Liver expression of hepcidin and other iron genes in two mouse models of beta-thalassemia

Background and Objectives. Homozygous b-thalassemia patients may develop iron overload even if untransfused, due to inappropriately high intestinal iron absorption. Reduction of hepcidin synthesis has been reported both in patients and in animal models. We have measured liver hepcidin and other iron gene transcripts in two different mouse models of b-thalassemia at different ages. Design and Methods. Mice Hbbth/th, characterized by spontaneous homozygous deletion of the major b1 globin gene were studied at 2 and 8 months. Mice Hbbth3/+, characterized by the heterozygous deletion of b1 and b2 globin genes were studied at 4 and 10 months. Hematologic data were obtained and iron overload estimated by Perls\u2019 staining of the liver. Expression of liver hepcidin, Tfr2, Hjv, Fpn and Hfe RNA was assessed by real-time polymerase chain reaction. Levels of serum cytokines (interleukin-6, IL-1b, IL-10, granulocyte-macrophage colony-stimulating factor) levels were assayed by enzyme-linked immunosorbent assay. Results. Hemoglobin, hematocrit and mean corpuscolar volume were significantly reduced in both b-thalassemia models, more significantly in Hbbth3/+, which have the greater, age-dependent, iron overload. Hepcidin RNA was not increased despite iron overload in both strains. Fpn RNA was increased and Tfr2 was decreased in older animals. Inflammatory cytokine levels were striking variable and unrelated to hepcidin levels. Interpretation and Conclusions. Although anemia is reported to inhibit hepcidin expression, normal hepcidin synthesis was maintained in both thalassemic models studied. However, hepcidin levels were inappropriate for the body iron, especially in Hbbth3/+ 10- month-old animals. As we previously reported in wild type mice after parenteral iron overload, Tfr2 is reduced and Fpn RNA increased in thalassemic mice. Inflammatory cytokines did not play a major role in increasing hepcidin levels or in modifying iron homeostasis in this stud

Optimization, refinement and reduction of murine in vivo experiments to assess therapeutic approaches for hemophilia A.

The tail cut bleeding model (CUT) is routinely used in factor VIII-deficient mice to assess pharmacodynamic effects of therapeutic strategies for haemophilia A. Results from this model are highly variable, many modifications to the model are reported and at times the animals' wellbeing may be compromised by recording survival as an endpoint. We therefore investigated if the ferric chloride carotid occlusion model (COM) used for thrombosis research can be applied to enhance data quality and animal welfare in haemophilia A research. Relative dose effects and relative dose variations were calculated for the CUT and COM. The requisite sample sizes were estimated and the importance of survival rates to assess rebleeds during recovery was evaluated by correlating initial blood loss to mortality. Relative dose effects increased with higher doses in both models. The COM was more sensitive at lower doses than the CUT, had up to 82% less variation across doses and clearly showed superior accuracy. Only 5% of the sample size required for the CUT would be needed to establish non-inferiority between a specific therapeutic dose in haemophilia A mice and healthy wild-type animals. A strong statistically significant correlation was found between initial blood loss and mortality within 24 h. Our findings clearly suggest that the COM is a valid tool for assessing haemophilia A treatment in vivo. The highly reproducible data means that significantly fewer animals are required and a more humane endpoint can be used by directly assessing clot stability instead of survival rate