Excitotoxicity. Experimental correlates to human epilepsy.

Neurochemical observations on cortical biopsies form 48 patients under surgical treatment for pharmacoresistant partial epilepsy showed a 70-80% increase in glutamate concentration when expressed in relation to neuron specific enolase. Intraperitoneal administration of one of its receptor agonists, kainic acid (KA), to the rat led to increased epileptogenic activity of the limbic type in a dose-dependent fashion. The KA injection also led to a neuronal cell death and a gliosis, closely correlated to the extent of seizure activity. In biopsies from human epileptogenic cortex, the concentration of neuron specific enolase correlated inversely to that of glial fibrillary acidic protein, a marker for astrocytic glial cells. Stimulation of the KA receptor decreased the extent of phosphorylation of the largest subunit of neurofilaments (NF-H) that have consequences for structural stability and axonal transport. Phosphorylated NF-H decreased also in human epileptic cortex, indicating either an overactivity of excitatory neurotransmitters or a loss of axonal compartments.

Bcl-2 expression regulates cell sensitivity to S100beta-mediated apoptosis.

The S100beta protein is overexpressed in the brain of patients with Alzheimer's disease and Down's syndrome and is able to induce apoptosis in neurons at high concentrations. The intracellular events that regulate the apoptotic effect are largely unknown. This study investigates the roles of the bcl-2 proto-oncogene, one of the best-defined apoptotic genes, on cell death induced by S100beta. Human neuronal precursor NT2/D1 cells showed a high degree of cell death by apoptosis after exposure to 2 microM S100beta in serum-free medium. Death was preceded by a down-regulation of the Bcl-2 protein. Gene transfer with a full-length bcl-2 cDNA under the control of a constitutive promoter in NT2 cells elevated Bcl-2 protein levels and repressed S100beta-mediated cell death. When exposed to retinoic acid, the NT2/D1 cells differentiated into a neuronal phenotype. The differentiated cells up-regulated their levels of Bcl-2 and became resistant to S100beta-induced cell death. Downregulation of Bcl-2 by an antisense oligonucleotide in the differentiated cells, however, increased their susceptibility to S100beta-related cytotoxicity. Therefore, apoptosis induced through S100beta signaling is subject to regulation by Bcl-2. A combined alteration such as up-regulation of S100beta together with down-regulation of Bcl-2 may be important in the pathogenesis of Alzheimer's disease and Down's syndrome.

Phosphorylated and non-phosphorylated neurofilament proteins: distribution in the rat hippocampus and early changes after kainic acid induced seizures.

The regional distribution of neurofilament proteins in the rat hippocampus and their early changes after kainic acid induced seizures were investigated immunocytochemically with antibodies against light weight neurofilament, phosphorylated and non-phosphorylated heavy weight neurofilament. The light weight and non-phosphorylated heavy weight neurofilaments were distributed more unevenly than the phosphorylated neurofilament. The perikarya and processes of pyramidal cells in the CA3 field contained the highest light weight and non-phosphorylated heavy weight neurofilaments, while the perikarya of granule cells contained only few light weight neurofilament and the perikarya of CA1 pyramidal cells were even devoid of immunoreactivity of both light and heavy weight neurofilaments. The fiber staining of the light weight and non-phosphorylated heavy weight neurofilaments, especially the former, was less in the CA1 field and molecular layer of dentate gyrus. The phosphorylated neurofilament immunoreactivity was identified only in axons. Mossy fibers, the axons of granule cells, contained the light weight and phosphorylated heavy weight neurofilaments, but not the non-phosphorylated neurofilament. Seven days after the kainic acid induced seizures, the phosphorylated neurofilament staining was greatly reduced in the CA1 and inner molecular layer of the dentate gyrus, probably resulting from the axonal degeneration of the Schaffer collaterals and the commissural/associational fibers. Furthermore, the nonphosphorylated neurofilament appeared in the mossy fibers of the CA3 stratum lucidum, which normally do not express such immunoreactivity. The results indicate that the neurofilaments are altered following the neuronal degeneration and postlesional plasticity caused by the kainic acid administration. Therefore, the examination of various phosphorylated neurofilaments may offer a comprehensive understanding of major hippocampal pathways, axonal plasticity and the possible roles of neurofilaments in the hippocampus following excitotoxic insults.

Blast exposure causes redistribution of phosphorylated neurofilament subunits in neurons of the adult rat brain.

There is little information on threshold levels and critical time factors for blast exposures, although brain damage after a blast has been established both clinically and experimentally. Moreover, the cellular pathophysiology of the brain response is poorly characterized. This study employs a rat model for blast exposure to investigate effects on the neuronal cytoskeleton. Exposure in the range of 154 kPa/198 dB or 240 kPa/202 dB has previously been shown neither to cause visual damage to the brain, nor to affect the neuronal populations, as revealed with routine histology. Here, the brains were investigated immunohistochemically from 2 h to 21 days after blast exposure. A monoclonal antibody was used which detects only the phosphorylated epitope of the heavy subunit of the neurofilament proteins (p-NFH). This epitope is normally restricted to axons, that is, not demonstrable in the perikarya. Eighteen hours after exposure in the 240-kPa/202-dB range, p-NFH immunoreactivity accumulated in neuronal perikarya in layers II-IV of the temporal cortex and of the cingulate and the piriform cortices, the dentate gyrus and the CA1 region of the hippocampus. At the same time, the p-NFH immunoreactivity disappeared from the axons and dendrites of cerebral cortex neurons. The most pronounced immunostaining of neuronal perikarya was found in the hemisphere, which faced the blast source. The perikaryal accumulation of p-NFH was present also at 7 days but the neuronal perikarya had become negative at 21 days, at which time the axons again displayed p-NFH immunoreactivity. Exposure in the range of 154 kPa/198 dB caused similar, although less marked accumulation of p-NFH immunoreactivity in the neuronal perikarya. The findings are interpreted to show a dephosphorylation of NFHs in axons and dendrites and a piling up of p-NFHs in the perikarya due to disturbed axonal transport.

Quantitative immunochemistry on neuronal loss, reactive gliosis and BBB damage in cortex/striatum and hippocampus/amygdala after systemic kainic acid administration.

Cell specific markers were quantified in the hippocampus, the amygdala/pyriform cortex, the frontal cerebral cortex and the striatum of the rat brain after systemic administration of kainic acid. Neuron specific enolase (NSE) reflects loss of neurons, glial fibrillary acidic protein (GFAP) reflects reactive gliosis, and brain levels of serum proteins measures blood-brain-barrier permeability. While the concentration of NSE remained unaffected in the frontal cerebral cortex and the striatum, their GFAP content increased during the first three days. In the hippocampus and amygdala, NSE levels decreased significantly. GFAP levels in the hippocampus were unaffected after one day and decreased in the amygdala/pyriform cortex. After that, GFAP increased strikingly until day 9 or, in the case of amygdala/pyriform cortex, even longer. This biphasic time course for GFAP was accompanied by a decrease of S-100 during days 1-9 followed by a significant increase at day 27 above the initial level. The regional differences in GFAP and S-100 could result from the degree of neuronal degeneration, the astrocytic receptor set-up and/or effects on the blood-brain barrier.

Induction of glucose regulated protein (grp78) and inducible heat shock protein (hsp70) mRNAs in rat brain after kainic acid seizures and focal ischemia.

Specific probes were obtained using PCR cloning from rat brain for the 78 kDa glucose regulated (grp78), inducible 72 kDa (hsp70) as well as constitutive 73 kDa (hsc73) heat shock mRNAs. Grp78 and hsc73 were expressed in normal rat brain whereas hsp70 was not. Subcutaneous injection kainic acid (10 mg/kg) produced seizures and induced all three mRNAs. The induction of grp78 and hsp70 mRNAs occurred within 2 h, peaked between 6-8 h, persisted for 48 h, and returned to control levels by 72 h. Expression of the grp78 and hsp70 mRNAs after focal ischemia progressively increased with occlusion durations from 15-120 min in the cerebral cortex. Though grp78 and hsp70 mRNAs were induced modestly in the striatum by 15 min of ischemia, longer durations of ischemia were characterized by little change in the grp78 mRNA levels and relatively lower levels of hsp70 expression. This result indicates that progressive increases in the duration of ischemia in brain, prior to infarction, may produce proportional increases in transcription of the heat shock genes. However, once the duration of ischemia is long enough to produce infarction, this severely limits the availability of ATP which blocks transcription of the heat shock genes. In conclusion, concurrent induction of the heat shock genes suggests that kainic acid seizures and focal ischemia induce several different stress responses in brain cells caused by denaturation of proteins, changes of protein synthesis, and changes of protein glycosylation.

Presence of S-100 beta in cholinergic neurones of the rat hindbrain.

The double staining of S-100 beta and choline acetyltransferase (ChAT) revealed that S-100 beta immunoreactivity was localized in most, but not in all, cholinergic neurones in the somatomotor nuclei of the cranial nerves and in the ambiguus nucleus. S-100 beta was present in almost all cholinergic neurones in the brain stem reticular, red, vestibular (excluding medial), mesencephalic trigeminal and cerebellar nuclei. However, S-100 beta immunoreactivity was lacking in cholinergic neurones in the parabrachial complex, the dorsal motor nucleus of the vagal nerve and most sensory nuclei. No S-100 beta-positive neurones lacked ChAT immunoreactivity. Taken together with the fact that the vulnerability of motoneurones to axotomy is markedly reduced in the first 3 postnatal weeks, during which period neuronal S-100 beta appears and increases, a possible effect of S-100 beta on the survival of cholinergic motoneurones may be suggested.

An acquired sensitivity to H2O2-induced apoptosis during neuronal differentiation of NT2/D1 cells.

Brief exposure of neuronally differentiated human NT2/D1 cells to hydrogen peroxide induced cell death by apoptosis with an ED50 of 30 microM, whereas a 70-fold higher concentration was required to obtain an ED50 effect in undifferentiated NT2/D1 neuronal precursor cells. This enhanced sensitivity in NT2/D1 neurons was correlated with an 8-fold lower level of intracellular glutathione. Pretreatment with N-acetyl-L-cysteine, an agent that is able to raise the levels of intracellular glutathione, promoted the survival of hydrogen peroxide-treated NT2/D1 neurons. Thus, the low glutathione level may contribute to the high sensitivity of NT2/D1 neurones to hydrogen peroxide-induced apoptosis. This study indicates that neuronal susceptibility to oxidative damage is developmentally regulated.

S-100 beta immunoreactivity in neurones of the rat peripheral sensory ganglia.

S-100 beta, which is capable of exerting neurotrophic effects on cultured neurones and promoting the survival of motor neurones in vivo, has recently been found in distinct neurones of the rat hindbrain. Here we report that S-100 beta, as well as being present in satellite and Schwann cells, is also present in neurones of sensory ganglia (dorsal root ganglion, trigeminal, petrosal, jugular and nodose ganglia) but absent from neurones of the superior cervical ganglion. In the sensory ganglia, many neurones were immunoreactive, while the staining intensity varied among the neurones. Neuronal S-100 beta appeared in developing rats as early as postnatal day 1. No immunoreactive neurones were observed in the superior cervical ganglion during development. The results are suggestive of selective neurotrophic effects of S-100 beta.

Structural and functional evidence for the denervation of human myometrium during pregnancy.

The neuronal influence of the contractility of the term-pregnant human uterus was studied by pharmacologic and histochemical methods. By the use of adrenergic and cholinergic antagonists or tetrodotoxin it was demonstrated that the contractile response of superfused myometrial strips from the term-pregnant uterus to electrofield stimulation was not influenced by any neuronal factor. Using an indirect immunofluorescence technique with an antiserum to the glial and Schwann cell specific S-100 protein, a marked paucity of nerve fibers was demonstrated in myometrial strips from the term-pregnant uterus as compared with the nonpregnant uterus. It is concluded that the described decrease of nerve fibers within the myometrium of the pregnant uterus may be of importance for the myometrial activity during pregnancy and parturition.

Plasticity of granule cell-mossy fiber system following kainic acid induced seizures: an immunocytochemical study on neurofilament proteins.

Abnormal reestablishment of mossy fibers with the CA3 pyramidal cells and granule cells is an important aspect of postlesional plasticity in epilepsy. However, basis for the structural reorganisation and functional consequences of the event remain uncertain. Therefore we have investigated alterations of neurofilaments, major cytoskeletal components of neurons, in the rat hippocampus after the kainic acid (KA) administration, an experimental model for the temporal lobe epilepsy. The immunoreactivity for phosphorylated heavy weight neurofilament (pNFH) and non-phosphorylated heavy weight neurofilament (npNFH), in particular the pNFH, decreased in the CA1 field and inner molecular layer of the dentate gyrus during 3 and 10 days after the KA administration. After 10 days, npNFH immunoreactivity appeared in the mossy fibers, in which it is normally absent, meanwhile the pNFH staining in the mossy fibers did not decrease. From day 21, the immunoreactivity of pNFH and npNFH was normal or above normal in the CA1 stratum lacunosum-moleculare, mossy fibers, hilus and inner molecular layer of the dentate gyrus. These alterations in the later phase remained at least to day 90. The reappearance and increase of the neurofilament immunoreactivity in the inner molecular layer of the dentate gyrus probably reflects a collateral extension of the granule cell axons known as mossy fiber sprouting. The results suggest that neurofilament changes in the granule cell-mossy fiber system may be a morphological basis for the structural reconstruction of granule cell axons, and neurofilaments are involved in the plasticity after the KA induced seizures.

A simple quantitative dot-immunobinding assay for glial and neuronal marker proteins in SDS-solubilized brain tissue extracts.

Immunoassays for quantitative determinations of the S-100 protein, the glial fibrillary acidic protein, the neuron specific enolase and the neurofilament proteins with molecular weight of 68 and 200 kDa in hot SDS sonicated rat brain extracts have been developed and characterized. The assays utilize a dot immunobinding technique, poly- or monoclonal antibodies and 125I-protein A. The SDS-sonication procedure was not found to affect the radioactivity recovery in the assay of the soluble S-100 protein or the neuron specific enolase. All 5 antigens can be measured with a within-assay variance below 10%. Even at a coefficient of variation less than or equal to 5%, the working ranges are approximately 30-100-fold with regard to the different antigens. It was found that gelatin-coated nitrocellulose membranes considerably increase the recovered radioactivity in the assay of the purified bovine S-100 protein, possibly by protein-protein interaction. This effect was not observed when SDS-sonicated rat brain extracts were assayed. The assay appears to be reproducible, convenient and rapid, and provides a high degree of precision in the determination of large number of samples.

A sensitive ELISA for glial fibrillary acidic protein: application in CSF of children.

In the present study we describe a sensitive ELISA for determination of glial fibrillary acidic protein (GFAP). To validate the method combined determinations of GFAP and S-100 protein were performed in cerebrospinal fluid (CSF) of normal children and children with autism. The GFAP ELISA is of sandwich type and uses the biotin-avidin system. Sensitivity was 16 pg/ml. Between-day precision was 0.079 (coeff. of variance). S-100 protein concentrations were measured using a commercially available ELISA kit. Normal CSF from children and young adults were analysed. The CSF levels of GFAP in normal children were low (16-163 pg/ml). Both GFAP and S-100 protein concentrations correlated with age (P < 0.01 and P < 0.05, respectively), but the GFAP increment was more pronounced, probably reflecting the age-dependent expansion of the fibrillary astrocytes in the central nervous system (CNS). GFAP levels in children with infantile autism were higher than those in normal children of the same age range. S-100 protein concentrations were similar in both groups. High levels of GFAP in combination with normal S-100 protein concentrations in CSF indicates reactive astrogliosis in the CNS. In conclusion, the sensitive ELISA described makes it possible to measure low levels of GFAP present in the CSF of children. Combined assays of GFAP and S-100 protein can be used to discriminate between acute and chronic brain disorders in children.

S-100 in the central nervous system of rat, rabbit and guinea pig during postnatal development.

The accumulation of the brain-specific S-100 protein has been studied during postnatal development of rat, rabbit and guinea pig quantitatively, using immunoelectrophoresis, and qualitatively, by immunoelectron microscopy. Newborn guinea pigs show high levels of S-100. The distribution was similar to that of adult animals with an enrichment of S-100 to the postsynaptic membranes and to the astrocytic filaments. The neuronal plasma membranes as well as the neuronal nuclear membranes, astrocytic and oligodendroglial plasma membranes, also showed a specific activity for S-100. The amount of S-100 increased linearly from birth until the 3rd and 4th postnatal week of rabbit and rat, respectively. During the 2nd and 3rd week rabbit and rat nervous systems showed an accumulation of S-100, especially in the postsynaptic membranes and in the astrocytic filaments. In this study we present evidence that the S-100 protein quantitatively and ultrastructurally appears according to a pattern which parallels the muturation of brain, showing adult characteristics already at birth in early developing brains (guinea pig) and a change towards adult pattern after birth in late developing brains (rat and rabbit). In the latter two species change towards an adult S-100 distribution pattern proceeds during the postnatal period concomitant with the enzymatic and electrophysiogical maturation of the brain.

S-100beta stimulates neurite outgrowth in the rat sciatic nerve grafted with acellular muscle transplants.

S-100beta promotes neurite extension in vitro and motoneuron survival in the chicken embryo. We demonstrate here that local administration of S-100beta stimulates the sciatic nerve regeneration into acellular muscle grafts. Normally there is a 8-10 day delay in the regeneration of axons into such grafts. Local administration of S-100beta (0.5-1.0 microg/h) significantly stimulated regeneration into the grafts. In S-100beta treated grafts, the regeneration distance was increased with a factor of about 2.3 times as compared to vehicle treated grafts. The distance of regeneration was monitored with pinch test which detects sensory axons. Regenerating axons were growing outside the necrotic muscle cells as revealed with immunohistochemistry for the neurofilament light weight polypeptide. S-100beta was demonstrated immunocytochemically in motor neurons of the rat lumbar spinal cord and in large and medium sized neurons of the dorsal root ganglia. The results suggest that S-100beta is a physiological growth factor for peripheral nerve axons.

S-100 beta has a neuronal localisation in the rat hindbrain revealed by an antigen retrieval method.

The localisation of S-100 in mammalian CNS neurons has been under debate for more than two decades. We address the question with two polyclonal and two new monoclonal antibodies. The specificity and the distribution in rat brain is based on an antigen retrieval method. We present evidence that aldehyde fixatives mask S-100 beta in neurons, and that the immunoreactivity is retrieved after trypsinisation. Neuronal S-100 beta is also detected in unfixed and ethanol fixed sections. The neuronal immunoreactivity is partly solubilised from unfixed tissue sections with 2.5 mM EDTA and is completely extracted with 2.5 mM EDTA and 1% Triton X-100. Most of the glial S-100 beta is washed out from unfixed tissue sections with saline. S-100 beta has distinct distribution in neurons of the hindbrain, i.e., the brainstem and cerebellum, but is not observed in the forebrain. One of the monoclonal antibodies immunostained neither neurons nor glia when it had been absorbed with S-100 crosslinked to nitrocellulose membranes. The distribution of neuronal S-100 beta differed from that of other neuronal calcium binding proteins, such as calbindin and parvalbumin. It was confined mainly to cholinergic neurons of the hindbrain. The presence of S-100 beta in distinct neuronal populations may indicate neurotrophic effects of S-100 beta. The notion is supported by the capability of S-100 to cause neurite outgrowth in vitro.

Comparison of rat brain membrane antigens by complement mediated release of neurotransmitters from brain fractions using antisera against S-100 and Thy-1.

Synaptosomal, glial and neuronal fractions were prepared from rat brain and incubated to accumulate radioactively labelled neurotransmitters. Treatment of these fractions with antisera and complement showed that anti-(Thy-1) serum gave good release (50-75% of total uptake) of all neurotransmitters tested from synaptosomal and glial fractions. GABA and glutamate were released from neuronal perikarya, but not norepinephrine or serotonin. Anti-(S-100) serum gave no significant release of any neurotransmitter tested from any of the fractions, although all of them had previously been shown to contain this protein. These results are compatible with the membrane nature of Thy-1 and the mainly soluble nature of S-100 protein. They permit a selection for membrane antigens and neurotransmitters from different brain fractions. Antigenic differences between neuronal and glial plasma membranes were suggested by results with antiserum raised against bulk-isolated rat neuronal perikarya.

The effect of an N-methyl-D-aspartate lesion in the hippocampus on glial and neuronal marker proteins.

The study employed an immunochemical quantification of brain cell marker proteins in addition to quantitative morphology in order to provide a more multifacetted and characterized model for an excitotoxic CNS lesion. The importance of the approach in the evaluation of the potential of neuroprotective agents is emphasized. The S-100 protein, the glial fibrillary acidic (GFA) protein, neuron specific enolase (NSE) and neuronal intermediary filament polypeptides (NF 68 and NF 200) were measured with a dot-immunobinding assay, 3-30 days after a unilateral injection of N-methyl-D-aspartate (NMDA) in the left dorsal hippocampus of the rat. After 3 days, the neuronal cell loss averaged 80% in the hippocampus. The S-100 content was reduced 3 days after injection, but was 150% of control at 30 days. GFA increased constantly from days 3 to 30. The neuronal marker proteins were all markedly reduced 7 days after injection. However, at 30 days, NF 68 and NF 200 were close to control (80%). Increasing content would reflect regeneration and sprouting of neurites. The content of the neuronal cytoplasmic marker, NSE, was significantly lower than control also at 10 and 30 days, although a gradual recovery could be traced.

Appearance of neuronal S-100 beta during development of the rat brain.

In addition to being an astroglial protein, S-100 beta is localised in distinct populations of neurons in the adult rat hindbrain. We report, here, the expression of S-100 beta in both neurons and glia of the rat brain during development. Prenatally, S-100 beta immunoreactivity was confined to glial cells close to the germinal zone. After birth, S-100 beta positive glial cells were seen mainly in the brainstem and cerebellum, while only a few were detected in cerebral cortex and hippocampus. The number of S-100 beta containing glial cells increased steadily during the first 2 postnatal weeks after which the adult pattern was attained. No S-100 beta containing neurons were present prenatally. The first S-100 beta containing neurons were seen in the mesencephalic trigeminal nucleus at postnatal day 1 (P1), and in the motor trigeminal nucleus at P3. Neuronal S-100 beta immunoreactivity in other nuclei was mostly attained from the 10th to the 21st postnatal day. The neuronal S-100 beta immunoreactivity was first detected in the cell nuclei during development, then increased in the cytoplasm with ages. A nuclear staining in many immunoreactive neurons persisted until the adult. It usually took 1 to 2 weeks for neuronal S-100 beta to attain the adult staining pattern, i.e., heavy staining of the cytoplasm and processes, after its appearance. The forebrain never contained S-100 beta positive neurons. The S-100 beta is first expressed in glial cells, suggesting it is primarily of the glial origin. Coupled with neurotrophic effects of the protein, the time course of neuronal S-100 beta expression during the critical period of neuronal development implies that it may be involved in neuronal differentiation and maturation.

Definition of a cell clone with astroglial characteristics derived from a chemically induced rabbit brain glioma.

Three-month-old rabbits were started on a fortnightly schedule of intravenous injections of N-methyl-N-nitrosourea. All but two of the central nervous system tumors induced in this manner were propagated in culture as permanent cell lines. On the 76RB-G-414-H line established from a grade 2 astrocytoma of this series of neoplasms, a cloning procedure was carried out using a laser microbeam. The clonal line originated in this way has been maintained in long-term culture and given the 76/RB-G-414-H-C designation. The cells of the clone display invariably a bipolar or multipolar configuration with long processes. Intermediate filaments are common and even abundant in some cells. Positivity for S-100 and GFA proteins is a regular finding in these cells. In addition, dibutyryl cyclic adenosine monophosphate treatment reduces cell division and stimulates cell process formation of these cells. Thus, it appears that we succeeded in establishing in vitro and maintaining in long-term culture a clone of tumor cells with astrocytic characteristics.