End-Structure of Afferent Axons Injured in the Peripheral and Central Nervous System

The end-structure of afferent axons chronically severed in the rat sciatic nerve or dorsal column (DC) was visualized by centrifugal transport of horseradish peroxidase (HRP) or wheatgerm agglutinin conjugated to HRP (WGA:HRP) injected into the L₄ or L₅ dorsal root ganglion. Nerve regeneration was prevented and neuroma formation encouraged by tightly ligating the cut nerve end. For the first few weeks postoperative, the time during which afferents trapped in a nerve-end neuroma generate their most intense ectopic impulse barrage, the developing neuroma was dominated by swollen terminal end-bulbs. There was some axonal dying-back, retrograde fiber growth, and terminal sprouting, but little preterminal branching. The rich tangle of fine preterminal branches usually thought of in relation to nerve-end neuromas did not elaborate until several months postoperative, a time when the neuroma is relatively quiescent electrically. Afferents cut in the DC, which never develop dramatic ectopic electrical activity, showed morphological peculiarities similar to nerve-end neuromas during the early postoperative period, including retrograde fiber growth and minimal sprouting. They did not, however, go on to form luxuriant branches. These data provide preliminary clues as to the structure of the ectopic impulse-generating mechanism thought to underlie paresthesias and pain associated with peripheral nerve injury.     

End-structure of afferent axons injured in the peripheral and central nervous system

The end-structure of afferent axons chronically severed in the rat sciatic nerve or dorsal column (DC) was visualized by centrifugal transport of horseradish peroxidase (HRP) or wheatgerm agglutinin conjugated to HRP (WGA:HRP) injected into the L4 or L5 dorsal root ganglion. Nerve regeneration was prevented and neuroma formation encouraged by tightly ligating the cut nerve end. For the first few weeks postoperative, the time during which afferents trapped in a nerve-end neuroma generate their most intense ectopic impulse barrage, the developing neuroma was dominated by swollen terminal end-bulbs. There was some axonal dying-back, retrograde fiber growth, and terminal sprouting, but little preterminal branching. The rich tangle of fine preterminal branches usually thought of in relation to nerve-end neuromas did not elaborate until several months postoperative, a time when the neuroma is relatively quiescent electrically. Afferents cut in the DC, which never develop dramatic ectopic electrical activity, showed morphological peculiarities similar to nerve-end neuromas during the early postoperative period, including retrograde fiber growth and minimal sprouting. They did not, however, go on to form luxuriant branches. These data provide preliminary clues as to the structure of the ectopic impulse-generating mechanism thought to underlie paresthesias and pain associated with peripheral nerve injury.     

Treatment of the painful neuroma by neuroma resection and muscle implantation

The successful treatment of the painful neuroma remains an elusive surgical goal. This report evaluates one approach to the management of this problem which entails neuroma excision and placement of the proximal end of the nerve away from denervated skin, away from tension, and into a well-vascularized environment: muscle. Seventy-eight neuromas in 60 patients with a mean follow-up of 31 months (range 18 to 43 months) were evaluated. Sixty-seven percent of these patients involved Workmen's Compensation and 57 percent had had at least one previous operation to treat their pain. The results demonstrated good to excellent results in 82 percent of the treated nerves in the entire group. Factors that were predictive of a poorer outcome were (1) digital neuroma (p less than 0.0005), (2) Workmen's Compensation (p less than 0.01), and (3) three or more previous operations for pain (p less than 0.01). Transposition of nerves into small superficial muscles or muscles with significant excursion resulted in treatment failures. The etiology and histopathology of treatment failures are reviewed. Treatment of radial sensory neuromas by transposition of the radial sensory nerve into the brachioradialis muscle when any associated injury to the lateral antebrachial cutaneous nerve was also treated, gave good to excellent relief of pain, and improved hand function in 88 percent of the patients.     

Treatment of painful hand neuromas by their transfer into bone

Painful neuromas in the hand are not only very disabling for the patient, but difficult to treat. We present the results of 20 painful neuromas treated by burying the neuroma in the bone. Eighteen of the 20 neuromas operated on had acceptable results, according to the criteria of Herndon et al. We present our technique and compare our results with other treatments in the literature.     

A quantitative evaluation of gross versus histologic neuroma formation in a rabbit forelimb amputation model: potential implications for the operative treatment and study of neuromas

Background

Surgical treatment of neuromas involves excision of neuromas proximally to the level of grossly "normal" fascicles; however, proximal changes at the axonal level may have both functional and therapeutic implications with regard to amputated nerves. In order to better understand the retrograde "zone of injury" that occurs after nerve transection, we investigated the gross and histologic changes in transected nerves using a rabbit forelimb amputation model.

Methods

Four New Zealand White rabbits underwent a forelimb amputation with transection and preservation of the median, radial, and ulnar nerves. After 8 weeks, serial sections of the amputated nerves were then obtained in a distal-to-proximal direction toward the brachial plexus. Quantitative histomorphometric analysis was performed on all nerve specimens.

Results

All nerves demonstrated statistically significant increases in nerve cross-sectional area between treatment and control limbs at the distal nerve end, but these differences were not observed 10 mm more proximal to the neuroma bulb. At the axonal level, an increased number of myelinated fibers were seen at the distal end of all amputated nerves. The number of myelinated fibers progressively decreased in proximal sections, normalizing at 15 mm proximally, or the level of the brachial plexus. The cross-sectional area of myelinated fibers was significantly decreased in all sections of the treatment nerves, indicating that atrophic axonal changes proceed proximally at least to the level of the brachial plexus.

Conclusions

Morphologic changes at the axonal level extend beyond the region of gross neuroma formation in a distal-to-proximal fashion after nerve transection. This discrepancy between gross and histologic neuromas signifies the need for improved standardization among neuroma models, while also providing a fresh perspective on how we should view neuromas during peripheral nerve surgery.     

Expression of AMPA receptor subunits at synapses in laminae I–III of the rodent spinal dorsal horn

Neuropathic pain may arise following peripheral nerve injury though the molecular mechanisms associated with this are unclear. We used proteomic profiling to examine changes in protein expression associated with the formation of hyper-excitable neuromas derived from rodent saphenous nerves. A two-dimensional difference gel electrophoresis (2D-DIGE) profiling strategy was employed to examine protein expression changes between developing neuromas and normal nerves in whole tissue lysates. We found around 200 proteins which displayed a >1.75-fold change in expression between neuroma and normal nerve and identified 55 of these proteins using mass spectrometry. We also used immunoblotting to examine the expression of low-abundance ion channels Nav1.3, Nav1.8 and calcium channel α2δ-1 subunit in this model, since they have previously been implicated in neuronal hyperexcitability associated with neuropathic pain. Finally, S³⁵methionine in vitro labelling of neuroma and control samples was used to demonstrate local protein synthesis of neuron-specific genes. A number of cytoskeletal proteins, enzymes and proteins associated with oxidative stress were up-regulated in neuromas, whilst overall levels of voltage-gated ion channel proteins were unaffected. We conclude that altered mRNA levels reported in the somata of damaged DRG neurons do not necessarily reflect levels of altered proteins in hyper-excitable damaged nerve endings. An altered repertoire of protein expression, local protein synthesis and topological re-arrangements of ion channels may all play important roles in neuroma hyper-excitability.     

A Silk Fibroin/Collagen Nerve Scaffold Seeded with a Co-Culture of Schwann Cells and Adipose-Derived Stem Cells for Sciatic Nerve Regeneration

As a promising alternative to autologous nerve grafts, tissue-engineered nerve grafts have been extensively studied as a way to bridge peripheral nerve defects and guide nerve regeneration. The main difference between autogenous nerve grafts and tissue-engineered nerve grafts is the regenerative microenvironment formed by the grafts. If an appropriate regenerative microenvironment is provided, the repair of a peripheral nerve is feasible. In this study, to mimic the body’s natural regenerative microenvironment closely, we co-cultured Schwann cells (SCs) and adipose-derived stem cells (ADSCs) as seed cells and introduced them into a silk fibroin (SF)/collagen scaffold to construct a tissue-engineered nerve conduit (TENC). Twelve weeks after the three different grafts (plain SF/collagen scaffold, TENC, and autograft) were transplanted to bridge 1-cm long sciatic nerve defects in rats, a series of electrophysiological examinations and morphological analyses were performed to evaluate the effect of the tissue-engineered nerve grafts on peripheral nerve regeneration. The regenerative outcomes showed that the effect of treatment with TENCs was similar to that with autologous nerve grafts but superior to that with plain SF/collagen scaffolds. Meanwhile, no experimental animals had inflammation around the grafts. Based on this evidence, our findings suggest that the TENC we developed could improve the regenerative microenvironment and accelerate nerve regeneration compared to plain SF/collagen and may serve as a promising strategy for peripheral nerve repair.     

An alternative to the classical nerve graft for the management of the short nerve gap

Reconstruction of a short nerve gap by a nerve graft produces donor-site scarring, loss of donor nerve function, and neuroma formation. This study compared the regeneration achieved after 1 year in 16 monkeys across a 3-cm upper arm ulnar nerve gap with a bioabsorbable polyglycolic acid nerve conduit with the regeneration achieved with a classical interfascicular interpositional sural nerve graft. The results demonstrated electrophysiologic and histologic evidence of neural regeneration across the gaps in all experimental groups. The bioabsorbable nerve conduit groups and the sural nerve graft group had mean fiber diameters, amplitudes, and conduction velocities each significantly less than those of normal control ulnar nerves. There was, however, no significant difference between any of the experimental groups. Electromyography demonstrated recovery of 19 of the 28 (68 percent) intrinsic muscles studied. These results demonstrate that the primate peripheral nerve can regenerate across short nerve gaps when guided by an appropriate nerve conduit, suggesting that a single-stage biodegradable polyglycolic acid conduit may be used as an alternative to a short interfascicular nerve graft.     

外科治疗囊性听神经瘤14例临床观察

目的：探讨囊性听神经瘤的临床特点及手术经验。方法：回顾分析2006年1月-2010年12月我院收治的14例囊性听经瘤的临床资料。结果：肿瘤全切除12例,不全切除2例,无死亡病例,术后4例病人面瘫加重,1例病人听力丧失。结论：与实质性听神经瘤相比,囊性听神经瘤体积较大,病程短,始发症状不典型,颅神经常被累及,可不发生内听道扩大,脑积水发生率低,手术效果差。     

Substance P-, CGRP- and NPY-immunoreactive nerve fibers in rat sciatic nerve-end neuromas

Substance P (SP)-, calcitonin gene-related peptide (CGRP)- and neuropeptide Y (NPY)-immunoreactive nerve fibers were examined in experimental sciatic nerve-end neuromas in the rat with immunohistochemical techniques. At 1-3 days after nerve ligation and section of the sciatic nerve there was an accumulation of SP-like immunoreactivity (SP-LI). Six days after the lesion there was, however, a marked reduction and the neuromas remained virtually depleted from SP-LI at survival times between 8 days and 3 months. CGRP-LI was strong at 1-5 days post-operatively. By 8 days, CGRP-LI was reduced, but a large number of axons were still immunoreactive, and remained immunolabelled up to 3 months. CGRP-LI nerve fibers formed endbulbs, and appeared to grow in both anterograde and retrograde directions. Fine fibers sprouts were first observed at 8 days, but preterminal branching in neuromas aged less than a month was uncommon. At early stages (1-3 days) after ligation, there was a marked accumulation of NPY-LI proximal to the nerve constriction. NPY-LI was reduced from 5 days and on, but many fibers remained NPY-positive and their growth pattern through proximal and distal neuroma segments could be determined. The present results thus may indicate a differential effect of nerve injury on neuropeptide expression: immunohistochemically detectable SP-LI rapidly disappears from sciatic nerve fibers trapped in nerve-end neuromas, but CGRP-LI and NPY-LI remain and are useful as neuroanatomical markers for two subclasses of sprouting axons. Furthermore, the findings suggest that both CGRP and NPY, but not SP, could be involved in ectopic electrical activity in experimental neuromas.     

Regulation of RhoA Signaling by the cAMP-dependent Phosphorylation of RhoGDIα

RhoA plays a pivotal role in regulating cell shape and movement. Protein kinase A (PKA) inhibits RhoA signaling and thereby induces a characteristic morphological change, cell rounding. This has been considered to result from cAMP-induced phosphorylation of RhoA at Ser-188, which induces a stable RhoA-GTP-RhoGDIα complex and sequesters RhoA to the cytosol. However, few groups have shown RhoA phosphorylation in intact cells. Here we show that phosphorylation of RhoGDIα but not RhoA plays an essential role in the PKA-induced inhibition of RhoA signaling and in the morphological changes using cardiac fibroblasts. The knockdown of RhoGDIα by siRNA blocks cAMP-induced cell rounding, which is recovered by RhoGDIα-WT expression but not when a RhoGDIα-S174A mutant is expressed. PKA phosphorylates RhoGDIα at Ser-174 and the phosphorylation of RhoGDIα is likely to induce the formation of a active RhoA-RhoGDIα complex. Our present results thus reveal a principal molecular mechanism underlying G_s/cAMP-induced cross-talk with G_q/G₁₃/RhoA signaling.     

STRETCHING OF THE SCIATIC NERVE—A Means of Relieving Postoperative Pain Following Removal of Ruptured Lumbar Intervertebral Discs

Stretching the sciatic nerve for the relief of “sciatica” was frequently employed before 1900 and was subsequently abandoned, probably because it was done without sufficient scrutiny of the indications. The procedure has recently been employed in cases in which “sciatica” remains following the operative removal of ruptured intervertebral discs, and it has been instrumental in relieving postoperative “sciatica” when the cause was the formation of adhesions about the lumbar nerve roots. If the nerve root is compressed by recurrent disc protrusion or by adjacent bone, the manipulation usually increases the pain, a phenomenon that has been helpful from a diagnostic standpoint.     

Mouse Macrophages Completely Lacking Rho Subfamily GTPases (RhoA,RhoB, and RhoC) Have Severe Lamellipodial Retraction Defects,but Robust Chemotactic Navigation and Altered Motility

RhoA is thought to be essential for coordination of the membrane protrusions and retractions required for immune cell motility and directed migration. Whether the subfamily of Rho (Ras homolog) GTPases (RhoA, RhoB, and RhoC) is actually required for the directed migration of primary cells is difficult to predict. Macrophages isolated from myeloid-restricted RhoA/RhoB (conditional) double knock-out (dKO) mice did not express RhoC and were essentially “pan-Rho”-deficient. Using real-time chemotaxis assays, we found that retraction of the trailing edge was dissociated from the advance of the cell body in dKO cells, which developed extremely elongated tails. Surprisingly, velocity (of the cell body) was increased, whereas chemotactic efficiency was preserved, when compared with WT macrophages. Randomly migrating RhoA/RhoB dKO macrophages exhibited multiple small protrusions and developed large “branches” due to impaired lamellipodial retraction. A mouse model of peritonitis indicated that monocyte/macrophage recruitment was, surprisingly, more rapid in RhoA/RhoB dKO mice than in WT mice. In comparison with dKO cells, the phenotypes of single RhoA- or RhoB-deficient macrophages were mild due to mutual compensation. Furthermore, genetic deletion of RhoB partially reversed the motility defect of macrophages lacking the RhoGAP (Rho GTPase-activating protein) myosin IXb (Myo9b). In conclusion, the Rho subfamily is not required for “front end” functions (motility and chemotaxis), although both RhoA and RhoB are involved in pulling up the “back end” and resorbing lamellipodial membrane protrusions. Macrophages lacking Rho proteins migrate faster in vitro, which, in the case of the peritoneum, translates to more rapid in vivo monocyte/macrophage recruitment.     

Comparison of neural histomorphology in tail tips from pigs docked using clippers or cautery iron

Tail docking of pigs is commonly performed to reduce the incidence of unwanted tail-biting behaviour. Two docking methods are commonly used: blunt trauma cutting (i.e. using side clippers), or cutting and concurrent cauterisation using a hot cautery iron. A potential consequence of tail amputation is the development of neuromas at the docking site. Neuromas have been linked to neuropathic pain, which can influence the longer-term welfare of affected individuals. To determine whether method of tail docking influences the extent of neuroma formation, 75 pigs were allocated to one of three treatments at birth: tail docked using clippers; tail docked using cautery iron; tail left intact. Tail docking was performed at 2 days of age and pigs were kept under conventional conditions until slaughter at 21 weeks of age. Tails were removed following slaughter and subjected to histological examination. Nerve histomorphology was scored according to the following scale: 1=discrete well-organised nerve bundles; 2=moderate neural proliferation and disorganisation affecting more than half of the circumference of the tail; 3=marked neural proliferation to form almost continuous disorganised bundles or non-continuous enlarged bundles compressing the surrounding connective tissue. Scores of 2 or 3 indicated neuroma formation. Scores were higher in docked pigs than undocked pigs (P<0.001), but did not differ between pigs docked using clippers and those docked using cautery (P=0.23). The results indicate that tail docking using either clippers or cautery results in neuroma formation, thus having the potential to affect long-term pig welfare.     

Lentiviral-mediated transfer of CDNF promotes nerve regeneration and functional recovery after sciatic nerve injury in adult rats

Peripheral nerve injury is often followed by incomplete and unsatisfactory functional recovery and may be associated with sensory and motor impairment of the affected limb. Therefore, a novel method is needed to improve the speed of recovery and the final functional outcome after peripheral nerve injuries. This report investigates the effect of lentiviral-mediated transfer of conserved dopamine neurotrophic factor (CDNF) on regeneration of the rat peripheral nerve in a transection model in vivo. We observed notable overexpression of CDNF protein in the distal sciatic nerve after recombinant CDNF lentiviral vector application. We evaluated sciatic nerve regeneration after surgery using light and electron microscopy and the functional recovery using the sciatic functional index and target muscle weight. HE staining revealed better ordered structured in the CDNF-treated group at 8 weeks post-surgery. Quantitative analysis of immunohistochemistry of NF200 and S-100 in the CDNF group revealed significant improvement of axonal and Schwann cell regeneration compared with the control groups at 4 weeks and 8 weeks after injury. The thickness of the myelination around the axons in the CDNF group was significantly higher than in the control groups at 8 weeks post-surgery. The CDNF group displayed higher muscle weights and significantly increased sciatic nerve index values. Our findings suggest that CDNF gene therapy could provide durable and stable CDNF protein concentration and has the potential to enhance peripheral nerve regeneration, morphological and functional recovery following nerve injury, which suggests a promising strategy for peripheral nerve repair.     

Different Roles for RhoA During Neurite Initiation, Elongation, and Regeneration in PC12 Cells

The goal of the present study was to characterize the effects of RhoA at different stages of nerve growth factor (NGF)-induced neuronal differentiation in the PC12 model. This comparative analysis was prompted by previous studies that reported apparently opposite effects for Rho in different models of neuronal differentiation and regeneration. PC12 cells were transfected with activated V14RhoA or dominant negative N19RhoA under the control of either a constitutive or a steroid-regulated promoter. Upon exposure to NGF, V14RhoA cells continued to proliferate and did not extend neurites; however, they remained responsive to NGF, as indicated by the activation of extracellular signal-regulated kinases. This inability to differentiate was reversed by C3 toxin and activation of cyclic AMP signaling, which inactivate RhoA. N19RhoA expression led to an increase in neurite initiation and branching. In contrast, when the RhoA mutants were expressed after NGF priming, only the rate of neurite extension was altered; V14RhoA clones had neurites approximately twice as long, whereas neurites of N19RhoA cells were approximately 50% shorter than those of appropriate controls. The effects of Rho in neurite regeneration mimicked those observed during the initial stages of morphogenesis; activation inhibited, whereas inactivation promoted, neurite outgrowth. Our results indicate that RhoA function changes at different stages of NGF-induced neuronal differentiation and neurite regeneration.     

Axonally transported proteins associated with axon growth in rabbit central and peripheral nervous systems

In an effort to determine whether the “growth state” and the “mature state” of a neuron are differentiated by different programs of gene expression, we have compared the rapidly transported (group I) proteins in growing and nongrowing axons in rabbits. We observed two polypeptides (GAP-23 and GAP-43) which were of particular interest because of their apparent association with axon growth. GAP-43 was rapidly transported in the central nervous system (CNS) (retinal ganglion cell) axons of neonatal animals, but its relative amount declined precipitously with subsequent development. It could not be reinduced by axotomy of the adult optic nerves, which do not regenerate; however, it was induced after axotomy of an adult peripheral nervous system nerve (the hypoglossal nerve, which does regenerate) which transported only very low levels of GAP-43 before axotomy. The second polypeptide, GAP-23 followed the same pattern of growth-associated transport, except that it was transported at significant levels in uninjured adult hypoglossal nerves and not further induced by axotomy. These observations are consistent with the “GAP hypothesis” that the neuronal growth state can be defined as an altered program of gene expression exemplified in part by the expression of GAP genes whose products are involved in critical growth-specific functions. When interpreted in terms of GAP hypothesis, they lead to the following conclusions: (a) the growth state can be subdivided into a “synaptogenic state” characterized by the transport of GAP-23 but not GAP-43, and an “axon elongation state” requiring both GAPs; (b) with respect to the expression of GAP genes, regeneration involves a recapitulation of a neonatal state of the neuron; and (c) the failure of mammalian CNS neurons to express the GAP genes may underly the failure of CNS axons to regenerate after axon injury.     

Structural Insights into the Activation of the RhoA GTPase by the Lymphoid Blast Crisis (Lbc) Oncoprotein

The small GTPase RhoA promotes deregulated signaling upon interaction with lymphoid blast crisis (Lbc), the oncogenic form of A-kinase anchoring protein 13 (AKAP13). The onco-Lbc protein is a hyperactive Rho-specific guanine nucleotide exchange factor (GEF), but its structural mechanism has not been reported despite its involvement in cardiac hypertrophy and cancer causation. The pleckstrin homology (PH) domain of Lbc is located at the C-terminal end of the protein and is shown here to specifically recognize activated RhoA rather than lipids. The isolated dbl homology (DH) domain can function as an independent activator with an enhanced activity. However, the DH domain normally does not act as a solitary Lbc interface with RhoA-GDP. Instead it is negatively controlled by the PH domain. In particular, the DH helical bundle is coupled to the structurally dependent PH domain through a helical linker, which reduces its activity. Together the two domains form a rigid scaffold in solution as evidenced by small angle x-ray scattering and ¹H,¹³C,¹⁵N-based NMR spectroscopy. The two domains assume a “chair” shape with its back possessing independent GEF activity and the PH domain providing a broad seat for RhoA-GTP docking rather than membrane recognition. This provides structural and dynamical insights into how DH and PH domains work together in solution to support regulated RhoA activity. Mutational analysis supports the bifunctional PH domain mediation of DH-RhoA interactions and explains why the tandem domain is required for controlled GEF signaling.     

Development of a bioartificial nerve graft. II. Nerve regeneration in vitro

A promising alternative for the repair of peripheral nerve injuries is the bioartificial nerve graft, or BNG, comprised of a tubular conduit preseeded with Schwann cells, which are an effective substrate for enhancing nerve regeneration. The physical properties of the conduit, porosity and wall thickness, as well as the Schwann cell seeding density, were tested for their effect on axon growth using rat dorsal root ganglia. These parameters can influence the amount of nutrients and growth factors made available to the neural tissue. Results show that a greater wall thickness and lower porosities have a detrimental effect on the growth of the axons. Over a four week period, axons extended 3.2 mm for the optimum case (DeltaR = 0.82 mm, epsilon = 0.75) compared to 1.8 and 1.6 mm for a lower porosity (0.55) and a greater wall thickness (1.4 mm), respectively. A maximum in the growth rate occurs at a porosity of 75% for Schwann cell seeded conduits but not for unseeded ones. When compared to mass transfer predictions, the results suggest that, at higher porosities, more growth factors diffuse out of the conduit, while at low porosities there is competition for nutrients. Increasing the Schwann cell seeding density enhances growth but also leads to an increase in the number of axons along the length of the conduit. This is indicative of branching of the axons, which requires additional resources to maintain and can lead to painful neuroma formation. Wall thickness and porosity were found not to have any significant effect on the axon number sprouting from the dorsal root ganglia and the mean diameter (p > 0.05). Considerations need to be made, not just on the polymer used, but also on its porosity, wall thickness, and Schwann cell seeding density. These parameters can be adjusted to create a bioartificial nerve graft that provides the optimal environment for nerve growth.