Rate of Red Cell Formation in Rats at 24°C and at 5OC’ N. B. EVERETT AND RUTH W. CAFFREY Department of Anatomy, University of Washington, Seattle, Washington It has been established that rats acclimatized to cold have an increased red cell volume. This increase is of the order of 20% after two or six weeks at 5°C (Everett and Matson, ’61). These observations raise the interesting fundamental question of what effect the stress of cold might have upon the turnover rate of the red cell population. Accordingly, studies were undertaken to determine the rate of erythrocyte formation and their circulating life span in rats exposed to 5°C for four to six weeks and in rats maintained at the animal room temperature of approximately 24 “C. Although a number of reports have appeared relative to the circulating life span of erythrocytes in rats under standard laboratory conditions, the results are not in very good agreement. As reviewed by Belcher and Harris (’59) the more recent estimations for the life span of the rat red cells range from 24-100 days. Most of these studies have been based upon the survival of radioisotopically labeled erythrocytes. Nevertheless, the specific methods employed have varied sufficiently to account for, at least in part, the differences in results. Two general methods have been employed for labeling erythrocytes to provide for following their percentage decrease with time. In one instance, the cells are labeled in vivo, usually with Fe5’, Ci4 or P32.In the other, the cells are labeled in vitro, commonly with Cr5’,and these are then introduced into a recipient. Counter methods have been more commonly employed for following the decrease in the activity of blood sampled at intervals for many days, and this decrease has been interpreted to be proportional to the loss of red cells from the circulation. Criticisms which have been made of these methods are the possible reutilization of the precursor substance, particularly Fe5’, and the fact that CP1 is slowly eluted from the labeled cells. Belcher and Harris (’59) employed both Cr51 and Fe5’ for labeling and have, in addition to the counter methods, employed radioautography in assessing the loss of erythrocytes labeled with Fe5’. Their studies covered a span of 75 days after labeling and showed the mean life span of red cells in the rat to be in the range of 49-55 days. Recently Everett and Yoffey (’59) employed a different approach in using Fe5’ and radioautography to determine the circulating life span of guinea pig erythrocytes. In these studies the rate at which distinctly labeled cells appeared in the peripheral blood after administering Fe5’ of high specific activity was determined. For the first seven days after iron administration there was a linear increase in the percentage of labeled cells and this rate was used to calculate the life span. This approach offered the advantage of making an accurate count of the newly-formed cells, since during this period only distinctly labeled cells were released into the circulation. In addition, the possible complication of Fe” reutilization was surmounted by limiting the time course of the studies to the first few days after radioiron injection, before there was any significant release of the label through cell destruction. In the studies reported here the method used for the guinea pig has been employed in determining the rate of erythrocyte formation in the rat. MATERIALS AND METHODS The animals used were male rats of the Sprague-Dawley strain. These were in 1 Supported by the, U. S. Air Force under Contract AF 41(657)-104,monitored by the Arctic Aeromedical Laboratory. 339 340 N. B. EVERETT AND RUTH W . C A F F R E Y two groups with respect to body weight for both controls and experimentals. One group was in the range of 150-200 gm and the other from 300-400 gm. Fourteen rats served as controls and these were kept at 24°C. The experimental animals, 17 in number, were individually caged and placed in the cold room (SOC) at a time which allowed them to reach the correct weight range by the end of the exposure period. As reported previously (Everett and Matson, '61), it was found that there was essentially no change in the weight of a rat during the first week of cold exposure and thereafter the weight gain was approximately 3.5 gm per day. Eleven rats were exposed to 5°C for four weeks, two for five weeks and four for six weeks before administering FeSY.After these periods at 5"C, rats are in a steady state with respect to total body red cell volume, since the increase in volume occurs during the first two weeks of cold exposure (Everett and Matson, '61). The Fe5' used was obtained from the Oak Ridge National Laboratories in a n acid solution as ferric chloride and ranged in specific activity from 7,000 to 23,000 mc/gm. Just prior to use the solution was buffered with a few crystals of sodium citrate and brought to pH six with sodium hydroxide. Using methylene blue as a n indicator and ascorbic acid as a reductant, the ferric ion was converted to the ferrous state. The final volume was adjusted to give a concentration of 1 mc/ml. Some of the animals were given a single intraperitoneal injection of the FeS9 solution, 1 w / g m body weight. The others received three separate injections of 0.5 w / g m at 24-hour intervals. The second and third injections were made immediately after taking blood for smears as described below. At 24 hours after each injection, and at 24-hour intervals thereafter, a small drop of blood was obtained from the tail of each rat. The blood was diluted in 0.2 cm' of homologous serum and thin smears were made on glass slides. After fixation in methanol, radioautographs were prepared using melted Eastman Kodak NTB3 emulsion as described previously (Everett et nl., '60). Subsequent to ex- posure for ten days and after photographic processing, the slides were stained with a Leishman Giemsa solution or with McNeal's tetrachrome. Approximately two thousand cells were counted i n the radioautographs of each blood sample in determining the percentage of labeled cells. Radioautographs were made of bone marrow smears from three rats at one-half hour, one hour and two hours respectively after FeS9 administration. The observed extensive labeling of the various erythrocyte precursors, including bone marrow reticulocytes, provided the basis for selecting the 24-hour post-injection interval for sampling the blood as described above. An additional control measure was to assess reticulocyte labeling in peripheral blood exposed to the FeS9. This was done by preparing radioautographs of blood after in uitro incubation with the FejS solution for one-half hour, one hour and two hours. RESULTS The radioautographs of the marrow smears revealed that at one hour after Fe'" administration essentially all of the erythroblasts were labeled as well as most of the marrow reticulocytes. After two hours the labeling intensity of these erythrocyte precursors had increased. These observations are in accord with those of Lamerton, Belcher and Harris ('59) who reported from 90 to 98% of the early-to-late erythroblasts to be labeled i n rat marrow at two hours after Fe59injection. I n contrast only rarely was a labeled reticulocyte observed in the peripheral blood after in vitro incubation with the FeS9 solution. I n addition, the labeling intensity was very low. Suit et al. ('57) similarly reported that in peripheral blood reticulocytes have a relatively small uptake of Fe5', but in the marrow a proportion of them show a n uptake similar i n magnitude to the late polychromatic normoblas ts. Figure 1 is representative of the radioautographs of blood from both the control and experimental animals. This figure illustrates clearly the separation that can be made between the newly-formed labeled cells and the older non-labeled erythro- 34 1 RED CELL FORMATION Fig. 1 Radioautograph of peripheral blood smear from control rat two days after FeS9 administration showing two labeled erythrocytes. x 1,200. cytes. With the dosages of iron used in these experiments, distinctly labeled cells appeared in the peripheral blood at a uniform rate in all animals for three days, figure 2. Although labeled cells continued to appear after three days, the labeling intensity in some of the preparations did not provide for reliable counts. Thus only the values for the first three days were used in calculating the rate of red cell formation and for estimating circulating life span. There were no significant differences in the percentages of lab,eled erythrocytes in animals of the two weight ranges. Accordingly, the data for the two are considered together at the respective time intervals within the control group as well as for the cold exposed animals. Additionally, all animals exposed to cold for four, five or six weeks are considered together, since there were no differences observed in their rates of red cell formation. It may be seen (fig. 2) that the rate at which newly-formed labeled cells appeared in the peripheral blood was approximately 3% of the total per day for both the control and experimental group. At three days the average percentage of labeled cells for controls was 8.86 and for experimentals the mean value was 9.38%. This slight difference in values is not statistically significant. It can be assumed that in the normal animal the rate of new red cell formation is equal to the rate of red cell destruction. Thus, since newly-formed erythrocytes daily entering the blood comprise 3% of the total, the circulating life span of the red cells would be 100/3 or approximately 33 days. DISCUSSION It is apparent that distinctly labeled erythrocytes begin to appear in the peripheral blood within a few hours after FeS9 administration and that their rate of entry into the blood is quite uniform as long as the reserve of labeled iron in marrow is sufficient to effect the labeling. Furthermore, it is apparent that the radioautographs of the peripheral blood for the first few days after FeS9administration provide for a clear-cut separation between newlyformed labeled cells and the older eryth- 342 N. B. EVERETT AND RUTH W. C A F F R E Y $2- rocytes. For these reasons it is believed that the approach employed here and previously for the guinea pig (Everett and Yoffey, '59) in assessing erythrocyte formation and circulating life span has advantages over the reciprocal method which determines the loss of labeled erythrocytes in time and begins with a high percentage of labeled cells. An additional advantage offered by the method here employed is in avoiding the problem of labeled iron reutilization which besets the longer term studies with radioiron. It is well known that a major fraction of the iron from destroyed red cells is reutilized in the synthesis of hemoglobin (Gibson et al., '47). In the studies of Bumvell, Brickley and Finch, '53; Davis, Alpen and Davis, '55; and Belcher and Harris, '59; in which the survival of Fe5$-labeled cells was determined, the difficulty of iron reutilization should have been overcome by giving repeated injections of excess non-radioactive iron after the initially labeled cells had appeared in the circulating blood. Questions still arise, however, particularly when employing counter methods only for assay, relative to some possible reutilization as well as to the extent of re-incorporation. It could be, too, as Belcher and Harris ('59) suggest that excess iron might affect the circulating red cells. It might be expected that the metabolic changes in the rat on cold acclimatization (Cottle and Carlson, '54) would be reflected in an increased turnover rate of certain cells or tissues. Although the present study provides no evidence that this might not be the case for other tissues, it appears that the circulating life span of erythrocytes is of the same order of magnitude for the cold acclimatized and control rats. The fact that the rate of red cell production in the cold acclimatized animals is increased only in proportion to the increase in red cell volume provides good evidence that the circulating life span of erythrocytes is not altered significantly by the cold exposure. It appears then that the increased and sustained red cell volume is due to an increase in erythropoiesis, supporting the greater metabolic needs induced by cold and there is neither an increase nor decrease in red cell life span. The data presented here provide no evidence relative to the specific cells of the erythropoietic series which are stimulated 343 RED CELL FORMATION by cold to account for the increase in red cell volume. The value reported here of approximately 33 days for the circulating life span of rat erythrocytes is somewhat less than the values obtained for the rat by determining the survival of Fe"-labeled cells. Burwell, Brickley and Finch ('53) found the mean life span to be 45-50 days; Davis, Alpen and Davis ('55) reported 51 days; and more recently, Belcher and Harris ('59) found the mean life span to be in the range of 49-55 days. On the other hand, most investigators who have used C P in red cell survival studies have reported values of less than 33 days for the mean life span of erythrocytes in the rat (Donohue et al., '55; Giblett et al., '56; Hall, Nash and Hall, '57; and Rudnick and Hollingsworth, '59). It is to be noted, however, that in the studies of Belcher and Harris ('59) using Cr5' the results were in good agreement with their results obtained with Fe5*. Furthermore, Smith, Ode1 and Caldwell ('59) found the survival of rat erythrocytes to be 65 days using Cr"-labeling and differential agglutination simultaneously. A mean survival time of approximately 60 days was found for rat red cells labeled with Ct4 (Fryers and Berlin, '52; Berlin, Van Dyke and Lotz, '53). A similar value was obtained by Van Putten ('58) from in. viuo labeling with DFP". It may be that the differences in reported values for the circulating life span of rat erythrocytes relate in part to the differences in the strains of animals studied. More likely, however, the disagreements in results of the various investigators are due in large measure to the differences in methods. Elution of the Crsi label from the cells is a complication of this method and reutilization of radioiron needs to be considered in studying the survival of Fe5'labeled cells. It is to be appreciated that without adequate corrections or precautions the Cr51 method would result in an underestimation of survival time whereas the Fe5' method would result in an overestimation. Another consideration to be made for the longer term studies in rats which has been too frequently overlooked is the continued growth of the animal and thus the increase in blood volume. Fur- thermore, the estimation of mean life span of cells by measuring cell loss is complicated by the need to consider random destruction versus senescence mechanisms in removing erythrocytes from the circulation (Belcher and Harris, '59). It is believed that the methods employed here for determining the rate of erythrocyte formation and for estimating circulating life span surmount many of the difficulties associated with the long-term survival studies. Finally, it is to be emphasized that the ability to observe and count the distinctly labeled cells after Fe5' administration provides for a meaningful and assuring interpretation of the hemodynamics of erythropoiesis. SUMMARY Radioautography of peripheral blood subsequent to Fe" administration was employed to determine the rate of erythrocyte formation in male Sprague-Dawley rats maintained at 24°C and after exposure to 5°C for four, five, and six weeks. The rate at which newly-formed labeled cells appeared in the blood was approximately 3% per day of the total circulating erythrocyte population for both control and experimental animals. This rate was the same for animals of two body weight ranges, 150-200 gm and 300-400 gm. Assuming that the rate of new red cell formation is equal to the rate of red cell destruction, the circulating life span of the rat erythrocytes is estimated to be approximately 33 days. LITERATURE CITED Belcher, E. H., and E. B. 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Ellis 1957 Studies on the Fe"9 uptake by normoblasts and the failure of X-irradiation to affect uptake. Brit. J. Haemat., 3: 165. Van Putten, L. M. 1958 The life span of red cells in the rat and the mouse as determined by labeling with DFP32 in vivo. Blood, 13: 789-793.