Endoneurial Space Research Articles

Research Progress of Neural Invasion in Pancreatic Cancer.

Pancreatic cancer is one of the highly malignant gastrointestinal tumors in humans, and patients suffer from cancer pain in the process of cancer. Most patients suffer from severe pain in the later stages of the disease. The latest studies have shown that the main cause of pain in patients with pancreatic cancer is neuroinflammation caused by tumor cells invading nerves and triggering neuropathic pain on this basis, which is believed to be the result of nerve invasion. Peripheral nerve invasion (PNI), defined as the presence of cancer cells along the nerve or in the epineurial, perineural, and endoneurial spaces of the nerve sheath, is a special way for cancer to spread to distant sites. However, due to limited clinical materials, the research on the mechanism of pancreatic cancer nerve invasion has not been carried out in depth. In addition, perineural invasion is considered to be one of the underlying causes of recurrence and metastasis after pancreatectomy and an independent predictor of prognosis. This article systematically reviewed the neural invasion of pancreatic cancer through bioinformatics analysis, clinical manifestations and literature reviews.

Bilateral facial palsy in cerebral venous sinus thrombosis – An uncommon association

Background: Cranial nerve palsy in cerebral venous sinus thrombosis (CVST) is not a very common presentation, its pathophysiology is not well understood. Case Description: We report a 27 year old young man with CVST having protein-S deficiency & hyperhomocystinemia, who during his initial days of hospitalization developed bilateral facial palsy. With conservative treatment of CVST, complete recovery of facial palsy occurred with recanalization of transverse sinus. Conclusions: Facial palsy in this patient was probably caused by transient neurapraxia in the intracranial segment of the nerve. It may be hypothesised that elevated venous transmural pressure in the nerve’s satellite vein, which belongs to the affected drainage territory of the transverse sinus, might have caused venous blood-brain barrier dysfunction in the intrinsic vascular system of the nerve, with leakage of fluids and ions into the endoneurial space and thus an increase in interstitial resistance causing conduction defects.

Evaluation of the Nose-to-Brain Transport of Different Physicochemical Forms of Uranium after Exposure via Inhalation of a Aerosol in the Rat.

Background:Health-risk issues are raised concerning inhalation of particulate pollutants that are thought to have potential hazardous effects on the central nervous system. The brain is presented as a direct target of particulate matter (PM) exposure because of the nose-to-brain pathway involvement. The main cause of contamination in nuclear occupational activities is related to exposure to aerosols containing radionuclides, particularly uranium dust. It has been previously demonstrated that instilled solubilized uranium in the rat nasal cavity is conveyed to the brain via the olfactory nerve.Objective:The aim of this study was to analyze the anatomical localization of uranium compounds in the olfactory system after in vivo exposure to a polydisperse aerosol of uranium tetraoxide () particles.Methods:The olfactory neuroepithelium (OE) and selected brain structures—olfactory bulbs (OB), frontal cortex (FC), hippocampus (HIP), cerebellum (Cer), and brainstem (BS)—were microdissected 4 h after aerosol inhalation via a nose-only system in adult rats. Tissues were subjected to complementary analytical techniques.Results:Uranium concentrations measured by inductively coupled plasma mass spectrometry (ICP-MS) were significantly higher in all brain structures from exposed animals compared with their respective controls. We observed that cerebral uranium concentrations followed an anteroposterior gradient with typical accumulation in the OB, characteristic of a direct olfactory transfer of inhaled compounds. Secondary ion mass spectrometry (SIMS) microscopy and transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy (TEM-EDX) were used in order to track elemental uranium in situ in the olfactory epithelium. Elemental uranium was detected in precise anatomical regions: olfactory neuron dendrites, paracellular junctions of neuroepithelial cells, and olfactory nerve tracts (around axons and endoneural spaces).Conclusion:These neuroanatomical observations in a rat model are consistent with the transport of elemental uranium in different physicochemical forms (solubilized, nanoparticles) along olfactory nerve bundles after inhalation of microparticles. This work contributes to knowledge of the mechanistic actions of particulate pollutants on the brain. https://doi.org/10.1289/EHP4927

Nerves and Pancreatic Cancer: New Insights into a Dangerous Relationship.

Perineural invasion (PNI) is defined as the presence of neoplastic cells along nerves and/or within the different layers of nervous fibers: epineural, perineural and endoneural spaces. In pancreatic cancer—particularly in pancreatic ductal adenocarcinoma (PDAC)—PNI has a prevalence between 70 and 100%, surpassing any other solid tumor. PNI has been detected in the early stages of pancreatic cancer and has been associated with pain, increased tumor recurrence and diminished overall survival. Such an early, invasive and recurrent phenomenon is probably crucial for tumor growth and metastasis. PNI is a still not a uniformly characterized event; usually it is described only dichotomously (“present” or “absent”). Recently, a more detailed scoring system for PNI has been proposed, though not specific for pancreatic cancer. Previous studies have implicated several molecules and pathways in PNI, among which are secreted neurotrophins, chemokines and inflammatory cells. However, the mechanisms underlying PNI are poorly understood and several aspects are actively being investigated. In this review, we will discuss the main molecules and signaling pathways implicated in PNI and their roles in the PDAC.

Immunohistochemestry for nerve morphometry?

Peripheral nerve biopsy is an invaluable aid to diagnosis carefully selected patients. The nerves histological evaluation gives clues for investigating diseases mechanisms and causes, as well as guides the therapeutic planning of inflammatory, infectious, demyelinating or degenerative lesions [1]. Nerves histological processing method that provides the best image quality is the epoxy resin embedding. Thus, nerve sections can be observed by light and/or transmission electron microscopy (TEM) and classes of nerve fibers can be morphometrically identified and studied. Epoxy resin embedding and TEM is considered the gold standard method for unmyelinated fibers identification and quantification. More recently, the immunohistochemistry technique (IHC) is being added to the inflammatory neuropathy investigation and the most important of the panel of antibodies employed are those needed to differentiate lymphocytes into T (CD3þve) and B cells (CD20þve). In addition, antibodies against macrophages, myelin basic protein, neurofilaments, and epithelial membrane antigen (EMA) (for perineurial cells) are included [1]. Neurotransmitters and their fiber “carrier” are much less explored. We suggest that it would be possible to use the IHC to identify nerve fiber classes as an alternative to TEM and morphometry, with the gain on functional information. To investigate this hypothesis we used IHQ and immunofluorescence (IF) to immunolabel small myelinated and unmyelinated fibers in sural nerves of rats. The nerves were surgically removed, immersed in paraformaldehyde 4% for 18 hours, and cryoprotected in increasing concentrations of sucrose solutions, before transversal cryosectioning (12μm). For IHC, sections were incubated in rabbit polyclonal antibody anti‐substance P (1:5.000, 18h) followed by anti‐rabbit IgG (1:500; 2h) and Extravidin‐HRP (1:1500, 2h). A reaction with diaminobenzidine intensified with nickel revealed immunoreactivity, observed by light microscopy. For IF, sections were incubated in rabbit polyclonal antibody anti‐substance P (1:1.000, 18h) followed by anti‐rabbit IgG (Alexa Fluor® 594) (1:200, 2h). The immunoreactivity was observed by fluorescence microscopy. Both protocols worked on transverse sections to identify substance P immunolabeled small myelinated and unmyelinated fibers. Results were compared with transverse sections prepared for TEM and the IHC results showed labeled small myelinated and unmyelinated axons intermingled with large unlabeled myelinated axons, with preservation of their myelin sheath. The IHC transverse sections were comparable to the epoxy resin embedded for small fiber localization in the endoneural space. Further studies on quantification of these fibers will be performed comparing both methods (IHC and IF) with the TEM images in order to show that IHC might also be a reliable method for quantifying axons.Support or Funding InformationFinancial support: FAPESP, CNPq, CAPES and FAEPAThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nf2 Mutation in Schwann Cells Delays Functional Neural Recovery Following Injury

Merlin is the protein product of the NF2 tumor suppressor gene. Germline NF2 mutation leads to neurofibromatosis type 2 (NF2), characterized by multiple intracranial and spinal schwannomas. Patients with NF2 also frequently develop peripheral neuropathies. While the role of merlin in SC neoplasia is well established, its role in SC homeostasis is less defined. Here we explore the role of merlin in SC responses to nerve injury and their ability to support axon regeneration. We performed sciatic nerve crush in wild-type (WT) and in P0SchΔ39–121 transgenic mice that express a dominant negative Nf2 isoform in SCs. Recovery of nerve function was assessed by measuring mean contact paw area on a pressure pad 7, 21, 60, and 90 days following nerve injury and by nerve conduction assays at 90 days following injury. After 90 days, the nerves were harvested and axon regeneration was quantified stereologically. Myelin ultrastructure was analyzed by electron microscopy. Functional studies showed delayed nerve regeneration in Nf2 mutant mice compared to the WT mice. Delayed neural recovery correlated with a reduced density of regenerated axons and increased endoneurial space in mutants compared to WT mice. Nevertheless, functional and nerve conduction measures ultimately recovered to similar levels in WT and Nf2 mutant mice, while there was a small (∼17%) reduction in the percent of regenerated axons in the Nf2 mutant mice. The data suggest that merlin function in SCs regulates neural ultrastructure and facilitates neural regeneration, in addition to its role in SC neoplasia.

Abstract 2355: Marrow-derived macrophages mediate invasion of pancreatic adenocarcinoma by RET activation

Abstract Introduction: Perineural invasion of cancer cells (CPNI) is found in most patients with pancreatic adenocarcinomas (PDAs). Although the presence of M2 macrophages in the perineural environment has been associated with shortened survival rates in PDA patients, the mechanism underlying this phenomenon has not been elucidated. It has been suggested that secretion of the RET ligand glial-derived neurotrophic factor (GDNF) by M2 macrophages causes cancerous RET-expressing cells to migrate along affected nerves. This concept, however, has not been demonstrated in vivo. This study was aimed at understanding the contribution of macrophages to perineural invasion for in vivo models of PDA, and to characterize the role of the GDNF-RET axis in this unique microenvironmental niche. Methods: Immunohistochemical analysis of PDA specimens of a novel Pdx-1-Cre/KrasG12D /p53R172H (KPC) mouse model, revealed that CD163+ (M2) endoneurial macrophages (EMΦs) predominate the endoneural space of invaded nerves. In a bone marrow transplant (BMT) from CX3CRGFP/+ mice, a 23- fold increase in the recruitment of GFP+ macrophages to the perineural space was witnessed as compared to GFP- cells, indicating recruitment of bone marrow- derived macrophages to the perineural niche, rather than resident tissue macrophages. A BMT from CCR2-/- mice lacking macrophage recruitment capacity resulted in a 6-fold decrease of the invasion severity score, and a 2.5-fold decrease in perineural invasion index. Analysis by fluorescence microscopy indicated that EMΦs expressed higher levels of GDNF compared to EMΦs in premalignant lesion and normal controls. In order to differentiate between the contributions of GDNF secreted by nerves to the invasion presses and its secretion from recruited M2 macrophages, we performed a BMT from GDNF+/- mice. The BMT led to a 1.5-fold improvement in both the invasion severity score and the perineural invasion index. Similarly, mice treated with PP1, a RET receptor inhibitor, caused a 1.5-fold decrease in the invasion severity score, and a 1.3-fold decrease in the perineural invasion index. Finally, xenografts of PDAC cells, in which RET was silenced using siRNA, resulted in a 3.5-fold decrease in the invasion severity score and a 3.2-fold decrease in the perineural invasion index. Discussion: Taken together, our results suggest a paracrine response between bone marrow-derived tumor-associated EMΦs and PDA cells. This loop probably orchestrates the formation of cancer nerve invasion. Further studies using novel transgenic animal models aimed at specifying the specific molecular signals that induce disease invasion and proliferation in the perineural microenvironment are needed. Citation Format: Moran Amit, Shorook Na'ara, Yoav Binenbaum, Ziv Gil. Marrow-derived macrophages mediate invasion of pancreatic adenocarcinoma by RET activation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2355. doi:10.1158/1538-7445.AM2015-2355

Axonal morphological changes following impulse activity in mouse peripheral nerve in vivo: the return pathway for sodium ions.

Conduction in myelinated axons involves substantial ion movements that must be reversed to restore homeostasis. The pathway taken by sodium ions returning to their original location and the potential osmotic consequences are currently unknown. We report striking morphological changes in axons following sustained impulse conduction that appear to result from osmosis and to indicate accumulation of ions in the periaxonal space followed by their release at the paranode. We conclude that the morphological changes illustrate a hitherto unrecognized part of normal axonal physiology that may also indicate the return pathway for the sodium ions involved in impulse formation. Myelinated axons can conduct sustained trains of impulses at high frequency, but this involves substantial ion movements that must be reversed to restore homeostasis. Little attention has been paid to the potential osmotic consequences of the ion movements or to the pathway taken by sodium ions returning to their original endoneurial location, given that the axolemmal Na(+)-K(+)-ATPase extrudes these ions into the periaxonal space beneath the myelin rather than into the endoneurium. Serial confocal imaging of fluorescent axons conducting at sustained physiological frequencies in vivo has revealed surprising morphological changes that may illuminate these problems. Saphenous nerves and spinal roots of anaesthetized transgenic mice expressing axoplasmic yellow fluorescent protein were stimulated electrically or pharmacologically (veratridine). Within 2 h, the axon herniated on one or both sides of the nodal membrane, displacing the paranodal myelin and widening the nodal gap. The herniated axoplasm became directed back towards the internode, forming a 'cap' up to 30 μm long. Concurrently, the fluid in the expanded periaxonal space accumulated into droplets that appeared to travel to the paranode, where they escaped. No such alterations occurred in axons treated with sodium channel or Na(+)-K(+)-ATPase inhibitors. Remarkably, impulse conduction continued throughout, and all these changes reversed spontaneously over hours or days. The morphological changes were verified ultrastructurally, and occurred in virtually all myelinated axons. The findings appear to reveal an overlooked part of the physiological repertoire of nerve fibres, and here they are interpreted in terms of osmotic changes that may illuminate the pathway by which sodium ions return to the endoneurial space after they have entered the axon during impulse conduction.

Neuropathies amyloïdes héréditaires : aspects cliniques et neuropathologiques

Transthyretin familial amyloid polyneuropathy (TTRFAP) is an autosomal dominant neuropathy that is fatal within about 10 years after symptom onset. TTRFAP is observed worldwide, albeit with a higher frequency of the most common variant, Val30met, in Portugal, Sweden and Japan. Various phenotypic differences are observed. TTRFAP should be considered in patients with a progressive axonal polyneuropathy of unknown origin, especially when associated with autonomic nervous system dysfunction. A positive family history is found in most cases when onset begins around 30 years of age, while late-onset FAP is often sporadic and may be confused with chronic inflammatory demyelinating polyneuropathy. Nerve biopsy is often used to confirm the presence of extracellular amyloid deposits in interstitial tissue of the endoneurial space, although amyloid can also befound in muscle, salivary gland and abdominal fat. It is important to stress that biopsy negativity does not rule out amyloidosis. Genetic testing for TTR gene mutations should be performed in case of progressive length-dependent axonal polyneuropathy predominantly involving small nerve fibers.

Diffuse large B-cell lymphoma presenting with neurolymphomatosis and intravascular lymphoma: a unique autopsy case with diverse neurological symptoms

A 78-year-old Japanese male noticed a difficulty in the beginning of standing up, followed by 7a progressive numbness of extremities with pain, Bell’s palsy, dysarthria, and difficulty in swallowing. A clinician had suspected cancer of unknown primary origin, accompanied by the diverse and elusive neurological symptoms, likely presenting as painful mononeuropathy simplex and cranial neuropathy. He developed dysbasia over weeks and died 1 month after the symptom onset. At autopsy, an ill-defined large and soft tumor mass in the right lobe of the liver with direct invasion into the right adrenal gland was observed. The left adrenal gland or right iliopsoas muscle was also involved. Microscopic findings showed a monotonous proliferation of medium-sized to large atypical lymphoid cells, which were diffusely positive for CD20 in immunohistochemistry, consistent with diffuse large B-cell lymphoma (DLBL). Furthermore, the lymphoma cells aggressively infiltrated endoneurial and subperineurial spaces not only in the peripheral nerves and plexuses, but partly in the spinal nerve roots, and intravascular spaces in various tissues. Therefore, systemic lymphoma (DLBL) complicated with neurolymphomatosis (NL) and intravascular lymphoma (IVL) was diagnosed. Very early diagnosis and treatment are necessary for the NL patients with poor prognosis.Virtual slidesThe virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5862472377020448.

Sampling method for morphometry of the myelinated fibers of sural nerves in adult rats

Morphometric analysis of peripheral nerves is time consuming, tedious, difficult to perform correctly and subject to many sources of errors. Researchers try to develop sampling methods to reduce the number of structures evaluated, particularly the myelinated fibers. Nevertheless, there is no consensus on which is the best sampling method or even if there is a method that reproduces with reliability the complete evaluation of the endoneural space of a determined nerve, investigated without any sampling method. We aimed to investigate a sampling method that is able to reproduce the data obtained by the integral morphometric analysis of the sural nerves in adult rats. Sural nerves were prepared for light microscopy and computer morphometry. Microscopic scanning of the endoneural space was completed by the automatically shifting of digitalized frames under the 100× lenses on the way that the sequenced digitalized frames, when put together, reproduce the complete image of the fascicle. Morphometric analysis was performed on 30%, 50% and 70%, and compared to 100% of the frames digitalized from the fascicle. The One Way ANOVA test was performed and no significant differences were found in any of the investigated parameters. The sampling methods were efficient in representing the evaluation of 100% of sural nerve fibers, being 30% enough on the morphometric evaluation of sural nerve in adult rats.Grant Funding Source : FAPESP, CNPq, CAPES and FAEPA

Abstract 1543: Potential role of Schwann cells and laminin adhesion in prostate cancer perineural invasion

Abstract Prostate tumor cell migration and invasion of prostatic nerves, a process known as perineural invasion (PNI), is a major route for extracapsular extension during prostate cancer metastasis. The clinical significance of perineural invasion as a predictor for poor patient outcome has been extensively documented. The laminin family of extracellular matrix glycoproteins lines the neural route and plays a prominent role in the development and maintenance of the peripheral nervous system. Evidence has shown that the laminin receptor integrin α6β1 promotes prostate tumor cell migration and invasion in aggressive disease. We hypothesize that α6β1 regulates prostate cancer perineural invasion by regulating laminin dependent adhesion and migration on nerves. In this study we demonstrate remarkable integrin α6 expression in prostate tumors undergoing active perineural invasion using immunohistochemical analysis of human prostate tumors. The expression of integrin α6 correlates with expression of the laminin subunits α5 and γ1 which comprise laminin-511, the major ligand for integrin α6β1. The laminin extracellular matrix on the peripheral nerves of the prostate is derived from Schwann cells during the myelination process. We demonstrate here that Schwann cells are highly expressed in prostatic nerves using immunohistochemical analysis of the Schwann cell specific marker S-100, suggesting that tumor cells invade myelinated nerves. We also demonstrate two specific patterns of prostate tumor nerve invasion along prostatic nerves. Invasion along the external surface of the nerve sheath or in the perineural space maintains nerve structure while invasion of the endoneural space disrupts nerve structure and displaces Schwann cells. This suggests that tumor interactions with nerves and Schwann cells may illicit nerve damage during the PNI process. Using a DU-145 prostate tumor and neuron/glial cell in vitro co-culture model we demonstrate that DU-145 cells (cytokeratin positive) migrate towards glial cells (GFAP positive) and neurons (Beta-III tubulin positive) isolated from dorsal root ganglia of male SCID mice. Mature cultures of the prostate tumor cells and neuron/glial cells demonstrate distinct islands of growth. The patterns of colony formation were non-random and interspersed colonies of tumor and nerve cells were not observed. Taken together, our immunohistochemical results suggest that perineural invasion of laminin coated nerves is regulated by the laminin adhesion receptor α6β1. Our in vitro model system demonstrates that tumor cells migrate to neuron/glial cells and dominate the co-culture, correlating with our immunohistochemical data. We expect that blocking α6β1 function in this system will impede prostate tumor cell migration and invasion in the nerve environment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1543. doi:10.1158/1538-7445.AM2011-1543

The effects of low-dose radiation in the treatment of sciatic nerve injury in rats

Peripheral nerve regeneration is often blocked by scar formation and misdirection of axon sprouts. The aim of this study is to evaluate electrophysiological and histopathological effects of low-dose radiation therapy on the prevention of intraneural scar formation in peripheral nerve injury. In this experimental study, twenty rats were randomly divided into two groups. Left sciatic nerves were exposed and clipped by temporary aneurysm clip for 5 minutes in both groups. In all animals, electrophysiological recordings were performed between 22-24 hours after sciatic nerve injury. The control group was not given any treatment. In the experimental group, 700 cGy low-dose radiation was administered on the left sciatic nerves 24 hours after clipping. Six weeks after injury, electrophysiological recordings were performed in both groups and animals were sacrificed to evaluate the injured nerves histopathologically. We observed that low-dose radiotherapy increased the amplitude and improved latency measurements in electrophysiological examinations. Histopathologically, more axonal degeneration and vacuolization was observed in the control group comparing with the experimental group. Endoneural space increased slightly more in the control group than the experimental group. It was observed that low-dose radiotherapy may prevent intraneural scar formation and may improve electrophysiological recovery in sciatic nerve injury performed in rats.

Two novel mutations of GJB1 gene associated with typical X-linked Charcot-Marie-Tooth disease

To analyze the relationship between phenotype and genotype and the role of immune cells in the pathogenesis of X-linked Charcot-Marie-Tooth disease (CMT1X). The probands of the two families with X-linked dominant inherited peripheral neuropathy were evaluated clinically, electrophysiologically, pathologically and genetically. The available family members were genetic analyzed and the novel mutations were compared with other known ones. (1) In both families, affected members presented progressive weakness and wasting of distal extremities and it seems that males suffered more severely than affected females with onset in the first decade of their life. Proband of family 1 showed moderately elevated CSF protein and marked increase of IgG-syn in CSF.(2) Nerve conduction velocity (NCV) of the peripheral nerves was intermediately slow in both motor and sensory nerves exhibiting the features of demyelination. Brain-stem auditory evoked potentials (BAEPs) was abnormal in the proband of family 1: delayed I-III interpeak intervals were recorded but with normal III-V interpeak intervals. (3) Sural nerve biopsy in the probands of the two families showed a prominent distinguished loss of myelinated fibers and a few clusters of regenerating axons without conspicuous onion-bulb formations. Thinly myelinated fibers was prominent in family 2 but not in family 1. Immunohistochemical staining showed that there were positive CD68 cells in the endoneurial space and lamellar sheath. (4) By genetic testing, we identified two novel missense mutations of GJB1 gene, which resulted in Ile127Phe amino acid substitution in family 1(located in the intracellular loop of connexin 32) and Asp178Gly amino acid substitution in family 2 (located in the 2(nd) extracellular loop of CX32), respectively. Both mutations were highly conserved in low species and were predicted to be possibly damaging through Polyphen prediction tool. The two novel GJB1 gene mutations cause a spectrum of clinical manifestations of CMT1X in both families. However, the mutations site of CX32 alone cannot predict these phenotypic variations in CMT1X fully. The immune system may be involved in the pathogenesis of the disease.

Diffuse intraneural leiomyoma in a case of sensorimotor neuropathy

Leiomyomas are benign smooth muscle tumors, usually characterized by well-deWned Wbrous pseudocapsule and intersecting fascicles of spindle cells [3]. Few cases of leiomyomas in the peripheral nervous system (PNS) causing external nerve compression have been described [1, 7]. We report the case of a 71-year-old man who was referred for a 2 years history of burning pain and paresthesias associated with slowly progressive weakness of the lower limbs. He underwent total colectomy for diVuse polyposis 2 years before. Neurological examination and nerve conduction studies revealed symmetric sensorimotor axonal neuropathy of the lower limbs. Since all immunological, hematological and cerebrospinal Xuid examinations were negative, a sural nerve biopsy was performed. On semithin plastic sections, a mild degree of endoneurial Wbrosis, reduction of myelinated Wbers and signs of acute axonal degeneration were evident. Some fascicles had greater myelinated Wber loss than others, primarily in the center of the fascicles. Interestingly, paraYn-embedded, hematoxylin and eosinstained sections showed in epineurial and endoneurial perivascular spaces atypical large cells with abundant eosinophilic cytoplasm. Upon electron microscopy, these cells exhibited a basement membrane and prominent pinocytotic vesicles at the periphery (Fig. 1). The cytoplasm was Wlled with actin and myosin Wlaments, aggregates of glycogen particles and few organelles. Some cells showed oval and centrally located nuclei. Immunohistochemically, they were positive for smooth muscle actin, desmin and calponin (Fig. 2), but negative for CD31, cytokeratin and S-100 protein, demonstrating that they were smooth muscle cells (SMC). These cells did not show proliferative activity (negativity for Ki-67). Neither necrosis nor a deWnite capsule were present. The morphological and immunohistochemical data suggested a diagnosis of leiomyoma. This is the Wrst description of a diVuse SMC inWltrate of the PNS, both in the endoneurium and epineurium, whereas well circumscribed and encapsulated leiomyomas have occasionally been reported in the PNS. Other potential tumors such as leiomyosarcoma, schwannoma and hemangioma have to be considered in the diVerential diagnosis, but can be excluded based on immunoproWle [2]. Several hypotheses can be envisaged for the possible cellular origin of this unique tumor in the PNS, including pluripotential mesenchymal cells, perivascular connective tissue elements or SMC of blood vessels [6]. Epineurial proliferation of SMC in sural nerve biopsies indicating inXammatory or non-inXammatory angiopathy has been described before [5]. However, our patient’s sural nerve biopsy revealed SMC also in the endoneurium without signs of angiopathy, suggesting a diVerent origin of SMC. How this inWltrate caused damage of peripheral nerve Wbers remains unclear. Our patient’s clinical presentation suggests a diVuse damage of the PNS, while nerve biopsy Wndings associated with F. Cerri (&) Department of Neurology and INSPE, San RaVaele ScientiWc Institute, Via Olgettina, 6

Distribution and tumor necrosis factor-alpha isoform binding specificity of locally administered etanercept into injured and uninjured rat sciatic nerve

Tumor necrosis factor-alpha (TNF) is a pro-inflammatory cytokine that is implicated in the initiation of neuropathic pain. Locally administered TNF antagonist etanercept offers a promising new treatment approach to target neuropathic pain. Here we evaluate the distribution and binding specificity for TNF isoforms of locally administered etanercept into injured and uninjured rat sciatic nerve. Distribution and co-localization of etanercept and TNF in the injured and uninjured nerve was evaluated at 1, 24, 48 and 96 h after etanercept local application using immunohistochemistry. In addition, binding specificity of etanercept for TNF isoforms was analyzed using immunoblot assay system in nerve lysates. A new observation was that locally administered etanercept reached the endoneurium of the injured but not the uninjured nerve 1 h after its application and mainly co-localized with TNF-positive structures, morphologically similar to Schwann cells and macrophages. We further noticed that immunoblot analyses for etanercept demonstrated its preferential binding to transmembrane and trimer TNF isoforms. Finally, locally administered etanercept inhibited pain-related behaviors in a rat sciatic nerve crush model. We conclude that locally administered etanercept reaches the endoneurial space in the injured nerve and preferentially binds to transmembrane and bioactive trimer TNF isoforms to modulate neuropathic pain. Locally administered etanercept has potential as a targeted immunomodulating agent to treat local pathogenesis in neuropathic pain after peripheral nerve injury.

Immunoglobulin M Deposition in Cutaneous Nerves of Anti-Myelin-Associated Glycoprotein Polyneuropathy Patients Correlates With Axonal Degeneration

Anti-myelin-associated glycoprotein (MAG) neuropathy is an antibody-mediated polyneuropathy. We correlated clinical features, immunoglobulin (Ig) M blood levels, IgM deposition and axonal degeneration in skin biopsies of anti-MAG neuropathy patients. By confocal microscopy, IgM deposits were found exclusively within perineurium-enclosed nerves; they were not found on single, non-perineurium-ensheathed myelinated axons. There was a linear correlation between IgM accumulation in nerve fascicles with IgM blood levels but not with anti-MAG antibody titer or disease duration. Axons with specific IgM deposits had signs of axonal damage, including neurofilament disintegration. Nodal structures were intact even at sites where the axons showed pathologic changes. Ultrastructural analysis revealed degeneration of myelinating Schwann cells. Taken together, these findings suggest that in anti-MAG neuropathy patients, IgM deposits are entrapped within cutaneous perineurium-ensheathed nerve bundles where they accumulate in the endoneurial space. High local IgM levels in the endoneurium may be required for IgM deposition on myelin and subsequent axonal injury and degeneration. This study underlines the importance of early, effective anti-B-cell treatments for preventing progression of this neuropathy.

The effect of ischemia on the permeability of the perineurium to protein tracers in rabbit tibial nerve.

The influence of ischemia on the permeability of the perineurium to protein tracers was studied in rabbit tibial nerve. Intravital ischemia was produced by a pneumatic cuff around the thigh and the effects of long-lasting permanent ischemia were investigated in postmortem experiments. The permeability was tested fluorescence microscopically by injections of albumin labelled with Evan's blue (EBA) around the nerve. The diffusion of EBA into the endoneurium of control tibial nerves was prevented by the perineurium. This “barrier” function of the perineurium resisted ischemia of even 24 hours’duration but after 48 hours of ischemia the tracer penetrated the perineurium and spread widely in endoneurial spaces. This diffusion barrier is therefore markedly resistant to ischemia and postmortem changes.

Histopathological analysis of gangliosides use in peripheral nerve regeneration after axonotmesis in rats

To analyze the action of gangliosides in peripheral nerve regeneration in the sciatic nerve of the rat. The sample was composed of 96 male Wistar rats. The animals were anaesthetized and, after identification of the anaesthesic plane, an incision was made in the posterior region of the thigh, followed by skin and muscle divulsion. The right sciatic nerve was isolated and compressed for 2 minutes. Continuous suture of the skin was performed. The animals were randomly divided into two groups: the experimental group (EG), which received subcutaneous injection of gangliosides, and the control group (CG), which received saline solution (0.9%) to mimic the effects of drug administration. No differences were observed between the experimental and control groups evaluated on the eighth day of observation. At 15 and 30 days the EG showed an decrease in Schwann cell activity and an apparent improvement in fibre organization; at 60 days, there was a slight presence of Schwann cells in the endoneural space and the fibres were organized, indicating nerve regeneration. At 15 and 30 days, the level of cell reaction in the CG had diminished, but there were many cells with cytoplasm in activity and in mitosis; at 60 days, hyperplastic Schwann cells and mitotic activity were again observed, as well as nerve regeneration, but to a lesser extent than in the EG. The administration of exogenous gangliosides seems to improve nerve regeneration.

Intranervous immunoglobulin deposits: An underestimated mechanism of neuropathy

There are several pathogenic mechanisms of peripheral nerve involvement in patients with monoclonal dysglobulinemia. Intranervous proliferation of malignant cells, immunoglobulin, or amyloid deposits in the endoneurial space can only be determined by examination of nerve biopsy specimens. We present clinical, electrophysiological, and histological data from seven patients whose polyneuropathy was induced by immunoglobulin deposits in the endoneurial space. As these lesions cannot be demonstrated on clinical and electrophysiological grounds, the indication for nerve biopsy derives from careful analysis of each patient presenting with a polyneuropathy and a monoclonal dysglobulinemia. To visualize and clearly characterize these deposits, electron microscopic examination is indispensable. Immunocytochemical methods using both light and electron microscopy for ultrastructural analysis are of great value. Demonstration of endoneurial immunoglobulin deposits may have major therapeutic consequences. Indeed, identification of these deposits prompted the use of aggressive treatment, which was quite effective in five of our seven patients.