Influencing axon regeneration after spinal cord injury

Spinal cord injury (SCI) results in a severing of axonal connections that leads to permanent sensorimotor deficits. In cases of incomplete injury, rehabilitation paradigms have been reported to be successful in improving behavioral outcome in humans with SCI. However, in cases of more anatomically complete injury, which leaves little or no spared axon projections below the injury, rehabilitation is ineffective. For decades, it has been realized that restoration of function will likely require the regrowth of severed axons through lesion sites. Unfortunately, axons in the mature mammalian central nervous system (CNS) fail to regenerate following injury due to a low intrinsic neuronal growth capacity as well as a non-supportive lesion environment. Although much progress has been made in the field of axon regeneration, current strategies produce only relatively little growth past SCI lesions. A more thorough understanding of the mechanisms governing growth of particular neuronal systems will be critical for developing therapeutic approaches to restore axon connectivity across lesion sites that may lead to behavioral improvement. This thesis is divided into three chapters. In chapter one, I provide a literature review of various reasons for axon regenerative failure and potential strategies to overcome these challenges. In chapter two, I describe two cell-grafting approaches that I investigated to bridge axons across SCI lesions. In the first approach, I replicated a promising cell grafting technique and performed a pilot experiment where we combined cell grafting with neuroprosthetic rehabilitation. While a subset of these rats had continuous bridges spanning the lesion site, we failed to observe an increase in behavioral recovery. This may have been the result of i) undirected and excessive growth of graft derived axons, or ii) cell masses which migrated to ectopic locations in the spinal cord, which may have compressed tissue. In chapter three, I describe the majority of my thesis work, in which I have been characterizing the requirements for propriospinal axon regeneration past severe SCI lesions in both mice and rats. We used a biomaterials approach to deliver protein growth factors which modulated the lesion environment into upregulating substantive amounts of growth supportive laminin. A chemotropic stimulatory cue in the form of glial derived neurotropic factor (GDNF) was delivered both within and caudal to SCI lesions, allowing some axons to grow into host tissue. In the absence of chemotrophic guidance cues, axons failed to grow out of SCI lesions. This further reinforces the idea that the absence of facilitating factors is a primary reason for the failure of CNS neurons to regenerate following injury. In order to enhance this growth, we tested viral manipulations which have been previously demonstrated to increase growth of other CNS neurons, and combined this with hydrogel depot delivery of growth factors. We found that downregulation of PTEN failed to increase growth of propriospinal neurons. In contrast, over expression of IGF1/CNTF/OPN resulted in robust growth of propriospinal axons into and through severe SCI lesions. Axons grew into and through the lesion site towards the chemotropic hydrogel depot, but did not grow past this. The results in this thesis are, to the best of our knowledge, the most extensive degree of host axon regeneration past anatomically complete SCI lesions to date.

Required growth facilitators propel axon regeneration across complete spinal cord injury

Mark Andrew Anderson, Sabry Leonardo Barlatey, Grégoire Courtine, Zhigang He, Nicholas David James, Michael V. Sofroniew, Cheng Wang

Transected axons fail to regrow across anatomically complete spinal cord injuries (SCI) in adults. Diverse molecules can partially facilitate or attenuate axon growth during development or after injury1,2,3, but efficient reversal of this regrowth failure remains elusive4. Here we show that three factors that are essential for axon growth during development but are attenuated or lacking in adults—(i) neuron intrinsic growth capacity2,5,6,7,8,9, (ii) growth-supportive substrate10,11 and (iii) chemoattraction12,13—are all individually required and, in combination, are sufficient to stimulate robust axon regrowth across anatomically complete SCI lesions in adult rodents. We reactivated the growth capacity of mature descending propriospinal neurons with osteopontin, insulin-like growth factor 1 and ciliary-derived neurotrophic factor before SCI14,15; induced growth-supportive substrates with fibroblast growth factor 2 and epidermal growth factor; and chemoattracted propriospinal axons with glial-derived neurotrophic factor16,17 delivered via spatially and temporally controlled release from biomaterial depots18,19, placed sequentially after SCI. We show in both mice and rats that providing these three mechanisms in combination, but not individually, stimulated robust propriospinal axon regrowth through astrocyte scar borders and across lesion cores of non-neural tissue that was over 100-fold greater than controls. Stimulated, supported and chemoattracted propriospinal axons regrew a full spinal segment beyond lesion centres, passed well into spared neural tissue, formed terminal-like contacts exhibiting synaptic markers and conveyed a significant return of electrophysiological conduction capacity across lesions. Thus, overcoming the failure of axon regrowth across anatomically complete SCI lesions after maturity required the combined sequential reinstatement of several developmentally essential mechanisms that facilitate axon growth. These findings identify a mechanism-based biological repair strategy for complete SCI lesions that could be suitable to use with rehabilitation models designed to augment the functional recovery of remodelling circuits

2018

Cortico-reticulo-spinal circuits mediate functional recovery after spinal cord contusion

Léonie Asboth

After a spinal cord injury (SCI), only half of affected individuals regain voluntary control of leg movements below the level of the lesion. Anatomical, electrophysiological and imaging data revealed that even the most severe forms of SCI usually spare a small bridge of tissue in the surrounding of the lesion cavity. These bridges contain residual fibers from brain projections that maintain a physical connection with the lumbar spinal cord, where the circuits coordinating leg movements reside. However, without therapeutic interventions, these spared fibers will fail to conduct nervous information from brain centers to the spinal circuits below injury, leading to permanent paralysis. It is accepted that spontaneous regeneration of lesioned fibers is limited in the adult nervous system, but hope for motor recovery lies in the capacity of the spared neural motor network to readapt and find compensational routes. In the past, a neuroprosthetic rehabilitation strategy combining electrochemical neuromodulation of the spinal cord during robot-assisted overground locomotion was developed for rats to restore volitional control of the paralyzed legs. Such rehabilitation platforms was developed for rats, enabling large-scale studies prior to pre-clinical trials in non-human primates. However, most innovative tools for the manipulation of specific populations of neurons are currently designed for mice, and these techniques are essential to establish causality between the anatomical reorganization and the observed functional recovery. In the first part of this thesis, we developed a robotic interface with end-effector modules that can be rapidly exchanged between experiments with rats or mice. The functionalities of the robot are tailored to the size and weight of the animal, to the personalized need of assistance and to the type of performed task. Using this platform, the next part of the thesis uncovers the mechanisms enabling motor recovery after a clinically relevant injury model â a severe spinal cord contusion. Such injury leads to permanent leg motor deficits with very little spontaneous recovery. Similar to humans, the modelled contusion injury induce highly variable white matter damage with undefined neural pathway sparing. For the first time, we show that the rehabilitation strategy enabled rats to regain supraspinal control of leg movements that persisted without any neuromodulation even during unpracticed motor tasks. We then investigated the reorganization of the spared circuitry that mediated this functional recovery after contusion. Despite interruption of direct corticospinal projections, we showed that the motor cortex regained adaptive control over the paralyzed legs during neuromodulation of lumbar circuits. We found that the cortical command was relayed through spared glutamatergic projection neurons in the reticular formation. Indeed, due to the distributed spatial topography of reticulospinal projections, the contusion systematically spares a subset of these projections. Extensive training promoted plasticity of the identified cortico-reticulo-spinal pathway mediating a motor cortex dependent recovery of voluntary leg movements. As the anatomy and function of reticulospinal pathways are well-conserved across mammals, this pathway may be recruited by similar therapeutic interventions to improve recovery in humans.

EPFL2017

Axon regeneration and circuit reorganization after complete spinal cord injury

Sabry Leonardo Barlatey

First human trials involving neuroprosthetic rehabilitation demonstrated recently that significant functional benefits can be achieved with lumbosacral neuromodulation and reorganized spared projections. However, complete spinal cord injuries (SCI) wholly isolate infralesional circuits from any supraspinal control, leading to irreversible motor deficits that neuroprosthetics still fails to address. As axons fail to regrow across the lesion site, neuroregenerative research after SCI has been dogmatically focusing attention on minimizing the environmental inhibitions to axon regeneration. In the present work, we demonstrate that spontaneous axon regeneration failure can be reversed, and that propriospinal axons are able to grow across complete non-neural lesion cores when the required facilitators are provided. We identified three mechanisms needed for propriospinal regrowth : i) enhancing neuronal intrinsic growth capacities using viral technologies, ii) remodeling the lesion core with growth factors, in order to densify permissive substrates, and iii) guiding axons chemically across the SCI site. Propriospinal neurons - that coordinate different spinal segments in healthy conditions - are known to support recovery of locomotion in incomplete models of spinal cord injury. However, restoring a robust descending bridge of propriospinal axons does not by itself promote any functional benefit. We hypothesize that a lack of somatosensory feedback to the motor cortex (M1), together with insufficient descending motor control, may prevent coherent exchange of information between lumbosacral and cortical motor centers. Therefore, we explored the neuroregenerative potential of reticulospinal axons - which are projected from the brainstem to the spinal cord - as a second relay for the descending motor cortical command. Together with axon guidance and lesion remodeling, our viral manipulation of intrinsic growth programs induced limited yet significant reticulospinal regeneration into the lesion core. In order to enhance this reticulospinal regrowth, we explored activity-dependent mechanisms of neurite growth control. As we observed that activity does not recruit growth programs after SCI, we revealed a possible divergence between the mechanisms that underly reticulospinal axon regrowth and neurite sprouting. In the mean time, we are developing a somatosensory interface that aims at restoring sensory feedback to the motor cortex after complete SCI. Based on hindlimb electromyographic activity, we linked locomotion to optogenetic neuromodulation of the motor thalamus, and were able to evoke responses in M1 by selectively activating thalamocortical projections, both before and after complete spinal lesion. Such technology enables a direct recruitment of thalamic nuclei that gate motor learning and motor planning at a cortical level. Coming experiments will test wether a strategy combining propriospinal regeneration with locomotion-dependent thalamocortical modulation is sufficient to restore voluntary stepping after complete SCI.

EPFL2019