SUMMARYDiminished erythrocyte count and erythropoiesis have been reported during hypothermia in some ectothermic animals. In this study, the African clawed frog, Xenopus laevis, was used to investigate the cause of hypothermia-induced anemia. We developed a new model of hypothermia at 5°C and monitored blood cell count and erythropoiesis on several days. Erythrocyte count declined by 30% on the first day following cold exposure (5°C) and mRNA expression of hemeoxygenase-1 was enhanced 10-fold; accumulation of iron as a result of heme degradation was observed in the liver. One day after low-temperature exposure, erythropoietin mRNA expression was elevated in the liver and lung compared with that at normal temperature (22°C) by qRT-PCR analysis. Examination of liver sections (i.e. the erythropoietic organ) showed an increase in o-dianisidine-positive erythrocytes in the hepatic sinusoid 5days after the onset of low-temperature exposure compared with normal liver. Peripheral erythrocyte count remained low, indicating that newly produced erythrocytes did not migrate from the liver to the circulation during hypothermia. In conclusion, this study reveals hypothermic anemia as being associated with hepatic erythrocyte destruction; prolonged anemia during low-temperature exposure is concomitant with newly produced erythrocytes being confined to the liver and may lead to new insights into vertebrate hematopoiesis.
SUMMARYHematopoietic responses to environmental factors are not fully characterized. Polycythemia has been reported during exposure to low temperatures in ectothermic animals. The relationship between the causes of polycythemia and erythropoiesis during low temperature exposure is not fully understood. In this study, we exposed C57BL/6 mice to 5°C and monitored the blood cell counts and erythropoiesis. The hematocrit level increased from 45.6 to 52.2% after 14days. Likewise, the hemoglobin concentration, initially 15.1gdl . The reticulocyte production index significantly increased from 4 to 8% after 7days. We examined the anatomy and cell composition of the spleens of the mice. On day5, the spleens were ~6mgg −1 of body mass, which was twofold greater than the spleens on day0. Flow cytometry showed fourfold more proerythroblasts on day5, compared with day0. Additionally, the number of late-stage mature erythroblasts increased on day14. Erythropoietin mRNA levels increased in the kidneys, and hypoxia-inducible genes were enhanced in the kidney. Our findings indicated that low ambient temperature is a novel erythropoietic stress, which induces polycythemia by enhanced erythropoiesis.
To survive, organisms must adapt to changes in the ambient environment. Here, we describe a new model of anemia based on exposure of African clawed frog, Xenopus laevis to low-temperature. Frogs exposed at low-temperature (5ºC) for five days had decreased numbers of peripheral blood erythrocytes, leukocytes, and thrombocytes as well as low hemoglobin levels. By contrast, spleen erythrocytes increased in number. Cell counts returned to normal in frogs re-warmed at ambient temperature (22ºC) for two days. To confirm these observations in vivo, we labeled peripheral blood cells with fluorescent reagent CFSE. During five days at 5ºC, labeled erythrocytes in peripheral blood decreased in number while those in spleen increased. When the temperature was raised to 22ºC, however, their numbers increased in peripheral blood. The findings suggested that exposure to low-temperature resulted in splenic pooling of peripheral erythrocytes. Accordingly, we looked at recovery from anemia induced by phenylhydrazine (PHZ) in this model. PHZ-treated frogs maintained at 22ºC decreased numbers of peripheral erythrocytes that were minimal on day 8, and increased gradually thereafter. In the liver, we found erythrocyte progenitors expressing erythropoietin receptor and GATA1-A detected by reverse transcription polymerase chain reactions and immunocytochemical staining but no mature forms. In PHZ-treated frogs exposed to 5ºC, peripheral erythrocyte counts remained minimal from day 8, and reversibly recovered when temperature returned to 22ºC. Erythrocyte progenitors were present in liver on day 8 but absent on day 12. Conversely, mature erythrocytes were absent in liver on day 8 but present on day 12. Finally, to learn whether the progenitors proliferate and differentiate without migrating from liver to peripheral blood, we treated frogs with thymidine analog bromodeoxyuridine (BrdU). In frogs kept at 22 ºC, BrdU-labeled erythrocytes were abundant in both liver and peripheral blood. However, frogs cooled at 5ºC had labeled cells in liver but few in peripheral blood. The findings suggest low-temperature exposure cause this anemia by impairing migration of mature/immature erythrocytes from the liver. In summary, this amphibian model offers a new perspective for investigating physiological effects of environmental temperature on vertebrate erythropoiesis.
4602 Erythropoietic stress such as hypoxia has been well described in mammals. These conditions decrease oxygen supply and then enhance production of erythropoietin (EPO) regulates production of red blood cells. Moreover, spleen becomes the main organ of erythropoiesis due to limited marrow space. Here, we describe a new erythropoietic stress, ambient low-temperature. It has been reported that peripheral blood cell counts are affected by ambient low-temperature in several vertebrates. Cold-acclimated rat and chicken exhibit polycythemia (Dveci et al, J Comp Physiol, 2001; Yahav S, Poult Sci, 1997). The response is considered to increase demand of tissues for oxygen, and then enhance metabolic rate and capacity for heat production to acclimate to ambient low-temperature. However, the physiological mechanisms had not been investigated. First, we examined peripheral erythrocyte levels in C57BL/6 mice putting into 5°C ambient. Hematocrits increased from 48% to a plateau of 53% after fourteen days. Likewise, hemoglobin concentration, initially 15 g/dl, rose to 17 g/dl. Reticulocyte production index significantly increased from 4% to 8% after seven days. These data suggested that mice exposed to low-temperature enhanced production of erythrocytes, so we next examined the anatomy and cell composition of their spleens. On day 5, spleens were about 6 mg/g of body weight, two-fold those on day 0. They gradually decreased to their initial weights on day 14. Flow cytometry showed 38% more Ter119+ splenocytes and four-fold more CD71 high Ter119+ early erythroblasts than normal. These values also gradually declined to their initial numbers by day 14. The results suggested the elevated red blood cell counts were due to an increase in production. To test erythropoietic activity in serum, we used an erythrocyte colony-forming assay. Serum from mice kept at low-temperature showed no ability to stimulate CFU-E colony formation in vitro. However, a combination of the serum with EPO (0.5 U/ml) increased CFU-E numbers 1.5 to 2 times that of a combination of normal serum plus EPO or EPO alone. Whether inducible factors account for these effects is the focus of future investigation. Our findings suggest a low-temperature environment is an erythropoietic stress that may offer insights into the adaptive physiology of red blood cell production in mice. Disclosures: No relevant conflicts of interest to declare.
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