Experimental uterotubovarian homotransplantation in the dog

Experimental

uterotubovarian

homotransplantation M.

B.

WINGATE,

F.R.C.S.(E),

M.D.(LoND.),

KARASEWICH,

L.

WINGATE,

M.D.(MAN.), B.Sc.

S. LAUCHIAN. RAY,

Winnipeg,

F.R.C.S.(C),

M.R.C.O.G.*

E.

M.

in the dog

F.R.C.S.(C)

(LEEDS)

M.B.,

CK.B.(EDIN.),

F.R.C.P.(C)

PH.D.(MAN.) Manitoba,

Canada

Using the dog as an experimental model, a technique is presented for homotransplantation of the uterus, Fallofiian tubes, and ovaries, together with their vascular pedicle. The pathologic findings are reported in untreated dogs, in those treated with arathioprine and corticosteroids, and in one dog treated with antithymocyte serum. Vascular thrombosis affecting large- and medium-sized vessels was a major finding in both treated and untreated dogs, and the possibility is put forward that this is a primary manifestation of rejection in this organ system. The technique of mixed leukocyte culture may be employed not only to test histocompatibility between donor and recipient, but as a monitoring procedure to deetrmine the effect of immunosu#Qressive therapy in the treatment of the rejection phenomenon.

rected to the replacement of organs in which absence or malfunction may interfere with the fulfillment of a normal life. This consideration has stimulated interest in the possibility of transplantation of the female reproductive tract as loss, disease, or gross damage of these organs may constitute a serious disability. There are no reports of clinical homotransplantation of the uterus and Fallopian tubes but the procedure has been studied experimentally.e Successful replantation of these organs in a variety of animals has been described by Zhordania and Gotsiridze,*”

THE MAJORITY OF surgeonswhopractice homotransplantation are presently concerned with those organs essential to maintaining life. When the problems of homograft rejection are overcome, attention may be diFrom the Medical College, University of Manitoba. Aided by a grant from the Upjohn Company of Canada. Presented at the Annual Meeting of the Royal College of Physicians and Surgeons of Canada, Vancouver, January, 1969. *Present address: Temple Hos ital Health Sciences Phr -tpadelphia, Pennsylvania

University Center, 19140. 1171

1172

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et al.

Amer.

and in dogs by Eraslan, Hamernik, and Hardy.3 Experimental and clinical ovarian homotransplantation has been reported by Castellanos and Sturgis,2 and Shaffer and Hulka. In earlier experiments the ovaries were enclosed in Millipore filters; later a capsule fashioned from cornea was employed. In both clinical and experimental studies there was evidence of endocrine function for up to one year, but ovulation and fertilization could not occur and the grafts were eventualIy rejected. In the field of experimental reproductive physiology, ovarian transplantation without rejection has been achieved in the mouse, which has a privileged ovarian site as this organ is enclosed in a capsular membrane. The graft has survived and maintained its endocrine and reproductive capabilities.5 Material

and

methods

Healthy, female weights ranged from

Fig.

1. The

mongrel dogs whose 13.6 to 23.3 kilograms

uterine vessels and their

entry

into

April 15, 1970 J. Obstet. Gym.

were immunized against distemper, dewormed, and quarantined for 2 weeks prior to the operation. No attempt was made to match donor and recipient dogs, and the mixed leukocyte culture technique, which is described later, was used only to familiarize the group with its possibilities, and to determine in vivo and in vitro efficacy of the immunosuppressive agents used. The donor dog was always killed, as removal of the graft entailed gross interference with the blood supply to the bladder and the lower limbs. The first experimental series was composed of 10 uterotubovarian transplants which were not treated with immunosuppressive agents. The second series reported here consisted of 7 uterotubovarian transplants, 6 of which were treated with azathioprine and corticosteroids, and one with antithymocyte serum prepared essentially after the method described by Shanfield and associates.8 The techniques developed were simple and little wastage of dogs occurred. In one experi-

the divisions

of the internal

iliac

artery

and vein.

VoIume Number

Uterotubovarian

106 8

ment the vasculature of the organ graft was damaged, requiring the use of a second donor; in another a second donor was required as the first was found to be pregnant. The dogs were anesthetized with intravenous Nembutal (30 mg. per kilogram) and maintained, following endotracheal intubation, with assisted respiration. Both donor and recipient received an intravenous infusion of 500 ml. of 5 per cent dextrose in water which contained 1,000 I.U. of heparin. Operative technique Donor. The abdomen was opened by a midline incision. The ovarian infundibular ligaments were divided and ligated. The mesosalpinx and broad ligament on both sides of the uterus and Fallopian tubes was divided lateral to the uterine vessels, allowing the ovary, Fallopian tubes,, and uterus to be swung down toward the pelvis. The vesical vessels on both sides were next divided and ligated, and the broad ligaments opened to expose the uterine vessels and their entry into the divisions of the internal iliac artery and vein (Fig. 1) . The internal iliac vein and the arterial branch below the uterine artery were divided and ligated. All other branches of the internal iliac vessels were divided and dissection proceeded cepha-

Fig. 2. Uterus, a monobloc.

Fallopian

tubes,

and

ovaries,

homotransplantation

1173

lad to the junction of the common iliac vessels with the aorta and inferior vena cava. The external iliac artery and vein on both sides were next divided and ligated, and attention turned to the vagina which was transected 2 cm. below the cervix. The lower part of the sigmoid colon was then clamped and divided to allow the graft and its vascular pedicle to be lifted out of the pelvis, and finally the lower 2 inches of aorta and inferior vena cava were dissected free. Division of these major vessels allowed the uterus, Fallopian tubes, and ovaries, together with their vascular pedicle, to be removed in a monobloc. The organ system was gently perfused with 50 ml. of normal saline containing 500 I.U. of heparin, weighed, and placed in normal saline at 4’ C. (Fig. 2). Recipient. The abdomen was opened by a midline incision. A total hysterectomy and bilateral salpingo-oophorectomy was performed. The lower 2 inches of the aorta and inferior vena cava were skeletonized and end-to-side anastomosis of graft inferior vena cava to recipient inferior vena cava was performed using 6-O arterial silk. This was followed by end-to-side aortic anastomosis between graft and recipient, which was carried out in a similar manner. The transplant vagina was sutured to the stump of the recipient’s VP-

together

with

their

vascular

pedicle,

removed

in

1174

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et al. Amer.

gina, and the ovaries and broad ligaments of the graft stitched orthotopically. The dogs were given 10 mg. of morphine postoperatively and 400,000 U. of penicillin and 0.5 Gm. of streptomycin intramuscularly for 5 days. Results

In the first series 2 dogs died on the first postoperative day from intraperitoneal hemorrhage and one (D) became febrile, anorexic, and lethargic, and was examined

Fig. 3. Intravascular venous.

thrombosis

involving

April 15, 1970 J. Obstet. Gym.

on the third postoperative day and found to have thrombosis of the aortic segment of the graft with accompanying gangrene of the transplanted genital tract. In the second series one dog died on the third postoperative day, again from intraperitoneal hemorrhage, and one became febrile, anorexic, began to pass bloody stools and was killed on the eighth postoperative day. It too was found to have had a thrombosis of the aortic graft vessel. In the remaining animaIs the postopera-

large- and medium-sized

vessels, both arterial

and

Volume Number

Uterotubovarian

106 8

tive course was uneventful. The first series of animals was killed at intervals up to 10 days following transplantation, and the graft and junctional vascular anastomotic area were weighed and submitted for histologic examination. Grossly, with each succeeding day after transplantation, adhesions became more numerous and more dense, the graft itself became edematous and hyperemic by the fourth or fifth day, and hemorrhagic by the sixth day. By the seventh postoperative day the organs were rigid with edema, the ovaries had developed marked cystic changes, and patchy necrosis was apparent. In the second series, 6 dogs were treated with 8 mg. per kilogram of azathioprine and 75 mg. of prednisolone (Solu-Medrol) intramuscularly from 2 days before the operation until the time they were killed. One dog (F) was treated with antithymocyte serum for 12 days prior to the operation. The dogs were killed 14 or 28 days In all of them there after transplantation.

Table

I. Series I. Untreated

Dog 1Day ) 3

7

Medium-sized ries predominantly

was thrombosis of the graft moderate to advanced necrosis

Arteritis of medium-sized vessels. Degenerating pregnancy

1 No

DogjDayl

Thrombosis

3

Aorta,

vena

cava

Aorta, vena cava, some medium-sized vessels

6

Aorta, vena cava, medium-sized vessels

Remarks

14

Medium-sized arteries and

B

28

Many medium-sized arteries and veins

Complete necrosis of tissue

C

28

Medium-sized

Tissues necrotic and almost unrecognizable

D

14

Inferior

E*

28

Medium-sized arteries

F

14

Aorta

G

28

Vena cava and medium-sized veins

Medium-sized arteries and

veins

Aorta, vena cava, medium-sized vessels

*Treated

Some inflammatory reaction suggestive of rejection, but with a heavy overlay of polymorph infiltration

with

A

1 No

10

vessels and of the graft.

Table II. Series II. Dogs treated azathioprine and corticosteroids

veins

veins

arte-

Aortic

1175

Pathology There was a rapid and substantial increase in the size and weight of the organ system due to marked interstitial edema of the tissues, accompanied in many cases by rupture of small vessels and extravasation of red blood cells. The inflammatory cell exudate consisted of a mixed population of neutrophil polymorphonuclear leukocytes, occasional plasma cells, and monocytoid cells. These findings were uniform throughout the various parts of the transplanted organs. The major pathologic finding was the presence of intravascular thrombosis involving large- and

dogs Remarks

Thrombosis

homotransplantation

with

vena

cava Tissues necrotic and almost unrecognizable

antithymocyte

Table III. Sampling culture in dogs

serum.

of mixed

leukocyte

Results Small CdlS

4 5 4* *Treated

Transformed I cells

I Mitosis

Total

55.7 75.5 78.0

0.0 0.0 0.0

900 300 200

and

corticasterdids.

44.3 24.5 22.0 with

azathioprinc

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et

al.

Amer.

medium-sized vessels, both arterial and venous (Fig. 3). Small-vessel thrombi either were not present or were intimately associated with areas of frank tissue necrosis. In one dog arteritis of medium-sized vessels was a feature (Tables I and II). Mixed

leukocyte

culture

Cultures of leukocytes from a potential donor and a recipient have been found to cause transformation of some leukocytes to easily identifiable large basophil cells which are capable of synthesizing desoxyribonucleic acid and undergoing mitosis. Studies on identical twins show the reaction to be related to the genetic differences between the 2 subjects and therefore to homograft immunity. The technique used was described by Bach and Hirschhorn,l and the changes can be measured by morphologic means (blast cell formation) or by the incorporation of a radioactive label ( H3-thymidine) and measured by autoradiography or a scintillation counter. A sample of the results obtained after 4 and 5 days’ culture, and after treatment with azathioprine and Solu-Medrol is given in Table III. Obvious transformation took place in all successful cultures and from the sampling given here it would appear that the dosage of immunosuppressive drugs employed was insufficient to suppress leukocyte transformation and, presumably, graft rejection. Comment The outstanding pathologic feature in these 2 series of canine homotransplants was the presence of vascular thrombosis involving large- and medium-sized vessels. There

REFERENCES

1.

Bach, F. H., and Hirschhorn, K.: Science 143: 813, 1964. 2. Sturgis, S. H., and Castellanos, H.: AMER. J. OBSTET. GYNEC. 76: 1132, 1958. 3. Eraslan, S., Hamernik, R. J., and Hardy, J. D.: Arch. Surg. 92: 9, 1966. J.: Transplantation 5: 870, 1967. 4. Hamburger, 5.

Krohn,

are several possible causes but the 2 most likely are that the thrombosis was associated with the surgical procedure, or that it was the primary manifestation of rejection in these viscera. Support for the second possibility exists. Porter,‘j in describing arteriolar and arterial lesions found during renal rejection, noted that gamma globulin appeared in the walls of afferent arterioles and interlobular arteries followed by the deposition of platelets and fibrin on the intima of these damaged vessels. He theorized that these vascular lesions might be due to the production of circulating antibody by host lymphoid tissue, and the formation of antigen antibody complexes in the vessel walls. Hamburger4 re-examined the concept of organ rejection in his study of 67 human and 63 canine renal homotransplants. He found that early rejection invariably took place toward the end of the first week and noted that many microscopic features described as typical of the classic picture of rejection were in fact due to a diminution of blood flow in these transplanted organs. The present state of knowledge does not allow a clear distinction between these 2 possibilities but it would be of value to supplement immunosuppressive therapy with a prolonged course of heparin in the hope that this would modify the tendency to thrombosis. It is suggested that the technique of mixed leukocyte culture may be employed not only as a test of histocompatibility between potential donors and recipients, but also as a monitoring procedure to determine the eficacy of immunosuppressive measures in the treatment of homograft rejection.

6. 7. 8.

9.

P. L.:

Brit.

Med.

Bull.

21: 2, 1965.

Porter, K. A.: Brit. Med.

Bull. 21: 2, 1965. and Hulka, J. F.: AMER. J. OBSTET. GYNEC. 103: 78, 1969. Shanfield, J., Ladaga, L. G., Wren, S. F., Blenerhassett, J. C., and McLean, L. D.: Surg. Gynec. Obstet. 126: 29, 1968. Yonemoto, R. H., DuSold, and Deliman, R.: Shaffer,

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