Pure peroneal intraneural ganglion cyst ascending along the sciatic nerve

Peroneal nerve entrapment is most commonly seen in the popliteal fossa. It is rarely caused by a ganglion. Intraneural ganglia, although uncommon and seldom cause serious complications, are well recognized and most commonly affect the common peroneal (lateral popliteal) nerve. Ganglionic cysts developing in the sheath of a peripheral nerve or joint capsule may cause compression neuropathy. The differential diagnosis should involve L5 root lesions, posttraumatic intraneural hemorrhage, nerve compression near the tendinous arch located at the fibular insertion of the peroneal longus muscle and nerve-sheath tumors. We present a unique case of a pure intraneural ganglion of the common peroneal nerve ascending along the sciatic nerve. This case underscores the importance of consideration of an intraneural ganglion cyst with sciatic nerve involvement.     

Visualization of spinal afferent innervation in the mouse colon by AAV8‐mediated GFP expression

Background Primary afferent neurons whose cell bodies reside in thoracolumbar and lumbosacral dorsal root ganglia (DRG) innervate colon and transmit sensory signals from colon to spinal cord under normal conditions and conditions of visceral hypersensitivity. Histologically, these extrinsic afferents cannot be differentiated from intrinsic fibers of enteric neurons because all known markers label neurons of both populations. Adeno‐associated virus (AAV) vectors are capable of transducing DRG neurons after intrathecal administration. We hypothesized that AAV‐driven overexpression of green fluorescent protein (GFP) in DRG would enable visualization of extrinsic spinal afferents in colon separately from enteric neurons. Methods Recombinant AAV serotype 8 (rAAV8) vector carrying the GFP gene was delivered via direct lumbar puncture. Green fluorescent protein labeling in DRG and colon was examined using immunohistochemistry. Key Results Analysis of colon from rAAV8‐GFP‐treated mice demonstrated GFP‐immunoreactivity (GFP‐ir) within mesenteric nerves, smooth muscle layers, myenteric plexus, submucosa, and mucosa, but not in cell bodies of enteric neurons. Notably, GFP‐ir colocalized with CGRP and TRPV1 in mucosa, myenteric plexus, and globular‐like clusters surrounding nuclei within myenteric ganglia. In addition, GFP‐positive fibers were observed in close association with blood vessels of mucosa and submucosa. Analysis of GFP‐ir in thoracolumbar and lumbosacral DRG revealed that levels of expression in colon and L6 DRG appeared to be related. Conclusions & Inferences These results demonstrate the feasibility of gene transfer to mouse colonic spinal sensory neurons using intrathecal delivery of AAV vectors and the utility of this approach for histological analysis of spinal afferent nerve fibers within colon.     

Genomic analysis reveals frequent TRAF7 mutations in intraneural perineuriomas

Intraneural perineuriomas are benign peripheral nerve sheath tumors that cause progressive debilitating focal extremity weakness. The etiology of perineuriomas is largely unknown. We utilized whole exome sequencing, copy number algorithm evaluation, and high‐resolution whole genome microarray to investigate for a genetic causal link to intraneural perineuriomas. Ten of 16 (60%) tumor cases had mutations in the WD40 domain of TRAF7, the same location for causal mutations of meningiomas. Two additional perineurioma cases had large chromosomal abnormalities in multiple chromosomes, including chromosome 22q. This study identifies a common cause for intraneural perineuriomas and an unexpected shared pathogenesis with intracranial meningiomas. Ann Neurol 2017;81:316–321     

Glial fibrillary acidic protein promoter determines transgene expression in satellite glial cells following intraganglionic adeno‐associated virus delivery in adult rats

Recombinant adeno‐associated viral (AAV)‐mediated therapeutic gene transfer to dorsal root ganglia (DRG) is an effective and safe tool for treating chronic pain. However, AAV with various constitutively active promoters leads to transgene expression predominantly to neurons, while glial cells are refractory to AAV transduction in the peripheral nervous system. The present study evaluated whether in vivo satellite glial cell (SGC) transduction in the DRG can be enhanced by the SGC‐specific GFAP promoter and by using shH10 and shH19, which are engineered capsid variants with Müller glia‐prone transduction. Titer‐matched AAV6 (as control), AAVshH10, and AAVshH19, all encoding the EGFP driven by the constitutively active CMV promoter, as well as AAV6‐EGFP and AAVshH10‐EGFP driven by a GFAP promoter (AAV6‐GFAP‐EGFP and AAVshH10‐GFAP‐EGFP), were injected into DRG of adult male rats. Neurotropism of gene expression was determined and compared by immunohistochemistry. Results showed that injection of AAV6‐ and AAVshH10‐GFAP‐EGFP induces robust EGFP expression selectively in SGCs, whereas injection of either AAVshH10‐CMV‐EGFP or AAVshH19‐CMV‐EGFP into DRG resulted in a similar in vivo transduction profile to AAV6‐CMV‐EGFP, all showing efficient transduction of sensory neurons without significant transduction of glial cell populations. Coinjection of AAV6‐CMV‐mCherry and AAV6‐GFAP‐EGFP induces transgene expression in neurons and SGCs separately. This report, together with our prior studies, demonstrates that the GFAP promoter rather than capsid tropism determines selective gene expression in SGCs following intraganglionic AAV delivery in adult rats. A dual AAV system, one with GFAP promoter and the other with CMV promoter, can efficiently express transgenes selectively in neurons versus SGCs.     

C-fiber structure varies with location in peripheral nerve

Recent advances in regeneration and pain research have revealed gaps in the understanding of normal C-fiber anatomy. In the rat PNS, C-fiber axons assemble into Remak bundles, but beyond this, features of C-fiber organization are not defined. Systematic sampling and quantitation reveals that Remak bundles exiting from the L5 dorsal root ganglion (DRG) contain large numbers of axons, for example, 56% of unmyelinated axons were in bundles of >20 axons. This is different from distal nerve segments such as the hindpaw plantar nerve where the median number of axons per bundle is 3. The cross-sectional area of unmyelinated axons in dorsal root was homogeneous near the DRG but variability in axonal area increased near the spinal cord (p = 0.00001) and the mean axonal area was unchanged. Unmyelinated axons in peripheral nerve were almost always isolated from one another by Schwann cell processes; however, in dorsal root 7% to 9% of unmyelinated axons were immediately adjacent within pockets containing 2 or more axons. Remak bundles in the distal peripheral nerve clustered with other Remak bundles. We observe that multiple unmyelinated axons are juxtaposed within the C-fiber/Remak bundle and that the close association of afferent axons may have important functional implications.     

Neuronal age influences the response to neurite outgrowth inhibitory activity in the central and peripheral nervous systems.

Axonal regeneration is abortive in the central nervous system (CNS) of adult mammals, but readily occurs in the injured peripheral nervous system (PNS). Recent experiments indicate an important role for both intrinsic neuronal features and extrinsic substrate properties in determining the propensity for axonal regrowth. In particular, certain components of adult mammalian CNS myelin have been shown to exert a strong inhibitory influence on neurite outgrowth. To determine whether the potent neurite outgrowth inhibitory activity found in CNS myelin may also be present in PNS myelin and to study the influence of neuronal age on neurite outgrowth, we used a cryoculture assay in which dissociated rat dorsal root ganglion (DRG) neurons of different ages were challenged to extend neurites on fractionated myelin and cryostat sections from the PNS (sciatic nerve and myelin-free degenerated sciatic nerve) and CNS (optic nerve) of adult rats. The CNS environment of the optic nerve did not support E17 to P8 DRG neurite adhesion or outgrowth. E17 DRG neurons, unlike their older counterparts, however, were able to attach and extend neurites onto normal sciatic nerve and onto purified PNS myelin. In contrast, a vigorous neurite outgrowth response from all the ages tested was observed on the myelin-free degenerated sciatic nerve. These results indicate that PNS myelin is a potent inhibitor of neurite outgrowth and that DRG neuronal age plays an important role in determining the propensity for neurite outgrowth and regenerative response on inhibitory PNS and CNS substrata.     

AAV8(gfp) preferentially targets large diameter dorsal root ganglion neurones after both intra-dorsal root ganglion and intrathecal injection

Adeno-associated viral vectors (AAV) are increasingly used to deliver therapeutic genes to the central nervous system (CNS) where they promote transgene expression in post mitotic neurones for long periods with little or no toxicity. In adult rat dorsal root ganglia (DRG), we investigated the cellular tropism of AAV8 containing the green fluorescent protein gene (gfp) after either intra-lumbar DRG or intrathecal injection and showed that transduced DRG neurones (DRGN) expressed GFP irrespective of the delivery route, while non-neuronal cells were GFP(-). After intra-DRG delivery of AAV8(gfp), the mean DRGN transduction rate was 11%, while intrathecal delivery transduced a mean of 1.5% DRGN. After intra-DRG injection, 2% of small DRGN (<30 μm in diameter) were GFP(+) compared with 32% of large DRGN (>60 μm in diameter). Axons of transduced DRGN were also GFP(+); no intra-spinal neurones were transduced. A small number of contralateral DRGN were transduced after intra-DRG injection, suggesting that AAV8 may diffuse from injected DRG into the spinal canal. Microglia and astrocytes were highly ramified with increased GFAP(+) immunoreactivity (i.e. activated) in the neuropil around GFP(+) DRG axon projections within the cord after intra-DRG injection. This study showed that after both intra-DRG and intrathecal delivery, strong preferential AAV8 tropism exists for large DRGN unassociated with cell death, but GFP(+) axons projecting in the spinal cord induced local glial activation. These results open up opportunities for targeted delivery of therapeutics such as neurotrophic factors to the injured spinal cord.     

Axonal transport of TNF-alpha in painful neuropathy: distribution of ligand tracer and TNF receptors

Tumor necrosis factor-alpha (TNF-alpha) is a key player in peripheral nerve injury. In the inflammatory chronic constriction injury (CCI) model of sciatic neuropathy, upregulation of TNF-alpha mRNA and protein at the site of nerve injury has been associated with pain. We now report the distribution of endogenous TNF-alpha protein and its receptors along normal and CCI-injured sciatic nerves, and within the corresponding lumbar dorsal root ganglia (DRG). Using Western blotting, TNF-alpha was found to be distinctly increased at the injury site, as well as in the axons just distal to the corresponding DRG. The TNF-alpha signal between the injury site and DRG (midaxonal) was induced between 2 and 5 days post-CCI, suggesting activation of TNF-alpha axonal transport. Endogenous TNF-alpha was localized in small-diameter, presumably nociceptive, and large-diameter, presumably mechanoceptive, DRG sensory neurons in both normal and CCI animals. Intraneural microinjection of biotin-labeled TNF-alpha showed specific axonal uptake at the injection site, as detected by avidin-biotin-peroxidase histochemistry, and confirmed by co-localization with neurobiotin tracer. In control animals, fast retrograde transport of biotinylated TNF-alpha to both L4 and L5 DRG neurons was apparent 6 h following injection. TNF receptors TNFRI and TNFRII co-localized with biotinylated TNF-alpha tracer along the nerve trunk, suggesting that TNF-alpha transport may be receptor-mediated. In animals with CCI neuropathy, uptake of biotinylated TNF-alpha by neuronal soma was inhibited. Instead, there was signal accumulation in the axons immediately distal to the DRG, and TNFRI and RII were increased at this same anatomic location. These findings highlight a dynamic process of TNF-alpha protein and receptor regulation throughout the peripheral neural axis that bears on both the normal function of DRG neurons and the pathogenesis of painful neuropathies.     

In vivo peripheral nervous system insulin signaling

Alterations in peripheral nervous system (PNS) insulin support may contribute to diabetic neuropathy (DN); yet, PNS insulin signaling is not fully defined. Here, we investigated in vivo insulin signaling in the PNS and compared the insulin responsiveness to that of muscle, liver, and adipose. Non‐diabetic mice were administered increasing doses of insulin to define a dose‐response relationship between insulin and Akt activation in the dorsal root ganglion (DRG) and sciatic nerve. Resulting EC50 doses were used to characterize the PNS insulin signaling time course and make comparisons between insulin signaling in the PNS and other peripheral tissues (i.e., muscle, liver, and adipose). The results demonstrate that the PNS is responsive to insulin and that differences in insulin signaling pathway activation exist between PNS compartments. At a therapeutically relevant dose, Akt was activated in the muscle, liver, and adipose at 30 min, correlating with the changes in blood glucose levels. Interestingly, the sciatic nerve showed a similar signaling profile as insulin‐sensitive tissues; however, there was not a comparable activation in the DRG or spinal cord. These results present new evidence regarding PNS insulin signaling pathways in vivo and provide a baseline for studies investigating the contribution of disrupted PNS insulin signaling to DN pathogenesis.     

Enhanced adenoviral gene delivery to motor and dorsal root ganglion neurons following injection into demyelinated peripheral nerves

Injection of viral vectors into peripheral nerves may transfer specific genes into their dorsal root ganglion (DRG) neurons and motoneurons. However, myelin sheaths of peripheral axons block the entry of viral particles into nerves. We studied whether mild, transient peripheral nerve demyelination prior to intraneural viral vector injection would enhance gene transfer to target DRG neurons and motoneurons. The right sciatic nerve of C57BL/6 mice was focally demyelinated with 1% lysolecithin, and the left sciatic nerve was similarly injected with saline (control). Five days after demyelination, 0.5 μl of Ad5‐GFP was injected into both sciatic nerves at the site of previous injection. The effectiveness of gene transfer was evaluated by counting GFP⁺ neurons in the DRGs and ventral horns. After peripheral nerve demyelination, there was a fivefold increase in the number of infected DRG neurons and almost a 15‐fold increase in the number of infected motoneurons compared with the control, nondemyelinated side. Focal demyelination reduced the myelin sheath barrier, allowing greater virus–axon contact. Increased CXADR expression on the demyelinated axons facilitated axoplasmic viral entry. No animals sustained any prolonged neurological deficits. Increased gene delivery into DRG neurons and motoneurons may provide effective treatment for amyotrophic lateral sclerosis, pain, and spinal cord injury. © 2010 Wiley‐Liss, Inc.     

Ultrasound-Guided Peripheral Nerve Stimulation of Cervical,Thoracic, and Lumbar Spinal Nerves for Dermatomal Pain: A Case Series

ObjectivesWith the development of percutaneously inserted devices, peripheral nerve stimulation (PNS) has been gaining attention within chronic pain literature as a less invasive neurostimulation alternative to spinal column and dorsal root ganglion stimulation. A majority of current PNS literature focuses on targeting individual distal nerves to treat individual peripheral mononeuropathies, limiting its applications. This article discusses our experience treating dermatomal pain with neurostimulation without needing to access the epidural space by targeting the proximal spinal nerve with peripheral nerve stimulation under ultrasound-guidance.Materials and MethodsA temporary, percutaneous PNS was used to target the proximal spinal nerve in 11 patients to treat various dermatomal pain syndromes in patients seen in an outpatient chronic pain clinic. Four patients received stimulation targeting the lumbar spinal nerves and seven patient received stimulation targeting the cervical or thoracic spinal nerves.ResultsThe case series presents 11 cases of PNS of the proximal spinal nerve. Seven patients, including a majority of the patients with lumbar radiculopathy, had analgesia during PNS. Four patients, all of whom targeted the cervical or thoracic spinal nerves, did not receive analgesia from PNS.ConclusionPNS of the proximal spinal nerve may be an effective modality to treat dermatomal pain in patients who are not candidates for other therapies that require access to the epidural space. This technique was used to successfully treat lumbar radiculopathy, post-herpetic neuralgia, and complex regional pain syndrome.     

Cyclooxygenase inhibition in nerve-injury- and TNF-induced hyperalgesia in the rat

After nerve injury, cyclooxygenase-2 (COX-2) is upregulated in spinal cord and peripheral nerve, the latter being dependent on tumor necrosis factor-alpha (TNF). Here we asked whether COX inhibitors attenuate pain behavior induced by chronic constrictive sciatic nerve injury (CCI) or intraneural injection of TNF (2.5 pg/ml). Rats received either 0.9% saline, the nonselective COX inhibitor ibuprofen (40 mg/kg) or the selective COX-2 inhibitor celecoxib (10 or 30 mg/kg) twice daily by gavage started 2 days before, 12 h or 7 days after surgery. Mechanical allodynia and thermal hyperalgesia induced by CCI was moderately, but consistently attenuated by early (day -2 or 12 h after CCI), but not late (7 days after CCI) ibuprofen and celecoxib treatment. Mechanical allodynia, but not thermal hyperalgesia induced by intraneural TNF, was reduced by ibuprofen, but not by celecoxib treatment 5 and 7 days after injection. Sciatic nerves, lumbar dorsal root ganglia (DRG) and spinal cords from rats with treatment started 12 h after surgery were analyzed for prostaglandin E2 (PGE2) levels 10 days after CCI. In injured nerves and ipsilateral DRG, PGE2 levels were increased. Ibuprofen treatment reversed PGE2 levels in injured nerves and DRG, whereas celecoxib blocked increased PGE2 levels only in nerves. In spinal cord, no change in PGE2 levels was observed. In contrast to the marked inhibition of nerve-injury-induced upregulation of PGE2 by COX inhibitors, the effect on pain behavior was modest. Nerve-injury- and TNF-induced pain-related behavior seem to be only partly dependent on peripheral prostaglandins.     

Adhesion molecule L1 overexpressed under the control of the neuronal Thy-1 promoter improves myelination after peripheral nerve injury in adult mice

L1 is an adhesion molecule favorably influencing the functional and anatomical recoveries after central nervous system (CNS) injuries. Its roles in peripheral nervous system (PNS) regeneration are less well understood. Studies using knockout mice have surprisingly revealed that L1 has a negative impact on functional nerve regeneration by inhibiting Schwann cell proliferation. To further elucidate the roles of L1 in PNS regeneration, here we used a novel transgenic mouse overexpressing L1 in neurons, but not in PNS or CNS glial cells, under the control of a neuron-specific Thy-1 promoter. Without nerve injury, the transgene expression, as compared to wild-type mice, had no effect on femoral nerve function, numbers of quadriceps motoneurons and myelinated axons in the femoral nerve but resulted in slightly reduced myelination in the sensory saphenous nerve and increased neurofilament density in myelinated axons of the quadriceps motor nerve branch. After femoral nerve injury, L1 overexpression had no impact on the time course and degree of functional recovery. Unaffected were also numbers of regenerated quadriceps motoneurons, precision of muscle reinnervation, axon numbers and internodal lengths in the regenerated nerves. Despite the lack of functional effects, myelination in the motor and sensory femoral nerve branches was significantly improved and loss of perisomatic inhibitory terminals on motoneurons was attenuated in the transgenic mice. Our results indicate that L1 is a regulator of myelination in the injured PNS and warrant studies aiming to improve function in demyelinating PNS and CNS disorders using exogenous L1.     

On the longevity of resident endoneurial macrophages in the peripheral nervous system: a study of physiological macrophage turnover in bone marrow chimeric mice

Macrophages are intimately involved in the pathogenesis of peripheral nervous system (PNS) disorders. Recently, we characterized a resident endoneurial macrophage population, which contributes rapidly to the endoneurial macrophage response in PNS diseases. Unlike microglial cells, resident macrophages undergo a physiological turnover of 50% in the sciatic nerve and 80% in dorsal root ganglia (DRG) within 12 weeks. Further information about the dynamics of this turnover is not available. This study examined the macrophage turnover in the sciatic nerve and DRGs over a longer period and addresses the question whether the turnover of resident macrophages is complete or whether there is a truly resident endoneurial macrophage population. We used chimeric mice carrying GFP(+) bone marrow and immunohistochemistry to detect hematogenous (GFP(+)) endoneurial macrophages after turnover. Non-exchanged, resident macrophages were GFP(-). Quantification of GFP(+) and GFP(-) macrophages revealed a maximal turnover of 75%, reached in DRGs after 12 weeks and in sciatic nerves after 36 weeks. GFP(-) long-term resident macrophages were further characterized after sciatic nerve injury, where they participated in the early macrophage response of Wallerian degeneration. Our results point toward a small but truly resident PNS macrophage population. These macrophages are an interesting target for further characterization and might have a distinct role in peripheral nerve disease.     

Familial amyloid polyneuropathy associated with transthyretin Gly42 mutation: a quantitative light and electron microscopic study of the peripheral nervous system

We performed extensive quantitative analyses of the peripheral nervous system (PNS) of two siblings with familial amyloid polyneuropathy (FAP) caused by a transthyretin (TTR) Gly42 mutation. Pronounced amyloid deposition was found in the sympathetic ganglia (SyG), dorsal root ganglia (DRG) and throughout the length of the peripheral nerve fibers with some accentuation in the more proximal portion. There was severe neuronal loss in the SyG and DRG together with nerve fiber depletion in the nerve trunk, while only a small amount of amyloid deposition with mild fiber loss was seen in the spinal roots. Sprouts of regenerating axons were very scanty even in the spinal nerves or roots. A teased fiber study mainly showed demyelinating fibers, but axonal degeneration was also present throughout peripheral nerves. An electron microscopic study showed fine amyloid fibrils in direct contact with the axoplasmic membrane of demyelinated axons and destruction of axons in some areas. Amyloid deposition within the PNS in this type of FAP resembled that in type I FAP (TTR Met30). However, direct axonal damage by amyloid fibrils appeared to be more prominent in our cases than in type I FAP. Lectin histochemistry using Ulex europaeus agglutinin I demonstrated preferential depletion of small neurons in the DRG and their primary afferent fibers in the spinal dorsal horn. Primary axonal degeneration and ganglionopathy due to amyloid deposition appear to be the pathogenetic mechanisms for peripheral neuropathy in this type of FAP.     

Reelin activates the small GTPase TC10 and VAMP7 to promote neurite outgrowth and regeneration of dorsal root ganglia (DRG) neurons

Axonal outgrowth is a fundamental process during the development of central (CNS) and peripheral (PNS) nervous system as well as in nerve regeneration and requires accurate axonal navigation and extension to the correct target. These events need proper coordination between membrane trafficking and cytoskeletal rearrangements and are under the control of the small GTPases of the Rho family, among other molecules. Reelin, a relevant protein for CNS development and synaptic function in the adult, is also present in the PNS. Upon sciatic nerve damage, Reelin expression increases and, on the other hand, mice deficient in Reelin exhibit an impaired nerve regeneration. However, the mechanism(s) involved the Reelin‐dependent axonal growth is still poorly understood. In this work, we present evidence showing that Reelin stimulates dorsal root ganglia (DRG) regeneration after axotomy. Moreover, dissociated DRG neurons express the Reelin receptor Apolipoprotein E‐receptor 2 and also require the presence of TC10 to develop their axons. TC10 is a Rho GTPase that promotes neurite outgrowth through the exocytic fusion of vesicles at the growth cone. Here, we demonstrate for the first time that Reelin controls TC10 activation in DRG neurons. Besides, we confirmed that the known CNS Reelin target Cdc42 is also activated in DRG and controls TC10 activity. Finally, in the process of membrane addition, we found that Reelin stimulates the fusion of membrane carriers containing the v‐SNARE protein VAMP7 in vesicles that contain TC10. Altogether, our work shows a new role of Reelin in PNS, opening the option of therapeutic interventions to improve the regeneration process.     

Expression of capsaicin receptor immunoreactivity in human peripheral nervous system and in painful neuropathies

We describe the expression of the capsaicin receptor (TRPV1) in human peripheral nervous system (PNS) and its changes in sural nerve and skin nerve fibers of patients with painful neuropathy. Dorsal root ganglion (DRG), root, and spinal cord autopsy specimens from subjects without PNS diseases were immunoassayed with anti-TRPV1 antibodies. Bright-field and confocal microscope studies using anti-TRPV1, protein gene product 9.5 (PGP 9.5), and unique-beta-tubulin (TuJ1) antibodies were performed in skin biopsies from 15 healthy subjects and 10 painful neuropathies. The density of intraepidermal nerve fiber (IENF) labeled by each antibody was quantified. Sural nerve biopsies from three patients with painful, one patient with nonpainful diabetic neuropathy, and two patients with multifocal motor neuropathy used as controls were immunoassayed with anti-TRPV1 antibodies and investigated by immunoelectron microscopy. TRPV1 strongly labeled laminae I and II of dorsal horns, most small-size and some medium-size DRG neurons, and small-diameter axons of dorsal roots. In sural nerve, TRPV1 was expressed within the cytoplasm of most unmyelinated and some small myelinated axons, in the muscular lamina of epineural vessels, and in the endothelium of endoneurial vessels. The density of IENF labeled by TRPV1, PGP 9.5, and TuJ1 did not differ. TRPV1 colocalized with TuJ1 in all IENF and dermal nerve bundles. Painful neuropathies showed a diffuse loss of TRPV1-positive axons both in the sural nerve and in the skin. Our findings demonstrated that TRPV1 is normally expressed throughout the nociceptive pathway of PNS and that TRPV1-positive peripheral nerve fibers degenerate in painful neuropathies.     

Plexiform-like neurofibromas develop in the mouse by intraneural xenograft of an NF1 tumor-derived Schwann cell line

Plexiform neurofibromas are peripheral nerve sheath tumors that arise frequently in neurofibromatosis type 1 (NF1) and have a risk of malignant progression. Past efforts to establish xenograft models for neurofibroma involved the implantation of tumor fragments or heterogeneous primary cultures, which rarely achieved significant tumor growth. We report a practical and reproducible animal model of plexiform-like neurofibroma by xenograft of an immortal human NF1 tumor-derived Schwann cell line into the peripheral nerve of scid mice. The S100 and p75 positive sNF94.3 cell line was shown to possess a normal karyotype and have apparent full-length neurofibromin by Western blot. These cells were shown to have a constitutional NF1 microdeletion and elevated Ras-GTP activity, however, suggesting loss of normal neurofibromin function. Localized intraneural injection of the cell line sNF94.3 produced consistent and slow growing tumors that infiltrated and disrupted the host nerve. The xenograft tumors resembled plexiform neurofibromas with a low rate of proliferation, abundant extracellular matrix (hypocellularity), basal laminae, high vascularity, and mast cell infiltration. The histologic features of the developed tumors were particularly consistent with those of human plexiform neurofibroma as well. Intraneural xenograft of sNF94.3 cells enables the precise initiation of intraneural, plexiform-like tumors and provides a highly reproducible model for the study of plexiform neurofibroma tumorigenesis. This model facilitates testing of potential therapeutic interventions, including angiogenesis inhibitors, in a relevant cellular environment.     

Spontaneous impulse generation in normal and denervated dorsal root ganglia: sensitivity to alpha-adrenergic stimulation and hypoxia

Previous experiments indicate that after peripheral nerve lesion, two sites of spontaneous ectopic impulse generation rapidly develop: the peripheral neuroma and the region of the dorsal root ganglion (DRG). In 30 adult Sprague-Dawley rats, microfilament recordings were made from either the dorsal root of L5 or the proximal sciatic nerve. The locus of the ectopic impulse generator, spontaneous firing patterns, and response to both adrenergic and hypoxic stimulation were observed in 200 spontaneously active isolated fibers. Results indicated that after sciatic transection the neuroma and the DRG behaved as independent sources of ectopic impulse generation. Spontaneous activity originating in the neuroma was responsive to adrenergic and hypoxic stimulation in 57% and 86% of fibers tested, respectively. Spontaneous activity originating in the DRG after chronic sciatic nerve transection demonstrated a response to adrenergic stimulation in 61% of fibers tested, and all fibers showed an increase in activity during hypoxic periods. Furthermore, after acute sciatic neurotomy in otherwise normal animals, spontaneous activity originating in the DRG could be recorded in a few fibers. Likewise, 48% of those fibers showed some response to topical or systemic epinephrine administration, and hypoxia produced some degree of excitation of firing in all fibers tested. Neither epinephrine administration nor hypoxic challenge produced excitation of firing in DRG neurons with intact receptive fields in normal animals. The pharmacology of adrenergic sensitivity of spontaneously active fibers from both the neuroma and the region of the DRG indicated alpha-adrenergic specificity. Furthermore, a number of fibers exhibiting spontaneous activity from both the region of the neuroma and the DRG showed either adrenergic or hypoxic sensitivity, but not both. Thus, the mechanisms of the largely excitatory actions of alpha-agonists and hypoxia on spontaneous discharges from these sites were felt to be different. These data indicate that adrenergic and/or hypoxic responsiveness is a property of (i) otherwise normal DRG neurons which demonstrate intrinsic spontaneous firing properties, (ii) neurons in chronically denervated ganglia which exhibit spontaneous activity, and (iii) some fibers within neuromas. Normal DRG neurons with intact receptive fields do not appear to increase their firing rate in response to either hypoxia or adrenergic stimulation. These findings may be relevant to the development of chronic pain in man following peripheral nerve injury.