Pathoarchitectonics of the cerebral cortex in chorea-acanthocytosis and Huntington's disease.

AIMS: Quantitative estimation of cortical neurone loss in cases with chorea-acanthocytosis (ChAc) and its impact on laminar composition. METHODS: We used unbiased stereological tools to estimate the degree of cortical pathology in serial gallocyanin-stained brain sections through the complete hemispheres of three subjects with genetically verified ChAc and a range of disease durations. We compared these results with our previous data of five Huntington's disease (HD) and five control cases. Pathoarchitectonic changes were exemplarily documented in TE1 of a 61-year-old female HD-, a 60-year-old female control case, and ChAc3. RESULTS: Macroscopically, the cortical volume of our ChAc cases (ChAc1-3) remained close to normal. However, the average number of neurones was reduced by 46% in ChAc and by 33% in HD (P = 0.03 for ChAc & HD vs. controls; P = 0.64 for ChAc vs. HD). Terminal HD cases featured selective laminar neurone loss with pallor of layers III, V and VIa, a high density of small, pale, closely packed radial fibres in deep cortical layers VI and V, shrinkage, and chromophilia of subcortical white matter. In ChAc, pronounced diffuse astrogliosis blurred the laminar borders, thus masking the complete and partial loss of pyramidal cells in layer IIIc and of neurones in layers III, V and VI. CONCLUSION: ChAc is a neurodegenerative disease with distinct cortical neurodegeneration. The hypertrophy of the peripheral neuropil space of minicolumns with coarse vertical striation was characteristic of ChAc. The role of astroglia in the pathogenesis of this disorder remains to be elucidated.

Comparison of low energy and high energy electron beam treatments on sensory and chemical properties of seeds.

Consumption of fresh and minimally processed foods such as seeds as a part of a healthy diet is a trend. Unfortunately, fat-rich seeds are often contaminated with pathogenic microorganisms and face frequent product recalls. Electron beams have been applied as a microbial decontamination measure for decades. Conventionally high energy electron beams (HEEB) are being used, whereas low energy electron beams (LEEB, <300 keV) have only recently been introduced to the food industry and more studies are needed. Electron beam treatment has several advantages over other decontamination technologies. The treatment is non-thermal, chemical-free, water-free, and does not use radioactive substances. The effect of electron beams on the sensory and chemical properties of seeds has not been widely studied. This study assessed LEEB and HEEB treated pumpkin and flax seeds immediately after treatments, and after three months of storage. The seeds' sensory profiles were altered after both treatments when compared with non-treated samples, with a higher dose leading to a greater level of alteration. However, the sensory profile of LEEB treated seeds was similar to the non-treated seeds whereas HEEB treated seeds differed from both. The storage period of three months further increased the observed differences between the samples. LEEB and HEEB treatments seemed to cause lipid degradation as the content of volatile aldehydes was increased. This effect was more profound in HEEB treated samples. The data presented in this study shows that LEEB as a microbial reduction solution has great potential to preserve the chemical and sensory properties of nutritious seeds.

Influence of hydrothermal treatment on the structural modification of spent grain specific carbohydrates and the formation of degradation products using model compounds.

Brewer's spent grain (BSG) constitutes various valuable carbohydrates that may contribute to a healthy diet. These components may be obtained from BSG via hydrothermal treatment (HT), a procedure for dissolving water-inextricable carbohydrates. The objective of this study was to investigate HT as an environmentally friendly technology for extracting high-molecular-weight fiber with proven beneficial effects on human health. Cellulose, ß-glucan, and arabinoxylan (AX) served as model substances and were subjected to auto-hydrolysis at different temperatures and reaction times. The results were evaluated in terms of structural and chemical characteristics. When the treatment temperature was increased, the original weight-average molar mass of AX (370 kDa) and ß-glucan (248 kDa) decreased gradually (<10 kDa), and the molar mass distribution narrowed. Further investigations focused on the heat-induced formation and elimination of monosaccharides and undesirable by-products. The concentrations of by-products were successfully described by kinetic models that can be used to optimize the hydrolysis process.

Characterization of genomic alterations associated with glioma progression by comparative genomic hybridization.

Genomic alterations associated with glioma progression were determined by comparative genomic hybridization (CGH) 30 tumors from 15 patients with primary gliomas of World Health Organization (WHO) grade II that on recurrence showed progression to malignant gliomas of WHO grades III or IV (five cases of astrocytoma grade II (A II) to grade III (AA III), five cases of A II to glioblastoma multiforme grade IV (GBM) and five cases of oligodendroglioma grade II (O II) to grade III (AO III)). All tumors were additionally screened for p53 mutations by single strand conformational polymorphism and heteroduplex analysis of exons 5-8, followed by direct sequencing. Mutations of p53 were found in the primary and recurrent tumors of all cases of A II progressing to GBM and three of five cases of A II recurring as AA III. Alterations identified by CGH in more than one primary A II included losses on Xp (3/10) and 5p (2/10), gains on 8q and 19p (2/10 each), and gain/amplification on 12p (2/10). Common progression associated changes found in AA III or GBM were losses on 4q, 9p, 10q, 11p, 13q (4/10 each) and gains on 1q, 6p, 20q (2/10 each). The most frequent amplification site was located on 12p13 (1/10 A II, 3/5 AA III, 1/5 GBM). Other amplified chromosomal regions were 13q32-q34 (1/10 AII, 2/5 GBM), 7q31-qter (1/5 AA III, 1/5 GBM), 12q22-qter and 18p (1/5 AA III). In contrast to the astrocytic gliomas, only one of five oligodendroglial cases showed a p53 mutation. Genetic abnormalities identified by CGH to occur more than once were restricted to four chromosomes (1, 4, 9 and 19). Our results provide a comprehensive overview of the genomic alterations associated with the progression of individual gliomas and substantiate the hypothesis that glioma progression is associated with a cumulative acquisition of multiple genetic changes.

Detection of complex genetic alterations in human glioblastoma multiforme using comparative genomic hybridization.

The aim of the present study was to detect complex genetic alterations in human glioblastoma multiforme (GBM) by comparative genomic in situ hybridization (CGH). Of the 24 GBM that were examined, increased fluorescence intensities indicating chromosomal polysomy of chromosome 7 and gene amplification at chromosome 7p were found in 42% of the tumors. In addition, signal enhancement of chromosome 19 was present in 29% and at 12q13-15 in 21% of the tumors. We also detected reduction of fluorescence intensities indicating gross deletions on chromosomes 10 (58%), 9p (46%), and 13 (29%). There was a close correlation of CGH results when compared with Southern analysis of the EGFR gene localized on chromosome 7 and loss of heterozygosity detection of chromosome 9 and 10 by microsatellite PCR. A close correlation was also observed between copy number changes of chromosome 7 and deletions of chromosome 10. Amplification of chromosome 12q and deletions of chromosomes 9p and 13 seemed to be complementary in the tumors investigated in the present study.

Immediate early gene expression in experimental epilepsy.

Neuronal excitation by experimentally induced seizures elicits the rapid induction of a set of genes called immediate early genes (IEGs). The gene products of fos, jun and Krox, multimember gene families that belong to the class of IEGs, participate in a fundamental biological control mechanism, the regulation of gene transcription. IEG encoded proteins act as third messengers in an intracellular signal transduction cascade between neural cell surface receptors, cytoplasmic second messenger systems and specific target genes in the nucleus, a process for which the term 'stimulus transcription coupling' has been given. Almost all types of seizures cause dynamic alterations of IEG expression in neurons of the limbic system, but also in non-limbic areas, such as the cortex, striatum and thalamus. IEG encoded transcription factors are thought to up- or down-regulate effector genes with preferential expression in the central nervous system, including genes for neurotransmitters, growth factors, receptors, synaptic and axonal proteins. If the concept holds true that IEGs act as molecular switches converting epileptic short-term excitation of neurons into alterations of the molecular phenotype, future research may help to explain hitherto unexplained phenomena in epileptogenesis including changes of synaptic efficacy, kindling and sprouting.

Stimulus-transcription coupling in focal cerebral ischemia.

Glutamate-mediated spreading depression is currently thought to be a key event in the pathogenesis of potential neuronal degeneration in the ischemic 'penumbra'. Glutamate receptor stimulation causes induction of transcription factors that belong to the class of immediate early genes (IEGs), thought to be involved in coupling neuronal excitation to target gene expression. Focal cerebral ischemia elicits a homogeneous expression of several IEGs, prominently in cortex. In the ischemic core, discrepancies are observed between mRNA and protein levels, due to a severe, persistent protein synthesis deficit, preventing the translation of IEG encoded mRNAs. Outside the ischemic core, widespread IEG expression occurs in the entire ipsilateral cortex at mRNA as well as at protein level. This homogeneous expression of transcription factors can be pinpointed to at least two different pathogenetic mechanisms by means of appropriate pharmacological antagonists. Prolonged IEG induction in the 'penumbra', an area in which neurons are metabolically compromised but not yet energy-depleted, cannot be suppressed by the administration of N-methyl-D-aspartate (NMDA) receptor antagonists. In contrast, short-lasting IEG induction in undamaged neurons remote from the ischemic territory, though also caused by ischemia-elicited spreading depression, can be blocked by NMDA receptor antagonists. In both areas, IEG expression identifies neurons destined to survive but is likely to be mediated by different signal transduction pathways, at the receptor, second messenger and/or the DNA level.

Selective c-JUN expression in CA1 neurons of the gerbil hippocampus during and after acquisition of an ischemia-tolerant state.

The selective delayed neuronal death of CA1 pyramidal cells after transient global ischemia in the gerbil brain can be prevented by preconditioning with a short sublethal period of ischemia 1-7 days prior to a subsequent, usually lethal ischemia of 5 min duration. Since changes of neuronal gene expression may play a crucial role in this tolerance induction, we investigated the postischemic expression profile of the fos, jun and Krox transcription factor families. We have previously reported that a single 5 min period of cerebral ischemia does not cause a de novo synthesis of immediate early gene (IEG) encoded proteins in CA1 neurons. In the present study, two experimental groups of Mongolian gerbils were investigated: one group was subjected to a single tolerance-inducing 2.5 min period of ischemia by bilateral occlusion of the common carotid artery. The second (combined ischemia) group was subjected to 2.5 min of ischemia, followed by 5 min of ischemia 4 days later. Post-ischemic expression of c-FOS, FOS B, c-JUN, JUN B, JUN D and KROX-24 was investigated by in situ hybridization and immunocytochemistry up to 48 h of recirculation. In contrast to a single 5 min period of ischemia, 2.5 min caused a postischemic expression of c-JUN protein, but no other IEGs, in CA1 neurons (peak at 6 h). Similarly, a selective but delayed c-JUN expression (peak at 18 h) was observed in animals subjected to combined ischemia. These results indicate that the induction of an endogenous neuroprotective state in CA1 neurons is associated with the activation of a genetic program which involves the expression of specific transcription factors.

Expression of synaptopodin, an actin-associated protein, in the rat hippocampus after limbic epilepsy.

Synaptopodin, a 100 kD protein, associated with the actin cytoskeleton of the postsynaptic density and dendritic spines, is thought to play a role in modulating actin-based shape and motility of dendritic spines during formation or elimination of synaptic contacts. Temporal lobe epilepsy in humans and in rats shows neuronal damage, aberrant sprouting of hippocampal mossy fibers and subsequent synaptic remodeling processes. Using kainic acid (KA) induced epilepsy in rats, the postictal hippocampal expression of synaptopodin was analyzed by in situ hybridization (ISH) and immunohistochemistry. Sprouting of mossy fibers was visualized by a modified Timm's staining. ISH showed elevated levels of Synaptopodin mRNA in perikarya of CA3 principal neurons, dentate granule cells and in surviving hilar neurons these levels persisted up to 8 weeks after seizure induction. Synaptopodin immunoreactivity in the dendritic layers of CA3, in the hilus and in the inner molecular layer of the dentate gyrus (DG) was initially reduced. Eight weeks after KA treatment Synaptopodin protein expression returned to control levels in dendritic layers of CA3 and in the entire molecular layer of the DG. The recovery of protein expression was accompanied by simultaneous supra- and infragranular mossy fiber sprouting. Postictal upregulation of Synaptopodin mRNA levels in target cell populations of limbic epilepsy-elicited damage and subsequent Synaptopodin protein expression largely co-localized with remodeling processes as demonstrated by mossy fiber sprouting. It may thus represent a novel postsynaptic molecular correlate of hippocampal neuroplasticity.

Intravenous brain-derived neurotrophic factor reduces infarct size and counterregulates Bax and Bcl-2 expression after temporary focal cerebral ischemia.

BACKGROUND AND PURPOSE: Pretreatment with intraventricular brain-derived neurotrophic factor (BDNF) reduces ischemic damage after focal cerebral ischemia. In this experiment we studied the effect of intravenous BDNF delivered after focal cerebral ischemia on neurological outcome, infarct size, and expression of proapoptotic and antiapoptotic proteins Bax and Bcl-2, respectively. METHODS: With the use of the suture occlusion technique, the right middle cerebral artery in rats was temporarily occluded for 2 hours. Thirty minutes after vessel occlusion, BDNF (300 microg/kg per hour in vehicle; n=12) or vehicle alone (n=13) was continuously infused intravenously for 3 hours. After 24 hours the animals were weighed and neurologically assessed on a 5-point scale. The animals were then killed, and brains underwent either 2,3,5-triphenyltetrazolium chloride staining for assessment of infarct volume or paraffin embedding for morphology and immunohistochemistry (Bax, Bcl-2). RESULTS: Physiological parameters (mean arterial blood pressure, PO(2), PCO(2), pH, body temperature, glucose) and weight revealed no difference between groups. Neurological deficit was improved in BDNF-treated animals versus controls (P:<0.05, unpaired, 2-tailed t test). Mean+/-SD infarct volume was 229.7+/-97.7 mm(3) in controls and 121.3+/-80.2 mm(3) in BDNF-treated animals (P:<0.05, unpaired, 2-tailed t test). Cortical infarct volume was 155.5+/-78.5 mm(3) in the placebo group and 69.9+/-50.2 mm(3) in the BDNF-treated group (P:<0.05, unpaired, 2-tailed t test). Subcortical infarct volume was 74.1+/-30.6 mm(3) in the placebo group and 51.1+/-26.8 mm(3) in the BDNF-treated group (P:=NS). Bax-positive neurons were significantly reduced in the ischemic penumbra in BDNF-treated animals (P:<0.05, unpaired, 2-tailed t test), whereas Bcl-2-positive neurons were significantly increased in this area (P:<0.001, unpaired, 2-tailed t test). CONCLUSIONS: This study demonstrates a neuroprotective effect of BDNF when delivered intravenously after onset of focal cerebral ischemia. As shown here, one possible mechanism of action of neuroprotection of BDNF after focal ischemia appears to be counterregulation of Bax/Bcl-2 proteins within the ischemic penumbra.

Effect of brain-derived neurotrophic factor treatment and forced arm use on functional motor recovery after small cortical ischemia.

BACKGROUND AND PURPOSE: Both the administration of growth factors and physical therapy such as forced arm use (FAU) are promising approaches to enhance recovery after stroke. We explored the effects of these therapies on behavioral recovery and molecular markers of regeneration after experimental ischemia. METHODS: Rats were subjected to photothrombotic ischemia: sham (no ischemia), control (ischemia), brain-derived neurotrophic factor (BDNF; ischemia plus BDNF, 20 microg), and FAU (ischemia plus FAU, 1-sleeve plaster cast ipsilateral limb). Animals survived 1 or 6 weeks and underwent behavioral testing (Rotarod, beam balance, adhesive removal, plantar test, neuroscore). After the rats were killed, brain sections were immunostained for semiquantitative analysis of MAP1B, MAP2, synaptophysin, GFAP expression, and quantification of infarct volumes. RESULTS: Infarct volumes were not different between the groups 1 or 6 weeks after ischemia. BDNF-treated animals had better functional motor recovery (Rotarod, beam balance, neuroscore) compared with all other groups (P<0.05). There was no significant adverse effect of early FAU treatment on motor recovery, although sensorimotor function (adhesive removal test) was impaired (P<0.05). There were no differences between groups as measured by nociception of the left and right forepaw (plantar test). BDNF treatment transiently induced MAP1B expression in the ischemic border zone and synaptophysin expression within the contralateral cortex 6 weeks after ischemia (P<0.05). Both BDNF and FAU reduced astrogliosis compared with controls (P<0.05). CONCLUSIONS: Postischemic intravenous BDNF treatment improves functional motor recovery after photothrombotic stroke and induces widespread neuronal remodeling. Early FAU treatment after stroke does not increase infarct size, impairs sensorimotor function, but leaves motor function unchanged. Postischemic astrogliosis was reduced by both treatments.

Selective gene expression in focal cerebral ischemia.

Regional patterns of protein synthesis were examined in rat cortex made ischemic by the occlusion of the right common carotid and middle cerebral arteries. At 2 h of ischemia, proteins were pulse labeled with intracortical injections of a mixture of [3H]leucine, [3H]isoleucine, and [3H]proline. Newly synthesized proteins were analyzed by two-dimensional gel fluorography, and the results correlated with local CBF, measured with [14C]iodoantipyrine as tracer. Small blood flow reductions (CBF = 50-80 ml 100 g-1 min-1) were accompanied by a modest inhibition in synthesis of many proteins and a marked increase in one protein (Mr 27,000). With further reduction in blood flow (CBF = 40 ml 100 g-1 min-1), synthesis became limited to a small group of proteins (Mr 27,000, 34,000, 73,000, 79,000, and actin) including two new polypeptides (Mr 55,000 and 70,000). Severe ischemia (CBF = 15-25 ml 100 g-1 min-1) caused the isoelectric modification of several proteins (Mr 44,000, 55,000, and 70,000) and induced synthesis of another protein (Mr 40,000). Two polypeptides (Mr 27,000 and 70,000) dominated residual protein synthesis in severe ischemia. The changes in protein synthesis induced by different grades of ischemia most likely comprise a variation of the so-called "heat shock" or "stress" response found in all eukaryotic cells subjected to adverse conditions. Since heat shock genes are known to confer partial protection against anoxia and a variety of other noxious insults, their induction may be a factor in limiting the extent of ischemic tissue damage.

Regional heterogeneity of L-[3-(3)H]tyrosine incorporation into rat brain proteins during severe hypoglycemia.

Regional protein synthesis was investigated during severe, insulin-induced hypoglycemia in anesthetized, artificially ventilated rats. Three minutes after the cessation of spontaneous EEG activity, animals received a single injection of L-[3H]tyrosine and were killed 30 min later. Autoradiographs revealed an almost complete inhibition of amino acid incorporation in the cerebral cortex, hippocampus, and caudate-putamen of hypoglycemic rats. Thalamus and spinal cord were less affected, and in hypothalamus, cerebellum, and brainstem the pattern of [3H]tyrosine incorporation was identical in control and experimental animals. The areas showing severely reduced protein biosynthesis during hypoglycemia coincide with those exhibiting extensive breakdown of energy metabolism, reduced cerebral metabolic rate for glucose, and high susceptibility to persistent cell damage. High-resolution autoradiography employing tritiated amino acids permits the assessment of regionally impaired protein biosynthesis and may thus be useful for the identification of neuronal populations at risk during hypoglycemia of varying degrees and of those cells most likely to recover after glucose infusion.

Focal brain ischemia in the rat: methods for reproducible neocortical infarction using tandem occlusion of the distal middle cerebral and ipsilateral common carotid arteries.

This article describes a 3-year experience with focal neocortical ischemia in three rat strains. Multiple groups of adult Wistar (n = 50), Fisher 344 (n = 31), and spontaneously hypertensive (n = 72) rats were subjected to permanent occlusion of the distal middle cerebral (MCA) and ipsilateral common carotid arteries (CCA). Twenty-four hours later the animals were killed, and frozen brain sections were stained with hematoxylin and eosin to demarcate infarcted tissue. The infarct volume for each section was quantified with an image analyzer, and the total infarct volume was calculated with an iterative program that summed all interval volumes. Neocortical infarct volume was the largest and most reproducible in the spontaneously hypertensive rats (SHR). Statistical power analysis to project the numbers of animals necessary to detect a 25 or 50% change in infarct volume with alpha = 0.05 and beta = 0.2 revealed that only the SHR model was practical in terms of requisite animals: i.e., less than 10 animals per group. Tandem occlusion of the distal MCA and ipsilateral CCA in the SHR strain provides a surgically simple method for causing large neocortical infarcts with reproducible topography and volume. The interanimal variability in infarct volume that occurs even in the SHR strain dictates that randomized, concomitant controls are necessary in each study to ensure the accurate assessment of experimental manipulations or pharmacologic therapies.

Synthesis of heat shock proteins in rat brain cortex after transient ischemia.

Cell-free protein synthesis and two-dimensional gel autoradiography were used to characterize early postischemic protein synthesis in rat neocortex. Severe forebrain ischemia was induced for 30 min (four-vessel occlusion model) and followed by 3 h of recirculation. Polysomes were isolated from the cerebral cortex, translated in vitro in a reticulocyte system, and analyzed by two-dimensional gel electrophoresis. The translation products of postischemic polysomes included a major new protein family (70 kDa) with multiple isoelectric variants that was found to comigrate with the 68- to 70-kDa "heat shock" protein synthesized from polysomes of hyperthermic rats. Two other stress proteins (93 and 110 kDa) also appeared to be synthesized in increased amounts after ischemia. A complement of proteins that was indistinguishable from that of controls was also synthesized after ischemia, indicating that messenger ribonucleic acid coding for most brain proteins is preserved after ischemia and is bound to polysomes.

Protein synthesis in postischemic rat brain: a two-dimensional electrophoretic analysis.

This study examined the pattern of protein synthesis in the neocortex, caudate-putamen, and the hippocampus following transient forebrain ischemia in rats. The animal model of temporary ischemia used in this study causes permanent damage to vulnerable neurons with a time course of injury that varies from hours (caudate nucleus) to days (hippocampus). To examine the spectrum of proteins synthesized in these regions at 3 and 18 h after recirculation, cerebral proteins were pulse-labeled in vivo by an intravenous injection of [35S]methionine. Newly synthesized (35S-labeled) and constitutive (unlabeled) proteins were analyzed by two-dimensional gel electrophoresis and fluorography. In all three brain regions, specific proteins underwent preferential synthesis (Mr approximately 27,000, approximately 65,000, approximately 70,000, approximately 110,000), while others showed decreased synthesis (neuron-specific enolase, alpha- and beta-tubulin). There was an early (3 h post ischemia) induction of the Mr approximately 70,000 mammalian "stress" protein; at 18 h post ischemia, its synthesis remained high in the hippocampus but was diminished in the neocortex and had largely subsided in the caudate-putamen. All regions at 18 h showed increased synthesis of an Mr approximately 50,000 protein, tentatively identified as glial fibrillary acidic protein. The results show that temporary forebrain ischemia induces changes in protein synthesis that include features similar to those observed in other eukaryotic cells subjected to injurious stress. These postischemic changes in protein synthesis are qualitatively similar in all brain regions examined despite regional differences in the severity of subsequent neuronal damage. The persistent synthesis of the Mr approximately 70,000 stress protein in the hippocampus, however, may reflect continued metabolic injury long after the ischemic episode has passed.

Cerebral protein synthesis during long-term recovery from severe hypoglycemia.

Regional protein synthesis was investigated in the rat brain during long-term recovery from insulin-induced hypoglycemia with 30 min of cerebral electrical silence. At various time intervals up to 14 days after glucose replenishment, animals received a single dose of L-[3,5-3H]tyrosine and were killed 30 min later. Brains were processed for autoradiography using the stripping film technique. Although hypoglycemia sufficiently severe to cause cessation of EEG activity leads to almost complete inhibition of amino acid incorporation in all "vulnerable" forebrain structures (cerebral cortex, hippocampus, caudoputamen), autoradiographs revealed a very specialized sequence with differential posthypoglycemic restoration of biosynthetic activity in certain neuronal cell types. Three major subpopulations could be distinguished: Neurons that fully regained their protein synthetic capacity within 6 h following hypoglycemia (cortical neurons of layer III-VI, large neurons in the caudoputamen, CA3 and CA4 pyramidal neurons, the majority of granule cells of the dentate gyrus) seemed to escape neuronal necrosis. Prolonged impairment of protein synthesis with only partial restoration during the early posthypoglycemic recovery period (CA1 neurons, most small- to medium-sized neurons of the caudoputamen) carried an increased risk of permanent cell damage. The large majority of these neurons, however, showed full recovery of protein synthesis as late as 7 days after hypoglycemia. Neurons with complete lack of amino acid incorporation after 6 h of recovery (granule cells at the crest of the dentate gyrus, small neurons of the dorsolateral caudoputamen) never resumed protein synthesis, regressed, and died. These studies in conjunction with morphological analysis indicate that the sequential recovery of protein synthesis reflects the extent to which neuronal populations are at risk during severe hypoglycemia.

NMDA and glycine receptor mRNA expression following transient global ischemia in the gerbil brain.

Excitotoxic activation of postsynaptic N-methyl-D-aspartate (NMDA) receptors is thought to be a key event for the molecular pathogenesis of postischemic delayed neuronal death in CA1 hippocampus. To assess a possible interference of ischemia with NMDA receptor expression, transcription of the NMDA receptor 1 (NMDA-R1) gene was examined by in situ hybridization in the gerbil brain after 5 min of global ischemia and various recirculation intervals. In normal gerbil brain, NMDA-R1 was strongly expressed and equally abundant in CA1 and CA3 neurons. After ischemia, expression remained unchanged for 24 h, followed by a selective decline in mRNA levels in CA1 neurons, resulting in the complete disappearance of hybridization signals after 4 days. NMDA-R1 expression in forebrain neurons less vulnerable or resistant to ischemia including CA3 and dentate granule cells remained unchanged. Similar in situ data were obtained for the beta subunit of the inhibitory glycine receptor (Gly-R). This subunit is also abundantly expressed in the pyramidal cell layer of the hippocampus, but not known to be involved in the mechanisms of post-ischemic excitotoxicity.

Hypoglycemia-elicited immediate early gene expression in neurons and glia of the hippocampus: novel patterns of FOS, JUN, and KROX expression following excitotoxic injury.

In the hippocampus there is a graded vulnerability of neuronal subpopulations to hypoglycemia-induced degeneration, most likely due to excitotoxic activation of glutamate receptors. The present study was conducted to investigate whether the induction of transcription factors of the immediate early gene (IEG) family after hypoglycemia reflects these different grades of neuronal vulnerability. We studied the expression profile of seven IEG-coded proteins in the rat hippocampus following severe insulin-induced hypoglycemia with 30 min of EEG isoelectricity and various survival periods for up to 42 h after glucose replenishment. Immunocytochemistry was performed on vibratome sections with specific polyclonal antisera directed against c-FOS, FOS B, c-JUN, JUN B, JUN D, KROX-24, and KROX-20. To unequivocally define the type of glial cells showing IEG induction, we investigated coexpression of c-FOS and glial marker proteins (glial fibrillary acid protein [GFAP], OX-42) by confocal laser scanning microscopy. Up to 3 h after glucose replenishment, differential temporospatial induction of IEG-coded transcription factors of the FOS, JUN and KROX families were observed in moderately injured neuronal subpopulations, including the majority of dentate granule cells and CA3 neurons. At later time points, however, a delayed and persistent c-JUN expression was found in severely, but reversibly, injured CA1 neurons and in neurons in the immediate vicinity of irreversibly damaged neurons in the crest of the dentate gyrus. Similar to the results with experimental models of central and peripheral axotomy, selective c-JUN induction in these neurons may represent an initial event in the regeneration process of sublethally injured neurons. In contrast to other models of excitotoxic injury such as ischemia and epilepsy, marked glial c-FOS expression was restricted to astrocytes, as assessed by confocal laser scanning microscopy.