Anti-neurofilament Antibody Research Articles

Classification of axonal subtypes based on cytoskeletal components.

BackgroundRetinal ganglion cells are often classified into different subtypes according to their morphology or physiological functions. The axons of RGCs contain three major cytoskeletal components: actin filaments (F-actin); microtubules; and neurofilaments (NFs). The contents of these components vary among axons. Our objective was to classify axons into subtypes based on the contents of cytoskeletal components and study their distributions across the retina in normal rodent retinas.MethodsWhole-mounted retinas of female Wistar rats were stained with phalloidin to label F-actin, anti-β-tubulin monoclonal antibody to mark microtubules, and antineurofilament antibody to label NFs. A confocal laser scanning microscope was used to provide en face images of retinal nerve fiber bundles with a resolution of 0.24 μm/pixel. Staining intensity profiles across axons were obtained for each cytoskeletal component. Axonal subtypes were then determined from the relative contents, indicated by the staining intensity, of these components. Linear density was used to investigate topographical distribution of each subtype across the retina.ResultsNormal axons could be classified into seven subtypes – FMN, FM, FN, and MN subtypes, (in which at least two or three cytoskeletal components were intensely stained), and F, M, and N subtypes, (in which only one cytoskeletal component was intensely stained within an axon). The FMN subtype was the most abundant subtype. There was no preferential distribution of subtypes around the optic nerve head. However, the densities of the axonal subtypes that contained NFs were found significantly different in the central and peripheral retinal regions. Axonal sizes were subtype-dependent.ConclusionAxons of retinal ganglion cells can be classified into different subtypes, based on the contents of axonal cytoskeletal components. The classified subtypes will provide a new means to study selective damage of axonal ultrastructures in ocular neuropathic diseases.

Sectorial loss of retinal ganglion cells in inherited photoreceptor degeneration is due to RGC death

AimsTo investigate the cause of retinal ganglion cell (RGC) loss in dystrophic aged Royal College of Surgeons (RCS) rats.MethodsRCS-p+ (dystrophic) female rats of postnatal times (P365, P450 and P540) and...

Isolation of Low Abundance Proteins and Cells Using Buoyant Glass Microbubble Chromatography

Conventional protein affinity chromatography relies on highly porous resins that have large surface areas. These properties are ideal for fast flow separation of proteins from biological samples with maximum yields, but these properties can also lead to increased nonspecific protein binding. In certain applications where the purity of an isolated protein is more important than the yield, using a glass solid phase could be advantageous as glass is nonporous and hydrophilic and has a low surface area and low nonspecific protein binding. As a proof of principle, we used protein A-conjugated hollow glass microbubbles to isolate fluorescently labeled neurofilament heavy chain spiked into serum and compared them to protein A Sepharose and protein A magnetic beads (Dynabeads) using an anti-neurofilament protein antibody. As expected, a greater volume of glass bubbles was required to match the binding capacity of the magnetic beads and Sepharose resins. On the other hand, nonspecific protein binding to glass bubbles was greatly reduced compared to the other resins. Additionally, since the glass bubbles are buoyant and transparent, they are well suited for isolating cells from biological samples and staining themin situ.

Recurrent Laryngeal Nerve Regeneration through a Silicone Tube Produces Reinnervation without Vocal Fold Mobility in Rats

We established an animal model of recurrent laryngeal nerve reinnervation with persistent vocal fold immobility following recurrent laryngeal nerve injury. In 36 rats, the left recurrent laryngeal nerve was transected and the stumps were abutted in a silicone tube with a 1-mm interspace, facilitating regeneration. The mobility of the vocal folds was examined endoscopically 5, 10, and 15 weeks later. Electromyography of the thyroarytenoid muscle was performed. Reinnervation was assessed by means of a quantitative immunohistologic evaluation with anti-neurofilament antibody in the nerve both proximal and distal to the silicone tube. The atrophy of the thyroarytenoid muscle was assessed histologically. We observed that all animals had a fixed left vocal fold throughout the study. The average neurofilament expression in the nerve both distal and proximal to the silicone tube, the muscle area, and the amplitude of the compound muscle action potential recorded from the thyroarytenoid muscle on the treated side increased significantly (p < 0.05) over time, demonstrating regeneration through the silicone tube. Recurrent laryngeal nerve regeneration through a silicone tube produced reinnervation without vocal fold mobility in rats. The efficacy of new laryngeal reinnervation treatments can be assessed with this model.

Conditions for quantitative evaluation of injured spinal cord by in vivo diffusion tensor imaging and tractography: Preclinical longitudinal study in common marmosets

Conventional magnetic resonance imaging (MRI) can detect hemorrhage, edema, syrinx, and spinal cord atrophy, but not axonal disruption after spinal cord injury (SCI). We previously demonstrated that diffusion tensor tractography (DTT) could depict axonal disruption after hemisection SCI in common marmosets. In the present study, to determine the relationship between DTT results and functional recovery after contusive SCI, we performed longitudinal DTT, behavioral, and histological analyses before and after contusive SCI in common marmosets. By comparing the tract fiber estimate depicted by DTT with neuronal fibers labeled with RT97 and SMI-31, anti-neurofilament antibodies, we determined the optimal fractional anisotropy (FA) threshold for fiber tracking to be 0.40. The ratio of the number of tract fiber estimates at the lesion site to the number before SCI, determined by DTT, was significantly correlated with the functional recovery after SCI. Moreover, comparison of the longitudinal pre- and post-SCI FA and axial diffusivity (λ(‖)) values revealed that they decreased after injury at the sites caudal to the lesion epicenter in the corticospinal tract and rostral to the lesion epicenter in the dorsal column. The FA values, then, showed partial recovery in the dorsal column. FA-value-oriented color DTT was used to represent axonal sparing or regeneration of the different tracts. These findings indicated that DTT analysis might be a versatile non-invasive tool for evaluating the axonal disruption after SCI.

Neurofilament a biomarker of neurodegeneration in autoimmune encephalomyelitis

Monitoring neuroaxonal loss in multiple sclerosis is an important objective to study the pathogenesis and response to treatment of the disease. The release of neurofilaments is a potential surrogate biomarker of neurodegeneration. One route to explore this aspect further is through the use of animal models that have well-defined, and predictable, disease courses. The release of neurofilaments into plasma across the course of relapsing-remitting and secondary progressive experimental autoimmune encephalomyelitis in Biozzi ABH was assessed, as well as measuring anti-neurofilament antibodies using ELISA. It was found that there was an immediate release in neurofilaments into the blood following the initial paralytic attack. Neurofilament release was continuous in relapse and remission but returned towards baseline in chronic disease, as animals entered the post-relapsing progressive phase of the disease. This was mirrored by a loss of neurofilament-specific antibodies. In contrast neurofilament levels increased dramatically as neurodegeneration and clinical disease occurred in the G93A SOD1 transgenic C57BL/6 x SJL mice, model of amyotrophic lateral sclerosis. These data further support neurofilament levels as a good surrogate measure of neurodegeneration and their potential use as a surrogate endpoint in neuroprotective studies.

Loss of neurofilaments in the neuromuscular junction in a rat model of proximal axonopathy

Rodents exposed to 3,3'-iminodipropionitrile (IDPN) develop an axonopathy similar to that observed in amyotrophic lateral sclerosis motor neurones, in which neurofilaments accumulate in swollen proximal axon segments. This study addressed the hypotheses that this proximal axonopathy is associated with loss of neurofilament proteins in the neuromuscular junctions and a progressive loss of neurofilaments advancing in a distal-proximal direction from the distal motor nerve. Adult male Long-Evans rats were exposed to 0 or 15 mM of IDPN in drinking water for 1, 3 or 5 weeks, and their distal axons and neuromuscular junction organization studied by immunohistochemistry. Quantitative data were obtained by confocal microscopy on whole mounts of the Levator auris longus. Muscles showed no change in the distribution of acetylcholine receptor labelling in the neuromuscular junctions after IDPN. In contrast, the amount of neurofilament labelling in the junctions was significantly reduced by IDPN, assessed with two different anti-neurofilament antibodies. In preterminal axons and in more proximal axon levels, no statistically significant reductions in neurofilament content were observed. The proximal neurofilamentous axonopathy induced by IDPN is associated with an abnormally low content of neurofilaments in the motor terminals, with a potential impact in the function or stability of the neuromuscular junction. In contrast, neurofilaments are significantly maintained in the distal axon.

Palisaded encapsulated neuroma of the upper lip

Palisaded encapsulated neuroma (PEN; solitary circumscribed neuroma) is a benign and hyperplastic lesion consisting of Schwann cells. PEN of the lower lip was reported by Tomich and Moll [1] 35 years ago. However, the accumulation of the information about PEN which occurred in the oral mucosa was not enough. This article describes a case of a PEN on the upper lip of a 41-year-old woman. The lesion with 0.7 cm diameter was performed excisional biopsy. Histologically, the tumor was almost circumscribed by thin fibrous capsule, and consisted of diffusely and dense proliferation of the spindle shape cells arranged in interlacing fascicles. Focal suggestions of nuclear pal-isaded growth were indicated within the tumor. Immunohistochemicallly, the fascicles of tumor cells were positive for S-100 protein, and vimentin and negative for α-actin and GFAP. A few numbers of axons were demonstrated by anti-neurofilament antibody in this lesion. Therefore, the definitive diagnosis was PEN.

Vitamin D3 improves respiratory adjustment to fatigue and H-reflex responses in paraplegic adult rats

We previously demonstrated that vitamin D₂ (ergocalciferol) triggers axon regeneration in a rat model of peripheral nerve transection. In order to confirm the regenerative potential of this neuroactive steroid, we performed a study in which vitamin D₃ (cholecalciferol) was delivered at various doses to paralytic rats. After spinal cord compression at the T10 level, rats were given orally either vehicle or vitamin D₃ at the dose of 50 IU/kg/day or 200 IU/kg/day. Three months later, M and H-waves were recorded from rat Tibialis anterior muscle in order to quantify the maximal H-reflex (H(max)) amplitude. We also monitored the ventilatory frequency during an electrically induced muscle fatigue known to elicit the muscle metaboreflex and an increase in respiratory rate. Spinal cords were then collected, fixed and immunostained with an anti-neurofilament antibody. We show here that vitamin D-treated animals display an increased number of axons within the lesion site. In addition, rats supplemented with vitamin D₃ at the dose of 200 IU/kg/day exhibit (i) an improved breathing when hindlimb was electrically stimulated; (ii) an H-reflex depression similar to control animals and (iii) an increased number of axons within the lesion and in the distal area. Our data confirm that vitamin D is a potent molecule that can be used for improving neuromuscular adaptive mechanisms and H-reflex responses.

Histologic study of age‐related change in the posterior pituitary gland focusing on abnormal deposition of tau protein

We studied histologic findings of age-related change in the posterior pituitary gland focusing specifically on abnormal deposition of tau protein. Posterior pituitary glands from a total of 201 patients with mean age of 72, range 15 to 100 years, were dissected at autopsy, and semiquantitative analysis of tau protein deposition in the posterior pituitaries was performed. We confirmed that tau protein deposition in the posterior pituitary appears histologically as either a 'thread-like' or 'dot' form. In double staining using an anti-neurofilament antibody and Gallyas-Braak staining, Gallyas-Braak-positive structures were located in the neurite. The grade and the frequency of tau protein deposition were increased in accord with aging. An interrelation was observed between tau protein deposition in the brain and that in the posterior pituitary. In tauopathy diseases, tau protein deposition in the posterior lobe is advanced compared to that in non-tauopathy diseases. The level of tau protein deposition in the hypothalamus was compared semi-quantitatively with that in the posterior pituitary, and the levels correlated well. We suggest that in the posterior pituitary of elderly people, high frequency of occurrence of deposition of abnormal tau protein in the neurites may cause dysfunction of the pituitary gland.

Overexpression of nerve growth factor in peritoneal fluid from women with endometriosis may promote neurite outgrowth in endometriotic lesions

To investigate the role of the nerve growth factor (NGF) in the endometriosis-associated innervation in the development of endometriosis-associated symptoms, 41 peritoneal fluid samples (PF) from patients with surgically and histologically proven endometriosis and 20 PF from patients with other gynecologic conditions were analyzed with Western blot and a novel in vitro model using dorsal root ganglia (DRG) to show neuronal outgrowth; endometrial cells also were analyzed. The results suggest that the PF of endometriosis patients and endometriotic lesions have neurotropic properties, because the Western blot analysis and the cell culture stainings showed NGF expression, and the neurite outgrowth of DRG treated with PF of patients with endometriosis was significantly higher than when treated with PF of patients without endometriosis. Furthermore, blocking NGF with both anti-NGF and K252a leads to a significant decrease in neurite outgrowth.

CNS-Resident Glial Progenitor/Stem Cells Produce Schwann Cells as well as Oligodendrocytes during Repair of CNS Demyelination

After central nervous system (CNS) demyelination-such as occurs during multiple sclerosis-there is often spontaneous regeneration of myelin sheaths, mainly by oligodendrocytes but also by Schwann cells. The origins of the remyelinating cells have not previously been established. We have used Cre-lox fate mapping in transgenic mice to show that PDGFRA/NG2-expressing glia, a distributed population of stem/progenitor cells in the adult CNS, produce the remyelinating oligodendrocytes and almost all of the Schwann cells in chemically induced demyelinated lesions. In contrast, the great majority of reactive astrocytes in the vicinity of the lesions are derived from preexisting FGFR3-expressing cells, likely to be astrocytes. These data resolve a long-running debate about the origins of the main players in CNS remyelination and reveal a surprising capacity of CNS precursors to generate Schwann cells, which normally develop from the embryonic neural crest and are restricted to the peripheral nervous system.

Modified paraffin-embedding method for the human cochlea that reveals a fine morphology and excellent immunostaining results

Conclusion: A modified paraffin-embedding method could be applied to histopathological and immunohistochemical studies of the human cochlea. The complementary use of molecular and immunohistochemical techniques by means of this method is thus considered to be a valuable tool for the future study of the human inner ear. Objective: To propose a new paraffin-embedding method for the morphological and immunohistochemical study of the human cochlea. Methods: Five human temporal bones were harvested at autopsy. The temporal bone specimens were fixed in 20% buffered formalin, decalcified with EDTA, cropped to a cube of approximately 15 mm, embedded in paraffin, and then cut into 6 μm thick sections. The sections were stained with hematoxylin and eosin, and immunostained with anti-prestin and anti-neurofilament antibodies. Results: Although paraffin-embedded sections cannot show the excellent morphology of the delicate membranous labyrinth obtained with celloidin, this technique successfully preserved the morphology of the cochlea, especially the organ of Corti, thereby enabling us to obtain excellent immunostaining results.

Linear Ordered Collagen Scaffolds Loaded with Collagen-Binding Brain-Derived Neurotrophic Factor Improve the Recovery of Spinal Cord Injury in Rats

Growth factors such as brain-derived neurotrophic factor (BDNF) are widely used in the recovery of spinal cord injury (SCI) for promoting axonal regeneration or improving neuron survival. However, the lack of efficient delivery approaches limits their clinical applications. In this study, we constructed an efficient delivery system including collagen-binding BDNF and linear ordered collagen scaffolds (LOCS). A collagen-binding domain (CBD) was utilized to construct collagen-targeting BDNF (CBD-BDNF) to allow its specific binding to the collagen. In vitro activity assay showed that CBD-BDNF had similar bioactivity in neurite outgrowth of dorsal root ganglia and the survival of PC12 cells. CBD-BDNF was shown to have specific binding activity to collagen. Using the rat hemisection SCI model, we found that LOCS loaded with CBD-BDNF significantly improved the SCI recovery evaluated by the Basso, Beattie, and Bresnahan scale and immunohistochemical staining with anti-neurofilament antibody. Thus, this targeting drug delivery system consisting of CBD-BDNF and LOCS could be an effective strategy for the repair of SCI.

The neurotoxicological effects of mastoparan Polybia-MPII at the murine neuromuscular junction: an ultrastructural and immunocytochemical study

Polybia-MPII (INWLKLGKMVIDAL-NH2), a mastoparan isolated from the crude venom of the swarming wasp Polybia paulista, was injected into the left hind limb of Swiss white mice. Between 3 h and 21 days later the mice were killed and the soleus muscles from both hind limbs were removed. Sections of the muscles were made for transmission electron microscopy and immunocytochemistry. Transmission electron microscopy showed that both the volume fraction occupied by synaptic vesicles and synaptic vesicle density was greatly reduced after exposure to Polybia-MPII, although there was no significant structural damage to the plasma membrane of the terminal boutons and mitochondria were indistinguishable from those in normal, control boutons. Immunocytochemistry revealed that in control muscles 99% of motor end plates identified by the positive labelling of acetylcholine receptors by TRITC-alpha-bungarotoxin co-labelled with anti-synaptophysin antibody, but this figure fell by 30% in muscles exposed to the toxin. These changes were transient. They were maximal at 6 h and fully reversed by 3 days. At no time was axonal labelling with anti-neurofilament antibodies affected by exposure to Polybia-MPII. We conclude that mastoparan Polybia-MPII is a minor neurotoxin and suggest that its neurotoxic activity is unlikely to be of clinical significance.

Safety Profile of Topical VEGF Neutralization at the Cornea

Bevacizumab eyedrops inhibit corneal neovascularization. The purpose of this study was to analyze the safety profile of VEGF-A neutralization at the ocular surface. Bevacizumab eyedrops (5 mg/mL) and an antimurine VEGF-A antibody (250 microg/mL) were applied to normal murine corneas five times a day for 7 and 14 days. Subsequently, corneas were analyzed for morphologic changes by light and electron microscopy. In a mouse model of corneal epithelial abrasion, the effects of topically applied anti-VEGF antibodies on epithelial wound healing were analyzed: the treatment group received bevacizumab (5 mg/mL) or the antimurine VEGF-A antibody (250 microg/mL) as eyedrops, and the control group received an equal volume of saline solution. After 12, 18, and 24 hours, corneas were photographed in vivo with and without fluorescein staining for morphometry. Afterwards the mice were killed, and eyes were removed for histology, immunohistochemistry with Ki67/DAPI, and electron microscopy. The effect of midterm anti-VEGF therapy on corneal nerve density was assessed by staining corneas treated with an FITC-conjugated anti-neurofilament antibody and morphometric analysis. Murine corneas treated with two different types of anti-VEGF antibody eyedrops did not show obvious corneal morphologic changes at the light and electron microscopic levels. Furthermore, anti-VEGF antibody eyedrops had no significant impact on the wound healing process after corneal epithelial injury or on normal murine corneal nerve fiber density. Topical neutralization of VEGF-A at the corneal surface does not have significant side effects on normal corneal epithelial wound healing, normal corneal integrity, or normal nerve fiber density. Therefore, anti-VEGF eyedrops seem to be a relatively safe option to treat corneal neovascularization.

Immunohistochemical analysis of neuron types in the mouse small intestine

The definition of the nerve cell types of the myenteric plexus of the mouse small intestine has become important, as more researchers turn to the use of mice with genetic mutations to analyze roles of specific genes and their products in enteric nervous system function and to investigate animal models of disease. We have used a suite of antibodies to define neurons by their shapes, sizes, and neurochemistry in the myenteric plexus. Anti-Hu antibodies were used to reveal all nerve cells, and the major subpopulations were defined in relation to the Hu-positive neurons. Morphological Type II neurons, revealed by anti-neurofilament and anti-calcitonin gene-related peptide antibodies, represented 26% of neurons. The axons of the Type II neurons projected through the circular muscle and submucosa to the mucosa. The cell bodies were immunoreactive for choline acetyltransferase (ChAT), and their terminals were immunoreactive for vesicular acetylcholine transporter (VAChT). Nitric oxide synthase (NOS) occurred in 29% of nerve cells. Most were also immunoreactive for vasoactive intestinal peptide, but they were not tachykinin (TK)-immunoreactive, and only 10% were ChAT-immunoreactive. Numerous NOS terminals occurred in the circular muscle. We deduced that 90% of NOS neurons were inhibitory motor neurons to the muscle (26% of all neurons) and 10% (3% of all neurons) were interneurons. Calretinin immunoreactivity was found in a high proportion of neurons (52%). Many of these had TK immunoreactivity. Small calretinin neurons were identified as excitatory neurons to the longitudinal muscle (about 20% of neurons, with ChAT/calretinin/+/- TK chemical coding). Excitatory neurons to the circular muscle (about 10% of neurons) had the same coding. Calretinin immunoreactivity also occurred in a proportion of Type II neurons. Thus, over 90% of neurons in the myenteric plexus of the mouse small intestine can be currently identified by their neurochemistry and shape.

Clinical and Pathological Heterogeneity of Neuronal Intermediate Filament Inclusion Disease

To report new cases of neuronal intermediate filament inclusion disease (NIFID). Case report. Pathologically proved NIFID was found in 2 patients from the Universitat de Barcelona-Hospital Clínic Brain Bank. The findings of a neuropathological examination in both patients revealed intracellular inclusions that were detected with hematoxylin-eosin and stained positive for antineurofilament and alpha-internexin antibodies, variably for ubiquitin, and negatively for tau, alpha-synuclein, and TAR-DNA binding protein 43. Medical records were retrospectively reviewed. The first patient developed progressive behavioral changes characterized by apathy and indifference at the age of 37 years, and frontotemporal dementia was diagnosed. The second patient developed progressive tremor and mild speech disturbances at the age of 70 years. Her neurological examination results showed mild dysarthria, hypomimia, a mild rigid-akinetic left-predominant parkinsonism, and bilateral rest and postural tremor. The clinical impression was atypical parkinsonism. No response was obtained with levodopa, and the disease progressed rapidly, with falls and frontal-subcortical cognitive impairment. Late-onset presentation may be the clinical debut of NIFID. These 2 cases confirm the clinical and pathological heterogeneity of NIFID and suggest its inclusion in the differential diagnosis of several neurodegenerative disorders, including frontotemporal dementia and atypical parkinsonism.

D-LSD-induced c-Fos expression occurs in a population of oligodendrocytes in rat prefrontal cortex

Induction of mRNA or protein for immediate–early genes, such as c- fos, is used to identify brain areas, specific cell types, and neuronal circuits that become activated in response to various stimuli including psychoactive drugs. The objective of the present study was to identify the cell types in the prefrontal cortex in which lysergic acid diethylamide (d-LSD) induces c-Fos expression. Systemic administration of d-LSD resulted in a dose-dependent increase in c-Fos immunoreactivity. Although c-Fos-positive cells were found in all cortical layers, they were most numerous in layers III, IV, and V. d-LSD-induced c-Fos immunoreactivity was found in cells co-labeled with anti-neuron-specific enolase or anti-oligodendrocyte Oligo1. The Oligo1-labeled cells had small, round bodies and nuclear diameters characteristic of oligodendrocytes. Studies using confocal microscopy confirmed colocalization of c-Fos-labeled nuclei in NeuN-labeled neurons. Astrocytes and microglia labeled with glial fibrillary acidic protein antibody and OX-42 antibody, respectively, did not display LSD-induced c-Fos expression. Pyramidal neurons labeled with anti-neurofilament antibody also did not show induction of c-Fos immunoreactivity after systemic d-LSD administration. The present study demonstrates that d-LSD induced expression of c-Fos in the prefrontal cortex occurs in subpopulations of neurons and in oligodendrocytes, but not in pyramidal neurons, astrocytes, and microglia.

Does Abnormal Expression of Acetylcholine Receptors in Hirschsprung's Disease Induce Acetylcholine Esterase Positive Nerve Fibres?

Alpha bungarotoxin (alpha-BTX) is a neurotoxin isolated from the venom of Bungarus multicinctus that binds specifically to the beta-subunits of nicotinic acetylcholine receptors (nAChR) on myotube membranes. The purpose of the present study was to investigate the distribution of alpha-BTX-sensitive nAChR in Hirschsprung's disease (HD) to understand the histopathological features of HD, especially the increase in acetylcholine esterase (AChE) positive nerve fibres. Confocal microscopy was used to study the expression of FITC (fluorescein isothiocyanate)-alpha-BTX, anti-synaptophysin (A-SY) antibody, and anti-neurofilament (A-NF) antibody to determine the distribution of nAChR and ganglion cell and nerve fibres in colon specimens from five cases of HD and three normal controls. Quantitative assessment of the immunoreactivity of colonic muscle and colonic mucosal epithelium from an aganglionic segment of HD bowel demonstrated markedly increased nAChR compared with colonic muscle and colonic mucosal epithelium from a ganglionic segment of HD bowel and normal bowel (p < 0.0001, respectively), both of which have only a few positive nAChR. In colonic muscle from aganglionic and transitional segments of HD, there were many nAChR around hypertrophic nerve trunks identified by A-NF and A-SY staining. We suggest that abnormal expression of nAChR in HD might be implicated in causing gastrointestinal dysmotility because of their localization around hypertrophic nerve trunks.