Permanent Damage

NSA’s FDA Neurotoxicity Presentation 2015

 

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 timing and scope 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 Fluro-Jade 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, the disintegrating cell bodies are clearly visible, and 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 Amino CuAg Disintegrative Degeneration–stained sections

Beta-Amyloid Precursor Protein (β-APP)

Beta-Amyloid Precursor Protein (ß-APP) has become a useful marker for axonal injury and commonly termed: ‘diffuse axon injury’.  ß-APP is carried out by fast anterograde transport vesicles to distal sites in the axon. Upon axonal injury, ß-APP pools in areas of impaired transport.

Alternative means of detecting axon damage use the disintegrative degeneration stains (most notably, the amino cupric silver (ACS) method of deOlmos). Adjacent sections stained with the ACS stain and for ß-APP reveal quite different degrees of staining: much more axonal injury/damage is shown by the ACS suggesting that the ß-APP staining is revealing a totally different form of damage or a subset of what is diplayed in the ACS stain.

Amino Cupric Silver Stain

Learn more about NSA and NeuroSafety:

Neurosafety Overview Approach

Chemical Changes Evaluation

Perturbations/Inflammation Evaluation

Neonate/Juvenile Safety Studies Approach

NSA NeuroSafety Protocols