The potential of electric field for promoting neurite guidance in spinal cord injury regeneration strategies

Anna Varone, University of Aberdeen

Authors*A. VARONE, Z. N. MUHAMAT, A. M. RAJNICEK, W. HUANG;
Lab Abstract:

Introduction Spinal cord injury, for which there is no cure, can lead to permanent loss of neurological function. Reasons for failed axon regrowth include poor intrinsic growth capacity of adult CNS neurons, inhibitory chemicals and physical barriers formed post injury. Electric field (EF) stimulation is a promising strategy for spinal repair because it promotes directional neurite growth in cultured non-mammalian or embryonic neurons. Here, we investigated the effects of EF exposure on postnatal rat cortical neurons and organotypic spinal cord slices to further explore its potential for spinal repair.

Methods Cortices and spinal cords of Sprague Dawley rats at postnatal days 0-3 were used to culture cortical neurons and organotypic spinal cord slices. Neurons were cultured directly in the EF migration chamber for 48h before applying EFs ranging from 50 to 350mV/mm for 3-6h. To examine if an EF could overcome the effects of inhibitory molecules present post-spinal injury, 10µg/ml Chondroitin Sulphate Proteoglycan-6 (CSPG6) was added to the culture medium during EF exposure. Spinal cord slices were prepared at 350µm thickness and lesioned with surgical blades to produce a 700µm lesion gap. Four days after lesion, slices were transferred to the migration chamber and 50mV/mm EF was applied for 24h.

Results Cortical neurons showed an increase in the proportion of neurites facing the anode and facing perpendicular to the EF vector compared to the random growth of controls without an EF. This bias increased as the EF strength increased, with 220mV/mm being optimal. However, EF stimulation did not increase neurite length compared to no-EF conditions at any field strength. Moreover, the EF stimulation overcame the inhibitory effects of CSPG6 on growth of anode facing cortical cell neurites. In spinal cord slice cultures, there was a substantial increase in the alignment of re-growing axons when an EF was applied at 50mV/mm for 24h compared to the non-EF condition. Alignment was abolished when an Epac antagonist or Rho agonist were present in the culture medium.

Summary This is the first demonstration that EF stimulation promotes directional growth of cultured postnatal rat cortical neurons and that an EF aligns growth of re-growing axons in an ex vivo model of spinal injury. Future studies will investigate its in vivo efficacy alone and in combinational with other spinal repair strategies.

Authors*A. VARONE, Z. N. MUHAMAT, A. M. RAJNICEK, W. HUANG;
Univ. of Aberdeen, Aberdeen, United KingdomDisclosures A. Varone: None. Z.N. Muhamat: None. A.M. Rajnicek: None. W. Huang: None.

Grant Support University of Aberdeen, Tenovus Scotland, Scottish Rugby Union

Program No. 213.23. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

Extraction and selective activation of muscle synergies through spinal stimulation for SCI

Authors: *R. CHENG, J. W. BURDICK;

Lab Abstract:

Epidural spinal stimulation (ESS) has been shown to enable recovery of motor control in patients with clinically complete spinal cord injury (SCI). It is hypothesized that this results from activation of postural and locomotor circuitry in the spinal cord, but the physiological mechanisms are still unknown. In this study, we extracted muscle synergies for standing in a complete SCI patient under ESS using a novel factorization algorithm, and compared them to muscle synergies in healthy subjects in order to better understand the physiological mechanisms enabling motor control under ESS.
Muscle synergies represent the coordinated recruitment of a group of muscles co-activated by a specific neural activation signal. Standard muscle synergy extraction algorithms (e.g. NNMF, PCA) fail when applied to SCI patients under ESS, because they do not compensate for the physiological delays of an electrically stimulated neural signal to reach different muscles (e.g. a signal takes longer to reach a thigh versus calf muscle). These delays are prevalent in SCI patients under ESS, since an activating signal with fixed frequency is externally induced at a specific area of the spinal cord. Therefore, we utilize a new algorithm — regularized ShiftNMF — that accounts for these delays when extracting muscle synergies. We find that muscle synergies extracted by this algorithm are significantly better at reconstructing EMG activity, they are much more reliable when cross-validated on other sections of the EMG, and their resulting features are more physiologically meaningful.
Using this algorithm, we examine muscle synergies for standing from SCI patients under different spinal stimulation conditions, and also compare them to muscle synergies in healthy subjects. We find that (1) SCI patients exhibit fewer muscle synergies than healthy subjects, (2) when stimulated with a fixed stimulation pattern during standing, the patient’s muscle activity is composed of only a single muscle synergy, and (3) ESS with certain stimulation conditions (interleaving of multiple stimulation patterns) can activate an additional, distinct muscle synergy that greatly enhances patient standing quality. We provide evidence suggesting that muscle synergies are encoded in the human spinal cord, remain intact but possibly dormant after SCI, and are critical to quiet standing. The results allow us to hypothesize that an important physiological mechanism enabling motor control under ESS is the activation of muscle synergies in the spinal cord.

*R. CHENG, J. W. BURDICK;
Caltech, Pasadena, CA. Extraction and selective activation of muscle synergies through spinal stimulation for SCI. Program No. 296.07. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

Early limb unloading elicits long-term motor deficits involving motorneuron hyperexcitability associated with persistent alterations in glutamatergic synaptic plasticity in spinal cord injury

Authors: *K. MORIOKA^1,2, T. TAZOE^3,2, J. HUIE^1,4, J. HAEFELI¹, C. A. ALMEIDA¹, J. A. SACRAMENTO¹, J. C. BRESNAHAN¹, M. S. BEATTIE¹, S. TANAKA⁵, T. OGATA², A. R. FERGUSON^1,4;

Lab Abstract:

Abnormal sensory afferent feedback from the lower extremities after spinal cord injury (SCI) has potential to induce neuronal dysfunction that contributes to long-term motor deficits. Here, we investigated the impact of aberrant afferent input after SCI using hindlimb unloading early after mild contusive thoracic injury in adult female SD rats (T9; 50 kdyn IH). Three days post-injury, subjects were randomized to two experimental groups: 1) hindlimb unloading (HU) by tail suspension for 2 weeks followed by normal-loading for 6 weeks, or 2) normal-loading control for 8 weeks. Outcome assessments included: i) BBB open-field scoring and kinematic gait analysis; ii) electrophysiological H-reflex testing; iii) biomolecular and automated high-resolution confocal microscopy analysis of plasticity-related changes in lumbar ventral horn motor neurons. The results demonstrated that HU worsened impairment of hindlimb coordination after unloading (BBB = 12 for HU vs 17 for normal-loading controls). H-reflex testing of hindlimb muscles at 8 weeks showed that HU induced chronic hyper-excitability of spinal reflex circuitry. Quantitative biochemistry of ventral spinal synaptoneurosomes revealed a chronic increase in AMPA receptor (AMPAR) subunit GluA1 serine 831 phosphorylation, while quantitative immunohistochemistry revealed a chronic increase in GluA1 at synaptic sites on spinal motoneurons, suggesting that HU induced maladaptive plasticity in the spinal cord. Data-driven multidimensional analysis identified strong association between AMPAR over-drive on motorneurons and time-dependent motor recovery, chronic motorneuron hyper-excitability after HU. Our findings suggest that early unloading-induced aberrant afferent input after SCI can worsen maladaptive plasticity undermining long-term recovery, and provide a mechanistic rationale for early post-SCI intervention with weight-bearing training for precision rehabilitation.

*K. MORIOKA^1,2, T. TAZOE^3,2, J. HUIE^1,4, J. HAEFELI¹, C. A. ALMEIDA¹, J. A. SACRAMENTO¹, J. C. BRESNAHAN¹, M. S. BEATTIE¹, S. TANAKA⁵, T. OGATA², A. R. FERGUSON^1,4;

¹Dept. of Neurolog. Surgery, Brain and Spinal Injury Ctr. (BASIC), UCSF, San Francisco, CA; ²Dept. of Rehabil. for the Movement Functions, Res. Institute, Natl. Rehabil. Ctr. for the Persons with Disabilities, Saitama, Japan; ³Neural Prosthesis Project, Tokyo Metropolitan Inst. of Med. Sci., Tokyo, Japan; ⁴San Francisco Veterans Affairs Med. Ctr., San Francisco, CA; ⁵Dept. of Orthopaedic Surgery, The Univ. of Tokyo, Tokyo, Japan.

Grant Support:NIH Grant NS067092 (ARF), NIH Grant NS069537 (ARF), NIH Grant NS088475 (ARF), Wings for Life Spinal Cord Research Foundation WFLUS013/13 (KM), Wings for Life Spinal Cord Research Foundation WFLUS008/12, WFLUS 006/14 (ARF), Craig H. Neilsen Foundation 224308 (ARF), Craig H. Neilsen Foundation 313739 (JH), UCSF Core Center for Musculoskeletal Biology and Medicine P30AR066262 (ARF/KM)

Program No. 213.27. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

The effects of a pro-angiogenic, RGD-functionalized, nanofiber composite biomaterial on mesenchymal stem cell-mediated repair of the injured spinal cord

Agnes Haggerty PhD

Abstract:

Spinal cord injury (SCI) results in nervous tissue loss and so far untreatable functional impairments. Preclinical studies have demonstrated that a transplant of bone marrow-derived mesenchymal stem cells (MSCs) elicits paracrine effects resulting in anatomical repair and partial functional recovery. The effects of transplanted MSCs on spinal cord repair depend on their survival. MSCs are anchorage-dependent cells that are susceptible to anoikis, i.e., programmed cell death due to lack of adherence to a substrate. Thus, MSC transplant-mediated repair may be affected by lack of a binding substrate. It is known that MSCs adhere via integrin receptors to the tripeptide, arginine-glycine-aspartic acid (RGD). We investigated the effects of an RGD-functionalized nanofiber hydrogel composite biomaterial (NHC) on MSC transplant survival and the effects on anatomical repair and functional recovery in a clinically relevant adult rat model of spinal cord contusion. NHC consists of pro-angiogenic hyaluronic acid and axon growth-promoting nanofibers which form an injectable composite gel that closely resembles the physical properties (i.e. stiffness/porosity) of the spinal cord nervous tissue. NHC could target a multitude of factors that influencing MSC transplant survival and tissue remodeling after SCI. Pilot data suggests NHC improves MSC transplant survival and anatomical repair of the damaged spinal cord.

Authors*A. E. HAGGERTY¹, X. LI³, Y. NITOBE^1,4, I. MALDONADO-LASUNCION^1,5, K. YAMANE^1,6, M. MARLOW¹, H.-Q. MAO³, M. OUDEGA^2,7,8,1;
¹The Miami Project, ²Dept. of Neurolog. Surgery, Univ. of Miami, Miami, FL; ³Materials Sci. and Engineering, Inst. for NanoBiotechnology, Johns Hopkins Univ., Baltimore, MD; ⁴Dept. of Orthopedic Surgery, Hirosaki Univ. Grad. Sch. of Med., Hirosaki, Japan; ⁵Netherlands Inst. for Neurosciences, Amsterdam, Netherlands; ⁶Dept. of Orthopedic Surgery, Okayama Univ., Okayama, Japan; ⁷Affiliated Cancer Hosp. and Inst., Guangzhou Med. Univ., Guangzhou, China; ⁸Bruce W. Carter Dept. of Veterans Affairs Med. Ctr., Miami, FL

Disclosures A.E. Haggerty: None. X. Li: None. Y. Nitobe: None. I. Maldonado-Lasuncion: None. K. Yamane:None. M. Marlow: None. H. Mao: None. M. Oudega: None.

This work is supported by the Miami Project, State of Florida, Maryland Stem Cell Research Fund (2018-MSCRFCO-4088), and Department of Veteran Affairs (I01BX007080).

Program No. 213.21. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

From wheelchair to walking after spinal cord injury

Several paraplegics can now walk again – thanks to precise electrical stimulation of their spinal cords via a wireless implant and intensive neurorehabilitation. Speaking at Science Unlimited 2019, Grégoire Courtine illustrated this incredible spinal implant technology, as well as the next steps to turn this proof-of-concept into commonly available treatments for spinal cord injury.

Grégoire Courtine, EPFL

Grégoire is a pioneer in radically new approaches for restoring motor function after spinal cord injury. In addition to developing multifaceted neuroprosthetic systems, robotic interfaces, and advanced neuroregenerative interventions and neurorehabilitation procedures, his work also seeks to uncover the neural mechanisms underlying locomotion control and the processes that reestablish motor functions after neuromotor disorders. Grégoire is Associate Professor in the Center for Neuroprosthetics and the Brain Mind Institute, EPFL.

Frontiers Forum LINK

ReNetX Bio, Inc. Announces U.S. FDA Authorization to Proceed for IND Application to Treat Patients with Chronic Spinal Cord Injury

NEW HAVEN, Conn., June 19, 2019 /PRNewswire/ — ReNetX Bio, Inc., a leading biotechnology company committed to reversing disease and damage for patients suffering from central nervous system disorders, has announced that the U.S. Food and Drug Administration (FDA) has allowed the Company’s Investigational New Drug (IND) application for its lead drug candidate, fusion protein AXER-204.

Recruitment will begin immediately for the Phase 1 first-in-human trial of AXER-204 for the treatment of chronic spinal cord injury (SCI). The “RESET” Trial will assess safety, tolerability, pharmacokinetics, and efficacy in patients at leading SCI treatment centers across the U.S. An estimated 300,000 people are currently living with chronic SCI in the U.S., and there is currently no approved therapeutic to restore sensory or motor function after injury. An effective therapeutic to promote recovery of function after damage would be the first of its kind to address a significant and completely unmet medical need.

See the Full Story at PRNewswire HERE

 

Regeneration of Spinal Cord Connectivity Through Stem Cell Transplantation and Biomaterial Scaffolds

Authors: Hiroyuki Katoh, Kazuya Yokota and Michael Fehlings

“In this review, we outline the pathophysiology of SCI that makes the spinal cord refractory to regeneration and discuss the research that has been done with cell replacement and biomaterial implantation strategies, both by itself and as a combined treatment. We will focus on the capacity of these strategies to facilitate the regeneration of neural connectivity necessary to achieve meaningful functional recovery after SCI.”

See the Full Article at Frontiers: HERE

Survey responses needed to help with SCI priorities … Please take a moment

We have questions!!

Included here is a survey link (https://www.surveymonkey.com/r/MCPT5GM) asking you to rank areas of SCI related research – this will hopefully help us begin the brainstorming process to identify your interests or needs in terms of research and our abilities and resources to begin working on future research applications/grants.

Please feel free to share with other people who have experienced spinal cord injury, the more input the better.  After gathering as many responses as possible, we’ll use this to help guide further discussions about new research projects.

OSU Model Systems

Putting 3D Printing to Work to Heal Spinal Cord Injury

“For people whose spinal cords are injured in traffic accidents, sports mishaps, or other traumatic events, cell-based treatments have emerged as a potential avenue for encouraging healing. Now, taking advantage of advances in 3D printing technology, researchers have created customized implants that may boost the power of cell-based therapies for repairing injured spinal cords.”

See the full article by Dr. Francis Collins in the NIH Directors Blog 

Interfering with Semaphorin3A in perineuronal nets to enhance plasticity

Authors: D. CARULLI, R. BROERSEN, F. DE WINTER, H.-J. BOELE, B. HOBO, C. CANTO, E. M. MUIR, C. I. DE ZEEUW, J. VERHAAGEN

Daniela Carulli, visiting Professor with Verhaagen Lab Group

Lab Abstract:
Neuronal plasticity is crucial for our brain to learn, adapt to the environment and recover from brain injury. Plasticity decreases with age, particularly after childhood/adolescence. Perineuronal nets (PNNs) play a crucial role in restricting plasticity in the adult central nervous system. However, it is not entirely known how they act. PNNs are macromolecular assemblies of extracellular matrix and are composed of hyaluronan and chondroitin sulfate proteoglycans, which are kept together by link proteins and tenascin-R. We recently identified the chemorepulsive axon guidance protein Semaphorin3A (Sema3A) as a prominent component of PNN, suggesting that it may be a prime candidate in the control of PNN-mediated plasticity. To test this hypothesis we investigated the effect of interfering with Sema3A in the PNN of the deep cerebellar nuclei (DCN) of adult mice, at both the synaptic and behavioral level. Given that the DCN have abundant PNNs and are essential for associative eyeblink conditioning, they form a brain structure that is uniquely suited to investigate the role of PNN-associated Sema3A in learning-associated plasticity. To interfere with Sema3A signaling, we used an adeno-associated viral vector (AAV) encoding a soluble form of the Sema3A receptor component Neuropilin-1 (NP1-Y297A-Fc). NP1-Y297A-Fc retains its ability to interact with Sema3A, while binding with its other ligand, vascular endothelial growth factor (VEGF), is abolished by the mutation Y297A. With this approach, NP1-Y297A-Fc would act locally as a scavenger for Sema3A in the nets. We found that treatment with NP1-Y297A-Fc induces a significant increase in the size of axon terminals of Purkinje cells (the main inhibitory input on DCN neurons), which retain their discrete distribution along the target neuron membrane. In contrast, the digestion of the whole PNNs by the enzyme chondroitinase results in a decreased partition between neighboring Purkinje terminals. During associative motor learning, i.e. in mice subjected to eyeblink conditioning, PNNs and their Sema3A content in the DCN are reduced, suggesting that Sema3A plays an inhibitory role in the formation of this type of memory. Notably, digesting the whole PNNs in the DCN leads to faster learning in the eyeblink conditioning paradigm. These data show that PNNs are crucial for cerebellar plasticity and cerebellum-dependent learning, and support the hypothesis that the chemorepulsive axon guidance cue Sema3A is an effector protein with a key role in PNN-mediated plasticity.

Abstract Citation
D. CARULLI1,3, R. BROERSEN2,4, *F. DE WINTER1, H.-J. BOELE4, B. HOBO1, C. CANTO2,4, E. M. MUIR5, C. I. DE ZEEUW2,4, J. VERHAAGEN1;
1Lab. for Neuroregeneration, 2Lab. for Cerebellar Coordination and Cognition, Netherlands Inst. for Neurosci., Amsterdam, Netherlands; 3Dept. of Neurosci. and Neurosci. Inst. Cavalieri-Ottolenghi, Univ. of Turin, Turin, Italy; 4Dept. of Neurosci., Erasmus MC, Rotterdam, Netherlands; 5Dept. of Physiology, Develop. and Neurosci., Univ. of Cambridge, Cambridge, United Kingdom. Interfering with Semaphorin3A in perineuronal nets to enhance plasticity. Program No. 201.13. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.