Spinal motor nerves are essential for relaying information between the central and peripheral nervous systems. Perturbations to cell types that comprise these nerves may impair rapid and efficient transmission of action potentials and alter nerve function. Identifying ultrastructural changes resulting from defects to these cellular components via transmission electron microscopy (TEM) can provide valuable insight into nerve function and disease. However, efficiently locating spinal motor nerves in adult zebrafish for TEM is challenging and time-consuming. Because of this, we developed a protocol that allows us to quickly and precisely locate spinal motor nerve roots in adult zebrafish for TEM processing.

To investigate the role of gravity in the sedimentation of lumbar spine nerve roots using magnetic resonance (MR) imaging of various body positions.

Electrical stimulation of peripheral nerves controlling the bladder is an alternative, nondestructive medical treatment for urinary incontinence and retention. In this study, we aimed to identify the most efficient sensory and motor spinal nerve roots involved in the micturition reflex. Unilateral L5-S2 dorsal and ventral roots were electrically stimulated, and bladder reflex contractions were recorded under isovolumetric conditions. Repeated stimulation of the L6 and S1 dorsal roots not only abolished bladder reflex contractions but also induced a poststimulation inhibitory effect, whereas repeated stimulation of the L5 and S2 dorsal roots had no effect. Only the L6 ventral root directly caused bladder contraction when ventral roots L5-S2 were stimulated in sequence. Upon retrograde tracing using pseudorabies virus (PRV), the sacral parasympathetic nucleus of the L6 segment had more PRV-positive cells than the other segments, though the S1 segment of the dorsal root ganglia had the highest density of PRV-positive neurons. These results suggest the L6 ventral root is most efficient in producing detrusor muscle contraction, and the S1 dorsal root best inhibits the micturition reflex.

The posterior rootlets in L6 and S1 spinal cord of adult male Sprague-Dawley rats underwent electrostimulation. The bladder pressure, urethral perfusion pressure and intracavernous pressure were recorded. When some posterior rootlets of L6 and S1 were electrostimulated, the intracavernous pressure peaked rapidly, but the bladder pressure and the urethral perfusion pressure curve did not show great change. When other rootlets were stimulated, the bladder pressure changed greatly, but the urethral perfusion pressure and the intracavernous pressure did not show great change. When different rootlets were stimulated, the urethral perfusion pressure changed maximally, but there were no great changes in bladder pressure or intracavernous pressure. Furthermore, stimulation of some rootlets produced simultaneous changes in two or three different pressure measures mentioned above. The results demonstrate that regulation by L6 and S1 posterior rootlets of the rat bladder detrusor, external urethral sphincter and penis cavernous body are significantly distinct. Different rootlets can be distinguished by electrostimulation.

A quantitative analytical method was used to measure myelinated axon morphometric parameters (e.g., axon area, ratio of axon area/fiber area, and index of circularity) in rat nervous tissue during intoxication with 2,5-hexanedione (HD). Parameters were assessed in nerve roots (dorsal and ventral) and in ascending (gracile fasciculus and spinocerebellar tract) and descending (corticospinal and rubrospinal tracts) spinal cord white matter tracts (L4-L5) of rats intoxicated with HD at two different daily dose-rates (175 or 400 mg HD/kg/day, gavage). For each dose-rate, tissue was sampled at four neurological endpoints: unaffected, slight, moderate, and severe toxicity, as determined by gait analysis and measurements of grip strength. Results indicate that, regardless of the HD dose-rate, axon atrophy (reduced axon area) was a widespread, abundant effect that developed in concert with neurological deficits. The atrophy response occurred contemporaneously in both ascending and descending spinal tracts, which suggests that loss of caliber developed simultaneously along the proximodistal axon axis. In contrast, swollen axons were a numerically small component and were present in nerve roots and spinal tracts only during subchronic intoxication at the lower HD dose-rate (i.e., 175 mg/kg/day). Intoxication at the higher dose-rate (400 mg/kg/day) produced neurological deficits in the absence of axonal swellings. These observations in conjunction with our previous studies of HD-induced peripheral neuropathy (Toxicol. Appl. Pharmacol. 135 (1995) 58; and Toxicol. Appl. Pharmacol. 165 (2000) 127) indicate that axon atrophy, and not axonal swelling, is a primary neuropathic phenomenon.

Up to 40% of patients diagnosed with lumbar spinal stenosis (LSS) show evidence of redundant nerve roots (RNR) of the cauda equina on their magnetic resonance images (MRI). The etiology of RNR is still unclear. Preoperative evidence of RNR is associated with a worse postsurgical outcome. Consequently, potential predictors of RNR could have a prognostic value. The aim was to test whether patient demographics and MRI-based measurements can predict RNR in LSS patients.

Introduction: Redundant nerve roots (RNRs) are common finding in lumbar spinal stenosis patients. Up to now, many relevant studies were carried out on the mechanism, pathogenic factors, and clinical features of redundant nerve roots. However, there are few studies on the surgical methods. In this study, posterior lumbar interbody fusion and internal fixations were used in 30 patients with RNRs in our hospital. Moreover, we also proposed new ideas about different types and subtypes of RNRs using patterns and their corresponding MRI images. Methods: Thirty patients with lumbar spinal stenosis and RNRs were enrolled in this study and underwent surgery between January 2009 and December 2014. Redundant nerve roots are identified as elongated, tortuous, or serpiginous nerve roots present in the subarachnoid space on sagittal T2-weighted magnetic resonance imaging (MRI) studies. Patients were treated with posterior decompression, intervertebral disc resection, and instrumented interbody fusion. The age, sex, disease course, operative time, intraoperative blood loss, operative segments were recorded. Outcome measures recorded to identify symptom improvement included pre-operative and post-operative visual analog scale (VAS), pre-operative and post-operative Oswestry Disability Index (ODI) and pre-operative and post-operative Japanese Orthopedic Association (JOA) scores. Results: VAS back pain, VAS leg pain VAS, ODI, and JOA with standard deviations were 6.4 ± 0.9, 7.1 ± 0.8, 43.0 ± 2.2, and 10.3 ± 2.6, respectively. At 3 months post-operatively, VAS back pain, VAS leg pain VAS, ODI, and JOA with standard deviations were 1.4 ± 0.5, 1.6 ± 0.6, 13.0 ± 1.6, and 25.0 ± 1.8, respectively. Nerve redundancy resolved in all cases on post-operative MRI. Conclusion: Posterior lumbar laminectomy and instrumented interbody fusion relieves low back and leg pain in patients with lumbar spinal stenosis and RNRs and can alleviate the tortuous appearance of the cauda equina in the decompressed segment.

Canine degenerative myelopathy (DM) is a progressive, adult-onset, multisystem degenerative disease with many features in common with amyotrophic lateral sclerosis (ALS). As with some forms of ALS, DM is associated with mutations in superoxide dismutase 1 (SOD1). Clinical signs include general proprioceptive ataxia and spastic upper motor neuron paresis in pelvic limbs, which progress to flaccid tetraplegia and dysphagia. The purpose of this study was to characterize DM as a potential disease model for ALS. We previously reported that intercostal muscle atrophy develops in dogs with advanced-stage DM. To determine whether other components of the thoracic motor unit (MU) also demonstrated morphological changes consistent with dysfunction, histopathologic and morphometric analyses were conducted on thoracic spinal motor neurons (MNs) and dorsal root ganglia (DRG) and in motor and sensory nerve root axons from DM-affected boxers and Pembroke Welsh corgis (PWCs). No alterations in MNs or motor root axons were observed in either breed. However, advanced-stage PWCs exhibited significant losses of sensory root axons, and numerous DRG sensory neurons displayed evidence of degeneration. These results indicate that intercostal muscle atrophy in DM is not preceded by physical loss of the motor neurons innervating these muscles, nor of their axons. Axonal loss in thoracic sensory roots and sensory neuron death suggest that sensory involvement may play an important role in DM disease progression. Further analysis of the mechanisms responsible for these morphological findings would aid in the development of therapeutic intervention for DM and some forms of ALS.

Diffusion tensor imaging (DTI) has shown promise in the measurement of peripheral nerve integrity, although the optimal way to apply the technique for the study of lumbar spinal nerves is unclear. The aims of this study are to use an improved DTI acquisition to investigate lumbar nerve root integrity and correlate this with functional measures using neurophysiology.

Brachial plexus injury (BPI) is a devastating type of nerve injury, potentially causing loss of motor and sensory function. Principally, BPI is either categorized as preganglionic or postganglionic, with the early establishment of injury level being crucial for choosing the correct treatment strategy. Despite diagnostic advances, the need for a reliable, non-invasive method for establishing the injury level remains. We studied the usefulness of in vivo magnetic resonance imaging (MRI) of the spinal cord for determination of injury level. The findings were related to neuronal and glial changes. Rats underwent unilateral L4 & L5 ventral roots avulsion or sciatic nerve axotomy. The injuries served as models for pre- and postganglionic BPI, respectively. MRI of the L4/L5 spinal cord segments 4 weeks after avulsion showed ventral horn (VH) shrinkage on the injured side compared to the uninjured side. Axotomy induced no change in the VH size on MRI. Following avulsion, histological sections of L4/L5 revealed shrinkage in the VH grey matter area occupied by NeuN-positive neurons, loss of microtubular-associated protein-2 positive dendritic branches (MAP2), pan-neurofilament positive axons (PanNF), synaptophysin-positive synapses (SYN) and increase in immunoreactivity for the microglial OX42 and astroglial GFAP markers. Axotomy induced no changes in NeuN-reactivity, modest decrease of MAP2 immunoreactivity, no changes in SYN and PanNF labelling, and a modest increase in OX42 and SYN labeling. Histological and radiological findings were congruent when assessing changes after axotomy, while MRI somewhat underestimated the shrinkage. This study indicates a potential diagnostic value of structural spinal cord MRI following BPI.

Redundant nerve root syndrome (RNRS) is characterized by tortuous, elongated, and enlarged nerve roots in patients with lumbar spinal stenosis. This study was performed to evaluate the effects of caudal block in patients with RNRS and assess factors associated with RNRS.

Following a spinal cord injury, there are usually a number of neural pathways that remain intact in the spinal cord. These residual nerve fibers are important, as they could be used to reconstruct the neural circuits that enable motor function. Our group previously designed a novel magnetic stimulation protocol, targeting the motor cortex and the spinal nerve roots, that led to significant improvements in locomotor function in patients with a chronic incomplete spinal cord injury. Here, we investigated how nerve root magnetic stimulation contributes to improved locomotor function using a rat model of spinal cord injury. Rats underwent surgery to clamp the spinal cord at T10; three days later, the rats were treated with repetitive magnetic stimulation (5 Hz, 25 pulses/train, 20 pulse trains) targeting the nerve roots at the L5-L6 vertebrae. The treatment was repeated five times a week over a period of three weeks. We found that the nerve root magnetic stimulation improved the locomotor function and enhanced nerve conduction in the injured spinal cord. In addition, the nerve root magnetic stimulation promoted the recovery of synaptic ultrastructure in the sensorimotor cortex. Overall, the results suggest that nerve root magnetic stimulation may be an effective, noninvasive method for mobilizing the residual spinal cord pathways to promote the recovery of locomotor function.

In neonatal animals, peripheral nerve axotomy induces cell death in the corresponding dorsal root ganglion neurons and motoneurons, indicating that trophic interactions between these neurons and their targets control neuronal survival at this age. However, axotomy-induced cell death masks the role of peripheral tissues in regulating the central connections between these neurons in neonates. Since we have shown in Bax-deficient mice (Bax-/-) that transection of the sciatic nerve at postnatal day (P) 0 rarely induced apoptosis in motoneurons, we examined whether peripheral nerve axotomy eliminates synaptic connections between group Ia afferents and motoneurons in Bax-/-. After the axotomy, we observed in P7 Bax-/- that many axons survived in the fourth lumber (L4) dorsal root and that primary afferent projections to L4 motor pools also remained. Sciatic nerve stimulation evoked synaptic responses in L4 ventral roots in these mice although the amplitudes were considerably smaller and the onset latencies longer compared with the controls. Our results suggest that the monosynaptic connection between group Ia afferents and motoneurons is morphologically and functionally preserved following axotomy. Peripheral tissues may modulate synaptic connectivity but do not contribute to the maintenance of primary afferent projections in the stretch reflex pathway at an immature stage.

Age-related degenerative changes in the lumbar spine frequently result in nerve root compression causing severe pain and disability. Given the increasing incidence of lumbar spinal disorders in the aging population and the discrepancies between the use of current diagnostic imaging tools and clinical symptoms, novel methods of nerve root assessment are needed. We investigated elderly patients with stenosis at L4-L5 or L5-S1 levels. Diffusion tensor imaging (DTI) was used to quantify microstructure in compressed L5 nerve roots and investigate relationships to clinical symptoms and motor neurophysiology. DTI metrics (i.e. FA, MD, AD and RD) were measured at proximal, mid and distal segments along compressed (i.e. L5) and intact (i.e. L4 or S1) nerve roots. FA was significantly reduced in compressed nerve roots and MD, AD and RD were significantly elevated in the most proximal segment of the nerve root studied. FA was significantly correlated with electrophysiological measures of root function: minimum F-wave latency and peripheral motor conduction time (PMCT). In addition, FA along the compressed root also correlated with leg pain and depression score. There was also a relationship between RD and anxiety, leg pain and disability score and AD correlated with depression score. Taken together, these data show that DTI metrics are sensitive to nerve root compression in patients with stenosis as a result of age-related lumbar degeneration. Critically, they show that the changes in microstructural integrity along compressed L5 nerve roots are closely related to a number of clinical symptoms associated with the development of chronic pain as well as neurophysiological assessments of motor function. These inherent relationships between nerve root damage and phenotype suggest that the use DTI is a promising method as a way to stratify treatment selection and predict outcomes.

Amyotrophic lateral sclerosis (ALS) is characterized by selective degeneration of motor neurons. Current imaging studies have concentrated on areas of the brain and spinal cord that contain mixed populations of sensory and motor neurons. In this study, ex vivo magnetic resonance microimaging (MRM) was used to separate motor and sensory components by visualizing individual dorsal and ventral roots in fixed spinal cords. MRM at 15μm in plane resolution enabled the axons of pure populations of sensory and motor neurons to be measured in the lumbar region of the SOD1 mouse model of ALS. MRM signal intensity increased by 38.3% (p<0.05) exclusively in the ventral motor nerve roots of the lumbar spinal cord of ALS-affected SOD1 mice compared to wildtype littermates. The hyperintensity was therefore limited to white matter tracts arising from the motor neurons, whereas sensory white matter fibers were unchanged. Significant decreases in ventral nerve root volume were also detected in the SOD1 mice, which correlated with the axonal degeneration observed by microscopy. These results demonstrate the usefulness of MRM in visualizing the ultrastructure of the mouse spinal cord. The detailed 3D anatomy allowed the processes of pure populations of sensory and motor neurons to be compared.

The rate of neuronal apoptosis increases after spinal cord injury (SCI). Anastomosing the normal nerve roots above the SCI level to the injured sacral nerve roots can enhance the functional recovery of neurons. Therefore, we evaluated the effect of sacral nerve root transfer after SCI on pontine neuronal survival.

The neuropeptide galanin is upregulated in primary afferent and sympathetic neurones and might be involved in the development of sympathetic perineuronal baskets ("rings") following nerve injury. Galanin, calcitonin gene-related peptide and tyrosine hydroxylase have been examined immunohistochemically in dorsal root ganglia and associated roots at times up to one year after transection of either sciatic or L5 spinal nerves in adult rats. Small diameter somata containing calcitonin gene-related peptide (with or without galanin) were reduced in number, whereas galanin (and, at later times, calcitonin gene-related peptide) appeared in medium to large diameter cells after both types of lesion. Galanin also appeared in axons in grey rami and somata in lumbar paravertebral ganglia. Within dorsal root ganglia, galanin-positive axons formed perineuronal rings of two types: (i) smooth coiled axons surrounded small (< 30 microm diameter) somata from which they probably arose; these were rare after 12 weeks, particularly after a spinal nerve lesion; and (ii) varicose terminals encircled medium to large galanin-positive somata; some arose from brightly immunofluorescent somata nearby and took nearly a year to disappear. About 30% of varicose galanin-positive rings had associated calcitonin gene-related peptide-positive terminals (partly colocalized) whereas nearly 45% had associated tyrosine hydroxylase-positive terminals (partly colocalized). Synaptophysin was present in swollen axons and in some varicosities of all types. We conclude that, after peripheral nerve lesions, varicose perineuronal rings around large diameter dorsal root ganglion cells may be formed by axotomized primary afferent neurones (some containing calcitonin gene-related peptide) and sympathetic neurones, both of which contain upregulated galanin. Exocytosis from the varicosities may modify the excitability of mechanosensitive somata. Small galanin-positive somata disappear over several months after both lesions as calcitonin gene-related peptide reappears in medium to large neurones.

The endoneurial extracellular matrix (ECM) molecules are involved in cell signalling during nervous system development and regeneration. Quantitative differences of immunofluorescence labelling for chondroitin sulfate proteoglycan (CSPG), fibronectin (FN), tenascin-C (TN-C), and thrombospondin (TSP) were evaluated in intact rat dorsal and ventral roots and dorsal and ventral roots 2 and 4 weeks after rhizotomy using image analysis. The distal stumps of spinal roots displayed increased immunolabelling for the molecules with higher immunofluorescence in dorsal than in ventral roots up to 2 weeks from transection. Four weeks after rhizotomy, the immunoreactivity for CSPG, TN-C and TSP decreased in dorsal and increased in ventral root stumps, although a higher level of immunofluorescence for FN remained in both dorsal and ventral root stumps 4 weeks after injury in comparison to 2 weeks after injury. We suggest that the amount of some ECM molecules changed differentially 2 and 4 weeks after rhizotomy to create an appropriate environment in the endoneurium for early and later regrowth of sensory and motor axons. The results presented here are the first report of differences between the endoneurial ECM content of damaged afferent and motor nerve fibers. In addition, the immunohistochemical detection of individual ECM molecules indicated that final extrinsic conditions stimulating the regrowth of regenerating axons probably arise from a balance of both growth-promoting and -inhibiting molecules in the endoneurium.

The expression of two vesicular glutamate transporters (VGLUTs), VGLUT1 and VGLUT2, was studied with immunohistochemistry in lumbar dorsal root ganglia (DRGs), the lumbar spinal cord and the skin of the adult mouse. About 12% and 65% of the total number of DRG neuron profiles (NPs) expressed VGLUT1 and VGLUT2, respectively. VGLUT1-immunoreactive (IR) NPs were usually medium- to large-sized, in contrast to a majority of small- or medium-sized VGLUT2-IR NPs. Most VGLUT1-IR NPs did not coexpress calcitonin gene-related peptide (CGRP) or bound isolectin B4 (IB4). In contrast, approximately 31% and approximately 42% of the VGLUT2-IR DRG NPs were also CGRP-IR or bound IB4, respectively. Conversely, virtually all CGRP-IR and IB4-binding NPs coexpressed VGLUT2. Moderate colocalization between VGLUT1 and VGLUT2 was also observed. Sciatic nerve transection induced a decrease in the overall number of VGLUT1- and VGLUT2-IR NPs (both ipsi- and contralaterally) and, in addition, a parallel, unilateral increase of VGLUT2-like immunoreactivity (LI) in a subpopulation of mostly small NPs. In the dorsal horn of the spinal cord, strong VGLUT1-LI was detected, particularly in deep dorsal horn layers and in the ventral horns. VGLUT2-LI was abundant throughout the gray spinal matter, 'radiating' into/from the white matter. A unilateral dorsal rhizotomy reduced VGLUT1-LI, while apparently leaving unaffected the VGLUT2-LI. Transport through axons for both VGLUTs was confirmed by their accumulation after compression of the sciatic nerve or dorsal roots. In the hind paw skin, abundant VGLUT2-IR nerve fibers were observed, sometimes associated with Merkel cells. Lower numbers of VGLUT1-IR fibers were also detected in the skin. Some VGLUT1-IR and VGLUT2-IR fibers were associated with hair follicles. Based on these data and those by Morris et al. [Morris JL, Konig P, Shimizu T, Jobling P, Gibbins IL (2005) Most peptide-containing sensory neurons lack proteins for exocytotic release and vesicular transport of glutamate. J Comp Neurol 483:1-16], we speculate that virtually all DRG neurons in adult mouse express VGLUTs and use glutamate as transmitter.

Voltage-gated Ca2+ (CaV) channels regulate multiple cell processes, including neurotransmitter release, and have been associated with several pathological conditions, such as neuropathic pain. Cdk5, a neuron-specific kinase, may phosphorylate CaV channels, altering their functional expression. During peripheral nerve injury, upregulation of CaV channels and Cdk5 in the dorsal root ganglia (DRG) and the spinal cord, has been correlated with allodynia. We recently reported an increase in the amplitude of the C component of the compound action potential (cAP) of afferent fibers in animals with allodynia induced by L5-6 spinal nerve ligation (SNL), recorded in the corresponding dorsal roots. This was related to an increase in T-type (CaV3.2) channels generated by Cdk5-mediated phosphorylation. Here, we show that CaV channel functional expression is also altered in the L4 adjacent intact afferent fibers in rats with allodynia induced by L5-6 SNL. Western blot analysis showed that both Cdk5 and CaV3.2 total levels are not increased in the DRG L3-4, but their subcellular distribution changes by concentrating on the neuronal soma. Likewise, the Cdk5 inhibitor olomoucine affected the rapid and the slow C components of the cAP recorded in the dorsal roots. Patch-clamp recordings revealed an increase in T- and N-type currents recorded in the soma of acute isolated L3-4 sensory neurons after L5-6 SNL, which was prevented by olomoucine. These findings suggest changes in CaV channels location and function in L3-4 afferent fibers associated with Cdk5-mediated phosphorylation after L5-6 SNL, which may contribute to nerve injury-induced allodynia.