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Ischemia/ Stroke
- Oxygen Glucose Deprivation (OGD) Induced Cell Lesion
This assay simulates the conditions occurring following brain ischemia. Isolated cortical neurons from chick embryos are maintained under oxygen free conditions in a hypoxia chamber, and then subjected to glucose deprivation.
The ionomycin cell lesion assay is a model for the damage to cell membranes and the cytoskeleton that results from ischemia. The assay is performed using primary cultures or cell lines.
The damage induced by glutamate mimics acute ischemia, where an increased release of glutamate followed by an overstimulation of glutamate receptors leads to apoptosis. With the L-Glutamate Cell Lesion Assay, effects of neuroprotective drugs on survival of cortical neurons as well as on the occurrence of apoptosis can be investigated. Cells are immunocytochemically stained with a MAP-2 antibody.
Apoptosis
Primary cultures of either chick embryo neurons, mouse or rat neurons, or cell lines (e.g., PC12, SH-SY5Y) are maintained in primary culture with a defined nutrition medium supplemented with 2% fetal calf serum. When the serum is withdrawn apoptosis results.
- Staurosporine Cell Lesion Assay
Apoptosis specific features as well as viability are emphasized this assay. The addition of staurosporine to primary cell cultures initiates a cascade of changes, from cytochrome C release to elevation of intracellular Ca++, that lead to protease mediated reactions resulting in apoptosis or, at higher staurosporine dosages, necrosis.
Neural Degeneration
- Cell Free Calpain Activity Assay
This biochemical assay addresses the potential inhibitory effect of a test substance on the Ca ++- dependent proteases µ- and m-calpain. These cysteine proteases play a significant role in ischemia, apoptosis, and cytoskeletal degradation in other neurodegenerative conditions. In this cell free assay a synthetic fluorescence labeled substrate and purified µ- and m-calpain are used. Each enzyme can be tested for interactions with potentially therapeutic substances. Evaluation is carried out in a fluorescence microplate reader.
NMDA (N-methyl-D-aspartic acid) induces a series of cytoplasmic and nuclear processes that promote neuronal cell death. The NMDA lesion assay is a straightforward method for studying the neuroprotective potential of drug candidates that might decrease NMDA excitotoxicity, a relevant event in ischemic stroke and post-traumatic lesions.
Alzheimer's disease
Fluofarma, one of NDI's partner companies, has developed a cellular model that mimics Alzheimer's disease. In this assay, primary cultures of mesencephalic neurons are grown in the presence of b-Amyloid peptide whose accumulation is known to contribute to the development of Alzheimer's disease. Test compounds are evaluated for their ability to reduce the neuronal degeneration that results from the b-Amyloid induced cell death. Furthermore, this model allows a distinction to be made between neurotrophic and neuroprotective effects.
- Aβ-induced neurotoxicity (NT2N cells)
This assay is useful for screening compounds that interfere with APP processing. The formation of intracellular Aβ is measured in NT2N cells that have differentiated into neurons. NT2N cells are used because they appear to process APP in ways similar to human neurons.
- Aβ-induced neurotoxicity (mesencephalic cells)
We have developed a novel in vitro assay for screening potential neuroprotective agents, that uses mesencephalic neurons and MAP2 (neuron-specific) quantification. An additional capability of this assay is detection of neurotrophic as well as neuroprotective effects. The cultures contain dopaminergic neurons taken from embryonic rat mesencephalon, which fully differentiate in vitro, and survive for at least 30 days.
Parkinson's disease
Primary cultures of embryonic mesencephalic dopaminergic neurons are treated with a neurodegeneration inducer, and then test compounds are evaluated for their ability to reduce the extent of this degeneration. The inducers are:
A botanical compound obtained from the roots of Derris sp., rotenone is an active ingredient in many pesticides. Rotenone blocks complex I of the mitochondrial respiratory chain and thereby induces nigrostriatal degeneration in rodents, thus reproducing the hallmark pathology of Parkinson’s disease.
The active metabolite of MPTP, MPP+ is a piperidine derivative which induces a Parkinsonian syndrome in mice and primates. MPP+ selectively enters dopamine neurons via the dopamine transporter and also blocks complex I of the mitochondrial respiratory chain.
Epoxymicin, a natural product isolated from Actinomyces sp., is a cell-permeable, potent, selective and irreversible proteasome inhibitor. Impairment of the ubiquitin-proteasome system replicates features of Parkinson’s disease.
The objective of these Parkinson's models is to identify compounds able to protect neurons and delay the neurodegenerative process.
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