Discovery and lead optimisation of a potent, selective and orally bioavailable RARß agonist for the potential treatment of nerve injury.

Oxadiazole replacement of an amide linkage in an RARα agonist template 1, followed by lead optimisation, has produced a highly potent and selective RARß agonist 4-(5-(4,7-dimethylbenzofuran-2-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (10) with good oral bioavailability in the rat and dog. This molecule increases neurite outgrowth in vitro and induces sensory axon regrowth in vivo in a rodent model of avulsion and crush injury, and thus has the potential for the treatment of nerve injury.

Retinoic acid synthesis by NG2 expressing cells promotes a permissive environment for axonal outgrowth.

Stimulation of retinoic acid (RA) mediated signalling pathways following neural injury leads to regeneration in the adult nervous system and numerous studies have shown that the specific activation of the retinoic acid receptor ß (RARß) is required for this process. Here we identify a novel mechanism by which neuronal RARß activation results in the endogenous synthesis of RA which is released in association with exosomes and acts as a positive cue to axonal/neurite outgrowth. Using an established rodent model of RARß induced axonal regeneration, we show that neuronal RARß activation upregulates the enzymes involved in RA synthesis in a cell specific manner; alcohol dehydrogenase7 (ADH7) in neurons and aldehyde dehydrogenase 2 (Raldh2) in NG2 expressing cells (NG2+ cells). These release RA in association with exosomes providing a permissive substrate to neurite outgrowth. Conversely, deletion of Raldh2 in the NG2+ cells in our in vivo regeneration model is sufficient to compromise axonal outgrowth. This hitherto unidentified RA paracrine signalling is required for axonal/neurite outgrowth and is initiated by the activation of neuronal RARß signalling.

Neuronal RARß Signaling Modulates PTEN Activity Directly in Neurons and via Exosome Transfer in Astrocytes to Prevent Glial Scar Formation and Induce Spinal Cord Regeneration.

Failure of axonal regeneration in the central nervous system (CNS) is mainly attributed to a lack of intrinsic neuronal growth programs and an inhibitory environment from a glial scar. Phosphatase and tensin homolog (PTEN) is a major negative regulator of neuronal regeneration and, as such, inhibiting its activity has been considered a therapeutic target for spinal cord (SC) injuries (SCIs). Using a novel model of rat cervical avulsion, we show that treatment with a retinoic acid receptor ß (RARß) agonist results in locomotor and sensory recovery. Axonal regeneration from the severed roots into the SC could be seen by biotinylated dextran amine labeling. Light micrographs of the dorsal root entry zone show the peripheral nervous system (PNS)-CNS transition of regrown axons. RARß agonist treatment also resulted in the absence of scar formation. Mechanism studies revealed that, in RARß-agonist-treated neurons, PTEN activity is decreased by cytoplasmic phosphorylation and increased secretion in exosomes. These are taken up by astrocytes, resulting in hampered proliferation and causing them to arrange in a normal-appearing scaffold around the regenerating axons. Attribution of the glial modulation to neuronal PTEN in exosomes was demonstrated by the use of an exosome inhibitor in vivo and PTEN siRNA in vitro assays. The dual effect of RARß signaling, both neuronal and neuronal-glial, results in axonal regeneration into the SC after dorsal root neurotmesis. Targeting this pathway may open new avenues for the treatment of SCIs. SIGNIFICANCE STATEMENT: Spinal cord injuries (SCIs) often result in permanent damage in the adult due to the very limited capacity of axonal regeneration. Intrinsic neuronal programs and the formation of a glial scar are the main obstacles. Here, we identify a single target, neuronal retinoic acid receptor ß (RARß), which modulates these two aspects of the postinjury physiological response. Activation of RARß in the neuron inactivates phosphatase and tensin homolog and induces its transfer into the astrocytes in small vesicles, where it prevents scar formation. This may open new therapeutic avenues for SCIs.

The loop diuretic bumetanide blocks posttraumatic p75NTR upregulation and rescues injured neurons.

Injured neurons become dependent on trophic factors for survival. However, application of trophic factors to the site of injury is technically extremely challenging. Novel approaches are needed to circumvent this problem. Here, we unravel the mechanism of the emergence of dependency of injured neurons on brain-derived neurotrophic factor (BDNF) for survival. Based on this mechanism, we propose the use of the diuretic bumetanide to prevent the requirement for BDNF and consequent neuronal death in the injured areas. Responses to the neurotransmitter GABA change from hyperpolarizing in intact neurons to depolarizing in injured neurons. We show in vivo in rats and ex vivo in mouse organotypic slice cultures that posttraumatic GABA(A)-mediated depolarization is a cause for the well known phenomenon of pathological upregulation of pan-neurotrophin receptor p75(NTR). The increase in intracellular Ca(2+) triggered by GABA-mediated depolarization activates ROCK (Rho kinase), which in turn leads to the upregulation of p75(NTR). We further show that high levels of p75(NTR) and its interaction with sortilin and proNGF set the dependency on BDNF for survival. Thus, application of bumetanide prevents p75(NTR) upregulation and neuronal death in the injured areas with reduced levels of endogenous BDNF.

Self-mutilation in patients after nerve injury may not be due to deafferentation pain: a case report.

OBJECTIVE: Animals with transected nerves may develop self-mutilating behavior (autotomy) directed at the denervated body part. Autotomy is often thought to be a response to deafferentation pain produced by pathological changes in the dorsal horn, and self-mutilation after dorsal rhizotomy has consequently been used as an outcome measure for the investigation of chronic pain in animal models. A less recognized hypothesis suggests that autotomy is simply an animal's efforts to remove the useless part. We report a case of self-mutilation of the thumb and fingers in a patient with loss of all sensory modalities in the arm after brachial plexus avulsion. CONCLUSION: Asking the patient about the reasons for his self-mutilation provides insights into the cause of autotomy which cannot be established from animal studies. We suggest that autotomy may not be a result of chronic pain, and discuss the human experience and alternative underlying pathological processes.

Novel strategies in brachial plexus repair after traumatic avulsion.

Clinical trials in spinal cord injury (SCI) can be affected by many confounding variables including spontaneous recovery, variation in the lesion type and extend. However, the clinical need and the paucity of effective therapies has spawned a large number of animal studies and clinical trials for SCI. In this review, we suggest that brachial plexus avulsion injury, a longitudinal spinal cord lesion, is a simpler model to test methods of spinal cord repair. We explore reconstructive techniques currently explored for the repair of brachial plexus avulsion and focus on the use of olfactory ensheathing cell transplantation as an adjunct treatment in brachial plexus repair.

Repair and rehabilitation of plexus and root avulsions in animal models and patients.

PURPOSE OF REVIEW: This review will discuss recent progress in experimental and translational research related to surgical repair of proximal nerve root injuries, and emerging potential therapies, which may be combined with replantation surgeries to augment functional outcomes after brachial plexus and cauda equina injuries. RECENT FINDINGS: Progress in experimental studies of root and peripheral nerve injuries has identified potential candidates for adjunctive therapies, which may be combined with surgical replantation of avulsed roots after brachial plexus and cauda equina injuries. We will discuss recent advances related to adjunctive neuroprotective strategies, neurotrophic factor delivery, and emerging cellular treatment strategies after extensive nerve root trauma. We will also provide an update on electrical stimulation to promote regenerative axonal growth and new insights on the recovery of sensory functions after root injury and repair. SUMMARY: In the light of recent advances in experimental studies, we envision that future repair of brachial plexus and cauda equina injuries will include spinal cord surgery to restore motor and sensory trajectories and a variety of adjunctive therapies to augment the recovery of neurological function.

Neuroregenerative effects of lentiviral vector-mediated GDNF expression in reimplanted ventral roots.

Traumatic avulsion of spinal nerve roots causes complete paralysis of the affected limb. Reimplantation of avulsed roots results in only limited functional recovery in humans, specifically of distal targets. Therefore, root avulsion causes serious and permanent disability. Here, we show in a rat model that lentiviral vector-mediated overexpression of glial cell line-derived neurotrophic factor (GDNF) in reimplanted nerve roots completely prevents motoneuron atrophy after ventral root avulsion and stimulates regeneration of axons into reimplanted roots. However, over the course of 16 weeks neuroma-like structures are formed in the reimplanted roots, and regenerating axons are trapped at sites with high levels of GDNF expression. A high local concentration of GDNF therefore impairs long distance regeneration. These observations show the feasibility of combining neurosurgical repair of avulsed roots with gene-therapeutic approaches. Our data also point to the importance of developing viral vectors that allow regulated expression of neurotrophic factors.

Root repair review: basic science background and clinical outcome.

Spinal nerve root injuries have a profound effect on the different parts (PNS and CNS) of the root itself as well as the pertinent spinal cord segment. A root avulsion from the spinal cord is a longitudinal spinal cord injury. There is degeneration of sensory and motor axons, loss of synapses, deterioration of local segmental connections, nerve cell death and reactions among non neuronal cells with scar formation, i.e. a cascade of events similar to those known to occur in any injury to the spinal cord. For function to be restored, nerve cells must survive and there must be regrowth of new nerve fibres along a trajectory consisting of CNS growth-inhibitory tissue in the spinal cord as well as PNS growth-promoting tissue in nerves. Problems in PNS regeneration such as non directional growths and unspecific reinnervation of target organs lead to unpredictable sensorimotor activity and conspires against a useful recovery of function. From the results of basic science experiments, a surgical strategy to treat root avulsion with spinal cord injury has been developed. In humans this technique is currently the most promising treatment of any spinal cord injury, with return of useful function together with pain alleviation in cases where all nerves to the extremity have been avulsed from the spinal cord. At present the shortcomings of this technique are proportionate to the delay before surgery, which leads to death of nerve cells and incomplete and unpredictable recovery. In order to improve this situation and achieve further recovery of useful function including sensory perceptions and to fully alleviate pain it is necessary to pursue research and development of both basic and clinical science.

Cell death after dorsal root injury.

Flow cytometry and terminal deoxynucleotidyl transferase-mediated biotinylated uridine triphosphate nick end-labelling (TUNEL) immunohistochemistry have been used to assess cell death in the dorsal root ganglia (DRG) or spinal cord 1, 2 or 14 days after multiple lumbar dorsal root rhizotomy or dorsal root avulsion injury in adult rats. Neither injury induced significant cell death in the DRG compared to sham-operated or naïve animals at any time point. In the spinal cord, a significant increase in death was seen at 1-2 days, but not 14 days, post injury by both methods. TUNEL staining revealed that more apoptotic cells were present in the dorsal columns and dorsal horn of avulsion animals compared to rhizotomised animals. This suggests that avulsion injury, which can often partially damage the spinal cord, has more severe effects on cell survival than rhizotomy, a surgical lesion which does not affect the spinal cord. The location of TUNEL positive cells suggests that both neuronal and non-neuronal cells are dying.

Intradural repair of lumbar nerve roots for traumatic paraparesis leading to functional recovery.

STUDY DESIGN: Case report and a literature review of intradural nerve graft repair of traumatic lacerations of lumbar nerve roots. OBJECTIVE: Describe the technique and results of recent approach to the surgical management of traumatic lumbar nerve root injuries. SUMMARY OF BACKGROUND DATA: Lumbar nerve root injuries are associated with a poor prognosis in terms of neurologic recovery and are generally managed conservatively. Animal studies and previous attempts at nerve root repair at both the level of the cervical and lumbar spine have demonstrated that some neurologic recovery is possible with the use of such methods. METHODS: The management of a 29-year-old man who sustained a traumatic fracture of his second lumbar vertebra resulting in the lacerations of his right second and third lumbar nerve roots with associated neurologic deficits is presented. The technique of nerve root repair is described with the outcome assessed by means of clinical examination, magnetic resonance imaging, and electromyography. RESULTS: At 8 years' clinical examination showed an absence of power in extension of his right knee with a complete recovery of motor function in hip flexion on the right side. Electromyography confirmed the clinical findings by demonstrating reinnervation of the right iliopsoas muscle. CONCLUSIONS: This case describes a surgical approach to the management of traumatic lumbar nerve root injuries that offers the possibility of neurologic recovery and challenges the traditional approach of conservative management.

Lentiviral vector-mediated reporter gene expression in avulsed spinal ventral root is short-term, but is prolonged using an immune "stealth" transgene.

PURPOSE: Spinal root avulsions result in paralysis of the upper and/or lower extremities. Implanting a peripheral nerve bridge or reinsertion of the avulsed roots in the spinal cord are surgical strategies that lead to some degree of functional recovery. In the current study lentiviral (LV) vector-mediated gene transfer of a green fluorescent protein (GFP) reporter gene was used to study the feasibility of gene therapy in the reimplanted root to further promote regeneration of motor axons. METHODS: A total of 68 female Wistar rats underwent unilateral root avulsion of the L4, L5 and L6 ventral lumbar roots. From 23 rats intercostal nerves were dissected before ventral root avulsion surgery, injected with a lentiviral vector encoding GFP (LV-GFP) and inserted between the spinal cord and avulsed rootlet. In the remaining 45 rats, the avulsed ventral root was injected with either LV-GFP or a lentiviral vector encoding a fusion between a GlyAla repeat and GFP (LV-GArGFP), and reinserted into the spinal cord. Expression of GFP was evaluated at 1,2, 4 and 10 weeks, and one group at 4 months. RESULTS: LV-GFP transduction of either nerve implants or reimplanted ventral roots revealed high GFP expression during the first 2 post-lesion weeks, but virtually no expression at 4 weeks. Since this reduction coincided with the appearance of mononuclear cells at the repair site, an immune response against GFP may have occurred. In a subsequent experiment reimplanted ventral roots were transduced with a vector encoding GFP fused with the GlyAla repeat of Epstein-Barr virus Nuclear Antigen 1 known to prevent generation of antigenic peptides from transgene products. Expression of this "stealth" gene persisted for at least 4 months in the reimplanted root. CONCLUSION: Thus persistent transgene expression can be achieved with non-immunogenic transgene products in reimplanted ventral roots. This demonstrates the feasibility of combining neurosurgical repair with LV vector-mediated gene therapy. The current approach will be used in future experiments with LV vectors encoding neurotrophic factors to enhance the regeneration of spinal motor neurons after traumatic avulsion of spinal nerve roots.

Corrigendum: New Treatments for Spinal Nerve Root Avulsion Injury.

[This corrects the article on p. 135 in vol. 7, PMID: 27602018.].

Surgical reconstruction of spinal cord circuit provides functional return in humans.

This mini review describes the current surgical strategy for restoring function after traumatic spinal nerve root avulsion in brachial or lumbosacral plexus injury in man. As this lesion is a spinal cord or central nervous injury functional return depends on spinal cord nerve cell growth within the central nervous system. Basic science, clinical research and human application has demonstrated good and useful motor function after ventral root avulsion followed by spinal cord reimplantation. Recently, sensory return could be demonstrated following spinal cord surgery bypassing the injured primary sensory neuron. Experimental data showed that most of the recovery depended on new growth reinnervating peripheral receptors. Restored sensory function and the return of spinal reflex was demonstrated by electrophysiology and functional magnetic resonance imaging of human cortex. This spinal cord surgery is a unique treatment of central nervous system injury resulting in useful functional return. Further improvements will not depend on surgical improvements. Adjuvant therapy aiming at ameliorating the activity in retinoic acid elements in dorsal root ganglion neurons could be a new therapeutic avenue in restoring spinal cord circuits after nerve root avulsion injury.

Long-Term Outcome of Brachial Plexus Reimplantation After Complete Brachial Plexus Avulsion Injury.

BACKGROUND: Complete brachial plexus avulsion injury is a severe disabling injury due to traction to the brachial plexus. Brachial plexus reimplantation is an emerging surgical technique for the management of complete brachial plexus avulsion injury. OBJECTIVE: We assessed the functional recovery in 15 patients who underwent brachial plexus reimplantation surgery after complete brachial plexus avulsion injury with clinical examination and electrophysiological testing. METHODS: We included all patients who underwent brachial plexus reimplantation in our institution between 1997 and 2010. Patients were assessed with detailed motor and sensory clinical examination and motor and sensory electrophysiological tests. RESULTS: We found that patients who had reimplantation surgery demonstrated an improvement in Medical Research Council power in the deltoid, pectoralis, and infraspinatous muscles and global Medical Research Council score. Eight patients achieved at least grade 3 MRC power in at least one muscle group of the arm. Improved reinnervation by electromyelography criteria was found in infraspinatous, biceps, and triceps muscles. There was evidence of ongoing innervation in 3 patients. Sensory testing in affected dermatomes also showed better recovery at C5, C6, and T1 dermatomes. The best recovery was seen in the C5 dermatome. CONCLUSIONS: Our results demonstrate a definite but limited improvement in motor and sensory recovery after reimplantation surgery in patients with complete brachial plexus injury. We hypothesize that further improvement may be achieved by using regenerative cell technologies at the time of repair.

Expression of Semaphorins, Neuropilins, VEGF, and Tenascins in Rat and Human Primary Sensory Neurons after a Dorsal Root Injury.

Dorsal root injury is a situation not expected to be followed by a strong regenerative growth, or growth of the injured axon into the central nervous system of the spinal cord, if the central axon of the dorsal root is injured but of strong regeneration if subjected to injury to the peripherally projecting axons. The clinical consequence of axonal injury is loss of sensation and may also lead to neuropathic pain. In this study, we have used in situ hybridization to examine the distribution of mRNAs for the neural guidance molecules semaphorin 3A (SEMA3A), semaphorin 3F (SEMA3F), and semaphorin 4F (SEMA4F), their receptors neuropilin 1 (NP1) and neuropilin 2 (NP2) but also for the neuropilin ligand vascular endothelial growth factor (VEGF) and Tenascin J1, an extracellular matrix molecule involved in axonal guidance, in rat dorsal root ganglia (DRG) after a unilateral dorsal rhizotomy (DRT) or sciatic nerve transcetion (SNT). The studied survival times were 1-365 days. The different forms of mRNAs were unevenly distributed between the different size classes of sensory nerve cells. The results show that mRNA for SEMA3A was diminished after trauma to the sensory nerve roots in rats. The SEMA3A receptor NP1, and SEMA3F receptor NP2, was significantly upregulated in the DRG neurons after DRT and SNT. SEMA4F was upregulated after a SNT. The expression of mRNA for VEGF in DRG neurons after DRT showed a significant upregulation that was high even a year after the injuries. These data suggest a role for the semaphorins, neuropilins, VEGF, and J1 in the reactions after dorsal root lesions.

Structural and Functional Substitution of Deleted Primary Sensory Neurons by New Growth from Intrinsic Spinal Cord Nerve Cells: An Alternative Concept in Reconstruction of Spinal Cord Circuits.

In a recent clinical report, return of the tendon stretch reflex was demonstrated after spinal cord surgery in a case of total traumatic brachial plexus avulsion injury. Peripheral nerve grafts had been implanted into the spinal cord to reconnect to the peripheral nerves for motor and sensory function. The dorsal root ganglia (DRG) containing the primary sensory nerve cells had been surgically removed in order for secondary or spinal cord sensory neurons to extend into the periphery and replace the deleted DRG neurons. The present experimental study uses a rat injury model first to corroborate the clinical finding of a re-established spinal reflex arch, and second, to elucidate some of the potential mechanisms underlying these findings by means of morphological, immunohistochemical, and electrophysiological assessments. Our findings indicate that, after spinal cord surgery, the central nervous system sensory system could replace the traumatically detached original peripheral sensory connections through new neurite growth from dendrites.

New Treatments for Spinal Nerve Root Avulsion Injury.

Further progress in the treatment of the longitudinal spinal cord injury has been made. In an inverted translational study, it has been demonstrated that return of sensory function can be achieved by bypassing the avulsed dorsal root ganglion neurons. Dendritic growth from spinal cord sensory neurons could replace dorsal root ganglion axons and re-establish a reflex arch. Another research avenue has led to the development of adjuvant therapy for regeneration following dorsal root to spinal cord implantation in root avulsion injury. A small, lipophilic molecule that can be given orally acts on the retinoic acid receptor system as an agonist. Upregulation of dorsal root ganglion regenerative ability and organization of glia reaction to injury were demonstrated in treated animals. The dual effect of this substance may open new avenues for the treatment of root avulsion and spinal cord injuries.

Laminin chains in rat and human peripheral nerve: distribution and regulation during development and after axonal injury.

During nerve growth, axons are dependent upon contact with matrix components, such as laminins, for elongation, guidance, and trophic support. Semiquantitative in situ hybridization histochemistry and immunohistochemistry (IHC) were used to identify laminin chains in normal peripheral nerves, during postnatal development, after sciatic nerve transection (SNT), and after sciatic nerve crush (SNC). Laminin alpha2, alpha4, beta1, beta2, and gamma1 chain mRNAs were all expressed at high levels in newborn rat sciatic nerves with declining levels during later developmental stages. At the adult stage, no laminin chain mRNA was detectable. Of interest, the mRNA levels for alpha4 chain declined faster than those for alpha2. After SNT, laminin alpha2, alpha4, beta1, and gamma1 mRNA levels were up-regulated at the site of the injury, with the most profound reaction in the proximal nerve stump. Laminin alpha2 and alpha4 chains differed in that the mRNA levels of alpha4 were up-regulated earlier and declined quicker, whereas alpha2 had a later onset, with high levels remaining even after 6 weeks. After SNC, there was an initial up-regulation of the same laminin chain mRNAs as after SNT in the nerve, however, less intense, and at 6 weeks after SNC, all laminin mRNA levels studied had returned to normal. IHC of adult human normal and transected peripheral nerves stained positive for laminin alpha2, alpha4, beta1, and gamma1 chains in close relation to neurofilament labeled axons. Laminin alpha3, alpha4, alpha5, beta1, beta2, and gamma1 chains were found in blood vessel-like structures and alpha3, alpha4, alpha5, beta2, and gamma1 in the perineurium. These results and a previously published description of integrin regulation in spinal motoneurons suggest that both laminin-2 (alpha2beta1gamma1) and laminin-8 (alpha4beta1gamma1) are important for the postnatal nerve development and axonal regeneration after injury and that laminin-8 may have important functions especially early postnatally and early after adult nerve lesion.