Search

Along with subplate neurons, Cajal-Retzius cells (CRc) are the first neurons to be generated in the cortical anlage. Studies of their chemical content, such as neurofilament and calcium-binding proteins, might give indications on their role in cortical development at midgestation in human fetuses (20-24 gestation weeks), when the CRc are morphologically mature. Cajal-Retzius cells were immunolabeled with antibodies to subunits of neurofilament proteins SMI31 and SMI32. The SMI32 antibodies (directed against the nonphosphorylated epitope) specifically labeled the CR cell bodies, dendrites, and proximal axons in a Golgilike fashion. Specific acetylcholinesterase activity is known to be typical of all the CRc, and double labeling for SMI32 immunoreactivity and acetylcholinesterase histochemistry demonstrated that all the CRc exhibited SMI32 immunoreactivity. The SMI31 antibodies (directed against the phosphorylated epitope) exclusively labeled the CRc axons, forming a dense positive network in the deep one-half of layer 1. This plexus was much denser than the one described with the Golgi method (Marin-Padilla, 1990: J. Comp Neurol 239:89-105). Calbindin D28k, parvalbumin, and calretinin immunoreactivities were visualized in the CRc. Double-labeling experiments showed that most of the CRc contained both calbindin and calretinin and sometimes parvalbumin. These colocalizations revealed a chemical heterogeneity within the CRc population even though they were described as morphologically homogeneous. These colocalizations of calcium-binding proteins in the CRc differed from the other nonpyramidal cortical neurons where calbindin, calretinin, and parvalbumin are contained in different (mutually exclusive) neuronal populations. Based on the morphological features and differential chemical contents described for the CRc, different hypotheses on their possible role and fate are discussed.

SMI-32, an antibody against a non-phosphorylated neurofilament epitope identifies a subpopulation of human cortical neurons preferentially lost in Alzheimer's or Huntington's disease. In murine cortical cultures SMI-32 labeled a small subset of neurons exhibiting enhanced vulnerability to kainate toxicity. Most SMI-32(+) neurons were GABAergic and exhibited kainate-activated Co2+ uptake. Thus expression of Ca2+ permeable AMPA or kainate receptor-gated channels likely underlies the heightened vulnerability of SMI-32(+) cortical neurons to kainate.

NB2/dl neuroblastoma cells acquire a neuronal phenotype in response to several differentiating agents, including dibutyryl cAMP (dbcAMP) and the withdrawal of serum. As shown previously, antibodies to the growth-associated protein, GAP-43, introduced intracellularly using a lipid carrier, blocked the differentiation induced by dbcAMP. Antibodies to GAP-43, at a low concentration, also blocked neurite outgrowth induced by serum withdrawal when cells were grown on a relatively unadhesive substrate. On more adhesive substrates such as poly-L-lysine and laminin, however, anti-GAP-43 antibodies had less of an effect on neurite outgrowth. Previous studies have shown that the increased adhesivity of laminin allows a small but significant population of neurites to grow from serum-deprived cells, even in the presence of the microtubule-depolymerizing drug, colchicine. The outgrowth of this population of neurites was blocked by antibodies to GAP-43. These results are in conformity with recent studies showing that the requirement for GAP-43 in neuritogenesis may be related to membrane adhesiveness, and may contribute to an understanding of some of the apparent discrepancies in the literature concerning the involvement of GAP-43 in neuronal differentiation.

The distribution of neurofilament proteins and peripherin in the pituitary gland of the rat was studied with a panel of monoclonal and polyclonal antibodies recognizing different neurofilament subunits. In the posterior lobe, a dense plexus of neurofilament- and peripherin-immunoreactive fibers was seen. In the intermediate lobe neurofilament- and peripherin-immunoreactivity was seen only in nerve fibers in the connective tissue septa, while no immunoreactivity was seen in parenchymal nerve fibers. Bilateral sympathetic ganglionectomy did not affect peripherin-immunoreactivity, indicating that the peripherin-immunoreactive fibers are of central origin. In the anterior lobe, a few solitary neurofilament- and peripherin-immunoreactive fibers were observed. Western blotting confirmed the presence of 150 kD and 200 kD neurofilament proteins in the posterior lobe. No neurofilament protein was detected in the intermediate and anterior lobes. Abundant intermediate filaments were seen with electron microscopy in the nerve fibers of the connective tissue septa in the intermediate lobe. In the parenchymal nerve fibers only microtubules were seen, indicating that the lack of neurofilament immunoreactivity is due to absence of neurofilaments.

A panel of monoclonal antibodies (MAbs) was produced against mouse brain proteins that bind to the tail domain of the neurofilament (NF) heavy (200-kDa) subunit (NF-H) in vitro. An in vivo association of the MAb ligands with NFs was confirmed by examining reactivity of the MAbs with immunoprecipitated NF-H complexes. Using this method we demonstrated association of the ligands of three of the MAbs with NFs. In contrast, glial fibrillary acidic protein and an unknown 97-kDa brain protein were not associated with NFs by this criterion. An 80-kDa doublet that coimmunoprecipitated with NF-H complexes, recognized by MAb 223, was shown by immunocytochemistry and immunoblotting to be synapsin Ia and Ib. Using a complementary approach, we confirmed an association of synapsin with NFs by demonstrating that immunoprecipitated synapsin I complexes contained NF-H and NF medium (160-kDa) subunits. MAbs 63 and 105 recognized a more complex set of proteins that had predominantly synaptic localizations. These data suggest that NFs may provide important support for attachment and/or transport of synaptic proteins in brain.

The main purpose of this study was to investigate the role of N-methyl-D-aspartate receptors in host-graft synaptic transmission in the neocortex. The effects of low extracellular magnesium, the glutamate agonist N-methyl-D-aspartate and N-methyl-D-aspartate antagonists on the synaptic activation of connections between embryonic neocortical graft tissue and the surrounding host tissue were studied in 17 perfused slices of rat neocortex. In standard artificial cerebrospinal fluid, stimulation of the host white matter evoked field potentials in four of 17 grafts. However, in Mg(2+)-free medium, the same stimulation evoked field potentials in an additional six grafts, with significant increases in the mean duration of the evoked responses in the 10 responsive grafts. In five of these slices stimulation of the graft also evoked field potentials in the host tissue, suggesting reciprocal interaction between graft and host. Simultaneous extracellular recordings from graft and host tissues in Mg(2+)-free medium showed that spontaneous epileptiform discharges developed in the graft and host tissue synchronously. In Mg(2+)-free medium, application of N-methyl-D-aspartate induced a shift of the baseline with superimposed epileptiform discharges in both graft and host. Application of the non-competitive N-methyl-D-aspartate antagonist ketamine and the competitive antagonist D,L-2-amino-5-phosphonovaleric acid attenuated or reversibly blocked both the spontaneous epileptiform discharges and the evoked field potentials. Our data provides evidence that N-methyl-D-aspartate receptors are present at synapses created between fetal graft and host neocortex, and that the N-methyl-D-aspartate-activated receptor-channel complex plays an active role in mediating excitatory synaptic transmission in host-graft circuitry.

To characterize neurons containing cortical Lewy bodies (LBs), vibratome sections of the superior temporal cortex from eight patients with the LB variant of Alzheimer's disease (LBV) were double-immunolabeled with anti-ubiquitin (a marker of LBs) and anti-nonphosphorylated neurofilament (SMI32; a marker of pyramidal cells) or parvalbumin (PV; a marker of interneurons) and were viewed with a laser-scanning confocal microscope. Almost all (96.1%) the LB-containing neurons were positive for SMI32, but not for PV. Furthermore, the average numbers of SMI32-immunoreactive neurons in layers 3 and 5 were 63% and 77% of those in controls, respectively. PV-immunoreactive neurons showed a greater than 40% decrease. These findings indicate that cortical LB-containing neurons are pyramidal cells and suggest that in LBV, there may be some differences in the degenerative processes effecting pyramidal cells and interneurons.

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 microns), 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.

The pathogenic mechanism underlying the vascular changes in Binswanger's encephalopathy (BE) is unknown. To test whether alterations of the humoral immunity may lead to endothelium damage, we analyzed serum levels of anti-brain endothelium antibodies (ABEA) (IgG and IgM) in 16 BE patients, 19 subjects with ischemic vascular disease without mental deterioration and 18 normal healthy subjects. ABEA IgM were found elevated in 1/16 (6%) BE patients and in 4/19 (21%) patients with cerebrovascular diseases; an increase in ABEA IgG was found in 6/16 (38%) BE patients and in 7/19 (37%) cerebrovascular patients. Association with anti-cardiolipin antibodies (IgG and/or IgM) was found in 50% of BE patients with elevated ABEA and only 10% of those with no increase, whereas high titres of anti-neurofilament antibodies (1:10,000) were detected in 40% and 71% respectively. In BE, ABEA IgG but not IgM showed a trend, although not significant, towards a correlation with the duration of the disease (rs = 0.47; p = 0.07) and significantly correlated with the cognitive function as assessed by the Mini mental state (MMS) score (rs = 0.56; p = 0.02). Higher mean values of the MMS score were found in BE patients with elevated ABEA than in those without (p = 0.04). This difference was not due to language disorders neither to an association with stroke risk factors or anti-neurofilament antibodies. However, there were no significant differences in MMS scores between cerebrovascular patients with ABEA and those without. A "neuro-protective" role is hypothesized for the ABEA in the development of dementia in BE.

A case of dysembryoplastic neuroepithelial tumour of the cerebellum occurring in a 28-year-old woman is presented. The lesion extended from the cortex of the inferior vermis upwards into the white matter. Histologically, it exhibited areas of microcystic cerebellar astrocytoma and glial regions with hamartomatous blood vessels as well as areas with oligodendrocyte-like cells (OLC) with a delicate, fibrillary stroma lying in a mucinous, often microcystic matrix. The OLC showed prominent rosette formation and immunohistochemical features suggesting neuronal, i.e. granule cell, differentiation.

The diagnosis of Alzheimer's disease (AD) neurodegeneration is based on histopathological detection of paired helical filament-associated lesions. Silver stains are routinely used but the results are fraught with intra- and interinstitutional variability. This study employed monoclonal antibodies to middle and high molecular weight neurofilament subunits in an immunohistochemical assay to assess the extent of paired helical filament-associated lesions in brains with AD, Down's syndrome plus AD lesions (AD+DN), Parkinson's disease dementia (PD), AD+PD, and normal aging changes. The densities of neurofilament-immunoreactive (NFI) cortical neurofibrillary tangles and plaques were significantly higher in AD and AD+DN than in PD and aged control brains (p < 0.001), and NFI neurofibrillary tangles and plaques were more abundant in AD and AD+DN compared with AD+PD and PD, yet all patients with AD, AD+PD, or PD died with end-stage dementia. In contrast, the densities of NFI dystrophic neurites (primarily dendrites) in cortical Layer 2 were similar among the AD, AD+DN, AD+PD, and PD groups, and all were significantly higher than control (p < 0.005). Stepwise multivariate regression analysis demonstrated significant correlations between AD diagnosis and high densities of NFI neurofibrillary tangles and plaques (p < 0.001) and between end-stage AD-type dementia and high densities of NFI dystrophic neurites (p < 0.001). This study demonstrates that the histopathological lesions correlated with AD dementia can be readily detected and quantified by immunostaining with monoclonal antibodies to phosphorylated and non-phosphorylated neurofilaments. Moreover, the findings suggest that NFI neurite pathology may be an important feature contributing to the clinically manifested AD-type dementia in individuals with Parkinson's disease.

The effects of the pH of solutions used in microwave-stimulated antigen retrieval were investigated for two antibodies commonly used in neurosciences, MAP-2 and SMI-32. A prolonged fixation time (longer than 1 week) prevents good immunohistochemical staining of these and many other antigens in paraffin and vibratome sections. On human brain tissue stored for 17 months in formaldehyde, the effects of different solutions during microwave treatment were compared, i.e., 4% aluminum chloride (pH 2.75), 0.1 N hydrochloric acid (pH 0.98), and citrate buffers (pH 2.5, 4.5, 6.0), together with the influence of temperature and microwave irradiation. We conclude that the pH and temperature of the solutions and the irradiation time are of great importance for the retrieval of antigens, but in a different way for the two antibodies studied. The SMI-32 staining results are optimal with citrate buffer at pH 2.5 and 2-hr irradiation at 90 degrees C, whereas MAP-2 staining results are optimal with citrate buffer at pH 4.5 and 10-min full-power irradiation. In the other conditions the staining is less intensive or less homogeneously distributed over the section.

In order to better characterize the autoantibodies induced by PC12 cells grafted into rat brain, we have tested sera from these animals by immunoblotting with several preparations, including phosphorylated and dephosphorylated neurofilaments, keratins, PC12 cells and proteins from various rat tissues, and by immunofluorescence of rat spinal cord neurons in culture. Sera from grafted rats reacted with several antigens present in all tissues tested and stained in cultured neurons not only NF but also cell bodies and membranous granular structures. These observations suggest either the polyreactivity of autoantibodies or the induction of a polyclonal B cell activation consecutive to the release of central nervous system antigens into the blood stream. These results are discussed with regard to the role of NF autoantibodies in neurodegenerative diseases.

The distribution and expression of developmentally regulated phosphorylation epitopes on the microtubule-associated protein 1B and on neurofilament proteins recognized by monoclonal antibody (mAb) 150 and mAb SMI-31 was investigated in the developing rat spinal cord. In the embryonic day 11 spinal cord, mAb 150 stained the first axons to appear, whereas mAb SMI-31 staining did not appear until embryonic day 12. At the start of axonogenesis, mAb 150 stained neuronal cell bodies and axons whereas at later times only the distal axon was stained, this is the first demonstration in vivo of a mAb 150 axonal gradient similar to that seen previously in vitro (Mansfield et al., 1991). During the postnatal period, axonal staining by mAb 150 dramatically declined so that by the third postnatal week, only the corticospinal tract, which contains axons that are still growing, was labelled. There was no evidence of dendritic staining except of adult primary motoneurons. In contrast, mAb SMI-31 staining of axons was not present as a gradient. Instead, mAb SMI-31 staining increased progressively throughout this period, persisted into adulthood and was shown by immunoblotting to be related to the increased phosphorylation of the medium and heavy neurofilament proteins. Axonal staining by mAb 150 re-appears in a sub-population of the SMI-31-labelled myelinated axons in the adult spinal cord and PNS and in the perikarya and dendrites of primary motoneurons, where it probably recognizes a phosphorylation epitope on heavy neurofilament proteins. This late appearing epitope has some similarities to that recognized by mAb SMI-31 on neurofilaments, but it is not identical. These cross-reactivities of mAbs that recognize phosphorylation epitopes on otherwise unrelated proteins dictate caution in interpreting immunohistochemical data. It may now be necessary in some cases to re-appraise published studies using these two antibodies.

This study was undertaken to investigate whether human sensory and sympathetic neurons contain phosphorylated neurofilament proteins, and whether they may be classified on the basis of this property, as in other mammalian species. The distribution of the phosphorylated 200-kDa neurofilament protein subunit (p200-NFP) was investigated in lumbar sympathetic and dorsal root ganglia by means of the RT97 monoclonal antibody (against p200-NFP). The intensity of immunostaining, and the size of neuronal body profiles were measured in order to define different neuron subclasses. In dorsal root ganglia, most of the neuronal profiles (96%) were p200-NFP immunoreactive, and the intensity of immunostaining was not related to neuronal perikarya size. In the lumbar paravertebral sympathetic ganglia, virtually all neurons displayed p200-NFP immunoreactivity, and the intensity of immunolabelling was also independent of the size of the neuronal somata. These results demonstrate heterogeneity in the expression of p200-NFP immunoreactivity in human sympathetic and sensory neurons. In contrast to other mammalian species, RT97 immunolabelling cannot be used as a discriminative marker for the two main types of human primary sensory neurons. On the other hand, our findings provide evidence for the occurrence of phosphorylated neurofilaments within peripheral neuron cell bodies.

Studies of peripheral nerves in two different lines of hypomyelinating transgenic mice support the hypothesis that myelinating Schwann cells exert a significant influence on key biological properties of axons. The mice contain transgenes combining the peripheral myelin protein zero gene (P0) promoter and either the diphtheria toxin A chain gene product or the SV40 (simian virus 40) large T antigen. The consequences of peripheral nerve hypomyelination on axon diameter, neurofilament (NF) density, and NF phosphorylation were analyzed. The sciatic nerves of the P0 diphtheria toxin A transgenic mice (DT) evidenced the most severe hypomyelination, and this was associated with a dramatic decrease in NF phosphorylation plus a marked increase in NF density. In contrast, the sciatic nerves in the P0 SV40 large T antigen transgenic mice (SV40) were not as severely hypomyelinated and there was a milder decrease in NF phosphorylation plus a more modest increase in NF density. Further, the sciatic nerves in both lines evidenced a decrease in axonal caliber without any change in NF content. Taken together, these studies provide strong evidence indicating that myelinating Schwann cells exert a significant influence on axon caliber by modulating NF phosphorylation and NF packing density in the axons of peripheral nerves. Thus, key biological properties of axons are modulated by signals transmitted from myelinating Schwann cells to axons of peripheral nerves.

To determine if hyperplastic and neoplastic lesions from medaka showed similar immunoreactivity to intermediate filament antibodies as the tissues of origin, two week old medaka were exposed to 10 or 20 mg/L of methylazoxymethanol acetate for two hours and transferred to clean water for up to six months. Using a streptavidin peroxidase method, paraffin embedded Bouins fixed neoplasms were incubated with cytokeratin, vimentin, or neurofilament antibodies. Like their nonneoplastic cellular counterparts, hepatocellular carcinoma, pancreatic acinar carcinoma and mesenchymal neoplasms including hemangioma and hemangiopericytoma reacted negatively to cytokeratin antibodies. Cholangiocarcinoma, mesothelioma, and proliferative lesions containing biliary epithelial cells reacted positively to cytokeratin antibodies. All neoplasms and proliferative lesions were negative with vimentin and neurofilament antibodies. These data indicate that while some epithelial neoplasms showed cytokeratin reactivity similar to the parent tissues, additional markers are needed to identify mesenchymal tissues and neoplasms.

Dissociated cell cultures of Xenopus laevis embryonic spinal cord have proved useful for studying the differentiation of neuronal ionic channels and membrane properties and for examining the dynamics of microtubules in developing neurons. To examine their usefulness for studying neurofilaments in developing neurites, we prepared similar cultures from stage 22 embryos. Between 3 and 55 h after plating, these cultures were fixed and immunostained with antibodies directed against various epitopes of neurofilament proteins from X. laevis. These antibodies were specific for nonphosphorylated epitopes of the two low molecular weight Xenopus neurofilament proteins (Xenopus NF-L and the Xenopus neuronal intermediate filament protein, XNIF), both phosphorylated and nonphosphorylated epitopes of the Xenopus middle molecular weight neurofilament protein (NF-M), and a nonphosphorylated epitope of the Xenopus high molecular weight neurofilament protein (NF-H). The emergence of these neurofilament proteins in culture was compared to the time course previously reported for them in Xenopus spinal cord neurons in situ. To facilitate the comparison of times in culture to developmental stages, the age of cultured neurons was converted to an equivalent Nieuwkoop and Faber normal stage using data presented here on the effect of changing temperature on developmental rates of X. laevis. With the exception of the nonphosphorylated epitope of NF-H, which is indicative of the most mature axons found in situ, the emergence of the other neurofilament protein antibody epitopes closely paralleled that previously reported for these antibodies in situ. Thus, with respect to XNIF, NF-M, and NF-L, the neurites of cultured neurons were typical of young, embryonic Xenopus laevis spinal cord axons. This system should prove useful for studying both the function of these neurofilament proteins during the early stages of axonal development and the dynamics of their transport.

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)

The expression of low-affinity nerve growth factor receptor (NGF-R) by primitive neuroectodermal tumors (PNETs) was analyzed in vivo and in vitro to investigate the relationship between NGF-R expression and cellular differentiation. NGF-R was expressed in one medulloblastoma cell line and two neuroblastoma cell lines. When these cells were induced to differentiate by treatment with dibutyryl cyclic adenosine monophosphate, NGF-R was overexpressed and there was increased expression of neurofilament proteins. Immunohistochemistry investigation of tumor tissues demonstrated that NGF-R was expressed by a subset of PNETs with a neuronal phenotype marked by neurofilament protein expression, but not by gliomas and PNETs without a neuronal phenotype. Growth inhibition assay demonstrated that NGF inhibited the growth of cells expressing NGF-R. These results indicate that NGF-R expression is a useful marker of neuronal differentiation by PNETs.

Connections between motor cortical neurons receiving somatosensory inputs from area 2 and large pyramidal cells in layer V were examined in the cat via intracellular injection of biocytin and immunohistochemistry of nonphosphorylated neurofilament proteins (npNFP). Biocytin was injected into pyramidal cells in layers II/III of the motor cortex that responded monosynaptically and polysynaptically to microstimulation of the somatosensory cortex and subsequently stained black by the avidin-biotinylated peroxidase complex method with diaminobenzidine (DAB) and nickel. By using a monoclonal antibody SMI-32 and a modified peroxidase-antiperoxidase method with Tris-aminophenyl-methane (TAPM) and p-cresol as a chromogen, pyramidal cells in layers III and V of the motor cortex were stained red for npNFP. In particular, all the large pyramidal cells in layer V, Betz cells, displayed intense npNFP immunoreactivity not only in the perikarya but also in the dendrites. Double staining with DAB/nickel and TAPM/p-cresol showed that biocytin-filled axon varicosities of the pyramidal cells, which were thought to receive monosynaptic inputs from area 2, made contacts with npNFP-positive dendrites in layers I-III around the biocytin-injected cell and in layers V-VI beneath the cell. The present results suggest that the corticocortical input from area 2 to pyramidal cells in layers II/III of the motor cortex is transferred to layer V pyramidal cells, including Betz cells, as well as to neighboring layer II/III pyramidal cells. Since tetanic stimulation of the somatosensory cortex reportedly produces long-term potentiation in layer II/III cells of the motor cortex, it seems reasonable to assume that a given area of the somatosensory cortex can produce a long-lasting change in the activity of a given group of output cells in the motor cortex.

Sera obtained from AIDS patients with cytomegalovirus (CMV) retinitis before and after treatment with foscarnet, AIDS patients with human immunodeficiency virus (HIV) retinopathy, AIDS patients without retinal disease, and normal healthy controls with and without positive CMV serologies were assayed for the presence of antibodies against the 200-kDa outer, 160-kDa middle, and 68-kDa core subunits of the neurofilament triplet. Additional studies were performed to determine the presence of antibodies reactive with proteins extracted from crude human retinal antigen preparations. Antibodies against the 200-, 260-, and 68-kDa proteins of the neurofilament triplet were detected in 15 of 15 AIDS patients with CMV retinitis. The expression of these antibodies was unaffected, qualitatively, by successful treatment with foscarnet. In contrast, only 30% of patients with HIV retinopathy unrelated to CMV, fewer than 35% of AIDS patients with positive CMV titers but without evident retinitis, and fewer than 25% of healthy controls with positive or negative CMV titers possessed antibodies against any of the triplet proteins (P < 0.001). Antibodies against several clusters of retinal antigens were also identified in the sera of patients with CMV retinitis. In summary, the data indicate that retinal elements damaged by CMV infection induce an antibody response against the 200-, 160-, and 68kDa components of the neurofilament triplet as well as other, as yet undefined retinal antigens.

Immunohistochemical labelling with the monoclonal antibody SMI32 to non-phosphorylated epitopes on neurofilament proteins of high molecular weight class was low in rat central optic fibers of controls. After unilateral transection of optic nerve, a strong, transient increase of labelling with SMI32 occurred in degenerating fibers of optic tract at 2 and 4 days, which then declined at 8 and remained low at 21 days. Consequently, immunostaining with SMI32 may serve as a positive marker for degenerating fibers in rat optic system.

When grafting human mesencephalic tissue to patients suffering from Parkinson's disease, the number of surviving dopamine (DA) neurons in the graft is probably crucial. It may be possible to increase the number of DA neurons available for grafting to a patient by pooling tissue from many human embryos collected over several days or by obtaining more DA neurons from each embryo. We have addressed these issues by cryopreserving human mesencephalic DA neurons prior to transplantation and also by grafting human embryonic diencephalic DA neurons. The effects of cryopreservation were assessed 4-15 weeks after xenografting ventral mesencephalic tissue into the DA-depleted striatum of immunosuppressed rats with unilateral 6-hydroxydopamine lesions of the mesostriatal pathway. Control rats grafted with fresh mesencephalic tissue displayed robust reductions in amphetamine-induced turning following transplantation. Functional effects of the cryopreserved mesencephalic grafts were only observed in the one rat out of nine which contained the largest graft in this group. The number of tyrosine hydroxylase immunoreactive neurons in animals transplanted with cryopreserved tissue was significantly reduced to 9% of fresh tissue control grafts. Morphological analysis showed that cryopreserved DA neurons were approximately 22% and 28% smaller regarding the length of the long and short axis, respectively, when compared to the neurons found in fresh grafts. In the second part of the study, the survival and function of human embryonic diencephalic DA neurons were examined following xenografting into the DA-depleted rat striatum. A reduction of motor asymmetry was observed in two out of seven diencephalon-grafted rats. This finding was consistent with a good graft survival in these particular rats, which both contained large grafts rich in tyrosine hydroxylase immunoreactive neurons. Moreover, there was immunopositive staining for graft-derived fibers in the rat striatum containing tyrosine hydroxylase and human neurofilament, both in rats grafted with mesencephalic and diencephalic DA neurons. These findings suggest that cryopreservation, using the current technique, is not a suitable storage method for use in clinical trials of DA neuron grafting in Parkinson's disease. On the other hand, the application of alternative sources of DA neurons may in the future develop into a strategy which can increase the number of neurons obtainable from each human embryo.

Type I human skin collagenase (HSC-1) was localized in developing embryonic and fetal skin ranging from 6 to 20 weeks estimated gestational age using an antigen-specific, affinity-purified, polyclonal antiserum to HSC-1 and an avidin-biotin alkaline phosphatase procedure. Double immunolabeling with monoclonal antibodies for Factor VIII-related antigen, type IV collagen, and the 68-kilodalton neurofilament subunit was performed using a direct peroxidase procedure. By 8 weeks estimated gestational age, HSC-1 localized to the periderm, the basal cell epidermal keratinocytes, dermal fibroblasts, and surrounding extracellular matrix. At 12 weeks estimated gestational age, HSC-1 immunolabeling showed a continued association with the epidermis and dermis. Dermal and subcutaneous blood vessels and the surrounding extracellular matrix were positive for HSC-1 labeling. HSC-1 staining was also found around developing nerves and in association with dermal fibroblasts. In the developing hair follicle, HSC-1 was present in keratinocytes of the pre-germ, germ, hair peg, and bulbous hair peg. HSC-1 immunoreactivity was also found in association with the hair canal, the bulge, and the dermal papillae, but was absent from the fetal sebaceous gland. These data demonstrate the association of HSC-1 with the development of interfollicular epidermis, the dermal collagenous matrix, the process of angiogenesis, the development of nerves, and hair follicle morphogenesis.

Recently we reported that a mouse foetal neural graft, when transferred into a lesioned juvenile neocortex, may induce cortical repair and stimulate proliferation of the host cells. The present study was focused on an immunohistochemical identification of neurones among these newly generated cells. Adjacent sections from the brains already used in our previous study were stained either with an antibody against the host-specific Thy-1 antigen, or with neurone-specific antibodies recognizing the microtubule-associated protein MAP2, the heavy subunit of neurofilaments and parvalbumin. Dividing cells, labelled repeatedly during the first three post-operative days with 3H-thymidine, were detected after 2 months by autoradiography. We found that in the repaired neocortical region newly formed host cells, whose distribution resembled the one found in an intact neocortex, also contained neurones. These new data corroborate our previous suggestion that a juvenile mammalian neocortex participates, after lesioning and under the presence of a foetal neural graft, in its own repair by the formation of new cells, including neurones.

We and other researchers have previously described the presence of axon-specific phosphorylated neurofilament epitopes in the cell bodies of three neuronal types in the rat: bipolar septofimbrial neurons and the large light A-type cells in the dorsal root ganglia and the mesencephalic nucleus of the Vth nerve. This spontaneous presence of phosphorylated neurofilaments at the level of the perikaryon contrasts with the induced appearance of these epitopes in axotomized neurons. We have undertaken a study of this phenomenon in rat, mouse, gerbil, rabbit, pig and chicken to analyse its species distribution. Phosphorylated neurofilament positive perikarya could be detected in the dorsal root ganglia and mesencephalic nucleus of the Vth nerve in all analysed species. Although this labelling has been shown to be specific for A-type cells in rat, in pig small cells were preferentially labelled, whereas the largest cells were mostly completely devoid of label. In the septofimbrial nucleus, phosphorylated neurofilament positive perikarya were seen in rat, mouse, gerbil and rabbit. In the pig, only a phosphatase-insensitive neurofilament antibody labelled these neurons. In the chicken, the labelling was completely absent. These observations establish the widespread species distribution of perikaryal phosphorylated neurofilament epitopes in the dorsal root ganglia and mesencephalic nucleus of the Vth nerve. In the septofimbrial nucleus however, this phenomenon seems to be restricted to rodents and lagomorphs. We discuss possible explanations for these cytoskeletal singularities in dorsal root ganglia, the mesencephalic nucleus of the Vth nerve and septofimbrial neurons.

Neurofilament proteins of mammalian axotomized peripheral axons, which regenerate effectively, resemble those of embryonic axons. However, injured centrally projecting mammalian axons, which fail to regenerate, have very different neurofilament compositions than during development. If changes in neurofilament composition after injury reflect the ability of axotomized neurons to regenerate effectively, then the neurofilaments of centrally projecting axons that can regenerate should more closely resemble those of developing axons. In this study, the neurofilament compositions of injured optic axons of the frog, Xenopus laevis, were examined, since these axons can regenerate a fully functional projection. Antibodies to phosphorylated and nonphosphorylated forms of neurofilament proteins that had been used previously to study the neurofilament composition of newly developing X. laevis optic axons were used in immunocytochemical studies to examine the return of neurofilaments to the optic nerve after an intraorbital nerve crush. Intraocularly injected wheat germ agglutinin conjugated to horseradish peroxidase was used to label the regenerating axons independently of their neurofilaments. Neurofilament immunoreactivities disappeared rapidly from crushed axons during the first week after surgery. By nine days after surgery, antibodies to nonphosphorylated forms of middle (NF-M) and low molecular weight (NF-L) neurofilament proteins and the Xenopus neuronal intermediate filament protein (XNIF) began to stain the nerve just beyond the lesion. By this time, however, growing axonal terminals had reached the optic chiasm. Antibodies to phosphorylated epitopes of NF-M began to stain axons at 15 days, just as growing axons began to arrive at the optic tectum. Nonphosphorylated high molecular weight neurofilament protein (NF-H) began to appear in axons between 18 and 21 days after surgery. Thus, the reappearance of neurofilaments during optic axon regeneration resembled the general pattern seen during development. The chief difference between development and regeneration was that neurofilament epitopes took longer to emerge during regeneration. One possibility is that cues encountered along the optic pathway influence the neurofilament composition of retinal ganglion cell axons. Then, the greater distances travelled by regenerating axons could account for the longer time taken for their neurofilament compositions to mature.

To find out what causes differences in phosphorylation states in neurofilaments (NF), we selected two types of dendrite, one provided with very few NFs (Purkinje cell) and the other with relatively many (anterior horn cell). We examined these with four monoclonal antibodies selected by the Western blot analysis, two (NE14 and SMI31) recognizing only phosphorylated, SMI32 recognizing only nonphosphorylated, and N52 recognizing phosphorylation-independent epitopes of NF-H. The immunoperoxidase labeling of dendrites, and also of perikarya, in both neurons was detectable with all four antibodies. After the tissue was treated with Triton X-100, the labeling was still detectable with SMI32 or N52, but undetectable with NE14 and SMI31. The brain homogenate Triton-extracted supernatant after centrifugation at 100,000g for 1 hr showed the staining of NE14, SMI31, and N52 but not that of SMI32. In Purkinje cell dendrite and perikaryon, NFs always appeared singly. In the immunogold labeling, they were labeled only with SMI32 or N52. Labeling by NE14 or SMI31 was distributed throughout the cytoplasm and hardly associated with NFs. In the anterior horn cell dendrite and perikaryon, NFs appeared both singly and in bundles. They were predominantly labeled with SMI32 or N52 when they were single, and with NE14, SMI31, or N52 when they were bundled. Even in one NF, portions that appeared single were labeled mostly with SMI32 or N52, while the remainder, to which other NFs approached closely, were labeled mostly with NE14, SMI31, or N52. Thus, when NFs appear singly, NF-H in their projections or cross-bridges with other organelles is not phosphorylated, while when NFs are bundled, NF-H is phosphorylated in crossbridges between NF core filaments. These data may explain why the NF-H is heavily phosphorylated in axons, where NFs are abundant, and not in dendrites and perikarya, where NFs are sparse.

Diffuse axonal injury (DAI) is observed commonly in traumatically brain injured humans. However, traditional histologic methods have proven of limited use in identifying reactive axonal change early (< 12 h) in the posttraumatic course. Recently, we have reported, in both humans and animals, that antibodies targeting neurofilament subunits are useful in the light microscopic recognition of early reactive change. In the present study, we extend our previous efforts in humans by analyzing the progression of traumatic brain injury (TBI)-induced axonal change at the ultrastructural level. This effort was initiated to follow the subcellular progression of reactive axonal change in humans and to determine whether this progression parallels that described in animals. Two commercially prepared antibodies were used to recognize reactive axonal change in patients surviving from 6 to 88 h. The NR4 antibody was used to target the light neurofilament subunit (NF-L), and the SMI32 antibody was used to target the heavy neurofilament subunit (NF-H). Plastic-embedded tissue sections were screened for evidence of reactive axonal change, and once identified, this reactive change was analyzed at the ultrastructural level. At 6 h survival, focally enlarged, immunoreactive axons with axolemmal infolding or disordered neurofilaments were seen within fields of axons exhibiting no apparent abnormality. By 12 h, some axons exhibited continued neurofilamentous misalignment, pronounced immunoreactivity, vacuolization, and, occasionally, disconnection. At later stages, specifically 30 and 60 h survival, further accumulation of neurofilaments and organelles had led to the further expansion of the axis cylinder, and clearly disconnected reactive swellings were recognized. These contained a dense core of disordered immunoreactive neurofilaments partially encompassed by a cap of less densely aggregated organelles. At 88 h, the reactive axons were larger and elongated, consistent with the continued delivery of organelles by axoplasmic transport. At the later time points, considerable heterogeneity was observed, with focally enlarged disconnected axons being observed in relation to axons showing less advanced reactive change. Our findings suggest that neurofilamentous disruption is a pivotal event in axonal injury.

The general structure of the enteric nervous system in the omasum of sheep was investigated by immunohistochemical techniques using antineurofilament protein antibody. The myenteric plexus consisted of polygonal ganglia and interganglionic nerve bundles. The meshwork of the plexus in the oral region was denser than the middle and aboral regions. Intraganglionic laminar endings were present in the myenteric ganglia. The submucosal plexus was divided into sublaminar and intralaminar plexuses. The latter was denser near the free border of the omasal laminae than in the region near the omasal wall. There were no ganglion cells showing neurofilament protein-like immunoreactivity in the submucosal and mucosal layers. Probable mucosal receptors were found in the horny omasal papillae near the reticulo-omasal orifice. The findings demonstrate a complicated structure for the enteric nervous system in the omasum, presumably reflecting its regulatory activities.

Spectroscopic methods have amply documented that small- and medium-sized peptides tend to assume unordered conformations in water. The conformational tendencies, however, manifest in halogenated alcohols, and the preferred secondary structures are apparent from the circular dichroism (CD) spectra. Here we report the results of immobilizing peptide and protein antigens from various mixtures of trifluoroethanol and water during enzyme-linked immunosorbent assays. The increased recognition by the appropriate monoclonal antibodies (mAbs) is correlated with the increase of the alpha helical, beta turn, or beta pleated sheet content of the peptides presented in the different solvent mixtures. Remarkably, the antibody binding can be detected at considerably lower antigen levels if the antigen is immobilized from trifluoroethanol. The antigens we used corresponded to fragments of normal human neurofilaments and tau protein found in the paired helical filaments of Alzheimer's disease, and the nucleoprotein of rabies virus. The conformation of myoglobin is as stable in water as in trifluoroethanol, and therefore acted as a negative control. Indeed, the recognition of myoglobin did not increase upon increasing the trifluoroethanol concentration in the solvent used to apply the antigen to the plate. The possibility of imperfect binding to the plastic carrier or nonspecific binding to irrelevant antibodies is excluded by using control experiments. We offer the first direct evidence that the mAbs recognize the secondary structure of epitopes, and that it is possible to correlate the binding conformation of the epitopes with CD measurements made in trifluoroethanol-water mixtures.

Protein kinase C (PKC) and growth-associated protein-43 (GAP-43) were investigated immunohistochemically in the dorsal motor nucleus of the vagus nerve and the hypoglossal nucleus after axotomy using monoclonal antibodies against type I, II and III PKC and GAP-43. In the control side of both nuclei, anti-type I and II PKC weakly stained neuronal cell bodies, while anti-type III PKC did not show any reaction with neurons. In the axotomized side of both nuclei, anti-type II PKC antibody intensely stained affected nerve cell bodies as well as plasma membrane. Some of the severed neurons showed intensified reactions for both anti-type II PKC and anti-GAP-43 antibodies in the serial sections. These findings suggest that axotomy increases the type II PKC of the severed neurons, and type II PKC seems to phosphorylate some protein, such as GAP-43, and plays some role in the retrograde neuronal reaction.

The autoreactivity of murine cytomegalovirus (MCMV)-neutralizing monoclonal antibody (mAb) AC1 was examined in vitro and in vivo. Both mAb AC1 and a human antiserum reactive with U1-small nuclear ribonucleoprotein (U1-snRNP) stained uninfected mouse embryo fibroblasts (MEF) in a speckled nuclear pattern and reacted with 70,000 molecular weight (MW) MEF nuclear antigens by immunoblotting, suggesting that mAb AC1 cross-reacted with the 70,000 MW component of U1-snRNP. However, only mAb AC1 cross-reacted with an additional epithelial cytoplasmic autoantigen present in cultured HEp2 cells. On tissue sections from uninfected mice, mAb AC1 predominantly reacted with a component of central and peripheral nervous systems, although cross-reactivity with the stratum spinosum of the skin and the outer sheath of hair follicles was also observed. Immunoblotting revealed that mAb AC1 reacted with phosphorylated epitopes present on a 98,000 MW MCMV structural protein and the 200,000 MW mouse neurofilament protein (NFP). Treatment of uninfected mice with mAb AC1 resulted in a severe interstitial pneumonia with greatly thickened and congested alveolar septa. Severe oedema of the hypodermis and a mild mesangial proliferative glomerulonephritis were also observed. These results demonstrate that a mAb reacting with a MCMV structural phosphoprotein which can protect mice against the dissemination of MCMV, can also promote the development of autoimmune disease. Therefore, the production of such cross-reactive antibodies may be an important mechanism in the development of autoimmunity following viral infection.

This study reports the detection of autoantibodies to myelin basic protein (MBP) and neurofilament proteins (NFP) in serum and cerebrospinal fluid (CSF) of Japanese encephalitis patients. The diagnosis of Japanese encephalitis was confirmed in 72 patients by the presence of virus specific antibodies to JEV in the CSF (28/72), viral antigen in the CSF (19/72) and simultaneous presence of both antigen and JEV antibodies in the CSF in 25/72 patients. Autoantibodies to either purified NFP (10) or MBP (8) or both (17) were detected in the CSF of 35 patients by ELISA in contrast with the control CSF samples. Amongst them 20 had similar antibodies in the serum as well. Correlation of immunological findings with the clinical outcome revealed that the presence of autoantibodies in the CSF especially to NFP was associated with a fatal outcome (P < 0.05).

Using two monoclonal antibodies, tau-1 and PHF-1, and a sequential staining method combining double-labeling immunofluorescence and Bielschowsky silver staining, we have demonstrated the presence of two populations of dystrophic neurites (DNs) with distinct immunocytochemical and argentophilic characteristics. Tau-1 and PHF-1 immunoreactivity were co-localized in many DNs. However, approximately 20% of the DNs were immunoreactive for PHF-1 only. PHF-1 single-labeled DNs were not visible or very weak with Bielschowsky silver stain. Of DNs continuous with neurofibrillary tangles (NFTs), tau-1/PHF-1 double-labeled DNs were continuous with intracellular NFTs only, while PHF-1 single-labeled DNs were continuous with extracellular NFTs only. Furthermore, the population of DNs that cluster around extracellular NFTs is different from those that cluster around or within senile plaques. The combined use of tau-1 and PHF-1 immunocytochemistry may provide a more accurate indication of the number of extracellular DNs and extracellular NFTs, which may aid in the diagnosis of severe and advanced AD cases.

The human-to-rat xenograft approach offers possibilities to study aspects of primate cortex development and function without monkeys. Human fetal cortical tissue was grafted to prepared cortical cavities of immunosuppressed host rats. Fetal tissue fragments were collected after routine low-pressure vacuum aspiration abortions performed in the first trimester of gestation. Human derived neurons and human nerve fiber outgrowth were visualized by immunohistochemistry with antibodies against human neurofilament protein 70 kD (hNFP70). Ingrowth from rat host striatum or cortex into the grafts was analyzed by immunohistochemistry with antibodies against tyrosine hydroxylase. Astrocytes were evaluated by immunohistochemistry with antibodies against glial fibrillary acidic protein. The grafts grew into different sizes (1-10 mm in diameter) and contained large numbers of hNFP70-positive nerve fibers. All grafts gave rise to outgrowth of hNFP70-positive fibers into the host with partly a cortical layering; layers III and IV received a majority of the human fibers. In several cases, the graft-derived nerve fibers entered the host brain at restricted areas, while there was no crossing over of nerve fibers at the rest of the graft-host interface. Tyrosine hydroxylase-positive fibers were usually not abundant in the grafts. Interestingly, cases of massive ingrowth occurred from host striatum into the graft in a pattern suggesting "permissive sites" at the graft-host interface in the same way as outgrowth from graft to host was found. Additionally, tyrosine hydroxylase-immunoreactive fibers from host cortex were found to grow into the transplant. Glial fibrillary acidic protein immunoreactivity was increased at the interfaces between graft and host cortex or host striatum. Immunohistochemistry using antibodies against rat IgG indicated the presence of rat IgG within the grafts, and in bordering areas of host brain, possibly indicating a defective graft-host barrier. Taken together, these results show that human cortical tissue pieces grafted to cortical cavities of immunosuppressed rats survive grafting and develop, and that reciprocal nerve fiber growth between grafts and hosts occur. Human cortical neurons can grow into the rat host brain in a pattern which is partly determined by host cortical architecture.

Organotypic cerebellar cultures were exposed to the mitotic spindle inhibitor, vinblastine, and examined by light and electron microscopy, and electrophysiologically. Purkinje cells in exposed cultures had stunted, varicose dendrites when observed in preparations reacted with antibody to nonphosphorylated neurofilament protein. Ultrastructurally, microtubules were selectively reduced in Purkinje cell dendrites, which contained disarrayed excess smooth endoplasmic reticulum, but no large accumulations of densely packed neurofilaments. Spontaneous cortical discharges were initially increased in vinblastine treated cultures and then ceased, at which time the cultures became unresponsive to electrical stimulation. The vinblastine-induced changes were attributed to impaired intracellular transport secondary to disruption of Purkinje cell dendritic microtubules.

A new model producing diffuse brain injury, without focal brain lesions, has been developed in rats. This has been achieved by allowing a weight of 450 gm to fall onto a metallic disc fixed to the intact skull of the animal which is supported by a foam bed. Two levels of injury were examined by adjusting the height of the falling weight to either 1 m or 2 m. Two groups of animals were studied. Group 1 animals were separated into three subgroups: 10 received a 1-m weight drop, 58 received a 2-m weight drop, and 13 served as controls; all were allowed to breathe spontaneously. Group 2 animals were separated into the same subgroups: four received a 1-m weight drop, six received a 2-m weight drop, and four served as controls; all of these were mechanically ventilated during the procedure. In Group 1, morphological studies using light and electron microscopy were performed at 1, 6, 24, or 72 hours, or 10 days after insult; all Group 2 rats were studied at 24 hours after injury. Results from Group 1 animals showed that no mortality occurred with the 1-m level injury, while 59% mortality was seen with the 2-m level injury. On the other hand, no mortality occurred in Group 2 animals regardless of the level of trauma induced. However, the morphological changes observed in both groups were similar. Gross pathological examination did not reveal any supratentorial focal brain lesion regardless of the severity of the trauma. Petechial hemorrhages were noticed in the brain stem at the 2-m level injury. Microscopically, the model produced a graded widespread injury of the neurons, axons, and microvasculature. Neuronal injury was mainly observed bilaterally in the cerebral cortex. Brain edema, in the form of pericapillary astrocytic swelling, was also noted in these areas of the cerebral cortex and in the brain stem. Most importantly, the trauma resulted in a massive diffuse axonal injury that primarily involved the corpus callosum, internal capsule, optic tracts, cerebral and cerebellar peduncles, and the long tracts in the brain stem. It is concluded that this model would be suitable for studying neuronal, axonal, and vascular changes associated with diffuse brain injury.

Since autoantibodies against neurofilaments (NF) were frequently found in neurodegenerative disorders, this work is an attempt to investigate whether the same phenomenon occurs after intracerebral grafting or lesioning. We have thus either grafted PC12 cells or injected culture medium alone into three sites of rat central nervous system (CNS): olfactory bulb (OB), olfactory anterior nucleus (OAN) and hippocampus (HC), all three sites being impaired in Alzheimer's disease. At day 15, rat sera were collected and tested against NF by western blotting. Sera from grafted rats recognized the H- and M-subunits of NF; we have then quantified the autoantibody response by using an ELISA technique. We show that, in all cases of grafts, the autoantibody response against NF significantly increased when compared to controls (normal rats without grafts or lesions) for total immunoglobulin (Ig) amount. In contrast, concerning the Ig isotypes, some differences appeared depending on the implantation site: for grafts into OB, the immune response was of both the IgG and IgM isotypes, into OAN it was mainly of the IgM isotype and into HC, the isotype of antibodies against NF was mostly IgG. In the case of lesions alone into OAN and HC, no significant enhancement of autoantibody response was observed; in contrast, lesions into OB induced an increase in autoantibody response against NF which significantly differed from controls for all Ig isotypes tested. These data point out the diversity of the autoantibody responses following lesions or grafts according to the rat brain areas.

Immunocytochemical analysis in fine-needle aspirates were performer in 46 cases of head and neck tumors. Immunocytochemical studies helped to establish the differential diagnosis of all diagnostically difficult cases. The application of 3 monoclonal antibodies against keratin, vimentin and LCA permitted to differentiate basic types of malignant tumors.

A method which demonstrates myelinated axons in the central and peripheral nervous systems will be described. Paraffin, frozen or semi-thin epoxy-embedded sections were immunostained first with a monoclonal antibody raised against a 200 kilo-Dalton neurofilament protein and then counter-stained with Luxol fast blue.

Myelin deposition and maintenance are critical to proper function of the mammalian nervous system. Previous investigations of myelination in the central nervous system (CNS) were hampered by the lack of an in vitro system that can faithfully reproduce in vivo events yet is amenable to biochemical investigation. We have developed a procedure, based on organotypic cultures, which permits efficient preparation of large numbers of cerebellar slice cultures that can be easily manipulated. Cultures have been examined to document myelination biochemically (by incorporation of [35S]sulfate into sulfolipids), immunohistochemically (by labeling the myelin components myelin basic protein and galactocerebroside), and morphologically (by both light and electron microscopy). We tested the effects of biologically active peptides and antibodies on myelination in the thin slices. The results indicate that the cultures provide an in vitro system that can be used to examine specific cellular events that occur during CNS myelination.

A cDNA library has been constructed in the expression vector lambda gt11 from mRNA isolated from squid (Loligo forbesi) optic lobes. The library was screened with antibodies generated against purified squid neurofilaments. A positive clone was isolated, which harboured a lambda gt11 recombinant having an insert size of 3.5 kb. Hybridization analysis by Southern and northern blotting showed that the corresponding protein is encoded by a single gene that gives rise to a transcript of 2.6 kb. Translation of the full nucleotide sequence of the gene revealed an open reading frame covering 557 amino acids. This squid-neurofilament-like protein, SNLK, bears the characteristic N-terminal head, rod and C-terminal tail domains present in all intermediate filament (IF) proteins. The rod has the classical heptad repeats indicating coiled-coil-forming ability, and the predicted lengths of the coils are similar to coils 1a, 1b and 2 of intermediate filaments. At the C-terminal end of the rod there is a strongly conserved IF epitope, and a fusion protein containing SNLK is recognised by the pan-specific intermediate filament antibody, IFA. A polyclonal antibody raised against SNLK has been used to show that the protein is present only in neuronal tissues and that it is immunologically related to neurofilaments from Myxicola but not from mammals.

The expression of vimentin and the phosphorylated variant of high molecular weight neurofilament protein (NF-H) was studied in developing human fetal dorsal root ganglia and spinal cord. The technique used for examination of cryosections was double-label fluorescence with monoclonal antibodies. Both proteins were present in the nerve fibres inside the ganglia of 6- and 8-week-old embryos. During further development the expression of vimentin continued to increase in the satellite cells, but was found to be decreasing in the ganglion cells. Phosphorylated NF-H was found in the processes of ganglion cells, as well as in the perikarya at all developmental stages. In the spinal cord of 6- and 8-week-old embryos, phosphorylated NF-H protein was found in the longitudinal fibres of the marginal layer and in processes of the mantle zone; some of the fibres also contained vimentin. Later the co-expression of the two proteins ceased and vimentin was found only in glial and mesenchymal derivatives. Phosphorylated NF-H was located, at all developmental stages, in the axons of both white and grey matter, but not in the neuronal perikarya. The results indicate that phosphorylation of the NF-H in human dorsal root ganglia starts in the perikarya of the ganglion cells while in the ganglion cells of the spinal cord it takes place in the axons.

Purpose: 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., Methods: 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., Results: 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., Conclusion: These findings indicate that a visceral neuropathy seems to be present in the majority of patients with severe, so-called idiopathic slow transit constipation.

Although specific patterns of cellular vulnerability have been identified in experimental models of cerebral ischemia, there is little data on the occurrence of similar abnormalities in human ischemia. We therefore used a variety of histochemical methods to define changes affecting specific classes of cells in post-mortem specimens from seven patients with hippocampal and neocortical ischemic lesions. In acute lesions, staining with SMI-32, an antibody directed against nonphosphorylated neurofilaments that labels pyramidal projection neurons, was prominently depleted even when conventional Nissl staining revealed only mild pyknosis. In contrast, staining for other markers such as microtubule-associated protein 2 (MAP-2), another cytoskeletal protein, or parvalbumin, a calcium-binding protein found in gamma-aminobutyric acid (GABA)-ergic interneurons, were relatively preserved. SMI-32 antibody also labeled dystrophic axons and axonal retraction balls in and around acute ischemic lesions. The pattern of differential changes in immunoreactivity was essentially the same in all acute ischemic injuries, including both diffuse lesions in the CA1 field (Sommer's sector) and discrete infarcts in CA1 and neocortex. In addition, immunoreactivity for the immediate early gene product c-fos was enhanced in and around the acute ischemic lesions that we studied. In some very acute lesions, immunoreactivity for glial fibrillary acidic protein (GFAP) was depleted in areas of severe ischemia and necrosis, but, as expected, GFAP immunoreactivity was increased in lesions more than a few days old. In contrast, the loss of SMI-32 immunoreactivity persisted in chronic lesions. These findings are consistent with those of experimental ischemia in animals and confirm the relevance of these studies for human cerebral ischemia. The pattern of selective changes also resembles that of injuries induced directly by excitatory amino acids, which may play a significant role in the pathogenesis of ischemic damage.

Adult rat telecephalon was surveyed for cells demonstrating immunopositivity for muscarinic receptor (M35 antibody), microtubule-associated proteins, neurofilaments, and brain-spectrin. Neurons immunostained for muscarinic receptor were found in frontal, parietal, temporal, and occipital isocortex where they accounted for approximately 15-16% of all neurons. This labeling involved a large proportion of layer V pyramidal cells, some layer III pyramidal cells and a small proportion of non-pyramidal cells in layers II-VI. In the hippocampus, pyramidal cells, non-pyramidal cells and granular cells were immunoreactive, as were many pyramidal cells in subicular and entorhinal cortices. In every cortical region examined, cells demonstrating muscarinic receptor were morphologically identical to cells stained lightly to moderately for acetylcholinesterase following pretreatment with diisopropylfluorophosphate, and they were found in similar numbers and in a similar laminar distribution. These characteristics further corresponded to those of cells whose somatodendritic compartments were intensely immunostained by antibodies to microtubule-associated proteins (MAP): MAP-1, MAP-2, MAP-5; neurofilament proteins (NF): NF-68kD, NF-160kD, NF-200kD; and brain-spectrin. Double immunostaining using a fluorescence method followed by an avidin-biotin staining procedure revealed that cortical cells which possessed immunoreactivity for muscarinic receptor demonstrated an 80-85% overlap with cells that were immunoreactive for MAP-2 (and tau) or NF-200kD. Following unilateral ibotenic acid lesions of the nucleus basalis, MAP-2 immunostaining was reduced in the ipsilateral isocortex. This significant reduction was most evident in the parietal cortex, exactly where maximal loss of acetylcholinesterase-containing fibers occurred. The same lesion produced no significant difference in immunodensity of muscarinic receptor, MAP-1, MAP-5 NF-68kD, NF-160kD and NF-200kD. Thus, cortical cholinoceptive cells are enriched with cytoskeletal components and cholinergic afferents modulate cortical MAP-2.

A polyclonal antibody against the high molecular weight neurofilament polypeptide (NF-H) obtained from cytoskeletal extracts of bovine spinal cord reacted with NF-H, with the middle molecular weight neurofilament polypeptide (NF-M) and with a M(r) 51,000 polypeptide, but not with the low molecular weight neurofilament polypeptide (NF-L). The M(r) 51,000 polypeptide corresponded to the glial fibrillary acidic protein (GFAP), which forms the intermediate filaments of astrocytes. The polyclonal antibody affinity-purified with GFAP, reacted with both purified GFAP and NF-H. Digestion of NF-H with alpha-chymotrypsin was used to determine the recognition site of the affinity-purified antibodies. Only fragments of the tail domain of NF-H reacted with the antibody. We propose that common epitopes exist between the variable C-terminal domains of NF-H and GFAP.

In the rat, intracerebral injection of bacterial hyaluronidase resulted in the almost complete disappearance of hyaluronic acid (HA) and glial hyaluronate-binding protein (GHAP) from cerebral hemispheres, brain stem, and cerebellum (but not from optic nerves and chiasm) starting 2-3 hr after the injection. HA and GHAP reappeared throughout the brain in characteristic patches 2-3 days after the injection. The patches gradually became confluent and after 12 days the brain appeared virtually normal. In normal rat optic nerve, staining for HA and GHAP ceased abruptly in the region of the lamina cribrosa. The retina was completely negative. HA and GHAP disappeared from hyaluronidase-injected optic nerve, chiasm, and contralateral optic nerve. In hyaluronidase-injected crushed optic nerves, regenerated axons were able to grow for short distances (about 500 microns) into the distal stump undergoing Wallerian degeneration. No such growth was observed in saline-injected controls.