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Prolonged survival of fragments of liver transplanted to an ectopic site in the mouse.

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Prolonged Survival of Fragments of Liver
Transplanted to a n Ectopic Site
in the Mouse
MARY J. SELLER
Paediatric Research Unit, Guy’s Hospital Medical School,
Loiidon, S . E . 1 .
Fragments of liver were successfully transplanted to an ectopic
site in the kidney cortex of the mouse. Survival was followed for varying periods
of time. In some cases both syngeneic grafts and allogeneic grafts placed in mice
previously made immunologically tolerant to the genotype of the donor were still
alive one year later. However, extensive amounts of liver tissue would not survive, at the best, strips of about 30 cells thick grew, and various measures tried
to promote an increase in this amount were not successful.
ABSTRACT
Transplantation of small grafts of liver
to ectopic sites has been easily achieved in
invertebrates such as the garden snail, and
in some of the lower vertebrates, for example the roach, toad, lizard (Grigorev,
’SS), the newt (Williams, ’61) and the
bullfrog (Koppanyi and Hsi Chung Chang,
’ 2 5 ) , but in mammals, such attempts usually have been unsuccessful. Indeed, as recently as 1970, Tavassoli and Crosby
stated categorically that “fragments of liver
tissue cannot be maintained in ectopic
sites” following a series of experiments in
the rat. They had transplanted autologous
liver to several sites: under the skin, in
the kidney cortex, and also back into the
liver, and had found a similar fate for each
transplant. There was necrosis of the liver
cells, a polyniorphonuclear leucocyte invasion and an eventual replacement by
connective tissue.
In this paper, I wish to report the results of experiments in the mouse, in
which, using a technique devised by
Wheeler, Corson and Dammin ( ’ 6 6 ) , I
have successfully transplanted liver tissue
to a n ectopic site, with survival in some
cases for one year - the longest period of
observation so far.
Wheeler et al. (’66) showed that a bed
cut in the kidney cortex of the mouse
proved to be an extremely satisfactory
site for the survival of syngeneic grafts of
spleen, lymph nodes, thymus and kidney.
They also transplanted liver to this posiANAT. REC., 172: 149-156.
tion, but only assessed its survival for 28
days, and although there were some viable
parenchyma cells present at that time,
there were also areas of necrosis, and so
there was no certain indication as to the
eventual fate of the graft.
For the purposes of other experiments
(the results of which will be published a t
a later date), I sought to get grafts of liver
fragments to survive for long periods, and
also to transplant successfully allogeneic
a s well as syngeneic tissue.
MATERIALS AND METHOD
Mice. For the syngeneic experiments,
the mice used were a pure line strain of
CBA/H mice. Each donor and recipient
pair were of the same sex, and in all, 56
mice were grafted. For the allogeneic
transplantation, the donors were the
CBA/H pure line mice, and the recipients
were mice of the W-series - either the
genotype Wv/+ which are slightly anaemic, or
completely normal. Donors
and recipients were matched for sex. There
are several ways of overcoming the immunological barrier in mice and one is to
render the recipient mice tolerant to the
genotype of the prospective donor before
grafting. In this experiment this was
achieved by injecting the W mice at birth
with 5 X lo6 CBA lymphoid cells (Seller,
’68). When these mice were adult, they
were challenged with a CBA skin graft,
+/+,
Received July 20, ’71. Accepted Sept. 20, ’71.
149
150
MARY J. SELLER
TABLE 1
and those which accepted the skin were
classified as having been rendered im- Results of transplanting syngeneic liver t o a n
ectopic site in the kidney cortex
munologically tolerant, and were then used
of the mouse
in the liver grafting experiments. Fifteen
Number with
of these mice were transplanted and will
viable liver tissue
Time after transplant
be referred to as “allogeneic tolerant” mice. Number transplanted
when graft was assessed
As controls, four completely untreated, and
28
therefore immunologically competent W
4 weeks
a
mice, also received a liver graft. These are
4
allogeneic grafts where no attempt has
5 weeks
6
been made to immunosuppress the recipients.
4
6 weeks
Thus there are three groups of mice,
4
the syngeneic group where there are no
5
8 weeks
immunological complications, and sur6
vival of the graft should depend on the
5
effectiveness of the transplantation tech6 months
6
nique ; the allogeneic tolerant series where
an attempt has been made to overcome the
4
1 year
4
immunological barrier, and graft survival
should be similar to the first group; and
the truly allogeneic group where persistTABLE 2
ence of the graft should be determined by
Results of transplanting allogeneic h e r to an
the immunological capabilities of the reectopic site in the kidney cortex of mice
cipient, regardless of the efficacy of the
previously made immunologically tolerant
transplantation technique.
Number with
Liver grafting. All the mice underwent
viable liver tissue
Time after transplant
when graft was assessed
a similar operation, and the technique was Number transplanted
exactly as described by Wheeler et al.
3
4 weeks
(’66). Under ether anaesthesia, and using
4
semi-sterile precautions, the recipient kid1
ney was exteriorised and the blood vessels
3 months
2
clamped. A graft bed was cut in the cortex.
1
A sliver of the donor liver, no more than
4 months
1
1 mm thick, was placed on the bed, and
held there for about thirty seconds for ad1
5 months
hesion to take place between the cut sur1
faces of the two tissues. The kidney and
3
graft were then gently restored to the
7 months
5
peritoneum, after removal of the clamp.
Assessment of graft survival. Initially,
2
1 year
mice were killed after four weeks, and the
2
kidney on which the grafting had been
performed was removed. It was inspected
macroscopically and then fixed in formalin,
Fig. 1 Healthy liver graft (left) on allogeneic
sectioned, and stained with haematoxylin kidney (right). The graft has survived for seven
and eosin and examined under the light months, the recipient previously having been
tolerant to the donor genotype. X 238.
microscope. As a result of good graft sur- rendered
Fig. 2 Syngeneic liver graft of two months
vival at this time, other mice were left duration on the kidney ( K ) . Two mitoses
for longer intervals before the condition of (arrowed) are visible in the liver ( L ) . X 467.
Fig. 3 Syngeneic liver graft ( L ) on kidney
their grafts was assessed. Good graft survival was continually achieved, and so ( K ) at one year. The macrophages in the liver
have engulfed particles (arrowed) from Indian
eventually i t was decided to terminate the ink
which was injected several hours before
experiment one year after the transplant. sacrifice. x 467.
LIVER FRAGMENTS TRANSPLANTED ECTOPICALLY
Figures 1-3
151
152
MARY J. SELLER
This would often extend much of the
lcngth of the kidney: the thickness varied
from about seven parenchyma cells to 3
maximum of about 30 cells. Unfortunately,
n m e of the measures resorted to, in an
attempt to encourage proliferation of the
liver cells, such as partial hepatectomy of
the recipient, was successful in increasing
the thickness or overall volume of the
graft.
The detailed microscopic picture of the
fate of the grafts with the passage of time
was the same in both the syngeneic and
“allogeneic tolerant” mice. One month
after grafting, there were many viable
liver cells, but also present were browncoloured, necrotic cells. These were found
especially along the cut edge of the liver
where it was apposed to the kidney (fig.
5). Small areas of these necrotic cells were
also within the graft. The grafts sometimes contained a few mononuclear cells,
and small foci of polymorphonuclear
leucocytes. At two to three months there
RESULTS
were fewer necrotic liver cells, which had
Tables 1 and 2 show the numbers of been further reduced by six to seven
mice in which there was healthy and viable months to a small cluster at the terminal
liver tissue in the kidney bed, when as- ends of the graft. However, by this time
sessed at various time intervals from four in some cases, they were completely absent.
weeks to one year after grafting, in both The junction between the kidney and liver
the syngeneic combination and when an was marked in some cases by a dense
allogeneic graft was introduced into mice band of mononuclear cells, which was repreviously made immunologically tolerant. duced as time went on, but they were nor
In all, 50 out of 56 (89% 1 syngeneic always present. By one year after the
grafts were successful, and 11 out of 15 transplant there was no necrosis of the
(73% ) “allogeneic tolerant” grafts sur- liver cells and no mononuclear cells where
vived. Thus, consistently in both combina- the liver and kidney cells were apposed.
tions there was a high degree of prolonged Occasional grafts had very small pockets
engraftment of fragments of liver tissue
Fig. 4 Allogeiieic liver grafted onto a n imin this one ectopic site (see fig. 1 ) . Bear- munologically
competent recipient one month
ing in mind the healthy appearance of previously. The graft is being rejected. The kidgrafts which had survived for one year ney bed ( K ) has a layer of connective tissue
(fig. 3 ) it is tempting to suggest that such ( C ) between it and the degenerating liver cells
(D). Mononuclear cells (arrowed) are present
grafts may last permanently.
the liver cells. X 467.
Macroscopically, on removal of the kid- zround
Fig. 5 The state of the liver graft a t one
ney and its graft from the animal, there month i n a syngeneic recipient. The liver tissue
was no definite sign of the presence of any ( L ) is healthy except at the interface with ihe
liver tissue. The site of the possible graft kidney ( K ) where there i s a line of degenerating
( D ) . X 297.
could be identified by a depression in the cells
Fig. 6 The picture one month after graftinq
kidney cortex where the bed had been cut, which was recorded as having been “unsuccessbut the presence or absence of grafted liver fully grafted.” It is thought that the graft became
could not be determined until microscopic separated from the kidney soon after the graftprocedure. There is no evidence on any decxamination. Then, it could be seen that ing
generating liver cells or cellular infiltration. Cona successful graft comprised a very thin nective tissue ( C ) h a s been laid down on the
strip of liver tissue apposed to the cortex. kidney bed ( K ) . x 467.
Other procedures. In view of the
acknowledged great proliferative ability of
the liver, attempts were made in some
mice to enlarge the size of grafts by various means. The measures tried were: ( 1 )
the liver graft was taken 72 hours after
the donor had been injected subcutaneously with 0.2 ml of 40% carbon tetrachloride in olive oil; ( 2 ) the donor liver
was taken from newborn, or 21 day old
juvenile mice; ( 3 ) the recipient was partially hepatectomised after i t was estimated
that the graft should have taken; (4) similar recipients were injected intravenously,
with serum from mice partially hepatectomised two days previously.
In addition, in order to see whether the
macrophage elements of the liver graft
were functioning normally, two mice
which had borne grafts for one year were
injected intravenously with a solution of
Indian ink in saline several hours before
sacrifice.
LIVER FRAGMENTS TRANSPLANTED ECTOPICALLY
Figures 4-6
153
154
MARY J. SELLER
of polymorphonuclear leucocytes at various
times after one month, but there was never
a large or consistent cellular infiltration
of the grafts.
These observations were in marked contrast to the allogeneic control mice which
were immunologically competent to reject
the tissue. After one month the whole graft
was densely invaded by mononuclear cells.
Nearly all the liver cells were degenerating,
although there were a very few parenchyma
cells which appeared to be viable. A layer
of connective tissue was present on both
sides of the graft (fig. 4).
In turn, this picture of rejection contrasted with the situation in several of the
syngeneic and “allogeneic tolerant” mice
which were recorded as being unsuccessrully grafted. For, in all cases examined,
at one month, the graft site bore no sign
of liver cells, degenerate or otherwise,
there was only a thin layer of connective
lissue present. Mononuclear cells were
present in the cut layer of the kidney cortex, but they did not extend into the connective tissue (fig. 6 ) . It is thought that
in these cases; the graft did not adhere
properly to the kidney, and was displaced
shortly after the kidney was restored to
the peritoneum. In one case, noted as a
failure in the “allogeneic tolerant” series
at three months, the whole kidney was
atrophied. Possibly, the blood supply had
not been effectively restored after clamping
for the grafting procedure.
Despite the fact that large scale proliferation of the graft could not be encouraged by any of the means tried, it was
common to observe at least one mitosis in
a graft (fig. 2). This was so, even a year
after the transplant.
Following the injection of Indian ink
into a mouse several hours before sacrifice,
black particles from the ink were found
in the cytoplasm of the macrophages of
both the recipient’s own liver and also in
those of the liver graft (fig. 3 ) .
DISCUSSION
Successful and prolonged survival of
fragments of liver tissue can be achieved
if they are transplanted in close apposition
to the cut surface of the renal cortex. The
extreme vascular nature of this area must
be of paramount importance in the success of grafts of tissues, such as the liver,
which require constant oxygenation. The
fact that so few cells survived compared
with the size of the initial graft may reflect this, as the survivors could be those
cells originally placed nearest to the blood
supply in the graft bed. Organs less vulnerable to anoxia fare better (Wheeler et al.,
’66; Tavassoli and Crosby, ’70; Seller, unpublished) : I have grafted spleen successfully and permanently many times, the size
of the graft is comparatively large, usually
about one-fourth the size of the recipient
kidney.
However, despite the fact that so few
liver cells survive, the ones which do seem
to be quite viable and healthy. The evidencc
for this in the present experiments is
taken from the regular observation of
parenchymal mitoses in the grafts, and the
fact that the macrophages phagocytose
particles from Indian ink quite normally.
There is no certainty whether the macrophages are the ones contained in the original graft or whether they have immigrated
from the host, since it is known that there
is a turnover of macrophages in the liver.
Nevertheless, whatever their derivation, the
fact that they are functioning normally
by phagocytosing particles suggests that
they are in a favourable environment.
The finding that none of the ways tried
would encourage the proliferation of the
liver graft was disappointing, and limits
certain possible applications for the technique in the future (Seller, in preparation). It does, however, emphasize how
complex the regulatory system is for liver
growth and regeneration.
The reason for the unsuccessful grafting
in a minority of cases in the syngeneic and
“allogeneic tolerant” mice was thought to
be due to the graft becoming detached
from the kidney soon after it was replaced
in the peritoneum, rather than other possibilities such as the whole graft dying
from anoxia, or a rejection process occurring. For, in each case, no necrotic liver
cells were seen in the graft bed - there
was only connective tissue present, and a
true rejection reaction with massive liver
cell necrosis and mononuclear cell infiltration was seen only in the allogeneic trans-
LIVER FRAGMENTS TRANSPLANTED ECTOPICALLY
plants to immunologically competent mice.
However, no observations were made during the early days after the transplant to
prove this point.
Leduc and Wilson ('63) also got small
syngeneic implants of liver tissue to survive in the mouse by transplanting them
to the spleen; however, unlike the present
work they were unsuccessful with adult
liver, succeeding only with foetal and
neonatal liver. They too obtained long lasting survival, but did not observe any proliferation of the grafts.
ACKNOWLEDGMENTS
This work was supported by The
Spastics Society and the M.R.C. Miss A. R.
Harcourt is thanked for able technical assistance, and Mr. L. Kelberman for help
with photography.
155
LITERATURE CITED
Grigorev, N. I. 1963 Changes in transplantcd
hepatic tissue in various vertebrate animals.
Trudy Ieningr. sanit. gig. med. Inst., 75:
141-156.
Koppanyi, T., Hsi Chung Chang 1925 Experiments on liver transplantation. Endocrinol.,
9: 513-514.
Leduc, E. H., J. W.Wilson 1963 Production of
transplantable hepatomas by intrasplenic implantation of normal liver i n the mouse. J. Natl.
Cancer Inst., 30: 85-95.
Seller, M. J. 1968 The transplantation of allogeneic haemopoietic tissue in adult anaemic
mice of the W-series previously made immunologically tolerant. Transplant., 6: 856860.
Tavassoli, M., and W. H. Crosby 1970 The
fate of fragments of livar implanted in ectopic
sites. Anat. Rec., 166: 143-152.
Wheeler, H. B., J. M. Corson and G. J. Dammin
1966 Transplantation of tissue slices i n mice.
Ann, N.Y. Acad. Sci. 129: 118-129.
Williams, D. D. 1961 Homografting of liver
in the newt Triturus viridescens. Transplant.
Bull., 27: 100-103.
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