Stalled Cerebral Capillary Blood Flow in Mouse Models of Essential Thrombocythemia
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1 SUPPLEMENTAL MATERIAL Stalled Cerebral Capillary Blood Flow in Mouse Models of Essential Thrombocythemia and Polycythemia Vera Revealed by In Vivo Two-Photon Imaging Thom P. Santisakultarm, Claire Q. Paduano, Tracy Stokol, Teresa L. Southard, Nozomi Nishimura, Radek C. Skoda, William L. Olbricht, Andrew I. Schafer, Richard T. Silver, Chris B. Schaffer Page 1 of 7
2 Supplemental Figures and Legends Figure S1. Leukocyte and platelet counts did not predict cortical capillary stall rate. To test whether leukocytosis or thrombocytosis influence the rate of capillary stalls in murine models of mixed MPN, ET, PV or secondary erythrocytosis, we investigated the relationship between leukocyte and platelet counts and the rate of capillary stalls. Stall rate was dependent on neither leukocytosis (A) nor thrombocytosis (B). Shaded areas represent normal leukocyte and platelet counts in C57/Bl6 mice [1]. Previous clinical studies have had mixed findings on the influence of leukocyte and platelet counts on cardiovascular events [2-5]. Page 2 of 7
3 remaining capillary stalls (% of baseline) remaining capillary stalls (% of baseline) A B 1 leukocyte plug platelet aggregate remaining capillary stalls (% of baseline) remaining capillary stalls (% of baseline) C 1 stagnant RBCs D 1 empty mixed MPN BMT ET Epo-inj BMT PV Figure S2. Capillary stalls associated with leukocyte plugs, platelet aggregates, stagnant RBCs, and empty vessels in MPN mouse models reestablished blood flow at unequal rates. Our data suggests that cortical capillary stalls in MPN mouse models are long-lived. We further scrutinized the stall survival time in stalled capillaries where (A) leukocyte plugs, (B) platelet aggregates, (C) only RBCs, or (D) only blood plasma (i.e. no blood cells) were present. Individual stalled capillaries of all causes were monitored until blood flow reestablished, or for ~2 hours, whichever was first. The proportion of persistent leukocyte plugs, platelet aggregates, RBC stalls, and empty vessels decay over time at unequal rates in BMT mixed MPN, BMT ET, Epo-inj, and BMT PV mice. Notably, in the BMT ET group, over % of the stalls caused by platelet aggregates resolved quickly in the first 1 minutes. Because platelet plugs caused about half of the stalled capillaries in BMT ET mice, this fast resolution was reflected the shorter persistence of all stall types over time (see Figure 3D). Page 3 of 7
4 Figure S3. Capillary density in ET, PV, mixed MPN, and secondary erythrocytosis mouse models. (A) Density of capillary segments shows a large variability among animals within each group but no significant difference between groups. Page 4 of 7
5 Supplemental Tables Table S1. Number of Subjects in Each Measurement Stall Stall Stall Type Diameter Speed Persistence Capillary Density wt BMT wt BMT mixed MPN BMT ET Epo-inj BMT PV Multiple types of measurements were frequently made in the same animal (e.g. rate of capillary stalling, stall type, stall persistence) Page 5 of 7
6 Supplemental References 1 Green EL. Biology of the Laboratory Mouse Tefferi A. Platelet count in essential thrombocythemia: the more the better? Blood. 28; 112: /blood Carobbio A, Thiele J, Passamonti F, Rumi E, Ruggeri M, Rodeghiero F, Randi ML, Bertozzi I, Vannucchi AM, Antonioli E, Gisslinger H, Buxhofer-Ausch V, Finazzi G, Gangat N, Tefferi A, Barbui T. Risk factors for arterial and venous thrombosis in WHOdefined essential thrombocythemia: an international study of 891 patients. Blood. 211; 117: /blood Barbui T, Carobbio A, Rambaldi A, Finazzi G. Perspectives on thrombosis in essential thrombocythemia and polycythemia vera: is leukocytosis a causative factor? Blood. 29; 114: /blood Gangat N, Strand JJ, Li C-Y, Wu W, Pardanani A, Tefferi A. Leucocytosis in polycythaemia vera predicts both inferior survival and leukaemic transformation. British Journal of Haematology. 27; 138: /j x. Page 6 of 7
7 Legends for the Video Files Video S1. 2PEF image stack from a BMT PV mouse. Capillaries with stalled blood flow are circled, with different colors indicating different causes of capillary stalls: stationary RBCs (red), platelet aggregate (green), leukocyte plug (blue), empty (white). The field of view of the image stack is 32 µm and the image frames are spaced by 1 µm along the z-axis. Page 7 of 7
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