When it comes to neuropathology studies, there is no one-size-fits-all approach. In this on-demand webinar, Senior Pathologist Lisa Mangus, DVM, PhD, DACVP, addresses the unique challenges that nervous tissues pose during sampling, preparation, and microscopic evaluation in non-clinical animal toxicity studies. Along the way, she also offers expert recommendations and best practices to increase your chances for a successful neuropathology study.

Watch the on-demand webinar for an in-depth look at this topic. Or, keep reading for a high-level overview of what Lisa discussed with the webinar attendees.

Overview of recommendations and best practices

Generally, guidelines for pharmaceutical safety testing in nonclinical species lack prescriptive detail regarding which sites throughout the nervous system to sample or how the tissues should be prepared.

While flexible regulatory guidance allows investigators a high degree of freedom to tailor their neuropath evaluations and the ability to focus their resources as they see fit, it also necessitates careful planning and decision-making in the early stages of study design. Otherwise, investigators may insufficiently characterize or fail to identify potential neurotoxic effects.

Fortunately, the Society of Toxicologic Pathology has published multiple papers that offer best practice recommendations for sampling and evaluating the nervous system for various types of nonclinical studies. As Lisa explains during the webinar, these are not hardline requirements; rather, they are recommendations that reflect a consensus among experts in the field. You can watch the on-demand webinar for more information on these papers, including their titles. Here is an introductory look at the topics discussed in the papers:

Types of studies from a neuropathology perspective

Studies are often referred to as tier one or tier two. Tier one includes general toxicity studies, as well as expanded neuropathology evaluations. Both study types under tier one include several non-neuro or non-histology endpoints that preclude specialized methods such as perfusion fixation or the use of certain fixative solutions.

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Dedicated neurobiology evaluations are classified as tier two studies. In these types of studies, optimal morphologic examination of the nervous system is a primary goal. You can learn more about each study type and their appropriate use cases here.

Fixation and processing of nervous system tissues

The overall objective of fixation is to preserve cells and tissue components in a “life-like” state for microscopic evaluation. Otherwise, without adequate fixation, the quality of morphologic evaluation will be limited. Correct fixation:

• Prevents autolysis, which begins immediately when tissues are deprived of blood supply
• Minimizes the degree of artifactual changes

Unfortunately, as critical as fixation is to accurate neuropathic evaluation, proper fixation of nervous system tissues is challenging compared to other organ systems. This is because:

• Nervous tissues have a high degree of lipid content, making them resistant to fixation
• There is a lack of connective tissue in the brain and spinal cord, making the tissues quite delicate
• A lot of nervous tissues, including the central nervous system, are encased in bone

Fixation procedures: Variables to consider

Every fixation procedure comes with three major variables to consider:

1. Which fixation solution(s) to use
2. What fixation method should be applied
3. Time (both under- and over-fixation can be problematic)

For an in-depth explanation of the advantages and disadvantages of various fixation solutions, along with a more detailed look at fixation methods and how time factors into the fixation process, watch the webinar. During the presentation, Lisa also detailed the steps, benefits, and disadvantages of these processing/embedding options:

• Paraffin
• Resin
• Gelatin
• Optimal cutting temperature (OCT) compound

Immunohistochemical labeling

Regarding immunohistochemical labeling, glial markers offer many advantages in neuropathology evaluation. They enable the detection of glial reactions that are inapparent or subtle in hematoxylin and eosin (H&E) stained tissues. This detection increases the sensitivity for identifying neuronal changes. Glial markers also aid in distinguishing true neuropathologic changes from artifacts.

Immunohistochemistry can be used to identify other cell types as well, such as neurons. Tyrosine hydroxylase is an IHC marker that highlights dopaminergic neurons and their processes. This is particularly useful in Parkinson’s Disease models.

Most of the routine IHC assays performed on nervous tissues at StageBio are robust and typically don’t require modification to the standard fixation protocols to be successful. However, if you intend to run a novel or uncommon IHC marker or in-situ hybridization (ISH) to label nucleic acids and tissues, you will want to plan accordingly and consider adjusting your fixation protocol. You can learn more here.

Considerations for molecular techniques: IHC and ISH

When it comes to preparing tissues for non-routine IHC and ISH, current recommendations for nervous system tissues are generally the same as those applied to other organs and tissues.

Some general recommendations include the use perfusion fixation, since delayed fixation can yield a lower signal. Additionally, probe manufacturer Advanced Cell Diagnostics (ACD) creates ISH probes optimized for 10% neutral-buffered formalin-fixed tissues and recommends a fixation period of 16 to 32 hours at room temperature.

Considerations and tips for quantitative analyses: Stereology and nerve histomorphometry

There are various types of quantitative analyses that can be performed on the nervous system. However, the two Lisa chose to focus on during the webinar are stereology and nerve histomorphometry.

Stereology versus nerve histomorphometry

Stereology is a discipline that combines the principles of geometry and statistics to generate 3D data based on 2D images of tissues. Meanwhile, nerve histomorphometry is a technique that quantifies the number, density, and average diameter of nerve fibers.  

Tips for achieving successful quantitative analyses
Both methods are quite resource and time intensive. Follow these tips to ensure your analysis with either method yields accurate and insightful results:

1. Plan ahead: Start as early in the study design as possible. These methods are often very challenging or impossible to perform retroactively.
2. Run a small pilot study: Sort out technical kinks prior to using irreplaceable study tissues and determine the number of animals needed per group.
3. Process and evaluate across all groups in the same timeframe: Maintain technical consistency and avoid batch effects. 

Specific questions regarding sampling and preparing nervous system tissues

At the end of the webinar, Lisa answered thought-provoking questions from the audience. Here are a few highlights:

How can you reduce dark neuron artifacts?

There are several factors that are thought to contribute to the generation of dark neurons. One of the major causes is handling the nervous tissues when they are fresh or partially fixed (i.e., immediately after perfusion fixation). Sometimes this is unavoidable, like when fresh tissue samples are being collected at necropsy. But whenever feasible, it’s helpful to handle and/or manipulate the tissues as little as possible and avoid applying pressure to the tissues with instruments like rongeurs and forceps.

Can transferring neuro tissues to 70% ethanol after fixation result in a white matter vacuolation artifact?

Yes. Transferring neuro tissues to 70% ethanol after fixation can lead to artifactual vacuolation of white matter, especially if the tissues are stored in ethanol for more than a few days. Typically, this isn’t a major problem since it’s a well-known artifact (i.e., it’s easy to explain based on what you know about the storage time and conditions) and having a good concurrent control group will help you feel confident in interpreting the vacuolation as an artifact. One situation in which it could be a larger concern is when there is a true vacuolar change that could be obscured or confounded by artifactual vacuolation. If vacuolar changes are something you expect to see based on the disease model or test article you’re working with, then it would be best to keep ethanol storage time to a minimum or avoid it all together.

Is cold fixative for perfusion recommended?

It’s typically recommended that flush and fixative solutions be at room temperature for perfusion fixation. Cold solutions can cause vasoconstriction, which leads to patchy areas where fixation is less optimal than others. When perfusing with a buffer solution only (i.e., when collecting fresh, non-fixed tissues at necropsy), this is less of a concern, and using chilled buffer can be helpful since it makes unfixed CNS tissues firmer and easier to cut.  

Watch the on-demand webinar and learn how to run a successful neuropathology study

To get a more detailed look at the recommendations and best practices for sampling and evaluating the nervous system, watch Lisa’s full webinar on demand here or contact our team.