Development and characterization of a human embryonic stem cell-derived 3D neural tissue model for neurotoxicity testing.

Alternative models for more rapid compound safety testing are of increasing demand. With emerging techniques using human pluripotent stem cells, the possibility of generating human in vitro models has gained interest, as factors related to species differences could be potentially eliminated. When studying potential neurotoxic effects of a compound it is of crucial importance to have both neurons and glial cells. We have successfully developed a protocol for generating in vitro 3D human neural tissues, using neural progenitor cells derived from human embryonic stem cells. These 3D neural tissues can be maintained for two months and undergo progressive differentiation. We showed a gradual decreased expression of early neural lineage markers, paralleled by an increase in markers specific for mature neurons, astrocytes and oligodendrocytes. At the end of the two-month culture period the neural tissues not only displayed synapses and immature myelin sheaths around axons, but electrophysiological measurements also showed spontaneous activity. Neurotoxicity testing - comparing non-neurotoxic to known neurotoxic model compounds - showed an expected increase in the marker of astroglial reactivity after exposure to known neurotoxicants methylmercury and trimethyltin. Although further characterization and refinement of the model is required, these results indicate its potential usefulness for in vitro neurotoxicity testing.

Multilobular tumor of the zygomatic bone in a dog.

Multilobular tumor of bone (MTB) (also known as Multilobular Osteochondrosarcoma) is an uncommon bone tumor frequently located on the skull of dogs, rarely on the ribs or pelvis. These neoplasms are slow growing, locally invasive, and have the potential to compress and invade the brain. A 10-year-old mixed breed dog was presented with a history of approximately 4 months of progressive growth of a left zygomatic mass. Radiographic investigation revealed a finely granular or stippled non homogeneous radiopaque mass involving the zygomatic arch. After surgery, grossly the neoplasm consisted of multiple, variably sized, grayish-white to yellow nodules separated by collagenous septa of different thickness. Histologically, the tumor was characterized by the presence of multiple lobules containing osteoid and cartilage, separated by a net of fibrous septae. This neoplastic pattern was consistent with a typical multilobular tumor of bone and based on clinical, radiographical, gross and light microscopic findings the definitive diagnosis was made. While reviewing veterinary literature only few cases of MTB were found in dogs.

Embryonic stem cell-based screen for small molecules: cluster analysis reveals four response patterns in developing neural cells.

Neural differentiation of embryonic stem cells (ESC) is considered a promising model to perform in vitro testing for neuroactive and neurotoxic compounds. We studied the potential of a dual reporter murine ESC line to identify bioactive and/or toxic compounds. This line expressed firefly luciferase under the control of the neural cell-specific tubulin alpha promoter (TUBA1A), and renilla luciferase under the control of the ubiquitous translation elongation factor 1-alpha-1 (EEF1A1) promoter. During neural differentiation, TUBA1A activity increased, while EEF1A1 activity decreased. We first validated our test system using the known neurotoxin methyl mercury. This compound altered expression of both reporter genes, with ESC-derived neural precursors being affected at markedly lower concentrations than undifferentiated ESCs. Analysis of a library of 1040 bioactive compounds picked up 127 compounds with altered EEF1A1 and/or TUBA1A promoter activity, which were classified in 4 clusters. Cluster 1 (low EEF1A1 and TUBA1A) was the largest cluster, containing many cytostatic drugs, as well as known neurodevelopmental toxicants, psychotropic drugs and endocrine disruptors. Cluster 2 (high EEF1A1, stable TUBA1A) was limited to three sulfonamides. Cluster 3 (high EEF1A1 and TUBA1A) was small, but markedly enriched in neuroactive and neurotoxic compounds. Cluster 4 (stable EEF1A1, high TUBA1A) was heterogeneous, containing endocrine disruptors, neurotoxic and cytostatic drugs. The dual reporter gene assay described here might be a useful addition to in vitro drug testing panels. Our two-dimensional testing strategy provides information on complex response patterns, which could not be achieved by a single marker approach.

Infection of organotypic slice cultures from rat central nervous tissue with Trypanosoma brucei brucei.

We recently described a new procedure to grow nervous tissue as organotypic culture. The main feature of these slice cultures is to maintain a well preserved, three-dimensional organisation of the central nervous tissue. As these cultures can be kept for several weeks (up to three months), we have used this in vitro approach to study the complex interactions between host tissue and parasites during late stages of cerebral African trypanosomiasis. Light and electron microscopical studies, as well as electrophysiological recordings demonstrate that the structure and function of the nervous tissue is not severely affected even after several weeks of trypanosome infection. The presence of a large number of parasites does not seem to be deleterious to neuronal survival. Secondly, most of the trypanosomes are located around the periphery of the nervous tissue, but many of them also penetrate into the nervous parenchyma. Thirdly, trypanosomes with well-conserved morphology are found within the cytoplasm of glial cells, which in some cases were identified as astrocytes. These "intracellular parasites" seem to actively invade the target cells. Our study demonstrates that the presence of proliferating trypanosomes does not per se interfere with the neural activity of CNS tissues. Secondly, it provides, to the best of our knowledge, the first in vitro demonstration of intracellular forms of African trypanosomes.

Coupling on-line brain microdialysis, precolumn derivatization and capillary electrophoresis for routine minute sampling of O-phosphoethanolamine and excitatory amino acids.

In previous papers, we described the analysis of excitatory amino acids (EAAs) and catecholamines in microdialysis samples using capillary electrophoresis with laser-induced fluorescence detection (CE-LIFD). In the present paper, we report that an automated analysis of such samples can be easily achieved by on-line coupling of the microdialysis probe with a continuous flow derivatization system and a commercially available CE-LIFD apparatus. Because of the short analysis time (less than 2 min) and high separation efficiency (100-200,000 theoretical plates), high temporal resolution of microdialysis (minute range) is preserved as compared to off-line systems, while both EAAs and O-phosphoethanolamine (PEA) can be simultaneously detected. This new method has been applied to the measurement of these compounds in microdialysis samples from hippocampal slice cultures and striatum of anesthetized rats. Extracellular concentrations of EAAs, but not PEA, increased during perfusion of a solution containing high K+ or a glutamate uptake inhibitor. However, after in vitro ischemia on hippocampal slices, both EAAs and PEA concentrations increased, but with different temporal patterns.

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.

Microelectrode arrays for electrophysiological monitoring of hippocampal organotypic slice cultures.

A three-dimensional platinum (Pt) microelectrode array embedded on a micromachined silicon (Si) substrate (porosity of 13%, via hole diameter of 40 microns) has been developed. Electrodes are 35-micron wide and 20-microns high, spaced 200 microns apart and arranged in an elliptic geometry. Integrated within a microperfusion chamber, the devices were used for stimulation and recording experiments of hippocampal slice cultures over a period of several days.

Staurosporine but not chelerythrine inhibits regeneration in hippocampal organotypic cultures.

A mechanical lesion in hippocampal organotypic cultures is followed by a recovery process involving scar formation, sprouting of fibres and formation of new functional synapses. Here we tested the effect of staurosporine and chelerythrine, two protein kinase C (PKC) inhibitors, on this lesion-induced neurite outgrowth of Shaffer collaterals. At a concentration of 1 microM, staurosporine delayed functional recovery assessed by measuring synaptic field potentials across the lesion, without altering synaptic transmission on nonlesioned cultures. Immunostaining carried out by using antibodies directed against neurofilament proteins showed that there was a marked reduction in the number of regenerating fibres crossing the lesion. In contrast to this, chelerythrine (50 microM) did not prevent functional recovery, although it affected synaptic transmission and plasticity at this concentration. We conclude that the inhibition of sprouting produced by staurosporine is independent of its blockade of PKC-mediated phosphorylation mechanisms.

Sprouting and functional recovery in co-cultures between old and young hippocampal organotypic slices.

We developed a model of lesion of Schaffer collaterals in hippocampal organotypic slice cultures to analyse the capacity for sprouting and functional recovery expressed in young (one week old) and old (four week old) slice cultures. Slice cultures were sectioned at different ages of maturation in two separate half-slices and maintained in co-culture. Functional recovery was assessed by measuring synaptic responses elicited across the lesion seven days after the lesion and sprouting was evaluated by biocytin labeling of the regenerating fibers seen under the same conditions. Sprouting and functional recovery were found to be markedly reduced and delayed in old vs young cultures. Preparation of co-cultures between young CA3 and old CA1 half-slices resulted in a significant reduction in the capacity for sprouting and regeneration of the young CA3 neurons. Conversely, co-cultures prepared between old CA3 and young CA1 half-slices showed a markedly enhanced capacity for sprouting and functional recovery of old CA3 neurons. These results indicate that the age-dependent impairment in sprouting and regeneration expressed in cortical regions can be improved by and depends upon the presence of a favourable environment.

Microdialysis monitoring of extracellular glutamate combined with the simultaneous recording of evoked field potentials in hippocampal organotypic slice cultures.

These experiments combined extracellular electrophysiological multirecordings from hippocampal organotypic slice cultures with application of drugs to and sampling of extracellular fluid from a restricted region of the slice using a microdialysis probe. Glutamate (Glu) concentrations were monitored in 0.5 or 2 min microdialysis samples, while evoked field potentials responses (EvFPR) in the CA1 region of the hippocampus (stimulation in the CA3 area) were simultaneously recorded using a multi-electrodes array (Physiocard). Glu was assayed by capillary electrophoresis with laser-induced fluorescence detection combined with a continuous flow derivatization of dialysates. The performance of this combined approach was demonstrated by monitoring extracellular Glu concentrations and EvFPR after K+ induced depolarisation, Glu uptake blockade by trans-pyrrolidine-2,4-dicarboxylic acid (PDC), and electrical stimulation. Such an approach allows a global monitoring of the neuronal functioning with a fine time resolution (up to 30 s) on a simple in vitro brain slice model, to be used as a complement to conventional in vivo microdialysis studies.