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Results for “Organotypic brain slices”

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At least 19 recordsLinked to original sources

PubMed2003

Increased TNFalpha production and Cox-2 activity in organotypic brain slice cultures from APPsw transgenic mice.

beta-Amyloid (Abeta) peptides are the principal component of senile plaques and vascular deposits in Alzheimer's disease and are derived from the proteolytic cleavage of the beta-amyloid precursor protein (APP). We have previously shown that synthetic Abeta can stimulate cyclooxygenase-2 (Cox-2) activity in brain organotypic slice cultures. In the present study, we used brain slices from transgenic APP Swedish (TgAPPsw) mice and control littermates of different age groups to determine the effect of APP overexpression on the levels of prostaglandin and tumor necrosis factor alpha (TNFalpha) release. The production of eicosanoid and TNFalpha was increased as a function of age in organotypic brain slice culture from TgAPPsw mice compared to age matched control littermates. We also showed that the selective Cox-2 inhibitor NS-398 reduces the production of eicosanoid and TNFalpha in organotypic brain slice cultures of TgAPPsw mice. In conclusion, our data suggest that either activity or expression of Cox-2 is increased in TgAPPsw mice brains as a function of age, contributing to an increased production of pro-inflammatory eicosanoids and TNFalpha.

Age Factors↗

PubMed2000

Excitatory amino acid neurotoxicity and modulation of glutamate receptor expression in organotypic brain slice cultures.

Using organotypic slice cultures of hippocampus and cortex-striatum from newborn to 7 day old rats, we are currently studying the excitotoxic effects of kainic acid (KA), AMPA and NMDA and the neuroprotective effects of glutamate receptor blockers, like NBQX. For detection and quantitation of the induced neurodegeneration, we have developed standardized protocols, including--a) densitometric measurements of the cellular uptake of propidium iodide (PI), --b) histological staining by Flouro-Jade, --c) lactate dehydrogenase (LDH) release to the culture medium, --d) immunostaining for microtubulin-associated protein 2, and --e) general and specific neuronal and glial cell stains. The results show good correlation between the different markers, and are in accordance with results obtained in vivo. Examples presented in this review will focus on the use of PI uptake to monitor the excitotoxic effects of --a) KA and AMPA (and NMDA) in hippocampal slice cultures, and --b) KA and AMPA in corticostriatal slice cocultures, with demonstration of differentiated neuroprotective effects of NBQX in relation to cortex and striatum and KA and AMPA. A second set of studies include modulation of hippocampal KA-induced excitotoxicity and KA-glutamate receptor subunit mRNA expression after long-term exposure to low, non-toxic doses of KA and NBQX. We conclude that organotypic brain slice cultures, combined with standardized procedures for quantitation of cell damage and receptor subunit changes is of great potential use for studies of excitotoxic, glutamate receptor-induced neuronal cell death, receptor modulation and related neuroprotection.

Animals↗

PubMed2000

Construction and application of a microprojectile system for the transfection of organotypic brain slices.

In this study we outline a method for constructing an inexpensive chamber used in the transfection of organotypic brain slices. This chamber differs from most commercially available chambers in that DNA-coated gold microcarriers are directly carried by a flow of helium at low pressure (26 psi). Most other chambers employ macrocarriers onto which DNA-coated gold is first loaded, and then released by a shock of helium onto the reverse side of the macrocarriers. This home constructed device has been successfully employed in the transfection of organotypic brain slices cultured using the air-medium interface method. Mammalian expression vectors containing cytomegalovirus (CMV) and simian virus (SV40) enhancers/promoters were used to express enhanced green fluorescence protein (EGFP). DNA was coated onto 0.6-microm gold microcarriers. Transfected cells were visualised under a fluorescence microscope and included identifiable neurones and oligodendrocytes. Also included in this study are step-by-step methods for the preparation of gold microcarriers and organotypic brain slices.

Animals↗

PubMed1998

A novel model of glioma cell invasion using organotypic brain slice culture.

A novel model of glioma cell invasion was established by using an organotypic culture of rat brains. Brain slices prepared from 2-day-old rat neonates were maintained in a culture at the interface between air and the culture medium. The slices were placed on double-layered membranes consisting of a polycarbonate membrane with 8-microm pores and a membrane with 0.4-microm pores. The organotypic cytoarchitecture of the cultured brain slices remained well preserved, and the neuronal viability was kept intact for over 2 months. When C6 glioma spheroids were cocultured with the brain slices, the tumor cells migrated in a scattered fashion around the spheroids. Exogenous L1 or glioma motility factor I strongly stimulated the cell migration, whereas fibronectin, tenascin, and glioma motility factor II had little or no effect. When C6 glioma cells placed on the brain slices were incubated while being stimulated by L1-transfected fibroblast cells for 2 days, many more tumor cells invaded and reached the bottom of the upper membrane. This L1-stimulated glioma cell invasion into brain slices was significantly inhibited by an anti-L1 antibody. Our novel invasion model, which mimics the in vivo conditions of the central nervous system, may make it possible to analyze actual events of glioma cell invasion in normal brains in situ.

Animals↗

PubMed2004

Organotypic brain slice cultures: an efficient and reliable method for neurotoxicological screening and mechanistic studies.

This paper reviews the current state of the use of organotypic brain slice cultures for neurotoxicological and neuropharmacological screening and mechanistic studies, as exemplified by excitotoxin application. At present, no in vitro systems have been approved by the regulatory authorities for neurotoxicity testing. For the evaluation of the slice culture method, organotypic hippocampal slice cultures were exposed to toxic doses of the excitotoxins, glutamate, N-methyl-D-aspartate (NMDA), kainic acid and 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and the glial toxin, DL-alpha-aminoadipic acid (DLAAA). Neuronal cell death was quantified by propidium iodide (PI) uptake, and visualised by Fluoro-Jade (FJ) staining. General cell death was monitored by lactate dehydrogenase (LDH) release into the culture medium. EC50 values for the different compounds, based on PI uptake after exposure for 48 hours in entire cultures, were: glutamate, 3.5 mM; DL-AAA, 2.3 mM; kainic acid, 13 microM; NMDA, 11 microM; and AMPA, 3.7 microM. In the slice cultures, the hippocampal subfields displayed the same differences in vulnerability as those observed in vivo. When subfield analysis was performed on the cultures, the CA1 subfield was most susceptible to glutamate, NMDA and AMPA, while CA3 was most susceptible to kainic acid. The amount of LDH release for DL-AAA was about four times that of L-glutamate, in accordance with the additional toxic effect on glial cells, which was also found by confocal microscopy to stain for FJ. In conclusion, it was found that organotypic brain slice culture, combined with standardised protocols and quantifiable markers, such as PI and FJ staining, is a relevant and feasible in vitro system for neurotoxicity testing. Considering the amount and quality of the available published data, it is recommended that the brain slice culture method could be subjected to pre-validation and formal validation for inclusion in a tiered in vitro neurotoxicity testing scheme to supplement and replace conventional animal tests.

Animal Testing Alternatives↗

PubMed2001

The invasive features of glial and non-central nervous system tumor cells are different on organotypic brain slices from newborn rats.

On radiograms, glial tumors are usually seen to invade in a finger-like fashion, while non-central nervous system (CNS) tumors expand in a mass-like fashion. We prepared organotypic brain slice cultures from newborn rats to investigate the invasive behavior of human brain tumors using glial tumor cell lines (U-87MG, U-373MG, U-251MG, and SF-126) and of non-CNS tumors using cell lines; HT-1080 (human malignant fibrosarcoma), RFRF (human lung carcinoma), MIA-PICA (human pancreatic carcinoma), and Colo38 (human malignant melanoma). We selected an area that was centered at a junction between deep cortical layers and the striatum, punched a hole measuring 0.5-0.7 mm in diameter in this area, implanted different rhodamine-labeled tumor cells at that site, and observed their invasive behavior under an inverted fluorescent microscope. Over the course of several days, all glioma cells invaded in a finger-like fashion; U-373 MG cells invaded farthest. Non-CNS tumors expanded in mass-like fashion into adjacent areas. Using the slice cultures, we also investigated the regulatory effect on tumor invasion of forced expression of glial fibrillary acidic protein (GFAP) after gene transfection. The forced expression of GFAP rendered U-87MG and HT-1080 cells less invasive. Our results show that organotypic brain slice cultures are an excellent medium for studying the invasive features of glial and non-CNS tumors.

Animals↗

PubMed2002

Transistor array with an organotypic brain slice: field potential records and synaptic currents.

Linear transistor arrays on a silicon chip were used to map evoked extracellular field potentials in organotypic brain slices of rat hippocampus. The shape and amplitude of the transients were similar to those from records with micropipette electrodes. The spatial resolution was 21 and 4.6 microm. The sampled profile of the field potential showed a wide and shallow trough of transients in the stratum radiatum and a narrow but higher ridge in the stratum pyramidale. Due to the high resolution, the profile could be interpreted quantitatively in terms of synaptic currents. Transistor chips may become a novel tool for neurophysiological and pharmacological studies in brain slices.

Animals↗

PubMed2005

A peptide preparation protects cells in organotypic brain slices against cell death after glutamate intoxication.

Cerebrolysin has been shown to have neurotrophic and neuroprotective potential similar to NGF or BDNF. In the present study organotypic brain slices were utilized to determine the neuroprotective effects of Cerebrolysin, in a glutamate lesion paradigm mimicking a key event in ischemia. The study focused on the effects of Cerebrolysin on both necrotic and apoptotic cell death. Two specific DNA intercalating dyes were used to distinguish the type of cell death. The drug effect was evaluated both microscopically and quantitatively before, 24 hours after and then again 8 days after the lesion. Cerebrolysin was added either before and after the lesion or after the lesion only. The most pronounced effect was seen with the drug added both prior to and after the glutamate lesioning. A treatment after the lesion only also counteracted necrosis and apoptosis. The results render the drug relevant for treating acute as well as chronic neurodegenerative diseases.

Amino Acids↗

PubMed1998

An in vitro blood-brain barrier model: cocultures between endothelial cells and organotypic brain slice cultures.

This communication describes a novel in vitro blood-brain barrier (BBB) model: organotypic slice cultures from the central nervous system were overlaid on endothelial cell monolayers grown on permeable membranes. Morphological, electrophysiological, and microdialysis approaches were carried out to characterize and validate this model. After 10 days in coculture, morphological studies reveal the presence of tight junctions. Electrophysiological recordings of neuronal activity performed on organotypic cultures with or without an endothelial cell monolayer show that amplitude of evoked responses were comparable, indicating good viability of cocultures after 2 weeks. Perfusion of known BBB permeable or nonpermeable molecules was used to test the coculture tightness in conjunction with electrophysiological or microdialysis approaches: application of glutamate (Glu), which doesn't easily cross the BBB, triggers off rhythmic activity only in control cultures, whereas epileptogenic activity was observed in both control cultures and cocultures during perfusions with picrotoxin, a molecule that can diffuse through the BBB. Finally, the microdialysis technique was used to determine the permeability of molecules coming from the perfusion chamber: L-dopa, dopamine, and Glu were employed to assess the selective permeability of the coculture model. Thus, these results indicate that the in vitro model described possesses characteristics similar to those of the BBB in situ and that cocultures of organotypic slices and endothelial cell monolayers have potential as a powerful tool for studying biochemical mechanisms regulating BBB function and drug delivery to the central nervous system.

Animals↗

PubMed2002

Nerve growth factor and cholinergic CNS neurons studied in organotypic brain slices. Implication in Alzheimer's disease?

Nerve growth factor (NGF) is a potent growth factor for cholinergic neurons. The aim of the present study was to investigate if NGF affects cholinergic neurons of the basal nucleus of Meynert (nBM) in organotypic brain slices. In single nBM slices cholinergic neurons rapidly degenerated when incubated without NGF. The number of remaining neurons was rescued by NGF application at any time point. When nBM slices were co-cultured with a cortex slice the number of cholinergic neurons was significantly increased pointing to a trophic influence of the cortex. Incubation with acetylcholine precursors did not affect the survival of cholinergic neurons. There was no significant difference when postnatal day 3 or day 10 nBM slices were cultured. In conclusion, NGF is the most potent growth factor for cholinergic neurons and is a promising candidate for treating Alzheimers disease, however, the delivery of NGF to the brain must the solved.

Acetyl Coenzyme A↗

PubMed2002

Granulocyte macrophage-colony stimulating factor activates microglia in rat cortex organotypic brain slices.

Cytokines play an important role in the regulation of proliferation and migration in the central nervous system. The aim of this study was to determine if granulocyte macrophage-colony stimulating factor (GM-CSF) activates cells in the cortex of organotypic brain slice cultures. Our data show that murine GM-CSF markedly stimulated the proliferation and migration of small round microglia from a cortex slice. These round cells were strongly positive for integrin CD11b (OX-42), isolectin B4-lectin-binding, the monocytic marker ED1 and partly expressed major histocompatibility complex (MHC) class II antigen (OX-6). Only some differentiated microglia were visible which expressed the integrin CD11c and MHCII. GM-CSF enhanced the proliferation as analyzed by bromodeoxyuridine incorporation. The number of migrated cells decreased during culturing and enhanced terminal dUTP nick-end labelling positive nuclei were found. Taken together, our data conclude that GM-CSF is an important cytokine, which regulates the proliferation and migration of cortical microglia.

Animals↗

PubMed2003

Brain capillaries and cholinergic neurons persist in organotypic brain slices in the absence of blood flow.

Angiogenesis plays an important role during development of the brain and under pathological conditions. The aim of the present study was to observe interaction of brain capillaries and cholinergic neurons in organotypic brain slices. Immunohistochemistry was used to visualize brain capillary-like structures (RECA-1 antigen) and cholinergic neurons (choline acetyltransferase). Under normal culture conditions, a very low number of brain capillaries was found in 2- and 4-week-old cortex brain slices. Treatment of slices with acidic medium (pH 6) or hyperthermia (42 degrees C) markedly enhanced the number of brain capillaries. Incubation with 10 ng/mL vascular endothelial growth factor only enhanced angiogenesis in more developed slices. Cholinergic neurons survived in slices of the basal nucleus of Meynert; however, hyperthermia but not acidosis markedly decreased their number. In coslices of the basal nucleus of Meynert and cortex (pretreated with acidic medium), a high number of RECA-1-positive capillaries and cholinergic neurons persisted and displayed strong nerve fibre growth of cholinergic fibres into the cortex. In conclusion, we have demonstrated that RECA-1-positive capillaries and cholinergic neurons can be studied in slice cultures in the absence of blood perfusion, and that this model could provide a system to study mechanisms involved in vascular dementia.

Acetylcholinesterase↗

PubMed1999

Coupling of organotypic brain slice cultures to silicon-based arrays of electrodes.

Fetal or early postnatal brain tissue can be cultured in viable and healthy condition for several weeks with development and preservation of the basic cellular and connective organization as so-called organotypic brain slice cultures. Here we demonstrate and describe how it is possible to establish such hippocampal rat brain slice cultures on biocompatible silicon-based chips with arrays of electrodes with a histological organization comparable to that of conventional brain slice cultures grown by the roller drum technique and on semiporous membranes. Intracellular and extracellular recordings from neurons in the slice cultures show that the electroresponsive properties of the neurons and synaptic circuitry are in accordance with those described for cells in acutely prepared slices of the adult rat hippocampus. Based on the recordings and the possibilities of stimulating the cultured cells through the electrode arrays it is anticipated that the setup eventually will allow long-term studies of defined neuronal networks and provide valuable information on both normal and neurotoxicological and neuropathological conditions.

Animals↗

PubMed2004

Silicon chip with capacitors and transistors for interfacing organotypic brain slice of rat hippocampus.

Probing projections between brain areas and their modulation by synaptic potentiation requires dense arrays of contacts for noninvasive electrical stimulation and recording. Semiconductor technology is able to provide planar arrays with high spatial resolution to be used with planar neuronal structures such as organotypic brain slices. To address basic methodical issues we developed a silicon chip with simple arrays of insulated capacitors and field-effect transistors for stimulation of neuronal activity and recording of evoked field potentials. Brain slices from rat hippocampus were cultured on that substrate. We achieved local stimulation of the CA3 region by applying defined voltage pulses to the chip capacitors. Recording of resulting local field potentials in the CA1 region was accomplished with transistors. The relationship between stimulation and recording was rationalized by a sheet conductor model. By combining a row of capacitors with a row of transistors we determined a simple stimulus-response matrix from CA3 to CA1. Possible contributions of inhomogeneities of synaptic projection, of tissue structure and of neuroelectronic interfacing were considered. The study provides the basis for a development of semiconductor chips with high spatial resolution that are required for long-term studies of topographic mapping.

Animals↗

PubMed2000

Dynamic mechanical stretch of organotypic brain slice cultures induces differential genomic expression: relationship to mechanical parameters.

Although the material properties of biological tissues are reasonably well established, recent studies have suggested that the biological response of brain tissue and its constituent cells may also be viscoelastic and sensitive to both the magnitude and rate of a mechanical stimulus. Given the potential involvement of changes in gene expression in the pathogenic sequelae after head trauma, we analyzed the expression of 22 genes related to cell death and survival and found that a number of these genes were differentially regulated after mechanical stretch of an organotypic brain slice culture. Twenty-four hours after stretch, the expression of BDNF, NGF, and TrkA was significantly increased, whereas that of bcl-2, CREB, and GAD65 was significantly decreased (MANOVA followed by ANOVA, p < 0.05). Expression of CREB and GAD65 was negatively correlated with strain, whereas expression of APP695 was negatively correlated with strain rate (all p < 0.05). This study demonstrates that a subset of genes involved in cell death and survival are differentially regulated after dynamic stretch in vitro and that the expression of specific genes is correlated with mechanical parameters of that stretch.

Analysis of Variance↗

PubMed2000

Traumatic injury induces differential expression of cell death genes in organotypic brain slice cultures determined by complementary DNA array hybridization.

The expression of a large panel of selected genes hypothesized to play a central role in post-traumatic cell death was shown to be differentially altered in response to a precisely controlled, mechanical injury applied to an organotypic slice culture of the rat brain. Within 48 h of injury, the expression of nerve growth factor messenger RNA was significantly increased whereas the levels of bcl-2, alpha-subunit of calcium/calmodulin-dependent protein kinase II, cAMP response element binding protein, 65,000 mol. wt isoform of glutamate decarboxylase, 1beta isoform of protein kinase C, and ubiquitin messenger RNA were significantly decreased. Because the expression levels of a number of other messenger RNAs such as the neuron-specific amyloid precursor protein, beta(2) microglobulin, bax, bcl(xl), brain-derived neurotrophic factor, cyclooxygenase-2, interleukin-1beta, interleukin-6, tumor necrosis factor-alpha, receptor tyrosine kinase A, and receptor tyrosine kinase B were unaffected, these selective changes may represent components of an active and directed response of the brain initiated by mechanical trauma. Interpretation of these co-ordinated alterations suggests that mechanical injury to the central nervous system may lead to disruption of calcium homeostasis resulting in altered gene expression, an impairment of intracellular cascades responsible for trophic factor signaling, and initiation of apoptosis via multiple pathways. An understanding of these transcriptional changes may contribute to the development of novel therapeutic strategies to enhance beneficial and blunt detrimental, endogenous, post-injury response mechanisms.

Animals↗

PubMed1998

Organotypic brain slice cultures for functional analysis of alcohol-related disorders: novel versus conventional preparations.

Assessment of long-term alterations in neural function and phenotype has usually involved culture techniques that utilize dissociated preparations. Recently, we have approached such topics in alcohol research by using brain slice cultures, also known as explant or organotypic preparations. In this symposium presentation, two preparations will be discussed, and examples of the particular advantages of these preparations will be presented in relation to alcohol research. First, we use the hippocampal explant preparation for assessment of long-term alterations in N-methyl-D-aspartate receptor (NMDAR) function due to chronic ethanol exposure and subsequent withdrawal. This preparation displays many synaptic, structural, and enzymatic phenotypes indicative of normal neural preparations. Patch clamp recordings reveal NMDAR-mediated excitatory postsynaptic current (EPSC) elicited upon stimulation of Schaffer collateral fibers and recorded from CA1 pyramidal cells. Long-term ethanol exposure followed by subsequent withdrawal resulted in a specific enhancement of NMDAR-mediated synaptic responses which preceded the expression of epileptiform events that occurred after prolonged withdrawal periods. Second, we describe a novel explant preparation, derived from horizontal slices of the entire forebrain and midbrain of the rat. These long-term explants displayed multiple normal phenotypes including Nissl, AChE, TH, and GFAP staining. Electrophysiologically, these explants displayed a functional corticostriatal pathway recorded with field and patch clamp techniques and elicited by synaptic stimulation. Taken together, these explant preparations display utility for long-term study of ethanol effects on neural systems, especially relating to withdrawal hyperexcitability as well as systems involved in drug-seeking behavior.

Alcoholism↗

PubMed2004

Correlation of in vitro infiltration with glioma histological type in organotypic brain slices.

Diffuse invasion of the brain, an intrinsic property of gliomas, renders these tumours incurable, and is a principal determinant of their spatial and temporal growth. Knowledge of the invasive potential of gliomas is highly desired in order to understand their behaviour in vivo. Comprehensive ex vivo invasion studies including tumours of different histological types and grades are however lacking, mostly because reliable physiological invasion assays have been difficult to establish. Using an organotypic rodent brain slice assay, we evaluated the invasiveness of 42 grade II-IV glioma biopsy specimens, and correlated it with the histological phenotype, the absence or presence of deletions on chromosomes 1p and 19q assessed by fluorescent in situ hybridisation, and proliferation and apoptosis indices assessed by immunocytochemistry. Oligodendroglial tumours with 1p/19q loss were less invasive than astrocytic tumours of similar tumour grade. Correlation analysis of invasiveness cell proliferation and apoptosis further suggested that grade II-III oligodendroglial tumours with 1p/19q loss grow in situ as relatively circumscribed compact masses in contrast to the more infiltrative and more diffuse astrocytomas. Lower invasiveness may be an important characteristic of oligodendroglial tumours, adding to our understanding of their more indolent clinical evolution and responsiveness to therapy.

Animals↗