A new model for diffuse brain injury by rotational acceleration: I model, gross appearance, and astrocytosis.

Rapid head rotation is a major cause of brain damage in automobile crashes and falls. This report details a new model for rotational acceleration about the center of mass of the rabbit head. This allows the study of brain injury without translational acceleration of the head. Impact from a pneumatic cylinder was transferred to the skull surface to cause a half-sine peak acceleration of 2.1 x 10(5) rad/s2 and 0.96-ms pulse duration. Extensive subarachnoid hemorrhages and small focal bleedings were observed in the brain tissue. A pronounced reactive astrogliosis was found 8-14 days after trauma, both as networks around the focal hemorrhages and more diffusely in several brain regions. Astrocytosis was prominent in the gray matter of the cerebral cortex, layers II-V, and in the granule cell layer and around the axons of the pyramidal neurons in the hippocampus. The nuclei of cranial nerves, such as the hypoglossal and facial nerves, also showed intense astrocytosis. The new model allows study of brain injuries from head rotation in the absence of translational influences.

A new model for diffuse brain injury by rotational acceleration: II. Effects on extracellular glutamate, intracranial pressure, and neuronal apoptosis.

The aim of this study is to monitor excitatory amino acids (EAAs) in the extracellular fluids of the brain and to characterize regional neuronal damage in a new experimental model for brain injury, in which rabbits were exposed to 180-260 krad/s2 rotational head acceleration. This loading causes extensive subarachnoid hemorrhage, focal tissue bleeding, reactive astrocytosis, and axonal damage. Animals were monitored for intracranial pressure (ICP) and for amino acids in the extracellular fluids. Immunohistochemistry was used to study expression of the gene c-Jun and apoptosis with the terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) technique. Extracellular glutamate, glycine, and taurine increased significantly in the hippocampus within a few hours and remained high after 24 h. Neuronal nuclei in the granule layers of the hippocampus and cerebellum were positive for c-Jun after 24 h. Little immunoreactivity was detected in the cerebral cortex. c-Jun-positive neuronal perikarya and processes were found in granule and pyramidal CA4 layers of the hippocampus and among the Purkinje cells of the cerebellum. Also some microglial cells stained positively for c-Jun. TUNEL reactivity was most intense at 10 days after trauma and was extensive in neurons of the cerebral cortex, hippocampus, and cerebellum. The initial response of the brain after rotational head injury involves brain edema after 24 h and an excitotoxic neuronal microenvironment in the first hour, which leads to extensive delayed neuronal cell death by apoptosis necrosis in the cerebral cortex, hippocampus and cerebellum.

Antisense inhibition of BCL-2 expression induces retinoic acid-mediated cell death during differentiation of human NT2N neurons.

Changes in expression of the proto-oncogene Bcl-2 are well known in the developing brain, with a high expression level in young post-mitotic neurons that are beginning the outgrowth of processes. The physiological significance of the Bcl-2 up-regulation in these neurons is not fully understood. We used a differentiation model for human CNS neurons to study the expression and function of Bcl-2. NT2/D1 human neuronal precursor cells differentiated into a neuronal phenotype in the presence of 10 microM retinoic acid for 3-5 weeks. This concentration of retinoic acid was not toxic to undifferentiated NT2/D1 cells but was sufficient to up-regulate the BCL-2 protein in 6 days. The BCL-2 levels increased further after 3 weeks, i.e. when the cells started to show neuronal morphology. Inhibition of the accumulation of endogenous BCL-2 with vectors expressing the antisense mRNA of Bcl-2 caused extensive apoptosis after 3 weeks of the retinoic acid treatment. The loss of neuron-like cells from differentiating cultures indicated that the dead cells were those committed to neuronal differentiation. Death was related to the presence of retinoic acid since withdrawal of retinoic acid after 16 days of treatment dramatically increased cell surviving. The ability of BCL-2 to prevent retinoic acid-induced cell death was also confirmed in undifferentiated NT2/D1 cells that were transfected with a vector containing Bcl-2 cDNA in sense orientation and exposed to toxic doses (40-80 microM) of retinoic acid. Furthermore, down-regulation of BCL-2 levels by an antisense oligonucleotide in neuronally differentiated NT2/D1 cells increased their susceptibility to retinoic acid-induced apoptosis. These results indicate that one function of the up-regulation of endogenous BCL-2 during neuronal differentiation is to regulate the sensitivity of young post-mitotic neurons to retinoic acid-mediated apoptosis.

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.

Antiserum against S-100 protein prevents long term potentiation through a cAMP-related mechanism.

Long term potentiation (LTP) was induced in the CA1 region of rat hippocampal slices by tetanization of the Schaffer collaterals. Local pretreatment of CA1 with serum of rabbits immunized against S-100 prevented the potentiation. However, treatment of the slices with a membrane permeant cAMP analogue, such as 8-Br-cAMP, could protect against the blocking effect of anti S-100 serum. We suggest that in the rat endogenous S-100b is involved in transduction mechanisms during LTP induction, via its ability to stimulate adenylate cyclase. Possible mechanisms of this action are discussed.

A new specific method for measuring S-100B in serum.

The S-100 family of proteins are acidic calcium and zinc binding low molecular weight proteins mainly present in astrocytes and in a population of oligodendrocytes of the CNS. S100b, an acidic low weight and zinc binding protein, has attracted considerable interest due to its release into the cerebrospinal fluid and blood from brain tissue following brain damage and from malignant melanomas. A new simple two-step incubation assay has now been elaborated in which two catcher and one tracer monoclonal antibodies are used. The specificity of this assay is high because all the MAbs used bind exclusively to S-100B, as shown by real-time biospecific interaction analyses. Moreover, the working range of the assay is 0.2-60 micrograms/L with a CV of less than 10%; the resulting high sensitivity has been confirmed by clinical studies. Time dependence, shaking conditions, lower limit of detection limits, effects of dilution, hook effect, recovery, impression as intra- and interassay variations, and crossreactivities with S-100A1 were tested in order to obtain a highly reproducible assay. Sera from healthy blood donors and patients undergoing cardiopulmonary bypass operations were tested with the assay. Several of the patients undergoing open heart surgery presented measurable values in this IRMA S-100 assay, indicating cerebral effects of open heart surgery. The test may be used for postoperative monitoring of these patients.

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.

Neuronal glycolytic pathway impairment induced by HIV envelope glycoprotein gp120.

Neurological impairment is a common feature of Acquired Immunodeficiency Syndrome (AIDS); functional alterations have been reported both in central and peripheral nervous system and the Human Immunodeficiency Virus (HIV) envelope glycoprotein gp120 has been proposed as a neurotoxin acting through a calcium-dependent mechanism. On the other hand it has been reported that gp120 treatment also induce about a 20% decrease in the cerebral glucose utilization and in the cellular ATP levels. The reported observations were performed on experimental system where also non-neuronal cells where present; in order to evaluate whether a direct interaction between HIV proteins and neuronal cells takes place, we used a neuroblastoma cultures where only neuronal cells are present. We analysed the effects of gp120 on the N18TG2 neuroblastoma clone. Treatments were performed both on growing and confluent cultures. Short time treatment with gp120 of confluent cultures causes a 25% reduction in the level of neuron-specific enolase, resulting in a similar decrease of oxygen consumption. Long time exposure of growing cells also causes a reduction in cell survival. Furthermore, using a membrane-specific fluorescent probe we observed that gp120 produces an increase of membrane trafficking. These observations suggest a direct interaction between the viral envelope protein and neuronal cells, which results in an alteration of glycolytic metabolism. This alteration may be related to the neurologic impairments observed in AIDS patients.

Elevated S100 blood level as an early indicator of intraventricular hemorrhage in preterm infants. Correlation with cerebral Doppler velocimetry.

The aim of this study was to assess the use of S100 protein in blood as a means of identifying preterm infants at risk of intraventricular hemorrhage. In 25 preterm newborns, S100 blood concentrations were measured by an immunoradiometric assay during the first 48 h. Cerebral Doppler velocimetry waveform patterns were also tested at the time the blood sample was taken, when clinical and cerebral ultrasound scanning were still normal. Of the 25 newborns studied, 14 were controls and 11 developed intraventricular hemorrhage as revealed by ultrasound scanning more than 72 h after birth, and clinically confirmed by neurological examination on the seventh day of follow-up. S100 blood concentrations were significantly higher (P<0.002) in infants with intraventricular hemorrhage than in control infants and also correlated significantly (r=0.81, P<0.003) with the grade of hemorrhage. A significant correlation (r=0.70, P<0.05) between the S100 blood concentration and the middle cerebral artery pulsatility index was also observed. The present data show that S100 blood concentrations offer a measurable parameter of brain lesion in preterm infants before a radiological assessment of hemorrhage can be performed, when clinical symptoms may be silent and preventive/therapeutic action could be especially useful.

Involvement of S-100 protein in anoxic long-term potentiation.

In in vitro rat hippocampal slices a short period (2 min) of hypoxia resulted in lasting potentiation of the population spike transynaptically evoked in CA1 by stimulation of Schaffer collaterals ("anoxic LTP"). Pretreatment of slices with antiserum against S-100 protein fully prevented this anoxic LTP. Since also "classical" (i.e., induced by high-frequency electrical stimulation) long-term potentiation is prevented by anti S-100 serum, this represents one more important similarity between these events.

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.

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.

Decreased levels of S-100 protein in non-ulcer interstitial cystitis.

Interstitial cystitis (IC) is a chronic debilitating condition which mainly affects women. Accumulated evidence indicates that IC is a heterogeneous syndrome. As compared to classic IC, the non-ulcer type of IC appears to be different concerning symptomatic, endoscopical and histological findings, as well as in response to various forms of treatment. S-100 is a neural protein considered to be located primarily in the axons. To explore further the neurogenic nature of the disease, we compared bladder wall S-100 content in controls and in patients with classic and non-ulcer IC. We noticed a decrease in S-100 content in non-ulcer IC as compared to controls. This may be an expression of altered peripheral innervation in non-ulcer IC, which, in turn, may be an indication of primary neurogenic etiology. The difference in S-100 content between classic and non-ulcer IC supports the hypothesis that they represent separate entities, which may explain differences in response to various treatments.

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.

Delayed decrease of calbindin immunoreactivity in the granule cell-mossy fibers after kainic acid-induced seizures.

Kainic acid (KA) administration induces an abnormal excitation and spontaneous recurrent seizures. Alterations of granule cell properties may be potential mechanisms. In this study, dynamic alterations of calbindin, a calcium binding protein particularly abundant in the granule cells, have been investigated immunocytochemically in the rat hippocampus after the KA-induced seizures. The calbindin immunoreactivity decreased slightly in the CA1/CA2 fields already after 1 and 3 days, and was lost partly or completely in the pyramidal layer after 10 days. From day 21, the calbindin immunoreactivity decreased in dendrites and soma of the granule cells and mossy fibers. The alterations remained at least to day 90, while no evident neuronal loss occurred in the granule cells. This may reflect a disturbance of calcium homostasis in the granule cells after seizures. The delayed decrease of calbindin has a time course similar to the occurrence of spontaneous recurrent seizures, suggesting a possible correlation between the two events.

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.

Eliprodil prevents expression of the 70 kDa heat shock protein in MK801-injured neurones.

The present study examined whether eliprodil (SL 82.0715), an N-methyl-D-aspartate (NMDA) receptor antagonist acting on the polyamine sites induced expression of the 70 kDa heat shock protein (HSP70) in the rat brain. Whereas the NMDA channel blocker MK801 consistently induced HSP70 in posterior cingulate and retrosplenial cortices, eliprodil had no such effects even at the highest dose (50 mg/kg, intraperitoneally), supporting the idea that injury to the cerebrocortical neurones by NMDA receptor antagonists is probably related to specific sites of the receptor. Furthermore, eliprodil, given immediately after injection of MK801, blocked the effects of MK801 on HSP70. The result is discussed in terms of high affinity of eliprodil for the sigma receptor.

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.

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.