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The effect of variation in the age of the donor on homologous grafts into the brain of the rat.

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The Effect of Variation in the Age of the Donor on
Homologous Grafts into the Brain of the Rat’
GAIL S. CROUSE
Division of A n a t o m y , H a h n e m a n n Medical College,
Philadelphia, Pennsylvania
In mammals homografts do not survive
permanently, although in certain transplantation sites they may flourish to a
greater degree (in terms of facility of
establishment and survival time) than in
others. The brain is such a favorable site.
Successful homotransplantations of embryonic tissue to the brain have been reported by Willis (’35,’36,’58), Clark (’40),
Glees (’41) and Tansley (’46). Slightly
less favorable results have been obtained
in the investigations of Siebert (’28), Sakurane (’29), Athias and Guiniarais ( ’ 3 3 ) ,
Medawar (’48) and Glees, Mohiuddin and
Smith (’49) with the utilization of adult
tissues. The author demonstrated (’56)
that a variety of embryonic tissues of the
rat (stomach, duodenum, lower small intestine, pancreas, gonads, limb bud) grew
and differentiated well when homotransplanted into the brain. In addition, many
grafts were still healthy at 90 days of age
and heterologous limb bud (mouse) grew
and differentiated with almost equal
facility.
These results were on transplants in
which the ages of the donor tissues and
host were constant, i.e., embryonic (usually 15 days) and young postnatal (usually 7 days), respectively. The effect of
variation in the age of the host and donor
tissue on the success of such transplants
was, therefore, of interest. Several series
of homotransplants to the brain were made
in which various age combinations of host
and donor were used.
MATERIALS AND METHODS
Non-inbred, unconditioned rats of the
Sherman strain were used. Embryonic
donor tissue of a given age was obtained
by observing the time of appearance of
spermatozoa in vaginal smears. Additional
donor tissue was taken from postnatal rats
of known age. Littermates were never used
as host and donor.
Since stomach was one of several organs which previously had grown and differentiated well as an implant to the brain,
it was selected arbitrarily as the constant
tissue type to be implanted. The stomach
was removed from the donor animal and
placed in Tyrode’s solution. Small pieces
(less than 1 mm3) were dissected free and
transplanted. After an incision had been
made in the scalp, one of the small stomach fragments was injected, by a number
22 needle, through the skull into the posterior portion of the right cerebral hemisphere of each host. In older hosts with
ossified skulls a hole was drilled in the
skull to accommodate the needle. The
scalp was then closed by an application
of thin celloidin.
One hundred thirty-four transplants were
made. The following basic combinations
of host-donor ages were used: young
donor ( 15 days embryonic)-old host (90
days postnatal); old donor (90 days postnatal)-young host ( 7 days postnatal);
young donor (selected progressive ages
beginning at 15 days embryonic)-young
host ( 7 days postnatal).
All animals were sacrificed by etherization on the 25th postoperative day. That
portion of the brain containing the graft
was fixed in Heidenhain’s “Susa” mixture.
After 24 hours of fixation the specimens
were dehydrated, cleared with amyl acetate and embedded in paraffin. Serial sections, 10-15 micra thick, were cut,
mounted and stained using the Masson trichrome technique.
Supported by the Pender Estate Fund and
an Institutional Research Grant, American Cancer Society.
215
216
GAIL S. CROUSE
Control slides, consisting of sections of
normal stomach taken from 15-day-oldembryonic and 7- and 75-day-old postnatal
rats, were prepared by the same staining
method.
RESULTS
Figures 1, 2 and 3 are photomicrographs
of sections of normal rat stomach of the
respective ages given above and are representative of the ages of the donor stomachs
used in the transplants. Such control
sections were useful not only in comparing
the morphology of the recovered grafts
with that of normal stomach but also in
determining the amount of growth and
differentiation which occurred in the tissue after grafting. Both adult and embryonic tissues were transplanted. Successful grafting of the former infers establishment, growth and maintenance; equal suc-
cess with the latter requires, in addition,
differentiation of the mural layers and
specialized cell types of the stomach.
Three criteria for designation of a graft
as positive or successful were used: ( 1 )
persistence for 25 days, ( 2 ) no evidence of
degeneration and ( 3 ) organization in the
graft. Thus, depending on whether the
donor tissue was adult or embryonic, positive grafts regenerated or differentiated the
mucosa, submucosa and muscularis which
were oriented in normal sequence. However, tube formation was not always present, in which case the mucosa was external
surrounding a core composed of the remaining layers. Illustration of sections
of transplants classified as positive are
presented in figures 4, 5 and 6.
Conversely, grafts were designated negative if degeneration was present, if the
TABLE 1
N u m b e r and p e r cent of positive results (“takes”) within cuch host-donor age combination
Host-donor age
combinations’
H.
7d.
90d.
90d.
15 d.2
D.
H.
D.
H.
D.
H.
7d.
15 d.2
7d.
17 d.2
D.
H.
7 d.
D. 19d.*
H.
7d.
D. 21 d.Z
H.
7d.
D. 22d.Z
Positive
Negative
Total
Per cent
positive
0
25
25
0.0
8
16
24
33.3
7
1
8
87.5
6
8
14
42.9
2
6
8
25.0
5
7
12
41.6
5
12
58.3
27
54
50.0
4
5
9
44.4
0
10
10
0.0
2
4
6
33.3
6
6
0.0
7
Totals: embnjonic donors-7
27
H.
D.
H.
D.
H.
D.
H.
D.
7d.
1 d.
7d.
2d.
7d.
5d.
7d.
7d.
0
Totals: postnatal donors-7
6
Grand tot a1
day hosts
day hosts
25
31
19.4
93
134
30.6
41
IAbbreviations: H., host; D., donor; d., days.
Embryonic.
2
VARIABLE DONOR AGE IN HOMOGRAFTING
constituent tissues were disoriented in
comparison with those of normal gut wall
or if the graft was a small focus of epithelial, connective or necrotic tissue. Figures 7, 8 and 9 exemplify negative grafts.
Data based on a total of 134 transplants
are summarized in table 1. Results of
0.0% positive were obtained with a n initial
group of transplants in which young hosts
( 7 days postnatal) received old donor tissue (90 days postnatal). In a reciprocal
group, young donor tissue ( 1 5 days embryonic) was transplanted into old hosts
(90 days postnatal); 33.3% of this group
was positive. When young donor tissue
( 1 5 days embryonic) was paired with
young hosts ( 7 days postnatal) the positive results reached 87.5%. This figure
verified the results of previous work
(Crouse, '56) in which the embryonic
donor-young postnatal host combination
was used exclusively in a larger number
of grafts with similar results. Since young
donor tissue was accepted by both young
and old hosts but old donor tissue did not
grow even in a young host, the possibility
arose that the age of the donor was more
important for successful transplants than
than of the host.
Consequently, a series of transplants
was carried out in which the host age
was always 7 days, while the age of the
donor tissue was progressively increased
from 15 days embryonic to 7 days postnatal. 'Table 1 shows that there was a n
initial drop in the positive results (87.525% ) as the donor age increased. However, with donor tissue of 21 and 22 days
embryonic and one day postnatal transplants were more successful (41.6-58.3% ) .
The remaining donor age groups yielded
the following results: at two and 7 days
postnatal-0.0% ; at 5 days postnatal33.3%.
Finally, table 1 shows totals and per
cent positive results on this series in two
collective groups : embryonic donors,
50.0% ; postnatal donors, 1 9 . 4 % .
DISCUSSION
Under standardized experimental conditions, the number of positive results
among homografts of rat stomach to the
brain varied with the ages of the host
217
and donor (table 1). The results indicated
that the optimum host-donor age combination was embryonic donor tissue (15 days)
paired with young postnatal hosts ( 7
days). The poorest combination was old
postnatal donor tissue (90 days) with
young postnatal hosts ( 7 days). Young
embryonic donor tissue (15 days) paired
with old postnatal hosts ( 9 0 days) was
a n intermediate age combination in respect to positive results. It is noteworthy
that young hosts ( 7 days) became more
refractory to grafting with increasing donor age ( 1 5 days embryonic to 7 days postnatal). The significance of age is even
more strongly emphasized by the collective
data shown in table 1. When embryonic donors were paired with 7 day hosts,
50.0% of the transplants were successful.
However, when postnatal donors were similarly paired with 7 day hosts, only 19.4%
were successful. If the one-day-old donors
were not included i n the postnatal group,
positive results were 9.09% in contrast to
49.2% for the group which then included
all embryonic and one day postnatal donors. This indicated that the first postnatal
day may be a critical time when factors
come into play that initiate a sharp drop
in the number of successful transplantations.
The decline in positive results with increasing donor age may be explained i n
terms of concomitant development of
transplantation antigens in the embryo.
Billingham, Brent and Medawar ( '56)
stated that such antigens are developed
well before birth. However, a period during which antigens are accumulated or
are undergoing activation or maturation
may postpone their effectiveness. 'This
would explain the more severe antagonism
after the first postnatal day (see table 1).
A gradual development of antigenic potency may mean that antagonism is not
at first total and thus account for the
positive results among the 5-day-old postnatal donors.
Since the donor tissue was injected, the
site of implantation was not controlled.
The tissue to be transplanted tended to
backwash along the pathway of the needle.
The route of injection often passed through
the cortex and hippocampus but some-
218
GAIL S . CROUSE
times went deeper to enter the diencephalon. In the first case, implants usually came
to rest in the needle tract between the
hippocampus and the cerebral cortex; in
the second case, between the diencephalon
and the hippocampus. In both cases, positive grafts grew into the adjacent parts
of the brain and, therefore, .were definitely
intracerebral. In a few cases the needle
did not pass completely through the
cerebral cortex; consequently, backwash
brought the implant to the surface where
it grew in subdural position. These grafts
did not differ in their behavior, within the
various age combinations, from those
which implanted in the hippocampal area.
Since the implantation of 15-day-old
embryonic donor tissue yielded 87.5% positive results with 7-day-old postnatal hosts
but only 33.3% with 90-day-old postnatal
hosts, it is obvious that host age is also a
factor in successful grafting. It is suggested that older hosts may have greater
reactivity potential than younger hosts.
Loefer and Gilles (’51) and Loefer (’52)
found successful grafting to be correlated
with both host and donor ages. Therefore,
these observations on intracerebral implantation of normal tissue are comparable to their results with subcutaneous
transplantation of rat fibrosarcomas.
Cannon, Weber and Longmire (’54) investigated the role of host-donor ages
in chick skin homografts. They found
that “ (1) . . . the tissue specificity or
antigenicity of chick skin is developing
at the time of hatching and is completely
formed by the 14th day and ( 2 ) the ability
to resist homografted tissue (immunity response) is also developing in the chick at
the time of hatching, but is absolute by
the seventh day post-hatching”. Our results, using a mammalian form, a “special” site for implantation and a visceral
donor tissue, conform, in principle, to their
conclusions.
Because the brain is known to be an
especially favorable transplantation site,
the possibility arose that immunizing factors were not involved in intracerebral
grafting. However, Eichwald, Rambo and
Henry (’52) have provided information
contrary to this belief. Working with mice
and guinea-pigs, they noted that hetero-
logous tumor grafts grew better in the
brain than in the eye; the latter was known
to participate in immunological events.
By appropriate experiments to explain this,
they found that the brain was capable of
initiating immunological reactions and,
furthermore, that previous systemic immunization influenced the grafts. Consequently, they conluded that factors other
than immunity caused or allowed the preferential growth of grafts in the brain.
This viewpoint offers further explanation
for the subtotal antagonism observed after
the establishment of presumptive antigenic maturity ( see table 1).
Finally, the overall decrease in successful homografts was observed during late
gestation and early postnatal life. It is
interesting that this period corresponds
to that in which acquired tolerance can be
induced in interstrain mice by injection
of foreign cells as shown by the work of
Billingham, Brent and Medawar (’53). In
a review publication, Medawar (’57) states
that acquired tolerance can be induced
up to about 7 days postnatal, with indifferent results during the period of 4-7
days. Hence, it would appear that foreign cells can induce tolerance when host
reactivity (ability to form antibodies)
against them is low and that at 7 days this
ability is much higher than during late
gestation and the first few postnatal days.
If such a gradual increase in reactivity
determines the time limits for the induction of tolerance, then in grafts to nonsensitized animals the age of the host may
be of equal importance with the age of the
donor.
SUMMARY
1. One hundred thirty-four homografts
of rat stomach to the brain were made.
Host animals were sacrificed 25 days postoperatively. Serial sections of the graft
were prepared using Masson’s tri-chrome
technique. On the basis of preestablished
criteria grafts were designated positive or
negative.
2. Table 1 shows host-donor age combinations which comprised the 134 grafts
and summarizes the results. Per cent positive results for three primary combinations used were: host, 7 days-donor, 90
days, 0.0%; host, 90 days-donor, 15 days
VARIABLE DONOR AGE IN HOMOGRAFTING
embryonic, 33.3% ; host, 7 days-donor,
15 days embryonic, 87.5%. Additional
combinations used consisted of 7-day-old
hosts paired with donors of increasing age
from 17 days embryonic through 7 days
postnatal. The combined per cent positive
results for all embryonic donors in this
series was 50.0% ; for all postnatal donors,
1 9 . 4 % . Antagonism sharply increased after the first postnatal day; if the one-dayold group was excluded from the postnatal donors the results dropped to 9.09%.
3 . These results suggest that during the
period of late gestation through the first
few days of postnatal life the ability to
elicit transplantation immunity (antigenic
potency) is progressively increasing. In
addition, it is likely that the age of the
host is correlated with its degree of ability
to react immunologically.
4. The results were discussed in relation to the use of the brain as a transplantation site, in relation to similar observations of other workers and in relation to acquired tolerance.
ACKNOWLEDGMENTS
The author wishes to thank Mr. Charles
Hummer, who aided in the technical work,
Miss Marjorie Stodgell and Mr. Steven
Gigliotti for their help with the illustrative
material, Dr. Edith Hurst for editing the
manuscript, Miss Irene Gamerman for
secretarial assistance and Mr. Louis Sunny,
who made the photomicrographs.
LITERATURE CITED
Athias, M., and A. Guimarais 1933 Greffe
ovarienne intracerebrale chez des Cobayes
mUes, entiers et prealablement chbtr6s. C . R.
SOC.Biol. Paris, 113: 733-735.
Billingham, R. E., L. Brent and P. B. Medawar
1953 ‘Actively acquired tolerance’ of foreign
cells. Nature, 172: 603-606.
1956 The antigenic stimulus in transplantation immunity. Ibid., 178: 514-519.
Cannon, J . A,, R. A. Weber and W. P. Longmire,
Jr. 1954 Factors influencing the survival of
219
successful skin homografts i n the chicken:
I. Effects of varying age of donor and recipient.
Ann. Surg., 139: 468-472.
Clark, W. E. Le Gros 1940 Neuronal differentiation i n implanted foetal cortical tissue. J.
Neurol. Psychiat., 3: 263-272.
Crouse, G. S. 1956 Differentiation of intracerebral implants of rudiments from rad and
mouse embryos in young rats. Anat. Rec.. 126:
369-394.
Eichwald, E. J., 0. N. Rambo and J. Henry
1952 Immunity to tumors transplanted in
the brain of heterologous hosts. Am. J. Path.,
28: 557, Abst.
Glees, P. 1941 The differentiation of the brain
and other tissues i n a n implanted portion of
an embryonic head. J. Anat. (Lond.), 75:
239-248.
Glees, P., A. Mohiuddin and A. G. Smith 1949
Transplantation of Pacinian bodies in the brain
and thigh of the cat. Acta Anat. (Basel), 7 :
2 13-224.
Loefer, J. B. 1952 Effect of age of the donor
on development of rat tumor grafts. Cancer, 5:
163-165.
Loefer, J. B., and N. G. Gilles 1951 Incidence
of a transplanted rat fibrosarcoma relative to
age of the host. Ibid., 4: 1259-1262.
Medawar, P. B. 1948 Immunity to homologous
grafted skin. 111. The fate of skin homografts
transplanted to the brain, to subcutaneous tissue and to the anterior chamber of the eye.
Brit. J. Exp. Path., 29: 58-69.
1957 The Immunology of Transplantation. Harvey Lectures, 1956-1957: 144-176.
Sakurane, Y. 1929 Experimentelle Untersuchungen iiber die Implantation der Hautstucke
mit besonderer Berucksichtigung derselben i n
das Gehirn. I. Mitteilunp: Versuche durch
Autoimplantation. Jap. J. Dermatol. Urol., 29:
51-52.
Siebert, W. J. 1928 Auto- and homoiotransplantation of thyroid gland into the brain of
guinea-pigs. Proc. SOC. Exp. Biol., 26: 236237.
Tansley, K. 1946 The development of the rat
eye in graft. J. Exp. Biol., 22: 221.
Willis, R. A. 1935 Experiments on the intracerebral implantation of embryo tissues in rats.
Proc. Roy. SOC.Lond. (B), 117: 400-412.
1936 The growth of embryo bones
transplanted whole in the rat’s brain. Ibid.,
120: 496-498.
1958 The growth of young embryos
transplanted whole into the brain i n rats. J.
Path. Bact., 76: 337-342.
PLATE 1
EXPLANATION O F FIGURES
All illustrations are photomicrographs of Masson tri-chrome stained material.
220
1
Normal stomach of 15-day-old rat embryo. Note, from lumen outward, simple epithelium
(only several layers thick, no glands) submucosal area and incipient smooth muscle
layer, x 80.
2
Normal stomach of 7-day-old rat. Well-defined muscularis externus at bottom partially
separated from submucosa. Mucosa fairly well differentiated with gastric pits and
specialized cell types. X 175.
3
Normal stomach of 75-day-old rat. Right lower portion shows longitudinal and circular
layers of muscularis externus. Left upper portion shows submucosa (next to the external musculature), muscularis mucosae, lamina propria and fully differentiated glandular epithelium. x 80.
4
Positive stomach graft i n brain, 25 days postoperative. Donor tissue, 15 days embryonic;
host, 7 days postnatal. Note inversion of layers of the gut wall with mucosa ( M )
oriented externally ( a common occurrence i n grafts of gastrointestinal tract) and
surrounding a core of the remaining layers of the gut wall. Note precipitated substance
( P ) intervening between mucosa and brain tissue. Compare degree of differentiation
of graft beyond that of normal 15-day-old embryonic stomach (fig. 1 ) . Adjacent brain
Structures: hippocampus ( H ) , corpus callosum (CC), cerebral cortex ( C ) , chorioid
plexus (CP), diencephalon ( D ) . X 10.
VARIABLE DONOR AGE IN HOR'IOGKAFTING
Gail S . Crouse
PLATE 1
22 1
PLATE 2
EXPLANATION OF FIGURES
222
5
Positive stomach graft in brain, 25 days postoperative. Donor tissue, 15 days embryonic;
host, 90 days postnatal. Layers of wall form tube. Note fully differentiated glandular
mucosa ( 6 ) and its transition with squamous epithelium ( S ) ; the latter is present in
a large area of the rat stomach. Other structures better seen i n high power view (fig. 6 ) .
Brain tissue surrounds graft. x 10.
6
Same as figure 5, high power of area outlined in white. Layers shown from top are:
brain (B), layers of muscularis externus ( M E ) , submucosa ( S ) , muscularis mucosae
and lamina propria ( M M ) , glandular epithelium ( G ) . Not seen at this magnification
are differentiated, specialized cell types of glands. Compare with normal adult rat
stomach (fig. 3). x 80.
7
Negative stomach graft in brain, 25 days postoperative. Donor tissue, 90 days postnatal;
host, 7 days postnatal. Graft remains only as disoriented connective tissue (CT) and
epithelial ( E ) elements embedded i n surrounding brain tissue. X 80.
8
Negative stomach graft in brain, 25 days postoperative. Donor tissue, 90 days Dostnatal; host, 7 days postnatal. Graft reduced to small connective tissue remnant (CT).
No epithelial elements found. For orientation: hippocampus ( H ) , callosal fibers (CC),
cerebral cortex ( C ) . X 80.
9
Negative stomach graft in brain, 25 days postoperative. Donor tissue, 15 days embryonic; host, 90 days postnatal. Negative grafts frequently formed large cysts encroaching
on the brain. Very small arc of cyst wall shown, with low epithelial layer (E:), cavity
of cyst ( C ) , connective tissue and smooth muscle ( C M ) , brain ( B ) . x 80.
VARIABLE DONOR AGE IN HOMOGRAFTING
Gail S. Crouse
PLATE 2
223
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