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Summary   Introduction  Muscle hematomas are the second most common complication of hemophilia and insufficient treatment may result in serious and even life‐threatening complications. Hemophilic dogs and rats do experience spontaneous muscle bleeding, but currently, no experimental animal model is available specifically investigating spontaneous muscle bleeds in a hemophilic setting.    Aim  The objective of this study was to develop a model of spontaneous muscle bleeds in hemophilia B mice. We hypothesized that treadmill exercise would induce muscle bleeds in hemophilia B mice but not in normal non‐hemophilic mice and that treatment with recombinant factor IX ( rFIX ) before treadmill exercise could prevent the occurrence of pathology.    Methods  A total of 203 mice (123 F9‐KO and 80 C57BL/6NTac) were included in three separate studies: (i) the model implementation study investigating the bleeding pattern in hemophilia B mice after treadmill exercise; (ii) a study evaluating the pharmacokinetics of recombinant FIX ( rFIX ) in hemophilia B mice and based on these data; (iii) the treatment study, which tested therapeutic intervention with  rFIX . At termination of the treadmill studies the presence of bleeds was evaluated.    Results  Treadmill exercise resulted in a high incidence of muscle bleeds in F9‐KO mice but not in C57BL/6NTac mice. Treating hemophilia B mice with  rFIX  before treadmill exercise prevented muscle bleeds.    Conclusion  A novel model of muscle bleeds in hemophilia B mice, responsive to  rFIX , has been developed.

Novel, high incidence exercise‐induced muscle bleeding model in hemophilia B mice: rationale, development and prophylactic intervention