Anti-neurofilament Antibody Research Articles

Fibrin sealant matrix supports outgrowth of peripheral sensory axons.

It has been suggested that fibrin-based matrix has an important role during the early stage of nerve regeneration. A fibrin sealant matrix, which was made by combining diluted human fibrinogen and thrombin, was used as a substrate for in vitro elongation of neurites and in vivo regeneration of axons. In the in vitro experiment, dissociated embryonic chick sensory neurons were cultured on dishes coated with fibrin sealant matrix and compared with the solution of thrombin/calcium chloride, or with poly-D-lysine (PDLctr). After 16 hours, cultures were stained immunohistochemically with a monoclonal antineurofilament antibody. The neurons survived well, and an abundant network of neurites, qualitatively similar to that on PDLctr, developed on the fibrin sealant matrix. The percentage of neurons that had outsprout at 16 hours was the same both in the fibrin sealant matrix and PDLctr groups. By contrast, all the neurons plated on the dishes treated with the solution of thrombin/calcium chloride were dead after 16 hours. Immunohistochemical staining of fibrinogen also showed an even distribution of fibrin matrix over the culture dishes. For the in vivo experiments, 48 rat sciatic nerves were cut and reconnected with two epineurial stitches. Fibrin sealant matrix or phosphate buffer solution was applied to the transsected and repaired region. Pinch reflex test showed that the regeneration of the leading sensory fibre was significantly faster in the fibrin sealant matrix group than in the control group at 3 and 4 days. These results indicate that fibrin sealant matrix accelerates regeneration of axons in vivo during the early phase, and also supports elongation of neurites in vitro.

Horizontal cells in the rabbit retina: Differentiation of subtypes at neonatal and postnatal stages

We are investigating the differentiation of the major subtypes of horizontal cell in the rabbit retina in order to learn more about developmental controls responsible for the variety of neuronal phenotypes. Immunohistochemistry with anti-neurofilament and anti-calbindin-D antibodies, followed by epoxy resin embedding, has facilitated study of these neurons. In the mature rabbit retina, axonless (A-type) horizontal cells reacted strongly in procedures using either antibody; short axon (B-type) somas did not show a reaction with anti-neurofilament antibodies and stained moderately using anti-calbindin antibodies. In the immature neonatal retina the somas of all the horizontal cells seemed to be similar with regard to general morphology, but two populations could be distinguished on the basis of immunostaining. Some, identified as A-type horizontal cells (by comparison with mature retina), were stained using either antibody. Interspersed among these were similar cells with no detectable immunoreactivity, identified as B-type horizontal cells. By the end of the first postnatal week, faint reactivity to anti-calbindin-D was present in the somas of B-type horizontal cells; they stained moderately throughout the rest of the period studied.Thus differences in immunostaining indicate that the two horizontal cell subpopulations are established early in the rabbit, though some other distinguishing characteristics emerge only gradually as the retina matures. These results suggest that in mammals the determination of phenotypic subtype occurs early, possibly at the time that the cell is specified as a horizontal neuron, or shortly thereafter.

Immunocytochemical properties of rat renal afferent neurons in dorsal root ganglia: A quantitative study

Immunocytochemical properties of dorsal root ganglion neurons innervating the kidney were studied with retrograde tracing of Fluorogold or Fast Blue dyes applied to the cut renal nerves in the rat. The proportions and sizes of renal afferent neurons labelled with a variety of markers were quantified in T9-L1 dorsal root ganglia from five rats. Compared with the overall size distribution in these ganglia, renal afferent neurons were mainly small with a few medium-sized neurons. The majority (79%) of renal afferent dorsal root ganglion neuronal somata were unlabelled by an anti-neurofilament antibody, RT79, and classified as neurofilament-poor with probable C-fibres. These had an approximately normal distribution of cell sizes. Only 21% were RT79-positive and classified as neurofilament-rich with probable A-fibres, and even these were small to medium sized cells, consistent with them being mostly Aδuˆfibre neurons. Percentages of renal afferent neurons showing labelling were as follows: peripherin-like immunoreactivity: 69%; calcitonin-gene related peptide: 93%; substance P: 37%; the lectins soybean agglutinin: 57% and peanut agglutinin: 68%; Calbindin D 28k-like immunoreactivity: 21% (only weak labelling); carbonic anhydrase like immunoreactivity: 0%. There were differences between probable C-fibre and probable A-fibre neurons, classified according to their labelling with RT97. The percentages of RT97-negative and RT97-positive neurons respectively labelled with the other markers were as follows: peripherin-like immunoreactivity: 82%, 25%; calcitonin gene-related peptide-like immunoreactivity: 99%, 79%; substance P-like immunoreactivity: 43%, 0%; soybean agglutinin: 69%, 24%; peanut agglutinin: 76%, 47%; calbindin-like immunoreactivity: 26%, 0%. Thus, the biggest differences between the probable A-and C-fibre renal afferent neurons were in their peripherin, substance P and calbindin contents. Thus, renal afferent neurons in the dorsal root ganglion are not homogeneous and it is suggested the differences may relate to the known different afferent receptor types within the kidney. It is suggested that the low proportion of neurons with substance P-like immunoreactivity in the renal afferent innervation compared to that of other viscera may relate to the role of the renal vasculature in urine formation.

Neuronal differentiation in vitro from precursor cells of regenerating spinal cord of the adult teleost Apteronotus albifrons.

This study documents neuronal differentiation in vitro from undifferentiated precursor cells of caudalmost regenerating spinal cord of the teleost Apteronotus albifrons. At 11 days in vitro, cells from the caudalmost tip of the regenerating cord are flat and polygonal in shape, lack neuronal processes and do not stain with antibody against neuron-specific filaments. At 15 days in vitro, some of the caudalmost cells have developed short, neurite-like processes; at 18 days in vitro, some cells react positively with antibody against neuron-specific filaments. At 26 days in vitro, many of the caudalmost cells have long branching neurites and react positively with anti-neurofilament antibody. Addition of insulin-like growth factor-I to the medium accelerates the process of neuronal differentiation from the caudalmost precursor cells in vitro. The source of these precursor cells is ultimately cells of the ependymal layer of adult spinal cord. Further investigation of the factors that control production and differentiation of these cells will be important in defining the developmental potential possible for vertebrate spinal cord cells and may aid in creating an optimal environment for regeneration of axons within mammalian spinal cord.

Cholera toxin beta subunit induces the differentiation of human medulloblastoma cell line DEV in a neuronal pathway.

Medulloblastomas are primitive neuroectodermal tumours that are thought to arise from multipotent precursor cells in the cerebellum. Medulloblastoma cells may be undifferentiated or exhibit glial, neuronal or ependymal characteristics, suggesting that they may conserve their ability to differentiate in appropriate circumstances. Medulloblastoma cell lines may thus provide models to study the commitment and differentiation of multipotent CNS progenitor cells. A human medulloblastoma cell line, DEV, has previously been shown to differentiate in an astrocytic pathway after infection by the retrovirus HTLV-1. In this study immunofluorescence flow cytometry shows that cholera toxin beta subunit (CT beta), which binds to the ganglioside GM1, induces a twofold increase in the number of DEV cells differentiating towards a neuronal pathway, as shown by the increased proportion and labelling intensity of cells stained by an anti-neurofilament antibody. Immunocytochemistry shows that after 3 days in culture with CT beta, DEV cells develop processes which stain positive for neurofilaments and MAP-1. This suggests that CT beta induces DEV cells to express a more neuronal phenotype.

Substance P, neurofilament, peripherin and SSEA4 immunocytochemistry of human dorsal root ganglion neurons obtained from post-mortem tissue: a quantitative morphometric analysis.

Immunocytochemical studies on lumbar dorsal root ganglia obtained at routine postmortem 24-36 h after death were carried out, and neuronal cross-sectional areas measured. The subjects were elderly (76-81 years), of both sexes, had died from heart attack or haemorrhage, and had no clinical evidence of clinical neuropathy or of disease known to be associated with neuropathy. The data were consistent between ganglia from the three subjects. There were striking similarities with data from other species. Two populations of cell profiles with overlapping size distributions were distinguished with an anti-neurofilament antibody, neurofilament-rich (45% of cell profiles) with a large mean area and neurofilament-poor with a smaller mean area. Anti-substance P and anti-peripherin antibodies both labelled a population with a small mean area, with extensive co-localization between them. There were also some differences between these human dorsal root ganglia and dorsal root ganglia from some other species. More neuronal profiles were labelled for substance P in humans (44%) than in rat (20%). More neuronal profiles were labelled for SSEA4 (stage specific embryonic antigen 4) in human (40.5%) than in rat dorsal root ganglia (10%), and the SSEA4-positive profiles were relatively smaller in human than in rat. No selective accumulation of lipofusin in profiles of large cells was apparent. This study also shows that quantitative morphometric analysis of immunocytochemically labelled dorsal root ganglion neuronal profiles can be carried out successfully on human sensory ganglia obtained at post-mortem. This is the first demonstration of the two main subgroups of dorsal root ganglia neurones with neurofilament-rich and poor somata in human tissue. The size distributions of neurons with neurofilament, substance P and peripherin are consistent with these neuronal populations having similar functional properties to those described in other species. From the known sensory and fibre loss with aging, it is speculated that the loss of some large diameter neurones with myelinated fibres and low mechanical thresholds, might account for the high percentage of neurones expressing substance P.

Confocal Microscopic Analysis of Integrin Expression on the Microvasculature and its Sprouts in the Neonatal Foreskin

Members of the integrin family of adhesion receptors are essential participants in blood vessel growth and remodeling. It is not known which integrins are involved in the initial stages of angiogenesis in vivo. In this study we determined the location of integrins on the blood vessels of a growing tissue, the neonatal foreskin, in which neovascularization is likely to occur. We used the confocal microscope to visually reconstruct vessels from the papillary dermis of the foreskin and to identify potential sprouts as narrow, tapering extensions from these vessels. Blood vessels were initially identified by their positive reaction with antibodies to von Willebrand factor or human platelet endothelial cell adhesion molecule and their negative response to anti-neurofilament antibodies. Later, vessels were identified by their shape and location. We screened vessels with anti bodies to integrin subunits alpha 1, alpha 2, alpha 3, alpha 5, alpha 6, alpha v, beta 1, beta 3 and beta 4. We found that integrin subunits alpha 6 and beta 4 were consistently found along the whole length of capillary loops and extended to the distal ends of presumed sprouts. The alpha 2 and alpha v integrin concentrations, which are normally low in the microvasculature, were increased on the sprouts. alpha 5 was either absent from vessels entirely or more concentrated on the body than on the sprout. alpha 1 was more commonly present on nerves than blood vessels. These studies suggest an important role for the alpha 6 beta 4 integrin in the initial stages of endothelial outmigration during new vessel growth.

Antibodies to different isoforms of the heavy neurofilament protein (NF-H) in normal aging and Alzheimer's disease.

Sera of normal controls and of patients with neurological diseases contain antineurofilament antibodies. Recent studies suggest that biochemically and immunologically distinct subclasses of neurofilaments occur in different types of neurons. Alzheimer's disease (AD), the major cause of dementia, is associated with a marked degeneration of brain cholinergic neurons. In the present work we characterized the repertoire and age dependence of antineurofilament antibodies in normal sera and examined whether the degeneration of cholinergic neurons in AD is associated with serum antibodies directed specifically against the neurofilaments of mammalian cholinergic neurons. This was performed by immunoblot assays utilizing neurofilaments from the purely cholinergic bovine ventral root neurons and from the chemically heterogeneous bovine dorsal root neurons. Antibodies to the heavy neurofilament protein NF-H were detected in normal control sera. Their levels were significantly higher in older (aged 70-79) than in younger (aged 40-59) subjects. These antibodies bound similarly to bovine ventral root and dorsal root NF-H and their NF-H specificity was unchanged during aging. In contrast, the levels of IgG in AD sera that are directed against ventral root cholinergic NF-H were higher than those directed against the chemically heterogeneous dorsal root NF-H. Immunoblot experiments utilizing dephosphorylated ventral root and dorsal root NF-H and chymotryptic fragments of these molecules revealed that AD sera contain a repertoire of antimamalian NF-H IgG. A subpopulation of these antibodies binds to phosphorylated epitopes that are specifically enriched in ventral root cholinergic NF-H and that are located on the carboxy terminal domain of this molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Antimyenteric neuronal antibodies in scleroderma.

The pathogenesis of gastrointestinal (GI) dysmotility in scleroderma is incompletely understood, although previous studies have proposed a neuropathic mechanism. We studied patients with scleroderma as compared with other connective tissue disease patients and normal controls for the presence of circulating antibodies to myenteric neurons. Serial dilutions of sera were overlaid on rat intestine, double-labeled with antineurofilament antibody as a myenteric plexus marker, and imaged using indirect immunofluorescence techniques. High titer sera (> or = 1:50) from 19 out of 41 scleroderma patients stained myenteric neurons, whereas none of 22 normals or 5 patients with idiopathic GI dysmotility were positive. Although 6 out of 20 SLE and 6 out of 10 mixed connective tissue disease patients' sera stained myenteric plexus neurons, when positive sera were absorbed with calf thymus extract to remove antinuclear antibody, 15 scleroderma sera, 0 SLE, and 2 mixed connective tissue disease patients retained positive staining of myenteric neurons. Western blotting using actin and neuronal intermediate filament preparations failed to show immunoreactivity with scleroderma sera containing antimyenteric neuronal antibodies. Paraneoplastic sera associated with GI dysmotility stained myenteric neurons in a different pattern than seen with scleroderma sera. A positive correlation between the presence of Raynaud's phenomenon and antimyenteric neuronal antibodies was observed in scleroderma patients. Our results indicate that IgG antibodies reacting with myenteric neurons are present in many patients with scleroderma. Although the neuronal antigen has not yet been identified, the presence of myenteric neuronal antibodies in patients with GI dysmotility and scleroderma suggests a neuropathic process.

Immunocytochemical and ultrastructural studies of the motor cortex in amyotrophic lateral sclerosis.

This report concerns an immunocytochemical and ultrastructural study of the motor cortices of 11 patients with amyotrophic lateral sclerosis (ALS). Specimens from 12 normal individuals served as controls. Antibodies against phosphorylated neurofilament (PNF; 200 kDa), ubiquitin, glial fibrillary acidic protein (GFAP) and phosphorylated tau protein were used. The pyramidal cells of layer III of all ALS patients were stained, with varying intensities, by the antibody to PNF. By contrast, Betz cells reacted less frequently with this antibody. Staining for GFAP was noted in numerous astrocytes in layer III and at the transition between white matter and motor cortex of most patients. Ubiquitin-positive inclusions were only occasionally seen in Betz cell and pyramidal cell of layer V. These observations indicate that alterations of the motor cortex occur first in the pyramidal cells of layer III rather than in Betz cells. Pyramidal cells and Betz cells were not stained by the antibody to phosphorylated tau protein. In controls, pyramidal cells and Betz cells were less frequently stained with the anti-neurofilament antibody than those from ALS patients. Immunoreactivity of GFAP in layer III and at the junction of white matter and motor cortex was observed in only one patient. Ultrastructural examination revealed that the Betz cells of some ALS patients had Bunina bodies (BB), Lewy body-like inclusions (LBI) and skein-like inclusions (SI), as well as bundles of filaments that were thicker than neurofilaments; some of these filaments appeared to be constricted. The incidence of these inclusions was lower than that seen in anterior horn neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Terminin (Tp 63/60), a novel cell senescence-related protein, is present in the aging human hippocampus

We have previously identified a cell senescence-related, detergent-insoluble cytoplasmic protein referred to as terminin, and have presented the development-dependent modification of terminin polypeptides in rat brain. In the present study, we characterize biochemically and immunocytochemically terminin expression in human hippocampus. By immunoblotting with a monoclonal antibody (mab 1.2) against terminin, we identified a 63 kDa polypeptide (Tp63) in the detergent-insoluble fraction of human brain proteins, which can be identified by either one- or two-dimensional electrophoresis. The 63 kDa protein in immunoprecipitates of human hippocampus was found to share a peptide pattern with the 60 kDa terminin (Tp60) previously found in rat brain, as demonstrated by in situ peptide mapping, suggesting that the human 63 kDa protein is a homologue of the rat Tp60. By immunocytochemistry, terminin immunoreactivity was demonstrated throughout the neuronal layers in the human hippocampus. The pyramidal cell layer in hippocampus proper and subiculum, as well as the granule cells in the dentate gyrus, gave rise to immunoreactivity for terminin. It was observed that in the cornu Ammonis, the highest terminin immunoreactivity was present in the pyramidal neurons of CA1. At the cellular level terminin was localized intraneuronally, as demonstrated by confocal microscopy of double staining with both mab 1.2 and an anti-neurofilament antibody. Taken together, the present study establishes that terminin is expressed in abundance in aging human CNS neurons.

Neuroendocrine carcinoma of the skin (Merkel cell carcinoma). An immunoelectron-microscopic case study.

An unusual tumor with a controversial name as well as histogenesis, the neuroendocrine carcinoma of the skin (also known as "Merkel cell carcinoma," "trabecular carcinoma of the skin") has previously been extensively studied by immunohistochemical methods at the light-microscopic level. Ultrastructural descriptions of this tumor have also been extensive, although immunocytochemical study of this neoplasm at the electron-microscopic level has been limited. In this report, we have used postembedding protein A-gold immunocytochemistry on thin sections from tumor embedded in Lowicryl K4M to investigate the expression and ultrastructural localization of a panel of commercially available, diagnostically useful antibodies. Antibodies associated with epithelial derivation included anti-keratin monoclonal antibody AE1/AE3, polyclonal anti-keratin, and monoclonal anti-cytokeratin cocktail (MAK-6), as well as a monoclonal antibody against epithelial membrane antigen (EMA). Antibodies associated with neuroendocrine derivation included monoclonal anti-chromogranin A and monoclonal anti-synaptophysin. Although staining with a polyclonal antibody directed against neuron-specific enolase (NSE) was equivocal, there was no labeling with a monoclonal anti-neurofilament antibody. The finding of positive keratin labeling of filaments arranged in paranuclear aggregates correlates well with the previously described immunohistochemical staining pattern at the light-microscopic level. Moreover, the presence of cytoplasmic synaptophysin and chromogranin positivity over dense-core granules exemplifies the neuroendocrine differentiation present in this fascinating tumor of the skin.

Co-twin control study on cerebral manifestations of systemic lupus erythematosus

All available twin pairs systemic lupus erythematosus (SLE) derived from the Finnish Twin Cohort were studied by clinical evaluation, magnetic resonance imaging (MRI), anticardiolipin (aCL), and antineurofilament (ANFA) antibodies. One of the five monozygotic and one of the eight dizygotic pairs were concordant for SLE. 10 of the 15 patients showed clinical neurological abnormalities, and 11 had abnormal MRI of the brain. Altogether, 12 patients were considered to have neuropsychiatric lupus (NPSLE). Seven of the 11 patients with long-term corticosteroid treatment had either central or cortical atrophy. High or moderate aCL level was found in eight patients and two co-twins. Of them, six patients had at least two manifestations of the antiphospholipid syndrome. ANFAs were found in five patients and four co-twins. Five co-twins fulfilled some of the SLE criteria. Of them, three MZ twins and one additional DZ co-twin with no ARA criteria had findings suggesting central nervous system (CNS) involvement. The results indicate that the majority of SLE patients has cerebral abnormalities either as a result of SLE, or concomitant risk factors. The co-twins without clinical SLE often have minor signs of SLE, and even they may have neurological and MRI abnormalities. However, their aCL and ANFA levels seem not to correlate with MRI abnormalities.

Visceral neuropathy in slow transit constipation

The aim of this study was to investigate neuropathologic changes in the colonic wall of patients with slow transit constipation using monoclonal antibodies raised against neurofilament. In a prospective study, 227 patients with severe, long-standing constipation and intractable defection disorders were analyzed according to a standard protocol. Slow transit constipation was diagnosed in 65 patients (29 percent). Forty-three patients (7 men and 36 women; mean age, 46 years; range, 16-76 years) underwent a partial (n = 20) or subtotal (n = 23) colectomy. In 39 patients (5 with megacolon and 34 with normal-sized colon) the cause of their constipation remained unexplained (idiopathic slow transit constipation). All resected colon specimens were investigated with the monoclonal antineurofilament antibody NF2F11 and compared with those of 20 control patients. In all controls the myenteric plexus revealed a moderate and diffuse axonal staining. In 29 of 39 patients with "idiopathic" slow transit constipation, the apparently normal axon bundles in the myenteric plexus stained markedly less than normal or failed to stain at all with the monoclonal antibody. In 17 patients this reduced or absent neurofilament expression was found along the entire length of the colon, whereas in 12 patients only a portion of the colon was affected. These findings indicate that a visceral neuropathy seems to be present in the majority of patients with severe, so-called idiopathic slow transit constipation.

Neurotrophic action of gliostatin on cocultured neurons with glial cells

Gliostatin is a polypeptide factor (apparent M r = 100k with a homodimeric structure comprising two 50 kDa subunits) acting on cortical neurons (neurotrophic action) as well as astrocytic cells (growth inhibition). Under the coculture system of cerebral cortical neurons and astrocytes from fetal rats (E15 or E16), the neurotrophic action of gliostatin was examined immunocytochemically. Immunostaining by an anti-neurofilament (NF) monoclonal antibody visualized a marked neurite-outgrowth and interconnecting bundles of neuritic processes induced by gliostatin in the coculture system. Neurons stimulated by gliostatin formed dense aggregates in clumps, while neurons in control coculture spread out. Gliostatin has also shown survival-promoting effects on neurons. Furthermore, it was shown that gliostatin induced the differentiation of protoplasmic astrocytes to fibrous astrocytes. These results further support our previous contention that gliostatin plays physiological roles on neuronal and glial development.

Tissue fixation methods alter the immunhistochemical demonstrability of neurofilament proteins, synpatophysin, and glial fibrillary acidic protein in human cerebellum

This study has examined the effect of postmortem autolysis, type, and duration of fixation on neurofilament, synaptophysin, and glial fibrillary acidic protein (GFAP) antigen decay as demonstrated by immunohistochemistry, using a streptavidin-biotin peroxidase method. The system used consisted of 5 normal cerebellar cortices. Time intervals, temperature, mode of fixation and storage, and staining technique were well controlled. Anti-neurofilament antibodies comprised SMI-31, MNF, and BF-10 against phosphorylated epitopes, and SMI-32 against a non-phosphorylated epitope. Bouin's and B5 fixative, and Sensofix gave best results, whereas formaldehyde and paraformaldehyde fixation gave much lower immunoreactivity. Phosphorylated neurofilament epitopes were less affected by aldehydes than unphosphorylated epitopes. GFAP staining was most consistent after Bouin fixation while the monoclonal antibody was much more sensitive to the fixative used than the polyclonal one. Aspecific background staining increased considerably after a postmortem interval of 24 hours. Synaptophysin immunoreactivity, as demonstrated by SY-38, proved very sensitive to prolonged fixation and was of poor quality following formaldehyde and paraformaldehyde fixation. Knowledge of antigen decay due to postmortem artifacts is essential for the correct evaluation of immunoperoxidase studies of autolyzed tissues that have been fixed and stored in different modes and for variable time interval.

Identification from a subtracted CDNA library of novel genes associated with differentiation of cytotrophoblast in vitro

Binding sites for iodinated endothelin (ET)-2, ET-3 and vasoactive intestinal contractor (VIC) were visualised in the adult rat brain using quantitative autoradiography and have a similar anatomical distribution to that of ET-1 and sarafotoxin S6b. Highest densities of binding sites for all 5 labelled peptides were present in the granular layer of the cerebellum. Cross-competition experiments show that at a concentration of 1 μM, unlabelled ET-1, ET-2, ET-3, VIC and sarafotoxin S6b were able to compete for the binding sites detected by each of the iodinated peptides. Binding sites for the ET isoforms were also present after 7–14 days in vitro in neurone-enriched primary cultures derived from embryonic rat cerebellum (16–18 days gestation) in which more than 90% of cells stained with an anti-neurofilament antibody. Using micro-autoradiography to detect the binding sites, an average of 14% of cells in these cultures with a diameter of9.2 ± 0.6 μm were associated with high silver grain densities (> 400grains/100 μm2). With some of these cells, silver grains were localised over cell bodies and branching processes characteristic of a neuronal phenotype. A second group of cells with high grain densities were more difficult to classify using morphological criteria and may be non-neuronal. The density of silver grains over the remaining cells was low(< 20grains/100 μm2) and was similar to that measured in nuclear emulsion overlying cultures used to assess non-specific binding. These results indicate that binding sites for all ET peptides are present in both adult rat brain and embryonic cerebellar cultures. In these cultures, binding does not appear to be confined to a single class of cells but may be present on both neuronal and non-neuronal cells. The results suggest that one or more of the ET isoforms could have actions in the rat CNS in addition to the proposed vasoactive role in the peripheral circulation.

Immunocytochemical analysis of a novel carbohydrate differentiation antigen (CDA‐3C2) associated with olfactory and otic systems during embryogenesis in the rat

Carbohydrate differentiation antigens are known to display specific patterns of expression during mammalian development and are thought to participate in significant morphogenetic events. In the present study, two monoclonal antibodies that react with a novel carbohydrate differentiation antigen (CDA-3C2) were used to analyze, by light microscopy, the spatiotemporal distribution of this unique high molecular weight antigen during embryogenesis in the rat. Correlative analysis of the development of peripheral neural structures, in which CDA-3C2 was expressed, was carried out with an anti-neurofilament antibody. Enzymatic digestion, combined with Western blots, reveal that the CDA-3C2 epitope is a carbohydrate which is carried on a high molecular weight glycoprotein with a mass of greater than 1 million Daltons. Characteristic of carbohydrate antigens, immunoreactivity was found in several distinct cellular patterns: only along the apical border of cells, along lateral and basal membranes of cells, and extracellular-like staining in the mesenchyme. During neurulation, CDA-3C2 showed differential staining in the ectoderm, distinguishing lateral from neural regions. Following closure of the neural tube, there was a striking specificity of expression of CDA-3C2 in the periphery, found almost exclusively in olfactory and otic epithelial structures. While CDA-3C2 is found in placode-derived tissues that subserve sensory transduction, it appears to be primarily associated with the supportive cells (and their secretions) in both otic and olfactory regions and less so with the sensory cells. The data suggest that a unique carbohydrate antigen on a large macromolecule may play a role in neurulation and/or morphogenesis of the placode-derived otic and olfactory structures.

Neuropathologic changes in the gerbil brain after chronic hypoperfusion.

An animal model has been developed to elucidate the pathological changes in brain cytoskeletal proteins during chronic hypoperfusion. Newly designed coiled clips were placed around both carotid arteries of Mongolian gerbils (n = 10) to cause stenosis without occlusion. Those gerbils showing impaired learning ability by the passive avoidance paradigm were killed for neuropathologic study after 12 weeks. The brains showed ventricular dilatation, cortical atrophy, and rarefaction of the white matter. Immunoreactivity to anti-microtubule-associated protein 2 antibody in the cerebral cortex and the hippocampus was diminished, indicating dendritic changes of neurons. In the thalamic axonal regions, staining with anti-neurofilament 200K protein antibody was increased, suggesting increased amounts of neurofilament proteins or increased phosphorylation of the protein. Increased immunoreactivity to anti-glial fibrillary acidic protein antibody was observed in a wedge-shaped configuration, corresponding to the border zone of perfusion by small vessels. These findings suggest that changes in the cytoskeletal proteins in dendrites, axons, and glial cells may cause neuronal death under conditions of chronic cerebral hypoperfusion.

Double-labeling immunohistochemical studies on canine senile plaques and cerebral amyloid angiopathy.

The relationship of senile plaques to neuronal cells, neurites, glial cells, or capillaries was examined using double labeling-immunostaining methods on the Bouin's solution-fixed serial brain sections from dogs. Compact deposits of beta protein (amyloid plaques) in the cerebral cortex always contained microvessels labeled by anti-collagen type IV antibody and some of them might be formed as the result of fusion of several perivascular beta amyloid deposits. In the periphery of those plaques swollen neurites recognized with anti-neurofilament antibody were sometimes present, but the relation between such plaques and neuronal cells or glial cells were unclear. Diffuse deposition of beta protein (diffuse plaques) was frequently developed beside neuronal cells, while most plaques did not contain glial cells. Some of those plaques were closely contact with microvessels, but some had no relation. Intact or irregularly arranged neurites were present in diffuse plaques. Such irregularity of the neurites were obvious in the plaques in the hippocampus as compared with those in the cerebral cortex. These results indicate the possibility that canine amyloid plaques would be formed as the result of amyloid degeneration of cortical capillaries, and diffuse parenchymal deposition of beta protein would originate from neuronal or neuritic processes.