Introduction | Scope of Stained Elements | Compare With H&E | Silver Degeneration Staining Evolution

Neonatal Cell Death (apoptosis) | Neurotoxicity Detection Paper | Planning a Neurotoxicity Experiment

The two goals of a neurotoxicity experiment are: 1) Assess whether a compound is neurotoxic or in fact 'neurosafe' and 2) If the compound is neurotoxic, at what dose levels neurotoxicity occurs. If neurotoxicity is observed at high doses, then a no effect level should also be established. The following discusses guidelines by which researchers can construct the most appropriate experiment for their needs. The guidelines are based on utilizing comprehensive staining techniques such as the deOlmos Amino Cupric Silver disintegrative degeneration stain for the reasons discussed in the paper: Neurotoxicity Detection. In order to assess neurotoxicity of a compound, the variables of dose schedule and survival times following dosing are used. Dosing level is the third variable which is introduced for the purpose of determining the level of a compound required to induce neurotoxicity.

Dosing Strategy

Neurotoxicity may occur after either a single (acute) dose of a compound, or only after repeated doses (subchronic). To initially determine neurotoxicity, the maximum tolerable dose (MTD) is used. The acute dose and the maximum effective dose (ED) should be used for daily dosing to elicit a chronic effect of neurotoxicity. Once the MTD and ED are known, different dose levels for acute and chronic dosing allow the neurotoxicity profile of the compound to be determined. (See the chart: Paradigm for Neurotoxicity Screening at right).

Survival Times

Using the disintegrative degeneration stain, neurodegeneration can be detected during a time period spanning 6+ days and may begin immediately following the dose. Since the day on which the cell death cycle begins is not known for a new compound, a group of animals should be sacrificed beginning on day 2 or 3, and then subsequent groups should be sacrificed at intervals of 5 days or less. For acute dosing, this sacrifice schedule should be maintained for a 14 day time period, and for subchronic dosing, the schedule should persist for at least 28 days. Dosing in the subchronic case may be discontinued two days prior to sacrifice for each group of animals. With chronic dosing strategies it is important to consider that damage may occur early in the dosing interval and that the resulting debris may no longer be visible at the end of the dosing period. For example, if the dosing interval is 6 weeks and damage is induced at 2 weeks, the associated debris is not likely to be present and stainable at the end of the 6 weeks.

Dosing Level Sequencing

Referring to the Paradigm chart to the right: If neurotoxicity is found using the MTD in the acute study, but not at the ED, then a 'safety factor' dose, some multiple of the ED, is administered. If no degeneration is found, then the repeated dosing series for 5 days and more begins. If no degeneration is found then the compound can be considered not neurotoxic. If degeneration is found then a risk benefit analysis must be performed.

Animal Numbers

A minimum of five animals in each group should receive the compound of interest (i.e. the test article). Ideally, a minimum of five animals of each gender should be used.

Controls

A positive control and a negative control of each gender should be used per MultiBrain^TM block.

Introduction | Scope of Stained Elements | Compare With H&E | Silver Degeneration Staining Evolution

Neonatal Cell Death (apoptosis) | Neurotoxicity Detection Paper | Planning a Neurotoxicity Experiment

 

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