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

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

In the assessment of neurotoxicity, selection of the most comprehensive detection technique is critical to achieve accurate results. Considerations for detection techniques are subject to interpretation of neurotoxicity definitions, the limitations for each detection method of revealing the full realm of neurotoxic events, and the reliability of positive and negative results. Ultimately, a detection method must be considered successful only if it is accurate in indicating neurotoxicity while avoiding false positives and false negatives.

Defining Neurotoxicity

Brain function depends on the integrity of individual neurons and the circuits they form. Loss of neurons is, then, the ultimate endpoint of the various measures of neurotoxicity because neurons do not regenerate. Additionally, exposure to a chemical or to external influences (such as ionizing or non-ionizing radiation, contaminated atmospheres) has the potential to induce changes in the brain that do not involve the death of neurons. Some of these changes may be reversible and others may not. The changes can be structural (e.g. "pruning' the branches of dendritic trees) or biochemical, affecting cell metabolism, neurotransmitter pathways, etc. Detecting these changes that do not cause the death of neurons poses the greatest challenge to the neurotoxicologist. Although most often seen as accompanying events to neuron cell death, even isolated destruction of synaptic terminals, dendrites and axons will render a neuronal cell ineffective. It is functionally appropriate then to consider neurons rendered ineffective through direct destruction or by accompanying events as indications of neurotoxicity.

Understanding the mechanisms of cell death is useful in assessing the best solution for detection of neurotoxicity. The particular form of an insult that ultimately causes the cell to die can be expected to determine the pre-mortem sequence of changes that take place. Death by mitochondrial poisoning, blockage of protein synthesis (e.g. puromycin, cycloheximide), blockage of transcription from DNA (e.g. actinomycin-D) or ion channel blockers, to suggest a few, can result in different sequences of chemical changes that can be markers of a particular route to death.

Detection Approaches

The wide range of activities surrounding neuronal cell death has spawned several methodologies of approach aimed at detecting neurotoxicity including: the detection of cellular events that may lead to cell death, observed behavioral changes, and the detection of endpoint pathology.

Predictive Methods

Leveraging in situ and immunohistochemical methods, technologies used to detect the individual cellular responses to trauma have been developed. There are numerous specific probes that detect changes that take place in neurons following a given level of perturbation. Some of these occur in succession to one another forming a sequence, the length of which may be dependent on the nature and degree of insult to the system. If the insult causes damage, transcription for molecular entities associated with repair will occur. In spite of these cellular responses the form and/or degree of insult may, or may not, overwhelm the cell beyond recovery resulting in its death and disintegration. Changes in cells other than neurons constitute indirect markers of neuronal injury that may lead to death. For example, proliferation of glia (gliosis) at the site of damage and increased synthesis of the astrocyte specific protein, glial fibrillic acid protein (GFAP) have been commonly used as evidence of neurotoxic effects. Often, the occurrence of either of these is coincidental with the destruction of neural elements. However, there are also examples of increased GFAP expression not accompanied by degeneration of neural elements. These methods are quite effective and specific in their detection capabilities; however, they are not detecting actual neuronal cell death or even the final pathway to cell death. Rather they detect symptoms that appear when a cell has been injured and have the potential to precede cell death. It is not possible using these techniques to determine whether the cell or other neuronal elements would eventually die, rendering this approach incomplete for determining neurotoxicity.

Various combinations of events are indicators that may lead
to the final common pathway defined as neurotoxicity.

 

Behavioral Approach to Detection

Behavioral observations are capable of identifying neurotoxicity. However, they are not able to rule out neurotoxicity. Persistent, behavioral neurologic symptoms would meet the criteria of having caused permanent damage to neurons and/or other neuronal elements. Whether or not behavioral symptoms are present as a result of the loss of neurons depends, however, on which system is involved and the extent of cell loss. For some systems, the loss of a fraction of their cells would be sub-symptomatic, meaning that the loss of cells falls within the system's capacity to functionally compensate. It is most important to realize, however, that because of this cell loss the capacity for compensation for future insults is diminished. Neurologic symptoms that occur later in life may be the deferred manifestation of an earlier exposure to a neurotoxic agent or condition. It is important, then, to identify any cell loss or destruction of neural components and consider it significant, regardless of the absence of behavioral signs. A positive assessment of neurotoxicity based on behavioral observations may be seen as valid, while a negative assessment is not possible strictly based on behavioral observations.

Direct Detection of Degenerating Elements

The final approach in the assessment of neurotoxicity is detection of endpoint pathology, or the detection of dead or dying neuronal elements and neurons. This approach has the potential to be the most accurate as it relies on the specific detection of the death of the structures in question. There is a variance in approach within this method as some techniques are designed to specifically detect dead/dying neurons, while other techniques expand the scope to include detection of other neuronal elements (synaptic terminals, dendrites and axons). The classic method, and still perhaps the most widely used, is the application of the Hematoxylin and Eosin (H&E) stain. In H&E stained sections, the term "red and dead" is an apt expression applied to the appearance of the cytoplasm in a cell that has just died, and "pink" if it is atrophic and near death. The next generation of endpoint pathology detection is disintegrative degeneration stains, of which the most recently evolved is the Amino Cupric Silver (ACS) stain. The ACS stain also detects dead and dying neurons (like H&E), but it also detects other dead/dying neuronal elements including synaptic terminals, dendrites and axons. With both the H&E and the ACS stains, positive indications for neurotoxicity are definitive. A negative indication for neurotoxicity when determined based on H&E could represent a false positive since the H&E stain is incapable of revealing evidence of the demise of synaptic terminals, dendrites and axons (especially unmyelinated axons). The ACS is the only detection method capable of providing a definitive positive or negative indication of neurotoxicity.

Signal to Noise of Detection

Two additional factors contribute to the advantage of the ACS stain over H&E: the signal to noise ratio of the stain and the chronologic window of detection for neurotoxic response. The positive sign for neurotoxicity for H&E is pink stained nucleoli against red stained neurons that represent normal healthy cells. If the dead cells are not coalesced and part of a massive lesion but instead comprise a dispersed population among normal appearing cells, detection is difficult since it requires microscopic examination at relatively high magnifications. By contrast, the ACS stains positive elements for neurotoxicity in black, while normal healthy cells do not stain and appear opaque or white (black on white). Signs of neurotoxicity with the ACS stain are definitive and more conspicuous increasing the chance of accurate results.

Time Window of Opportunity for Detection

The implications of the window of detection differences between H&E and ACS yield increased accuracy and efficiency for the ACS stain over H&E. The chronologic window of opportunity for neurodegeneration detection refers to the amount of time a dying cell exists in a state that is detectable by the histology stains. The cell body itself remains in this detectable state of death for 2 to 3 days; however the chronologic time frame during which one or more of the synaptic terminals, dendrites, axons and cell bodies are able to be detected extends for over 6 days.

Time Based Presence of Disintegrating Elements

This fact has implications to detection as well as experiment design. Since there are more elements to detect in the ACS stain over a longer period of time, the chance of accurately identifying a neurotoxic event is increased. When designing an experiment, groups must be sacrificed at intervals not to exceed the detection window so that a neurotoxic event is not missed. When designing an experiment with the ACS stain, groups can be spaced up to 6 days apart without the risk of having a gap in the detection window. For H&E, the spacing of the groups can not exceed 3 days. Therefore, in order to have comparable coverage for a period of time, twice as many groups (twice as many animals, histology, analysis. etc.) would be required when designing for H&E vs. ACS.

Detection Range of H&E vs. Disintegration Stain

The neuronal elements that are impregnated by the degeneration-sensitive silver stains are the products of a disintegrative degeneration process. Typically, degenerating axon terminals are the first to disintegrate and to which the silver is first attracted while the impregnation of dendrites and cell bodies follows soon after. Early disintegrative changes in axons can be seen with the disintegrative degeneration staining methods at about the same time that cell bodies can become impregnated by silver. Axons initially can be seen intact but then become progressively fragmented. Argyrophilia of axon fragments and proteolysed debris can persist for weeks and even months after insult, long after the debris of terminals, cell bodies and dendrites have been cleared away. In humans the debris can persist for years.

Conclusion

The increasing variety of detection techniques for neurotoxicity as well as of the events preceding and potentially predicting neurotoxicity offer an increased potential for accuracy when conducting a neurotoxicity experiment. The various techniques discussed that act as markers and predictors of eventual neuron death are valuable tools when used for predictive purposes and are followed by verification of neurotoxicity itself. The H&E stain, the only other detection method fully capable of indicating a positive result for neurotoxicity, is deficient in its ability to detect the degeneration of all neuronal elements that define a neurotoxic response. The added implications of increased animal usage and more difficult analysis (with the potential for error), make H&E an inefficient, outdated and potentially ineffective tool when compared to the ACS stain. The ACS stain features conclusive positive results, eliminates the risk of negative results, is easy to interpret due to excellent signal to noise staining, and is capable of adding efficiencies to experiment design, making the ACS stain the clear choice for neurotoxicity assessment.

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

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

For futher reference, please see: Stains

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