Circulating Rat Platelets in Lethally X-radiated
Mice Given Rat Bone Marrow
L. H. SMITH,T. MAKINODAN,
ANDC. C. CONGDON
(Biology Division,
Oak Ridge National
By the use of immunohematological technics it
has been established that erythropoietic bone mar
row cells of the rat implant, proliferate, and func
tion hematopoietically when infused into lethally
irradiated animals (6, 7, 9). Furthermore, a cytochemical technic has been used to show that
granulopoietic elements of the rat likewise implant
and proliferate in irradiated, marrow-injected
mice (7, 8). Ford et al. (2) have observed rat
chromosomes in the bone marrow of irradiated
mice given injections of rat bone marrow. These
same workers also identified a distinctive chromo
some in dividing cells of bone marrow of irradiated
CBA mice given injections of a cell suspension of
spleen from mice heterozygous for an unequal re
ciprocal translocation. The present experiments
were designed to determine whether platelet pre
cursors of rat bone marrow would also implant and
proliferate in lethally irradiated mice.
MATERIALS AND METHODS
Preparation of antisera.—Platelets were ob
tained from the whole blood of C3H X lOlFi mice
and Sprague-Dawley rats by a modification of a
method described by Cronkite et al. (1). For the
antimouse platelet serum preparation, pooled
mouse platelets were washed 3 times in a NaClSequestrene1 solution (1 per cent Sequestrene in
0.7 per cent NaCl). A 1 per cent suspension of the
washed platelets was emulsified with an adjuvant
composed of Bayol F, Falba, and killed TB or
ganisms (3). Aliquots of this emulsion were in
jected subcutaneously into two Sprague-Dawley
rats. Thirty days later the rats were bled, and the
sera obtained were inactivated by heat (56°C. for
30 minutes). Merthiolate (1:10,000) was added
before the sera were stored at 2°C.
Similarly, antirat platelet sera were obtained
from C3H X lOlFi mice given injections of
Sprague-Dawley platelets, except that the donorrecipient ratio was 1:4.
1Disodium-ethylenediamine-tetraacetic
Received
for publication
December
acid.
10, 1956.
Laboratory,
Oak Ridge, Tennestee)
Agglutination test method.—Platelets were ob
tained as described from the blood of one mouse or
one rat. One drop of the platelet suspension (usu
ally about a 0.05 per cent suspension) was placed
on a glass microscope slide. Fifteen /il. of the antiserum was added directly to the suspension and
the slide tilted back and forth several times to in
sure good mixing. A cover slip was placed over the
mixture and the preparation examined microscopi
cally for agglutination of the platelets. Degrees of
agglutination were judged by the number of free
platelets and the number and size of the clumps.
Preparations were graded as 0, +,+-)-, or + ++.
When it occurred, agglutination was immediate,
although, upon standing for several minutes, the
clumps usually became larger. Beyond 5 minutes,
no further increase in clump size was observed.
Concomitant controls, in which normal rat and
mouse sera were used, were run on all platelet
suspensions.
Irradiation and bone marrow injection.—Male or
female C3H X lOlFi mice 3-4 months old were ex
posed to a single dose (950 r) of whole-body x-rays.
The mice were irradiated in groups of twelve
in a revolving lucite cage. Radiation factors were
as follows: 250 kv at 15 ma; rate in air about 160
r/min; target-skin distance, 60.0 cm.; inherent fil
tration, 1.0 mm. Al; added filtration, 1.0 mm. Al;
and a HVL of 0.5 mm. of Cu.
Within 2 hours after irradiation, each mouse re
ceived by tail vein the combined bone marrow
from one femur and one humérusof a SpragueDawley rat in 0.5 ml. of 0.9 per cent NaCl. Two
mice of each irradiated group served as uninjected
controls. The mice were caged individually or in
two's.
Tests performed.—At various intervals after ir
radiation, platelets were obtained and tested as
previously described against rat antimouse plate
let and mouse antirat platelet sera and against
normal mouse and rat sera. Peripheral erythrocytes were also tested with species-specific antisera
according to a method described by Makinodan
(7). Gomori (4) and Wachstein (10) reported that
367
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Cancer Research
368
rat granulocytes are alkaline phosphatase-positive, while mouse granulocytes are alkaline phosphatase-negative. Blood smears were obtained
from each mouse, therefore, to test granulocytes
for the presence of alkaline phosphatase (5). Each
mouse was autopsied, and hematoxylin and eosin
sections were prepared from the bone marrow and
spleen in all but two animals.
RESULTS (Table 1)
Platelets.—Rat platelets were present 7 days
after treatment in the blood of irradiated mice
given injections of rat bone marrow. Residual
kinodan (7). On the 20th and 30th days after ir
radiation, the six mice tested had rat platelets
only, although only 24 and 37 per cent, respec
tively, of the erythrocytes were those of a rat. At
points 60 days and beyond, the RBC and platelets
paralleled each other in species type, with one ex
ception. Mouse 22, 92 days after irradiation, con
tained a preponderance of mouse RBC but no
mouse platelets.
Alkaline phosphatase.—Thepresence of alkaline
phosphatase was detected in granulocytic elements
of the blood of treated mice at all postirradiation
intervals where rat RBC and platelets were de-
TABLE 1
THE APPEARANCE
OFTHEFORMEDELEMENTSOFRATBLOODIN THEPERIPHERAL
CIRCULATION
OFX-RADIATEDMICE GIVENINJECTIONSOFRATBONEMARROW
MOUSE
•¿o.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
DATS AFTER
IC-BAT
EBTTHBOCTTESRBC
(PEBRat6749951610232826184336321001002+t1001000lit100CENT)Mouae9493969191958490777
PLATELETS
Rat
MOUM
7
7
7
11
11
11
14
14
14
20
20
20
30
:»o
30
60
60
0
0
0
0
0
0
0
0
0
0
60
0
69
0
69
82
92
114
* + sign indicates presence and 0 sign the absence of alkaline phosphatase-positive cells. No
attempt was made to determine the rat : mouse ratio.
t Results of gross agglutination tests. Gross agglutination was graded from 1±to 4±.
mouse platelets were also present in the same mice
at^this time. Some mice in the 11- and 14-day
groups showed evidence of mouse platelets ; but in
five of the six animals in these groups, there was a
preponderance of rat platelets. At 20 and 30 days
after irradiation, all circulating platelets were of
rat origin. The blood of treated mice tested at sev
eral periods thereafter up to 114 days contained
only rat platelets, with two exceptions. An 82-day
animal had mouse platelets only, and one 60-day
mouse had a mixture of mouse and rat platelets.
Erythrocytes.—Theappearance rate of rat eryth
rocytes and the disappearance rate of mouse
erythrocytes in the circulation of x-radiated mice
given injections of rat bone marrow followed a pat
tern similar to that previously reported by Ma-
tected. In one mouse, 82 days after irradiation
only mouse peripheral blood elements were de
tected.
Bone marrow and spleen histology.—Thesections
of bone marrow and spleen showed a nearly nor
mal amount of blood cell-forming tissue in all the
animals examined from day 7 through 114. Erythropoietic, granulopoietic, and megakaryocytic
cell formation was present in the red pulp of the
spleen and in the bone marrow of all animals. The
relative amounts of each varied considerably, how
ever, from animal to animal, as seen in the histo
logie sections. The white pulp of the spleen did not
regenerate normally in any of the mice. The granu
locytic cells were often intensely eosinophilic.
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SMITHet al.—Rat Platelets in Irradiated Mice
There was no evidence of hemorrhage in any of the
mice at autopsy.
DISCUSSION
The results of these experiments show that rat
platelets appear and persist in the peripheral blood
of lethally x-radiated mice given injections of rat
bone marrow. Since it is generally accepted that
platelets are derived from megakaryocytes, this
observation implies that megakaryocyte precur
sors of the rat implant and proliferate rat platelets
in irradiated mice given injections of rat bone mar
row. It was observed that, in all but one of the
mice tested between 14 and 30 days after irradia
tion, 100 per cent of the circulating platelets were
those of a rat. At 30 days, however, only 37 per
cent of the erythrocytes were rat erythrocytes.
The 100 per cent rat erythrocyte level is not at
tained until the 60th day (7). The results of the
present studies show that the appearance rate of
foreign platelets is more rapid than that of the for
eign erythrocytes and, conversely, the disappear
ance rate of mouse platelets is greater than that of
the mouse erythrocytes. The latter observation is
probably related to the life span of the two formed
elements of mouse origin, because the life span of
circulating platelets is shorter than that of the
erythrocytes. On this basis, one would expect to
find residual mouse erythrocytes after mouse
platelets were no longer present. It may be, how
ever, that some mouse platelets continue to be
produced after irradiation, because megakaryo
cytes persist in the bone marrow of mice for sev
eral days after irradiation.
The absence of hemorrhage in all mice autopsied
suggests that the rat platelets were functioning in
the mouse at least in the capacity to prevent
hemorrhage.
Mouse platelets were present in the blood of
two mice out of eight at later periods, viz., 60 and
82 days after irradiation. The progressive increase
in the proportion of rat platelets between 7 and 20
days, together with the persistence of 100 per cent
rat platelets exclusively in twelve of fourteen mice
between 20 and 114 days, suggests that regression
369
of rat platelet precursors and reestablishment of
mouse platelet precursors occurred in these two
animals.
SUMMARY
Circulating platelets of lethally irradiated mice
given injections of rat bone marrow were tested
with species-specific antiplatelet sera. Rat plate
lets were detected in the peripheral blood of
treated mice at various intervals from 7 to 114
days after treatment. The 100 per cent rat platelet
level was attained in some mice by the 14th day
and in all mice by the 20th day. The results indi
cate that platelet precursors implant and produce
functional platelets in irradiated mice given injec
tions of rat bone marrow.
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Downloaded from cancerres.aacrjournals.org on July 12, 2017. © 1957 American Association for Cancer Research.
Circulating Rat Platelets in Lethally X-radiated Mice Given Rat
Bone Marrow
L. H. Smith, T. Makinodan and C. C. Congdon
Cancer Res 1957;17:367-369.
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