Polycythemic Mice Research Articles

ERYTHROPOIETIC EFFECT OF TESTOSTERONE IN THE POLYCYTHEMIC MOUSE.

SummaryTestosterone has been demonstrated to stimulate erythropoiesis in the polycythemic mouse. A response can be observed following a single injection of 0.5 mg of testosterone. One mg is as effective as larger single doses. A greater erythropoietic response was observed with 10 daily injections. Possible mechanisms of action of testosterone on erythropoiesis are discussed.

Pluripotent stem cell function of the mouse marrow “lymphocyte.”

Bone marrow from normal and polycythemic mice was filtered through glasswool columns to remove cells other than lymphocytes. For a given number of nucleated cells, filtered marrow was more efficient than the original marrow in repopulating the spleen of an isogenic recipient previously exposed to lethal irradiation. The proliferative capacity of both the filtered and unfiltered marrow suspensions appeared to be a constant function of the number of small and medium lymphocytes present and not of any other cell type.

RESPONSE OF THE POLYCYTHEMIC MOUSE AND POLYCYTHEMIC GUINEA PIG TO HUMAN ERYTHROPOIETIN.

Fe59 RBC incorporation was increased in the hypertransfused guinea pig following administration of an extract of urine from a patient with chloramphenicol-induced hypoplastic anemia. This extract also had erythropoietic activity in the polycythemic mouse. Maximum Fe59 incorporation into erythrocytes of guinea pigs occurred when the Fe59 C13 was given 48 hours after administration of the erythropoietically active human urine extract.

IN VITRO STUDIES OF CELL TYPES RESPONSIVE TO ERYTHROPOIETIN.

Bone marrow cells derived from rats that had been exposed to X rays, treated with phenylhydrazine to induce anemia, or starved; peripheral white and red cells from rats; and spleen cells from normal and polycythemic mice have been tested for the effect of erythropoietin on glucosamine-1-14C incorporation. Only in hemopoietic cells was this uptake stimulated by the hormone. The relative proportion of normoblasts in the marrow was shown not to affect the response to erythropoietin; possibilities for the nature of erythropoietin target cells are discussed. (Endocrinology 74: 968, 1964)

Pluripotent Stem Cell Function of the Mouse Marrow "Lymphocyte"

Bone marrow from normal and polycythemic mice was filtered through glasswool columns to remove cells other than "lymphocytes." For a given number of nucleated cells, filtered marrow was more efficient than the original marrow in repopulating the spleen of an isogenic recipient previously exposed to lethal irradiation. The proliferative capacity of both the filtered and unfiltered marrow suspensions appeared to be a constant function of the number of small and medium "lymphocytes" present and not of any other cell type.

Hematopoiesis and Cerebellar Hemangioblastoma

ABSTRACT To the Editor:— The data of Race et al in the Jan 11 issue of The Journal (187:150) may be interpreted differently. The occurrence of reticulocytosis after injection of tumor fluid into two normal rabbits is not necessarily evidence of a true erythropoietic effect,1 but rather may reflect the development of a hemolytic state in those animals because the foreign protein extract contained heterologous antibodies to their red blood cells (RBC). It is unfortunate that no information on the RBC count, or hemoglobin or hematocrit levels was given to permit interpretation of the reticulocyte data. In addition, normal animals are poor subjects for the demonstration of erythropoietin2 because of their insensitivity to administered hormone. Starved or polycythemic mice would have been better test animals for assay of any hormonal activity.3 In view of the reported thrombocytosis which developed in the rabbits, it would have been of

The Use of Mice for the Assay of Erythropoietin in Sheep Plasmas

In a series of preliminary observations in which sheep and rabbit recipients were used, it was found impossible to demonstrate conclusively the presence of Erythropoietin (.ESF) in the plasma of sheep rendered anaemic by bleeding or by injection of phenylhydrazine. Both anaemic and normal plasma produced a fall in the recipients’ red cell count, and this could have produced the reticulocytosis which invariably followed. However, mice were used successfully to demonstrate ESF in anaemic sheep plasma, and the results obtained following the use of 2 simple assay techniques on a variety of plasmas and plasma extracts are described. A polycythaemic mouse assay gave encouraging results, but was a little cumbersome. An assay based on the production of a reticulocytosis in intact mice was simpler but the interpretation of results depended to a large extent on the control of variance in the colonies of mice used. Extracts producing a good measure of response to the above tests were injected into mice daily for 21 days to investigate their erythrocyte-stimulating ability. A concentrated extract of anaemic-by-phenylhydrazine sheep plasma produced a polycythaemia in mice without altering the mean corpuscular volume or mean corpuscular haemoglobin concentration.

ERYTHROID HOMEOSTASIS IN NORMAL AND GENETICALLY ANEMIC MICE: REACTION TO INDUCED POLYCYTHEMIA.

After hypertransfusion, normal ww and anemic WW(T) mice show the same increase in red-cell volume, decrease in reticulocytes, and temporary cessation of blood formation. The time required for the red cell volume to return to the value observed before treatment is the same for both groucps of inice. The cell volume at which new erythrocytes are again released into the circulation depends upon the genotype of functioning blood-forming tissue. Injections of erythropoietin stimulate red cell formation in polycythemic ww mice, but have much less (if any) effect upon polycythemic WW(T) mice.

ANGIOTENSIN II AND ERYTHROPOIESIS.

1. Polycythemic mice and hypophysectomized rats were used as assay animals to measure the erythropoietic stimulating activity of synthetic angiotensin II (A-S) and of angiotensin II made from a crude extract of the hog kidney (A-CE). 2. No increase in erythropoietic activity, as measured by the red blood cell incorporation of Fe59, was found following injection of these substances. The assay animals were responsive to a concentrated sheep plasma erythropoietin. 3. These results suggest that the reninangiotensin system does not encompass erythropoietin but do not rule out the possibility that a second material is released by the juxtaglomerular cells.

Factors Stimulating Erythropoiesis in Frogs

Abstract 1. Sublethal levels of hypoxia do not stimulate erythropoiesis in the frog as measured by the incorporation of thymidine-2-C14 into peripheral erythrocytes, whereas bleeding of approximately one-third of the blood volume increases erythropoiesis 70-fold as judged by this criterion. This is taken as evidence that for the frog, the fundamental stimulus for erythropoiesis may not be hypoxia. 2. Cobaltous ion administration does not increase erythropoiesis in the frog. 3. The serum of bled frogs increases the incorporation of thymidine-2-C14 into the erythrocytes of frogs into which it is injected. This serum does not stimulate erythropoiesis in the polycythemic mouse. Serum from an anemic patient which contains large amounts of erythropoietin as measured in the polycythemic mouse does not stimulate erythropoiesis in the frog.

Immunologic Studies on the Mechanism of Action of Erythropoietin.

SummaryInjections into normal mice of serum obtained from rabbits previously immunized with human urinary erythropoietin results in a progressive daily decrease in the relative numbers of erythroid cells in the bone marrow. Injections of this serum into polycythemic mice 24 to 44 hours after initiation of a wave of erythropoiesis with erythropoietin has no effect on the magnitude of the erythropoietic response, although injections of the serum one hour before or at the same time that exogenous erythropoietin is administered completely abolish the erythropoietic response. These results support the concepts that erythropoietin is involved in normal regulation of red cell production and that this regulation is the result of the effect of erythropoietin on the differentiation of stem cells. Some speculative interpretations of the effect of injections of the immune serum in polycythemic mice during the first 18 hours after injection of exogenous erythropoietin are also presented.

Response to Erythropoietin.

Alpen and Cranmore (1) and Erslev (2) have concluded that erythropoietin increases red cell output by accelerating the rate of stem cell differentiation into erythroid precursors. Observations on erythropoiesis in polycythemic mice by Gurney, et al.(3) have supported this conclusion. However, observations by several investigators (4-7) seem to indicate that the action of the erythropoietic humoral factor (s) is not limited to regulation of stem cell differentiation. Experiments reported here tested the effect of erythropoietin on later phases of erythroid formation and the results suggest an action here as well as in the early stem cell phase. Methods. Female Sprague-Dawley rats, weighing 180 to 200 g, were made polycythemic by one or several transfusions of packed homologous red blood cells. Each transfusion equaled 50% of the animal's original red cell mass. At various intervals following the initial transfusion the polycythemic rats were given a single injection of human urine erythropoietin (8) or purified sheep erythropoietin (9). Subsequent changes in erythropoiesis were assessed by reticulocyte counts (10) and measurement of the 18-hour erythrocyte radio-iron incorporation(8). Erythroid elements per 1000 nucleated cells were enumerated in smears of marrow from control polycythemic animals. In 3 experiments, polycythemic rats were given single intraperitoneal injections of 2 ml of urine concentrate on either the 5th, 15th or 31st day after their first transfusion. Control polycythemic rats were injected with saline. At the end of each successive 24-hour period following erythropoietin or saline injection, representative animals from each group were killed and the reticulocyte counts, hematocrits and erythrocyte radio-iron incorporation were determined. The radio-iron was injected 18 hours before the rats were killed. All groups given urine concentrate included at least 5 rats.

Erythropoietin assays using iron59 incorporation into blood and spleen of the polycythemic mouse.

SummaryIncorporation of Fe59 with blood and spleen of transfused polycythemic mice at various intervals has been used to find sensitive assay systems for erythropoietin. Transfusion polycythemia markedly reduces erythropoiesis as judged by low levels of Fe59 uptake into the spleen and into the blood after 16 hours. In polycythemic mice receiving injections of erythropoietin, incorporation of Fe59 is maximal in the spleen at 8 hours and in the blood at 48 hours after injection of the isotope. Incorporation of Fe59 into the blood of the polycythemic mouse 48 hours after giving the isotope was found to be the most sensitive assay when compared to the starved rat assay and other polycythemic mouse assays.

Immunochemical studies of human urinary erythropoietin.

SummaryAn immunochemical study of human urinary erythropoietin has been presented. The experiments indicate that the erythropoietic activity as assayed in the polycythemic mouse of erythropoietically active human urinary concentrates can be neutralized in vitro by addition of sera obtained from rabbits previously immunized with such concentrates. A significant depression of erythropoiesis was observed after injection of such sera into normal mice. It is concluded that neutralization is the result of an antigen-antibody reaction.

Studies on Erythropoiesis. XVII. Some Quantitative Aspects of the Erythropoietic Response to Erythropoietin

Abstract The transfusion-induced polycythemic mouse is an excellent subject in which to study the physiology of erythropoiesis because endogenous erythropoiesis is reduced in a manner that does not appear to impair, to any recognizable degree, the animal’s ability to respond to an erythropoietic stimulus. Using a purified glycoprotein, erythropoietin, obtained from the plasma of anemic sheep, a number of experiments was carried out on polycythemic mice. Morphological evidence of erythropoiesis in the spleen was observed following a single subcutaneous injection of erythropoietin. A cumulative response to multiple injections was noted. For a given total dose of erythropoietin, a greater response was produced when this amount was fractioned than when it was administered as a single dose. Animals made polycythemic by transfusion were maintained in a polycythemic state by daily injections of erythropoietin. Finally, a true erythrocytosis was produced by a prolonged course of injections of erythropoietin in normal mice.

Studies on Erythropoiesis. XII. The Effect of Transfusion-Induced Polycythemia in the Mother on the Fetus

Abstract Erythropoiesis is reduced to zero in the mouse by repeated homologous red cell transfusion. Mating of transfusion-induced polycythemic mice was allowed, and observations were made on maternal and fetal erythropoiesis. It was observed that erythropoiesis in the pregnant, polycythemic mouse was suppressed throughout pregnancy, this observation suggesting that erythropoietin production was concomitantly suppressed. Erythropoiesis in the fetuses of polycythemic mothers was initiated by 10 days and maintained in an active state throughout fetal life. Hematologic values of these fetuses at birth were higher than those of the newborn normal pregnant mice of the same strain. It is possible that the fetus initiates and maintains erythropoietin independently of erythropoietin from the maternal circulation. The higher hematologic values of the newborn of polycythemic mothers are probably the result of the greater availability of iron from the breakdown of transfused cells, since it was found that comparable hematologic values were achieved in the newborn when iron-dextran (Imferon) was injected into the maternal tissues during pregnancy.

Studies on Erythropoiesis. XI. Reticulocyte Response of Transfusion-Induced Polycythemic Mice to Anemic Plasma from Nephrectomized Mice and to Plasma from Nephrectomized Rats Exposed to Low Oxygen

Abstract Mice with transfusion-induced polycythemia have been used to assay erythropoietic activity in plasma derived from mice and rats subjected to various stimuli and experimental procedures. 1. Anemic, anoxic, or cobalt mouse plasma increased erythropoiesis from the zero baseline to about the normal range after four 0.5 cc. injections. No significant effect is observed from the same number of injections of normal rat or mouse plasma or normal saline. Similarly, plasma from rats starved for 4 days had no erythropoietic activity, but erythropoietic activity was present in the plasma of pregnant mice during the last trimester of pregnancy. 2. Plasma from mice made anemic by phenylhydrazine and then subjected to bilateral nephrectomy contained slight erythropoietic activity (less than 0.2 per cent reticulocytes). The plasma of sham-operated anemic animals had considerably more erythropoietic activity (2.0 per cent). The plasma was collected for assay 10 hours after the operative procedures. 3. Plasma from nephrectomized rats exposed to hypoxic anoxia for 8, 16, or 24 hours, had slight erythropoietic activity when measured by the peripheral reticulocyte response of the recipients (0.18, 0.11, and 0.00 per cent reticulocutes, respectively). The same plasma had no erythropoietic activity when judged by the Fe59 red cell incorporation response in starved rats. Plasma from rats subjected to bilateral ureter ligation and similarly exposed had an erythropoietic activity (0.7, 1.38, and 1.26 per cent reticulocytes, respectively), comparable with that of the plasma from normal rats exposed to hypoxic anoxia (0.9, 1.71, and 1.78 per cent, respectively). The low and inconstant erythropoietic activity in the plasma of nephrectomized mice and rats subjected to anemic or hypoxic anoxia is more or less comparable with the response occasionally produced by normal plasma. We are, therefore, disinclined to consider the data as evidence against the renal origin of erythropoietins, but the alternative possibilities are discussed.

Studies on erythropoiesis. IV. Reticulocyte response of hypophysectomized and polycythemic rodents to erythropoietin.

Summary and Conclusions1. Following hypophysectomy of rats, reticulocytes in the peripheral blood fall to a minimal level within a period of 3 to 4 weeks. This reduction represents a 5- to 10-fold decrease from the prehypophysectomy control. This reticulocyte decline is more rapid in 8-week-old rats than in younger animals. 2. Administration of anemic plasma to hypophysectomized rats produces a reticulocyte rise that is many fold greater than that produced by normal plasma or saline. 3. Erythropoiesis, as measured by the number of reticulocytes in peripheral blood, falls to zero in 6 days in mice made polycythemic by repeated intraperitoneal injections of homologous red cells. Anemic plasma obtained from rats or rabbits, when injected intravenously or intra-peritoneally into polycythemic mice, produces a reticulocytosis many times greater than that observed from normal plasma. Normal saline produces no increase in reticulocytes. 4. In polycythemic mice and hypophysectomized rats, reticulocyte determinatio...

Erythrocyte life span in small animals; comparison of two methods employing radioiron.

Erythrocyte life span in small animals; comparison of two methods employing radioiron.

Direct determinations of plasma, cell, and organ-blood volumes in normal and hypervolemic mice.

SummaryDirect single and combined cell- and plasma-volume determinations were made on normal and polycythemic mice with 32P-tagged red cells and 131I-tagged plasma. The data indicate that: (a) The Evans-blue plasma volumes in mice are above the probably more correct 131I-plasma volumes by a factor of 1.2 and in rabbits by a factor of 1.08. (b) The so-called mixing time of Evans blue is a withdrawal time of this dye by Evans blue-affin cells, most of which are probably macrophages. (c) The cell volume calculations based on Evans-blue values are higher than those obtained by direct determinations with the 32P technic by a factor of 1.56 in normal and 1.51 in tumor-bearing mice tested. (d) The average body hematocrit in mice is approximately 0.88 times the venous hematocrit. (e) On the basis of these conversion values a calculation of all values can be made if only one technic is employed (Fig. 1). (f) Hypervolemia caused by estrogen-secreting tumors is attributable to an almost selective plasma-volume rise ...