FIG. 10.

In vitro modeling of thyroid hormone deiodination and transport in the brain. (A) Schematic representation of the Transwell System in which an insert is placed on a six-well plate and cells (D2-expressing H4 glial cells) are seeded inside the insert; D3-expressing neuroblastoma cells (SK-N-AS) are seeded at the bottom of the six-well plate. After cells are seeded, both cell types are kept separated overnight and then placed together in the same multiwell plate as indicated. (B) At the end of the incubation medium samples are collected, extracted and processed through a UPLC or HPLC connected to the flow gamma counter to separate and quantify the activity of each iodothyronine. The red arrow indicates the pathway completed by the column eluate through the gamma counter; courtesy Dr. Antonio Bianco. (C) Chromatograms of H4 cell medium at the indicated times after addition of ¹²⁵I-T₄. Typical peaks of ¹²⁵-T₃ and ¹²⁵I are shown after 24 hours. (D) Same as in (C), except that ¹²⁵I-T₃ was added to cultures of SK-N-AS cells and ¹²⁵I-T₂ and ¹²⁵I-T₁ peaks are visualized. (E) Same as in (C), except that ¹²⁵I-T₄ was added to H4 and SK-N-AS cocultures and the indicated peaks are visualized. UPLC, ultrahigh performance liquid chromatography; HPLC, high performance liquid chromatography; T₁, 5′-monoiodothyronine. Reproduced with permission from Freitas et al. (173).