Citation:

O'Shaughnessy, Katherine, A. Sasser, AND K. Bell. A Novel Histological Method to Detect Abnormal Thyroid Hormone Action in the Developing Rat Brain. Annual Society of Toxicology Meeting, Salt Lake City, UT, March 10 - 14, 2024.

Impact/Purpose:

Many industrial chemicals are tested for potential thyroid hormone (TH) interference in vivo, as exposure to environmental thyroid disrupting chemicals can affect children¿s health. Over the years, we have shown that perinatal maternal TH insufficiency leads to the formation of a periventricular heterotopia in rodent offspring and this phenotype could be a marker of developmental hypothyroidism. However, this extreme morphological defect is currently assessed around postnatal day 14 in the rat. Given our mechanistic knowledge of how the heterotopia forms, we set out to develop a new histological method to rapidly detect abnormal TH action in the brain. This assay quantifies radial glial cell morphology, which is crucial for normal neuronal migration in the brain. For this study, we induced moderate maternal hypothyroidism from gestational day 6 to postnatal day 14 using propylthiouracil. This exposure significantly reduced pup serum T4, and pup and brain T4 and T3. On PN8, pup forebrains were processed for vimentin immunohistochemistry to visualize radial glial cells in the developing telencephalon. A representative group of radial glial cells were measured on both hemispheres of each section, for each animal. We found a statistically significant decrease in radial glial cell length. Control rat pups (N=5) averaged a length of 181.1 um, while PTU exposed animals (N=5) were 122.5 um. Radial glial cell angles were also measured. The PTU pups consisted of extreme angles, while control animals were consistently closer to 90°. When quantified as a deviation from 90-degrees°, there was a significant change as control pups averaged 13.4° and treated averaged 29.8°. In this study we have shown that the quantification of radial glial cell morphology can detect abnormal thyroid hormone action in the developing brain. This histopathology assay will help the Agency¿s interest in protection children¿s health and aid in rapidly identifying chemicals that may alter brain TH action.  

Description:

Assaying for thyroid interference is of increasing importance in standardized developmental and reproductive toxicity studies. While serum T4 quantification is an endpoint assessed in developing rats, it is unclear how this measure may correlate to adverse neurodevelopmental outcomes. Previously, we have shown that developmental thyroid hormone (TH) insufficiency leads to the formation of a periventricular heterotopia in rats, and this phenotype can be assayed as a readout of abnormal TH action. However, the heterotopia is assessed around postnatal day 14 (PN14), and is considered a severe morphological defect. Here, we have developed a novel histological method to detect abnormal TH action in the brain during the neonatal period. This assay quantifies radial glial (RG) cell morphology, which are progenitor cells that mediate neuroblast migration and are known targets of TH action. When RG cells are abnormal, this can lead to later heterotopia formation, representing an upstream key event. As a proof-of-concept study, we exposed pregnant rats to 0 or 3 ppm propylthiouracil (PTU), from gestational day 6 until PN14. This exposure significantly reduced pup serum T4, and pup and brain T4 and T3 during the first 2 postnatal weeks. On PN8, pup brains were fixed, sectioned, and radial glial cells were visualized by vimentin immunohistochemistry. Sections were then imaged and analyzed using Aperio Slide Scanner and ImageScope, tools commonplace in pathology laboratories. A representative population of RG cells were measured on both cortical hemispheres of each section; 4 sections were measured for each animal. First, RG cell length was quantified. Control rat pups (N=5) averaged 181.1 µm, while PTU-exposed animals (N=5) were 122.5 µm, demonstrating a statistically significant decrease in RG cell length. The angle of RG orientation from the ventricular zone was then measured. RG cells of PTU-exposed pups consisted of extreme acute and obtuse angles, while control animals were consistently closer to 90°, as expected. When quantified as a deviation from 90°, control pups averaged 13.4° and PTU-exposed pups averaged 29.8°, a significantly significant change. In summary, these data show that quantification of RG cell morphology can detect abnormal TH action in the developing brain. This assay can be performed in young animals, can be detected in minimal sections, and could be automated in the future. This histopathology assay represents a potentially rapid and sensitive method to identify thyroid disrupting chemicals in vivo. This work does not necessarily reflect US EPA policy.

Record Details: