A shape-adjusted ellipse approach corrects for varied axonal dispersion angles and myelination in primate nerve roots.

Segmentation of axons in light and electron micrographs allows for quantitative high-resolution analysis of nervous tissues, but varied axonal dispersion angles result in over-estimates of fiber sizes. To overcome this technical challenge, we developed a novel shape-adjusted ellipse (SAE) determination of axonal size and myelination as an all-inclusive and non-biased tool to correct for oblique nerve fiber presentations. Our new resource was validated by light and electron microscopy against traditional methods of determining nerve fiber size and myelination in rhesus macaques as a model system. We performed detailed segmental mapping and characterized the morphological signatures of autonomic and motor fibers in primate lumbosacral ventral roots (VRs). An en bloc inter-subject variability for the preganglionic parasympathetic fibers within the L7-S2 VRs was determined. The SAE approach allows for morphological ground truth data collection and assignment of individual axons to functional phenotypes with direct implications for fiber mapping and neuromodulation studies.

LINGO-1 shRNA Loaded by Pluronic F-127 Promotes Functional Recovery After Ventral Root Avulsion.

Spinal root avulsion typically leads to massive motoneuron death and severe functional deficits of the target muscles. Multiple pathological factors such as severe neuron loss, induction of inhibitory molecules, and insufficient regeneration are responsible for the poor functional recovery. Leucine-rich repeat and immunoglobulin-like domain-containing Nogo receptor-interacting protein 1 (LINGO-1), a central nervous system (CNS)-specific transmembrane protein that is selectively expressed on neurons and oligodendrocytes, serves as a potent negative mediator of axonal regeneration and myelination in CNS injuries and diseases. Although accumulating evidence has demonstrated improvement in axonal regeneration and neurological functions by LINGO-1 antagonism in CNS damage, the possible effects of LINGO-1 in spinal root avulsion remain undiscovered. In this study, a LINGO-1 knockdown strategy using lentiviral vectors encoding LINGO-1 short hairpin interfering RNA (shRNA) delivered by the Pluronic F-127 (PF-127) hydrogel was described after brachial plexus avulsion (BPA). We provide evidence that following BPA and immediate reimplantation, transplantation of LINGO-1 shRNA lentiviral vectors encapsulated by PF-127 rescued the injured motoneurons, enhanced axonal outgrowth and myelination, rebuilt motor endplates, facilitated the reinnervation of terminal muscles, improved angiogenesis, and promoted recovery of avulsed forelimbs. Altogether, these data suggest that delivery of LINGO-1 shRNA by a gel scaffold is a potential therapeutic approach for root avulsion. Impact Statement In this study, we attempted transplantation of lentivirus (LV)/leucine-rich repeat and immunoglobulin-like domain-containing Nogo receptor-interacting protein 1 (LINGO-1)-short hairpin interfering RNA (shRNA) encapsulated by the Pluronic F-127 (PF-127) hydrogel into a brachial plexus avulsion (BPA)-reimplantation model. We found that administration of LV/LINGO-1 shRNA facilitates neuron survival and axonal regeneration, attenuates muscle atrophy and motor endplate (MEP) loss, enhances neovascularization, and promotes functional recovery in BPA rats. Co-transplantation of LV/LINGO-1 shRNA and gel reinforces the survival-promoting effect, axonal outgrowth, and angiogenesis in comparison with LV/LINGO-1 shRNA application alone. Our research provides evidence that LV /LINGO-1 shRNA delivered by PF-127 represents a new treatment strategy for BPA repair.

Morphological study of myelinated and unmyelinated fibres in the sacrococcygeal dorsal roots of the rat.

BACKGROUND: The number and calibre of myelinated and unmyelinated fibres of the sacrococcygeal dorsal roots innervating the tail of rats were studied by means of light and electron microscopy. MATERIALS AND METHODS: There were an estimated total of 12,500 myelinated and 25,500 unmyelinated dorsal root fibres innervating the tail of a rat. RESULTS: The results showed that from the second sacral (S2) to the fourth sacral (S4) segment, the fibre diameter spectrum of myelinated fibres within each dorsal root was bimodal with two peaks at 5 microns and 10 microns, respectively. The first sacral (S1) segment was composed of numerous smaller-size myelinated fibres, thus forming a right-skewed distribution. The coccygeal (Co) segments showed a unimodal distribution peaking at 10 microns for the first (Co1) segment and gradually shifting to 7 microns for the third (Co3) segment. Overall, there was a continuous relative increase of the larger vs. the smaller myelinated fibres from the sacral to coccygeal segments. The fibre diameter of unmyelinated fibres of all these roots was unimodal with a single peak at 0.5 microns. The ratio of unmy- elinated to myelinated fibre numbers was on average 2.83 for the S1-S2 roots, 1.66 for the S3-S4 roots, and 1.24 for the coccygeal roots. CONCLUSIONS: The comparison of the left- and right-side nerve fibres show that there was no significant difference, thus implying a symmetrical sensory innervation of the rat's tail.

Alterations in the spinal cord and ventral root after cerebral infarction in non-human primates.

BACKGROUNDS: Cerebral infarction does not only cause focal injury in the ischemic site, but also secondary non-ischemic damage at the remote areas of nervous system associated with the primary focus. OBJECTIVE: This study investigated the changes in the spinal cord and ventral root after middle cerebral artery occlusion (MCAO) in cynomolgus monkeys (Macaca fascicularis). METHODS: Adult male cynonolgus monkeys (4-5 years, 5.5-7.5 kg) were subjected to MCAO (n = 6) or sham surgery (n = 4). After 12 weeks, spinal cords and the ventral roots were harvested. Morphometric alterations in the spinal cord were detected at C5 and L5 levels via immunofluorescence. The profiles of C5 and L5 ventral roots were displayed by toluidine blue staining and transmission electron microscopic examination. RESULTS: Significant axonal loss in the contralateral corticospinal tract and abnormally enlarged axons in the ipsilateral were observed in monkeys with MCAO. The number of neurons in the contralateral ventral horn got declined while that in the ipsilateral was almost unaffected after MCAO compared with sham controls. Glial activation post-MCAO was observed in the bilateral corticospinal tract and the ventral horn. Aberrant nerve fibers appeared frequently in the contralateral ventral roots of MCAO monkey but rarely in the ipsilateral. CONCLUSIONS: These results indicate that focal cerebral infarction leads to pathological alterations in the spinal cord and ventral roots in non-human primates.

Survival among children with "Lethal" congenital contracture syndrome 11 caused by novel mutations in the gliomedin gene (GLDN).

Biallelic GLDN mutations have recently been identified among infants with lethal congenital contracture syndrome 11 (LCCS11). GLDN encodes gliomedin, a protein required for the formation of the nodes of Ranvier and development of the human peripheral nervous system. We report six infants and children from four unrelated families with biallelic GLDN mutations, four of whom survived beyond the neonatal period into infancy, childhood, and late adolescence with intensive care and chronic respiratory and nutritional support. Our findings expand the genotypic and phenotypic spectrum of LCCS11 and demonstrate that the condition may not necessarily be lethal in the neonatal period.

Transmission electron microscopy of zebrafish spinal motor nerve roots.

BACKGROUND: 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. RESULTS: Following fixation, a transverse slab of adult zebrafish dissected from the trunk region was mounted in embedding media, sectioned, and secondary fixation with osmium tetroxide performed. Transverse sections containing motor nerves were selected for TEM ultrathin sectioning and imaging. CONCLUSIONS: We developed an efficient protocol for locating spinal motor nerves in adult zebrafish to allow for ultrastructural characterization. Although our work focuses on spinal motor nerves, this protocol may be useful for efficiently identifying other discrete, repeated structures within the developing and mature nervous system that are difficult to find via traditional, whole organism TEM processing. Developmental Dynamics 246:956-962, 2017. © 2017 Wiley Periodicals, Inc.

Microstructural changes are coincident with the improvement of clinical symptoms in surgically treated compressed nerve roots.

Diffusion tensor imaging (DTI) has been widely used to visualize peripheral nerves, but the microstructure of compressed nerve roots can be assessed using DTI. However, there are no data regarding the association among microstructural changes evaluated using DTI, the symptoms assessed using the Oswestry Disability Index (ODI) and the duration of symptoms after surgery in patients with lumbar disc herniation (LDH). Thirty patients with unilateral radiculopathy were investigated using DTI. The changes in the mean fractional anisotropy (FA) and the apparent diffusion coefficient (ADC) values as well as the correlation between these changes and the severity and duration of the clinical symptoms were investigated before and at least one month after surgery. The FA values were significantly increased after surgical treatment (p < 0.0001). Both the ADC and ODI values were noticeably decreased (p < 0.0001). A strong positive correlation between the preoperative and postoperative DTI parameters (p < 0.0001) as well as between the preoperative ODI and postoperative ODI/ODI changes (p < 0.0001) were found. In addition, there was a significant positive correlation between the changes in the DTI parameters and changes in the ODI (p < 0.0001). This preliminary study suggests it may be possible to use DTI to diagnose, quantitatively evaluate and follow-up patients with LDH.

Ultrasound of the cervical roots and brachial plexus in neonates.

INTRODUCTION: In this exploratory study we investigated whether ultrasound can visualize the neonatal cervical roots and brachial plexus. METHODS: In 12 healthy neonates <2 days old, the neck region was studied unilaterally with ultrasound using a small-footprint 15-7-MHz transducer. RESULTS: The C5-C8 nerve roots and brachial plexus could be imaged with sufficient delineation of the root exits to assess their integrity. The brachial plexus was more difficult to discern from the surrounding area in neonates compared with adults, especially in the interscalene region because of the smaller amount of connective tissue in and surrounding muscles and nerves. In addition, the large deposits of brown fat make for a different ultrasound appearance of the neonatal neck compared with adults. CONCLUSIONS: Ultrasound of the neonatal cervical nerve roots is feasible and may be used as a non-invasive screening technique to assess nerve root integrity in obstetric brachial plexus injury.

Spinal cord organotypic slice cultures for the study of regenerating motor axon interactions with 3D scaffolds.

Numerous in-vitro techniques exist for investigating the influence of 3D substrate topography on sensory axon growth. However, simple and cost-effective methods for studying post-natal motor axon interactions with such substrates are lacking. Here, spinal cord organotypic slice cultures (OSC) from post-natal day 7-9 rat pups were presented with spinal nerve roots, or blocks of fibrin hydrogel or 3D microporous collagen scaffolds to investigate motor axon-substrate interactions. By 7-14 days, axons from motor neuronal pools extended into the explanted nerve roots, growing along Schwann cell processes and demonstrating a full range of axon-Schwann cell interactions, from simple ensheathment to concentric wrapping by Schwann cell processes and the formation of compact myelin within a basal lamina sheath. Extensive motor axon regeneration and all stages of axon-Schwann interactions were also supported within the longitudinally orientated microporous framework of the 3D collagen scaffold. In stark contrast, the simple fibrin hydrogel only supported axon growth and cell migration over its surface. The relative ease of demonstrating such motor axon regeneration through the microporous 3D framework by immunofluorescence, two-photon microscopy and transmission electron microscopy strongly supports the adoption of this technique for assaying the influence of substrate topography and functionalization in regenerative bioengineering.

Effects of epidural capsaicin on nociceptive threshold and neurological functions in rabbits.

UNLABELLED: Capsaicin, as a principle active component of Chili peppers, is popularly consumed by many people around the world. Whether capsaicin-induced neuropathy alters the function of sensory neurons is still unknown. OBJECTIVE: The objectives of this study were to determine the effects of epidural capsaicin on nociceptive threshold and neurological functions in a rabbit model. DESIGN: An intrathecal injection system was set up using a rabbit model. Rabbits were treated with capsaicin at doses of 0.04, 0.10, and 0.20 mg/kg once. The changes in neurological functions and morphology of the spinal cord and spinal nerve roots were determined within 24 hours. Changes in the nociceptive threshold in the hind limbs of the rabbits were observed for 30 days. METHODS: Capsaicin's effect on the changing neurological functions was evaluated by the neurological functional scores. The structural changes of spinal cord and spinal nerve roots were observed by hematoxylin and eosin staining and transmission electron microscopy. The nociceptive threshold changes in the rabbits were measured by the responding time for pain induced by a thermostimulation. RESULTS: The results showed that capsaicin reversed changes in the neurological function of rabbit hindlimbs. In the 0.10 and 0.20 mg/kg groups, structural abnormalities were found in the rabbit's spinal nerves. Capsaicin also significantly increased the pain threshold in rabbits when compared with the control group (P < 0.05 or P < 0.01). The maximum values of pain threshold were found in the 0.10 mg/kg capsaicin group after 3 days of capsaicin treatment. CONCLUSION: With the exception of a potential toxicity, capsaicin may be a potential candidate agent for providing pain relief of both neuropathic and nociceptive conditions.

Direct injection into the dorsal root ganglion: technical, behavioral, and histological observations.

Direct injection of agents into the dorsal root ganglia (DRGs) offers the opportunity to manipulate sensory neuron function at a segmental level to explore pathophysiology of painful conditions. However, there is no described method that has been validated in detail for such injections in adult rats. We have found that 2 µl of dye injected through a pulled glass pipette directly into the distal DRG, exposed by a minimal foraminotomy, produces complete filling of the DRG with limited extension into the spinal roots. Injection into the spinal nerve required 3 µl to achieve comparable DRG filling, produced preferential spread into the ventral root, and was accompanied by substantial leakage of injected solution from the injection site. Injections into the sciatic nerve of volumes up to 10 µl did not reach the DRG. Transient hypersensitivity to mechanical stimulation at threshold (von Frey) and noxious levels (pin) developed after 2 µl saline injection directly into the DRG that was in part attributable to the surgical exposure procedure alone. Only minimal astrocyte activation in the spinal dorsal horn was evident after DRG saline injections. Injection of adeno-associated virus (AAV) vector conveying green fluorescent protein (GFP) transgene resulted in expression as soon as 1 day after injection into the DRG, including fibers in the spinal dorsal horn and columns. AAV injection into the DRG produced additional thermal hypersensitivity and withdrawal from the stroke of a brush and compromised motor performance. These findings demonstrate a method for selective injection of agents into single DRGs for anatomically restricted actions.

Morphologic study of nerve root and types of needle used in transforaminal injections.

BACKGROUND: The bevel type and location of the distal orifice of the needle may have relevance for potential complications occurring during transforaminal epidural injection. METHODS: We examined by scanning electron microscopy the structural aspects of spinal nerve root cuffs of 3 human cadavers, and 3 needle types used in transforaminal injections: 22-gauge Quincke spinal needles, 22-gauge blunt nerve block needles, and 20-gauge radiofrequency blunt needles. We made punctures in vitro in the spinal nerve root cuffs, and we studied the structures affected. RESULTS: There is fat tissue within the nerve root with irregular distribution. In needles with a round tip, the distal orifice maintained an extraneural location after puncture of the nerve root cuff. The length of the needle required to introduce the distal orifice completely inside the nerve root cuff was variable, depending on the type: shortest for a Quincke needle (1.8 mm), 4.1 mm for the Epimed, and longest for radiofrequency needles (5.7 mm). CONCLUSIONS: The layer of fat around nerve roots may prevent the contact of the needle tip with axons. The sharp needle tip entered the nerve root cuff more easily than the blunt tip in the cadaveric nerve root samples, measured in a qualitative manner. There is a need for clinical studies to ascertain if blunt needles may be safer than sharp needles for transforaminal injections.

In vivo imaging of dorsal root regeneration: rapid immobilization and presynaptic differentiation at the CNS/PNS border.

Dorsal root (DR) axons regenerate in the PNS but turn around or stop at the dorsal root entry zone (DREZ), the entrance into the CNS. Earlier studies that relied on conventional tracing techniques or postmortem analyses attributed the regeneration failure to growth inhibitors and lack of intrinsic growth potential. Here, we report the first in vivo imaging study of DR regeneration. Fluorescently labeled, large-diameter DR axons in thy1-YFPH mice elongated through a DR crush site, but not a transection site, and grew along the root at >1.5 mm/d with little variability. Surprisingly, they rarely turned around at the DREZ upon encountering astrocytes, but penetrated deeper into the CNS territory, where they rapidly stalled and then remained completely immobile or stable, even after conditioning lesions that enhanced growth along the root. Stalled axon tips and adjacent shafts were intensely immunolabeled with synapse markers. Ultrastructural analysis targeted to the DREZ enriched with recently arrived axons additionally revealed abundant axonal profiles exhibiting presynaptic features such as synaptic vesicles aggregated at active zones, but not postsynaptic features. These data suggest that axons are neither repelled nor continuously inhibited at the DREZ by growth-inhibitory molecules but are rapidly stabilized as they invade the CNS territory of the DREZ, forming presynaptic terminal endings on non-neuronal cells. Our work introduces a new experimental paradigm to the investigation of DR regeneration and may help to induce significant regeneration after spinal root injuries.

The border between the central and the peripheral nervous system in the cat cochlear nerve: a light and scanning electron microscopical study.

The transition between the central (CNS) and peripheral nervous system (PNS) in cranial and spinal nerve roots, referred to here as the CNS-PNS border, is of relevance to nerve root disorders and factors that affect peripheral-central regeneration. Here, this border is described in the cat cochlear nerve using light microscopical sections, and scanning electron microscopy of the CNS-PNS interfaces exposed by fracture of the nerve either prior to or following critical point drying. The CNS-PNS border represents an abrupt change in type of myelin, supporting elements, and vascularization. Because central myelin is formed by oligodendrocytes and peripheral myelin by Schwann cells, the myelinated fibers are as a rule equipped with a node of Ranvier at the border passage. The border is shallower and smoother in cat cochlear nerve than expected from other nerves, and the borderline nodes are largely in register. The loose endoneurial connective tissue of the PNS compartment is closed at the border by a compact glial membrane, the mantle zone, of the CNS compartment. The mantle zone is penetrated by the nerve fibers, but is otherwise composed of astrocytes and their interwoven processes like the external limiting membrane of the brain surface with which it is continuous. The distal surface of the mantle zone is covered by a fenestrated basal lamina. Only occasional vessels traverse the border. From an anatomical point of view, the border might be expected to be a weak point along the cochlear nerve and thus vulnerable to trauma. In mature animals, the CNS-PNS border presents a barrier to regrowth of regenerating nerve fibers and to invasion of the CNS by Schwann cells. An understanding of this region in the cochlear nerve is therefore relevant to head injuries that lead to hearing loss, to surgery on acoustic Schwannomas, and to the possibility of cochlear nerve regeneration.

Stimulation of the human lumbar spinal cord with implanted and surface electrodes: a computer simulation study.

Human lumbar spinal cord networks controlling stepping and standing can be activated through posterior root stimulation using implanted electrodes. A new stimulation method utilizing surface electrodes has been shown to excite lumbar posterior root fibers similarly as with implants, an unexpected finding considering the distance to these target neurons. In the present study we apply computer modeling to compare the depolarization of posterior root fibers by both stimulation techniques. We further examine the potential for additional direct activation of motoneurons within the anterior roots. Using an implant, action potentials are initiated in the posterior root fibers at their entry into the spinal cord or along the longitudinal portions of the fiber trajectories, depending on the cathode position. For transcutaneous stimulation low threshold sites of the same fibers are identified at their exits from the spinal canal in addition to their spinal cord entries. In these exit regions anterior root fibers can also be activated. The simulation results provide a biophysical explanation for the electrophysiological findings of lower limb muscle responses induced by posterior root stimulation. Efficient excitation of afferent spinal cord structures with a simple noninvasive method can become a promising modality in the rehabilitation of people with motor disorders.

Autotaxin and lysophosphatidic acid1 receptor-mediated demyelination of dorsal root fibers by sciatic nerve injury and intrathecal lysophosphatidylcholine.

BACKGROUND: Although neuropathic pain is frequently observed in demyelinating diseases such as Guillain-Barré syndrome and multiple sclerosis, the molecular basis for the relationship between demyelination and neuropathic pain behaviors is poorly understood. Previously, we found that lysophosphatidic acid receptor (LPA1) signaling initiates sciatic nerve injury-induced neuropathic pain and demyelination. RESULTS: In the present study, we have demonstrated that sciatic nerve injury induces marked demyelination accompanied by myelin-associated glycoprotein (MAG) down-regulation and damage of Schwann cell partitioning of C-fiber-containing Remak bundles in the sciatic nerve and dorsal root, but not in the spinal nerve. Demyelination, MAG down-regulation and Remak bundle damage in the dorsal root were abolished in LPA1 receptor-deficient (Lpar1-/-) mice, but these alterations were not observed in sciatic nerve. However, LPA-induced demyelination in ex vivo experiments was observed in the sciatic nerve, spinal nerve and dorsal root, all which express LPA1 transcript and protein. Nerve injury-induced dorsal root demyelination was markedly attenuated in mice heterozygous for autotaxin (atx+/-), which converts lysophosphatidylcholine (LPC) to LPA. Although the addition of LPC to ex vivo cultures of dorsal root fibers in the presence of recombinant ATX caused potent demyelination, it had no significant effect in the absence of ATX. On the other hand, intrathecal injection of LPC caused potent dorsal root demyelination, which was markedly attenuated or abolished in atx+/- or Lpar1-/- mice. CONCLUSIONS: These results suggest that LPA, which is converted from LPC by ATX, activates LPA1 receptors and induces dorsal root demyelination following nerve injury, which causes neuropathic pain.

Dicer in Schwann cells is required for myelination and axonal integrity.

Dicer is responsible for the generation of mature micro-RNAs (miRNAs) and loading them into RNA-induced silencing complex (RISC). RISC functions as a probe that targets mRNAs leading to translational suppression and mRNA degradation. Schwann cells (SCs) in the peripheral nervous system undergo remarkable differentiation both in morphology and gene expression patterns throughout lineage progression to myelinating and nonmyelinating phenotypes. Gene expression in SCs is particularly tightly regulated and critical for the organism, as highlighted by the fact that a 50% decrease or an increase to 150% of normal gene expression of some myelin proteins, like PMP22, results in peripheral neuropathies. Here, we selectively deleted Dicer and consequently gene expression regulation by mature miRNAs from Mus musculus SCs. Our results show that in the absence of Dicer, most SCs arrest at the promyelinating stage and fail to start forming myelin. At the molecular level, the promyelinating transcription factor Krox20 and several myelin proteins [including myelin associated glycoprotein (MAG) and PMP22] were strongly reduced in mutant sciatic nerves. In contrast, the myelination inhibitors SOX2, Notch1, and Hes1 were increased, providing an additional potential basis for impaired myelination. A minor fraction of SCs, with some peculiar differences between sensory and motor fibers, overcame the myelination block and formed unusually thin myelin, in line with observed impaired neuregulin and AKT signaling. Surprisingly, we also found signs of axonal degeneration in Dicer mutant mice. Thus, our data indicate that miRNAs critically regulate Schwann cell gene expression that is required for myelination and to maintain axons via axon-glia interactions.