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Avascular transplantation of small bowel in newborn piglets
Avascular Transplantation of Small Bowel in Newborn Piglets By lngrid Klos responsible for this. Homografts behave like autografts in the beginning, their structures being restored, but are all rejetted by the sixth week. Immaturity of the immunologic system in the newborn may be the reason for this late rejection.
l Segments of small bowel of newborn piglets were isolated and wrapped in omentum as free autotransplants. After initial necrosis, they regenerate either completely, or at least their muscular layer is restored. As there is a lot of inflammatory reaction in most of the gmfts, the conclusion is drawn that either preexisting infection, or autoaggressive reactions are
INDEX WORDS: intestine
transplantation.
I
NTESTINAL TRANSPLANTATION is still an unsolved problem. While in other organs, like the kidney or skin the immunologic problems can usually be kept under adequate control, this is not the case in the gut. It appears that by some little understood reaction the lymphatic tissue of the transplanted intestine itself starts to destroy the graft. This is suggested by the findings of Behelak and Richter,’ who believe that these intestinal “killer cells” have already been sensitized against the microorganisms of the gastrointestinal tract. Since in pediatric surgery the short bowel syndrome usually occurs in newborn infants, we have tried to investigate the possibility of transplanting small bowel of newborns without vascular anastomoses, assuming that the immunological system of newborns might be less competent than that of older individuals, MATERIALS
AND METHODS
Normal farmbred piglets, between a few hr and two days of age, were anesthetised and a laparotomy was performed. In a first group of animals, a small bowel segment of 5 cm length was isolated and the vessels ligated. It was rinsed with an antibiotic solution and tucked into the omentum. An end-to-end anastomosis of the remaining gut was performed. In some of the animals, the ends of the intestinal segment were brought out of the abdominal wall, and a thin plastic catheter with multiple perforations was introduced into the transplanted loop. During the first postoperative days the bowel was rinsed with antibiotics through this tube until it was lost. The stomata closed spontaneously. When we found out that there was no difference in results we made no stomas in the rest, but just tried to cover the graft completely with omentum, the open ends included. In a second group of animals, we transplanted small bowel segments in the same way to other newborn piglets of the same breed.
RESULTS
Group I: Autotransplants In some of the animals we removed the transplants after 3 and 4 days respectively (Fig. 1). In these grafts the intestinal wall shows an ischemic necrosis From the Kinderchirurgische Presented Surgeons,
Shejield.
Address for Karlsruhe.
Klinik
before the XXIII
Annual
England,
reprints:
Dr.
Krankenanstalten
Congress
Ju1.v 7-9, I976 and Warsaw, Ingrid
Klos.
Kinderchirurgische
Karlsruhe.
oj the British
Poland,
German!.
Association
oj Paediatric
July I I -I 3, 1976.
Klinik.
Karl- Wilhelmstrasse
I .D-7500
Germany.
E 1977 b_v Grune & Stratton,
Journal
der Staedtischen. International
of Pediatric
Surgery,
Inc.
Vol. 12, No. 1 (February), 1977
43
INGRID
KLOS
Fig. 1.
of all layers, with desquamation of the mucosa, and loss of nuclei of the epithelial cells. There is cellular infiltration in the muscular layers and subserosa, with proliferation of mesenchymal cells and capillaries in these layers. Capillary endothelium can clearly be seen. These findings are the same on the third and fourth day. Results after 3, 4, 5, and 8 wk: All transplants had shrunk considerably, some to half the original size; some were tiny yellowish stripes in a somewhat thickened but otherwise unchanged omentum. This explains why in two cases we found only fatty tissue, histologically. All layers were present in 7 cases, muscular layer and some connective tissue alone in 9 cases, which means that of the 19 grafts in this series 16 survived either partially or totally. In 3 cases only there were no signs of inflammation, while there was granulomatous inflammation in 10, lymphatic infiltration in 2, and foam cells as signs of resorption in 2. The intestinal lumen was obstructed by connective tissue in 2 of the grafts, in which no mucosa had been preserved. Group 2: Homotransplants The transplants were removed after 3,4, 5, and 9 wk. Mucosa was preserved to some extent in only 4 grafts, but only up to 4 wk. Muscular layers were present in 9. In 4 cases we found only fatty tissue with signs of inflammation, and a foam cell granuloma in 2 (after 4 and 9 wk) as signs of total destruction and absorption of the grafts. There was bleeding in all layers of the grafts twice after 5 wk. Patches of foam cells in all parts of the grafts were found 4 times after 4 and 5 wk. Figure 2 shows autotransplants after 7 and 5 wk: a general view on the left,
AVASCULAR
TRANSPLANTATION
Fig. 2.
with normal muscular layers; an almost normal mucosa; and in the periphery the omentum, acting as a well vascularized new mesentery. A closer view of the mucosa in the middle shows the normal structure with goblet cells, just like the one on the right, in which the villi are much shorter. Figure 3 gives some examples of homotransplants, all after 4 wk; normal muscular layer on the left (A),
INGRID
46
KLOS
Fig. 3.
but cellular infiltration and beginning rejection of the mucosa; lymphatic infiltration also of the muscular layer in the middle (B), and a foam cell granuloma after complete resorption of the transplant on the right (C). CONCLUSIONS
Avascular free transplantation of small bowel is possible, but as our results in the group of autografts show, obviously depends largely on the sterility of the
AVASCULAR
TRANSPLANTATION
47
tissue to be transplanted. As we already know from the avascular transplantation of skeletal muscle,4 the graft becomes completely necrotic in the first few days after transplantation. The mucosa is rejected, its epithelial cells and the cells of the muscular layer losing their nuclei. But there is very high activity of mesenchymal cells and fibroblasts, and capillaries growing in from the surrounding tissues can be distinguished on the third day. After that, all layers are regenerated, and after 3 wk, a normal appearance of even the mucosa is restored. Sometimes the villi are short and sometimes there is only one row of cuboid epithelial cells without villi. Yoshida’s findings on intestinal transplantation’ suggest that these are just different stages of regeneration, and that a normalization will take place over a period of 4 mo. In all but three of the transplants we found different degrees of inflammation in all layers, but sometimes only in the surrounding tissues. We think that lack of sterility of the transplanted gut may be the source of these inflammatory reactions, the inflammation again being responsible for a secondary rejection of the graft. Obviously, rinsing of the bowel with antibiotics is not sufficient to stop inflammation. A means of sterilizing the gut will have to be found. On the other hand, the findings of Behelak and Richter’ show that in the unsensitized rabbit SAPP cells (cells of sacculus rotundus, appendix, and Payer’s patches) are able to kill practically loo”,; of autologous lymphocytes. These “killer cells” were probably presensitised by microorganisms of the gastrointestinal tract, or, as we would prefer to put it, against all kinds of heterogenous proteins passing the bowel. This seems to be another possible answer to signs of rejection in our autotransplants. The killer qualities of the SAPP cells could be set free by the abnormal conditions under which the graft lives. In one of the transplants we found a well preserved myenteric plexus, which means that the nerve function will be restored. Having found such a considerable degree of inflammation in the autotransplants, we think it difficult to distinguish between simple inflammation and graft rejection in the homotransplants. Surprisingly, even in the homotransplant the normal structure of the gut is restored before rejection takes place. No mucosa could be found later than 4 wk after transplantation, and in those grafts in which parts of the muscular layer were preserved there were strong infiltrations of foam cells, lymphatic cells, or granulation tissue. There is no doubt that without immunosuppression all homotransplants are rejected after 6 wk. As Deutsch et al.,j using fetal intestine, found a complete rejection by the eighth day in untreated animals, the longer survival of grafts in our series may be explained by immaturity of the immunological system of newborns. ACKNOWLEDGMENTS We would like to thank Prof. K. Vossius for his kind permission to use the theater of the Institut fuer Biokybernetik at the Technical University of Karlsruhe for our experiments. We also thank Mr. P. Bauer for his valuable assistance, and Dr. Ii. Wolfmiiller for the histological consultation.
REFERENCES 1. Behelak Y, Richter M: Cells involved in cellLmediated and transplantation immunity in the rabbit. VIII. The killer cell activity of the
lymphocytes in the gastrointestine-associated lymphoid organs of the normal unsensitized adult rabbit. Transplantation l&229, 1974
INGRID
48
2. Yoshida testinal
H: Experimental
transplantation: of the free autotransplant.
studies
on in-
Physiological changes Mich Med J 1X:319.
1969 3. Deutsch AA, Arensman The effect of antilymphocyte
R, Levy R, et al.: serum on fetal rat
KLOS
intestine transplanted as free subcutaneous homografts. J Pediat Surg 9:29, 1974 4. Thompson N: Autogenous free grafts of skeletal muscle: A preliminary experimental and
clmical
I I. 1971
study.
Plast
Reconstr
Surg
4X:
l Segments of small bowel of newborn piglets were isolated and wrapped in omentum as free autotransplants. After initial necrosis, they regenerate either completely, or at least their muscular layer is restored. As there is a lot of inflammatory reaction in most of the gmfts, the conclusion is drawn that either preexisting infection, or autoaggressive reactions are
INDEX WORDS: intestine
transplantation.
I
NTESTINAL TRANSPLANTATION is still an unsolved problem. While in other organs, like the kidney or skin the immunologic problems can usually be kept under adequate control, this is not the case in the gut. It appears that by some little understood reaction the lymphatic tissue of the transplanted intestine itself starts to destroy the graft. This is suggested by the findings of Behelak and Richter,’ who believe that these intestinal “killer cells” have already been sensitized against the microorganisms of the gastrointestinal tract. Since in pediatric surgery the short bowel syndrome usually occurs in newborn infants, we have tried to investigate the possibility of transplanting small bowel of newborns without vascular anastomoses, assuming that the immunological system of newborns might be less competent than that of older individuals, MATERIALS
AND METHODS
Normal farmbred piglets, between a few hr and two days of age, were anesthetised and a laparotomy was performed. In a first group of animals, a small bowel segment of 5 cm length was isolated and the vessels ligated. It was rinsed with an antibiotic solution and tucked into the omentum. An end-to-end anastomosis of the remaining gut was performed. In some of the animals, the ends of the intestinal segment were brought out of the abdominal wall, and a thin plastic catheter with multiple perforations was introduced into the transplanted loop. During the first postoperative days the bowel was rinsed with antibiotics through this tube until it was lost. The stomata closed spontaneously. When we found out that there was no difference in results we made no stomas in the rest, but just tried to cover the graft completely with omentum, the open ends included. In a second group of animals, we transplanted small bowel segments in the same way to other newborn piglets of the same breed.
RESULTS
Group I: Autotransplants In some of the animals we removed the transplants after 3 and 4 days respectively (Fig. 1). In these grafts the intestinal wall shows an ischemic necrosis From the Kinderchirurgische Presented Surgeons,
Shejield.
Address for Karlsruhe.
Klinik
before the XXIII
Annual
England,
reprints:
Dr.
Krankenanstalten
Congress
Ju1.v 7-9, I976 and Warsaw, Ingrid
Klos.
Kinderchirurgische
Karlsruhe.
oj the British
Poland,
German!.
Association
oj Paediatric
July I I -I 3, 1976.
Klinik.
Karl- Wilhelmstrasse
I .D-7500
Germany.
E 1977 b_v Grune & Stratton,
Journal
der Staedtischen. International
of Pediatric
Surgery,
Inc.
Vol. 12, No. 1 (February), 1977
43
INGRID
KLOS
Fig. 1.
of all layers, with desquamation of the mucosa, and loss of nuclei of the epithelial cells. There is cellular infiltration in the muscular layers and subserosa, with proliferation of mesenchymal cells and capillaries in these layers. Capillary endothelium can clearly be seen. These findings are the same on the third and fourth day. Results after 3, 4, 5, and 8 wk: All transplants had shrunk considerably, some to half the original size; some were tiny yellowish stripes in a somewhat thickened but otherwise unchanged omentum. This explains why in two cases we found only fatty tissue, histologically. All layers were present in 7 cases, muscular layer and some connective tissue alone in 9 cases, which means that of the 19 grafts in this series 16 survived either partially or totally. In 3 cases only there were no signs of inflammation, while there was granulomatous inflammation in 10, lymphatic infiltration in 2, and foam cells as signs of resorption in 2. The intestinal lumen was obstructed by connective tissue in 2 of the grafts, in which no mucosa had been preserved. Group 2: Homotransplants The transplants were removed after 3,4, 5, and 9 wk. Mucosa was preserved to some extent in only 4 grafts, but only up to 4 wk. Muscular layers were present in 9. In 4 cases we found only fatty tissue with signs of inflammation, and a foam cell granuloma in 2 (after 4 and 9 wk) as signs of total destruction and absorption of the grafts. There was bleeding in all layers of the grafts twice after 5 wk. Patches of foam cells in all parts of the grafts were found 4 times after 4 and 5 wk. Figure 2 shows autotransplants after 7 and 5 wk: a general view on the left,
AVASCULAR
TRANSPLANTATION
Fig. 2.
with normal muscular layers; an almost normal mucosa; and in the periphery the omentum, acting as a well vascularized new mesentery. A closer view of the mucosa in the middle shows the normal structure with goblet cells, just like the one on the right, in which the villi are much shorter. Figure 3 gives some examples of homotransplants, all after 4 wk; normal muscular layer on the left (A),
INGRID
46
KLOS
Fig. 3.
but cellular infiltration and beginning rejection of the mucosa; lymphatic infiltration also of the muscular layer in the middle (B), and a foam cell granuloma after complete resorption of the transplant on the right (C). CONCLUSIONS
Avascular free transplantation of small bowel is possible, but as our results in the group of autografts show, obviously depends largely on the sterility of the
AVASCULAR
TRANSPLANTATION
47
tissue to be transplanted. As we already know from the avascular transplantation of skeletal muscle,4 the graft becomes completely necrotic in the first few days after transplantation. The mucosa is rejected, its epithelial cells and the cells of the muscular layer losing their nuclei. But there is very high activity of mesenchymal cells and fibroblasts, and capillaries growing in from the surrounding tissues can be distinguished on the third day. After that, all layers are regenerated, and after 3 wk, a normal appearance of even the mucosa is restored. Sometimes the villi are short and sometimes there is only one row of cuboid epithelial cells without villi. Yoshida’s findings on intestinal transplantation’ suggest that these are just different stages of regeneration, and that a normalization will take place over a period of 4 mo. In all but three of the transplants we found different degrees of inflammation in all layers, but sometimes only in the surrounding tissues. We think that lack of sterility of the transplanted gut may be the source of these inflammatory reactions, the inflammation again being responsible for a secondary rejection of the graft. Obviously, rinsing of the bowel with antibiotics is not sufficient to stop inflammation. A means of sterilizing the gut will have to be found. On the other hand, the findings of Behelak and Richter’ show that in the unsensitized rabbit SAPP cells (cells of sacculus rotundus, appendix, and Payer’s patches) are able to kill practically loo”,; of autologous lymphocytes. These “killer cells” were probably presensitised by microorganisms of the gastrointestinal tract, or, as we would prefer to put it, against all kinds of heterogenous proteins passing the bowel. This seems to be another possible answer to signs of rejection in our autotransplants. The killer qualities of the SAPP cells could be set free by the abnormal conditions under which the graft lives. In one of the transplants we found a well preserved myenteric plexus, which means that the nerve function will be restored. Having found such a considerable degree of inflammation in the autotransplants, we think it difficult to distinguish between simple inflammation and graft rejection in the homotransplants. Surprisingly, even in the homotransplant the normal structure of the gut is restored before rejection takes place. No mucosa could be found later than 4 wk after transplantation, and in those grafts in which parts of the muscular layer were preserved there were strong infiltrations of foam cells, lymphatic cells, or granulation tissue. There is no doubt that without immunosuppression all homotransplants are rejected after 6 wk. As Deutsch et al.,j using fetal intestine, found a complete rejection by the eighth day in untreated animals, the longer survival of grafts in our series may be explained by immaturity of the immunological system of newborns. ACKNOWLEDGMENTS We would like to thank Prof. K. Vossius for his kind permission to use the theater of the Institut fuer Biokybernetik at the Technical University of Karlsruhe for our experiments. We also thank Mr. P. Bauer for his valuable assistance, and Dr. Ii. Wolfmiiller for the histological consultation.
REFERENCES 1. Behelak Y, Richter M: Cells involved in cellLmediated and transplantation immunity in the rabbit. VIII. The killer cell activity of the
lymphocytes in the gastrointestine-associated lymphoid organs of the normal unsensitized adult rabbit. Transplantation l&229, 1974
INGRID
48
2. Yoshida testinal
H: Experimental
transplantation: of the free autotransplant.
studies
on in-
Physiological changes Mich Med J 1X:319.
1969 3. Deutsch AA, Arensman The effect of antilymphocyte
R, Levy R, et al.: serum on fetal rat
KLOS
intestine transplanted as free subcutaneous homografts. J Pediat Surg 9:29, 1974 4. Thompson N: Autogenous free grafts of skeletal muscle: A preliminary experimental and
clmical
I I. 1971
study.
Plast
Reconstr
Surg
4X: