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

Abstract

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.

Procoagulant activity induced by vascular injury determines contribution of elevated factor VIII to thrombosis and thrombus stability in mice

“…22 17 has comparable cofactor activity as murine FVIII, 17 and promotes coagulation after tail clipping and vessel injury in hemophilic mice. 17,[24][25][26][27] In addition, previous studies 28,29 and our findings (FVIII activity assay, see "FVIII activity assay") show FVIII has sufficient half-life in murine circulation for these experiments. The endogenous FVIII concentration in mice (1 U/mL, 100%) 30 was raised by infusing human FVIII to 285% (total murine plus human FVIII) of normal, consistent with levels associated with thrombosis in humans.…”

Maintenance and break of immune tolerance against human factor VIII in a new transgenic hemophilic mouse model

“…35 At the end of the observation period of 60 minutes, mice were killed by cervical dislocation before recovery from anesthesia.…”

“…(B) Blood loss after tail cut was determined over a period of 60 minutes using the tail-cut model. 35 Presented are box plots representing the results obtained with groups of 16 mice. Compared are transgenic mice expressing the human F8 cDNA (huF8), conventional hemophilic E17 F8-knockout mice (E17 F8 ko), and wild-type mice (C57BL/6).…”

“…We used the tail-cut model 35 and determined the resulting amount of blood loss. Both conventional hemophilic E17 mice and human F8 transgenic mice of subline E expressed a hemophilic phenotype.…”

Effect of Aloe Vera (Aloe barbadensis) on Thrombosis in Mice

“…Bleeding time in mice was evaluated by the method of Dejana et al 11 to assess the bleeding time in comparison to heparin and vitamin-K. Mice were anaesthetized by sodium pentobarbital (70 mg kgG 1 , i.p.) and placed on a pad at room temperature 12 . Bleeding time in mice was measured by two ways: via filter paper method and λ max method.…”