RESUMO
In mammalian peripheral nerves, unmyelinated C-fibers usually outnumber myelinated A-fibers. By using transmission electron microscopy, we recently showed that the saphenous nerve of the naked mole-rat (Heterocephalus glaber) has a C-fiber deficit manifested as a substantially lower C:A-fiber ratio compared with other mammals. Here we determined the uniqueness of this C-fiber deficit by performing a quantitative anatomical analysis of several peripheral nerves in five further members of the Bathyergidae mole-rat family: silvery (Heliophobius argenteocinereus), giant (Fukomys mechowii), Damaraland (Fukomys damarensis), Mashona (Fukomys darlingi), and Natal (Cryptomys hottentotus natalensis) mole-rats. In the largely cutaneous saphenous and sural nerves, the naked mole-rat had the lowest C:A-fiber ratio (â¼1.5:1 compared with â¼3:1), whereas, in nerves innervating both skin and muscle (common peroneal and tibial) or just muscle (lateral/medial gastrocnemius), this pattern was mostly absent. We asked whether lack of hair follicles alone accounts for the C-fiber paucity by using as a model a mouse that loses virtually all its hair as a consequence of conditional deletion of the ß-catenin gene in the skin. These ß-catenin loss-of function mice (ß-cat LOF mice) displayed only a mild decrease in C:A-fiber ratio compared with wild-type mice (4.42 compared with 3.81). We suggest that the selective cutaneous C-fiber deficit in the cutaneous nerves of naked mole-rats is unlikely to be due primarily to lack of skin hair follicles. Possible mechanisms contributing to this unique peripheral nerve anatomy are discussed.
Assuntos
Doenças Desmielinizantes , Ratos-Toupeira/anatomia & histologia , Fibras Nervosas Amielínicas/fisiologia , Nervo Fibular/citologia , Pele/inervação , Nervo Sural/citologia , África , Animais , Doenças Desmielinizantes/genética , Feminino , Folículo Piloso/citologia , Folículo Piloso/inervação , Folículo Piloso/fisiologia , Masculino , Ratos-Toupeira/classificação , Fibras Nervosas Amielínicas/classificação , Nervo Fibular/química , Nervo Fibular/fisiologia , Pele/citologia , Especificidade da Espécie , Nervo Sural/química , Nervo Sural/fisiologiaRESUMO
The extracellular matrix of peripheral nerve plays a vital role in terms of normal nerve fibre function and also in the regenerative response following nerve injury. Nerve fibre loss is a major feature of diabetic neuropathy; however, the regenerative response is limited and this may be associated with changes in the composition of the extracellular matrix. Glycoproteins and collagens are major components of the extracellular matrix and are known to be important in terms of axonal growth. This work has therefore examined whether changes in the expression of two major glycoproteins, laminin and tenascin, and three collagen types (IV, V and VI) occur in the endoneurial and perineurial connective tissue compartments of human diabetic nerve. Despite being known to have a positive effect in terms of axonal growth, laminin levels were not elevated in the diabetic nerves. However, the pattern of tenascin expression did differ between the two groups being found in association with axon myelin units in the diabetic samples only. The pattern of collagen IV expression was the same in both tissue groups and was not found to be up-regulated. However, levels of collagen V and VI were both significantly increased in the endoneurium and for collagen VI also in the perineurium.
Assuntos
Axônios/patologia , Neuropatias Diabéticas/patologia , Matriz Extracelular/patologia , Regeneração Nervosa , Axônios/química , Axônios/metabolismo , Biomarcadores/análise , Estudos de Casos e Controles , Colágeno Tipo IV/análise , Colágeno Tipo V/análise , Colágeno Tipo VI/análise , Matriz Extracelular/química , Humanos , Imuno-Histoquímica , Laminina/análise , Bainha de Mielina/metabolismo , Coloração e Rotulagem , Estatísticas não Paramétricas , Nervo Sural/química , Nervo Sural/patologia , Tenascina/análiseRESUMO
For proteomic analysis, sample preparation plays a crucial role in two-dimensional gel electrophoresis (2DE), since, very often, each tissue or cell culture requires specific treatments. In the present paper, we report a sample preparation procedure suitable for 2DE that was done on peripheral nerve using bovine sciatic nerves and human sural nerve biopsies. We obtained an appreciable reduction of tissue heterogeneity using protein extracts obtained from nerve-fiber bundles instead of the entire nerve. In addition, we optimized 2DE protein separation using a combination of CHAPS, Triton X-100, and SB3-10 detergents in an isoelectric-focusing (IEF) buffer. The reported experimental procedures appear to be essential for 2DE separation of peripheral nerve proteins for the establishment of a reference map.
Assuntos
Proteínas do Tecido Nervoso/análise , Nervos Periféricos/química , Animais , Bovinos , Detergentes , Eletroforese em Gel Bidimensional , Géis , Humanos , Fibras Nervosas/química , Mapeamento de Peptídeos , Manejo de Espécimes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Nervo Sural/químicaRESUMO
Mutations in the gene encoding for myelin protein zero (MPZ) cause inherited demyelinating peripheral neuropathies of different severity. The molecular and cellular mechanisms by which the MPZ mutations cause neuropathy are incompletely understood. We investigated MPZ, myelin basic protein, and peripheral myelin protein 22 (PMP22) protein expression levels in a nerve biopsy of a Charcot-Marie-Tooth type 1B patient heterozygous for the Val 102 frame-shift mutation. We demonstrate by quantitative immunohistochemical as well as by Western blot analyses that MPZ expression levels were not reduced in myelin membranes, a finding that is in accordance with the mild phenotype of this patient. Our data show that heterozygous 'loss-of-function' of MPZ may not necessarily lead to reduced protein levels. In conclusion, we demonstrate that careful analysis of protein expression levels in peripheral nerve tissues provides important information with respect to the understanding of the molecular basis of these neuropathies.
Assuntos
Doenças Desmielinizantes/genética , Doenças Desmielinizantes/metabolismo , Proteína P0 da Mielina/biossíntese , Proteína P0 da Mielina/genética , Nervo Sural/metabolismo , Adulto , Biópsia , Western Blotting , Doenças Desmielinizantes/patologia , Feminino , Mutação da Fase de Leitura , Humanos , Imuno-Histoquímica , Proteínas da Mielina/biossíntese , Linhagem , Fenótipo , Nervo Sural/química , Nervo Sural/patologiaRESUMO
Binding of ligands to the receptor for advanced glycation end products (RAGE) results in activation of the transcription factor nuclear factor kappa B (NF-(kappa)B) and subsequent expression of NF-(kappa)B-regulated cytokines. This has been shown to be a relevant pathomechanism in diabetic polyneuropathies (PNP). To determine whether this pathway may contribute to the pathogenesis of PNP due to impaired glucose tolerance (IGT) we performed a pilot study to demonstrate the presence of the RAGE ligand N (epsilon)-(Carboxymethyl)lysine (CML), the receptor itself and N-(kappa)B in sural nerve biopsies of 4 patients with IGT-related PNP. Biopsies of either 4 patients with diabetic PNP and with Charcot-Marie-Tooth disease (CMT) I and II served as positive and negative controls, respectively. In IGT-related PNP and diabetic PNP, CML, RAGE, and NF-(kappa)B was found in the perineurium, epineurial vessels and in part in endoneurial vessels. CMT patients showed, if any, only weak staining for one or the other antigen. These data suggest that activation of the RAGE pathway may be one of the first steps in the pathogenesis of PNP even before chronic hyperglycemia occurs.
Assuntos
Neuropatias Diabéticas/etiologia , Intolerância à Glucose/complicações , Produtos Finais de Glicação Avançada/fisiologia , NF-kappa B/fisiologia , Receptores Imunológicos/fisiologia , Idoso , Biópsia , Doença de Charcot-Marie-Tooth/patologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/patologia , Neuropatias Diabéticas/patologia , Humanos , Lisina/análogos & derivados , Lisina/análise , Pessoa de Meia-Idade , NF-kappa B/análise , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/análise , Nervo Sural/química , Nervo Sural/patologiaRESUMO
Autosomal recessive demyelinating Charcot-Marie-Tooth disease (CMT4), Dejerine-Sottas disease and congenital hypomyelinating neuropathy are variants of hereditary demyelinating neuropathy of infancy, a genetically heterogeneous group of disorders. To explore the spectrum of early-onset demyelinating neuropathies further, we studied the clinicopathological and genetic aspects of 20 patients born to unaffected parents. In 19 families out of 20, consanguinity between the parents or presence of an affected sib suggested autosomal recessive transmission. Screening of various genes known to be involved in CMT4 revealed six mutations of which five are novel. Four of these novel mutations occurred in the homozygous state and include: one in GDAP1, one in MTMR2, one in PRX and one in KIAA1985. One patient was heterozygous for a novel MTMR2 mutation and still another was homozygous for the founder mutation, R148X, in NDRG1. All patients tested negative for mutations in EGR2. Histopathological examination of nerve biopsy specimens showed a severe, chronic demyelinating neuropathy, with onion bulb formation, extensive demyelination of isolated fibres and axon loss. We did not discern a specific pattern of histopathology that could be correlated to mutations in a particular gene.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Adolescente , Adulto , Idade de Início , Sequência de Bases , Biópsia , Doença de Charcot-Marie-Tooth/patologia , Doença de Charcot-Marie-Tooth/fisiopatologia , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Genes Recessivos , Humanos , Masculino , Proteínas de Membrana/análise , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação , Linhagem , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases não Receptoras , Nervo Sural/química , Nervo Sural/ultraestruturaRESUMO
BACKGROUND: Recently, immunoreactivity of transcription factor nuclear factor kappaB (NF-kappaB) was found in peripheral nerves from patients with Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), and familial amyloidotic polyneuropathy (FAP), suggesting a role in their pathogenesis. OBJECTIVE: To investigate expression and activation of NF-kappaB in nerve biopsy specimens from patients with peripheral neuropathies of different origins. PATIENTS: Nerve biopsies from 17 patients (5 with CIDP, 3 with vasculitis, 4 with Charcot-Marie-Tooth disease, and 5 with FAP) and 3 normal sural nerves were studied by immunocytochemistry and Western blot of nuclear extracts for the activated form of NF-kappaB. Nuclear factor kappaB DNA-binding activity was studied by electrophoretic mobility shift assay. RESULTS: Immunobinding for the activated form p65 of NF-kappaB was found in 2% to 5% of endoneurial vessel walls, in the external myelin of 5% to 10% of fibers, and in a few axons in CIDP specimens. It was also found in 5% to 15% of epineurial and endoneurial vessels in vasculitis specimens and at the level of amyloid deposits in FAP nerves. Nuclear factor kappaB immunoreactivity was not correlated to type of inflammatory cells, but it often corresponded to the deposition of the terminal complement complex C5b9. Western blot analysis of nuclear extracts showed a single band corresponding to 65 kDa in all affected nerves. Nuclear factor kappaB DNA-binding activity was revealed by electrophoretic mobility shift assay in specimens from patients with CIDP, vasculitis, and FAP. CONCLUSION: Our novel findings suggest a crucial role of NF-kappaB in inflammatory neuropathies and FAP.
Assuntos
Neuropatias Amiloides Familiares/metabolismo , NF-kappa B/metabolismo , Doenças do Sistema Nervoso Periférico/metabolismo , Adulto , Idoso , Doença Crônica , Síndrome de Guillain-Barré/metabolismo , Humanos , Immunoblotting , Imuno-Histoquímica , Inflamação/metabolismo , Macrófagos/química , Macrófagos/imunologia , Macrófagos/metabolismo , Pessoa de Meia-Idade , NF-kappa B/imunologia , Nervo Sural/química , Nervo Sural/imunologia , Nervo Sural/metabolismoRESUMO
BACKGROUND: The authors previously reported that immunoglobulin G (IgG) antibody to the ganglioside N-acetylgalactosaminyl GD1a (GalNAc-GD1a) is associated with the pure motor variant of Guillain-Barré syndrome (GBS). Elucidation of the localization of GalNAc-GD1a in human peripheral nerve tissue may lead to understanding of the pathogenetic role of anti-GalNAc-GD1a antibody in GBS. METHODS: IgG anti-GalNAc-GD1a-monospecific antibody was purified from anti-GalNAc-GD1a antibody-positive rabbit sera through an affinity column. Anti-neurofilament-200 monoclonal and anti-HNK-1 monoclonal antibodies were used as the markers for axon and myelin. Immunohistochemical study using double fluorescence labeling technique was conducted in human ventral roots (VR), dorsal roots (DR), intramuscular nerves, and sural nerves. Human teased ventral fibers also were studied. RESULTS: Anti-GalNAc-GD1a antibody immunostained an inner part of compact myelin and additionally a periaxonal-axolemma-related portion in the VR, small-diameter DR fibers, and IM nerves. In sural nerves, small fibers were selectively stained. In VR, the staining was localized in the paranodal region. CONCLUSION: Anti-GalNAc-GD1a antibodies in patients' sera may bind to those regions in the VR and IM nerves where GalNAc-GD1a is localized, and may function in the pathogenesis of pure motor type GBS. Further investigation is needed to explain the discrepancy between the immunolocalization of GalNAc-GD1a in sensory nerves and the absence of sensory disturbance in patients with GBS with IgG anti-GalNAc-GD1a antibodies.
Assuntos
Autoanticorpos/imunologia , Autoantígenos/imunologia , Epitopos/imunologia , Gangliosídeos/imunologia , Nervos Periféricos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Reações Antígeno-Anticorpo , Autoantígenos/análise , Doenças Autoimunes do Sistema Nervoso/imunologia , Epitopos/análise , Feminino , Gangliosídeos/análise , Síndrome de Guillain-Barré/imunologia , Humanos , Imunização , Imunoglobulina G/imunologia , Microscopia de Fluorescência , Músculo Esquelético/inervação , Fibras Nervosas/química , Fibras Nervosas/imunologia , Nervos Periféricos/química , Coelhos , Raízes Nervosas Espinhais/química , Raízes Nervosas Espinhais/imunologia , Nervo Sural/química , Nervo Sural/imunologiaRESUMO
Tie-2, a tyrosine kinase receptor, is essential for vascular integrity by regulating cellular adhesion between pericytes and endothelial cells. The aim of this study was to identify sites of expression of Tie-2 other than the vasculature. Tie-2 expression was first detected in human colon by Western blotting and reverse-transcription-polymerase chain reaction (RT-PCR) in tissue extracts. The presence of the Tie-2 mRNA and protein was detected by immunohistochemistry and in situ hybridization in cells of the colon myenteric and submucosal plexus, in both neuronal and Schwann cells. Tie-2 protein was also found in the nervous system of the female urogenital tract. In the human sciatic nerve and schwannoma, RT-PCR, Western blotting and immunohistochemistry analysis further confirmed the presence of Tie-2 mRNA and protein in non-autonomic peripheral nervous tissue. In conclusion, using several approaches and tissues we have demonstrated the presence of Tie-2 in human peripheral and autonomic nervous tissue, suggesting a role for Tie-2 in neural tissue. Thus, attempts to disrupt the tumour vessels by manipulation of the Tie-2 system in tumours may result in side-effects in peripheral nerves.
Assuntos
Plexo Mientérico/fisiologia , Receptores Proteína Tirosina Quinases/genética , Plexo Submucoso/fisiologia , Nervo Sural/fisiologia , Colo/química , Colo/citologia , Colo/inervação , Humanos , Imuno-Histoquímica , Hibridização In Situ , Plexo Mientérico/química , Plexo Mientérico/citologia , Neurônios/química , Neurônios/fisiologia , RNA Mensageiro/análise , Receptores Proteína Tirosina Quinases/análise , Receptor TIE-2 , Células de Schwann/citologia , Células de Schwann/fisiologia , Plexo Submucoso/química , Plexo Submucoso/citologia , Nervo Sural/química , Nervo Sural/citologia , Sistema Urogenital/química , Sistema Urogenital/citologia , Sistema Urogenital/inervaçãoRESUMO
BACKGROUND: Thirty-nine percent of permanent altitude dwellers in the Andes experience acral paresthesias. METHODS: Clinical examinations, sural nerve biopsies, and electrodiagnostic studies on peripheral nerves were performed on 15 men. Ten Cerro de Pasco (CP) natives living at 4,338 meters were biopsied. Three of these subjects had no burning feet/burning hands (BF/BH); three had BF/BH; and four had chronic mountain sickness (CMS), a maladaptation syndrome resulting from living in the Andes, all with BF/BH. Three patients with CMS were biopsied in Lima within hours after leaving CP. Two normal Lima natives were biopsied in Lima. Symptom scores for BF/BH and CMS score ratings were used. The nerves were assayed for Na+, K+ adenosine triphosphatase (ATPase), cytochrome oxidase (CO), substance P (SP), and endothelin (ET). RESULTS: Low ATPase was inversely related to symptom scores and CMS scores (p < 0.001). Patients with CMS biopsied in normoxia (Lima) had ATPase levels similar to those of controls. Nerve motor conduction velocities and sensory action potentials were normal. CO was inversely related to age (p < 0.03) and no relation of SP to any variable was found. ET levels were lower in sea level natives (p = 0.04). CONCLUSIONS: Acral paresthesias are associated with low ATPase in peripheral nerves. Lower ET levels of sea level natives likely reflect lowered release from vasa nervorum.
Assuntos
Altitude , Parestesia/fisiopatologia , Adulto , Doença da Altitude/enzimologia , Doença da Altitude/metabolismo , Biópsia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Endotelinas/metabolismo , Humanos , Masculino , Condução Nervosa/fisiologia , Parestesia/enzimologia , Parestesia/metabolismo , Peru , ATPase Trocadora de Sódio-Potássio/metabolismo , Substância P/metabolismo , Nervo Sural/química , Nervo Sural/metabolismo , Nervo Sural/fisiopatologiaRESUMO
BACKGROUND: Mutations in P0, the major protein of the myelin sheath in peripheral nerves, cause the inherited peripheral neuropathies Charcot-Marie-Tooth disease type 1B (CMT1B), Dejerine-Sottas syndrome (DSS) and congenital hypomyelination (CH). We reported earlier a de novo insertional mutation c.662_663GC (Ala221fs) in a DSS patient. The c.662_663GC insertion results in a frame shift mutation Ala221fs altering the C-terminal amino acid sequence. The adhesion-relevant intracellular RSTK domain is replaced by a sequence similar to Na+/K+ ATPase. To further clarify the molecular disease mechanisms in this sporadic patient we constructed wild type P0 and the c.662_663GC mutant expression cassettes by site-specific mutagenesis and transfected the constructs into insect cells (S2, High5). To trace the effects in live cells, green fluorescent protein (GFP) has been added to the carboxyterminus of the wild type and mutated P0 protein. RESULTS: In contrast to the membrane-localized wild type P0-GFP the Ala221fs P0-GFP protein was detectable almost only in the cytoplasm of the cells, and a complete loss of adhesion function was observed. CONCLUSIONS: The present study provides evidence that GFP is a versatile tool to trace in vivo effects of P0 and its mutations. Not only a loss of adhesion function as a result of the loss of the RSTK domain, but also altered intracellular trafficking indicated by a loss of membrane insertion are possible consequences of the Ala221fs mutation.
Assuntos
Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteína P0 da Mielina/genética , Proteína P0 da Mielina/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Vias Aferentes/patologia , Vias Aferentes/fisiopatologia , Substituição de Aminoácidos/genética , Animais , Adesão Celular/genética , Adesão Celular/fisiologia , Linhagem Celular , Pré-Escolar , Citoplasma/química , Proteínas de Fluorescência Verde , Neuropatia Hereditária Motora e Sensorial/genética , Neuropatia Hereditária Motora e Sensorial/patologia , Neuropatia Hereditária Motora e Sensorial/fisiopatologia , Humanos , Indicadores e Reagentes , Insetos , Masculino , Microscopia de Fluorescência , Mutagênese Insercional/genética , Proteína P0 da Mielina/fisiologia , Condução Nervosa/genética , Condução Nervosa/fisiologia , Proteínas Recombinantes de Fusão/síntese química , Nervo Sural/química , Nervo Sural/metabolismo , Nervo Sural/patologia , Nervo Sural/fisiopatologia , TransfecçãoRESUMO
Increased oxidative stress and advanced glycosylation are important factors in the development of diabetic neuropathy. In non-diabetic neuropathies their influence has not been investigated in detail so far. We studied the localisation of N(epsilon)-carboxymethyllysine (CML) - a biomarker for oxidative stress - by immunohistochemistry in sural nerve biopsies of 31 patients with different polyneuropathies [diabetic polyneuropathy (n=5), alcohol-associated polyneuropathy (n=4), vitamin B12-deficient polyneuropathy (n=6), chronic inflammatory demyelinating polyneuropathy (CIDP) (n=6), vasculitic neuropathy (n=6), Charcot-Marie-Tooth disease type I (CMT I) (n=4)] and 4 normal controls. CML was detected in the perineurium of patients with diabetic, alcohol-associated, vitamin B12-deficient and vasculitic polyneuropathies. Epineurial, perineurial and endoneurial vessels were CML positive in diabetic, vitamin B12-deficient and vasculitic polyneuropathies. CML was also found in mononuclear inflammatory cells in vasculitic neuropathy. In CIDP and normal controls there was only marginal perineurial CML deposition in 2/6 and 1/4 cases. In CMT I no CML was detected. Immunohistochemical results were confirmed by immunoblot. Our data suggest a role of oxidative stress in the pathogenesis not only of diabetic but also of alcohol-associated, vitamin B12-deficient and vasculitic polyneuropathies. It may be a minor pathogenetic factor in CIDP and may not be involved in CMT I. Underlying causes for increased oxidative stress may be an elevated production of reactive oxygen species and an impairment of antioxidative defences. Therefore, an antioxidative treatment should be considered in alcohol-associated, vitamin B12-deficient and vasculitic polyneuropathy.
Assuntos
Neuropatias Diabéticas/metabolismo , Lisina/análise , Adulto , Idoso , Biópsia , Western Blotting , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Neuropatias Diabéticas/patologia , Feminino , Humanos , Imuno-Histoquímica , Lisina/análogos & derivados , Lisina/metabolismo , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/metabolismo , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/patologia , Nervo Sural/química , Nervo Sural/metabolismo , Nervo Sural/patologiaRESUMO
Familial amyloidotic polyneuropathy (FAP) is a neurodegenerative disorder characterized by extracellular deposition of transthyretin (TTR) amyloid fibrils, particularly in the peripheral nervous system. No systematic immunohistochemical data exists relating TTR deposition with FAP progression. We assessed nerves from FAP patients in different stages of disease progression (FAP 0 to FAP 3) for TTR deposition by immunohistochemistry, and for the presence of amyloid fibrils by Congo Red staining. The nature of the deposited material was further studied by electron microscopy. We observed that early in FAP (FAP 0), TTR is already deposited in an aggregated nonfibrillar form, negative by Congo Red staining. This suggested that in vivo, preamyloidogenic forms of TTR exist in the nerve, in a stage before fibril formation. Cytotoxicity of nonfibrillar TTR was assessed in nerves of different FAP stages by immunohistochemistry for macrophage colony-stimulating factor. FAP 0 patients already presented increased axonal expression of macrophage colony-stimulating factor that was maintained in all other stages, in sites related to TTR deposition. Toxicity of synthetic TTR fibrils formed in vitro at physiological pH was studied on a Schwannoma cell line by caspase-3 activation assays and showed that early aggregates but not mature fibrils are toxic to cells. Taken together, these results show that nonfibrillar cytotoxic deposits occur in early stages of FAP.
Assuntos
Neuropatias Amiloides Familiares/patologia , Pré-Albumina/metabolismo , Adolescente , Adulto , Amiloide/metabolismo , Neuropatias Amiloides Familiares/metabolismo , Animais , Caspase 3 , Caspases/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular , Progressão da Doença , Ativação Enzimática/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Fator Estimulador de Colônias de Macrófagos/metabolismo , Microscopia Eletrônica , Pessoa de Meia-Idade , Pré-Albumina/farmacologia , Pré-Albumina/ultraestrutura , Nervo Sural/química , Nervo Sural/patologia , Nervo Sural/ultraestruturaRESUMO
The NF-kappaB family of transcription factors is critically involved in the immune response. The activity of these proteins is under strict control of an inhibitory molecule called IkappaB. The present study investigated the expression and distribution pattern of NF-kappaB and IkappaB in sural nerve biopsies obtained from patients with Guillain-Barré syndrome, chronic inflammatory demyelinating polyradiculoneuropathy, and various non-inflammatory neuropathies. In inflammatory demyelinating as well as non-inflammatory neuropathies, NF-kappaB was primarily expressed by macrophages, as determined by immunohistochemistry. IkappaB, however, could be localized to macrophages as well as T cells in inflammatory demyelinating neuropathies, whereas in non-inflammatory controls Schwann cells were found to be the primary cell type expressing this inhibitor. Quantitation of immunoreactivity revealed a statistically significant increase of NF-kappaB expression in inflammatory demyelinating cases compared to controls. Our results suggest an important function of the NF-kappaB pool in the genesis of inflammatory demyelination in the peripheral nervous system.
Assuntos
Síndrome de Guillain-Barré/imunologia , Proteínas I-kappa B/biossíntese , NF-kappa B/biossíntese , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/imunologia , Síndrome de Guillain-Barré/metabolismo , Humanos , Proteínas I-kappa B/imunologia , Imuno-Histoquímica , Macrófagos/imunologia , Macrófagos/metabolismo , NF-kappa B/imunologia , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/metabolismo , Nervo Sural/química , Nervo Sural/imunologiaRESUMO
In vitro and in vivo models have implicated numerous cytokines as major modulators of inflammation, destruction and repair in the peripheral nervous system (PNS). The in situ production of cytokines in human peripheral nerve disorders is still poorly documented. We studied the expression of interleukin (IL)-1 beta, tumor necrosis factor (TNF)-alpha, IL-6, IL-10, IL-4, IL-3 and nerve growth factor (NGF) in 35 human sural nerve biopsies using immunohistochemistry; additional reverse transcription-polymerase chain reaction and mRNA in situ hybridization were performed for IL-4 and NGF. Expression of IL-1 beta and TNF-alpha was shown in both morphologically normal nerves and various neuropathies, and macrophages appeared as their predominant source. Levels of IL-1 beta and TNF-alpha expression were significantly correlated (P < 0.01) with each other and with expression of NGF. Multiple endoneurial sources were suggested for IL-6 and IL-10 with low immunoreactivity in the vast majority of cases. Conversely, IL-4 and IL-3 expression were found in neuropathies of various etiologies and Schwann cells appeared to be a predominant source of IL-4 in double-labeling immunofluorescence studies. IL-3 immunoreactivity correlated with IL-1 beta, TNF-alpha and IL-6. In this retrospective study, no specific cytokine profile of expression could be assigned to a precise subgroup of neuropathies. This is the first report of IL-4 and IL-3 expression in human neuropathies, and it may be important given the potential role of these cytokines in modulating macrophage activity in the PNS.
Assuntos
Citocinas/análise , Proteínas do Tecido Nervoso/análise , Doenças do Sistema Nervoso Periférico/metabolismo , Nervo Sural/química , Adolescente , Adulto , Idoso , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Biópsia , Criança , Pré-Escolar , Citocinas/genética , Citocinas/imunologia , DNA Complementar/genética , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Perfilação da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Hibridização In Situ , Interleucinas/análise , Interleucinas/genética , Interleucinas/imunologia , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Fatores de Crescimento Neural/análise , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/imunologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/imunologia , Doenças do Sistema Nervoso Periférico/patologia , Nervo Sural/patologia , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
AIM: The relationship between perineurial collagen IV content and fascicle size was determined in diabetic and control human peripheral nerve. METHODS AND RESULTS: Age-matched diabetic and control sural nerve samples were immunostained using antibodies to collagen IV. The number of cell layers and the perimeter of the fascicle were measured and the collagen IV content of the perineurium determined. Using this method, a comparison could be made of collagen IV content in the perineuria of fascicles of different size. A positive linear relationship was found between fascicle size and the amount of collagen IV per unit of perineurium. The number of perineurial cell layers and the collagen IV content of the diabetic nerve did not differ from control values. CONCLUSIONS: The linear relationship between fascicle size and perineurial collagen IV content per unit of perineurium underlines the importance of taking fascicle size into account when determining changes in basement membrane components associated with neuropathy. The results indicate that increased deposition of collagen IV is not involved in the early changes in the perineurial cell basement membrane and is thus not the primary factor involved in altered nerve function associated with diabetic neuropathy.
Assuntos
Colágeno/análise , Complicações do Diabetes , Neuropatias Diabéticas/metabolismo , Nervo Sural/química , Idoso , Biópsia , Neuropatias Diabéticas/etiologia , Neuropatias Diabéticas/patologia , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Nervo Sural/patologia , Nervo Sural/ultraestruturaRESUMO
Anti-GM2 IgM antibodies have been reported in some patients with dysimmune neuropathy or lower motor neuron syndrome, in whom they were often associated with a concomitant reactivity with GM1. To investigate the possible clinical and pathogenetic relevance of these antibodies we measured serum anti-GM2 IgM titers by ELISA in 224 patients with different neuropathies and motor neuron disease and examined their binding to SK-N-SH neuroblastoma cells by indirect immunofluorescence (IIF). High titers of anti-GM2 IgM antibodies were found in eight patients with dysimmune neuropathies including two with multifocal motor neuropathy (MMN), two with purely motor demyelinating neuropathy without conduction block (MN) and four with Guillain-Barré syndrome (GBS). In two MMN patients reactivity with GM2 was associated with anti-GM1 reactivity and in one MN patient with anti-GM1, -GD1a and -GD1b reactivity. All but one patient had a concomitant reactivity with GalNAc-GD1a. Serum IgM from all positive patients intensely stained by IIF the surface of SK-N-SH neuroblastoma cells. This reactivity was blocked by serum pre-incubation with GM2, was not observed with sera from patients without anti-GM2 antibodies including those with high anti-GM1 or other anti-glycolipid antibodies, and correlated with the presence of GM2 in the SK-N-SH neuroblastoma cells. These findings indicate that anti-GM2 antibodies, though infrequent, are strictly associated with dysimmune neuropathies and suggest that SK-N-SH neuroblastoma cells can be a suitable in vitro model to study the functional and biological effects of these antibodies.
Assuntos
Autoanticorpos/sangue , Gangliosídeo G(M2)/imunologia , Imunoglobulina M/sangue , Neuroblastoma/imunologia , Adolescente , Adulto , Autoanticorpos/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Gangliosídeo G(M2)/análise , Humanos , Immunoblotting , Imunoglobulina M/imunologia , Masculino , Pessoa de Meia-Idade , Doença dos Neurônios Motores/imunologia , Bainha de Mielina/química , Bainha de Mielina/imunologia , Polirradiculoneuropatia/imunologia , Nervo Sural/química , Nervo Sural/imunologia , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/imunologiaRESUMO
Cisplatin is a valuable antineoplastic drug which as a dose-limiting side-effect causes sensory neuropathy, and which therefore is often combined with less neurotoxic carboplatin. It has not been possible to reproduce cisplatin neuropathy in experimental animals, and the neurotoxic mechanism in man is disputed. We investigated post-mortem material from 12 patients and 15 control subjects. Half of the fibres with diameters of > or = 9 microns, or more than 15% of all fibres (P < 0.02), had disappeared in the sural nerves of patients. Signs of axonal regeneration were lacking. The dorsal root ganglia D12 and L2 of some but not of all patients contained necrotic neurons and nodules of Nageotte. The mean volume of the somata was reduced by 18% (P < 0.03). A relation between cumulated doses, treatment free interval and changes in nerve or ganglia was not found. The platinum content was high in all tissues except in the spinal cord when the patient had died shortly after treatment, and it decreased with increasing interval, least so in liver, sensory ganglia and sural nerves. The results support the hypothesis that cisplatin neuropathy is a neuroneopathy rather than a dying-back axonopathy.
Assuntos
Antineoplásicos/efeitos adversos , Carboplatina/efeitos adversos , Cisplatino/efeitos adversos , Gânglios Espinais/efeitos dos fármacos , Platina/análise , Nervo Sural/efeitos dos fármacos , Adolescente , Adulto , Autopsia , Relação Dose-Resposta a Droga , Feminino , Gânglios Espinais/química , Gânglios Espinais/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Nervo Sural/química , Nervo Sural/patologiaRESUMO
The steady-state mRNA levels of glial cell line-derived neurotrophic factor (GDNF), GDNFR-alpha and RET were examined in various human peripheral neuropathies to determine the relationship with myelinated fiber pathology, and T cell and macrophage invasions in the diseased nerves. GDNF and GDNFR-alpha mRNA levels were elevated to variable extent in the diseased nerves, although they were not specific to the type of diseases. The increase of GDNFR-alpha mRNA levels was correlated with the extent of the nerves with axonal pathology, and was proportional to the extent of invasion of the nerves by T cells and macrophages. The GDNF mRNA levels were not related to axonal, demyelinating pathology, or inflammatory cell invasions. RET mRNA expression was not detected in normal nor diseased nerves. The GDNF and GDNFR-alpha expression in the diseased human nerves is regulated by an underlying pathology-related process, and could play a role in peripheral nerve repair.
Assuntos
Doenças Desmielinizantes/metabolismo , Proteínas de Drosophila , Fatores de Crescimento Neural , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Axônios/química , Axônios/metabolismo , Axônios/patologia , Doença de Charcot-Marie-Tooth/imunologia , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Doenças Desmielinizantes/imunologia , Doenças Desmielinizantes/patologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Humanos , Macrófagos/imunologia , Macrófagos/patologia , Fármacos Neuroprotetores/metabolismo , Poliarterite Nodosa/imunologia , Poliarterite Nodosa/metabolismo , Poliarterite Nodosa/patologia , Polirradiculoneuropatia/imunologia , Polirradiculoneuropatia/metabolismo , Polirradiculoneuropatia/patologia , Proteínas Proto-Oncogênicas c-ret , RNA Mensageiro/análise , Síndrome de Sjogren/imunologia , Síndrome de Sjogren/metabolismo , Síndrome de Sjogren/patologia , Nervo Sural/química , Nervo Sural/metabolismo , Nervo Sural/patologia , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
Two distinct populations of Na+ channels (kinetically fast and slow) are present on the cell bodies and axons of cutaneous afferent neurons; the fast current is increased and the slow current reduced in amplitude following nerve injury. The present study was undertaken to determine if similar changes occur on the axons of these neurons following peripheral nerve injury. The compound action potentials from rat sural nerves were recorded in a sucrose gap chamber. Following application of 4-aminopyridine, a prominent and well-characterized depolarization (the delayed depolarization) followed the action potential. This potential, only present on cutaneous afferent axons, has been correlated with activation of a slow Na+ current. The delayed depolarization was reduced after nerve transection. The refractory period of transmission of the action potential was shortened in the transected nerves, but that of the delayed depolarization was prolonged. The changes were largest when the sural nerve was cut and ligated [control: 38.1 +/- 1.7% (n = 5); injury: 24.5 +/- 2.8% (n = 5), P < 0.05], which prevented reconnection to its peripheral target. When the nerve was crushed and allowed to reestablish peripheral target connections, the delayed depolarization was minimally effected. These results indicate that the changes in Na+ channel organization following peripheral target disconnection observed on cutaneous afferent cell bodies also occur on their axons.