In Vitro Neurotoxicology: Methods and Protocols


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In this field, the selection of the most appropriate in vitro neuronal system relies on specific endpoints that are of particular relevance for the neurotoxicological phenomena that will be studied. Furthermore, application of specific endpoints to various neuronal cellular models should be done in a careful way to build reliable and feasible testing strategies. This review addresses the use of in vitro models for neurotoxicity research, aiming to contribute to a better understanding and guidance of in vitro neurotoxicological studies. As such, subcellular systems, namely isolated mitochondria and synaptosomes, and cellular models, including immortalized cell lines, primary cultures, co-cultures, organotypic cultures, neural stem cells and blood—brain barrier models, as well as their inherent advantages and limitations, are discussed.

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Therefore, long-term survival of low-density hippocampal neu- ronal cultures is not routinely possible. If microscopic techniques, which are difficult to apply to cells growing in plastic culture dishes, are to be used, neurons should be plated on glass coverslips coated with PLYS. After the overnight incubation, wash dishes or glass cover- slips twice with sterile water and once with HBSS. Leave the last wash in the plates to ensure that no water is left behind as water is toxic to neurons. Place the papain and the DNAse stock solutions in the water bath until needed. Minimize the time, making sure that all needed materials are ready before starting the procedure.

The dissection should not take more than 2. Euthanize the pregnant rat using carbon dioxide. Perform all the following steps under a laminar flow hood. During the dissection, the brain is visualized best if kept in dish filled with cold CMF-HBSS under a dissection micro- scope illuminated from above. Using a spatula, remove the entire brain and place it on sterile filter paper.

Keep brain moist to prevent the tissues from dying. Separate the two cerebral hemispheres from the brainstem. Cut the two cerebral hemispheres along the midline using a sharp scalpel. Lay each hemisphere on its side. With the diencephalon side facing up, place the spatula between the diencephalon and the cerebral hemisphere and spread the diencephalon away from the cerebral hemisphere to see the hippocampus.

Being careful not to damage the hippocampus, separate the hemisphere completely from the diencephalon using a fine curved-tip forceps. When viewed from this posi- tion the hippocampus is a well-defined C-shape area, which is contiguous to surrounding area of the brain delineated by blood vessels. If parts of the thalamus remain attached to the hippocampus, gently remove them using a fine forceps. Remove meninges and choroid plexus rolling the tissue on filter paper. Mince the tissue into small chunks with the fine-curved for- ceps.

Transfer the tissue to a mL polypropylene conical tube using a disposable pipette. Gently triturate the hippocampal tissue ten times with a Pasteur pipette to dissociate larger aggregates. Remove the supernatant cell suspension and filter through a mm filter into a mL sterile tube.

Methods of in vitro toxicology.

Mix gently using a mL pipette. Wait a few seconds. Count cells using a hemocytometer, and seed them at a den- sity of 0. Preparation The striatum is a subcortical area of the brain, which represents of Striatal Neuronal the major component of the basal ganglia and processes the inputs Cultures involved in voluntary movements.

✅ Long Protocol for IVF

All cortical areas send excitatory glutaminergic projections to specific portions of the striatum. The striatum also receives excitatory inputs from the intralaminar nuclei of the thalamus, dopaminergic projections from the mid- brain, and serotoninergic input from the raphe nuclei.