Eur. surg. Res. 7: 242-248 (1975)

Active Enhancement of Heart Transplants in the Inbred Rat with Donor Strain Blood M anuele D i P aola and Sergio C olizza1 Istituto Patología Chirurgica II, Universita di Roma, Policlinico Umberto I«, Rome

Key Words. Active enhancement • Rat heart transplant Abstract. Prolongation of survival time of heart transplants in inbred rats fol­ lowing intravenous injection of donor strain blood according to various time-dose schedules is described. The immunological status of long-term survivors shows de­ tectable antidonor antibodies, normal graft-versus-host reactions and rejection of skin grafts in the usual time. Lymphocytotoxic activity is well related to the survival time of the heart in those rats which respond to the treatment.

Prolonged survival of both kidney and heart transplants has been re­ ported in inbred rats after intravenous injection of donor strain blood or blood constituents [8, 10, 12]. Active enhancement was thought to be the mechanism involved, but antidonor antibodies were detected only recent­ ly in some animals [8]. We have previously reported on the lymphocyto­ toxic activity in rats receiving heterotopic heart transplants following treatment with donor strain blood [5]. Lymphocytotoxic activity appeared to be related to the survival time of the transplant. This study was under­ taken to evaluate the mechanism of prolonged survival of heart trans­ plants in inbred rats treated with donor strain blood according to various time-dose schedules.

Received: October 16, 1974, accepted: February 28, 1975.

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1 We wish to thank Miss Paola Paggi for the statistical evaluation of all the data and Ethicon SpA for kindly supplying suture material.

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Animals. Inbred male F/344, BUF and F, (F/344XBUF) rats maintained in our laboratories were used. F/344 and BUF rats differ at the major Ag-B histocompati­ bility locus [14], Transplants were done from F/344 (weight 150-200 g) to BUF (weight 200-250 g). Heart transplantation. Heterotopic heart transplantation was performed as de­ scribed [1, 13] with some minor changes [3]. Briefly, anesthesia was induced with ether and maintained with halothane. No heparin was used either in donor or in re­ cipient. Hearts were perfused with normal saline at 5 C via inferior cava, aorta and pulmonary artery (respectively, 4, 8, and 8 ml), and immersed in saline at this tem­ perature until transplanted. Mean cold ischemia time was 42 min (range 36-50). The recipient abdominal vessels were occluded with an S-shaped clamp for an aver­ age of 20-25 min. Continuous sutures of 9/0 Ethilon monofilament (Ethicon) were used both for aorta-aorta and pulmonary artery-cava anastomoses. No magnifying glasses were used. All the recipients had a standard subcutaneous hydration with 15 ml of warmed saline (38 °C), i.e. 5 ml before laparotomy, 5 ml after aortic anas­ tomosis, 5 ml at the end of the operation. This was increased to 20-30 ml in case of bleeding. On opening of the clamp, hearts started to beat vigorously. The mean op­ eration time for the recipient was 49 min (range 37-60). Evaluation of graft function. Graft function was assessed daily by palpation and graft rejection was taken as absence of any pulsation even in presence of electric ac­ tivity of the heart. Sometimes a laparotomy was performed to be sure of the state of the heart. Skin grafting. Skin grafting was performed according to the technique and graft evaluation described by Balner [2], except for the use of 4-6 stay sutures. Experimental design. The animals were divided into four groups: 10 rats without any treatment (controls; group A); 10 rats given an intravenous injection of 2 ml do­ nor strain blood 14 days before heart transplantation (group B); 10 rats given an in­ travenous injection of 2 ml donor strain blood 14 days and 7 days before heart tran­ splantation (group C); 10 rats given an intravenous injection of 2 ml donor strain blood 14 days before and 7 days after heart transplantation (group D). Donor strain blood was collected by cardiac puncture, anticoagulated with 20 units/ml of hepar­ in, and administered in a tail vein of the recipient a few minutes after collection. Follow-up studies. Humoral immunity was assessed 7 days before transplanta­ tion, studying hemagglutinating, hemolytic and Iymphocytotoxic antibodies. A hemolytic test was performed on A: *U’ plates of the same microtiter as (Cooke Engineering Co., Alexandria, Va.) with serial dilutions (25 /tl of donor red blood cells suspensions in 2-percent saline were added for 1 h at room temperature to the serial 25-trl dilutions of recipient serum). A hemolytic test was performed on group A: ‘U’ plates of the same microtiter as above (25 «1 of donor red blood cell suspensions in 1-percent Veronal buffer added for 10 min at room temperature to the serial 25-/il dilutions of recipient serum; fi­ nally 25 nl of fresh guinea pig complement was added for an incubation time of 30 min). For the Iymphocytotoxic activity, F/344 lymphocytes were separated from peri­ pheral blood, suspended in 1-ul aliquots containing 1X10® cells to which 1 /tl of re­

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Materials and Methods

244

Di Paola/C olizza

cipient serum was added and incubated for 30 min. Then 1 ¡A of fresh guinea pig complement was added for 30 min and finally the viability of the lymphocytes was tested by trypan blue exclusion. Lymphocytotoxic activity was expressed as a per­ centage of killed F/344 lymphocytes. Appropriate controls were performed for all the humoral tests. The local graftversus-host reaction (GVHR) was evaluated on the longest survivors with the Elkins test, using 4-week-old F,(F/344XBUF) [6]. The recipients to be tested for GVHR underwent splenectomy. Lymphoid cells were obtained from the spleen with a via­ bility of greater than 90°/o on testing by trypan blue exclusion, and suspended in fresh Hanks’ solution so as to yield 50X106 cells in 0.1 ml. Aliquots were injected under the capsule of the right kidneys of F,(F/344XBUF) rats under light halothane anesthesia. Animals were anesthetized again 7 days later [7], the kidney re­ moved, trimmed of fat, blood vessels and ureter, and then weighed and examined macroscopically.

Statistical analyses. The significance of all comparisons was assessed by Student’s t-test. Results The results are given in table I. The mean survival time of controls is consistent with previously reported data [3-5]. Significant prolongation

Table /. Effect of different intravenous treatments with donor strain blood on heart graft survival Group Number Treatment or rats

Allograft survival, days individual values

5, 5,6,6,6,6, 7, 7, 8,9

p value mean± SD

A

10

none (controls)

B

10

2 ml on day —14 14,20!, 24, 30, 38,44, 51,60,70', 85 43 ±23

6± 1

C

10

2 ml on day -1 4 7,7, 7, 8,8, 8,8, 15,30,301 2 2 ml on day —7

13 ± 9

p

Active enhancement of heart transplants in the inbred rat with donor strain blood.

Eur. surg. Res. 7: 242-248 (1975) Active Enhancement of Heart Transplants in the Inbred Rat with Donor Strain Blood M anuele D i P aola and Sergio C...
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