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Rate of red cell formation in rats at 24┬░C and at 5┬░C.

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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. Harris 1959 Studies
of red cell life span i n the rat. J. Physiol., 246:
217-234.
Berlin, N. I., D. C. Van Dyke and C. Lotz 1953
Life span of the red blood cell in the hypophysectomized rat. Proc. SOC. Exp. Biol., N. Y.,
82: 287-288.
Burwell, E. L., B . A. Brickley and C. A. Finch
1953 Erythrocyte life span in small animals:
comparison of two methods employing radioiron. Amer. J. Physiol., 172: 718-724.
Cottle, W., and L. D. Carlson 1954 Adaptive
changes i n rats exposed to cold. Caloric exchange. Ibid., 178: 305-308.
Davis, W. M., E. L. Alpen and A. K. Davis 1955
Studies of radioiron utilization and erythrocyte
life span i n rats following thermal injury. J.
Clin. Invest., 34: 67-74.
Donohue, D. M., A. G. Motulsky, E. R. Giblett,
G. Pirzio-Biroli, V. Viranuvatti and C. A. Finch
344
N. B. EVERETT AND RUTH W. CAFFREY
1955 The use of chromium as a red cell tag.
Brit. J. Haemat., 1: 249-263.
Everett, N. B., and L. Matson 1961 Red cell
and plasma volumes of the rat and of tissues
during cold acclimation. J. Appl. Physiol., 16:
557-561.
Everett, N. B., and J. M. Yoffey 1959 Life of
guinea pig circulating erythrocyte and its relation to erythrocyte population of bone marrow.
Proc. SOC.Exp. Biol. and Med., 101: 318-319.
Everett, N. B., W. 0. Rieke, W. 0. Reinhardt and
J. M. Yoffey 1960 Radioisotopes in the study
of blood cell formation with special reference
to lymphopoiesis. Ciba Found. Symp. "Haemopoiesis: Cell Production and Its Regulation,"
pp. 43-69. J. and A. Churchill, London.
Fryers, G . R., and N. I. Berlin 1952 Mean red
cell life of rats exposed to reduced barometric
pressure. Amer. J. Physiol., 171: 465-470.
Giblett, E. R., A. G . Motulsky, F. Casserd, B.
Houghton and C. A. Finch 1956 Studies on
the pathogenesis of splenic anemia. Blood,
11: 1118-1131.
Gibson, J. G., J. C. Aub, R. D. Evans, W. C. Peacock, J. W. Irvine and T. Sack 1947 The
measurement of post-transfusion survival of
preserved stored human erythrocytes by means
of' two isotopes of radioactive iron. J. Clin.
Invest., 26: 704-714.
Hall, C. E., J. B. Nash and 0. Hall 1957 Erythrocyte survival and blood volume in the rat
as determined by labelling the red cells with
C F . Amer. J. Physiol., 190: 327-329.
Lamerton, L. F., E. H. Belcher and E. B. Harris
1959 Blood uptake of Fe59 i n studies of red
cell production. The Kinetics of Cellular Proliferation, pp. 301-311. Grune and Stratton, New
York.
Rudnick, P., and J. W. Hollingsworth 1959
Lifespan of rat erythrocytes parasitized by Bartonella muris. J. Infect. Dis., 104: 24-27.
Smith, L. H., T. T. Odell, Jr. and B. Caldwell
1959 Life span of rat erythrocytes as determined by Cr5I and differential agglutination
methods. Proc. SOC.Exp. Biol. and Med., 100:
29-3 1.
Suit, H. D., L. G . Lajtha, R. Oliver and F. 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.
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