Evaluation of Permanent Damage
Many alterations to the brain can be negative and potentially long-lasting, but not all can be considered permanent. Some injuries can be treated or will resolve after time if the source of the injury is removed (i.e., a harmful compound ceases to be administered), while other injuries lead to permanent implications. The most definitive and easy way to interpret evidence of permanent damage is the destruction of neurons. Even though the brain has amazing functional compensatory power, the loss of irreplaceable neurons creates a permanent deficit. All comprehensive safety evaluations at some point need to evaluate the risk of permanent damage and the mechanism of that evaluation is to observe neurodegeneration as it occurs during the cell death cycle.
There are scope and timing considerations in designing the safety study and in selecting a detection method.
Timing Detection of neurodegeneration requires critical timing considerations. Depending on the detection method chosen, a dying cell can be viewed for only ~2–6 days from the time it begins to disintegrate, after which there is no debris remaining to observe. Therefore, the timing of sacrifice following exposure to a compound is a critical element of study design. The graphic below shows the timeline of the detectability of degenerating neurons for some known neurotoxins. The days indicate the amount of time following a single acute dose on day 0. The bars represent the most probable timing opportunities for detection.
Scope of Elements
The neuron has an extended temporal and spatial footprint when the dendrites, axon terminals and axons are evaluated in addition to the neuron cell body itself. The class of stains called the disintegrative degeneration stains is able to detect disintegration of each of these elements, while other methods capable of detecting cell death only reveal pathology of the cell body itself. Disintegrative degeneration stains include various FluroJade and Cupric Silver methods. NSA routinely performs the disintegrative degeneration stains as well as methods such as H&E.
Features visible in a Full Scope (Amino CuAg) Disintegrative Degeneration Stain vs. a Cell Body–Only Degeneration Stain (H&E)
In the Amino Cupric Silver–stained section, not only are the disintegrating cell bodies clearly visible, but the degenerating synaptic terminals, dendritic debris and axons are also conspicuous. In the H&E-stained section, the damage is far less obvious and the characteristic eosinophilia of the cytoplasm and pyknotic nuclei of affected cells requires higher magnification to be distinct, as shown on the right. While it is possible to witness cell death with either category of stain, the degeneration stains provide a higher contrast signal leading to cost and time efficiencies during analysis.
Features visible in the Disintegrative Degeneration–stained sections are shown magnified below.
Of all four degenerated elements shown on this page, only the cell bodies in the picture above (without the dendrites) are conspicuous with a cell body stain such as H&E.
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