Murine Hippocampal Slice Cultures Research Articles

Fluorescence detection of Europium-doped very small superparamagnetic iron oxide nanoparticles in murine hippocampal slice cultures.

Fluorescence detection of Europium-doped very small superparamagnetic iron oxide nanoparticles in murine hippocampal slice cultures.

The Controlled Generation of Functional Basal Forebrain Cholinergic Neurons from Human Embryonic Stem Cells

An early substantial loss of basal forebrain cholinergic neurons (BFCN) is a constant feature of Alzheimer's disease and is associated with deficits in spatial learning and memory. The ability to selectively control the differentiation of human embryonic stem cells (hESCs) into BFCN would be a significant step toward a cell replacement therapy. We demonstrate here a method for the derivation of a predominantly pure population of BFCN from hESC cells using diffusible ligands present in the forebrain at developmentally relevant time periods. Overexpression of two relevant human transcription factors in hESC-derived neural progenitors also generates BFCN. These neurons express only those markers characteristic of BFCN, generate action potentials, and form functional cholinergic synapses in murine hippocampal slice cultures. siRNA-mediated knockdown of the transcription factors blocks BFCN generation by the diffusible ligands, clearly demonstrating the factors both necessary and sufficient for the controlled derivation of this neuronal population. The ability to selectively control the differentiation of hESCs into BFCN is a significant step both for understanding mechanisms regulating BFCN lineage commitment and for the development of both cell transplant-mediated therapeutic interventions for Alzheimer's disease and high-throughput screening for agents that promote BFCN survival.

Tumor Necrosis Factor Receptor-1 is Essential for LPS-Induced Sensitization and Tolerance to Oxygen—Glucose Deprivation in Murine Neonatal Organotypic Hippocampal Slices

Inflammation and ischemia have a synergistic damaging effect in the immature brain. The role of tumor necrosis factor (TNF) receptors 1 and 2 in lipopolysaccharide (LPS)-induced sensitization and tolerance to oxygen-glucose deprivation (OGD) was evaluated in neonatal murine hippocampal organotypic slices. Hippocampal slices from balb/c, C57BL/6 TNFR1(-/-), TNFR2(-/-), and wild-type (WT) mice obtained at P6 were grown in vitro for 9 days. Preexposure to LPS immediately before OGD increased propidium iodide-determined cell death in regions CA1, CA3, and dentate gyrus from 4 up to 48 h after OGD (P<0.001). Extending the time interval between LPS exposure and OGD to 72 h resulted in tolerance, that is reduced neuronal cell death after OGD (P<0.05). Slices from TNFR1(-/-) mice showed neither LPS-induced sensitization nor LPS-induced tolerance to OGD, whereas both effects were present in slices from TNFR2(-/-) and WT mice. Cytokine secretion (TNFalpha and interleukin-6) during LPS exposure was decreased in TNFR1(-/-) slices and increased in TNFR2(-/-) as compared with WT slices. We conclude that LPS induces sensitization or tolerance to OGD depending on the time interval between exposure to LPS and OGD in murine hippocampal slice cultures. Both paradigms are dependent on signaling through TNFR1.

The temperature dependence and involvement of mitochondria permeability transition and caspase activation in damage to organotypic hippocampal slices following in vitro ischemia

The aggravating effect of hyperglycemia on ischemic brain injury can be mimicked in a model of in vitro ischemia (IVI) using murine hippocampal slice cultures. Using this model, we found that the damage in the CA1 region following IVI in the absence or presence of 40 mm glucose (hyperglycemia) is highly temperature dependent. Decreasing the temperature from 35 to 31 degrees C during IVI prevented cell death, whereas increasing the temperature by 2 degrees C markedly aggravated damage. As blockade of the mitochondrial permeability transition (MPT) is equally effective as hypothermia in preventing ischemic cell death in vivo, we investigated whether inhibition of MPT or of caspases was protective following IVI. In the absence of glucose, the MPT blockers cyclosporin A and MeIle4-CsA but not the immunosuppressive compound FK506 diminished cell death. In contrast, following hyperglycemic IVI, MPT blockade was ineffective. Also, the pan-caspase inhibitor Boc-Asp(OMe)fluoromethyl ketone did not decrease cell death in the CA1 region following IVI or hyperglycemic IVI. We conclude that cell death in the CA1 region of organotypic murine hippocampal slices following IVI is highly temperature dependent and involves MPT. In contrast, cell death following hyperglycemic IVI, although completely prevented by hypothermia, is not mediated by mechanisms that involve MPT or caspase activation.

Glucose but not lactate in combination with acidosis aggravates ischemic neuronal death in vitro.

Hyperglycemia aggravates brain damage in clinical stroke and in experimental in vivo models of cerebral ischemia. Elevated preischemic glucose levels, lactate production, and intracerebral acidosis correlate with increased brain damage. We have developed a murine hippocampal slice culture model of in vitro ischemia (IVI), suitable for studies of the mechanisms of neuronal death. In this model we investigated the individual contribution of glucose, pH, lactate, and combinations thereof as well as ionotropic glutamate receptor activation to the development of hyperglycemic ischemic cell death. Murine organotypic hippocampal slice cultures were exposed to IVI in a medium with an ionic composition similar to that of the extracellular fluid in the brain during ischemia in vivo. Cell death was assessed by propidium iodide uptake. Ionotropic glutamate receptor blockade was accomplished by D-2-amino-5-phosphonopentanoic acid (D-APV) or 2,3-dihydro-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX). The combination of high glucose levels and acidosis (pH 6.8), but not acidosis per se or the combination of lactate and acidosis during IVI, exacerbated damage. Cell death after hyperglycemic IVI was not diminished by blockade of ionotropic glutamate receptors. Aggravation of brain damage by hyperglycemia in vivo can be reproduced in hippocampal slice cultures in vitro. Our results demonstrate that glucose per se, but not lactate, in combination with acidosis mediates the detrimental hyperglycemic effect through a mechanism independent of ionotropic glutamate receptors.

Temporal characterization of microglia, II-1β-like immunoreactivity and astrocytes in the dentate gyrus of hippocampal organotypic slice cultures

These studies demonstrate that murine hippocampal slice cultures possess neural-immune elements that show responses parallel to comparable in vivo models of neural-immune activation. Using immunocytochemical techniques, this study characterized the phenotypes of specific glial elements and the expression of the cytokine, interleukin-1 (IL-1β), in the hippocampal dentate gyrus over a period of 10days in vitro (DIV). Preparation of organotypic slice cultures of neonatal mouse hippocampus produced cellular damage including axotomy of afferent fibers within the molecular layer of the dentate gyrus. This form of lesion-induced injury caused activation of neural-immune elements in the slice cultures. Staining with the microglial specific, biotinylated Griffonia simplicifolia B4-isolectin revealed reactive microglia were most prevalent at 2DIV and decreased in number from 4 to 10DIV, whereas the initial population of resting microglia at 2DIV increased approximately four-fold from 4 to 10DIV. The presence of a round IL-1β-like immunophenotype closely paralleled the temporal and spatial distribution of the reactive form of microglia observed in the dentate gyrus. In addition, between 4 and 10DIV, some IL-1β-like immunoreactive cells exhibited a stellate-like morphology with numerous branching processes, similar to resting microglia. At 2DIV, astrocytes showed minimal labeling with antibodies directed against glial fibrillary acidic protein (GFAP), while from 4 to 10DIV, a dramatic hypertrophic astrocytic response occurred, resulting in a gliotic scar forming over the entire dentate gyrus. We conclude that neural-immune activation in the hippocampal organotypic slice culture preparation closely parallels similar responses observed in vivo and thus slice cultures represent an excellent model for further studies of neural-immune interactions resulting from lesion-induced injury in the central nervous system.