A Novel Chemically Induced Fischer F344 Rat Model for Thrombotic Sequelae of Hemolytic Anemias.

In hemolytic diseases like sickle cell disease (SCD) and thalassemia, the mechanisms of thrombosis are poorly understood. Appropriate animal models would help increase our understanding of the pathophysiology of thrombosis, and in the development of specific disease modifying agents. We recently demonstrated that gavage exposure to a toxin, 2-butoxyethanol (2-BE), in Fischer F344 rats leads to the development of a unique chemically induced rat model of hemolytic anemia and microvascular thrombi. Using in vitro red blood cell (RBC) flow analysis of aggregability, adherence and deformability, we demonstrated that microthrombi formation in rats exposed to 2-BE was predominantly due to enhanced RBC adherence, possibly secondary to the recruitment of cellular adhesion molecules at the endothelial cell (EC)-RBC interface (Arch Toxicol. 2003,77(8):465–69). To validate our previous findings, and to further elucidate the mechanisms of thrombosis, we exposed Fischer F344 rats to 250mg/kg of 2-BE for up to 4 days. The exposure and laboratory outcome analysis was performed on 8 groups (5 animals/group) of rats, 4 control (treated with vehicle carrier) and 4 treatment groups. One control and one treatment group were terminated each day, after the 1^st, 2^nd, 3^rd, and 4^th daily dose. Blood collection for laboratory testing was performed on each day prior to euthanasia. Multiple rat tissue sites were evaluated for histopathologic evidence of thrombosis and infarction. In addition, ocular expression of vascular cell adhesion molecule-1 (VCAM-1), endothelial intercellular adhesion molecule-1 (ICAM-1), and P-selectin was determined using immunohistochemical testing. Consistent with our previous findings, all 2-BE exposed rats developed acute hemolysis as evidenced by anisopoikilocytosis, fragmented RBCs, an increased reticulocyte count, and a > 50% drop in hemoglobin (Hgb) after the first dose. The mean pre- and post-2-BE exposure Hgb was 15.0 gm/dl (range: 14.7–15.3 gm/dl) and 5.4 gm/dl (range: 3.5–10.3 gm/dl) respectively (p < 0.01; Mann-Whitney). In addition, gross and histopathologic evidence of microvascular thrombosis in multiple organs including brain, lungs, and bone were identified by day 4, in the rats treated with 2-BE. In contrast, serial platelet counts, prothrombin times, activated plasma thromboplastin times, fibrinogen, antithrombin-III levels, and D-dimers, were unchanged between the treated and control rats. Tissue expression of VCAM-1 correlated strongly with the presence and extent of thrombosis, but no change in the expression of ICAM-1 and P-selectin was seen. Hence, from our studies, it is suggested that the RBC/EC interaction appears to be a more potent catalyst for thrombosis seen in hemolytic disorders, in contrast to abnormalities involving platelets or coagulation factors. This is consistent with the observations of increased RBC/EC interaction, and increased VCAM-1 expression observed in human studies during acute sickle cell crises, and provides an explanation for the relative failure of anticoagulant/antiplatelet strategies during acute thrombotic episodes in SCD. Thus, with further characterization, our chemically induced rat model may serve as an ideal model to characterize thrombotic sequelae of SCD and thalassemia. Future studies using our model may also lead to the development of unique therapeutic strategies in SCD, aimed at decreasing the RBC/EC interaction (e.g. targeting VCAM-1), and ultimately thrombosis.