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The use of chondroitin sulfate in prolonging survival of adult skin allografts
JOURNAL
OF
SURGICAL
RESEARCH,
11,
350-355
Preliminary THE
USE
MARSHALL
OF CHONDROITIN ADULT W.
ASHBY,
M.D.,
A.
SULFATE IN PROLONGIXG SKIN ALLOGRAFTS
RODERICK
THOMSOlr;,
AND
YUKIHIKO
Report
SURVIVAL
NOS6,
M.D.,
OF
PH.D.
feet the development of survival of the grafts. Rylka et al. [2] f ound antitrophoblastic antibodies in the human postpartum interestingly. Potts [5] showed that when a fertilized mouse egg is implanted in the uterus, there is not yet any appreciable fibrinoid material surrounding the egg, and when implantation takes place in an extrauterine site, the embryo is susceptible to a preexisting state of immunity. Kirby and associates [3] noted that 2 clays after implantation of a fertilized mouse egg, a fibrinoid layer developed and from then on the trophoblast could be transplanted without the development of a reaction. The fibrinoid material elaborated from the trophoblasts is composed of mucopolysaccharide rich in hyaluronic acid and sialic acids of which chondroitin-4-sulfate is a conspicuous component. These mucopolysaccharides are believed to produce the immunologic inertness of the fetus to the mot’hcr. It is known that the layer of Nitabuch is conspicuously thicker at the maternal fetal junction in the placenta of a hybrid fetus than when mother and fetus are antigenically identical [l]. Examples supporting the belief of chondroitin-4-sulfate as the chemical imparting immunologic inertness are found in the hamster’s cheek pouch, the anterior chamber of the eye and cartilage, all of which contain high amounts of chondroitin-4-sulfate. The aforementioned facts have given support to the belief that the administration of chondroitin sulfate should prolong the acceptance of allogenic grafts. To test this thesis, a series of experiments was designed.
PREGNANCY REPRESENTS the union of two genetically different individuals and is a true, tolerant, transplantation phenomenon that by its very existence has caused the profound interest of immunologists because it readily defies the principle of transplantation rejection. The ovum after eruption from the ovary carries with it ovarian follicle cells which seemingly are a loose strata and afford no barrier to penetration by the sperm. Once sperm and egg are combined the conceptus is antigenitally different from the mother and may be rejected [6]. When the conceptus has entered the uterus, its outer syncytial layer propagates into a cellular layer that surrounds and protects the fetus in multilayers. These syncytial cells (syncytio-trophoblasts) differentiate into the trophoblasts which permeate the prepared decidua of the uterus. Vascular fetal spaces intimate with the maternal vasculature of the deciduae are lined with trophoblasts. It is here that an immunologic buffer zone is established. Simmons and Russell [7] transplanted the proliferative cells of the trophoblastic layers of the mouse placenta into a kidney of the maternal strain. There was no induced sensitivity of the growing trophoblasts to the paternal antigens, nor did trophoblastic cells from a mouse of different strain with a preexisting state of immunity against paternal tissue afFrom Artificial Organs Research Laboratory, Division of Research, The Cleveland Clinic Foundation, Cleveland. Ohio 44106. Supported by Grant HE-12716 from the Xational Institutes of Health with Dr. Y. No& as Principal Investigator. 350
MATERIALS
ASHBY,
THOMSON,
AND
METHODS
AND
NOSti:
Adult male and female inbred strains of C57 and CBA mice” were used as test animals because of their known histoincompatibility [4]. From 2 to 2.5 inch skin grafts were exchanged between the C57 and CBA mice. Males were paired against males and females paired against females. Five groups of animals were set up for observation wit’h 10 males and 10 females in each group. Group I-Control animals. Group IIA-Heparini (aqueous) (excessive dosage) 10 mg. (or 1000 USP units) intraperitoneally daily. Group IIB-Heparin (aqueous) (low dosage) 1.0 mg. ior 100 USP units) intraperitoneally daily. Group IIC$--Heparin (aqueous) (low dosage) 0.5 mg. (or 30 USP units) intraperitoneally daily. Group IIIA&-Chondroitin sulfate (low dosage) 10 mg. intraperitoneally initially and 5 mg. int’raperitoneally daily. Group IIIB-Chondroitin sulfate (high dosagei 100 mg. intraperitoneally initially and 10 mg. intraperitoneally daily. Anesthesia was initiated with ether and was maintained by injection of sodium pentobarbital (Dibutal). Males were given 1.8 mg. and females 1.1 mg. int’raperitoneally. The graft area was shaved wit.h electric clippers and swabbed with Zephiran solution. Grafts were taken from the backs of the mice with the anterior edge over the midthoracic vertrbrae. Tissue forceps were then used to lift, the graft, which was cut in a posterior to anterior direction with scissors. Grafts were 1.5-2.5 cm. in diameter. The graft was then placed on a section of Styrofoam cup and pinned out cpidermal side down. The same procedure was done on the other mouse. When skins of both animals had the subcutaneous loose strata re* Through the courtesy of Jackson Laboratories, Harbor, Maine 04609. -t Through the courtesy of Riker Laboratories. Korthridge, Calif. 91324. $ Subsequent group added after deaths noted from hemorrhages in IIB and IIA. $Through the courtesy of Mat,hrson Scientific (an admit,tedly impure extract). Bar
ADULT
SKIN
ALLOGRAFTS
3.‘31
moved via gross planing with a number 1 I scalpel, the graft,s were punctured at multiple areas for effective drainage of serous exudates postoperatively. The CBA skin was put on the C57 mouse and vice versa. Grafts were sutured to the mouse’s own skin at five point’s on the perimeter with 6-O silk. A semicircular Tygon cover was put over the graft by t,he wrapping of adhesive tape around the body and the cover. Covers for the grafts were made by cutting 0.75-inch segments of 0.5-inch diameter Tygon t,ubing and bisecting each longitudinally. The resulting semicircular cover was punctured in the center, forming a 0.25-inch diameter hole for ventilation. ,4 hole was cut in t,he t,ape to coincide with the air hole. To aid recovery each mouse was given 0.22-0.40 ml. of 5% dextrose in saline intraperitoneally. The dose varied depending on the weight and depth of anesthesia of the animal at t,he end of surgery. The mice were examined daily when given the injections. Skin biopsies were at’tempted on the 5th, lOth, 16th, and 21st days when the graft survived that, long, and every 7 days thereafter. Sections of the graft and its bed were rectangular pieces taken by cutting from the mouse’s own skin into the graft which yielded a block of tissue 1.5 x 0.25 x 0.10 cm. Grafts were considered sloughing when they were edematous, discolored, or grossly necrotic [4]. As long as the graft appeared soft and pliable, it was considered a take. A slough usually began with raised dark edges and became wet with a clear exudate. If the graft became infected (based on a strictly visual interpretation) Neosporin ointment was applied. Later the groups were redone with Neosporin applied initially at grafting on all grafts. When the animal died, an autopsy was done, including procurement of the graft plus recipient skin and graft bed, lung, stomach, spleen, liver, intestine, and kidneys. All tissues taken for study were fixed in Zenker’s solution. RESULTS The survival times of animals in each group are shown in Table 1. The rejection of the transplanted skin graft. is designated in Table 2 as survival days.
332
JOURNAL
Table
SURGICAL
1. Statistics Comp&cl on fhe Days Survival in Each Group Xumbera
GKlllp
20 18 20 19 20
1 2a 2b 3a 3b
that
OF
a The number are natural
Rejection
REHEIRCH,
of
Mean 7.0 7.05 1.8 13.25 18.65
7.0 7.0 2.0 15.0 21.0 deaths
Time
Group I. As predicted from incompatibility studies by the breeding laboratory farm all t,est animals disclosed rejections of allografts from 5 to 8 days. Group IIA. The excessive dose of heparin, 1000 USP units/19-22 gm. mouse resulted in death of the animal in 1 to 8 days from intraperitoneal hemorrhage, solid viscous hematomas, or large hematomas of the abdominal wall. All grafts at the time of death were hemorrhagic and edematous. Total slough of the grafts was noted in each animal. Group IIB. A dosage of 100 USP units of aqueous heparin resulted in hemorrhagic complications which caused deaths of eight mice before the fifth day. Petechial hemorrhages grossly persisted in the mice with surviving grafts. Group IIC. With a dosage of 30 USP units of aqueous heparin there was gross survival of all grafts and animals with no gross rejection evident until day 21-24, after which slough was grossly evident. Chrondroitin sulfate group. Prolongation of allograft survival with the crude preparation of chondroitin sulfate was viewed from two dosageranges. Table
2. Number
of Mice
Rejecting
11,
7,
NO.
JULY
Microscopic fkudy hTotes on Specimens Taken Results of skin biopsies and autopsy slides are summarized in Tables 3 and 4. The results are as follows: Group I-control. All skin biopsy sections on the sixth day were necrotic with severe inflammation. Neutrophilic reactions were graded as 4f (14 scale with a No. 1 grade showing the least, inflammation). Group II (heparin). Grafts were biopsied and studied. Results of all three heparin dosage groups are as follows: Group IIA (1000 USP units). ,411 grafts Grafte
at Specijic
Intervals
of Day.3 Interval
Group
Medication
TotalN
1
None
20
-
2a 2b 3a
Heparin, 100 units daily Heparin, 1000 units daily mg. + 5 Chondroitin Sod,50 mg. daily Chondroitin SO*, 100 mg. -+ 10 mg. daily
18 20
7 19 -
3b __I__-
20
-----
Of Days
__ 04
19
1971
Group ZIZA. Crude chondroitin sulfate, 50 mg.. was given intraperitoneally postgrafting initially and then 5 mg. int’raperitoneally daily thereafter. One animal died of over dosage of anesthesia before the fifth day; three animals had total infection of their grafts, which led t#o rejection before the eighth day. One animal died from overdosage of anesthesia on the 18th day, though its graft was still pliable and soft. Fifteen animals had graft survival up to the 16th day before gross signs of rejection became evident. Group IIZB. When 100 mg. of crude chondroitin sulfate was given intraperitoneally initially, followed by 10 mg. intraperitoneally daily thereafter, t’he results were further improved. One animal, despite Neosporin ointment, had total rejection of his graft before the eighth day after infection. Two animals succumbed t’o overdosages of anesthesia on the 12th day, and another similarly on t’he 21st day, hut, all grafts were grossly intact and viable. Fifteen animals, as listed in Table 2 showed the first signs of rejection in from 21 to 24 days, at this dosage.
Grajl
.~___ Median
is based on nonaccident,al or direct drug-induced deaths.
VOL.
5-8
20 2 1 3 1
9-12
13-16
-
G
3 -
-
15 2
2
17-20
21-24
-
-.
-
1
_
15
ASHBY,
THOMSON,
Table
AND
NO&:
3. Autopsy Number
Autopsies Number
Gr0tlp
Graft Bleeding
Lung
Kidneys
1
5
2:t 2b 3a
10 16 4
-5
4 1 -
-
3b
10
-'
la
-
* These refrigerated sied within
Day
353
Summary of Observed
- __-.-~-Xbnormalities
in Each Group Hematoma
Intestinal Region
Spleen
--___Large
Red at Nails and Other Effects
__Small
_.4 5
3 7
1 7
l:,
8 9
1
4. Histologic
Changes
on Skin
T
Groul
Slide NO.
after Transplant
T
-278C -2781
6 6
F F
Sex of Mouse
-2773 -2774 -27x -277E -28732 -2948 -2945 -2948 -2928 -3051 -3m -3363 -3183 -3184
3 3 3 3 7 7 7 7 7 9 13 16 14 14
F F M M F M F M F M M F M M
IIB
-2925
1
IIIA
-3155 -315f -315i -3432
Hornlayer
--
2s
Tissue
____
-I-
+I
+ ++ +++ + ++ ++ f + + f
++ ++ ++ ++ + + f ++ + +++ ++++ Sf++-
F
++
f
-
+
4 4 4 12
31 F M M
++ ++ ++ ++
+ + + -
+ ++ + ++
++ -
-3433 -3434 -3184 -3185 -3672 -3673
12 12 5 5 19 19
F F F M F M
+ + + + +-I+
+ + ++ -
+ +++ +++ + -
+ ++ + -
f
++
+
-3880 -4468 -5148 -5149
7 21 24 24
M M F F
+ ++ ++
+ + ++
+ -
+ + __
-~~_-
+ t t t
I Commenl
+ +
-
+
++
+ f + -t+ + ++ + ++ + + + ++ ++
bodies were and autop-
-
t
-
Both killed
Grafts
+++++
I
+ - Nothing
Baseml ent Celh Cell I(Atrop: hY) ][ntiltration --
6 10 -.
5 .-_
l:,
Subcutaneous
__
i
ALLOGRAiE’T.5
data are from two mice whose deaths were due to needle punct,ure of the liver. for 24 hours before autopsy, and the data are of questionable accuracg. Six mice 1 hour of death showed no abnoramlities. Table
IIIB
SKIN
-
--
IIA
ADULT
+ ++ +++ +++
Subcut +I+ + ++
-
-
+
~ ~.-
Nccrot ir Necrotic:
I+’
f i+ ++ t-f + + + 1-f +++ ++ + + +++ +++ +++ +++ +I+ ++
+++ + ++ __- -
Good new tion Defect Necrotic Partially Partially Partially Partially
granttla-
of skin necrotic necrotic necrotic necrotic
Relatively good Partially destroyed Relatively good Partially replaced by grantdation Excellent Partially defective
Excellent Partially Total
defertive necrosis
Partial necrosis Relatively good
354
JOURNAL
OF
SURGICAL
RESEARCH,
were alive but with considerable hemorrhage in the graft bed. Neutrophilic infiltrate density was difficult to grade. Group IIB (100 USP units). Third day and seventh day skin grafts showed a living graft with grade I neutrophilic infiltrate. The 10th and 16th day skin biopsies showed an increasing neutrophilic and lymphocytic cellular infiltration with neutrophils predominating. Group UC (SO USP units). Disclosed similar findings as in Group IIB, but the graft microscopically appeared viable until the 16th day, after which it was necrotic with prominent neutrophilic infiltrates. Group IIIA (50 mg. chondroitin sulfate followed by 5 mg. daily). Skin biopsy sections were similar to those in heparin group although neutrophilic infiltrate was noted to be increased on the 10th day to 4+, in contrast to that of the heparin group on the 12th day. Group IIIB (100 mg. chondroitin sulfate followed by 10 mg. daily). This dosage was more favorable according to microscopic evidence with neutrophilic infiltrate not graded to 4+ until the 16th day. At 21 days the skin graft microscopically was infiltrated with neutrophils and the inflammatory reaction in the graft bed was severe. COMMENT Chondroitin sulfate in the dosages employed proved effective in prolonging the survival of skin allografts in mice. Likewise heparin proved effective in subhemorrhagic doses. Concanavalin A as reported by Markowitz et al. [4] in appropriate dosages has been effective in prolonging the survival of skin allografts likewise, but only up to 22 days. Chondroitin sulfat’e, though a crude extract, would therefore seem to be a superior drug in this respect, and even more superior when it is noted that concanavalin A produced peritoneal inflammatory reactions and hard exudates, whereas chondroitin sulfate did not. It is interesting that heparin and chondroitin sulfate are mucoplysaccharides. Concanavalin A is only a polysaccharide derived from jackbean. Of interest is that polymorphonuclear (or neutrophil) infiltrate was delayed by heparin
VOL.
11,
NO.
7,
JULY
1971
and by chondroitin sulfate. Perhaps these polyamino acids delay chemotaxis of the polymorphonuclear cells and thus delay the onslaught of the lymphocytes. This viewpoint will be explored in future experiments. It can be surmised from our microscopic work that the decreased neutrophilic infiltration may express the increased negativity imparted to the grafted tissue by chondroitin sulfate as well as heparin. The established fact that an ischemic tissue is positively charged and therefore chemotactic to neutrophils cannot be taken lightly. Reestablishment of a high negative charge to the lipoprotein cell membrane by the sulfate groups of heparin and chondroitin sulfate would seemingly explain the latency of neutrophils appearing in allotransplants and thus delayed rejection. The hemorrhagic properties of heparin, though more highly negatively charged would negate its value in being an effective medication in prolonging allograft survival. Chondroitin sulfate appears to be the superior medication of the two as the evidence here seems to document.
The placenta supplies immunologic protection for the fetus. Chondroitin sulfate, which is in t’rophoblastic cells in the placenta, and its immunosuppressive effects were investigated in regard to survival of skin allografts. Heparin, an antithrombogenic agent like chondroitin sulfate, showed its immunosuppressive capability and was used as a control agent. CBA mice and C57 mice served as skin allograft models, with groups divided into controls; heparin high and low dosage groups ; and chondroitin sulfate high and low dosage groups. Heparin and chondroitin sulfate were given 2-6 hours postgrafting and daily till total slough of the graft or until death of the animal. The control allografts sloughed as predicted after an average of 7 I 3 days but in the high-dosage group 22 * 2 days. REFERENCES 1. Bradbury, S., Billington, W. D., and Kirby, D. S. A histochemical and electron microscopica study of the fibrinoid of the mouse placenta. Roy. Micr. Sot. 84:199. 1965.
R. J.
ASHBY,
THOMSON,
AND
NO&+:
2. Hylks, J. F., Brinton, V., Schaaf, J., and Baney, C. Appearance of antibodies to trophoblasts during the postpartum period in normal human pregnancies. Nature London 198501, 1963. 3. Kirby, D. R. S., Billington, W. D., and James, D. A. Transplantat.ion of eggs to the kidney and uterus of immunized mice. Transplantation 4 :713, 1966. 4. Markowitz, H., et al. Immunosuppressive activity of concanavalin il. Science 163:1, 1969.
ADULT
SKIN
ALLOGRAFTS
355
5. Potts, D. M. Implantation. An electron microscopical study with special reference to the mouse. Ph.D. thesis, Cambridge University, 1965. 6. Simmons, R. L., and Russell, P. S. Histocompatibility antigens in transplanted mouse eggs. Nature London 208:698, 1965. 7. Simmons, R. L., and Russell, P. S. Mechanisms of trophoblast nonantigenicity. Fed. Proc. 25 :356, 1966.
OF
SURGICAL
RESEARCH,
11,
350-355
Preliminary THE
USE
MARSHALL
OF CHONDROITIN ADULT W.
ASHBY,
M.D.,
A.
SULFATE IN PROLONGIXG SKIN ALLOGRAFTS
RODERICK
THOMSOlr;,
AND
YUKIHIKO
Report
SURVIVAL
NOS6,
M.D.,
OF
PH.D.
feet the development of survival of the grafts. Rylka et al. [2] f ound antitrophoblastic antibodies in the human postpartum interestingly. Potts [5] showed that when a fertilized mouse egg is implanted in the uterus, there is not yet any appreciable fibrinoid material surrounding the egg, and when implantation takes place in an extrauterine site, the embryo is susceptible to a preexisting state of immunity. Kirby and associates [3] noted that 2 clays after implantation of a fertilized mouse egg, a fibrinoid layer developed and from then on the trophoblast could be transplanted without the development of a reaction. The fibrinoid material elaborated from the trophoblasts is composed of mucopolysaccharide rich in hyaluronic acid and sialic acids of which chondroitin-4-sulfate is a conspicuous component. These mucopolysaccharides are believed to produce the immunologic inertness of the fetus to the mot’hcr. It is known that the layer of Nitabuch is conspicuously thicker at the maternal fetal junction in the placenta of a hybrid fetus than when mother and fetus are antigenically identical [l]. Examples supporting the belief of chondroitin-4-sulfate as the chemical imparting immunologic inertness are found in the hamster’s cheek pouch, the anterior chamber of the eye and cartilage, all of which contain high amounts of chondroitin-4-sulfate. The aforementioned facts have given support to the belief that the administration of chondroitin sulfate should prolong the acceptance of allogenic grafts. To test this thesis, a series of experiments was designed.
PREGNANCY REPRESENTS the union of two genetically different individuals and is a true, tolerant, transplantation phenomenon that by its very existence has caused the profound interest of immunologists because it readily defies the principle of transplantation rejection. The ovum after eruption from the ovary carries with it ovarian follicle cells which seemingly are a loose strata and afford no barrier to penetration by the sperm. Once sperm and egg are combined the conceptus is antigenitally different from the mother and may be rejected [6]. When the conceptus has entered the uterus, its outer syncytial layer propagates into a cellular layer that surrounds and protects the fetus in multilayers. These syncytial cells (syncytio-trophoblasts) differentiate into the trophoblasts which permeate the prepared decidua of the uterus. Vascular fetal spaces intimate with the maternal vasculature of the deciduae are lined with trophoblasts. It is here that an immunologic buffer zone is established. Simmons and Russell [7] transplanted the proliferative cells of the trophoblastic layers of the mouse placenta into a kidney of the maternal strain. There was no induced sensitivity of the growing trophoblasts to the paternal antigens, nor did trophoblastic cells from a mouse of different strain with a preexisting state of immunity against paternal tissue afFrom Artificial Organs Research Laboratory, Division of Research, The Cleveland Clinic Foundation, Cleveland. Ohio 44106. Supported by Grant HE-12716 from the Xational Institutes of Health with Dr. Y. No& as Principal Investigator. 350
MATERIALS
ASHBY,
THOMSON,
AND
METHODS
AND
NOSti:
Adult male and female inbred strains of C57 and CBA mice” were used as test animals because of their known histoincompatibility [4]. From 2 to 2.5 inch skin grafts were exchanged between the C57 and CBA mice. Males were paired against males and females paired against females. Five groups of animals were set up for observation wit’h 10 males and 10 females in each group. Group I-Control animals. Group IIA-Heparini (aqueous) (excessive dosage) 10 mg. (or 1000 USP units) intraperitoneally daily. Group IIB-Heparin (aqueous) (low dosage) 1.0 mg. ior 100 USP units) intraperitoneally daily. Group IIC$--Heparin (aqueous) (low dosage) 0.5 mg. (or 30 USP units) intraperitoneally daily. Group IIIA&-Chondroitin sulfate (low dosage) 10 mg. intraperitoneally initially and 5 mg. int’raperitoneally daily. Group IIIB-Chondroitin sulfate (high dosagei 100 mg. intraperitoneally initially and 10 mg. intraperitoneally daily. Anesthesia was initiated with ether and was maintained by injection of sodium pentobarbital (Dibutal). Males were given 1.8 mg. and females 1.1 mg. int’raperitoneally. The graft area was shaved wit.h electric clippers and swabbed with Zephiran solution. Grafts were taken from the backs of the mice with the anterior edge over the midthoracic vertrbrae. Tissue forceps were then used to lift, the graft, which was cut in a posterior to anterior direction with scissors. Grafts were 1.5-2.5 cm. in diameter. The graft was then placed on a section of Styrofoam cup and pinned out cpidermal side down. The same procedure was done on the other mouse. When skins of both animals had the subcutaneous loose strata re* Through the courtesy of Jackson Laboratories, Harbor, Maine 04609. -t Through the courtesy of Riker Laboratories. Korthridge, Calif. 91324. $ Subsequent group added after deaths noted from hemorrhages in IIB and IIA. $Through the courtesy of Mat,hrson Scientific (an admit,tedly impure extract). Bar
ADULT
SKIN
ALLOGRAFTS
3.‘31
moved via gross planing with a number 1 I scalpel, the graft,s were punctured at multiple areas for effective drainage of serous exudates postoperatively. The CBA skin was put on the C57 mouse and vice versa. Grafts were sutured to the mouse’s own skin at five point’s on the perimeter with 6-O silk. A semicircular Tygon cover was put over the graft by t,he wrapping of adhesive tape around the body and the cover. Covers for the grafts were made by cutting 0.75-inch segments of 0.5-inch diameter Tygon t,ubing and bisecting each longitudinally. The resulting semicircular cover was punctured in the center, forming a 0.25-inch diameter hole for ventilation. ,4 hole was cut in t,he t,ape to coincide with the air hole. To aid recovery each mouse was given 0.22-0.40 ml. of 5% dextrose in saline intraperitoneally. The dose varied depending on the weight and depth of anesthesia of the animal at t,he end of surgery. The mice were examined daily when given the injections. Skin biopsies were at’tempted on the 5th, lOth, 16th, and 21st days when the graft survived that, long, and every 7 days thereafter. Sections of the graft and its bed were rectangular pieces taken by cutting from the mouse’s own skin into the graft which yielded a block of tissue 1.5 x 0.25 x 0.10 cm. Grafts were considered sloughing when they were edematous, discolored, or grossly necrotic [4]. As long as the graft appeared soft and pliable, it was considered a take. A slough usually began with raised dark edges and became wet with a clear exudate. If the graft became infected (based on a strictly visual interpretation) Neosporin ointment was applied. Later the groups were redone with Neosporin applied initially at grafting on all grafts. When the animal died, an autopsy was done, including procurement of the graft plus recipient skin and graft bed, lung, stomach, spleen, liver, intestine, and kidneys. All tissues taken for study were fixed in Zenker’s solution. RESULTS The survival times of animals in each group are shown in Table 1. The rejection of the transplanted skin graft. is designated in Table 2 as survival days.
332
JOURNAL
Table
SURGICAL
1. Statistics Comp&cl on fhe Days Survival in Each Group Xumbera
GKlllp
20 18 20 19 20
1 2a 2b 3a 3b
that
OF
a The number are natural
Rejection
REHEIRCH,
of
Mean 7.0 7.05 1.8 13.25 18.65
7.0 7.0 2.0 15.0 21.0 deaths
Time
Group I. As predicted from incompatibility studies by the breeding laboratory farm all t,est animals disclosed rejections of allografts from 5 to 8 days. Group IIA. The excessive dose of heparin, 1000 USP units/19-22 gm. mouse resulted in death of the animal in 1 to 8 days from intraperitoneal hemorrhage, solid viscous hematomas, or large hematomas of the abdominal wall. All grafts at the time of death were hemorrhagic and edematous. Total slough of the grafts was noted in each animal. Group IIB. A dosage of 100 USP units of aqueous heparin resulted in hemorrhagic complications which caused deaths of eight mice before the fifth day. Petechial hemorrhages grossly persisted in the mice with surviving grafts. Group IIC. With a dosage of 30 USP units of aqueous heparin there was gross survival of all grafts and animals with no gross rejection evident until day 21-24, after which slough was grossly evident. Chrondroitin sulfate group. Prolongation of allograft survival with the crude preparation of chondroitin sulfate was viewed from two dosageranges. Table
2. Number
of Mice
Rejecting
11,
7,
NO.
JULY
Microscopic fkudy hTotes on Specimens Taken Results of skin biopsies and autopsy slides are summarized in Tables 3 and 4. The results are as follows: Group I-control. All skin biopsy sections on the sixth day were necrotic with severe inflammation. Neutrophilic reactions were graded as 4f (14 scale with a No. 1 grade showing the least, inflammation). Group II (heparin). Grafts were biopsied and studied. Results of all three heparin dosage groups are as follows: Group IIA (1000 USP units). ,411 grafts Grafte
at Specijic
Intervals
of Day.3 Interval
Group
Medication
TotalN
1
None
20
-
2a 2b 3a
Heparin, 100 units daily Heparin, 1000 units daily mg. + 5 Chondroitin Sod,50 mg. daily Chondroitin SO*, 100 mg. -+ 10 mg. daily
18 20
7 19 -
3b __I__-
20
-----
Of Days
__ 04
19
1971
Group ZIZA. Crude chondroitin sulfate, 50 mg.. was given intraperitoneally postgrafting initially and then 5 mg. int’raperitoneally daily thereafter. One animal died of over dosage of anesthesia before the fifth day; three animals had total infection of their grafts, which led t#o rejection before the eighth day. One animal died from overdosage of anesthesia on the 18th day, though its graft was still pliable and soft. Fifteen animals had graft survival up to the 16th day before gross signs of rejection became evident. Group IIZB. When 100 mg. of crude chondroitin sulfate was given intraperitoneally initially, followed by 10 mg. intraperitoneally daily thereafter, t’he results were further improved. One animal, despite Neosporin ointment, had total rejection of his graft before the eighth day after infection. Two animals succumbed t’o overdosages of anesthesia on the 12th day, and another similarly on t’he 21st day, hut, all grafts were grossly intact and viable. Fifteen animals, as listed in Table 2 showed the first signs of rejection in from 21 to 24 days, at this dosage.
Grajl
.~___ Median
is based on nonaccident,al or direct drug-induced deaths.
VOL.
5-8
20 2 1 3 1
9-12
13-16
-
G
3 -
-
15 2
2
17-20
21-24
-
-.
-
1
_
15
ASHBY,
THOMSON,
Table
AND
NO&:
3. Autopsy Number
Autopsies Number
Gr0tlp
Graft Bleeding
Lung
Kidneys
1
5
2:t 2b 3a
10 16 4
-5
4 1 -
-
3b
10
-'
la
-
* These refrigerated sied within
Day
353
Summary of Observed
- __-.-~-Xbnormalities
in Each Group Hematoma
Intestinal Region
Spleen
--___Large
Red at Nails and Other Effects
__Small
_.4 5
3 7
1 7
l:,
8 9
1
4. Histologic
Changes
on Skin
T
Groul
Slide NO.
after Transplant
T
-278C -2781
6 6
F F
Sex of Mouse
-2773 -2774 -27x -277E -28732 -2948 -2945 -2948 -2928 -3051 -3m -3363 -3183 -3184
3 3 3 3 7 7 7 7 7 9 13 16 14 14
F F M M F M F M F M M F M M
IIB
-2925
1
IIIA
-3155 -315f -315i -3432
Hornlayer
--
2s
Tissue
____
-I-
+I
+ ++ +++ + ++ ++ f + + f
++ ++ ++ ++ + + f ++ + +++ ++++ Sf++-
F
++
f
-
+
4 4 4 12
31 F M M
++ ++ ++ ++
+ + + -
+ ++ + ++
++ -
-3433 -3434 -3184 -3185 -3672 -3673
12 12 5 5 19 19
F F F M F M
+ + + + +-I+
+ + ++ -
+ +++ +++ + -
+ ++ + -
f
++
+
-3880 -4468 -5148 -5149
7 21 24 24
M M F F
+ ++ ++
+ + ++
+ -
+ + __
-~~_-
+ t t t
I Commenl
+ +
-
+
++
+ f + -t+ + ++ + ++ + + + ++ ++
bodies were and autop-
-
t
-
Both killed
Grafts
+++++
I
+ - Nothing
Baseml ent Celh Cell I(Atrop: hY) ][ntiltration --
6 10 -.
5 .-_
l:,
Subcutaneous
__
i
ALLOGRAiE’T.5
data are from two mice whose deaths were due to needle punct,ure of the liver. for 24 hours before autopsy, and the data are of questionable accuracg. Six mice 1 hour of death showed no abnoramlities. Table
IIIB
SKIN
-
--
IIA
ADULT
+ ++ +++ +++
Subcut +I+ + ++
-
-
+
~ ~.-
Nccrot ir Necrotic:
I+’
f i+ ++ t-f + + + 1-f +++ ++ + + +++ +++ +++ +++ +I+ ++
+++ + ++ __- -
Good new tion Defect Necrotic Partially Partially Partially Partially
granttla-
of skin necrotic necrotic necrotic necrotic
Relatively good Partially destroyed Relatively good Partially replaced by grantdation Excellent Partially defective
Excellent Partially Total
defertive necrosis
Partial necrosis Relatively good
354
JOURNAL
OF
SURGICAL
RESEARCH,
were alive but with considerable hemorrhage in the graft bed. Neutrophilic infiltrate density was difficult to grade. Group IIB (100 USP units). Third day and seventh day skin grafts showed a living graft with grade I neutrophilic infiltrate. The 10th and 16th day skin biopsies showed an increasing neutrophilic and lymphocytic cellular infiltration with neutrophils predominating. Group UC (SO USP units). Disclosed similar findings as in Group IIB, but the graft microscopically appeared viable until the 16th day, after which it was necrotic with prominent neutrophilic infiltrates. Group IIIA (50 mg. chondroitin sulfate followed by 5 mg. daily). Skin biopsy sections were similar to those in heparin group although neutrophilic infiltrate was noted to be increased on the 10th day to 4+, in contrast to that of the heparin group on the 12th day. Group IIIB (100 mg. chondroitin sulfate followed by 10 mg. daily). This dosage was more favorable according to microscopic evidence with neutrophilic infiltrate not graded to 4+ until the 16th day. At 21 days the skin graft microscopically was infiltrated with neutrophils and the inflammatory reaction in the graft bed was severe. COMMENT Chondroitin sulfate in the dosages employed proved effective in prolonging the survival of skin allografts in mice. Likewise heparin proved effective in subhemorrhagic doses. Concanavalin A as reported by Markowitz et al. [4] in appropriate dosages has been effective in prolonging the survival of skin allografts likewise, but only up to 22 days. Chondroitin sulfat’e, though a crude extract, would therefore seem to be a superior drug in this respect, and even more superior when it is noted that concanavalin A produced peritoneal inflammatory reactions and hard exudates, whereas chondroitin sulfate did not. It is interesting that heparin and chondroitin sulfate are mucoplysaccharides. Concanavalin A is only a polysaccharide derived from jackbean. Of interest is that polymorphonuclear (or neutrophil) infiltrate was delayed by heparin
VOL.
11,
NO.
7,
JULY
1971
and by chondroitin sulfate. Perhaps these polyamino acids delay chemotaxis of the polymorphonuclear cells and thus delay the onslaught of the lymphocytes. This viewpoint will be explored in future experiments. It can be surmised from our microscopic work that the decreased neutrophilic infiltration may express the increased negativity imparted to the grafted tissue by chondroitin sulfate as well as heparin. The established fact that an ischemic tissue is positively charged and therefore chemotactic to neutrophils cannot be taken lightly. Reestablishment of a high negative charge to the lipoprotein cell membrane by the sulfate groups of heparin and chondroitin sulfate would seemingly explain the latency of neutrophils appearing in allotransplants and thus delayed rejection. The hemorrhagic properties of heparin, though more highly negatively charged would negate its value in being an effective medication in prolonging allograft survival. Chondroitin sulfate appears to be the superior medication of the two as the evidence here seems to document.
The placenta supplies immunologic protection for the fetus. Chondroitin sulfate, which is in t’rophoblastic cells in the placenta, and its immunosuppressive effects were investigated in regard to survival of skin allografts. Heparin, an antithrombogenic agent like chondroitin sulfate, showed its immunosuppressive capability and was used as a control agent. CBA mice and C57 mice served as skin allograft models, with groups divided into controls; heparin high and low dosage groups ; and chondroitin sulfate high and low dosage groups. Heparin and chondroitin sulfate were given 2-6 hours postgrafting and daily till total slough of the graft or until death of the animal. The control allografts sloughed as predicted after an average of 7 I 3 days but in the high-dosage group 22 * 2 days. REFERENCES 1. Bradbury, S., Billington, W. D., and Kirby, D. S. A histochemical and electron microscopica study of the fibrinoid of the mouse placenta. Roy. Micr. Sot. 84:199. 1965.
R. J.
ASHBY,
THOMSON,
AND
NO&+:
2. Hylks, J. F., Brinton, V., Schaaf, J., and Baney, C. Appearance of antibodies to trophoblasts during the postpartum period in normal human pregnancies. Nature London 198501, 1963. 3. Kirby, D. R. S., Billington, W. D., and James, D. A. Transplantat.ion of eggs to the kidney and uterus of immunized mice. Transplantation 4 :713, 1966. 4. Markowitz, H., et al. Immunosuppressive activity of concanavalin il. Science 163:1, 1969.
ADULT
SKIN
ALLOGRAFTS
355
5. Potts, D. M. Implantation. An electron microscopical study with special reference to the mouse. Ph.D. thesis, Cambridge University, 1965. 6. Simmons, R. L., and Russell, P. S. Histocompatibility antigens in transplanted mouse eggs. Nature London 208:698, 1965. 7. Simmons, R. L., and Russell, P. S. Mechanisms of trophoblast nonantigenicity. Fed. Proc. 25 :356, 1966.