From www.bloodjournal.org by guest on March 6, 2016. For personal use only.
Influence
of Cobalt,
Sheep
Erythropoietin
and
Hormones
on Erythropoiesis
in Bone
Marrows
Isolated
Perfused
Hind
Limbs
of Dogs
By
E
W.
JAMES
RYTHROPOIESIS
shown
Cobalt
ROH,
CHARLES
is known
ACTH,12
stances.
hormones,1’5
to
hormone3
found
to
cobalt’s
was
bleeding
directly
and
release
bled,
from
rats.
of the
Some
early
isolated
perfusion
of
perfusion
fluid
little
of the
if cobalt,
erythropoietin
and
stimulate
erythropoiesis
in
of
the
cobalt,
was
in
compared
the
with
Hind
limb
12
for
the
were
Kg.
blood
in
carried
moved
through
foam
venous
This
the
elements
from
the
an
in
column
was
pressures
An abstract
ventricle
monitored
tvas
clog
20
the
that
rats
slim-
hind
concerning
the
limb.
In
limbs
of
in
the
the
changes
order
ml.
to
in
deter-
by
and
these
plasma.
studied
and
A
the
All
Grant
at Fed.
to
a
Proc.
was
re-
perfusion
the
blood
with
Saran
anti-
The
arterial
Grass
polygraph
the
from
Develoinnent
25:666,
10
donors
circulate
system.
A-2973(C4)
Career
blood
heart-lung
used
of
as
procedures
ml.
oxygenator
use
used
surgical
Davol
perfusion
the
Research
publication
Instruments
was
between
were
1000
bubble
in
weighing
Kg.
studies.
Actuator#{176}
USPHS
was also presented
1, 1963; accepted
for
International
Medical
growth
sheep
were
dogs
15-25
Approximately
with
USPHS
bovine
from
mongrel
between
Pulmo-Pak
blood
(4)
limbs
defibrinated.
occasionally
supported
of this work
female
and
Abbott
oxygenate
Diseares
the
shown
METHODS
throughout
and
An
were
through
limbs.
anesthesia.
donor
tb
Metabolic
Submitted
May
#{176}Obtained from
system
used
hyoxia,
et al.13 concerning
with
changes
perfused
weighing
pentobarbital
system.
investigation
Arthritis
and
GM-K3-5990-C2.
in
dogs
perfusion
single-ended
the
AND
performed
female
anesthetized
perfusion
perfused
perfused
contralateral
were
and
under
OUt
with
pump
male
cobalt
as
have
) 3,5,3’-triiodothyronine,
( 6 ) purified
erythropoietin
nonperfused
perfusions
Both
isolated
rats.
its
were
kidney
when
such
erythropoiesis
given
in the
MAmRIAIs
and
observed
(3
or
erythroid
the
and
some
of the hormonal
agents
mentioned
the bone
marrow
directly,
the isolated
hind
with
normal
blood
and blood
containing
(1)
perfused
) hydrocortisone,
( 5 ) testosterone
(2
hormone,
Changes
dog
been
the kidneys
release
of
co-workers12
of
was
marrow
mine
thyroid
have
or phenyihydrazine-treated
information
sub-
and
that
when
the
been
and
marrows
above
limb
has
to stimulate
exposed
bone
hormonal
animals.1#{176} Stimuli
Cordon
bone
NIGHTINGALE
hormones
classical
experiments
by Drinker
dog
hind
limbs
dealt
primarily
and
morphology
several
reduced
is through
effect
appear
in the
0.
intact
or hypophysectomized
to stimulate
erythropoiesis7
nephrectomized
low-pressure
by
are
in either
decades
erythropoietin.11
erythropoiesis
ulates
W.
thyrotrophic
testosterone
ferrikinetic
in
AND
stimulated
be significantly
primary
effect
phenyihydrazine
kidney
of
plasma
slight
a
assayed
COUCH
steroids,34
and
to stimulate
erythropoiesis
has been
known
for several
ervthropoietin,8’#{176}
be
adrenocortical
growth
effects
have
been
removed.8
Although
the
B. L.
FIsmR,
Several
of
and
and
Institute
Award
of
Grant
1963.
Aug.
14, 1963.
Corp.,
Stoneham,
Mass.
87
BLOOD,
VOL.
23,
No.
1
(JANUARY),
1964
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88
ROH,
FISHER,
AND
COUCH
NIGHTINGALE
-SCHEMATIC DIAGRAMOF DOG HIND LIMB PERFUSIONSYSTEM-
AIR
Fig.
1.-A
Saran
iliac
6
femoral
10
13
Statham
in
=
pressure
towels
heat
mesenteric
transducers.
at
37
saturated
C.
with
8
nutrient
was
warmed
temperature
physiologic
saline
to
in
water
and
D
external
femoral
9
femur;
artery;
hypogastric
12
nutrient
vein
artery.
a
bottom
bath.
warmed
2
deep
vein;
midsacral
=
filters;
artery;
5
femoral
=
artery
14
nylon
iliac
vein;
artery;
blood
a constant
external
=
iliolumbar
=
C
pressure;
1
artery;
The
in
venous
=
reservoir;
7
inferior
maintained
B
blood
=
artery;
4
=
femoral
artery;
11 =
vein;
hypogastric
=
to femur;
E
iliolumbar
3
deep
vein;
pressure;
column;
vein;
=
and
arterial
=
antifoam
LINE
drain
The
throughout
bottle
limb
hind
the
reservoir
was
wrapped
experiment
with
a
lamp.
Figure
1 is
artery
and
anterior
in
on
the
abdominal
the
following
hypogastric
femoral
artery
to
vein.
the
mately
2-3
bone
vein
through
marrow
above
initiated
aorta
and
vena
cava
completely
limb
microscopic
artery
and
isolate
was
of
point
with
then
vein
branch.
it from
for
The
the
and
the
upper
shaft
inserted
deep
at
ligated
heavy
remainder
of
of
of
the
the
perfusion
extemal
exposed
limb
hind
artery,
inferior
to
bladder.
deliver
a portion
the
femur
through
external
iliac
femoral
artery
rate
of
1
cm.
carcass.
marrow
system
nutrient
and
veins
minute.
above
then
At
a
per
this
smears,
was
by
and
approxi-
The
per-
abdominal
around
the
deep
circula-
artery
vein
point
suspension
tested
The
the
The
the
an
ligated
mesenteric
branch.
placed
point
was
of
artery
and
48
were
the
iliac
through
circulation
cords
bone
right
the
a stroke
The
were
mid-sacral
approximately
Three
the
branches
of
system.
limb,
to
veins,
into
the
alone
preparation
integrity
perfusion
perfused
complete
terminal
where
the
circulation
their
blood
were
removed
sections.
the
limb
hind
was
and
were
the
the
arteries
vein
cannulae
then
left
collateral
and
was
to
The
iliolumbar
fusion
iliolumbar
which
artery
steel
cm.
of
limb,
incision.
and
Stainless
diagram
right
order:
artery,
lion
a schematic
vein
femur
where
the
the
limb
from
the
counts
adding
and
a
sus-
From www.bloodjournal.org by guest on March 6, 2016. For personal use only.
BONE
MARROW
pension
of
per
was
mg.
helpful
time
could
be
twenty
added
for
to
for
the
at
of
Bone
the
the
60 mg.
with
N NaoH
of
the
cobaltous
supplied
the
plasma
was
the
by
in
re-
plasma
remove
cobalt
out
bone
was
the
to
for
by
of
16,491A),
prepared
the
cobalt
a modifica-
the
use
for
This
of
the
upper
rat
fasted
a bone
end
of
of
the
opens
to
saw,
preparation
foramen
artery
a hemacytometer
the
shaft
the
Into
bone
marrow.
on
They
for
a
chamber.
these
Giemsa
of bone
stain.
marrow
Five
each
smear.
The
were
stained
with
qualitative
Total
hemacytometer
60 mg.
with
differential
used
from
approximately
stained
decalcification.
were
counting
calculated
from
and
added
further
to
the
Sodium
hundred
bone
marrow
Giemsa
comparison
hexahydrate
lead
of
0.005
per
stain
between
the
)
by
being
5
mixed
A
Upjohn
of
Co.
plasma
the
Hormone
method
employing
is equivalent,
was
in
in effect,
derived
per
blood.
Section,
lyophilized
especially
Growth
unit
1 U.S.P.
pH to
the
3,5,3’-triiodothyronine
7310)
dissolved
unitsf
Study
batch
gain
One
system.
RM
was
from
cobalt
with
Hematology
al.17
the
of
USP
weight
approximately
by adjusting
added
to the perfusion
Coleman
and
Bell
cent
prepared
activity
NIH
et
body
No.
of
dissolved
0.01
in
system.
Analytical
Lot No.
332188)
0.85
per
cent
saline
system.
before
day
a potency
dissolved
perfusion
Lot
being
( Matheson,
K 103-277A
the
#{176}
having
for
protein
The
sheep
was
prepared
these
was
diluted
Standard
hypophysectomized
of
was
was
Lot
dissolved
in
which
according
sodium
to
succinate
dissolved
( 1 unit/mg).
using
the
(U-
in
No. 7254,
prepared
with 0.85 per cent
female
CoCL,
phenyihydrazine-
erythropoietin,
studies,
( Upjohn
Reference
to 5 micromoles
from
mg.
testosterone
to the perfusion
system.
Hydrocortisone
infusion)
in 50 per cent ethyl
alcohol
with
the
used
nutrient
were
to 9.0 before
for
White
#{176}Equipotent
be
foramen.
were
marrow
being
a specific
saline
to
the
with
Liothyronine
adjusted
( Lot
procedure
added
intravenous
hours
carried
perfused
and
from
each femur
was removed,
quantitatively
diluted
with
1 ml. of 2 per cent acetic
acid,
Smears
sections
perfusion
with
cent
and
24
from
nutrient
counted
and
before
chloride
sheep
was
of
studies,
with
marrow
removed
of fresh marrow
were prepared
a bone
French
impurity
to
per
were
removed
sample
analyzed
was
limbs
was
perfusion
for
being
hind
limbs.
pH
maximum
0.85
A
was
delivers
marrow
fixation
The
the
homogenizer,
plasma.
as
and
and
added
limb.
longitudinally,
bone
was
and
and
dog
after
Erythropoietin
10
of
NaoH
Kline
treated
side
of bone
nonperfused
Mill)
(Smith,
grade
ml.
split
growth
hormone
( NIH-GH-B6)
was mixed
in physiologic
saline
Bovine
unit/mg.
femur
assay
of
mg.
femur
glass
mounted.
and
with
and
the
resin
sample
agents
All
units
One hundred
The papavthe perfusion
studied
were
al.14
A sample
diluted
made
being
perfused
a vise
materials
400,000
hydrochloride.
limb
exchange
in
the
ground
enumerated
were
before
hind
the
following
sulfate,
cannula.
terminated
plasma
et
the
streptomycin
erythropoietic
Erythropoietin
counts
per mg.
marrow
smears
0.3
were
ion
Fried
either
of
slightly,
sections
The
of the
of
cannulation,
contralateral
some
60
sample
a
cell
Bone
with
cells
on
shaft
to
mixed
papavarine
perfusion
the
Each
length.
1 cm.
medial
nucleated
counts.16
In
approximately
homogenized
and
placed
entire
approximately
transferred
mg.
Cr’
sacrificed
system
of
Technic
were
weighing
A second
of
Almond.15
procedure
their
posterior
that
assay.
to
mg.
None
The dog was then
from
the perfusion
perfusion
system
every
20 minutes.
blood
vessels
dilated
in order
that
was
Dowex-50
of
the
femurs
throughout
8-9
against
Marrow
smears
as
perfusions.
system.
removed
500
60
arterial
perfusion
assay.
technic
to
The
same
control
Prior
as 6 hours.
of the
erythropoietin
according
rat.14
and
as long
the
several
perfusate:
heparin
arm
When
4 C.
to the
tion
for
side
erythropoietin
dialyzed
prior
fasted
blood
mg.
extended
treated
moved
the
the
40
6 hours.
marrow
to
the perfusion
sample
was
glucose
was added
to the
in keeping
the hind limb
through
4
in
to
penicillin-G,
anne
and
liter
cells
to escape
from
A plasma
assay
added
procaine
red
were found
exsanguination.
erythropoietin
were
89
IN DOGS
chromium-51-labeled
labeled
RBC
by complete
for
STUDIES
saline
for
saline
Assayed
by
rats.
the
fasted
rat
assay.
From www.bloodjournal.org by guest on March 6, 2016. For personal use only.
90
FISHER,
ROH,
COUCH
NIGHTINGALE
AND
.
(.24150T)
)
L(N)
The
standard
heavy
error
horizontal
of
the
lines
mean
within
from
the
CONTROLS
(2MG)
Fig. 2.-Influence
of sheep
erythropoietin,
cobalt,
alone on the nucleated
ervthroid
cells in the femoral
limbs.
The
total
height
of each
bar represents
the
limbs.
MEAN
- - - -
(2MG)
HYDROcORTISONE
(.1 UNIT)
.
each
bar
contralateral
several
hormones
and
marrows
of perfused
mean
value
for the
represent
the
nonperfused
mean
limb
blood
dog hind
perfused
value
with
the
same
from
dog. Number
enclosed
at the bottom
in that particular
group.
The vertical
error
of
from
the
is
the
the
and
mean.
nonperfused
mean
perfused
of each bar represents
the number
of animals
lines at the top of each bar indicate
standard
Two
dots
at the top
of the
bars indicate
significantly
different
contralateral
limbs at the 1 per cent level. The broken
line
value
obtained
added
to
determining
by
pooling
the
ervthroid
cell
counts
from
the
46
non-
limbs.
control
the
perfusion
significant
The
system.
differences
technic
of
experimental
between
analysis
of
variance
was
used
for
groups.15
RESULTS
1.
Effects
Cell
of Several
Counts
Figure
Hormones,
in the
Bone
2 demonstrates
femurs
for 4-6
hours
(0.24
units/ml.
poietin
(0.1
unit/ml.
),
ml.)
and
testosterone
(332
±
roid
cell
32.5)
counts
Cobalt
Marrow
the
with
),
results
blood
cobalt
of
alone
chloride
(0.5
3,5,3’-triiodothyronine
and
sodium
cobalt
within
and
Sheep
of Isolated
succinate
(252
± 21)
the femoral
Erythropoietin
Perfused
perfusion
or
hind
of
the
on Erythroid
Limbs
isolated
itg./ml.
), hydrocortisone
(0.2
mg./ml.).
Both
produced
marrows
hind
blood
containing
sheep
( 1 smole/ml.
) , growth
limb
erythrohormone
(0.2 mg./
erythropoietin
significant
increases
in erythof the perfused
limbs
when
From www.bloodjournal.org by guest on March 6, 2016. For personal use only.
BONE
MARROW
91
IN DOGS
STUDIES
750
700
650
.
600
C,,
550
C-,
_
500
I
_;-
MEAN
NONPERFUSED
-----
450
CONTROLS
400
___a______JL____
CONTROL
ERYTHROPOIET)
N
COBALT
GROWTH
HOR
MONE
Fig.
3.-Total
nucleated
cell counts
within
femoral
marrows
perfused
with blood
alone
or blood
containing
sheep
erythropoietin,
cobalt
or growth
hormone.
Top of
each bar indicates
mean
values
for each perfused
limb. The heavy
horizontal
lines
within
each
bar represent
the mean
values
with
standard
errors
of the mean
for
contralateral
nonperfused
limbs.
Standard
errors of the mean are represented
by the
vertical
lines at the top of each bar. Two dots at the top of the bar representing
the
erythropoietin
perfusions
contralateral
the pooled
compared
and
with
± 19.8 )
pared
with
alone,
or blood
one
did
pared
result
their
these
substances
counts
in the
or
perfused
change
marrow
value
cell
42
growth
hormone
cells
when
cornof limbs
in erythroid
for
( 176 ±
controls
Perfusion
Cobalt
and Growth
of Isoliited
Perfused
growth
nonperfused
mean
with
or
counts
blood
testoster-
when
corn-
controls.
Hormone
on
Hind
Limbs
cell counts
alone
and
hormone.
a moderate
bone
.
the
is the
3,5,3’-triiodothyronine
nonperfused
produced
)
23.8
the total
nucleated
perfused
with
blood
cobalt
line
nonperfused
( 194 ±
in a significant
from
broken
with
blood
containing
increase
in erythroid
hydrocortisone,
respective
Figure
3 compares
dog
hind
limbs
different
The
contralateral
of Erythropoietin,
Counts
in I%farrows
erythropoietin,
level.
of femurs
a moderate
controls
containing
significantly
cent
respective
Perfusion
resulted
in
nonperfused
not
with
2. Influence
ated Cell
of
their
± 15.4).
198
( 229
indicate
limbs at the 1 per
nonperfused
controls.
As
to
marked
preparations.
within
blood
previously
increase
Perfusion
Total
Nude-
femoral
containing
noted
in
in
marrows
sheep
figure
2,
erythroid
cell
of marrows
with
From www.bloodjournal.org by guest on March 6, 2016. For personal use only.
92
ROH,
FISHER,
blood
alone
cell counts.
in
resulted
Perfusion
a significant
in a slight
of femurs
increase
in
the contralateral
limbs
is almost
equivalent,
absolute
increase
blood
containing
were
slightly
but
limbs
has
with
in
cell
parable
ratio.
the
marrows
testosterone
the
rnyeloid:
after
respective
of
Erythroid
order
changing
the
cells
change
normoblasts
and
within
no
crease
the
increase
erythropoietin
erythroid
in
a corn-
of the M : E
total
nude-
the
mean
by
a
in
counts
and
values
of
cell
on
Various
cent
)
in
the
the
marked
,
the
and
basophilic
other
bone
the
their
predominantly
four
basic
of
cell
perfused
in
(68.5
a marked
the
into
( 120
cent)
and
per
inabso-
may
suggest
a more
mature
( 68.5
cent )
basophilic
demon-
The
pronormoblasts
cell compartment
a pronounced
per
As
in
compartment.
to differentiate
with
of the marnormoblasts
types.
resulted
orthochromatic
stem
in
cell
nude-
Figure
4 shows
polychrornatic
marrow
three
marrow
numbers
stem
increase
indicate
that
the
cycle.
Cobalt
resulted
cent)
)
cent
were
of the
perfusions
the
would
least
per
which
made
within
in the
within
stimulates
The
was
of the perfused
limbs.
basophilic
normoblasts,
occurred
per
types
analysis
erythropoietin
types
Hormone
or growth
hormone.
Perfusion
in a slight
increase
in basophilic
( 132
per
increase
without
Marrows
norrnoblasts
polychromatic
(75
slight
3,5,3’-triiodothyronine
Growth
Perfused
an
resulted
cell
precursor.
certainly
one cell
after
series,
stimulates
erythropoiesis
Total
nucleated
cell
from
and
particular
cobalt
4, sheep
( 52
lute
that
the
change
in all four
The
perfusion,
hydrocortisone,
orthochromatic
figure
ratio.
counts
leukocytic
has also resulted
in a shift
to significantly
change
the
within
the rnarrows
in pronormoblasts,
appreciable
in
cell
in the
hormone
different
perfusions,
alone,
erythropoietin,
with
blood
alone
strated
blasts
erythroid
Cobalt
to determine
ated
erythroid
the per cent
erythroid
controls.
Types
during
blood
rows
with
Erythropoietin,
Cell
in
change
growth
significantly
nonperfused
Effects
In
not
with
with
which
control
suggest
that this hormone
than
sheep
erythropoietin.
perfused
were
compared
cells.
Femoral
marrows
perfused
had total
nucleated
cell counts
than
the mean
value
for the
elevation
change
in the leukocytic
series,
The failure
of growth
hormone
femoral
when
nucleated
resulted
)
counts
to the
a corresponding
noted
count
may
mechanism
but
in
in total
± 38.1
( 636.7
counts
NIGHTINGALE
increase
in total
nucleated
of the
counting
procedure,
limits
erythroid
± 19.9)
greater
without
a shift
ated
cell
different
3.
the
Therefore,
.
decrease
cell
.
within
counts
nucleated
AND
± 48.4 ) The
in nucleated
cobalt
(481.7
not significantly
cobalt,
resulted
erythroid
total
(443.3
± 17.8)
(472.3
perfusion
but non-significant
with
erythropoietin
COUCH
has
increase
per
cent),
normoblasts
undergone
in pronormo-
polychromatic
(35
at
per
cent)
normoblasts,
but
very
little
change
occurred
in the
orthochromatic
normoblasts.
An increase
in the numbers
of orthochromatic
normoblasts
may
have
occurred
if the perfusion
time
could
have
been
extended
for a longer
period
of time.
more
mature
pronormoblasts.
erythropoiesis
or the
growth
Growth
nucleated
This
by
hormone
inducing
of a more
perfusions
erythroid
may
indicate
the
mature
stem
produced
cells
but failed
that
growth
cell
erythroid
to differentiate
cell.
a slight
to cause
hormone
but
increase
in the
an increase
in the
does
not stimulate
may
effect
mitosis
From www.bloodjournal.org by guest on March 6, 2016. For personal use only.
BONE
MARROW
IN
STUDIES
93
DOGS
c::::
PRONORMOBLASTS
BASOPHILIC
llii
POLYCNROMAT)C
-
240
(PR)
NORMORLASTS)RN)
ORThOONRONATC
NORMOSLASTS
(OR-N)
2t0
too
(SO
o
ISO,
(40
ISO
-
-
(00
-
--
-
-
CONTROL
10f11
CONTROL
Fig.
or
blood
of perfusing
containing
major
broken
for
ERYTHROPOI(T)N
4.-Influence
sheep
each
cell
type
4. Effects
in
group
Table
1 shows
the
nonperfused
represents
Samples
in RBC
by
the
addition
of
L1
the
incorporation
saline
controls.
containing
the dialyzed
in
RBC
that
lating
All
which
As
their
of the
plasma
represented
respective
erythropoiesis
enclosed
perfused
System
slightly
but
by
X-time
the
four
number
to
in each
group.
on Radioactive
values
Iron
were
assayed
were
not
controls.
except
for
to
values,
of
Therefore,
to
have
greater
z\2
testosterone,
sam-
prior
found
significantly
and
plasma
of the erythropoietic
differences
are mdi-
with
the exception
in significantly
greater
0 time
fasted
that
substances
studied,
in the
The
in RBC
samples
agents,
resulted
substances
alone
the
marrow
compartment.
The
line for the values
obtained
system
prior
to addition
of the perfusion.
The
were
blood
on
Rat
erythropoietic
the erythropofetic
cobalt
sample,
than
all
values.
various
values
Perfusion
with
hormone
of limbs
Fe5#{176}
incorporation
pies removed
from
the perfusion
stimulant
and at the termination
cated
the
Fasted
HORMONE
dog
growth
controls.
the number
from
of the
mean
or
of the
within
the bone
taken
as the base
respective
of bars
GROWTh
limb
cobalt
precursors
cent value
the
of Plasma
Incorporation
hind
erythropoietin,
nucleated
erythroid
line is the 100 per
the left of each
COBALT
the isolated
the
Fe59
than
all
the
plasmas
testosterone
Fe59 incorporation
it
are
seems
capable
and
apparent
of
stimu-
animal.
DIsCUSSION
The
are
the
present
capable
femoral
utilizing
study
indicates
of stimulating
marrow
of
absolute
direct
stimulatory
anemic
hypoxia
counts
effect
when
that
the
the
of
nucleated
of blood
such
erythropoietin,
bone
isolated
blood
marrow
hind
from
cobalt
directly
limb
of
erythroid
donor
is perfused
cells,
animals
through
and
growth
hormone
when
perfused
the dog.
Kuna
have
demonstrated
subjected
the
through
et al.,’9
to hypoxic
isolated
of the rat. Gordon
et al.2#{176}
have also shown
that a human
urinary
factor
produces
an absolute
increase
in nucleated
red
cells
hind
limb
preparations.
Our studies,
which
show
that
a more
hind
a
or
limb
erythropoietic
in perfused
rat
highly
purified
From www.bloodjournal.org by guest on March 6, 2016. For personal use only.
94
FISHER,
Table
Fe’9
Control
plasma
Cobalt
Sheep
Incorporation
AND
NIGHTINGALE
System
on
in RBC
X Time
0 Time
Saline
COUCH
from Hind
Limb
Perfusion
in RBC of the Fasted
Rat
1.-influence
of Plasinas
Fe5#{176}
incorporation
Treatment
ROH,
j.2
5.50
±
.22
8.26
±
.82
2.76
8.40
±
1.42
8.10
±
1.17
2.60
10.90
±
1.30
17.74
± 1.95#{176}
ND
.14
D
9.64
2.80
erythro-
poietin
Growth
honnone
Hydrocortisone
6.67
±
.61
1.17
11.90
±
2.25*
5.23
8.03
± .79
2.53
12.54
± .92#{176}
4.51
6.62
±
1.12
11.67
±
1.75#{176}
5.05
4.81
15.88
±
1.08#{176}
5.57
±
.99
.72
3,5,3’-Triiodothyronine
Plasma
9.49
samples
erythropoietic
were
substance
taming
the
plasma
sample.
4-6
± 1.01
± 1.63
10.31
Testosterone
test
3.99
removed
from
(0
and
time)
substances,
and
Numbers
of
the
at
assayed
dog
perfusion
system
the
termination
the
fasted
in
plasmas
9.70
of
rat.
assayed
prior
the
Five
may
be
.21
to
the
perfusion
rats
(X
were
found
in
of
addition
used
time)
to
figure
2.
before
assay
by
the
termination
the
con-
assay
each
time
was
dialyzing
at
X
hours.
difference
from the saline
controls.
difference
between
the 0 time
and
D =
cobalt
was removed
from
a portion
4 C. against
Dowex-50
ion exchange.
ND = plasma
samples
containing
cobalt
fusion
experiment
without
dialysis.
L’
=
2
=
±
standard
=
error
of
the
#{176}In&cates significantly
control
samples.
of the
were
plasma
assayed
at
of the
per-
mean.
different
at
the
5 per
cent
level
from
the
respective
0 time
plasma.
extracted
erythropoietin
the
X time
numbers
of the
from
of erythroid
dog,
indicate
sheep
plasma
precursors
that
produces
in the
erythropoietin
an
isolated
stimulates
absolute
perfused
the
increase
femoral
bone
marrow
in
marrow
by
a direct
action.
This
on
is apparently
in the
lion,
stimulates
by this organ.8’9
bating
bone
concluded
either
the kidney
Barron
et
However,
directly
or
cobalt.
In addition,
lion
which
does
synthesis
in vitro.
view
poietin
of
the
have
bone
marrow
directly,
one
Our
first
report
marrow
of a direct
elements.
Warren
et
in
in an inhibition
on the
bone
al.22
were
respiration
in
shown
cultures
would
that
vitro
findings
to
with
stimulate
in vitro.
expect
to
cobalt
that
unable
to
marrow
Therefore,
see some
stimulates
cobalt.
heme
the
Plasmas
if cobalt
change
in
the
bone
the
use
mitotic
erythrorates25
stimulates
erythropoiesis
bone
marrow
cultures
marrow
of
in a concentradepresses
heme
with
cobalt
in
containing
and
and
anoxic
a depression,
with
cobalt,
actually
our results
synthesis24
respiration,
by an
produce
that
in addi-
erythropoietin
added
to incu-
was
of cell
marrow
of cobalt
cobalt,
of
of
Laforet
et al.23 demonstrated
not influence
cellular
respiration,
It is very
difficult
to interpret
been
influence
clear
directly
to increase
production
al.2’ found
that
when
cobalt
indirectly,
above
finding
stimulatory
It seems
marrow
in vitro,
it resulted
that
cobalt
acted
directly
mechanism.
vitro.
the
erythropoiesis
directly
in the
in
in
hind
From www.bloodjournal.org by guest on March 6, 2016. For personal use only.
BONE
MARROW
limb
perfusion
other
factors
STUDIES
IN
system
in
95
DOGS
may
plasma
be
related
used
in
upon
erythropoietin
itself
or upon
erythropoiesis
directly.
It would
erythropoiesis
plasma
in
cofactors
that cobalt
erythropoietin
the
isolated
are
absent.
merely
seems
clear
is well
documented,
that
cobalt
A second
action,
on erythroid
effect
Growth
sectomized
with
addition
isolated
the
hind
that
growth
the
isolated
that
has been
found
but does
not
of
the
for
the
in body
that
This
growth
the
production.
maturation
metabolic
action
onset
may
which
is its direct
stimula-
in the
peripheral
hypophyanemia
also
of these
hormones
and as a result
necessary
is necessary
for
growth
intake
thyroid
hormone
isolated
mediated
Another
through
is that their
their
activities
and
on
to stimulate
substrate
testosterone
to
limb
may
blood
detected
that
help
indicate
organ
in the
they
to
ever,
it is interesting
of
action
previously
by
action
other
blasts.
marrows
poietin
The
mature
to suggest
of the
other
than
substances
workers,28
an
effect
from
found
our
data
to be
erythropoietin
on
the
differentiation
several
active
may
in our
induce
explain
formation
in such
have
of stem
one
cells
or
more
in
counts,
mechaHowas
As
into
the
is slow
short-term
possibilities
system.
inbone
signifi-
may
perfusion
experiments.
Very
few
definite
conclusions
may
be drawn
from
erythroid
cell
such
as those
used
in our hind
limb perfusion
system,
concerning
the
nism
and sites of action
of erythropoietin,
cobalt
and growth
hormone.
sites
to
hypothesis
growth
hormone
in the perfused
alterations
in
hormone
the
more
hind
which
be
counts
other
intermediary
of these
hormones
the
influence
cannot
growth
hormone
that
in
finding
a pre-existing
support
perfused
possibility
increase
cell
provides
found
the
Our
that
for
not
food
an
erythroid
indicates
on the kidney
or some
production.
The
action
be
rate.5’27
in
dogs
does
in the
act
increase
in
in
marrows.
It seems
more
likely
of red cell precursors
erythropoiesis
substances
erythropoietin
animal
action,
erythropoietin.
resulted
perfused
finding
increase
stimulate
rat
is not
hyperplasia.
cantly
the
studies
co-workers2#{176} have
also
being
perfused
through
the
normal
as hypophysectomy,
by direct
stimulation.
failure
of hydrocortisone,
intact
and
such
first
report,
and
blood
within
of
limb
marrows
The
in
cells
a slight
red cell
rate
of
that
these
to enhance
The
erythropoiesis
eliminate
the
hypophysectomized
red
produces
or
from
to elaborate
present
Gordon
hormone
to
hind
an increase
erythropoietin
show
actions.
to stimulate
appear
to
hormone
erythropoiesis
depend
sensitive
to a titer of plasma
erythroid
hyperplasia.
It
kidney
in the
or
may
in order
to stimulate
if cobalt
stimulates
when
to
more
produce
the
perfused
such
limb
possible
erythropoietin
Cobalt
cofactor
to know
well-defined
is described
hypophysectomy.
of growth
erythroid
material
creases
two
it stimulates
of nucleated
deficiency,
induce
be
of
system.
tissue.
limb
numbers
presence
hind
may
the bone
marrow
not
otherwise
has
which
hormone
rat’26’27
associated
that
the
perfused
is that
tory
the
perfusion
some
plasma
be interesting
It
renders
would
that
to
the
to
the
outlined
sites
of
pronormo-
increase
seen in the absolute
numbers
of pronormoblasts
in femoral
in our hind
limbs
perfused
with erythropoietin
indicates
that erythrostimulates
the stem
cell to differentiate.
The increase
seen in the more
erythroid
precursors
within
a 4-6-hour
perfusion
time
may
also
mdi-
From www.bloodjournal.org by guest on March 6, 2016. For personal use only.
96
FISHER,
cate that erythropoietin
from
the stage
of
possibly
on through
of more
precise
directly
on
all
growth
may
be
ing
four
finding
and
basophilic
cobalt
seen
cell
to
differentiate
cobalt,
than
on
maturation
effect
in the
numbers
hormone
with
the
primarily
only
but after
elements.
24 hours
Maximum
to explain
after
rapid
why
a slight
an
the
increase
erythroid
may
be
the
bone
explained
creted
by
marrow
in erythroid
the
rats
elements
fact
that
this
removed
can
very
erythropoietin
of
the
is being
time
and
not
that
growth
effect
with
on
erythro-
after
injection,
the erythroid
It is difficult
in the
dog
limbs
The
more
of the dog
recirculated
being
its
change
little
seen
of
cobalt
little
containing
erythropoietmn.
in the isolated
hind
limb
period
from
be
of
indicate
12 hours
in
to have
injected
in erythropoiesis
with
blood
erythropoietin
during
or otherwise
hypertransfused
increase
effect
very
with
an
stem
of action
seems
may
rate
an
an
with
observation
has
the
onset
was noted
which
involved
all of
activity
was observed
at 48 hours.
increase
4-6
hours
perfusion
onset
of action
of
see
of
may
abso-
cobalt
causes
hormone
maturation
increase
to
precursors
This
to accelerate
showed
in polychromat-
that
a lack
Growth
erythroid
of pronormoblasts.
acts
or
intermediate
normoblasts,
tissue.
The
of a slower
cell,
cells.
mature
rate of stem cell differentiation.
Stohiman
et al.29 reported
that
poietin
stem
of
erythroid
cells.
pronormoblasts
which
failure
result
acted
a shorten-
or more
indicates
tissue
The
the
of erythroid
more
growth,
increase
cobalt
erythroid
be
on
rate
on the
has
enhancement
of one
a moderate
on
may
erythropoietin,
apparent
change
in orthochromatic
of cobalt
on the erythroid
pronormoblasts.
normoblasts
greatest
produces
secondary,
into
orthochromatic
the
and
or
erythroid
cells
normoblast
and
studies
with
the use
erythropoietin
This
omission
NIGHTINGALE
of the
of non-mitotic
the
in pronormoblasts
direct
that
AND
mitosis
in the more
mature
increases
the numbers
of
with
very
little
the site of action
either
reveal
types.
times,
COUCH
in growth
orthochromatic
Further
acceleration
additional
markedly
normoblasts
increase
action,
of an
an
may
cell
generation
or
ic normoblasts,
help
to explain
lute
tools
result
that
acceleration
to the
erythrocyte.
erythroid
the
divisions,
The
causes
an
pronormoblast
to the mature
erythrokinetic
of intermitotic
mitotic
the
the
ROH,
through
inactivated,
ex-
system.
SUMMARY
A technic
dog
by
has
been
perfusing
marrows
were
containing
developed
the
perfused
sheep
for
erythroid
taming
bone
marrows
sion
system.
cobalt
in the
and
bone
4-6
counts
erythropoietin
from
It may
growth
marrow
and
limbs
be
hormone
directly.
are
disveloppate
un
technica
femoral
the
donor
growth
cobalt.
growth
capable
was
of stimulating
INTERLINGUA
pro
studiar
le
seen
with
also
in nudeblood
con-
noted
in the
added
to the
that
erythropoietin,
erythroid
medulla
bone
alone
and blood
hydrocortisone,
was
experiments
of the
method,
were
increase
IN
marrow
this
perfused
hormone
these
bone
of
increases
marrows
A slight
from
use
dog blood
hormone,
Significant
femoral
in which
concluded
the
With
hours
with
cobalt,
within
SUMMA.RIO
Esseva
studying
limb.
or testosterone.
cell
sheep
hind
erythropoietin,
3,5,3’-triiodothyronine
ated
for
isolated
del
perfu-
hyperplasia
osso
femoral
From www.bloodjournal.org by guest on March 6, 2016. For personal use only.
BONE
MARROW
del
can
per
methodo,
IN DOGS
perfusionar
medullas
guine
de
ovin,
0
STUDIES
osso
canin
hormon
testosterona.
intra
al
posterior.
durante
o con
augmentos
femoral
perfusion.
A
de
erythroide
le
iste
san-
numerationes
sanguine
a contento
esseva
etiam
de crescentia
iste
de
in le
essite
il pote
crescentia
es
le medulla
in
de
notate
habeva
experimentos
e hormon
directemente
de
con
erythropoietina
in
con
augmento
hormon
base
uso
3,5,3’-tri-iodothyronina,
significative
cobalt,
le
continente
perfusionate
Un leve
in que
erythropoietina,
hyperplasia
sanguine
Con
4 a 6 horas
hydrocortisona,
constatate
de
que
tal
crescentia,
ovin
o cobalt.
ab extremitates
systema
stimular
extremitate
perfusionate
le medullas
erythropoietina
medullas
ossee
concludite
isolate
esseva
sol
de
Esseva
erythroidee
addite
le
de
donatores
cobalt,
97
esser
capace
a
ossee.
ACKNOWLEDGMENTS
The
Mr.
authors
Dan
supply
gratefully
Knight.
of
We
are
for
3,5,3’-triiodothyronine;
the
form
bovine
the
indebted
extracted
Section
soluble
also
erythropoietin
for
the
acknowledge
from
growth
of testosterone
D.
A.
A.
succinate
assistance
USPHS
F.
to
the
Siele,
Mrs.
Judy
Study
USPHS
Smith,
Shepherd,
and
of
Hematology
plasma;
to
Dr.
sodium
the
sheep
hormone;
and
technical
to
and
for
Endocrinology
Kline
The
Parker
Section
and
Upjohn
Co.,
the
Study
French
Labs.,
the
aqueous
for
hydrocortisone.
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From www.bloodjournal.org by guest on March 6, 2016. For personal use only.
1964 23: 87-98
Influence of Cobalt, Sheep Erythropoietin and Several Hormones on
Erythropoiesis in Bone Marrows of Isolated Perfused Hind Limbs of Dogs
JAMES W. FISHER, B. L. ROH, CHARLES COUCH and W. O. NIGHTINGALE
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