(P-CB-3) A Mouse Model for Severe Thrombocytopenia Causing Spontaneous Gastrointestinal Bleeding and Worse Outcomes in Intracerebral Hemorrhage

Background/Case Studies: Sufficient numbers of functioning platelets are required for adequate hemostasis. Many medical conditions cause severe thrombocytopenia and spontaneous bleeding, often manifesting as gastrointestinal (GI) bleeding. In addition, these patients may have worse outcomes from intracerebral hemorrhage (ICH), the deadliest form of stroke. Here we present a reproducible mouse model for severe thrombocytopenia with GI bleeding and our results from transfusion and ICH experiments.

Study

Design/Methods: To generate thrombocytopenic mice, a commercially available, conditional transgenic mouse model was used. Inducible diphtheria toxin receptor (iDTR) is expressed in megakaryocytes and platelets driven by PF4-Cre (“PF4-iDTR”); diphtheria toxin (DT) administration leads to platelet depletion. PF4-iDTR mice and iDTR controls were dosed with 400 ng diphtheria toxin (DT) every other day by intraperitoneal injection for a total of 4 doses. Platelet counts were assessed by flow cytometry. For transfusions, experimental mice were transfused with 8x10^7-2x10^8 fresh platelets from transgenic mouse lines expressing either green fluorescent protein (GFP) or red fluorescent protein (RPF) on platelets. To detect GI bleeding, fecal occult blood testing (FOBT) was performed on stool collected from each mouse. To induce ICH, a stereotactic apparatus with a micro-infuser was used to infuse 0.5 µL type VII collagenase (0.5 U/animal) into the right striatum at specific coordinates. Hemorrhage volumes were monitored over time by MRI and measured using a semi-automated quantitative measurement software (MIPAV, NIH).

Results/Findings: Pre-DT treatment, platelet counts between PF4-iDTR and iDTR controls were not significantly different. Platelet counts in PF4-iDTR mice begin to fall approximately 4 days and nadir between days 5 and 7 after DT the first DT injection, with the lowest platelet counts ranging from 1-15x10³ platelets/µL. Thrombocytopenic PF4-iDTR mice had GI bleeding for 2-3 days, resolving as platelet counts began to rebound; iDTR controls never had GI bleeding. Platelet transfusions given to thrombocytopenic PF4-iDTR mice as GI bleeding was detected caused total resolution of bloody stools for most mice. Preliminary results from ICH transfusion experiments using a mixed-effects model suggest that thrombocytopenic mice that were not transfused had larger hematoma volumes after ICH induction (P=0.004) and higher mortality compared to controls (P=0.02, Figure. 1). Survival of transfused thrombocytopenic mice was not significantly different from controls (P=0.3, Figure. 1). Platelet count was inversely correlated with hemorrhage size 1 hour after ICH (P=0.008).

Conclusions: Taken together, we present a reproducible mouse model for severe thrombocytopenia that causes spontaneous GI bleeding and worse outcomes after ICH. Platelet transfusion ameliorated GI bleeding reduced mortality after ICH for thrombocytopenic mice.