Neurofilament Immunostaining Research Articles

Differential expression of triiodothyronine receptors in schwannoma and neurofibroma: role of Schwann cell-axon interaction.

Regulation of gene expression in Schwann cells may be determined, at least in part, by the interaction of these cells with axons. Two peripheral nerve tumors, neurofibroma and schwannoma, represent good tools for studying Schwann cell activity in the presence or absence of axon action. In the present work we studied the expression of triiodothyronine receptors (T3R) by Schwann cells in these two tumors and also in adult normal sciatic nerve. Confirming the results of the histological examination, immunostaining of the neurofilaments showed the presence of fascicles or scattered axons in all neurofibroma sections studied. In these neurofibromas, Schwann cells did not express T3R immunoreactivity. Furthermore, in adult normal sciatic nerve, Schwann cells which ensheathed axons were devoid of any T3R expression. In contrast, in schwannoma, the complete absence of axons was demonstrated by the lack of neurofilament immunostaining. Here, Schwann cells deprived of axonal interaction displayed clear T3R immunoreactivity. In schwannoma cell cultures, Schwann cells continued to express T3R, even in cultures treated with medium that had been conditioned with rat sensory neurons. On the basis of these results, we suggest that, beside the possible regulatory mechanisms for T3R, the synthesis of T3R is regulated, at least in part, by Schwann cell-axon interaction.

Neuronal activity in primate visual cortex assessed by immunostaining for the transcription factor Zif268.

It is now well established that environmental signals mediated via neurotransmitters and hormones can induce responses in cells which involve a cascade of receptors, G proteins, and second messengers. These in turn can induce transcription factors which regulate long-term changes in gene expression. It has been proposed that the stimulus-transcription coupling properties of these DNA-binding proteins can be exploited to visualize activated neurons by way of immunostaining. We have used standard immunohistochemical techniques to detect the expression of one specific transcription factor, Zif268, in the visual cortex (area 17, V1) of vervet monkeys (Cercopithecus aethiops). Immunopositive neurons were present in large numbers throughout the visual cortex of the normal animal, being concentrated in layers 2/3 and 6 and at moderate levels in 4C beta and 5. To determine if Zif268 expression was affected by visual stimulation in the monkey, we restricted light input to one eye with the aim of revealing ocular-dominance columns in striate cortex. We found that short-term monocular deprivation induced either by enucleation, intravitreal TTX injection, or eyelid suturing resulted in dramatic changes in Zif268 levels, revealing vertically oriented columns of reduced Zif268 staining interdigitated with columns of normal expression. Furthermore, these columns were discernible after just 2 h of monocular blockade. A comparison of the ocular-dominance pattern obtained with Zif268 immunostaining and cytochrome oxidase histochemistry in long-term monocularly deprived animals showed a coincident reduction of both markers along columns that were precisely aligned in adjacent sections, indicating that Zif268 expression is restricted to cortical regions of high metabolic activity. Simultaneous immunostaining for Zif268 and the calcium-binding proteins calbindin and parvalbumin showed a negative correlation, suggesting that the Zif268 protein may be expressed selectively within excitatory neurons. A similar approach with immunostaining for neurofilament and microtubule-associated proteins (SMI-32 and MAP2) revealed pyramidal neurons which were regularly found to contain a Zif268-positive nucleus. Furthermore, confocal images of lucifer yellow filled neurons possessing Zif268-positive nuclei all showed pyramidal morphology. Taken together, these results point to activity-dependent expression of Zif268 within a subset of excitatory neurons.

Development of human fetal ventral mesencephalic grafts in rats with 6-OHDA lesions of the nigrostriatal pathway

Neuronal transplantation is an approach that can be exploited to study the development of the human central nervous system as well as being used in attempts to restore neurological function. In the present study, we have examined cellular events that appear to precede the development of dopamine nerve fiber extension by neurons from the human fetal ventral mesencephalon. These cellular events were examined using neuronal cell suspensions from human fetal ventral mesencephalic tissue (gestational ages 7–10 weeks) transplanted into the striatum of unilaterally lesioned 6-hydroxydopamine (6-OHDA) rats. Animals were sacrificed for immunohistochemistry 9–10 weeks after the transplantation prior to the manifestation of behavioral recovery. Histological analysis revealed tyrosine hydroxylase (TH) immunoreactive neurons in the grafts. The majority of these neurons had very short TH positive processes (60–70 um), indicating that the maturation of grafted dopaminergic neurons was still incomplete. Immunostaining for the human specific intermediate neurofilament (hNF, clone: BF-10) showed dense neuronal fibers in the grafts. These fibers extended deeper into the host brain than the TH positive neuronal processes. The whole striatum, particularly the medial part of the striatum, exhibited long NF positive processes. Glial fibrillary acidic protein (GFAP) immunohistochemistry revealed fine astrocytic processes inside the grafts, which were clearly different from host reactive glial cells surrounding the grafts. These graft-derived glial processes tended to extend into the host brain deeper than the TH positive neuronal processes from the grafts. These early histological findings of the grafted human fetal ventral mesencephalon suggest that the graft-derived NF positive neuronal processes, as well as the glial processes, radiate from the grafted tissue and extend into the host brain prior to the extension of TH positive processes. These results further suggest that human-to-rat xenografts can be used to study the neural development of human fetal brain tissue.

Image analysis of neuritic regeneration by adult rat dorsal root ganglion neurons in culture: quantification of the neurotoxicity of anticancer agents and of its prevention by nerve growth factor or basic fibroblast growth factor but not brain-derived neurotrophic factor or neurotrophin-3

Peripheral neuropathies are a common side effect of chemotherapeutic agents, particularly antineoplastic drugs such as taxol, cisplatin, or vinca-alkaloids (vincristine, vinblastine, vindesine). Using dissociated cultures of adult rat dorsal root ganglion (DRG) neurons and video image analysis after neurofilament immunostaining, we have designed a system that allows: (i) rapid screening of potential neurotoxic agents, with the establishment of dose-response curves and the calculation of IC 50; (ii) quantification of neurotrophic effects; and (iii) demonstration of neuroprotection by trophic factors. In particular, we show that nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) stimulate in vitro neuritic regeneration by adult rat DRG neurons, while brain-derived neurotrophic factor and neurotrophin-3 lack such effects. Furthermore, 24 h of pretreatment by NGF or bFGF drastically decreases the neurotoxic effect of vincristine and cisplatin.

The pathological spectrum of diffuse axonal injury in blunt head trauma: assessment with axon and myelin stains

Although diffuse axonal injury (DAI) has been described as a major form of primary damage to the brain in blunt head injury, there has been no systematic study of the pathological changes in different regions of the brain. In this study, 22 cases of DAI were comprehensively examined histologically in the following areas : corpus callosum, internal capsule, superior cerebellar peduncles, cerebral white matter, fornix, rostral brain stem and globus pallidus, with a total of 17 standard blocks in each case. Sections were stained for axons with Glees and Marsland and neurofilament immunostaining and myelin with luxol fast blue and myelin basic protein immunostaining, and axonal retraction balls and myelin globoids were counted. Neurofilament immunostaining was superior to Glees and Marsland in both the positivity rates and the actual scores. Small myelin globoids were identified by the myelin stains, probably as a form of myelin damage secondary to axonal disruption. Such acute myelin damage was previously undescribed. There was no significant difference in both positivity rates and the scores obtained for luxol fast blue and myelin basic protein. Of all the regions of the brain examined, the internal capsule, corpus callosum and superior cerebellar peduncles yielded the highest counts of axonal balls as well as the highest incidences. It is recommended that in cases of DAI, these three regions of the brain should be examined most profitably with neurofilament immunostaining supplemented with a myelin stain.

Neurofilament reorganisation and neurofilament antigen redistribution in spinal motoneurones following retrograde axonal transport of diphtheria toxin.

Single unilateral injections of diphtheria toxin (DTX) into the external anal sphincter muscle or internal intercostal nerve of cat induced characteristic ultrastructural lesions in corresponding ipsilateral spinal motoneurones 6-8 days later. The chief neuronal lesion was a progressive disruption of Nissl body composition and organisation, which between days 8-19 post injection was accompanied by a progressive accumulation of neurofilaments in motoneuronal perikarya and dendrites. Some axons in the ipsilateral ventral horn became hypertrophied due to neurofilamentous accumulation. Related immunocytochemical investigations 6-35 days after injection of DTX revealed abnormal immunoreactivity intoxicated motoneurones for 200-kDa and 160-kDa phosphorylated neurofilament proteins, but not in contralateral motoneurones. By day 35 abnormal neurofilament immunostaining also occurred in ipsilateral and some contralateral interneurones but not contralateral motoneurones. Abnormalities of Nissl body endoplasmic reticulum, neurofilament organisation, and neurofilament protein immunostaining were identical after either intraneural and intramuscular injections of DTX, indicating abnormalities were attributable to toxicity and not injection-related axonal damage. Since DTX acts specifically in the soma to inhibit protein synthesis, neurofilament abnormalities are secondary to cytotoxicity and probably result from deficits in transference of existing partially phosphorylated neurofilaments to the axonal transport system, or axonal transport per se.

Expression of neurofilament proteins in the rat cerebellar cortex as a function of age: an immunohistochemical study

The present study was designed to assess changes in the expression of cytoskeletal proteins in the cerebellar cortex of rats of different ages using immunohistochemical techniques associated with image analysis. Male Wistar rats of 3 months (young), 12 months (adult) and 24 months (old) were used. Neuronal cytoskeleton was investigated using monoclonal antibodies to phosphorylated neurofilament (NF) proteins of high (H), medium (M) and low+H+M molecular weight (NF triplet). In young and adult rats in which the expression of phosphorylated NF immunostaining was similar, a dark-brown immunoreactivity was observed primarily in axons of the white matter and of basket neurons which surround Purkinje neurons and enter in the molecular layer. In adult rats a NF-H immunoreactivity was sometimes observed within the dendritic tree of Purkinje neurons. A significant decrease in the density of NF immunoreactivity involving the three different subunits investigated was found in the cerebellar cortex of old rats. This suggests that cytoskeletal abnormalities occur not only in senile dementia, but also in the so called ‘physiological aging’. The cerebellar cortex may represent an interesting model for evaluating age-dependent changes of cytoskeleton.

Unexpected presence of neurofilaments in axon-bearing horizontal cells of the mammalian retina

In several mammals only one of the two types of retinal horizontal cell, the axonless A-type, appears to express neurofilaments. Neurofilament immunostaining of rodent retinas reveals a horizontal cell plexus that has previously been interpreted as belonging to A-type cells. Our intracellular Lucifer yellow injections strongly suggest that there are no A-type horizontal cells in rat and gerbil. Counterstaining of dye-injected cellular structures with a neurofilament antibody directly shows that the axon terminal systems of the axon-bearing B-type horizontal cells contain neurofilaments. These unexpected findings explain and reinterpret the neurofilament plexus in rodent retinas. In contrast, Lucifer yellow injections in guinea pig retina reveal both A- and B-type horizontal cells, showing that horizontal cell types are not uniform among rodents. In the guinea pig retina both A-type cells and B-type axon terminal systems contain neurofilaments.

Disturbed myelinogenesis and recovery in hyperphenylalaninemia in rats: An immunohistochemical study

Chronic hyperphenylalaninemia (HPA) in rats has been used as an experimental model of the human inborn error of metabolism phenylketonuria (PKU). Impaired brain development in PKU and HPA is reflected in reduced myelin formation. We have used immunohistochemistry, with antibodies to cell-specific antigenic markers, to investigate the cellular basis of the hypomyelination in the corpus callosum and cerebral cortex of rats made hyperphenylalaninemic from Postnatal Days 3–17. The rats were then allowed to recover until Day 59. No effects were seen on the number and differentiation pattern of ganglioside G D3-expressing glial progenitors. Myelin basic protein and 2′3′-cyclic nucleotide 3′-phosphohydrolase (CNP) immunostaining demonstrated a reduction in myelin formation in the corpus callosum and subcortical white matter at 12 and 17 days postnatal. However, numbers of CNP + oligodendrocytes appeared normal throughout development. No reactive astrogliosis was seen at any stage. The intensity of axonal neurofilament immunostaining was reduced in the corpus callosum at 17 days. In layers II and III of the cortical gray matter there was an increase in the cell packing density and a concomitant decrease in cell body size. Myelination in the corpus callosum was rapid during the recovery period with no difference noted at Day 59. Axonal neurofilament staining also returned to normal in the corpus callosum. However, recovery became increasingly incomplete away from the corpus callosum into the cortical gray matter. Our data suggest a primary effect of HPA on axonal maturation with hypomyelination consequential upon this.

Cerebellar Ganglioglioma

A 14-year-old boy presented with cerebellar ganglioglioma manifesting as severe headache and confusion. Computed tomographic scans showed a huge, partly enhanced cystic cerebellar tumor. The tumor was totally removed. Histological examination disclosed glial cells and mature ganglion cells. The latter were identified by Nissl's staining and immunostaining for neurofilaments. Ganglion cells were present in the cerebellum and the surrounding subarachnoid space. This heterotopic growth of ganglion cells enabled a firm diagnosis of cerebellar ganglioglioma.

Abnormal perikaryal immunoreactivity to the phosphorylated heavy neurofilament unit in intracerebral basal forebrain transplants

Grafts of Embryonic Day 14–15 basal forebrain tissue (medial septal/diagonal band nuclei) were transplanted into an aspirative fimbria-fornix cavity or the hippocampus of young adult rats. After extended periods of survival (1 and 2 years) the grafts were examined with immunocytochemical probes to identify specific types of neurons and assess the (spatial) distribution of the phosphorylated heavy neurofilament protein. Subpopulations of the long-term transplanted neurons expressed immunoreactivity to choline acetyltransferase (CAT) and the low-affinity nerve growth factor receptor (192-IgG). Axons from the grafted neurons, visualized with the monoclonal antibody RT97 to the M r 200,000 phosphorylated neurofilament unit, were observed to extend over the surfaces of the brain and connect with the host hippocampus. In subgroups of neurons without apparent axonal connections to the hippocampus, a change from axonal to cell body RT97 immunoreactivity was evident. A population of these neurons with abnormal neurofilament immunostaining in the soma was simultaneously identified as cholinergic with the CAT antibody. These studies indicate that abnormal changes can develop in the cytoskeleton of neurons in long-term intracerebral septal transplants. Although the reasons for this type of neurofilament modification in the grafted neurons are unknown, inappropriate terminal connections may be an important factor in the expression of this cytoskeletal change.

Endocrine Cell Hyperplasia of the Uterine Cervix: A Precursor of Neuroendocrine Carcinoma of the Cervix?

A 33-year-old woman who presented with vaginal bleeding was diagnosed to have neuroendocrine small cell carcinoma based on cervical smear and biopsy. Hysterectomy was performed, and a tumor measuring 5.5 X 2 mm was found at the squamocolumnar junction of the uterine cervix. In the immediate vicinity of the tumor, there was proliferation of cytologically benign endocrine cells in the normal endocervical glands and in the glands showing intraepithelial glandular neoplasia. Both the hyperplastic endocrine cells and the invasive tumor cells showed argyrophilia and immunostaining for neuron-specific enolase, neurofilament, and chromogranin. The topographical relationship suggests that endocrine cell hyperplasia may represent a precursor of neuroendocrine carcinoma of the cervix.

Immunohistochemical Spectrum of Rhabdomyosarcoma and Rhabdomyosarcoma-like Tumors

Twenty-five rhabdomyosarcomas (RMSs), including 12 alveolar and 13 embryonal types, were immunohistochemically studied for the presence of different classes of intermediate filament proteins and muscle actins (MAs). For the most part, formaldehyde-fixed and paraffin-embedded tissue was used in immunostaining. All RMSs showed desmin and MAs, usually in a major portion of tumor cells. The number of MA-positive cells was sometimes higher than that of desmin-positive cells. Vimentin was present in all tumors studied in frozen sections. Eight of 12 alveolar RMSs showed small number of cytokeratin-positive neoplastic cells. Cytokeratin-positive cells were present less commonly in embryonal RMS (3/13 cases). The 68-kD neurofilament protein was found in frozen sections of two embryonal RMSs. The cytokeratin and neurofilament immunostaining could be reproduced by immunofluorescence technique. In addition, we studied three childhood sarcomas, which showed abundant desmin and MA immunostaining but did not conform to the ultrastructural criteria of RMS. Scattered cytokeratin-positive cells were found in two of these tumors, and neurofilaments were found in the two cases for which frozen sections were available. The results show that typical RMS may demonstrate immunohistological pleomorphism with cytokeratin and neurofilament immunoreactivity suggesting the presence of multidirectional differentiation. In addition, there are tumors that by morphology look like RMS and have muscle cell markers but cannot be verified as RMS by electron microscopy; also, these tumors seem to show immunohistological pleomorphism. The presence of nonmyoid markers in RMS should be considered when making immunohistological diagnosis of soft tissue sarcomas.

Microglial and neural differentiation in human teratomas.

Ten human teratomas arising outside the central nervous system (CNS) were studied using a panel of immunohistochemical, and lectin histochemical stains to determine the relationship of the presence of microglia to markers of neural maturity or differentiation. Microglia, identified by silver carbonate, Ricinus communis agglutinin-1 (RCA-1), or both were found in eight out of ten cases. They were common in mature areas which also had S-100, glial fibrillary acidic protein, vimentin, neurofilament, and synaptophysin immunostaining. Microglia were distinguished from macrophages in necrotic foci. Cells which were RCA-1 positive and silver carbonate positive were found in immature neural tissues but these lacked all typical features of microglia. These observations indicate that true microglia are frequent in nonCNS teratomas and that they are found in association with other indicators of neural maturation. The presence of possible precursors in immature areas suggests that microglia undergo maturation concurrent with neural differentiation in these tumors.

Neurons cultured from developing rat brain attach and spread preferentially to laminin.

Neurons, mechanically dissociated from newborn rat brain and identified by immunostaining for neurofilaments, attached preferentially to laminin-coated coverslips without need of an underlying glial monolayer. The most extensive neurite outgrowth was seen when 20-30 micrograms/ml of laminin was used to coat the coverslips. Higher concentrations of laminin (greater than 30 micrograms/ml) supported single neurons to spread on the coverslips. Fibronectin coating of the coverslips allowed glial cells to attach more rapidly than on uncoated surfaces, but it did not support neuronal spreading or neurite outgrowth. Spreading of neurons and neurite outgrowth were completely inhibited by preincubation of laminin-coated coverslips with laminin antibodies but were unaffected by fibronectin antibodies. These results indicate that laminin is an attachment and spreading factor for central neurons in culture and suggest the presence of a laminin receptor on the neuronal cell surfaces.