Severe postpartum haemorrhage model
We designed the severe PPH model based on the previous model21 and the actual clinical treatment after delivery. After lower midline laparotomy, we performed caesarean section and delivered foetuses in the right side of the rabbit bicornate uterus (Figure 1A). Thereafter, uncontrolled haemorrhage was induced by transecting the right uterine artery (Figure 1B). The bleeding was wiped off using a high-absorbent gauze (BEMCOT M-1, Asahikasei, Tokyo, Japan), and we counted the absorbed blood volume every 5 min. To compensate the blood loss, an isovolemic infusion of 6% HES (Voluven 6%; Fresenius Kabi Deutschland, Bad Homburg, Germany) was repeated every 5 min via the femoral vein until 30 min or the bleeding volume reached 100 mL. The reason for setting the bleeding volume at 100 mL is as follows. The need for blood transfusion was determined when the shock index (heart rate/systolic blood pressure) exceeded 1.5 based on a previous study, with a shock index of 1.5 corresponding to a blood loss of approximately 2.5 L25. In pregnant women weighing 60 kg, the circulating blood is approximately 4.6 L, and thus, the blood loss rate in the severe PPH is estimated at approximately 54% of circulating blood. According to the guidelines of the European Federation of Pharmaceutical Industries Associations and the European Centre for the Validation of Alternative Methods26, rabbit circulating blood is estimated at 56 mL/kg. Considering that the pregnant rabbits weighed about 3.5 kg, the bleeding volume corresponding to severe PPH was calculated at 106 mL (3.5 kg×56 mL/kg×0.54). Thus, we set the bleeding volume of severe PPH at 100 mL.