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The time of origin of the parabiotic anastomosis.

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The Time of Origin of the Parabiotic Anastomosis'
MARVIN SODICOFF AND ROBERT T. BINHAMMER
DepaTtment of Anatomy, University of Cincinnati, College of Medicine,
Cincinnati, Ohio
ABSTRACT
The course of development of parabiotic anastomosis was followed
with respect to time in Holtzman ( Sprague-Dawley) parabionts. Comparison of nonirradiated pairs was made to pairs in which one of the partners had received 800r.
The development of the anastomosis was studied by determining the per cent transfer
of CR51 labeled erythrocytes (which represented a cellular element) and radio-iodinated
serum albumin (which represented a molecular element) from one partner to the
other. Erythrocytes were found in the non-injected animal in small numbers at 22
hours after pairing; at 47 hours the rate of transfer became much more rapid. RISA
was detected in the non-injected partner as early as five hours after pairing and accumulated steadily thereafter. Irradiation had no effect on the course of development of
the parabiotic anastomosis as evidenced by similarity of accumulation rates of Cr51
labeled erythrocytes and RISA when compared to the non-irradiated pairs.
There is no exact information in the
literature concerning the time of development of the parabiotic vascular anastomosis. The present study was designed to
provide more precise information regarding the early phase in the development of
the parabiotic union.
That an actual vascular anastomosis
does develop was suggested by the work of
Hill ( ' 3 2 ) using a colorimetric determination of the plasma of parabionts after injection of brilliant vital red into the vascular system of one partner. Van Dyke et al.
('48) demonstrated vascular continuity between partners using Fe59tagged erythrocytes. Several investigators have indicated
that the anastomosis becomes functional
between two and four days after union
Anderson ('54) demonstrated the presence
of capillary connections as early as 3 to 4
days after pairing. Van Dyke et al. ('48)
reported almost no exchange of FeS9labeled
erythrocytes until 2 to 3 days after pairing.
The present study' determines more precisely the time of origin of vascular anastomosis with respect to cellular elements, as
represented by erythrocytes, and non-cellular elements, as represented by the protein
serum albumin. It was also of interest to
determine whether or not irradiation of
one animal of a pair would alter the development of the anastomosis.
MATERIALS AND METHODS
The experimental animals were female
rats of the Sprague-Dawley strain (Holtz-
man) weighing 160-180 gm. Animals
were maintained at a temperature of 24°C
and fed a diet of Purina laboratory chow
pellets and tap water ad libitum.
Rats of similar weight were united surgically under pentobarbital anesthesia using
the technique of Bunster and Meyer ('33).
In those experiments in which one of the
partners was irradiated, the animals were
exposed two hours prior to pairing. Three
or four unanesthetized rats were placed in
a ventilated plastic box 15 X 15 X 15 cm
for irradiation. Animals received a single
800 r dose directed to their dorsums which
in this laboratory represents an LD 60/30
days. Radiation was administered with a
250 KVP machine using 250 KV, 15 ma,
0.5 mm Cu and 1.0 mm Al, HVL 1.7 mm
Cu, STD 70 cm, at a dose rate of about
67 r/min. The machine was calibrated before each exposure using a 100 r Victoreen
ionization chamber in a paraffin phantom.
Cross-circulation was studied by following NaCP04 tagged red blood cells in
some pairs and radioiodinated serum albumin (RISA) in different pairs. Erythrocytes from donor rats were tagged in vitro
after separation by centrifugation. Approximately 8 wc NaCrS1O4/mlwhole blood was
added to the erythrocytes and incubation
was carried out at room temperature for
30-45 minutes using gentle agitation. At
the end of the incubation period 1 ml of
1 This inyestigation was supported by a Public
Health Service Fellowship (GPM 19,030) from Natlonal
Institute of General Medical Sciences and by grant
CA-03390from the National Canc
Cancer Institute.
625
MARVIN SODICOFF A N D ROBERT T. BINHAMMER
BINHAMMER
626
50
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2.
30-
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c
r
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10-
-
0
0
10
20
1
30
I
I
I
40
60
50
Hours after pairing
I
I
I
i
70
80
90
100
Fig. 1 Erythrocyte and pIasma transfer at various sampling intervals after pairing.
Vertical lines on the curves denote standard error. Label was injected at time of pairing.
ascorbic acid (50 mg/ml) was added to the
incubation mixture. Cells were washed
twice with normal saline and resuspended
in saline; 1.0 ml of this preparation was
injected into the tail vein of one member
of each pair immediately after pairing. In
those pairs in which one animal had been
irradiated the non-irradiated partner was
injected. In those pairs in which RISA was
used, 7-8 vc diluted in 0.5 ml saline was
injected into the tail vein immediately
after pairing. It was assumed that immediately after injection 100% of the activity
resided in the injected animal.
Development of the cross-circulation
was determined by sampling the blood of
each animal of a pair at several intervals
after pairing and determining the per cent
of available label that had transfused into
the non-injected animal. This was done
by determining the radioactivity present in
equal volumes of blood from each animal
corrected for hematocrit differences. No
correction for blood volume was applied
since the paired animals were about of
equal weight and the blood volumes were
therefore considered to be similar. The
per cent of activity transferred was therefore represented by the CPM in the blood
sample of the non-injected animal divided
by the total CPM of both samples. Approximatelv 0.3 ml of blood was drawn into
heparhized syringes from cardiac punctures. Aliquots were then taken for micro-
10
30 50
70
Hours after pairing
90
Fig. 2 Erythrocyte and plasma transfer at
various sampling intervals after pairing plotted
on a probability scale.
627
PARABIOTIC ANASTOMOSIS
Hours after pairing
Fig. 3 Comparison of erythrocyte and plasma transfer in the irradiated (one partner
received 800 r) and non-irradiated pairs.
hematocrits and 0.1 ml or 0.2 ml samples
of the blood were used for determinations
of radioactivity present. The activity of
each sample was determined in a deep well
chamber using a Nuclear-Chicago Scaler
Model 186. Cross-circulation was followed
with CrS1tagged erythrocytes in 15 nonirradiated pairs and in 14 pairs in which
one member was irradiated. Transfer of
RISA was followed in 15 non-irradiated
pairs and in nine pairs in which on? partner had received radiation.
RESULTS
The accumulation of tagged erythrocytes
and serum albumin in the non-injected
partner of non-irradiated pairs is shown in
figure 1. CrS' was f m t detected at 22 hours
after pairing in very low amounts (0.04 t
0.01% ). Cr"' label accumulated slowly, so
that at 47 hours after pairing only 2.1 t
0.46% of the label had been transferred.
From this point on the label transferred
to the non-injected animal at a faster rate,
as indicated by the steeper slope, 8.4 t
1 . 5 % , 27.5 2 2.9%, 34.0 -+- 4.0%, and
47.4 2 1.5% at 54, 70, 77 and 95 hours,
respectively.
At the earliest sampling interval after
pairing, five hours, Ii3' was found in small
quantities in the non-injected animal (0.27
2 0.06% ). The label thereafter accumulated steadily, 3.7 2 0.4%, 8.3 t- 0.8%,
15.9 0.9%, 17.9 2 1.3%, 30.6 2.1%,
and 44.2 2 1.5% at 22, 31, 46, 53, 70 and
94 hours, respectively. When the data were
*
plotted on a normal probability scale (fig.
2 ) , the points in the case of erythrocytes
as well as RISA appeared to have a linear
distribution. The differences in accumulation of labeled erythrocytes and labeled
serum albumin were statistically significant at 22, 32, 47 and 54 hours ( P <
0.001).
In those pairs where one partner was
irradiated, the pattern of the transfer of
the label was not significantly different
from that exhibited by those which received no irradiation (fig. 3).
DISCUSSION
The shape of the curve showing the
accumulation of the Cr5' labeled erythrocytes (fig. 1 ) into the non-injected animal
was clearly sigmoid indicating that development of the vascular anastomosis followed a normal growth pattern. Although
the shape of the curve for RISA accumulation was not as clearly sigmoid (fig. l ) ,
the fact that both curves showed a linear
distribution when plotted on a probability
scale indicated that transfer of RISA as
well as tagged erythrocytes described a
similar pattern of vascular inosculation.
Transfer of the CrS1label began later
after pairing than did RISA, which was
found in the non-injected partner as early
as five hours after surgical union. Appearance of RISA in the non-injected animal
at such a short time after pairing suggested
that its early transfer, at least, may not
have been by va.scular anastomosis but
628
MARVIN SODICOFF AND ROBERT T. B I N H A M M E R
rather by diffusion. Schiff and Plagge (‘55)
have suggested that relatively small molecules might diffuse from one animal to
the other rather than cross via blood vessels. Jacobsohn (’48) also maintained that
parabiotic exchange was by non-vascular
pathways and suggested diffusion into the
lymphatics. Albumin, the smallest of the
plasma proteins, escaped through capillaries in large quantities (Wasserman and
Mayerson, ’ 5 2 ) , and was returned to the
blood via lymphatic vessels. Injury to
capillaries increased their permeability and
allowed increased protein leakage. Incision
of the skin and lateral body wall at the
time of surgical parabiotic union certainly
severed lymphatics, capillaries and larger
blood vessels and allowed blood and lymph
to escape into the tissue spaces. Since it
has been shown that cut lymphatics may
remain open for 24-48 hours (McMaster
and Hudock, ’34), protein could have
continued to enter the interstitial fluid
for this period of time. It was this fluid,
at the areas of surgical union, which
was probably absorbed by the uninjured
lymphatics of the other animal, thus
resulting in transfer of the labeled protein at earlier time intervals than tagged
erythrocytes. Eichwald et al. (’59) using
injections of Evans blue immediately after
surgery found that the dye passed to the
non-injected animal one and two days after
surgery. However, on these days the dye
was limited to the region of the suture line
and did not gain entrance to the vascular
system. He found it wasn’t until the third
day after pairing that the dye could be
transmitted systemically. In contrast, the
present work with tagged albumin indicated that it entered the vascular system
within hours after surgery.
Erythrocyte transfer proceeded very
slowly until approximately 48 hours after
union when a more rapid phase was initiated. These results suggested that during
the first 47 hours after pairing only a few
and/or small vascular channels were present, and after 47 hours the vessels of the
capillary anastomosis had increased sufficiently in number and size to permit a
rapid transfer. RISA did not give such a
clear illustration of the development of the
anastomosis, for it was not possible to
detect any point in the transfer of RISA
in which there was a sudden marked increase in transfer rate. The difference in
transfer of erythrocytes and RISA was
attributed to the physical characteristics
of the labeled particle. The erythrocyte
requires a vascular channel of greater size
than does the albumin molecule. On the
basis of the work of Clark (’18), who described the formation of new capillaries as
originating as solid endothelial sprouts in
which a lumen forms and then widens
enough to permit circulation of blood cells,
one would have anticipated earlier transfer
of the small particle through the new anastomoses. The rather abrupt rise in rate of
transfer of erythrocytes after 48 hours can
thus be interpreted as indicating the time
in the development of the capillary anastomosis when lumens of the newly developed
capillaries have attained sufficient size to
permit passage of erythrocytes. RISA
showed no similar abrupt increase in rate
probably due to its having a virtually nonexistent “threshold size.” In the later phase
of the development of the anastomosis any
diffusion of RISA via tissue fluid to lymphatics of the non-injected partner would
probably be insignificant as compared to
the vascular capillary transfer. This is supported by the fact that equal cross-circulation times for erythrocytes and albumin
were found in pairs with well established
capillary anastbmosis (Binhammer and
Hull, ’62).
Irradiation of one animal of each pair
did not significantly change the development of the parabiotic anastomosis. The
accumulation of Cr51 labeled erythrocytes
and RISA in the non-injected (non-irradiated) partner followed much the same pattern as in those pairs where no irradiation
was administered. This might support the
work of Pohle et al. (’49) who found that
skin irradiated with less than 1,000 r prior
to its incision healed at a normal rate.
ACKNOWLEDGMENT
The authors gratefully acknowledge the
fine technical assistance of Mrs. Marvin
Sodicoff and Mrs. Paul Joyce.
LITERATURE CITED
Andresen, R. H. 1954 Cross circulation and tissue reaction in parabiosis. Arch. Path., 58:
455474.
PARABIOTIC ANASTOMOSIS
Binhammer, R. T., and J. K. Hull 1962 Cross
circulation in normal and intoxicated parabionts. Proc. SOC.Exper. Biol. and Med., 2 2 :
134-139.
Bunster, E., and R. K. Meyer 1933 A n improved method of parabiosis. Anat. Rec., 57:
339-343.
Clark, E. R. 1918 Studies on the growth of
blood vessels in the tail of the frog larva. Am.
J. Anat., 23: 37-88.
Eichwald, E. J., E. C. Lustgraaf and M. Strainer
1959 Genetic factors in parabiosis. J. Nat.
Cancer Inst., 23: 1193-1213.
Hill, R. T. 1932 Blood exchange and hormonic
reactions in parabiotic rats. J. Exp. Zool., 63:
203-234.
Jacobsohn, D. 1948 On the mode of action of
ovarian hormones on growth and development
629
of the mammary gland. Acta Physiol. Scandinav., 17: Supp. 57.
McMaster, P. D., and S. Hudock 1934 The participation of skin lymphatics in repair of the
lesion due to incision and burns. J. Exper.
Med., 84: 473494.
Pohle, E. A., G. Ritchie and W. W. Moir 1949
Studies of the effect of roentgen rays on healing
of wounds. Radiology, 52: 707-713.
Schiff, G. J., and J. C. Plagge 1955 Use of
fluorescein in testing the union of parabiotic
rats. Proc. SOC. Exper. Biol. and Med., 88:
559-561.
Van Dyke, D. C., R. L. Huff and H. M. Evans
1948 The efficiency of the vascular union i n
parabiosis. Stanford M. Bull., 6: 271-275.
Wasserman, K., and H. S. Mayerson 1952 Dynamics of lymph and plasma protein exchange.
Cardiologia, 21: 296-307.
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