Experimental purpura and related hematological studies in the hamster.
Desai, Rajendra G.
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Hemorrhagic disorders designated by the term "purpura" are usually associated with the appearance of petechiae, ecchymoses, and lowered platelet counts. However, the mechanism of bleeding and the etiology remain unsolved. The cheek pouch of the hamster was used in an attempt to determine the cause of the bleeding under two conditions: first, in experimental purpura produced by antiplatelet serum prepared by repeated injections of hamster platelets into rabbits, and second, following infusion with dextran. Peripheral blood counts, bone marrow examinations, coagulation tests, and special tests for vascular fragility and hemostatic function (negative pressure, snake venom, and microelectrode) have been applied to normal hamsters as a basis for interpretation in hemorrhagic diathesis. Microscopic observations were made in vivo and recorded on Kodachrome motion picture film by means of cinephotomicrographic equipment. The results of previous blood counts and bone marrow examinations have been confirmed and analyzed more critically. Detailed observations have been made on the morphology of the blood cells in the stained smear and reticulocyte counts have been reported for the first time in the hamster. The red cell count in the hamster (6.8 ± 1.2 million per cu. mm.) is slightly higher than that in man, and the reticulocyte value is also higher (2.5 ± 1.2 per cent). The polymorphonuclear and lymphocytic values were reversed in the hamster (polymorphs 30 ± 6 per cent, lymphocytes 61 ± 7.5 per cent). The peripheral blood smear showed moderate polychromatophilia and occasional target cells. The bone marrow of the hamster was more cellular than that of man, with a ratio of white cells to red cells of 8:1. The coagulation tests were within limits comparable with those for man. Three in vivo tests for vascular fragility and hemostatic function in the hamster cheek pouch were utilized: namely, the moccasin venom test (Fulton, Lutz, Shulman, and Arendt, in press); the negative pressure test (Shulman, Mode, Kagan, and Fulton, Anat. Rec. 118:408, 1954); and the microelectrode test (Fulton, Akers, and Lutz. Blood 8:140, 1953). The snake venom test applied to the cheek pouch of the normal hamster produced 71 ± 6 petechiae at one hour intervals and 138 ± 20 at the end of two hours. The negative pressure test produced from 0 to 4 petechiae at one minute Nith a negative pressure of 20 mm of mercury. The microelectrode test was used to evaluate the fragility of blood vessels by stimulation of the wall with single faradic shocks. Vasoconstriction occurred at low voltages (threshold, 4 to 10 volt single shock) in muscular arterioles and venules. The sphincters were more sensitive than other portions of the vascular network. White cell sticking and platelet thrombosis occurred at higher voltages (10 to 3O volts). The vessel wall of normal hamsters was resistant even to very high voltages (150 volts). If normal vessels were ruptured, the broken ends were sealed immediately, probably by "electrocoagulation". The results of the microelectrode test were compared in young and "old age" hamsters. No significant differences were obtained except for an increase in white cell adhesiveness in the "old age" group. Hamsters of either sex and 75 to 100 grams in weight were splenectomized. The platelet counts increased daily for 4 to 6 days after splenectomy and gradually returned to normal on the 14th to 18th day. The smear of peripheral blood exhibited increased polychromatophilia, target cells, and clumps of platelets, particularly on the 4th to 8th day. Howell-Jolly bodies were absent. Changes in the bone marrow were not remarkable. The microelectrode test for thrombus susceptibility revealed a marked tendency for thrombosis, while the fragility threshold did not differ from that in normal hamsters. Experimental thrombocytopenic purpura in hamsters, caused by injection of antiplatelet serum, produced spontaneous purpura, ecchymoses and bleeding manifestations in the cheek pouch and in almost all organs of the body. The platelet counts were decreased and the bone marrow showed immature-like amorphic megakaryocytes. Bleeding times and clot retraction determinations were abnormal. Values for the bleeding time in excess of 240 seconds v-rere not uncommon (normal bleading time, 109 ± 19). The snake venom test produced profuse numbers of petechiae within one half hour. Stimulation of the blood vessel wall with low voltage produced platelet thrombi in normal hamsters. In thrombocytopenic hamsters, thrombus formation and leukocytic sticking did not occur even with high voltages. However, the vessel walls were fragile and rupture occurred at electric thresholds lower than 150 volts (60 to 140). These changes were more marked at 24 hours after treatment with antiplatelet serum than at two hours. Small repeated injections of antiplatelet serum produced similar results. In control experiments, hamsters were injected with serum obtained from normal rabbits and with saline solution. The test procedures showed no appreciable deviations from normal values. The fragility thresholds were normal. The splenectomized hamsters with elevated platelet counts were given antipletelet serum. The fragility threshold to electrical stimulation of the vessel wall was lowered and hemorrhage occurred at low voltages. Consequently, splenectomy and the concomitant increase in numbers of circulating platelets shed no protective effect in experimental purpura. Recently, detrimental reactions to plasma substitutes have been critically evaluated and the dextrans have been reported to cause a hemostatic defect in human beings. Dextran increased the bleeding time in normal volunteers as well as in patients with shock. The exact nature of the bleeding phenomenon has not been determined. The precise techniques available for the study of hemorrhage in the hamster cheek pouch have been used to evaluate the hemostatic defect caused by dextran and to observe in vivo the changes occurring in the formed elements of the blood end in the characteristics of the vessel wall. Hematological changes in the hamster after infusion with dextran were insignificant except for the effects of hemodilution and the clumping of platelets. The results of the various coagulation tests (clotting time, prothrombin time, clot retraction, and fibrinolysis test) were within normallimits. The bleeding time was prolonged significantly as shown by values of more than 240 seconds as compared with values of 109 ± 19 seconds in normal hamsters. All dextrans used in this study (Lares, Cutter) produced an increased bleeding time except Expandex. The Lares dextran seemed to be more detrimental than other types. After injection of Lares, increased numbers of leukocytes were adherent to the endothelium at 24 hours and during a period of 3 to 4 weeks. The thrombosis threshold was increased. The formation, canalization, and embolization of thrombi were recorded on Kodachrome motion picture film. The frag ility threshold, as tested with microelectrode and also snake venom, was lowered. The vascular defect was greater with Lares than with other dextrans. In conclusion, experimental purpura was produced in hamsters by antiplatelet serwn for the first time. The resultant bleeding phenomenon and hemostatic defect was related to lowered platelet counts and decreased frag ility of the vessel wall. Splenectomy accompanied by increased platelet counts was not remedial. The effects of dextran infusions in hamsters have been critically evaluated by routine hematological tests and by special tests for vascular fragility and hemostatic function. A defect in the vessel wall was shown by the lowered fragility to faradic stimulation. This finding provided the basis for a new explanation for the hemostatic defect, namely, damage to the vessel wall. This is in addition to the previously known factors of prolonged bleeding time and unexplained clumping of the platelets.
Thesis (Ph. D.)--Boston University
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