to-low thoracic injuries or uninjured controls[6]. Further, high level SCI is commonly accompanied by persistent orthostatic intolerance[2, 3], along with transient episodes of hypertension, known as ‘autonomic dysreflexia’, which are often accompanied by disturbances in heart rate and rhythm[5]. Recent evidence suggests that autonomic completeness of injury, that is the degree of disruption to the descending spinal autonomic pathways, also plays a critical role in resting cardiovascular control, whereby those with an autonomic complete injuries exhibit the most severe disruption in resting cardiovascular function independent of injury level[7]. Athletes with SCI have also been documented to self-induce autonomic dysreflexia prior to competition with a view of increasing blood pressure and improving their performance, a technique known as ‘boosting’[1]. For health safety reasons, boosting is officially banned by the International Paralympics Committee. The purpose of this chapter is to review the present literature available on the complex relationship between autonomic function and aerobic exercise performance/exercise capacity in athletes with SCI, specifically focusing on the cardiovascular response to exercise.

We should recognize that exercise performance in individuals with SCI is an extremely complex issue and multiple additional factors should also be considered. The unique cardio-autonomic profiles of athletes with SCI present a variety of challenges to coaches, medical practitioners, classifiers and the athletes themselves. Despite a growing body of literature demonstrating the importance of autonomic integrity on athletic performance no sporting classification currently accounts for between-athlete differences in autonomic function. Such an addition would provide a valuable opportunity for collaboration between athletes, scientists, clinicians and governing bodies with the overall goal to level the sporting playing field and ensure athletes with a similar degree of cardio-autonomic dysfunction can compete fairly against one another. References 1. Blauwet CA, Benjamin-Laing H, Stomphorst J, Van d, V, Pit-Grosheide P, Willick SE. Testing for boosting at the Paralympic games: policies, results and future directions. Br.J.Sports Med. 2013; 47:832-837 2. Claydon VE, Krassioukov AV. Orthostatic hypotension and autonomic pathways after spinal cord injury. J Neurotrauma 2006; 23:1713-1725 3. Illman A, Stiller K, Williams M. The prevalence of orthostatic hypotension during physiotherapy treatment in patients with an acute spinal cord injury. Spinal Cord 2000; 38:741-747 4. Mills PB, Krassioukov A. Autonomic function as a missing piece of the classification of paralympic athletes with spinal cord injury. Spinal Cord. 2011; 49:768-776 5. Wan D, Krassioukov AV. Life-threatening outcomes associated with autonomic dysreflexia: a clinical review. J.Spinal Cord.Med. 2014; 37:2-10 6. West CR, Mills P, Krassioukov AV. Influence of the neurological level of spinal cord injury on cardiovascular outcomes in humans: a meta-analysis. Spinal Cord. 2012; 50:484-492 7. West CR, Romer LM, Krassioukov A. Autonomic function and exercise performance in elite athletes with cervical spinal cord injury. Med.Sci.Sports Exerc. 2013; 45:261-267 Supported by CIHR; C. Nielsen Foundation; Rich Hansen Institute

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Pharmacological management of autonomic dysreflexia: Effects on intraspinal plasticity and inflammation after complete spinal cord injury Alexander G. Rabchevsky Department of Physiology and Spinal Cord & Brain Injury Research Center (SCoBIRC), University of Kentucky, Lexington, KY USA

AGRab@uky.edu

Autonomic dysreflexia (AD) is an abnormal hypertensive syndrome that affects the majority of individuals who have a spinal cord injury (SCI) above the sixth thoracic (T6) level. It is typically elicited by unperceived noxious somatosensory or visceral stimuli below the injury level which then activate uninhibited spinal sympathetic neurons to elicit widespread vasoconstriction that rapidly elevates blood pressure creating a potential life-threatening syndrome. Importantly, AD events often occur on a daily basis because full bladders and constipated bowels are common triggers of this aberrant viscero-spinal-sympathetic reflex after SCI. Remarkably, there is currently no prophylactic treatment to prevent the onset of AD in susceptible individuals, only anti-hypertensive agents to relieve the cardiovascular crises, but which do not target the causative factor(s). In our search for palliative therapeutics, we first documented post-traumatic maladaptive intraspinal plasticity of nociceptive afferent fibers and subsequent activation and sprouting of ascending propriospinal neuron pathways that potentiate uninhibited sympathetic preganglionic neuron responses (i.e., hypertension) to noxious colorectal distension (CRD) (Cameron, Smith et al. 2006; Hou, Duale et al. 2008; Hou, Duale et al. 2009). Subsequently, employing blood pressure telemetry we found that the neuropathic pain medication, gabapentin (GBP), reduces the incidence of spontaneous AD (sAD) when given once daily and significantly diminishes the severity of CRD-induced AD (iAD) when given acutely (Rabchevsky, Patel et al. 2011; Rabchevsky, Patel et al. 2012). Since acute GBP treatment effectively reduces the severity of AD, likely by impeding excitatory neurotransmission, we have begun investigating whether it abrogates the expression of sprouting transcription factors and inflammatory cytokines in dorsal root ganglia (DRG) and spinal cord that contribute to maladaptive plasticity underlying the temporal development of AD. We have preliminary evidence that prolonged repeated iAD is correlated with increased expression of inflammation/sprouting genes (e.g. ATF-3/IL-1b) in DRGs, their corresponding spinal cord segments, and also visceral organs they innervate compared to SCI alone. Moreover, such elevated gene expression profiles in central and peripheral tissues were partially reversed by a single acute GBP administration that effectively mitigated repeated iAD. Accordingly, ongoing studies are testing whether continuous prophylactic GBP treatment beginning after complete T4 SCI will prevent the development of sAD by abrogating maladaptive plasticity as measured by 1) sprouting/synaptogenesis of primary afferent and ascending propriospinal fibers and 2) attenuation of inflammatory cytokine and sprouting transcription factor expression in the spinal cord and DRG. References 1. Cameron, A.A., Smith, G.M., Randall, D.C., Brown, D.R. & Rabchevsky, A.G. Genetic manipulation of intraspinal plasticity after spinal cord injury alters the severity of autonomic dysreflexia. J Neurosci 26, 2923-2932 (2006). 2. Hou, S., et al. Plasticity of lumbosacral propriospinal neurons is associated with the development of autonomic dysreflexia after thoracic spinal cord transection. J Comp Neurol 509, 382-399 (2008). 3. Hou, S., Duale, H. & Rabchevsky, A.G. Intraspinal sprouting of unmyelinated pelvic afferents after complete spinal cord injury is correlated with autonomic dysreflexia induced by visceral pain. Neuroscience 159, 369-379 (2009). 4. Rabchevsky, A.G., et al. Gabapentin for spasticity and autonomic dysreflexia after severe spinal cord injury. Spinal cord 49, 99-105 (2011). 5. Rabchevsky, A.G., et al. Effects of gabapentin on muscle spasticity and both induced as well as spontaneous autonomic dysreflexia after complete spinal cord injury. Front Physiology 3, 329 (2012). Supported by Kentucky Spinal Cord and Head Injury Research Trust (KSCHIRT), NIH/NINDS R01NS049901; 2P30NS051220

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Regulation of autonomic control of bladder voiding after a complete spinal cord injury Parag Gad Terasaki Life Sciences Building, UCLA, Los Angeles, CA, USA

paraggad@gmail.com

The inability to control bladder emptying after a complete spinal cord injury is one of the most dangerous functional deficits that occurs after complete paralysis. Maintenance of bladder health and avoiding retro-flow of urine to the kidneys and urinary tract infections are of utmost importance. The inability to hold urine (incontinence) and to void when desired is extremely socially humiliating. Having demonstrated that electrodes placed epidurally on the dorsum of the spinal cord can be used in animals and humans to recover postural and locomotor function after complete paralysis, we hypothesized that a similar approach could be used to recover bladder function after paralysis. Also knowing that posture and locomotion can be initiated immediately with a specific frequency-dependent stimulation pattern and that with repeated stimulation-training sessions these functions can improve even further, we reasoned that the same two strategies could be used to regain bladder function. Herein, we show that both of these mechanisms. First, we have observed that daily, chronic stimulation leads to spontaneously improved bladder function over a period of a few weeks even without the presence of stimulation. Secondly immediate voiding can be initiated and completed with seconds of the onset of a specific pattern of stimulation in awake, completely paralyzed rats. The clinical implications of these results are substantial in that this intervention has the potential to have significant impact on the quality of life and longevity of patients, while simultaneously reducing ongoing health maintenance after a spinal cord injury. References Edgerton et al., 2001, Courtine et al.. 2009, Harkema et al.,2011,Angeli et al., 2014 This research was supported by the National Institute of Biomedical Imaging and Bioengineering (NIBIB) R01EB007615, the National Institute of Health (NIH) R01NS062009, Christopher and Dana Reeve Foundation, the Walkabout Foundation, the Russian Foundation for Basic Research Grants 13-04-0109a, 13-04-01091 and 13-0412030 ofi-m. The work is performed according to the Russian Government Program of Competitive Growth of Kazan Federal University.

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Infections as ‘outcome modifying riskfactor’ after spinal cord injury (SCI) – phenotype and underlying mechanisms: a bed-side to bench approach

M.A. Koppa, B. Brommer

a, H. Prüss

a, V. Failli

a, T. Liebscher

b, M.J. DeVivo

c, Y. Chen

c, Meisel

a, U.,

Dirnagla, Jan M. Schwab

a,b

aDepartment of Neurology and Experimental Neurology, Clinical and Experimental Spinal Cord Injury Research

(Neuroparaplegiology), Charite Universitaetsmedizin Berlin, D-10117 Berlin, Germany, b

Spinal Cord Injury Center, Trauma Hospital Berlin, D-12683 Berlin, Germany,

cNSCISC, University of Alabama, AL 35233, USA

jan.schwab@charite.de

Spinal cord injury (SCI) causes a systemic immunodeficiency, which increases mortality and worsens outcome by increasing the susceptibility to infections, such as pneumonia and sepsis. It is known that immunosuppression is differentially modulated by lesion localization, suggesting a critical role for central and peripheral neuronal circuits as well as of immune organ innervation. SCI induces a disturbance of the normally well-balanced interplay between the immune system and the CNS (SCI-induced immunodeficiency syndrome, SCI-IDS). We hypothesized that infections operate as disease modifying factors after SCI in human. In order to address this we investigated the association of infections (i.e. pneumonia and/or postoperative wound infections) with functional neurological outcome after acute severe traumatic spinal cord injury.

We screened data sets of 24 762 patients enrolled in a prospective cohort study (National Spinal Cord Injury Database, Birmingham, AL, USA). The group with pneumonia and/or postoperative wound infections (n = 855) revealed significantly less ASIA impairment scale upward conversions after 1 year than the control group (n = 855). ASIA motor score gain [median (interquartile range)] was significantly lower in patients with infections.

Infections associated with spinal cord injury, such as pneumonia and/or postoperative wound infections, qualify as independent risk factors for poor neurological outcome after motor complete human spinal cord injury. Within a bed-to-benchside approach we established an experimental model of controlled pneumonia in order to investigate the neurogenic origin of SCI-IDS and the underlying patho-neurobiology of infections as prevalent co-morbidity, disease modifying factor and neurobiological rehabilitation confounder. Infections constitute a clinically relevant target for protecting the limited endogenous functional regeneration capacity. Upcoming interventional trials might gain in efficacy with improved patient stratification and might benefit from complementary protection of the intrinsic recovery potential after spinal cord injury. SCI-IDS might serve as a model to study the mechanisms and mediators of CNS control over immunity. More importantly, understanding SCI-IDS will allow us to work on developing effective therapeutic strategies to protect the outcome ‘at risk’ after SCI by eliminating a significant negative denominator of poor motor recovery. References Failli V et al. Functional neurological recovery after spinal cord injury is impaired in patients with infections. Brain. 2012 135:3238-50 Glass CK, et al. Mechanisms underlying inflammation in neurodegeneration. Cell. 2010 140:918-34 Lucin KM, Sanders VM, Popovich PG. Stress hormones collaborate to induce lymphocyte apoptosis after high level spinal cord injury. J Neurochem. 2009 110:1409-21 Meisel C et al., Central nervous system injury-induced immune deficiency syndrome. Nat Rev Neurosci. 2005 10:775-86 Moreno B et al. Systemic inflammation induces axon injury during brain inflammation. Ann Neurol. 2011 70:932-42 Schwab JM et al., The paradox of chronic neuroinflammation, systemic immune suppression, autoimmunity after traumatic chronic spinal cord injury. Exp Neurol. 2014 258C:121-129 Zhang et al., Autonomic dysreflexia causes chronic immune suppression after spinal cord injury. J Neurosci. 2013 33:12970-81 Supported by the German Research Council (DFG; Cluster of Excellence NeuroCure), the Berlin-Brandenburg Center for Regenerative Therapies (BCRT; # 81717034) and the Wings for Life Spinal Cord Research Foundation (# WfL-DE-006/1)

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Session IV: NATHALIE ROSE BARR PHD STUDENTSHIP PRESENTATION Chair: Professor Ann Logan

Promoting neuroplasticity after spinal cord injury by over-expressing polysialic acid Louise Adams, Yi Zhang, Ping Yip, Adina Michael-Titus, John Priestley, Xuenong Bo Centre for Neuroscience & Trauma, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, UK L.Adams@qmul.ac.uk

Polysialic acid (PSA) is a linear homopolymer of alpha 2,8-linked sialic acid. It is abundantly expressed in the CNS during development, where it is found closely associated with axon growth and synapse formation. Previous results from our laboratory noted the appearance of perineuronal nets (PNN) in the spinal cord was correlated with diminished expression of PSA. We postulate that over-expression of PSA in neurons around the spinal cord lesion site may modify the PNN structure and promote neuroplasticity and behavioural improvement in a rat cervical spinal cord injury model. To engineer expression of PSA in the spinal cord, we used a lentiviral vector to express polysialyltransferase under the control of the neuronal specific promoter human synapsin-1 (LV/PST). Lentiviral vector expressing GFP controlled by the same promoter (LV/GFP) was used as a control. Adult rats received a mid-cervical lateral hemisection, which was immediately followed by four injections of either LV/PST or LV/GFP rostral and caudal to the injury site. Motor recovery was assessed for six weeks using the Montoya staircase test, open field, grid exploration test and the Catwalk XT system. Anterograde tracing of the corticospinal tract was performed by injection of BDA into the motor cortex. LV/PST injection resulted in strong PSA expression on the surface of transduced neurons. Behavioural tests showed a tendency towards improved function in the LV/PST group compared with LV/GFP; however this did not reach significance due to the large variance among the animals. Whether PSA over-expression promotes sprouting of the corticospinal tract and the formation of new synaptic connections with the neurons on the lesioned side is still being examined. Supported by the International Spinal Research Trust (ISRT)

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Day 2 Saturday, 6th September 2014

Session V: ELECTRONIC INTERFACES WITH THE NERVOUS SYSTEM

Chair: Professor Peter Ellaway

Recent progress with a high-performance brain-computer interface Andrew Schwartz Systems Neuroscience Institute, University of Pittsburgh, Pittsburgh, PA, USA

abs21@pitt.edu

A better understanding neural population function would be an important advance in systems neuroscience. Neurons encode many parameters simultaneously, but the fidelity of encoding at the level of individual neurons is weak. However, because encoding is redundant and consistent across the population, extraction methods based on multiple neurons are capable of generating a faithful representation of intended movement. The realization that useful information is embedded in the population has spawned the current success of brain-controlled interfaces. Since multiple movement parameters are encoded simultaneously in the same population of neurons, we have been gradually increasing the degrees of freedom (DOF) that a subject can control through the interface. Our early work showed that 3-dimensions could be controlled in a virtual reality task. We then demonstrated control of an anthropomorphic physical device with 4 DOF in a self-feeding task.

Currently, monkeys in our laboratory are using this interface to control a very realistic, prosthetic arm with a wrist and hand to grasp objects in different locations and orientations. Our recent data show that we can extract 10-DOF to add hand shape and dexterity to our control set. This technology has now been extended has been extended to a paralyzed patient who cannot move any part of her body below her neck. Based on our laboratory work and using a high-performance “modular prosthetic limb” she has been able to control 10 degrees-of-freedom simultaneously. The control of this artificial limb is intuitive and the movements are coordinated and graceful, closely resembling natural arm and hand movement. This subject has been able to perform tasks of daily living-- reaching to, grasping and manipulating objects, as well as performing spontaneous acts such as self-feeding. Current work is progressing toward making this technology more robust and extending the control with tactile feedback to sensory cortex. Supported by DARPA (972352, N66001-12-C-4027) and NIH (R01NS0256-05S1)

14

Improvements in nerve-electrode integration for closed-loop control of neuroprostheses Daniel Chew John Van Geest Centre for Brain Repair, University of Cambridge, Cambridge, UK

Dc501@cam.ac.uk

The applications of neuroprostheses have currently a modest integrative capacity with the

nervous system. This is in part due to biological incompatibility leading to foreign body reactions and scar tissue formation. Many implantable devices also suffer from physiological incompatibility, in that they are not able to adequately deconstruct and translate recorded neurological activity from the milieu of compounded activity and electrical interference. Neural activity can be stimulated robustly; but to electrically inhibit erroneous neural activity in cases of neuropathic pain, neurogenic bladder (hyper reflexia and over-activity), and other neurological disorders is a challenging task.

Here we present data that describes a surgical manipulation of peripheral nerves and dorsal roots to provide robust improvements in recordable extracellular signal through a neuroprosthetic device. These ‘rootlets’ or ‘nervelets’ are small enough in diameter to grow into, or be surgically placed into, electrically insulated micro-cuffs (microchannels). These microchannels are fabricated to include nanometer thin gold electrode contact sites for optimal recording.

In the case of the peripheral nerve, sciatic ‘nervelets’ of 100um in diameter can grow after transection into a 3 dimensional microchannel network. This can occur as early as 3 days post implantation, and the axons survive anatomically and functionally up to 9 months. Recordings from the device enable individual action potential spikes to be detected, providing a potential robust recording system for use in a closed loop neuroprosthesis in amputee patients.

Additionally dorsal roots can be surgical micro-dissected into 100um diameter ‘rootlets’, enabling direct implantation into a linear array of microchannels. These rootlets remain anatomically and functionally viable up to 3 months post implantation. This technique could have direct application as a bladder fullness indicator, in combination to the clinically marketed bladder nerve stimulators, such as the Medtronic Interstim or Finetech Brindley devices, used as treatment for overactive or neurogenic bladder respectively.

Further; refining stimulation parameters, such as applying low frequency (10Hz) stimulation of the dorsal roots or high frequency (20KHz) stimulation of the ventral roots, one can neuromodulate the local bladder circuitry, to retain bladder continence. Combined with ‘traditional’ stimulation parameters (30Hz) of the ventral roots in cases of bladder areflexia, this neuromodulation and improved recording dynamics provides the basis for the development of a reactive and intelligent neuroprosthesis.

References: Chew DJ, Zhu L, Delivopoulos E, Minev IR, Musick KM, Mosse CA, Craggs M, Donaldson N, Lacour SP, McMahon SB, Fawcett JW (2013). A microchannel neuroprosthesis for bladder control after spinal cord injury in rat. Sci Trans Med 6;5(210) Delivopoulos E, Chew DJ, Minev IR, , Fawcett JW, Lacour SP (2012). Concurrent recording of bladder afferents from multiple nerves using a microfabricated PDMS microchannel electrode array. Lab Chip 21;12(14);2540-51 Minev IR, Chew DJ, Delivopoulos E, Fawcett JW, Lacour SP (2012). High sensitivity recording of afferent nerve activity using ultra-compliant microchannel electrodes: an acute in vivo validation. J Neural Eng. 9(2):026005 FitzGerald JJ, Lago N, Benmerah S, Serra J, Watling CP, Cameron RE, Tarte E, Lacour SP, McMahon SB, Fawcett JW (2012). A regenerative microchannel neural interface for recording from and stimulating peripheral axons in vivo. J Neural Eng 9(1):016010 Supported by a grant from the EPSRC

15

Brain machine interface controlled functional electrical stimulation therapy Cesar Marquez-Chin

2, Kathryn Atwell

1,2, Steve McGie

1,2, Milos R. Popovic

1,2

(1) Institute of Biomaterials and Biomedical Engineering, University of Toronto, Canada (2) Toronto Rehabilitation Institute, University Health Network, Toronto, Canada

milos.popovic@utoronto.ca

In recent years, through a number of clinical trials, our team has demonstrated that functional electrical stimulation (FES) therapy can be used to improve upper limb function in C4 to C7 spinal cord injured (SCI) individuals. We have demonstrated that the therapy is effective in both sub-acute and chronic patients. This therapy essentially promotes neuroplasticity in the spinal cord and the brain, that ultimately results in improved upper limb function. In parallel with these activities we have carried out a series of experiments evaluating which control strategy is more effective in further promoting neuroplasticity when it is coupled with the FES therapy. The outcome of this study was that out of 3 different control strategies applied, the brain machine interface controlled FES therapy produced the most profound neuroplastic changes. For the purpose of that experiment we have used fairly ineffective brain machine interface, and yet the results achieved surpassed electromyography and push button controlled methods for delivering FES therapy. This further motivated us to develop a real-time electroencephalography-based (EEG-based) brain machine interface that is able to reliably and robustly detect different grasps, such as palmar, pinch, and lumbrical grasps, from EEG recordings alone. In this lecture, we will present the results of these three independent activities, and we will present the road map for development of the brain machine interface controlled FES therapy for improving upper limb function in SCI individuals.

16

Session VI: NOVEL TECHNIQUES FOR SCI RESEARCH Chair: Professor Jerry Silver

Systemic administration of Epothilone B promotes axon regeneration and functional recovery after spinal cord injury Frank Bradke

1, Jörg Ruschel

1, Farida Hellal

1†, Kevin C Flynn

1‡, Sebastian Dupraz

1, Margaret Bates

2,

Christopher Sliwinski3, Kristina Dobrint

4, Michael Peitz

4, Oliver Brüstle

4, Armin Blesch

3, Norbert Weidner

3,

Mary Bartlett Bunge2, John L. Bixby

2

1) Axonal Growth and Regeneration, German Center for Neurodegenerative Diseases, Bonn, Germany

2) The Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, USA 3) Spinal Cord Injury Center, Heidelberg University Hospital, Heidelberg, Germany 4) Institute of Reconstructive Neurobiology, Life&Brain Center, University of Bonn and Hertie Foundation, Bonn, Germany † Current address: Institute for Stroke and Vascular Dementia Research, University of Munich Medical Center, Munich, Germany ‡ Current address: Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, Germany

Frank.Bradke@dzne.de

After central nervous system (CNS) injury, axon regeneration is prevented by growth inhibitory factors in the lesion scar as well as by poor intrinsic axon growth potential. Microtubule stabilization controls scar formation and axon growth. However, the action of microtubule stabilization on these processes has remained unclear. Here, systemic and post-injury administration of a blood-brain barrier permeable microtubule stabilizing drug, epothilone B, decreased scarring in spinal cord injured rodents by disrupting cell polarity of meningeal fibroblasts, which abrogated directed cell migration. Further, epothilone B propelled axon growth through an inhibitory environment by enabling microtubule protrusion into the axon tip. Finally, epothilone B treatment improved walking disabilities after spinal cord injury. As epothilones received recently clinical approval, they hold promise for clinical translation in enabling axon regeneration and functional recovery after CNS injury. This work was supported by NIH, IRP, WfL and DFG

17

Successful spinal cord regeneration in the zebrafish Catherina G. Becker, Thomas Becker, Gianna Maurer, Jochen Ohnmacht, Antón Barreiro-Iglesias, Yujie Yang Centre for Neuroregeneration, The University of Edinburgh, Edinburgh, UK

catherina.becker@ed.ac.uk

In contrast to mammals, the adult zebrafish can successfully repair injury to the spinal cord leading to full functional recovery (1,2). During repair, axons recross the lesion site and motor- and interneurons are generated from progenitor cells at the central canal (3). We have used the embryonic fish to screen for compounds enhancing this neurogenesis and have found that during development, dopamine and serotonin augment the number of motor neurons. This mechanism is also active during regeneration of the spinal cord in adult fish. Furthermore, application of dopamine to human ES cell derived neural stem cells enhances the number of motor neurons generated in vitro, underscoring the relevance of fish findings for mammalian spinal cord development, and potentially, repair (4).

We have now begun to use mechanical lesion of the larval spinal cord in zebrafish (Ohnmacht et al., unpublished). Lesions as early as 3 days post fertilisation lead to a macrophage/microglial response and regeneration of motor neurons in the proximity of the lesion site. This can be enhanced by application of dopaminergic drugs, similar to adults. We are now further refining mechanic and genetic lesions paradigms in the post-embryonic/larval zebrafish to investigate signalling mechanisms directing neurogenesis and to allow compound screens on the regenerating spinal cord.

References 1. Becker T, Becker CG (2014) Axonal regeneration in zebrafish. Curr Opin Neurobiol. 27C:186-191 2. Becker T, Wullimann MF, Becker CG, Bernhardt RR, and Schachner M (1997) Axonal regrowth after spinal cord transection in adult zebrafish. J Comp Neurol 377:577-595 3. Reimer

, MM, Sörensen I, Frank

RE, Liu

C, Becker

CG*, Becker

T* (2008) Motor neuron regeneration in adult zebrafish, J Neurosci

28: 8510-8516 4. Reimer MM, Norris A, Ohnmacht J, Patani R, Zhong Z, Dias TB, Kuscha V, Scott AL, Chen Y, Rozov S, Frazer SL, Wyatt C, Higashijima S, Patton EE, Panula P, Chandran S, Becker T*, Becker CG* (2013) Dopamine signaling from the brain augments spinal motor neuron generation during development and adult regeneration via hedgehog pathway activation, Dev Cell 25(5): 478-491 Supported by the BBSRC, the National Centre for the 3Rs and the Motor Neurone Disease Association

18

Visualization of neuronal networks in the mouse brain, spinal cord and mouse embryos by ultramicroscopy Hans-Ulrich Dodt, C. Hahn, N. Jährling, S. Saghafi, K. Becker Department of Bioelectronics, Institute of Solid State Electronics, TU Vienna, Vienna, Austria Section of Bioelectronics, Center for Brain Research, Medical University Vienna, Vienna, Austria

dodt@tuwien.ac.at

It would be very helpful for the analysis of neuronal networks of the brain, if one could visualize these networks in 3 dimensions. Up to now this was only possible with limited resolution by sequential slicing and reconstruction of the brain. This time consuming attempt is easily hampered by artifacts as shrinkage and distortion induced by standard histological procedures.

To overcome these problems we used a microscopy based on extreme darkfield illumination with a light sheet, once called ultramicroscopy. This microscopy allows optical sectioning of whole mouse brains and was combined with an approach to clear fixed neuronal tissue: Mouse brains were made completely transparent by immersion in oil of the same refractive index as protein. By illuminating the brains with blue light (λ = 488 nm), neurons labeled with GFP were visualized by fluorescence. This way we could detect single neurons in hippocampi inside whole brains.

By surface rendering the shape and position of hippocampi relative to the brain surface could be depicted. In complete excised hippocampi subcellular resolution was obtained by 3D reconstruction from several hundred optical sections. The dendritic trees of CA1 hippocampal neurons with dendrites and dendritic spines could be visualized.

Many proteins can be labelled in transgenic mice with genetically encoded fluorescent markers. Using these markers our approach will represent a high-throughput screening method for protein expression in 3 D. This expression can be monitored with µm resolution and should allow the elucidation of complex neuronal networks in the brain and spinal cord.

We show that ultramicroscopy allows also optical sectioning and detailed 3D reconstruction of whole mouse embryos by imaging autofluorescent structures. Especially the circulatory system in the body and brain became apparent as blood remaining in the preparation showed strong fluorescence. Also other applications like e.g. visualization of nerve bundles in whole embryos and visualization of plaques in brains of mice with Alzheimers disease will be shown. In general the method is well suited for high-throughput phenotype screening of transgenic mice and thus will benefit the investigation of disease models.

References 1. H.U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C.P. Mauch, K. Deininger, J.M. Deussing , M. Eder, W. Zieglgänsberger, and K. Becker, Nat. Meth., 2007, 4, 331-336 2. A. Ertürk, C.P. Mauch, F. Hellal, F. Förstner, T. Keck, K. Becker, N. Jährling, H. Steffens, M. Richter, M. Hübener, E. Kramer, F. Kirchhoff, H.U. Dodt, and F. Bradke, Nat. Med., 2012, 18, 166-171 3. A. Ertürk, K. Becker, N. Jährling, C.P. Mauch, C.D. Hojer, J.G. Egen, F. Hellal, F. Bradke, M. Sheng, and H.U. Dodt, Nat. Protoc., 2012, 7, 1993-95 4. C. Schönbauer, J. Distler, N. Jährling, M. Radolf, H.U. Dodt, M. Frasch, F. Schnorrer, Nature, 2011, 479, 406-409

19

A transgenic approach to permanently labeling stressed or damaged neurons Matt S. Ramer

1,2, Franziska Denk

2, Leanne M. Ramer

2, Mohammed A. Nassar

3, Yury Bogdanov

4,

Massimo Signore5, John N. Wood

4, Stephen B McMahon

2

1. International Collaboration on Repair Discoveries, the University of British Columbia, Vancouver, Canada 2. Wolfson Centre for Age-Related Diseases, King’s College London, UK 3. Department of Biomedical Science, University of Sheffield, UK 4. Molecular Pain Laboratory, University College London, UK 5. Institute of Child Health, London, UK

ramer@icord.org

A major challenge in spinal cord injury research, particularly in clinically-relevant contusive injuries, is in distinguishing injured axons from spared fibres. This is an important issue since experimental therapies targeting injured axons may in fact be better suited to harnessing spared systems (and vice versa).

Here we describe a new transgenic mouse knock-in which was designed to exploit upregulation of the injury-induced transcription factor Activating in Transcription Factor 3 (ATF3) in order to effect genomic recombination. An ATF3 - Cre-ERT2 construct, flanked by an ATG start codon and the ATF3 3’UTR and SV40 stop signal was inserted into exon 2 of the native ATF3 gene. Mice carrying the knock-in mutation were then bred with lox-stop-lox Rosa26-tdtomato mice. The expectation was that upregulation of ATF3 gene expression in the offspring, along with tamoxifen treatment to allow Cre translocation to the nucleus, would cause excision of the floxed stop signal and induction of reporter expression. In naïve adult mice, occasional neurons underwent recombination in the olfactory bulb (OB) granular layer, in the dentate gyrus and in trigeminal and dorsal root ganglion (DRG) neurons. This is in line with the known expression patterns of ATF3 in the adult mouse. In the OB and dentate gyrus, the few neurons labeled had Golgi-like morphology. The central projections of primary afferent axons were likewise completely filled.

Peripheral nerve injury induced recombination in approximately 50% of DRG neurons and in a smaller fraction of sympathetic neurons by four days post-lesion, indicating that the Cre construct is “leaky”. Lateral spinal hemisection induced reporter expression in DRG neurons close to the lesion, in midline neurons below the injury (in area X) which send ascending axons into the lateral funiculus, in magnocellular reticular neurons, in vestibular nuclei (lateral and spinal), in the red nucleus, and in the descending part of the paraventricular hypothalamic nucleus. Treatment of uninjured animals with tamoxifen induced extensive recombination (i.e. ATF3 expression) in the OB, in the dentate gyrus, in vascular endothelium and arteriolar smooth muscle, and in primary sensory (and sympathetic) neurons. We show that tamoxifen-induced ATF3 induction in the brain occurs via the antiestrogen binding site (rather than via estrogen receptors), and can be blocked by alpha-tocopherol (vitamin E). In the DRG, tamoxifen induces ATF3 via anti-estrogenic and non-estrogenic mechanisms. These mice may be useful for labeling specific neuronal populations following injury in the absence of tamoxifen treatment, or knocking out floxed constructs with or without tamoxifen in addition to injury. This work was carried out by the M.S. Ramer while on sabbatical in the laboratory of Prof. SB McMahon. Dr. Franziska Denk designed the mouse.

20

Session VII: CELL THERAPIES FOR SCI Chair: Professor Sue Barnett

Transplantation of corticospinal motor neurons derived from human iPS to repair spinal cord cervical injuries

Giles W. Plant Department of Neurosurgery and Stanford Institute for Neuroscience, Stanford University, Stanford, CA, USA

gplant@stanford.edu

Regeneration of corticospinal tract (CST) axons is a major goal in the successful repair of the adult spinal cord following an injury. Determining how severed axons that regrow can produce functional and anatomical benefits is vital to creating future treatments for patients. Following injury to the spinal cord CST axons fail to regenerate, and retract rostrally from the original lesion site. Therefore an effective therapeutic intervention needs to provide both a positive growth milieu, and meaningful connections to the limbs once innervated. Many current stem cell therapies have not been defined well making any direct correlation of repair with a specific cell type difficult to assess. Stem cell strategies can also involve the use of undifferentiated or differentiated embryonic and adult neural stem cells, which cannot be obtained from the injured patient. Our development of human induced pluripotent stem cells (iPSC) differentiated into neuronal lineages gives us the potential to immunologically match grafts without the need for immunosuppression and avoids the ethical issues arising from embryonic tissue use. We employ a rat cervical unilateral cut or contusion model of spinal cord injury at the C5 level to measure anatomical and functional outcomes after transplanting human iPS derived corticospinal motor neurons (CSMN). In addition we have electrophysiologically mapped host CST neurons, which infer specific forelimb movements. Previous studies have indicated that rehabilitative strategies have additive benefits when combined with therapeutic interventions. For this reason, our animals are undergoing rehabilitation and functional testing paradigms following CSMN transplantation.

We hypothesize that our treatment with CSMN transplants will improve functional and anatomical outcomes after a cervical spinal cord injury. We propose that any functional improvement observed will arise from long tract growth of the transplanted CSMN neurons forming synaptic connections to host spinal cord circuitry. In addition we propose that host CST axons will form synaptic connections to those neurons from the CSMN transplants via the injured ipsilateral or uninjured contralateral CST. Functional tests encouraging the use of the injured forelimb and assessment will also be undertaken in groups to ascertain improved growth and target specificity. Supported by the International Spinal Research Trust (ISRT), Klein Family Research Fund, and Wings for Life

21

Intravenous multipotent adult progenitor cell treatment for acute spinal cord injury: promoting recovery through immune modulation Marc DePaul

1, Marc Palmer

2, Rochelle Cutrone

2, Jason A. Hamilton

2, Bradley T. Lang

1, Robert J. Deans

2,

Robert W. Mays2, Jerry Silver

1, Sarah A. Busch

2

1Dept. of Neurosciences, Case Western Reserve Univ., Cleveland, OH, USA

2Athersys, Inc. Regenerative Medicine, Cleveland, OH, USA

sbusch@athersys.com

Adult bone marrow-derived stem cells are known to have immunomodulatory capabilities, but their potential to alter inflammatory processes and promote regeneration after spinal cord injury (SCI) has not been thoroughly studied. We have previously demonstrated that multipotent adult progenitor cells (or MAPCs) prevent axonal dieback and promote re-growth of injured axons in vivo in a dorsal column crush model (Busch et al., 2011). In the current study, we sought to determine the optimal window of administration, dosing, and the biodistribution of human multipotent adult progenitor cells (MAPCs) in a spinal cord contusion model (250 kdyn, Infinite Horizon Impactor injury at T8). A clinical grade variant of MAPC therapy, known as MultiStem

®, is currently in clinical evaluation for treatment of ischemic stroke.

Rats received saline or 4x106 cells via intravenous (iv) injection immediately following or 1 day post injury

(DPI). We performed locomotor testing through 10 weeks post injury (WPI) and found significant and sustained improvements in BBB scores, subscores, and the Catwalk regularity index. We monitored urination every two weeks using metabolic cages and found the void volume significantly reduced 10 weeks post injury in cell treated animals. At the study endpoint, rats underwent urodynamic assessment. Bursting activity of the external urethral sphincter was seen in correlation with a void in several treated animals, whereas in untreated animals bursting was rare, sporadic, and uncoordinated. Treated animals urinated at a smaller bladder volume, had less residual volume, showed improved return to baseline pressure following a void, and had a decrease in bladder weight. Increasing the dose to 8x10

6 cells did

not lead to additional improvements in locomotor recovery, but did improve bladder function. Cell distribution was determined using CryoViz technology by iv infusing Qdot-labeled MAPCs into either SCI or laminectomy control animals 1 DPI. Lungs, liver, spleen, and spine were collected 24 or 48 hours after treatment. MAPCs were found in the lungs, liver, and spleen at 24 hours, amounting to <5% of administered cells, and cell numbers decreased at 48 hours. Normalizing cell counts to tissue weight showed a preferential homing to the spleen, while few cells were found in the spinal column. Microarray analysis of the lesion, blood, and spleen suggests MAPCs administered 1 day post injury alter many injury-induced pathways including those involved in recruitment, activation and migration of immune cells, which we confirmed using qPCR. In support of this data, we found a decrease of ED1

+ macrophages at

the lesion site 4 DPI in treated animals. These data suggest that MAPCs, when administered iv in an acute model of SCI, are more likely to exert benefit through peripheral organ systems than via homing and direct interaction with the site of injury.

References S.A. Busch, J.A. Hamilton, K.P. Horn, F.X. Cuascut, N. Lehman, A.E. Ting, R.J. Deans, R.W. Mays, J. Silver. Multipotent Adult Progenitor Cells Prevent Macrophage-Mediated Axonal Dieback and Promote Regrowth after Spinal Cord Injury. Journal of Neuroscience. 2011 31: 944-953 Supported by a grant from the Ohio Third Frontier to J. Silver / R.W. Mays through the National Center for Regenerative Medicine

22

Role of endogenous neural stem cells in spinal cord injury Moa Stenudd Jonas Frisén lab, Department of Cell and Molecular Biology, Karolinska Institutet, Sweden

moa.stenudd@ki.se

Spinal cord injury is followed by the formation of a glial scar, which has both positive and negative effects on recovery from the injury (1). The astrocytes in the glial scar are generated from both self-duplicating resident astrocytes and neural stem cells. Ependymal cells are the neural stem cells in the adult spinal cord, and after spinal cord injury they proliferate vigorously and generate both oligodendrocytes and more than half of the astrocytes in the glial scar (2).

A good understanding of the separate functions of astrocyte-derived and neural stem cell-derived astrocytes is important to develop therapies modulating the contribution to the glial scar. By blocking the neural stem cells’ contribution to the scar, we have shown that neural stem cell progeny is necessary to maintain tissue integrity and prevent neuronal death after spinal cord injury (3). These results identify neural stem cells as key players in protective mechanisms following spinal cord injury, making them an interesting target for future non-invasive treatments for spinal cord injury. References (1) Burda JE, Sofroniew M V. Reactive gliosis and the multicellular response to CNS damage and disease. Neuron. 2014;81(2):229–48 (2) Barnabé-Heider F, Göritz C, Sabelström H, et al. Origin of new glial cells in intact and injured adult spinal cord. Cell Stem Cell. 2010;7(4):470–82 (3) Sabelström H, Stenudd M, Réu P, et al. Resident neural stem cells restrict tissue damage and neuronal loss after spinal cord injury in mice. Science. 2013;342(6158):637–40

Work in the lab of Dr. Jonas Frisén is supported by Swedish Research Council, the Swedish Cancer Society, the Karolinska Institute, Tobias Stiftelsen, AFA Försäkringar, the Strategic Research Programme in Stem Cells and Regenerative Medicine at Karolinska Institutet (StratRegen), Torsten Söderbergs Stiftelse and Knut och Alice Wallenbergs Stiftelse

23

Session VIII: CLINICAL SESSION

Chair: Professor James Guest

Inclusive SCI clinical trials: Predicting homogeneous trial participants and modeling outcome measures for incomplete SCI participants John D. Steeves

1, Lorenzo Tanadini

2, Dirk Haupt

1, Armin Curt

2

1

ICORD, University of British Columbia, Vancouver, Canada 2 Uniklinik Balgrist, University of Zurich, Zurich, Switzerland

steeves@icord.org

Never before have there been so many clinical trials and human studies examining a wide range of therapeutic approaches for the treatment of spinal cord injury (SCI). It is desirable to recruit and enroll as many participants as possible to a trial. However, we know this is burdened by ethical and safety concerns. In order to minimize possible harm, many Phase I SCI trials begin by enrolling volunteers with a thoracic sensorimotor complete (AIS A) injury. If there are no adverse events and risks seem minimal, the trial program often advances to a Phase II trial involving participants with cervical sensorimotor complete (AIS A) SCI. This sequential recruitment approach is safe, but slow and an inefficient utilization of equipment, staff and participants. After safety has been preliminarily established in a small Phase I study, an alternative approach would recruit a broader range of participants with either complete or incomplete SCI (iSCI) to a Phase II trial. Such a strategy has some justification since many of the experimental treatments were developed using animals models with iSCI (AIS B-D).

Nevertheless, people living with iSCI exhibit highly variable patterns of functional recovery. Such heterogeneity in trial participants makes it difficult to detect statistically valid treatment effects or clinical benefits. Some trials have recruited iSCI subjects and used a common clinical endpoint for all SCI types, hoping the therapeutic would have a large enough effect size to overcome any participant variability in the patterns of recovery. But, this approach has not been successful and will likely miss detecting subtle, but significant treatment effects. Thus, how can we create more homogeneous study cohorts in an objective and unbiased manner, while identifying those participants who should be excluded from participation because they may recover to extensively to detect a therapeutic benefit (e.g. ceiling effect)? Finally, what outcome measures (trial endpoints) should we use for different cohorts in a phase II study?

We require predictive data-driven algorithms that will accurately stratify participants based on early neurological and functional values. In addition, we need to model (test) reasonable cohort-specific outcomes for iSCI participants. We have been employing the EMSCI database and a recently developed multivariate statistical method, unbiased recursive partitioning, to identify homogeneous cohorts at an early time point after SCI, as well as to model different cohort-specific outcome measures. If such a recruitment strategy could be implemented, it would mean that trials could be completed faster and more efficiently, as well as identify which types of SCI respond best to the therapeutic being examined.

24

The SCIentinel study - prospective multicenter study to define the spinal cord injury-induced immune depression syndrome (SCI-IDS)': aiming at protection of the endogenous recovery potential after SCI Marcel A. Kopp

1,2, Claudia Druschel

2,3, Christian Meisel

4,5,6, Thomas Liebscher

7, Erik Prilipp

7, Ralf Watzlawick

1,2,

Paolo Cinelli8, Andreas Niedeggen

7, Klaus-Dieter Schaser

3, Guido A. Wanner

8, Armin Curt

9, Gertraut Lindemann

9,

Natalia Nugeva10

, Michael G. Fehlings10

, Peter Vajkoczy11

, Mario Cabraja11

, Julius Dengler11

, Wolfgang Ertel12

, Axel Ekkernkamp

13, Peter Martus

14, Hans-Dieter Volk

4,5, Nadine Unterwalder

5, Uwe Kölsch

5, Benedikt Brommer

1,2, Rick C.

Hellmann1,2

, Ramin Raul Ossami Saidi1,2

, Ines Laginha1,2

, Harald Prüss

1,15, Vieri Failli

1,2, Ulrich Dirnagl

1,16, Jan M.

Schwab1,2,7

1Dept. of Neurology and Experimental Neurology,

2 Spinal Cord Injury Research (Neuroparaplegiology),

3Dept. of Musculoskeletal

Surgery, 4Institute of Medical Immunology,

5 Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité -

Universitätsmedizin Berlin, Germany; 6Dept. of Immunology, Labor Berlin – Charité Vivantes GmbH, Berlin, Germany;

7Treatment

Centre for Spinal Cord Injuries, Trauma Hospital Berlin, Germany; 8Division of Trauma Surgery, University Hospital of Zürich,

9Spinal

Cord Injury Center, University Hospital Balgrist, Zurich, Switzerland; 10

Dept. of Neurosurgery, University of Toronto, Canada; 11

Dept. of Neurosurgery,

12Centre for Trauma- and Reconstructive Surgery, Charité - Universitätsmedizin Berlin, Germany;

13Trauma

Surgery and Orthopedics Clinic, Trauma Hospital Berlin, Germany; 14

Dept. of Clinical Epidemiology and Applied Biostatistics, Eberhard Karls Universität Tübingen, Germany;

15German Center for Neurodegenerative Diseases (DZNE),

16Center for Stroke

Research Berlin, Charité - Universitätsmedizin Berlin, Germany

marcel.kopp@charite.de or jan.schwab@charite.de

Infections are the leading cause of death following acute spinal cord injury (SCI). Furthermore, infections represent a ‘disease modifying factor’ independently associated with poor neurological outcome

1. The increased susceptibility for infections is not sufficiently explained by the risk of aspiration,

bladder dysfunction, or high-dose methylprednisolone treatment. Experimental and clinical pilot datae.g.2,3,4

suggest that SCI disturbs the signaling pathways between the central nervous system and the immune system and shifts homeostasis towards an acute and chronic state of suppressed immune functions. The objectives of the SCIentinel trial are to characterize the dysfunction of the innate and adaptive immune system after SCI and to explore its proposed ‘neurogenic’ origin by analyzing its correlation with lesion height and severity. Decreased HLA-DR (MHC II) expression on monocytes serves as a key surrogate parameter. Secondary hypotheses are that the Spinal Cord Injury-induced Immune Depression Syndrome (SCI-IDS)

5 causes transient lymphopenia and triggers qualitative functional leukocyte deficits,

which may complicate the post-acute phase after SCI. SCIentinel is a prospective, international, multicenter study aiming to recruit about 120 patients

with acute SCI or control patients with acute vertebral fracture without neurological deficits scheduled for spinal surgery. The assessment points are: <31 hours, 31-55 hours, 7 days, 14 days, and 10 weeks post-trauma. Neurological classification includes American Spinal Injury Association impairment scale and neurological level. Laboratory analyses comprise haematological profiling, immunophenotyping, cytokines and gene expression of immune modulators. Preliminary results reveal a SCI severity dependent immune depression in terms of reduced HLA-DR molecule numbers on monocytes occurring within the first days after SCI and extending into the sub-acute phase in the group of complete SCI. Better definition of the SCI-IDS provides a basis to prevent infectious complications in order to attenuate the impact of ‘disease modifying factors’ on neurological outcome. This putatively results in improved SCI care. References 1 Failli V, Kopp MA, Gericke C, Martus P, Klingbeil S, Brommer B, Laginha I, Chen Y, DeVivo MJ, Dirnagl U, Schwab JM:

Functional neurological recovery after spinal cord injury is impaired in patients with infections. Brain 2012, 135(Pt 11):3238-3250 2 Cruse JM, Lewis RE, Jr., Bishop GR, Kliesch WF, Gaitan E, Britt R: Decreased immune reactivity and neuroendocrine alterations

related to chronic stress in spinal cord injury and stroke patients. Pathobiology 1993, 61(3-4):183-192 3 Campagnolo DI, Keller SE, DeLisa JA, Glick TJ, Sipski ML, Schleifer SJ: Alteration of immune system function in tetraplegics. A

pilot study. Am J Phys Med Rehabil 1994, 73(6):387-393 4 Riegger T, Conrad S, Schluesener HJ, Kaps HP, Badke A, Baron C, Gerstein J, Dietz K, Abdizahdeh M, Schwab JM:

Immunedepression syndrome following human spinal cord injury (SCI): a pilot study. Neuroscience 2009, 158(3):1194-1199 5 Meisel C, Schwab JM, Prass K, Meisel A, Dirnagl U: Central nervous system injury-induced immune deficiency syndrome. Nat

Rev Neurosci 2005, 6(10):775-786 Supported by the German Research Council (DFG; Cluster of Excellence NeuroCure), the Berlin-Brandenburg Center for Regenerative Therapies (BCRT; # 81717034) and the Wings for Life Spinal Cord Research Foundation (# WfL-DE-006/1)

25

Session IX: DISCUSSION FORUM / DEBATE Chair: Professor Wolfram Tetzlaff

Cell transplantations for SCI – will we really need them? Panel members: Sue Barnett Susan.Barnett@glasgow.ac.uk Frank Bradke frank.bradke@dzne.de Armin Blesch Armin.Blesch@med.uni-heidelberg.de Simone Di Giovanni s.di-giovanni@imperial.ac.uk Karim Fouad karim.fouad@ualberta.ca Jim Guest jguest@med.miami.edu Dana McTigue dana.mctigue@osumc.edu Adina Michael-Titus a.t.michael-titus@qmul.ac.uk Giles Plant gplant@stanford.edu Phil Popovich Phillip.Popovich@osumc.edu Jerry Silver jxs10@po.cwru.edu Wolfram Tetzlaff tetzlaff@icord.org

26

Table of Contents – Poster Abstracts

Promoting neuroplasticity after spinal cord injury by over-expressing polysialic acid

Louise Adams ........................................................................................................................................ 29

Regaining over-ground locomotor function following severe contusion injury with epidural

stimulation and treadmill training

Yazi D. Al’joboori ................................................................................................................................... 30

Remyelination of spinal axons by Schwann cells following spinal cord injury is a Neuregulin-1

dependent endogenous repair mechanism

Katalin Bartus ........................................................................................................................................ 31

Assessing functional recovery in the injured corticospinal tract: an optogenetic approach

Murray G. Blackmore ............................................................................................................................ 32

Investigating functional plasticity and synaptogenesis following experimental spinal cord injury

Emily R. Burnside .................................................................................................................................. 33

Alpha9 integrin activation in neurite outgrowth and axon regeneration

Menghon Cheah .................................................................................................................................... 34

Olfactory ensheathing cell transplants improve vertical climbing in rats after cervical level dorsal root

rhizotomy

Andrew Collins ...................................................................................................................................... 35

Transcriptomic changes evoked by culturing Dorsal Root Ganglion neurons overwhelm those evoked

by axonal injury

Matthew C. Danzi .................................................................................................................................. 36

Nanotechnology in neuroregeneration

Suradip Das ........................................................................................................................................... 37

Transduction of cells with a lentiviral vector encoding mammalian chondroitinase ABC in an in vitro

model of neurite outgrowth: enhanced neurite outgrowth is accompanied by a drop in PTEN levels

Priscilla Day ........................................................................................................................................... 38

Regulation of IL10 by chondroitinase ABC promotes a distinct immune response following spinal cord

injury

Athanasios Didangelos .......................................................................................................................... 39

The role of PKA in translating rehabilitative training after SCI into neuroplasticity

Karim Fouad .......................................................................................................................................... 40

MicroRNA-155 deletion restricts inflammatory signaling in macrophages and enhances axon growth

capacity: implications for spinal cord repair

Andrew D. Gaudet................................................................................................................................. 41

27

Artificial Intelligence in Medical Systems Neurobiology: Finding a Treatment for Paralysis

Barbara Grimpe ..................................................................................................................................... 42

Does overexpression of Fibroblast Growth Factor Receptor 1 (fgfr1) in CNS neurons enhance axon

regeneration and recovery after spinal cord injury in rats?

Barbara Haenzi ...................................................................................................................................... 43

AAV9-IL4 exacerbates a pathogenic systemic immune response that impairs functional recovery after

contusive spinal cord injury

Jodie C.E. Hall ........................................................................................................................................ 44

Human neural progenitors transplanted to the deaffarented murine spinal cord promote

regeneration of functional sensory fibers

Jan Hoeber ............................................................................................................................................ 45

Do severity and duration of compression impact on recovery after severe acute spinal cord injury in

dogs?

Hilary Hu................................................................................................................................................ 46

Sympathetic and sensory sprouting after spinal cord injury: peripheral consequences of central

injuries

Diana V. Hunter ..................................................................................................................................... 47

Chondroitinase gene therapy as a treatment for spinal cord injury

Nicholas D. James.................................................................................................................................. 48

Combinatorial treatment with GSK3β inhibitors and chondroitinase ABC to regulate glial scar

formation and promote axon regeneration in the spinal cord

Ashik Kalam ........................................................................................................................................... 49

Optimizing and understanding the use of intracellular sigma peptide as a translatable therapeutic for

spinal cord injury

Bradley T. Lang ...................................................................................................................................... 50

Early intravenous delivery of mesenchymal progenitor cells modulates the secondary inflammatory

response after cervical spinal cord injury leading to behavioral and pathological amelioration

Seok Voon Lee ....................................................................................................................................... 51

Facilitating reproducibility and data integration for SCI research with MIASCI and RegenBase

Vance P. Lemmon ................................................................................................................................. 52

Expression of a hyperactive transcription factor increases axon growth and regeneration

Saloni T. Mehta ..................................................................................................................................... 53

Daily acute intermittent hypoxia following cervical spinal cord injury

Kristiina Negron .................................................................................................................................... 54

A novel role for Wnt signalling in regulating astrogliosis in adult white matter

Andrea Rivera ........................................................................................................................................ 55

28

Wnts: more than an axonal growth inhibitor in the adult spinal cord

F. Javier Rodríguez. ............................................................................................................................... 56

Characterization of a novel axon growth repellent and its role in spinal cord injury

Julia Schaeffer ....................................................................................................................................... 57

Transplantation of neural progenitors to improve respiration following spinal cord injury

Victoria M. Spruance ............................................................................................................................. 58

Investigating neuroprotection by carbon nanotubes following spinal cord injury

Merrick Strotton ................................................................................................................................... 59

Intramuscular Tibialis Anterior coherence and subacute spinal cord injury: mechanisms of

neuroplasticity underlying SCI

Julian Taylor .......................................................................................................................................... 60

Oral administration of the p38α MAPK inhibitor, UR13870, inhibits anterior cingulate microglial

expression and affective pain behaviour following spinal cord injury

Julian Taylor .......................................................................................................................................... 61

The fate of boundary cap neural crest stem cells following transplantation to the surface of avulsed

or uninjured spinal cord

Carl Trolle .............................................................................................................................................. 62

Early transplantation of mesenchymal stem cells after spinal cord injury relieves pain

hypersensitivity through suppression of pain-related signaling cascades and reduced inflammatory

cell recruitment

Kenzo Uchida…………………………………………………………………………………………………………………………………..63

Effects of pudendal and cortical paired associative stimulation on reflex and cortico-spinal control of

anal sphincter responses in patients with incomplete spinal cord injury: a feasibility study

Natalia Vásquez ..................................................................................................................................... 64

Chondroitinase ABC rescues complete respiratory motor activity following cervical contusion injury

Philippa M. Warren ............................................................................................................................... 65

Extensive recovery of respiratory motor function at chronic and super-chronic time points following

cervical spinal cord injury

Philippa M. Warren ............................................................................................................................... 66

29

Promoting neuroplasticity after spinal cord injury by over-expressing polysialic acid Louise Adams, Yi Zhang, Ping Yip, Adina Michael-Titus, John Priestley, Xuenong Bo Centre for Neuroscience & Trauma, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, UK

L.Adams@qmul.ac.uk

Polysialic acid (PSA) is a linear homopolymer of alpha 2,8-linked sialic acid. It is abundantly expressed in the CNS during development, where it is found closely associated with axon growth and synapse formation. Previous results from our laboratory noted the appearance of perineuronal nets (PNN) in the spinal cord was correlated with diminished expression of PSA. We postulate that over-expression of PSA in neurons around the spinal cord lesion site may modify the PNN structure and promote neuroplasticity and behavioural improvement in a rat cervical spinal cord injury model. To engineer expression of PSA in the spinal cord, we used a lentiviral vector to express polysialyltransferase under the control of the neuronal specific promoter human synapsin-1 (LV/PST). Lentiviral vector expressing GFP controlled by the same promoter (LV/GFP) was used as a control. Adult rats received a mid-cervical lateral hemisection, which was immediately followed by four injections of either LV/PST or LV/GFP rostral and caudal to the injury site. Motor recovery was assessed for six weeks using the Montoya staircase test, open field, grid exploration test and the Catwalk XT system. Anterograde tracing of the corticospinal tract was performed by injection of BDA into the motor cortex. LV/PST injection resulted in strong PSA expression on the surface of transduced neurons. Behavioural tests showed a tendency towards improved function in the LV/PST group compared with LV/GFP; however this did not reach significance due to the large variance among the animals. Whether PSA over-expression promotes sprouting of the corticospinal tract and the formation of new synaptic connections with the neurons on the lesioned side is still being examined. Supported by the International Spinal Research Trust (ISRT)

30

Regaining over-ground locomotor function following severe contusion injury with epidural stimulation and treadmill training

Yazi D. Al’joboori, Ronaldo M. Ichiyama School of Biomedical Sciences, University of Leeds, Leeds, UK

bsydaj@leeds.ac.uk

Spinal contusion injuries result in a loss of motor, sensory and autonomic function. Electrical epidural stimulation (ES) of the lumbar spinal cord (L2 to S1) has previously been shown to improve locomotor function in complete transection models of rat spinal cord injury in conjunction with monoaminergic and serotonergic agonists and bipedal locomotor training; however, ES has never been assessed in incomplete contusion lesions where some descending and ascending pathways remain. Here we demonstrate that the use of epidural stimulation (40 Hz; L2) and locomotor training following severe spinal contusion injury (T9/10) leads to improved locomotor function.

Adult Sprague-Dawley rats received a severe spinal contusion injury (T9/10) and epidural implantation at segmental levels L2 and S1 and were randomly assigned to one of four groups: cage control, training only, ES only or ES+training. Rats in either trained group stepped bipedally on a body weight supported treadmill (5-16 cm/s) (5 days/week, 20 mins/day) for 8 weeks. By the end of the 8-week period rats in the ES+training group showed improvements not only in supported treadmill stepping ability but also in open field locomotion (BBB), with combination treated animals achieving the highest overall increase in mean BBB score (12.5±1.5), significantly different from the improvement seen in cage control animals (8.2±0.6; *p-value 0.016). Therefore these results suggest that a combination of step training and epidural stimulation in an incomplete model of SCI successfully improved locomotor function further than either therapy administered alone; with animals not only improving in treadmill step performance but were also able to transfer this skill to an open field task which has not previously been observed in complete transection models. Support provided by the International Spinal Research Trust (ISRT) and Medical Research Council (MRC)

31

Remyelination of spinal axons by Schwann cells following spinal cord injury is a Neuregulin-1 dependent endogenous repair mechanism Katalin Bartus

1, Jorge Galino

2, John M. Dawes

2, Nicholas D. James

1, Florence R. Fricker

2, Andrew

N. Garratt3,4

, Matt S. Ramer5, Carmen Birchmeier

3,4, David L. H. Bennett

2 & Elizabeth J. Bradbury

1

1The Wolfson Centre for Age-Related Diseases, Regeneration Group, King’s College London, Guy’s Campus,

London Bridge, London, UK; 2Nuffield Department of Clinical Neurosciences, West Wing John Radcliffe Hospital,

Oxford, UK; 3Max Delbrück Center for Molecular Medicine, Berlin, Germany;

4Charité Universitätsmedizin Berlin,

Charitéplatz, Berlin, Germany; 5International Collaboration on Repair Discoveries, The University of British

Columbia, Vancouver, Canada

katalin.bartus@kcl.ac.uk

One of the spontaneous intrinsic regenerative responses following traumatic spinal contusion, which is the most common form of human spinal cold injury (SCI), is remyelination that is triggered by acute demyelination of spinal axons. However, this endogenous repair response is suboptimal and may account for the persistently compromised function of some surviving axons. This remyelination is largely mediated by Schwann cells, a phenomenon commonly observed in the chronically injured human spinal cord, where injured demyelinated axons become associated with peripheral myelin. This phenomenon is particularly prominent in the dorsal columns which contain long tracts of ascending myelinated axons that are known to undergo severe demyelination after SCI, followed by a degree of remyelination. It remains unknown what governs this Schwann cell-mediated remyelination of injured spinal axons. The growth factor neuregulin-1 (Nrg1) is known to play a key role of in virtually every phase of Schwann cell development and myelination within the peripheral nervous system (PNS), via signaling through ErbB tyrosine kinase receptors. Here we used a tamoxifen inducible Nrg1mutant mouse to determine the mechanisms controlling remyelination after spinal contusion injury. We examined whether Nrg1 is required for Schwann cell-mediated remyelination of dorsal column axons after spinal contusion injury, and whether Nrg1 ablation influences the degree of spontaneous remyelination and functional recovery. Conditional ablation of Nrg1 was associated with a complete absence of Schwann cells within the spinal cord and profound demyelination of spinal axons as determined by immunohistochemistry and electronmicroscopy. This was associated with a significant deficit in spontaneous locomotor recovery following spinal contusion as assessed by using the Basso Mouse Scale for hindlimb locomotor function. There was no compensatory oligodendrocyte remyelination and the majority of remyelinating Schwann cells appeared to originate from within the spinal cord. These findings provide novel mechanistic insight into the molecular signalling that governs spontaneous Schwann cell remyelination and repair after SCI. Supported by the International Spinal Research Trust (ISRT), Wings for Life, Medical Research Council, Wellcome Trust

32

Assessing functional recovery in the injured corticospinal tract: an optogenetic approach

Naveen Jayaprakash

1, Brian Hoeynck

1, Zimei Wang

1, Murray G. Blackmore

1

1Department of Biomedical Sciences, Marquette University, Milwaukee, WI, USA

murray.blackmore@marquette.edu

Spinal Cord Injury (SCI) impairs motor function as a consequence of axon transection and lost synaptic connections. A major goal in SCI research is to increase axon growth and restore synaptic connectivity. Axons in the spinal cord possess some endogenous capacity for compensatory plasticity, particularly in situations of partial injury, and various therapeutics have succeeded in evoking additional regenerative axon growth. For instance, we have shown that forced overexpression of two genes, Sox11 and KLF7, can increase corticospinal tract (CST) sprouting into contralateral spinal cord denervated by pyramidotomy, as well as regenerative growth after cervical hemisection. Behavioral changes, both positive and negative, have been correlated with this regenerative growth, but remain difficult to interpret. For instance, what are the contributions of direct CST connectivity by regenerated axons in the spinal cord, as opposed to alterations in upstream relays? A promising diagnostic tool in this regard is the use of optogenetics, which allows highly specific activation of neuronal populations that express light-sensitive Channelrhodopsin proteins. Accordingly, we are exploring optogenetic stimulation of murine CST neurons transduced with viral Channelrhodopsin (rAAV9/CaG-ChR2-EYFP). Electrophysiology has confirmed light-evoked cell firing in sensorimotor cortex that emerges one week after viral injection. By two weeks post-injection, direct stimulation of Channelrhodopsin-expressing terminals in the spinal cord evokes post-synaptic activity in spinal neurons. Importantly, because CST fibers are the only terminals in the spinal cord to express light-sensitive channels, this approach distinguishes direct cortical drive from potential relays. As expected, spinal responses to cortical stimulation are abolished acutely after dorsal hemisection of the spinal cord, which severs descending cortical axons. Moreover, spinal responses occur predominantly in tissue located contralateral to the treated cortex, and this laterality is maintained after a pyramidotomy injury. We are now assessing whether sprouting CST axons in KLF7- or Sox11-treated animals acts to restore direct synaptic connectivity in cervical hemisection or pyramidotomy injury models. Our preliminary data suggest that optogenetics may be a powerful approach to monitor the restoration of synaptic connections from specific neuronal populations after spinal injury. Supported by grants from the International Spinal Research Trust (ISRT), the Craig H. Neilsen Foundation, the Bryon Riesch Paralysis Foundation, and NINDS R01NS083983

33

Investigating functional plasticity and synaptogenesis following experimental spinal cord injury Emily R. Burnside

1, Federico Grillo

2, Karen D. Bosch

1, Stephen B. McMahon

1, Jonathon S. Carp

3,

Juan Burrone2, Elizabeth J. Bradbury

1 1Neurorestoration Group, Wolfson Centre for Age-Related Diseases, King’s College London, UK

2 Medical Research Council Centre for Developmental Neurobiology, Kings College London, UK

3 Wadsworth Center, New York State Department of Health, Albany, New York, USA

emily.burnside@kcl.ac.uk

The CNS has a poor intrinsic capacity for regeneration, although some functional recovery does occur. This is mainly in the form of sprouting, dendritic remodeling and changes in neuronal coding, firing and synaptic properties; elements collectively known as plasticity. Following spinal cord injury (SCI), a fundamental approach to repair the injured CNS is therefore to harness, promote and refine plasticity. This is partly limited by some components of the extracellular matrix, important inhibitory molecules which may be manipulated by therapeutics such as chondroitinase ABC. The corticospinal tract (CST) is an important descending motor pathway involved in locomotion, posture and voluntary skilled movements. Therefore regeneration and anatomical reorganisation of this projection is often examined in studies of experimental SCI. Techniques such as anterograde tracing have been combined with immunolabeling for synaptic proteins or electron microscopy to elucidate connectivity; however whether active synaptogenesis occurs following SCI and potential therapeutic manipulations has not been studied. Genetically encoded reporters of presynaptic activity represent novel tools to assess synaptogenesis and gain insight into the anatomical and functional status of new connections. Here we use an adeno-associated viral (AAV) vector encoding SynaptopHluorin (SpH), as a tracing tool before exploring its potential for measuring vesicular release and functional synaptogenesis. We present an ex-vivo acute cervical spinal slice preparation from adult rats, in which the CST has been transduced using AAV vectors encoding fluorescent probes reporting presynaptic activity or neurotransmitter release. We electrophysiologically stimulate the CST and use two-photon microscopy to real-time image fluorescence indicative of activity. We aim to perform this ex vivo preparation on rats which have undergone a unilateral pyramidotomy lesion, and in which the intact contralateral CST is transduced with the functional reporter. This will allow us to investigate functional synaptogenesis resulting from plasticity of a known, spared population of fibres following injury and furthermore how this may change following therapeutic delivery of plasticity-inducing therapeutics such as chondroitinase gene therapy. This work is supported by the MRC

34

Alpha9 integrin activation in neurite outgrowth and axon regeneration Menghon Cheah

1, Melissa Andrews

2, Daniel Chew

1, Joost Verhaagen

3, Reinhard Fässler

4, Andreas

Faissner5, James Fawcett

1

1John van Geest Centre for Brain Repair, University of Cambridge, Cambridge, UK

2School of Medicine, University of St Andrews, North Haugh, St Andrews, UK

3Netherlands Institute for Neuroscience, Amsterdam, NL

4Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, DE

5Department of Cell Morphology and Molecular Neurobiology, Ruhr University Bochum, Bochum, DE

mc747@cam.ac.uk

Spinal cord injury is a debilitating condition which results in serious neurological consequences due to the failure of axon regeneration in the central nervous system. Two recent separate studies in our laboratory have shown that α9 integrin (a cell adhesion receptor subunit) promotes neurite outgrowth on growth inhibitory tenascin-C, and kindlin-1 (a cytoplasmic integrin activator) overcomes the inhibition of chondroitin sulfate proteoglycans (CSPG) such as aggrecan by activating integrins to enhance axon growth. This study aims to combine the beneficial effects of both α9 integrin and kindlin-1 to promote better neurite outgrowth and axon regeneration. Adult rat dorsal root ganglion (DRG) was used as a model of study for both in vitro and in vivo. Significant neurite outgrowth was observed from neurons co-expressing both α9 integrin and kindlin-1 in cell culture. For the in vivo study investigating sensory axon regeneration, the virus AAV5-fGFP, AAV5-α9-V5 and AAV5-kindlin1-GFP were used to transduce DRGs in the lower cervical region after a hemilaminectomy and dorsal root crush. This was followed by ladder-walking, Randall-Siletto and Hargreave’s behavioural testing for 12 weeks post-injury. Electrophysiological recording showed significant recovery of axonal activity for the group of animals receiving both α9 integrin and kindlin-1 treatment for 12 weeks. Anatomical analysis also confirmed axon regeneration into dorsal root entry zone, dorsal horn and dorsal column; this was coupled with recovery in behavioural testing. Alpha9 integrin activation results in better neurite outgrowth and axon regeneration in the presence of both tenascin-C and CSPG after an axonal injury as α9 integrin is a receptor for tenascin-C and kindlin-1 overcomes the inhibition of CSPG. References Andrews et al, 2009. Alpha9 integrin promotes neurite outgrowth on tenascin-C and enhances sensory axon regeneration. J Neurosci. 2009 Apr 29;29(17):5546-57 Tan et al, 2012. Kindlin-1 enhances axon growth on inhibitory chondroitin sulfate proteoglycans and promotes sensory axon regeneration. J Neurosci. 2012 May 23;32(21):7325-35 Supported by Christopher and Dane Reeve Foundation, Cambridge Trusts, Yousef Jameel Academic Program

35

Olfactory ensheathing cell transplants improve vertical climbing in rats after cervical level dorsal root rhizotomy Andrew Collins, Sara Bowie, Daqing Li, Ying Li Spinal Repair Unit, Institute of Neurology, University College London, London, UK

Andrew.Collins@ucl.ac.uk

A brachial plexus injury (BPI) involves damage to spinal roots at the cervical level of the spinal cord. Such injuries most often result from road traffic accidents and can lead to sensory or motor impairment. Up to 90% of BPI patients also face permanent pain, described by some as a “burning and crushing” sensation on their arm

1. A lack of relevant preclinical models is one factor

behind the lack of effective treatments. Transplants of olfactory ensheathing cells (OECs) have evoked long distance axon

regeneration in thoracic level lesions and restored breathing in a high cervical injury model2,3

. A matrix method of transplantation was developed to ensure retention of the OEC transplant at the dorsal root entry zone

4. We sought to establish a rat model of dorsal root injury (DRI) which mimics both the

sensory impairment and pain aspect of a BPI. The effect of OEC transplants on these parameters would then be assessed.

Unilateral transection of C6, C7, C8 and T1 dorsal roots at the dorsal root entry zone (DREZ) led to a long-term forelimb sensory deficit. Proprioceptive function and forepaw tactile sensitivity were measured using vertical cage climbing and adhesive tape tests, respectively. Those rats which received an acute transplant of GFP-labelled OECs climbed better than controls at 7- and 8-weeks post injury, despite maintaining a deficit in forepaw tactile sensitivity. In terms of pain, a separate cohort of (control) rats showed no increased sensitivity to mechanical, thermal or cold stimuli after C6-T1 rhizotomy.

Immunohistochemical staining of the DREZ and dorsal horn revealed histological differences between ipsilateral and contralateral sides after C6-T1 rhizotomy. Markers for GFAP, laminin, CGRP, neurofilament and VGLUT were identified on transverse and longitudinal sections at various time points up to 8 weeks post-injury. Clear differences were apparent between intact and injured regions but further quantitative analysis is required to determine any potential effect of OECs.

A C7C8 dorsal root avulsion study is ongoing which is more likely to induce forepaw sensitivity to cold, mechanical or thermal stimuli due to greater vascular damage and cell loss at the dorsal horn

5. Specific preclinical models of cervical injury tailored to study either sensory impairment

or pain should allow us to assess the efficacy of OECs and optimize their use in patients. References 1. Waikakul S. et al. 2000 J Med Assoc 2 .Ramon-Cueto A. et al. 1992 J Neurosci 3. Li Y et al. 2003 J Neurosci 4. Li et al. 2004 Exp Neur 5. Chew DJ et. al 2008 Neurosci Lett Supported by Medical Research Council

36

Transcriptomic changes evoked by culturing Dorsal Root Ganglion neurons overwhelm those evoked by axonal injury Matthew C. Danzi

1, Dario Motti

1, John L. Bixby

1,2,3, and Vance P. Lemmon

1,3

1The Miami Project to Cure Paralysis, Departments of

2Molecular and Cellular Pharmacology,

3Neurological

Surgery, Miller School of Medicine, University of Miami, Miami, FL, USA

m.danzi@med.miami.edu

A necessary step toward functional recovery after spinal cord injury is axon regeneration. Unfortunately, Central Nervous System (CNS) axons regenerate poorly. In contrast, injured axons of the Peripheral Nervous System (PNS), such as those of Dorsal Root Ganglia (DRG), often succeed in regenerating long distances. For this reason, the regenerative capabilities of DRG neurons are commonly studied both in vitro and in vivo. We performed RNA-Seq on DRG neurons isolated from mice that had undergone a sciatic nerve crush, DRG neurons from mice that had undergone a sham surgery, and DRG neurons grown in culture, and characterized the transcriptomic similarities and differences among these three DRG sample groups. This characterization included ontological analysis of the pathways and biological processes overrepresented by gene isoforms differentially expressed by each of the samples relative to the others, analysis of the diversity of transcription start sites and examination of isoform expression frequency distributions. Additionally, we predicted the relative overrepresentation of different transcription factor binding sites among the isoforms differentially expressed in pairwise comparisons of each of the sample groups. Our analysis revealed that placing DRG neurons in culture had a significantly greater effect on their transcriptome than did a nerve crush carried out in vivo. Since DRGs in culture bear so little resemblance to their regeneration-competent or actively regenerating counterparts in vivo, our findings call into question the validity of using cultured DRG neurons as a model for gene expression by regeneration-competent neurons. This work was supported by the National Institutes of Health grants HD057521 (to V.P.L and J.L.B.), and NS059866 (to J.L.B.), DOD grant W81XWH-05-1-0061 (to V.P.L. and J.L.B.), State of Florida Specific Appropriation 538, the Buoniconti Fund and the Walter G. Ross Distinguished Chair in Developmental Neuroscience (to V.P.L)

37

Nanotechnology in neuroregeneration Suradip Das

1,, Manav Sharma

1, Dhiren Saharia

2, Kushal Konwar Sharma

3, Utpal Bora

1,4*

1Department of Biotechnology, Indian Institute of Technology Guwahati;

2 Saharia’s Path Lab & Blood Bank;

3 Department of Surgery and Radiology, College of Veterinary Sciences, Khanapara;

4Mugagen Laboratories

Private Limited, Technology Incubation Centre, IIT Guwahati, Guwahati, Assam, India

*Corresponding author – ubora@iitg.ernet.in, ubora@rediffmail.com

The application of nanotechnology for the development of advanced functional scaffolds for

neural regeneration has been predominantly restricted to the fabrication of nanofiber based scaffolds. Although forming nano-dimensional fibers through electrospinning remains the preferred fabrication method, some notable work has also been reported using self assembly of peptides to form nanofibers. Self assempbled peptide nanofibers (SAPNs) having sequence arginine, alanine, aspartate, and alanine (RADA)16 have been shown to support adhesion of neuronal cells (PC12) in vitro, enhancing neurite outgrowth as well as facilitating functional synapse formation (Holmes et al, 2000). In vivo administration of this peptide has shown success in brain lesion repair by promoting axonal regeneration and knitting the brain tissues together by forming nanofibers (Ellis-Behnke et al, 2006). The injectable sol-gel nature of SAPNs make them ideal for CNS interventions whereas electrospun nanofibrous scaffolds have been mostly used in fabricating implantable nerve growth conduits to facilitate peripheral nerve regeneration. Several natural materials like chitosan, laminin, silk fibroin, as well as synthetic polymers like PGA, PLA and composites like laminin-PLLA, collagen-PCL etc have been used as biomaterials to form nano-scaffolds by electrospinning technique. Electrically conductive nanomaterials like grapheme films and foams have been reported to support growth of mouse hippocampal cells as well as promote differentiaition of neural stem cells (NSCs) towards astrocytes and neurons (nf4, nn6). Carbon nanotube based scaffolds have also emerged as potential platforms promoting neural regeneration due to their ability to interface with neuronal circuits, synapses and facilitating conduction of nerve impulse. On the other hand, the use of nanoparticles in neurology has been largely limited to applications in drug delivery and diagnostics. There are very few reports on the use of nanoparticles for enhancing nerve regeneration.

In the present poster, we attempt to review the extent of nanotechnology interventions in solving neuro-regeneration problems as well as indicate the untapped potential of the field where future research could be directed.

38

Transduction of cells with a lentiviral vector encoding mammalian chondroitinase ABC in an in vitro model of neurite outgrowth: enhanced neurite outgrowth is accompanied by a drop in PTEN levels Priscilla Day

1, Joao Alves

2, James Fawcett

2, Roger Keynes

1, Elizabeth Muir

1

1Department of Physiology Development and Neuroscience, University of Cambridge, Cambridge, UK

2 John Geest Centre for Brain Repair, University of Cambridge, Cambridge UK

It is known that chondroitin sulphate proteoglycans (CSPGs) bind to some receptors also bound by myelin inhibitory proteins, and it is likely that they use some of the same signalling pathways to bring about inhibition of neurite outgrowth. Using SH-SY5Y cells differentiated with retinoic acid and DMEM containing 1% FCS and CSA at 75µg/ml, we show here that, in common with myelin-associated glycoprotein, digestion of chondroitin-4-sulphate (CSA) with chondroitinase via transduction of the cells with a recombinant lentivirus (LVChABC) encoding the modified chondroitinase transgene, results in a drop in cellular Phosphatase and tensin homolog (PTEN) mRNA and protein levels. This is accompanied by enhanced neurite outgrowth on an inhibitory substrate (CSA) compared to untransduced controls. Addition of the PTEN-specific inhibitor VO(OH)pic resulted in a similar increase in neurite outgrowth, an effect that was not further enhanced by transducing the cells with the ChABC vector, consistent with a role for PTEN in CSPG-based inhibition of neurite outgrowth. Staining of the cells for beta1 integrin showed that it is up-regulated in cells containing the ChABC transgene, suggesting that CSPGs, like myelin inhibitors, may negatively remodel integrins at the cells surface in a PTEN-dependent manner.

39

Regulation of IL10 by chondroitinase ABC promotes a distinct immune response following spinal cord injury Athanasios Didangelos, Michaela Iberl, Elin Vinsland, Katalin Bartus and Elizabeth J. Bradbury Wolfson Centre for Age Related Diseases, Guy’s Campus, King’s College London, London, UK

athanasios.didangelos@kcl.ac.uk

Abstract Chondroitinase ABC (ChABC) has striking effects on neuronal plasticity after spinal cord injury (SCI) but little is known about its involvement in other pathological mechanisms. Recent work from our lab showed that ChABC might also modulate the immune response by promoting M2 macrophage polarization (Bartus et al. 2014, Journal of Neuroscience, 34: 4822-36). Here we investigate in detail the immunoregulatory effects of ChABC following clinically-relevant SCI in rats. Initially, we examined the gene expression profile of 16 M1/M2 macrophage polarization markers at 3 hours and 7 days post-injury. ChABC treatment had a clear effect on the immune signature after SCI. More specifically, ChABC increased the expression of the anti-inflammatory cytokine IL10, accompanied by a reduction in the pro-inflammatory cytokine IL12B in injured spinal tissue. These effects were associated with a distinct, IL10-mediated anti-inflammatory response in ChABC-treated spinal cords. Mechanistically, we show that IL10 expression is driven by tissue injury and macrophage infiltration, while the p38 MAP kinase is the central regulator of IL10 expression in vivo. These findings provide novel insights into the effects of ChABC in the injured spinal cord and explain its immunoregulatory activity. Supported by Medical Research Council UK and Rosetrees Trust

40

The role of PKA in translating rehabilitative training after SCI into neuroplasticity Karim Fouad, C Hurd, D Wei, D Galleguillos, S Sipione University of Alberta, Edmonton, Alberta, Canada

Karim.fouad@ualberta.ca

It is well accepted that rehabilitative training promotes neuroplasticity and functional recovery following injuries of the central nervous system. One mechanism that could be involved in translating training and the subsequent neuronal activity into plasticity and recovery of function is the frequently reported up-regulation of BDNF. Another possible mechanism is via an activity induced up-regulation of cAMP levels, which would promote neurite outgrowth possibly via the activation of PKA. Earlier studies supported this idea by showing that electrical stimulation of a peripheral nerve increased intracellular cAMP levels and promoted neurite outgrowth in sensory fibers. We also reported that rehabilitative training can counteract (at least partially) the drop in PKA activation in cortical neurons following spinal cord injury. In our quest to show that PKA is a key player in translating training into neuroplasticity we intended to inhibit PKA activation in the motor cortex during a rehabilitative phase (i.e., single pellet reaching) in rats with a cervical spinal cord injury. However, as so often, the hypothesis was quickly disproven when we found increased CST plasticity and improved training induced functional recovery. It is noteworthy that we were able to repeat this result. Currently we are attempting to confirm that the PKA inhibitor rp-cAMP actually did inhibit PKA activation. This however is proofing to be a greater challenge than anticipated. If anything we are finding an increase of CREB phosphorylation, which is the opposite of the predicted effect. In conclusion, the current view on how neuronal activity translates into recovery is likely over simplified and other components of the pathway have to be explored. The promoted recovery may occur via other pathways that get strengthened by PKA inhibition or a potentially pro-inflammatory effect of the treatment. This study was supported by a grant of the Canadian Health Research Council

41

MicroRNA-155 deletion restricts inflammatory signaling in macrophages and enhances axon growth capacity: implications for spinal cord repair Andrew D. Gaudet

1,2, Philipp J. Schmitt

1,2, Xinyang Xu

1,2, Amelia Hargrove

1,2, David R. Sweet

1,2,

Zhen Guan1,2

, Mireia Guerau-de-Arellano2,3

, Phillip G. Popovich1,2

1 Center for Brain and Spinal Cord Repair,

2 Department of Neuroscience,

3 Health and Rehab Sciences,

Wexner Medical Center, The Ohio State University, Columbus, OH, USA

andrew.gaudet@osumc.edu

MicroRNAs (miRs) bind to various target mRNAs limiting their expression, and consequently their ability to encode proteins. Modifying expression of relevant miRs could elicit improved repair after spinal cord injury (SCI) by altering responses extrinsic and intrinsic to the neuron. Our lab showed that an intense and prolonged macrophage-dominant inflammatory response contributes to SCI pathology

1; therefore, discovering miRs that regulate macrophage phenotype could alter tissue

repair. In macrophages, miR-155 is a switch that initiates inflammatory cascades. miR-155 is upregulated by inflammatory signals (e.g., toll-like receptor activation) and can regulate translation of multiple target mRNAs to amplify inflammation.

We hypothesized that miR-155 deletion would improve macrophage-elicited axon growth and limit pathology. In addition, we explored whether miR-155 deletion from neurons would impact intrinsic neurite outgrowth capacity. First, we characterized the phenotype of cultured WT and miR-155 knockout (KO) mouse macrophages. miR-155 KO macrophages expressed lower inflammation-induced iNOS and higher levels of the anti-inflammatory marker Ym1. When WT or miR-155 KO macrophages were co-cultured with WT dorsal root ganglion (DRG) neurons, miR-155 KO macrophages enhanced neurite extension and the percent of neurons bearing neurites. miR-155 also had a neuron-intrinsic role; miR-155 deletion improved growth potential of DRG neurons. After contusive SCI, smaller lesions in the rostral extension of the contusion sites of miR155 KO mice correlated with reduced inflammatory macrophage density, although functional recovery was not improved in these mice.

These data indicate that miR-155 deletion could improve tissue repair through both neuron-extrinsic (macrophage phenotype) and –intrinsic (neuron growth capacity) mechanisms. Ongoing studies will determine whether miR-155 deletion impacts axon plasticity in vivo after SCI. References 1. Kigerl KA, Gensel JC, Ankeny DP, Alexander JK, Donnelly DJ, Popovich PG (2009) Identification of two distinct macrophage subsets with divergent effects causing either neurotoxicity or regeneration in the injured mouse spinal cord. J Neurosci 29: 13435-13444 Supported by a Canadian Institutes of Health Research (CIHR) Postdoctoral Fellowship (ADG), The Ray W. Poppleton Endowment (PGP), National Institutes of Health Grant R21NS081413 (PGP/MG), and International Foundation for Research in Paraplegia (IRP) Grant #P129 (PGP/MG).

42

Artificial Intelligence in Medical Systems Neurobiology: Finding a Treatment for Paralysis Baumgartner W. Jr.*

1, Waldera-Lupa D.M.*

2, Pape D.

3, Georgiev I.

1, Grichtchenko I.

1, Hunter L.

1,

Stühler K.#2

, Cohen K.#1

, Grimpe B.#3

1 Computational Bioscience Program, School of Medicine, University of Colorado at Denver, USA

2 Molecular Proteomics Laboratory, Biological-Medical Research Center, Heinrich Heine University Düsseldorf,

Germany 3 Applied Neurobiology, Medical Center of the Heinrich Heine University Düsseldorf, Germany

* = equal experimental performance # = equally supervising students

William.Baumgartner@ucdenver.edu, Daniel.Waldera@uni-duesseldorf.de, Daniel.Pape@med.uni-duesseldorf.de, ivogeorg@gmail.com, irina.grichtchenko@gmail.de, Larry.Hunter@ucdenver.edu, Kai.Stuehler@uni-duesseldorf.de, Kevin.Cohen@gmail.com, Barbara.Grimpe@med.uni-duesseldorf.de

Spinal cord injury (SCI) affects approximately three million people worldwide, and in spite of

over a century of research, no treatment is available. A potential solution to this problem lies in capturing the complexity of the underlying processes that lead to regeneration failure.

To address this problem we modify an existing systems biology tool, called Hanalyzer, to work within the SCI domain. We use this tool in conjunction with quantitative proteomics data obtained from contused rat spinal cords tissue collected at different time points after trauma in comparison to sham or unlesioned animals. The contusion injury model is justified because it is the most common trauma to the spine leading to paralysis in humans. The results are consolidated in a data network and associated with the knowledge network build within the Hanalyzer. This knowledge network is derived from a wide variety of biological data sources including natural language text. To facilitate text mining we defined a controlled vocabulary in order to extract the information contained in the SCI literature. Here, a carefully selected list of words and phrases, which consists of basic scientific as well as clinical vocabulary from the area of SCI and regeneration are used to tag units of information so that they are more easily retrieved by a computer. The next step is the generation of the first SCI ontology with the aim to annotate publications and databases from that domain.

The Hanalyzer is an example of a novel software tool, which deals with the problems of high throughput data analysis, as it combines reading, reasoning, and reporting (3R) methods to facilitate knowledge-based analysis of experimental data. The goal of the 3R system is to assist scientists in forming explanations of the phenomena in genome and proteome-scale data, and to generate significant hypotheses that can influence the design of future experiments. Supported by the German Federal Ministry of Education and Research (BMBF, 01GQ1206, BG), the National Science Foundation (NSF, LH) and National Institute of Health (NIH, 2T15LM009451, LH)

43

Does overexpression of Fibroblast Growth Factor Receptor 1 (fgfr1) in CNS neurons enhance axon regeneration and recovery after spinal cord injury in rats? Barbara Haenzi, Thomas Hutson, Sean Menezes, Claudia Kathe, Denise Duricki, Kat Gers-Barlag, Mary Bartlett Bunge, Lawrence Moon King’s College London, University of London, London, UK

barbara.haenzi@kcl.ac.uk

We have identified 500 genes which were up-regulated in adult spinal neurons that had regenerated an axon into a Schwann cell transplant placed in the injured rat spinal cord. These genes were screened to identify those which promote neurite outgrowth when overexpressed in CNS neurons cultured on inhibitory substrates. Fifteen regeneration associated genes (RAGs) were identified.

We are currently overexpressing one of the identified RAGs, fgfr1, in corticospinal axons in a model of rat spinal cord injury. Adeno-associated viral vectors encoding fgfr1 (or a control gene) linked via 2A to EGFP were injected into cortex and axonal regeneration and sensorimotor recovery will be measured after unilateral pyramidotomy. In vitro: I will overexpress fgfr1 in postnatal CNS neurons and investigate the cell signaling pathway in these cells in detail. I hope to identify candidates for co-overexpression to increase regenerative performance of spinal axons in vitro and in vivo. I will stimulate the pathway with different ligands and identify the ligand with the best effect. The most potent ligands will be used for in vivo stimulation of CNS axon regeneration. Furthermore, we have mutated important tyrosine residues of the fgfr1 receptor to dissect the downstream pathway most important for axon regeneration.

In summary, we aim to improve repair after spinal cord injury by overexpression of the fgfr1 receptor in corticospinal neurons. Supported by the Swiss National Foundation (BH), the Christopher and Dana Reeve Foundation (MBB) the Henry Smith Charity (TH and LM), the International Spinal Research Trust (ISRT) (CK and LM), and Wings For Life (LM)

44

AAV9-IL4 exacerbates a pathogenic systemic immune response that impairs functional recovery after contusive spinal cord injury Jodie C.E. Hall

1, 2; Kristina A. Kigerl

1, 2; Kevin D. Foust

1, 2; Alexander D. Roszman

1; Brian K. Kaspar

2,

3, Phillip G. Popovich

1, 2

1

Center for Brain and Spinal Cord Repair, 2Department of Neuroscience, Wexner Medical Center,

The Ohio State

University, Columbus, Ohio, USA 3

Center for Gene Therapy, The Research Institute at Nationwide Children’s Hospital, Columbus, Ohio, USA

Jodie.Hall@osumc.edu

Spinal cord injury (SCI) elicits both a local and systemic immune response, which results in macrophage accumulation at the injury site. The composition of activation stimuli at sites of inflammation determines their phenotype and function. Classically activated ‘M1’ macrophages predominate after SCI and have neurotoxic effector functions. Conversely, alternatively activated ‘M2’ macrophages are not toxic and augment regenerative growth in adult sensory axons, but these cells occupy the lesion site only transiently

1. Interleukin-4 (IL4) is a cytokine that causes newly activated

macrophages to differentiate into M2 cells1. We have created a viral vector engineered to produce

IL4, a non-invasive tool that reprograms macrophages at the injury site. Our hypothesis is that AAV9-IL4 will promote differentiation of intraspinal macrophages toward an alternatively activated phenotype, limiting intraspinal pathology and promoting functional recovery.

Two hours after a moderate (75 kDyn) or mild (60kDyn) mid-thoracic contusion SCI, female C57BL/6 mice were injected (i.v.) with an adeno-associated viral (AAV9) vector engineered to produce IL4 (AAV9-IL4) or GFP (AAV-GFP) under the control of a cytomegalovirus (CMV) promoter (1 x 10

9-11 vg/mouse). Open field locomotor function was assessed regularly, blood was collected and

mice were perfused and tissues processed for western blot or histology. Our data show that AAV9-IL4 causes widespread intraspinal transduction and a significant

shift in macrophage phenotype at the injury site (measured using M1 and M2 markers). However, counter to our hypothesis, lesion pathology is exacerbated and functional recovery is impaired relative to mice injected with AAV9-GFP. Similar results were obtained after titrating the dose or severity of SCI. We suspected that systemic complications caused by SCI were synergizing with the immune-modulatory effects of IL4. Indeed, innate and adaptive immune responses elicited by SCI arise in secondary lymphoid tissues (outside the CNS) and the effects of i.v. AAV9 will manifest throughout the body (non-specific tropism). New data show that AAV9-IL4 stimulates IL4R expression on circulating leukocytes and causes leukocytosis. Previously, we found that contusive SCI activates pathogenic autoreactive B cells

2. Since IL4 is a B cell growth factor, AAV9-IL4 could exacerbate a

systemic autoimmune response elicited by SCI. Our data show that IL4 dependent signaling via AAV9-IL4 is enhanced after SCI and exacerbates SCI-induced splenomegaly. These changes in the periphery correlate with increased numbers of intraspinal B cells and anti-CNS antibodies found in AAV9-IL4 SCI mice. Thus, post-injury intravenous injection of AAV9-IL4 redirects the intraspinal inflammatory environment to an alternative macrophage phenotype, but it also enhances pathogenic autoantibody synthesis and exacerbates functional recovery.

Future work will explore intraspinal IL4 injection, use of a second-generation AAV9 vector in which IL4 production is controlled by a GFAP promoter (limit synthesis to CNS) and evaluate axon/glial interactions within the M2 dominant lesion foci of AAV9-IL4 injected mice. References 1. Kigerl KA, Gensel JC, Ankeny DP, Alexander JK, Donnelly DJ, Popovich PG (2009) Identification of Two Distinct

Macrophage Subsets with Divergent Effects Causing either Neurotoxicity or Regeneration in the Injured Mouse Spinal Cord. J Neurosci 29: 13435-13444

2. Ankeny DP, Guan Z, Popovich PG (2009) B cells produce pathogenic antibodies and impair recovery after spinal cord injury in mice. J Clin Invest 119(10): 2990-9

Supported by the International Spinal Research Trust (ISRT) and the Craig. H Neilsen Foundation

45

Human neural progenitors transplanted to the deaffarented murine spinal cord promote regeneration of functional sensory fibers Jan Hoeber

1, Carl Trolle

1, Niclas König

1, Allessandro Gallo

2, Emmanuel Hermans

2, Ronald

Deumens2, Elena Kozlova

1

1Regenerative Neurobiology, Department of Neuroscience, Biomedical Centre, Uppsala University, Sweden

2Neuropharmacology, Pole Cellular and Molecular, Institute of Neuroscience, Université Catholique de Louvain,

Belgium

jan.hoeber@neuro.uu.se

Dorsal root avulsion (DRA) leads to a loss of sensorimotor functions due to deaffarentation and direct injury of the dorsal horn (Carlstedt 2008). Therapies for the recovery of sensorimotor functions in DRA need to address two major issues, first avulsed dorsal root axons fail to regenerate across the CNS-PNS border and second to prevent the loss of second-order neurons in the dorsal horn (Chew et al. 2008). Human embryonic cell derived neuronal progenitors (hNPE) have been shown to promote regeneration of axons when transplanted into the transected spinal cord (Erceg et al. 2010). Recently, we showed that murine neural progenitors are able to survive, migrate and differentiate into different neuronal subtypes when transplanted to the junction between avulsed dorsal root and injured dorsal root transitional zone (DRTZ) (König et al. 2014). By combining our stem cell transplantation approach in a model of DRA of lumbar spinal roots 3-5 (L3-L5 DRA) with the regenerative abilities of hNPE, we aimed to test the ability of hNPE to support regeneration of sensory fibers across the CNS-PNS border and their potential for the recovery of sensorimotor functions. hNPE were generated from human embryonic stem cells and expressed markers typical for a spinal neuronal lineage of neuronal progenitors. 3 month after transplantation to the site of spinal cord injury caused by L3-L5 DRA, hNPE were found to differentiate into interneurons and inhibitory nerve cells. Further, transplantation of hNPE led to the ingrowth of myelinated sensory axons. Sensory axons regenerated across the region of hNPE transplantation and grew back into the dorsal horn. In long term behavioral experiments, L3-L5 DRA animals showed a severe loss of hind limb sensitivity and grip strength. In contrast, hNPE treated animals showed both improved sensitivity and strength. Rhizotomy peripheral to the region of hNPE transplantation completely abolished the observed improvement. Taken together, hNPE transplantation to the site of L3-L5 DRA resulted in good survival and differentiation of transplanted cells and induced regeneration of functional sensory axons, resulting in the recovery of sensorimotor functions in the dorsal root avulsion model. References Carlstedt, T. (2008). Root repair review: basic science background and clinical outcome. Restorative neurology and neuroscience, 26(2), 225-241 Chew, D. J., Leinster, V. H., Sakthithasan, M., Robson, L. G., Carlstedt, T., & Shortland, P. J. (2008). Cell death after dorsal root injury. Neuroscience letters, 433(3), 231-234 Konig, N., Trolle, C., Kapuralin, K., Adameyko, I., Mitrecic, D., Aldskogius, H., Shortland P. J. & Kozlova, E. N. (2014). Murine neural crest stem cells and embryonic stem cell‐derived neuron precursors survive and differentiate after transplantation in a model of dorsal root avulsion. Journal of tissue engineering and regenerative medicine. [Epub ahead of print] Erceg, S., Ronaghi, M., Oria, M., Roselló, M. G., Aragó, M. A. P., Lopez, M. G., Radojevic, I., Moreno-Manzano, V., Rodríguez-Jiménez, F. J., Bhattacharya, S. S., Cordoba, J. & Stojkovic, M. (2010). Transplanted oligodendrocytes and motoneuron progenitors generated from human embryonic stem cells promote locomotor recovery after spinal cord transection. Stem Cells, 28(9), 1541-1549 This work was supported by the Swedish Research Council, proj no. 20716, Stiftelsen Olle Engkvist Byggmastare, Signhild Engkvist’s Stiftelse and the Swedish Institute’s Visby program Dnr 00613/2011

46

Do severity and duration of compression impact on recovery after severe acute spinal cord injury in dogs? Hilary Hu, Nick Jeffery College of Veterinary Medicine, Iowa State University, Ames, USA

hilaryhu@iastate.edu

Evidence from laboratory experiments suggests that recovery after spinal cord injury (SCI) is adversely affected by more prolonged and severe compression.

1 This suggests that it would be

beneficial for human SCI patients to undergo decompressive surgery as early as possible, but this suggestion remains controversial.

2,3 SCI is very common in domestic pet dogs (because intervertebral

disc herniation occurs with high prevalence), providing an ideal model in which effects of various variables that impact on SCI in human patients can be investigated.

In this study we prospectively recruited dogs that had incurred thoracolumbar SCI that had abolished all motor and sensory function to the hindquarters (i.e. equivalent to ASIA ‘A’ patients). We recorded historical data that indicated the duration of cord injury and the dogs then underwent routine imaging and decompressive surgery (which in dogs is routinely carried out as soon as possible for this category of SCI). Maximal severity of cord compression in each dog was measured on cross sectional MR and CT images Dogs were followed up until they recovered independent ambulation or for three months from surgery (which in dogs is equivalent to ~12 months in humans in terms of functional recovery). Logistic regression was used to evaluate the relationship of recovery with duration and severity of compression and Cox regression used to analyze variable relationships with rapidity of recovery.

78 cases were available for full analysis. Our data revealed that the delay from onset of paraplegia till surgical intervention had no detectable effect on the likelihood of recovery. There was weak evidence that more severe compression was associated with greater likelihood of recovery – and even weaker evidence that slower onset of clinical signs prior to onset of paraplegia also had the same effect. There was no evidence for an interaction between severity of compression and delay between onset of paraplegia and surgery. In the second series of analyses we determined that, in animals that recovered after surgery, more severe compression and longer duration of clinical signs prior to onset of paraplegia were associated with more rapid recovery.

We conclude it is likely that, in this type of SCI in which an impact injury is followed by prolonged spinal cord compression of varying severity, the prognosis appears to be largely defined by the magnitude of the initial mechanical injury and that early decompressive surgery has no detectable benefit. However, for cases in which the initial injury has not caused cross sectionally complete destruction of the spinal cord, decompressive surgery within the first few days after injury may play a role in reversing the clinical signs and accelerating functional recovery. References 1. Dimar JR 2nd, Glassman SD, Raque GH, et al. The influence of spinal canal narrowing and timing of decompression on neurolgic recovery after spinal cord contusion in a rat model. Spine 1999;24:1623-33 2. Fehlings MG, Vaccaro A, Wilson JR, et al. Early versus delayed decompression for traumatic cervical spinal cord injury: results of the Surgical Timing in Acute Spinal Cord Injury Study (STASCIS). PLoS One 2012;7:e32037 3. van Middendorp JJ. Letter to the editor regarding: "Early versus delayed decompression for traumatic cervical spinal cord injury: results of the Surgical Timing in Acute Spinal Cord Injury Study (STASCIS)”. Spine J 2012;12:540 Supported by Hilary Hu is a graduate student funded through a grant from the International Spinal Research Trust (ISRT) to investigate the effect of intralesional chondroitinase ABC on functional outcome after severe SCI in dogs.

47

Sympathetic and sensory sprouting after spinal cord injury: peripheral consequences of central injuries Diana V. Hunter, L.M. Ramer, M.A. Crawford, P.A. van Stolk, S.B. McMahon, M.S. Ramer

ICORD, University of British Columbia, Vancouver, Canada and Wolfson Centre for Age-Related Diseases, King’s College London, London, UK dianavhunter@gmail.com

Though motor system deficits may be the most outwardly apparent challenge after spinal cord injury (SCI), dysfunction of the sensory and autonomic systems can result in an equal if not greater impact on quality of life. Numerous studies have described injury-induced changes in sympathetic and sensory pathways within the spinal cord itself, however less is known about how the peripheral components of these pathways change and impact function after SCI. This work characterizes the peripheral changes that occur within sensory (dorsal root ganglia; DRGs) and sympathetic ganglia, with specific interest in the peripheral sprouting of these two neuronal populations. To examine the changes in these two systems after SCI, a complete transection at the third thoracic (T3) level was performed in adult male Wistar rats and both sensory and sympathetic ganglia were collected and examined after one, two or four weeks. The density of sympathetic fibers within the DRGs rostral (T1), caudal (T5, T10), and far distal (L1-S1) to the injury was analyzed immunohistochemically using antibodies against tyrosine hydroxlase (TH). Sympathetic ganglionic axons invaded the cell layer of the DRG within one month of SCI. The density of the TH-expressing axons in DRGs was significantly increased in the SCI animals at one month when compared to sham-injured controls. Interestingly, sympathetic sprouting was most pronounced in DRGs far distal to SCI. To examine sensory sprouting in sympathetic ganglia, major pelvic ganglia, mixed sympathetic/parasympathetic ganglia of the pelvic regions, were also collected and processed for markers of sensory axons. The changes occurring within these two types of peripheral ganglia indicate that dysfunction after SCI may be mediated by factors outside of spinal plasticity and the central nervous system. Further study into the crossing of the sympathetic and sensory systems may shed light into both sensory and autonomic dysfunction such as neuropathic pain and autonomic dysreflexia. Supported by the International Spinal Research Trust (ISRT), ICORD and NSERC

48

Chondroitinase gene therapy as a treatment for spinal cord injury Nicholas D. James, Katalin Bartus, Karen D. Bosch, John H. Rogers, Bernard L. Schneider, Joost Verhaagen, Elizabeth M. Muir, Elizabeth J. Bradbury King’s College London, Regeneration Group, The Wolfson Centre for Age-Related Diseases, London, UK

nicholas.d.james@kcl.ac.uk

Spinal cord extracellular matrix is densely packed with growth inhibitory chondroitin sulphate proteoglycans (CSPGs), which become more abundant after injury. Thus, matrix modification has become a leading experimental strategy for promoting repair following spinal cord injury. Despite the beneficial effects that have been achieved by digesting CSPGs with the bacterial enzyme chondroitinase ABC (ChABC), the potential for achieving long term efficacy in traumatic injuries that mimic a human spinal cord injury has been limited, due to suboptimal delivery methods and issues of enzyme instability. However, we have recently demonstrated that gene therapy, using a mammalian compatible ChABC gene, offers a route to achieving stable and continuous delivery of ChABC, resulting in a dramatic reduction in pathology and significant improvements in functional recovery when used to treat a clinically relevant spinal contusion injury model in adult rats

1. Following on from

these findings we now demonstrate the efficacy of chondroitinase gene therapy in contusion injury models at differing spinal levels (cervical and thoracic) and of differing severities. When used to treat a contusion injury at either C5 or T10 spinal level, ChABC gene therapy resulted in increased spinal conduction through the injury epicenter, improved functional performance in skilled locomotion, significant neuroprotection and enhanced plasticity of intact spinal circuitry. Further to this, we present findings from recent experiments in which we have assessed the efficacy of different viral vectors (both adeno-associated viral vectors and lentiviral vectors) containing the modified ChABC gene in order to determine the optimal vector structure to be used for ChABC gene therapy. We find that the use of different promoters results in differing patterns of ChABC expression, due to which cell types are transduced. Furthermore, the timing and pattern of expression affects the efficacy of ChABC gene therapy. The use of a PGK promoter primarily leads to transduction of neuronal cells and axons, resulting in widespread CSPG degradation throughout the spinal cord and the most dramatic improvements in functional and anatomical outcome measures. Thus, we demonstrate the therapeutic potential of ChABC gene therapy to treat clinically relevant injury models at different spinal levels and present findings on optimizing the delivery of chondroitinase gene therapy. References Bartus, K, James, ND, Didangelos, A, et al. Large-Scale Chondroitin Sulfate Proteoglycan Digestion with Chondroitinase Gene Therapy Leads to Reduced Pathology and Modulates Macrophage Phenotype following Spinal Cord Contusion Injury. J Neurosci. 2014; 34:4822-4836 Supported by the U.K. Medical Research Council, the International Spinal Research Trust (ISRT), the Henry Smith Charity and the International Foundation for Research in Paraplegia

49

Combinatorial treatment with GSK3β inhibitors and chondroitinase ABC to regulate glial scar formation and promote axon regeneration in the spinal cord Ashik Kalam

1,2, Athanasios Didangelos

2, Katalin Bartus

2, Andrea Rivera

1, Nicholas James

2,

Elizabeth Bradbury2, Arthur Butt

1

1 Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, Portsmouth, UK

2 Wolfson CARD, Guy’s Campus, King’s College London, London, UK

ashik.kalam@myport.ac.uk

The glial scar is the key reason for the failure of axonal regeneration in the adult CNS and acts as a physical and biochemical barrier for growing axons.

1 Functional regeneration in the spinal

cord is thus limited by the glial scar inhibiting axonal growth. Therefore, modification of the glial scar to a more growth permissive one is necessary for any regeneration to occur. Lithium and small molecule inhibitors of GSK3β has been shown to trigger profound changes in the astroglial cytoarchitecture in the optic nerve and promote neuronal survival and neurite outgrowth in vitro and axonal sprouting following spinal cord injury (SCI).

2,3 Hence, GSK3β is a

potential target for regulating astrogliosis and stimulating the formation of a potential scaffold for regenerating axons. Moreover, the glial scar contains chondroitin sulphate proteoglycans (CSPGs) that inhibit axon growth following SCI. Enzymatic digestion CSPGs with chondroitinase ABC (ChABC) has been shown to disrupt the glial scar and promote axon sprouting and partial recovery of function.

4,5 Therefore, the gliomorphic effects of GSK3β inhibitors combined with enzymatic removal

of CSPGs could have a synergistic effect on promoting regrowth of axons, reformation of connections and recovery of function in SCI. The aim of this collaborative project is to determine the effects of a combinatorial therapy of GSK3β inhibition and ChABC on glial scar formation and axon regeneration, using multiple techniques and models, including an in vitro scratch assay as a model for astroglial scar formation, ex vivo organotypic cultures of spinal cord and optic nerve, established rodent models of SCI, and genomic, biochemical, histological, electrophysiological and behavioural analyses. References 1. Sandvig et al. (2004) Glia. 46(3):225-51 2. Azim K and Butt AM. (2011) Glia. 59(4):540-53 3. Dill J et al (2008). J Neurosci. 28(36):8914-28 4. Bradbury EJ et al. (2002) Nature. 416(6881):636-40 5. Bartus K et al. (2014) J Neurosci. 34(14):4822-36 Supported by the Nathalie Rose Barr studentship award from the International Spinal Research Trust (ISRT)

50

Optimizing and understanding the use of intracellular sigma peptide as a translatable therapeutic for spinal cord injury Bradley T. Lang

1, Jared M. Cregg

1, Marc DePaul

1, Amanda Tran, Benjamin Brown

1-2, Sarah A.

Busch1, Yingjie Shen

3, Jerry Silver

1

1

Case Western Reserve University, Department of Neurosciences, Cleveland, OH, USA 2

Baldwin Wallace University, Berea, OH, USA 3

The Ohio State University, Center for Brain and Spinal Cord Repair, Department of Neuroscience, Columbus, OH, USA

BTL21@case.edu

Regeneration and sprouting following spinal cord injury is curtailed by several processes, with

the inhibitory chondroitin-sulfate proteoglycan (CSPG)-rich glial scar and perineuronal net being major impediments. We designed a small membrane-permeable peptide modulator of the CSPG receptor PTPσ (Intracellular Sigma Peptide, ISP), which was capable of blocking CSPG-mediated inhibition in vitro

1-3. Delivered systemically over several weeks, ISP treatment restored coordinated walking and

urinary function following contusive SCI in a large percentage of animals. We sought to further characterize the mechanism by which ISP regulates PTPσ function in order to devise strategies toward optimizing treatment. We previously determined that ISP binds to the intracellular domain of PTPσ. Using in silico techniques, we identified a potential binding pocket for ISP near the wedge domain of PTPσ. ISP truncation and alanine substitution analysis using an in vitro CSPG gradient assay provided experimental evidence for this in silico finding. We are currently performing the necessary point mutation studies to confirm this binding site.

Interestingly, in addition to binding rat and mouse PTPσ, ISP also bound human recombinant PTPσ. Therefore, we tested whether ISP treatment could overcome CSPG inhibition in more clinically relevant human neurons. Human induced pluripotent neurons had severely diminished outgrowth on a CSPG rich substrate vs a laminin only control substrate. Importantly, treatment with Ch’ABC as well as ISP restored outgrowth of human neurons to that of laminin levels.

Finally, we sought to optimize the dose of ISP in vivo. Following a T8 contusion injury (Infinite Horizon Impactor, 250kDyne), we treated animals daily with increasing doses of ISP, from 3.3μg to 44μg, for 7 weeks (n=5/dose). Escalating ISP to 44μg/day (4x of previous dose) led to a dramatic improvement in urinary function, with all animals recovering function. This suggests that increasing ISP efficacy, either through increased concentration or possibly via more direct delivery paradigms, may further enhance urinary recovery. Interestingly, locomotor recovery, as measured by open field BBB and gridwalk tests, was not dose dependent in our small sample of animals. A larger number of animals may be required to show a dose response in these locomotor behaviors. We hypothesize that combinatorial therapies in conjunction with ISP, including a variety of neuroprotective and rehabilitation strategies, will be necessary to promote maximal recovery. Our data provide strong verification of the role of CSPGs and PTPσ in regeneration/sprouting failure following neurological trauma. References 1. Shen, Y., A. P. Tenney, et al. (2009). "PTPsigma is a receptor for chondroitin sulfate proteoglycan, an inhibitor of neural regeneration." Science 326(5952): 592-6 2. Lang, B.T., Cregg, J.M, et al (2014). “Systemic modulation of the proteoglycan receptor PTPσ promotes functional recovery after spinal cord injury” Under Review 3:.Gardner, R. T., L. Wang, et al. (2014). "Targeting protein tyrosine phosphatase σ after myocardial infarction restores cardiac sympathetic innervation and prevents arrhythmias." Under Review Supported by NINDS NS25713 (JS); Case Western Reserve University Council to Advance Human Health (CAHH), Unite to Fight Paralysis, The Brumagin Memorial Fund, Spinal Cord Injury Sucks (SCIS), and United Paralysis Found

51

Early intravenous delivery of mesenchymal progenitor cells modulates the secondary inflammatory response after cervical spinal cord injury leading to behavioral and pathological amelioration Seok Voon Lee

1,3, Chris Czisch

1, Yingxiang Huang

2, May H. Han

2, Alan R. Harvey

3, Giles W. Plant

1

1Department of Neurosurgery, Stanford University, Stanford, CA, USA

2Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA, USA

3School of Anatomy, Physiology and Human Biology, The University of Western Australia, Perth, Australia

svlee@stanford.edu

Cellular transplantation strategies utilizing mesenchymal progenitor cells (MPCs) have previously been reported to be beneficial in spinal cord injury (SCI). Methods of transplantation include intraspinal, intrathecal and intravenous (IV) injection. Our previous data have shown efficacy of intraspinal injection of human MPCs into rat thoracic SCI

1, 2. However, intraspinal injection is

technically challenging, especially in an unstable environment after an injury, thus we have investigated the feasibility and benefits of IV injection of MPCs in a cervical contusion SCI injury model. Compact bone MPCs were isolated from GFP-luciferase transgenic mice

3. Co-culture data of

the MPCs with splenocytes indicate that MPCs enhance anti-inflammatory and decrease pro-inflammatory cytokine expression. SCI (unilateral C5, 30kdy, 3s dwell; IH impactor, NY) was performed on adult female FVB mice and 1x10

6 MPCs in 300μL HBSS or 300μL HBSS were IV

injected via tail vein at D1, D3, D7, D10 or D14 after injury (n=5 per group). After injection, MPCs were tracked using bioluminescence. Live in vivo imaging data showed that 24hrs after IV injection, MPCs tracked to the lungs and remained there. After 72hrs, minimal luciferase signal was detected in the lungs and did not appear elsewhere. This was irrespective of when the MPCs were injected following injury. Terminal bioluminescence tracking of the MPCs confirmed that cells tracked to the lungs and no other major organ, and were cleared within 7 days. Comparison of naïve versus injured mice showed that MPCs were cleared more rapidly within 24hrs in the injured animals. Behavioral testing by cylinder test recorded at D7, D21, D35 and D49 showed that animals receiving IV injection of MPCs at D1 and D3 after injury had significant amelioration compared to their control counterparts (p<0.05). No improvement was seen at the other injection time points. Mice were sacrificed at 8 weeks after injury. Tissue analysis showed that both D1 and D3 IV injection of MPCs resulted in smaller lesion size compared to their controls but no difference was observed at the other time points. Immunohistochemical analysis revealed less scarring and vascularization only at the D1 and D3 IV MPC injection time points. An additional study where SCI mice received IV injection of MPCs or HBSS at D1 after injury and sacrificed at 6hrs following IV injection showed that the beneficial effect of the IV MPC injection occurred almost immediately. These findings suggest positive modulation of secondary inflammatory responses after the initial mechanical injury. In summary, IV injection of MPCs is neuroprotective in terms of clinical amelioration shown by behavioral testing and histopathological changes in the damaged spinal cord. The timing of MPC delivery is crucial to achieve this benefit. References 1Hodgetts SI, Simmons PJ, Plant GW (2013). Exp Neurol 248:343-59

2Hodgetts SI, Simmons PJ, Plant GW (2013). Cell Transplant 22:393-413

3Short BJ, Brouard N, Simmons PJ (2009). Methods Mol Biol 482:259-82

This work is supported by the Saunders Family Neuroscience Fund, James Doty Neurosurgery Fund, Stanford Neuroscience Institute, and an International Postgraduate Research Scholarship from The University of Western Australia

52

Facilitating reproducibility and data integration for SCI research with MIASCI and RegenBase Vance P. Lemmon

1, Alison Callahan

2, Kunie Sakurai

1, Saminda W. Abeyruwan

3, Adam R.

Ferguson4, Phillip G. Popovich

5, Ubbo Visser

3, John L. Bixby

1,

1Miami Project to Cure Paralysis, University of Miami School of Medicine, Miami, FL, USA

2Stanford Center for Biomedical Informatics Research, Stanford University, USA

3Department of Computer Science, University of Miami, Coral Gables, FL, USA

4Brain and Spinal Injury Center (BASIC), Department of Neurological Surgery, University of California, San

Francisco, San Francisco, CA, USA 5Center for Brain and Spinal Cord Repair and the Department of Neuroscience, The Ohio State University,

Columbus, OH, USA

VLemmon@med.miami.edu

The lack of reproducibility in many areas of science, including spinal cord injury (SCI) research, is due in part to the lack of common reporting standards. Over the past three years an ad hoc consortium of scientists has developed a minimum information reporting standard for SCI, called Minimum Information About an SCI Experiment (MIASCI, J Neurotrauma. 2014 Jul 11. PMID: 24870067). Version 1.0 of the MIASCI contains 11 sections: investigator, organism, surgery, perturbagen, cell transplantation, biomaterials, histology, immunohistochemistry, imaging, behavior, and data analysis and statistics. Each section has a number of data elements to be filled in that detail essential metadata about the project, materials and methods. Depending on a particular study, not all sections will apply. The purpose of MIASCI is to improve transparency of reporting and to encourage the use of best practices. A secondary benefit is to facilitate the aggregation and automated interrogation of diverse datasets using a formal standard language. Thus, a parallel effort is underway to develop an ontology about SCI: the RegenBase ontology. Expanding RegenBase by incorporating MIASCI concepts facilitates paper curation and knowledge creation. We present MIASCI concepts, show integration with the RegenBase Ontology and present different approaches to paper annotation. Querying the RegenBase knowledgebase using the integrated ontology will also be illustrated. Acknowledgments NINDS NS080145 and NICHD HD057632

53

Expression of a hyperactive transcription factor increases axon growth and regeneration Saloni T. Mehta

1, Xueting Luo

1, Tatiana I. Slepak

1, Kevin K. Park

1,3, John L. Bixby

1,2,3, Vance P.

Lemmon1,3

1The Miami Project to Cure Paralysis, Departments of

2Molecular and Cellular Pharmacology,

3Neurological

Surgery, University of Miami Miller School of Medicine, University of Miami, Miami, FL, USA

s.mehta8@med.miami.edu

Axonal regeneration after spinal cord injury (SCI) is intrinsically and extrinsically inhibited by multiple factors. One major form of intrinsic inhibition of axon regeneration is the altered expression of regeneration-associated transcription factors in mature neurons of the central nervous system (CNS); these factors fail to be activated post-injury. A gene expression study of regeneration-capable peripheral nervous system (PNS) neurons

1 identified candidate transcription factors that could

potentially, if expressed in CNS neurons, promote axon growth and regeneration. Of these, signal transducer and activator of transcription 3 (Stat3) showed a significant upregulation in PNS neurons compared to CNS neurons. To maximize gene transcription and, potentially, neurite outgrowth and axon regeneration, a constitutively active variant of Stat3 (Stat3CA) was fused with a viral transcriptional activation domain (VP16). VP16 “hyper-activates” transcription factors by recruiting transcriptional co-factors to the DNA binding domain. This VP16-Stat3CA chimera significantly increased neurite outgrowth when expressed in rat cortical neurons in vitro. Furthermore, when virally transduced into retinal ganglion cells (RGCs) in vivo, VP16-Stat3CA led to increased axon regeneration in the optic nerve. These findings indicate that hyperactivation and overexpression of transcription factor Stat3, which is downregulated in the adult CNS, can promote axon regeneration after injury. References 1Smith RP, Lerch-Haner JK, Pardinas JR, Buchser WJ, Bixby JL, Lemmon VP. Mol Cell Neurosci. 2011;46:32–44

This work was supported by the National Institutes of Health grants HD057521 (to V.P.L), and NS059866 (to J.L.B.), DOD grant W81XWH-05-1-0061 (to V.P.L. and J.L.B.), State of Florida Specific Appropriation 538, the Buoniconti Fund and the Walter G. Ross Distinguished Chair in Developmental Neuroscience (to V.P.L)

54

Daily acute intermittent hypoxia following cervical spinal cord injury Kristiina Negron, Tanya Bezdudnaya, Victoria Spruance, Timothy Whelan, Michael Lane Department of Neurobiology, Drexel University College of Medicine, PA, USA

Kmn67@drexel.edu

Among the wide range of motor and sensory deficits that arise following cervical spinal cord injuries (SCIs), impaired breathing remains one of the most devastating. Cervical SCIs typically result in ventilator-dependence, thus increasing the risk of life-threatening secondary complications and greatly impairing quality of life. Respiratory dysfunction primarily results from a compromise to the phrenic motor system which controls the diaphragm – the primary respiratory muscle. While there is some spontaneous functional neuroplasticity, the extent of recovery is limited and significant respiratory deficits persist. The goal of the present research is to develop rehabilitative strategies capable of harnessing respiratory plasticity and improving long-term functional recovery.

Previous work has identified that phrenic motor activity and respiratory plasticity can be enhanced by repeated, intermittent exposures to hypoxia. While hypoxia stimulates increases in respiratory activity, repetitive exposures to hypoxia can elicit phrenic motor facilitation. Several studies have shown that intermittent hypoxia can be used to promote recovery of phrenic motor function following high cervical hemisection. Whether such increases in respiratory activity can be used to therapeutically enhance phrenic recovery following cervical contusion injury is not well defined. The present work begins to address this gap in knowledge by testing whether daily acute intermittent hypoxia (dAIH) can amplify respiratory plasticity and enhance recovery one week following experimental contusion injury.

Adult female Srague Dawley rats received a lateralized mid-cervical (C3-4) contusion injury (Infinite Horizon pneumatic impactor; intended impact force of 200 kilodynes). These injuries disrupt descending bulbospinal respiratory pathways and result in loss of spinal phrenic inter- and motoneurons, leading to diaphragm paresis. In addition, the diaphragmatic response to increased respiratory demands – which can be stimulated with hypoxia or hypercapnia - is attenuated post-contusion. One week post-SCI animals underwent five days of respiratory challenge via dAIH, which consisted of a total of 50, 5 minute hypoxic exposures over the course of five days (10 per day). Implanted telemetric EMG electrodes were used to assess diaphragm function pre- and post- injury, and daily during and following IH treatment. At the end of the 5-day IH therapy, retrograde neuroanatomical tracers (pseudorabies virus or cholera toxin beta subunit) were used to trace the phrenic circuit ipsilateral to injury. Terminal phrenic nerve recordings were used to assess phrenic motor output. Animals were then perfuse-fixed with paraformaldehyde for histological analyses. Transverse spinal cord sections (40 micron, frozen sections) were immunolabeled for the presence of 5HT, c-fos and neuronal tracers. Preliminary results from these ongoing studies have suggested that IH may elicit only a very modest effect on respiratory plasticity and phrenic function following cervical contusion injury.

55

A novel role for Wnt signalling in regulating astrogliosis in adult white matter Andrea Rivera, Arthur M. Butt University of Portsmouth, Southsea, UK

andrea.rivera@port.ac.uk ; arthur.butt@port.ac.uk

Astrocytes perform multiple functions that are essential for CNS function. Following a CNS

insult, astrocytes undergo a characteristic injury response, termed ‘reactive astrogliosis’ and form a chemo-physical barrier called the glial scar, which is inhibitory to axonal regeneration. The mechanisms regulating the astrocyte injury response are unresolved, but Wnt signalling is implicated in a number of different pathologies, including injury, Parkinson Disease, Amyotrophic Lateral Sclerosis and Alzheimer’s Disease. Wnt acts through the “canonical pathway”, or GSK3b/β-catenin pathway, and the “non-canonical” pathway, which is independent of GSK3b. Here, we have examined canonical Wnt/β-catenin signalling in adult white matter astrocytes, using a specific Wnt3a activator. All procedures were in accordance with the Animal Scientific Procedures Act (1986). Optic nerves (ONs) from adult mice were isolated intact and maintained in organotypic culture for 3 days in vitro. Wnt3a significantly increased the number of astrocytes compared to controls (p<0.05, unrelated t-tests) and induced the generation of novel astrocytes with a “simple” stellate morphology. To characterise the genotype of these novel astrocytes we compared the transcriptome of Wnt3a treated optic nerves with control and acutely isolated tissue. We identified a number of genes specifically altered by Wnt3a which are involved in regulation of planar cell polarity (Ctnnd1 and Cpne1). Pathway analysis (IOA, Ingenuity Systems) identified the Axon-Guidance and NGF pathways amongst the top pathways regulated by Wnt3a. The results identify a novel role for Wnt signalling in regulating astrogliosis and indicate that stimulation of Wnt/β-catenin in astrocytes may provide an environment that supports axonal growth. Supported by the Anatomical Society and IBBS

56

Wnts: more than an axonal growth inhibitor in the adult spinal cord Pau H. González

1, Carlos González-Fernández

1, Carmen María Fernández-Martos

1-2, Ernest

Arenas3, F. Javier Rodríguez

1.

1Laboratory of Molecular Neurology, Hospital Nacional de Parapléjicos (HNP), Toledo, SPAIN

2Actual address:

Wicking Dementia Research and Education Centre, University of Tasmania Hobart, TAS, Australia 3Molecular

Neurobiology Unit, MBB, Karolinska Institute, Stockholm, Sweden

fjrodriguez@sescam.jccm.es

Wnt proteins are a large family of molecules that are critically involved in CNS development. Interestingly, although initially reported a lack of Wnt expression in the adult spinal cord of mice with an acute reinduction after injury of few Wnt ligands and receptors [1], subsequent reports have shown that most Wnt ligands, modulators and receptors are constitutively expressed in mice and rats with altered expression patterns after injury [2-5]. Furthermore, Wnts have been involved in a variety of physiological (angiogenesis, adult neurogenesis, myelination, sensory function or activity-induced synapse formation) and pathophysiological processes (neuroinflammation, cancer and neurodegeneration). This is driven by a vast array of Wnt signalling effects, which are elicited by a tightly regulated pattern of expression of Wnt ligands, modulators and receptors at each cell type involved in a particular physiological/ pathophysiological mechanism. In this regard, SCI is a multifaceted pathophysiology with different spatio-temporal actors and requirements post-injury, and by extension Wnt patterns and signalling events. However, little is known regarding their specific expression patterns at cellular level in the healthy and injured adult spinal cord, which will be essential to understand their roles in the physiology and pathophysiology of CNS and, eventually, allow for the development of novel Wnt-targeted therapies for SCI without secondary adverse effects. Therefore, we decided to analyse the spatio-temporal mRNA and protein expression patterns of Wnt ligands, modulators and receptors in the spinal cord of adult rats and mice after SCI using quantitative RT-PCR and single and double IHQ. In brief, our results show that:

The mRNA encoding most Wnt ligands, soluble inhibitors and Frizzled receptors are constitutively expressed in the healthy spinal cord of adult rats. Strikingly, contusion spinal cord injury induced a time-dependent increase in Wnt mRNA expression from 6 hours until 28 days post-injury, and a narrow peak in the expression of soluble Wnt inhibitors between 1 and 3 days post-injury.[2, 3].

Analysis of cellular Frizzled 5 expression pattern showed that, in the uninjured rat spinal cord, this receptor was expressed in neurons, oligodendrocytes, astrocytes, microglia and NG2+ glial precursors. After injury, Frizzled 5 was also found in axons at all evaluated time points and in reactive microglia/macrophages from 3 to 14 days post-injury [3].

In uninjured adult rats, Ryk is expressed in neurons, astrocytes, and blood vessels. Following SCI, we observed an increase in Ryk expression from 24h until 14 dpi in the damaged tissue, where it was observed in reactive astrocytes and microglia/macrophages, NG2+ glial precursors, fibronectin+ cells, oligodendrocytes, and axons [4].

The mRNAs of most Wnt proteins are constitutively expressed in the uninjured spinal cord of adult mice, where IHC revealed a differential expression of Fz1 by neurons and oligodendrocytes and Fz4 by astrocytes. After dorsal hemisection, we found significant time-dependent variations with a prominent Wnt downregulation besides an upregulation of Wif1 [5].

Our results provide compelling evidences of constitutive expression of Wnts in both rats and mice, as well as that SCI induces dramatic changes in their spatio-temporal profiles with striking differences between the two rodent species analyzed. Remarkably, our results also show cell-specific expression patterns of Wnt receptors and thus suggestive for different physiological and pathophysiological functions. Future research interests include further characterization of the roles of each Wnt receptor in SCI with a special focus in inflammation and the development of specific peptides for their modulation.

References [1] Liu et al. J Neurosci 2008, 28: 8376-8382; [2] Fernandez-Martos et al. PLoS One 2011; 6(11):e27000; [3] Gonzalez et al. PLoS One 2012; 7(12): e50793; [4] Gonzalez

et al. J Neurotrauma 2013, 30(10):806-17; [5] González-

Fernández et al. J Neurotrauma 2014, 31(6):565-81 Financial support FISCAM (Grant PI2008-39) and FIS (Grants PI08/1475 and PI12/2895 with FEDER co-funding)

57

Characterization of a novel axon growth repellent and its role in spinal cord injury Julia Schaeffer, Geoffrey Cook, Roger Keynes Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK

js2064@cam.ac.uk

During the development of the peripheral nervous system, migrating neural crest cells and outgrowing motor and sensory axons follow a segmented pattern, the mesodermal somites. Growth cone repulsion is an important mechanism controlling axon growth, and ensuring that the peripheral nervous system develops without obstruction by the developing vertebral column. During development it guides axons by excluding them from “no-go” areas in the embryo

1. Following injury to

the adult brain and spinal cord it may also block regeneration, with serious clinical consequences. Among the candidate molecules, PNA-binding glycoproteins, chondroitin sulphate proteoglycans (CSPGs) and semaphorin 3A, a member of the semaphorin family, have been proposed as repellent molecules acting on different receptor systems expressed by primary sensory axon growth cones

2.

Recent work in the lab has identified a protein in embryonic somites that generates spinal nerve segmentation by contact-repulsive axon guidance. The protein is expressed selectively on the surface of somite cells, and cause collapse of axon growth cones when applied to cultured sensory neurons. It has also been shown previously that detergent extracts of mammalian (rat) grey matter and of a cultured line of human astrocytes possess growth cone collapse-inducing activity

3. Further

experiments have indicated that this CNS-derived activity has molecular properties closely similar to that in somites, so it is possible that this contact-repulsive system has been co-opted in the CNS to play an important role in regulating connectivity and plasticity. The overall aim of the project is to examine this novel CNS-derived system in more detail to confirm its molecular identity, elucidate how it is related to the somite-derived axon repellent, and assess its role in spinal cord injury.

To date, I have shown that the protein is expressed at the cell surface of a human astrocyte cell line by live cell immunostaining and 2D gel electrophoresis and immunoblotting. Detergent extracts of these astrocytes have been shown to cause growth cone collapse, and a lectin, jacalin, is currently being used as an affinity reagent to further analyse this molecular system.

The experimental objectives are: 1) to test whether inhibition of the somite protein blocks growth cone collapse induced by

detergent extracts of cultured human astrocytes; 2) to identify the protein responsible for this activity in human astrocytes extracts, using

biochemical techniques (2D gel electrophoresis, Western blotting and proteomics) and molecular cloning;

3) to assess the sites and level of expression of the protein in normal and injured adult rat CNS; 4) to test the most effective in vitro inhibitor of the biologically active protein for its ability to

promote axon regeneration and functional recovery using an in vivo rat model of spinal cord injury (in collaboration with Prof James Fawcett, Cambridge Centre for Brain Repair).

References 1. Keynes, R. et al. Surround repulsion of spinal sensory axons in higher vertebrate embryos. Neuron 18, 889–897 (1997) 2. Kuan, C.-Y. K., Tannahill, D., Cook, G. M. W. & Keynes, R. J. Somite polarity and segmental patterning of the peripheral nervous system. Mech. Dev. 121, 1055–1068 (2004) 3. Fok-Seang, J. et al. An analysis of astrocytic cell lines with different abilities to promote axon growth. Brain Res. 689, 207–223 (1995) Supported by the International Spinal Research Trust (ISRT), Nathalie Rose Barr Studentship, and the Rosetrees Trust

58

Transplantation of neural progenitors to improve respiration following spinal cord injury Spruance V.M., Sanchez D.E., Bezdudnaya T., Negron K.M., Whelan T.J., Reier P.J., Lane M.A.

Drexel University, Department of Neurobiology & Anatomy, Philadelphia, PA, USA

Victoria.spruance@gmail.com

Spinal cord injury (SCI) at nearly any level can result in some degree of respiratory deficiency, and impaired breathing remains one of the leading causes of morbidity and mortality following cervical injury. Over 50% of cervical SCI patients require assisted ventilation at some point during their care, incurring high costs of healthcare and significantly affecting quality of life. Given the majority of SCIs occur at the cervical level, there is an urgent need for improved therapeutic treatments targeting respiratory function following spinal injury. In recent years, there has been a growing appreciation for the endogenous, spontaneous neuroplasticity that can occur following injury contributing to limited functional recovery of respiratory systems. Recent experimental studies have suggested that interneurons may modulate phrenic motor function and diaphragm activity following a cervical SCI. The central hypothesis of the following work is that transplantation of fetal spinal cord (FSC), inherently rich in interneuronal progenitors, will promote the establishment of a novel circuitry capable of enhancing respiratory recovery following injury.

Adult, female Sprague-Dawley rats received lateralized C3/4 contusions using the Infinite Horizons Impactor Device at a preset force of 200 kilodynes. One week later, mechanically dissociated fetal spinal cord tissue (obtained from age E13.5 Sprague-Dawley or Fischer 344-GFP rats) was injected directly into the lesion cavity. Animals were allowed to recover for one month, at which time a retrograde, transynaptic tracer (pseudorabies virus, PRV) was applied to the ipsilateral hemi diaphragm or injected into the transplant site. Tracing studies have revealed anatomical connectivity between the transplanted cells and host phrenic neurons, and innervation of donor tissue from neurons throughout the brainstem, cervical and thoracic spinal cord. Immunohistochemical analysis has also revealed of a variety of neuronal phenotypes within transplanted tissue, including GABAergic, glutamatergic, cholinergic and catecholaminergic neurons. Furthermore, serotonergic and catecholaminergic projections were seen within outermost regions of donor tissue. Extensive c-fos immunoreactivity was observed throughout donor neurons, suggesting that transplanted cells were active. This was confirmed with multiunit recordings from within transplanted tissue, which revealed neurons with phasic respiratory and non-respiratory activity. Terminal electrophysiological recording of diaphragm and phrenic nerve revealed improved function during normal (eupneic) breathing and in response to respiratory challenge (hypoxia), suggesting that respiratory recovery was enhanced in transplant recipients. The results from these ongoing studies suggest that transplantation of neural progenitors can facilitate improved respiratory function following cervical spinal cord injury. Supported by National Institute of Health, NINDS; R01 NS081112

59

Investigating neuroprotection by carbon nanotubes following spinal cord injury Merrick Strotton

1, Noelia Rubio Carrero

2, Khuloud Al-Jamal

2, Elizabeth Bradbury

1

1King’s College London, Regeneration Group, The Wolfson Centre for Age-Related Diseases, London, UK

2King’s College London, Drug delivery group, Institute of Pharmaceutical Science, London, UK

merrick.strotton@kcl.ac.uk

In spinal cord injury (SCI) research, biomaterials are often thought of as growth permissive scaffolds. However, a class of particulate nanomaterials also exists, which have the advantage of being injectable. Carbon nanotubes (CNTs) are an example of such a material, but they have a number of additional unique properties that could be exploited for studying SCI, such as a large surface area for protein/genetic species delivery, infra-red absorption properties enabling in vivo imaging, and ultrahigh conductivity which may modify neuronal properties by lowering action potential firing thresholds (Cellot et al., 2009). Furthermore, functionalising (solubilising) carbon nanotubes through the covalent addition of positively charged amine groups to CNT side walls (forming α-CNTs) has been demonstrated to create an agent that when delivered to various stroke models leads to reduced levels of cell death (Al-Jamal et al., 2011, Lee et al., 2011, Moon et al., 2012).

We explored the therapeutic potential of α-CNTs as a neuroprotective agent in a rat model of SCI with bilateral intraspinal injections of α-CNTs rostral and caudal to a dorsal column crush injury at the cervical level. Tissue sparing at the injury epicentre and the survival and spread of CNTs were examined at various post injury time points. In a separate group, positively charged α-CNTs were co-injected with chondroitinase ABC to evaluate if cleaving negatively charged glycosaminoglycans in the extracellular matrix potentiated the spread of α-CNTs through the lesion. Using a battery of forelimb dependent behaviours, electrophysiological assessments of the descending corticospinal tract and histological analyses of the lesion epicenter, we demonstrate a slightly reduced lesion volume in the presence of α-CNTs, but no functional improvement (or deficit). Further analysis of behaviour and electrophysiological data is ongoing, and electron microscopy studies are currently underway to evaluate those cell types which uptake α-CNTs. This will be important information for future studies where we aim to explore the use of α-CNTs as a non-viral vector for siRNA mediated knockdown of targets of interest.

References Al-Jamal, K. T., Gherardini, L., Bardi, G., Nunes, A., Guo, C., Bussy, C., Herrero, M. A., Bianco, A., Prato, M., Kostarelos, K. & Pizzorusso, T. 2011. Functional motor recovery from brain ischemic insult by carbon nanotube-mediated siRNA silencing. Proc Natl Acad Sci U S A, 108, 10952-7 Cellot, G., Cilia, E., Cipollone, S., Rancic, V., Sucapane, A., Giordani, S., Gambazzi, L., Markram, H., Grandolfo, M., Scaini, D., Gelain, F., Casalis, L., Prato, M., Giugliano, M. & Ballerini, L. 2009. Carbon nanotubes might improve neuronal performance by favouring electrical shortcuts. Nat Nanotechnol, 4, 126-33 Lee, H. J., Park, J., Yoon, O. J., Kim, H. W., Lee Do, Y., Kim Do, H., Lee, W. B., Lee, N. E., Bonventre, J. V. & Kim, S. S. 2011. Amine-modified single-walled carbon nanotubes protect neurons from injury in a rat stroke model. Nat Nanotechnol, 6, 121-5 Moon, S. U., Kim, J., Bokara, K. K., Kim, J. Y., Khang, D., Webster, T. J. & Lee, J. E. 2012. Carbon nanotubes impregnated with subventricular zone neural progenitor cells promotes recovery from stroke. Int J Nanomedicine, 7, 2751-65 This work was supported by a studentship funded by Guy’s and St Thomas hospital through the King’s Bioscience Initiative

60

Intramuscular Tibialis Anterior coherence and subacute spinal cord injury: mechanisms of neuroplasticity underlying SCI Bravo-Esteban E.

1,2 Taylor J.

1 Aleixandre M.

2 Simon-Martínez C.

1 Torricelli D.

2 Pons J.L.

2 Avila-Martin

G., Galan-Arriero I., Gómez-Soriano J.1,3

elisabethb@sescam.jccm.es

1 Sensorimotor Function Group, Hospital Nacional de Parapléjicos, Toledo, Spain

2 Spanish National Research Council (CSIC), Madrid, Spain

3 Nursing and Physiotherapy School, Castilla La Mancha University, Toledo, Spain

Tibialis Anterior (TA) coherence estimation is assumed to reflect common supraspinal descending input spinal motoneurons, related to corticospinal tract activity (1-4). This study documented residual voluntary motor recovery at 2 week intervals during subacute spinal cord injury (SCI) with intramuscular TA coherence estimation within the 10-60Hz bandwidth, assessed during controlled maximal isometric and isokinetic dorsiflexion. Several clinical and functional lower limb measures (muscular testing, dorsiflexion maximal voluntary torque and gait function measured with the WISCI II) and neurophysiological measures (TA motor evoked potentials, MEPs) were also recorded.

Total and TA muscle strength, voluntary torque generation and gait function improved during subacute SCI, in addition to 40-60Hz, but not 15-30Hz intramuscular TA coherence. TA MEPs failed to reflect significant recovery of function in this cohort. The SCI spasticity syndrome non-specifically reduced 15-30Hz TA coherence and was detected as high TA coherence values during fast isokinetic movement in all frequency bands.

To conclude, longitudinal assessment of adaptive and maladaptive motor plasticity during subacute SCI can be detected with TA EMG coherence estimation during controlled movement, providing orientative diagnostic information during neurorehabilitation. References 1. Bravo-Esteban E, Taylor J, Aleixandre M, Albu S,Cristina Simon ,Torricelli D, J. L. Pons, Gómez-Soriano J. Muscle

coherence during controlled voluntary movement in healthy subjects and patients with spinal cord injury: contraction and velocity dependence. Journal of NeuroEngineering and Rehabilitation (In press)

2. Farina D, Negro F, and Jiang N: Identification of common synaptic inputs to motor neurons from the rectified electromyogram. J Physiol 2013, 591(10): 2403-18

3. Gómez-Soriano, J, Bravo-Esteban E, Aleixandre M, Taylor J, Pons JL. Method for the measurement of muscle coherence for neurological disorders.PATENT. January 12, 2014 with application nº EP14150864

4. Halliday DM, Conway BA, Christensen LO, Hansen NL, Petersen NP, and Nielsen JB: Functional coupling of motor units is modulated during walking in human subjects.J Neurophysiol 2003, 89: 960-8

This project is funded by the Spanish Ministry of Science and Innovation CONSOLIDER-INGENIO, project HYPER (Hybrid NeuroProsthetic and NeuroRobotic Devices for Functional Compensation and Rehabilitation of Motor Disorders, CSD2009-00067)

61

Oral administration of the p38α MAPK inhibitor, UR13870, inhibits anterior cingulate microglial expression and affective pain behaviour following spinal cord injury Iriana Galan-Arriero

1, Gerardo Avila-Martin

1, Agueda Ferrer-Donato

1, Julio Gomez-Soriano

1,2,

Elisabeth Bravo-Esteban1,3

, Julian Taylor1

igalan@jccm.es

1Sensorimotor Function Group, Hospital Nacional de Parapléjicos, SESCAM, Toledo, Spain

2E.U. E. Fisioterapia de Toledo, Universidad de Castilla la Mancha, Toledo, Spain

3IAI, Consejo Superior de Investigaciones Científicas (CSIC), Arganda del Rey, Spain

The p38α MAPK cell signalling pathway is a key mechanism of microglia activation and has been intensively studied as a potential target for neuropathic pain (NP) control (2). The effect of UR13870 (4), a demonstrated p38α MAPK inhibitor, on the modulation of anterior cingulate cortex (ACC) and spinal dorsal horn microglia reactivity was addressed in this study following T9 contusion spinal cord injury (SCI) in the rat, in addition to behavioural testing of pain-related aversion and anxiety.

Administration of UR13870 (1mg/kg i.v.) and Pregabalin (30mg/kg i.v.) reduced place escape avoidance paradigm (PEAP) behaviour during chronic SCI at 42 days after injury. Animals treated with UR13870, administered daily (10mg/kg p.o.) demonstrated reduced PEAP, but not anxiety behaviour, at 28 days after SCI. Although administration of UR13870 (10mg/kg p.o.) failed to reduce either microglia or astrocyte reactivity within the spinal dorsal horn following SCI, a reduction in the total volume of damaged tissue was identified at 28 days after injury. In the ACC, an increase in microglia reactivity, in addition to an upregulation of the metabotropic glutamate type 5 receptor (mGluR5) expression, were identified after SCI. While UR13870 (10mg/kg p.o.) treatment significantly reduced OX-42 expression, mGluR5 and NR2B in the ACC, no change in astrocyte reactivity was observed.

To conclude, oral treatment with a p38α MAPK inhibitor reduces the affective cognitive component of pain component following SCI, mediated preferentially by the inhibition of microglia reactivity within the ACC remote from the injury site (1). This study supports a central role of ACC glia reactivity for affective pain behaviour after SCI (3, 5, 6). References 1. Detloff MR, Fisher LC, McGaughy V, Longbrake EE, Popovich PG, Basso DM. Remote activation of microglia and pro-

inflammatory cytokines predict the onset and severity of below-level neuropathic pain after spinal cord injury in rats. Exp Neurol 2008;212(2):337-347

2. Ji RR, Suter MR. p38 MAPK, microglial signaling, and neuropathic pain. Mol Pain 2007;3:33 3. Lu Y, Zhu L, Gao YJ. Pain-related aversion induces astrocytic reaction and proinflammatory cytokine expression in the

anterior cingulate cortex in rats. Brain Res Bull 2010;84(2):178-182 4. Mihara K, Almansa C, Smeets RL, Loomans EE, Dulos J, Vink PM, Rooseboom M, Kreutzer H, Cavalcanti F, Boots AM,

Nelissen RL. A potent and selective p38 inhibitor protects against bone damage in murine collagen-induced arthritis: a comparison with neutralization of mouse TNFalpha. Br J Pharmacol 2008;154(1):153-164

5. Widerstrom-Noga E, Pattany PM, Cruz-Almeida Y, Felix ER, Perez S, Cardenas DD, Martinez-Arizala A. Metabolite concentrations in the anterior cingulate cortex predict high neuropathic pain impact after spinal cord injury. Pain 2012;154(2):204-212

6. Zhao P, Waxman SG, Hains BC. Modulation of thalamic nociceptive processing after spinal cord injury through remote activation of thalamic microglia by cysteine cysteine chemokine ligand 21. J Neurosci 2007;27(33):8893-8902

This work has been supported by the following founding sources: Fundación Mutua Madrileña, 2013, INNPACTO (Ministerio de Ciencia e Innovación, IPT-010000-2010-016), Consorcio “Dendria-Draconis Pharma S.L.” (Centro para el Desarrollo Tecnológico Industrial), Instituto de Salud Carlos III PI11/00592. UR13870, was manafactured and kindly donated by Heidi Sisniega of PalauPharma, S.A. (Barcelona, Spain)

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The fate of boundary cap neural crest stem cells following transplantation to the surface of avulsed or uninjured spinal cord Carl Trolle*, Niclas König*, Ninnie Abrahamsson, Svitlana Vasylovska, Elena N. Kozlova *Equal contribution Regenerative Neurobiology, Dept of Neuroscience, Biomedical Center, Uppsala University, Uppsala, Sweden

Carl.Trolle@neuro.uu.se, Niclas.Konig@neuro.uu.se

Spinal root avulsion results in loss of motor and sensory function as well as often chronic intractable neuropathic pain. Although surgical restoration of the ventral root may improve motor function, methods to improve sensory function still needs to be developed.

We have previously described the ability of boundary cap neural crest stem cells (BCs) to form elongated bands, associated with regenerating sensory fibers, after transplantation to the avulsed dorsal roots of rodents

1. Our previous findings also show that BCs remaining outside the

spinal cord express the glial marker GFAP whereas the BCs that migrate into the spinal cord as single cells may adopt a neuronal phenotype

2. Here we investigate the fate of transplanted BCs in different

injury models (durectomy, dorsal root rhizotomy and dorsal root avulsion). Our results indicate that the BCs survive in all the above mentioned conditions but at a lower extent following durectomy when the nervous system is left intact. While the BCs form band-like structures when transplanted to either cut or avulsed dorsal roots, they tend to remain in small clusters when transplanted to intact dorsal roots. Furthermore, following dorsal root rhizotomy as well as dorsal root avulsion, single BCs migrate into the superficial laminae of the spinal cord. In all the three different conditions, the BCs outside the spinal cord tend to be positive for the glial marker GFAP. We believe that the inflammatory processes initiated after nerve injury are beneficial for the survival of BCs and that the BCs are able to respond to the injury by organizing into band-like structures possibly supporting regenerating fibers. References 1. Konig N, Trolle C, Kapuralin K, Adameyko I, Mitrecic D, Aldskogius H, Shortland PJ, Kozlova EN (2014) Murine neural crest stem cells and embryonic stem cell-derived neuron precursors survive and differentiate after transplantation in a model of dorsal root avulsion. J Tissue Eng Regen Med (Epub ahead of print) 2.Trolle C, Konig N, Abrahamsson N, Vasylovska S, Kozlova EN (2014) Boundary cap neural crest stem cells homotopically implanted to the injured dorsal root transitional zone give rise to different types of neurons and glia in adult rodents BMC Neurosci 5;15:60

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Early transplantation of mesenchymal stem cells after spinal cord injury relieves pain hypersensitivity through suppression of pain-related signaling cascades and reduced inflammatory cell recruitment Kenzo Uchida

1, Hideaki Nakajima

1, Shuji Watanabe MD

1, Kazuya Honjoh

1, William E.B. Johnson

3,

Hisatoshi Baba1

1Department of Orthopaedics and Rehabilitation Medicine, Faculty of Medical Sciences, University of Fukui,

Eiheiji, Fukui, Japan

2Life & Health Sciences, Aston University, Aston Triangle, Birmingham, UK

kuchida@u-fukui.ac.jp

Bone marrow-derived mesenchymal stem cells (BMSC) modulate inflammatory/immune responses and promote motor functional recovery after spinal cord injury (SCI).

1) 2) However, the

effects of BMSC transplantation on central neuropathic pain and neuronal hyperexcitability after SCI remain elusive. This is of importance because BMSC-based therapies have been proposed for clinical treatment. We investigated the effects of BMSC transplantation on pain hypersensitivity in GFP-positive bone marrow-chimeric mice subjected to a contusion SCI, and the mechanisms of such effects. BMSC transplantation at day 3 post-SCI improved motor function and relieved SCI-induced hypersensitivities to mechanical and thermal stimulation. The pain improvements were mediated by suppression of PKC-γ and p-CREB expression in dorsal horn neurons. BMSC transplants significantly reduced levels of p-p38 MAPK and p-ERK1/2 in both hematogenous macrophages and resident microglia, and significantly reduced the infiltration of CD11b and GFP double-positive hematogenous macrophages without decreasing the CD11b-positive and GFP-negative activated spinal-microglia population. BMSC transplants prevented hematogenous macrophages recruitment by restoration of the blood-spinal cord barrier, which was associated with decreased levels of (i) inflammatory cytokines (TNF-α, IL-6); (ii) mediators of early secondary vascular pathogenesis (MMP-9); (iii) macrophage recruiting factors (CCL2, CCL5, CXCL10), but increased levels of a microglial stimulating factor (GM-CSF). These findings support the use of BMSC transplants for SCI treatment; further, they suggest that BMSC reduce neuropathic pain through a variety of related mechanisms that include neuronal sparing and restoration of the disturbed blood-spinal cord barrier, mediated through modulation of the activity of spinal-resident microglia and the activity and recruitment of hematogenous macrophages. References 1. Nakajima H, Uchida K, Guerrero AR, et al. Transplantation of mesenchymal stem cells promotes an alternative pathway of macrophage activation and functional recovery after spinal cord injury. J Neurotrauma 2012;29:1614-25 2. Tan Y, Uchida K, Nakajima H, et al. Blockade of interleukin 6 signaling improves the survival rate of transplanted bone marrow stromal cells and increases locomotor function in mice with spinal cord injury. J Neuropathol Exp Neurol 2013;72:980-93

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Effects of pudendal and cortical paired associative stimulation on reflex and cortico-spinal control of anal sphincter responses in patients with incomplete spinal cord injury: a feasibility study Vásquez N.

1, Knight S.

1, Susser J.

1, Gall, A.

1 Ellaway P.H.

1,3, Craggs M.D.

1,2

1The London Spinal Cord Injury Centre, Royal National Orthopaedic Hospital,

2University College London,

3Imperial College, London, UK

Natalia.Vasquez@rnoh.nhs.uk

Incomplete spinal cord injury (iSCI) frequently impacts on spino-bulbo-spinal pathways causing major disruption to the control of the pelvic organs and sphincter muscles. Restoration of bladder and bowel control are top priorities for those with iSCI, especially in cases of paraplegia (Anderson, 2004). In iSCI subjects who have retained a pudendal anal reflex (PAR), a surrogate marker for the bladder guarding reflex, the reflex can be facilitated by conditioning transcranial magnetic stimulation of the motor cortex in approximately 50% of cases (Vásquez et al, 2014). This raised the question as to whether the ability of paired associative stimulation (PAS) to induce plasticity in neural circuits, based on paired pudendal nerve and cortical stimulation, could be shown to induce plasticity in the cortico-spinal circuits controlling the PAR. A long-term aim would be to see whether development of the technique might lead to changes in neural circuitry accompanied by functional restoration of continence.

Aims: To assess whether repetitive, associative paired stimulation of the dorsal penile nerve and the motor cortex produces changes in cortico-spinal circuitry controlling anal sphincter muscle responses, in iSCI.

Methods: Eighteen male subjects with incomplete, supra-sacral spinal cord injuries and symptoms of a neuropathic bladder were recruited. Incontinence was assessed using the International Consultation on Incontinence Modular Questionnaire (ICIQ). Electromyographic activity of the external anal sphincter was recorded. The PAR was elicited by electrical stimulation of the dorsal penile nerve (DPN). Motor cortical excitation was achieved using transcranial magnetic stimulation (TMS). A PAS protocol (DPN and TMS, interval 40ms) was applied for 8 min at 0.25Hz using either real or sham TMS (randomised order) of the motor cortex. Pudendal (DPN) somatosensory evoked potentials (pSSEPs) were recorded.

Results: A PAR could be recorded in all subjects and an anal sphincter MEP in 12 of the 18 subjects. In all but one subject the PAR could be facilitated by prior (30 ms) conditioning TMS. Group mean amplitudes of the PAR, the conditioned PAR (cPAR) and MEP showed no significant change immediately after or 20’ minutes after either real or sham PAS. There was no change in the group mean ICIQ scores. 13 subjects individually showed significant changes (10 increases, 11 decreases) in one or more anal sphincter responses to either real or sham PAS. These individual responses were not correlated with the presence or latency of either pSSEPs or the anal sphincter MEP.

Conclusions: Paired associative stimulation has the potential to alter the excitability of cortico-spinal and reflex circuitry controlling the anal sphincter in certain iSCI individuals. The basis for the variable nature of the responses to this particular PAS protocol is not known but could not be explained by differences in afferent or efferent spinal conduction pathways. References Anderson KD. J Neurotrauma 2004;21:1371–83 Vasquez N, Balasubramaniam V, Kuppuswamy A, Knight S, Susser J, Gall, A Ellaway PH, Craggs MD. Neurourology and Urodynamics 2014 E-Pub Supported by the INSPIRE Foundation (Registered Charity No. 296284 UK) and the RNOH Charity (Registered Charity No. 226955 UK)

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Chondroitinase ABC rescues complete respiratory motor activity following cervical contusion injury P.M. Warren

1; B.I. Awad

1,2; W.J. Alilain

1

1 MetroHealth Medical Centre, Case Western Reserve University, Cleveland, OH, USA

2 Dept. of Neurological Surgery, Mansoura University School of Medicine, Mansoura, Egypt

pmw45@case.edu

Chondroitinase ABC mediates functional recovery of damaged respiratory motor pathways following both acute and chronic C3 cervical contusion (C3C). This auspicious treatment evokes functional improvement in multiple muscle groups associated with inspiratory activity (diaphragm, external intercostals, genioglossus) and suggests that the modification of the extracellular matrix (ECM) causes plasticity at multiple levels and motoneuron pathways throughout the spinal cord. Relatively few studies have been conducted studying the treatment of respiratory motor paralysis and dysfunction following cervical spinal cord contusion. One of the major impairments to functional respiratory motor recovery following C3C is the development of the astro-glial scar. This CSPG (chondroitin sulphate proteoglycan) rich matrix acts as a barrier to plasticity and regeneration. Application of chrondroitiase ABC (ChABC) catabolises the CSPGs within the ECM, increasing functional plasticity. We have assessed the therapeutic efficacy of delayed ChABC application (4 concurrent injections totaling 0.015 U) upon multiple respiratory motor systems at both acute and chronic stages following severe lateral cervical (C3) contusion (LC3C). Diaphragmatic, intercostal, and genioglossus electromyography were used to assess functional restoration of spared and damaged tracts following LC3C while anatomical alterations were investigated through immunohistochemistry. Control animals showed severely compromised respiratory motor activity ipsilateral to the injury at 3 and 6 weeks post contusion with minimal endogenous recovery. Indeed, the damaged/contused pathways were unable to maintain respiratory motor activity alone. However, application of ChABC at both 1 and 4 weeks post LC3C caused significant restoration in respiratory motor function and repair of these damaged pathways which, alone, were able to maintain complete inspiratory activity. These data suggest that ChABC treatment, at both acute or chronic time points, is sufficient to evoke recovery of respiratory function. This possibly occurs through plastic mechanisms including anatomical reorganization and an increase in serotonergic signaling. Further, we demonstrate that the treatment can positively affect multiple systems that collectively act to mediate inspiratory activity. However, this functional change in respiratory motor activity is neither absolute nor completely robust when applied at acute stages suggesting an optimal time course for treatment application must be determined to mediate sustained recovery. These data support the use of ECM modification to mediate total respiratory motor system recovery following acute and chronic cervical contusion. Supported by the International Spinal Research Trust (ISRT), the Craig H. Neilson Foundation, Wings for Life, and the Egyptian Governmental Scholarship

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Extensive recovery of respiratory motor function at chronic and super-chronic time points following cervical spinal cord injury P.M. Warren

1; P.M. MacFarlane

2; J. Silver

3; W.J. Alilain

1

1 MetroHealth Medical Centre, Case Western Reserve University, Cleveland, OH, USA

2 Dept. of Pediatrics, Case Western Reserve University, Rainbow Babies & Children’s Hospital, Cleveland, USA

3 Dept. of Neurosciences, Case Western Reserve University, Cleveland, OH, USA

pmw45@case.edu

Treatments to restore respiratory function following chronic cervical spinal cord injury (SCI) have not been extensively studied. We demonstrate that a pharmacological agent and rehabilitative training may provide the key for recovery of diaphragm activity following chronic trauma. The ablation of respiratory function is caused by disruption of motoneuron pathways, formation of the chondroitin sulphate proteoglycan (CSPG) rich astroglial scar, and a reduction in interneuron, motoneuron and synaptic density. Following acute cervical SCI, CSPG breakdown by application of chrondroitiase ABC (ChABC) can restore functional diaphragm activity while intermittent hypoxia (IH) training increases respiratory drive and synaptic strength. We now provide evidence for the recovery of robust functional respiratory motor activity at both chronic (3 month) and super-chronic (1.5 year) time points following LC2H through a combination of IH training and ChABC. We used diaphragmatic electromyography (diaEMG) and phrenic nerve recordings to demonstrate that a single application of ChABC (0.005 U) can recover extensive respiratory motor function following chronic and super-chronic cervical SCI. Control treated animals showed no endogenous recovery of diaphragm function. While having limited effect upon diaEMG patterns, IH training alone was shown to enhance maximal phrenic nerve activity. However, the combined treatment of IH and ChABC was shown to substantially enhance diaEMG and maximal phrenic nerve activity beyond that demonstrated by either group alone. Interestingly, in a subpopulation of animals the muscle activity in this combination group can become unstructured, demonstrated by degraded patterned activity on the lesioned side. This tonic/chaotic activity is governed by a serotonergic (5-HT) mechanism and suggests considerable remodeling of spinal cord circuitry below the level of the lesion at chronic stages. Indeed, ChABC and IH treated animals which recover normal breathing patterns following treatment can be made chaotic by giving exogenous 5-HT, while those that are already chaotic can be normalized by blocking certain 5-HT receptors. These data demonstrate the significant restoration of diaphragm function and nerve activity at chronic and super-chronic time points following cervical SCI due to matrix modification, induction of plasticity and facilitation of drive. Yet, the potential emergence of chaos is indicative of the complications inherent in repairing the chronically injured spinal cord and suggests the need for tight mechanistic and environmental control. Supported by the International Spinal Research Trust (ISRT) and the Craig H. Neilson Foundation

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Delegate list

First Name Last Name Company Email Address

Louise Adams Queen Mary University of London louise_adams24@hotmail.co.uk

Hakan Aldskogius Uppsala University Hakan.Aldskogius@neuro.uu.se

Warren Alilain Case Western Reserve University wja4@case.edu

Yazi Al'Joboori University of Leeds bsydaj@leeds.ac.uk

David Allan National Spinal Injuries Unit for Scotland davidballan@btinternet.com

Mark Bacon ISRT research@spinal-research.org

Sue Barnett University of Glasgow Susan.Barnett@glasgow.ac.uk

Katalin Bartus King's College London katalin.bartus@kcl.ac.uk

Catherina Becker University of Edinburgh catherina.becker@ed.ac.uk

Murray Blackmore Marquette University murray.blackmore@marquette.edu

Armin Blesch UniversitätsKlinikum Heidelberg Armin.Blesch@med.uni-heidelberg.de

Xuenong Bo Queen Mary University of London x.bo@qmul.ac.uk

Sara Bowie UCL Institute of Neurology sara.bowie@ucl.ac.uk

Liz Bradbury King's College London elizabeth.bradbury@kcl.ac.uk

Frank Bradke DZNE Bonn frank.bradke@dzne.de

Tom Brushart Johns Hopkins tbrusha@jhmi.edu

Srinivasa Budithi Midlands Centre for Spinal Injuries, Oswestry Srinivasa.budithi@rjah.nhs.uk

Emily Burnside King's College London emily.burnside@kcl.ac.uk

Sarah Busch Athersys, Inc. sab37@case.edu

Arthur Butt University of Portsmouth arthur.butt@port.ac.uk

Menghon Cheah University of Cambridge mc747@cam.ac.uk

Daniel Chew University of Cambridge dc501@cam.ac.uk

David Choi UCL Institute of Neurology d.choi@ucl.ac.uk

Andrew Collins UCL Institute of Neurology Andrew.Collins@ucl.ac.uk

Sylvie Coupaud National Spinal Injuries Unit for Scotland sylvie.coupaud@strath.ac.uk

Michael Craggs UCL Institute of Neurology michael.craggs@ucl.ac.uk

Graham Creasey Stanford University gcreasey@stanford.edu

Matt Danzi University of Miami m.danzi@med.miami.edu

Suradip Das Indian Institute of Technology Guwahati suradip@iitg.ernet.in

Priscilla Day University of Cambridge pd380@cam.ac.uk

Simone Di Giovanni Imperial College London s.di-giovanni@imperial.ac.uk

Athanasios Didangelos King's College London athanasios.didangelos@kcl.ac.uk

Hans-Ulrich Dodt University of Technology Vienna hans-ulrich.dodt@meduniwien.ac.at

Wagih El Masry Midlands Centre for Spinal Injuries, Oswestry bellstonehse@btinternet.com

Peter Ellaway Imperial College London p.ellaway@imperial.ac.uk

Karim Fouad University of Alberta karim.fouad@ualberta.ca

Robin Franklin University of Cambridge rjf1000@cam.ac.uk

Matthew Fraser National Spinal Injuries Unit for Scotland matthewfraser@nhs.net

Parag Gad University of California, Los Angeles paraggad@gmail.com

Andrew Gaudet The Ohio State University Andrew.Gaudet@osumc.edu

Isabella Gavazzi King's College London isabella.gavazzi@kcl.ac.uk

Marieta Georgieva University of Aberdeen marieta.georgieva.10@aberdeen.ac.uk

Ioana Goganau Heidelberg University Hospital Ioana.Goganau@med.uni-heidelberg.de

68

Alfredo Gorio University of Milan alfredo.gorio@unimi.it

Andy Greenhalgh McGill University andrew.greenhalgh@mail.mcgill.ca

Barbara Grimpe Heinrich Heine University Duesseldorf Barbara.Grimpe@med.uni-duesseldorf.de

James Guest University of Miami jguest@med.miami.edu

Barbara Haenzi King's College London barbara.haenzi@kcl.ac.uk

Jodie Hall The Ohio State University jodie.hall@osumc.edu

Jan Hoeber Uppsala University jan.hoeber@neuro.uu.se

Kazuya Honjoh University of Fukui kazuya@u-fukui.ac.jp

Hilary Hu Iowa State University hilaryhu@iastate.edu

Wenlong Huang University of Aberdeen w.huang@abdn.ac.uk

Diana Hunter University of British Columbia ICORD dianavhunter@gmail.com

Ronaldo Ichiyama University of Leeds R.M.Ichiyama@leeds.ac.uk

Nicholas James Kings College London nicholas.d.james@kcl.ac.uk

Nick Jeffery Iowa State University njeffery@iastate.edu

Eustace Johnson Aston University w.e.johnson@aston.ac.uk

Linda Jones Craig H. Neilsen Foundation linda@chnfoundation.org

Ksenija Jovanovic National Hospital for Paraplegics kjovanovic@sescam.jccm.es

Ashik Kalam University of Portsmouth ashik.kalam@myport.ac.uk

Naomi Kleitman Craig H. Neilsen Foundation naomi@chnfoundation.org

Niclas König Uppsala University niclas.konig@neuro.uu.se

Timea Konya ISRT timea@spinal-research.org

Marcel Kopp Charité-Universitätsmedizin Berlin marcel.kopp@charite.de

Elena Kozlova Uppsala University elena.kozlova@neuro.uu.se

Andrei Krassioukov University of British Columbia ICORD krassioukov@icord.org

Michael Lane Drexel University mlane.neuro@gmail.com

Bradley Lang Case Western Reserve University btl21@case.edu

Kaythi Latt RNOH, Southport and Ormskirk kaythi.latt@nhs.net

Stuart Law UCL Institute of Neurology stuart.law@ucl.ac.uk

Rosi Lederer Wings for Life rosi.lederer@wingsforlife.com

Seok Voon Lee Stanford University svlee@stanford.edu

Vance Lemmon University of Miami vlemmon@med.miami.edu

Daqing Li UCL Institute of Neurology daqing.li@ucl.ac.uk

Ying Li UCL Institute of Neurology ying.li@ucl.ac.uk

Ann Logan University of Birmingham a.logan@bham.ac.uk

Elisa Lopez-Dolado National Hospital for Paraplegics lamidolado@gmail.com

Stephen McMahon King's College London stephen.mcmahon@kcl.ac.uk

Dana McTigue The Ohio State University dana.mctigue@osumc.edu

Claire Meehan Copenhagen University claire@sund.ku.dk

Saloni Mehta University of Miami s.mehta8@med.miami.edu

Madge Miah UCL Institute of Neurology m.miah@ucl.ac.uk

Adina Michael-Titus Queen Mary University of London a.t.michael-titus@qmul.ac.uk

Gordon Mitchell University of Wisconsin - Madison Mitchell@svm.vetmed.wisc.edu

Lawrence Moon King's College London lawrence.moon@kcl.ac.uk

Liz Muir University of Cambridge emm1@mole.bio.cam.ac.uk

Hans Werner Müller University of Düsseldorf hanswerner.mueller@uni-duesseldorf.de

Tiina Negron Drexel University TiinaM.Negron@gmail.com

69

Raymond Onders University Hospitals Case Medical Center Raymond.onders@uhhospitals.org

Karen Oprych UCL Institute of Neurology k.gladwin@ucl.ac.uk

Aheed Osman Midlands Centre for Spinal Injuries, Oswestry aheed.osman@rjah.nhs.uk

James Phillips UCL Institute of Neurology jb.phillips@ucl.ac.uk

Giles Plant Stanford University gplant@stanford.edu

Milos Popovic University of Toronto milos.popovic@utoronto.ca

Phil Popovich The Ohio State University Phillip.Popovich@osumc.edu

John Priestley Queen Mary University of London J.V.Priestley@qmul.ac.uk

Mariel Purcell National Spinal Injuries Unit for Scotland margaret.purcell@ggc.scot.nhs.uk

Alexander Rabchevsky University of Kentucky agrab@uky.edu

Matthew Ramer University of British Columbia ICORD ramer@icord.org

Peter Richardson Queen Mary University of London p.richardson@qmul.ac.uk

John Riddell University of Glasgow j.s.riddell@bio.gla.ac.uk

Andrea Rivera University of Portsmouth andrea.rivera@port.ac.uk

Javier Rodriguez National Hospital for Paraplegics fjrodriguez@sescam.jccm.es

Gordana Savic RNOH, Stoke Mandeville Hospital Gordana.Savic@buckshealthcare.nhs.uk

Dimitry Sayenko University of Louisville DimitrySayenko@KentuckyOneHealth.org

Julia Schaeffer University of Cambridge julia.schaeffer13@gmail.com

Jan Schwab Charité-Universitätsmedizin Berlin jan.schwab@charite.de

Andrew Schwartz University of Pittsburgh abs21@pitt.edu

Derryck Shewan University of Aberdeen d.shewan@abdn.ac.uk

Jerry Silver Case Western Reserve University jxs10@po.cwru.edu

Victoria Spruance Drexel University Victoria.spruance@gmail.com

John Steeves University of British Columbia ICORD steeves@icord.org

Moa Stenudd Karolinska Institutet moa.stenudd@ki.se

Merrick Strotton King's College London merrick.strotton@kcl.ac.uk

Julian Taylor National Hospital for Paraplegics juliantaylorgreen@yahoo.es

Veronica Tom Drexel University veronica.tom@drexelmed.edu

Carl Trolle Uppsala University carl.trolle@neuro.uu.se

Kenzo Uchida University of Fukui kuchida@u-fukui.ac.jp

Anna Varone University of Aberdeen varoneanna@gmail.com

Natalia Vasquez RNOH, Stanmore nataliavasquezp@yahoo.com

Joost Verhaagen Netherlands Institute for Neuroscience J.Verhaagen@nin.knaw.nl

Laurent Vinay CNRS & Aix Marseille Université laurent.vinay@univ-amu.fr

Philippa Warren Case Western Reserve University pmw45@case.edu

Claudia Wheeler-Kingshott UCL Institute of Neurology c.wheeler-kingshott@ion.ucl.ac.uk

Edward Wirth Asterias Biotherapeutics, Inc. ewirth@asteriasbio.com

Ping Yip Queen Mary University of London p.yip@qmul.ac.uk