Bjarne Winther Kristensen

Markers for neuronal degeneration in organotypic slice cultures.

This protocol describes ways of monitoring spontaneous or induced neuronal degeneration in organotypic brain slice cultures. Hippocampal cultures (4-week-old) are grown in normal serum-free control medium, or exposed to the neurotoxin trimethyltin (TMT) (0.5-100 microM) for 24 h or the excitotoxic glutamate agonist kainic acid (KA) (5-25 microM) for 48 h followed by 24 h or 48 h, respectively, in normal medium. Corticostriatal slice cultures (also 4-week-old) are exposed to KA (6-24 microM) for 48 h and normal medium for control. The resulting neurodegeneration is estimated by (a) propidium iodide (PI) uptake, (b) lactate dehydrogenase (LDH) efflux to the culture medium, (c) ordinary Nissl cell staining, (d) staining by the neurodegenerative marker Fluoro-Jade (FJ), (e) neuronal microtubule degeneration by immunohistochemical staining for microtubule-associated protein 2 (MAP2), and (f) Timm sulphide silver staining for heavy metal alterations. Both hippocampal and corticostriatal slice cultures show a dose- and time-dependent increase in PI uptake and LDH efflux after exposure to TMT and KA. The mean PI uptake and the LDH efflux into the medium correlate well for both types of cultures. Both TMT and KA exposed hippocampal cultures display in vivo patterns of differential neuronal vulnerability as evidenced by PI uptake, FJ staining and MAP2 immunostaining. Corticostriatal slice cultures exposed to a high dose of KA display extensive striatal and cortical degeneration in FJ staining as suggested by a high PI uptake. A change in Timm sulphide silver staining in deep central parts of some control cultures, corresponds to areas with loss of cells in cell staining, loss of MAP2 staining, PI uptake, and FJ staining. We conclude that organotypic brain slice cultures, in combination with appropriate markers in standardized protocols, represent feasible means for studies of excitotoxic and neurotoxic compounds.

Organotypic Hippocampal Slice Cultures for Studies of Brain Damage, Neuroprotection and Neurorepair

Slices of developing brain tissue can be grown for several weeks as so-called organotypic slice cultures. Here we summarize and review studies using hippocampal slice cultures to investigate mechanisms and treatment strategies for the neurodegenerative disorders like stroke (cerebral ischemia), Alzheimers disease (AD) and epilepsia. Studies of non-excitotoxic neurotoxic compounds and the experimental use of slice cultures in studies of HIV neurotoxicity, traumatic brain injury (TBI) and neurogenesis are included. For cerebral ischemia, experimental models with oxygen-glucose deprivation (OGD) and exposure to glutamate receptor agonists (excitotoxins) are reviewed. For epilepsia, focus is on induction of seizures with effects on neuronal loss, axonal sprouting and neurogenesis. For Alzheimers disease, the review centers on the use of beta-amyloid (Abeta) in different models, while the section on repair is focused on neurogenesis and cell migration. The culturing techniques, set-up of models, and analytical tools, including markers for neurodegeneration, like the fluorescent dye propidium iodide (PI), are reviewed and discussed. Comparisons are made between hippocampal slice cultures and other in vitro models using dispersed cell cultures, experimental in vivo models, and in some instances, clinical trials. New techniques including slice culturing of hippocampal tissue from transgenic mice as well as more mature brain tissue, and slice cultures coupled to microelectrode arrays (MEAs), on-line biosensor monitoring, and time-lapse fluorescence microscopy are also presented.

Comparison of excitotoxic profiles of ATPA, AMPA, KA and NMDA in organotypic hippocampal slice cultures

The excitotoxic profiles of (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propionic acid (ATPA), (RS)-2-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainic acid (KA) and N-methyl-D-aspartate (NMDA) were evaluated using cellular uptake of propidium iodide (PI) as a measure for induced, concentration-dependent neuronal damage in hippocampal slice cultures. ATPA is in low concentrations a new selective agonist of the glutamate receptor subunit GluR5 confined to KA receptors and also in high concentrations an AMPA receptor agonist. The following rank order of estimated EC(50) values was found after 2 days of exposure: AMPA (3.7 mM)>NMDA (11 mM)=KA (13 mM)>ATPA (33 mM). Exposed to 30 microM ATPA, 3 microM AMPA and 10 microM NMDA, CA1 was the most susceptible subfield followed by fascia dentata and CA3. Using 8 microM KA, CA3 was the most susceptible subfield, followed by fascia dentata and CA1. In 100 microM concentrations, all four agonists induced the same, maximal PI uptake in all hippocampal subfields, corresponding to total neuronal degeneration. Using glutamate receptor antagonists, like GYKI 52466, NBQX and MK-801, inhibition data revealed that AMPA excitotoxicity was mediated primarily via AMPA receptors. Similar results were found for a high concentration of ATPA (30 microM). In low GluR5 selective concentrations (0.3-3 microM), ATPA did not induce an increase in PI uptake or a reduction in glutamic acid decarboxylase (GAD) activity of hippocampal interneurons. For KA, the excitotoxicity appeared to be mediated via both KA and AMPA receptors. NMDA receptors were not involved in AMPA-, ATPA- and KA-induced excitotoxicity, nor did NMDA-induced excitotoxicity require activation of AMPA and KA receptors. We conclude that hippocampal slice cultures constitute a feasible test system for evaluation of excitotoxic effects and mechanisms of new (ATPA) and classic (AMPA, KA and NMDA) glutamate receptor agonists. Comparison of concentration-response curves with calculation of EC(50) values for glutamate receptor agonists are possible, as well as comparison of inhibition data for glutamate receptor antagonists. The observation that the slice cultures respond with more in vivo-like patterns of excitotoxicity than primary neuronal cultures, suggests that slice cultures are the best model of choice for a number of glutamate agonist and antagonist studies.

CD133 identifies perivascular niches in grade II–IV astrocytomas

The aim of the present study was to investigate the localization and distribution of the putative brain tumour stem cell marker CD133 in formalin fixed paraffin embedded astrocytomas. A retrospective analysis of 114 grade II, III and IV astrocytomas was undertaken. The immunohistochemical expression of CD133 in paraffin sections was analysed using morphometry. In all grades, CD133 was expressed on tumour and endothelial cells. Tumour cells were found in perivascular niches, as dispersed single cells and in pseudopalisade formations around necrosis. There was no correlation between the mean volume fraction of CD133+ niches and all CD133+ tumour cells and tumour grade. However, the volume fraction of CD133+ blood vessels increased significantly from 0.4% in diffuse astrocytomas to 2.2% in glioblastomas. Neither of them was related to patient survival. Double immunofluorescence stainings showed that the CD133+ niches both contained CD133+ cells with and without co-expression of the intermediate filament protein marker nestin, and only few CD133+/MIB-1+ proliferating cells were found. In conclusion, a CD133+ perivascular stem cell-like entity exists in astrocytomas. CD133+ tumour vessels may play an important role in a brain tumour stem cell context, while CD133 alone does not appear to be a specific tumour stem cell marker related to patient survival.

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

Summary. 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.

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.

Inconsistent Immunohistochemical Expression Patterns of Four Different CD133 Antibody Clones in Glioblastoma

The putative tumor stem cell marker CD133 is the marker of choice for identifying brain tumor stem cells in gliomas, but the use of different CD133 antibody clones possibly recognizing different CD133 splice variants with epitopes of different glycosylation status confuses the field. The aim was to investigate if current inconsistent CD133 observations could be a result of using different CD133 antibodies for immunohistochemical identification of CD133. Ten glioblastomas were immunohistochemically stained with four different CD133 antibody clones (AC133, W6B3C1, C24B9, and ab19898) and analyzed by quantitative stereology. Moreover, the CD133 staining pattern of each antibody clone was investigated in kidney, pancreas, and placenta tissue as well as in glioblastoma and retinoblastoma cultures and cell lines. All antibody clones revealed CD133+ niches and single cells in glioblastomas, but when using different clones, their distribution rarely corresponded. Morphology of identified single cells varied, and staining of various tissues, cultures, and cells lines was also inconsistent among the clones. In conclusion, the authors report inconsistent CD133 detection when using different primary CD133 antibody clones. Thus, direct comparison of studies using different antibody clones and conclusions based on CD133 immunohistochemistry should be performed with caution.

Symptoms after Ingestion of Pig Whipworm Trichuris suis Eggs in a Randomized Placebo-Controlled Double- Blind Clinical Trial

Symptoms after human infection with the helminth Trichuris suis have not previously been described. Exposure to helminths has been suggested as immune therapy against allergy and autoimmune diseases. We randomized adults with allergic rhinitis to ingest a dose of 2500 T. suis eggs or placebo every 21 days for 168 days (total 8 doses) in a double-blind clinical trial. In a previous publication, we reported a lack of efficacy and a high prevalence of adverse gastrointestinal reactions. The aim of the present study was to present a detailed description of the adverse event data and post-hoc analyses of gastrointestinal reactions. Adverse events and severity (mild, moderate, severe) were recorded daily by subjects, classified by organ using MedDRA 10.0, and event rates compared between subjects on T. suis treatment vs. subjects on placebo. T. suis-specific serum IgG antibodies were measured by a fluoroenzymeimmunoassay (Phadia ApS). During 163 days complete follow-up, subjects ingesting T. suis eggs (N = 49) had a three to 19-fold higher rate of events (median duration, 2 days) with gastrointestinal reactions (moderate to severe flatulence, diarrhea, and upper abdominal pain) compared with placebo subjects (N = 47). The highest incidence of affected subjects was seen from the first few days and until day 42 (3rd dose): 63% vs. 29% for placebo; day 163: 76% vs. 49% for placebo. Seroprevalences increased concurrently in the T. suis group: Day 59, 50%; day 90, 91%; day 170, 93%. The combined duration of episodes with onset before day 42 was ≤14 days in 80% of affected subjects. Age, gender, total IgE, and recent intestinal symptoms at baseline did not predict gastrointestinal side effects. In conclusion, during the first 2 months, repeated ingestions of 2500 T. suis eggs caused frequent gastrointestinal reactions lasting up to 14 days, whereas 4 months further treatment mainly provoked a subclinical stimulation. Trial registration University hospital Medical Information Network trial registry Reg. no. R000001298, Trial ID UMIN000001070.

Biocompatibility of silicon-based arrays of electrodes coupled to organotypic hippocampal brain slice cultures

In this study we examined the passive biocompatibility of a three-dimensional microelectrode array (MEA), designed to be coupled to organotypic brain slice cultures for multisite recording of electrophysiological signals. Hippocampal (and corticostriatal) brain slices from 1-week-old (and newborn) rats were grown for 4-8 weeks on the perforated silicon chips with silicon nitride surfaces and 40 microm sized holes and compared with corresponding tissue slices grown on conventional semiporous membranes. In terms of preservation of the basic cellular and connective organization, as visualized by Nissl staining, Timm sulphide silver-staining, microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP) immunostaining, the slice cultures grown on chips did not differ from conventionally grown slice cultures. Neither were there any signs of astrogliosis or neurodegeneration around the upper recording part of the 47-microm-high platinum-tip electrodes. Slice cultures grown on a separate set of chips with platinum instead of silicon nitride surfaces also displayed normal MAP2 and GFAP immunostaining. The width of the GFAP-rich zone (glia limitans) at the bottom surface of the slice cultures was the same ( approximately 20 microm) in cultures grown on chips with silicon nitride and platinum surfaces and on conventional insert membranes. The slice cultures grown on chips maintained a normal, subfield differentiated susceptibility to the glutamate receptor agonist N-methyl-D-aspartate (NMDA) and the neurotoxin trimethyltin (TMT), as demonstrated by the cellular uptake of propidium iodide (PI), which was used as a reproducible and quantifiable marker for neuronal degeneration. We conclude that organotypic brain slice cultures can grow on silicon-based three-dimensional microelectrode arrays and develop normally with display of normal subfield differentiated susceptibilities to known excito- and neurotoxins. From this it is anticipated that the set-up, designed for recording of electrophysiological parameters, can be used for long-term studies of defined neuronal networks and provide valuable information on both normal, neurotoxicological and neuropathological conditions.

Excitotoxic effects of non-NMDA receptor agonists in organotypic corticostriatal slice cultures

The excitotoxic effects of the glutamate receptor agonists kainic acid (KA) and 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and the corresponding neuroprotective effects of the AMPA/KA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) were examined in corticostriatal slice cultures. The purpose was to examine the feasibility of these cultures for excitotoxic studies, and to demonstrate possible differential excitotoxic effects of KA and AMPA on striatal and cortical neurons. Slices of dorsolateral striatum with overlying neocortex were obtained from neonatal rats and grown on semiporous membranes in serum-free medium for 3-4 weeks before exposure to KA or AMPA for 48 h. The uptake by injured cells of the fluorescent dye propidium iodide (PI) added to the culture medium was used as a quantifiable measure for neuronal degeneration and compared with efflux of the cytosolic enzyme lactate dehydrogenase (LDH) into the culture medium and loss of glutamic acid decarboxylase (GAD) activity in the tissue. Histological sections were also stained by the fluorescent dye Fluoro-Jade (FJ), for degenerating neurons and by immunocytochemical staining for gamma-aminobutyric acid (GABA). Digitized images showed a dose (0-24 microM KA, 0-6 microM AMPA) and time (0-48 h) dependent increase in PI uptake in both striatum and cortex. In other cultures exposed to KA (24 microM) or AMPA (6 microM) together with NBQX (0.1-9 microM), NBQX was found to exert a differential neuroprotective effect on striatum and cortex at low doses. NBQX was thus more protective against KA in the cortex than in the striatum, while the opposite was seen in relation to AMPA. Regarding neurodegenerative markers, PI uptake was significantly correlated with (1) LDH release into the culture medium, (2) optical density of Fluoro-Jade staining, (3) loss of GAD-activity in tissue homogenates, and (4) loss of GABA-immunostained neurons. We conclude that both differences between compounds (AMPA vs. KA) and brain areas (striatum vs. cortex) can be demonstrated in corticostriatal slice cultures, which in conjunction with an established set of markers for neuronal cell damage appears to be a feasible model for studies of the neurotoxic and neuroprotective effects of glutamate receptor agonists and antagonists.