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.

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.

Corrigendum: New Treatments for Spinal Nerve Root Avulsion Injury.

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

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.

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.

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.

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 effects of minocycline or riluzole treatment on spinal root avulsion-induced pain in adult rats.

UNLABELLED: Spinal root avulsion produces tactile and thermal hypersensitivity, neurodegeneration, and microglial and astrocyte activation in both the deafferented and the adjacent intact spinal cord segments. Following avulsion of the fifth lumbar spinal root, immediate and prolonged treatment with riluzole or minocycline for 2 weeks altered the development of behavioral hypersensitivity. Riluzole delayed the onset of thermal and tactile hypersensitivity and partially reversed established pain behavior. Minocycline effectively prevented and reversed both types of behavioral change. Histologic analysis revealed that both drugs reduced microglial staining in the spinal cord, with minocycline being more effective than riluzole. Astrocyte activation was ameliorated to a lesser extent. Surprisingly, neither drug provided a neuroprotective effect on avulsed motoneurons. PERSPECTIVE: Immediate treatment of spinal root avulsion injuries with minocycline or riluzole prevents the onset of evoked pain hypersensitivity by reducing microglial cell activation. When treatment is delayed, minocycline, but not riluzole, reverses pre-established hypersensitivity. Thus, these drugs may provide a new translational treatment option for chronic avulsion injury pain.

Segmental spinal root avulsion in the adult rat: a model to study avulsion injury pain.

Road traffic accidents are the most common cause of avulsion injury, in which spinal roots are torn from the spinal cord. Patients suffer from a loss of sensorimotor function, intractable spontaneous pain, and border-zone hypersensitivity. The neuropathic pains are particularly difficult to treat because the lack of a well-established animal model of avulsion injury prevents identifying the underlying mechanisms and hinders the development of efficacious drugs. This article describes a hindlimb model of avulsion injury in adult rats where the L5 dorsal and ventral spinal root are unilaterally avulsed (spinal root avulsion [SRA]), leaving the adjacent L4 spinal root intact. SRA produced a significant ipsilateral hypersensitivity to mechanical and thermal stimulation by 5 days compared with sham-operated or naïve rats. This hypersensitivity is maintained for up to 60 days. No autotomy was observed and locomotor deficits were minimal. The hypersensitivity to peripheral stimuli could be temporarily ameliorated by administration of amitriptyline and carbamazepine, drugs that are currently prescribed to avulsion patients. Histological assessment of the L4 ganglion cells revealed no significant alterations in calcitonin gene-related peptide (CGRP), IB4, transient receptor potential cation channel subfamily V member 1 (TrpV1), or N52 staining across groups. Immunohistochemistry of the spinal cord revealed a localized glial response, phagocyte infiltration, and neuronal loss within the ipsilateral avulsed segment. A comparable response from glia and phagocytes was also found in the intact L4 spinal cord, supporting the role for central mechanisms within the L4-5 spinal cord in contributing to the generation of the pain-related behavior. The SRA model provides a platform to investigate possible new pharmacological treatments for avulsion injuries.

Metabolic changes in the spinal cord after brachial plexus root re-implantation.

OBJECTIVE: To investigate metabolic changes within the spinal cord using proton magnetic resonance spectroscopy ((1)H-MRS) and determine their relationship with clinical function in patients with complete brachial plexus avulsion who underwent reimplantation of the ventral roots. METHODS: Single-voxel (1)H-MRS of the cord between C1 and C3 was performed in 10 patients with normal spinal cord on MRI, who underwent reimplantation of C5 to T1 ventral roots on average 5.5 years earlier, and 19 healthy controls. The ratios of the concentrations of the following main metabolites, with respect to total creatine levels, were obtained: total N-acetyl-aspartate, choline-containing compounds, creatine and phosphocreatine (Cr), and myo-inositol (m-Ins). Patient disability was assessed using upper limb scales. Differences in metabolite concentration ratios and their correlations with disability were investigated. RESULTS: Patients showed increased m-Ins/Cr ratio compared with controls, which was associated with the level of function of the affected arm and time from injury. CONCLUSIONS: The finding of increased m-Ins/Cr in patients suggests that reactive gliosis, perhaps in response to the degeneration of avulsed fibers, may occur in the spinal cord above the site of injury and be relevant to motor dysfunction. However, this pathological process appears to diminish with time. These insights underline the need to integrate metabolic imaging with structural and functional magnetic resonance imaging to obtain a complete view of spinal cord plasticity. Last, this study provides the first steps toward identifying markers to serve as outcome measures for trials comparing strategies of plexus repair following avulsion injury.

The longitudinal spinal cord injury: lessons from intraspinal plexus, cauda equina and medullary conus lesions.

Spinal nerve root avulsion injury interrupts the transverse segmental spinal cord nerve fibers. There is degeneration of sensory, motor, and autonomic axons, loss of synapses, deterioration of local segmental connections, nerve cell death, and reactions among non neuronal cells with central nerve system (CNS) scar formation, i.e., a cascade of events similar to those known to occur in any injury to the spinal cord. This is the longitudinal spinal cord injury (SCI). For function to be restored, nerve cells must survive and there must be regrowth of new nerve fibers along a trajectory consisting of CNS growth-inhibitory tissue in the spinal cord as well as peripheral nervous system (PNS) growth-promoting tissue in nerves. Basic science results have been translated into a successful surgical strategy to treat root avulsion injuries in man. In humans, this technique is currently the most promising treatment of any spinal cord injury, with return of useful muscle function together with pain alleviation. Experimental studies have also identified potential candidates for adjunctive therapies that, together with surgical replantation of avulsed roots after brachial plexus and cauda equina injuries, can restore not only motor but also autonomic and sensory trajectories to augment the recovery of neurological function. This is the first example of a spinal cord lesion that can be treated surgically, leading to restoration of somatic and autonomic activity and alleviation of pain.

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.

Return of spinal reflex after spinal cord surgery for brachial plexus avulsion injury.

Motor but not sensory function has been described after spinal cord surgery in patients with brachial plexus avulsion injury. In the featured case, motor-related nerve roots as well as sensory spinal nerves distal to the dorsal root ganglion were reconnected to neurons in the ventral and dorsal horns of the spinal cord by implanting nerve grafts. Peripheral and sensory functions were assessed 10 years after an accident and subsequent spinal cord surgery. The biceps stretch reflex could be elicited, and electrophysiological testing demonstrated a Hoffman reflex, or Hreflex, in the biceps muscle when the musculocutaneous nerve was stimulated. Functional MR imaging demonstrated sensory motor cortex activities on active as well as passive elbow flexion. Quantitative sensory testing and contact heat evoked potential stimulation did not detect any cutaneous sensory function, however. To the best of the authors' knowledge, this case represents the first time that spinal cord surgery could restore not only motor function but also proprioception completing a spinal reflex arch.

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.

An overture to basic science aspects of nerve injuries.

Does the lack of improvement in surgical treatment of nerve injury despite thousands of years of research disturb you? Do you think that basic science has not really contributed to any advancement in the treatment of nerve injury? Have you contributed? Do you think that new molecular biology knowledge in nerve injury and repair is important? Knowing from basic science that the immature nervous system is more fragile would you agree with the view that to be 'aggressive' in surgery of the newborn with a brachial plexus injury could be unscrupulous? As molecular biology of the nervous system has demonstrated that the best conditions for regeneration occur immediately after an injury do you find the approach of postponing surgery until at least 3 months after a closed nerve injury to be ignorant and even negligent? Taking into account the normal occurrence of inhibitory molecules in the uninjured peripheral nerve do you think that functional improvement from end to side nerve repair is a myth? Are the recent attempts to artificially enhance nerve regeneration for instance in synthetical conduits like nature seen 'through a glass darkly'? Do you agree that new concepts in surgical treatment of nerve injury are timely? Do you have the time?

Karolinska institutet 200-year anniversary. Symposium on traumatic injuries in the nervous system: injuries to the spinal cord and peripheral nervous system - injuries and repair, pain problems, lesions to brachial plexus.

The Karolinska Institutet 200-year anniversary symposium on injuries to the spinal cord and peripheral nervous system gathered expertise in the spinal cord, spinal nerve, and peripheral nerve injury field spanning from molecular prerequisites for nerve regeneration to clinical methods in nerve repair and rehabilitation. The topics presented at the meeting covered findings on adult neural stem cells that when transplanted to the hypoglossal nucleus in the rat could integrate with its host and promote neuron survival. Studies on vascularization after intraspinal replantation of ventral nerve roots and microarray studies in ventral root replantation as a tool for mapping of biological patterns typical for neuronal regeneration were discussed. Different immune molecules in neurons and glia and their very specific roles in synapse plasticity after injury were presented. Novel strategies in repair of injured peripheral nerves with ethyl-cyanoacrylate adhesive showed functional recovery comparable to that of conventional epineural sutures. Various aspects on surgical techniques which are available to improve function of the limb, once the nerve regeneration after brachial plexus lesions and repair has reached its limit were presented. Moreover, neurogenic pain after amputation and its treatment with mirror therapy were shown to be followed by dramatic decrease in phantom limb pain. Finally clinical experiences on surgical techniques to repair avulsed spinal nerve root and the motoric as well as sensoric regain of function were presented.

On acute gene expression changes after ventral root replantation.

Replantation of avulsed spinal ventral roots has been show to enable significant and useful regrowth of motor axons in both experimental animals and in human clinical cases, making up an interesting exception to the rule of unsuccessful neuronal regeneration in central nervous system. Compared to avulsion without repair, ventral root replantation seems to rescue lesioned motoneurons from death. In this study we have analyzed the acute response to ventral root avulsion and replantation in adult rats with gene arrays combined with cluster analysis of gene ontology search terms. The data show significant differences between rats subjected to ventral replantation compared to avulsion only. Even though number of genes related to cell death is similar in the two models after 24 h, we observed a significantly larger number of genes related to neurite growth and development in the rats treated with ventral root replantation, possibly reflecting the neuroregenerative capacity in the replantation model. In addition, an acute inflammatory response was observed after avulsion, while effects on genes related to synaptic transmission were much more pronounced after replantation than after avulsion alone. These data indicate that the axonal regenerative response from replantation is initiated at an earlier stage than the possible differences in terms of neuron survival. We conclude that this type of analysis may facilitate the comparison of the acute response in two types of injury.