Developmental regulation of gene expression and astrocytic processes may explain selective hippocampal vulnerability

The hippocampus plays a central role in the brain network that is essential for memory function. Paradoxically, the hippocampus is also the brain structure that is most sensitive to hypoxic‐ischemic episodes. Here, we show that the expression of genes associated with glycolysis and glutamate metabolism in astrocytes and the coverage of excitatory synapses by astrocytic processes undergo significant decreases in the CA1 field of the monkey hippocampus during postnatal development. Given the established role of astrocytes in the regulation of glutamate concentration in the synaptic cleft, our findings suggest that a developmental decrease in astrocytic processes could underlie the selective vulnerability of CA1 during hypoxic‐ischemic episodes in adulthood, its decreased susceptibility to febrile seizures with age, as well as contribute to the emergence of selective, adultlike memory function. © 2009 Wiley‐Liss, Inc.     

Colchicine induces apoptosis in organotypic hippocampal slice cultures

The microtubule-disrupting agent colchicine is known to be particular toxic for certain types of neurons, including the granule cells of the dentate gyrus. In this study we investigated whether colchicine could induce such neuron-specific degeneration in developing (1 week in vitro) and mature (3 weeks in vitro) organotypic hippocampal slice cultures and whether the induced cell death was apoptotic and/or necrotic. When applied to 1-week-old cultures for 48 h, colchicine induced primarily apoptotic, but also a minor degree of necrotic cell death in the dentate granule cells, as investigated by cellular uptake of the fluorescent dye propidium iodide (PI), immunostaining for active caspase 3 and c-Jun/AP-1 (N) and fragmentation of nuclei as seen in Hoechst 33342 staining. All four markers appeared after 12 h of colchicine exposure. Two of them, active caspase 3 and c-Jun/AP-1 (N) displayed a similar time course and reached a maximum after 24 h of exposure, 24 h ahead of both PI uptake and Hoechst 33342 staining, which together displayed similar time profiles and a close correlation. In 3-week-old cultures, colchicine did not induce apoptotic or necrotic cell death. Attempts to interfere with the colchicine-induced apoptosis in 1-week-old cultures showed that colchicine-induced PI uptake and formation of apoptotic nuclei were temporarily prevented by coapplication of the protein synthesis inhibitor cycloheximide. Application of the pancaspase inhibitor z-VAD-fmk almost completely abolished the formation of active caspase 3 protein and apoptotic nuclei induced by colchicine, but the formation of necrotic nuclei increased correspondingly and the PI uptake was unaffected. We conclude that colchicine induces caspase 3-dependent apoptotic cell death of dentate granule cells in hippocampal brain slice cultures, but the apoptotic cell death is highly dependent on the developmental stage of the cultures.     

Dehydroepiandrosterone and its sulfated derivative reduce neuronal death and enhance astrocytic differentiation in brain cell cultures

Human studies of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEA-S) have shown age-related changes in serum levels of these two sex hormone precursors. The levels of both DHEA and DHEA-S are characterized by monotonic decreases after puberty in females and after 20-24 yr of age in males. Further studies have shown that DHEA and DHEA-S levels are significantly low or close to minimal at ages when the incidence of senile dementia of Alzheimer's type (SDAT) begins to increase. We propose that DHEA and DHEA-S play a significant role in normal function of neuronal cells and that supplementation with them may prevent neuronal loss and/or damage. In the present study, using methods of immunocytochemistry, autoradiography, and scanning electron microscopy, we show that a supplement of as little as 10(-8) M DHEA or DHEA-S greatly increases neuronal survival and differentiation and reduces astroglial proliferation rates in mouse brain cells in cultures. These results suggest that correcting the DHEA and the DHEA-S deficit may prevent and/or improve the SDAT condition in humans.     

bFGF、VEGF在大鼠创伤海马脑组织中的表达

目的 研究碱性成纤维细胞生长因子(bFGF)和血管内皮细胞生长因子(VEGF)在大鼠创伤海马组织中不同时间的表达及其它们之间的关系，从分子水平探讨颅脑损伤后的病理机制，为临床治疗脑损伤的新途径提供实验基础。方法 改进Marmarou大鼠加速弥漫性脑损伤模型，取海马区创伤脑组织免疫组化染色观察bFGF、VEGF基因表达情况。结果 海马CAl区伤后6h，bFGF基因表达增加，12h达高峰；VEGF基因表达伤后24h达高峰，72h回复到对照水平。结论 bFGF、VEGF基因表达与脑损伤密切相关，作为生长因子，bFGF、VEGF可能参与颅脑损伤后神经元保护及损伤后修复过程。     

Potentiation of 3-hydroxyglutarate neurotoxicity following induction of astrocytic iNOS in neonatal rat hippocampal cultures

Neuronal damage in glutaryl-CoA dehydrogenase deficiency (GDD) has previously been addressed to N-methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity of the accumulating neurotoxic metabolite 3-hydroxyglutarate. However, acute encephalopathic crises in GDD patients are typically precipitated by febrile illness or even routine vaccinations, suggesting a potentiating role of inflammatory cytokines. In the present study we investigated the effect of interleukin-1beta and interferon-gamma on 3-hydroxyglutarate toxicity in rat cortical astrocyte cultures and neonatal rat hippocampal cultures. A cotreatment of both culture systems with interleukin-1beta and interferon-gamma induced the protein expression of astrocytic inducible nitric oxide synthase (iNOS), resulting in increased nitric oxide (NO) production. Cytokine pretreatment alone had no effect on cell viability but potentiated 3-hydroxyglutarate neurotoxicity. NOS inhibition by aminoguanidine and L-NAME prevented an iNOS-mediated potentiation of 3-hydroxyglutarate neurotoxicity but failed to protect neurons against 3-hydroxyglutarate alone. In contrast, superoxide dismutase/catalase as well as MK-801 prevented toxicity of 3-hydroxyglutarate alone as well as its potentiation by iNOS, supporting a central role of NMDA receptor stimulation with subsequently increased superoxide anion production. It is concluded that the potentiation of 3-hydroxyglutarate neurotoxicity is most probably due to an induction of astrocytic iNOS and concomitantly increased NO production, enabling enhanced peroxynitrite formation. Thus, we provide evidence for a neuroimmunological approach to the precipitation of acute encephalopathic crises in GDD by inflammatory cytokines.     

Localized gene transfer into organotypic hippocampal slice cultures and acute hippocampal slices

Viral vectors derived from herpes simplex virus, type-1 (HSV), can transfer and express genes into fully differentiated, post-mitotic neurons. These vectors also transduce cells effectively in organotypic hippocampal slice cultures. Nanoliter quantities of a virus stock of HSVlac, an HSV vector that directs expression of E. coli β-galactosidase (β-gal), were microapplied into stratum pyramidale or stratum granulosum of slice cultures. Twenty-four hours later, a cluster of transduced cells expressing β-gal was observed at the microapplication site. Gene transfer by microapplication was both effective and rapid. The titer of the HSVlac stocks was determined on NIH3T3 cells. Eighty-three percent of the β-gal forming units successfully transduced β-gal after microapplication to slice cultures. β-Gal expression was detected as rapidly as 4 h after transduction into cultures of fibroblasts or hippocampal slices. The rapid expression of β-gal by HSVlac allowed efficient transduction of acute hippocampal slices. Many genes have been transduced and expressed using HSV vectors; therefore, this microapplication method can be applied to many neurobiological questions.     

胶质瘤bFGF基因表达与增殖活性的相关性研究

目的探讨碱性成纤维细胞生长因子（ｂＦＧＦ）的异常表达与胶质瘤的恶性程度和增殖活性的关系。方法采用Ｎｏｒｔｈｅｒｎ杂交和免疫组化法对５７例人脑胶质瘤进行ｂＦＧＦ基因表达及Ｋｉ－６７标记指数检测及相关性分析。结果各组胶质瘤均有ｂＦＧＦｍＲＮＡ表达，且表达水平以恶性胶质瘤为高。Ｋｉ－６７标记指数在胶质瘤中升高显著。ｂＦＧＦｍＲＮＡ表达水平与胶质瘤恶性度及Ｋｉ－６７标记指数呈正相关。结论ｂＦＧＦ与胶质瘤的恶性进展有关，可作为判定胶质瘤恶性度的指标之一。     

Free radicals induce gene expression of NGF and bFGF in rat astrocyte culture.

Hydrogen peroxide (H2O2) is a type of active oxygen species produced mainly in blood by inflammation, ischemia or anoxia. Treatment of rat neonatal cortical astrocytes in culture with 0.2-1.0 mM H2O2 which is lethal for hippocampal neurons, increases nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) mRNA content in a time dependent manner. H2O2 also increases c-fos mRNA expression, which is probably involved in the gene regulation of both NGF and bFGF. Maximal induction was reached after 6 h of incubation (5.7-fold increase in NGF and 2.4-fold induction of bFGF mRNA). Hydrogen peroxide induced bFGF and NGF gene expression suggests that neurotrophic factors in astrocytes could be induced by lesion, consistent with their protective function in the CNS.     

Multiplexed cytokine protein expression profiles from spreading depression in hippocampal organotypic cultures.

Cytokines are involved in ischemic tolerance, including that triggered by spreading depression (SD), yet their roles in neuroprotection remain incompletely defined. The latter may stem from the pleiotropic nature of these signaling molecules whose complexities for interaction might be better deciphered through simultaneous measurement of multiple targeted proteins. Accordingly, the authors used microsphere-based flow cytometric immunoassays and hippocampal organotypic cultures (HOTCs) to characterize the magnitude, time course, and diversity of cytokine (interleukin [IL] 1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-10, granulocyte-macrophage colony-stimulating factor [GM-CSF], interferon-gamma [IFN-gamma], and tumor necrosis factor-alpha [TNF-alpha]) response to SD. GM-CSF was not detected in HOTCs or media. However, SD triggered a significant, generalized increase in seven cytokines evident in HOTCs 6 hours later, with the remaining cytokine, IL-1beta, becoming significantly different at 1 and 3 days. Additionally, these changes extended to include surrounding media for IL-6 and TNF-alpha by 1 and 3 days. This increase was localized to microglia via immunostaining for IL-1alpha, IL-1beta, and interferon-y. IL-10, although significantly more abundant in HOTCs 6 hours after SD, was significantly less abundant in surrounding media at that time and at 1 day. Finally, the generalized early increase in tissue cytokines later settled to a pattern at 3 days of recovery centering on changes in IL-1alpha, IL-1beta, and TNF-alpha, cytokines capable of modulating ischemic injury.     

Glucocorticoids worsen excitotoxin-induced expression of pro-inflammatory cytokines in hippocampal cultures

Glucocorticoids (GCs), the adrenal steroid hormones released during stress, have well-known anti-inflammatory actions. Despite that, there is increasing evidence that GCs are not uniformly anti-inflammatory in the injured nervous system and, in fact, can be pro-inflammatory. The present report continues this theme. Primary hippocampal cultures were treated with GC concentrations approximating basal, acute (1 h) stress or chronic (24 h) stress conditions and were then exposed to the excitotoxin kainic acid (KA). KA induced expression of the pro-inflammatory cytokines IL-1 beta and TNF-alpha, and chronic high dose GC exposure excacerbated this induction. In a second study, cultures were exposed to the physiological range of GC concentrations for 24 h prior to KA treatment. Low- to mid-range GC concentrations were anti-inflammatory, decreasing expression of IL-1 beta and TNF-alpha, while the highest GC doses either failed to be anti-inflammatory or even potentiated expression further. These findings add to the growing picture of these classically anti-inflammatory hormones potentially having pro-inflammatory effects in the injured CNS.     

Development and selective neurodegeneration in cell cultures from different hippocampal regions

Previous studies have shown that pyramidal neurons in hippocampal regions CA1 and CA3 are selectively vulnerable in several neurodegenerative disorders and that a subpopulation of pyramidal neurons in cell cultures of embryonic hippocampus are sensitive to glutamate neurotoxicity. In order to determine whether the patterns of cell loss seen in situ correlate with intrinsic differences in neuronal sensitivities to glutamate-induced degeneration acquired during development, we characterized cultures established from different regions of postnatal rat hippocampus and then examined neuronal sensitivity to glutamate. Tissue corresponding to the dentate gyrus (DG) and regions CA1, CA2 and CA3 of Ammon's horn was removed by microdissection from transverse hippocampal slices and was used to establish cultures of dissociated cells. Cultures from all 4 regions contained 3 major morphological classes of neurons; pyramidal-like, bipolar and stellate. Pyramidal-like neurons comprised the majority of neurons in all cultures; these neurons extended one long and branching axon, and one or more short dendrites. Immunocytochemistry showed that all neurons possessed high levels of glutamate-like and gamma-aminobutyric acid (GABA)-like immunoreactivity when grown in isolation. In contrast, when bipolar and pyramidal neurons were cultured in contact with glial cells, glutamate and GABA immunoreactivity were selectively reduced in the bipolar and pyramidal cells, respectively, suggesting that cell interactions influence neurotransmitter phenotype. Subpopulations of hippocampal neurons from each hippocampal region were vulnerable to glutamate-induced neurotoxicity. Bipolar and stellate cells were resistant to glutamate, while pyramidal-like neurons showed varying degrees of sensitivity to glutamate depending upon which region they were taken from. Experiments with specific glutamate receptor agonists and antagonists demonstrated that both non N-methyl-D-aspartic acid (NMDA) receptors and NMDA receptors mediated glutamate-induced degeneration. There were clear differences in the vulnerability of the pyramidal-like neuron populations in cultures from the different hippocampal regions. The rank order of the vulnerability of pyramidal-like neurons to glutamate-induced neurodegeneration between regions in culture was: DG less than CA2 less than CA3 less than CA1. This pattern of selective vulnerability in cell culture corresponds directly to the pattern of selective cell loss seen in situ in Alzheimer's disease, epilepsy, and stroke suggesting that intrinsic neuronal differences in glutamate sensitivity may be involved in these disorders.     

Morphological study of hippocampal development in cell and explant cultures]

Cell-and explantatcultures from hippocampal region of embryonal rats and newborn rats were cultivated up to 4 weeks. Pyramidal neurons, multiformed neurons and granule cells were observed in the explantatcultures. In the cellcultures were observed small and big Neurons after 4 weeks in vitro. Mantenance and differentiation of cellcultures are possible only by showing in at least 60 000 cells/ml. The Morphology of neurons in vitro was related to in vivo.     

Kainic acid induces rapid cell death followed by transiently reduced cell proliferation in the immature granule cell layer of rat organotypic hippocampal slice cultures

Brain injury due to seizures results in transiently increased cell proliferation and neurogenesis in the subgranular zone of the adult dentate gyrus. In contrast, the immature postnatal brain appears to be more resistant to cell death after seizure-induced brain injury and paradoxically reacts to seizures by reducing SGZ proliferation. Organotypic hippocampal slice cultures are a useful paradigm for modelling the early postnatal hippocampus. We have investigated the temporal relationship between cell death and cell proliferation after kainate in the granule cell layer of rat organotypic hippocampal slice cultures equivalent to post natal day 11 animals. We found stable numbers and densities of mature thionine stained cells in the granule cell layer over 72 h in control cultures grown in defined medium. We also found a slowly declining cell proliferation rate over the same time period under control conditions. We report evidence of early cell death in the granule cell layer after just 2 h exposure to 5 microM kainate, followed by a significant decrease in cell proliferation in the granule cell layer at 24 h. In contrast to control conditions, cell proliferation rose significantly in the kainate exposed cultures by 72 h back to levels seen at 2 h. There were no significant changes in cell labelling with antibody to activated caspase-3 between kainate treated and control cultures at any time point examined. Our results suggest that kainate-induced injury in the early postnatal hippocampus damages precursor cells contributing to a reduction in granule layer cell proliferation.     

A beta 25-35 induces presynaptic changes in organotypic hippocampal slice cultures

Memory loss in Alzheimer's disease (AD) may be related to synaptic defects in damaged hippocampal neurons. We investigated the relationship between amyloid peptide A beta 25-35-induced neuronal death pattern and presynaptic changes in organotypic hippocampal slice cultures. In propidium iodide (PI) uptake and annexin V labeling, A beta 25-35-induced neuronal damage dramatically increased in a concentration dependent manner, indicating both types of cell death. In ultrastructural analysis, apoptotic features in CA1 and CA3 area and synaptic disruption in stratum lucidum were detected in A beta 25-35-treated slices. Immunofluorescence and Western blot analysis for caspase-3 showed A beta 25-35 concentration dependently induced caspase-3 activation. Immunofluorescence and Western blot analysis to determine changes in presynaptic marker proteins demonstrated that expression of synaptosomal-associated protein-25 (SNAP-25) and synaptophysin were reduced by A beta 25-35 in CA1, CA3 and DG area at concentrations >2.5 microM. In conclusion, A beta 25-35-induced apoptotic cell death and caspase-3 activation at relatively low concentration, and induced synaptic disruption and loss of synaptic marker protein at concentrations >2.5 microM in organotypic hippocampal slice cultures. These suggest that A beta 25-35-induced apoptosis via triggering caspase-3 activation and lead to synaptic dysfunction in organotypic hippocampal slice cultures.     

The relationship between oxygen and adenosine in astrocytic cultures

Brain tissue oxygenation affects cerebral function and blood flow (CBF). Adenosine (Ado), a purine nucleoside, moderates neuronal activity, and arterial diameter. The cellular source of Ado in brain remains elusive; however, astrocytes are a logical site of production. Using astrocytic cultures, we tested the hypothesis that astrocytic derived Ado reflects cerebral oxygenation. We found that during alterations in pO₂, extracellular levels of Ado [Ado]_e changed rapidly. Graded reductions of oxygen tension revealed that[Ado]_e reached 10⁻⁷ M to 10⁻⁶ M with a pO₂ of 30–10mmHg, comparable with [Ado]_e and oxygen levels found in brain tissue during normoxemia. Higher O₂ levels were associated with a depression of [Ado]_e. Under conditions of low pO₂ (pO₂ ≤ 3 mmHg), inhibition of extracellular catabolism of adenosine monophosphate (AMP) prevented an increase of [Ado]_e and resulted in a rise in [AMP]_e. The rise in [AMP]_e preceded the increase in [Ado]_e. In the presence of nucleoside transporter inhibitors, accumulation of [Ado]_e persisted. On the basis of our studies in culture we conclude that astrocytes are a significant source of Ado and that during hypoxia, the changes in [Ado]_e are in a range to affect both neuronal activity as well as CBF. © 2010 Wiley‐Liss, Inc.