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Fetal rat intestinal transplantation: Cryopreservation and cyclosporin A
Fetal Rat Intestinal Transplantation: Cryopreservation and Cyclosporin A By Frank M. Guttman, Luong T. Nguyen, Jean-Martin Laberge, Ngoc-Van Nguyen, Thomas A. Seemayer, and Laurie Gibbons Montreal, Canada
9 Successful preservation of small bowel by cryobiologic techniques would increase the feasibility of intestinal transplants. Immunosuppression by Cyclosporin A (CyA) has also increased interest in intestinal transplantation. W e have investigated the effect of cryopreservation and immunosupprassion in fetal rat intestinal transplantation. Segments of fetal bowel implanted isogeneically into the paravertebral gutter of young rats were found to grow in a high percentage of animals (53% to 100%). Segments frozen to --20~ or - 4 0 ~ at two rates of cooling, grew isogeneically (50% to 89%), demonstrating the feasibility of cryopreservation. Histologic examination of this bowel showed preservation of structure. When these segments were cooled and implanted allogeneically, no immunosuppressive effect was found. Segments protected by daily CyA administration grew. No synergistic effect was seen by associating CyA and cryopreservation. These experiments suggest the possibility of creating fetal small bowel long-term banking. 9 1985
by Grune
& Stratton, Inc.
INDEX WORDS: Transplantation small intestine; Cyclosporin A; cryopreservation.
O PEDIATRIC SURGEONS, the need for intes-
tinal transplantation is quite apparent. Massive T bowel resection, secondary to necrotizing enterocolitis or volvulus in neonates or from inflammatory bowel disease later on, has been treated by total or partial parenteral nutrition. This form of therapy has helped enormously, changing early mortality. Nevertheless, it is associated with high morbidity. Over a lifetime, the attractiveness of a bowel transplant is therefore evident. In the late 1950s and early 1960s, laboratory investigation, led by Lillehei, established the technical feasibility of bowel transplantation in the dog I and the rat. 2 Nevertheless, graft v host disease (GVHD) was thought to be the explanation of sudden death in some of these experiments. 2~sThe knowledge obtained in the laboratory was applied clinically to nine patients in the early 1970s with no long-term success, as outlined in Kirkman's recent review. 7 The availability of the new immunosuppressive drug, Cyclosporine A (CyA) has reawakened interest in bowel transplantation experimentally."s9 The first intestinal transplant in the CyA era has just (April 1985) been carried out with the implantation of the bowel of a ten-year-old into a 26-year-old woman in Toronto) ~ In previous experiments, we succeeded in cryopreJournal of Pediatric Surgery, Vo120, No 6 (December),1985: pp 747-753
serving about 50% of canine bowel segments frozen to - 8 0 ~ and thawed. 1H3 With isolated cultures of cells, cryopreservation has been reported to alter the immunogenicity of the tissue. TM The aim of this paper is to report on our efforts to test this hypothesis using rat-fetal bowel and to investigate the possible synergistic effect of cryopreservation and immunosuppression. MATERIALS AND METHODS Pregnant CD Fischer or Lewis rats (Charles River, Boston, Mass) were anesthetized with ketamine (60 mg/kg) and nembutal (20 mg/kg) at 19 days of gestation. The fetii were removed and their intestinal tract mobilized, then placed into a cold (10 ~ solution of perfusion media or media plus dimethylsulfoxide (Me2SO). The composition of the perfusion media was as previously described in kidney freezing experiments (Table 1).1~A total of 635 rats were the subjects of these experiments. In the first set of experiments, there were two control and two experimental groups: (1) Control: the intestine (6 cm) was placed into cold media (40 rain). (2) Control: placed into media plus a concentration of 1.5 mol/L Me2SO for 20 minutes and then 3 M Me2SO media for 20 minutes. (3) Experimental: as in (2) then placed into freezing chamber (Controlled Rate Freezer, FTS Systems Inc, Stone Ridge, NY, model CRF-4E) and cooled to - 2 0 ~ at 0.5 ~ The low temperature was maintained for 1 hour. The intestine was then placed into a saline bath at 25 ~ and warmed. When thawed, the Me2SO was washed out with hyperosmolar mannitol solution in a stepwise fashion as previously described)6 (600 mosm - 5 0 0 mosm - 4 0 0 mosm). (4) Experimental: cooled to - 4 0 ~ at 0.5 ~ and treated as above. The thawed segments were then implanted either isogeneically or allogeneically into a prepared subcutaneous paravertebral pocket in young rats (150 g). One half of the allograft experiments were Lewis fetal intestine into Fisher young rats, and half implanted in the other direction, ie, Fisher to Lewis. The segments were marked at each end with silk sutures and the wound closed with Michel clips. All the rats were killed after six weeks. Intestinal segments that have both lumen and mucosa were measured. Analysis was also carried out nonparametrically, with 1 to 2 cm deemed fair growth, and 2 to 5 cm good growth. Histologic studies of the bowel and disaccharidase activity were carried out, in random segments.
From the Departments of Pediatric Surgery and Pathology, The Montreal Children's Hospital, McGill University, and The Montreal Children's Hospital-Research Institute, Montreal. Supported by the Medical Research Council of Canada MA7077 and The Montreal Children's Hospital-Research Institute. Presented before the 16th Annual Meeting of the American Pediatric Surgical Association, Kohala Coast, Hawaii, May 1-4, 1985. Address reprint requests to Frank M. Guttman, MD, The Montreal Children's Hospital, 2300 Tupper, Suite C-1129, Montreal, Quebec, H3H 1P3, Canada. 9 1985 by Grune & Stratton, Inc. 0022-3468/85/2006-0037503.00/0 747
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GU-I-I'MAN ET AL
(89% to 75%) but not allogeneically. Cryopreservation, alone, did not allow for successful allotransplantation
Table 1. Composition of the Hypothermic Perfusion Medium (per Liter)
Glucose KCI K2HPO4 NaHCO3 Glutathione-reduced MgCI2-6H20 CaCI2 Adenine Hepes Mannitol Chlorpromazine
(0/21, 0/] 0).
180.0 mmol 28.2 mEq 7.2 10.0 5.0 2.0 1.0 1.0 25.0 110.0 0.025 g
In a second series of experiments using allotransplanted bowel, CyA was given to the rats orally in oil base (15 mg/kg/d). All were killed at 6 weeks after receiving CyA for 2 weeks. Group 1) control-intestine was immersed in cold media. Group 2) control was immersed in Me2SO. Group 3 as in 2, segments were cooled to - 4 0 ~ at 0.5 ~ In a third series of experiments, treatment with CyA was continued until death at 6 weeks: (1) control media; (2) control Me2SO; (3) as in (2) with cooling to 20 ~ at 0.5 ~ (4) -20~ at 1 ~ (5) - 4 0 ~ at 0.5 ~ (6) - 4 0 ~ at 1~ Random segments were removed en bloc and fixed in 10% formalin. Paraffin-embedded sections were stained with hematoxylin-phloxine-saffran. Other segments were submitted for disaccharidase activity (Dahlquist Method). 17
RESULTS Successful fetal bowel transplantation is evident from these experiments when the segments are transplanted isogeneically (Table 2A). When the segments are bathed in perfusion media, or media plus the cryoprotectant Me2SO, segments will grow isogenically (53% to 100%). When these segments are transplanted allogeneically with no immunosuppression, they do not survive (0/4, 0/7). Segments cooled to - 2 0 ~ or - 4 0 ~ at a rate of 0.5 ~ and kept at that temperature for one hour, will grow isogenically
In the second set of experiments when CyA was given for 2 weeks (Table 2B), allotransplanted segments did not survive when analyzed at 6 weeks (Controls 75% to 80%). Segments cooled to - 4 0 ~ at 0.5 ~ again showed good growth 15/30 (50%) when transplanted isogenically but no growth when allotransplanted. The third series of experiments again demonstrated (Table 2C) good growth in isogeneic transplants whether the segments were in control media, control Me2SO, or cooled to - 2 0 oC or - 4 0 oC at 0.5 ~ or l ~ (71% to 93%). As expected, no segments grew when allotransplanted without imrnunosuppression. However, a good result was obtained when allotransplanted bowel was implanted into rats receiving CyA for 6 weeks, whether the segments were controls or cooled to - 2 0 ~ or - 4 0 ~ at 0.5 ~ or 1.0 ~ (23% to 48%). When our results are analyzed parametrically (Table 3), good growth is obtained when the segments, transplanted isogeneically, are control, cooled to - 2 0 ~ or - 4 0 ~ at 0.5 ~ or 1.0 ~ When the segments are transplanted allogeneically with CyA, there is a significant difference in growth in all groups when compared to syngeneic controls. A two-way Anova statistical analysis demonstrated no difference in all the syngeneic treatment groups, ie, freezing at - 2 0 ~ or - 4 0 ~ at 0.5 ~ or at 1~ for one hour gives as good results as the controls, both as the measured length and as nonparametric analysis. In the first set of experiments (syngeneic group), serial transverse sections of bowel from three experimental groups, media control, Me2SO, - 2 0 ~
Table 2. Growth (Nonparametric 2 to 5 cm) Treatment
Media Me2SO --20 ~ -40 ~ Media Me2SO --40 ~ Media Me2SO -20 ~ --20 ~ --40 ~ -40 ~
~ ~
0/4 (0)* 0/7 (0)* 0/21 (0)* 0/10(0)*
~
12/15 (80%) 12/16 (75%) 15/30 (50%)
0/6 (0)* 0/11 (0)* 0/13 (0)*
28/30 45/50 12/14 16/19 20/28 19/24
O/11 (O)* 7/30 (23%)? 0/23 (0)* 10/28 (36%) t 0/3 (0)* 14/35 (40%) t 14/29 (48%) t 10/35 (29%) t 12/35 (34%) t
~ ~ ~
Treatment
8/15 (53%) 6/6 (100%) 25/28 (89%) 9/12 (75%)
~
Table 3. Measurement of Growth (Parametric)
AIIo TX Syngen TX (%) AIIo TX (%) With CyA (%) Weeks
(93%) (90%) (86%) (84%) (71%) (79%)
* P < 0.0001, compared to syngeneic control. t P < 0.05, compared to syngeneic control.
0/6 (0) 0/11 (0) 0/16 (0)
2 2 2 6 6 6 6 6 6
Media Me2SO -20 ~ -40 ~ Media Me,SO --40 ~ Media Me2SO -20 ~ --20 ~ -40 ~ -40 ~
Svngen (mm length)
AIIogen Without CyA
~ ~
21.09 24.09 23.09 15.87
_+ 2.24 _+ 1.89 • 2.24 _+ 1.85
0 0 0 0
~
21.09 _+ 2.24 24.09 • 1.89 15.87 • 1.85
0 0 O
~ ~ ~ ~
21.09 24.09 23.33 28.95 15.87 30.50
0 0 0
• 2.24 • 1.89 _+ 2.24 _+ 3.89 • 1.85 +_ 3.90
*P < .0001, compared to syngeneic control.
AIIogenWith CyA (mm length)
0 0 0 5.55 6.61 8.99 15.00 8.05 7.57
+- 2.65* -+ 21.9" -+ 2.19" -+ 3.20* -+ 2.25* _+ 2.05*
FETAL RAT INTESTINAL TRANSPLANTATION
749
Table 4. Specific Activity of Disaccharidase (total activity/rag protein/g tissue) Control n~2 Sucrase Trelalase
Maltase Lactase
109.5 291.0 720.5 34
• • • •
2.5 24.0 7.5 0,0
AIIotx (Growth 3 cm) --20~ n=3 35.7 7.0 0.8 0.6
_+ 10.6" • 1.9 t • 0.81 t • 0.37 t
*P < 0.01, compared to control. t P < 0.005, compared to control.
Fig 1. Photomicrograph of syngeneic intestinal segment originally cooled to --20 ~ Note well-preserved morphology (HPS x 200).
revealed similar findings that include marked distension with intraluminal mucous, mucosal preservation with variable degrees of villus formation, and normal development of muscularis propria and myenteric plexus (Fig 1). Neither a cellular infiltrate nor necrosis was recognized. Sections from the fourth group ( - 40 ~ revealed less dilatation of the bowel, reduced intraluminal mucous, and modest numbers of mononuclear cells in the lamina propria. From the allogeneic group (treated for 6 weeks with CyA), sections from the media control group revealed transmural necrosis in each segment. In one, the necrotic bowel demonstrated a modest lymphocytic infiltrate. Sections from the Me2SO treated group revealed intense transmural mononuclear infiltration associated with necrosis in two rats. In one, multifocal transmural necrosis was noted, although many portions of the bowel appeared normal. In the - 2 0 ~ group, all four rats demonstrated variable degrees of transmural necrosis with mononuclear cell infiltration (Fig 2). One segment, frozen to - 4 0 ~ showed much of the bowel well preserved, although multifocal seg-
Fig 2. Photomicrograph of allogeneic intestinal segment originally cooled to --20 ~ illustrating intense mononuclear cell infiltration of muscular wall (HPS x 200).
ments of transmural necrosis were noted. Disaccharidase activity was found to be markedly diminished in allotransplanted segments (Table 4). DISCUSSION
Experimental transplantation of the bowel in animals is clearly established. The dramatically improved status of transplantation of some organs with CyA has evoked a new interest in small bowel transplantation since immunologic reactions have been thought to be one of the major limiting factors. 2'7-9 Richard Lillehei was the first to establish the limits of viability of autotransplanted small bowel. ~'3 He then showed that with immunosuppression (prednisone and azothiaprine) allotransplants were feasible in the dog. 3 Without immunosuppression, the dogs died at 6 to 9 days without a satisfactory explanation of the cause of death. There was gross enlargement of the mesenteric lymph nodes, but without cellular infiltration of the bowel wall. The suggestion was that severe G V H D was responsible. When immunosuppression was given, some of the dogs died of rejection, with the typical changes of rejection; thickening of the bowel wall, mononuclear cell infiltration, mucosal necrosis, bleeding, sloughing, villous atrophy, and occasional perforation. Cohen et al studied the balance between G V H D and HVGD (rejection). 6 Control animals died at about nine days with enlarged lymph nodes. If the bowel was irradiated with 150 rads prior to the transplant, the mesenteric nodes were not enlarged but the bowel was rejected with sloughing and mononuclear cell infiltration. When the bowel was irradiated with a lower dose, 50 rads, only minimally enlarged nodes were found with longer survival times and normal bowel. This study suggests that G V H D is mediated by the lymphatic load in the allograft and when the load is sufficient it will result in a GVHD. When the lymphatic tissue is completely eliminated, typical rejection will occur. A more immunologically elegant study by Monchik and Russel using microvascular technique demonstrated the feasibility of small bowel transplantation in the rat. 2 Using highly inbred and F1 hybrid rats, they showed that when the transplant was in the direction of
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Lewis to Hybrid Lewis Brown Norway (and rejection could not take place), a G V H D occurred in 12 to 20 days. This GVHD could be prevented by prior irradiation. When the transplant was done in the opposite direction (Hybrid LBN to Lewis), typical rejection changes were found. Thus, when a two-way reaction is possible rejection did occur. By mechanically reducing the lymphatic load in a bowel transplant, Deltz et al demonstrated the reduction of GVHD mortality, is They did this by transplanting 50% v 100% of small bowel, or by surgically removing the mesenteric lymph nodes prior to transplantation. 19 Again, if competent T lymphocytes are not transferred with the graft, G V H D will not take place. 7 In the rat, intestinal irradiation will prevent GVHD. 2't8"~9 In outbred dogs the explanation for the GVHD is not as readily apparent, nor is it clear that the experimental work on inbred rats has clinical relevance in man. In 1971, Zinzar, Leitina, Tumyan, et al demonstrated the feasibility of transplanting segments of fetal bowel subcutaneously into a syngeneic adult, which grew progressively forming organ-like cavities, z~ The intestinal segment initially devoid of blood supply would become vascularized and remain as differentiated intestine. In 1974, Leapman, Deutsch, Grand, et al z! confirmed that fetal rat intestine transplanted into a syngeneic host would establish blood supply, grow progressively, and exhibit normal histology. Absorption studies showed that glucose, glycine, and oleic acid were absorbed. Peristalsis was present and the brush border enzymes (disaccharidase activity) was normal. This same group led by Folkman showed that normal intestine was present in the recipients for as long as 1 year after transplantation, zz Using rabbit antirat lymphocyte serum (RALS) they also could delay rejection for up to 26 days or more. The suggestion was then made that in short bowel syndrome, this subcutaneous intestinal loop could serve as an accessory gut enabling discontinuing intravenous alimentation. In time, the patients own short gut might adapt to progressive enteral feedings of elemental food. The subcutaneous loop could then be removed. Alternatively, the same group have demonstrated that the loop might be placed into the intestinal circuit, if the loop was introduced in stages near the proximal end of the recipient's small bowel. The advent of CyA has clearly changed the transplantation world. It would seem to be an ideal immunosuppressive agent since it is effective in GVHD as well as in rejection. Schwartz, Flye, and Storozuk have reported on experiments whereby CyA prevented rejection of fetal small bowel transplanted into the back of allogeneic rats, across a major histocompatibil-
GU-I-FMAN ET AL
ity barrier (Fisher-Wistar). 23 The function of the transplanted fetal bowel was good, as measured by substrate absorption, D N A content, and disaccharidase activity. Bass, Tai, Schweltzer, et al noted that the neogut in this model has structural and electrophysiologic characteristics similar to those of normal rat intestine, z4 Montgomery, Sybicki, and Grand z5 have also demonstrated autonomous biochemical and morphologic differentiation in the fetal rat intestine transplanted at 17 and 20 days of gestation. Reznick, Craddock, Langer, et al first reported on the use of CyA to allotransplant successfully small bowel in the dog. 8 This Toronto group led by Langer and Cohen has fully reported on several aspects of the morphology and function of canine small bowel transplants and on the absorption of CyA from the gut. 6'8 During this same period, Starzl's group has also explored the limits of intestinal transplantation showing successful transplants with CyA and prednisone. 9 Two of their animals were living at 550 and 555 days. Maintenance of nutrition and absorption of D-Xylose and fat were better than in control animals with a short gut, but worse than that of normal dogs. Using minipigs, Stauffer, Becker, Karamehmedovic, et al have demonstrated the usefulness of biopsy of ostomy sites to predict rejection and allow for the removal of irreversibly rejecting grafts, saving the life of the recipient, z6 In these experiments, we have shown that even across a minor histocompatibility locus, fetal bowel will be rejected, or at least fail to grow. Evidently, not enough lymphatic tissue is transplanted to make any change on the young adult rat recipient's growth or skin. Cryopreservation is feasible with a high percent of segments transplanted isogeneically growing. Cryopreservation in the manner described has no immunosuppressive effect. The 6-week period for this experiment is probably the cause of the distension of the implants. With no egress, after 6 weeks atrophy has been found in some segments. Some of our control segments showed ischemic necrosis on histologic examination. It is entirely possible that had we chosen a 4-week experimental period, these changes would not have been found. The increase in mucous secretion has been noted on previous electron microscopic studies we have carried out in dog bowel freezing experiments, z7 In our experiment, when CyA was used for 2 weeks, the allogeneically-transplanted fetal bowel did not survive. Possibly it may have grown and then at death, the rejection process resulted in no visible trace of the transplant. Some experiments with rat intestine allografting have demonstrated prolonged survival with only seven days of CyA treatment) However, with
FETAL RAT INTESTINAL TRANSPLANTATION
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immunosuppression of C y A for 6 weeks, a large n u m ber of segments grew when allotransplanted whether they were controls or cooled to - 2 0 ~ or - 4 0 ~ at either 0.5 ~ or 1.0 ~ The hypothesis that cryopreservation will induce some immunosuppressive effect is not found to be valid in this study. However, when t r a n s p l a n t e d syngeneically, cryopreservation has been shown to be effective in producing viable bowel that grows when frozen down to - 2 0 ~ or - 4 0 ~ Cryopreservation a n d immunosuppression together have not been found to have a synergistic effect. The low levels of all disaccharidases, but especially maltase and lactase have been noted by all authors in this model. 21'23'z5One likely explanation is the lack of substrate, well known to be directly related to the levels of enzyme found. In addition, we have expressed our levels in terms of a c t i v i t y / m g p r o t e i n / g of tissue, which in this type of traumatised bowel m a y be increased (collagen a n d fibrosis).
This study leads to an extension of F o l k m a n ' s original suggestion in 1974, that fetal intestine could be i m p l a n t e d as an accessory gut p e r m i t t i n g t e m p o r a r y or p e r m a n e n t feeding and nutrition. T h e extension of that suggestion would be to have fetal intestinal segments b a n k e d at low temperatures. L o n g - t e r m cryopreservation has been r e m a r k a b l y successful with RBC, spermatozae, ova, embryos, a n d clumps of tissue (parathyroid, thyroid, islets). T h e sporadic success reported in whole organ freezing has been inconsistant probably because of a multiplicity of factors; geometry, multiplicity of cell types, each with an o p t i m u m rate of freeze a n d thaw, o p t i m u m c o n c e n t r a t i o n of cryoprotectant, and washout technique. T h e fetal bowel because of its size would thus seem to be a feasible organ to store and m a k e fetal small bowel b a n k i n g a reality. In the C y A era, the first intestinal t r a n s p l a n t in m a n has been carried out. More clinical application will doubtless be done in the near future.
REFERENCES
1. Lillehei RC, Goot B, Miller FA: The physiologicalresponse of the small bowel of the dog to ischemia including prolonged in vivo preservation of the bowel with successfulreplacement and survival. Ann Surg 15:543-559, 1959 2. MonchikGJ, Russel PS: Transplantation of small bowel in the rat: Technical and immunologicalconsiderations. Surgery 70:693702, 1971 3. Ruiz JO, Uchida H, Schultz LS, et al: Problems in absorption and immunosuppressionafter entire intestinal allotransplantation. Am J Surg 123:297-303, 1972 4. Hardy MA, Quint J, State D: Effect of antilymphocyte serum and other immunosuppressiveagents on canine jejunal allografts. Ann Surg I71:51-60, 1970 5. Kao GF, Lerner KG: The histopathologyof human intestinal graft-versus-hostdisease (I-GVHD). Gastroenterology66:888, 1974 (abstr) 6. Cohen Z, MacGregor AB, Moore KTH, et al: Canine small bowel transplantation. A study of the immunological responses. Arch Surg 111:248-253, 1976 7. Kirkman RL: Small bowel transplantation. Transplantation 37:429-433, 1984 8. Reznick RK, Craddock GN, Langer B, et al: Structure and function of small bowel allografts in the dog: Immunosuppression with CyclosporinA. Can J Surg 25:51-55, 1982 9. Diliz-Perez HS, McClure J, Bedetti C, et al: Successful small bowel allotransplantation in dogs with cyclosporineand prednisone. Transplantation 37:126-129, 1984 10. Cohen Z, Langer B: personalcommunication, April 1985 11. Guttman FM, Khalessi A, Huxley BW, et al: Whole organ preservation. I. A technique for in vivo freezing canine intestine using intraarterial helium and ambient nitrogen. Cryobiology6:3236, 1969 12. Guttman FM, Khalessi A, BerdnikoffG: Whole organ preservation. II. A study of the protective effect of glycerol, dimethyl sulfoxide and both combined, while freezing canine intestine employingan in vivotechnique. Cryobiology6:339-344, 1970
13. Guttman FM, BerdnikoffG, Sangbhundhu K, et al: Preservation of canine small bowel by freezing. Arch Surg 111:260-262, 1976 14. Wodinsky I, Meaney KF, Kensler CJ: The stain specificityof transplantable neoplasm after freezing and storage in liquid nitrogen. Cryobiology2:44-246, 1965 15. Segal NB, Guttman FM: Kinetics of permeation and intracellular events associated with Me2So permeation of rabbit kidneys during perfusion at 10 ~ Cryobiology19:41-49, 1982 16. Segal NB, Guttman FM: Function of rabbit kidneys in vitro at normothermia following equilibriation with 3.0M Me2SO and removal by hypertonic washout at 10 ~ Cryobiology 19:50-60, 1982 17. Dahlquist A: Method for assay of intestinal disaccharidases. Ann Biochem 70:18-25, 1964 18. Deltz E, Muller-Hermelink HK, Ulrichs K, et al: Development of graft-versus-host reaction in various target organs after small intestinal transplantation. Transplant Proc 13:1215-1216, 1981 19. Thiede A, Deltz E, Schack T, et al: Successful manipulation of GVH-reaction in semi allogeneic intestinal transplantation in rat by treatment of the donor recipient. Seventh International Congress in Microsurgery, Lyon, 1982 (abstr) 20. Zinzar SN, Leitina BI, Tumyan BC, et al: Large organ-like structures formed by syngeneic foetal alimentary tract transplanted as a whole or in parts. Rev Eur Etudes Clin Biol 16:455, 1971 21. Leapman SB, Deutsch AA, Grant R J, et al: Transplantation of fetal intestine: Survival and function in a subcutaneous location in adult animals. Ann Surg 1:109-114, 1974 22. Deutsch AA, Arensman R, Levey R, et al: The effect of antilymphocyte serum of fetal rat intestine transplanted as free subcutaneous homografts. J Pediatr Surg 9:29-34, 1974 23. Schwartz MZ, Flye MW, Storozuk RB: Effect of Cyclosporin A on growth and function of transplanted fetal rat intestine. Surg Forum 33:341-343, 1982
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24. Bass BL, Tai Y-H, Schweltzer E J, et al: Neogut: Anatomic and physiologic properties of the transplanted fetal intestine. Surg Forum 34:181-183, 1983 25. Montgomery RK, Sybieki MA, Grand R J: Autonomous biochemical and morphological differentiation in fetal rat intestine
transplanted at 17 and 20 days of gestation. Dev Biol 87:76-84, 1981 26. Stauffer UG, Beeker M, Karamehmedovie H, et al: Transplantations of small intestines. J Pediatr Surg 9:21-28, 1974 27. Makita T, Khalessi A, Guttman FM, et al: The ultrastructure of small bowel epithelium during freezing. Cryobiology 8:25-45, 1971
Discussion Dennis Lund (Boston): For the past two years, I've had the privilege of working in the surgical research laboratory of the Boston Children's Hospital, where some of the early work on fetal gut transplantation was done by Leapman, Deutsch, and Folkman. We continue to be very interested in this problem. In work from our laboratory, recently submitted to the Surgical Forum, by Joseph Friedberg, a Harvard medical student, and Dr Daniel Ryan, human fetal intestine has been successfully transplanted into athymic animals. Very briefly, fresh human fetal intestine was obtained from 19 to 23-week-old abortuses from the Pathology Department of the Brigham and Women's Hospital, with the full informed consent of the women aborted. This fetal intestine was immediately transported to our laboratory, where it was implanted either subcutaneously, as described by Dr Guttman, or intraperitoneally into homozygous nude rats or mice. Of the 41 transplants examined to date, 34 or 83%, have shown vascularization by 1 week and subsequent growth. Preliminary results showed these intestinal transplants to have the capacity to absorb both intraluminal glucose and glycine. Some of the transplants showed visible peristalsis, and 15 of 16 examined histologically showed viable ganglion cells. Dr Guttman's success with cryopreservation and Cyclosporin A in fetal intestinal transplants is very exciting. Dr Schwartz and his colleagues have also had excellent success with Cyclosporin A in the same model, as published in the most recent issue of the journal, Surgery. Now, with the success of Friedberg and Ryan with human fetal intestinal transplants in nude animals, plus the proven effectiveness of Cyclosporin A in immunosuppression, and cryopreservation, we may very rapidly be approaching the point where the application of this technology will begin to solve the problem of the short gut syndrome. Marshall Schwartz (Sacramento): The specific questions asked by Dr Guttman were: Can cryopreservation of fetal intestine result in a functioning graft and will cryopreservation have an immunosuppressive effect, either by itself or in conjunction with Cyclosporin? The authors did demonstrate that cryopreservation was an effective means of preserving rat fetal small intestine. However, the grafts were somewhat
shorter than had been shown initially by Folkman and in our previous study. This study did not demonstrate any immunosuppressive effect of cryopreservation, either alone or in conjunction with Cyelosporin. Three years ago I reported at the Surgical Forum, a study which asked the questions; would Cyclosporin be an effective immunosuppressive agent and/or would it have a toxic effect on fetal intestine when transplanted across major histocompatibility loci. We showed that Cyclosporin, at least in this model, was an effective immunosuppressive agent and did not have a toxic effect on the development of the fetal intestine. Now, what does all this mean? There has been recent interest with whole-organ small intestine transplantation because of the availability of Cyclosporin. However, experimental studies have shown that Cyclosporin is not the panacea that it has been for liver and heart transplantation. Therefore, using a fetal model may have more significance. With the availability of Cyclosporin and, now, cryopreservation as an effective means of preserving fetal intestine, this model may have some merit as a clinical tool. I have attempted to incorporate the transplanted intestine into the host's own intestine, with very limited success. I have two questions for Dr Guttman. One, he did not report on whether long-term cryopreservation might effect the function of the intestine. Also, I'm concerned about the disaccharidase data for two reasons: it is based on small numbers and there was a dramatic difference between the controls and experimental group. I am wondering if this is due to chronic rejection. Frank Guttman (closing): The low levels of disaccharidase activity does also concern me. Perhaps, it's that we're just learning to do this, but I think there is a significant difference that we have to explain, and I think chronic rejection may be part of the explanation. Long-term preservation--we're working on that now. We have 24-hour and 1-week conservation that seems to be successful in our most recent experiments. Dr Schwartz mentioned the Cyclosporin toxicity. That's well known, and we have done control studies showing that these young adult rats grow and all their organs are unaffected by this dose of Cyclosporin that we're
FETAL RAT INTESTINAL TRANSPLANTATION
giving, which is not high, it's on the low side (15 rag). On the question of reintegrating the gut into the circuit, Dr Folkman, ie, Deutsch, Leapman, and Folkman, did that originally in rats, and tried various ways of reconnecting the bowel into the circuit in a staged manner. They were able, by putting the gut in proximally and staging it, to have some survivals in this model. But, of course, that may not be really necessary. Dr Folkman originally suggested 12 to 14 years ago,
753
that one possibility was not reintegrating the gut into the circuit but using it as an accessory gut just subcutaneously, as an infusion port. We have been working, also, with Drs Goldman and Colle on pancreatic islet--human islets--for preservation and for culture. They have succeeded in culturing islets for over five months; and this is, indeed, an area of further research.
9 Successful preservation of small bowel by cryobiologic techniques would increase the feasibility of intestinal transplants. Immunosuppression by Cyclosporin A (CyA) has also increased interest in intestinal transplantation. W e have investigated the effect of cryopreservation and immunosupprassion in fetal rat intestinal transplantation. Segments of fetal bowel implanted isogeneically into the paravertebral gutter of young rats were found to grow in a high percentage of animals (53% to 100%). Segments frozen to --20~ or - 4 0 ~ at two rates of cooling, grew isogeneically (50% to 89%), demonstrating the feasibility of cryopreservation. Histologic examination of this bowel showed preservation of structure. When these segments were cooled and implanted allogeneically, no immunosuppressive effect was found. Segments protected by daily CyA administration grew. No synergistic effect was seen by associating CyA and cryopreservation. These experiments suggest the possibility of creating fetal small bowel long-term banking. 9 1985
by Grune
& Stratton, Inc.
INDEX WORDS: Transplantation small intestine; Cyclosporin A; cryopreservation.
O PEDIATRIC SURGEONS, the need for intes-
tinal transplantation is quite apparent. Massive T bowel resection, secondary to necrotizing enterocolitis or volvulus in neonates or from inflammatory bowel disease later on, has been treated by total or partial parenteral nutrition. This form of therapy has helped enormously, changing early mortality. Nevertheless, it is associated with high morbidity. Over a lifetime, the attractiveness of a bowel transplant is therefore evident. In the late 1950s and early 1960s, laboratory investigation, led by Lillehei, established the technical feasibility of bowel transplantation in the dog I and the rat. 2 Nevertheless, graft v host disease (GVHD) was thought to be the explanation of sudden death in some of these experiments. 2~sThe knowledge obtained in the laboratory was applied clinically to nine patients in the early 1970s with no long-term success, as outlined in Kirkman's recent review. 7 The availability of the new immunosuppressive drug, Cyclosporine A (CyA) has reawakened interest in bowel transplantation experimentally."s9 The first intestinal transplant in the CyA era has just (April 1985) been carried out with the implantation of the bowel of a ten-year-old into a 26-year-old woman in Toronto) ~ In previous experiments, we succeeded in cryopreJournal of Pediatric Surgery, Vo120, No 6 (December),1985: pp 747-753
serving about 50% of canine bowel segments frozen to - 8 0 ~ and thawed. 1H3 With isolated cultures of cells, cryopreservation has been reported to alter the immunogenicity of the tissue. TM The aim of this paper is to report on our efforts to test this hypothesis using rat-fetal bowel and to investigate the possible synergistic effect of cryopreservation and immunosuppression. MATERIALS AND METHODS Pregnant CD Fischer or Lewis rats (Charles River, Boston, Mass) were anesthetized with ketamine (60 mg/kg) and nembutal (20 mg/kg) at 19 days of gestation. The fetii were removed and their intestinal tract mobilized, then placed into a cold (10 ~ solution of perfusion media or media plus dimethylsulfoxide (Me2SO). The composition of the perfusion media was as previously described in kidney freezing experiments (Table 1).1~A total of 635 rats were the subjects of these experiments. In the first set of experiments, there were two control and two experimental groups: (1) Control: the intestine (6 cm) was placed into cold media (40 rain). (2) Control: placed into media plus a concentration of 1.5 mol/L Me2SO for 20 minutes and then 3 M Me2SO media for 20 minutes. (3) Experimental: as in (2) then placed into freezing chamber (Controlled Rate Freezer, FTS Systems Inc, Stone Ridge, NY, model CRF-4E) and cooled to - 2 0 ~ at 0.5 ~ The low temperature was maintained for 1 hour. The intestine was then placed into a saline bath at 25 ~ and warmed. When thawed, the Me2SO was washed out with hyperosmolar mannitol solution in a stepwise fashion as previously described)6 (600 mosm - 5 0 0 mosm - 4 0 0 mosm). (4) Experimental: cooled to - 4 0 ~ at 0.5 ~ and treated as above. The thawed segments were then implanted either isogeneically or allogeneically into a prepared subcutaneous paravertebral pocket in young rats (150 g). One half of the allograft experiments were Lewis fetal intestine into Fisher young rats, and half implanted in the other direction, ie, Fisher to Lewis. The segments were marked at each end with silk sutures and the wound closed with Michel clips. All the rats were killed after six weeks. Intestinal segments that have both lumen and mucosa were measured. Analysis was also carried out nonparametrically, with 1 to 2 cm deemed fair growth, and 2 to 5 cm good growth. Histologic studies of the bowel and disaccharidase activity were carried out, in random segments.
From the Departments of Pediatric Surgery and Pathology, The Montreal Children's Hospital, McGill University, and The Montreal Children's Hospital-Research Institute, Montreal. Supported by the Medical Research Council of Canada MA7077 and The Montreal Children's Hospital-Research Institute. Presented before the 16th Annual Meeting of the American Pediatric Surgical Association, Kohala Coast, Hawaii, May 1-4, 1985. Address reprint requests to Frank M. Guttman, MD, The Montreal Children's Hospital, 2300 Tupper, Suite C-1129, Montreal, Quebec, H3H 1P3, Canada. 9 1985 by Grune & Stratton, Inc. 0022-3468/85/2006-0037503.00/0 747
748
GU-I-I'MAN ET AL
(89% to 75%) but not allogeneically. Cryopreservation, alone, did not allow for successful allotransplantation
Table 1. Composition of the Hypothermic Perfusion Medium (per Liter)
Glucose KCI K2HPO4 NaHCO3 Glutathione-reduced MgCI2-6H20 CaCI2 Adenine Hepes Mannitol Chlorpromazine
(0/21, 0/] 0).
180.0 mmol 28.2 mEq 7.2 10.0 5.0 2.0 1.0 1.0 25.0 110.0 0.025 g
In a second series of experiments using allotransplanted bowel, CyA was given to the rats orally in oil base (15 mg/kg/d). All were killed at 6 weeks after receiving CyA for 2 weeks. Group 1) control-intestine was immersed in cold media. Group 2) control was immersed in Me2SO. Group 3 as in 2, segments were cooled to - 4 0 ~ at 0.5 ~ In a third series of experiments, treatment with CyA was continued until death at 6 weeks: (1) control media; (2) control Me2SO; (3) as in (2) with cooling to 20 ~ at 0.5 ~ (4) -20~ at 1 ~ (5) - 4 0 ~ at 0.5 ~ (6) - 4 0 ~ at 1~ Random segments were removed en bloc and fixed in 10% formalin. Paraffin-embedded sections were stained with hematoxylin-phloxine-saffran. Other segments were submitted for disaccharidase activity (Dahlquist Method). 17
RESULTS Successful fetal bowel transplantation is evident from these experiments when the segments are transplanted isogeneically (Table 2A). When the segments are bathed in perfusion media, or media plus the cryoprotectant Me2SO, segments will grow isogenically (53% to 100%). When these segments are transplanted allogeneically with no immunosuppression, they do not survive (0/4, 0/7). Segments cooled to - 2 0 ~ or - 4 0 ~ at a rate of 0.5 ~ and kept at that temperature for one hour, will grow isogenically
In the second set of experiments when CyA was given for 2 weeks (Table 2B), allotransplanted segments did not survive when analyzed at 6 weeks (Controls 75% to 80%). Segments cooled to - 4 0 ~ at 0.5 ~ again showed good growth 15/30 (50%) when transplanted isogenically but no growth when allotransplanted. The third series of experiments again demonstrated (Table 2C) good growth in isogeneic transplants whether the segments were in control media, control Me2SO, or cooled to - 2 0 oC or - 4 0 oC at 0.5 ~ or l ~ (71% to 93%). As expected, no segments grew when allotransplanted without imrnunosuppression. However, a good result was obtained when allotransplanted bowel was implanted into rats receiving CyA for 6 weeks, whether the segments were controls or cooled to - 2 0 ~ or - 4 0 ~ at 0.5 ~ or 1.0 ~ (23% to 48%). When our results are analyzed parametrically (Table 3), good growth is obtained when the segments, transplanted isogeneically, are control, cooled to - 2 0 ~ or - 4 0 ~ at 0.5 ~ or 1.0 ~ When the segments are transplanted allogeneically with CyA, there is a significant difference in growth in all groups when compared to syngeneic controls. A two-way Anova statistical analysis demonstrated no difference in all the syngeneic treatment groups, ie, freezing at - 2 0 ~ or - 4 0 ~ at 0.5 ~ or at 1~ for one hour gives as good results as the controls, both as the measured length and as nonparametric analysis. In the first set of experiments (syngeneic group), serial transverse sections of bowel from three experimental groups, media control, Me2SO, - 2 0 ~
Table 2. Growth (Nonparametric 2 to 5 cm) Treatment
Media Me2SO --20 ~ -40 ~ Media Me2SO --40 ~ Media Me2SO -20 ~ --20 ~ --40 ~ -40 ~
~ ~
0/4 (0)* 0/7 (0)* 0/21 (0)* 0/10(0)*
~
12/15 (80%) 12/16 (75%) 15/30 (50%)
0/6 (0)* 0/11 (0)* 0/13 (0)*
28/30 45/50 12/14 16/19 20/28 19/24
O/11 (O)* 7/30 (23%)? 0/23 (0)* 10/28 (36%) t 0/3 (0)* 14/35 (40%) t 14/29 (48%) t 10/35 (29%) t 12/35 (34%) t
~ ~ ~
Treatment
8/15 (53%) 6/6 (100%) 25/28 (89%) 9/12 (75%)
~
Table 3. Measurement of Growth (Parametric)
AIIo TX Syngen TX (%) AIIo TX (%) With CyA (%) Weeks
(93%) (90%) (86%) (84%) (71%) (79%)
* P < 0.0001, compared to syngeneic control. t P < 0.05, compared to syngeneic control.
0/6 (0) 0/11 (0) 0/16 (0)
2 2 2 6 6 6 6 6 6
Media Me2SO -20 ~ -40 ~ Media Me,SO --40 ~ Media Me2SO -20 ~ --20 ~ -40 ~ -40 ~
Svngen (mm length)
AIIogen Without CyA
~ ~
21.09 24.09 23.09 15.87
_+ 2.24 _+ 1.89 • 2.24 _+ 1.85
0 0 0 0
~
21.09 _+ 2.24 24.09 • 1.89 15.87 • 1.85
0 0 O
~ ~ ~ ~
21.09 24.09 23.33 28.95 15.87 30.50
0 0 0
• 2.24 • 1.89 _+ 2.24 _+ 3.89 • 1.85 +_ 3.90
*P < .0001, compared to syngeneic control.
AIIogenWith CyA (mm length)
0 0 0 5.55 6.61 8.99 15.00 8.05 7.57
+- 2.65* -+ 21.9" -+ 2.19" -+ 3.20* -+ 2.25* _+ 2.05*
FETAL RAT INTESTINAL TRANSPLANTATION
749
Table 4. Specific Activity of Disaccharidase (total activity/rag protein/g tissue) Control n~2 Sucrase Trelalase
Maltase Lactase
109.5 291.0 720.5 34
• • • •
2.5 24.0 7.5 0,0
AIIotx (Growth 3 cm) --20~ n=3 35.7 7.0 0.8 0.6
_+ 10.6" • 1.9 t • 0.81 t • 0.37 t
*P < 0.01, compared to control. t P < 0.005, compared to control.
Fig 1. Photomicrograph of syngeneic intestinal segment originally cooled to --20 ~ Note well-preserved morphology (HPS x 200).
revealed similar findings that include marked distension with intraluminal mucous, mucosal preservation with variable degrees of villus formation, and normal development of muscularis propria and myenteric plexus (Fig 1). Neither a cellular infiltrate nor necrosis was recognized. Sections from the fourth group ( - 40 ~ revealed less dilatation of the bowel, reduced intraluminal mucous, and modest numbers of mononuclear cells in the lamina propria. From the allogeneic group (treated for 6 weeks with CyA), sections from the media control group revealed transmural necrosis in each segment. In one, the necrotic bowel demonstrated a modest lymphocytic infiltrate. Sections from the Me2SO treated group revealed intense transmural mononuclear infiltration associated with necrosis in two rats. In one, multifocal transmural necrosis was noted, although many portions of the bowel appeared normal. In the - 2 0 ~ group, all four rats demonstrated variable degrees of transmural necrosis with mononuclear cell infiltration (Fig 2). One segment, frozen to - 4 0 ~ showed much of the bowel well preserved, although multifocal seg-
Fig 2. Photomicrograph of allogeneic intestinal segment originally cooled to --20 ~ illustrating intense mononuclear cell infiltration of muscular wall (HPS x 200).
ments of transmural necrosis were noted. Disaccharidase activity was found to be markedly diminished in allotransplanted segments (Table 4). DISCUSSION
Experimental transplantation of the bowel in animals is clearly established. The dramatically improved status of transplantation of some organs with CyA has evoked a new interest in small bowel transplantation since immunologic reactions have been thought to be one of the major limiting factors. 2'7-9 Richard Lillehei was the first to establish the limits of viability of autotransplanted small bowel. ~'3 He then showed that with immunosuppression (prednisone and azothiaprine) allotransplants were feasible in the dog. 3 Without immunosuppression, the dogs died at 6 to 9 days without a satisfactory explanation of the cause of death. There was gross enlargement of the mesenteric lymph nodes, but without cellular infiltration of the bowel wall. The suggestion was that severe G V H D was responsible. When immunosuppression was given, some of the dogs died of rejection, with the typical changes of rejection; thickening of the bowel wall, mononuclear cell infiltration, mucosal necrosis, bleeding, sloughing, villous atrophy, and occasional perforation. Cohen et al studied the balance between G V H D and HVGD (rejection). 6 Control animals died at about nine days with enlarged lymph nodes. If the bowel was irradiated with 150 rads prior to the transplant, the mesenteric nodes were not enlarged but the bowel was rejected with sloughing and mononuclear cell infiltration. When the bowel was irradiated with a lower dose, 50 rads, only minimally enlarged nodes were found with longer survival times and normal bowel. This study suggests that G V H D is mediated by the lymphatic load in the allograft and when the load is sufficient it will result in a GVHD. When the lymphatic tissue is completely eliminated, typical rejection will occur. A more immunologically elegant study by Monchik and Russel using microvascular technique demonstrated the feasibility of small bowel transplantation in the rat. 2 Using highly inbred and F1 hybrid rats, they showed that when the transplant was in the direction of
750
Lewis to Hybrid Lewis Brown Norway (and rejection could not take place), a G V H D occurred in 12 to 20 days. This GVHD could be prevented by prior irradiation. When the transplant was done in the opposite direction (Hybrid LBN to Lewis), typical rejection changes were found. Thus, when a two-way reaction is possible rejection did occur. By mechanically reducing the lymphatic load in a bowel transplant, Deltz et al demonstrated the reduction of GVHD mortality, is They did this by transplanting 50% v 100% of small bowel, or by surgically removing the mesenteric lymph nodes prior to transplantation. 19 Again, if competent T lymphocytes are not transferred with the graft, G V H D will not take place. 7 In the rat, intestinal irradiation will prevent GVHD. 2't8"~9 In outbred dogs the explanation for the GVHD is not as readily apparent, nor is it clear that the experimental work on inbred rats has clinical relevance in man. In 1971, Zinzar, Leitina, Tumyan, et al demonstrated the feasibility of transplanting segments of fetal bowel subcutaneously into a syngeneic adult, which grew progressively forming organ-like cavities, z~ The intestinal segment initially devoid of blood supply would become vascularized and remain as differentiated intestine. In 1974, Leapman, Deutsch, Grand, et al z! confirmed that fetal rat intestine transplanted into a syngeneic host would establish blood supply, grow progressively, and exhibit normal histology. Absorption studies showed that glucose, glycine, and oleic acid were absorbed. Peristalsis was present and the brush border enzymes (disaccharidase activity) was normal. This same group led by Folkman showed that normal intestine was present in the recipients for as long as 1 year after transplantation, zz Using rabbit antirat lymphocyte serum (RALS) they also could delay rejection for up to 26 days or more. The suggestion was then made that in short bowel syndrome, this subcutaneous intestinal loop could serve as an accessory gut enabling discontinuing intravenous alimentation. In time, the patients own short gut might adapt to progressive enteral feedings of elemental food. The subcutaneous loop could then be removed. Alternatively, the same group have demonstrated that the loop might be placed into the intestinal circuit, if the loop was introduced in stages near the proximal end of the recipient's small bowel. The advent of CyA has clearly changed the transplantation world. It would seem to be an ideal immunosuppressive agent since it is effective in GVHD as well as in rejection. Schwartz, Flye, and Storozuk have reported on experiments whereby CyA prevented rejection of fetal small bowel transplanted into the back of allogeneic rats, across a major histocompatibil-
GU-I-FMAN ET AL
ity barrier (Fisher-Wistar). 23 The function of the transplanted fetal bowel was good, as measured by substrate absorption, D N A content, and disaccharidase activity. Bass, Tai, Schweltzer, et al noted that the neogut in this model has structural and electrophysiologic characteristics similar to those of normal rat intestine, z4 Montgomery, Sybicki, and Grand z5 have also demonstrated autonomous biochemical and morphologic differentiation in the fetal rat intestine transplanted at 17 and 20 days of gestation. Reznick, Craddock, Langer, et al first reported on the use of CyA to allotransplant successfully small bowel in the dog. 8 This Toronto group led by Langer and Cohen has fully reported on several aspects of the morphology and function of canine small bowel transplants and on the absorption of CyA from the gut. 6'8 During this same period, Starzl's group has also explored the limits of intestinal transplantation showing successful transplants with CyA and prednisone. 9 Two of their animals were living at 550 and 555 days. Maintenance of nutrition and absorption of D-Xylose and fat were better than in control animals with a short gut, but worse than that of normal dogs. Using minipigs, Stauffer, Becker, Karamehmedovic, et al have demonstrated the usefulness of biopsy of ostomy sites to predict rejection and allow for the removal of irreversibly rejecting grafts, saving the life of the recipient, z6 In these experiments, we have shown that even across a minor histocompatibility locus, fetal bowel will be rejected, or at least fail to grow. Evidently, not enough lymphatic tissue is transplanted to make any change on the young adult rat recipient's growth or skin. Cryopreservation is feasible with a high percent of segments transplanted isogeneically growing. Cryopreservation in the manner described has no immunosuppressive effect. The 6-week period for this experiment is probably the cause of the distension of the implants. With no egress, after 6 weeks atrophy has been found in some segments. Some of our control segments showed ischemic necrosis on histologic examination. It is entirely possible that had we chosen a 4-week experimental period, these changes would not have been found. The increase in mucous secretion has been noted on previous electron microscopic studies we have carried out in dog bowel freezing experiments, z7 In our experiment, when CyA was used for 2 weeks, the allogeneically-transplanted fetal bowel did not survive. Possibly it may have grown and then at death, the rejection process resulted in no visible trace of the transplant. Some experiments with rat intestine allografting have demonstrated prolonged survival with only seven days of CyA treatment) However, with
FETAL RAT INTESTINAL TRANSPLANTATION
751
immunosuppression of C y A for 6 weeks, a large n u m ber of segments grew when allotransplanted whether they were controls or cooled to - 2 0 ~ or - 4 0 ~ at either 0.5 ~ or 1.0 ~ The hypothesis that cryopreservation will induce some immunosuppressive effect is not found to be valid in this study. However, when t r a n s p l a n t e d syngeneically, cryopreservation has been shown to be effective in producing viable bowel that grows when frozen down to - 2 0 ~ or - 4 0 ~ Cryopreservation a n d immunosuppression together have not been found to have a synergistic effect. The low levels of all disaccharidases, but especially maltase and lactase have been noted by all authors in this model. 21'23'z5One likely explanation is the lack of substrate, well known to be directly related to the levels of enzyme found. In addition, we have expressed our levels in terms of a c t i v i t y / m g p r o t e i n / g of tissue, which in this type of traumatised bowel m a y be increased (collagen a n d fibrosis).
This study leads to an extension of F o l k m a n ' s original suggestion in 1974, that fetal intestine could be i m p l a n t e d as an accessory gut p e r m i t t i n g t e m p o r a r y or p e r m a n e n t feeding and nutrition. T h e extension of that suggestion would be to have fetal intestinal segments b a n k e d at low temperatures. L o n g - t e r m cryopreservation has been r e m a r k a b l y successful with RBC, spermatozae, ova, embryos, a n d clumps of tissue (parathyroid, thyroid, islets). T h e sporadic success reported in whole organ freezing has been inconsistant probably because of a multiplicity of factors; geometry, multiplicity of cell types, each with an o p t i m u m rate of freeze a n d thaw, o p t i m u m c o n c e n t r a t i o n of cryoprotectant, and washout technique. T h e fetal bowel because of its size would thus seem to be a feasible organ to store and m a k e fetal small bowel b a n k i n g a reality. In the C y A era, the first intestinal t r a n s p l a n t in m a n has been carried out. More clinical application will doubtless be done in the near future.
REFERENCES
1. Lillehei RC, Goot B, Miller FA: The physiologicalresponse of the small bowel of the dog to ischemia including prolonged in vivo preservation of the bowel with successfulreplacement and survival. Ann Surg 15:543-559, 1959 2. MonchikGJ, Russel PS: Transplantation of small bowel in the rat: Technical and immunologicalconsiderations. Surgery 70:693702, 1971 3. Ruiz JO, Uchida H, Schultz LS, et al: Problems in absorption and immunosuppressionafter entire intestinal allotransplantation. Am J Surg 123:297-303, 1972 4. Hardy MA, Quint J, State D: Effect of antilymphocyte serum and other immunosuppressiveagents on canine jejunal allografts. Ann Surg I71:51-60, 1970 5. Kao GF, Lerner KG: The histopathologyof human intestinal graft-versus-hostdisease (I-GVHD). Gastroenterology66:888, 1974 (abstr) 6. Cohen Z, MacGregor AB, Moore KTH, et al: Canine small bowel transplantation. A study of the immunological responses. Arch Surg 111:248-253, 1976 7. Kirkman RL: Small bowel transplantation. Transplantation 37:429-433, 1984 8. Reznick RK, Craddock GN, Langer B, et al: Structure and function of small bowel allografts in the dog: Immunosuppression with CyclosporinA. Can J Surg 25:51-55, 1982 9. Diliz-Perez HS, McClure J, Bedetti C, et al: Successful small bowel allotransplantation in dogs with cyclosporineand prednisone. Transplantation 37:126-129, 1984 10. Cohen Z, Langer B: personalcommunication, April 1985 11. Guttman FM, Khalessi A, Huxley BW, et al: Whole organ preservation. I. A technique for in vivo freezing canine intestine using intraarterial helium and ambient nitrogen. Cryobiology6:3236, 1969 12. Guttman FM, Khalessi A, BerdnikoffG: Whole organ preservation. II. A study of the protective effect of glycerol, dimethyl sulfoxide and both combined, while freezing canine intestine employingan in vivotechnique. Cryobiology6:339-344, 1970
13. Guttman FM, BerdnikoffG, Sangbhundhu K, et al: Preservation of canine small bowel by freezing. Arch Surg 111:260-262, 1976 14. Wodinsky I, Meaney KF, Kensler CJ: The stain specificityof transplantable neoplasm after freezing and storage in liquid nitrogen. Cryobiology2:44-246, 1965 15. Segal NB, Guttman FM: Kinetics of permeation and intracellular events associated with Me2So permeation of rabbit kidneys during perfusion at 10 ~ Cryobiology19:41-49, 1982 16. Segal NB, Guttman FM: Function of rabbit kidneys in vitro at normothermia following equilibriation with 3.0M Me2SO and removal by hypertonic washout at 10 ~ Cryobiology 19:50-60, 1982 17. Dahlquist A: Method for assay of intestinal disaccharidases. Ann Biochem 70:18-25, 1964 18. Deltz E, Muller-Hermelink HK, Ulrichs K, et al: Development of graft-versus-host reaction in various target organs after small intestinal transplantation. Transplant Proc 13:1215-1216, 1981 19. Thiede A, Deltz E, Schack T, et al: Successful manipulation of GVH-reaction in semi allogeneic intestinal transplantation in rat by treatment of the donor recipient. Seventh International Congress in Microsurgery, Lyon, 1982 (abstr) 20. Zinzar SN, Leitina BI, Tumyan BC, et al: Large organ-like structures formed by syngeneic foetal alimentary tract transplanted as a whole or in parts. Rev Eur Etudes Clin Biol 16:455, 1971 21. Leapman SB, Deutsch AA, Grant R J, et al: Transplantation of fetal intestine: Survival and function in a subcutaneous location in adult animals. Ann Surg 1:109-114, 1974 22. Deutsch AA, Arensman R, Levey R, et al: The effect of antilymphocyte serum of fetal rat intestine transplanted as free subcutaneous homografts. J Pediatr Surg 9:29-34, 1974 23. Schwartz MZ, Flye MW, Storozuk RB: Effect of Cyclosporin A on growth and function of transplanted fetal rat intestine. Surg Forum 33:341-343, 1982
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24. Bass BL, Tai Y-H, Schweltzer E J, et al: Neogut: Anatomic and physiologic properties of the transplanted fetal intestine. Surg Forum 34:181-183, 1983 25. Montgomery RK, Sybieki MA, Grand R J: Autonomous biochemical and morphological differentiation in fetal rat intestine
transplanted at 17 and 20 days of gestation. Dev Biol 87:76-84, 1981 26. Stauffer UG, Beeker M, Karamehmedovie H, et al: Transplantations of small intestines. J Pediatr Surg 9:21-28, 1974 27. Makita T, Khalessi A, Guttman FM, et al: The ultrastructure of small bowel epithelium during freezing. Cryobiology 8:25-45, 1971
Discussion Dennis Lund (Boston): For the past two years, I've had the privilege of working in the surgical research laboratory of the Boston Children's Hospital, where some of the early work on fetal gut transplantation was done by Leapman, Deutsch, and Folkman. We continue to be very interested in this problem. In work from our laboratory, recently submitted to the Surgical Forum, by Joseph Friedberg, a Harvard medical student, and Dr Daniel Ryan, human fetal intestine has been successfully transplanted into athymic animals. Very briefly, fresh human fetal intestine was obtained from 19 to 23-week-old abortuses from the Pathology Department of the Brigham and Women's Hospital, with the full informed consent of the women aborted. This fetal intestine was immediately transported to our laboratory, where it was implanted either subcutaneously, as described by Dr Guttman, or intraperitoneally into homozygous nude rats or mice. Of the 41 transplants examined to date, 34 or 83%, have shown vascularization by 1 week and subsequent growth. Preliminary results showed these intestinal transplants to have the capacity to absorb both intraluminal glucose and glycine. Some of the transplants showed visible peristalsis, and 15 of 16 examined histologically showed viable ganglion cells. Dr Guttman's success with cryopreservation and Cyclosporin A in fetal intestinal transplants is very exciting. Dr Schwartz and his colleagues have also had excellent success with Cyclosporin A in the same model, as published in the most recent issue of the journal, Surgery. Now, with the success of Friedberg and Ryan with human fetal intestinal transplants in nude animals, plus the proven effectiveness of Cyclosporin A in immunosuppression, and cryopreservation, we may very rapidly be approaching the point where the application of this technology will begin to solve the problem of the short gut syndrome. Marshall Schwartz (Sacramento): The specific questions asked by Dr Guttman were: Can cryopreservation of fetal intestine result in a functioning graft and will cryopreservation have an immunosuppressive effect, either by itself or in conjunction with Cyclosporin? The authors did demonstrate that cryopreservation was an effective means of preserving rat fetal small intestine. However, the grafts were somewhat
shorter than had been shown initially by Folkman and in our previous study. This study did not demonstrate any immunosuppressive effect of cryopreservation, either alone or in conjunction with Cyelosporin. Three years ago I reported at the Surgical Forum, a study which asked the questions; would Cyclosporin be an effective immunosuppressive agent and/or would it have a toxic effect on fetal intestine when transplanted across major histocompatibility loci. We showed that Cyclosporin, at least in this model, was an effective immunosuppressive agent and did not have a toxic effect on the development of the fetal intestine. Now, what does all this mean? There has been recent interest with whole-organ small intestine transplantation because of the availability of Cyclosporin. However, experimental studies have shown that Cyclosporin is not the panacea that it has been for liver and heart transplantation. Therefore, using a fetal model may have more significance. With the availability of Cyclosporin and, now, cryopreservation as an effective means of preserving fetal intestine, this model may have some merit as a clinical tool. I have attempted to incorporate the transplanted intestine into the host's own intestine, with very limited success. I have two questions for Dr Guttman. One, he did not report on whether long-term cryopreservation might effect the function of the intestine. Also, I'm concerned about the disaccharidase data for two reasons: it is based on small numbers and there was a dramatic difference between the controls and experimental group. I am wondering if this is due to chronic rejection. Frank Guttman (closing): The low levels of disaccharidase activity does also concern me. Perhaps, it's that we're just learning to do this, but I think there is a significant difference that we have to explain, and I think chronic rejection may be part of the explanation. Long-term preservation--we're working on that now. We have 24-hour and 1-week conservation that seems to be successful in our most recent experiments. Dr Schwartz mentioned the Cyclosporin toxicity. That's well known, and we have done control studies showing that these young adult rats grow and all their organs are unaffected by this dose of Cyclosporin that we're
FETAL RAT INTESTINAL TRANSPLANTATION
giving, which is not high, it's on the low side (15 rag). On the question of reintegrating the gut into the circuit, Dr Folkman, ie, Deutsch, Leapman, and Folkman, did that originally in rats, and tried various ways of reconnecting the bowel into the circuit in a staged manner. They were able, by putting the gut in proximally and staging it, to have some survivals in this model. But, of course, that may not be really necessary. Dr Folkman originally suggested 12 to 14 years ago,
753
that one possibility was not reintegrating the gut into the circuit but using it as an accessory gut just subcutaneously, as an infusion port. We have been working, also, with Drs Goldman and Colle on pancreatic islet--human islets--for preservation and for culture. They have succeeded in culturing islets for over five months; and this is, indeed, an area of further research.