Chemotropic guidance facilitates axonal regeneration and synapse formation after spinal cord injury.

A principal objective of spinal cord injury (SCI) research is the restoration of axonal connectivity to denervated targets. We tested the hypothesis that chemotropic mechanisms would guide regenerating spinal cord axons to appropriate brainstem targets. We subjected rats to cervical level 1 (C1) lesions and combinatorial treatments to elicit axonal bridging into and beyond lesion sites. Lentiviral vectors expressing neurotrophin-3 (NT-3) were then injected into an appropriate brainstem target, the nucleus gracilis, and an inappropriate target, the reticular formation. NT-3 expression in the correct target led to reinnervation of the nucleus gracilis in a dose-related fashion, whereas NT-3 expression in the reticular formation led to mistargeting of regenerating axons. Axons regenerating into the nucleus gracilis formed axodendritic synapses containing rounded vesicles, reflective of pre-injury synaptic architecture. Thus, we report for the first time, to the best of our knowledge, the reinnervation of brainstem targets after SCI and an essential role for chemotropic axon guidance in target selection

Depolarization and electrical stimulation enhance in vitro and in vivo sensory axon growth after spinal cord injury

Activity dependent plasticity is a key mechanism for the central nervous system (CNS) to adapt to its environment. Whether neuronal activity also influences axonal regeneration in the injured CNS, and whether electrical stimulation (ES) can activate regenerative programs in the injured CNS remains incompletely understood. Using KCl-induced depolarization, in vivo ES followed by ex-vivo neurite growth assays and ES after spinal cord lesions and cell grafting, we aimed to identify parameters important for ES-enhanced neurite growth and axonal regeneration. Using cultures of sensory neurons, neurite growth was analyzed after KCl-induced depolarization for 1-72h. Increased neurite growth was detected after short-term stimulation and after longer stimulation if a sufficient delay between stimulation and growth measurements was provided. After in vivo ES (20Hz, 2× motor threshold, 0.2ms, 1h) of the intact sciatic nerve in adult Fischer344 rats, sensory neurons showed a 2-fold increase in in vitro neurite length one week later compared to sham animals, an effect not observed one day after ES. Longer ES (7h) and repeated ES (7days, 1h each) also increased growth by 56-67% one week later, but provided no additional benefit. In vivo growth of dorsal column sensory axons into a graft of bone marrow stromal cells 4weeks after a cervical spinal cord lesion was also enhanced with a single post-injury 1h ES of the intact sciatic nerve and was also observed after repeated ES without inducing pain-like behavior. While ES did not result in sensory functional recovery, our data indicate that ES has time-dependent influences on the regenerative capacity of sensory neurons and might further enhance axonal regeneration in combinatorial approaches after SCI

An Inducible Tyrosine Kinase Receptor for Axonal Regeneration

The prevention or reduction of neuronal degeneration remains a challenge in neurotrophins therapy. An inducible trkA (ItrkA) system has been shown to regulate embryonic dorsal root ganglion (DRG) neuronal survival and neurite outgrowth in vitro. A new ItrkA plasmid ItrkA-membrane (ItrkAmemb) with one adenine at 3’ terminal was established by correcting the sequence of the original plasmid ItrkA-cytosol (ItrkAcyto). Adult DRGs were dissected from adult Fischer 344 rats (8-14 weeks) for the treatment with AP20187 (membrane-permeable small-molecule ligand), vehicle or NGF (Nerve Growth Factor). Neurite outgrowth assessments were done by manually tracing the longest neurite of each neuron. Cell diameters were also measured and averaged for each well. Protein expression after ItrkAmemb transfection and trkA downstream signaling were investigated by Western-blotting. Neurite length of ItrkAmemb transfected DRGs was not influenced by AP20187 or NGF but cells displayed shorter neurites compared to GFP control groups. While ItrkAcyto transfected DRGs cultured with AP20187 had the longest neurite growth compared to ItrkAmemb transfected neurons and ItrkAcyto transfected cells treated with vehicle or NGF, no significant difference to GFP controls was detected. Quantification of the mean diameter of transfected DRGs demonstrated that ItrkAmemb electroporation significantly increased cell diameter, while the diameter of ItrkAcyto transfected neurons and GFP controls were almost the same as naïve neurons. In contrast to electroporated adult DRG neurons, ItrkAmemb virus transfection did not affect the diameter of infected adult DRG Neurons. No obvious difference was observed between the ItrkAmemb and GFP electroporated cells, and only cells transduced with ItrkAmemb treated with AP20187 seemed to show higher phosphorylation both of Akt and Erk1/2. The effect of adult DRG neurons after ItrkA transfection differs, which depends on the change of cell soma size and/or neurite growth, gene delivery technique, expression level and the localization of ItrkA

Systemic epothilone D improves hindlimb function after spinal cord contusion injury in rats

Following a spinal cord injury (SCI) a growth aversive environment forms, consisting of a fibroglial scar and inhibitory factors, further restricting the already low intrinsic growth potential of injured adult central nervous system (CNS) neurons. Previous studies have shown that local administration of the microtubule-stabilizing drug paclitaxel or epothilone B (Epo B) reduce fibrotic scar formation and axonal dieback as well as induce axonal growth/sprouting after SCI. Likewise, systemic administration of Epo B promoted functional recovery. In this study, we investigated the effects of epothilone D (Epo D), an analog of Epo B with a possible greater therapeutic index, on fibrotic scarring, axonal sprouting and functional recovery after SCI. Delayed systemic administration of Epo D after a moderate contusion injury (150 kDyn) in female Fischer 344 rats resulted in a reduced number of footfalls when crossing a horizontal ladder at 4 and 8 weeks post-injury. Hindlimb motor function assessed with the BBB open field locomotor rating scale and Catwalk gait analysis were not significantly altered. Moreover, formation of laminin positive fibrotic scar tissue and 5-HT positive serotonergic fiber length caudal to the lesion site were not altered after treatment with Epo D. These findings recapitulate a functional benefit after systemic administration of a microtubule-stabilizing drug in rat contusion SCI

Targeted tissue engineering: hydrogels with linear capillary channels for axonal regeneration after spinal cord injury

Spinal cord injury (SCI) frequently results in the permanent loss of function below the level of injury due to the failure of axonal regeneration in the adult mammalian central nervous system (CNS). The limited intrinsic growth capacity of adult neurons, a lack of growth-promoting factors and the multifactorial inhibitory microenvironment around the lesion site contribute to the lack of axonal regeneration. Strategies such as transplantation of cells, delivery of bioactive compounds and gene transfer have been investigated as a means to promote axonal regrowth through the lesion, to form new synaptic connections and to improve functional outcomes. Although growth of some axonal populations can be robustly enhanced by cellular implants alone or in combination with neurotrophic factors, axons usually extend in random orientation and even reverse growth direction in the lesion site (Figure 1A) (Gros et al., 2010; Günther et al., 2015). Thus, regenerating axons often fail to approach the distal edge of the lesion site, a pre-requisite for proper contact with spared host neurons. The lack of a 3-dimensional organization in the injury site is therefore an additional barrier for successful axonal bridging. Two approaches, physical guidance through structured scaffolds and chemical guidance by growth factor gradients, have emerged as potential means to provide directional cues for axonal growth through the lesion

Transient growth factor delivery sustains regenerated axons after spinal cord injury

Growth factors influence the topography of axonal projections during nervous system development and facilitate axonal sprouting and regeneration after injury in the adult. However, in the absence of target reinnervation and reestablishment of synaptic activity, we hypothesized that continuing delivery of neurotrophins would be required to sustain regenerating axons for prolonged times points after neurotrophin-induced axon growth after spinal cord injury ( SCI) in the adult. Using tetracycline-inducible expression of brain-derived neurotrophic factor by genetically modified fibroblasts, we were able to extensively and significantly turn growth factor expression "on" or "off" in vitro and in vivo within sites of SCI. Notably, we find that transient growth factor delivery is sufficient to sustain regenerated axons for prolonged time periods within spinal cord lesion sites. Immunohistochemical analysis demonstrated an absence of neuronal targets or synapses within transient growth factor expressing grafts but the persistent presence of Schwann cells. Thus, the adult CNS appears capable of sustaining axons that have extended after transient growth factor delivery, an effect potentially attributable to the persistence of Schwann cells in lesion/graft sites

Gene Therapy and Cell Transplantation for Alzheimer's Disease and Spinal Cord Injury

The targeted delivery of genes and the transplantation of suitable cell types into the adult nervous system have received considerable interest over the last years. The development of improved vector systems for in vivo gene delivery and the discovery of neural stem cells in the adult nervous system have opened new venues for potential therapeutic intervention in progressive neurodegenerative disease and nervous system injury. Thus, strategies have evolved for the delivery of potentially neuroprotective molecules, such as neurotrophic factors, and the replacement of cells and tissue lost due to CNS injury and degeneration

Gene therapy and cell transplantation for Alzheimer's disease and spinal cord injury

The targeted delivery of genes and the transplantation of suitable cell types into the adult nervous system have received considerable interest over the last years. The development of improved vector systems for in vivo gene delivery and the discovery of neural stem cells in the adult nervous system have opened new venues for potential therapeutic intervention in progressive neurodegenerative disease and nervous system injury. Thus, strategies have evolved for the delivery of potentially neuroprotective molecules, such as neurotrophic factors, and the replacement of cells and tissue lost due to CNS injury and degeneration