The detailed work flow of the study has been graphically illustrated in Fig. 1.
Institutional Review and Board
This study was approved by the animal research ethics committees of the City University of Hong Kong, the University of Hong Kong, and the Department of Health of the Hong Kong Special Administrative Region.
Further this study is reported in accordance with ARRIVE guidelines.
2.1 Animal Subjects
Pregnant female Sprague Dawley (SD) rats (N = 12, 350–450 g) were selected to enter the study approximately one week prior to giving birth. The University of Hong Kong's Laboratory Animal Unit (AAALAC) provided the animals for the experiments. The rats were housed at Laboratory Animal Research Unit, City University of Hong Kong under a constant temperature of 25 degrees with chow food and drinking water in separate cages. The birth of the baby rats (referred to as pups or subjects) from each dam, was recorded as P1.
On the fourth day postpartum P4, all pups from each mother were weighed and the twelve heaviest pups (n = 12) were chosen for breast-feeding, with equal numbers of males and females. This study analyzed three time-points of lactation followed by postpartum: P4, P11, and P18. Total 96 (n = 8×12) pups from 8 dams (N = 2 control, N = 2 lithium, N = 2 lithium + 0.025%iodine and N = 2 lithium + 0.05% iodine in drinking water) were chosen for P18 to demonstrate the effect of iodine supplement on lithium medicated dams and N = 4 control dams with total of 48 pups were chosen to demonstrate the controlled maternal hormones effect on pups (control = 6 x 4 = 24 and lithium = 4 x 6 = 24) for P11. Subjects underwent a range of analyses at the time points, including blood testing and analytical spectroscopy.
2.2 Lithium preparation and administration
Lithium carbonate Li2CO3 with the product number (62470-100G-F) was purchased from Sigma Aldrich, United States of America (USA). Lithium with iodine-supplemented groups of dams were administered 900 mg/50kg/12hours of lithium carbonate using gavage till P18. The process of body weight lithium administration has been adopted from the reference 1. This way, the pups receive lithium through breast milk 1.
To further decouple the lithium effects on lactating pups from controlled hormones of the dam, lithium with a dosage of 900 mg/50kg/24 hours was gavage to half number of pups as four lithium groups (n = 6x4 = 24) from each 4 dam till P11 as per the body weight. The rest of the pups as four controlled group (n = 4x6 = 24) from the same 4 dams were gavaged with water. On P4 each pup was labeled as control and lithium group and was lactated from their respective mothers along with body weight measurement.
2.3 Iodine supplement preparation and administration
Molecular iodine (I2, 326143-100G) was purchased from Sigma Aldrich, USA. Two solutions of iodine supplement named 0.025% and 0.05% iodine were prepared by combining 0.25 and 0.5 g of crystal molecular iodine with each 1000 ml of distilled drinking water. A light, sensitive and tightly sealed beaker was magnetically stirred for a total of 48 hours at room temperature to ensure thoroug dissolution of solute in the solvent. The 0.025% and 0.05% iodine supplement solutions were prepared and then placed into 500 milliliter sealed drinking bottles as a fluid intake for lactating dams administered with lithium.
2.4 Blood analysis and Pups Thyroids extraction
Eight batches of pups (n = 96) from 8 dams (N = 2 controls, N = 2 lithium, N = 2 lithium + 0.025% iodine and N = 2 lithium + 0.05% iodine) at P18 were sacrificed using surgical procedure for the extraction of blood and thyroid along with their mothers. Xylazine was injected intraperitoneally at a rate of one milliliter per kilogram of body weight to put the subjects to sleep for the experiment after their body weights were measured at time points P18. Similarly, four batches of pups as four lithium (n = 6x4 = 24) and control groups (n = 6x4 = 24) from 4 dams were also sacrificed along with their dams at time point P11. The subjects at time points P11 and P18 were dissected to draw blood from their hearts using 1 ml syringes. To assess the blood's levels of lithium, total thyroxine (T4), and blood urea nitrogen (BUN), the blood was collected in 5ml tubes. ELISA was utilized to analyze total T4 and BUN. Their ELISA sandwich kits were acquired from Cusabio United States. At P11 and P18 postnatal days, all animals’ (pups and dams) total T4 and BUN were measured as per the ELISA protocol defined in reference 1.
Following blood extraction, thyroid glands were extracted from the pups, the protocols of excising thyroid were extracted as per surgery protocols defined in references 1,19,20. The thyroids of the pups were removed, and they were sent for the ICP-MS to be analyzed for trace elements such as Li, I, Na, K, and Ca.
For thyroid elemental measurements and blood lithium, ICP-MS (Perkin Elmer DRC II) was used in line with the methods supplied by AOAC INTERNATIONAL. Blood lithium at P11 and P18 from dams and pups was collected, and the serum was obtained by centrifuging one milliliter of blood at a rate of 2,500 revolutions per minute for one minute following the protocol defined in [BD]. For the thyroid content of Li, I, Na, K, and Ca, the digestion and measurement protocol was adapted from]21for pups at P11 and P18.
2.5 Statistical Analysis
At P11 and P18, measures of body weight, blood lithium, total T4, BUN and intra-thyroidal elements were statistically compared across the groups using a one-way analysis of variance (ANOVA). The Tukey test was selected as the statistical method of interest for use in the post-hoc analysis that was carried out. For the results to be regarded as having statistical significance, a p-value threshold of 0.05 was chosen.