JOURNAL OF NUCLEAB MEDICINE 7:416-423, 1966 Homotransplantation Studies in Dogs Following Selective Radioisotopic Lymphatic Ablation1 H. S. Winchell, M. Pollycove, W. D. Loughman, V. Richards, L. Kim, and J. H. Lawrence Berkeley, California In previous communications we described a technique for relatively selec tive radiation of lymphatic structures using internally administered Y-90 chelated with diethylene triamine penta-acetic acid—DTPA (1,2,3). This technique results in radiation doses to lymph nodes approximately two and one-half times greater than the radiation dose to bone marrow. A second method outlined in this paper results in further selectivity in radiating lymphatic tissue. Since the homograft rejection response appears to be a function of cells residing in lym phatic structures, we used these techniques in preparing dogs for tissue homo- grafting. MATERIALS AND METHODS All dogs were mongrels of unspecified type between the ages of six months and two years. In most cases the irradiated animals were males, while the donors of the tissue homotransplant were unrelated females. Following irradia tion they were given daily injections of penicillin with streptomycin (600,000 units penicillin, 0.75 gm streptomycin) for a four week period. All counts were performed on venous blood. Duplicated counts of platelets were made by phase contrast microscopy and those of WBC by routine tech niques. At post mortem examination, tissues were fixed in Bouin's solution, em bedded in paraffin or celloidin and stained with hematoxylin and eosin or Pollak's trichrome. The Yttrium-90-DTPA intravenous urine recycling procedure was the same as that previously described (1,3). Intralymphatic injection of colloidal chromic :|-P phosphate (Abbott ) was performed on each of the four legs of the dog after the lymphatic channels had been rendered visable by the intracutaneous and subcutaneous injection of Evans* blue dye into the foot pad. The mesenteric 'University of California, Doner Laboratory, Berkeley 4, California. 416 HOMOTRANSPLANTATION STUDIES IN DOGS 417 nodes were injected directly with colloidal chromium :i-P phosphate at the time of laparotomy using a number 27 needle. X-irradiation of the anterior medias tinum was performed using a supervoltage x-ray machine set at 250 Kvp, 15 milliamps, with an absorber of 0.6 mm tin, 0.25 mm copper and 1.0 mm alumi num. Tube to target distance was 50 centimeters. The field was 15 X 20 cm and extended from above the xyphoid to the cricoid cartilage and laterally to include the medial one-half of both lung fields. Radiation flux rate was approximately 40 r per minute. The radiation was given in two exposures of 1000 r each (air dose). Splenectomy was performed in a routine manner. Renal homotransplanta- tions were performed utilizing an end-to-end anastomosis of the renal artery with either the hypogastric or common iliac artery, and an end-to-side anastomo sis of the renal vein with the common iliac vein. The ureter was implanted in the bladder through a submucosal tunnel. Surgical transplantation of the spleen was performed with vascular anastomosis similar to that outlined for the kidney. Bone marrow was obtained utilizing multiple aspirations with Bierman needles from the femoral, tibial, iliac and humoral bone marrow sites. Spleen cell suspensions were made by chopping the donor spleen into fine particles and then passing them through graded steel mesh filters and through a nylon mesh filter prior to its intraperitoneal administration. The animals receiving the col loidal chromic :i-P phosphate intralymphatic injections and splenectomy, re ceived Y-90-DTPA radiation as a single dose, without subsequent urinary re cycling. Dosimetry estimates were obtained using the data previously pub lished (3) and the plasma Y-90-DTPA clearance curves obtained on these dogs. "Clubbed neutrophiles" were scored in the male dogs receiving female bone marrow according to the technique described by Porter (4). Karyotypes were obtained on peripheral blood leukocytes following short-term culture by the tech nique of Loughman (5), which is a modification of the technique used in humans by Hastings et al (6). All samples containing an isotope were appropriately diluted and plated on stainless steel planchéisand counted in a Nuclear-Chicago gas flow counter fitted with a micromil window of 150 ¿ig/cm-surface density. RESULTS Figure 1. demonstrates changes in peripheral blood cell values which occur in animals given lethal doses Y-90-DTPA by the intravenous urine re cycling technique (3), with and without subsequent treatment with bone marrow homotransplants. The ordinate of the graph represents the mean value of the given parameter expressed as per cent of its mean value on the day of radia tion. The broken lines represent mean values obtained on seven dogs who had lethal doses of Y-90-DTPA but no marrow transplant ( 1); the solid lines repre sent values on 14 male dogs given lethal doses of Y-90-DTPA and subsequently given homologous bone marrow from unrelated female donors. In the lethally irradiated animals, which did not receive marrow, the lymphocyte and granulo- cyte values fell to low levels between one and two weeks following radiation. The mean time of death of these dogs was the fourteenth day following radia- 418 WINCHELL, POLLYCOVE, LOUGHMAN, RICHARDS, KIM, LAWRENCE tion with a range of eleven to twenty-four days. Between the 14th and 18th days following radiation the animals in this group had the following ranges of blood values, expressed as a percentage of their preirradiation values: Granu- locytes 0.7^-5.0%; lymphocytes Q.Q%-14%;platelets ll%-30% reticulocytes O.Of-1.2%. Dogs given homologous bone marrow showed depressions in mean peripheral lymphocyte values comparable to that obtained in the lethally irradiated dogs not receiving bone marrow, however, the mean granulocyte levels of these dogs remained generally within the normal range as compared to the marked de pression seen in animals not receiving bone marrow transplants. Between the 14th and 18th days following radiation the animals in this group had the fol lowing ranges of blood values, expressed as a percentage of their preirradiation values: Granulocytes 20%-705%;lymphocytes 0.0%-46£;platelets 12^-79%; reticu locytes 0.0%-1700%.We did not see the marked rebound granulocytosis seen by others following successful marrow transplantation (7 ). Figure 2 demonstrates changes in the peripheral blood platelet and reticu- locyte levels of these animals. In the animals given homologous bone marrow, there was a rise in peripheral reticulocytes (solid lines) following bone marrow transplantation as compared to zero values in the lethally irradiated nontrans- planted group (broken lines). Following radiation, platelet levels fell in animals 200 LYMPHOCYTES 100 O 400 \ GRANULOCYTES Y90 + Horn, bone marrow (14 dogs) 300 Y90 No bone marrow (7 dogs) o> o. 200 O) u_ 0) o. 100 10 20 30 40 50 60 Days after irradiation MUB-3737 Fig. 1. Peripheral blood lymphocyte and granulocyte changes following Y-90 DTPA irradiation in control dogs (broken lines), and in dogs given homologous bone marrow (solid lines) (ordinate expressed as per cent of value on day of radiation). HOMOTRANSPLANTATION STUDIES IN DOGS 419 given homologous bone marrow (solid lines) but not to the low level reached in the lethally irradiated nontransplanted group (broken lines). In the 14 animals given lethal radiation plus homologous bone marrow, 11 survived over 25 days. Of these 11, three died in the 25-60 day period following radiation; one secondary to surgery, one of gastrointestinal bleeding, and one of a syndrome resembling "secondary disease" of the mouse. Of the eight animals surviving over 60 days following radiation, five lived over one and one-half years. In the long term survivors five renal homotransplants were attempted. Two of these were technical failures, two appeared to be rejected and one renal homo- transplant survived 36 days following the homotransplant procedure. Several of the male dogs given lethal doses of radiation and homologous marrow transplantation from female donors had increased numbers of "female type" clubbed neutrophiles in the peripheral blood 12 to 25 days following bone marrow homotransplantation. The finding of these "clubbed" neutrophiles in the peripheral blood of irradiated animals is difficult to interpret relative to the "success" of marrow homotransplantation in our experiments. We have noted an increase in nuclear projections on male neutrophiles which could have been mis taken for female neutrophiles in animals given sub-lethal doses of radiation with out transplantation of homologous bone marrow (8). 1500 + Hom. bone marrow (14 dogs) 20 30 40 Days after irradiation MUB-3738 Fig. 2. Peripheral blood reticulocyte and platelet changes following Y-90 DTPA irradia tion in control dogs (broken lines), and in dogs given homologous bone marrow (solid lines). (Ordinate expressed as per cent of value on day of radiation.) 420 WINCHELL, POLLYCOVE, LOUGHMAN, RICHARDS, KIM, LAWRENCE In two of the long term survivors, peripheral blood leukocyte cultures ob tained one-to-two years following the radiation procedure failed to show dividing cells of the donor sex karyotype. One animal, not represented on this graph, received a lethal dose of Y-90- DTPA radiation and in addition had splenectomy and 2000 r of x-rays delivered to the area of its thymus. This animal subsequently received homologous bone marrow and a renal homotransplant which functioned for 34 days. Evidence that the Y-90-DTPA radiation suppressed the homograft rejection response may be summarized as follows; homologous bone marrow transplanta tion saved the lives of lethally irradiated animals and some of these animals developed findings suggestive of "secondary disease"; renal homografts had prolonged survival in some irradiated animals. That the suppression of the homo- graft rejection response was incomplete or temporary was evidenced by eventual rejection of renal homografts.