Erythropoietic Response of Hypertransfused Neonatal Rats Suckled by Anemic Mothers ROBERT D. C A R M I C H A E L , ' ALBERT S. GORDON AND JOSEPH L O B U E The A . S. Gordon Laboratory of Experimental Hematology, Graduate School of A r t s and Science, Department of Biology, New York University, New York

ABSTRACT

Twelve-day-old hypertransfused neonatal rats nursed for four days by a twice-bled mother exhibited higher 48-hour RBC-"Fe incorporation than control neonates nursed by a normal mother. Erythropoietin (Ep) in plasma of 12-day-old hypertransfused neonates suckled for four days by twicebled mothers was initially equivalent to approximately 0.5 IU/day. This calculation was based on the observation t h a t reticulocytosis induced in these animals was similar to t h a t produced in neonates of the same age injected intraperitoneally with 0.5 IU Ep for four days while nursing from normal mothers. The reticulocyte maturation curve was shifted to the left in 12-day-old hypertransfused neonates suckled by anemic mothers, and in 12-day-old normal neonates rendered anemic by bleeding, while nursing from normal mothers. This left-shift of the reticulocyte maturation curve was also evident in 12-dayold hypertransfused neonates injected with Ep. Decreases in relative percentages of nucleated RBC was evident in spleens of 12-day-old neonates nursed by anemic mothers and spleens of 12-day-olds injected with Ep. Significant reduction in nucleated RBC were noted in both spleen and marrow of 12-day-old anemic neonates. These results suggest: (1) Ep, present in increased amounts in t h e anemic mother, is transmitted through milk to nursing neonates thereby stimulating erythropoiesis in these animals; (2) Ep may not stimulate stem cell differentiation towards the erythroid compartment but rather acts on already differentiated erythroid cells by influencing their r a t e of maturation.

Few studies have been made on the relations of maternal-fetal and maternal-neonatal erythropoiesis in the rodent. Jacobson e t al. ('59) observed t h a t transfusion of a pregnant mouse resulted in suppression of erythropoiesis in t h e mother but not in the fetus. Schooley e t al. ('68) reported t h a t after injection of anti-Ep into pregnant mice, erythropoiesis was significantly depressed in t h e mother and in some but not all of the fetuses. The effects of pertubation of erythropoiesis in t h e neonatal a s compared with the adult r a t are contrasting. I t has been intensively reported t h a t in t h e adult rodent anemia or hypoxia will stimulate red cell production, whereas hypertransfusion, nephrectomy and starvation suppress erythropoiesis (Lucarelli et al., '68). These effects are thought to be the result of changes in Ep production by t h e ANAT. REC. (1978)191: 227-238

kidney, which serves a s a principal regulator of erythropoiesis in t h e adult. In contrast, erythropoiesis in the newborn animal is relatively little affected by these procedures (Lucarelli e t al., '68). Exposure of rats to hypoxia does not result in a n increase in erythropoiesis until after the twenty-fourth day of life (Carmena e t al., '66; Contopoulos e t al., '56; Garcia, '57). Moreover, starvation suppresses the production of red cells in the adult but not in t h e newborn r a t (Lucarelli e t al., '68). G r a n t ('551, in experiments employing young rats and mice suckled by hypoxic mothers, reported t h a t Ep is transmitted to the newborn via maternal milk. In this regard, Received Aug. 9, '77. Accepted Dec. 9, '77. I All communicat~onsshould be directed to: Dr. Robert D. Carmichael. Department of Biological Sciences, Herbert H. Lehman College of the City University of New York. Bedford Park Boulevard West, Bronx, New York 10468. Phone (212) 960-8410.

227

228

R. D. CARMICHAEL. A. S. GORDON AND J O S E P H LOBUE

Lucarelli et al. ('64) and Stohlman et al. ('64) noted little effect on erythropoiesis in neonates suckled by mothers whose Ep production had been suppressed by transfusion or nephrectomy. Evidence now available indicates t h a t Ep is present in the milk of lactating sheep (Hyzy, '69) and in woman's milk (Bielecki e t al., '72) during the first week of lactation. The purpose of this article is to describe in greater detail: (1) the evidence for the maternal transfer of Ep to the suckling r a t via the milk, with consequent stimulation of their erythropoiesis; and, (2) the hypothesis t h a t in newborn rats, regulation of red cell production differs in several respects from t h a t observed in the adult.

MCV MCH MCHC

hematocrit (!XI x 10 erythrocytesiwl

=

=

=

hemoglobin (gX) x 10 erythrocytedpl

hemoglobin (gX) x 100. hematocrit ( X ) )

The percent of erythroid cell precursors in the bone marrow and spleen was determined from benzidine-Wrights-Giesma-stained slide preparations. These values were obtained from a differential count employing a minimum of 1,000 nucleated cells per slide. Iron utilization was studied using 59FeC13 (Abbott Laboratories, Oak Ridge, Tennessee, Sp. Act. 18 pc/mg Fe). Neonatal rats were given 0.5 p c "FeC1, intraperitoneally and sacrificed 48 hours later. Subsequent to isoMATERIALS AND METHODS tope administration blood was sampled by Long-Evans strain lactating rats (290-300 cardiac puncture, under light ether anesthegm) and their suckling offspring were used in sia, and peripheral erythrocyte 5YFeincorporathese experiments. The mother, while nursing tion measurements on 0.5 ml aliquots were their young, were fed Purina Laboratory Chow made using a well-type gamma scintillation and tap water ad libitum. The age of the neo- counter (Baird-Atomic Model 530 Spectromnates was determined by the method of eter) and expressed a s percent of injected Schribner-Seeley et al. ('71): rats 0 to 24 hours dose: bv (ml) X net cpm/ml blood were classed as 1-day-old;25 to 48 hours a s 2X 5yFeInc. = x 100 total cpm 5YFeinjected day-old, etc. Hematological determinations were made on blood in a heparinized syringe Here, total blood volume (bv, ml) used was (1-ml tuberculin syringe with a 23-gauge nee- 7.3% body weight (gm) for hypertransfused dle) drawn by cardiac puncture from neonates neonates as reported by Butturini e t al. ('71). placed under light ether anesthesia. Experiment I. Radioiron incorporation T h e hematological p a r a m e t e r s studied studies in 12-day-old hypertransfused were: numbers of circulating erythrocytes/pl neonates suckled by anemic mothers of blood, determined by t h e hemocytometer method; percent reticulocytes determined by Two groups consisting of one mother and the reticule method from smears stained with her neonates (pups) per group were estabNew Methylene Blue (Brecher, '49) employing lished and the litter size was reduced to six a minimum count of 1,000 erythrocytes; pups per mother. The pups of both groups were hematocrit; and hemoglobin concentration transfused intraperitoneally with 0.5 ml of using the cyanornethemoglobin technique. Re- whole blood from adult female rats on day 5 ticulocytes were classified according to their and 0.5 ml on day 7 of life rendering them stage of maturation a s described by Heilmeyer plethoric and thereby more sensitive to Ep ('31) and by Cantor and Gordon ('73); class 5 stimulation. The mother of group one, the ex(oldest cells), one to three blue granules; class perimental group, was bled (8-9 ml) via car4, more than three granules; class 3, granules diac puncture on day 8, and 5.0 ml on day 10 interspersed with thread-like material; class post-partum. The mother of group 2 (control) 2, loose clumps of materials; class 1 (youngest was not bled. All suckling neonates received cells), one dense clump. The data were ex- 0.5 p c 59FeC13intraperitoneally on the tenth pressed as percentages of reticulocytes per day of life and radioiron incorporation values 1,000 red cells counted. Mean corpuscular were obtained 48 hours later. Determinations volume (MCV), mean corpuscular hemoglobin were made of hematocrits, and a differential (MCH), and mean corpuscular hemoglobin count of reticulocytes to reveal their stage concentration (MCHC) were calculated a s of maturation. The radioiron incorporation follows: values of peripheral erythrocytes (previously

ERYTHROPOIETIC RESPONSE OF NEONATAL RATS

described) were determined. I n addition, radioiron incorporation values for spleen and bone marrow, expressed a s percent of injected dose, were calculated from t h e equation: (net cpmltissue) X 59FeInc. ~x 100. (mg tissue x cpm 5gFeinjected) mg tissue

O N

4'4

m 0

tI

0 0

99

0 0

Experiment ZI. Comparison o f the erythropoietic effects i n 12-day-old hypertransfused neonates nursed by anemic mothers with those in hypertransfused neonates injected with E p To compare the effects of exogenous Ep from two different sources in erythropoiesis stimulation in neonates, three groups were employed. The groups consisted of one mother and her pups per group and t h e litter size was reduced to five or six pups per mother. All neonates in each of the three groups were transfused intraperitoneally with 0.5 ml whole blood from adult female rats on day 5 and 0.5 ml on day 7 of life rendering them more sensitive to Ep stimulation. The neonates in group 1were injected intraperitoneally once per day with 0.5 IU Ep for four days beginning on t h e eighth day of life through day 11. The mother of this group was not treated. The mother of group 2 was bled (8-9 ml) via cardiac puncture on day 8 and 5.0 ml on day 10 of neonatal life. Group 3 served as t h e control and the mother of this group was not bled. All suckling pups in each of t h e three groups were sacrificed on t h e twelfth day of life and the erythropoietic parameters as previously described were examined to assess their erythropoiesis.

tl

zz

N 0 0 3

tl

W d

To!

m rN

tI

mIn

'oc

N m(D 3

tl mu3

2: 3

tI

-

d

99 3 3

tl

m o

22 d

+I m d

4 9

Experiment ZZZ. Erythropoietic response of 12-day-old normal neonates to anemia induced by phlebotomy Attempts to mimic the erythropoietic response of 12-day-old neonates to exogenous Ep was made in this experiment. Two groups of animals were established. The groups consisted of one mother and her pups per group and t h e litter size was reduced to four or five pups per mother. Neonates in the experimental group (group 1) were bled (0.3-0.35 ml) via cardiac puncture on t h e eighth day of life. The mother of this group was not treated. The control group of neonates (group 2) was not treated, neither was their mother. All pups were sacrificed on t h e twelfth day of life and t h e erythropoietic parameters previously described were examined.

2O tI

W

-a

230

R. D. CARMICHAEL, A. S. GORDON AND JOSEPH LOBUE

MATURATION STAGES

OF RETICULOCYTES IN THE 12-DAY-OLD TRANSFUSED

RAT

SUCKLED BY ANEMIC (BLED) MOTHER SUCKLED BY NORMAL (NON-BLED) MOTHER

0 4.0-

3.53.02.52 .o1.51.o-

0.5I

II

Ill

IV

V

STAGES OF MATURATION

Fig. 1 Percentages of peripheral blood reticulocytes in neonatal rats classified according t o stage of maturation. The black and white vertical lines at the top of the bars depict ? one standard error for six neonates per group. Stage I , the youngest reticulocytes; stage 11, the next youngest, etc.; Stage V , the oldest reticulocytes.

The use of one mother per group of five to six neonates used in all experiments was considered appropriate since in a separate group of similar experiments (Carmichael e t al., unpublished) in which three mothers per group were employed, all mothers responded in like fashion to t h e experimental manipulation. Moreover, data from the three separate groups of neonates nursed by three anemic mothers was also closely similar. RESULTS

Experiment I. Radioiron incorporation studies in 12-day-old hypertransfused neonates suckled by anemic mothers The effect of m a t e r n a l blood loss on erythropoiesis in hypertransfused neonatal rats is shown in table 1 and figure 1.I t is seen (table 1) t h a t the percent RBC-59Feincorporation is significantly higher in neonates suckled by the anemic (bled) mother (experimental group) than t h a t noted for neonates nursed by their normal (non-bled) mother (control group (p < 0.001). The level of 59Feincorporation per mg spleen in the experimental

group is also significantly higher than t h a t observed in the control group (p < 0.001). No radioiron incorporation values in the bone marrow of either t h e experimental or control group was observed. The low 59Feincorporation values observed (as compared to adults) in spleens of both experimental and control groups, and t h e lack of detectable 59Feincorporation in t h e bone marrow of both groups may be due, in part, to a high r a t e of erythropoiesis in these young animals resulting in increased rates of iron utilization by the developing red cells followed by their prompt release. The percent incorporation of the injected dose of 59Fe by these tissues had already fallen to very low or non-detectable levels by t h e time these data were obtained. A decrease in splenic weight in neonates suckled by t h e anemic mother is observed, whereas the weight of the tibiae and femora appears to increase. The percentage of reticulocytes in the control group is higher than t h a t seen in the experimental group, but when this percentage is corrected for the deviation from normal in hematocrit (Hillman and Finch, '67) no significant difference is seen. The corrected reticu-

23 1

ERYTHROPOIETIC RESPONSE OF NEONATAL RATS

MATURATION STAGES OF RETICULOCYTES IN THE 12-DAY-OLD TRANSFUSED RAT

4.5

S U C K L E D BY A N E M I C ( B L E D ) MOTHER

-I

SUCKLED BY N O R M A L (NON-BLED) MOTHER

I

+ SUCKLED mill Ep-INJECTED BY N O R M A L (NON-BLED)

I

P'E

4.0

I

I

II

Ill

MOTHER

IV

V

STAGES OF MATURATION

Fig. 2 Percentages of peripheral blood reticulocytes in neonatal rats classified according to stage of maturation. The black and white vertical lines at the top of the bars depict ? one standard error for five to six neonates per group. Stage I, the youngest reticulocytes; stage 11, the next youngest, etc.; Stage V , the oldest reticulocytes

locytes, employing hematocrit = percent re- mitted to t h e nursing neonates thereby stimuticulocytes x hypertransfused neonate hema- lating their erythropoiesis. I t is further suggested t h a t Ep escapes inactivation, at least to tocrit/non-transfused neonate hematocrit (method of Hillman and Finch, '67) are indi- some extent, in t h e gastro-intestinal tract of cated in table 1. A differential count of reticu- the neonatal rat. locytes, classified according to their stage of maturation is shown in figure 1. I t is seen t h a t Experiment II. Comparison o f the erythropoietic effects i n 12-day-old hypertransfused the number of younger reticulocytes (stages I neonates nursed by anemic mothers and 11) in neonates suckled by bled and nonwith those in hypertransfused bled mothers a r e in the same range, whereas, neonates injected w i t h E p t h e numbers of older reticulocytes (stages IIITable 2 and figure 2 show the peripheral V). are considerably less in neonates of t h e bled mother. This causes a shift to the left of blood values of 12-day-old hypertransfused the reticulocyte maturation curve of pups neonates suckled by anemic a n d normal nursed by the bled mother, suggesting a de- mothers as compared to those injected intracrease in t h e maturation time of reticulocytes peritoneally with Ep. It is apparent (table 2) t h a t the MCV, MCH and MCHC for neonates which probably resulted from Ep stimulation. The results of this experiment demonstrate nursed by t h e anemic mother parallel those of t h a t hypertransfusion depresses erythropoie- pups injected with Ep, and all a r e significantsis in t h e neonatal r a t to some extent and ly higher than those seen in pups suckled by t h a t when suckled by a n anemic mother, the normal mother. The hematocrit of all erythropoiesis in these pups is stimulated, a s three groups when compared to normal (table judged by increased levels of RBC-59Feincor- 4) are elevated as a result of transfusion, and poration. Since bleeding t h e mother results in appear highest in the Ep-injected group, alt h e appearance of increased levels of Ep in her though t h e same amount of blood was given to plasma, i t is suggested t h a t this Ep is trans- all neonates in t h e three groups. This is true

232

R. D. CARMICHAEL. A. S. GORDON AND JOSEPH LOBUE

2 2 coo 2: UYw 2: N O

.- tl

ti

e w

cow

3

tI

c C

-

2 2 m2 2w

4

wo

ti

tl

tl

for t h e hemoglobin values also for which a significant difference is seen only in the Ep-injected group. While hematocrits of neonates nursed by the anemic mother were slightly lower t h a n those of either Ep-treated and controls, the elevated MCH and MCHC of these pups paralleled those of neonates injected with Ep, and both a r e significantly higher t h a n those of pups suckled by the normal mother. The relative number of reticulocytes in neonates suckled by t h e anemic mother is significantly higher t h a n those from the normal mother (p < 0.01), but no significant difference is seen in their absolute numbers. The absolute reticulocyte count was calculated from the percent reticulocyte and the number of circulating red celldpl. The relative percent of reticulocytes in neonates injected with Ep (which parallel those in pups suckled by the anemic mother) differs significantly from those in pups nursed by the normal mother (p < 0.05). The absolute number of reticulocytes in the Ep-injected neonates is significantly higher than in neonates suckled by the normal mother (p < 0.051, but not in neonates derived from anemic mother (p > 0.10). Thus, the trend was in t h e direction of increased reticulocytes in neonates of t h e anemic mother. However, the coefficients of variation are too dissimilar to allow firm conclusions to be drawn. A differential count of reticulocytes (fig. 2) shows a shift to the left of the curve for neonates nursed by the anemic mother, and those injected with Ep suckled by a normal mother, demonstrating a greater number of younger reticulocytes in the peripheral blood of these two groups. Variations in percentage of reticulocytes may indicate different degrees of early release of these cells from the hematopoietic tissue, and/or altered maturation time for these cells in the peripheral blood. Table 3 shows t h a t t h e number of erythroid precursors in the bone marrow of neonates suckled by the anemic mother, and those injected with Ep, do not differ significantly from those in the bone marrow of neonates of the normal mother. On the other hand, the numbers of red cell precursors in the spleens of neonates nourished by t h e anemic mother, and those which received Ep are significantly less t h a n in spleens of normally fed neonates (p < 0.05; p < 0.001 respectively). A differential count of erythroid precursors indicate t h a t the reduction in the overall percent of

+

@?z 22 o m

%X O N C

O

tl

m

N

(DO N

tl

m

(

tl

D

233

ERYTHROPOIETIC RESPONSE OF NEONATAL RATS

MATURATIONI 1STAGES OF RETICULOCYTES IN THE 12-DAY-OLD RAT ANEMIC {BLED] NEONATE

0 NORMAL INON-BLED1 NEONATE

i

Fig. 3 Percentages of peripheral blood reticulocytes in neonatal rats classified according to stage of maturation. The black and white vertical lines a t the top of the bars depict % one standard error for four or five neonates per group. Stage I, the youngest reticulocytes; stage 11, the next youngest, etc.; stage V, the oldest reticulocytes.

TABLE 3

Effects of maternal blood loss on erythroid cellprecursors in the bone marrow and spleen of 12-day-old transfused neonates: suckled by anemic (bled) mother; suckled by normal (non-bled)mother; Ep-injected isuckled by normal (non-bled)mother. Means 5 S.E.M. Treatment

Number of animals

RCP %

Pro-E

Baso-E

Suckled by anemic mother Bone marrow Spleen

5 5

21.6622.44 39.4822.36

3.38e0.34 2.8850.29

Suckled by normal mother Bone marrow Spleen

5 5

27.0822.41 47.5021.55

2.58%00.26 9.84e1.38 3.7020.25 22.26e1.14

Ep-injected (0.5I U Ep X 4) + suckled by normal mother Bone marrow Spleen

6 6

26.851 1.40 32.0322.53

1.3520.16 1.28*0.20

Poly-E

7.72e1.17 9.7420.92 16.5451.23 19.8621.38 13.88e0.95 20.8820.68

Ortho-E

0.32-tO.08 0.2030.03 0.7820.09 0.66-tO.13

9.01-t0.67 15.7821.44 0.5330.14 10.4121.08 18.11+-2.17 0.5520.12

RCP. red cell precursors in spleen and bone marrow smears; Pro-E, proerythroblasts; Baso-E, basophilic erythroblasts; Poly-E, polychromatophilic erythroblast; Ortho-E. orthochromatic erythroblasts.

these cells in spleens of pups suckled by the anemic mother, and those injected with Ep, is due to a decrease in number of cells in the basophilic erythroblast compartments. The similarity in results seen in t h e periphera1 blood, bone marrow and spleen of neonates injected with Ep, and those suckled by

t h e anemic mother, suggest t h a t erythropoiesis is being stimulated by approximately the same quantity of Ep. That is t o say, t h e amounts of Ep transmitted to suckling neonates by anemic mother, via t h e milk, is similar to the amounts of t h a t which had been injected intraperitoneally into pups nursed by

234

R. D. CARMICHAEL, A. S. GORDON AND JOSEPH LOBUE TABLE 4

PeriDheral blood values o f 12-day-old anemic (bled)and normal (non-bled)suckling neonatal rats. Means Body weight

Treatment neonates

Number of animals

Bled (anemic)

4

Non-bled (normal)

5

28.82 '-0.52

MC,"

x 10-6/p1

Hgb g i l 0 0 ml

3.80 20.20

7.74 20.19

3.74 20.15

7.38 20.12

'-3.00

Hct

RBC

g

X

33.0 fO.22

21.75 20.47 25.60 20.67

S.E.M.

Retics

Absolute retics

x,

%:

x 10-51p1

35.38 k0.53

45.67 22.64

17.3 kO.85

29.74 eO.97

11.1 20.67

MCH Pg

MCHC

57.71 '-3.18

20.50 '-0.98

68.85

19.80 20.50

28.91 20.97

fi

2

Hct, hematocrit; Hgb. hemoglobin: MCV. mean corpuscular hemoglobin; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; Retics. reticulocytes.

TABLE 5

Effects of phlebotomy on erythroid cell precursors i n the bone marrow and spleen o f 12-day-old suckling neonatal rats. Means f S. E. M. Treatment of neonates

Number Of animals

'y

Pro-E

Baso-E ~

Po1y.E

Ortho-E

~~~

Bled (anemic) Bone marrow Spleen

4 4

20.7721.09 19.42k2.67

1.3520.10 1.3720.14

9.0520.85 7.6221.63

10.3520.49 10.22kO.94

0.2520.02 0.2020.12

Non-bled (normal) Bone marrow Spleen

5 5

26.3021.78 29.5021.72

1.6820.16 1.45k0.15

9.5221.31 9.4820.78

15.0220.57 18.2821.34

0.0820.03 0.30f0.09

RCP, red cell precursors in spleen and bone marrow smears: Pro-E. proerythroblasts; Baso-E. basophilic erythroblasts; Poly-E, polychromatophilic erythroblasts; Ortho-E. orthochromatic erythroblasts.

the normal mother. These results indicate t h a t erythropoiesis stimulation has occurred in these young rats as judged by changes in their peripheral blood values, and the response of erythroid precursors in the spleen to Ep. The similarity in erythropoietic response of Ep-injected animals and those suckled by the anemic mother is best seen in: (1)t h e shift t o the left of the reticulocyte maturation curve; (2) a significant increase in MCH and MCHC; (3) a reduction in relative numbers of red cell precursors in the spleen.

Experiment ZZZ.Erythropoietic response of 12-day-old normal neonates to anemia induced by phlebotomy The effect of phlebotomy on erythropoiesis in 12-day-oldneonatal rats is shown in tables, 4,5 and figure 3. I t is apparent (table 4) t h a t bleeding the neonate results in a lowered hematocrit, but t h e numbers of circulating RBC are not significantly different from those of non-bled neonates. On t h e other hand, the MCV of neonates subjected t o bleeding is significantly lower t h a n t h a t of non-bled neonates (p < 0.05);however, the MCH is significantly higher in bled than in non-bled neo-

nates (p < 0.001). This trend towards smaller sized red cells has also been observed in neonatal rats suckled by anemic mothers or fed cow milk to which Ep had been added (Carmichael e t al., unpublished). I t is also noted (table 4)t h a t both relative and absolute numbers of reticulocytes a r e significantly higher in pups t h a t were bled than in those t h a t were not bled (p < 0.001; p < 0.001 respectively). The MCH does not differ significantly in the two groups. A differential count of reticulocytes (fig. 3) revealed a n increased number of stage 11, 111 and IV cells in the peripheral blood of t h e bled neonates. Table 5 shows t h a t t h e percentages of red cell precursors in t h e bone marrow and spleen a r e significantly less in anemic neonates t h a n in the bone marrow and spleen of normal controls (p < 0.05; p < 0.05, respectively). The numbers of proerythroblasts and basophilic erythroblasts in t h e bone marrow and spleen of anemic neonates parallel those in t h e bone marrow and spleen of normal neonates, and a r e not different significantly. On the other hand, the numbers of polychromatophilic erythroblasts in the bone marrow and spleen are considerably reduced in anemic neonates.

ERYTHROPOIETIC RESPONSE OF NEONATAL RATS

Failure to observe an increase in the number of proerythroblasts (the earliest recognizable precursor of the erythroid series) and the basophilic erythroblast, the next in the proliferating differentiating erythroblast compartment, together with a marked reduction in number of cells of the polychromatophilic erythroblast compartment, suggest that Ep in the bled neonate may not stimulate stem cell differentiation towards the erythroid compartment. I t seems rather to exert its action predominantly at the level of differentiated erythroid cells by influencing their rate of maturation and hemoglobin synthesis. This results in a shortening of the mean transit time of the proliferating erythroblast compartment, involving particularly a marked reduction of the transit time of the non-proliferating orthochromatic and reticulocyte compartment. This type of regulation of red cell production may also characterize normal neonatal erythropoiesis - but due to the increased production of Ep in the bled neonate, i t is more readily apparent in these animals.

235

stimulation may be seen from the work of Garcia and Van Dyke ('61), who reported that although 14-day-oldnormal rats did not exhibit an increase RBC-radioiron uptake, when these animals' red cell production was suppressed by hypertransfusion, a subsequent injection of Ep did evoke an increase in RBC-59Feincorporation. Stimulation of erythropoiesis in the hypertransfused newborn r a t has also been demonstrated following exposure to hypoxia or the administration of Ep (Zaizov and Matoth, '71). In the RBC-59Feincorporation experiment a lower percentage of reticulocytes was seen in neonates nursing from the anemic mother, but when the percentages of both groups were corrected for their respective hematocrit values, no significant difference was found (table 1). The finding of a considerably smaller number of older reticulocytes in the peripheral blood of neonates suckled by the bled mother suggests a decreased rate of maturation of these cells as a result of Ep stimulation. In other words, the reticulocytes seen in pups nursed by the unstimulated (non-bled) mother were more DISCUSSION mature. These observations are reminiscent of Grant ('55) reported stimulation of eryth- those of Butturini et al. ('711, who reported ropoiesis in neonatal rats and mice previously that the reticulocyte counts in peripheral suckled by hypoxic mothers, suggesting ma- blood of 10-day-old normal and starved rats ternal transfer of Ep through the milk. The were in the same range, but a differential detection of Ep in the milk of anemic lactating count of these cells classified according to sheep (Hyzy, '69) and in woman's milk (Bie- stage of maturation showed t h a t reticulocytes lecki et al., '72) lends further credence to the of the starved rat were more mature. presence of Ep in the milk of hypoxic lactating The quantity of Ep in the 12-day-old hyrats and to the fact that the hormone is pertransfused rat nursing for four days from a acquired through their milk by the nursing twice-bled mother, as judged by changes in peyoung. Thus, a maternal-fetal andlor mater- ripheral blood values (e.g., reticulocytosis), is nal-neonatal erythropoietic relation seems to equivalent to approximately 2.0 IU. This calexist in that, stimulation of Ep formation in culation is based on the observation that four the mother by hypoxic hypoxia (Grant, '55) or daily intraperitoneal injections of 0.5 IU Ep anemic hypoxia (Carmichael e t al., unpub- into hypertransfused neonates of the same age lished) appears to augment neonatal red cell suckled by a normal mother induced a similar production in the rat most likely through the intensity of effect. The erythropoietic paralpassage of maternal Ep via the milk from the lelism of these two groups was evident when erythropoietic stimulated mother into the compared to hypertransfused pups not treated with Ep and suckled by non-bled mother connursing neonates. Further support for this contention is pro- trols. The prompt release of a younger populavided in the present study by the RBC- tion of reticulocytes (stages I and 11) in neoradioiron incorporation experiment in 12-day- nates nursed by the anemic mother or injected old hypertransfused neonatal rats suckled by with Ep, is compatible with the work of Cantor an anemic mother. The percent 59Feincorpo- and Gordon ('73) who demonstrated that ration into red blood cells was significantly injection of Ep into hypertransfused neonatal higher in the neonates of the anemic mother rats results in an early release of reticulothan in those suckled by the normal mother. cytes followed by a true stimulation of That this effect is actually the result of Ep erythropoiesis. In this connection, it has been

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R. D. CARMICHAEL, A. S. GORDON AND JOSEPH LOBUE

shown by Gordon et al. ('62) t h a t Ep induces bers of nucleated cells in the marrow reprompt reticulocyte release from the bone mained at about normal levels. marrow of isolated perfused hind legs of rats. The results obtained by these workers clearI t is interesting t h a t t h e shift to the left of t h e ly show t h a t stimulation of erythropoiesis in reticulocyte maturation curve in experimen- the adult rodent involves a n increase in nucletal groups of neonates may be due to a reduc- ated RBC in the spleen, which is in contrast to tion in the later stages of reticulocytes, or a n t h a t of the neonate, in which, as shown in the increase in early forms. The interpretation of present study, a decrease in splenic red cell these data is based on a number of observa- precursors accompanies t h e stimulation of tions employing 12-day-old hypertransfused erythropoiesis. Observed also in the present neonates orally-fed Ep for four days, 14-day- study was a reduction in splenic weight in the old hypertransfused pups suckled by a n face of increased red cell production. The reanemic mother, and 12-day-old normal (non- duction in spleen weight associated with detransfused) animals nursed by a n anemic creased numbers of splenic nucleated RBC mother. Here, a n increase in newly-formed re- noted in t h e stimulated neonate is the antiticulocytes (stage I or 11) and a decrease in thesis of t h a t occurring in t h e adult in which older reticulocytes (stages 111, IV and V) were i t has been shown by Fruhman ('70) t h a t both seen in all experimental groups (Carmi- hypoxia induces a n increase in t h e numbers of splenic nucleated erythroid cells accompanied chael et al., unpublished). Since hemorrhagic anemia in t h e neonatal by a n increase in splenic weight. The response of the marrow of neonates rat evoked a decrease in the percentage of RBC precursors in the spleen and in the bone injected with Ep or suckled by a n anemic marrow, i t appears t h a t endogenous Ep exerts mother appears similar to t h a t of the erythstimulatory effects on erythropoiesis similar ropoietic stimulated adult. However, when to those of exogenous Ep. These findings also neonatal rats are hypertransfused for four suggest t h a t regulation of red cell production days t h e number of nucleated erythroid cells in t h e neonatal r a t is different, in some re- in their bone marrow or spleen do not despects, from t h a t of t h e adult. Fruhman ('70) crease. This is unlike the situation in the reported t h a t hypobaric hypoxia, anemia, and adult in which a diminution in the numbers of the injection of Ep constitute effective stimu- these cells does occur. Moreover, there was a lators of splenic erythropoiesis in the adult dramatic increase in early RBC precursors in r a t , but their effects upon marrow red cell pro- t h e spleen following transfusion (tables 3, 5). duction are less obvious. Bruce and McCulloch These results are similar to those of Lucarelli ('64) demonstrated t h a t after four days of e t al. ('68) who found t h a t starvation suphypoxia, adult mice displayed histologic evi- presses erythropoiesis in t h e adult r a t but not dence of massive splenic erythropoiesis but in the neonate. These investigators reported showed no qualitative changes in t h e marrow t h a t not only does there appear to be no suppicture. On t h e other hand, Turner et al. ('67), pression of red cell production after starvawho employed q u a n t i t a t i v e morphological tion, but in a substantial number of neonates techniques to study the effect of hypoxia on starved prior to t h e tenth to fifteenth day of the marrow in adult mice, found t h a t the ab- life, the percentage of erythroid elements in solute numbers of marrow nucleated erythroid the bone marrow appeared to be somewhat cells were increased by hypoxia and reached a greater than in litter mate controls. This peak at the same time a s t h e increase in sple- again suggests a different control mechanism nic weight. Morphologic and ferrokinetic stud- for red cell production in t h e bone marrow as ies performed by Kubanek e t al. ('68) indi- well as the spleen of t h e neonatal rat. According to Hodgson ('701, Ep in the adult cated t h a t Ep injected into adult mice acted primarily to stimulate erythropoiesis in the acts on a morphologically unrecognizable spleen, whereas, the marrow was not influ- erythropoietin responsive cell (ERC) t h a t is enced beyond normal levels. Radioiron uptake derived from a multi-potential stem cell was shown to be increased manyfold in t h e (CFU-S). Ep and perhaps other regulatory facspleen a t a time when 59Feuptake in t h e mar- tors, possibly microenvironmental, are rerow was reduced. In addition, Kubanek et al. quired for t h e differentiation of stem cells into ('68) demonstrated t h a t the numbers of nucle- t h e pathway leading t o RBC production ated cells in t h e spleen rose and paralleled the (Hodgson, '70). However, evidence from severradioiron uptake values. However, t h e num- al studies performed with Ep in adult animals

ERYTHROPOIETIC RESPONSE OF NEONATAL RATS

(Fischer, '62; Hodgson and Eskuche, '62; Gallagher et al., '63) have shown that the fundamental action of Ep is not only to trigger ERC differentiation into proerythroblasts, but that it also acts on recognizable RBC precursors. Goldwasser ('76) also reported that there is a reasonable amount of evidence indicating that Ep can act on a t least two types of cells: an as yet unidentified primitive cell with no erythroid characteristics and a n erythroid cell that is present during normal erythropoiesis. Butturini e t al. ('711, from experimental data obtained in the starved newborn rat, suggested t h a t Ep effects the maturation rate of erythroid cells in the newborn rat while it only moderately influences stem cell differentiation toward the erythroid compartment during neonatal life. Evidence presented in the present study lends support t o this hypothesis, and further indicates that the regulation of erythropoiesis in the neonatal rat differs in several respects from that observed in the adult. The decrease in nucleated RBC in erythroid tissues (most noticeable in the spleen) and the shift t o the left of the reticulocyte maturation curve in the peripheral blood suggest that Ep exerts its influence a t the level of the more differentiated elements of the erythroid series, probably by influencing their rate of maturation and hemoglobin synthesis, and by causing a prompt release of young reticulocytes which mature rapidly in the peripheral blood. That Ep also acts on the stem cell compartment or alters the proliferation rates of erythroblasts cannot be precluded. Findings in the present work further indicate t h a t Ep is transmitted to neonatal rats via maternal milk, and escapes inactivation, at least t o some extent, in the process of absorption from the gastro-intestinal tract with consequent stimulation of erythropoiesis in these animals. ACKNOWLEDGMENTS

This work was supported by a grant (5-RO1HL03357-19) from the National Heart, Lung and Blood Institute, NIH, USPHS. Robert D. Carmichael was a trainee of the National Heart, Lung and Blood Institute, NIH, USPHS (Training Grant 5-T01-HL05645-08). The erythropoietin used in these experiments was of human urinary origin, procured by the Department of Physiology, University of the Northeast Corrientes, Argentina, and processed by the Hematology Research Laboratories, Childrens Hospital of Los Angeles,

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under Research Grant HE10880 (National Heart, Lung and Blood Institute). Authorized for distribution by the Erythropoietin Committee of the National Heart, Lung and Blood Institute. LITERATURE CITED Bielecki, M., F. Przala and M. Lazewska 1972 Level of erythropoietin in the woman milk. Acta Physiol. Pol.,23: 435-439. Brecher, G. 1949 New methylene blue as a reticulocyte stain. Amer. J. Clin. Path., 19: 895-896. Bruce, W. R., and E. A. McCulloch 1964 The effect of erythropoietic stimulation on the hemopoietic colony-forming cells of mice. Blood, 23: 216-232. Butturini, U., C. Carnevali, V. Rizzali, R. Tarbutt and G. Lucarelli 1971 Stem cell and erythropoietin in the new born rat. In: Regulation of Erythropoiesis. First International Conference on Erythropoiesis, Capri 13-14,pp. 183-187. Cantor, L. N., and A. S. Gordon 1973 Erythropoietic response of the neonatal rat to hypertransfusion and erythropoietin (Ep). J. Surg. Oncology, Alan R. Liss, Inc., New York City, pp. 85-88. Carmena, A., G. Lucarelli, C. Carnevali and F. Stohlman, J r . 1966 Regulation of erythropoiesis. XIX. Effect of hypoxia on erythropoiesis in the new born animal. Proc. SOC.Exp. Biol. Med., 121: 652-655. Contopoulos, A. N., D. C. Van Dyke, M. E. Simpson, J. H. Lawrence and H. M. Evans 1956 Failure of new-born rats to respond to hypoxia with increased erythropoiesis. Proc. SOC.Exp. Biol. Med., 86: 713-715. Fischer, S. 1962 Studies on the mechanism and site of action of erythropoietin. In: Erythropoiesis. L. Jacobson and M. Doyle, eds. Grune and Stratton, New York, pp. 204-215. Fruhman, G. J. 1970 Splenic erythropoiesis. In: Regulation of Hematopoiesis. A. S. Gordon, ed. Appleton-Century-Crofts, New York, Vol. 1, pp. 339-368. Gallagher, N., C. Seifert, J. Lallinan, A. Maes and R. Lange 1963 Influence of erythropoietin on erythropoiesis in polycythemic rats. J. Lab. Clin. Med., 61: 258-265. Garcia, J. F. 1961 Erythropoietic response to hypoxia as function of age in the normal male rat. Amer. J. Physiol., 190: 25-30. Garcia, J. F., and D. C. Van Dyke 1961 Response of rats of Exp. Biol. Med., various ages to erythropoietin. Proc. SOC. 106: 585-588. Goldwasser, E. 1976 Erythropoietin. Blut Band, 33: 135-140. Gordon, A. S.,J. LoBue, B. S. Dornfest and G. W. Cooper 1962 Reticulocyte and leukocyte release from isolated perfused r a t legs and femurs. In: Erythropoiesis. L. Jacobson and M. Doyle, eds. Grune and Stratton, New York, pp. 321-327. Grant, W. C. 1955 The influence of axonia of lactating rats and mice on blood of their normal offspring. Blood, 10: 334-340. Heilmeyer, L. 1931 Probleme, Methoden une Kritik der Whippleschen Theorie. Deut. Arch. Klin. Med., 171: 123-153. Hillman, R. S., and C. A. Finch 1967 Erythropoiesis: normal and abnormal. Seminars in Hematol., 4: 327-336. Hodgson, G. 1970 Mechanism of action of erythropoietin. In: Regulation of Hematopoiesis. A. S. Gordon, ed. Appleton-Century-Crofts, New York, Vol. 1, pp. 459-469. Hodgson, G.,and I. Eskuche 1962 Time course effects of

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Lucarelli 1968 Effect0 della policitemia da transfusione sulla erithropoiesi re1 ratto neonato. Bioch. Biol. Sperim., 4: 1. Schooley, J. C., J. L. Garcia, L. N. Cantor and V. N. Havens 1968 A summary of some studies on erythropoietin using anti-erythropoietin immune serum. Ann. N. Y. Acad. Sci., 149: 266-280. Scribner-Seeley, V.,L. N. Cantor and A. S. Gordon 1971 Response of the neonatal r a t to erythropoietin (ESF). Biol. Neonate, 19: 108-117. Stohlman, F., Jr., G. Lucarelli, D. Howard, B. Morse and B. Leventhal 1964 Regulation of erythropoiesis. XVI. Cytokinetic patterns in disorders of erythropoiesis. Medicine, 43: 651-660. Turner, M.S., J. M. Hurst and J. M. Yoffey 1967 Studies on hypoxia. VIII. Effect of hypoxia and post-hypoxic polycythemia (rebound) on mouse marrow and spleen. Brit. J. Haematol., 13: 942-948. Zaizov, R., and Y. Matoth 1971 Regulation of erythropoiesis in the newborn rat. Israel J. Med. Sci., 7: 846-849.

Erythropoietic response of hypertransfused neonatal rats suckled by anemic mothers.

Erythropoietic Response of Hypertransfused Neonatal Rats Suckled by Anemic Mothers ROBERT D. C A R M I C H A E L , ' ALBERT S. GORDON AND JOSEPH L O B...
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