A Novel Histological Method to Detect Abnormal Thyroid Hormone Action in the Developing Rat Brain
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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.