Thyroxine t4

The purified RGCs were seeded at the desired density on the PDL- and laminin-coated coverslips in 24-well or six-well plates in prewarmed RGC growth medium and maintained in a 37°C cell culture incubator with a humidified atmosphere containing 5% CO₂ and 95% air. The RGC growth medium was improved from the formulation described in the Cold Spring Harbor Protocols, based on our repeated experiments, and contained Neurobasal medium, BSA (0.1 mg/mL; Sigma), transferrin (0.1 mg/mL; Sigma), progesterone (60 ng/mL; Sigma), putrescine (16 µg/mL; Sigma), selenium (40 ng/mL; Sigma), 3,5,3-triiodothyronine T3 (40 ng/mL; Sigma), thyroxine T4 (40 ng/mL; Sigma), B27 (20 µl/mL; Invitrogen), sodium pyruvate (1 mM; Gibco), glutamine (2 mM; Gibco), N-acetyl-l-cysteine (NAC, 5 µg/mL; Sigma), insulin (5 µg/mL; Sigma), forskolin (5 µM; Sigma), brain-derived neurotrophic factor (BDNF, 50 ng/mL; PeproTech, Rocky Hill, NJ), ciliary neurotrophic factor (CNTF, 10 ng/mL; PeproTech), basic fibroblast growth factor (bFGF, 10 ng/mL; PeproTech), and penicillin-streptomycin (100 U/mL; Gibco). Half of the medium was replenished every 3 days. However, although a high level of purity was obtained with FACS, the level of cell survival was low. The causes of this low survival are unclear.

Mice (C57 BL/6J) were maintained under conditions of controlled temperature (23 ± 1 °C), relative humidity (50 ± 1%), and lighting (07:00–19:00 h). The control mice were fed laboratory chow (Labo MR Breeder; Nosan, Yokohama, Japan) with ad libitum tap water. To generate hypothyroid mice model, pregnant mice were administered antithyroid agents (1% potassium perchlorate and 0.05% methimazole in tap water) from gestational day 17 to postnatal day 14. From postnatal day 15 onward, the dams and pups were given normal water. To observe the effects of exogenous thyroid hormone on the expression of PV mRNA, the mice were injected with thyroxine (T₄; 20 ng/g body weight; Sigma-Aldrich Co., MO, USA) from PD 0 to PD 14.
All animal experiments were conducted in accordance with international standards on animal welfare according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, the ARRIVE guidelines, and the Animal Experiment Guidelines of the Institutes for Animal Experimentation at Tohoku University. The protocol for animal experiments was approved by the Center for Laboratory Animal Research, Tohoku University (reference number: 2016IsA-004-1).