RESUMEN
BACKGROUND: Immunohistochemical staining of entire nerve fibres allows for studying the molecular composition of functional fibre subunits and may add to the diagnostic value of nerve fibre teasing. NEW METHOD: In this study, we established a sealed-slide method for reproducible immunostaining of deep axoplasmic proteins in permanently straightened nerve fibres. RESULTS: Immunostaining of teased nerve fibres very much is facilitated by tip-fixation with biocompatible glass adhesives. Antibody penetration in fresh nerves can be achieved by thermic and chemical permeabilisation while enzymatic digestion allows for sufficient permeability after aldehyde fixation. COMPARISON WITH EXISTING METHODS: The methods recommended herein are easy to perform and represent a reliable and reproducible way to whole mount immunostaining. CONCLUSIONS: Sealed-slide immunostaining of tip-fixed and permeabilised nerve biopsies will help to validate neurophysiological abnormalities and to screen for target molecules and predictive markers of peripheral nerve disorders such as in inherited neuropathies and Guillain-Barré syndrome.
Asunto(s)
Inmunohistoquímica/métodos , Fibras Nerviosas , Fijación del Tejido/métodos , Animales , Vidrio , Mamíferos , Vaina de Mielina/química , Fibras Nerviosas/química , Nervio Peroneo/química , Nervio Peroneo/citología , Reproducibilidad de los Resultados , Manejo de Especímenes/métodos , Adhesivos Tisulares , Nervio Cubital/química , Nervio Cubital/citologíaRESUMEN
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.
Asunto(s)
Enfermedades Desmielinizantes , Ratas Topo/anatomía & histología , Fibras Nerviosas Amielínicas/fisiología , Nervio Peroneo/citología , Piel/inervación , Nervio Sural/citología , África , Animales , Enfermedades Desmielinizantes/genética , Femenino , Folículo Piloso/citología , Folículo Piloso/inervación , Folículo Piloso/fisiología , Masculino , Ratas Topo/clasificación , Fibras Nerviosas Amielínicas/clasificación , Nervio Peroneo/química , Nervio Peroneo/fisiología , Piel/citología , Especificidad de la Especie , Nervio Sural/química , Nervio Sural/fisiologíaRESUMEN
Stereological techniques have been increasingly employed for assessment and characterization of neuromuscular diseases in humans and animals. As an adjunct to histopathology, morphometrical algorithms provide quantitative evidence of the peripheral nerve composition, thereby shedding light on its fibre characteristics and basic electrophysiological properties. In the horse, stereological investigations already have focussed on the recurrent laryngeal, deep peroneal and lateral palmar nerves (LPN). Of these, only the latter is suitable for taking biopsies in clinical settings, however, it does not contain any motor fibres and Ia-afferents. On account of its virtually mixed fibre qualities, most researchers today recommend the cervical branch of the equine accessory nerve (AN) for harvesting diagnostic samples. Thus, the present study was carried out to gain morphometrical proof of the AN composition and to obtain stereological base values in healthy individuals using state-of-the-art technology. All parameters were compared to the common peroneal nerve (CPN), known to harbour all myelinated fibre classes. As this second biopsy site is located farther distally to the neuro-axis, attention was paid to possible length-dependent features. Taken together, digital image analysis could be accurately applied on all AN samples. Stereology supported the histological and clinical evidence that the AN contains all myelinated fibre types. The huge range and scatter of fibre counts and density (3351-17,812/mm(2)) per fascicle were comparable to that measured in the equine common peroneal, deep peroneal, lateral palmar and recurrent laryngeal nerves. Similar to those, fibre diameter distribution was bimodal with slow Abeta- and Agamma-mechanoceptor afferents outnumbering large myelinated Aalpha-fibres by a factor of about 1.5. With a g-ratio at 0.55 +/- 0.001, the overall degree of myelination in the AN is highly consistent and insignificantly ranges between that of the equine common peroneal and LPNs. Apart from this subtle deviation, a statistically relevant difference between the more proximal AN and the distal CPN could not be documented. By obtaining morphometrical standard parameters and even more sophisticated distribution indices, stereology is a valuable tool for detection of subtle changes that are likely to escape from the investigators' eyes. The AN serves as a reliable source for advanced peripheral nerve research and should be accompanied by farther distal nerve probes for assessment of neuropathies that present with a proximodistal gradient.
Asunto(s)
Nervio Accesorio/anatomía & histología , Caballos/anatomía & histología , Nervios Periféricos/anatomía & histología , Nervio Peroneo/anatomía & histología , Nervio Accesorio/química , Animales , Enfermedades de los Caballos/patología , Fibras Nerviosas Mielínicas/fisiología , Fibras Nerviosas Mielínicas/ultraestructura , Enfermedades Neuromusculares/patología , Enfermedades Neuromusculares/veterinaria , Nervios Periféricos/química , Nervio Peroneo/químicaRESUMEN
This report presents a case of neural fibrolipoma arising from the superficial peroneal nerve in the ankle. A 28-year-old woman was referred with a soft tissue mass in the anterior aspect of the right ankle, which had been gradually enlarging for the past 10 years. Magnetic resonance imaging showed a mass lesion, measuring approximately 8 x 3 x 2 cm, with high to partially low signal intensity on both T1- and T2-weighted images. A band of low signal intensity within the lesion, which is indicative of coexistence with the tumor and the superficial peroneal nerve, could be detected on both T1- and T2-weighted images. The patient underwent an excisional biopsy. The specimen microscopically consisted of nerve bundles and fibro-fatty proliferation with abundant collagen fibers. Immunoreactivity for CD34 antigen antibody was detected in fibrous spindle cells. This is the first report to present an immunohistochemical profile of neural fibrolipoma. Neural fibrolipoma should be considered as a differential diagnosis when a lipomatous lesion is encountered in the foot or ankle as well as in the upper extremities.
Asunto(s)
Lipoma/patología , Neoplasias del Sistema Nervioso Periférico/patología , Nervio Peroneo/patología , Neuropatías Peroneas/patología , Neoplasias de los Tejidos Blandos/patología , Adulto , Tobillo/patología , Tobillo/cirugía , Antígenos CD34/análisis , Biomarcadores de Tumor/análisis , Femenino , Humanos , Técnicas para Inmunoenzimas , Lipoma/química , Lipoma/cirugía , Imagen por Resonancia Magnética , Neoplasias del Sistema Nervioso Periférico/química , Neoplasias del Sistema Nervioso Periférico/cirugía , Nervio Peroneo/química , Nervio Peroneo/cirugía , Neoplasias de los Tejidos Blandos/química , Neoplasias de los Tejidos Blandos/cirugía , Resultado del TratamientoRESUMEN
We report laminin alpha 2 (merosin) deficiency associated with muscular dystrophy and demyelinating neuropathy in two cats. The cats developed progressive muscle weakness, and atrophy. Either hypotonia or contractures resulted in recumbency, necessitating euthanasia. Muscle biopsies showed dystrophic changes including marked endomysial fibrosis, myofiber necrosis, variability of fiber size, and perimysial lipid accumulation. Immunohistochemistry showed that laminin alpha 2 chain was absent or reduced, while dystrophin and all the components of the dystrophin-associated glycoprotein complex were present and normal. One cat was examined in detail. Motor nerve conduction velocity (MNCV) was decreased, and ultrastructurally the peripheral nerves showed Schwann cell degeneration and demyelination. Brain imaging was not performed, but white matter changes were not apparent in the brain at necropsy. The disease in these cats is similar to primary or secondary merosin (laminin alpha 2)-deficient congenital muscular dystrophy (CMD) in humans and to dystrophia muscularis in mice.
Asunto(s)
Enfermedades de los Gatos/genética , Enfermedades Desmielinizantes/veterinaria , Laminina/deficiencia , Proteínas Musculares/deficiencia , Distrofia Muscular Animal/genética , Proteínas del Tejido Nervioso/deficiencia , Animales , Enfermedades de los Gatos/patología , Gatos , Contractura/etiología , Enfermedades Desmielinizantes/complicaciones , Enfermedades Desmielinizantes/genética , Enfermedades Desmielinizantes/patología , Femenino , Laminina/genética , Proteínas Musculares/análisis , Proteínas Musculares/genética , Rigidez Muscular/etiología , Músculo Esquelético/química , Músculo Esquelético/patología , Atrofia Muscular/etiología , Distrofia Muscular Animal/complicaciones , Distrofia Muscular Animal/patología , Proteínas del Tejido Nervioso/genética , Conducción Nerviosa , Nervio Peroneo/química , Nervio Peroneo/patologíaRESUMEN
To learn more about the mechanisms of nerve lesions in POEMS syndrome, nerve specimens from four patients were studied with an immunogold method at the ultrastructural level to detect and localise the M protein in the different nerve compartments. An indirect immunolabelling technique was applied on 4% PFA fixed and LR White embedded nerve specimens. Antisera against IgG, IgA, IgM, and lambda and kappa light chains were used as primary antisera. Morphological studies disclosed an important axonal loss in association with the demyelinative process. Endoneurial deposits of immunoglobulins were found in all cases. In the patient with the most severe form of neuropathy, diffuse deposits were present in the endoneurial space, especially in the subperineurial area. In the other patients, occasional deposits of the M protein were found in the myelin sheath (n=2); or between cells (n=1). No deposit was found in the axons. The class of the M protein labelled in the nerve corresponded to that detected in the serum in three of four patients, with labelling of two heavy chains in one patient. Immunolabelling of the M protein on the myelin sheath, Schwann cells, and in the endoneurial space favour a direct role of the M component in the lesions of nerve fibres, and justify active treatment of the plasmacytic proliferation.
Asunto(s)
Proteínas Musculares , Proteínas de Mieloma/análisis , Síndrome POEMS/diagnóstico , Nervio Peroneo/química , Nervio Peroneo/ultraestructura , Biopsia , Proteínas Sanguíneas/inmunología , Conectina , Enfermedades Desmielinizantes/patología , Humanos , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Síndrome POEMS/inmunología , Estudios RetrospectivosRESUMEN
BACKGROUND: Reflex sympathetic dystrophy (RSD) (recently reclassified as complex regional pain syndrome type I) is a syndrome occurring in extremities and, when chronic, results in severe disability and untractable pain. RSD may be accompanied by neurologic symptoms even when there is no previous neurologic lesion. There is no consensus as to the pathogenic mechanism involved in RSD. To gain insight into the pathophysiology of RSD, we studied histopathology of skeletal muscle and peripheral nerve from patients with chronic RSD in a lower extremity. METHODS: In eight patients with chronic RSD, an above-the-knee amputation was performed because of a nonfunctional limb. Specimens of sural nerves, tibial nerves, common peroneal nerves, gastrocnemius muscles, and soleus muscles were obtained from the amputated legs and analyzed by light and electron microscopy. RESULTS: In all patients, the affected leg showed similar neurologic symptoms such as spontaneous pain, hyperpathy, allodynia, paresis, and anesthesia dolorosa. The nerves showed no consistent abnormalities of myelinated fibers. In four patients, the C-fibers showed electron microscopic pathology. In all patients, the gastrocnemius and soleus muscle specimens showed a decrease of type I fibers, an increase of lipofuscin pigment, atrophic fibers, and severely thickened basal membrane layers of the capillaries. CONCLUSION: In chronic RSD, efferent nerve fibers were histologically unaffected; from afferent fibers, only C-fibers showed histopathologic abnormalities. Skeletal muscle showed a variety of histopathologic findings, which are similar to the histologic abnormalities found in muscles of patients with diabetes.
Asunto(s)
Músculo Esquelético/patología , Nervios Periféricos/patología , Distrofia Simpática Refleja/patología , Adulto , Capilares/patología , Femenino , Humanos , Lipofuscina/análisis , Masculino , Microscopía Electrónica , Persona de Mediana Edad , Neuronas Motoras/patología , Neuronas Motoras/ultraestructura , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/patología , Fibras Musculares Esqueléticas/ultraestructura , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/química , Necrosis , Fibras Nerviosas/patología , Fibras Nerviosas/ultraestructura , Neuronas Aferentes/patología , Neuronas Aferentes/ultraestructura , Nervios Periféricos/irrigación sanguínea , Nervios Periféricos/química , Nervio Peroneo/irrigación sanguínea , Nervio Peroneo/química , Nervio Peroneo/patología , Nervio Sural/irrigación sanguínea , Nervio Sural/química , Nervio Sural/patología , Nervio Tibial/irrigación sanguínea , Nervio Tibial/química , Nervio Tibial/patologíaRESUMEN
The IgM monoclonal autoantibodies of patients with demyelinating paraproteinaemic polyneuropathy recognize a carbohydrate structure present on both myelin-associated glycoprotein (MAG) and protein zero (P0). These autoantibodies are sufficient to cause the disease but the mechanism of demyelination remains unclear. We have analysed nerve biopsies from eight patients with polyneuropathy and anti-MAG antibodies by quantitative immunohistochemistry and find a concordant pattern of reduced expression of myelin markers with the loss of myelinated fibres. We report here novel features of this disease, in particular a selective lack of detectable MAG in a large proportion of myelinated-fibres containing P0, myelin basic protein (MBP) and periaxin. There is also an inverse correlation of the distribution of MAG in peripheral nerve myelin with the serum anti-MAG antibody titres but no correlation of these titres with the loss of myelinated fibres. Double immunofluorescence staining of paraproteinaemic polyneuropathy (PPN) nerves shows anti-MAG IgM deposited on the periphery of myelinated fibres associated with or lacking MAG staining. These data suggest that the binding of anti-MAG antibodies to MAG and/or other myelin component(s) results in MAG downregulation and may have an essential role in the molecular mechanisms leading to demyelination and partial regeneration in this disease.