Astrogliosis in different zones of the spinal cord gray matter after sciatic nerve axotomy in the newborn rat: a morphometric and immunohistochemical study

Astrocytic response following unilateral sciatic nerve axotomy was examined in the spinal gray matter of newborn rats. Using an antiserum to glial fibrillary acidic protein (GFAP), immunoreactive astrocytes were studied in the ventral, dorsal and transitional region between the dorsal and ventral gray matters (TDVG) at intervals of one day, one week, two weeks and one month postaxotomy. The axotomized side showed an obvious increase in the number of immunoreactive astrocytes at one week, two weeks and one month after surgery. The numerical density per area of the glial cells (N(a)) was determined in all groups on both the intact and axotomized sides, and it increased in all groups at the axotomized sides. The percentage of glial cell increase (Pgi) was also determined. At the ventral horn Pgi increased at day one and continued to increase in all groups, while the increase in TDVG and the dorsal horn occurred at later time points. The total motoneuron count in the ventral horn at the axotomized and intact sides was done at all time points, and the percentage of motoneuron reduction (Pmr) was calculated, the highest Pmr being noticed at one month (41%). A nonlinear regression for Pmr and Pgi showed that the rate of Pgi was approximately double that of Pmr. The rate of glial cell increase at each time point (one day, one week, two weeks and one month groups) was calculated, and the highest rate of glial cell increase in the ventral horn occurred one week after axotomy, while the highest rate in the dorsal horn and TDVG occurred at the second week. The conclusion of the study is that there may be an initial post-axotomic proliferative phase of the glial cells, which was followed by a differentiation phase. Also a gradient of an increase in the rate glial cell proliferation was noticed from the ventral horn toward the dorsal horn, maybe due to stimulation by a paracrine factor.     

In vivo and in vitro differentiation of neurons and astrocytes in the rat embryo. Immunofluorescence study with neurofilament and glial filament antisera

Antisera raised against neurofilament (NF) peptides and glial fibrillary acidic protein (GFA) (subunit of glial filaments) have been used to identify neurons and astrocytes in order to study their development and differentiation in rat embryo. In vivo observations showed that NF-positive cells first appeared in 12-day-old embryos, whereas GFA-positive cells appeared in brain and spinal cord on the 18th day. In vitro observations showed that NF-positive cells could be obtained only in cultures from 12-day embryos onwards. The further differentiation of neurons involved neurite elongation, aggregation of cell bodies to form islets, and emergence of very brightly staining prominent neurons with large cell bodies and long neurites which took part in complicate pattern formation. GFA-positive cells appeared in vitro on the 16th day and they could be observed even in cultures obtained from 10-day-old embryos. As the culture aged, the GFA staining became highly fibrillary. There was no physical interaction between neuronal and glial processes.     

Acetylcholinesterase distribution in chick spinal cord cultures

Summary Explants of 10–12 day chick embryo spinal cord were cultured by coverslip-roller tube method for 3–80 days. The cellular and subcellular localization of acetylcholinesterase activity in cultured neurons was studied by the thiocholine techniques of Karnovsky and Roots and Lewis and Shute.At the light microscopic level, acetylcholinesterase was demonstrated in the neurons of both ventral and dorsal horn regions. Occasionally neurons migrated in the outgrowth zone exhibited strong intracellular activity.At the electron microscopic level, acetylcholinesterase activity was found in the nuclear envelope, granular endoplasmic reticulum and the Golgi apparatus of the neurons. No enzyme reaction was detected in the glial cell cytoplasm.     

Phosphorylation and dephosphorylation of distinct isoforms of the heavy neurofilament protein NF-H

Summary 1. Previous immunohistochemical studies led to the suggestion that distinctly phosphorylated neurofilament isoforms exist in different types of neurons. We have recently examined this hypothesis by direct biochemical experiments, which revealed that the heavy neurofilament protein NF-H of bovine ventral root cholinergic neurons is more acidic and markedly more phosphorylated than that of bovine dorsal root neurons.2. In the present study we employed this system to study the degree to which distinctly phosphorylated NF-H isoforms differ in the extents to which they can be phosphorylated and dephosphorylatedin vitro. This was performed utilizing alkaline phosphatase and protein kinase PK40^ERK, which is specific to serines of Lys-Ser-Pro (KSP) repeats. The results obtained reveal that:3. The more extensively phosphorylated ventral root NF-H is dephosphorylated more rapidly than dorsal root NF-H.4. Ventral root NF-H and dorsal root NF-H in their native form are both poor substrates of PK40^ERK.5. Following dephosphorylation, ventral root and dorsal root NF-H are phosphorylated extensively and differentially by this kinase. Under these conditions, PK40^ERK catalyzes the incorporation of, respectively, 4.2±1.3 and 2.8±0.6 mol of phosphate per molecule of ventral root NF-H and dorsal root NF-H. The ratio of phosphates incorporated into ventral root NF-H to those incorporated into dorsal root NF-H is 1.46±0.17.6. These findings support the hypothesis that different classes of neurons contain distinctly phosphorylated neurofilaments and show that ventral root and dorsal root neurons are a useful model system for studying the distinct characteristics of neurofilament phosphorylation in different types of neurons.     

Ultrastructural and quantitative motoneuronal changes after ventral root avulsion favor early surgical repair

This study assesses qualitative and quantitative morphological changes that occur in motoneurons after ventral root avulsion. The motoneuronal perikaryal changes in the ventral horn of the cat's C7 cord segment were studied after survival times of 2, 8, 14, 30, 60 and 90 days. Generally, large motoneurons showed a light type of reaction, and the small ones either light or dark. In addition, neurons with a normal ultrastructural appearance were found. These latter are considered to be in a 'steady state', which may be associated with regenerative potency. All these types of neuron reactions were present at all survival times, but the number of cells marked by a specific reaction depends on the time of survival. Qualitative and quantitative evidence is given for cell death in 36% of the motoneuronal population between 2 and 14 days after avulsion. This reduction primarily concerns large, presumably alpha motoneurons with the light type reaction. Small, presumably gamma motoneurons become seriously affected after 14 days. These findings suggest that early surgical repair may have the better chances for clinical recovery.     

Distinctly Phosphorylated Neurofilaments in Different Classes of Neurons

Abstract: Recent immunohistochemical experiments revealed that specific anti-neurofilament monoclonal antibodies yield distinct patterns in different types of neurons. This led to the suggestion that neurofilaments are a family of heterogeneous molecules whose occurrence and distribution are a function of cell type. In the present study we examined the hypothesis that this heterogeneity is due to differences in the extent of phosphorylation of neurofilament proteins in distinct types of neurons. In view of the large number of potential phosphorylation sites on the heavy neurofilament protein (NF-H), we focused on this protein and examined its extent of phosphorylation in different types of neurons. This was performed using neurofilaments isolated from axons of the cholinergic bovine ventral root motor neurons and of the chemically heterogeneous bovine dorsal root neurons. Two-dimensional gel electrophoresis revealed that the isoelectric point of ventral root NF-H (pl 5.10) was ∼0.2 pl units more acidic than that of dorsal root NH-F. This difference was abolished by treating the neurofilaments with alkaline phosphatase, suggesting that the excess negative charge of ventral root NF-H is due to increased levels of phosphorylation. Amino acid analysis confirmed that the phosphoserine content of ventral root NF-H (27.2 ± 2.5% of the serines) is markedly higher than that of dorsal root NF-H (15.5 ± 6.2% of the serines). These findings provide a novel system for studying the biochemistry and function of distinctly phosphorylated neurofilaments in different types of neurons.     

Characterisation of transverse slice culture preparations of postnatal rat spinal cord: preservation of defined neuronal populations

Spinal cord injury induces degenerative and regenerative processes and complex interactions of neurons with non-neuronal cells. In order to develop an in vitro tool for the investigation of such processes, we prepared and characterised spinal cord slice cultures (SCSC) from Wistar rats (p0–12). SCSC were sustained in vitro up to 12 days and characterised by immunohistochemistry. Calbindin⁺ neurons, distributed across the entire gray matter, were visible also after longer culture periods. NeuN⁺ neurons were best preserved in the dorsal horn whereas large NeuN⁺ and choline acetyltransferase⁺ motoneurons in the ventral horn vanished after 3 days in vitro. Nestin immunoreactivity was found in animals of all age groups, either in cells interspersed in the ependymal lining around the central canal or in cells resembling protoplasmic astrocytes. Glial fibrillary acidic protein⁺ astrocytes, initially restricted to the white matter, invaded the gray matter of SCSC early during the culture period. Microglial cells, stained by Griffonia simplicifolia isolectin B₄, were rapidly activated in the dorsal tract and in the gray matter but declined in number with time. SCSC derived from p0 or p3 animals showed a better preservation of the cytoarchitecture than cultures derived from older animals. In summary, SCSC undergo degenerative changes, but they contain defined neuronal populations, the cytoarchitecture is partially preserved and the glial reaction is limited.     

The 70-kDa Heat Shock Cognate Protein (HSC70) Is a Major Constituent of the Central Nervous System and Is Up-Regulated Only at the mRNA Level in Acute Experimental Autoimmune Encephalomyelitis

Abstract: The expression of the 70-kDa heat shock cognate (HSC70) and stress-inducible (HSP70) proteins, and their mRNAs, was examined in experimental autoimmune encephalomyelitis, a model of inflammatory demyelination in the CNS. This study was undertaken as an extension of previous work demonstrating an abrupt decline in mRNA levels of both glial fibrillary acidic protein and the low-molecular-weight neurofilament subunit in experimental autoimmune encephalomyelitis spinal cord at 12 days after inoculation, the height of inflammation and clinical signs. Using the same total RNA preparations as our previous study, we report here that mRNA levels for HSC70 increased approximately sixfold over control values at the same time that glial fibrillary acidic protein and low-molecular-weight neurofilament subunit messages decreased and were similar to controls by 21 days after inoculation. In situ hybridization experiments showed that HSC70 mRNA was predominantly expressed in neurons and that the influx of inflammatory cells into the CNS was not responsible for the large increase in HSC70 message. Despite this elevation in mRNA, only small (if any) increases in protein levels for HSC70 were detected by both western blotting and in vitro cell-free translation systems. However, by quantitative immunoblotting, we determined that constitutive levels of HSC70 comprised a substantial portion of CNS proteins, representing 2–3% of the total protein content of spinal cord. Immunohistochemical staining illustrated that the distribution of HSC70 was consistent with that of its message. In contrast, no HSP70 mRNA or protein was detected in either control or experimental animals.     

Combined immunostaining of neurofilaments, neuron specific enolase, GFAP and S-100. A possible means for assessing the morphological and functional status of the enteric nervous system

Neurofilaments, part of the cytoskeletal network, and neuron specific enolase, a major enzyme in glycolysis, are both present in central and peripheral neurons. Glial fibrillary acidic protein and S-100, on the other hand, are soluble proteins which are found exclusively in the supportive cells of the nervous system, i.e. the glial cells. Examination was made, using immunocytochemistry, of all main areas of the gastrointestinal tract of three mammalian species, rat, pig and man. By applying serial tissue sectioning, it was possible to study the relative occurrences of the two neuronal markers in the same cell bodies and to examine the relationships of the neurons with the glial cells as revealed by the antibodies to glial fibrillary acidic protein and S-100. Both neurofilaments and neuron specific enolase were localised to an extensive system of enteric nerves, with the level of neuron specific enolase-immunoreactivity showing greater variability than that observed using antibodies to neurofilaments. Comparison of the occurrence of neuron specific enolase and neurofilament immunoreactivity in serially sectioned neuronal cell bodies revealed that a minor population stained only with antibodies to neurofilaments. The equivocal or absent neuron specific enolase-immunoreactivity in some perikarya may reflect variations in functional status within the nervous system. Glial fibrillary acidic protein- and S-100-immunoreactivities were confined to glial cells which, in this normal tissue, were always in close association with the neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibodies to neurofilament protein and other brain proteins reveal the innervation of peripheral organs

Summary Monoclonal and polyclonal antibodies to neurofilament proteins, neuron-specific enolase, glial fibrillary acidic protein and S-100 have been used to demonstrate nerves, ganglion cells and the supportive glial system of the innervation of various organs. The female genitalia, the urinary tract, the respiratory system, the pancreas, the heart and the skin of several mammalian species, including rat, mouse, guinea pig, cat, pig, monkey and man were fixed in parabenzoquinone and portions of each organ were snap frozen. Serial or free-floating thick cryostat sections were stained using indirect immunofluorescence and peroxidase anti-peroxidase immunocytochemistry. In addition, the newly described and highly sensitive immunogold-silver staining technique was used on Bouin's-fixed and wax-embedded tissues.Antibodies to neurofilament proteins seemed to react with neuronal structures in all the species studied. Alternately stained serial sections showed a similar distribution of neurofilament proteins and neuron-specific enolase-containing nerves. Neuron-specific enolase staining had a diffuse appearance and was found to be highly variable, indicating that the neuron-specific enolase content might be related to the physiological state of the nerves and ganglion cells, whereas antibodies to neurofilament protein gave a consistently intense and very clear picture of the ganglion cells and nerve fibres. Antibodies to S-100 stained supportive elements of the peripheral nervous system in all tissues examined, whereas antibodies to glial fibrillary acidic protein were more selective.Abbreviations GFAP glial fibrillary acidic protein - NSE neuron-specific enolase - PBS phosphate-buffered saline - PAP peroxidase anti-peroxidase - FITC fluorescein-isothiocyanate     

Proteolysis of filament proteins in glial and neuronal cells afterIn vivo stimulation of hippocampal NMDA receptors

An intrahippocampal injection of N-methyl-D-aspartate induced the appearance of degradation products of both the 68 kiloDalton neurofilament protein and the glial fibrillary acidic protein, as revealed by immunoblot techniques. The degradation of these two filament proteins was maximal at 10 days after the lession. The degradation patterns were similar to those induced with calpains or calcium in vitro. There were no degradation effects on the 200 kD neurofilament protein as tested with both mono- and polyclonal antibodies. Consequently, the neuronal degeneration after excessive activation of NMDA receptors appears to involve calcium activation of proteolytic enzymes. The effects on the glial proteins are probably secondary to neuronal damage but could be related to calcium dependent processes.     

Nimodipine maintains in vivo the increase in GFAP and enhances the astroglial ensheathment of surviving motoneurons in the rat following permanent target deprivation

Facial and hypoglossal nerves were resected unilaterally in a total of 108 rats. Rats were divided into two groups; one group received standard food pellets (placebo), the other received food pellets containing the Ca2+-blocking agent nimodipine. The expression of glial fibrillary acidic protein was examined in paraffin sections of the brainstem using light microscopical immunocytochemistry, and the degree of glial process ensheathment of the surviving neuronal perikarya in the hypoglossal and facial nuclei quantified on electron micrographs. Up to 28 days post-axotomy no differences in glial fibrillary acidic protein-immunoreactivity were observed between placebo and nimodipine-treated animals. By 42–days, glial fibrillary acid protein-immunoreactivity was stronger in the nimodipine treated animals and by 112 days, glial fibrillary acid protein-immunoreactive astrocytes occured only in nimodipine-treated animals. Thin astrocytic processes were seen to ensheath neurons in both placebo and nimodipine-treated animals. By 28 days post axotomy, lesioned neurons in nimodipine treated animals were covered by a mean of 2.6 (hypoglossal) and 2.9 (facial nucleus) astrocytic lamellae, compared with 1.7 lamellae in the placebo group. This relatively greater ensheathment of hypoglossal and facial neurons was maintained up to 112 days post-lesion, but reduced in the placebo-treated group to ～ 1.4 lamellae. It is concluded that nimodipine enhances the formation of astrocytic lamellae on lesioned neurons and that this process may be associated with a protective role for activated astrocytes directed towards motoneurons suffering from permanent target-deprivation.     

Expression of cytoskeletal proteins in glial cells of dorsal root ganglia

The localization of S-100 protein-, glial fibrillary acidic protein- and vimentin-like immunoreactivity has been studied in dorsal root ganglia of the rat using monoclonal antibodies. A positive reaction for both S-100 protein-like and vimentin-like was found in satellite and Schwann cells. In addition, some large and intermediate sized neurons also result S-100 protein-like immunoreactivity. No positive reaction for glial fibrillary acidic protein-like was observed. The authors discuss these results.     

Cell cultures derived from early zebrafish embryos differentiate in vitro into neurons and astrocytes

The zebrafish is a polular nonmammalian model for studies of neural development. We have derived cell cultures, initiated from blastula-stage zebrafish embryos, that differentiate in vitro into neurons and astrocytes. Cultures were initiated in basal nutrient medium supplemented with bovine insulin, trout serum, trout embryo extract and fetal bovine serum. After two weeks in culture the cells exhibited extensive neurite outgrowth and possessed elevated levels of acetylcholinesterase enzyme activity. Ultrastructural analysis revealed that the neurites possessed microtubules, synaptic vessicles and areas exhibiting growth cone morphology. The cultures expressed proteins recognized by antibodies to the neuronal and astrocyte-specific markers, neurofilament and glial fibrillary acidic protein (GFAP). Poly-D-lysine substrate stimulated neurite outgrowth in the cultures and inhibited the growth of nonneuronal cells. Medium conditioned by the buffalo rat liver line, BRL, promoted the growth and survival of the cells in culture. Mitotically active cells were identified in cultures that had undergone extensive differentiation. The embryo cell cultures provide an in vitro system for investigations of biochemical parameters influencing zebrafish neuronal cell growth and differentiation.     

Antibodies to neurofilament protein and other brain proteins reveal the innervation of peripheral organs

Monoclonal and polyclonal antibodies to neurofilament proteins, neuron-specific enolase, glial fibrillary acidic protein and S-100 have been used to demonstrate nerves, ganglion cells and the supportive glial system of the innervation of various organs. The female genitalia, the urinary tract, the respiratory system, the pancreas, the heart and the skin of several mammalian species, including rat, mouse, guinea pig, cat, pig, monkey and man were fixed in para-benzoquinone and portions of each organ were snap frozen. Serial or free-floating thick cryostat sections were stained using indirect immunofluorescence and peroxidase anti-peroxidase immunocytochemistry. In addition, the newly described and highly sensitive immunogold-silver staining technique was used on Bouin's-fixed and wax-embedded tissues. Antibodies to neurofilament proteins seemed to react with neuronal structures in all the species studied. Alternately stained serial sections showed a similar distribution of neurofilament proteins and neuron-specific enolase-containing nerves. Neuron-specific enolase staining had a diffuse appearance and was found to be highly variable, indicating that the neuron-specific enolase content might be related to the physiological state of the nerves and ganglion cells, whereas antibodies to neurofilament protein gave a consistently intense and very clear picture of the ganglion cells and nerve fibres. Antibodies to S-100 stained supportive elements of the peripheral nervous system in all tissues examined, whereas antibodies to glial fibrillary acidic protein were more selective.     

Anomalous placement of introns in a member of the intermediate filament multigene family: an evolutionary conundrum.

The origin of introns and their role (if any) in gene expression, in the evolution of the genome, and in the generation of new expressed sequences are issues that are understood poorly, if at all. Multigene families provide a favorable opportunity for examining the evolutionary history of introns because it is possible to identify changes in intron placement and content since the divergence of family members from a common ancestral sequence. Here we report the complete sequence of the gene encoding the 68-kilodalton (kDa) neurofilament protein; the gene is a member of the intermediate filament multigene family that diverged over 600 million years ago. Five other members of this family (desmin, vimentin, glial fibrillary acidic protein, and type I and type II keratins) are encoded by genes with six or more introns at homologous positions. To our surprise, the number and placement of introns in the 68-kDa neurofilament protein gene were completely anomalous, with only three introns, none of which corresponded in position to introns in any characterized intermediate filament gene. This finding was all the more unexpected because comparative amino acid sequence data suggest a closer relationship of the 68-kDa neurofilament protein to desmin, vimentin, and glial fibrillary acidic protein than between any of these three proteins and the keratins. It appears likely that an mRNA-mediated transposition event was involved in the evolution of the 68-kDa neurofilament protein gene and that subsequent events led to the acquisition of at least two of the three introns present in the contemporary sequence.     

Colonization of the avian hindgut by cells derived from the sacral neural crest

Studies were done to test the hypothesis that the chick hindgut is colonized by emigrés from the sacral region of the neural crest. Crest-derived cells were identified immunocytochemically with the monoclonal antibody, NC-1, and by their ability to give rise to neurons or glia in the bowel. Neurons were recognized by demonstrating acetylcholinesterase activity, neurofilament immunoreactivity, or the immunoreactivity of a neurofilament-associated protein, NAPA-73, with a monoclonal antibody, E/C8. The visualization of glial fibrillary acidic protein immunoreactivity was employed to detect enteric glia. Separate rostral and caudal populations of NC-1-immunoreactive cells were detected in stage 21 embryos (Day E3.5) that extended in continuous streams from the sacral crest to the hindgut. The rostral group, coexpressed neural markers, while the caudal population did not. The rostral, dually labeled cells appeared to become embedded in the mesenchyme of the dorsal bowel by Day E4 and then to enter the mesentery by Day E5 to give rise to the ganglion of Remak. The caudal NC-1-immunoreactive group, which did not express neural markers, appeared to ascend within the colorectum and, in contrast to the rostral cells, fully encircled the gut. NC-1-immunoreactive neurons and glia developed in organotypic tissue cultures and chorioallantoic membrane grafts of both dorsal and ventral halves of the postumbilical bowel explanted at Days E4 and 5, ages known to precede the colonization of the hindgut by cells from the vagal crest. These observations are consistent with the view that NC-1-immunoreactive cells, which do not express neural markers, migrate from the sacral crest to the hindgut. A subset of these cells appears to be capable of giving rise to neurons in vitro, explaining the development of neurons in the explants of the ventral halves of the gut; however, the fate of the sacral crest-derived cells in situ remains to be established.     

Differential Changes in Neuronal Excitability in the Spinal Dorsal Horn After Spinal Nerve Ligation in Rats

Previous studies demonstrated that peripheral nerve injury induced excessive neuronal response and glial activation in the spinal cord dorsal horn, and such change has been proposed to reflect the development and maintenance of neuropathic pain states. The aim of this study was to examine neuronal excitability and glial activation in the spinal dorsal horn after peripheral nerve injury. We examined noxious heat stimulation-induced c-Fos protein-like immunoreactivity (Fos-LI) neuron profiles in fourth-to-sixth lumbar (L4–L6) level spinal dorsal horn neurons after fifth lumbar spinal nerve ligation (L5 SNL). Immunofluorescence labeling of OX-42 and GFAP was also performed in histological sections of the spinal cord. A significant increase in the number of Fos-LI neuron profiles in the spinal dorsal horn at the L4 level was found at 3 days after SNL, but returned to a level similar to that in sham-operated controls by 14 days after injury. As expected, a decrease in the number of Fos-LI neuron profiles in the spinal dorsal horn at the L5 level was found at 3 days after SNL. However, these profiles had reappeared in large numbers by 14 and 21 days after injury. Immunofluorescence labeling of OX-42 and GFAP indicated sequential activation of microglia and astrocytes in the spinal dorsal horn. We conclude that nerve injury causes differential changes in neuronal excitability in the spinal dorsal horn, which may coincide with glial activation. These changes may play a substantial role in the pathogenesis of neuropathic pain after peripheral nerve injury.