Td 80394

Twelve male CD1 mice (3 wk of age) were made Fe deficient through use of a low-iron diet of ∼3-mg Fe/Kg diet based on the modified AIN-76A purified rodent diet (24 (link)) (TD.80396; Harlan Teklad) for 3 wk (i.e., until they were 6 wk of age). Four mice were also placed on a normal iron-sufficient diet (48-mg Fe/Kg diet) (TD.80394; Harlan Teklad) to serve as control. The diets were of identical composition except that the iron-sufficient diet contained iron added as ferric citrate. After this, blood was withdrawn from the tails to determine the initial Hb concentrations of the mice. The Fe-deficient mice were then divided into 3 treatment groups based on similar Hb concentrations. These 12 mice were maintained on the low-Fe diet in groups of 4, of which 1 group did not receive any iron supplementation (low-iron diet); the 2 other groups were gavaged daily with 150-μg Fe as nano Fe(III) compound or FeSO₄ for 7 d (until mice were 7 wk of age). After the 7 d, mice were weighed, anesthetized, and blood samples were taken for Hb and serum iron determinations. The mice were then killed by isoflurane anesthesia followed by neck dislocation, and the spleen, duodenum, kidney, and liver samples were excised, snap frozen in liquid nitrogen, and stored at −80°C until further analysis.

CD1 mice were used for all experiments except the hypotransferrinaemic (Hpx) mice that are of Balb/c background. Mice were placed in a hypobaric chamber at 0.5 atm for 72 h to effect hypoxia. Controls were kept at room air pressure. Dietary iron deficiency was induced by feeding 4-week old CD1 male mice with a low-iron diet (Formula TD. 80396, elemental iron concentration: 3–6 mg/kg or control diet containing 48 mg/kg iron, TD. 80394; Harlan-Teklad; Madison, WI, USA) for 3 weeks. Enhanced erythrocyte turnover was induced by intraperitoneal injections of neutralized phenylhydrazine (PHZ) at 60 mg/kg body weight; (Sigma-Aldrich, UK) on 2 consecutive days. All mice were maintained on standard commercial diet (Rodent Maintenance diet; RM1, Special Diet Services, UK) feed and water ad libitum. Mice were sacrificed after anaesthesia and neck dislocation, after which tissues were collected. Femurs were excised and flushed with Dulbecco's minimum essential medium (DMEM; Sigma-Aldrich, UK), to collect the hemopoeitic progenitor cells from the bone marrow of control and PHZ-treated CD1 mice. All procedures were approved and conducted in accordance with the UK. Animals (Scientific Procedures Act, 1986).