The potential for differentiation and radiosensitivity of the stem hemopoietic cells (KOE) under conditions of initial and later hypokinesia is examined. It is established that in the initial period of hypokinesia (3 days) when a stress reaction prevails, changes occur in the erythroid differentiation and radiosensitivity of KOE. This effect is associated with redistribution of T-lymphocytes that increase in number in the bone marrow of mice during hypokinesia. At later periods of hypokinesia (30 days) when changes in the organism are related to hypokinesia proper, differentiation and radiosensitivity of KOE were normalized

Shvets, V. N.

English

NASA Technical Reports Server

  
 
 
N O T I C E 
 
THIS DOCUMENT HAS BEEN REPRODUCED FROM 
MICROFICHE. ALTHOUGH IT IS RECOGNIZED THAT 
CERTAIN PORTIONS ARE ILLEGIBLE, IT IS BEING RELEASED 
IN THE INTEREST OF MAKING AVAILABLE AS MUCH 
INFORMATION AS POSSIBLE 
https://ntrs.nasa.gov/search.jsp?R=19800009409 2020-03-21T20:01:23+00:00Z
NASA TE:'HNICAL MEMORANDUM
	
NASA TM-7E066
DIFFERENTIATION AND RADIOSENSITIVITY OF HEMOPOIETIC
STEM CELLS OF MICE DURING HYPOKINESIA
V. N. Sheets
Translation of "Di fferentsirovka i radiochuvstvitel'nost' stvolovykh
krovetvorn^^kh kletok rgyshey pri gipokinezii", Radiobiologiyn, Vol. 19,
is	 No. 2, 1979, FP 199-203
(NASA -TH-'I60," 6)	 DIFFERENTIATION AND
	 'NAU-17671
FADIOSENSITIYITY OF HRMOPOIFTIC STEM CELLS
OF !l ICE DORIIIG HYPORINESIA (National
Aeronautics and Space Administration) 10 p	 tlnclas
HC A02 /!! P A01
	 CSCL 06C GS/51 47206
,..
^a	
`9,^
^' ', ^^,1E^,
``	
.^FS^^ pF^'
.	 P	 .^^
NATIONAL AERONAUTICS AND SPACE ADMINISTRATION
;dASHING`1'ON, D. C. 2546	 FEBRUARY 19°0
DIFFERENTIATION AND RADIOSENSITIVITY OF HEMOPOIETIC
STEM CELLS OF MICE DURING HYPOKINESIA
by
V. N. Shvets
Currently the problem of hypokinesia is acquiring general biological /199
and medical importance due to scientific and technical progress resulting
in a reduction of muscle loads in daily human activity; in clinical prac-
tice when the patient is strictly confined to bed for long periods; in
space medicine where hypokinesia is an integral part. The main etiological
factor of hypokinesia is the sharp reduction in the volume of muscle acti-
vity (^1] leading to a decrease in the oxygen demand by the tissues [2].
A change in the respiration by the muscle tissue during hypokinesia can
affect the erythropoietic and compensatory function of the bone marrow. It
is generally acknowledged that the process of hemopoiesis is based on the
proliferation and differentiation of ^;he stem a:^:mopoietic cells. The reaction
of the latter under conditions of. hypokinesia ^^: the effect of ionizing
radiation is not known. In addition, hypokinesia (as a unique form of
immobilization) can serve as a model of stress which, as a rule, occurs
in the first two weeks of hypokinesia [1]. Taking into consideration the
Numbers in margin indicate pagination in original foreign text.
1
..	 :.	
,.	 r
T . ^
^^'.	 ^^
described peculiarities of hypokinesia the task was set of studying the
potential for differentiation and radiosensitivity of the stem hemopoietic
cells (KOE^ under conditions of initial hypokinesia when the changes in
the blood system are characteristic f'or stress, and latex hypokinesia when
the changes in many ;issues of th; R organism are determined by hypokinesia
strictly [].]. For this purpose tli^ method of exogenous cloning of the stem
cells in the organism of lethally-irradiated recipient mice [3].
Material and Technique
Hypokinesia was created (non-rigid) by placing mice of the strain (SVA x
S 57 Vl^ Fl , females, 2-3 months old in • small-sized box cages. On the third
and 30th days of hypokinesia bone marrow was removed from the mice and
suspended. Part of the cellular bone marrow suspension was irradiated
in vitro with Y-rays of 137Cs (power of dose 35-37 R^min). The standard
number of cells irradiated in different doses ( 100-600 R) were injected
intravenously to lethally irradiated (950 R) recipients of the same strain.
The control was bone marrow of mice maintained under vivarium conditions,
irradiated in the same doses as the bone marrow of experimental mice, then
transplanted to the lethally irradiated recipients. On the ninth day after
transplanting the cells of the experimental and control animals the spleen
and femur were removed from the recipients. After fixing in Bouin's fluid
the macroscopically distinguishable cell colonies were counted in the
spleen. The femurs after decalcination in 5^ nitric acid were cast in 	 ^00
paraffin. On series histological sections of the femoral bone marrow 5 ^,m
thick stained with hematoxylin-eosin the colonies of cells of different
types were counted, eryt'^droid, myeloid, megakaryocytic, nondifferentiated,
and mixed. The last two types of colonies were not entered into the table.
The method of least squares i:7as used to compute the magnitude of dose DO
and the value of the extrapolation number n.
Tn part of the expel:;+.ments conducted in the N. F. Gamaleya Institute
of Epidemiology and Microbiology of the USSR Academy of Medical Sciences^-
an analysis was made of the number of T-lymphocytes in the thymus and
The author is deeply grateful to N. V. T.atsinik for help ire setting
up the cytotoxic reaction.
2
C
1
_	 ^	 -..o-,--^.,^ ...-..
bone marrow of mice who spent three days under conditions of hypokinesia.
The T-lymphocytes were identified according to the test of cytotoxicity of
the standard anti-T-serum whose index is the magnitude of the cytotoxic
index (CI) in perconts, computed according to the formula [4•]: CI= Nl-N2
100-N2
wheys NZ -percentage of dead cells with anti-T-serum, N 2--percentage of dead
cells with normal serum.
Results and Discussion
Q^s follows from the data given in the table the intact and irradiated
cells of the bone marrow of normal mice primarily form myeloid colonies
in the bone marrow. The ratio of the number of erythroid colonies to the
number of myeloid (E!M) equals 0.6-0.7. ^,t the same time the maintenance
'
	
	
of mice under conditions of 3-day hypokinesia results in the primary forma-
tion in the bone marrow of the recipients of erythroid colonies (E^M^1.3-
1.6) regardless of the magnitude of the dose of irradiation of tha trans-
planted cells. On the 30th day of hypokinesia the nature of the KOE dif-
ferentiation in• the direction of the erythro- and myelopoiesis significantly
varies (E^M=0.7-2.0^. The deviation from the usual type of KOE differenti-
ation on the third day of the experiment, apparently, can be linked to
the affect of stress developing in the mice in these periods [1]. This is
indicated by the data on the development of atrophy of the thymico-lymphatic
apparatus, and the increase in the level of corticosteroi.ds in the blood
plasma [l, 2]. One can think about the two paths for i:he hormone affecting
the KOE population. It ca.n be direct or mediated through the other cell-
targets necessary for KOE proliferation, and primarily through the T-lympho-
cytes. It seems to us that intensification in the formation of erythroid
colonies in the bone marrow of the recipient after transp3.anting of
cells of hypokinetic mice is linked to the r®distribution under conditions
of hypokinesia of the T-lymphocytes that occurs during hypercorticism [5-
'^	 7]. Based on this one can hypothesize that at the early periods of hypo-
,	 kinesia redistribution of the lymphocytes occurs as in the exogenous
^,
	
	 introduction of corticosteroids [^-7]. The evaluation of the T-system of
immunity with the help of anti-T-serum showed the increase in the number
of T-cells in the bone marrow of the mice during immobilization [8]. The
3
f
s,.
	
.
-^	 ,^	 <
...^
l
u
G0
c]
'O
h
C
U
J
rJ
G
C
O
.,.tJ^
S,1
0
u
C J
-r+	 U
^ ^
^	 e..
^,	 •r•/
J C!
VL
`.:..^
	 w
O O
^ Q
:!	 }+
G ~'
f,
i.l
:n
u
u
u
0
c
J
^J
0
J
7N
u^
^ -. i
-.-f rl W .
G :3 U n
C	 (3 e-1
U ^^	 !^ ^J ^J J r.
.-1 C. h ^ !.: : ^,] C] L- r_CJ -_ C^ -- - Gam'	 = O OC ^ Ql C`. .• OQO C'_C CC CC'C COO 0:7
`	 -^ ii _`
	 t
=^ -^
^w.T
^ ^!	  ++ ' ^^
-
t^
 C O 1-t ^I. L-]	 ofON-' r ^? .^;1 cl -` .^GO -- c/000 _^. G O...CO G O ^OC	 '
J ^ ^_ COG GOO 000 Cv'J
^
OCO QCQ O,O
';.;, c v -! - - I -1-I -I ,tyi.L..I I .	 , ^i_f^-	 I ^^	 p it'.-7 -1	 , !^-t ^-1	 I i I rf 1i	 r+l .C1 "A N O n O e0 ^; ... ^,; ^ .-. h cry ^ ^ 1 r.._ ^'^ •s+ O . .
t	 .^ ^^. L:
^ J M - - ^ "•^^	 :J ^ C.1 --/0C CCO C OQO 00'	 > U ^-!
•^;	 Q	 ._
.^ h^'!C aL7C CV7ti [^Ch 3 r: yV .n ^C x ^. .']
7	 ^ Cam-' C-^ N C^^ C-'O OHO O^."7. C-ri	 t^
• -- ^
E^
•r
t i;
^ •c^ tf c v ^co a_!o cal.: ..^ca! qc^-, .^
^. 1 ^ ^ -t_I 'LI_I T I Ti T^ ^I -	 ; : _ - _ i !_ r I ^i_^
i n L_ (^ ^1
_
...
^.
'^ :•J --• C•7
-
^ ^ ^
t
C t i^
i	 ^' r - to l.:O C-:. t CI ^_ L_ - _^ :1^.	 .... •	 j-
:J T• i ^ - _ _ 'i iTi'iI ^ i i I•^I ^i_ _ ^i -.l- 1t 1`_t ll_
I T c: ^; :
i^.
o ' _!=
0 J {
1	 •-' ^ ^ C-C OOC17 CNC =I=': L: CCt L. .. ^d :7 .-
J.J	 U
^a^zu ;uazda^ `
a	
^	 ,,
•rl	 1	 U M! ^ ^ ^ w ^
.. 4•+	 O •r.l ^.. : .	 :3 C? ..	 ..
Ti O	 w
^ t-- --
U ^ J ^^tJ r•! C	 O	 O
G O •^^ u	 ;t
I
_. _	 ^ . -•_ . __-- _ --	 __. • ..- —__
r :!
^^
m
c!
G
G
.•-1
O
11
'7
O
U
.,;
C
S+
.Ji'1
u
0
t!
J
^r
E -f
-.
ORIGINAL PAQE IS
OF POOR QC^ALI'FY
4•	 Y	 x. _	 •
^d'_:_J2.M"^'~	 _'^'^^	 .....w^.<^,...+.Y^T	
^.^	
- .nom.__....	 _...,...	 _	
. 	
- _
same also occurs in the mice who spent three days in a state of hypokinesia.
Thus, in the control mice the cytotoxic index for the bone marrow is 0.08
(8^), and in the hypokinetic mice it equals 0.26-0.34 (with thinning of the
anti-T-serum 7.: 8), i.e., the T-lymphocytes comprise 26-34%. 1n the thymus
of normal and hypokinetic mice respectively 98-100 and 77-84^ T-cells are
found. The results of these experiments indicate that an intensification
of KOE differentiation in the direction cf erythropoiesis during hypokinesia
is linked to the redistribution of T-lymphocytes. Fronf of this can also
be the experiments with addition of corticoresistant T-lymphocytes to the
transfusion of the bone marrow of normal mice C9]. In this case in the
bone marrow of the recipient colonies of the erythroid type are primarily
formed in the same way as in the transplanting of bone marrow from the hypo-
_	 kinetic mice. The described changes in the differentiation of KOE in
hypokir,^esia are more clearly pronounced in the period of development of the
stress reaction and do not depend on the dose of irradiation. ^t the later 	 202
periods of hypokinesia the direction of KOE diffe:Y:^^n^:iation approaches •'the
level of the control.
The data we obtained with the help of the method of cloning KOE in the
spleen on the radi^ensitivity of the stem cells of normal mice whose DO
magnitude was equal to 112 R, and n=1.5 d,id not differ fr..om those established
in the literature. During cloning in the bone marrow the values of these
parameters significantly differ: D 0=208 R, and n=0.8. At the same time the
stay of the animals under conditions of 3-day hypokinesia results in the
change in KOE radiosensitivity. The values of DO and n in this case are
respectively 113 R and 1.8 as compared to 208 R and 0.8 in the control.
According to the spleen test the,.value of these parameters is not altered
in relation to the control. On the 30th day of hypokinesia the KOE radio-
sensitivity remains on the 1eve.1 of the norm; DD 180 R, n=0.6 and D^ 115 R,
n=1.2 according to the bone marrow and spleen tests respectively. The
moa'^.fication of KOE radiosensitivity with 3
-day hypokinesia, evidently, in
the same way as KOE differentiation is linked to the effect of the T-lympho-
cytes. As follows from the figure, depending on the conditions for con-
ducting the experiment (6 variants of experiments and the cloning method
(in the spleen or bone marrow different values are recorded for the
5
_	 r `'	 r	 y
m^	 .
^•	 O,>-0,6xR	 0,91xR
^' , 
X^KM ::. —'^' ^
^j^ 1ff
^ 1617-^00
'
'
lj5{5-1/0
PcO 135
4
`	 ^	 A'OfirM o.• _'' ^
195
^ ^} 100
^}T-lymph.
	
^
.
4. XOIEXM .. —n- ^ •
-	 ^^^ T-lymph.
<:	 _-
11 fyo
^ ^ 100
kI
. ^ 100
5. XOExM ^ ^• —"-
'
^ i} 100
l71' ^
m
-4^	 ^
6.^ XOEp . o
	
_^.
o.o
^ 100
^ 11S
Change in Values of Dose DO
 Depending on Conditions of Irradiation and Method
of Cloning
Kf^E^ KOE from bone marrow; KOE c
--KOE from spleen; T-lymph'.--thymocytes;
1-6--variants of experiment. Colonies were studied in the spleen and bone
marrow of the femur on the 8-9th day after +,ransplantation of thA cells.
parameter DO
 [10-12]. Thus,acco^ding to the cloning test in the spleen tl^ie
amount DO
 for the KOE from the bone marrow or spleen (first and second
variants equals 80-115 R; according to the cloning test in the bone marrow
	
20
the amount DO
 for the same KOE significantly differs from that in cloning
9n the spleen (D0=160-?^JO R for KOE from the bone marrow and 120-135 R for
the KOE from the spleen). .Based on the fact that the cellular suspension
from the spleen is distinguished from that of the bone marrow by the presence
of T-lymphocytes one can think about the modifying effect of T-cells on
the KOE radiosensitivity from the spleen. The latter can be verified
6
^.^-_.-
	
.-..	 y	 _	 ...,, T
_„^,^. ,^:... ^'m.._.^:...rc,.u__._ ^ . _^,...^ l .'riJL^.s.rat^.^ -^	 - '.,+Y^^14^^EI^+I^^^r_.
	 ...__._..	 «........ _
	 _.
experimentally if T-lymphocytes are added to the bone marrow cells. Such
a mixture as if simulates the population of spleen cells of normal mice.
Asa result of the corresponding experiments it was established that under
the influence of the exogenous thymocytes the amount D O for the KOE from
the bone marrow is modified regardless of whether the T-cells were irradiated
jointly with the KOE (third variant of experiment} or they were added later
to the irradiated bone marrow cells (fourth variant of experiment}. 5a.nce
under conditions of stress redistribution of the T-lymphocytes occurs, the
number^of whici: increases in the bone marrow [5-7] experiments were under-
.
taken to introduce into the mice hydrocortisone (fifth and sixth variants
of the experiment . As a result the endogenous T-lymphocytes that migrated
into the bone marrow after injection of the hydrocortisone also modify the
radicsensitivity of the KOE from the bone marrow like the exogenous T-
lymphocytes. A similar pat^ern is observed with the injection of bone
marrow from mice who spent three days under conditions of hypokinesia. 'Phus,
in a comparison of the results of the experiments given both in this work
and in the previous studies [10-12] one can consider th^.t the T-lymphocytes
modify the reaction of the KOE to irradiation.
References
1. Portugalov, V. V.; Gazenko, 0. G.; et al., Kosmich. biol., 1, 5 (1967)
18-25.
2. Kovalenko, Ye. A.; Maylyan, E. S.; et al., Uspekhi fiziol. nauk, 6, 3
(1975), 110-136.
3. Till, J. E.; and McCulloch, E. A. Radiation Res. 14, 2 (1861), 213-222.
4. Raff, M. C.; Nase, S.; and Mitchison, N. A. Nature New Bio1., 230, 1
(1971), 50-51.
5. Cohen, J. J. J. Immunology, 108, 3 (1972), 841-844.
6. Moorhead, J. W.; and Claman, H. N. Ce11. Immunol., 5, 1 (1972}, 74-86.
7. Fauci, A. A. Immunology, 2$, ^ ( 1975), 669-680.
8. Zimin, Yu. I.
	 and Khaitov, R. M. Byul. eksperim. biol. i med., 80,
12 (1975}, 68-70.'	 ^'
g. Shvets, V. N. Radiobiologiya, XVS, 5 (1.976), 707- "111.
7
s
^ l
10. Shvets, V. N. Byul. eksperim. bio1. i med. 80, 5 ( 1975), ^0-^.
11. Shvets, V. N. Byul. eksperim. biol. i med., 81, 1 (1976 , 7^-76.
l2. Shvets, V. N. Tsitologiya, 18, to (1976), 1z5^-^58.
COPYRIGHT HOLDER: Izdatel°tsvo "Nauka", "Radiobiologiya" 1979
e,
.:
8
n
^,..T
	
..^
JO
C
7
cJ
C
UJ
GCrl
C
O
ya
O
^l
C
O
^ J
^ V
-
O^^^. ^_
J U
—
C.^	 ^..^
O O
a
r+ O
.--1 	:.
J	 f+U ^
r)
L
u
,..
L
;J
0
.J
J
7
1
I'
u .^ c `
ti ^ :JC U ^ c] _	 ^_ 'mil-1 ' r 7_ _
U U
.=
— — 1 _ —i 1'— —i IT 7! 1.1 — i T l ^^— -^ —y^y
J —O J ^j Ca 07 00
^,
^.	 C r
^	
U	
•-1
i c p .	 .
►^^^ n ^^-
i ^. ^! — CEO ....j O^
i	 J v , COG ,.CO v^ . ^Val •.• ^'U vCJ ^JC CC7
_ Ci U ^ — — ^^ -i —'^^ -1 — 	1 —1 ^'I t^_i ^'I	 i -y	 ^—ice — 1^	 ^	 '
:J	 L C ^ ^^ C^I^1 r. --. CC C1C .' CC C J OO 00
G
^ •^
e-^-o c^^:o ^^^- .=cam - ter, ticr. =n^
.	 ^
-	 -	 -C^ ^ -	 -C — ^l -	 -	 -C-- -	 -	 -C — O -	 -	 -O^J -	 -	 -0--O -	 -	 ; IC-^
i
^	 4 L 1	 C1
r
^1 -7— 1^ ='.'H!11 L`—^ T I ^-1--I '7 -1 — 1 --7 —' --1T _ _^ -1 - ....	 -	 '
.	
_ = 1
C 1 iI
_^^^ '7 'C CI .1 •,1 ^: CI .^ - 7- _lamr
- 7. ^^
'	 O
it	
c	 t
O i i
-
1 ^ c -__ coca C=10 - 1 -'^^._ c	 :_^. =-^a =	 ,
^ W ^	 C -	
_
--^n^
^
-` _
-1 - - -'----
L U
i
ao- •^ :sacda^
c 1
- C ''
y	 ++	 1 U ++
-
v T= ^
^
w
v O ^ Cl
_ ^—^- ^ ^
U CJ W
_ : f ^ ^ _ ^_ J O
C Cl r- C ^ _ -. -- CI ''
^ V U :J ^ i
.
f
_.	 _.__	 _.__ _ —_____. __ _ —___ ^	 __. _ ^_ _--.
v
r' J
I	 L -: _- - ^^ ^_
ORK3INAL PAQE IS
OF POOR QUALRY
4
d:
J
C
C
U
U
T
J
L
J
f..
U
J
+y^	 ^/.
	
-
..
same also occurs in the mice who spent three days in a state of hypokinesia.
Thus, in the control mice the cytotoxic index for the bone marrow is 0.08
(BJ^, and in the hypokinetic mice it equals 0.26-0.3^ (with thinning of the
anti-T-serum 1:8^, i.e., the T-lymphocytes comprise 26-3^. In the thymus
of normal and hypokinetic mice respectively 98-100 and 77-84J T-cells are
found. The results of these experiments indicate that an intensification
of KOE differentiation in the direction of erythropoiesis during hypokinesia
is linked to the redistribution of T-lymphocytes. Fronf of this can also
be the experiments with addition of corticoresistant T-lymphocytes to the
transfusion of the bone marrow of normal mice [9]. In this case in the
tiane marrow of the recipient colonies of the erythroid type are primarily
formed in the same way as in the •transplanting of bone marrow from the hypo-
:	 kinetic micem The described changes in the differentiation of KOE' in
'hypokinesia are more clearly pronounced in the period of development of the
stress reaction and do not depend on the dose of irradiation. 4^t the later	 202
periods of hypokinesia the direction of KOL differ®ntiation approaches the
level of the control.
TYie data we obtained with the help of the method of cloning KOE in the
splae;n on the radissensitivity of the stem cells of normal mice whose DO
magni'cude was equal to 112 R, and n =1o5 did not differ from those established
in the literature. During cloning in the bone marrow the values of these
parameters significantly differ: D^ 208 R, and n=0.8. At the same time the
stay of the animals under conditions of 3-day hypokinesia results in the
change in KOE radiosensitivity. The values of DO
 and n in this case are
respectively 113 R and 1.8 as compared to 208 R and 0.8 in the control.
According to the spleen test the,.value of these parameters is not altered
in relation to the control. On the 30th day of hypokinesia the KOE radio-
sensitivity remains on the level of the norm; DO=180 R, n=0.6 and DQ 115 R,
n=1.2 according to the bone marrow and spleen tests respectively. The
modification of KOE radiosensitivity with 3-day hypokinesia, evidently, in
the same way as KOE differentiation is linked to the effect of the T-lympho-
.,
cytes. As follows from the figure, depending on the conditions for con-
ducting the experiment (6 variants of experiments and the cloning method
(in the spleen or bone marrow different values are recorded for the
5
	
•	 Y
^... _	 _	 ^	 •
.X	 r'^':^>.+.dR:...w,:/liEr `.:^x]itr^IRi i I	 _ ,.^.,.a^... 	 ...	 , _


Differentiation and radiosensitivity of hemopoietic stem cells of mice during hypokinesia

Abstract

Similar works

Full text

Available Versions

NASA Technical Reports Server